MXPA00009663A - Nucleic acid encoding hyaluronan synthase and methods of use - Google Patents

Abstract

The present invention relates to a nucleic acid segment having a coding region segment encoding enzymatically active bacterial multocida hyaluronate synthase (PmHAS), and to the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its hyaluronic acid product. Hyaluronate is also known as hyaluronic acid or hyaluronan. The present invention also relates to the use of the PmHAS in constructing"knock-out"mutant strains of P. multocida for use in vaccinations. The present invention further relates to the use of the PmHAS in diagnostic tests in the field determinations of livestock P. multocida infection.

Description

NUCLEIC ACID THAT CODIFIES FOR HIALURONAN SYNTHASE AND METHODS OF USE BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a DNA sequence encoding hyaluronan synthase from Pasturella multocida. More particularly, the present invention relates to a DNA sequence encoding hyaluronan synthase from Pasturella multocida which is capable of being placed in a recombinant construct so that it is capable of expressing hyaluronan synthase in a foreign host. The present invention also relates to methods for using a DNA sequence encoding hyaluronan synthase from Pasturella multocida to: (1) produce hyaluronan polymers of varying size distribution; (2) producing hyaluronan polymers that incorporate sugars of substitute or additional bases; (3) develop novel and novel Aryan vaccines; and (4) develop new and novel diagnostic tests for the detection and identification of animal pathogens.

Ref: 123685 2. Brief Description of the Background of the Technique The polysaccharide hyaluronic acid ("HA") or hyaluronan is an essential component of higher animals that serve in structural and recognition roles. In mammals and birds, HA is present in large quantities in the skin, the synovial fluid of the joints and the vitreous humor of the eye. Certain pathogenic bacteria, specifically Streptococcus of gram-positive groups A and C and Gram-negative pasturella Type A Carter, produce extracellular capsules containing HA with the same chemical structure as the HA molecule found in their vertebrate hosts. This "molecular imitation" causes failed attempts to mount a strong antibody response to the capsular polysaccharide. In contrast, capsular polysaccharides with different structures produced by other bacteria are often highly antigenic. The HA capsule also apparently helps pathogens evade host defenses including phagocytosis. Historically, researchers in the field have not succeeded in cloning or identifying hyaluronan synthase ("HAS") from Pasturella. Bacterial enzymes of HAS of Streptococcus groups A and C have been identified and cloned. HasA of Streptococcus pyogenes was the first HAS definitively identified. This integral membrane protein utilizes intracellular UDP-GlcA and UDP-GlcNAc as substrates. The nascent HA chain is extruded through the membrane to form the extracellular capsule. It has also been determined that a Xenopus protein, DG42, is a HAS. Several human and murine DG42 homologs, designated HAS1, HAS2 and HAS3, have also been identified. There is considerable similarity between these mammalian enzymes molecularly cloned at the amino acid level, but they reside on different chromosones. The unique HAS of P. multocida has a primary structure that does not strongly resemble previously cloned enzymes of Streptococcus, PBVC-1 virus or higher animals. A viral HAS, with an ORF (open reading frame) called A98R, has been identified as 28-30% identical to streptococcal and vertebrate enzymes. PCV-1 (Chorella virus from Paramecium bursaria) produces an authentic HA polysaccharide shortly after infection of its host green algae similar to clórela. A98R is the first virally encoded enzyme identified as producing a carbohydrate polymer. P. mul-type A Carter, the causative agent of cholera in poultry, is responsible for large economic losses in the poultry industry in the United States. Acamphoid mutants of P. multocida do not thrive in the bloodstream of turkeys after intravenous injection, where the encapsulated parental strains multiply rapidly and cause death within 1 to 2 days. A mutant strain that arises spontaneously which is acapsular, which is also 105 times less virulent than the wild type, but the nature of the genetic defects in all cases before the described mutant (as described below) are not known. Pasturella's bacterial pathogens cause widespread losses in US agriculture. It has been proposed that the extracellular polysaccharide capsule of P. mul tocida is a major virulence factor. The type A capsule is composed of a polysaccharide, specifically HA, which is identical to the normal polysaccharide in the host body and is therefore invisible to the immune system. This "molecular imitation" also blocks host defenses such as phagocytosis and complement-mediated lysis. In addition, HA is not a strong immunogen since the polymer is a normal component of the host body. Capsules of other bacteria that are made up of different polysaccharides, however, are usually major targets of the immune response. Antibodies raised against capsular polymers are often responsible for clearance of microorganisms and long-term immunity. Knowledge of the factors responsible for the virulence of a pathogen provides clues to the way in which the disease is overcome intelligently and effectively. In P. multocida type A, one of the virulence factors is the protective cover of non-immunogenic HA, an almost insurmountable barrier for host defenses. Some strains do not seem to be based on the HA capsule for protection, but use other unknown factors to resist host mechanisms. Alternatively, these strains may possess much smaller capsules that are not detected by classical tests. For chickens, and especially turkeys, the cholera of poultry can be devastating. Up to an amount of 1,000 cells of some encapsulated strains can kill a turkey in 24-48 hours. Poultry cholera is an economically important disease in the United States. Studies conducted in the late 1980s show some of the effects of poultry cholera in the turkey industry: (i) poultry cholera causes 14.7 to 18% of all diseases, (ii) ) in a state only, the annual losses is $ 600,000, (iii) it costs $ 0.40 / bird to treat a chick with antibiotics, and (iv) it costs $ 0.12 / bird in treatment to avoid infection. Certain strains of P. multocida type A cause pneumonic lesions and displacement fever in cattle submerged under tension. The subsequent reduction in weight gain and loss of feeding causes greater losses. Strain coils are different from poultry cholera strains, but the molecular basis for these differences in host range preference is not yet clear. Type A also causes half of the pneumonia in swine. P. mulócida type D is well known for its relationship in atopic rhinitis, a disease of high priority in porcine. The capsular polymer of type D has an unknown structure that appears to be of some type of glycosaminoglycan; This is the same polymer family that includes HA. This disease is also precipitated by Bordetella bronchiseptica, but the condition is worse when both species of bacteria are present. It is estimated that Type F causes approximately 10% of cases of cholera in poultry. In this case, the capsular polymer is not HA, but a related polymer called chondroitin. Currently, the prevention of the disease in the group of poultry is mediated by two elements: vaccines and antibiotics, as well as a strict hygienic condition. The utility of the first option is limited, since there are many serotypes in the field and the vaccines are only effective against a limited subset of the entire spectrum of pathogens. Vaccines of dead or inactivated cells are given by injection, which requires extensive work, and the protection that is obtained is not high. Therefore, this route is usually reserved for animals on farms. The most effective live cell vaccines can be delivered by means of a water supply, but it is difficult to dose thousands of chicks uniformly. Additionally, live "avirulent" vaccines sometimes cause disease in themselves if the birds are stressed or otherwise ill. The most common reason for this inability to predict is that these avirulent strains arise from spontaneous mutations in unknown or uncharacterized genes. Protocols that use repeated alternating exposure to live and dead vaccines can protect birds only against exposure with the same serotype. The second option of disease prevention are antibiotics. These are used either at sub-therapeutic doses to avoid infection or at high doses to convert the cholera of poultry into infected birds. The percentage of birds with disease may decrease with treatment with medication, but adequate treatment is necessary in time and length. Subsequent doses or a premature withdrawal of the antibiotic often results in a cholera of chronic poultry and sick birds with abscesses or lesions leading to their destruction and losses in sales. In addition, since P. multocida resistant strains arise continuously and the costs of medications are high, this solution is not attractive in the long term. In addition, P. mul tocida type FP. it can cause 5-10% of poultry cholera in the United States. A vaccine directed against type A strains may not fully protect against this other capsular type if it emerges as a major pathogen in the future. In the livestock and pig industries, there has not been a vaccine that is totally satisfactory. Prophylactic treatment with antibiotics is used to avoid losses in weight gain, but this option is expensive and is subject to the issue of microbial resistance. In the present invention, enzymes involved in the production of the protective bacterial HA capsule have been identified at the gene / DNA level. The identification of these enzymes will lead to the intervention of the disease by blocking the synthesis of capsule of pathogens with specific inhibitors that prevent the biosynthesis of HA of the host. For example, a medicament limits the substrates used to produce HA or an HA-synthase regulator of P. multocida stops the production of the bacterial HA polysaccharide, and thus blocks the formation of the capsule. This is a direct analogy with many current antibiotics which have different effects on the microbial and host systems. This solution is preferred because the P. multocida HA synthase and the vertebrate HA synthase are very different at the protein level. Therefore, it is likely that the enzymes also differ in the reaction mechanism or at the substrate binding sites.

The P. multocida bacterium, once lacking its protective cover capsule, is significantly more vulnerable and can be targeted by host defenses. Phagocytes engulf and easily destroy the acapsular microbes. The complement complex of the host reaches and breaks the sensitive outer membrane of the bacterium. Antibodies against the newly exposed immunogens, such as the lipopolysaccharides and the surface proteins that determine the somatic serotype in P. multocida, are more easily generalized. These antibodies are better able to bind to the acapsular cells later in the immune response. Therefore, the immune response from vaccinations are more effective and are also more cost effective. Medications that inhibit the capsule are substantial additions to the treatment of poultry cholera. The present invention and the use of capsule biosynthesis of P. mul-type type A aid in the understanding of other capsular serotypes. DNA probes for the type A capsule genes have been used to establish that D and F types have similar homologs. HA of high molecular weight also has a wide variety of useful applications - ranging from cosmetic to eye surgery. Due to its high viscosity potential and its high biocampatibility, HA finds particular application in ocular surgery as a substitute for vitreous fluid. HA has also been used to treat racing horses with traumatic arthritis by intraarticular injections of HA in shaving creams as a lubricant and in various cosmetic products due to their high viscosity physicochemical properties and their ability to retain moisture for extended periods of time. In fact, in August 1997 the Food and Drug Agency of the United States approved the use of HA of high molecular weight in the treatment of severe arthritis by injecting HA of high molecular weight directly into the affected joints. In general, higher molecular weight HAs are best used. This is because the viscosity of the HA solution increases with the average molecular weight of the individual HA polymer molecules in the solution. Unfortunately, HAs with very high molecular weight, ranging from up to 107, are difficult to obtain because of the isolation procedures currently available. To solve these and other difficulties, there is a need for new methods and constructs that can be used to produce HA having one or more improved properties such as greater purity or ease of preparation. In particular, there is a need to develop methodology for the production of relatively large amounts of relatively high molecular weight and relatively pure HA than is currently commercially available. There is also another need to be able to develop methodology for HA production that has a modified size distribution (HA? Size) as well as HA that has a modified structure (HA? Mod). Therefore, the present invention functionally characterizes the genes of P. mul Tocida type A involved in the biosynthesis of the capsule, determines the role of the capsule as a virulence factor in the cholera of poultry, and has obtained genes homologs involved in the biosynthesis of capsules type D and F. With this information, vaccines have been developed that use genes of P. mul tocida "agénicos" or inactivated that do not produce HAS. These avirulent strains have the ability to act as vaccines for poultry cholera or transport fever.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to novel HAS that produces HA. Using a variety of molecular biology techniques, a new HAS gene has been found in Pasturella multocida type A pathogen of poultry cholera. This new HAS of Pasturella mul tocida ("PmHAS"), has been cloned and has been shown to be functional in other species of bacteria.

Therefore, a new source of HA has been identified. The DNA sequence of PmHAS has also been used to generate strains of potential attenuated vaccines of P. multocida bacteria after inactivation of the normal microbial gene by homologous recombination with a broken version. Additionally, the DNA sequence for PmHAS allows the generation of bacterial typing probes for diagnosis, for related types of P. multocida which are agricultural pathogens of poultry, cattle, sheep and pigs.

BRIEF DESCRIPTION OF THE DIFFERENT VIEWS OF THE DRAWINGS Figure 1 is a partial sequence alignment of PmHAS of P. ipultocida and other glycosyltransferases of other bacteria. The sequence of PmHAS in Figure 1 is SEC. FROM IDENT. NO: 10; the sequence of EpsI in Figure 1 is SEC.

FROM IDENT. NO: 11; the sequence of Cpsl4E in figure 1 is the SEC. FROM IDENT. NO: 12; the sequence of LgtD in Figure 1 is SEC. FROM IDENT. NO: 13; the sequence SpHasA in Figure 1 is SEC. FROM IDENT. NO: 14; and the consensus sequence in Figure 1 is SEC. FROM IDENT. NO: 15. Figure 2 is a sequence alignment of residues 342-383 of PmHAS (SEQ ID NO: 16) as compared to residues 362-404 of UDP-GalNAc: mammalian GalNA-transferase polypeptide (FIG. ID SECTION N0: 17). The consensus sequence in Figure 2 is SEC. FROM IDENT. NO: 18. Figure 3 is an autoradiogram representation of a photoaffinity labeling study with UDP sugar analogues of PmHAS. Figure 4 is an autoradiogram showing the labeling of reduced photoaffinity to absent PmHAS in various Tn mutants of PmHAS. Figure 5 shows photomicrographs demonstrating the production of HA in recombinant E. coli. Figure 6 shows graphically the construction of PmHAS and its subcloning into an expression vector. Figure 7 shows the pH dependence of the PmHAS activity. Figure 8 shows the metal dependence of HAS activity. Figure 9 shows the dependence of HAS activity on the concentration of UDP-GlcNAc. Figure 10 shows the activity dependence of HAS in the concentration of UDP-GlcA. Figure 11 is a Hanes-Woolf plot of the estimate of V ^ K ,,,. Figure 12 is a Southern blot mapping of Tn mutants.

Figure 13 shows templates of chimeric or recombinant DNA for sequence analysis of the Tn cleavage sites. Figure 14 is a diagrammatic representation of a portion of HA biosynthesis locus of P. multocida type A. Figure 15 is a Southern blot analysis of various types of P. multocida capsule with capsule gene probes type A. Figure 16 is an electrophoretogram of PCR of type A DNA and heterologous DNA with various type A primers Figure 17 is a partial sequence comparison of type A homologs (SEQ ID NO: 5) and F (SEC DE IDENT NO: 4) of KfaA and KfaA of E. coli (SEQ ID NO: 6). Figure 18 is a schematic of the wild type HAS gene versus an inactivated mutant gene. Figure 19 is the biological molecular confirmation of the inactivated mutant to be encapsulated by Southern blot and PCR analysis. Figure 20 is a sequence comparison of P. mul-type type A and F. The sequence PmHAS in Figure 20 is SEC. FROM IDENT. NO: 7; the sequence PmCS in Figure 20 is SEC. FROM IDENT. NO: 8; and the consensus sequence in Figure 20 is SEC. FROM IDENT. NO: 9 Figure 21 is a Western blot analysis of native and recombinant PmHAS proteins.

DETAILED DESCRIPTION OF THE INVENTION Before explaining at least one embodiment of the invention in detail, it should be understood that the invention is not limited to its application to the details of construction and arrangement of the components set forth in the following description or illustrated in the drawings. . The invention is capable of other modalities or of being carried out or carried out in various ways. further, it should be understood that the phraseology and terminology used herein are for the purpose of description and should not be considered as limiting. As used herein, the term "nucleic acid segment" and "DNA segment" are used interchangeably to refer to a DNA molecule which has been isolated free from total genomic DNA of a particular species. Therefore, a "purified" DNA or nucleic acid segment as used herein, refers to a DNA segment which contains the sequence encoding hyaluronate synthase ("HAS") although it has been isolated and removed from, or which is found in purified free form, of the unrelated genomic DNA, for example, total muriate turella mulotida or, for example, genomic DNA of a mammalian host.

Within the term "DNA segment" are included segments of DNA and smaller fragments of such segments, and also recombinant vectors including, for example, plasmids, cosmids, phages, viruses and the like. Similarly, a DNA segment comprising an isolated or purified PmHAS gene refers to a DNA segment that includes sequences encoding HAS isolated substantially away from other naturally occurring genes or sequences encoding proteins. In this regard, the term "gene" is used for simplicity to refer to a functional protein, polypeptide or unit encoding peptide. As will be understood by those familiar with the art, this functional term includes genomic sequences, cDNA sequences or combinations thereof. The term "substantially isolated" away from other coding sequences "means that the gene of interest, in this case PmHAS, forms the significant part of the coding region of the DNA segment and that the DNA segment does not contain large portions of coding DNA that occur Of course, this refers to the DNA segment as originally isolated, and does not exclude genes or coding regions that are subsequently added to, or that are leave intensionally in the segment by the hand of man.

