WO2004033633A2 - Compatible host/vector systems for expression of dna - Google Patents

Abstract

The present invention provides shuttle vectors compatible with a panel of different bacterial hosts and capable of replicating in evolutionarily divergent bacterial species. In various embodiments the shuttle vectors of the present invention can integrate into the genomes of and/or replicate in both Gram-negative and Gram-positive organisms of both high and low GC content. The vectors of the present invention therefore make possible the expression of environmental DNA in varied families of bacterial hosts using a single vector that is transferable between hosts by a variety of methods. In some embodiments, the vector can replicate in a genera of bacteria from each of the following groups: Escherichia or Klebsiella; Pseudomonas, Xanthomonas, or Acetobacter; Bacillus or Clostridia, and Corynebacteria, Streptomycete, or Rhodococcus. In various embodiments, the vectors are cosmid, plasmids, fosmids, or bacterial artificial chromosomes (BACs).

Description

COMPATIBLE HOST/VECTOR SYSTEMS FOR EXPRESSION OF DNA

Related Applications

[0001] This application claims the benefit of U.S. Provisional Application No. 60/421,120, filed October 4, 2002, the contents of which are incorporated herein in their entirety.

Field of the Invention

[0002] The invention relates to host/vector systems for the expression of DNA.

Introduction

[0003] The following discussion of the background of the invention is merely provided to aid the reader in understanding the invention and is not admitted to describe or constitute prior art to the present invention.

[0004] The bacterial kingdom is a highly heterogeneous world of species that have diverged from one another over the past 4.5 billion years. Apart from the Archeae realm, most characterized bacterial species belong to the Gram-positive or Gram-negative families, with these families containing members having either high or low GC content in their DNA. These four classes reflect the major physiological and genetic differences between bacteria.

[0005] These different classes of bacteria use different transcription and translation mechanisms to convert their genes into functional enzymes and structural constituents. Therefore, in many cases DNA from a given species will only be recognized and usefully expressed in cloning hosts which are evolutionarily related. Furthermore, expression of DNA from a given species in a species of a different class often has lethal effects on the host organism due to inappropriate or untimely gene expression and/or protein toxicity. Consequently, the development of a single host organism able to efficiently express DNA from diverged and dormant species has been extremely difficult to achieve.

Summary of the Invention

[0006] The present invention is broadly directed to shuttle vectors compatible with a panel of different bacterial hosts. [0007] The present invention provides in one aspect a recombinant shuttle vector comprising (a) a first origin of replication or region of integration (OR/RI) functional in low GC content Gram-positive (G+) bacteria, (b) a second OR/RI functional in a first bacterial type, (c) a third OR/RI functional in a second bacterial type, (d) at least one cos site, and (e) at least one antibiotic selection marker, wherein the first bacterial type is different from the second bacterial type, and the first and second bacterial types are selected from the group consisting of high GC content Gram-positive (G+) bacteria, high GC content Gram-negative (G-) bacteria and low GC content Gram-negative (G-) bacteria. The first OR/RI may be an origin of replication functional in low GC content G+ bacteria selected from the group consisting of pAM/31, pHT1030, pT181, pC194, pE194, pSN2, pTB19, pWVOl, and pIP404. In an alternative embodiment, the first OR/RI may be a region of integration which is a region of homology with low GC content G+ bacteria. The second OR/RI may be selected from the group consisting of (i) an origin of replication functional in high GC content G+ bacteria, (ii) an origin of replication functional in high GC content G- bacteria, (iii) an origin of replication functional in low GC content G- bacteria; and (iv) a region of integration functional in high GC content G+ bacteria.

[0008] In some embodiments, the second OR/RI is the origin of replication functional in high GC content G+ bacteria, and the third OR/RI may be selected from the group consisting of (i) an origin of replication functional in high GC content G- bacteria, (ii) an origin of replication functional in low GC content G- bacteria, and (iii) a region of integrationfunctional in high GC content G+ bacteria.

[0009] In certain embodiments, the second OR/RI is the origin of replication functional in high GC content G+ bacteria, and the third OR/RI is the origin of replication functional in high GC content G- bacteria. The vector may further comprise a region of integration functional in low GC content bacteria, thereby allowing replication or integration of the vector in the four types of bacteria.

[0010] In certain embodiments, the second OR/RI is the origin of replication functional in high GC content G+ bacteria, and the third OR/RI is the origin of replication functional in low GC content G- bacteria. The vectors can further comprise a region of integration functional in high GC content G- bacteria, thereby allowing replication or integration of the vector in the four types of bacteria. [0011] In one embodiment, the vector of the present invention comprises (a) a RK2 or a ColEl replicon, (b) an amyE homologous region, (c) a pHM1519 origin of replication, (d) two cos sites; and (e) at least two resistance markers. The vector may comprise a chloramphenicol (Cm^R) resistance marker, an ampicillin (Amp^R) resistance marker, a spectinoniycin (Spc^R) resistance marker, a kanamycin (Km^R) resistance marker, or combinations of any two or more thereof. The vectors may be SS2000, SS2002 or SS2003.

[0012] In an alternative embodiment, the vector of the present invention comprises (a) an F factor replicon, (b) an amyE homologous region, (c) a pHM1519 origin of replication, (d) an øπT origin of transfer, (e) an oriV origin of replication, (f) at least two cos sites, and (g) at least two resistance markers. The F factor replicon may comprise a repE locus and one or more partition locus. The one or more partition locus may comprise a par A locus, aparB locus, and aparC locus. The vector may comprise a chloramphenicol (Cm^R) resistance marker, an ampicillin (Amp^R) resistance marker, a spectinomycin (Spc^R) resistance marker, a kanamycin (Km^R) resistance marker, or combinations of any two or more thereof. The vector maybe CCSS2003.

[0013] In some embodiments, the second OR/RI is the origin of replication functional in high GC content G- bacteria, and the third OR/RI is selected from the group consisting of (i) a region of integration functional in low GC content G- bacteria, and (ii) a region of integration functional in high GC content G+ bacteria.

[0014] In certain embodiments, the second OR/RI is the origin of replication functional in high GC content G- bacteria, and the third OR/RI can be the region of integration functional in high GC content G+ bacteria. The vector can further comprise a region of integration functional in low GC content G-, thereby allowing replication or integration of the vector in four bacterial types.

[0015] In some embodiments, the second OR/RI is the origin of replication functional in low GC content G- bacteria, and the third OR/RI is a region of integration functional in high GC content G+ bacteria. The vector may further comprise a region of integration functional in high GC content G- bacteria, thereby allowing replication or integration of the vector in the four bacterial types.

[0016] In one embodiment, the vector of the present invention comprises (a) an origin of replication that is an RK2 or a ColEl replicon, (b) an amyE homologous region, (c) an attP-int region, (d) two cos sites, and (e) at least two resistance markers. The vector may further comprise an oriT origin of transfer. The vector may comprise a Cm^R an ampicillin (Amp ) resistance marker, a spectinomycin (Spc^R) resistance marker, a kanamycin (Km ) resistance marker, or combinations of any two or more thereof. The vector may be SS3000. The vector may be inducible to high copy number replication.

[0017] The present invention provides in another aspect a recombinant shuttle vector comprising (a) an origin of replication functional in low and high GC content G- bacteria, (b) a first OR/RI functional in low GC content G+ bacteria, (c) at least one cos site; and (d) at least one antibiotic selection marker. The vector may further comprise a second OR/RI functional in high GC content G+ bacteria, thereby allowing replication or integration of the vector in four types of bacteria.

[0018] The present invention also provides a method of expressing DNA, which comprises (a) inserting a DNA into the vectors of the present invention, (b) transfecting a host cell with the vector; and (c) expressing the DNA in a host.

[0019] The invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, as well as from the claims.

Brief Description of the Drawings

[0020] Figure 1 provides a schematic illustration of an embodiment of the vectors of the present invention and an illustration of methods of expressing heterologous DNA.

[0021] Figure 2 provides a schematic illustration of a vector of the present invention, SS2002.

[0022] Figure 3 provides a schematic illustration of a vector of the present invention, vector SS3000.

[0023] Figure 4 provides a schematic illustration of a vector of the present invention, vector CCSS2003. This vector can maintain environmental libraries at a single copy and is inducible when desired to higher copy number. Detailed Description of the Invention

[0024] The present invention provides shuttle vectors compatible with a panel of different bacterial hosts and capable of replication and expression in at least three families of microbes of evolutionarily divergent species. The shuttle vectors of the present invention can integrate or replicate in at least one organism from at least three of the following four types of bacteria: high GC content Gram-negative (G-) organisms, low GC content Gram-negative (G-) organisms, high GC content Gram-positive (G+) organisms, and low GC content Gram- positive (G+) organisms. The vector has an origin of replication or a region of integration (inf) that is functional in at least three of the four types of bacteria. The vectors of the present invention therefore make possible the expression of an exogenous DNA by applying a single vector that is transferable between hosts of the three or four of the four bacterial types.

[0025] By "high GC content" is meant that 52% or greater of the base pairs in the bacterial genome are GC pairs. By "low GC content" is meant that fewer than 52% of the base pairs in the bacterial genome are GC pairs. Persons of ordinary skill in the art understand that Gram- positive organisms such as those of Bacillus and Clostridium contain 40% or fewer GC pairs in the genome (and are therefore low GC content Gram-positive organisms). Conversely, Actinomycetes contain 60% or greater GC content in their genome (and are therefore high GC content Gram-positive organisms). Gram-negative organisms such as Pseudomonas contain 67% GC content (and are therefore G- high GC content organisms), and E. coli and enteric bacteria contain about 50% GC content in the genome (and are therefore G- low GC content organisms).

[0026] Gram-negative and Gram-positive organisms are determined according to the well- known Gram staining procedure. Gram-positive organisms are those that assume a violet color under standard Gram staining. Gram-negative organisms incorporate the counter stain rather than the primary Gram stain. Gram-negative bacteria are high in lipid content and low in peptidoglycan content. In Gram-negatives, the primary crystal- violet escapes from the cell when the decolorizer is added because the primary stains favor binding with peptidoglycan, which Gram-negative organisms have very little of.

[0027] By "replication" is meant the vector is multiplied within the cell. When the vector "integrates," the vector enters the cell and becomes covalently bonded to the host genome at both ends of the vector. [0028] The "origin of replication" is the specific site in the DNA where DNA replication begins. In some embodiments, DNA replication proceeds bidirectionally from the origin of replication. The "region of integration" is an area on the vector having a sequence that enables the vector to integrate into the genome of an organism, e.g. , a Bacillus. Regions of integration are specific for particular genera of bacteria. Integration is the recombination process which inserts a small DNA molecule (usually by homologous recombination) into a larger one. If the molecules are circular, integration involves only a single crossing-over; if linear, then two crossings-over are required. A well known example is the integration of phage λ (lambda) DNA into the E. coli genome.

[0029] An origin of replication that is "functional" means that the orign of replication serves as the starting position of DNA replication for the vector, which replication proceeds to completion. A region of integration that is functional means that the region of integration is a sequence that enables the vector to integrate into the genome of an organism. By "compatible" is meant that the vector can be replicated and/or integrated into the host cell chromosome. Compatible vectors are also maintained in the host cell and transferred to future generations of host cells, as opposed to being degraded or expelled from the host cell, or genetically rearranged by the host such that they no longer perform an important function or are no longer able to be transferred to progeny cells.

[0030] Origins of replication functional in low GC content G+ bacteria include, but are not limited to pHT1030 (Lereclus and Arantes, Mol. Microbiol. 6:35-46, 1992), functional, e.g., in Bacillus subtilis and Bacillus thuringiensis; pAMβl (Clewell et al., J. Bacteriol. 117:283-289, 1974), functional, e.g., in most low GC content Gram-positive (Enter ococcus, Streptococcus, Bacillus, Clostridium and Lactic Acid Bacteria (Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, and Propionibacterium)); pT181 (Novick, Annu. Rev. Microbiol. 43:537-565, 1989), functional, e.g., in Bacilli, Staphylococcus, and lactic acid bacteria; pC194 and ρE194 (Grass and Ehrlich, Microbiol. Rev. 53:231-241, 1989), functional, e.g., in abroad range of lowGC content Gram-positives; pSN2 (Grass and Ehrlich, supra), functional, e.g., in Staphylococci, and Bacilli; pTB19 (Imanaka et al, J. Bacteriol. 146:1091-1097, 1981), functional, e.g., in Bacilli; pWVOl (Seegers et al, Mol. Gen. Genet. 249:43-50, 1995), functional, e.g., in lactic acid bacteria, Clostridium, Bacilli, and Streptococci; and finally pIP404 (Brefort et al, Plasmid 1:52-66, 1977), functional, e.g., in Clostridium spp. [0031] Origins of replication functional in low GC content G- bacteria include, but are not limited to, ColEl (Twigg and Sherratt, Nature 283:216-218, 1980), functional, e.g., in low- medium GC content G- such as enteric bacteria; pi 5 A (Chang and Cohen, J Bacteriol. 134:1141-1156, 1978), functional, e.g., in enteric acteήa;pSC101 (Hashimoto-Gotoh et αl, Gene 16:227-235, 1980), functional e.g., in E. coli and close relatives (Klebsiella and Salmonella); oriF from F-like plasmids (Mulec et αl, Curr. Op. Microbiol. 44:231-235, 2002), which are found in about 80 different isolates belonging to the enteric bacteria family, and oriF homologs.

[0032] In certain embodiments, an example of which is illustrated in Figure 4, the F factor replicon is included on the vector, including the repE locus, and αrA,pαrB, and pαrC loci. These vectors are designed to allow one to build and maintain environmental libraries at a single copy, and then induce the clones to high copy number as desired. ParA, ParB, and ParC are partition proteins. Partition proteins are named after the function of partitioning chromosomes into daughter cells after replication. Bacteria generally have one protein of type ParA and one of type ParB. ParB binds to specific sequences around the origin of replication and there ensues condensation producing foci that are visible microscopically. ParA associates both with the cell membrane and with the parB DNA complexes and causes the ori regions of the two daughter chromosomes to be positioned at opposite poles in the replicating cell. "RepE" is the replication initiator protein, which enables the vector containing the F factor replicon to initiation DNA replication.

[0033] Origins of replication functional in high GC content G+ bacteria include, but are not limited to Actinomycete origins of replication which include, but are not limited to pSAl.l (Yokoyama et αl, FEMS Microbiol. Lett. 169:103-109, 1998), functional, e.g., in Streptomyces; pHM1519 (Miwa et αl., Agric. Biol. Chem. 48:2901-2903, 1985), functional e.g., in Corynebαcterium and some Rhodococcus species; pLTlOl (Kieser et αl., Mol. Gen. Genet. 185:223-238, 1982), functional, e.g., in broad host range of Streptomyces spp; pSRl (Archer and Sinskey, J Gen Microbiol. 139:1753-1759, 1993), functional, e.g., in Corynebαcterium and Rhodococcus families; pAL5000, (De Mot et αl., Microbiology 143:3137-3147, 1997), functional, e.g., i Mycobαcterium (pAL5000), Corynebαcterium (pXZ10142), Brevibαcterium (pRBLl), Bifidobαcterium (pMBl) and Neisseriα (pJDl); and pMFl (Bachrach et αl., Microbiology 146:297-303, 2000), functional, e.g., in Mycobacteria.

[0034] Origins of replication functional in high GC content G- bacteria include, but are not limited to pRO1600 (Schweizer, Curr. Op. Biotechnol. 12:439-445, 2001), functional, e.g., in Pseudomonads; pNIlO (Ito et al, Appl. Microbiol. Biotechnol. 61:240-246, 2003); and PP8- 1 (Holtwick et al, Microbiology 147:337-344, 2001), functional, e.g., in Pseudomonads.

[0035] Origins of replication functional in high and low GC content G- bacteria include, but are not limited to RK2 (Keen et al, Gene 70:191-197, 1988) and RSF1010 (Scholz et al, Gene 75:271-288), both of which are functional in broad host range.

[0036] A region of integration within a vector allows the vector of the present invention to be integrated into the host cell's genome and replicate with the host cell's chromosome (e.g., in Bacillus and Corynebacterium).

