WO1995023226A1 - Gene for mycobacterial diaminopimelic acid - Google Patents

Gene for mycobacterial diaminopimelic acid Download PDF

Info

Publication number
WO1995023226A1
WO1995023226A1 PCT/US1995/002455 US9502455W WO9523226A1 WO 1995023226 A1 WO1995023226 A1 WO 1995023226A1 US 9502455 W US9502455 W US 9502455W WO 9523226 A1 WO9523226 A1 WO 9523226A1
Authority
WO
WIPO (PCT)
Prior art keywords
mycobacterial
dapb
ala
enzyme
val
Prior art date
Application number
PCT/US1995/002455
Other languages
French (fr)
Inventor
William R. Jacobs
Jeffrey D. Cirillo
Original Assignee
Albert Einstein College Of Medicine Of Yeshiva University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Albert Einstein College Of Medicine Of Yeshiva University filed Critical Albert Einstein College Of Medicine Of Yeshiva University
Priority to AU19722/95A priority Critical patent/AU1972295A/en
Publication of WO1995023226A1 publication Critical patent/WO1995023226A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/35—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Mycobacteriaceae (F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies

Definitions

  • This invention relates to a polynucleotide which encodes the mycobacterial enzyme DapB and to the use of said polynucleotide and said enzyme to treat bacterial and mycobacterial infection, including infection caused by M. tuberculosis, M. avium, M. fortuitum, M. ⁇ ordoneae, M. haemoilphilum.
  • the inventors have identified, cloned, sequenced and characterized a polynucleotide which encodes an enzyme which is involved in mycobacterial synthesis of diaminopimelic acid, an essential component of mycobacterial cell walls. Utilizing the nucleic acid sequence of the polynucleotide, antibiotic drugs and vaccines are produced which are useful in the treatment and prevention of bacterial and mycobacterial infection.
  • antibiotic drugs and vaccines Utilizing the nucleic acid sequence of the polynucleotide, antibiotic drugs and vaccines are produced which are useful in the treatment and prevention of bacterial and mycobacterial infection.
  • Mycobacteria-related diseases such as tuberculosis and leprosy, currently cause the highest number of deaths of any infectious disease throughout the world, and are responsible for one in four avoidable deaths in developing countries. Further, there has been a recent rise in the incidence of antibiotic-resistant tuberculosis.
  • mycobacterial infection is on the rise, a need exists to develop methods for treating and preventing bacterial and mycobacterial infection, including infection caused by M. tuberculosis. M. avium. M. fortuitum. M. ⁇ ordoneae. M. haemoilphilum. M. paratuberculosis. M. bovis and M. leprae.
  • One means of treating and preventing mycobacterial infection is to interrupt biosynthetic functions of bacteria and mycobacteria, thereby killing the infecting bacteria and mycobacteria.
  • Diaminopimelic acid is an essential component of the peptidoglycan layer of mycobacterial cell walls.
  • N-glucolylmuramyl-L-alanyl-D-isoglutaminyl- meso-diaminopimelate purified from the other mycobacterial cell components produces enhanced cellular and humoral responses in guinea pigs when administered with an antigen.
  • the peptidoglycan layer of mycobacteria has unusual inter-DAP linkages as well as DAP content in the mycobacterial cell wall.
  • coli is thought to synthesize DAP solely via the succinylase pathway, whereas the dehydrogenase and acetylase pathways are utilized by Bacillus species.
  • biosynthetic pathways are utilized to synthesize DAP.
  • B. sphaericus uses solely the dehydrogenase pathway
  • B. me ⁇ aterium uses the acetylase pathway
  • B. macerans uses both the dehydrogenase and acetylase pathways.
  • C. ⁇ lutamicum appears to utilize all three of the possible biosynthetic pathways for the production of DAP. Although it may seem inefficient for bacteria to maintain more than one pathway for synthesis of DAP this fact may indicate the importance of the pathway for bacterial survival.
  • mycobacterial infection remains one of the greatest causes of death throughout the world, a strong need exists to develop methods of treating and preventing mycobacterial infections, as well as bacterial infections.
  • interruption of biosynthetic functions of mycobacteria is an effective way of killing both mycobacteria and bacteria, it is desirable to develop methods of treating and preventing mycobacterial and bacterial infection by interrupting biosynthetic pathways and functions of mycobacteria.
  • This invention relates to a polynucleotide which encode the mycobacterial enzyme DapB, which enzyme is involved in mycobacterial synthesis of diaminopimelic acid, and to the use of said polynucleotide and said enzyme encoded by the polynucleotide to treat and prevent mycobacterial and bacterial infection.
  • the nucleotide sequence of the polynucleotide which encodes mycobacterial enzyme DapB is utilized to produce compounds and vaccines useful in the treatment and prevention of bacterial and mycobacterial infection, including infection caused by M. tuberculosis. M. avium. M. fortuitum. M. ⁇ ordoneae, M. haemoilphilum, M.
  • Figure 1 represents the three possible pathways for biosynthesis of DAP.
  • the abbreviations utilized are as follows: ASP, L-aspartate; ASP-P, L-aspartyl-phosphate; ASA, L-aspartate semialdehyde; DHDP, L-2,3-dihydrodipicolinate; THDP,
  • L-N-acetyl-2-amino-6-ketopimelate NS-DAP, LL-N-succinyl-2,6-diaminopimelate; NA-DAP, LL-N-acetyl- 2,6-diaminopimelate; 11-DAP, LL-diaminopimelate;
  • Figure 2 represents physical maps of BCG DAP complementing regions. The size of the fragments are shown to scale with the length in kb indicated to the right. Positions of Xhol and Pstl sites are noted;
  • Figure 3 represents the nucleotide and deduced amino acid sequence of the 1791 bp BCG DNA fragment that was shown to complement the E. coli dapB mutation.
  • Direction of translation of the proteins is shown by small arrows. Underlined sequences indicate potential ribosomal binding sites and • • • symbols represent termination codons;
  • Figure 4 represents maps of the BCG dapB complementing fragments and the deletions into this region. Bars without arrowheads shown below the dapB construct indicate the regions retained in the Pstl and Sall deletion constructs. As shown, the dapB-Pstl construct contains both putative genes and the dapB-Sall construct only contains open reading frame ORFz. The Sail and Pstl restriction sites (other than those present in the polylinker of KSII+) used for the construction of these deletions are indicated on the dapB construct. Direction and length of the dapB gene and ORFz are indicated by arrows; and
  • Figure 5 represents amino acid alignment of the deduced BCG DapB protein (enzyme) and DapB proteins (enzymes) from other species.
  • the * indicates residues that are identical 100% and # indicates residues that are identical in over 50% of the DapB proteins. Amino acid identity and conservation is indicated as compared to the deduced BCG dapB sequence.
  • DAP biosynthetic enzymes are essential for bacteria and mycobacteria in vivo due to the absence of DAP in mammalian cells. Therefore, bacteria and mycobacteria must be able to synthesize DAP in vivo. For this reason, DAP biosynthetic genes are useful as targets for anti-mycobacterial and anti-bacterial agents as well as for the design of in vivo selection systems.
  • mycobacterial dapB polynucleotide provides a useful tool in the treatment and prevention of mycobacterial infection and in the development of in vivo marker systems.
  • mycobacterial infection can be treated by administering a pharmaceutically effective amount of an oligonucleotide which inhibits the mRNA activity of the mycobacteria.
  • oligonucleotides can be prepared utilizing the nucleic acid sequence of the dapB gene (polynucleotide) of the invention, which is depicted in Figure 3.
  • Compounds which can block the activity of the mycobacterial enzyme DapB can be produced by overexpressing mycobacterial DapB enzyme, purifying the overexpre ⁇ sed enzyme, performing x-ray crytallography on the purified enzyme so as to obtain the molecular structure of the enzyme, and then creating a compound with a similar molecular structure to the enzyme. This compound can be administered so as to inhibit the activity of the enzyme, thereby causing cell death.
  • vaccines useful in the treatment and transmission prevention of mycobacterial infection can be produced. Because the inventors have determined the sequence of the mycobacterial dapB gene, it is possible to determine the existence of a mutated mycobacterial dapB gene. A mutated mycobacterial dapB gene can be administered in vaccine form to treat and prevent mycobacterial infection.
  • vaccines can be formed which comprise a mycobacterial strain having a deleted dapB gene, wherein the mycobacterial strain is grown on artificial media containing dioaminopimelic acid and then administered in vaccine form.
  • a recombinant vaccine can be prepared by deleting dapB gene from a mycobacterial strain and cloning into the mycobacterial strain a plasmid which expresses dapB.
  • antibodies specific for polynucleotides having the nucleic acid sequence of the dapB gene of the invention can produced. These antibodies can be passively administered to treat and prevent mycobacterial infection.
  • the polynucleotides of the invention can also be used to produce research tools capable of identifying virulence genes and drug targets in mammals.
  • a research tool is prepared by deleting dapB gene from a mycobacterial strain and inserting therein a plasmid containing a promoterless dapB gene and a genomic mycobacterial nucleotide sequence so as to produce a library. This library is then passed through mammalian cells and used to identify virulence genes and drug targets.
  • any polynucleotides which are involved in mycobacterial biosynthesis of diaminopimelic acid can be used to treat and prevent both bacterial and mycobacterial infection.
  • polynucleotides which can be used to treat and prevent bacterial and mycobacterial infection include mycobacterial dapA, mycobacterial dapD, mycobacterial dapE, mycobacterial ddh, mycobacterial asd and mycobacterial ask.
  • promoters and translation initiation sequences for the mycobacterial dapB gene are useful in the treatment and prevention of bacterial and mycobacterial infection.
  • the bacterial strains used by the inventors are described in Table 1, below. E. coli strains were grown in Luria Broth (LB) or on LB agar with supplements.
  • Ampicillin was added when necessary at a final concentration of 25 ⁇ g/ml, isopropylthio- ⁇ -D-galactoside (IPTG) at 2 mM and 5-bromo-4- chloro-3-indolyl- ⁇ -D-galactoside (X-gal) at 40 ⁇ g/ml.
  • IPTG isopropylthio- ⁇ -D-galactoside
  • X-gal 5-bromo-4- chloro-3-indolyl- ⁇ -D-galactoside