Due to certain advantages associated with the use of prokaryotic sources, one probably realizes that most of the advantages are in isolating the HAS gene from prokaryotic P. mulotocida. One such advantage is that, typically, eukaryotic enzymes may require significant post-translational modifications that can only be obtained in a eukaryotic host. This will tend to limit the applicability of any eukaryotic HA synthase gene that is obtained. In addition, those currently skilled in the art will also realize the additional advantages in terms of time and ease of genetic manipulation when looking to use a gene for a prokaryotic enzyme. These additional advantages include: (a) ease of isolation of a prokaryotic gene due to the relatively small size of the genome and, therefore, a reduced amount of analysis of the corresponding genomic library, and (b) ease of handling due to the size Total coding region of a prokaryotic gene is significantly lower due to the absence of introns. In addition, if the product of the PmHAS gene (ie, the enzyme) requires post-translational modifications, these can be carried out in a better manner in a similar prokaryotic cell environment (host) from which the gene is derived. Preferably, the DNA sequences according to the present invention will further include regions of genetic control which allow the expression of the sequence in a selected recombinant host. Of course, the nature of the control region used will generally vary depending on the particular use desired (eg cloning of a host). In particular embodiments, the invention relates to isolated segments of DNA and recombinant vectors that incorporate DNA sequences which encode a PmHAS gene, which includes within its amino acid sequence an amino acid sequence according to SEQ. FROM IDENT. NO: 1. In addition, in other particular embodiments, the invention relates to isolated segments of DNA and recombinant vectors that incorporate DNA sequences which code for a gene that includes within its amino acid sequence the amino acid sequence of a gene or DNA for HAS and in particular a gene or cDNA for HAS, corresponding to HAS of Pas turella multocida. For example, when the DNA segment or vector encoding full-length HAS protein, or is designed for use in the expression of the HAS protein, preferred sequences are those which are essentially set forth in SEC. FROM IDENT. NO: l. Truncated PmHAS are also within the definition of the preferred sequences that are established in the SEC. FROM IDENT. NO: 1. For example, in the c-terminal part you can remove approximately 270-272 amino acids from the sequence and still have a functional HAS. Those of ordinary skill in the art will appreciate that simple removal of amino acids from either end of the PmHAS sequence can be performed. The truncated versions of the sequence simply must be verified to determine their HAS activity in order to determine if such truncated sequences are still capable of producing HAS. Segments of nucleic acid having HA synthase activity can be isolated by methods described herein. The term "a sequence essentially as set forth in SEQ ID NO: 1" means that the sequence corresponds substantially to a portion of SEQ. FROM IDENT. NO: 1 and having relatively few amino acids which are not identical to, or biologically functionally equivalent to, the amino acids of SEC. FROM IDENT. NO: 1. The term "biologically functionally equivalent" is well understood in the art and is further defined in detail herein, as a gene having a sequence essentially as set forth in SEC. FROM IDENT. NO: 1 which is associated with the ability of prokaryotes to produce HA or a hyaluronic acid coating. The technique is replete with examples of researchers able to make structural changes to a segment of nucleic acid (that is, they code for amino acid substitutions conserved or are drained) and still retain their enzymatic or functional activity. See for example; (1) Risler et al. "Amino Acid Susbtitutions in Structurally Related Proteins, A Pattern Recognition Approach." J. Mol. Biol. 204-1019-1029 (1988) ["... according to the observed exchange capacity of the amino acid side chains, only four groups can be delineated: (i) lie and Val; (ii) Leu and Met , (iii) Lys, Arg and Gln and (iv) Tyr and Phe "]; (2) Niefind et al,. "Amino Acid Similarity Coefficients for Protein Modeling and Sequence Alignment Derived from Main-Chain Folding Anoles." J. Mol. Biol. 219: 481-497 (1991) [similarity parameters allow amino acid substitutions to be designed]; and (3) Overington et al. "Environment -Specific Amino Acids Substitution Tables: Tertiary Templates and Prediction of Folds Protein," Protein Science 1: 216-226 (1992) ["the analysis of the observed pattern of substitutions as a function of local environment shows that there are different patterns. . "compatible changes can be made]. These references and many others indicate that a person usually skilled in the art, given a nucleic acid sequence, can make substitutions and changes to the nucleic acid sequence without changing its functionality. In addition, a segment of substituted nucleic acid can be highly identical to retain its enzymatic activity with respect to its unadulterated parent structure, and still not hybridize thereto.

The invention describes nucleic acid segments that encode an enzymatically active hyaluronate synthase of P. mul tocida-PmHAS. A person ordinarily skilled in the art will appreciate that substitutions can be made to the nucleic acid segment of PmHAS included in SEQ. FROM IDENT. NO: 2 without deviating or departing from the scope of the claims of the present invention. Table A shows the substitutions of standardized and accepted functionally equivalent amino acids.

TABLE A Another preferred embodiment of the present invention is a purified segment of nucleic acid encoding a protein according to SEQ. FROM IDENT. NO: 1, further defined as a recombinant vector. As used herein, the term "recombinant vector" refers to a vector that has been modified to contain a segment of nucleic acid encoding a HAS protein or a fragment thereof. The recombinant vector can be further defined as an expression vector comprising a promoter operably linked to the nucleic acid segment encoding HAS. A further preferred embodiment of the present invention is a host cell, which becomes recombinant with a recombinant vector comprising a .gen HAS. The preferred recombinant host cell can be a prokaryotic cell. In another embodiment, the recombinant host cell is a eukaryotic cell. As used herein, the terms "engineered" or "recombinant" cell are intended to refer to a cell into which a recombinant gene has been introduced, such as the gene encoding HAS. Therefore, the engineered cells are differentiable from naturally occurring cells, which do not contain a gene introduced recombinantly. The cells undergo engineering and therefore the cells that have a gene or genes introduced through man. The recombinantly introduced genes may be in the form of a cDNA gene, a copy of a genomic gene or may include genes placed adjacent to a promoter that is not naturally associated with the particular introduced gene. In preferred embodiments, the DNA segments encoding HA synthase also include DNA sequences known in the art functionally as origins of replication or "replicons" which allow the replication of contiguous sequences by the particular host. Such origins allow the preparation of extrachromosomally located and replicating chimeric segments or plasmids, to which the DNA sequences for HA synthase are ligated. In more preferred examples, the origin used is one capable of replication in bacterial hosts suitable for biotechnology applications. However, for greater versatility of the cloned DNA segments, it may be desirable or alternatively, even additionally, to use origins recognized by other host systems whose use is contemplated (such as a conductive vector). Isolation and use of other origins of replication such as SV40, origins of bovine papilloma or polyoma virus, which can be used for cloning or expression in many higher organisms, are well known to those ordinarily skilled in the art. In certain embodiments, the invention can be defined in this manner in terms of a recombinant transformation vector which includes the sequence of the gene encoding HA synthase together with an appropriate origin of repliciation and under the control of selected control regions. Therefore, it will be appreciated by those skilled in the art that other means may be used to obtain the HAS gene or cDNA, in light of the present disclosure. For example, DNA fragments can be obtained by polymerase chain reaction or produced by RT-PCR containing complete complements of genes or cDNAs from numerous sources, including other strains of Pasturellas or other eukaryotic sources, such as cDNA libraries. Virtually, any molecular cloning approach can be used for the generation of DNA fragments according to the present invention. Therefore, the only limitation generally is with respect to the particular method used for DNA isolation is that the isolated nucleic acids must code for a biologically functionally equivalent HA synthase. Once the DNA has been isolated, it is ligated together with the selected vector. Virtually any cloning vector can be used to carry out the advantages according to the invention. Typical utility vectors include plasmids and phages for use in prokaryotic organisms and even viral vectors for use in eukaryotic organisms. Examples include pKK223-3, pSA3, recombinant lambda, SV40, polyoma, adenovirus, bovine papilloma virus and retroviruses. However, it is considered that the particular advantages will finally be carried out where vectors capable of replication are used in both strains of Lactococcus or Bacillus and in E. coli. Vectors such as these, exemplified by vector pSA3 and Dao and Ferretti or vector pAT19 of Trieu-Cuot, et al., Allow one to make a selection of clonal colonies in an easily manipulated host such as E. coli, followed by Subsequent retrotransfer within a food grade strain of Lactococcus or Bacillus for HA production. These are benign and well-studied organisms used in the production of certain foods and biotechnology products. It is advantageous if one can increase the ability of the Lactococcus or Bacillus strains to synthesize HA by gene dosage (i.e., providing additional copies of the HA synthase gene by amplification) or by inclusion of additional genes to increase the availability of precursors ha. The inherent ability of a bacterium to synthesize HA can also be increased through the formation of additional copies, or amplification, of the plasmid presenting the gene for HA synthase. This amplification can explain up to a tenfold increase in plasmid copy number and, therefore, in the gene copy number for HA synthase.

Another method that can further increase the copy number of the gene for HA synthase is the insertion of multiple copies of the gene into the plasmid. Another technique may include integrating the HAS gene into the chromosomal DNA. This additional amplification may be especially feasible, since the size of the bacterial HA synthase gene is small. In some scenarios, the vector linked to chromosomal DNA is used to transfect the host that is selected for clonal analysis purposes, such as E. coli by using a vector that is capable of expressing DNA inserted into the chosen host. In some other embodiments, the invention relates to isolated segments of DNA and recombinant vectors that include within their sequence a nucleic acid sequence essentially as set forth in SEQ. FROM IDENT. NO: 2. The term "essentially as set forth in SEQ ID NO: 2" is used in the same sense as described above and means that the nucleic acid sequence substantially corresponds to a portion of SEC DF IDENT. NO: 2 and has relatively few codons which are not identical, or functionally equivalent, to the codons of the SEC. FROM IDENT. NO: 2. The term "functionally equivalent codon" is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine, as set forth in Table A, and also refers to to codons that code for biologically equivalent amino acids. It will also be understood that the amino acid and nucleic acid sequences may include additional residues, such as additional N or C terminal amino acids, or 5 'or 3' nucleic acid sequences and still essentially be as set forth in one of the sequences described herein. , to the extent that the sequence satisfies the criteria established above, including the maintenance of biological protein activity when related to protein expression and enzymatic activity. The addition of terminal sequences is particularly applicable to nucleic acid sequences which may include, for example, several non-coding sequences flanking the 51 or 3 'portions of the coding region, or they may include internal sequences, which are known that occur within genes. In addition, the residues of the terminal amino acids N or C can be removed and still be essentially as established in one of the sequences described here, to the extent that the sequence satisfies the criteria established above, as well. Allowing the degeneracy of the genetic code as well as the conserved and semi-preserved substitutions, the sequences which have between approximately 40% and approximately 80%; or more preferably, between about 80% and about 90%; or even more preferably, between about 90% and about 99% nucleotides which are identical to the nucleotides of SEQ. FROM IDENT. NO: 2, will be sequences which are "essentially as stated in SECTION ID NO: 2". The sequences which are essentially the same as those established in the SEC. FROM IDENT. NO: 2 can also be functionally defined as sequences which are capable of hybridizing with a nucleic acid segment containing the complement of the SEC. FROM IDENT. NO: 2 under standard hybridization conditions or less astringent. Suitable standard hybridization conditions are known to those skilled in the art and can be clearly established here. As used herein, the term "standard hybridization conditions" is used to describe those conditions under which substantially complementary nucleic acid segments will form a standard Watson-Crick base pairing. Many factors are known that determine the specificity of binding or hybridization, such as pH, temperature, salt concentration, presence of agents such as formamide and dimethyl sulfoxide, the length of the segments that hybridize, and the like. When it is contemplated that shorter segments of nucleic acid will be used for hybridization, eg, fragments between about 14 and about 100 nucleotides, the preferred conditions of saline concentration and temperature for hybridization will include 1.2-1.8 x HPB at 40-50. ° C. Naturally, the present invention also encompasses DNA segments which are complementary, or essentially complementary to, the sequence set forth in the SEC. FROM IDENT. NO: 2. The nucleic acid sequences which are "complementary" are those which are capable of base matching according to the Watson-Crick standard complementarity rules. As used herein, the term "complementary sequences" means nucleic acid sequences which are substantially complementary, which can be determined by the same nucleotide comparison as set forth above, or which are defined as the ability to hybridize with the segment of nucleic acid of the SEC. FROM IDENT. NO: 2. The nucleic acid segments of the present invention, regardless of the length of the coding sequence itself, can be combined with other DNA sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, sites of multiple cloning, epitopic labels, polyhistidine regions or other coding segments and the like, so that their total length can vary (considerably.) Therefore, it is contemplated that a nucleic acid fragment of almost any length can be used, and the total length is preferably limited by the ease of preparation and use of the proposed recombinant DNA protocol Naturally, it will also be understood that this invention is not limited to the particular amino acid and nucleic acid sequences of SEQ ID NO. ly 2. Recombinant vectors and isolated segments of DNA can therefore variably include regions HAS encoders themselves, coding regions that exhibit selected alterations or modifications in the basic coding region, or that can encode larger polypeptides which nevertheless include regions encoding HAS or that can encode biologically functional proteins or peptides which store variant amino acid sequences. The DNA segments of the present invention encompass biologically functionally equivalent HAS proteins and peptides. Such sequences may arise as a consequence of the codon redundancy and functional equivalence which are known to occur naturally within the nucleic acid sequences and the proteins encoded in this manner. Alternatively, functionally equivalent proteins or peptides can be generated via the application of recombinant DNA technology, in which changes in the structure of the protein can be engineered, based on considerations of amino acid properties. that they change. Human-designated changes can be introduced by the application of site-directed mutagenesis techniques, for example to introduce improvements in the enzymatic activity or the antegenicity of HAS protein or to test HAS mutants in order to examine the activity of HA synthase at the molecular level. In addition, specific changes in the sequence encoding HAS may result in the production of HA having a modified size distribution or structural configuration. A person ordinarily skilled in the art can appreciate that the sequence encoding HAS can be manipulated so as to produce an altered hyaluronate synthase which in turn is capable of producing hyaluronic acid having polymer sizes or functional capacities, or both, that differ. For example, the sequence encoding HAS can be altered in such a way that the hyaluronate synthase has an altered specificity for sugar substrate so that the hyaluronate synthase generates a novel hyaluronic acid-like polymer incorporating a different structure such as a sugar or sugar derivative not previously incorporated. This new incorporated new sugar can result in a modified hyaluronic acid having different functional properties, a hyaluronic acid having a polymer size or lower or higher molecular weight, or both. As will be appreciated by those ordinarily skilled in the art given the HAS coding sequences, changes or substitutions, or both, can be made in the sequence encoding HAS so that these properties or desired size modifications can be carried out. . The term "modified structure", as used herein, denotes a polymer of hyaluronic acid containing a sugar or derivative that is not normally found in a naturally occurring HA polysaccharide. The term "modified size distribution" refers to the synthesis of hyaluronic acid molecules of a size distribution that is not normally found with the native enzyme; The size under engineering can be much smaller or larger than normal. Different products of hyaluronic acid of different size have application in areas of drug supply and the generation of an enzyme of altered structure can be combined with a hyaluronic acid of different size. Applications in angiogenesis and wound healing are potentially large if polymers of hyaluronic acid of about 20 monosaccharides can be made in large quantities. Another particular application for small oligosaccharides of hyaluronic acid is in the stabilization of recombinant human proteins used for medical purposes. A major problem with such proteins is their clearance of blood and their short biological life. A present solution to this problem is to attach to a small molecule that prevents the protein from being cleared from the circulation too fast. Hyaluronic acid of very small molecular weight is suitable for this role and may be non-immunogenic and biocompatible. A larger molecular weight hyaluronic acid bound to a drug or protein can be used to target the system of endothelial reticular cells which have endocytic receptors for hyaluronic acid. A person ordinarily skilled in the art, given this description, can appreciate that there are several ways in which the size distribution of the hyaluronic acid polymer manufactured by the hyaluronate synthase can be regulated to provide different sizes. First, the kinetic control of the product size can be altered by decreasing the temperature, decreasing the time of action of the enzyme or by decreasing the concentration of one or more sugar nucleotide substrates. By decreasing any or all of these variables, it provides smaller amounts and smaller sizes of hyaluronic acid product. The disadvantages of these solutions are that the performance of the product also decreases and it may be difficult to obtain reproducibility from one day to another or from one batch to another. Second, the alteration of the intrinsic ability of the enzyme to synthesize a large hyaluronic acid product. Changes to the protein can be engineered by recombinant DNA technology, which includes substitution, deletion and addition of specific amino acids (or even the introduction of prosthetic groups through metabolic processing). Such changes result in an intrinsically slower enzyme which can then allow a more reproducible control of the size of the hyaluronic acid by a kinetic means. The final size distribution of hyaluronic acid is determined by certain characteristics of the enzyme, which are based on the particular amino acids in the sequences. Among the 20% of residues conserved absolutely between streptococcal enzymes and eukaryotic hyaluronate synthases, there is a set of amino acids in unique positions that control or that greatly influence the size of the hyaluronic acid polymer that the enzyme can make. Specific changes in any of these residues can produce a modified HAS that produces an HA product that has a modified size distribution. Engineering changes to spHAS, pmHAS or cvHAS that decrease the intrinsic size of hyaluronic acid that the enzyme can make before hyaluronic acid is released will provide a powerful means to produce a hyaluronic acid product of smaller or potentially larger size than the native enzyme. Finally, the larger molecular weight hyaluronic acid can be degraded with specific hyaluronidases to produce lower molecular weight hyaluronic acid. This practice, however, is very difficult to carry out in a reproducible manner and one must thoroughly re-purify hyaluronic acid to remove hyaluronidase and unwanted digestion products. Structurally modified hyaluronic acid is not conceptually different from altering the size distribution of a hyaluronic acid product by changing particular amino acids in the desired HAS or spHAS. UDP-GlcNAc derivatives, in which the N-acetyl group (UDP-GlcN) is lost or replaced with another chemically useful group, they are expected to be particularly useful. The strong substrate specificity should be based on a particular subset of amino acids among the 20% that is conserved. Specific changes to one or more of these residues that generate a functional synthase that interact less specifically with one or more of the substrates compared to the native enzyme. This altered enzyme can then be used to alternate with special natural sugar nucleotides to incorporate sugar derivatives designed to allow different chemicals to be used for the following purposes: (i) covalently coupling specific drugs, proteins or toxins to structurally modified hyaluronic acid to generalized or targeted medication supplies, radiological procedures, etc. (ii) covalently cross-linking the hyaluronic acid itself or other supports to obtain a gel, or other three-dimensional biomaterial with stronger physical properties, and (iii) covalently binding hyaluronic acid to a surface to create a biocompatible film or monolayer. The present invention relates to novel HAS for producing HA. Using a variety of molecular biology techniques, a gene for the new HAS is found in Pas aurella multocida type A of the poultry cholera pathogen. This new HAS of Pasturella multocida or PmHAS has been cloned and shown to be functional with other species of bacteria. The PmHAS protein is polymerized for the authentic HA polysaccharide. The carbohydrate produced by recombinant E. coli transformed with PmHAS is recognized by the HA-binding protein of cartilage and is sensitive to HA-lyase digestion. Both reagents are considered by those usually skilled in the art as specific for the HA polysaccharide. In addition, both UDP-GlcA and UDP-GlcNAc are required for the synthesis of HA in vi tro. Azido-UDP-GlcA and azido UDP-GlcNAc, but not azido-UDP-Glc, is photocolorporated specifically in PmHAS. As in the case of HasA streptococcus and Xenopus DG42, it seems that a polypeptide species, PmHAS, transfers two different sugar groups to the nascent HA chain. Many encapsulated Gram-negative bacteria that include E. coli, Neisseria meningi tidis and Hemophilus influenzae possess groups of genes responsible for capsule biosynthesis organized in operons. These operons often have genes that code for: (i) enzymes necessary for the synthesis of sugar nucleotide precursors, (ii) glycosyltransferases to polymerize the exopolysaccharide, and (iii) proteins involved in the export of polysaccharides. The HA capsule operon of P. multscida type A contains: (i) an analog of KfaA, (ii) HA synthase, and (iii) a putative UDP-Glc dehydrogenase. The Tn916 elements in the H and L mutants of P. multocida are not directly integrated into the HAS gene, but rather are located in the KfaA homologous gene. Since PmHAS exists at a locus of at least several genes essential for making the polysaccharide, a lesion or defect in any of the capsule genes can alter HA production and capsule formation in Pasturella. Therefore, by breaking an adjacent gene, a vaccine can also be made. For example, if UDP-Glc dehydrogenase is removed or broken, a precursor sugar for HA synthase is not available and HA can not be made. Furthermore, if Kfa or another gene associated with transport is inactivated, there is no HA on the surface prepared by the microbe. Therefore, the HA synthase product can be stopped in the natural Pasturella microbe, ie, an HA capsule by: (a) interrupting the precursor formation, or (b) interrupting the polymerization machinery, or ( c) interrupt the transport machinery. At the amino acid level, PmHAS is not as similar as other HAS cloned, as would be expected by a person usually skilled in the art. Two potential short motifs, DGS (S / T) (SEQ ID NO: 19) in residues 477-480 and DSD in residues 527-529 of PmHAS are present in HasA. Another similar reason that contains DGS is repeated in residues 196-198 of PmHAS. The DG of the first reason and the DSD are kept in all the HAS. However, there are several absolute conserved motifs ((S / G) GPLXXY (SEQ ID NO: 20), GDDRXLTN (SEQ ID NO: 21) and LXQQXRWXKS (Y / F / W) (F / ORE (SEQ ID NO: 22)) found in all HAS previously cloned are absent in PmHAS, instead a variety of bacterial glycosyltransferases are more closely aligned with the sequence in the central portion of the protein HAS of P. mul.sup.-These enzymes, which have been shown or predicted to transfer GlcNAc, galactose or GalNAc groups, are generally one third the size of PmHAS and their terminal amino acid sequences are aligned together with the PmHAS polypeptide media, residues 430-540 The sections of the first 420 PmHAS residues show some similarity to portions of UDP-GalNAc: mammalian polypeptide-GalNAc-transferase These observations may be a reflection of the possible domain structure within of PmHAS, the last 340 waste PmHAS samples are not significantly similar to the other entries in the sequence databases. Therefore, the HAS of mulostida is unique and is very likely to be the prototype of a completely new HAS class. PmHAS is generally twice the size of streptococcal, viral or vertebrate HAS - 972 residues versus 417-588, respectively. In addition, the hydropathy plots of PmHAS and other known HASs are different. Using the TMPRED program, which is readily available and known to those ordinarily skilled in the World Wide Web art, PmHAS is predicted to have only two candidate transmembrane helices (centered at residues 170 and 510) and both terminal portions of the protein can be located in the cytoplasm. Topologically, these assumptions imply that one third of the P. multocida polypeptide (approximately 340 residues) is located outside the cytoplasm. On the other hand, a different topology is predicted for other HAS classes. An analysis of the function of an indicator enzyme for streptococcal HAS confirms that a different topological arrangement exists in this enzyme, consisting of: (i) two transmembrane helices near the terminal part of amino acids, (ii) a putative cytoplasmic domain, followed by ( iii) three membrane-associated regions in the carboxyl half of the protein. The connection curls between the membrane-associated regions are rather short (4-10 residues); therefore, the vast majority of the polypeptide chain is probably not exposed extracellularly. The following detailed experimental steps and discussion of the results confirm that the present invention relates to a novel and unique PmHAS. 1. Molecular cloning of PmHAS First the insertional mutagenesis Tn916 and the generation of probes are completed. Tn326 'is used to break and label the locus of HA biosynthesis of P. multocida. The Tn element in a non-replicating plasmid, pAM150, is introduced into the wild type encapsulated P. multocida strain (ATCC number 15742) by electroporation. The morphology of the altered colony is initially analyzed by visual examination with oblique illumination. The wild-type strain forms large mucoid colonies ("wet" appearance) that appear iridescent (red and green coloration). The smaller and more "dry" colonies lack iridescence when chosen and streaked. Indian ink staining and optical microscopy are used as a secondary analysis to determine the state of encapsulation. The position of the Tn elements in the mutant chromosome is mapped by Southern analysis. The DNA sequences at the Tn-broken sites of various independently selected mutants are obtained by dideoxy sequence analysis of the labeled chromosomal DNA. Briefly, a chimeric DNA fragment consisting of a 12-kb portion of the Tn916 element and a short region of the P. mulotida DNA generated by H digestion of the mutant chromosomal DNA is purified by agarose gel electrophoresis (the all genomic fragments of wild-type Hhal are less than or equal to 7 kb). The chimeric fragment serves as the template in the cycle sequencing reactions using 33P terminators and a primer of the right arm end part Tn916 (51-GACCTTGATAAAGTGTGATAAGTCC-3 '(SEQ ID NO: 23)). Sequence data are used to design PCR primers. The gel purified PCR products are labeled with digoxigenin using the High Prime system manufactured by Boehringer Mannheim and is well known to those ordinarily skilled in the art. The next step is the isolation of a functional HAS locus. Is a library created? of wild-type DNA partially digested with 5au3A using the Expr? vector system Zap separated by BamHl, produced by Stratagene. The raised portions of the plate are analyzed to determine hybridization with a digoxigenin-labeled PCR product. Is Escherichia coli XLl-Blue MRF 'co-infected with clones? positive purified individual and with the auxiliary phage ExAssist to generate phagemids. The resulting phagemids are transfected into E. coli XLOLR cells to recover the plasmids. The plasmids are transformed into a more suitable host for the production of polysaccharide HA, E. coli K5 (strain BÍ8337-41). This strain produces UDP-GlcA, a substrate necessary for the biosynthesis of HA that is not found at significant levels in most laboratory strains. Additionally, K5 possesses many other genes essential for the capsular transport of polysaccharide in E. coli. Another host used for expression studies is E. coli EV5, a capsular derivative of a Kl strain which produces a capsule of polysialic acid and which also possesses all the general capsular polysaccharide transport machinery that K5, but has no high levels of UDP-Glc dehydrogenase. Cultures of the E. coli transformants with the candidate plasmids growing in fully defined medium are tested for HA polysaccharide production as previously described, except that the cell pellets are extracted with 8 M urea, 0.1% SDS. 95 ° C for 2 minutes. The test of the HA test produced by Pharmacia Biotech Inc., which is well known to those of ordinary skill in the art, uses a specific protein that binds HA to detect HA at concentrations greater than or equal to 0.1 μg / ml . Multiple determinations of HA levels are averaged. The concentration of HA in bacterial cultures is normalized to determine the differences in the number of cells by measuring the A600 value and present the data as μg of HA / ml / A600 of bacteria. A plasmid, pPm7A, with a 5.8 kb insert, gives E. col i K5 the ability to produce HA; HA is not produced by the cells solely with the plasmid vector. A truncated derivative of pPm7A contains an insert of approximately 3.3 kb, termed pPm? 6e, can direct HA biosynthesis when transformed into E. coli K5. Therefore, the sequences of both strands of plasmid pPm7A corresponding to the DNA of pPm? 6e are determined. An ORF of 972 unique complete residues, which we call PmHAS, is found and demonstrated in the SEC. FROM IDENT. NO: 1. In the SEC.