[0037] An "att site" is a DNA sequence at which site-specific recombination occurs during integration of a DNA molecule into the chromosomes of its host. "attP" represents a phage DNA attachment site and "attB" represents an attachment site on a bacterial chromosome. Attachment sites are the specific sequences on phage (attP) and bacterial (attB) chromosomes between which site-specific recombination occurs in order to integrate the phage genome into the bacterial chromosome. Insertion and excision requires both bacterial and phage gene products and occurs through homologous recombination at the small att sites. Thus, att sites are regions of integration. The integration site is the portion of bacteriophage lambda (λ) DNA that enables bacteriophase lambda (λ) DNA to be inserted into a specific site in the E. coli chromosome and to be excised from this site. "Integrase" (inf) refers to an enzyme that allows exogenous DNA to be spliced into the host cell's DNA.

[0038] Regions of integration functional in low GC content G+ bacteria (e.g., Bacillus subtilis, Lactic Acid Bacteria) include any chromosomal sequence, e.g., a region of homology of 500pb to lkb. The region of homology may be amyE, e.g. , amyE of Bacillus subtilis or other loci, e.g., in Lactic Acid Bacteria.

[0039] Regions of integration functional in low GC content G- bacteria can be attP sites (e.g., for integration into enteric bacteria). The sequences for attP sites vary between species.

[0040] Regions of integration functional in high GC content G+ bacteria can be attP sites (e.g., used as integration sites in Streptomyces and Corynebacterium), although their sequences vary slightly between species.

[0041] Regions of integration functional in high GC content G- bacteria can be attP sites (e.g., used in Pseudomonas species), although their sequences vary slightly between species. [0042] In certain embodiments, the vector has an origin of replication or a region of integration functional in all four of the following genera of bacteria: Escherichia, Pseudomonas, Bacillus, and Corynebacteria.

[0043] The vectors of the present invention can also comprise one or more cos sites. "Cos sites" are the cohesive ends between concatemeric genomes and are important in phage DNA cleavage and packaging. The phage makes specific nucleolytic cleavages at the cos sites, thus releasing the phage genome in unit length molecules for packaging.

[0044] Additionally, the vectors of the present invention include one or more resistance markers. Persons of ordinary skill will realize that the precise marker selected is not critical and that various markers can be chosen that provide the selectivity required to identify clones that are carrying the vector.

[0045] A "vector" or "expression vector" is an autonomously replicating DNA molecule into which foreign DNA fragments are inserted and then propagated in a host cell. By "shuttle vector" is meant an expression vector wherein the inserted fragment of DNA can be efficiently expressed in more than one of the above described four bacterial families.

[0046] In various embodiments, the vectors of the present invention are plasmids, cosmids, fosmids, or bacterial artificial chromosomes (BAC) vectors. "Fosmids" are based on vector DNA derived from the F-plasmid of E. coli, which is a vector used to clone DNA fragments in E. coli cells (35- to 40-kb insert size). Fosmids carry one cos site and the E. coli F factor origin of replication, and therefore are present at only one copy per cell. "BAC vectors" are based on naturally occurring F-factor plasmid found in E. coli and are a cloning vector capable of carrying between 100 and 300 kilobases of target sequence. They are propagated as a mini- chromosome in a bacterial host. A "cosmid" is a hybrid plasmid that contains cos sites at each end, which are recognized during head filling of lambda phages.

[0047] Referring to Figure 1, in various embodiments the vectors can have one or more features selected from the following: an RK2 or ColΕl origin of replication for replicating in Gram-negative species such as Pseudomonas and E. coli, cos sites for packaging by lambda phage, an amyE homologous region for integration in Bacillus (a G+ low GC content organims), an attP-int region for integration in Corynebacterium, an ori pHM1519 origin of replication for Actinomycetes (such as Corynebacterium or Rhodococcus) replication, and ampicillin (Amρ^R), spectinomycin (Sρc^R), and kanamycin (Kan^R) resistance markers, or any combination of the above structures.

[0048] The person of ordinary skill in the art will realize that any of the above structures can be substituted with an origin of replication or region of integration for another species from the same family. Thus, instead of a Bacillus, a Clostridium can be used as a representative of the low GC content G+ organism family and an origin of replication or a region of integration for Clostridium included on the vector. Or a Streptomyces can be substituted for a Corynebacterium to represent the G+ high GC content family. Similar substitutions can be made for any (or all) of the other three families. Therefore, in various embodiments the vectors of the present invention can replicate or integrate in a genera of bacteria selected from each of the three or four of the following four groups: an Escherichia or Klebsiella; a Pseudomonas, Xanfhomonas, or Acetobacter; a Bacillus or Clostridium, and a Corynebacteria, Streptomyces, or Rhodococcus. Thus, in various embodiments any combination of bacteria can be used, selecting any one or more bacterial genera from each of the three or four of the four groups.

[0049] In various embodiments, the vectors of the present invention can be carried as a stable, single copy vector and be made inducible to a multi-copy vector when higher levels of expression are desired. Thus the vectors of the present invention can have a single copy origin of replication and a high copy origin of replication. In other embodiments, the gene encoding the replication initiation protein has been integrated in the host's chromosome under the control of an inducible promoter (e.g., E. coli) or on an autonomous plasmid instead of present on the vector. An embodiment of these vectors is illustrated in Figure 4. In certain embodiments, the vector is inducible to high copy replication with a simple sugar as the inducer. In one embodiment, the high copy origin of replication is ori V and is present as part of the RK2 replicon, which includes the loci trfA, oriV, Amp^R, and oriT. The trfA gene has been integrated into the chromosome of the host under the control of the αrαBAD promoter and is made inducible by adding arabinose to the medium. The vector has a pHM1519 origin of replication for replicating in Corynebαcterium and Actinomycetes, two cos sites, an αmyE homologous region, and Spc^R Km^R, and Amp^R as resistance markers. A "replicon" is meant a DNA molecule or portion thereof that possesses an origin of replication and which is therefore capable of being replicated in a suitable cell. The RK2 plasmid is very similar or identical to plasmids R18, R68, RP4, and RPl. The replicons or origins of replication from these plasmids can be substituted for RK2 in any of the vectors of the invention. [0050] In another embodiment illustrated in Figure 3, the resistance markers have been substituted with markers for Km^R and Spc^R. The pHM1519 origin of replication for Corynebacterium has been substituted with the attP and integrase protein. This will therefore give the user the option of switching to integration of the vector into Corynebacterium if instability is found in carrying multiple copies.

[0051] A "single copy origin of replication" is an origin of replication on a vector that is not present at greater than one or two (before division) copies per cell. An example of a single copy origin of replication is the F-factor of E. coli, which can be present either integrated into the E. coli chromosome or as an autonomous plasmid. A "high copy origin of replication is an origin of replication of a vector that is present at more than 50 copies per cell. For example, the RK2 replicon is a high copy origin of replication. Cloning systems with inducible high copy origins can be designed by placing the sequences of replication initiation proteins on a cell's chromosome under the control of an inducible promoter, so that the proteins will be made by the cell in response to an inducer. Such modified organisms can be created, and are also commercially available. "Low copy" vectors are present at 2-9 copies per cell, and "medium copy" vectors are present at 10-50 copies per cell. Copy numbers are measured according to any suitable method, for example, the method described in Example 4 below.

[0052] In another aspect, the present invention provides a bacterial cell comprising a vector of the present invention. The bacterial cell may be transformed or transfected, and can include spore-forming Gram-positives (e.g., Bacilli, Clostridiales), lactic acid bacteria, enteric bacteria, Pseudomonales (e.g., Pseudomonas species), and Actinomycetes (e.g., Streptomyces spp, Coryneform bacteria Rhodococcus spp and Mycobacteria). The transformed or transfected bacterial host cell of the present invention can also include, but are not limited to Bacillus subtilis, E. coli and enteric bacteria, Pseudomonas aeruginosa, P. putida, Xanthomonas campestris, Acinetobacter calcoaceticus, Azotobacter vinelandii, Acetobacter calcoaceticus, Streptomyces lividans, Streptomyces coelicolor, Rhodococcus rhodochrous, R. erythropolis, Corynebacterium glutamicum, and Mycobαcteriurn smegmαtis.

[0053] In various embodiments, the vectors of the invention efficiently express the protein in one of the four families of bacteria above. By "efficiently expressed" is meant that a sufficient quantity of the proteins coded for by the inserted fragment of DNA is expressed for the expressed protein to be harvested in an amount necessary to determine the biological activity performed by the expressed protein. [0054] In another aspect, the present invention provides methods of expressing exogenous DNA. The methods involve inserting the DNA into a vector of the invention, transfecting a host cell with the vector; and expressing the exogenous DNA in the host. The exogenous DNA can be in the form of a DNA library created from the exogenous DNA source. In some embodiments the exogenous DNA is DNA from a dormant or nonculturable species. By "exogenous DNA" is meant DNA produced outside of the host cell. "Dormant species" are species that are alive but are not readily culturable on standard laboratory media. These species are sometimes referred to as "viable but nonculturable species." These species are present in many samples, especially environmental samples. These species are detectable by means known to those of ordinary skill, such as fluorimetrically or with a microscope, but do not grow on standard laboratory media. Since a variety of bacterial hosts are available for expression, it is much more likely that at least one of the available hosts will be able to express the DNA from dormant or nonculturable species at a useful expression level.

[0055] The fragment of DNA is preferably from an environmental source. The vector systems of the present invention offer the ability to replicate environmental DNA (eDNA) in evolutionarily or genetically divergent bacterial hosts using the same vector in the hosts. "Environmental DNA" (eDNA) is DNA derived from an environmental sample. Environmental samples include samples having their source in the environment. Examples of environmental samples include, but are not limited to, lakes, ponds, terrestrial soil, and marine samples, and even samples produced by filtering atmospheric air. The vector system is designed to be transferable from source organisms (e.g., E. coli) to the divergent hosts by various methods, such as transfection, transformation, electroporation, and conjugative matings. Thus, the present invention provides the ability to express exogenous DNA in a laboratory host, leading to much greater access to the genetic diversity of the source organisms or eDNA.

[0056] High-copy cloning sometimes leads to undesirable "artifacts" and genetic rearrangements that result from the instability of the cloned inserts at high copy number. This is even more common when screening for genes and pathways having antimicrobial activity. The use of single copy bacterial artificial chromosomes (BACs) and fosmid vectors increases the likelihood of cloning DNA fragments that carry genes producing products that are toxic or detrimental to the growth of the cloning hosts. But single copy vectors produce only low levels of expression of cloned DNA and low DNA yields that can be harvested for analysis. Thus, these vectors may not lead to efficient expression of the DNA fragment in host organisms. The present invention therefore offers the stability afforded by single copy cloning combined with the high expression levels achieved by multi-copy vectors.

[0057] Since the multi-copy replication of the vectors of the present invention can be made inducible, the user can make and maintain libraries or clones of genomic DNA, cDNA, or PCR products at a single copy, and induce the clones to high copy number (e.g., 10-50+ copies per cell) when desired. The vectors therefore combine the advantages of high yields with the stability afforded by single copy cloning.

[0058] The present invention provides a solution to the dual problems of the toxicity of foreign proteins and poor expression of foreign genes in heterologous hosts. A scale of toxicity was created for comparing the cloning efficiencies of a given DNA into single-copy, low-copy, and high-copy vectors. Without wanting to be bound by any particular theory, it is believed by Applicant that an observed decreasing cloning efficiency with increasing copy number indicates toxicity of the particular DNA to the cloning host. In the present invention it was discovered that (i) transcription efficiency correlates to GC content, leading to poor transcription of high GC content DNA in low GC content hosts (whether from Gram-positive or Gram-negative species), and (ii) toxicity effects are most significant between Gram-negative and Gram-positive organisms with the same GC content (consistent with expected efficient expression). For example, cloning of Bacillus subtilis or Staphylococcus aureus (low GC content Gram-positives) genomic DNA into E. coli (a low GC content Gram-negative) with substantial expression is the most toxic combination. The vectors of the present invention enable the expression of foreign DNA or DNA from a dormant or nonculturable species in multiple classes of hosts, ensuring that useful and efficient expression is obtained in at least one of the host classes. The present vectors and methods therefore enable access to a broad . range of genetic diversity from dormant species.

[0059] In some embodiments, four classes of hosts can be created: (i) the low GC content Gram-negative enteric family with E. coli and Klebsiella oxytoca as model organisms, (ii) the high GC content Gram-negative family with Pseudomonas aeruginosa as a model organism, (iii) the low GC content Gram-positive family as Bacillus subtilis as a model organism, and (iv) the high GC content Gram-posive family as Actinomycetes (e.g., Corynebacterium glutamicum and some Rhodococcus species) as model organisms. The vectors of the present invention can replicate in at least three, and in some embodiments, all four of the above classes of organisms. [0060] In certain embodiments, such as that depicted in Figure 4, the vectors of the present invention has two origins of replication ~ the broad host range RK2 replicon, which contains the oriV origin, and the single copy E. coli F-factor replicon, which is present in E. coli as an autonomous F factor plasmid. The oriV origin has been engineered and made inducible with arabinose, and is commercially available as the COPYCONTROL™ cloning system using EPI300™ E. coli as the cloning host (Epicentre, Madison, WI). Initiation of replication from oriV requires the "trfA" gene product. But the trfA gene is absent in the vectors and in most laboratory strains of E. coli. A strain of E. coli can therefore be produced that contains a mutant trfA gene under tight control of an inducible promoter , which can be a simple sugar (e.g., as the COPYCONTROL™ EPI300™). In the absence of the trfA gene induction agent (e.g., arabinose or another simple sugar) replication of the vector is controlled by the F-factor replicon and the vector is present at one copy per cell. Addition of the simple sugar (or other inducer) to the growth medium induces expression of TrfA and subsequent amplification of the clone to high copy number (e.g., 10-50+ copies per cell) to facilitate purification of microgram amounts of DNA. Persons of ordinary skill in the art will realize that the oriV origin can be engineered to respond to other inducing agents, for example, other simple sugars. For example, the lactose or xylose responsive promoters are also available in the art and can be fused to the TrfA replication protein for the vector to be maintained in multiple copies in the cell. Of course other inducible systems responsive to non-sugar inducing agents can also be engineered into the vectors.

[0061] In one embodiment the vector of the present invention is a fosmid, which contains the single copy E. coli F-factor replicon. This embodiment is desirable for constructing libraries of cosmid-sized clones (e.g., 40 kb). The stability of inserts cloned into fosmid vectors is greater than that in high copy vectors (Kim et al, Nucl. Acids Res. 20:1083, 1992). Fosmid vectors containing both the ori V high copy origin of replication and the E. coli F-factor replicon therefore provide the user with the clone stability of single-copy fosmid cloning and the high yields of DNA that can be realized from cosmid clones. In certain embodiments the present invention uses randomly-sheared, end-repaired and 5'-phosphorylated DNA fragments. Shearing the DNA to approximately 40 kb generates highly random DNA fragments, as opposed to more biased libraries that result from partial restriction endonuclease digestion.

[0062] In certain embodiments as depicted in Figure 3, the vector system of the present invention is used in E. coli or any enteric species (low GC content, Gram-negative bacteria), or in Pseudomonas, Xanfhomonas or Acetobacter. [0063] Several origins of replication or replicons are suitable for plasmid replication in a broad range of Gram-negative bacteria, whether of high or low GC content content. These include pRO1600 (Schweizer, Curr. Op. Biotechnol. 12:439-445, 2001); pNIlO (Itoh et al, Appl Microbiol. Biotechnol 61:240-246, 2003); ρPP8-l (Holtwick et al, supra); RK2 (Keen et al.Gene 70:191-197, 1988); and RSF1010 (Scholz et al, Gene 75:271-288, 1989). Another set of replicons are known to function in enteric bacteria which belong to the low/medium GC content Gram-negative family. These include colEl (Twigg and Sherratt, Nature 283:216-218, 1980), pl5A (Chang and Cohen, J. Bacteriol. 134:1141-1156, 1978), and pSClOl (Hashimoto- Gotoh et al, Gene 16:227-235, 1981).