  • BCG chromosomal DNA was isolated as described by Jacobs et al.. Methods Enzvmol.. Vol. 204, pp. 537-555 (1991). Approximately 2 to 4 kb size fragments were generated by Sau3A partial digestion. The agarose gel purified fragments were ligated to BamHI digested dephosphorylated pBlueScript II KS+ (KSII+) DNA (Stratagene) . The resulting ligation was then transformed by electroporation into DH5 ⁇ and plated on LB agar containing a picillin, IPTG and X-gal.
  • the ligation was transformed in the same manner and plated on LB plates containing ampicillin in the absence of IPTG so as to allow propagation of potentially lethal clones. Greater than 100,000 colonies produced in this manner were pooled and a plasmid was isolated from them for use in the following experiments.
  • the library was transformed into various mutant bacterial strains by electroporation.
  • the vector plasmid KSII+ was used as a negative control. These transformations were then plated on two types of selective mediums. The first was LB agar containing ampicillin and DAP to determine the number of transformants. The second medium was LB agar containing ampicillin and IPTG to allow expression of recombinant proteins. The colonies present in the absence of DAP above that obtained with KSII+ were assumed to contain plasmids carrying mycobacterial genomic fragments that complemented the DAP mutation present in the strain. Plasmid was then isolated from at least 10 clones that complemented each individual E.
  • each complementing fragment contained more than one mycobacterial gene
  • the complementation profile for each mycobacterial DAP complementing region was determined by testing each clone that complemented an E. coli mutation for its ability to complement the other E. coli mutations as well. This procedure was accomplished in the same manner as described above except that all E. coli DAP mutants were tested simultaneously.
  • a physical map of a representative clone that complemented each mutation was determined. Physical maps were constructed with the restriction enzymes Pstl and Xhol. Sizes of restriction fragments were determined by analytical agarose gel electrophoresis.
  • the mycobacterial dapB region was then analyzed and sequenced. Two deletions were constructed in the dapB complementing fragment in order to determine the locations of the relevant complementing genes present (see Figure 5).
  • the first deletion was constructed by digestion of the dapB complementing plasmid with Pstl and purifying the resulting large DNA fragment by agarose gel electrophoresis. This DNA fragment was then self ligated, transformed with DH5 ⁇ and the appropriate clone containing an approximately 600 bp deletion in the 5' end of the mycobacterial DNA was selected.
  • the complementation profile for this construct, designated dapB-Pstl was then determined in the same manner as described above.
  • dapB-Sall A further deletion of this construct was made by digesting dapB-Pstl with Sail and isolation of the resulting large DNA fragment by agarose gel electrophoresis. The large DNA fragment was then self ligated, transformed into DH5 ⁇ and the appropriate clone containing a further deletion of approximately 1.1 kb into the 5' end of the fragment selected. The complementation profile for this construct, designated dapB-Sall, was then determined in the same manner as described above.
  • Sequence analysis was performed on the construct dapB-Pstl in order to determine the nucleotide sequence of the mycobacterial dapB region. Sequencing was carried out on double stranded DNA as described by Kraft et al., Bio Techniques. Vol. 6, pp. 544-547 (1988) using Sequenase (USB) . The complete sequence was determined on both strands using synthetic oligonucleotides . The resulting sequences were compared for overlaps using Fasta (see Pearson et al., Proc. Natl. Acad. Sci. USA. Vol. 85, pp. 2444-2448 (1988)). Protein sequence alignments were constructed using Fasta and Pileup from the Genetics Computer Group (Wisconsin) software package. Analysis for the presence of open reading frames (ORF) was also accomplished using the Genetics Computer Group software package.
  • Wisconsin Genetics Computer Group
  • BCG DAP biosynthetic genes were isolated. Although the mycobacterial asd gene was isolated previously, the inventors tested the ability of the KSII+ expression library to complement the asd mutation contained in the E. coli strain X6118. This experiment allowed the inventors to test the quality of the library using a well characterized system. The results of this complementation experiment are shown in Table 2, below. TABLE 2
  • Frequencies are calculated as the number of colonies that grow in the absence of DAP as compared to the transformation frequency from the number of colonies that grow in the presence of DAP.
  • the DAP biosynthetic pathway present in BCG was elucidated by complementing E. coli mutations in four additional steps in this pathway.
  • the steps that were chosen are catalyzed by the products of the E. coli genes dapA, dapB, dapD, and dapE.
  • E. coli strains containing mutations in each of these genes were transformed with the expression library. The results of these experiments are shown in Table 2.
  • the frequency at which complementing clones were obtained was highly variable. However, the level of complementation of the plasmids isolated from each of the complemented clones was high. Several clones were isolated from the library that appeared to complement all five of the E. coli mutations tested. A single clone for each gene was purified for further characterization.
  • each set of clones was tested for its-ability to complement the other steps in the DAP biosynthetic pathway.
  • the results of this analysis are shown in Table 3, below.
  • the overlapping clones that complemented that asd, dapA, dapD, and dapE mutations did not exhibit the ability to complement any other DAP mutation.
  • the dapB complementing clones however, exhibited complementation of the dapD and dapE mutations as well.
  • the frequencies obtained were in the same range as for the secondary complementation experiments described previously.
  • Codon usage analysis of this region revealed the presence of two large ORFs that display the expected codon preference for mycobacteria.
  • a potential translation start for the first ORF occurs at nucleotide 312 and may encode a protein of 271 amino acids in length.
  • the region upstream of the putative translational start was examined for the presence of ribosomal binding site. No sequences were found that fit the E. coli consensus exactly, however, at a distance of 8 nucleotides upstream there was a sequence of AGG that corresponds to that which would be expected.
  • the second ORF begins and has a potential translational start at nucleotide 1151, and would allow a protein of 177 amino acids to be produced. The presence of a potential ribosomal binding site was detected 10 bp 5' of the putative translational start (see Figure 3) .
  • the deduced protein sequence from the first gene was compared to the amino acid sequence of other dapB genes (see Figure 5) .
  • Significant sequence conservation was observed between this ORF and the DapB proteins from B. lactofermentum and E. coli. There are several regions in these proteins that display a high level of conservation across species. In particular, a five amino acid region, PSGTA, is completely conserved in all three species.
  • PSGTA a five amino acid region
  • the inventors found evidence for the ability to synthesize DAP via the dehydrogenase pathway.
  • the BCG dapB complementing fragment was found to complement both the dapD and dapE E . coli mutations. Although this result may be explained by linkage between the BCG dapB and ddh genes, it was unexpected since none of the original dapD and dapE complementing clones displayed the ability to complement both mutations. This discrepancy may be explained by the observation that the dapB complementing fragment complements these mutations at a 5 to 10 fold lower frequency than the dapD and dapE genes themselves. Thus, this observation may be explained by the fact that the inventors examined only a few clones for each gene.
  • the BCG DapB protein is significantly different from the E. coli DapB protein, making it plausible for it to have acquired a new function. There is, however, a higher degree of similarity in the structure of the Brevibacterium and BCG DapB proteins that may indicate that they may have the same function. This possibility has not been tested with the Brevibacterium gene. Brevibacterium species have been shown to express ddh activity (see Misono et al., J. Bacteriol.. Vol. 137, pp. 22-27 (1979)). The reactions catalyzed by the dihydrodipicolinate reductase and diaminopimelate dehydrogenase are similar. Both the BCG and Brevibacterium DapB proteins have a conserved amino terminal region reminiscent of the amino terminal domain of NADP+-dependent dehydrogenases,
  • the dapB gene of mycobacteria provides an extremely useful tool for the development of new antibiotics specific for bacteria and for mycobacteria, and for use in the construction of a marker system that may have applications for studies in vivo.
  • the dapB gene product catalyzes a key step in the biosynthetic pathway of DAP whether one or all three of the potential pathways is utilized.
  • An inhibitor of the DapB enzyme would be lethal in the absence of the ability to acquire DAP from an exogenous source. Since the dapB gene has been cloned by the inventors, substrate analogues which inhibit the activity of the purified recombinant protein can be determined. Future studies will allow the crystal structure of this protein to be determined. This information combined with the above-described demonstration of highly conserved regions will further contribute to strategies for the design of inhibitors. Inhibitors of DapB will have a broad applicability, similar to other antibiotics that inhibit cell wall biosynthesis.
  • an ideal marker system for the maintenance of recombinant DNA in bacteria during mammalian infection or vaccination would require the use of auxotrophic marker systems.
  • the use of antibiotic markers is disadvantageous due to possible further dissemination of antibiotic resistance and due to the inability to maintain selective pressure in vivo. Since DAP is not present in mammalian cells, a dapB mutant would provide a suitable host strain for maintenance of recombinant DNA carrying the dapB gene in vivo.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

This invention is related to a mycobacterial diaminopimelic acid gene and enzyme, and to the use of said gene and enzyme to treat and prevent both bacterial and mycobacterial infection, including mycobacterial infection caused by M. tuberculosis, M. avium, M. fortuitum, M. gordonae, M. haemoilphilum, M. paratuberculosis, M. bovis and M. leprae.