FROM IDENT. NO: 2 the corresponding nucleotide sequence is shown. The expression of recombinant HAS of P. multocida is then carried out. The PmHAS ORF in the pPm7A insert is amplified by 13 cycles of PCR with Tag polymerase and the primers corresponding to the sequence near the amino endpoint and deduced carboxyl (codons with capitals: direct, 5 '-gcgaatJtcaaaggacagaaaATGAAcACATTATCACAAG-3 '(SEQ ID NO: 2 4), and antisense, 5' -ggqaa11ctgeaqttaTAGAGTTATACTATTAATAATGAAC-3 '(SEQ ID NO: 25), start and stop codons, respectively, in bold type. > C) is altered (italic small letter) to increase protein production in E. coli The primers also contain EcoRI and PstI restriction sites (underlined letters) to facilitate cloning in the expression plasmid pKK223-3 (promoter tac, Pharmacia) The resulting recombinant construct, pPmHAS, is transformed into SURE E. coli cells (Stratagene) and this strain is used as the source of membrane preparations for HAS assays in vitro. induce cultures in logarithmic phase (LB broth, 30 CC) with 0.5 mM isopropylthiogalactoside for 3 hours before harvesting. The plasmid is also transformed into E. coli K5; the resulting strain is examined for the presence of capsule by light microscopy and floating density centrifugation. K5 bacterial cultures are not introduced systematically because the addition of isopropylthiogalactopyranoside does not increase HA levels in LB or in a significantly defined medium. Then a photoaffinity labeling of HAS of native P. multocida is carried out. Radiolabeled UDP sugar analogues [32 P] azido-UDP-GlcA (3 mCi / μMol) and [3J2P] azido-UDP-GlcNAc (2.5 mCi / μMol) are prepared and purified as described in the literature and as is known by a person usually skilled in the art. Membrane preparations for P. multocida wild type in 50 mM Tris, 20 mM MgCl2, pH 7, are incubated either with a probe (final concentration, 20 μM) for 30 seconds on ice before irradiation with ultraviolet light (254 nm, 90 seconds). Proteins are precipitated with 5% trichloroacetic acid before analysis by SDS-polyacrylamide gel electrophoresis. Radiolabels are not incorporated if the irradiation stage is omitted. As a specificity control, a 10-fold molar excess of normal UDP sugar is co-incubated with the probe and the membranes. It is also used as another control [32P] azido-UDP-Glc (3 mCi / μMol). Approximately 8 x 104 transformants containing Tn are analyzed by several rounds of mutagenesis to determine differences in colony morphology. By optical spectroscopy with Indian ink, the cells of small non-iridescent colonies (n = 4) do not possess detectable capsule (they are acapsular), while the cells of iridescent colonies of medium size (seem to have a capsule of approximately 10-25% of diameter compared to the wild type (they are microcapsular) Two of the acapsular mutants, designated H and L, which have Tn elements that are mapped into the same HiiadlII or BstXI genomic fragments are reverted to wild-type colony morphology at about 10"3. The element Tn in each revert has been cut from the original position and reinserted into a new and different position, judging by the Southern analysis, on the one hand, all the acapsular subclones retain the element Tn in the original position No significant HAS activity is detected in membrane preparations for mutant H cells, while substantial activity is detected ial of HAS for wild-type cells (less than or equal to 0.7 versus 120 pmol of GlcA transfer / mg protein / h, respectively). These findings suggest that the Tn elements in the H and L mutants are actually responsible for breaking the locus of HA biosynthesis. In order to unite the separation between the Tn insertion sites of two acapsular mutants, PCR is carried out using the chromosomal DNA template of the L mutant with a primer derived from the sequence at the cleavage site of the H mutant, PmHF (5 '-CTCCAGCTGTAAATTAGAGATAAAG-3' (SEQ ID NO: 26) ) and a primer corresponding to the left terminal part of T n 9 1 6, TnL2 (5 '- GCACATAGAATAAGGCTTTACGAGC-3' (SEQ ID NO: 27)). A specific PCR product of approximately 1 kb is obtained; alternatively, no product is formed if PmHR (reverse complement of PmHF) or the right arm primer of TN916 is replaced. The PCR product is used as a hybridization probe to obtain a functional copy of HAS of P. multocida. Six plates are found that hybridize positively after an analysis of approximately 104 plates and these phages become plasmids. It is found that a plasmid, pPm7A, can direct E. coli K5 to produce HA in vivo (20 μg HA / ml / A600 bacterium). E. coli K5 with control plasmids does not produce HA '(less than or equal to 0.05 μg HA / ml / A600). E. coli XLOLR or E. coli EV5 cells (which lack UDP-Glc dehydrogenase activity) do not produce HA even if they contain pPm7A plasmid (less than or equal to 0.05 μg HA / ml / A600). This genetic evidence implies that the pPm7A insert does not code for a functional UDP-Glc dehydrogenase enzyme. A truncated derivative of plasmid pPm7A with the smallest insert capable of directing HA biosynthesis (85 μg HA / ml / A600 of K5 bacteria), pPm? 6e, contains a single complete ORF coding for a protein of 972 residues, as shown in the SEC. FROM IDENT. NO: 1. There is no obvious promoter present in the SEC. FROM IDENT. NO: 1, but there is a predicted binding site for ribosome labeled in bold "centered at nucleotides -10 to -7 and in the two putative transmembrane regions predicted by TMPRED underlined (residues 162-182, and 503-522) The PmHAS of SEQ ID NO: 1 is twice as large as the streptococcal HasA.This protein is the P. multocida HA synthase, PmHAS.The Mr predicted is 111.923 and the calculated isoelectric point is 6.84 SEQ ID NO: 2 is the nucleotide sequence for PmHAS This PmHAS is used as the key in the BLASTP searches of the protein sequence database The central portion of PmHAS (residues 436-536) is more homologous to bacterial glycosyltransferases from a wide variety of genera including Streptococcus, Vibrio, Neisseria, and Staphylococcus, which form exopolysaccharides or the lipopolysaccharide carbohydrate moieties (the smallest sum of probabilities, 10"22-10" 10). shows on the fig ura 1. Figure 1 graphically shows the HAS sequence alignment of P. mul-tocida and other glycosyltransferases. The MULTALIN alignment illustrates that the central region of PmHAS (residues 436-536) are more similar to the amino-terminal portions of several enzymes that produce other polysaccharides (Streptococcus thermophilus EpsI, type 14, S. pneumoniae Cpsl4J) or the carbohydrate moiety. lipopolysaccharides (homology with H. influenzae LgtD). Figure 1 shows only some possible examples. HasA from S. pyogenes (residues 61-168) has limited similarity to this region shown from PmHAS. The most striking sequence similarities are the DGSTD motifs (ID SECTION NO: 28) and DXDD (ID SECTION NO: 29). Unexpectedly, there is not a significant total similarity of PHmAS with HAS of streptococcal, viral or vertebrate HAS having a smaller sum probability of 0.33. Only a short region of HAS1 esptreptococcal is aligned with PmHAS in a convincing manner, and is shown in Figure 1. Some segments of the first half of PmHAS are also similar to the portions of UDP-GalNAc: mammalian GalNAc-transferase polypeptide, an enzyme that initiates O-glycosylation of mucin-like proteins with the smallest sum probability of approximately 10"3, Figure 2. As shown in Figure 2, the sequence alignment of residues 342-383 of PmHAS is More similar to residues 362-404 of UDP-GalNAc: mammalian GalNAc-transferase polypeptide For both figures 1 and 2, the identical residues are in bold and underlined, and the consensus symbols are:!, either I or V; #, any of N, D, E or Q;%, either of F or Y. Acid residue groups are well conserved across the sequences.