[0064] In one embodiment, the broad host range RK2 replicon (Doran et al, J. Biol Chem. 273: 8447-8453, 1998) includes the Amp^R selection marker, which confers resistance to ampicillin in E. coli as well as to carbenicillin and other β-lactams in hosts that are intrinsically resistant to ampicillin. The vector also contains two cos sites, which mediate the in vitro packaging in λ-phage, a highly efficient cloning system for all λ-sensitive hosts (Wahl et al., Proc. Natl Acad. Sci. USA 84: 2160-2164, 1987). For use in Bacillus subtilis, the amyE homologous region is included (which directs chromosomal integration into this low GC content Gram-positive host), and the spectinomycin resistance marker (Spc^R).

[0065] Any chromosomal locus found in the host can be used for integration in Bacilli and Lactic acid bacteria. A region of homology of bp to lkb is standard. While amyE is the paradigm for Bacillus subtilis and other Bacilli, different loci can be used, particularly in Lactic Acid Bacteria and other low GC content Gram-positive hosts. As an alternative to chromosomal integration, the vectors of the present invention may include an origin of replication functional in lowGC content Gram-positive hosts such as pHT1030 which is functional in Bacillus subtilis and Bacillus thuringiensis; pAMβl which is functional in most low GC content Gram-positive (Enterococcus, Streptococcus, Bacillus, Clostridium and Lactic Acid Bacteria (Lactobacillus, Lactococcus, Leuconostoc, Listeria, Pediococcus, and Propionibacterium)); pT181 which is functional in Bacilli, Staphylococcus, and Lactic Acid Bacteria; pC194 and pE194 which are functional in a broad range of lowGC content Gram- positives; pSN2 which is functional in Staphylococci, and Bacilli; pTB19 which is functional in Bacilli; pWVOl which is functional in Lactic Acid Bacteria, Clostridium, Bacilli, and Streptococci; and finally ρIP404 which is functional in Clostridium spp. For use in Corynebacterium, the attP-int region, which allows chromosomal integration, is added (Moreau et al, Microbiology 145:539-548, 1999). [0066] Another embodiment is illustrated in Figure 2. This example includes the pHM1519 origin of replication for multi-copy maintenance into this high GC content Gram- positive host (Schafer et al, J. Bacteriol. 172: 1663-1666, 1990). Other origins of replication are functional in Actinomycetes and can be used instead of pHM1519, such as pSAl.l functional in Streptomyces species (Yokoyama et al, FEMS Microbiol. Lett. 169:103-109, 1998); pIJlOl (Kieser et al, Mol. Gen. Genet. 185:223-238, 1982), a broad host range Streptomyces replicon; pSRl (Archer and Sinskey, J. Gen Microbiol. 139:1753-1759, 1993), functional in Corynebacterium and Rhodococcus families; pAL5000 (De Mot et al, Microbiology 143:3137-3147, 1997), functional in Mycobacterium, Corynebacterium, Brevibacterium, Bifidobacterium and Neisseria species; and finally the single copy replicon pMFl (Bachrach et al, Microbiology 146:297-303, 2000) functional in Mycobacteria. More than one origin of replication can be combined in any given vector of the present invention as long as only one of them is functional in each host. Replication initiation taking place simultaneously at more than one origin would create genetic instability and ultimately deletion or rearrangement in the plasmid. The kanamycin resistance marker (Kan^R) can be used in most Gram-positive and Gram-negative hosts.

[0067] Yet another embodiment is illustrated in Figure 4. This vector contains the F factor replicon on the vector. The vector also contains the RK2 replicon (which has the oriV origin of replication) and is inducible to high copy expression. The oriT origin of transfer is present to provide for conjugative matings between E. coli and other species. And the origin of replication from pHM1519 provides an origin of replication for Corynebacterium and other Coryneforms. Various restriction sites are also present on all the vectors, which allows for easy manipulation and combination of loci as needs require.

[0068] Thus, it can be seen that the structures described can be combined in many embodiments to produce vectors with great versatility, and that can be engineered to suit a variety of purposes. These variations are also contemplated within the scope of the invention. For example, depending on the genes and pathways to be cloned, lower copy numbers, and/or larger or smaller DNA fragments to be inserted in the vector can be desirable. A fosmid vector can be designed according to the present invention that allows for building and maintaining environmental libraries at a single copy level and then, whenever desired, also can be induced to a high copy number (e.g., 10-50⁺ copies per cell). Single copy vectors often perform better when cloning not only large DNA fragments, but also low molecular weight DNA, such as cDNA or PCR products, and often result in more complete and more unbiased clone libraries. [0069] The host spectrum can also be advantageously expanded to marine model organisms, such as Vibrio harveyi (Czyz et al, Appl Env. Microbiol. 66: 599-605, 2000) or to photosynthetic and nitrogen-fixing cyanobacteria, such as Synechocystis as the model organism. Using the above described strategies and techniques known to persons of ordinary skill in the art, features from these organisms can be incorporated into the vectors to allow the vectors to replicate in these organisms. Thus, DNA can be efficiently expressed from the broad species of marine symbiotic bacteria. Diverse soil environments harbor bacterial kingdoms that are largely uncultivated and widely distributed. These include the abundant, but little-known, bacterial divisions of Acidobacterium and Verrucomicrobia, which are associated with neither the Gram-positive nor the Gram-negative bacterial families (Hugenholtz et al, J. Bacteriol. 180: 4765-4774, 1998). Vectors can be created that will be replicated and efficiently expressed in these organisms as well, thereby further expanding the genetic diversity available.

Example 1 - Isolation of DNA From Dormant and Nonculturable Species

[0070] DNA was isolated from a dormant species from soil according to the following procedure.

[0071] 100 g of soil sample and 150 ml of 0.1M sodium phosphate buffer (pH 5.0) supplemeted with 1% acid- washed PVPP was combined and placed in a blender. The sample was whipped in the blender for 1 minute and then placed on ice for 2 additional minutes to remove heat generated by the blending. This step was repeated two more times to ensure that bacteria are separated from soil particles at high yield. The liquid was equally distributed between two centrifuge bottles and centrifuged at 2,000 rpm for 10 minutes at 10 °C to remove large soil particles. The supernatant, which contains the bacteria, was removed and placed in a clean centrifuge bottle. 75 ml of 0.1 M sodium phosphate buffer (pH 5.0) was added to each bottle containing the soil pellet, and the bottles were shaken to resuspend the pellets and the samples place back into the blender.

[0072] The steps above were repeated two more times to maximize the yield of bacteria from the soil particles. To pellet the bacteria, the bottles containing the supernatant were centrifuged at high speed (8,000 rpm) for 10 minutes at 10 °C, and the supernatant removed. The bacterial pellet was washed with DNA wash buffer (1.5 M NaCl; 1% hexadecylmethylammonium bromide (CTAB), 0.2 M Na₃PO₄ buffer, pH 8.0 at 37 °C from 1 hour to overnight. This step is important for removing a large quantity of humic acid from the sample (more than 50%) since humic acid dissolves well in this buffer. 10 ml of DNA extraction buffer was added (1.5 M NaCl, 1% CTAB, 0.2 M Na₃PO₄ buffer, 0.1 M EDTA, pH 8.0) to 5g of bacterial fraction, which was obtained from the pellet wash of the previous step. Alternatively, 0.1M Tris, 0.1 M EDTA, pH 8.0 can also be used instead of the phosphate buffer.

[0073] 2 g of glass beads were added to the bottle, and the sample vortexed briefly (15 seconds) to disrupt the bacterial cells. Repetitive freeze and thaw cycles may also be used for this purpose. 80 μl of proteinase K (20 mg/ml) was added and the sample incubated at 37 °C for 30 minutes to 1 hour to facilitate disruption of the bacterial cells and also to digest contaminating proteins. 1.5 ml 20% SDS was added, the sample mixed and incubated at 70 °C for 1 hour, with mixing accomplished by inverting the tube about every 15 minutes. This step further completes the disruption of the bacterial cells and releases bacterial DNA into the medium. The sample was then centrifuged at 3000 rpm for 15 minutes, with the supernatant retained for later use, in order to remove cell debris and remaining soil particles. 0.53 volumes of isopropanol was added to the pellet to precipitate the DNA.

[0074] When a DNA pellet is formed at this step, the clump is scooped up with a glass pipette. If no obvious DNA pellet is formed, the sample is centrifuged at 4000 rpm for 20 minutes. The DNA was washed twice with 70% ethanol to remove any salts that co- precipitated with the DNA, and dissolve the sample in 1 ml of Tris-EDTA buffer, as above. If the color of the DNA solution was still dark brown at this step, the sample was extracted once with phenol and twice with chlorofornr.iso-amyl alcohol (24:1) to remove remaining humic acid. 1/10 volume of 3 M sodium acetate was added and the DNA in the sample precipitated with 2 volumes of cold 100% ethanol to remove additional humic acid that may still be present. The pellet was resuspended in 1 ml of Tris-EDTA buffer. The sample was further purified by pulse field electrophoresis on a low melting agarose gel (1%), using a 5 kb ladder as a marker (Parameters: 4 V/cm, switch time 1-6 seconds, running time: 3 hours). This step serves to remove the DNA from any remaining humic acid and other contaminants that may be present, as well as to size fractionate the DNA. Many instruments for conducting pulse field electrophoresis are known in the art and can be used for this purpose, for example, CHEF-DR II™ by Bio-Rad Laboratories (Hercules, CA) or equivalent. The electrophoresis buffer used was lx TAE (0.04 M Tris-Acetate, O.OOIM EDTA pH 8.0). The resulting gel was stained with ethidium bromide for 30 min (5 μg/ml Et-Br in water), and de-stained for 45 min in water. [0075] DNA bands larger than 30 kb were cut under UV-light, and the DNA purified on a /5-agarose gel. Various gels are commercially available that are suitable and often are sold in the form of kits, such as the 3-agarase DNA purification kit available from BioLabs or equivalent. Other kits are also available for the purification, for example, the GELase™ kit from Epicentre can also be used, as can Nal solution. These kits contain a /3-agarose digesting enzyme, and a low melting point agarose gel. The DNA is now ready for further analysis, such as cutting with restriction enzymes and cloning into a library.

Example 2 - Vector and Cosmid Preparation

[0076] In one embodiment, vectors and cosmids of the invention are conveniently prepared using the Qiagen MIDIPREP^® kit (Qiagen, Valencia, CA) or equivalent. Shuttle vectors and environmental DNA inserts are prepared and ligated according to standard procedures such the SUPERCOS^® cloning kit (Strategene, La Jolla, CA) or the COPYCONTROL^® Fosmid Library Production Kit (Epicentre, Madison, WI).

[0077] Shuttle vector arms SS2000 (Fig. 1), SS2002 (Fig. 2), and SS3000 (Fig. 3) were prepared for ligation with Sau3A-digested inserts according to the following procedure:

[0078] Ten micrograms of plasmid DNA was digested with Nhel in a total volume of 100 μl in the presence of the appropriate restriction buffer for 2 hours at 37 °C. The restriction mixture was incubated at 65 °C for 3 minutes and the linearized DNA was dephosphorylated in the presence of 0.25 unit of Calf Intestine Alkaline Phosphatase (CLAP, Invitrogen, Carlsbad, CA). The reaction mixture was extracted once with phenol: chloroform and once with chloroform, and the DNA was precipitated overnight at -20 °C in the presence of 0.3 M NaAcetate and 2.5 volumes of ethanol. After centrifugation, the DNA pellet was dried, resuspended and digested with BamHI a total volume of 100 μl for 2 hours at 37 °C. The reaction mixture was extracted again with phenol: chloroform and chloroform, precipitated with ethanol as described above, centrifuged and the pellet was resuspended in 10 μl water. One microliter of vector arms was ligated with 0.25 to 0.5 μg of soil DNA digested with Sau3A.

[0079] Shuttle vector arms (e.g. , SS2000, SS2002, SS2003) can also be prepared for ligation with end-repaired inserts by substituting Nrul for BamHI the procedure above.

[0080] Vector CCSS2003 (Figure 4) of the invention was prepared according to the following procedure. Ten micrograms of plasmid DNA was digested with BamHI in a total volume of 100 μl in the presence of the appropriate restriction buffer for 2 hours at 37 °C. The restriction mixture was incubated at 65 °C for 3 minutes and the linearized DNA was dephosphorylated in the presence of 0.25 unit of Calf Intestine Alkaline Phosphatase (CLAP, Invitrogen, Carlsbad, CA). The reaction mixture was extracted once with phenol: chloroform and once with chloroform, and the DNA was precipitated overnight at -20 °C in the presence of 0.3M NaAcetate and 2.5 volume ethanol. After centrifugation, the pellet was resuspended in lOμl water. One microliter of vector was ligated with 0.25 to 0.5 μg of soil DNA digested with Sau3A and prepared as described above. In cases where soil DNA inserts were end- repaired, the vector was digested with Eco 721 instead of BamHI.

[0081] In certain embodiments, the clone libraries are pre-amplified in E. coli before transfer into other hosts. After in vitro packaging of the ligation mixture using a commercial packaging kit, the libraries were transfected into E. coli strain ΕPI300 and amplified at 30 °C overnight. The E. coli were then stored in different formats, depending on their future use.

Construction of the SS3000 Vector

[0082] A 1.5 kb fragment in the 3 ' region of Bacillus subtilis amyE gene was amplified by

PCR using oligonucleotides:

<SEQ LD NO. 1> 5'-CATAGGATCCGCTTTAAAGAATCGTAATCTGG-3', and

<SEQ ID NO. 2> S'-GTTTATCGATAATTAATCATCCTTGCAGGGTATGTTT-S'.

The PCR product was digested with BamHI and Clal and ligated with BamHI/Clal-digested cosmid. The cosmid is a 7.9 kb vector containing a pUCori, an Amp^R site, a BamHI restriction site, two cos recognition sequences and two cos sites, a PSV40 site, and a Neo^R site.

Suitable cosmids are commercially available, e.g., the SUPERCOS^® vector from Stratagene

(La Jolla, CA) and equivalent cosmids.

[0083] The spectinomycin resistance gene from the mini-TnlO transposon (e.g., Steinmetz and Richter (1994), "Easy cloning of mini-TnlO insertions from the Bacillus subtilis chromosome," J. Bacteriol. 176:1761-1763) was amplified by PCR using oligonucleotides: <SEQ ID NO. 3> 5'-TTTAGCATGCCTACGGGGTCTGACGCTC-3', and <SEQ ID NO. 4> S'-TTTAGGATCCCAAAAATTGTTCAATATTTATCAAT-S', digested with Sphl, filled-in with DNA polymerase (Klenow fragment) in the presence of 0.2mM dNTP and digested with BamHI. The PCR product was ligated with the vector from Step 1, previously digested with Sail, and filled-in and digested with BamHI. The resulting intermediary construct was designated SBCos and is suitable for cloning and expression in E. coli, K. oxytoca and B. subtilis. [0084] The integrase gene (intA) and attachment site (attP) from Corynebacterium glutamicum temperate phage l6 (e.g., Moreau et al, Microbiology 145:539-548, 1999) was amplified by PCR using ATCC strain 21792, oligonucleotides:

<SEQ ID NO. 5> 5'-TTAAGGATCCGGAGCAGGCACAGTACGCTCT-3', and

<SEQ ID NO. 6> 5'-AATTGAATTCCAGCTCCGCAACCAGCGCGTC-3'.

The PCR product was digested with EcoRI and BamHI. In parallel, the origin of transfer (ori T) from the conjugative plasmid RP4 was amplified by PCR using oligonucleotides:

<SEQ ID NO. 7> 5'-AATTGAATTCGGCGCTCGGTCTTGCCTTGCT-3', and

<SEQ ID NO. 8> 5'-AATTGAATTCGCTTTTCCGCTGCATAACCCTG-3', and digested with EcoRI. A three-way ligation reaction was set between (i) SBCos digested with EcoRI and BamHI, (ii) the digested intA-attP PCR product and (iii) the digested oriT PCR product. The resulting shuttle was designated SSCos and is suitable for cloning, transfer and expression in E. coli, K. oxytoca, B. subtilis, and C. glutamicum.

[0085] The ColEl replicon was replaced with the broad host range RK2 minimal replicon from plasmid pJB137. DNA from pJB137 was digested with Tthllll restriction enzyme, filled-in, digested with EcoRI, and ligated with plasmid SSCos (from above) digested with Stul and EcoRI. The ligation mixture was transformed into a suitable E. coli strain (e.g., XLlBlueMR from Stratagene, La Jolla, CA) and plated onto LB spectinomycin media to counterselect self-ligated ColEl or RK2 replicons. The resulting construct was designated SS3000 and is suitable for cloning, transfer and expression into E. coli, K. oxytoca and all enteric bacteria, Xanthomonas, Acetobacter, Pseudomonas, B. subtilis, and C. glutamicum where it integrates into the chromosome at the attB site (Moreau et al. 1999).