Description

GENE FOR MYCOBACTERIAL DIAMINOPI ELIC ACID
Statement of Government Interest
This invention was made with government support under NIH Grant Number AI27160. As such, the government has certain rights in the invention.
FIELD OF THE INVENTION
This invention relates to a polynucleotide which encodes the mycobacterial enzyme DapB and to the use of said polynucleotide and said enzyme to treat bacterial and mycobacterial infection, including infection caused by M. tuberculosis, M. avium, M. fortuitum, M. αordoneae, M. haemoilphilum.
M. paratuberculosis, M. bovis, and t±. leprae.
Specifically, the inventors have identified, cloned, sequenced and characterized a polynucleotide which encodes an enzyme which is involved in mycobacterial synthesis of diaminopimelic acid, an essential component of mycobacterial cell walls. Utilizing the nucleic acid sequence of the polynucleotide, antibiotic drugs and vaccines are produced which are useful in the treatment and prevention of bacterial and mycobacterial infection. BACKGROUND OF THE INVENTION Mycobacteria-related diseases, such as tuberculosis and leprosy, currently cause the highest number of deaths of any infectious disease throughout the world, and are responsible for one in four avoidable deaths in developing countries. Further, there has been a recent rise in the incidence of antibiotic-resistant tuberculosis. Because mycobacterial infection is on the rise, a need exists to develop methods for treating and preventing bacterial and mycobacterial infection, including infection caused by M. tuberculosis. M. avium. M. fortuitum. M. αordoneae. M. haemoilphilum. M. paratuberculosis. M. bovis and M. leprae. One means of treating and preventing mycobacterial infection is to interrupt biosynthetic functions of bacteria and mycobacteria, thereby killing the infecting bacteria and mycobacteria.
Diaminopimelic acid (DAP) is an essential component of the peptidoglycan layer of mycobacterial cell walls. N-glucolylmuramyl-L-alanyl-D-isoglutaminyl- meso-diaminopimelate purified from the other mycobacterial cell components produces enhanced cellular and humoral responses in guinea pigs when administered with an antigen. The peptidoglycan layer of mycobacteria has unusual inter-DAP linkages as well as DAP content in the mycobacterial cell wall. The importance of the DAP molecule has been further suggested through work in Bordetella pertussis where DAP has been shown to play a key role in the structure of a cytotoxin (see Luker et al., Proc. Natl. Acad. Sci. USA. Vol. 90, pp. 2365-2369 (1993)). The inventors hypothesized that the unusual structure of the cell wall of mycobacteria may contribute to the virulence of different mycobacterial species. There are three known pathways for biosynthesis of DAP utilized by bacteria, which pathways are depicted in Figure 1. The inventors have designated these the succinylase, dehydrogenase and acetylase pathways. E. coli is thought to synthesize DAP solely via the succinylase pathway, whereas the dehydrogenase and acetylase pathways are utilized by Bacillus species. There is a great deal of variability among bacteria as to which biosynthetic pathways are utilized to synthesize DAP. For example, B. sphaericus uses solely the dehydrogenase pathway, whereas B. meαaterium uses the acetylase pathway and B. macerans uses both the dehydrogenase and acetylase pathways. C. αlutamicum appears to utilize all three of the possible biosynthetic pathways for the production of DAP. Although it may seem inefficient for bacteria to maintain more than one pathway for synthesis of DAP this fact may indicate the importance of the pathway for bacterial survival.
Because mycobacterial infection remains one of the greatest causes of death throughout the world, a strong need exists to develop methods of treating and preventing mycobacterial infections, as well as bacterial infections. In addition, because interruption of biosynthetic functions of mycobacteria is an effective way of killing both mycobacteria and bacteria, it is desirable to develop methods of treating and preventing mycobacterial and bacterial infection by interrupting biosynthetic pathways and functions of mycobacteria.
It is therefore an object of this invention to provide methods of treating bacterial and mycobacterial infection.
It is another objection of this invention to provide methods of producing compounds useful in the treatment of bacterial and mycobacterial infection. It is a further objection of this invention to provide vaccines useful in the treatment and prevention of bacterial and mycobacterial infection.
It is a still further object of this invention to provide polynucleotides which can be used in the treatment and prevention of bacterial and mycobacterial infection.
SUMMARY OF THE INVENTION This invention relates to a polynucleotide which encode the mycobacterial enzyme DapB, which enzyme is involved in mycobacterial synthesis of diaminopimelic acid, and to the use of said polynucleotide and said enzyme encoded by the polynucleotide to treat and prevent mycobacterial and bacterial infection. The nucleotide sequence of the polynucleotide which encodes mycobacterial enzyme DapB is utilized to produce compounds and vaccines useful in the treatment and prevention of bacterial and mycobacterial infection, including infection caused by M. tuberculosis. M. avium. M. fortuitum. M. αordoneae, M. haemoilphilum, M. paratuberculosis. M. bovis and M. leprae. BRIEF DESCRIPTION OF THE DRAWINGS The above brief description, as well as further objects and features of the present invention, will be more fully understood by reference to the following detailed description of the presently preferred, albeit illustrative, embodiments of the present invention when taken in conjunction with the accompanying drawings wherein:
Figure 1 represents the three possible pathways for biosynthesis of DAP. The abbreviations utilized are as follows: ASP, L-aspartate; ASP-P, L-aspartyl-phosphate; ASA, L-aspartate semialdehyde; DHDP, L-2,3-dihydrodipicolinate; THDP,
L-Δ -tetrahydrodipicolinate; NS-AKP, L-N-succinyl-2- amino-6-ketopimelate; NA-AKP,
L-N-acetyl-2-amino-6-ketopimelate; NS-DAP, LL-N-succinyl-2,6-diaminopimelate; NA-DAP, LL-N-acetyl- 2,6-diaminopimelate; 11-DAP, LL-diaminopimelate;
Figure 2 represents physical maps of BCG DAP complementing regions. The size of the fragments are shown to scale with the length in kb indicated to the right. Positions of Xhol and Pstl sites are noted;
Figure 3 represents the nucleotide and deduced amino acid sequence of the 1791 bp BCG DNA fragment that was shown to complement the E. coli dapB mutation. Direction of translation of the proteins is shown by small arrows. Underlined sequences indicate potential ribosomal binding sites and • • • symbols represent termination codons;
Figure 4 represents maps of the BCG dapB complementing fragments and the deletions into this region. Bars without arrowheads shown below the dapB construct indicate the regions retained in the Pstl and Sall deletion constructs. As shown, the dapB-Pstl construct contains both putative genes and the dapB-Sall construct only contains open reading frame ORFz. The Sail and Pstl restriction sites (other than those present in the polylinker of KSII+) used for the construction of these deletions are indicated on the dapB construct. Direction and length of the dapB gene and ORFz are indicated by arrows; and
Figure 5 represents amino acid alignment of the deduced BCG DapB protein (enzyme) and DapB proteins (enzymes) from other species. The * indicates residues that are identical 100% and # indicates residues that are identical in over 50% of the DapB proteins. Amino acid identity and conservation is indicated as compared to the deduced BCG dapB sequence.
DETAILED DESCRIPTION OF THE INVENTION Disruption of biosynthesis of DAP results in cell death, most probably by lysis due to instability of the peptidoglycan. DAP biosynthetic enzymes are essential for bacteria and mycobacteria in vivo due to the absence of DAP in mammalian cells. Therefore, bacteria and mycobacteria must be able to synthesize DAP in vivo. For this reason, DAP biosynthetic genes are useful as targets for anti-mycobacterial and anti-bacterial agents as well as for the design of in vivo selection systems.