The partial ORF (27 residues) towards the 3 'end of PmHAS is very similar to the amino terminal part of several UDP-Glc dehydrogenases of bacteria including E. coli, Salmonella typhimurium and Streptococcus pneumoniae (67-74% identity) . Severe truncation in the original pPm7A clone can be expected to result in complete loss of dehydrogenase activity. The other ORF (623 residues) towards the 5 'end of PmHAS is highly homologous to the KfaA protein of E. coli K5 with a smaller sum probability of 10"52, a protein putatively involved in the transport of a capsular polysaccharide outside The predicted size of 972 residues (112 kDa) for PmHAS is confirmed by photoaffinity labeling of membrane preparations from P. multocida wild type, both probes [32P] azido-UDP-GlcA and [32P] azido-UDP-GlcNAc is photoincorporated into a protein of approximately 110 kDa, in a UV-dependent manner, Figure 3 is a photoaffinity labeling of PmHAS with UDP sugar analogs. [32P] azido-UDP-GlcA and [32P] azido-UDP-GlcNAc are incubated with membrane preparations (45 μg of protein) isolated from P. multocida wild type and irradiated with UV light In figure 3 the autoradiograms (5 days exposure) of 10% gels are shown SDS-PAGE Both probes photomark an approximate protein a.k.a. 110 kDa in a UV-dependent manner (lanes "-"). In order to determine the specificity of photoincorporation, a parallel sample is treated identically, except that the reaction mixtures include a 10-fold excess of the unlabeled competitor (UDP-GlcNAC or UDP-GlcA, respectively; marked by the lanes "+ "). The band intensities are reduced compared to the "-" lanes. The standards are marked in kDa. Competition with the corresponding unlabeled natural UDP sugar precursors decreases the extent of photoincorporation of the probe. In parallel experiments, [32 P] azido-UDP-Glc, an analog of normal HA precursors, does not label this 110 kDa protein. In addition, membranes derived from Tn mutants have zero or very low amounts of azido-UDP-GlcA incorporation in this protein. As shown in Figure 4, membrane preparations (60 μg of protein) of wild-type Tn mutants (W) or various tn capsular mutants (A, G, or H) are photo-tagged with [32 P] azido-UDP- GlcA. The region of the autoradiogram in the vicinity of the protein of approximately 110 kDa is shown in Figure 4. No photoincorporation is observed in samples A and G. The small degree of photo-labeling in sample H is due to the low inversion rate observed in this particular mutant. The size of the protein labeled by photoaffinity in sample W corresponds well with the predicted Mr of the cloned PmHAS ORF.

Membranes derived from E. coli SURE cells containing the plasmid pPmHAS, but not the cell samples with the pKK223-3 vector alone, synthesize HA in vi tro when supplied with both UDP-GlcA and UDP-GlcNAc ( 25 versus less than or equal to 1.5 pMol of GlcA transfer / mg protein / hour, respectively). No incorporation of [1 C] GlcA is observed if UDP-GlcNAc is omitted or if the divalent metal ions are chelated with EDTA. The activity of HAS derived from recombinant HAS is similar to the enzyme obtained from wild-type P. multocida membranes because Mn2 + stimulates at least 10 times more activity compared to Mg2 +. Recombinant E. coli cultures are also tested for the presence of HA polysaccharide with a radiometric assay using labeled HA binding protein. E. coli K5 with pPmHAS produces 460 μg HA / ml / A600. K5 cells with the pKK223-3 vector only do not produce HA (less than or equal to 0.05 μg HA / ml / A600). For comparison, wild-type P. multocida, which grows in the same medium, produces 1,100 μg / HA / ml / A600. E. coli K5 with pPmHAS produces high levels of HA so that the cells are encapsulated. As shown in figure 5, Panel A, photomicrographs of recombinant E. coli stained with Indian ink (magnification 1000 x) shows that E. coli K5 cells with pPmHAS produce a substantial capsule that appears as a white halo around the cells. The radius of the capsule of the recombinant strain is approximately 0.2-0.5 μm (establishing the assumption that the width of the bacterial cell is 0.5 μm). This capsule can be removed by treatment with either testicular oval hyaluronidase, or with HA lyase of Streptomyces. As shown in Figure 5, panel B, the capsular material is removed from the E. coli K5 cells (pPmHAS) by a brief treatment with HA lyase from Streptomyces. Therefore, PmHAS directs the polymerization of the HA polysaccharide. Neither the native K5 host strain nor the transformants containing the pKK223-3 vector possess an easily observable capsule, as determined by optical spectroscopy. K5 cells with pPmHAS also appear encapsulated by floating density centrifugation. The recombinant cells float on the top of the 58% Percoll mattress, while the vector control cells or the recombinant cells treated with hyaluronidase are pelleted through the Percoll mattress. Plasmid p / PmHAS in E. coli K5 is the first generation system for making recombinant HA with PmHAS; other optimized vectors or hosts may provide higher yields, and these other optimized vectors or hosts are contemplated herein for use with the present invention. A person usually skilled in the art, given the description, will be able to optimize such vectors or hosts. 2. Enzyme characterization of PmHAS The protein is determined by the Coomassie dye staining assay using a standard bovine serum albumin. Wild type mulifecida (American Type Culture Collection 15742) is maintained, a highly virulent strain in turkeys, which forms very mucoid colonies, in a brain / heart drive medium under aerobic conditions at 37 ° C. A capsular mutant of the strain, which forms smaller and more "dry" colonies called TnA, is generated by the newly described insertional mutagenesis method Tn916, mentioned here. The total membranes of P. multocida are prepared by modifying the method to produce HA synthase from E. coli with recombinant plasmids containing hasA. The cells are grown with vigorous agitation to a semilogarithmic phase (0.4-0.8 A600) and then bovine testicular hyaluronidase (Sigma type V, final 20 units / ml) is added to remove the capsule. After 40 min, the cells are cooled in ice and harvested by centrifugation (2000Xg for 15 min). The cells are washed twice with PBS by repeated suspension and centrifugation, and the cell pellet can be stored at -80 ° C. All the following stages are performed on ice unless otherwise indicated. The cells are resuspended by pipetting at 1/400 the original culture volume of 20% sucrose and 30 mM Tris, pH 8.0 containing protease inhibitors pepstatin and leupeptin. Cell lysis is carried out using lysozyme digestion (addition of 1/10 of the suspended volume of 4 mg / ml of enzyme in 0.1 M EDTA, 40 minutes of incubation) followed by ultrasonic rupture (energy establishment 3, three cycles of 30 s on / off, Heat Systems W-380 with microprobe). Before the ultrasonification step, sodium thioglycolate (final concentration of 0.1 mM) is added to the mixture followed by the addition of phenylmetanesulfonyl fluoride. In all the remaining manipulations, the PBS also contains thioglycollate newly added at the same concentration. The lysate is treated with DNase and RNase (1 μg / ml each, 10 minutes at 4 ° C) and the cell debris is removed by low speed centrifugation (100 ° C for 1 hour). The supernatant fraction is diluted 6 times with PBS and the membrane fraction is harvested by ultracentrifugation (100OOXg for 1 hour). The pellet is washed twice by repeated suspension in PBS containing 10 mM MgCl2, followed by ultracentrifugation. To generate membrane preparations used in metal specificity studies, Mg l2 is omitted and replaced with 0.2 mM EDTA during the washing steps. The membrane preparations are suspended in 50 mM Tris, pH 7 and 0.1 mM thioglycolate, at a concentration of 1-3 mg / ml protein, and stored at -80 ° C.

The HA synthase activity is detected systematically by incorporation of the radiolabel derived from the nucleotide precursor with UDP- [14C] GlcA sugar (0.27 Ci / mmol, ICN), within higher molecular weight products. The various assay buffers, described in the legends in the figure, also contain 0.3 mM DTT. The assays (100 μl final volume) are initiated by adding membranes to the reaction mixture and incubation at 37 ° C. After 1 hour, the reactions are terminated by the addition of SDS (final 2%) and mixed. For kinetic studies, the product and the precursors are separated by descending paper chromatography (Whatman 3M with 65:35 ethanol / 1 M ammonium acetate, pH 5.5). The HA polysaccharide at the origin of the paper chromatogram is eluted with water before liquid scintillation counting. These assays are typically performed under conditions in which up to 5% of the precursors are consumed by limiting amounts of enzyme. Controls to verify the incorporation of authentic HA include the omission of the nucleotide precursor of the required second sugar or digestion using Streptomyces hyalurolyticus-specific hyaluronidase. Gel filtration chromatography with Sephacryl S-200 (Pharmacia) in PBS is used to determine the molecular weight of the radiolabeled polymer formed in vi tro under optimized assay conditions. These samples are treated as for paper chromatography except that, after completion, they are heated at 95 ° C for 2 minutes and rinsed by centrifugation 15000Xgr for 7 minutes) before application to the column. EDTA (0.2 mM) is used to chelate any metal ion present in the assay mixtures to verify the metal dependence of HAS activity. Various divalent metals are tested including Mg, Mn, Cu, Co and Ni, as well as their chloride salts. The Km values of the substrates are estimated by titration of the nucleotide concentration of sugar while maintaining the other radiolabelled precursor at a constant concentration and in saturation. For these studies, UDP- [3H] GlcNAc (30 Ci / mmoles, NEN) is used as well as the precursor UDP- [14C] GlcA. The P. multocida cells produce an easily visible extracellular HA capsule, and since HasA streptococcus is a transmembrane protein, the membrane preparations of the poultry cholera pathogen are tested. In the first tests, the raw membrane fractions derived from ultrasonication in themselves possess very low levels of UDP- [1 C] GlcA dependent on UDP-GlcNac, incorporated in HA [approximately 0.2 pmoles of GlcA transfer (μg of proteins) " 1h "1] when tested under conditions similar to those for measuring streptococcal HAS activity. The E. coli enzyme with the recombinant HAS plasmid is also recalcitrant for isolation the first time. These results contrast with the easily detectable amounts obtained from Streptococcus by similar methods. An alternative preparation protocol using a treatment with ice-cooled lysozyme in the presence of protease inhibitors together with ultrasonication allows substantial recovery of HAS activity from both species of Gram-negative bacteria. Activities of 5-10 pmol of transfer GlcA (μg of protein) ^ h "1 for crude membranes of wild-type P. multocida with the new method are obtained in the absence of UDP-GlcNac. incorporates radioactivity (less than 1% identical assay with both sugar precursors) from UDP- [14C] GlcA in the higher molecular weight material.Membranes prepared from the acapsular mutant, TnA, have no detectable HAS activity when they are supplemented with both sugar nucleotide precursors, analysis by gel filtration using a Sephacryl S-200 column indicates that the molecular mass of most of the 14C labeled product synthesized in vi tro is = 8 x 104 Da since the material elutes in the empty volume, such volume corresponds to an HA molecule consisting of at least 400 monomers.This product is also sensitive to digestion by hyaluronidase by Streptomyces, but resistant to treatment by pronasa. The assay parameters for HAS are varied to maximize the incorporation of UDP sugars within the polysaccharide by P. mul-tocida membranes. HasA streptococcal requires Mg2 + and therefore this metallic ion is included in the initial tests of P. multocida membranes. HAS mulostida is relatively active at pH 6.5 to 8.6 in Tris-type buffers with an optimum at pH 7, figure 7. Figure 7 shows the pH dependence of the HAS activity of P. mul-tocida. The incorporation of [14C] GlcA into membrane-catalyzed HA polysaccharide (38 μg protein) is measured in buffered reactions at various pH values (50 mM Tris / 2- (N- (morpholino) ethanesulfonic acid, bis-Tris / HCl or tris / Hcl; no specific effects of principal buffer ion are observed. The incubation mixture also contains 20 mM MgCl 2, 120 μM UDP-GlcA (4.5 x 10 4 DPM / assay) and 300 μM UDP-Glc α AC. The incorporation of the assay using the optimal buffer, Tris pH 7, is established at 100% activity. An optimum of wide pH around neutrality is observed. The HAS activity is linear with respect to the incubation time at neutral pH for at least 1 hour. Apparently the P. multocida enzyme is less active at higher ionic strengths due to the addition of 10 M NaCl to the reaction containing 50 mM Tris, pH 7 and 20 mM MgCl2 reduces the incorporation of sugar by approximately 50%. The specificity of the metal ion of Has multiplied to pH 7, figure 8 is determined. Figure 8 shows metallic dependence of HAS activity. HA production is measured in the presence of increasingly higher concentrations of Mg (circles) or Mn (squares) ion. The membranes (46 μg of protein), are previously washed with 0.2 mM EDTA and incubated in a mixture of metal ion in 50 mM Tris, pH 7, UDP UDP 120 μM (4.5 x 104 dpm / assay), and UDP Glc -Nac 300 μM for 1 hour. The metal-free background present (22 dpm) is subtracted from each point. Mn is more effective than Mg. Under metal free conditions in the presence of EDTA, there is no detectable incorporation of radiolabeled precursor into the polysaccharide (<0.5% of the maximum signal). The Mn2 + ion provides the highest rates and incorporation at lower ion concentrations for the metals tested (Mg, Mn, Co, Cu, and Ni). The Mg2 + ion provides approximately 50% of the stimulation of Mn2 +, but at 10 times higher concentrations.

The Co2 + or Ni2 + ions at 10 mM, support low levels of activity (20% or 9%, respectively, of Mn2 * 1 mM assays), but the membranes supplied with 10 mM Cu2 + are inactive. In fact, mixing 10 mM Cu and 20 mM Mg2 + with the membrane preparation results in almost no label incorporation in the polysaccharide (< 0.8% Mg only value). The initial characterization of HAS of P. mul tocida is carried out in the presence of Mg2 +. The binding affinity of the enzyme for its sugar nucleotide precursors is determined by measuring the apparent K ^ value. The incorporation of [14C] GlcA or [3H] GlcNAc within the polysaccharide are monitored at varying concentrations of UDP-GlcNAc or UDP-GlcA, respectively, Figures 9 and 10, respectively. Figure 9 shows activity dependence of HAS in the concentration of UDP-GlcNAc. Membranes (20 μg of proteins) are incubated with increasing concentrations of UDP-GlcNAc in buffer containing 50 mM Tris, pH 7, 20 mM MgCl2 and 800 μM UDP-GlcA (1.4 x 105 dpm of 14 C) for 1 hour. The background reductivity is subtracted from each point (identical test but without UDP-GlNAc added). The highest specific incorporation rate in HA (average of approximately 780 dpm / hour) in the titration is defined as Vmax for 100% normalization. Figure 10 shows the dependence of HAS activity on the concentration of UDP-GlcA. In experiments parallel to those described in Figure 9, increasing amounts of UDP-GlcA are incubated with 1 mM UDP-GlcNAc (2.7 x 105 dpm of 3H) under the same general buffer and assay conditions. The background radioactivity is subtracted from each point (assay without adding UDP-GlcA). The data is presented in figure 9. The specific incorporation to averaged Vmax is approximately 730 dpm / hour. In buffers containing Mg2 +, the apparent KM values ~ 20 μM for UDP-GlcA and ~75 μM for UDP-GlcNAc are determined using the Hanes-Woolf plots ([S] ¡v versus [S]) of the titration data shown in Figure 11. Figure 11 shows the estimation of the graph of Hanes-Woolf of Vmax and KM. The specific incorporation data used to generate Figure 9 (tables) and Figure 10 (circles) are plotted as [S] / v versus [S]. The parallel slopes, which correspond to l / Vmax, indicate that the maximum speeds of sugar nucleotide precursors are equivalent. The intercept with the x-axis, which means -KM, provides KM values of 75 and 20 μM for UDP-GlcNAc and UDP-GlcA, respectively. The values of Vmax for both sugars are the same because the slopes are equivalent. In comparison with the results of the Mg2 + assays, the KM value for UDP-GlcNAc is increased approximately 25-50% to ~105 μM, and the Vmax increases by a factor of 2-3 times in the presence of Mn2 +. These values are shown in Table I. TABLE I As previously stated, the HA capsules of the pathogens of P. multocida and S. pyogenes are virulence factors that help in the evasion of host defenses. The HA synthase enzyme from any bacterial source uses UDP-sugars, but it has a slightly different kinetic optimum with respect to pH and metal ion dependence as well as KM values. Both enzymes are more active at pH 7; however, PmHAS works best on the alkaline side of the optimum pH until at least pH 8.6. On the other hand, spHAS continuously shows more activity at slightly acidic pH and is relatively inactive at pH above 7.4. The enzymes of P. multocida use Mn2 + more efficiently than Mg2 + under the conditions of in vitro testing. PmHAS binds to UDP sugars more tightly than HasA streptococcal. The iCM values measured for pmHAS in crude membranes are approximately 2-3 times lower for each substrate compared to those obtained from HAS that is found in streptococcal membranes. 3. Use of PmHAS for vaccinations The DNA sequence of PmHas can also be used to generate potential attenuated vaccine strains of P. multocida bacteria after deletion of the normal microbial gene by homologous recombination with a broken version. Additionally, the PmHAS DNA sequence allows the generation of diagnostic bacterial typing for related P. multocida types that are pathogenic agricultural poultry, cattle, sheep and pigs. There are at least five different types of P. multocida bacterial pathogen with different capsular antigens. The cholera of poultry or avian pasteurellosis, which is caused mainly by type A strains, is a widespread disease that economically damages commercial poultry. An acute onset or attack of poultry cholera is usually detected only when the birds suddenly collapse as the symptoms often seem just a few hours before death. Although little is known about the molecular basis for the virulence of P. multocida, apparently one of the virulent strains of the pathogen possesses a polysaccharide capsule, its colonies show a mucoid or "wet" morphology on agar plates. The white blood cells have difficulty in surrounding and inactivating the bacteria and the complement complex can not make contact with the bacterial membrane to cause lysis. The main component of the capsule of P. mul Tocida Type A Carter, which is responsible perhaps for 90-95% of the cholera disease of poultry is the polysaccharide HA and HA does not induce an immune response in virtually all the members of the animal kingdom. Even if the immune response occurs, it would present a problem for the bird due to the repercussions of autoimmune reactions. Strains of P. multocida type A are also prominent causes of porcine and bovine pneumatic pasteurellosis and displacement fever in cattle. Two other capsular types of P. mul tocida, the type D and type F have been studied to a lesser degree, but they are prevalent pathogens in the United States. Type F is isolated in approximately 5-10% of cases of poultry cholera. Type D also causes pneumonia in cattle, sheep and pigs. The isolates of pneumonic lesions of these domestic animals are analyzed for the type of capsule, and approximately 25-40% are type D and the rest are type A. Additionally, the type D strain is intimately involved in porcine atrophic rhinitis. The capsules of these type D and type F microbes are composed of different polysaccharides with unknown structures, but apparently they are similar to chondroitin, a molecule prevalent in the body of vertebrates. The general main structure of chondroithane, repeated units (ßl, 4) GlcA (ßl, 3) GalNAc, is very similar to that of HA. Therefore, it is not surprising that type D and F polysaccharides are poorly immunogenic. Typically, antibodies against bacterial surface components derived from previous infections (or vaccinations) are an important means for soft blood cells to adhere to bacteria during phagocytosis; this trait usually makes the immune system extremely effective in fighting the disease. Therefore, the presence of capsules composed of non-immunogenic polymers, such as HA or chondroitano-like sugars, compromise the efficiency of all phases of host defenses. Streptococcus pyogenes, a human pathogen, also uses an HA capsule as a molecular "imitation" to protect against host defenses. The mutants of S. pyogenes acapsulares can not survive in blood and are 100 times less virulent than the wild type in mice. Many strains of virulent E. coli possess capsules made of other polysaccharides that mimic host molecules and help the cell bypass the immune system. The capsules of all these pathogenic bacteria are intelligent evolutionary adaptations that must be solved in order to eliminate the disease. Previous research has focused on the capsule of P. multocida and its role in virulence. As for poultry strains type A, encapsulated wild type bacteria and the various acapsular forms are tested for their ability to survive exposures by isolated defenses of the host (white blood cells and complement) or to cause infection and death of live poultry. Acapsular bacteria typically are: (a) mutants that arise spontaneously; (b) chemically induced mutants; or (c) wild-type bacteria treated with hyaluronidase [HAase], an enzyme that specifically degrades HA. In general, type A bacteria deficient in capsule are more likely to be destroyed by isolated host defenses in vi tro. The turkey serum destroys both mutant and HAase-treated cells, while the wild-type cells multiply. The complement system is involved, since the destruction capacity is lost by heat or a calcium chelation treatment of the serum before the incubation with the bacteria. The fact that the encapsulated wild-type cells consume or reduce the level of complement in serum without inactivation indicates that the complex binds but can not lyse the cells. Pay and heterophile macrophages phagocytose acapsular variants and cells treated with HAase more avidly than wild-type cells. In tests on live animals, the spontaneous mutants are 103 to 105 times less virulent than the corresponding wild type parent strain, determined by LDS0 (ie, the fatal dose for 50% of the animals tested).