Sequence of SS3000 <SEQ ID NO. 9>

GGTGGCTGCT GAACCCCCAG CCGGAACTGA CCCCACAAGG CCCTAGCGTT TGCAATGCAC

CAGGTCATCA TTGACCCAGG CGTGTTCCAC CAGGCCGCTG CCTCGCAACT CTTCGCAGGC

TTCGCCGACC TGCTCGCGCC ACTTCTTCAC GCGGGTGGAA TCCGATCCGC ACATGAGGCG

GAAGGTTTCC AGCTTGAGCG GGTACGGCTC CCGGTGCGAG CTGAAATAGT CGAACATCCG

TCGGGCCGTC GGCGACAGCT TGCGGTACTT CTCCCATATG AATTTCGTGT AGTGGTCGCC

AGCAAACAGC ACGACGATTT CCTCGTCGAT CAGGACCTGG CAACGGGACG TTTTCTTGCC

ACGGTCCAGG ACGCGGAAGC GGTGCAGCAG CGACACCGAT TCCAGGTGCC CAACGCGGTC

GGACGTGAAG CCCATCGCCG TCGCCTGTAG GCGCGACAGG CATTCCTCGG CCTTCGTGTA

ATACCGGCCA TTGATCGACC AGCCCAGGTC CTGGCAAAGC TCGTAGAACG TGAAGGTGAT

CGGCTCGCCG ATAGGGGTGC GCTTCGCGTA CTCCAACACC TGCTGCCACA CCAGTTCGTC

ATCGTCGGCC CGCAGCTCGA CGCCGGTGTA GGTGATCTTC ACGTCCTTGT TGACGTGGAA

AATGACCTTG TTTTGCAGCG CCTCGCGCGG GATTTTCTTG TTGCGCGTGG TGAACAGGGC

AGAGCGGGCC GTGTCGTTTG GCATCGCTCG CATCGTGTCC GGCCACGGCG CAATATCGAA

CAAGGAAAGC TGCATTTCCT TGATCTGCTG CTTCGTGTGT TTCAGCAACG CGGCCTGCTT

GGCCTCGCTG ACCTGTTTTG CCAGGTCCTC GCCGGCGGTT TTTCGCTTCT TGGTCGTCAT

AGTTCCTCGC GTGTCGATGG TCATCGACTT CGCCAAACCT GCCGCCTCCT GTTCGAGACG

ACGCGAACGC TCCACGGCGG CCGATGGCGC GGGCAGGGCA GGGGGAGCCA GTTGCACGCT GTCGCGCTCG ATCTTGGCCG TAGCTTGCTG GACCATCGAG CCGACGGACT GGAAGGTTTC GCGGGGCGCA CGCATGACGG TGCGGCTTGC GATGGTTTCG GCATCCTCGG CGGAAAACCC CGCGTCGATC AGTTCTTGCC TGTATGCCTT CCGGTCAAAC GTCCGATTCA TTCACCCTCC TTGCGGGATT GCCCCGGAAT TAATTCCCCG GATCGATCCG TCGATCTTGA TCCCCTGCGC CATCAGATCC TTGGCGGCAA GAAAGCCATC CAGTTTACTT TGCAGGGCTT CCCAACCTTA CCAGAGGGCG CCCCAGCTGG CAATTCCGGT TCGCTTGCTG TCCATAAAAC CGCCCAGTCT AGCTATCGCC ATGTAAGCCC ACTGCAAGCT ACCTGCTTTC TCTTTGCGCT TGCGTTTTCC CTTGTCCAGA TAGCCCAGTA GCTGACATTC ATCCGGGGTC AGCACCGTTT CTGCGGACTG GCTTTCTACG TGGCTGCCAT TTTTGGGGTG AGGTCGTTCG CGGCCGAGGG GCGCAGCCCC TGGGGGGATG GGGTGCCGCG TTAGCGGGCC GGGAGGGTTC GAGAAGGGGG GGCACCCCCC TTCGGCGTGC GCGGTCACGC GCCAGGGCGC AGCCCTGGTT AAAAACAAGG TTTATAAATA TTGGTTTAAA AGCAGGTTAA AAGACAGGTT AGCGGTGGCC GAAAAACGGG CGGAAACCCT TGCAAATGCT GGATTTTCTG CCTGTGGACA GCCCCTCAAA TGTCAATAGG TGCGCCCCTC ATCTGTCATC ACTCTGCCCC TCAAGTGTCA AGGATCGCGC CCCTCATCTG TCAGTAGTCG CGCCCCTCAA GTGTCAATAC CGCAGGGCAC TTATCCCCAG GCTTGTCCAC ATCATCTGTG GGAAACTCGC GTAAAATCAG GCGTTTTCGC CGATTTGCGA GGCTGGCCAG CTCCACGTCG CCGGCCGAAA TCGAGCCTGC CCCTCATCTG TCAACGCCGC GCCGGGTGAG TCGGCCCCTC AAGTGTCAAC GTCCGCCCCT CATCTGTCAG TGAGGGCCAA GTTTTCCGCG TGGTATCCAC AACGCCGGCG GCCCTACATG GCTCTGCTGT AGTGAGTGGG TTGCGCTCCG GCAGCGGTCC TGATCCCCCG CAGAAAAAAA GGATCTCAAG AAGATCCTTT GATCTTTTCT ACGGGGTCTG ACGCTCAGTG GAACGAAAAC TCACGTTAAG GGATTTTGGT CATGAGATTA TCAAAAAGGA TCTTCACCTA GATCCTTTTA AATTAAAAAT GAAGTTTTAA ATCAATCTAA AGTATATATG AGTAAACTTG GTCTGACAGT TACCAATGCT TAATCAGTGA GGCACCTATC TCAGCGATCT GTCTATTTCG TTCATCCATA GTTGCCTGAC TCCCCGTCGT GTAGATAACT ACGATACGGG AGGGCTTACC ATCTGGCCCC AGTGCTGCAA TGATACCGCG AGACCCACGC TCACCGGCTC CAGATTTATC AGCAATAAAC CAGCCAGCCG GAAGGGCCGA GCGCAGAAGT GGTCCTGCAA CTTTATCCGC CTCCATCCAG TCTATTAATT GTTGCCGGGA AGCTAGAGTA AGTAGTTCGC CAGTTAATAG TTTGCGCAAC GTTGTTGCCA TTGCTACAGG CATCGTGGTG TCACGCTCGT CGTTTGGTAT GGCTTCATTC AGCTCCGGTT CCCAACGATC AAGGCGAGTT ACATGATCCC CCATGTTGTG CAAAAAAGCG GTTAGCTCCT TCGGTCCTCC GATCGTTGTC AGAAGTAAGT TGGCCGCAGT GTTATCACTC ATGGTTATGG CAGCACTGCA TAATTCTCTT ACTGTCATGC CATCCGTAAG ATGCTTTTCT GTGACTGGTG AGTACTCAAC CAAGTCATTC TGAGAATAGT GTATGCGGCG ACCGAGTTGC TCTTGCCCGG CGTCAACACG GGATAATACC GCGCCACATA GCAGAACTTT AAAAGTGCTC ATCATTGGAA AACGTTCTTC GGGGCGAAAA CTCTCAAGGA TCTTACCGCT GTTGAGATCC AGTTCGATGT AACCCACTCG TGCACCCAAC TGATCTTCAG CATCTTTTAC TTTCACCAGC GTTTCTGGGT GAGCAAAAAC AGGAAGGCAA AATGCCGCAA AAAAGGGAAT AAGGGCGACA CGGAAATGTT GAATACTCAT ACTCTTCCTT TTTCAATATT ATTGAAGCAT TTATCAGGGT TATTGTCTCA TGAGCGGATA CATATTTGAA TGTATTTAGA AAAATAAACA AATAGGGGTT CCGCGCACAT TTCCCCGAAA AGTGCCACCT GGGTCGATCG ACGGATCTTT TCCGCTGCAT AACCCTGCTT CGGGGTCATT ATAGCGATTT TTTCGGTATA TCCATCCTTT TTCGCACGAT ATACAGGATT TTGCCAAAGG GTTCGTGTAG ACTTTCCTTG GTGTATCCAA CGGCGTCAGC CGGGCAGGAT AGGTGAAGTA GGCCCACCCG CGAGCGGGTG TTCCTTCTTC ACTGTCCCTT ATTCGCACCT GGCGGTGCTC AACGGGAATC CTGCTCTGCG AGGCTGGCCG TAAGCTCTAA GAAACCATTA TTATCATGAC ATTAACCTAT AAAAATAGGC GTATCACGAG GCCCTTTCGT CTTCAAGAAT TAATTCACTG GCCGTCGTTT TACAACGTCG TGACTGGGAA AACCCTGGCG TTACCCAACT TAATCGCCTT GCAGCACATC CCCCTTTCGC CAGCAGATCC GTCAACTGCA TCCAGCCCGT TTTCACACAA CCTGGGGCGA GAGGCGACGA CCTCCAAAAA AGCCTCCTCA CTACTTCTGG AATAGCTCAG AGGCCGAGGC GGCCTCGGCC TCTGCATAAA TAAAAAAAAT TAGTCAGCCA TGGGGCGGAG AATGGGCGGA ACTGGGCGGA GTTAGGGGCG GGATGGGCGG AGTTAGGGGC GGGACTATGG TTGCTGACTA ATTGAGATGC ATGCTTTGCA TACTTCTGCC TGCTGGGGAG CCTGGGGACT TTCCACACCT GGTTGCTGAC TAATTGAGAT GCATGCTTTG CATACTTCTG CCTGCTGGGG AGCCTGGGGA CTTTCCACAC CCTAACTGAC ACACATTCCA CAGCCGGATC TGCAGGACCC AACGCTGCCC GAGATGCGCC GCGTGCGGCT GCTGGAGATG GCGGACGCGA TGGATATGTT CTGCCAAGGG TTGGTTTGCG CATTCACAGT TCTCCGCAAG AATTGATTGG CTCCAATTCT TGGAGTGGTG AATCCGTTAG CGAGGTGCCG CCGGCTTCCA TTCAGGTCGA GGTGGCCCGG CTCCATGCAC CGCGACGCAA CGCGGGGAGG CAGACAAGGT ATAGGGCGGC GCCTACAATC CATGCCAACC CGTTCCATGT GCTCGCCGAG GCGGCATAAA TCGCCGTGAC GATCAGCGGT CCAATGATCG AAGTTAGGCT GGTAAGAGCC GCGAGCGATC CTTGAAGCTG TCCCTGATGG TCGTCATCTA CCTGCCTGGA CAGCATGGCC TGCAACGCGG GCATCCCGAT GCCGCCGGAA GCGAGAAGAA TCATAATGGG GAAGGCCATC CAGCCTCGCG TCGCGAACGC CAGCAAGACG TAGCCCAGCG CGTCGGCCGC CATGCCGGCG ATAATGGCCT GCTTCTCGCC GAAACGTTTG GTGGCGGGAC CAGTGACGAA GGCTTGAGCG AGGGCGTGCA AGATTCCGAA TACCGCAAGC GACAGGCCGA TCATCGTCGC GCTCCAGCGA AAGCGGTCCT CGCCGAAAAT GACCCAGAGC GCTGCCGGCA CCTGTCCTAC GAGTTGCATG ATAAAGAAGA CAGTCATAAG TGCGGCGACG ATAGTCATGC CCCGCGCCCA CCGGAAGGAG CTGACTGGGT TGAAGGCTCT CAAGGGCATC GGTCGAGGAA CTTTCGGCGG CTTTGCTGTG CGACAGGCTC ACGTCTAAAA GGAAATAAAT CATGGGTCAT AAAAATTATC ACGTTGTCGG CGCGGCGACG GATGTTCTGT ATGCGCTGTT TTCCGTTGGC CGTTGCTGTC TGGTGATCTG CCTTCTAAAT CTGCACAGCC GAATTGCGCG AGCTTGGTTT TGCTGAAACC GACACACAGC AACTGAATAC CAGAAAGAAA ATCACTTTGC CTTTCTGACA TCAGAAGGGC AGAAATTTGC CGTTGAACAC CTGGTCAATA CGCGTTTTGG TGAGCAGCAA TATTGCGCTT CGATGAGCCT TGGCGTTGAG ATTGATACCT CTGCTGCACA AAAGGCAATC GACCGAGCTG GACCAGCGCA TTCGTGACAC CGTCTCCTTC GAACTTATTC GCAATGGAGT GTCATTCATC AAGGACGCCT GATCGCAAAT GGTGCTATCC ACGCAGCGGC AATCGAAAAC CCTCAGCCGG TGACCAATAT CTACAACATC AGCCTTGGTA TCCTGCGTGA TGAGCCAGCG CAGAACAAGG TAACCGTCAG TGCCGATAAG TTCAAAGTTA AACCTGGTGT TGATACCAAC ATTGAAACGT TGATCGAAAA CGCGCTGAAA AACGCTGCTG AATGTGCGGC GCTGGATGTC ACAAAGCAAA TGGCAGCAGA CAAGAAAGCG ATGGATGAAC TGGCTTCCTA TGTCCGCACG GCCATCATGA TGGAATGTTT CCCCGGTGGT GTTATCTGGC AGCAGTGCCG TCGATAGTAT GCAATTGATA ATTATTATCA TTTGCGGGTC CTTTCCGGCG ATCCGCCTTG TTACGGGGCG GCGACCTCGC GGGTTTTCGC TATTTATGAA AATTTTCCGG TTTAAGGCGT TTCCGTTCTT CTTCGTCATA ACTTAATGTT TTTATTTAAA ATACCCTCTG AAAAGAAAGG AAACGACAGG TGCTGAAAGC GAGCTTTTTG GCCTCTGTCG TTTCCTTTCT CTGTTTTTGT CCGTGGAATG AACAATGGAA GTCAACAAAA AGCAGACGTA TCTAGACACG TCTGAAGCTA GCTTCGAGGA ACTTTCGGCG GCTTTGCTGT GCGACAGGCT CACGTCTAAA AGGAAATAAA TCATGGGTCA TAAAAATTAT CACGTTGTCG GCGCGGCGAC GGATGTTCTG TATGCGCTGT TTTCCGTTGG CCGTTGCTGT CTGGTGATCT GCCTTCTAAA TCTGCACAGC CGAATTGCGC GAGCTTGGTT TTGCTGAAAC CGACACACAG CAACTGAATA CCAGAAAGAA AATCACTTTG CCTTTCTGAC ATCAGAAGGG CAGAAATTTG CCGTTGAACA CCTGGTCAAT ACGCGTTTTG GTGAGCAGCA ATATTGCGCT TCGATGAGCC TTGGCGTTGA GATTGATACC TCTGCTGCAC AAAAGGGAAT CGACCGAGCT GGACCAGCGC ATTCGTGACA CCGTCTCCTT CGAACTTATT CGCAATGGAG TGTCATTCAT CAAGGACGCC TGATCGCAAA TGGTGCTATC CACGCAGCGG CAATCGAAAA CCCTCAGCCG GTGACCAATA TCTACAACAT CAGCCTTGGT ATCCTGCGTG ATGAGCCAGC GCAGAACAAG GTAACCGTCA GTGCCGATAA GTTCAAAGTT AAACCTGGTG TTGATACCAA CATTGAAACG TTGATCGAAA ACGCGCTGAA AAACGCTGCT GAATGTGCGG CGCTGGATGT CACAAAGCAA ATGGCAGCAG ACAAGAAAGC GATGGATGAA CTGGCTTCCT ATGTCCGCAC GGCCATCATG ATGGAATGTT TCCCCGGTGG TGTTATCTGG CAGCAGTGCC GTCGATAGTA TGCAATTGAT AATTATTATC ATTTGCGGGT CCTTTCCGGC GATCCGCCTT GTTACGGGGC GGCGACCTCG CGGGTTTTCG CTATTTATGA AAATTTTCCG GTTTAAGGCG TTTCCGTTCT TCTTCGTCAT AACTTAATGT TTTTATTTAA AATACCCTCT GAAAAGAAAG GAAACGACAG GTGCTGAAAG CGAGCTTTTT GGCCTCTGTC GTTTCCTTTC TCTGTTTTTG TCCGTGGAAT GAACAATGGA AGTCAACAAA AAGCAGAGCT TATCGATCAT TAATCATCCT TGCAGGGTAT GTTTCTCTTT GATGTCTTTT TGTTTGTGAA GTATTTCACA TTTATATTGT GCAACACTTC ACAAACTTTT GCAAGAGAAA AGTTTTGTCT GATTTATGAA CAAAAAAGAA ACCATCATTG ATGGTTTCTT TCGGTAAGTC CCGTCTAGCC TTGCCCTCAA TGGGGAAGAG AACCGCTTAA GCCCGAGTCA TTATATAAAC CATTTAGCAC GTAATCAAAG CCAGGCTGAT TCTGACCGGG CACTTGGGCG CTGCCATTAT TAAAAATCAC TTTTGCGTTG GTTGTATCCG TGTCCGCAGG CAGCGTCAGC GTGTAAATTC CGTCTGCATT TTTAGTCATT GGTTTTCCAG GCCAAGATCC GGTCAATTCA ATTACTCGGC TCCCATCATG TTTATAGATA TAAGCATTTA CCTGGCTCCA ATGATTCGGA TTTTGATAGC CGATGGTTTT GGCCGACGCT GGATCTCTTT TAACAAAACT GTATTTCTCG GTCCTCGTTA CACCATCACT GTTCGTTCCT TTTAACATGA TGGTGTATGT TTTGCCAAAT TGGATCTCCT TTTCCGATTG TGAATTGATC TCCATCCTTA AACGCCTGTC GTCTGGTCCA TTATTGATTT GATAAACGGC TTTTGTTGTA TTCGCATCTG CACGCAAGGT AATCGTCAGT TGATCATTGA AAGAATGTGT TACACCTGTT TTGTAATTCT CAAGGAAAAC ATGAGGCGCT TTTGCAATAT CATCAGGATA AAGCACAGCT ACAGACCTGG CATTGATCGT GCCTGTCAGT TTACCATCGT TCACTTGAAA TGAACCCGCT CCAGCTTTAT TGTCATACCT GCCATCAGGC AATTTTGTTG CCGTATTGAT AGAGACAGAG GATGAACCTG CATTTGCCAG CACAACGCCA TGTGAGCCGC GCTGATTCAT AAATATCTGG TTGTTTCCAT TCGGGTTCGA GAGTTCCTCA GGCTGTCCAG CCATCACATT GTGAAATCTA TTGACCGCAG TGATAGCCTG ATCTTCAAAT AAAGCACTCC CGCGATCGCC TATTTGGCTT TTCCCCGGGA ACCTCACACC ATTTCCGCCT CCCTCAGGTC TGGAAAAGAA AAGAGGCGTA CTGCCTGAAC GAGAAGCTAT CACCGCCCAG CCTAAACGGA TATCATCATC GCTCATCCAT GTCGACCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTGAC TCTAGAGGAT CGATCTGTAT AATAAAGAAT AATTATTAAT CTGTAGACAA ATTGTGAAAG GATGTACTTA AACGCTAACG GTCAGCTTTA TTGAACAGTA ATTTAAGTAT