Prior hereto, the mycobacterial DAP biosynthetic pathway had not been determined, and there has been little information available concerning the genes involved in mycobacterial DAP biosynthetic pathways. The importance of the DAP biosynthetic pathway in mycobacteria has been illustrated by data indicating that mutations in the aspartate semialdehyde (asd) gene are lethal even in the presence of exogenous DAP (see Cirillo et al., J. Bacteriol.. Vol. 173, pp. 7772-7780 (1991)). Using complementation analysis in E. coli, the inventors determined which pathways are utilized in BCG for synthesis of DAP. Complementing clones, which complemented five of seven steps in the DAP pathway, were isolated after further analysis indicated the presence of at least two biosynthetic pathways for synthesis of DAP in BCG. The nucleotide sequence of the dapB polynucleotide complementing fragment was determined. Complementation analysis using this polynucleotide fragment provided evidence that it contains the putative BCG dihydrodipicolinate reductase (dapB) and DAP dehydrogenase (ddh) genes. The presence of a novel bifunctional dihydrodipicolinate reductase/DAP dehydrogenase in mycobacteria was postulated. This isolated, cloned, sequenced and characterized mycobacterial dapB polynucleotide (gene) provides a useful tool in the treatment and prevention of mycobacterial infection and in the development of in vivo marker systems. For example, mycobacterial infection can be treated by administering a pharmaceutically effective amount of an oligonucleotide which inhibits the mRNA activity of the mycobacteria. These oligonucleotides can be prepared utilizing the nucleic acid sequence of the dapB gene (polynucleotide) of the invention, which is depicted in Figure 3.
In addition, compounds which bind to the mycobacterial enzyme DapB can be produced and administered. These compounds would inhibit the activity of the DapB enzyme. Because DAP is a necessary component of cell walls, the inability of the mycobacteria to synthesize DAP would result in cell death.
Compounds which can block the activity of the mycobacterial enzyme DapB can be produced by overexpressing mycobacterial DapB enzyme, purifying the overexpreεsed enzyme, performing x-ray crytallography on the purified enzyme so as to obtain the molecular structure of the enzyme, and then creating a compound with a similar molecular structure to the enzyme. This compound can be administered so as to inhibit the activity of the enzyme, thereby causing cell death.
In addition, vaccines useful in the treatment and transmission prevention of mycobacterial infection can be produced. Because the inventors have determined the sequence of the mycobacterial dapB gene, it is possible to determine the existence of a mutated mycobacterial dapB gene. A mutated mycobacterial dapB gene can be administered in vaccine form to treat and prevent mycobacterial infection.
In addition, vaccines can be formed which comprise a mycobacterial strain having a deleted dapB gene, wherein the mycobacterial strain is grown on artificial media containing dioaminopimelic acid and then administered in vaccine form. Alternatively, a recombinant vaccine can be prepared by deleting dapB gene from a mycobacterial strain and cloning into the mycobacterial strain a plasmid which expresses dapB.
Further, because the sequence of the mycobacterial dapB gene is now known, antibodies specific for polynucleotides having the nucleic acid sequence of the dapB gene of the invention can produced. These antibodies can be passively administered to treat and prevent mycobacterial infection.
The polynucleotides of the invention can also be used to produce research tools capable of identifying virulence genes and drug targets in mammals. A research tool is prepared by deleting dapB gene from a mycobacterial strain and inserting therein a plasmid containing a promoterless dapB gene and a genomic mycobacterial nucleotide sequence so as to produce a library. This library is then passed through mammalian cells and used to identify virulence genes and drug targets.
In addition, any polynucleotides which are involved in mycobacterial biosynthesis of diaminopimelic acid can be used to treat and prevent both bacterial and mycobacterial infection. Specifically, polynucleotides which can be used to treat and prevent bacterial and mycobacterial infection include mycobacterial dapA, mycobacterial dapD, mycobacterial dapE, mycobacterial ddh, mycobacterial asd and mycobacterial ask. In addition, promoters and translation initiation sequences for the mycobacterial dapB gene are useful in the treatment and prevention of bacterial and mycobacterial infection. The bacterial strains used by the inventors are described in Table 1, below. E. coli strains were grown in Luria Broth (LB) or on LB agar with supplements. Ampicillin was added when necessary at a final concentration of 25 μg/ml, isopropylthio-β-D-galactoside (IPTG) at 2 mM and 5-bromo-4- chloro-3-indolyl-β-D-galactoside (X-gal) at 40 μg/ml.
TABLE 1
Bacterial strains
STRAIN GENOTYPE SOURCE
BCG
Pasteur BCG vaccine strain WHO reference center, Copenhagen
E. coli
DH5α O80d, lacZΔM15, endAl, recAl, Bethesda Research hsdR17, supE44, thi-1, gyrA96, Laboratories Δ(lacZγA-argf)U169 vo
X6118 lacγl, glnV44, galK2, galT22, tyrT58, R. Curtiss, III etBl, hsdR514, trpR55, asd-4 ec2904 dapA: :Cm C. Richaud
AT999 dapB17::Mu, relAl, spoTl, thi-1 (6)
AT986 dapD8, relAl, spoTl, thi-1 (6) ec2927 dapE: :Km, thrB1004, pro, thi, strA C. Richaud hsdS, lacZΔM15
In order to construct a BCG expression library, BCG chromosomal DNA was isolated as described by Jacobs et al.. Methods Enzvmol.. Vol. 204, pp. 537-555 (1991). Approximately 2 to 4 kb size fragments were generated by Sau3A partial digestion. The agarose gel purified fragments were ligated to BamHI digested dephosphorylated pBlueScript II KS+ (KSII+) DNA (Stratagene) . The resulting ligation was then transformed by electroporation into DH5α and plated on LB agar containing a picillin, IPTG and X-gal. The total number of non-recombinants, as judged by the percentage of blue colonies on this medium, was less than 10%. To prepare a large quantity of the library for use in complementation analysis, the ligation was transformed in the same manner and plated on LB plates containing ampicillin in the absence of IPTG so as to allow propagation of potentially lethal clones. Greater than 100,000 colonies produced in this manner were pooled and a plasmid was isolated from them for use in the following experiments.
In order to perform complementation of DAP mutations, the library was transformed into various mutant bacterial strains by electroporation. The vector plasmid KSII+ was used as a negative control. These transformations were then plated on two types of selective mediums. The first was LB agar containing ampicillin and DAP to determine the number of transformants. The second medium was LB agar containing ampicillin and IPTG to allow expression of recombinant proteins. The colonies present in the absence of DAP above that obtained with KSII+ were assumed to contain plasmids carrying mycobacterial genomic fragments that complemented the DAP mutation present in the strain. Plasmid was then isolated from at least 10 clones that complemented each individual E. coli mutation and retransfor ed into the strain to confirm the ability of the plasmid to confer the complementing activity. Mycobacterial DAP regions were then characterized. In order to determine whether each complementing fragment contained more than one mycobacterial gene, the complementation profile of the clones was also determined. The complementation profile for each mycobacterial DAP complementing region was determined by testing each clone that complemented an E. coli mutation for its ability to complement the other E. coli mutations as well. This procedure was accomplished in the same manner as described above except that all E. coli DAP mutants were tested simultaneously. To further compare the regions of mycobacterial DNA that complemented the E. coli DAP mutations, a physical map of a representative clone that complemented each mutation was determined. Physical maps were constructed with the restriction enzymes Pstl and Xhol. Sizes of restriction fragments were determined by analytical agarose gel electrophoresis.
The mycobacterial dapB region was then analyzed and sequenced. Two deletions were constructed in the dapB complementing fragment in order to determine the locations of the relevant complementing genes present (see Figure 5). The first deletion was constructed by digestion of the dapB complementing plasmid with Pstl and purifying the resulting large DNA fragment by agarose gel electrophoresis. This DNA fragment was then self ligated, transformed with DH5α and the appropriate clone containing an approximately 600 bp deletion in the 5' end of the mycobacterial DNA was selected. The complementation profile for this construct, designated dapB-Pstl, was then determined in the same manner as described above.