This huge difference shows the importance of the capsule in the pathogenesis of type A strains. The fate of bacteria in live turkeys also depends on the encapsulation; only the wild-type cells survive in the liver. Fifteen to twenty-four hours after the injection, it is found that the wild type cells in blood are 105 times higher in concentration compared to the unencapsulated mutants. Another role for the HA capsule is adhesion and colonization. Certain cells in the body of vertebrates have specific proteins that bind to HA on their surface; and bacteria can potentially adhere to the host via this interaction between the protein and HA.

Similarly, capsules of P. multocida and type F are implicated as virulence factors that give the microbes resistance to phagocytosis. When type D or F cells are treated with chondroitinase, the microbe loses its capsule and is more readily phagocytosed in vi tro. In addition, these polymers are not strongly immunogenic. For in vivo tests, encapsulated type D strains produce more severe nasal lesions in pigs and have a much lower LD50 in mice, compared to non-encapsulated variants (102 versus 107"8 cells, respectively). mutants type A and D studied before, however, the genetic nature of their defects is unknown, and there is no easy method to map mutations, particularly with chemical mutagenesis, it is likely that there is more than one mutation in a "mutant" Furthermore, it has not been demonstrated that the production of HA has been completely eradicated in the "acapsular" mutants, the thin capsules are not detectable by colony morphology, optical microscopy or chemical tests that may exist, more sensitive radiometric tests are required. and of floating density for the detection of even small capsules.With the use of these new methods, it has been determined that a strain used In several virulence tests that have been reported to be encapsulated, it actually has a very thin HA capsule. Therefore, it is important to determine the effects of a truly acapsular strain. Historically, the genes involved in the production of the capsule of P. mul tocida are not known. Several genes residing in the capsule locus of other bacteria in a related genus, Haemophilus influenzae, have been mapped and sequenced, but the molecular details of the biosynthetic apparatus are not available. Even in E. coli, a well-studied gram-negative organism, the exact role of each putative gene product is not well understood, although the loci of capsule formation have been mapped in a general manner and the DNA sequences have been obtained for Various types of capsule. The cloning of the sequence of the HA biosynthesis locus of Streptococcus pyogenes has been reported. This microbe, like P. multocida, uses an HA capsule to evade human defenses. The HA operon contains three genes arranged in a battery of approximately 4 kb of DNA. The first gene, hasA, codes for 45.1 kDa of HA synthase that polymerase the two sugar nucleotide precursors, UDP-GlcA and UDP-GlcNAc, to form the HA polysaccharide. The second gene, hasB, codes for a UDP-glucose dehydrogenase of 45.5 kDa, which converts UDP-glucose (UDP-Glc) into UDP-GlcA for HA biosynthesis. The third gene, hasC, codes for a UDP-glucose pyrophosphorylase of 34 kDa, which forms UDP-Glc from UTP and glucose-1-phosphate. There is an auxiliary enzyme dedicated to form UDP sugars for capsule biosynthesis; another "constitutive" gene residing elsewhere in the chromosome supplies UDP-Glc for the normal metabolic pathways of the bacterium. UDP-GlcNac is present in all eubacteria due to its role in cell wall synthesis. Thus, HA synthase and dehydrogenase are the only two exogenous proteins that are needed to direct the synthesis of HA polysaccharide in heterologous bacteria. The HA synthase of S. pyogenes, which is predicted to be a membrane protein with transmembrane helices, probably polymerizes HA and transports the growing polysaccharide chain to the outside of the cell. As previously discussed before, the gene responsible for the production of HA in P. multocida has been isolated and sequenced. (See, for example, SEQ ID NOS: 1 and 2). This gene is described and claimed as part of the present invention. As also discussed previously, polymers of P. mul-tocida capsules have a dilemma for host defenses. Using the sequence information of the PmHAS gene, recombinantly produced P. multocida strains having the "inactivated" HA synthesis gene will break the bacterial capsular synthesis of P. multocida. Using the "inactivated" strain as a vaccine will allow the host organism to repel exposures by the pathogen in the field. As discussed earlier, Tn916, a versatile and proven mutagen, is inserted into the chromosome of P. multocida in various apparently almost random positions. Tn is introduced into the cells in a "suicidal" plasmid - that is, it can not be replicated in P. multocida - via electroporation. Tn immobilized or driven outside the plasmid into the genome at a frequency of approximately 4,000 events / microgram of DNA. The resulting progeny possesses the? 916 tetracycline resistance gene and is easily selectable by the drug. It is also discussed before that a panel of independent transposon mutants defective in the biosynthesis of the capsule of the virulent parental strain are generated. After using a combination of visual and biochemical analysis of approximately 105 transfected colonies, two classes of capsule defects have been found that result in microcapsular or acapsular mutants. The first class (seven independent strains) have a very small HA capsule, and are therefore called microcapsular. The encapsulated wild-type strain produces large mucoid iridescent colonies on plates in medium and the individual cells form a capsule with a thickness approximately equal to the diameter of the body of the cell, as measured by optical microscopy. In comparison, microcapsular strains form smaller iridescent colonies that appear a little drier on medium plates; capsule thicknesses of the individual cell are in the order of a quarter (or less) of the wild type. Four mutants (four independent strains) appear as truly acapsular forming small, dry colonies on medium plates. No capsule is detected by optical microscopy. The floating density of the acapsular strain, which depends on the state of encapsulation, is equivalent to the wild-type cell that are extracted from its capsule by treatment by hyaluronidase. These strains also lack HA synthase activity; the exogenous radiolabeled UDP-sugar precursors are added to the preparations derived from these mutants but HA polysaccharide is not formed. Two of the acapsular mutants, TnH and TnL possess the interesting property that, at a frequency of approximately 10"3, occasions with the phenotype of the wild-type capsule appear on the plates of half-reversions. wild type and are considered by optical microscopy and radiometric assay for HA polysaccharide. The molecular explanation is that occasionally, Tn purely cuts the capsular gene (not added or base deleted) and integrates elsewhere in the chromosome. The resulting encapsulated progeny in medium plate is easily observable.This phenomenon of reversion is a classical genetic test that Tn, in these two strains, is responsible for mutating an important site necessary for the synthesis of the capsule. this relative instability, a mutant derived from Tn is not suitable for an attenuated vaccine strain, since virulent forms can be generated with certain frequency Southern blotting is used to map the position of Tn in both the original and the encapsulated reversal mutants. TnH and TnL have a Tn element in the same locus as considered by the pattern after digestion by HipdlII or BstXI, as shown in Figure 12. Figure 12 is a Southern blot mapping of the Tn mutants. Chromosomal DNA from a classification of capsule mutants, encapsulated reversals and controls, are digested with HipdlII. The DNA is separated by gel electrophoresis and subjected to Southern blot analysis. The Tn probe recognizes two bands for each transposon due to an internal restriction site (forming a large arm of 10 kb and a small arm of 5 kb). The Tn probe does not hybridize with DNA of the parental strain without a Tn (lane 0). Multiple DNA preparations derived from separate colonies of the TnH (H) and TnL (L) acapsular mutants of the individual encapsulated reversals (indicated with underlined lines) are processed. All mutants have a unique Tn insertion element, except for TnL, which usually has 2 copies of Tn (one of the subcultured strains has 3 copies). TnW (W) is a control strain that contains mucoid Tn. The positions of the HindI I I lambda (?) Markers for 23.1, 9.4 and 6.6 kb (from the top to the bottom) are those that are marked. The element Tn and the acapsular mutants H and L (which have no HA synthase activity, and the representative microcapsular mutant, TnD (D), are mapped in the same position.) Upon the reversal of the mucoid phenotype, the relative element Tn is moves to a new position in each case.The broken DNA of the mutants is isolated at this locus and probes are generated for the capsule genes.The subsequent analysis of the sequence determines that the TnH and TnL insertions are separated by approximately 1 kb. In all cases, reversals of these mutants lose Tn in the original position and gain a new Tn at different sites (ie, figure 12, lanes with underlined letters) Alternatively, there has never been an observed reversion of any of the microcapsular mutants The Tn responsible for the totality of the microcapsular mutants (typed by TnD) mapped to the same HindIII fragment of 17 kilobases in the TnH and TnL mutants. ocalization is also confirmed by mapping with BstXI.

In the other acapsular mutants, TnA and TnG, the Tn elements are located in other irrelevant genes and the locus of the HA capsule becomes nonfunctional due to spontaneous mutations. Occasional spontaneous mutation is expected; In fact, in similar studies of the locus of the esptreptococcal capsule, 12 of 13 strains are the result of spontaneous mutations. TnSIS insertional mutagenesis is used to identify and clone the DNA involved in the biosynthesis of the capsule HA of P. mul tocida. In carrying out this task, three stages are used: (i) sequencing of host DNA at a Tn insertion site using a primer corresponding to DNA at the terminal part of Tn916, (ii) designing PCR primers for the Tn916 gene. capsule based on new sequences to amplify the DNA segment between the two elements of Tn; and (iii) analyze the wild type genomic libraries in lambda virus for a functional clone using the capsule locus-specific PCR product, as a probe of hybridization. The key step to obtain P. multocida DNA adjacent to Tn is the use of a recently formulated direct sequencing technique which has been described fully in DeAngelis, P.L. (1998) "Transposon Tn916 insertional mutagenesis of Pasturella multocida and direct sequencing of the disruption site", Microbial Pathogenesis, which is incorporated herein by reference. The P. multocida genome of all capsular types contains many sites for the Hhal restriction enzyme; therefore, almost every DNA fragment in the digested less than 7 kilobases (kb) and is shown in figure 13, lane "0". Figure 13 shows templates of chimeric DNA for sequence analysis of Tn cleavage sites. Through this method, the DNA sequence of any gene interrupted by the element Tn 916 can be obtained quickly and directly. The method capitalizes the differential sensitivity of the element Tn and the genome of P. multocida type A for the restriction enzyme Hhal. The Tn element of 16 kb is the only one with a Hhal site that results in 12 and 4 kb fragments when digested. Therefore, any gene interrupted by the element Tn will have an additional 12 kb of DNA. The increase in the size of the Hhal fragment allows the easy resolution of the gene labeled Tn of the rest of the chromosomal DNA by electrophoresis in conventional agarose. This 0.7% gel shows that the Hhal-digested pattern of the chromosomal DNA of the parental strain without a Tn (lane 0), and several mutants containing Tn (lanes with mutant Tn). The lambda / HindIII markers (lane S) are indicated in kb. The fragments of Chimeric Tn / genomic DNA that migrate to approximately 13-17 kb (marked with the arrow) are found only in the Tn mutants. Note that lane L has three chimeric bands, this particular mutant has three elements Tn (see figure 12). The chimeric DNA can be isolated and used directly as a sequencing template; no cloning or PCR is required. The resulting large chimeric DNA molecule, which is easily separated from the rest of the small genomic fragments by agarose gel electrophoresis, serves as the template in the cycle sequencing reactions. A sequencing primer corresponding to the right N-terminal part of Tn916 directs the elongation outward within the broken DNA. Therefore, the sequence data at this mutant DNA cleavage site can be obtained systematically without PCR amplification or cloning of the template DNA. The new sequence information is used to design primers for PCR for amplification of the DNA region between the TnL and TnH mutants. A specific product of 1 kb is used as a hybridization probe to obtain a 5.8 kb portion of the biosynthesis operon of the P. mul-type A capsule, as indicated in Figure 14, which shows the locus scheme of HA biosynthesis of P. multocida type A. As shown in figure 14, the insert of a clone of genomic DNA type A that can direct the biosynthesis of HA in E. coli is the one that is sequenced. It is found that open reading frames encode for: two proteins similar to E. coli molecules involved in the transport of polysaccharides, Kps and KfaA; an HA synthase that polymerizes for the polysaccharide HA; and a precursor of the forming enzyme, UDP-glucose dehydrogenase. The suppression analysis of the original plasmid shows that an intact HA synthase is essential for the production of HA in heterologous bacteria. The location of the original Tn inserts corresponding to TnH and TnL are marked with stars. The Tn insertion events apparently cause polar mutations that stop the expression of the HA synthase toward the 3 'end and the dehydrogenase genes. Therefore, by using sequence analysis, the intact open reading frames of novel PmHAS and a putative polysaccharide transport homolog, similar to E. coli KfaA are those found. The data also show that a UDP-Glc dehydrogenase homologue which constitutes a precursor of UDP-GlcA, and another transporter protein, and the homologue of the kps gene of E. coli are present near the HA synthase. The unique 110-kDa protein of P. mul-tocida, PmHAS, directs the production of the HA capsule in E. coli. The capsule of the recombinant cells produced with PmHAS in the plasmid is coarse as that of the virulent wild type strain. The capsular material is considered authentic HA, due to its susceptibility to specific digestion with HA lyase and its reactivity with protein that binds selectively to HA. Interestingly, PmHAS is not very similar to other HAS at the amino acid level. In order to make stable isogenic mutants of P. multocida, a modification of a mutagenesis method used with P. haemolytica is used. An inactivated cassette is produced for the inactivation directed by a double crossing of the HA synthase. A promoter-free chloramphenicol resistance gene (cat) is inserted into the middle part of the entire open reading frame for PmHAS (at the XHol site) and cloned into a plasmid (pKK223-3) that does not replicate stable way in P. mul tocida. The plasmid is transformed into the encapsulated wild-type strain and the cells plated in medium with chloramphenicol. When integrated into the target gene, the intact PmHAS protein is no longer formed. The 'cat gene is transcribed by endogenous capsule gene promoter; the gene towards the 5 'end, UDP-glucose dehydrogenase does not seem to be affected. Therefore, true isogenic mutants are formed. Three isogenic, acapsular mutants have been isolated. None of these mutants is detected with capsules under optical microscopy and Indian ink staining. HA synthase breaks down to both DNA and biochemical levels. See, for example, Table II. By Western blot analysis using an antibody directed against a portion of the PmHAS enzyme, the inactive acapsular mutant is lost from the approximately 110 kDa band, the PmHAS enzyme, which is found in the wild type parent strain. In combination with the data in Table II (the lack of polysaccharide production), functional PmHAS is not found in the inactivated strain. Certain regions are common or very similar between genes of various capsular types. This is shown in the Southern blot analysis of type D or F DNA shown in Figure 15. As shown in Figure 5, the chromosomal DNA of Type A, F or D strains is digested with either HindIII or EcoRI (right or left lane, respectively, for each probe) and subjected to Southern blotting. Probes of PCR product labeled with digoxigenin that correspond to the regions of either the kfaA (K) homologue or the HA synthase (H) type A genes are used to detect homologous sequences in bacteria or other types of capsules (bands marked with stars). The homologs of KfaA are evident in both F and D types. A very similar synthase homology is found in type F, but not in type D. The probes are suitable for analyzing libraries. The lambda / HindIII standards are marked in kilobases. Type F has regions that are similar for both probes, whereas type D is only similar for the transporter protein probe. PCR is used with several sets of primers corresponding to type A sequences to amplify type D or F genomic DNA, as shown in Figure 16. PCR of heterologous DNA with type A primers is shown in Figure 16. Several pairs of primers are used that correspond to the homolog gene kfaA (panel A) or the gene of HA synthase (panel B) of the strain type A, to amplify the genomic DNA isolated from several additional strains of P. multocida with different types of capsule. 40 cycles are performed (94 ° C, 30 sec, 42 ° C, 30 sec, 72 ° C, 60 sec) polymerase chain reaction with the enzyme Taq. The reaction mixtures are separated on a 1% agarose gel and stained with ethidium (lanes: A, Type A, D, Type D; F, Type F; 0, without template control). The standards (S) are a 100 bp ladder, the 1 and 0.5 kb bands are marked with arrows. The pairs of P-I primers show products of all three capsule types, but the D-type product is smaller than that of other products. The P-II and P-III pairs only amplify DNA type A and F. In contrast, the pairs P-IV and PV amplify only type A. It seems that the capsule loci type A and F are more similar to each other than to type D. The PCR products of the PI primer pair will serve as good hybridization probes for the capsule locus of other types. Not all primer combinations provide PCR products with the heterologous template DNA. The 0.2-1 kb portions of the F-type genes encoding the HA synthase of the capsule polysaccharide transporter analog are amplified. A 1-kb region of the D-type genome encoding the carrier protein is also amplified. Sequence analysis of several PCR products shows homologous but distinct sequences. In general, these data suggest that type A and F strains are more related to each other and not as similar as with type D. Sequence comparison of KfaA type A and F homologs and KfaA of E. coli is shown in Figure 17. The PCR product that is generated by F-type amplification with the PI primer set (see Figure 16) is gel purified and sequenced with one of the original primers. It is found that the type A and F sequences are very similar at the amino acid level; this partial alignment of the protein sequences shows that in this region, the sequences are mostly identical with some poor matches (the differences are underlined in Figure 17). In general, the sequences of P. multocida are very homologous to the KfaA protein of E. coli, which is involved in the transport of polysaccharides (the identical residues are with bold in Figure 17). These PCR products are also useful as hybridization probes to obtain functional capsule loci from genomic libraries of type A or F. The cloned DNA also allows the construction of inactive gene plasmids: in which the resulting mutant strains are useful for virulence tests or for vaccines. The production of the bacterial capsule of P. multocida involves at least the following steps: (i) synthesis of sugar nucleotide precursors; (ii) polymerization of precursors to form the capsular polysaccharide; and (iii) exporting or transporting the polysaccharide to the extracellular space where capsule assembly occurs, of course, there are potential regulatory genes or factors that control enzyme levels or enzymatic activity, but the focus is on the main structural enzymes of the pathway. In E. coli, the candidate type II capsule genes encoding enzymes for this process are located together at a single site on the bacterial chromosome. The strains of E. coli that produce capsules with different structures have different enzymes for the stages (i) and (ii) above, but all seem to share a common transport / export machinery for step (iii). It has been found that in S. pyogenes, a single integral membrane enzyme polymerizes precursor sugars and transports HA polysaccharide through the membrane. P. mul-type A has four different genes that are involved in each of the three biosynthetic stages for the production of bacterial capsules. (See Figure 14). The similarity of the P. multocida polysaccharide transporter to the E. coli homolog at the protein level suggests that the general functions of some other capsule genes may also be similar to these two species. The role of the capsule has been determined as a virulence factor in poultry cholera. In order to avoid chinks and gaps found in bacterial capsule studies and in virulence, the defined mutants are compared with wild-type microbes. Isogenic mutants type A have broken capsule genes that are tested for their ability to avoid pre-existing or pre-immune host defenses in vi tro, as well as to infect live poultry in vivo. Stable isogenic mutants are produced according to the methods described in the foregoing. Using a broken version of the PmHAS gene in a plasmid (see Figure 18) and homologous recombination, a recombinant P. mul tocida strain is generated which has lost the ability to produce a hyaluronan capsule. The strain is further analyzed at both DNA and biochemical levels. We found that the functional gene for HA synthase is replaced with a defective gene containing a cassette of interruption by both Southern blotting and PCR analysis (See Figure 19). Figure 19 shows the confirmation of the gene break. Panel A is a Southern blot analysis. The chromosomal DNA of various strains is digested with HindIII, separated on a 0.7% agarose gel and transferred to nitrocellulose. The blot is hybridized with a HAS gene probe for P. mul-tocida. Two bands are detected due to an internal restriction site HindIII in the PmHAS gene. Lane M is the mucoid transformant; the KO lane is the inactive mutant to encapsulate; Lane P is the parental strain. The addition of a 670 bp cat cassette causes a displacement of the upper band size to the KO rail (marked with an arrow). Panel B of Figure 19 is a PCR analysis. The DNA in the cell lysates of various strains is amplified by 35 cycles of PCR with a pair of oligonucleotide primers flanking the Xhol site of PmHAS. The length of the amplicon from the normal wild-type gene is 650 base pairs. PCR reactions are separated on a 1% agarose gel and visualized with ethidium bromide. Lane M is the mucoid transformant; the KO lane is the inactive mutant to encapsulate; lane P is the parent strain; lane C is the cloned PmHAS plasmid control; and lane S are the size standards. The PCR product produced by the inactive mutant template is approximately 1,300 bp (marked with an arrow); This band is composed of a cat cassette of 670 bp and 650 bp derived from PmHAS. A wild-type amplicon is not detected in the reaction of the inactive strain, therefore, homologous recombination mediated by a double-crossing event occurred.