ATGTCCAATC TAGGGTAAGT AAATTGAGTA TCAATATAAA CTTTATATGA ACATAATCAA

CGAGGTGAAA TCATGAGCAA TTTGATTAAC GGAAAAATAC CAAATCAAGC GATTCAAACA

TTAAAAATCG TAAAAGATTT ATTTGGAAGT TCAATAGTTG GAGTATATCT ATTTGGTTCA

GCAGTAAATG GTGGTTTACG CATTAACAGC GATGTAGATG TTCTAGTCGT CGTGAATCAT

AGTTTACCTC AATTAACTCG AAAAAAACTA ACAGAAAGAC TAATGACTAT ATCAGGAAAG

ATTGGAAATA CGGATTCTGT TAGACCACTT GAAGTTACGG TTATAAATAG GAGTGAAGTT

GTCCCTTGGC AATATCCTCC AAAAAGAGAA TTTATATACG GTGAGTGGCT CAGGGGTGAA

TTTGAGAATG GACAAATTCA GGAACCAAGC TATGATCCTG ATTTGGCTAT TGTTTTAGCA

CAAGCAAGAA AGAATAGTAT TTCTCTATTT GGTCCTGATT CTTCAAGTAT ACTTGTCTCC

GTACCTTTGA CAGATATTCG AAGAGCAATT AAGGATTCTT TGCCAGAACT AATTGAGGGG

ATAAAAGGTG ATGAGCGTAA TGTAATTTTA ACCCTAGCTC GAATGTGGCA AACAGTGACT

CTGGTGAAA TTACCTCGAA AGATGTCGCT GCAGAATGGG CTATACCTCT TTTACCTAAA

GAGCATGTAA CTTTACTGGA TATAGCTAGA AAAGGCTATC GGGGAGAGTG TGATGATAAG

TGGGAAGGAC TATATTCAAA GGTGAAAGCA CTCGTTAAGT ATATGAAAAA TTCTATAGAA

ACTTCTCTCA ATTAGGCTAA TTTTATTGCA ATAACAGGTG CTTACTTTTA AAACTACTGA

TTTATTGATA AATATTGAAC AATTTTTGGG ATCCGGAGCA GGCACAGTAC GCTCTCGATA

ACCTGTAAAA CACAAAAAAA TTCCCCCTGT GCACGCCCGT CGCCAAACGA TATGCACAGA

GGGAATCTTA AGACATTAAA AACAGATATC CAGATTTTCT GAATCCAGAC AGGACCAGTG

TACCAATGGG TGTTCAACGA CGGCCAAAAA AAGGCTCAGC GCAAGATAAA GGTCAAAAAC

CTCGGTGGGT AGGGCGCTAC CGAGACCACT CGGGGAAAGA ATATTCCAAA ACTTTCCATA

CCGAAAAAGA AGCCAAAAGC GTGGGGTTGG TGAAAAAGAG CGGTCACTAC GCGATGGATT

CGATGGGTCA GCCCAGAGGA GGAGCAGGCA CAGCTTTGCT TGATCTTGCC AAAGAGTGGA

AAGACGAAGC GACTAAGCCT AATACGATCG CTAATCGAAA AGCATTAGTC GATAATCTTG

GGAAGCTCGG TGGCATCCCA CTCAATAAAA TTATTCCAGG CGATATCAGC GCCTGGAATA

GAACGCTTTT AGAGGGACGC CCATGGAAAG ACGGAAAGCC GATGTCCTCA TCGACCGTCG

CAGTCATGAC CGGACAAGTT GCCGGCCTAC TGAACCGTGC ACACCAGGAC CGCATTCTCA

CCCATATCCC AAGAATCTCA GCACCTAAAG CCCCACCAAA AATGGCGGTC TCAAGGCAAG

ACCTTGCAAC GCCTCAAGAA TTAGCCCAGA TCCTAGATCT AGCCCACGAA GGTAAGAAGA

GAAAGACCGG CGCTCCTACC CCGCCGAGGA TTTGGCTTGA GCACATGATC CAAGTAGCGC

TCGGCACCGG CATGCGTGTC AGTGAGATTG GCGCACTCTT CCCTGAAGAT CTCGATGTGC

TCAAGCTTCA ATTATCGGTC GTGCGCCAGG TGCTCCCCGG AGGTCGAAAA ACTGGGCCGC

TGAAAACCGA GAGACCCAGA ACCATTCCGA TTCCGAAGAG CGTGTCAGAT ATTCTTGAAT

CAAGGATCAA GGATCTTAAT GTAGGCCCTG GAGAGCCGAT CTTCCCATAT ACGGGTGAGT

CAAAATTCGA GGATCGTATT TACCACGACC GAAACTCGAC GGGGCGCACG CTCGCCCGCA

TCATTGAGGT TTTAGAAATG CGGGAAATCA CTTTCCATGA TTTCCGCCAC TACTATGCAT

CCGTCCTCAT CGCCGAAGGT GCCGCCGTCG CCGATGTCCA GGAAGCCCTT GGACACGCCA

ACGCATCCAC GACTTTAAAT ACTTACAGCA CCTTTTCGGC AACCACGATG AGCGCACGGG

CGAGCCGCAA ATTCTGCGTC GACTACGTGC GGGCAGCGCG CTCGGGCAAA TCACCGCTAG

TGAAGCTGGT GATCAACTCG ACAAGAAGCG TCGAGCCAGG TCACAGGCTT AGCTAAACTC

TGGATCTTCA GTGCGGCTGC GCTTGATTTC AAAGAAATGA CAATAAAGCC CCGCCCAGGG

GTTTTACATC GTTGCAGGCC AAGGCGATTA TGTCGATTCT GTAAATTCTG CATTTTCCGC

AAATTATGTG GGCTGCAAAG CGCTTTGCGG GCAAATTGCG GGCAAAAAGG CTTGCCCGGC

GCGAGCTTTT TGACGCGCTG GTTGCGGAGC TGGAATT

Construction of the Super Shuttle Vector SS2000

[0086] A 3-way ligation was set up between (i) the intermediate shuttle construct from Step 2 above, SBCos, digested with Stul and BamHI, (ii) the pJB137 TthlllI_{mtά xi}-PstI fragment containing the broad host range RK2 minimal replicon, and (iii) the origin of replication from Corynebacterium plasmid pHMl 519 (Tauch, A., Kirchner, O., Wehmeier, L., Kalinowski, J., and A. Puhler, (1994) "Corynebacterium glutamicum DNA is subjected to methylation-restriction in Escherichia coli," FEMS Microbiol. Lett. 123:343-348) amplified by PCR using oligonucleotides: <SEQ ID NO. 10> 5'-ACTGGGATCCTTTACGGCGTCCTCGTGGAAG-3', and <SEQ ID NO. 11> 5'-GCATCTGCAGCACAATCGCGTGGATCGCCTA-3' and digested with BamHI and Pstl. The resulting construct was designated SS2000 (Figure 1) and is suitable for cloning, transfer and expression into E. coli, K. oxytoca and all enteric bacteria, Xanthomonas, Acetobacter, Pseudomonas, B. subtilis, and C. glutamicum where it replicates autonomously.

Construction of super shuttle vectors SS2002 and SS2003

[0087] SS2002 (Figure 2) is the same vector as SS2000, except that a kanamycin resistance gene was added at the Smal site between 'amyE and the spectinomycin resistance gene.

[0088] SS2003 is the same vector as SS2002, except that the spectinomycin resistance gene has been deleted.