A further deletion of this construct was made by digesting dapB-Pstl with Sail and isolation of the resulting large DNA fragment by agarose gel electrophoresis. The large DNA fragment was then self ligated, transformed into DH5α and the appropriate clone containing a further deletion of approximately 1.1 kb into the 5' end of the fragment selected. The complementation profile for this construct, designated dapB-Sall, was then determined in the same manner as described above.
Sequence analysis was performed on the construct dapB-Pstl in order to determine the nucleotide sequence of the mycobacterial dapB region. Sequencing was carried out on double stranded DNA as described by Kraft et al., Bio Techniques. Vol. 6, pp. 544-547 (1988) using Sequenase (USB) . The complete sequence was determined on both strands using synthetic oligonucleotides . The resulting sequences were compared for overlaps using Fasta (see Pearson et al., Proc. Natl. Acad. Sci. USA. Vol. 85, pp. 2444-2448 (1988)). Protein sequence alignments were constructed using Fasta and Pileup from the Genetics Computer Group (Wisconsin) software package. Analysis for the presence of open reading frames (ORF) was also accomplished using the Genetics Computer Group software package.
BCG DAP biosynthetic genes were isolated. Although the mycobacterial asd gene was isolated previously, the inventors tested the ability of the KSII+ expression library to complement the asd mutation contained in the E. coli strain X6118. This experiment allowed the inventors to test the quality of the library using a well characterized system. The results of this complementation experiment are shown in Table 2, below. TABLE 2
Frequencies of complementation
Percent Complenta ion3
Mutation E. coli strain Library Secondary0 KSII+
asd X6118 0.02 36 0 dapA ec2904 1 .8 x 10~5 19 1 x 10-6 dapB AT999 2 .0 x 10~4 15 0 dapD AT986 2 .1 X 10~5 10 5 X 10-6 c dapE ec2927 0.025 57 2 X 10~6
Frequencies are calculated as the number of colonies that grow in the absence of DAP as compared to the transformation frequency from the number of colonies that grow in the presence of DAP.
Secondary complementation frequencies are obtained from the mean range of complementation observed when re-transformed into the same strain
.
-14-
Once the quality of the expression library was determined, the DAP biosynthetic pathway present in BCG was elucidated by complementing E. coli mutations in four additional steps in this pathway. The steps that were chosen are catalyzed by the products of the E. coli genes dapA, dapB, dapD, and dapE. E. coli strains containing mutations in each of these genes were transformed with the expression library. The results of these experiments are shown in Table 2. The frequency at which complementing clones were obtained was highly variable. However, the level of complementation of the plasmids isolated from each of the complemented clones was high. Several clones were isolated from the library that appeared to complement all five of the E. coli mutations tested. A single clone for each gene was purified for further characterization.
In order to perform complementation profiles of the BCG DAP regions, each set of clones was tested for its-ability to complement the other steps in the DAP biosynthetic pathway. The results of this analysis are shown in Table 3, below. The overlapping clones that complemented that asd, dapA, dapD, and dapE mutations did not exhibit the ability to complement any other DAP mutation. The dapB complementing clones, however, exhibited complementation of the dapD and dapE mutations as well. The frequencies obtained were in the same range as for the secondary complementation experiments described previously. TABLE 3
Complementation profiles
Percent Complentationa of Clone for
Mutation Strain as dapA dapB dapD dapE
asd X6118 42 0 0 0 0 dapA ec2904 0 23 0 0 0 dapB AT999 0 0 14 0 0 dapD AT986 0 0 1.5 9.2 0 dapE ec2927 0 0 20 0 53
Complementation is expressed as the mean percentage of transformants that complement each mutation
To perform physical mapping of the BCG DAP regions, the positions of Xhol and Pstl sites in the BCG DAP complementing regions were determined. The resulting physical maps are shown in Figure 2. No similarity was observed between the physical maps of any of the DNA fragments from BCG that complemented the E. coli DAP mutations. The estimated size of the DNA fragments correlated well with that expected for this expression library, which was constructed using 2-4 kb fragments. There are a higher number of Pstl and Xhol than would be expected from the random DNA segments. This fact is consistent with the high guanine + cytosine (G+C) content of BCG DNA.
Nucleotide sequencing and ORF analysis of the BCG dapB region was performed. The sequence of the dapB complementing DNA fragment was determined from the Pstl site downstream. The resulting sequence was 1792 bp in length (see Figure 3) . The total G+C content of the DNA fragment was 71%. When the nucleotide sequence of this region was used to search Genbank for similar sequences, none were found that had significant similarity at the nucleotide level.
Codon usage analysis of this region revealed the presence of two large ORFs that display the expected codon preference for mycobacteria. A potential translation start for the first ORF occurs at nucleotide 312 and may encode a protein of 271 amino acids in length. The region upstream of the putative translational start was examined for the presence of ribosomal binding site. No sequences were found that fit the E. coli consensus exactly, however, at a distance of 8 nucleotides upstream there was a sequence of AGG that corresponds to that which would be expected. The second ORF begins and has a potential translational start at nucleotide 1151, and would allow a protein of 177 amino acids to be produced. The presence of a potential ribosomal binding site was detected 10 bp 5' of the putative translational start (see Figure 3) .
In order to determine the location of the regions that confer the DapB+ and ddh+ phenotype, two deletions were made into the dapB complementing fragment (see Figure 4). The complement construct and these deletions were tested for their ability to complement each of the E. coli mutations. The resulting complementation profiles for these constructs is shown in Table 4, below. The original construct and the dapB-Pstl deletion were able to complement the dapB, dapD, and dapE mutations. However, the dapB-Sall deletion, that contains only the last ORF from the sequenced region, was unable to complement any of these mutations. These results indicate that the first ORF conferred the ability to complement the E. coli dapB, dapD, and dapE mutations. In order to determine the identity of the second ORF on this fragment, its deduced amino acid sequence was compared to the protein sequence databases of EMBL and SwissProt. No similarity was observed between this ORF and sequences in these databases.
TABLE 4 Complementation profiles of dapB fragment deletions
Percent Complentation for Mutation Strain dapB dapB-Pstl dapB-Sall KSII+
oo dapB AT 99 7.4 17 0 0 dapD AT986 0.49 1.3 2 x lo-5 4 X 10~5 dapE ec2927 5.8 2.0 0 0
The deduced protein sequence from the first gene was compared to the amino acid sequence of other dapB genes (see Figure 5) . Significant sequence conservation was observed between this ORF and the DapB proteins from B. lactofermentum and E. coli. There are several regions in these proteins that display a high level of conservation across species. In particular, a five amino acid region, PSGTA, is completely conserved in all three species. When the deduced amino acid sequence of this ORF was compared to the only ddh protein sequence available (from C. αlutamicum) no significant sequence similarity was observed. These results indicate that the BCG dapB gene encodes a novel bifunctional enzyme with the ability to catalyze both the dihydrodipicolinate reductase and diaminopimelate dehydrogenase reactions involved in biosynthesis of DAP.
These results indicate that BCG is able to synthesize DAP by at least two of three possible pathways. Using complementation analysis in E. coli. the inventors isolated the BCG genes encoding asd, dapA, dapB, dapD, and dapE involved in the succinylase pathway. Other than the asd and ask genes, which have been shown to be in an operon, none of the DNA fragments that carry these genes appear to be linked, as judged by physical mapping. This apparently unlinked arrangement of genes is not entirely unexpected due to the dispersed arrangement of genes in E. coli. In addition, the BCG dapD and dapE complementing fragments were unable to complement the dapE and dapD mutations, respectively, in E. coli. These results indicate the presence of two separable genes in BCG that encode proteins involved in the succinylase pathway for biosynthesis of DAP.