In addition, using the sensitive radiochemical assay for the HA polysaccharide, it has been found that the mutant strain does not produce HA, and is shown in Table II, which includes HA production of various strains.

Table II The strains included in Table II are overnight cultures of several strains which are tested for the presence of HA polysaccharide using the specific radiometric assay indicated above. The cultures are normalized by spectrophotometry and the data are presented as the concentration of HA in a culture with an absorbance of 1.0 at 600 nm. The wild type parent or a mucoid encapsulated transformant synthesize substantial amounts of HA.

In contrast, detectable HA is not produced by the inactive acapsular mutant (KO). Therefore, the role of the capsule in virulence can be determined. The methodology used can also be used to construct other mutants of P. multocida and a person ordinarily skilled in the art, given the description of the present invention, can carry out such a task. Animal tests have compared the in vivo pathogenicity of the mutants with the complemented mutant controls and with wild-type P. multocida type A. The inactive strain of Pasturella multocida type A ATCC 15742 (which causes cholera in poultry) is sent to the USDA Research Station in Ames, Iowa for virulence testing. Using an objective homologous recombination, the biosynthesis of the capsule of the inactivated cell has been broken, and the inactivated strain is predicted to be 1,000 times less virulent. A virulence test is carried out to verify the safety of the KO strain as a vaccine strain. Turkey eggs are harvested in clean conditions and raised until the age of two weeks. The chicks are injected with various concentrations of bacteria (either parental wild type or inactive strain). The bacterial count is enumerated by spectroscopy and colony counts after sowing and plating. The animals are injected intramuscularly and placed in a biological confinement cage. Inoculated chicks (groups of 6 or 7 per microbial dose ranging from approximately 80 to 107 bacteria in 10-fold stages) are observed. The general appearance, activity level and morbidity are verified for 6 days. Dead or dying birds are autopsied and tests are performed to verify the presence of lesions, abscesses and organ failure. The results of the in vivo experiments are summarized in Table III.

Table III The point of this type of test is to determine the general tendencies of infection with respect to the encapsulation of the pathogen. For each determination, white turkeys are inoculated with a titrated amount of bacteria, IM. The symptoms and death of the turkeys are measured and tabulated in order to compare the relative virulence of the mutants. Protective assays will be carried out in order to determine whether immunized turkeys can survive exposure to virulent wild-type organisms. Inactivated type A strains that infect cattle and rabbits have also been prepared. The tests will be carried out in vivo in order to determine both the pathogenicity of these inactivated strains as well as the protection assays to determine if immunized animals can survive exposure to virulent wild-type organisms. Two main types of protection experiments will be carried out. The first is performed with passive immunization. A chicken is infected with the KO strain of potential vaccine and a sample of its serum is taken (with protective antibodies) approximately 1-2 weeks after inoculation. This serum or the purified purified antibodies are injected into an unexposed chicken. The unexposed chicken is exposed with the wild type strain. The birdIf you receive the protective antibody, you will survive exposure to the wild type bacteria that is otherwise deadly. Second, active immunization is carried out. In this case, the same chicken is infected sequentially with the KO strain of potential vaccine, and a few weeks later, the bird is exposed with a normally lethal dose of wild-type bacteria. In this case, antibody-mediated and cell-mediated immunity is tested. Using the present invention, it is predicted that there are similarities in the capsule loci of the various encapsulated types of P. multocida due to the close structural similarity of the polysaccharides. The present invention also relates to the gene for P. mul tocida type F homolog ("PmCS"). The information of the PmCS sequence is provided in the SEC. FROM IDENT. NO: 3. The type F gene is approximately 85% identical to the type A gene and the sequence comparison is shown in figure 20. It is found that this DNA-level homology between the cloned type A capsule genes and certain regions of type D and F genomes by Southern blotting and PCR, as shown in Figures 15, 16 and 17. The F-type genomic DNA libraries in lambda phages are analyzed to isolate loci from analogous capsules. Genomic D-type DNA libraries in lambda phage are analyzed to isolate the loci from homologous capsules and a person ordinarily skilled in the art will appreciate and understand that the D-type capsule loci can be determined in exactly the same manner as with type A and F. The PmHAS sequences of type A and F are 89% similar. The gene for type F polysaccharide synthase is obtained by using a PCR product hybridization probe, Figure 16, joining the HAS homologue and the Kfa homologue. A 3 kb amplicon is produced using genomic DNA from the F type strain and the appropriate primers from Kfa and the synthase regions. This material is labeled with digoxigenin and used to obtain a clone and subsequently a plasmid from a genomic F-type DNA library in a library that expresses ZAP lambda (described for type A cloning.) The hybridization clone is positively sequenced.As in the case of the HA synthase type A gene, PmHAS, the functionality is verified by expression in the vector pKK223-3 (Pharmacia) in E. coli This enzyme is found to incorporate UDP-GalNAc and UDP-GlcA within the high molecular weight polymer, as expected for a chondroitin molecule.Sypsas of capsular polysaccharides are monitored with antibodies and transfer analysis Western.Antibodies are generated against a synthetic peptide corresponding to a shared homologous region (amino acid residues 12-20) of the synthase enzymes.The tests by Western blot confirm that P. mulotida type A and type F have an immunoreactive protein of 110 kDa, by SDS-PAGE Figure 21 is a Western blot analysis of native and recombinant PmHAS proteins, native PmHAS and various proteins derived from truncated PmHAS. ecombinants manufactured in E. coli are compared in SDS-PAGE gels. For the recombinant samples, the total lysate (T), the membranes (M) and the cytoplasm (C) are subjected to Western blot, with antibody against PmHAS. The original protein found in native mulleted Pasturella (Pm, lane W, marked with an arrow) migrates to approximately 110 kDa; the inactive vaccine strain (KO lane) has lost this band. The native PmHas and the recombinant version lose a portion of the carboxyl terminal portion (Pm? CC) has HA synthase activity. The other truncated constructs are inactive. 4. Use of PmHAS in diagnostic applications The present invention also relates to the generation of useful probes because they facilitate the identification of P. multocida type A, D and F or P. haemolytica in the field. The diagnosis from which the particular strain is present in animals is currently determined by serology, agglutination or DNA fingerprints after restriction analysis. The first two methods can be problematic, often provide false identification and vary depending on the source of typed antiserum. The capsular serology of Cárter types A, D and F does not even use an antibody because these polymers are very poor immunogens. Instead, laborious trials involving enzymatic digestion or flocculation of cells with acriflavine are routinely used. The fingerprint of DNA is accurate, but it is based on extensive knowledge of numerous strains of types on file. Capsule-specific primer sets will be used to easily perform these epidemiological studies, especially by using rapid and easy PCR analysis to identify pathogenic isolates in half a day with minimal handling and no subculture. Once the pathogen is identified, a more informed decision can be made regarding the choice of antibiotic or vaccine. The utility of using DNA capsule information to quickly determine the type of P. multocida is obvious in light of the problems with current typing methods. Whether hybridization or PCR-based typing are considered as practical, sensitive and rapid. A specific embodiment would be to place a labeled or appropriately labeled DNA synthase probe (or by extension a capsule locus gene which differs between the type of capsule) which, by virtue of its unique condition, can be differentiated under appropriate hybridization conditions (for example, complementarity of genes and probes will hybridize to provide a signal, while the non-identical genus of another capsular type will not hybridize and therefore there will be no signal). Another specific antibody would be to design PCR primers that can differentiate capsule types. An amplicon of the correct size can mean a particular type of capsule; The lack of amplicon means another type of different capsule. In the current state of the art, several pairs of PCR primers provide bands of differentiable size and different in a single reaction and can be considered. Such a multiple method can allow many reactions to be carried out simultaneously. Knowledge of the DNA sequence of the various capsule biosynthesis loci, in particular, the synthases, allows these tests to quickly differentiate the various pathogenic strains. Therefore, it will be apparent that, according to the invention, isolated and sequenced PmHAS has been provided, as well as methods for making and using PmHAS and inactive P. multocida strains that fully satisfy the objectives and advantages set forth above. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to encompass all such alternatives, modifications and variations that are within the spirit and broad scope of the appended claims. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.LIST OF SEQUENCES +1? 1 KCQEKL AHP s s s AHLSVNKEE VN 4 KVNV c D s l PLDIATQLL SNVKKL 7 VLSD s EKNTLKNK w K ^ s E LLTEKK NAEVRAVALVPKDFPDLVLAP L 0 r 3 PDHVNDFTWYKKRKKRLGIKP F. 6 QKVGLSIIVTTFNRPAIL s lTLA 9 CLVNQKTHYPF and VIVTDDGSQED 2 LSPIIRQYENKLDIRYVRQKD NG 5 FQASAARNMGLRLAKYDFIGL LD 8 CDMAPNPLWVHSYVA and LL and DDDL 1 TII 'GPRKYIDTQHIDPKDFLNNA 4 s LLE s LPEVKTNN s VAAKG and GTV 7 s LDWRL and QFEKT and NLRLSD s PFR 0 FFAAGNVAFAKK w LNKSGFFD and e 63 FNKWGGEDV and FGYRLFRYGSFFK 86 TIDGIMAYHQ and PPGKEN and TDREA 9 GKNITLDIMREKVPYIYRKLL PI 2 EDSKINRVPLV s IYIPAYNCANY 55 IQR c VDSALNQTVVDL e VCICND 78 GSTDNTL e VINKLYGNNPRVRIM 01 SKPNGGIASASNAAVS F A K G Y Y I 24 G Q L- D S D D Y L e P D A V e L C K E F L ~ K 47 D K T L A C V Y T T N R N V N P D G S L I N N 70 G Y N W P E F s R e K L T T M M A H H F R 93 F T I R A W H L T D? FNEKIENAVDYD 16 MFLKLSEVGKFKHLNKICYNR VL 39 HGDNT s IKKLGIQKKNHFVVVNQ 62 SLN n qgi t YYNYD and FDDLD and SRK 85 YIFNKTAEYQ ee IDILKDIKIIQ 08 NKDAKIAVSIFYPNTLNGLVK KL 31 NNI 1 EYNKNIFVIVLHVDKNHL t 54 PDIKKEILAFYHKH 0 VNILLNND 77 ISYYT s NRLIKTEAHLSNINKL s OO QLNLN c EYIIFDNHDSLFVKND s 23 YAYMKKYDVGMNFSALTHDWI EK 46 INAHPPFKKLIKTYFNDNDKS M 69 NVKGA s QGMFTYALAHELLTI r 92 KE v IT s CQSID s VPEYNTEDI w F 15 QFALLIL e KKTGHVFNKTS t LTY 38 MPWERKLQ w TNEQI e SAKRG and NI 61 PVNKFI 1 N s ITL * +1? -18 ATTTTTTTAAGGACAGAAAATGAATACATTATCACAAGCAATAAAAGCATATAACAGCAATGACT? TCAA 52 TTAGCACTCAAATTATTTGAAAAGTCGGCGGAAATCTATGGACGGAAAATTGTTGAATTTCAAATTACC 121 AAATGCCAAGAAAAACTCTCAGCACATCCTTCTsTTAATTCAGCACATCTTTCTGTAAATAAAGAAGAA 190 AAAGTCAATGTTTGCGATAGTCCGTTAGATATTGCAACACAACTGTTACTTTCCAACGTAAAAAAAT7A 259 GTACTTTCTGACTCGGAAAAAAACACGTTAAAAAATAAATGGAAATTGCTCACTGAGAAGAAATCTGAA 32? AATGCGGAGGTAAGAGCGGTCGCCCTTGTACCAAAAGATTTTCCCAAAGATCTGGTTTTAGCGCCtTTA 397 CCTGATCATGTTAATGATT TACATGGTACAAAAAGCGAAAGAAAAGACTTGGCATAAAACCtGAACAT CAACATGTrGGTCTTTCTATTATCGTrACAACATTCAATCGACCAGCAATTtTATCGAtTACATtAGCC TGTTTAGTAAACCAAAAAACACATTACCCGTTTGAAGTTATCGTGACAGATGATGGTAGTCAGGAAGAT 466 535 673 60 CTATCACCGATCATTCGCCAATATGAAAATAAATTGGATATTCG TACGTCAGACAAAAAGATAACGGT TTTCAAGCCAGTGCCGCTCGGAATATGGGATTACGCTTAGCAAAATATGACTTTAtTGGCTTACTCGAC TGTGATATGGCGCCAAATCCATTATGGGTTCATTCTTATGTTGCAGAGCTATTAGAAGATGATGATTTA 742 811 ACAATCATTGGTCCAAGAAAATACATCGATACACAACATATTGACCCAAAAGACTTCtTAAATAACGCG 8 B0 AGTTTGCTTGAATCATTACCAGAAGTGAAAACCAATAATAGTGTTGCCGCAAAAGGGGAAGGAACAGTT September 9 TCTCTGGATTGGCGCTTAGAACAAtTCGAAAAAACAGAAAATCTCCGCTTATCCGATTCGCCTTTCCGT 1018 TTTTTTGCGGCGGGTAATGTTGCTrTCGCTAAAAAATGGCTAAATAAATCCGGTTTCTTTGATGAGGAA 1087 TTTAATCACTGGGGTGGAGAAGATGTGGAATTTGGATATCGCTTATTCCGTTACGGTAGTTTCTTTAAA 1156 ACTATTGATGGCATTATGGCCTACCATCAAGAGCCACCAGGTAAAGAAAATGAAACCGATCGTGAAGCG 1225 GGAAAAAATATTACGCTCGATATTATGAGAGAAAAGGTCCCTTATATCTATAGAAAACTTTTACCAATA 129 GAAGATTCGCATATCAATAGAGTACCTTTAGTTTCAATTTATATCCCAGCTTATAACTGTGCAAACTAT 1363 ATTCAACGTTGCGTAGATAGTGCACTGAATCAGACTGTTGTTGATCTCGAGGTTTGTATTTGTAACGAT 1 32 GGTTCAACAGATAATACCTTAGAAGTGATCAATAAGCTTTATGGTAATAATCCTAGGGTACGCATCATG 1501 TCTAAACCAAATGGCGGAATAGCCTCAGCATCAAATGCAGCCGTTTCTTTTGCTAAAGGTTATTACATT 1570 GGGCAGTTAGATtCAGATGATTATCTTGAGCCTGATGCAGTTGAACTGTGTTtAAAAGAATTTTtAAAA 1639 GAT? AACGCT? GCTtGTGTTTAtACCACTAAT? GAAACGTCAATCCGGATGGTAGCTTAATCGCTAAT 1708 GGTTACAATTGGCCAGAATTTTCACGAGAAAAACTCACAACGGCTATGATTGCTCACCACTTTAGAATG 1777 TTCACGATTAGAGCTTGGCATTTAACTGATGG? TTCAATGAAAAAATTGAAAATGCCGTAGACTATGAC 1846 ATGTTCCTCAAACTCAGTGAAGTTGGAAAATTTAAACATCTTAATAAAATCTGCTATAACCGTCT? TT? 1915 CATGGTGATAACAC? TCAATTAAGAAACTTGGCAtTCAA? AGAAAAACCATtTTGTTGTAGTC ?? TC? G 1984 TCATTAAATAGACAAGGCATAACTTATTATAATTATGACGAATTTGATGATTTAGATGAAAGTAGAAAG 2053 TATATTTTCAATAAAACCGCTGAATATCAAGAAGAGATTGATATCTT? AAAGATATTAAAATCATCC? G 2122 AATAAAGATGCCAAAATCGCAGTCAGTATTTTTTATCCCAATACATTAAACGGCTTAGTGAAAAA? CT? 2191 AACAATATTATTGAATATAATAAAAATATATTCGTTATTGTTCTACATGTTGATAAGAATC? TCTTACÁ 2260 CCAG? TATC? AA ??? GAAATACT? GCCTTCT? TCAT? AAC? TCA? GTG ?? T? TTTT? CT? A? TA? TCAT 2329 ATCtCATATTACACGAGTAATAGATTAATAAAAACTGAGGCGCATTTAAGtAATATTAATAAATTAAGt 2398 CAGTTAAATCTAAATtGTGAATACATCATTTTtGATAATCATGACAGCCTATTCGTTAAAAATG? CAGC 2467 TATGCTTATATGAAAAAATATGATGTCGGCATGAATTTCTCAGCATTAACACATGATTGGATCGAGAAA ATCAATGCGCATCCACCATTTAAAAAGCTCATTAAAACTTATtTtAATGACAATGACTTAAAAAGTATG 2536 2674 2605 AATGTGAAAGGGGCATCACAAGGTATGTTTATGACGTATGCGCTAGCGCATGAGCTTCTGACGATTATT AAAGAAGTCATCACATCTTGCCAGTCAATTGATAGTGTGCCAGAATATAACACTGAGGATATTTGGTTC 2743 CAATTTGCACTTTTAATCTTAGAAAAGAAAACCGGCCATGTATTTAATAAAACATCGACCCTGACTTAT 2812 ATGCCTTGGGAACGAAAATTACAATGGACAAATGAACAAATTGAAAGTGCAAAAAGAGGAGAAAATATA 2881 CCTGTTAACAAGTTCATTATTAATAGTATAACTCTATAA high sec. of Ident N °: 3 _ type F PmCS for PM condriotina sinasa 1 MNTLSQAIKAYNCNDYE TO KLFEKSAETYGRKIVEFQIIKCKEKLSTNSYVSEDNSYVS 61 EDKKNSVCDSATQLLISNVKKLTLSESEKNSLKNKWKSITGKKSENAEIRKVELVP 121 KDFPKDLVLAPLPDHVNDFTWYKNRKKRLGIKPVNKNTGLSIIIPTFNRSRILDITLACL 181 VNQKTNYPFEVWADDGSKENLLTIVQKYeQKLDIKYVRQKDYGYQLCAVRNLGLRTAKY 241 DFVSILDCDMAPQQLWVHSYLTELLEDIDIV IGPRKYVDTHNITAEQFLNDPYLIESLP 301 ETATNNNPSITSKGNIRLeHFKKTDNLRLCDSPFRYFVAGNVAFSKEWLNKVGWFD 361 eeFNHWGGEDVEFGYRLFPKGCFFRVIDGGMAYHQEPPGKENETEREAGKSITLKIVKEK 421 VPYIYRK PIEDSHIHRIPLVSIYIPAYNCANYIQRCVDSA NQTWDLEVCICNDGST 481 DNTIEVINKLYGNNPRVRIMSKPNGGIASASNAAVSFAKGYYIGQLDSDDYVEPDAVELC 541 LKEFLKDKTLACVYTTNRNVNPDGSLIANGYNWPEFSREKLTTAMIAHHFRMFTIRA HL 601 TDGFNeNIENAVDYDMF SEVGKFKH NKICYNRVLHGDNTSIKKLGIQKK HFWVK 661 QSLNRQGINYYNYDKFDDLDESRKYIFNKTAeYQeEIDMLKDLKLIQNKDAKIAVSIFYP 721 NTLNGLVKKLNNIIEYNKNIFVIILHLDKNHLTPDIKKEIIAFYHKHQVNILLNNDISYY 781 TSNRLIKTEAHLSNINKLSQLNLNCEYIIFDNHDSLFVKNDSYAYM KYDVGM FSALTH 841 DWIEKINAHPPFKK IKTYFNDNDLRSMNVKGASQGMFMKYALRHALLTIIKEVITSCQS 901 IDSVPEYNTEDIWFQFAL ILEKKTGHVFNKTSTLTYMPWERKLQWTNEQIQSAK GENI 961 PVNKFIINSITL 972

Claims (115)

- 97 - CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A purified segment of nucleic acid, characterized in that it comprises a coding region encoding the enzymatically active hyaluronate synthase of P. multocida.

2 . The purified nucleic acid segment according to claim 1, characterized in that the purified segment of nucleic acid codes for the P. mul-tocida hyaluronate synthase of SEC. FROM IDENT. N0: 1

3. The purified segment of nucleic acid, according to claim 1, characterized in that the purified segment of nucleic acid comprises a nucleotide sequence according to SEQ. FROM IDENT. NO: 2

4. A purified segment of nucleic acid, characterized in that it has a coding region encoding enzymatically active hyaluronate synthase, wherein the purified segment of nucleic acid is capable of hybridizing to the nucleotide sequence of SEQ. FROM IDENT. NO: 2

5. A purified segment of nucleic acid, characterized in that it has a coding region encoding enzymatically active hyaluronate synthase, wherein the purified nucleic acid segment has semi-conservative or conservative amino acid codon changes when compared to the SEC nucleotide sequence. FROM IDENT. NO: 2

6. A recombinant vector, characterized in that it is selected from the group consisting of a plasmid, cosmid, phage or viral vector, and wherein the recombinant vector further comprises a purified segment of nucleic acid having a coding region encoding enzymatically active hyaluronan synthase of P. mul tocida.

7. The recombinant vector according to claim 6, characterized in that the purified segment of nucleic acid codes for the P. mulotidated hyaluronan synthase of SEQ. DE IDENT: 1.

8. The recombinant vector according to claim 6, characterized in that the purified -99-nucleic acid segment comprises a nucleotide sequence according to SEQ. FROM IDENT: NO: 2.

9. The recombinant vector according to claim 6, characterized in that the plasmid further comprises an expression vector.

10. The recombinant vector according to claim 9, characterized in that the expression vector comprises a promoter operably linked to a coding region for P. multocida enzymatically active hyaluronan synthase.

11. A recombinant host cell, characterized in that the recombinant host cell is a prokaryotic cell transformed with a recombinant vector comprising a purified segment of nucleic acid having a coding region encoding the enzymatically active hyaluronan synthase of P. multocida.

12. The recombinant host cell according to claim 11, characterized in that the purified segment of nucleic acid encodes the P. multocida hyaluronan synthase of SEQ. FROM IDENT. NO: 1. - 100 -

13. The recombinant host cell, according to claim 11, characterized in that the purified segment of nucleic acid comprises a nucleotide sequence according to SEQ. FROM IDENT. NO: 2

14. The recombinant host cell, according to claim 13, characterized in that the host cell produces hyaluronic acid.

15. The recombinant host cell according to claim 11, characterized in that the enzymatically active hyaluronan synthase is capable of producing a polymer of hyaluronic acid having a modified structure.

16. The recombinant host cell according to claim 11, characterized in that the enzymatically active hyaluronan synthase is capable of producing a hyaluronic acid polymer having a modified size distribution.

17. A recombinant host cell, characterized in that the recombinant host cell is a eukaryotic cell transfected with a recombinant vector comprising a purified segment of nucleic acid having a 101-encoding region encoding the enzymatically active hyaluronan synthase of P. multocida.

18. The recombinant host cell according to claim 17, characterized in that the purified segment of nucleic acid codes for P. mul-tocida hyaluronan synthase, of SEQ. FROM IDENT. NO: 1.

19. The recombinant host cell according to claim 17, characterized in that the purified segment of nucleic acid codes for a nucleotide sequence according to SEQ. FROM IDENT. NO: 2

20. The recombinant host cell, according to claim 19, characterized in that the host cell produces hyaluronic acid.

21. The recombinant host cell according to claim 17, characterized in that the enzymatically active hyaluronan synthase is capable of producing a polymer of hyaluronic acid having a modified structure.

22. The recombinant host cell according to claim 17, characterized in that the enzymatically active hyaluronan synthase is capable of producing a hyaluronic acid polymer-102 having a modified size distribution.

23. A recombinant host cell, characterized in that the recombinant host cell is electroporated to introduce a recombinant vector into the recombinant host cell, wherein the recombinant vector comprises a purified segment of nucleic acid having a coding region encoding hyaluronan synthase enzymatically active of P. mul tocida.

24. The recombinant host cell according to claim 23, characterized in that the purified segment of nucleic acid codes for the P. mulotidated hyaluronan synthase of SEQ. FROM IDENT. NO: 1.

25. The recombinant host cell according to claim 23, characterized in that the purified nucleic acid segment comprises a nucleotide sequence according to SEQ. FROM IDENT. NO: 2

26. The recombinant host cell, according to claim 25, characterized in that the host cell produces hyaluronic acid. - 103 -

27. The recombinant host cell according to claim 23, characterized in that the enzymatically active hyaluronan synthase is capable of producing a polymer of hyaluronic acid having a modified structure.

28. The recombinant host cell according to claim 23, characterized in that the enzymatically active hyaluronan synthase is capable of producing a polymer of hyaluronic acid having a modified size distribution.

29. A recombinant host cell, characterized in that the recombinant host cell is translated with a recombinant vector comprising a purified segment of nucleic acid having a coding region encoding the enzymatically active hyaluronan synthase of P. multocida.

30. The recombinant host cell according to claim 29, characterized in that the purified segment of nucleic acid codes for the P. mul-tocida hyaluronan synthase of SEQ. FROM IDENT. NO: 1.

31. The recombinant host cell, according to claim 29, characterized in that the -purified nucleic acid segment-104 encodes a nucleotide sequence in accordance with SEQ. FROM IDENT. NO: 2

32. The recombinant host cell, according to claim 31, characterized in that the host cell produces hyaluronic acid.

33. The recombinant host cell according to claim 29, characterized in that the enzymatically active hyaluronan synthase is capable of producing a polymer of hyaluronic acid having a modified structure.

34. The recombinant host cell, according to claim 29, characterized in that the enzymatically active hyaluronan synthase is capable of producing a polymer of hyaluronic acid having a modified size distribution.

35. A purified composition, characterized in that the purified composition comprises an enzymatically active hyaluronan synthase polypeptide of P. mul tocida.

36. A purified composition, characterized in that the purified composition comprises a polypeptide having an amino acid sequence according to SEQ. FROM IDENT. NO: l.

37. A method for detecting a DNA species, characterized in that it comprises the steps of: obtaining a DNA sample; contacting the DNA sample with a purified segment of nucleic acid according to SEC. FROM IDENT. NO: 2; hybridizing the DNA sample and the purified segment of nucleic acid whereby a hybridized complex is formed; and detect the complex.

38. A method for detecting a bacterial cell that expresses mRNA coding for P. mulotidated hyaluronan synthase, the method is characterized in that it comprises the steps of: obtaining a bacterial cell sample; contacting at least one nucleic acid of the bacterial cell sample with a purified nucleic acid segment, in accordance with SEQ. FROM IDENT. NO: 2; hybridizing at least one nucleic acid and the purified segment of nucleic acid, whereby a hybridized complex is formed; and - 106 - detecting the hybridized complex, wherein the presence of the hybridized complex is indicative of a bacterial strain expressing mRNA encoding P. mul-tocida hyaluronan synthase.

39. A method for producing hyaluronic acid, characterized in that it comprises the steps of: introducing a purified segment of nucleic acid having a coding region encoding the enzymatically active hyaluronan synthase of P. mul tocida in a host organism, wherein the host organism contains nucleic acid segments encoding enzymes which produce UDP-GlcNAc and UDP-GlcA; to grow the host organism in a medium to secrete hyaluronic acid; and recover the secreted hyaluronic acid.

40. The method according to claim 39, characterized in that the step of recovering hyaluronic acid comprises extracting the secreted hyaluronic acid from the medium.

41. The method according to claim 40, characterized in that it also comprises the step of purifying the extracted hyaluronic acid. - 107 -

42. The method according to claim 39, characterized in that in the step of growing the host organism, the host organism secretes a structurally modified hyaluronic acid.

43. The method according to claim 39, characterized in that, in the step of growing the host organism, the host organism secretes a hyaluronic acid having a modified size distribution.

44. A pharmaceutical composition, characterized in that it comprises a preselected pharmaceutical medicament and an effective amount of hyaluronic acid produced by P. mul-tocida hyaluronan synthase.

45. The pharmaceutical composition according to claim 44, characterized in that the hyaluronic acid is produced by the P. mul tocida hyaluronan synthase of SEC. FROM IDENT. NO: 1.

46. The pharmaceutical composition according to claim 44, characterized in that the molecular weight of the hyaluronic acid is modified so that a pharmaceutical composition with a modified molecular weight capable of evading an immune response is produced.

47. The pharmaceutical composition according to claim 44, characterized in that the molecular weight of the hyaluronic acid is modified so that a pharmaceutical composition with modified molecular weight capable of targeting a tissue or specific cell type within a patient is produced, and has affinity for the modified molecular weight pharmaceutical composition.

48. A purified and isolated nucleic acid sequence encoding enzymatically active hyaluronan synthase, the nucleic acid sequence is characterized in that it is selected from the group consisting of: (a) the nucleic acid sequence according to SEQ. FROM IDENT. NO: 2; (b) Nucleic acid complementary sequences for the nucleic acid sequence according to SEQ. FROM IDENT. NO: 2; (c) nucleic acid sequences which hybridize with the nucleic acid according to SEQ. FROM IDENT. NO: 2; and - 109 - (d) the nucleic acid sequences which will hybridize with the complementary nucleic acid sequences of SEQ. FROM IDENT. NO: 2

49. A purified and isolated segment of nucleic acid, characterized in that it consists essentially of a nucleic acid segment encoding the enzymatically active hyaluronan synthase of P. multocida.

50. A prokaryotic or eukaryotic host cell transformed or transfected with an isolated segment of nucleic acid, according to claim 1, 2 or 3, in a manner that allows the host cell to express hyaluronic acid.

51. An isolated segment of nucleic acid, characterized in that it consists essentially of a nucleic acid segment encoding P. mul-tocida hyaluronan synthase having a nucleic acid segment sufficiently duplicative of the nucleic acid segment according to SEQ. FROM IDENT. NO: 2 to allow the position of the biological coding property for hyaluronan synthase of P. multocida. - 110 -

52. A cDNA sequence, characterized in that it is in accordance with claim 51.

53. A prokaryotic or eukaryotic host cell, characterized in that it is transformed or transfected with a nucleic acid segment, according to claim 51, so as to allow the host cell to express hyaluronic acid.

54. A purified segment of nucleic acid, characterized in that it has a coding region encoding the enzymatically active hyaluronan synthase of P. mul tocida wherein the purified segment of nucleic acid is capable of hybridizing with the nucleotide sequence, in accordance with SEQ. FROM IDENT. NO: 2

55. The purified segment of nucleic acid, characterized in that it has a coding region encoding the enzymatically active hyaluronate synthase of P. multocida, the purified segment of nucleic acid is selected from the group consisting of: (A) the nucleic acid segment of compliance with the SEC. FROM IDENT. NO: 1; (B) the nucleotide sequence according to SEC. FROM IDENT. NO: 2; - 111 - (C) nucleic acid segments which hybridize with the nucleic acid segments defined in (A) or (B) or fragments thereof; (D) segments of nucleic acids which, except for the degeneracy of the genetic code, or which encode or which are functionally equivalent amino acids, would hybridize with the nucleic acid segments defined in subparagraphs (A), (B) and (C) .

56. A method for creating a P. mulotocida vaccine, characterized in that it comprises the steps of: producing a cassette of broken capsule genes on a non-replicating plasmid; introduce the broken gene cassette into a strain of Pas turella having a chromosomal gene; recombining the chromosomal gene of the Pasturella strain with the cassette of the broken gene, generating a mutant of Pasturella strains; perform an analysis to determine the mutant strains of Pasturella; and selecting an appropriate mutant strain for use as a vaccine. - 112 -

57. A live attenuated vaccine for Pasturella multocida, characterized in that it is selected from the group consisting of: a strain of Pasturella having at least one broken capsular gene; a strain of Pastμrella with a broken capsular locus; a strain of Pasturella with a broken polysaccharide biosynthesis gene; a strain of Pasturella with a broken polysaccharide synthase gene; and a strain of Pasturella with a broken HA synthase gene.

58. A method for detecting a mullein Pasturella infection in live cattle, characterized in that it comprises the steps of: providing a capsule gene probe or a mixture of probes capable of hybridizing to a DNA extract of a bacterial sample taken from an animal; incubating the capsule gene probe and the bacterial sample in a hybridization reaction chamber; and providing a hybridization detection system capable of differentiating complementarity between the bacterial sample. - 113 -

59. A diagnostic test to detect P. multocida in live cattle, characterized in that it comprises: a pair of PCR primers of a capsule gene or a mixture of pairs of primers capable of amplifying a DNA extract of a bacterial sample from a sample derived from an animal; a means for performing thermal cyclisation PCR with the capsule gene PCR primer and bacterial samples, whereby amplicons are provided; and - a means for detecting the amplicons; and a means for separating the amplicons into specific capsule groups.

60. The purified segment of nucleic acid, according to claim 1, characterized in that the purified nucleic acid segment codes for the P. muloformed chondroitin synthase of SEC. FROM IDENT. NO: 3

61. A segment of nucleic acid, characterized in that it comprises a coding region coding for hyaluronate synthase.

62. A segment of nucleic acid, characterized in that it has a coding region encoding enzymatically active hyaluronate synthase. - 114 -

63. A purified segment of nucleic acid, characterized in that it has a coding region encoding enzymatically active hyaluronate synthase.

64. A recombinant vector, characterized in that the recombinant vector further comprises a nucleic acid segment having a coding region encoding hyaluronan synthase.

65. A recombinant host cell, characterized in that the recombinant host cell is transformed with a recombinant vector comprising a nucleic acid segment having a coding region encoding hyaluronan synthase.

66 A recombinant host cell, characterized in that the recombinant host cell is transfected with a recombinant vector comprising a nucleic acid segment having a coding region encoding hyaluronan synthase.

67. A recombinant host cell, characterized in that the recombinant host cell is electroporated to introduce a recombinant vector into the recombinant host cell, wherein the recombinant vector-115-comprises a nucleic acid segment having a coding region encoding hyaluronan synthase

68. A recombinant host cell, characterized in that the recombinant host cell is subjected to transduction with a recombinant vector comprising a nucleic acid segment having a coding region encoding hyaluronan synthase.

69 A composition, characterized in that the composition comprises a hyaluronan synthase polypeptide.

70. A composition, characterized in that the composition comprises a polypeptide having an amino acid sequence in accordance with SEQ. FROM IDENT. NO: 1.

71. A method for detecting a DNA species, characterized in that it comprises the steps of: contacting a sample with a nucleic acid segment; and hybridizing the DNA sample and the nucleic acid segment to thereby form a hybridized complex. - 116 -

72. A method for detecting bacterial cells expressing mRNA encoding hyaluronan synthase, characterized in that it comprises the steps of: contacting at least one nucleic acid of a bacterial cell sample with a nucleic acid segment; and hybridizing at least one nucleic acid and the nucleic acid segment to thereby form a hybridized complex.

73. A method for producing hyaluronic acid, characterized in that it comprises the steps of: introducing a nucleic acid segment having a coding region encoding hyaluronan synthase, into a host organism; and growing the host organism in a medium to secrete hyaluronic acid.

74. A pharmaceutical composition, characterized in that it comprises a pharmaceutical medicament and an effective amount of hyaluronic acid produced by hyaluronan synthase. - 117 -

75. A nucleic acid sequence encoding hyaluronan synthase, the nucleic acid sequence is selected from the group consisting of: (a) the nucleic acid sequence according to SEQ. FROM IDENT. NO: 2; (b) Nucleic acid sequences complementary to the nucleic acid sequence, according to SEQ. FROM IDENT. NO: 2; (c) nucleic acid sequences which will hybridize with the nucleic acid according to SEC. FROM IDENT. NO: 2; Y (d) nucleic acid sequences which will hybridize with the complementary nucleic acid sequences of SEQ. FROM IDENT. NO: 2

76. A nucleic acid segment, characterized in that it comprises a nucleic acid segment encoding hyaluronan synthase.

77. A host cell transformed or transfected with a nucleic acid segment, according to claims 1, 2 or 3, characterized in that it is in a manner that allows the host cell to express hyaluronic acid. - 118 -

78. A nucleic acid segment, characterized in that it comprises a nucleic acid segment encoding hyaluronan synthase having a nucleic acid segment sufficiently duplicative of the nucleic acid segment according to SEQ. FROM IDENT. NO: 2 to allow the position of the biological property of coding for hyaluronan synthase.

79. A host cell transformed or transfected with a nucleic acid segment, according to claim 78, in a manner that allows the host cell to express hyaluronic acid.

80. A nucleic acid segment, characterized in that it has a coding region encoding hyaluronan synthase, wherein the nucleic acid segment is capable of hybridizing to the nucleotide sequence, in accordance with SEQ. FROM IDENT. NO: 2

81. A segment of nucleic acid, having a coding region encoding hyaluronate synthase, the nucleic acid segment is selected from the group consisting of: (A) the nucleic acid segment according to SEQ. FROM IDENT. N0: 1; - 119 - (B) the nucleotide sequence according to SEC. FROM IDENT. NO: 2; (C) nucleic acid segments which hybridize with the nucleic acid segments defined in subparagraphs (A) or (B) or fragments thereof; and (D) segments of nucleic acids which, except for the degeneracy of the genetic code, or which encode or which are functionally equivalent amino acids, would hybridize with the nucleic acid segments defined in subparagraphs (A), (B) and (C) ).

82. A method for creating a vaccine, characterized in that it comprises the steps of: making a cassette of broken capsule genes on a non-replicating plasmid; introducing the broken gene cassette into a bacterial strain having a chromosomal gene; recombining the chromosomal gene of the bacterial strain with the cassette of the broken gene so that mutant bacterial strains are generated; perform an analysis to determine the mutant strains; and selecting an appropriate mutant strain for use as a vaccine. - 120 -

83. A vaccine characterized in that it is selected from the group consisting of: a bacterial strain with at least one broken capsular gene; - a bacterial strain with a broken capsular locus; a bacterial strain with a broken polysaccharide biosynthesis gene; a bacterial strain with a broken polysaccharide synthase gene; and - a bacterial strain with a broken HA synthase gene.

84. A method for detecting a bacterial infection, characterized in that it comprises the steps of: providing a capsule gene probe or a mixture of probes capable of hybridizing to a DNA extract of a bacterial sample taken from an animal; incubating the capsule gene probe and the bacterial sample in a hybridization reaction chamber; and providing a hybridization detection system capable of differentiating complementarity between the bacterial sample.

85. A diagnostic test for detecting bacteria, characterized in that it comprises: - a pair of PCR primers of a capsule gene or a mixture of pairs of primers capable of amplifying a DNA extract of a bacterial sample from a derived sample of an animal; - a means for performing cyclization PCR with the capsule gene PCR primer and bacterial samples, in order to thereby provide amplicons; and a means to detect the amplicons; and a means for separating the amplicons into specific capsule groups.

86. The purified segment of nucleic acid, according to claim 1, characterized in that the purified nucleic acid segment encodes from position 45 to position 972 in SEQ. FROM IDENT. NO: 1.

87. The purified segment of nucleic acid, according to claim 1, characterized in that the purified segment of nucleic acid comprises a nucleotide sequence according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2

88. A purified segment of nucleic acid, characterized in that it has a coding region encoding enzymatically activated hyaluronate synthase, wherein the purified segment of nucleic acid is capable of hybridizing to the nucleotide sequence from position 135 to position 2,919 in SEC. FROM IDENT. NO: 2

89. A purified segment of nucleic acid, characterized in that it has a coding region encoding enzymatically activated hyaluronate synthase, wherein the purified nucleic acid segment has semi-conservative or conservative amino acid codon changes compared to the nucleotide sequences of position 135 to position 2,919 in the SEC. FROM IDENT. NO: 2

90. The recombinant vector according to claim 6, characterized in that the purified segment of nucleic acid encodes from position 45 to position 972 in SEC. FROM IDENT. NO: 1.

91. The recombinant vector according to claim 6, characterized in that the purified segment of nucleic acid comprises a nucleotide sequence according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2

92. The recombinant host cell, according to claim 11, characterized in that the purified nucleic acid-123 segment encodes from position 45 to position 972 in SEQ. FROM IDENT. NO: 1.

93. The recombinant host cell, according to claim 11, characterized in that the purified segment of nucleic acid comprises a nucleotide sequence according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2

94. The recombinant host cell according to claim 17, characterized in that the purified segment of nucleic acid encodes from position 45 to position 972 in SEC. FROM IDENT. N0: 1

95. The recombinant host cell, according to claim 17, characterized in that the purified segment of nucleic acid comprises a nucleotide sequence according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2

96. The recombinant host cell, according to claim 23, characterized in that the purified segment of nucleic acid encodes from position 45 to position 972 in SEQ. FROM IDENT. N0: 1 - 124 -

97. The recombinant host cell, according to claim 23, characterized in that the purified segment of nucleic acid comprises a nucleotide sequence according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2

98. The recombinant host cell, according to claim 29, characterized in that the purified segment of nucleic acid encodes from position 45 to position 972 in SEC. FROM IDENT. NO: l.

99. The recombinant host cell, according to claim 29, characterized in that the purified segment of nucleic acid comprises a nucleotide sequence according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2

100. A purified composition, characterized in that the purified composition comprises a polypeptide having an amino acid sequence according to position 45 to position 972 in SEQ. FROM IDENT. NO: 1.

101. A method for detecting a DNA species, characterized in that it comprises the steps of: obtaining a DNA sample; - 125 - contacting the DNA sample with a purified segment of nucleic acid, according to position 135 to position 2919 in SEC. FROM IDENT. NO: 2; hybridizing the DNA sample and the purified segment of nucleic acid whereby a hybridized complex is formed; and detect the complex.

102. A method for detecting a bacterial cell that expresses mRNA coding for P. mulotidated hyaluronan synthase, the method is characterized in that it comprises the steps of: obtaining a bacterial cell sample; contacting at least one nucleic acid of the bacterial cell sample with a purified segment of nucleic acid, according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2; hybridizing at least one nucleic acid and the purified segment of nucleic acid, whereby a hybridized complex is formed; and detecting the hybridized complex, wherein the presence of the hybridized complex is indicative of the bacterial strain expressing mRNA encoding the hyaluronan synthase of P. mul tocida. - 126 -

103. A purified and isolated nucleic acid sequence, characterized in that it encodes for enzymatically active hyaluronan synthase, wherein the nucleic acid sequence is selected from the group consisting of: (a) the nucleic acid sequence according to position 135 to position 2,919 in the SEC. FROM IDENT. NO: 2; (b) complementary sequences of nucleic acid with the nucleic acid sequence according to positions 135 to position 2,919 in SEQ. FROM IDENT. NO: 2; (c) nucleic acid sequences which will hybridize with the nucleic acid according to position 135 to position 2,919 in SEQ. FROM IDENT. N0: 2; and (d) nucleic acid sequences which will hybridize with the complementary nucleic acid sequences from position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2

104. An isolated segment of nucleic acid, characterized in that it consists essentially of a segment of nucleic acid encoding P. mul-tocida hyaluronan synthase having a nucleic acid segment sufficiently duplicative of the nucleic acid segment, according to position 135 to the nucleic acid segment. position 2,919 of the SEC. FROM IDENT. NO: 2 to allow the position of the biological property of encoding P. mul-tocida hyaluronan synthase. - 127 -

105. A cDNA sequence characterized in that it is in accordance with claim 104.

106. A prokaryotic or eukaryotic host cell, characterized in that it is transformed or transfected with a nucleic acid segment, according to claim 104, in a manner that allows the host cell to express hyaluronic acid.

107. A purified segment of nucleic acid, characterized in that it has a coding region encoding the enzymatically active hyaluronan synthase of P. mul tocida, wherein the purified segment of nucleic acid is capable of hybridizing with the nucleotide sequence according to position 135 to position 2,919 in the SEC. FROM IDENT. NO: 2

108. A purified segment of nucleic acid, characterized in that it has a coding region encoding the enzymatically active hyaluronate synthase of P. multocida, the purified segment of nucleic acid is selected from the group consisting of: (A) the nucleic acid segment of according to position 45 to position 972 in the SEC. FROM IDENT. NO.-l; - 128 - (B) the nucleotide sequence according to position 135 to position 2,919 in SEC. FROM IDENT. NO: 2; (C) nucleic acid segments which hybridize with the nucleic acid segments defined in subparagraphs (A) or (B) or fragments thereof; and (D) nucleic acid segments which, except for the degeneracy of the genetic code, or which code for functionally equivalent amino acids, would hybridize with the nucleic acid segments defined in subsections (A), (B) and (C).

109. A composition, characterized in that the composition comprises a polypeptide having an amino acid sequence according to position 45 to position 972 in SEQ. DE 'IDENT. NO: l.

110. A nucleic acid sequence, characterized in that it encodes for hyaluronan synthase, wherein the nucleic acid sequence is selected from the group consisting of: (a) the nucleic acid sequence according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2; (b) complementary sequences of nucleic acid with the nucleic acid sequence according to the positions 135 to position 2,919 in the SEC. FROM IDENT. N0: 2; - 129 - (c) nucleic acid sequences which will hybridize with the nucleic acid according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2; and (d) nucleic acid sequences which will hybridize with the complementary nucleic acid sequences from position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2

111. A nucleic acid segment, characterized in that it comprises a nucleic acid segment encoding hyaluronan synthase having a sufficiently duplicative nucleic acid segment of the nucleic acid segment according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2 to allow the position of the biological coding property for hyaluronan synthase.

112. A host cell transformed or transfected with a nucleic acid segment, according to claim 111, in a manner that allows the host cell to express hyaluronic acid.

113. A nucleic acid segment, characterized in that it has a coding region encoding hyaluronan synthase, wherein the nucleic acid segment is capable of hybridizing with the nucleotide sequence according to 130 with position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2

114. A segment of nucleic acid, characterized in that it has a coding region encoding hyaluronate synthase, the nucleic acid segment is selected from the group consisting of: (A) the nucleic acid segment according to position 45 to position 972 in SEC. FROM IDENT. N0: 1; (B) the nucleotide sequence according to position 135 to position 2,919 in SEQ. FROM IDENT. NO: 2; (C) nucleic acid segments which hybridize with the nucleic acid segments defined in subparagraphs (A) or (B) or fragments thereof; and (D) 'nucleic acid segments which, except for the degeneracy of the genetic code, or which code for functionally equivalent amino acids, would hybridize with the nucleic acid segments defined in subparagraphs (A), (B) and (C) .

115. The pharmaceutical composition according to claim 44, characterized in that hyaluronic acid is produced by P. mul hyaluronan synthase from position 45 to position 972 in SEC. FROM IDENT. NO: 1. - 131 - SUMMARY OF THE INVENTION The present invention relates to a segment of nucleic acid having a segment of a coding region that encodes enzymatically active bacterial muliticidal hyaluronate synthase (PmHAS), and with the use of this nucleic acid segment in the preparation of recombinant cells which produce hyaluronate synthase and its product hyaluronic acid The hyaluronate is also known as hyaluronic acid or hyaluronan The present invention also relates to the use of PmHAS to construct mutagenic strains of "P. multocida" for the use in vaccines The present invention is further related to the use of PmHAS in diagnostic tests in the field of determination of infection in cattle by P. multocida.