Sequence of SS2003 <SEQ LO NO. 12>

AGGCCTTTAT CCATGCTGGT TCTAGAGAAG GTGTTGTGAC AAATTGCCCT TTCAGTGTGA

CAAATCACCC TCAAATGACA GTCCTGTCTG TGACAAATTG CCCTTAACCC TGTGACAAAT

TGCCCTCAGA AGAAGCTGTT TTTTCACAAA GTTATCCCTG CTTATTGACT CTTTTTTATT

TAGTGTGACA ATCTAAAAAC TTGTCACACT TCACATGGAT CTGTCATGGC GGAAACAGCG

GTTATCAATC ACAAGAAACG TAAAAATAGC CCGCGAATCG TCCAGTCAAA CGACCTCACT

GAGGCGGCAT ATAGTCTCTC CCGGGATCAA AAACGTATGC TGTATCTGTT CGTTGACCAG

ATCAGAAAAT CTGATGGCAC CCTACAGGAA CATGACGGTA TCTGCGAGAT CCATGTTGCT

AAATATGCTG AAATATTCGG ATTGACCTCT GCGGAAGCCA GTAAGGATAT ACGGCAGGCA

TTGAAGAGTT TCGCGGGGAA GGAAGTGGTT TTTTATCGCC CTGAAGAGGA TGCCGGCGAT

GAAAAAGGCT ATGAATCTTT TCCTTGGTTT ATCAAACGTG CGCACAGTCC ATCCAGAGGG

CTTTACAGTG TACATATCAA CCCATATCTC ATTCCCTTCT TTATCGGGTT ACAGAACCGG

TTTACGCAGT TTCGGCTTAG TGAAACAAAA GAAATCACCA ATCCGTATGC CATGCGTTTA

TACGAATCCC TGTGTCAGTA TCGTAAGCCG GATGGCTCAG GCATCGTCTC TCTGAAAATC

GACTGGATCA TAGAGCGTTA CCAGCTGCCT CAAAGTTACC AGCGTATGCC TGACTTCCGC

CGCCGCTTCC TGCAGGTCTG TGTTAATGAG ATCAACAGCA GAACTCCAAT GCGCCTCTCA

TACATTGAGA AAAAGAAAGG CCGCCAGACG ACTCATATCG TATTTTCCTT CCGCGATATC

ACTTCCATGA CGACAGGATA GTCTGAGGGT TATCTGTCAC AGATTTGAGG GTGGTTCGTC

ACATTTGTTC TGACCTACTG AGGGTAATTT GTCACAGTTT TGCTGTTTCC TTCAGCCTGC

ATGGATTTTC TCATACTTTT TGAACTGTAA TTTTTAAGGA AGCCAAATTT GAGGGCAGTT

TGTCACAGTT GATTTCCTTC TCTTTCCCTT CGTCATGTGA CCTGATATCG GGGGTTAGTT

CGTCATCATT GATGAGGGTT GATTATCACA GTTTATTACT CTGAATTGGC TATCCGCGTG

TGTACCTCTA CCTGGAGTTT TTCCCACGGT GGATATTTCT TCTTGCGCTG AGCGTAAGAG

CTATCTGACA GAACAGTTCT TCTTTGCTTC CTCGCCAGTT CGCTCGCTAT GCTCGGTTAC

ACGGCTGCGG CGAGCGCTAG TGATAATAAG TGACTGAGGT ATGTGCTCTT CTTATCTCCT

TTTGTAGTGT TGCTCTTATT TTAAACAACT TTGCGGTTTT TTGATGACTT TGCGATTTTG

TTGTTGCTTT GCAGTAAATT GCAAGATTTA ATAAAAAAAC GCAAAGCAAT GATTAAAGGA

TGTTCAGAAT GAAACTCATG GAAACACTTA ACCAGTGCAT AAACGCTGGT CATGAAATGA

CGAAGGCTAT CGCCATTGCA CAGTTTAATG ATGACAGCCC GGAAGCGAGG AAAATAACCC

GGCGCTGGAG AATAGGTGAA GCAGCGGATT TAGTTGGGGT TTCTTCTCAG GCTATCAGAG

ATGCCGAGAA AGCAGGGCGA CTACCGCACC CGGATATGGA AATTCGAGGA CGGGTTGAGC

AACGTGTTGG TTATACAATT GAACAAATTA ATCATATGCG TGATGTGTTT GGTACGCGAT

TGCGACGTGC TGAAGACGTA TTTCCACCGG TGATCGGGGT TGCTGCCCAT AAAGGTGGCG

TTTACAAAAC CTCAGTTTCT GTTCATCTTG CTCAGGATCT GGCTCTGAAG GGGCTACGTG

TTTTGCTCGT GGAAGGTAAC GACCCCCAGG GAACAGCCTC AATGTATCAC GGATGGGTAC

CAGATCTTCA TATTCATGCA GAAGACACTC TCCTGCCTTT CTATCTTGGG GAAAAGGACG

ATGTCACTTA TGCAATAAAG CCCACTTGCT GGCCGGGGCT TGACATTATT CCTTCCTGTC

TGGCTCTGCA CCGTATTGAA ACTGAGTTAA TGGGCAAATT TGATGAAGGT AAACTGCCCA CCGATCCACA CCTGATGCTC CGACTGGCCA TTGAAACTGT TGCTCATGAC TATGATGTCA TAGTTATTGA CAGCGCGCCT AACCTGGGTA TCGGCACGAT TAATGTCGTA TGTGCTGCTG ATGTGCTGAT TGTTCCCACG CCTGCTGAGT TGTTTGACTA CACCTCCGCA CTGCAGTTTT TCGATATGCT TCGTGATCTG CTCAAGAACG TTGATCTTAA AGGGTTCGAG CCTGATGTAC GTATTTTGCT TACCAAATAC AGCAATAGTA ATGGCTCTCA GTCCCCGTGG ATGGAGGAGC AAATTCGGGA TGCCTGGGGA AGCATGGTTC TAAAAAATGT TGTACGTGAA ACGGATGAAG TTGGTAAAGG TCAGATCCGG ATGAGAACTG TTTTTGAACA GGCCATTGAT CAACGCTCTT CAACTGGTGC CTGGAGAAAT GCTCTTTCTA TTTGGGAACC TGTCTGCAAT GAAATTTTCG ATCGTCTGAT TAAACCACGC TGGGAGATTA GATAATGAAG CGTGCGCCTG TTATTCCAAA ACATACGCTC AATACTCAAC CGGTTGAAGA TACTTCGTTA TCGACACCAG CTGCCCCGAT GGTGGATTCG TTAATTGCGC GCGTAGGAGT AATGGCTCGC GGTAATGCCA TTACTTTGCC TGTATGTGGT CGGGATGTGA AGTTTACTCT TGAAGTGCTC CGGGGTGATA GTGTTGAGAA GACCTCTCGG GTATGGTCAG GTAATGAACG TGACCAGGAG CTGCTTACTG AGGACGCACT GGATGATCTC ATCCCTTCTT TTCTACTGAC TGGTCAACAG ACACCGGCGT TCGGTCGAAG AGTATCTGGT GTCATAGAAA TTGCCGATGG GAGTCGCCGT CGTAAAGCTG CTGCACTTAC CGAAAGTGAT TATCGTGTTC TGGTTGGCGA GCTGGATGAT GAGCAGATGG CTGCATTATC CAGATTGGGT AACGATTATC GCCCAACAAG TGCTTATGAA CGTGGTCAGC GTTATGCAAG CCGATTGCAG AATGAATTTG CTGGAAATAT TTCTGCGCTG GCTGATGCGG AAAATATTTC ACGTAAGATT ATTACCCGCT GTATCAACAC CGCCAAATTG CCTAAATCAG TTGTTGCTCT TTTTTCTCAC CCCGGTGAAC TATCTGCCCG GTCAGGTGAT GCACTTCAAA AAGCCTTTAC AGATAAAGAG GAATTACTTA AGCAGCAGGC ATCTAACCTT CATGAGCAGA AAAAAGCTGG GGTGATATTT GAAGCTGAAG AAGTTATCAC TCTTTTAACT TCTGTGCTTA AAACGTCATC TGCATCAAGA ACTAGTTTAA GCTCACGACA TCAGTTTGCT CCTGGAGCGA CAGTATTGTA TAAGGGCGAT AAAATGGTGC TTAACCTGGA CAGGTCTCGT GTTCCAACTG AGTGTATAGA GAAAATTGAG GCCATTCTTA AGGAACTTGA AAAGCCAGCA CCCTGATGCG ACCACGTTTT AGTCTACGTT TATCTGTCTT TACTTAATGT CCTTTGTTAC AGGCCAGAAA GCATAACTGG CCTGAATATT CTCTCTGGGC CCACTGTTCC ACTTGTATCG TCGGTCTGAT AATCAGACTG GGACCACGGT CCCACTCGTA TCGTCGGTCT GATTATTAGT CTGGGACCAC GGTCCCACTC GTATCGTCGG TCTGATTATT AGTCTGGGAC CACGGTCCCA CTCGTATCGT CGGTCTGATA ATCAGACTGG GACCACGGTC CCACTCGTAT CGTCGGTCTG ATTATTAGTC TGGGACCATG GTCCCACTCG TATCGTCGGT CTGATTATTA GTCTGGGACC ACGGTCCCAC TCGTATCGTC GGTCTGATTA TTAGTCTGGA ACCACGGTCC CACTCGTATC GTCGGTCTGA TTATTAGTCT GGGACCACGG TCCCACTCGT ATCGTCGGTC TGATTATTAG TCTGGGACCA CGATCCCACT CGTGTTGTCG GTCTGATTAT CGGTCTGGGA CCACGGTCCC ACTTGTATTG TCGATCAGAC TATCAGCGTG AGACTACGAT TCCATCAATG CCTGTCAAGG GCAAGTATTG ACATGTCGTC GTAACCTGTA GAACGGAGTA ACCTCGGTGT GCGGTTGTAT GCCTGCTGTG GATTGCTGCT GTGTCCTGCT TATCCACAAC ATTTTGCGCA CGGTTATGTG GACAAAATAC CTGGTTACCC AGGCCGTGCC GGCACGTTAA CCGGGCTGCA TCCGATGCAA GTGTGTCGCT GTCGACGAGC TCGCGAGCTC GGACATGAGG TTGCCCCGTA TTCAGTGTCG CTGATTTGTA TTGTCTGAAG TTGTTTTTAC GTTAAGTTGA TGCAGATCAA TTAATACGAT ACCTGCGTCA TAATTGATTA TTTGACGTGG TTTGATGGCC TCCACGCACG TTGTGATATG TAGATGATAA TCATTATCAC TTTACGGGTC CTTTCCGGTG ATCCGACAGG TTACGGGGCG GCGACCTCGC GGGTTTTCGC TATTTATGAA AATTTTCCGG TTTAAGGCGT TTCCGTTCTT CTTCGTCATA ACTTAATGTT TTTATTTAAA ATACCCTCTG AAAAGAAAGG AAACGACAGG TGCTGAAAGC GAGCTTTTTG GCCTCTGTCG TTTCCTTTCT CTGTTTTTGT CCGTGGAATG AACAATGGAA GTCCGAGCTC ATCGCTAATA ACTTCGTATA GCATACATTA TACGAAGTTA TATTCGATGC GGCCGCAAGG GGTTCGCGTC AGCGGGTGTT GGCGGGTGTC GGGGCTGGCT TAACTATGCG GCATCAGAGC AGATTGTACT GAGAGTGCAC CATATGCGGT GTGAAATACC GCACAGATGC GTAAGGAGAA AATACCGCAT CAGGCGCCAT TCGCCATTCA GCTGCGCAAC TGTTGGGAAG GGCGATCGGT GCGGGCCTCT TCGCTATTAC GCCAGCTGGC GAAAGGGGGA TGTGCTGCAA GGCGATTAAG TTGGGTAACG CCAGGGTTTT CCCAGTCACG ACGTTGTAAA ACGACGGCCA GTGAATTGTA ATACGACTCA CTATAGGGCG AATTCGAGCT CGGTACCCGG GGATCCCACG TGGGATCCTC TAGAGTCGAC CTGCAGGCAT GCAAGCTTGA GTATTCTATA GTGTCACCTA AATAGCTTGG CGTAATCATG GTCATAGCTG TTTCCTGTGT GAAATTGTTA TCCGCTCACA ATTCCACACA ACATACGAGC CGGAAGCATA AAGTGTAAAG CCTGGGGTGC CTAATGAGTG AGCTAACTCA CATTAATTGC GTTGCGCTCA CTGCCCGCTT TCCAGTCGGG AAACCTGTCG TGCCAGCTGC ATTAATGAAT CGGCCAACGC GAACCCCTTG CGGCCGCCCG GGCCGTCGAC CAATTCTCAT GTTTGACAGC TTATCATCGA ATTTCTGCCA TTCATCCGCT TATTATCACT TATTCAGGCG TAGCAACCAG GCGTTTAAGG GCACCAATAA CTGCCTTAAA AAAATTACGC CCCGCCCTGC CACTCATCGC AGTACTGTTG TAATTCATTA AGCATTCTGC CGACATGGAA GCCATCACAA ACGGCATOAT GAACCTGAAT CGCCAGCGGC ATCAGCACCT TGTCGCCTTG CGTATAATAT TTGCCCATGG TGAAAACGGG GGCGAAGAAG TTGTCCATAT TGGCCACGTT TAAATCAAAA CTGGTGAAAC TCACCCAGGG ATTGGCTGAG ACGAAAAACA TATTCTCAAT AAACCCTTTA GGGAAATAGG CCAGGTTTTC ACCGTAACAC GCCACATCTT GCGAATATAT GTGTAGAAAC TGCCGGAAAT CGTCGTGGTA TTCACTCCAG AGCGATGAAA ACGTTTCAGT TTGCTCATGG AAAACGGTGT AACAAGGGTG AACACTATCC CATATCACCA GCTCACCGTC TTTCATTGCC ATACGAAATT CCGGATGAGC ATTCATCAGG CGGGCAAGAA TGTGAATAAA GGCCGGATAA AACTTGTGCT TATTTTTCTT TACGGTCTTT AAAAAGGCCG TAATATCCAG CTGAACGGTC TGGTTATAGG TACATTGAGC AACTGACTGA AATGCCTCAA AATGTTCTTT ACGATGCCAT TGGGATATAT CAACGGTGGT ATATCCAGTG ATTTTTTTCT CCATTTTAGC TTCCTTAGCT CCTGAAAATC TCGATAACTC AAAAAATACG CCCGGTAGTG ATCTTATTTC ATTATGGTGA AAGTTGGAAC CTCTTACGTG CCGATCAACG TCTCATTTTC GCCAAAAGTT GGCCCAGGGC TTCCCGGTAT CAACAGGGAC ACCAGGATTT ATTTATTCTG CGAAGTGATC TTCCGTCACA GGTATTTATT CGCGATAAGC TCATGGAGCG GCGTAACCGT CGCACAGGAA GGACAGAGAA AGCGCGGATC TGGGAAGTGA CGGACAGAAC GGTCAGGACC TGGATTGGGG AGGCGGTTGC CGCCGCTGCT GCTGACGGTG TGACGTTCTC TGTTCCGGTC ACACCACATA CGTTCCGCCA TTCCTATGCG ATGCACATGC TGTATGCCGG TATACGACGT CGTCGCCTCT CGCCCCAGGT TGTGTGAAAA CGGGCTGGAT GCAGTTGACG GATCTGCTGG CGAAAGGGGG ATGTGCTGCA AGGCGATTAA GTTGGGTAAC GCCAGGGTTT TCCCAGTCAC GACGTTGTAA AACGACGGCC AGTGAATTAA TTCTTGAAGA CGAAAGGGCC TCGTGATACG CCTATTTTTA TAGGTTAATG TCATGATAAT AATGGTTTCT TAGAGCTTAC GGCCAGCCTC GCAGAGCAGG ATTCCCGTTG AGCACCGCCA GGTGCGAATA AGGGACAGTG AAGAAGGAAC ACCCGCTCGC GGGTGGGCCT ACTTCACCTA TCCTGCCCGG CTGACGCCGT TGGATACACC AAGGAAAGTC TACACGAACC CTTTGGCAAA ATCCTGTATA TCGTGCGAAA AAGGATGGAT ATACCGAAAA AATCGCTATA ATGACCCCGA AGCAGGGTTA TGCAGCGGAA AAGATCCGTC GATCGACCCA GGTGGCACTT TTCGGGGAAA TGTGCGCGGA ACCCCTATTT GTTTATTTTT CTAAATACAT TCAAATATGT ATCCGCTCAT GAGACAATAA CCCTGATAAA TGCTTCAATA ATATTGAAAA AGGAAGAGTA TGAGTATTCA ACATTTCCGT GTCGCCCTTA TTCCCTTTTT TGCGGCATTT TGCCTTCCTG TTTTTGCTCA CCCAGAAACG CTGGTGAAAG TAAAAGATGC TGAAGATCAG TTGGGTGCAC GAGTGGGTTA CATCGAACTG GATCTCAACA GCGGTAAGAT CCTTGAGAGT TTTCGCCCCG AAGAACGTTT TCCAATGATG AGCACTTTTA AAGTTCTGCT ATGTGGCGCG GTATTATCCC GTGTTGACGC CGGGCAAGAG CAACTCGGTC GCCGCATACA CTATTCTCAG AATGACTTGG TTGAGTACTC ACCAGTCACA GAAAAGCATC TTACGGATGG CATGACAGTA AGAGAATTAT GCAGTGCTGC CATAACCATG AGTGATAACA CTGCGGCCAA CTTACTTCTG ACAACGATCG GAGGACCGAA GGAGCTAACC GCTTTTTTGC ACAACATGGG GGATCATGTA ACTCGCCTTG ATCGTTGGGA ACCGGAGCTG AATGAAGCCA TACCAAACGA CGAGCGTGAC ACCACGATGC CTGTAGCAAT GGCAACAACG TTGCGCAAAC TATTAACTGG CGAACTACTT ACTCTAGCTT CCCGGCAACA ATTAATAGAC TGGATGGAGG CGGATAAAGT TGCAGGACCA CTTCTGCGCT CGGCCCTTCC GGCTGGCTGG TTTATTGCTG ATAAATCTGG AGCCGGTGAG CGTGGGTCTC GCGGTATCAT TGCAGCACTG GGGCCAGATG GTAAGCCCTC CCGTATCGTA GTTATCTACA CGACGGGGAG TCAGGCAACT ATGGATGAAC GAAATAGACA GATCGCTGAG ATAGGTGCCT CACTGATTAA GCATTGGTAA CTGTCAGACC AAGTTTACTC ATATATACTT TAGATTGATT TAAAACTTCA TTTTTAATTT AAAAGGATCT AGGTGAAGAT CCTTTTTGAT AATCTCATGA CCAAAATCCC TTAACGTGAG TTTTCGTTCC ACTGAGCGTC AGACCCCGTA GAAAAGATCA AAGGATCTTC TTGAGATCCT TTTTTTCTGC GGGGGATCAG GACCGCTGCC GGAGCGCAAC CCACTCACTA CAGCAGAGCC ATGTAGGGCC GCCGGCGTTG TGGATACCAC GCGGAAAACT TGGCCCTCAC TGACAGATGA GGGGCGGACG TTGACACTTG AGGGGCCGAC TCACCCGGCG CGGCGTTGAC AGATGAGGGG CAGGCTCGAT TTCGGCCGGC GACGTGGAGC TGGCCAGCCT CGCAAATCGG CGAAAACGCC TGATTTTACG CGAGTTTCCC ACAGATGATG TGGACAAGCC TGGGGATAAG TGCCCTGCGG TATTGACACT TGAGGGGCGC GACTACTGAC AGATGAGGGG CGCGATCCTT GACACTTGAG GGGCAGAGTG ATGACAGATG AGGGGCGCAC CTATTGACAT TTGAGGGGCT GTCCACAGGC AGAAAATCCA GCATTTGCAA GGGTTTCCGC CCGTTTTTCG GCCACCGCTA ACCTGTCTTT TAACCTGCTT TTAAACCAAT ATTTATAAAC CTTGTTTTTA ACCAGGGCTG CGCCCTGGCG CGTGACCGCG CACGCCGAAG GGGGGTGCCC CCCCTTCTCG AACCCTCCCG GCCCGCTAAC GCGGCACCCC ATCCCCCCAG GGGCTGCGCC CCTCGGCCGC GAACGACCTC ACCCCAAAAA TGGCAGCCAC GTAGAAAGCC AGTCCGCAGA AACGGTGCTG ACCCCGGATG AATGTCAGCT ACTGGGCTAT CTGGACAAGG GAAAACGCAA GCGCAAAGAG AAAGCAGGTA GCTTGCAGTG GGCTTACATG GCGATAGCTA GACTGGGCGG TTTTATGGAC AGCAAGCGAA CCGGAATTGC CAGCTGATCG ATCATTAATC ATCCTTGCAG GGTATGTTTC TCTTTGATGT CTTTTTGTTT GTGAAGTATT TCACATTTAT ATTGTGCAAC ACTTCACAAA CTTTTGCAAG AGAAAAGTTT TGTCTGATTT ATGAACAAAA AAGAAACCAT CATTGATGGT TTCTTTCGGT AAGTCCCGTC TAGCCTTGCC CTCAATGGGG AAGAGAACCG CTTAAGCCCG AGTCATTATA TAAACCATTT AGCACGTAAT CAAAGCCAGG CTGATTCTGA CCGGGCACTT GGGCGCTGCC ATTATTAAAA ATCACTTTTG CGTTGGTTGT ATCCGTGTCC GCAGGCAGCG TCAGCGTGTA AATTCCGTCT GCATTTTTAG TCATTGGTTT TCCAGGCCAA GATCCGGTCA ATTCAATTAC TCGGCTCCCA TCATGTTTAT AGATATAAGC ATTTACCTGG CTCCAATGAT TCGGATTTTG ATAGCCGATG GTTTTGGCCG ACGCTGGATC TCTTTTAACA AAACTGTATT TCTCGGTCCT CGTTACACCA TCACTGTTCG TTCCTTTTAA CATGATGGTG TATGTTTTGC CAAATTGGAT CTCCTTTTCC GATTGTGAAT TGATCTCCAT CCTTAAACGC CTGTCGTCTG GTCCATTATT GATTTGATAA ACGGCTTTTG TTGTATTCGC ATCTGCACGC AAGGTAATCG TCAGTTGATC ATTGAAAGAA TGTGTTACAC CTGTTTTGTA ATTCTCAAGG AAAACATGAG GCGCTTTTGC AATATCATCA GGATAAAGCA CAGCTACAGA CCTGGCATTG ATCGTGCCTG TCAGTTTACC ATCGTTCACT TGAAATGAAC CCGCTCCAGC TTTATTGTCA TACCTGCCAT CAGGCAATTT TGTTGCCGTA TTGATAGAGA CAGAGGATGA ACCTGCATTT GCCAGCACAA CGCCATGTGA GCCGCGCTGA TTCATAAATA TCTGGTTGTT TCCATTCGGG TTCGAGAGTT CCTCAGGCTG TCCAGCCATC ACATTGTGAA ATCTATTGAC CGCAGTGATA GCCTGATCTT CAAATAAAGC ACTCCCGCGA TCGCCTATTT GGCTTTTCCC CGGGAACCTC ACACCATTTC CGCCTCCCTC AGGTCTGGAA AAGAAAAGAG GCGTACTGCC TGAACGAGAA GCTATCACCG CCCAGCCTAA ACGGATATCA TCATCGCTCA TCCATGTCGA CCTACGGGGT CTGACGCTCA GTGGAACGAA AACTCACGTT AAGGGATTTT GGTCATGAGA TTATCAAAAA GGATCTTCAC CTAGATCCTT TTGACTCTAG AGGATCGATC TGTATAATAA AGAATAATTA TTAATCTGTA GACAAATTGT GAAAGGATGT ACTTAAACGC TAACGGTCAG CTTTATTGAA CAGTAATTTA AGTATATGTC CAATCTAGGG TAAGTAAATT GAGTATCAAT ATAAACTTTA TATGAACATA ATCAACGAGG TGAAATCATG AGCAATTTGA TTAACGGAAA AATACCAAAT CAAGCGATTC AAACATTAAA AATCGTAAAA GATTTATTTG GAAGTTCAAT AGTTGGAGTA TATCTATTTG GTTCAGCAGT AAATGGTGGT TTACGCATTA ACAGCGATGT AGATGTTCTA GTCGTCGTGA ATCATAGTTT ACCTCAATTA ACTCGAAAAA AACTAACAGA AAGACTAATG ACTATATCAG GAAAGATTGG AAATACGGAT TCTGTTAGAC CACTTGAAGT TACGGTTATA AATAGGAGTG AAGTTGTCCC TTGGCAATAT CCTCCAAAAA GAGAATTTAT ATACGGTGAG TGGCTCAGGG GTGAATTTGA GAATGGACAA ATTCAGGAAC CAAGCTATGA TCCTGATTTG GCTATTGTTT TAGCACAAGC AAGAAAGAAT AGTATTTCTC TATTTGGTCC TGATTCTTCA AGTATACTTG TCTCCGTACC TTTGACAGAT ATTCGAAGAG CAATTAAGGA TTCTTTGCCA GAACTAATTG AGGGGATAAA AGGTGATGAG CGTAATGTAA TTTTAACCCT AGCTCGAATG TGGCAAACAG TGACTACTGG TGAAATTACC TCGAAAGATG TCGCTGCAGA ATGGGCTATA CCTCTTTTAC CTAAAGAGCA TGTAACTTTA CTGGATATAG CTAGAAAAGG CTATCGGGGA GAGTGTGATG ATAAGTGGGA AGGACTATAT TCAAAGGTGA AAGCACTCGT TAAGTATATG AAAAATTCTA TAGAAACTTC TCTCAATTAG GCTAATTTTA TTGCAATAAC AGGTGCTTAC TTTTAAAACT ACTGATTTAT TGATAAATAT TGAACAATTT TTGGGATCCT TTACGGCGTC CTCGTGGAAG TTCAATGCCC GCAGACTTAA GTGCTCTATT CACGGTCTGA CGTGACACGC TAAATTCAGA CATAGCTTCA TTGATTGTCG GCCACGAGCC AGTCTCTCCC TCAACAGTCA TAAACCAACC TGCAATGGTC AAGCGATTTC CTTTAGCTTT CCTAGCTTGT CGTTGACTGG ACTTAGCTAG TTTTTCTCGC TGTGCTCGGG CGTACTCACT GTTTGGGTCT TTCCAGCGTT CTGCGGCCTT TTTACCGCCA CGTCTTCCCA TAGTGGCCAG AGCTTTTCGC CCTCGGCTGC TCTGCGTCTC TGTCTGACGA GCAGGGACGA CTGGCTGGCC TTTAGCGACG TAGCCGCGCA CACGTCGCGC CATCGTCTGG CGGTCACGCA TCGGCGGCAG ATCAGGCTCA CGGCCGTCTG CTCCGACCGC CTGAGCGACG GTGTAGGCAC GCTCGTAGGC GTCGATGATC TTGGTGTCTT TTAGGCGCTC ACCAGCCGCT TTTAACTGGT ATCCCACAGT CAAAGCGTGG CGAAAAGCCG TCTCATCACG GGCGGCACGC CCTGGAGCAG TCCAGAGGAC ACGGACGCCG TCGATCAGCT CTCCAGACGC TTCAGCGGCG CTCGGCAGGC TTGCTTCAAG CGTGGCAAGT GCTTTTGCTT CCGCAGTGGC TTTTCTTGCC GCTTCGATAC GTGCCCGTCC GCTAGAAAAC TCCTGCTCAT AGCGTTTTTT AGGTTTTTCT GTGCCTGAGA TCATGCGAGC AACCTCCATA AGATCAGCTA GGCGATCCAC GCGATTGTGC TGCAG