In addition to the presence of the succinylase biosynthetic pathway in BCG, the inventors found evidence for the ability to synthesize DAP via the dehydrogenase pathway. The BCG dapB complementing fragment was found to complement both the dapD and dapE E. coli mutations. Although this result may be explained by linkage between the BCG dapB and ddh genes, it was unexpected since none of the original dapD and dapE complementing clones displayed the ability to complement both mutations. This discrepancy may be explained by the observation that the dapB complementing fragment complements these mutations at a 5 to 10 fold lower frequency than the dapD and dapE genes themselves. Thus, this observation may be explained by the fact that the inventors examined only a few clones for each gene.
Deletion analysis of the dapB complementing region indicated that a very small region of DNA was responsible for the dapB and ddh activities. This region was too small to encode a normal dapB gene as well as a ddh gene. Sequence analysis and comparison of the deduced amino acid sequence of the protein encoded by this region indicated significant homology to DapB proteins from other bacteria but no homology to ddh from C. αlutamicum. These observations indicate that the BCG dapB gene encodes a bifunctional protein.
The BCG DapB protein is significantly different from the E. coli DapB protein, making it plausible for it to have acquired a new function. There is, however, a higher degree of similarity in the structure of the Brevibacterium and BCG DapB proteins that may indicate that they may have the same function. This possibility has not been tested with the Brevibacterium gene. Brevibacterium species have been shown to express ddh activity (see Misono et al., J. Bacteriol.. Vol. 137, pp. 22-27 (1979)). The reactions catalyzed by the dihydrodipicolinate reductase and diaminopimelate dehydrogenase are similar. Both the BCG and Brevibacterium DapB proteins have a conserved amino terminal region reminiscent of the amino terminal domain of NADP+-dependent dehydrogenases,
(V/I) (A/G) (V/I)XGXXGXXG.
A sequence similar to this consensus is also found in the amino terminus of the C. αlutamicum ddh protein. This domain is expected to be present in ddh ad DapB proteins since they both require NADPH. Since the DapB enzyme has the ability to bind NADPH, dihydrodipicolinate and tetrahydrodipicolinate, a two step reaction where the DapB protein would utilize two molecules of NADPH may occur. The first molecule would be utilized to reduce dihydrodipicolinate and the second to reduce tetrahydrodipicolinate producing DAP. An alternative explanation may be that overexpression of this protein in E. coli confers an abnormal phenotype under non-physiological conditions. The use of biochemical assays may be necessary to determine whether the acetylase pathway is present for synthesis of DAP in BCG.
The dapB gene of mycobacteria provides an extremely useful tool for the development of new antibiotics specific for bacteria and for mycobacteria, and for use in the construction of a marker system that may have applications for studies in vivo. The dapB gene product catalyzes a key step in the biosynthetic pathway of DAP whether one or all three of the potential pathways is utilized. An inhibitor of the DapB enzyme would be lethal in the absence of the ability to acquire DAP from an exogenous source. Since the dapB gene has been cloned by the inventors, substrate analogues which inhibit the activity of the purified recombinant protein can be determined. Future studies will allow the crystal structure of this protein to be determined. This information combined with the above-described demonstration of highly conserved regions will further contribute to strategies for the design of inhibitors. Inhibitors of DapB will have a broad applicability, similar to other antibiotics that inhibit cell wall biosynthesis.
Further, an ideal marker system for the maintenance of recombinant DNA in bacteria during mammalian infection or vaccination would require the use of auxotrophic marker systems. The use of antibiotic markers is disadvantageous due to possible further dissemination of antibiotic resistance and due to the inability to maintain selective pressure in vivo. Since DAP is not present in mammalian cells, a dapB mutant would provide a suitable host strain for maintenance of recombinant DNA carrying the dapB gene in vivo.
Although the invention herein has been described with reference to particular embodiments, it is to be understood that these embodiments are merely illustrative of various aspects of the invention. Thus, it is to be understood that numerous modifications may be made in the illustrative embodiments and other arrangements may be devised without departing from the spirit and scope of the invention.
SEQUENCE LISTING
(1) GENERAL INFORMATION
(i) APPLICANT: Jacobs et al
(ii) TITLE OF INVENTION: GENE FOR MYCOBACTERIAL
DIAMINOPIMELIC ACID
(iii) NUMBER OF SEQUENCES: 4
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Amster, Rothstein & Ebenstein
(B) STREET: 90 Park Avenue
(C) CITY: New York
(D) STATE: New York
(E) COUNTRY: U.S.A.
(F) ZIP: 10016
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 inch 1.44 Mb storage diskette
(B) COMPUTER: IBM PC Compatible
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: Word Processor (ASCII)
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: Not Yet Assigned
(B) FILING DATE: Not Yet Assigned
(C) CLASSIFICATION: Not Yet Assigned
(vii) PRIOR APPLICATION DATA: none
(A) APPLICATION NUMBER: none
(B) FILING DATE: none
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: George, Kenneth P.
(B) REGISTRATION NUMBER: 30,259
(C) REFERENCE/DOCKET NUMBER: 96700/358
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (212) 697-5995
(B) TELEFAX: (212) 286-0854 or 286-0082
(C) TELEX: TWX 710-581-4766 (2) INFORMATION FOR SEQ ID NO: 1
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1801
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(A) DESCRIPTION: dapB gene
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: BCG
(A) ORGANISM: BCG
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME SEGMENT:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION: none
(A) AUTHORS:
(B) __ ± ±. —£..
(C) JOURNA :
(D) VOLUME:
(F) PAGES :
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
CTGCAGGTAC AGATGCTTGA CCTGCTACCG CACGCCCGCC GGTCCGTCCT CGACCCTGCG 60
GATGTCCCGC GATGTCCCGC CGCCGCACCC GCATCCGCCC CGTCGTCGTC GACGAGCACG 120
AGAAAGAGCC GGCCGGCGGC GTGAAGACGG ATGCCGCGGG GAAGACGGGA CGGTACGCCT 180
GCCCCTGAGG GAACGCGTCG GCACGCATCT GGAAGTACTC CTCCAGCAGG GCATGTGCGC 240
GCGGGTCGTC GGCGGCGAGG GGGTCGAGCA GCACCATCCC TCGAGCGTAA CCGTGTCGGC 300
CGCACGGCCT AGGCTGGAAC C ATG ACG ACG AGA GTG GCC CTC GTG GGC GGC 351
Met Thr Thr Arg Val Ala Leu Val Gly Gly 5 10
ACC GGC AAG CTC GGT GCG ATC ATC GCC GGC GTG ATC GAC GAG CTC GAC 399 Thr Gly Lys Leu Gly Ala He He Ala Gly Val He Asp Glu Leu Asp 15 20 25
GAC TTC GAG ACG GTG GCC GTT CTG GGG TCG GAC GAG CGA TCT CGC GAG 447 Asp Phe Glu Thr Val Ala Val Leu Gly Ser Asp Glu Arg Ser Arg Glu 30 35 40
ATC GAC GCG GCC GAT CTC GTC GTC GAT GCG TCG ACG CCC GGT GTG TCG 495 He Asp Ala Ala Asp Leu Val Val Asp Ala Ser Thr Pro Gly Val Ser 45 50 55
ATC GAC GTC GTC CGC GCA GCG ATC GAA CGC GGG AAG AAC GTG CTC GTC 543 He Asp Val Val Arg Ala Ala He Glu Arg Gly Lys Asn Val Leu Val 60 65 70
GGC ACC TCG GGC TGG TCG ACC GAA CGG ATC GCG CTG GTG CGC CCC CTC 591 Gly Thr Ser Gly Trp Ser Thr Glu Arg He Ala Leu Val Arg Pro Leu 75 80 85 90
GTC GAA GCG GCG GGT ACG GGC GCG GTC TTC ATT CCC AAC TTC TCC CTG 639 Val Glu Ala Ala Gly Thr Gly Ala Val Phe He Pro Asn Phe Ser Leu 95 100 105
GGC TCG GTC GTC GCG ACG GCA CTG GCG GCG GCG GCG GCA CCT CTG TTC 687 Gly Ser Val Val Ala Thr Ala Leu Ala Ala Ala Ala Ala Pro Leu Phe 110 115 120
CCC TCG ATC GAG ATC GTC GAG ACG CAT CGC GAG ACG AAA GTC GAC TCG 735 Pro Ser He Glu He Val Glu Thr His Arg Glu Thr Lys Val Asp Ser 125 130 135
CCG AGT GGC ACG GCG GTG CGC ACC GCC GAA CTC ATC GCC GAC GCT CGC 783 Pro Ser Gly Thr Ala Val Arg Thr Ala Glu Leu He Ala Asp Ala Arg 140 145 150 GTC GGG GTC GGG CCG GTC GAG TCC CCG CAT GTC GAT CAG CGG GCG CGC 831 Val Gly Val Gly Pro Val Glu Ser Pro His Val Asp Gin Arg Ala Arg 155 160 165 170