Construction of the super shuttle vector CCSS2003

[0089] The F' single copy replicon is recovered from a plasmid containing the replicon (e.g., CClFos^®, Epicentre, Madison WI) by a Eco72I/ Stul digest and ligated with the Tthllllmed-m-PvuII fragment from pJB137 which harbors the RK2 minimal replicon lacking the TrfA gene. The chloramphenicol resistance gene is replaced with an ampicillin resistance gene and the origin of tranfer (oriT) from pJB137 is added to allow conjugative transfer between E. coli and Pseudomonas or Corynebacterium. The resulting plasmid can be controlled with respect to copy number in E. coli when used with a strain containing the inducible trfA gene on its chromosome (e.g., in EPI300 strain from Epicentre, Madison, WI). As such, this intermediate is most effectively used in E. coli. Copy control use in other enteric bacteria and in Pseudomonas requires that the trfA gene be first integrated into the host's chromosome.

[0090] The Nhel/Pstl fragment from SS2000, encompassing the 'amyE truncated gene, spectinomycin resistance gene, and a Corynebacterium origin of replication (e.g., from pHMl 519) is recovered and ligated with Hpal/Hindlll digested CCSCos. The resulting super shuttle is designated CCSS2003 and is suitable for building single-copy libraries in E. coli (e.g., EPI300) and subsequent transfer into all enteric bacteria, Xanthomonas, Acetobacter, Pseudomonas, B. subtilis, and C. glutamicum where it replicates autonomously. For use in Gram-negative species, preliminary cloning and chromosomal integration of the trfA gene encoding replication initiation protein of the RK2 replicon is required. Common techniques for chromosomal gene integration in those species are described in the literature (e.g., Schweize, Curr. Opin. Biotechnol. 12:439-445, 2001; Hamilton et al, J. Bacteriol. 171:4617- 4622, 1989; Ried and Collmer, Gene 57:239-246, 1987).

Example 3 - Cosmid Transfer Between Species

[0091] For transfer into Bacillus, transfected cells were harvested after amplification on selective agar media and cosmid DNA were bulk-extracted. This can be conveniently accomplished using a suitable commercial kit (e.g., the "Large Construct Kit" [Qiagen, Valencia, CA] or equivalent). The cosmid preparation is transformed into B. subtilis mutant strain JH892, which exhibits increased transformation efficiency. The following protocol was used:

[0092] B. subtilis strain JH892 was streaked on a LB or TBAB plate and placed at 37°C overnight. A 2-3 ml of liquid LB culture was inoculated and allowed to shake at 37 °C for 3 hours. The culture was diluted 1 :20 in GE medium (see below), and shaken at 37 °C for 4 hours. 300 μl of cells were mixed with an appropriate amount of DNA (adjust to optimize the number of colonies per plate). The mixture was shaken at 37 °C for 30 minutes. 100 μl of regeneration buffer (see below) was added. The mixture was shaken at 37 °C for 1 hour. 100 μl of the sample was plated on an LB/Spectinomycinl50. The sample was incubated at 37 °C overnight. [0093] GE medium contains:

50% Glucose 200 μl

10% Potassium Glutamate 200 μl

1M Potassium Phosphate buffer pH 7.0 1.0 ml

1M Na₃ citrate 30 μl

1M MgSO₄ 30 μl

FeNH₄ citrate (22mg/ml) 10 μl

Tryptophan 250 μl ddH₂0 8.28 ml

[0094] Regeneration buffer contains 2.5% casamino acids and 2.5% yeast extract.

[0095] For transfer of environmental libraries into Pseudomonas aeruginosa, cells were harvested after amplification on solid selective media and used in triparental conjugative matings between (i) the amplified environmental library in EPI300, (ii) P. aeruginosa strain PAO615, and (iii) E. coli strain MM294 containing the helper vector pRK2013, according to published procedures (Zhao et al, Antimicrob. Agents Chemotherapy, 42:225-2231, 1998).

[0096] For transfer into Corynebacterium glutamicum, libraries are first amplified in E. coli strain SI 7-1 and introduced into C. glutamicum strain RM3 (Res^") by interspecies conjugative matings according to published procedures (Schafer et al, J. Bacteriol. 172:1663- 1666, 1990).

[0097] Once the CCSS2003 vector is built and used to build environmental libraries, amplification in E. coli can be performed in the absence of CopyControl inducer in order to maintain cosmids in single copy and avoid loss of clones due to toxicity.

Example 4 - Copy Number Determination

[0098] Copy number determinations were made using data provided by the manufacturer (Strategene for SUPERCOS^® derivatives, Epicentre for CClFos). Copy numbers of pFosl were performed using the method of Kim et al, Nucl. Acid Res. 20:1083-1085, 1992. For the RK2 replicon (~7 copies per cell for the wild-type, 20-25 copies per cell for the copy-up trfA mutant that was used to build some super-shuttle derivatives), the method of Durland was used (Durland et al, J. Bacteriol 172:3859-3867, 1990). Example 5 - Copy Control Shuttle Vector

[0099] Referring to Figure 4, a vector of the present invention is illustrated, which is an example of an inducible vector for use in E. coli. The vector is inducible to high copy number, and therefore useful for minimizing the loss of clones due to toxicity of the expressed DNA fragment. The vector contains an F factor replicon, which contains loci for RepE, ParA, ParB, and ParC; cos sites; an origin of transfer (oriT) for conjugative matings between E. coli and other species such as Pseudomonas, enteric bacteria and Actinomycetes; a broad host range origin of replication (oriV) functional in most Gram-negative species; a 5'-truncated amyE gene from B. subtilis for chromosomal integration by homologous recombination; origin of replication for Actinomycete species (ori pHM1519); ampicillin and chloramphenicol resistance markers for selection in Gram-negatives; and a spectinomycin resistance marker as a selection marker in all Gram-positives.

Example 6 - Other Vectors of the Present Invention

[0100] The vector described below is derived from the vector SS2002 described previously. The example illustrates how variations on the vectors presented herein can be obtained to meet particular requirements.

[0101] The SS3000 shuttle vector (Figure 3) is an embodiment of the present invention that integrates into the chromosomes of high-GC content Gram-positive species, as opposed to other embodiments discussed earlier that replicate autonomously in Corynebacterium.

[0102] The region encompassing trfA-oriV-ampR-oriT is the RK2 minimal replicon that provides plasmid replication and selection in a broad range of Gram-negative species including E. coli and all enteric bacteria, including Pseudomonas, Xanthomonas, and Acetobacter. This region was recovered from pJB137 (Blatny et al, Appl Environ. Microbiol. 63:370-379, 1997).

[0103] The two cos sites of the vector mediate packaging into lambda particles. A 5'- truncated copy of B. subtilis amyE gene was inserted into the vector to allow chromosomal integration via homologous integration into the B. subtilis chromosome (low-GC content Gram-positive). The spectinomycin resistance gene can be selected for in all Gram-positive species. [0104] The int-attP module was isolated from Corynebacterium phage φl6 (Moreau et al, Microbiol. 145: 539-548, 1999) and inserted into the vector to provide for chromosomal integration into Corynebacterium glutamicum and other Actinomycetes (high GC content Gram-positive).

Example 7 - Screening an Environmental Library

[0105] This example illustrates the screening of an environmental library using a shuttle vector of the present invention for the production of a small bioactive molecule.

1. Primary screening for bioactive compounds

[0106] Libraries constructed in a shuttle vector of the present invention were pre-amplified in E. coli or in Klebsiella oxytoca by plating 5,000 to 10,000 clones per plate on LB solid medium supplemented with the appropriate antibiotic and growing overnight at 30 °C. Colonies (up to 5 million per library) were scraped off the plates, pooled together, and stored at -80 °C after the titer was determined.

[0107] When screening for antibacterial compounds produced by the environmental clones, libraries were plated on solid LB media containing the appropriate antibiotic, and inducer in the case of libraries built in CClFos derivatives. Clones were plated at a density of 500 to 800 per 100 mm Petri dish to allow for isolated colonies to form for two days at 30 °C. Each plate was then subjected to UV radiation for 2 minutes to kill bacteria present on the surface of each colony and prevent them from interfering with subsequent steps. Each plate was overlayed with 6 ml of soft LB agar (0.7% agar) inoculated with 10⁶ cells/ml of an indicator species such as Staphylococcus aureus or Bacillus subtilis. Plates were incubated overnight at 37 °C and screened for clearing zones in the overlay around clones which produce a compound that kills the indicator strain. The average rate of confirmed hits which reproducibly prevent growth of Bacillus subtilis as the indicator strain was 1/28,000.

[0108] Alternatively, an environmental library is transferred from E. coli into Pseudomonas, Bacillus or Corynebacterium using the methods described previously. Primary screening is then achieved using the method described above, with one of the alternate species as the cloning host, except that the UV irradiation step is 10 minutes for Bacillus and 5 minutes for Pseudomonas and Corynebacterium. 2. Secondary screening of positive or bioactive clones

[0109] Positive clones were retrieved from underneath the soft layer and purified. After confirming their ability to prevent growth of the primary indicator strain, they were tested against a panel of clinically relevant pathogens including Methicillin resistant Staphylococcus aureus, Streptococcus pneumoniae, Vancomycin resistant Enterococcus, Pseudomonas aeruginosa, E. coli 0157.

3. Purification of bioactive compounds produced by library clones

[0110] In order to grow a library clone, a large plastic tray containing 2 liters of medium (LB with 0.75% agar and 25 mg/L kanamycin) was prepared. The tray was inoculated with a culture of a bioactive clone (grown over night at 37°C in LB containing 25 mg/1 kanamycin) using a 96-pronge replicator and incubated for 3 days at 30 °C. To harvest the bacterial broth the media was freeze/thawed by moving the tray in and out of a -20 °C freezer. The freezing process disrupts the structure of the agar and thereby allows the recovery of ~1.2 liters of the liquid broth by filtrating the agar containing media through 4 layers of cheesecloth. For the organic solvent extraction the pH of the broth was lowered to 3-4 by the addition of 0.5% TFA. The broth was then extracted twice with 300 ml of 1-butanol. The resulting butanol phases were recovered and dried in a rotation evaporator. The remaining oil (~1.5 g) was resuspended in 15 ml methanol, centrifuged at 30,000xg for 15 minutes, and loaded onto a reversed phase (RP) column (60 ml C18-silica). The column was developed with 350 ml solvent using a 0 to 100% gradient of methanol in water, both containing 0.1% TFA. Ten ml fractions were collected. 1% of each fraction was transferred into a well of a 96-well plate, dried and then tested for biological activity by adding 0.04 ml of media containing the tester strain. After over night incubation growth or non-growth of the tester strain was observed by the presence or absence of a cell pellet at the bottom of the each well. The lack of a cell pellet indicates that the corresponding fraction of the reversed phase column separation contains one or more compounds that inhibit cell growth. The active fractions were dried and the remaining substance was resuspended in 0.4 ml methanol. The active substance (or substances) present in these samples was further purified by two identical successive chromatographic separations using an HPLC reversed phase column (20 ml C18-silica) which was developed with 100 ml of solvent using a 0 to 70 % gradient of acetonitrile in water (both solvents containing 0.1% TFA). Both chromatographic separations resulted in 48 fractions each, which were tested for biological activity as described above. The active fractions of the first of the two separations were used for the second separation. The active fractions of the second HPLC separation were tested for purity by mass spectroscopy. If the purity was not satisfactory an additional chromatographic separation was performed using a Synergi Polar™-RP HPLC column (Phenomenex^®, Torrance, CA) under the same conditions described above for the HPLC reversed phase chromatography.

[0111] The invention illustratively described herein may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by certain embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

[0112] The contents of the articles, patents, and patent applications, and all other documents and electronically available information mentioned or cited herein, are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to physically incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.

[0113] The inventions illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

Thus, for example, the terms "comprising", "including," containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by certain embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention. [0114] The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0115] i addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

[0116] Other embodiments are set forth within the following claims.

Claims

That which is claimed is:

1. A recombinant shuttle vector comprising:

(a) a first origin of replication or region of integration (OR/RI) functional in low GC content Gram-positive bacteria,

(b) a second OR/RI functional in a first bacterial type,

(c) a third OR/RI functional in a second bacterial type,

(d) at least one cos site; and

(e) at least one antibiotic selection marker; wherein the first bacterial type is different from the second bacterial type, and the first and second bacterial types are selected from the group consisting of high GC content Gram- positive bacteria, high GC content Gram-negative bacteria and low GC content Gram-negative bacteria.

2. A vector according to claim 1, wherein said vector comprises at least two cos sites.

3. A vector according to claim 1, further comprising an oriT origin of transfer.

4. A vector according to claim 1, wherein said vector is inducible to high copy number replication.

5. A vector according to claim 1, wherein said vector is selected from the group consisting of a cosmid, a plasmid, a fosmid, and a bacterial artificial chromosomes (BAC).

6. A vector according to claim 5, wherein said vector is a cosmid.

7. A vector according to claim 1, wherein said vector is functional in Bacillus subtilis, B. licheniformis, B. cereus, B. purnilus, B. anthracis, Clostridium acetobutylicum, Lactobacillus lactis, L. plantarum, or Enterococcus feacalis.

8. A transformed bacterial host cell, said host cell comprising a vector according to claim 1.

9. A transformed bacterial host cell according to claim 8, wherein said host cell is selected from the group consisting of spore-forming Gram-positive bacteria, lactic acid bacteria, enteric bacteria, Pseudomonales, and Actinomycetes.

10. A transformed bacterial host cell according to claim 8, wherein said host cell is selected from the group consisting of Bacillus subtilis, E. coli, enteric bacteria, Pseudomonas aeruginosa, P. putida, Xanthomonas campestris, Acinetobacter calcoaceticus, Azotobacter vinelandii, Acetobacter calcoaceticus, Streptomyces lividans, Streptomyces coelicolor, Rhodococcus rhodochrous, R. erγthropolis, Corynebacterium glutamicum, and Mycobacterium smegmatis.

11. A vector according to claim 1 , wherein said first OR/RI is an origin of replication functional in low GC content Gram-positive bacteria selected from the group consisting of pAM/31, pHT1030, pT181, pC194, pE194, pSN2, pTB19, pWVOl, and pIP404.

12. A vector according to claim 1, wherein said first OR/RI is a region of integration which is a region of homology with low GC content Gram-positive bacteria.

13. A vector according to claim 12, wherein said region of homology comprises an amyE locus.

14. A vector according to claim 13, wherein said amyE locus is a B. subtilis amyE locus.

15. A vector according to claim 1, wherein said second OR/RI is selected from the group consisting of: an origin of replication functional in high GC content Gram-positive bacteria, an origin of replication functional in high GC content Gram-negative bacteria, an origin of replication functional in low GC content Gram-negative bacteria, and a region of integration functional in high GC content Gram-positive bacteria.

16. A vector according to claim 15, wherein said origin of replication functional in high GC content Gram-positive bacteria is selected from the group consisting of pSAl.l, pHM1519, pIJlOl, pSRl, pAL5000, and pMFl; further wherein said origin of replication functional in high GC content Gram-negative bacteria is selected from the group consisting of RK2, RSF1010, pRO1600, pNHO, and pPP8.1; further wherein said origin of replication functional in low GC content Gram-negative bacteria is selected from the group consisting of ColEl, pl5A, SC101, and oriF; and further wherein said region of integration functional in high GC content Gram-positive bacteria is an attP attachment site functional in high GC content Gram-positive bacteria.

17. A vector according to claim 15, wherein said second OR/RI is said origin of replication functional in high GC content Gram-positive bacteria, and further wherein said third OR RI is selected from the group consisting of (i) an origin of replication functional in high GC content Gram-negative bacteria, (ii) an origin of replication functional in low GC content Gram-negative bacteria, and (iii) a region of integration functional in high GC content Gram-positive bacteria.

18. A vector according to claim 17, wherein said origin of replication functional in high GC content Gram-positive bacteria is an Actinomycete origin of replication.

19. A vector according to claim 18, wherein said Actinomycete origin of replication is selected from the group consisting of pSAl.l, pHM1519, pIJlOl, pSRl, pAL5000, and pMFl.

20. A vector according to claim 19, wherein said Actinomycete origin of replication is pHM1519.

21. A vector according to claim 20, wherein said pHM 1519 comprises an ori 1519.

22. A vector according to claim 18, wherein said first OR/RI comprises an amyE region of integration.

23. A vector according to claim 17, wherein said third OR/RI is said origin of replication functional in high GC content Gram-negative bacteria.

24. A vector according to claim 23, wherein said origin of replication functional in high GC content Gram-negative bacteria is selected from the group consisting of pRO1600, pNIlO, pPPδ.l, RK2, and RSF1010.

25. A vector according to claim 23, wherein said origin of replication functional in high GC content Gram-negative bacteria is an RK2 replicon.

26. A vector according to claim 25, wherein said RK2 replicon comprises an oriV origin of replication.

27. A vector according to claim 26, further comprising a sequence encoding trfA.

28. A vector according to claim 27, further comprising an Amp^R resistance marker.

29. A vector according to claim 24, wherein said origin of replication functional in high GC content Gram-positive bacteria is an Actinomycete origin of replication.

30. A vector according to claim 29, wherein said first OR/RI comprises an amyE region of integration.

31. A vector according to claim 23, wherein said vector further comprises a region of integration functional in low GC content Gram-negative bacteria.

32. A vector according to claim 17, wherein said third OR/RI is said origin of replication functional in low GC content Gram-negative bacteria.

33. A vector according to claim 32, wherein said origin of replication functional in low GC content Gram-negative bacteria is selected from the group consisting of ColEl, pl5A, RK2, RSF1010, F factor, and SC101 replicons.

34. A vector according to claim 32, wherein said origin of replication functional in low GC content Gram-negative bacteria is an RK2 replicon.

35. A vector according to claim 34, wherein said RK2 replicon comprises an oriV origin of replication.

36. A vector according to claim 35, further comprising a sequence encoding trfA.

37. A vector according to claim 36, wherein said vector further comprises an Amp^R resistance marker.

38. A vector according to claim 32, wherein said origin of replication functional in low GC content Gram-negative bacteria is an F factor replicon.

39. A vector according to claim 38, wherein said F factor replicon comprises a repE locus and one or more partition locus.

40. A vector according to claim 39, wherein said one or more partition locus comprises a par A locus, aparB locus, aparC locus, or a combination of any two or more thereof.

41. A vector according to claim 33, wherein said origin of replication functional in high GC content Gram-positive bacteria comprises an Actinomycete origin of replication.

42. A vector according to claim 41, wherein said first OR/RI comprises an amyE homologous region.

43. A vector according to claim 32, wherein said first OR/RI comprises an amyE homologous region; said second OR/RI comprises a pHM1519 origin of replication; said third OR/RI comprises an RK2 or a ColEl replicon; and wherein said vector comprises two cos sites and at least two resistance markers.

44. A vector according to claim 43, wherein said resistance markers comprise a Km^R resistance marker and a Sρc^R resistance marker.

45. A vector according to claim 44, wherein said vector is selected from the group consisting of SS2000, SS2002, and SS2003.

46. A vector according to claim 32, wherein said first OR/RI comprises an amyE homologous region; said second OR/RI comprises a pHM1519 origin of replication; said third OR/RI comprises an F factor replicon; and wherein said vector further comprises an oriT origin of transfer and an oriV origin of replication.

47. A vector according to claim 46, wherein said F factor replicon comprises a repE locus and one or more partition locus.

48. A vector according to claim 47, wherein said one or more partition locus comprises a par A locus, aparB locus, aparC locus, or a combination of any two or more thereof.

49. A vector according to claim 48, wherein said resistance markers comprise a Cm^R resistance marker, a Amp^R resistance marker, and a Spc^R resistance marker.

50. A vector according to claim 49, wherein said vector is CCSS2003.

51. A vector according to claim 32, further comprising a region of integration functional in high GC content Gram-negative bacteria.

52. A vector according to claim 15, wherein said second OR/RI is said origin of replication functional in high GC content Gram-negative bacteria, and further wherein said third OR/RI is selected from the group consisting of (i) a region of integration functional in low GC content Gram-negative bacteria, and (ii) a region of integration functional in high GC content Gram-positive bacteria.

53. A vector according to claim 52, wherein said third OR/RI is said region of integration functional in high GC content Gram-positive bacteria.

54. A vector according to claim 53, further comprising a region of integration functional in low GC content Gram-negative.

55. A vector according to claim 15, wherein said second OR/RI is said origin of replication functional in low GC content Gram-negative bacteria, and further wherein said third OR/RI is a region of integration functional in high GC content Gram-positive bacteria.

56. A vector according to claim 55, wherein said origin of replication functional in low GC content Gram-negative bacteria is selected from the group consisting of pi 5 A, colEl, RK2, RSF1010, oriF homologs, and SCI 01 origins of replication.

57. A vector according to claim 55, wherein said origin of replication functional in low GC content Gram-negative bacteria is an RK2 replicon.

58. A vector according to claim 57, wherein said RK2 replicon comprises an oriV origin of replication.

59. A vector according to claim 58, further comprising a sequence encoding trfA.

60. A vector according to claim 59, further comprising an Amp^R resistance marker.

61. A vector according to claim 60, wherein said origin of replication functional in low GC content Gram-negative bacteria is an F factor replicon.

62. A vector according to claim 61, wherein said F factor replicon comprises an oriF.

63. A vector according to claim 63, wherein said F factor replicon comprises a repE locus and one or more partition locus.

64. A vector according to claim 63, wherein said one or more partition locus comprises a parA locus, a parB locus, a parC locus, or a combination of any two or more thereof.

65. A vector according to claim 56, wherein said first OR/RI comprises an amyE locus.

66. A vector according to claim 55, wherein said region of integration functional in high GC content Gram-positive bacteria is an attP-int fragment.

67. A vector according to claim 66, wherein said attP-int fragment is derived from an Actinomycete attP locus and comprises an int integrase gene functional in high GC content Gram-positive bacteria.

68. A vector according to claim 66, wherein said origin of replication functional in low GC content Gram-negative bacteria is an RK2 replicon or an F factor replicon.

69. A vector according to claim 68, wherein said first OR/RI comprises an Amy E.

70. A vector according to claim 55, wherein said first OR/RI comprises an amyE homologous region; said second OR/RI comprises an RK2 or ColEl replicon; said third OR/RI comprises an attP-int region; and wherein said vector comprises at least two cos sites and at least two resistance markers.

71. A vector according to claim 70, further comprising an oriT origin of transfer.

72. A vector according to claim 71, wherein said at least two resistance markers are a Amp^R resistance marker and a Spc^R resistance marker.

73. A vector according to claim 72, wherein said vector is SS3000.

74. A vector according to claim 72, wherein said resistance markers further comprise a Kan^R resistance marker.

75. A vector according to claim 70, wherein said vector is inducible to high copy replication.

76. A vector according to claim 55, further comprising a region of integration functional in high GC content Gram-negative bacteria.

77. A recombinant shuttle vector comprising:

(a) an origin of replication functional in low and high GC content Gram-negative bacteria,

(b) a first OR/RI functional in low GC content Gram-positive bacteria,

(c) at least one cos site; and

(d) at least one antibiotic selection marker.

78. A vector according to claim 77, further comprising a second OR/RI functional in high GC content Gram-positive bacteria.

79. A vector according to claim 77, wherein said vector comprises at least two cos sites.

80. A vector according to claim 77, wherein said origin of replication of (a) is an RK2 replicon or an RSF1010 replicon.

81. A vector according to claim 80, wherein said vector is functional in one or more bacteria selected from the group consisting of Bacillus subtilis, B. licheniformis, B. cereus, B. purnilus, B. anthracis, Clostridium acetobutylicum, Lactobacillus lactis, L. plantaru, and Enterococcus feacalis.

82. A vector according to claim 77 or 80, wherein said first OR/RI is an origin of replication functional in low GC content Gram-positive bacteria selected from the group consisting of pHT1030, pAMbl, pC194, pE194, pTB19, pT181, pWVOl, and pIP404; or a region of homology with low GC content Gram-positive bacteria.

83. A vector according to claim 82, wherein said region of homology comprises an amyE locus.

84. A vector according to claim 83, wherein said amyE locus is a B. subtilis amyE locus.

85. A vector according to claim 78 or 80, wherein said second OR/RI is an Actinomycete origin of replication or an attP attachment site.

86. A vector according to claim 85, wherein said first OR/RI comprises a an amyE locus.

87. A vector according to claim 85, wherein said attP attachment site is an attP-int fragment mediating chromosomal integration.

88. A vector according to claim 85, wherein said Actinomycete origin of replication is selected from the group consisting of pSAl.l, pHM1519, pIJlOl, pSRl, ρAL5000, andpMFl.

89. A vector according to claim 88, wherein said Actinomycete origin of replication is PHM1519.

90. A vector according to claim 80, wherein said vector comprises said RK2 replicon and an amyE locus.

91. A vector according to claim 90, further comprising an attP attachment site.

92. A vector according to claim 91, wherein said vector is SS3000.

93. A vector according to claim 78, wherein said vector is functional in Bacillus subtilis, enteric bacteria, Pseudomonales, or Corynebacteria.

94. A vector according to claim 90, further comprising an pHM1519 ori.

95. A vector according to claim 94, further comprising an Amp^R resistance marker and a Spc^R resistance marker.

96. A vector according to claim 95, wherein said vector is SS2000.

97. A vector according to claim 96, further comprising a Kan^R resistance marker.

98. A vector according to claim 97, wherein said vector is SS2002.

99. A vector according to claim 94, further comprising an Amp^R resistance marker and a Kan^R resistance markers.

100. A vector according to claim 99, wherein said vector is SS2003

101. A vector according to claim 94, wherein said vector is functional in Bacillus subtilis, enteric bacteria, Pseudomonales, or Corynebacteria.

102. A vector according to any one of claims 31, 51, 54, 76 or 78, wherein said vector replicates in at least one genera of bacteria selected from each of the following groups consisting of:

Escherichia or Klebsiella;

Pseudomonas, Xanthomonas, or Acetobacter;

Bacillus or Clostridium, and

Corynebacteria, Streptomyces, or Rhodococcus.

103. A method of expressing DNA, said method comprising:

(a) inserting a DNA into the vector of claim 1 or 77;

(b) transfecting a host cell with said vector; and

(c) expressing said DNA in a host.