GGC CAG CAG GTC GCG AGT GTT CCC ATC CAT TCG TTG CGC CGG CCG GGC 879 Gly Gin Gin Val Ala Ser Val Pro He His Ser Leu Arg Arg Pro Gly 175 180 185
GTC ATC GCG AAC GAG GAG ACG ATC CTG TCG GGG CCG GGG GAG TCG CTG 927 Val He Ala Asn Glu Glu Thr He Leu Ser Gly Pro Gly Glu Ser Leu 190 195 200
ACC ATC GTC CAC GAC ACG ATC GAG CCG GCC CGC GCG TAC GCG CCC GGC 975 Thr He Val His Asp Thr He Glu Pro Ala Arg Ala Tyr Ala Pro Gly 205 210 215
ATC CGC ATC GCC CTT GCG GCG CGC GCG GAC GCG CGC GGG GTG ACG ATC 1023 He Arg He Ala Leu Ala Ala Arg Ala Asp Ala Arg Gly Val Thr He 220 225 230
GGC CTC GAC GCG CTC ATC GAT CTC GGT CTC GCG CCG CGC CCC GCA GCG 1071 Gly Leu Asp Ala Leu He Asp Leu Gly Leu Ala Pro Arg Pro Ala Ala 235 240 245 250
CCG GTC ATC GAG GTC GTC GAC GAG GGC TCC GTT CCC GGG CAA GTC GCC 1119 Pro Val He Glu Val Val Asp Glu Gly Ser Val Pro Gly Gin Val Ala 255 260 265
CGC GTC ACC GGG GCA TGAAGGCCCG CGCGGGCGTC GCCGTC ATG GCG GCG 1169 Arg Val Thr Gly Ala Met Ala Ala
270
CTG CTC GTC CTC TAC ATC GTT CTC GTC GCG CAG CGC GCG TGG CTG TTG 1217 Leu Leu Val Leu Tyr He Val Leu Val Ala Gin Arg Ala Trp Leu Leu 275 280 285 290
CTG ATC TCG GGG CAG ATC ATC GGC GTC GCC ATG GGC GTG GCG CTG ATC 1265 Leu He Ser Gly Gin He He Gly Val Ala Met Gly Val Ala Leu He 295 300 305
GTC CTG CCC GTC ATC GCG GGG TGG GCG CTC TGG CGG GAG CTT GCT TTC 1313 Val Leu Pro Val He Ala Gly Trp Ala Leu Trp Arg Glu Leu Ala Phe 310 315 320
GGG CGC AGC GCC GAG CGC CTC GCC AGG CCG GCT GGA GGC CGA AGG CCG 1361 Gly Arg Ser Ala Glu Arg Leu Ala Arg Pro Ala Gly Gly Arg Arg Pro 325 330 335
CCG CTC CCG AGC GAG GAG ATG GAT GTC CGC GTC AGC GGC CGT CCC GAC 1409 Pro Leu Pro Ser Glu Glu Met Asp Val Arg Val Ser Gly Arg Pro Asp 340 345 350 CGT GCG CAG GCC GAT GCG GTG TTC CCG CAC TAC CGG CAC GAC GTC GAG 1457 Arg Ala Gin Ala Asp Ala Val Phe Pro His Tyr Arg His Asp Val Glu 355 360 365 370
GCG CAC CCC ACC GAT TGG CGC GCC TGG TTC CGG CTG GGG CTG GCC TAC 1505 Ala His Pro Thr Asp Trp Arg Ala Trp Phe Arg Leu Gly Leu Ala Tyr 375 380 385
GAC GGC GCC GGC GAT CGA CGC CGC GCC CGC GAA GCC ATC CGA CGG GCG 1553 Asp Gly Ala Gly Asp Arg Arg Arg Ala Arg Glu Ala He Arg Arg Ala 390 395 400
ATC ACG CTC GAG AAG AAC CCG CCG ACG CGC TGG ATC AGG CCG GCC ACC 1601 He Thr Leu Glu Lys Asn Pro Pro Thr Arg Trp He Arg Pro Ala Thr 405 410 415
GCG GCG GCC ACG GCA TCC TCG ACC GTC GGA TGC GAG AAG ACG AAG CCG 1649 Ala Ala Ala Thr Ala Ser Ser Thr Val Gly Cys Glu Lys Thr Lys Pro 420 425 430
GAT GCC GTG AGC GCC TCG GGG ACC ACC ATT CAT GTC GCA CCG 1691
Asp Ala Val Ser Ala Ser Gly Thr Thr He His Val Ala Pro 435 440 445
TGAGCCAGTC CCTCTGCCGC GTCGCGCCCG AGGACCAGCT TCAGCGCCCA CTCCGGTACG 1751
CGCAGCAGGT AGGGGCGATT CATCCGCCTC GCGAGAGCGA ACCCGAGATC 1801
(3) INFORMATION FOR SEQ ID NO: 2
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 271
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(A) DESCRIPTION: dapB protein
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE:
(vi) ORIGINAL SOURCE: BCG
(A) ORGANISM: BCG
(B) STRAIN:
(C) INDIVIDUAL ISOLATE: (D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME SEGMENT:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(x) PUBLICATION INFORMATION: none
(A) AUTHORS:
(B) TITLE:
(C) JOURNAL:
(D) VOLUME:
(F) PAGES:
(G) DATE:
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2
Met Thr Thr Arg Val Ala Leu Val Gly Gly Thr Gly Lys Leu Gly Ala
5 10 15
He He Ala Gly Val He Asp Glu Leu Asp Asp Phe Glu Thr Val Ala 20 25 30
Val Leu Gly Ser Asp Glu Arg Ser Arg Glu He Asp Ala Ala Asp Leu 35 " 40 45
Val Val Asp Ala Ser Thr Pro Gly Val Ser He Asp Val Val Arg Ala 50 55 60
Ala He Glu Arg Gly Lys Asr. Val Leu Val Gly Thr Ser Gly Trn Ser 65 70 75 " 80
Thr Glu Arg He Ala Leu Val Ala Ala Pro Ser Ser Lys Arg Arg Arg
85 90 95 Ala Ala Val Phe He Pro Asn Phe Ser Leu Gly Ser Val Val Ala Thr 100 105 110
Ala Leu Ala Ala Ala Ala Ala Pro Leu Phe Pro Ser He Glu He Val 115 120 125
Glu Thr His Arg Glu Thr Lys Val Asp Ser Pro Ser Gly Thr Ala Val 130 135 140
Arg Thr Ala Glu Leu He Ala Asp Ala Arg Val Gly Val Gly Pro Val 145 150 155 160
Glu Ser Pro His Val Asp Gin Arg Ala Arg Gly Gin Gin Val Ala Ser
165 170 175
Val Pro He His Ser Leu Arg Arg Pro Gly Val He Ala Asn Glu Glu 180 185 190
Thr He Leu Ser Gly Pro Gly Glu Ser Leu Thr He Val His Asp Thr 195 200 205
He Glu Pro Ala Arg Ala Tyr Ala Pro Gly He Arg He Ala Leu Ala 210 215 220
Ala Arg Ala Asp Ala Arg Gly Val Thr He Gly Leu Asp Ala Leu He 225 230 235 240
Asp Leu Gly Leu Ala Pro Arg Pro Ala Ala Pro Val He Glu Val Val
245 250 255
Asp Glu Gly Ser Val Pro Gly Gin Val Ala Arg Val Thr Gly Ala 260 265 270
(4) INFORMATION FOR SEQ ID NO: 3
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 248
(B) TYPE: arr.ir.o acid
(C) STRANDEDNESS : sinσle
(D) TOPOLOGY: linear'
(ii) MOLECULE TYPE:
(A) DESCRIPTION: dapB protein
( iii) HYPOTHE ICA : no
(iv) ANTI-SENSE:
(v) FRAGMENT TYPE: (vi) ORIGINAL SOURCE: B lactofermentum
(A) ORGANISM: B lactofermentum
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE:
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE: (H) CELL LINE: (I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME SEGMENT:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(X) PUBLICATION INFORMATION:
(A) AUTHORS: Pisabarro et al
(B) TITLE:
(C) JOURNAL: J Bacteriol
(D) VOLUME: 174
(F) PAGES: 2743-2749
(G) DATE: 1993 (H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RESIDUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3
Met Gly He Lys Val Gly Val Leu Gly Ala Lys Gly Arg Val Gly Gin
5 10 15
Thr He Val Ala Ala Val Asn Glu Ser Asp Asp Leu Glu Leu Val Ala 20 25 30
Glu He Gly Val Asp Asp Asp Leu Ser Leu Leu Val Asp Asn Gly Ala 35 * 40 45
Glu Val Val Val Asp Phe Thr Thr Pro Asn Ala Val Met Gly Asn Leu 50 55 60
Glu Phe Cys He Asn Asn Gly He Ser Ala Val Val Gly Thr Thr Gly 65 70 75 80 Phe Asp Asp Ala Arg Leu Glu Gin Val Arg Ala Trp Leu Glu Gly Lys
85 90 95
Asp Asn Val Gly Val Leu He Ala Pro Asn Phe Ala He Ser Ala Val 100 105 110
Leu Thr Met Val Phe Ser Lys Gin Ala Ala Arg Phe Phe Glu Ser Ala 115 120 125
Glu Val He Glu Leu His His Pro Asn Lys Leu Asp Ala Pro Ser Gly 130 135 140
Thr Ala He His Thr Ala Gin Gly He Ala Ala Ala Arg Lys Glu Ala 145 150 155 160
Gly Met Asp Ala Gin Pro Asp Ala Thr Glu Gin Ala Leu Glu Gly Ser
165 170 175
Arg Gly Ala Ser Val Asp Gly He Pro Val His Ala Val Arg Met Ser 180 185 190
Gly Met Val Ala His Glu Gin Val He Phe Gly Thr Gin Gly Gin Thr 195 200 205
Leu Thr He Lys Gin Asp Ser Tyr Asp Arg Asn Ser Phe Ala Pro Gly 210 215 220
Val Leu Val Gly Val Arg Asn He" Ala Gin His Pro Gly Leu Val Val 225 230 235 240
Gly Leu Glu His Tyr Leu Gly Leu
245
(5) INFORMATION FOR SEQ ID NO: 4
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 272
(B) TYPE: amino acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE:
(A) DESCRIPTION: dapB protein
(iii) HYPOTHETICAL: no
(iv) ANTI-SENSE: (v) FRAGMENT TYPE:
( i) ORIGINAL SOURCE: E coli
(A) ORGANISM: E coli
(B) STRAIN:
(C) INDIVIDUAL ISOLATE:
(D) DEVELOPMENTAL STAGE
(E) HAPLOTYPE:
(F) TISSUE TYPE:
(G) CELL TYPE:
(H) CELL LINE:
(I) ORGANELLE:
(vii) IMMEDIATE SOURCE:
(viii) POSITION IN GENOME:
(A) CHROMOSOME SEGMENT:
(ix) FEATURE:
(A) NAME/KEY:
(B) LOCATION:
(C) IDENTIFICATION METHOD:
(D) OTHER INFORMATION:
(X) PUBLICATION INFORMATION:
(A) AUTHORS: Bouvier et .
(B) TITLE:
(C) JOURNAL: J Biol Chem
(D) VOLUME: 259
(F) PAGES: 14829-14834
(G) DATE: (1984)
(H) DOCUMENT NUMBER:
(I) FILING DATE:
(J) PUBLICATION DATE:
(K) RELEVANT RES 11DUES:
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4
Met His Asp Ala Asn He Arg Val Ala He Ala Gly Ala Gly Gly Arg
5 10 15
Met Gly Arg Gin Leu He Gin Ala Ala Leu Ala Leu Glu Gly Val Gin 20 25 30
Leu Gly Ala Ala Leu Glu Arg Glu Gly Ser Ser Leu Leu Gly Ser Asp 35 40 45
Ala Gly Glu Leu Ala Gly Ala Gly Lys Thr Gly Val Thr Val Gin Ser 50 55 60 Ser Leu Asp Ala Val Lys Asp Asp Phe Asp Val Phe He Asp Phe Thr 65 70 75 80
Arg Pro Gly Thr Leu Asn His Leu Ala Phe Cys Arg Gin His Gly Lys
85 90 95
Gly Met Val He Gly Thr Thr Gly Phe Asp Glu Ala Gly Lys Gin Ala 100 105 110
He Arg Asp Ala Ala Ala Asp He Ala He Val Phe Ala Ala Asn Phe 115 120 125
Ser Val Gly Val Asn Val Met Leu Lys Leu Leu Glu Lys Ala Ala Lys 130 135 140
Val Met Gly Asp Tyr Thr Asp He Glu He He Glu Ala His His Arg 145 150 155 160
His Lys Val Asp Ala Pro Ser Gly Thr Ala Leu Ala Met Gly Glu Ala
165 170 175
He Ala His Ala Leu Asp Lys Asp Leu Lys Asp Cys Ala Val Tyr Ser 180 185 190
Arg Glu Gly His Thr Gly Glu Arg Val Pro Gly Thr He Gly Phe Ala 195 200 205
Thr Val Arg Ala Gly Asp He Val Gly Glu His Thr Ala Met Phe Ala 210 215 220
Asp He Gly Glu Arg Leu Glu He Thr His Lys Ala Ser Ser Arg Met 225 230 235 240
Thr Phe Ala Asn Gly Ala Val Arg Ser Ala Leu Trp Leu Ser Gly Lys
245 250 255
Glu Ser Gly Leu Phe Asp Met Arg Asp Val Leu Asp Leu Asn Asn Leu 260 265 270

Claims

WE CLAIM :
1. A polynucleotide which encodes an enzyme required for mycobacterial biosynthesis of diaminopimelic acid.
2. A polynucleotide which encodes the mycobacterial enzyme DapB.
3. A polynucleotide which encodes the mycobacterial enzyme DapB, said polynucleotide having a nucleic acid sequence depicted in Figure 3.
4. A method of treating bacterial or mycobacterial infection comprising the administration of a compound which binds to the mycobacterial enzyme DapB, thereby inhibiting the activity of said enzyme.
5. A method of treating bacterial or mycobacterial infection comprising the administration of a compound which blocks the activity of the mycobacterial enzyme encoded by the mycobacterial dapB gene.
6. The method of Claim 5 wherein the mycobacterial dapB gene has a nucleic acid sequence depicted in Figure 3.
7. The method of Claim 5 wherein the mycobacterial infection is caused by a mycobacterium selected from the group consisting of M. tuberculosis. M. avium. M. fortuitum. M. αordoneae. M. haemoilphilum. M. paratuberculosis. M. bovis and M. leprae.
8. A method of treating bacterial or mycobacterial infection comprising: (a) preparing anti-DNA or anti-RNA oligonucleotides capable of inhibiting the mRNA activity of the bacteria or mycobacteria causing said infection utilizing the DNA sequence of the dapB mycobacterial gene; and
(b) administering a pharmaceutically effective amount of said oligonucleotide, either alone or in combination with other compositions.
9. The method of Claim 8 wherein the mycobacterial dapB gene has a nucleic acid sequence depicted in Figure 3.
10. A method of producing a compound useful in the treatment of bacterial or mycobacterial infection, which compound blocks the activity of the mycobacterial enzyme DapB comprising: (a) overexpressing the mycobacterial DapB enzyme;
(b) purifying said overexpressed enzyme;
(c) performing x-ray crystallography on said purified enzyme so as to obtain the molecular structure of said enzyme; and
(d) creating a compound with a similar molecular structure to said enzyme.
11. A compound made by the method of Claim 10.
12. A vaccine comprising a mycobacterial strain which contains a mutated mycobacterial dapB gene such that the mycobacterial strain is incapable of synthesizing diaminopimelic acid in vivo.
13. A vaccine comprising a mycobacterial strain having a deleted dapB gene such that the mycobacterial strain is incapable of synthesizing diaminopimelic acid in vivo.
14. A method of producing a vaccine comprising deleting dapB gene from a mycobacterial strain.
15. The method of Claim 14 wherein the dapB gene has a nucleic acid sequence depicted in Figure 3.
16. Antibody which is immunoreactive with a polynucleotide which encodes the mycobacterial enzyme
DapB.
17. The antibody of Claim 16 wherein the polynucleotide has a nucleic acid sequence depicted in
Figure 3.
18. A method of preventing and treating bacterial or mycobacterial infection comprising passively administering antibody which is immunoreactive with a polynucleotide which encodes the mycobacterial enzyme DapB.
19. The method of Claim 18 wherein the polynucleotide has a nucleic acid sequence depicted in Figure 3.
20. A research tool comprising a mycobacterial strain having a dapB gene deletion and containing a plasmid, which plasmid contains a promoterless dapB gene into which a genomic mycobacterial nucleotide sequence can be inserted so as to produce a library which can be passed through mammalian cells and used to identify virulence genes or drug targets.
21. A method of producing a research tool comprising deleting the dapB gene from a mycobacterial strain and inserting therein a plasmid containing a promoterless dapB gene and a genomic mycobacterial nucleotide sequence so as to produce a library which can be passed through mammalian cells and used to identify virulence genes and drug targets.
PCT/US1995/002455 1994-02-28 1995-02-27 Gene for mycobacterial diaminopimelic acid WO1995023226A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU19722/95A AU1972295A (en) 1994-02-28 1995-02-27 Gene for mycobacterial diaminopimelic acid

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US20319094A 1994-02-28 1994-02-28
US08/203,190 1994-02-28

Publications (1)

Publication Number Publication Date
WO1995023226A1 true WO1995023226A1 (en) 1995-08-31

Family

ID=22752889

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1995/002455 WO1995023226A1 (en) 1994-02-28 1995-02-27 Gene for mycobacterial diaminopimelic acid

Country Status (2)

Country Link
AU (1) AU1972295A (en)
WO (1) WO1995023226A1 (en)

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BIOCHEMISTRY, Volume 13, No. 17, issued 1974, WIETZERBIN et al., "Occurrence of D-Alanyl-(D)-Meso-Diaminopimelic Acid and Meso-Diaminopimelyl-Meso-Diaminopimelic Acid Interpeptide Linkages in the Peptidoglycan of Mycobacteria", pages 3471-3476. *
JOURNAL OF BACTERIOLOGY, Volume 176, No. 14, issued July 1994, CIRILLO et al., "Genetic Determination of the Meso-Diaminopimelate Biosynthetic Pathway of Mycobacteria", pages 4424-4429. *
MOLECULAR MICROBIOLOGY, Volume 11, No. 4, issued 1994, CIRILLO et al., "Isolation and Characterization of the Aspartokinase and Aspartate Semialdehyde Dehydrogenase Operon from Mycobacteria", pages 629-639. *
THE JOURNAL OF BIOLOGICAL CHEMISTRY, Volume 259, No. 23, issued 10 December 1984, BOUVIER et al., "Nucleotide Sequence and Expression of the Escherichia Coli dapB Gene", pages 14829-14834. *

Also Published As

Publication number Publication date
AU1972295A (en) 1995-09-11

Similar Documents

Publication Publication Date Title
Blomfield et al. Integration host factor stimulates both FimB‐and FimE‐mediated site‐specific DNA inversion that controls phase variation of type 1 fimbriae expression in Escherichia coli
Putkey et al. Bacterial expression and characterization of proteins derived from the chicken calmodulin cDNA and a calmodulin processed gene.
Liu et al. Genetics and sequence analysis of the pcnB locus, an Escherichia coli gene involved in plasmid copy number control
Rosteck Jr et al. Homology between proteins controlling Streptomyces fradiae tylosin resistance and ATP-binding transport
Arias et al. Characterization and modelling of VanT: a novel, membrane‐bound, serine racemase from vancomycin‐resistant Enterococcus gallinarum BM4174
Varon et al. Bacillus subtilis gtaB encodes UDP-glucose pyrophosphorylase and is controlled by stationary-phase transcription factor sigma B
Bartlett et al. Nucleotide sequence of IS492, a novel insertion sequence causing variation in extracellular polysaccharide production in the marine bacterium Pseudomonas atlantica
Donohue et al. Cloning and expression of the Rhodobacter sphaeroides reaction center H gene
WO1998040394A1 (en) Vector constructs for the selection and identification of open reading frames
JPH09107981A (en) Use of phosphinotrisyn-resistant gene
JP2006068019A (en) EXPRESSION PLASMIDS REGULATED BY osmB PROMOTER
Stephenson et al. Comparison of the nucleotide and amino acid sequences of the RsrI and EcoRI restriction endonucleases
Cirillo et al. Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria
Buchet et al. Regulation of the carnitine pathway in Escherichia coli: investigation of the cai-fix divergent promoter region
JPH0838184A (en) Kanamycin resistant gene derived from rhodococcus bacterium
US6565855B1 (en) Mycobacteria functional screening and/or expression vectors
Hinton et al. Cloning, expression and sequencing the molybdenum-pterin binding protein (mop) gene of Clostridium pasteurianum in Escherichia coli
Xu et al. Positive and negative regulation of sequences upstream of the form II cbb CO2 fixation operon of Rhodobacter sphaeroides
JP2002320494A (en) Polypeptide implicated in expression of resistance to, particularly, glycopeptide in gram-positive bacterium, nucleotide sequence encoding the polypeptide, and use in diagnosis
WO1995023226A1 (en) Gene for mycobacterial diaminopimelic acid
Goosen et al. Genes involved in the biosynthesis of PQQ from Acinetobacter calcoaceticus
EP0292984A2 (en) Cloned rhizobium meliloti ntrA (rpoN) gene
Callahan et al. Identification and characterization of the Escherichia coli rbn gene encoding the tRNA processing enzyme RNase BN
JP2007505616A (en) Non-antibiotic resistance selection marker-containing selection system
JPH05506996A (en) Protease-deficient bacterial host

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AM AT AU BB BG BR BY CA CH CN CZ DE DK ES FI GB GE HU JP KE KG KP KR KZ LK LT LU LV MD MG MN MW MX NL NO NZ PL PT RO RU SD SE SI SK TJ TT UA UZ VN

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): KE MW SD SZ UG AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA