# Patent

## Title
Actinobacillus succinogenes shuttle vector and methods of use

## Abstract

An Actinobacillus succinogenes plasmid vector which provides a means to overexpress proteins in A. succinogenes. The plasmid can be transformed efficiently by electroporation, and replicates in a stable manner in A. succinogenes. The plasmid comprises at least one marker gene, operably linked to a first promoter functional in Actinobacillus succinogenes, an origin of replication functional in Actinobacillus succinogenes, a second promoter isolated from Actinobacillus succinogenes, and a cloning site downstream from the second promoter. Plasmids pLGZ901, pLGZ920, pLGZ921, and pLGZ922 are disclosed. The pckA gene polypeptide sequence and nucleic acid sequence of Actinobacillus succinogenes, including the promoter and ribosome binding site, is disclosed. Furthermore, a method for producing a recombinant Actinobacillus succinogenes is described, including a method of transformation. Additionally, a recombinant Actinobacillus succinogenes is disclosed and a method for producing succinate utilizing this recombinant Actinobacillus succinogenes is described.

## Background
This application is a divisional of copending application(s) application Ser. No. 10/911,961 filed on Aug. 5, 2004
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to Provisional application Ser. No. 60/492,804, filed Aug. 6, 2003.


STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
This research was supported by grants from the USDA (#00-34189-9045) and from the National Science Foundation NSF (#BES-0224596). The U.S. government has certain rights to this invention.


BACKGROUND OF THE INVENTION
(1) Field of the Invention
The present invention relates generally to plasmid vectors, and more particularly to Actinobacillus succinogenes plasmid vectors. The present invention further relates to overexpression of proteins in A. succinbgenes and to engineered A. succinogenes strains which have the plasmids introduced into them.
(2) Description of the Related Art
Succinate has many industrial fine chemical uses. Succinate can also be used as an intermediary commodity chemical feedstock for producing bulk chemicals. Its greatest market potential might lie in its use as feedstock to produce stronger-than-steel plastics, biodegradable chelators, and green solvents. Most of the 17,000 tons (15,400 metric tons) of succinate sold per year are produced petrochemically from maleic acid. Succinate is also produced as a fine chemical by fermentation from glucose. For fermentation to be competitive in producing succinate as a commercial chemical, the overall production cost should be lowered from one (1) dollar per pound ($2.20 per kg) to approximately twenty (20) cents per pound (44 cents per kg).
U.S. Pat. No. 5,143,833 to Datta et al. teaches a method for producing succinic acid by growing a succinate producing Anaerobiospirillum succiniciproducens microorganism under specific conditions.
U.S. Reissued Patent No. RE37,393 to Donnelly et al. teaches a method for isolating succinic acid producing bacteria by increasing the biomass of an organism which lacks the ability to catabolize pyruvate and then growing the biomass in glucose-rich medium under an anaerobic environment to enable pyruvate-catabolizing mutants to grow. By using this method, Donnelly provides a mutant E. coli that produces high amounts of succinic acid. The mutant E. coli was derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase.
U.S. Pat. No. 6,455,284 and U.S. Pat. Application Publication No. 2003/0087381, both to Gokarn et al. teach metabolic engineering to increase the carbon flow toward oxaloacetate to enhance production of bulk biochemicals, such as lysine and succinate, in bacterial and industrial fermentations. The carbon flow is redirected by genetically engineering bacteria to overexpress the enzyme pyruvate carboxylase.
U.S. Pat. Application Publication No. 2003/0113885 to Lee et al. teaches a novel microorganism, Mannheimia sp. 55E, capable of producing organic acids and a process using the microorganism for producing organic acids through anaerobic and aerobic incubations.
U.S. Pat. No. 6,420,151 to Eikmanns, et al. teaches an isolated nucleic acid from coryneform bacteria which encodes a phosphoenolpyruvate carboxykinase which is involved in production of succinate.
While the above methods can be used to produce succinate, Actinobacillus succinogenes is still the best succinate producer known. Actinobacillus succinogenes is a gram-negative capnophilic, anaerobic bacillus that belongs to Pasteurellaceae. A. succinogenes produces up to one hundred (100) grams per liter of succinate in optimized conditions. Much effort has been spent on engineering Escherichia coli strains to produce high succinate amounts, however none of the engineered E. coli in strains surpassed A. succinogenes for succinate production. Carbon flow is stringently regulated in microorganism metabolism, including carbon flux towards oxaloacetate. Overcoming this control of carbon flux will possibly improve the yields of desirable products, such as succinate.
Previously, no genetic tools had been tested or developed that could be used for engineering A. succinogenes into an industrial succinate-producing strain. Therefore, it would be desirable to have a means to genetically engineer A. succinogenes to overproduce succinate. However, there is not yet a means for constructing recombinant A. succinogenes. The present invention provides a means for constructing recombinant A. succinogenes using a plasmid for the expression of proteins in A. succinogenes. 
SUMMARY OF THE INVENTION
The present invention provides a plasmid comprising at least one marker gene operably linked to a first promoter functional in Actinobacillus succinogenes, an origin of replication functional in Actinobacillus succinogenes, a second promoter isolated from Actinobacillus succinogenes, and a cloning site downstream from the second promoter.
In further embodiments, the marker gene confers antibiotic resistance to Actinobacillus succinogenes. In further embodiments, the antibiotic is selected from the group consisting of ampicillin, chloramphenicol, tetracycline, and erythromycin.
In further embodiments, the second promoter is from the pcka gene of Actinobacillus succinogenes. In some embodiments, the second promoter comprises substantially the nucleic acid sequence set forth in SEQ ID NO: 21 from between about nucleotide 25 and nucleotide 255. In further still embodiments, the second promoter provides a ribosome binding site comprising the nucleotide sequence AGGTG. In further still embodiments, the plasmid further comprises a ColE1 origin of replication. In further still embodiments, the cloning site comprises one or more restriction endonuclease cleavage sites.
The present invention also provides a plasmid which is pLGZ901 deposited under the terms of the Budapest Treaty at the ATCC, Manassas, Va. on Dec. 22 2005, with accession number PTA-7297, and a plasmid which is pLGZ920 (ATCC PTA-6140 deposited on Aug. 3, 2004).
The present invention further provides a polypeptide which comprises an amino acid sequence substantially similar to the amino acid sequence set forth in SEQ ID NO. 22. The present invention still further provides a nucleic acid which comprises a nucleotide sequence substantially similar to the nucleotide sequence set forth in SEQ ID NO: 21.
The present invention further provides a method for producing a recombinant Actinobacillus succinogenes comprising providing a plasmid comprised of at least one marker gene operably linked to a first promoter functional in Actinobacillus succinogenes, an origin of replication for the plasmid functional in Actinobacillus succinogenes, a second promoter isolated from Actinobacillus succinogenes, and a cloning site for a nucleic acid downstream of the second promoter, transforming an Actinobacillus succinogenes with the plasmid, and selecting the recombinant Actinobacillus succinogenes from non-transformed Actinobacillus succinogenes. 
In some embodiments of the method, the transformation is electroporation. In some embodiments of the method, the marker gene confers antibiotic resistance to the recombinant Actinobacillus succinogenes. In further embodiments of the method, the antibiotic is ampicillin, tetracycline, or chloramphenicol. In some embodiments of the method, the recombinant Actinobacillus succinogenes is selected from non-transformed Actinobacillus succinogenes by culturing in the presence of the antibiotic. In some embodiments of the method, the second promoter is from the pcka gene of Actinobacillus succinogenes. In some embodiments of the method, the second promoter comprises substantially the nucleic acid sequence set forth in SEQ ID NO: 21 from between about nucleotide 25 and nucleotide 255. In some embodiments of the method, the second promoter provides a ribosome binding site comprising the nucleotide sequence AGGTG. In some embodiments of the method, the plasmid further includes a ColE1 origin of replication. In some embodiments of the method, the plasmid is pLGZ901 or pLGZ920.
The present invention further provides a recombinant Actinobacillus succinogenes comprising a plasmid capable of autonomous replication in Actinobacillus succinogenes which comprises at least one selectable marker gene and the Actinobacillus succinogenes pckA gene. In some embodiments of the recombinant Actinobacillus succinogenes, the pckA gene comprises the nucleic acid sequence set forth in SEQ ID NO: 21. In some embodiments of the recombinant Actinobacillus succinogenes, the plasmid is pLGZ902. In some embodiments of the recombinant Actinobacillus succinogenes, the marker gene confers ampicillin, tetracycline, or chloramphenicol resistance to the recombinant Actinobacillus succinogenes. 
The present invention further provides a method for producing succinate comprising providing a recombinant Actinobacillus succinogenes comprising a plasmid capable of autonomous replication in Actinobacillus succinogenes which comprises at least one selectable marker gene and a recombinant gene expressed under the control of the Actinobacillus succinogenes pcka promoter, providing a growth medium for culturing the recombinant Actinobacillus succinogenes, and culturing the recombinant Actinobacillus succinogenes in the growth medium to produce the succinate.
In some embodiments of the method the promoter comprises substantially the nucleic acid sequence set forth in SEQ ID NO: 21 from between about nucleotide 25 and nucleotide 255. In some embodiments of the method the plasmid is pLGZ901, and in further embodiments the plasmid is pLGZ920. In some embodiments of the method the marker gene confers ampicillin, tetracycline, or chloramphenicol resistance to the recombinant Actinobacillus succinogenes. In some embodiments of the method the growth medium comprises ampicillin, tetracycline, or chloramphenicol.
In further embodiments of the method the recombinant Actinobacillus succinogenes is a mutant strain in which an Actinobacillus succinogenes gene which inhibits succinate production comprises a deletion. In further embodiments of the method the Actinobacillus succinogenes gene further comprises a selectable marker under the control of the Actinobacillus succinogenes pckA promoter. In further embodiments of the method the selectable marker gene confers resistance to an antibiotic selected from the group consisting of chloramphenicol and tetracycline to the mutant Actinobacillus succinogenes strain.
The present invention further provides a recombinant microorganism comprising the nucleic acid which comprises a nucleotide sequence substantially similar to the nucleotide sequence set forth in SEQ ID NO: 21.
The present invention further provides a plasmid which is pLGZ921, and a plasmid which is pLGZ922, each deposited under the terms of the Budapest Treaty at the ATCC, Manassas, Va. on Dec. 22, 2005, with accession numbers PTA-7296 and PTA-7298, respectively.
OBJECTS
Therefore, it is an object of the present invention to provide a method for producing recombinant Actinobacillus succinogenes. 
It is a further object of the present invention to provide a plasmid which replicates in A. succinogenes. 
It is further still an object of the present invention to provide a recombinant A. succinogenes which overexpresses proteins.
These and other objects will become increasingly apparent by reference to the following description and the drawings.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 illustrates the nucleotide sequence SEQ ID NO: 21 of A. succinogenes pckA and deduced amino acid sequence SEQ ID NO: 22 of PEPCK. Two pairs of putative −35 and −10 promoter regions are underlined. The putative ribosome binding site (RBS) is in bold. The stop codon is indicated by three asterisks. The GenBank accession number for the nucleotide sequence is AY308832.
FIG. 2 illustrates β-Galactosidase activity in A. succinogenes 130Z grown in medium A.
FIG. 3 illustrates β-Galactosidase activity in A. succinogenes 130Z grown in TS broth.
FIG. 4 illustrates the stability of plasmids pLGZ901 and pLGZ920 in A. succinogenes and E. coli. 
FIG. 5 illustrates the physical map of the pGZRS-1 plasmid. The dashed line represents the minimum region required for replication in A. pleuropneumoniae. 
FIG. 6 illustrates the physical map of the pLGZ901 plasmid. The origin of replication functional in A. succinogenes, the cloning site (MCS), the A. succinogenes promoter, and the bla-Tn3 marker gene are shown.
FIG. 7 illustrates the physical map of the E. coli-A. succinogenes shuttle vector pLGZ920. The ColE1 origin of replication, the origin of replication functional in A. succinogenes, the cloning site (MCS), the A. succinogenes promoter, and the bla-Tn3 marker gene are shown.
FIG. 8 illustrates the physical map of the pLGZ920 plasmid with an inserted pckA gene. The ColE1 origin of replication, the origin of replication functional in A. succinogenes, the cloning site (MCS), the A. succinogenes promoter, the pckA gene, and the bla-Tn3 marker gene are shown.
FIG. 9 illustrates the time for plasmid-harboring A. succinogenes to form colonies on TS agar at 370 Celsius in the presence of tetracycline or chloramphenicol.



DETAILED DESCRIPTION OF THE INVENTION
All patents, patent applications, government publications, government regulations, and literature references cited in this specification are hereby incorporated herein by reference in their entirety. In case of conflict, the present description, including definitions, will control. The plasmids pLGZ901, pLGZ920, pLGZ921, and pLGZ922 are available upon request from Michigan State University.
Definitions for the following terms are provided to promote a further understanding of the present invention.
The term “marker” refers to sequences which encode a gene product, usually an enzyme, that inactivates or otherwise detects or is detected by a compound in the growth medium. For example, the inclusion of a marker sequence can render the transformed cell resistant to an antibiotic, or it can confer compound-specific metabolism on the transformed cell. Marker genes can confer resistance to antibiotics including, but not limited to, ampicillin, chloramphenicol, tetracycline, and erythromycin.
The term “pLGZ901” refers to the plasmid construct as represented by FIG. 6. The plasmid is available upon request from Michigan State University.
The term “pLGZ920” refers to the plasmid construct as represented by FIG. 7. The plasmid is available upon request from Michigan State University, and has been deposited under the terms of the Budapest Treaty at the ATCC, Manassas, Va. on Aug. 3, 2004, with accession number PTA-6140.
The term “promoter” refers to a DNA fragment to which ribonucleic acid polymerase binds to initiate the transcription of nucleic acid sequences linked to the promoter.
A promoter is “operably linked” to a nucleic acid sequence if it can be used to control or regulate transcription of the nucleic acid sequence.
The term “origin of replication” refers to a nucleic acid sequence which is necessary to allow replication of a plasmid within an organism.
The term “cloning site” refers to a region which allows for the insertion of desired nucleic acid sequences. Typically, the cloning site comprises one or more restriction endonuclease recognition sites. Cloning sites include, but are not limited to, multiple cloning sites or polylinkers.
The term “ribosome binding site” refers to a short nucleotide sequence to which the ribosome binds upon the transcribed ribonucleic acid.
The term “transformation” means the process of introducing DNA into an organism which changes the genotype of the recipient organism.
The term “PEPCK” refers to phosphoenolpyruvate carboxykinase.
The term “vector” refers to a deoxyribonucleic acid which is capable of replication and also capable of incorporating desired deoxyribonucleic acid fragments for cloning. Vectors include plasmids, cosmids, phages, and yeast artificial chromosomes.
The term “shuttle vector” refers to a vector able to replicate in two different organisms.
A required step toward A. succinogenes metabolic engineering is developing genetic tools. It is necessary to be able to increase the expression level of certain enzymes, probably by introducing them on stable, multicopy plasmids, and to shut down some pathways by generalized or targeted mutagenesis. One method for producing succinate can comprise a mutant strain of A. succinogenes in which a selected gene which minimizes succinate production contains a deletion. Another method for producing succinate can comprise both increasing expression levels of some enzymes by introducing them on plasmids, and shutting down a pathway which minimizes succinate production in which a selected gene contains a deletion. The tools needed to achieve this include (i) selection markers, (ii) a transformation and/or conjugation system(s), (iii) a complementation system, and (iv) targeted and generalized mutagenesis systems.
A plasmid was developed suitable for use in A. succinogenes to express proteins. First, a replicon that stably replicates in A. succinogenes was found. A. succinogenes can be transformed by electroporation at reasonably high efficiency by pGZRS-19, pGZRS-19 replicates in a stable manner in A. succinogenes, and the ampicillin resistance gene carried by pGZRS-19 is expressed in A. succinogenes. These properties made pGZRS-19 an excellent starting point to develop an E. coli-A. succinogenes shuttle vector to be used for expressing foreign proteins in A. succinogenes. The plasmid pGZRS-19 was constructed from pGZRS-1, illustrated in FIG. 5, which is a member of the H2 class of Apl plasmids. The pGZRS-1 plasmid is an endogenous Actinobacillus pleuropneumoniae (Apl) 4.3 kilobase plasmid found in the serotype 7 strain EL312. (West, S. E., et al., Gene 160: 81–86(1995)). The plasmid pGZRS-19 carries the multiple cloning site from pUC19, and the bla gene from Tn3 under the control of a putative Apl promoter. The plasmid pGZRS-19 replicates in Apl, Escherichia coli, Pasteurella haemolytica, and Haemophilus (Actinobacillus) actinomycetemcomitans. West et al. demonstrated that there is a minimal region required for replication in both Apl and E. coli, which is within the 1.5 kB PstI-HaeII fragment of pGZRS-1. We demonstrated that A. succinogenes is transformed by electroporation at reasonably high efficiency, that pGZRS-19, with this fragment, replicates in a stable manner in A. succinogenes, and that the ampicillin resistance gene carried by pGZRS-19 is expressed in A. succinogenes. 
Three steps were required to develop this new vector. (i) A gene that we knew was constitutively expressed at high levels in A. succinogenes (i.e., the pckA gene) was cloned and sequenced. (ii) Its promoter region and ribosome binding site were subcloned into pGZRS-19. A unique XbaI site was included immediately downstream of the pckA ribosome binding site to facilitate the cloning of foreign genes under control of the pckA promoter. (iii) Finally, the ColE1 origin of replication was added to the vector to increase its stability in E. coli. High levels of β-galactosidase and PEPCK activities detected in cultures of recombinant A. succinogenes strains confirmed that both A. succinogenes and foreign proteins could be expressed in A. succinogenes under control of the A. succinogenes pckA promoter carried by our pGZRS-19-derived vector, pLGZ920.
EXAMPLE 1
Bacterial strains and plasmids used in this study are listed in Table 1.









TABLE 1





Plasmid
Relevant characteristics
References







pCR TM II
TOPO TA cloning vector
Invitrogen,




Carlsbad, CA


pCR2.1
TOPO TA cloning vector
Invitrogen


pUC19
AmpR, lacZα, ColE1 multicopy cloning vector
Invitrogen


pProEx-1
AmpR, ColE1 multicopy cloning vector
[Bolivar, 1978




#3323]


pBR325
AmpR, CmR, TetR, ColE1 cloning vector
Laboratory




collection


pGZRS-19
pGZRS-1 replicon, AmpR (Tn3), replicates in Aple,
(30)



Phae, Aact, Replicates in E. coli, pUC18/19 multiple



cloning site, lacZα-complementation


pGZRS-30
pGZRS-1 replicon, KmR (Tn903), CmR (Tn9)
(30)


UB214
Mannheimia haemolytica pMHT1 replicon, TetR
(17)


pLGZ901
pGZRS-19 derivative, A. succinogenes pckA promoter
This study


pLGZ902
pGZRS-19 derivative, A. succinogenes pckA gene
This study


pLGZ903
pGZRS-19 derivative, E. coli lacZα under control of
This study



the A. succinogenes pckA promoter


pLGZ920
pGZRS-19 derivative, A. succinogenes pckA promoter,
This study



pGZRS-1 and ColE1 replicons


pLGZ921
pLGZ920 derivative, pBR325 CmR under control of
This study



the A. succinogenes pckA promoter


pLGZ922
pLGZ920 derivative, pBR325 TetR under control of
This study



the A. succinogenes pckA promoter


pGZRS-19/UB214
Fusion of pLGZ901 and UB214 plasmids
This study


UB214-AmpR
UB214 derivative containing AmpR from pGZRS-19
This study









E. coli DH5α and JM110 were used for plasmid construction. E. coli strains were grown in LB medium (Sambrook, J., et al., Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)) supplemented with antibiotics (at 50 μg/ml) when necessary. A. succinogenes 130Z (ATCC 55618) and its derivative FZ6 were obtained as gifts from MB1 International (Lansing, Mich.). Strain FZ6 is a succinate-overproducing mutant that was selected by resistance to fluoroacetate (Guettler, M. V., et al., U.S. Pat. No. 5,573,931 (1996)). FZ6 produces only pyruvate and succinate as fermentation products from glucose. FZ6 lacks pyruvate-formate lyase activity (Park, D. H., et al., J. Bacteriol. 181: 2403–2410 (1999)). A. succinogenes strains were grown in Trypticase-Soy (TS) broth (Becton-Dickinson, Cockeyville, Md.) or medium A (per liter: 9 g glucose, 10 g sodium bicarbonate, 5 g yeast extract, 8.5 g NaH2PO4.H2O, and 15.5 g K2HPO4). The pH was adjusted to 7.5 with NaOH before autoclaving, leading to a pH of 7.0 after autoclaving. The glucose was added aseptically after sterilization. A. succinogenes cultures were grown in butyl-rubber-stopper 18-ml anaerobic tubes and 158-ml serum vials containing 10 ml and 100 ml medium, respectively. When grown on plates, A. succinogenes strains were grown in an anaerobic jar under CO2 atmosphere.
EXAMPLE 2
Antibiotic minimum inhibitory concentrations. To determine the sensitivity of A. succinogenes 130Z to antibiotics, the growth of A. succinogenes 130Z was tested in medium A (liquid medium) containing a series of antibiotics commonly used in bacteriology. Antibiotics and antibiotic concentrations used are listed in Table 2. One ml of 130Z preculture was inoculated into 9 ml of medium A containing each antibiotic and incubated at 37° C. for 7 days. Cell growth was followed by measuring OD660.







TABLE 2







Antibiotics and antibiotic concentrations used in


this study.













Concentration used




Concentrations
in plates after




tested in liquid
transformation



Antibiotic
cultures (μg/ml)
(μg/ml)















Ampicillin
5, 10, 25, 50
25



Chloramphenicol
1, 2.5, 5, 10
2.5



Tetracyclin
5, 10, 20, 30
10



Erythromycin
5, 10, 20
10



Streptomycin
30
Not used



Kanamycin
50
Not used










Resistance of A. succinogenes to antibiotics. Growth of A. succinogenes 130Z was tested in the presence of antibiotics at the concentrations listed in Table 2. A. succinogenes 130Z grew perfectly well in the presence of 50 μg/ml kanamycin or 30 μg/ml streptomycin. Very slow growth was observed in the presence of 5 μg/ml ampicillin or 5 μg/ml erythromycin (OD660 of approximately 0.05 after 7 days culture). No growth was detected in the presence of 10 μg/ml ampicillin, 1 μg/ml chloramphenicol, 5 μg/ml tetracycline, or 10 μg/ml erythromycin. A. succinogenes is sensitive to low concentrations of ampicillin, chloramphenicol, erythromycin, and tetracycline, so the corresponding resistance genes (i.e., AmpR, CmR, TetR, and EryR) could be used as selection markers on plasmids used to transform A. succinogenes. We discovered that the Tn3 AmpR gene carried by pGZRS-19 is naturally expressed in A. succinogenes. 
EXAMPLE 3
A. succinogenes 130Z (ATCC 55618) was used as the host strain. One hundred ml of actively growing cells in TS broth (OD660 of 0.3) were chilled on ice and harvested by centrifugation at 4,000 xg for 10 minutes at 4° C. Cells were washed twice with 5 ml of ice-cold 15% glycerol and resuspended in 0.2 ml of ice-cold 15% glycerol. Fifty microliters (4) of this cell suspension was mixed with 0.5-1 microgram (μg) plasmid in a chilled, 2.0 mm Gene pulser cuvette (BioRad, Richmond, Calif.). Electroporation was performed at 2.5 kV, 200 Ω, and 25 μF. One milliliter (ml) of TS broth was immediately added to the electroporated cells. The suspension was incubated for 1 hour (hr) at 37° C. and then spread (under air) on TS agar plates containing ampicillin, chloramphenicol, or tetracycline (concentrations listed in Table 2). Plates were incubated in an anaerobic jar under CO2 atmosphere at 37° C. for 24–48 hr. A. pleuropneumoniae-E. coil shuttle vectors, pGZRS-19 and pGZRS-30 were provided by Dr. Susan West (Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wiss. 53706) Shuttle vector UB214 was provided by Dr. Stefan Schwarz (für Tierzucht and Tierverhalten der Bundesforschungsantalt für Landwirtschaft Braunschweig (FAL), Dörnbergstr 25–27, 29223 Celle, Germany).
Introduction of plasmids into bacteria can be done using any means known in the art, including, but not limited to, electroporation, chemical transformation, conjugation, liposome mediated gene transfer, and particle bombardment. Actinobacillaceae are naturally non-transformable. Lalonde et al. (Am. J. Vet. Res. 50: 1957–1960 (1989)) were the first to develop electroporation in A. pleuropneumoniae. Electroporation has since been used extensively to transform various Actinobacillus, Haemophilus, and Pasteurella strains with efficiencies of up to 3.106–107 (Brogan, J. M., et al., Gene 169: 141–142 (1996); Frey, J., Res. Microbiol. 143: 263–269 (1992)). Conditions typically used include a capacitance set at 25 μF, a pulse controller at 200–1000 Ω, and a pulse amplitude of 2.5 to 6.5 kV/cm (Craig, F. F., et al., J. Gen. Microbiol. 135: 2885–2890 (1989); Dixon, L. G., et al., Plasmid 32: 228–232 (1994); Frey, J., Res. Microbiol. 143: 263–269: (1992); Lalonde, G., et al., Am. J. Vet. Res. 50: 1957–1960 (1989); Oswald, W., et al., FEMS Microbiol. Lett. 179: 153–160 (1999); West, S. E., et al., Gene 160: 81–86 (1995); Wright, C. L., Plasmid 37: 65–79 (1997)). The electroporation conditions (i.e., 2.5 kV, 200 Ω, and 25 μF) that are standard for E. coli, and that are among the typical conditions used for Actinobacillaceae. 
Because the organization of Actinobacillaceae promoters is different from that of E. coli promoters (Doree, S. M., et al. J. Bacteriol. 183: 1983–1989 (2001)), most antibiotic resistance genes that have been successfully used in Pasteurellaceae shuttle vectors originate from transposons or from Actinobacillus indigenous plasmids. We first tested ampicillin, chloramphenicol, and tetracyclin resistance (AmpR, CmR, and TetR, respectively) genes that are known to be expressed in Pasteurellaceae species (Craig, F. F., et al., J. Gen. Microbiol. 135: 2885–2890 (1989); Kehrenberg, C., et al., Antimicrob. Agents Chemother. 42: 2116–2118 (1998); West, S. E., et al., Gene 160: 87–88 (1995); West, W. E., et al., Gene 160: 81–86 (1995); Wright, C. L., et al., Plasmid 37: 65–79(1997)).
A number of plasmids have been isolated from Pasteurellaceae or have been constructed as vectors for Pasteurellaceae species (Craig, F. F., et al., J. Gen. Microbiol. 135: 2885–2890 (1989); Dixon, L. G., et al., Plasmid 32: 228–232 (1994); Frey, J., Res. Microbiol. 143: 263–269 (1992); Galli, D. M., et al., Plasmid 36: 42–48 (1996); Ishii, H., et al., Nippon Juigaku Zasshi 52: 1–9 (1990); Kehrenberg, C., et al., Antimicrob. Agents Chemother. 42: 2116–2118 (1998); Lalonde, G., et al., Gene 85: 243–246 (1989); Nakano, Y., et al., Gene 169: 139–140 (1996); West, S. E., et al., Gene 160: 87–88 (1995); West, S. E., et al., ene 160: 81–86 (1995); Wright, C. L., et al., Plasmid 37: 65–79 (1997)). We tested three replicons for their ability to replicate in A. succinogenes. 
Efficiency of electroporation of A. succinogenes with plasmids pGZRS-19, pGZRS-30, and UB214 was used to determine the ability of these plasmids to replicate and to express their antibiotic resistance genes in A. succinogenes. Electroporation of A. succinogenes with pGZRS-19 gave an average of 5.4×104 CFU/μg plasmid (Table 4). No significant difference was observed in the transformation yields obtained using pGZRS-19 DNA purified from E. coli and A. succinogenes strains (Table 4), suggesting that A. succinogenes does not have a restriction system inhibiting transformation.







TABLE 4







Efficiency of electroporation of A. succinogenes and E. coli with pGZRS-19 and


its derivatives









CFU/μg plasmid











PGZRS-19
pLGZ901
pLGZ903













Host
aEco
bAcs
Eco
Acs
Eco
Acs





A. succinogenes








130Z
6.2 × 104
5.6 × 104
5.1 × 104
7.2 × 104
2.4 × 104
4.2 × 104


FZ6
5.9 × 104
3.9 × 104
3.5 × 104
3.6 × 104
2.0 × 104
2.5 × 104


E. coli


DH5α
5.6 × 106
6.3 × 106
7.2 × 106
7.3 × 106
4.7 × 106
5.4 × 106


JM110
1.8 × 106
4.2 × 106
2.6 × 106
1.8 × 106
1.7 × 106
1.8 × 106





aEco: The plasmid DNA used for transformation was purified from E. coli;


bAcs: The plasmid DNA used for transformation was purified from A. succinogenes.






A. succinogenes transformants containing pGZRS-19 did not show any colonies after 48 hours on plates containing 100 μg/ml ampicillin, so ampicillin at 25 μg/ml was used in the selection medium. No colonies were obtained after transformation of A. succinogenes with pGZRS-30, using the antibiotic concentrations listed in Table 2. While the A. pleuropneumonlae-E. coil shuttle vector pGZRS-19 was stably maintained and its Tn3 AmpR gene was expressed in A. succinogenes, attempts at identifying other replicons that are maintained and other antibiotic resistance genes that are expressed in A. succinogenes were not. The M. haemoiytica pMHT1 replicon (tested using the UB214-AmpR construct) was unable to replicate in A. succlnogenes. In contrast to the Tn3 AmpR gene, the Tn9 CmR gene carried by pGZRS30 and the TetR gene carried by UB214 (tested in the pGZRS-19/UB214 construct) were not expressed from their own promoters in A. succlnogenes. Once expressed under a functional promoter (i.e. the pckA promoter), though, we determined that CmR and TetR were functional and that they could be used as selection markers in A. succlnogenes. These selection markers can be used to label knock-out mutations.
Because transformation of A. succinogenes with pGZRS-19 was successful, our lack of success with pGZRS-30 most probably comes from the inability of the CmR gene of Tn9 to be expressed in A. succinogenes. In the case of plasmid UB214, the absence of transformants could be due either to the inability of the plasmid to replicate in A. succinogenes, or to the inability of its antibiotic resistance gene to be expressed in A. succinogenes. To determine whether UB214 (i.e., the M. haemolytica pMHT1 replicon) can replicate in A succinogenes, we constructed the UB214-AmpR derivative. In this plasmid, the 1.4 kb EcoRI fragment carrying the AmpR gene of pGZRS-19 was subcloned into the unique EcoRI site of UB214. UB214AmpR conferred ampicillin resistance to E. coli, but A. succinogenes transformed with UB214-AmpR did not show any colonies after 48 hours on ampicillin (25 μg/ml) plates. To determine whether the TetR gene carried by UB214 is expressed in A. succinogenes, the whole UB214 plasmid was cloned into the unique BamHI site of pGZRS-19. The pGZRS-19/UB214 construct conferred both ampicillin and tetracycline resistance to E. coli. A. succinogenes transformed with pGZRS-19/UB214 showed many colonies after 48 hours on ampicillin (25 μg/ml) plates, but none on tetracycline (5 μg/ml) plates. These results indicate that not only is the M. haemolytica pMHT1 replicon unable to replicate in A. succinogenes, but its TetR gene is also not expressed from its own promoter in A. succinogenes. 
EXAMPLE 4
Plasmid DNA purification, PCR amplification, restriction analysis, subcloning, transformation, and bacterial culture utilized methods generally described in Sambrook, J., Fritsh, E. F., and Maniatis, T., Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring Harbor Laboratory Press, N.Y. (1989); and Ausubel, F. M., et al., Current Protocols in Molecular Biology Volumes 1–3, John Wiley & Sons, Inc., N.Y. (1993–1998). Oligonucleotides used for PCR reactions and for sequencing were synthesized by the Michigan State University Macromolecular Structure, Sequencing, and Synthesis Facility. DNA sequencing was performed by the Michigan State University Genomics Technology Support Facility. DNA was recovered from agarose gels with the Geneclean II kit (BIO 101, La Jolla, Calif.).
The A. succinogenes pckA gene was cloned in 4 steps. In step (i), an alignment of E. coli, Anaerobiospirillum succiniciproducens, Haemophilus influenzae, Rhizobium sp. NGR234, and Vibrio cholerae PEPCks sequences (Genbank accession Nos. F65135, U95960, NP—438969, S18606, and Q9KNK0, respectively) indicated that sequences YGGEMKK, SEQ ID NO: 23, and NTGWNG, SEQ ID NO: 24, were highly conserved. Degenerate oligonucleotides SEQ ID NO: 1 and SEQ ID NO: 2 (Table 3) were used as primers to amplify a pckA internal fragment using A. succinogenes 130Z chromosomal DNA as the template. The 0.7 kb PCR product was cloned into pCRTMII (Invitrogen, Carlsbad, Calif.) and sequenced. For step (ii), A. succinogenes PEPCK was purified to homogeneity as described (Shen, T. L., et al., J. Mass Spectrom. 34: 1154–1165 (1999)) and submitted to N-terminal sequencing (performed by the Michigan State University Macromolecular Structure, Sequencing, and Synthesis Facility). Oligonucleotides SEQ ID NO: 3 (encoding the N-terminal sequence TDLNKLV, SEQ ID NO: 25) and SEQ ID NO: 4 (in the center of the pcka gene) (Table 3) were used as primers to amplify the 5′-end of pckA using A. succinogenes 130Z chromosomal DNA as the template. The 1.0 kb PCR product was cloned into pCRTMII and sequenced. In step (iii), pckA's 3′-end was amplified by an extender PCR approach (Brown, A. J. H., et al., Biotechniques 26: 804–806 (1999)) as follows: A. succinogenes 130Z chromosomal DNA was digested by EcoRI, EcoRI extremities were annealed to oligonucleotides SEQ ID NO: 5 and SEQ ID NO: 6 (Table 3), 3′-ends were blocked with ddATP, and two successive amplifications were performed, first with primers SEQ ID NO: 7 and SEQ ID NO: 8, then with primers SEQ ID NO: 9 and SEQ ID NO: 10 (Table 3). The final, 2.3-kb PCR product was cloned into pCRTMII and sequenced. In step (iv), pckA's promoter region was amplified by extender PCR. The EcoRI-digested, annealed, and blocked A. succinogenes 130Z chromosomal DNA from step (iii) was used as the template in two successive amplifications, first with primers SEQ ID NO: 8 and SEQ ID NO: 11, then with primers SEQ ID NO: 10 and SEQ ID NO: 12 (Table 3). The final, 2.0-kb PCR product was cloned into pCRTMII and sequenced.










TABLE 3





SEQ ID





NO:
Primer sequence (5′ to 3′)a
Properties


















1
GGTAYGGNGGNGARATGAARAARG
pckA non-coding strand, encodes YGGEMKK



 


2
CCRTTCCANCCNGTRTT
pckA non-coding strand, encodes NTGWNG


 


3
ATGCANGAYYTNAYAARYT
pckA non-coding strand, encodes the N-terminal




sequence TDLNKLV


 


4
TCCGCGGTTAAGAAAATCACTTT
pckA coding strand, encodes KVIFLT


 


5
AATTTGCATGTC
Extender PCR EcoR1 linker


 


6
TGCGAGTAAGGATCCTCACGCAAGGA
Extender PCR EcoR1 adaptor



ATTCCGACCAGACATGCA


 


7
CACATCGACAACATCGTTCGTC
pckA non-coding strand, encodes HIDNIVR


 


8
TGCGAGTAAGGATCCTGACGCA
Extender PCR primer P1


 


9
GTATTGCCGCCGGTTTCAAAACTG
pckA non-coding strand, encodes LPPVSKL


 


10
CGCAAGGAATTCCGACCAGACA
Extender PCR primer P2


 


11
CGACCGGTAAAAATCCCCGTATCG
pckA coding strand, encodes DTGIFTGR


 


12
CCAAACCCGGTTTGGTTTCTTCC
pckA coding strand, encodes LGLTDVKE


 


13
CTTTAATCGGAAGCTTTCGATAAATTG
Non-coding strand, upstream of pckA, sequence



AAAATGCAG
AAGCTT creates a HindIII site


 


14
GTCAGTTCTAGATCACCTCATTGATAAT
Coding strand, upstream of pckA, sequence TCTAGA



TTAAAATTAAA
creates an XbaI site


 


15
CAATGAGGTCTAGAATGACTGACTTAAAC
pckA non-coding strand, sequence TCTAGA creates an



AAACTCG
XbaI site upstream of the ATG start codon


 


16
CAAAAGCCCGGGTGGAAATACTCAGCCTT
Coding strand, downstream of pckA, sequence CCCGGG



ATTTTTC
creates an XmaI site


 


17
TTTCTAGAATGCTGGCCGTCGTTTTACAAC
In pUC19, sequence TCTAGA creates an XbaI site



GTCGTGACTACTGG
upstream of laZα's ATG start codon


 


18
CCACATTTACCCGGGACCCAAA AAAGAC
In pUC19, sequence CCCGGG creates an XmaI site



TTAC
downstream of laZα


 


19
AAGCTTTCTGCTAATCCTGTTACCAGTGGG
In pPROEX-1, sequence AAGCTT creates a HindIII site


 


20
AAGCTTCCGCATCAGGCGCTCTTCGCGTTC
In pPROEX-1, sequence AAGCTT creates a HindIII site


 


31
TCTAGAATGAAATCTAACAATGCGCTC
In pBR325, creates an XbaI site upstream of TetR's




start codon


 


32
GAGCTCTCAGGT CGAGGTGGCCCGG
In pBR325, creates a SacI site downstream of TetR


 


33
TCTAGAATGGAGAAAAAAATCACTGG
In pBR325, creates an XbaI site upstream of CmR's




start codon


 


34
GAGCTCTTACGCCCCGCCCTGCCA C
In pBR325, creates a SacI site downstream of CmR





aWhere N is A, C, G, or T; R is A or G; and Y is T or C.






The pckA promoter region was amplified using oligonucleotides SEQ ID NO: 13 and SEQ ID NO: 14 (Table 3) as primers and A. succinogenes chromosomal DNA as the template. The 230 bp PCR product was cloned into the HindIII and XbaI sites of pGZRS19 to yield plasmid pLGZ901 (4.8 kb). The pckA gene was amplified using oligonucleotides SEQ ID NO: 15 and SEQ ID NO: 16 (Table 3) as primers and A. succinogenes chromosomal DNA as the template. The 1.6 kb PCR product was cloned into the XbaI and XmaI sites of pLGZ901 to yield plasmid pLGZ902 (6.4 kb). The plasmid pLGZ902 has been deposited under the terms of the Budapest Treaty at the ATCC, Manassas, Va. on May 19, 2005 with accession number PTA-6720. The lacZα fragment was amplified using oligonucleotides SEQ ID NO: 17 and SEQ ID NO: 18 (Table 3) as primers and pUC19 as the template. The 0.9 kb PCR product was cloned into the XbaI and XmaI sites of pLGZ901 to yield plasmid pLGZ903 (5.7 kb). The ColEl origin of replication was amplified using oligonucleotides SEQ ID NO: 19 and SEQ ID NO: 20 (Table 3) as primers and pProEx-1 (Invitrogen, Carlsbad, Calif.) as the template. The PCR product (0.55 kb) was cloned into pCRTMII. After sequence verification, the HindIII PCR fragment was subcloned into the HindIII site of pLGZ901, yielding plasmid pLGZ920 (5.4 kb). The Tn10 tetracycline resistance gene (TetR) was amplified using oligonucleotides SEQ ID NO: 31 and SEQ ID NO: 32 as primers and plasmid pBR325 as the template. The 1.2 kb PCR fragment was cloned into pCR2.1. After sequence verification, the PCR fragment was subcloned between the XbaI and SacI sites of pLZG920, yielding plasmid pLGZ921. The Tn9 chloramphenicol resistance gene (CmR) was amplified using oligonucleotides SEQ ID NO: 33 and SEQ ID NO: 34 as primers and plasmid pBR325 as the template. The 0.65 kb PCR fragment was cloned into pCR2.1. After sequence verification, the PCR fragment was subcloned between the XbaI and SacI sites of pLZG920, yielding plasmid pLGZ922.
The pckA gene is constitutively expressed at high levels in A. succinogenes (van der Werf, M. J., et al., Arch. Microbiol. 167: 332–342 (1997)), and PEPCK is a key enzyme in succinate production by A. succinogenes. For these reasons, and because we needed a strong A. succinogenes promoter that could be used in an expression vector, we decided to clone A. succinogenes pckA. The complete sequence of A. succinogenes pckA and its promoter region is shown in FIG. 1. The 2 kb DNA fragment contained a single, 1, 623 bp open reading frame, encoding a 538-residue protein. In BLAST search, this protein showed 85% and 74% identity (91% and 84% similarity) to the H. influenzae and E. coli PEPCK sequences, respectively (not shown), confirming that we indeed cloned the A. succinogenes pckA gene. As expected, the consensus sequences involved in ATP, Mg2+, phosphoenolpyruvate, and oxaloacetate binding that are conserved in all ATP/ADP-dependent PEPCKs (Laivenieks, M., et al., Appl. Environ. Microbiol. 63: 2273–2280 (1997)) are present in A. succinogenes PEPCK (not shown). A few nucleotides upstream of the ATG start codon is the sequence AGGTG that could act as pckA's ribosome binding site. Two pairs of sequences located 83 nt and 24 nt upstream of pckA's start codon, respectively, match the A. pleuropneumoniae consensus promoter (Doree, S. M., et al., J. Bacteriol. 183: 1983–1989 (2001)) sequences TTRAA (−35) and TATAAT (−10) (FIG. 1). The distances separating the −35 and −10 sequences in these two potential promoter regions (i.e., 15 and 18 nucleotides) are in agreement with the short spacing identified between the −35 and −10 elements of A. pleuropneumoniae promoters (Doree, S. M., et al., J. Bacteriol. 183: 1983–1989 (2001)).
EXAMPLE 5
A. succinogenes 13OZ and E. coil DH5α cells harboring pLGZ901 and pLGZ920 were inoculated in TS (A. succinogenes) and LB (E. coil) media without ampicillin and incubated at 37° C. Culture samples were removed at one-hour intervals for numeration. Total cell number was counted on TS-agar (A. succinogenes) and LB-agar (E. coil), and plasmid-containing cells were counted on TS-agar-ampicillin (25 μg/ml, A. succinogenes) and LB-agar-ampicillin (50 μg/ml, E. coil).
The stability of pLGZ901 was tested in A. succinogenes 130Z and in E. coli DH5α. As shown in FIG. 4, pGLZ901 is much less stable in E. coli than in A. succinogenes. After five generations of growth in the absence of antibiotic, 70% of the A. succinogenes cells still contained the plasmid, whereas fewer than 20% of the E. coli cells did. To increase the stability of our vector in E. coli, we subcloned the ColE1 origin of replication into pLGZ901's unique HindIII site, yielding plasmid pLGZ920. The stability of pLGZ920 was tested in A. succinogenes 130Z and in E. coli DH5α (FIG. 4). As expected (ColE1 plasmids are typically not stable in Actinobacillaceae), the presence of the ColE1 origin of replication in pLGZ920 did not affect the stability of the plasmid in A. succinogenes. In contrast, pLGZ920 was significantly more stable than pLGZ901 in E. coli: after 8 generations, while only 10% of E. coli cells still contained pLGZ901, 90% still contained pLGZ920.
EXAMPLE 6
β-Galactosidase activity was measured as follows: A. succinogenes recombinant strains were grown either in TS broth or in medium A. Thirty microliter aliquots were harvested during the growth time course. The cells were disrupted by vortexing after adding 0.97 ml Z-buffer (60 mM Na2HPO4.7H2O, 40 mM NaH2PO4.H2O, 10 mM KCl, 1 mM MgSO4.7H2O, and 50 mM β-mercaptoethanol, pH 7.0), 20 μl chloroform, and 20 μl of 0.1% SDS. The β-galactosidase reaction was initiated by adding 0.2 ml of ONPG solution (4 mg/ml) to the cell lysate. After a thirty minute incubation at 37° C., the reaction was stopped by adding 0.5 ml of 1 M sodium carbonate. The activity was estimated from the absorbance at 420 nm.
To test PEPCK activity, A. succinogenes strains were grown in medium A, cells were harvested in the exponential phase, and they were disrupted in a French press as described (van der Werf, M. J., et al., Arch. Microbiol. 167: 332–342 (1997)). The PEPCK activity was measured by following the consumption of NADH in a coupled assay at 37° C. The 1 ml reaction mixture consisted of 100 mM Tris (pH 6.6), 35 mM NaHCO3, 16 mM MgCl2, 0.3 mM NADH, 2 U phosphoglycerate phosphokinase/glyceraldehyde phosphate dehydrogenase (Sigma Diagnostics 366–2), 1 mM DTT, 10 mM ADP, 1.8 mM 3-phosphoglycerate (Sigma Diagnostics 366–1), 5 mM PEP, and the cell extract. The extinction coefficient for NADH was 6.22 cm−1 mM−1 at 340 nm. Protein concentrations were determined using the BioRad Protein Assay kit (Richmond, Calif.) using bovine serum albumin (BSA) as the standard.
Since we could reproducibly introduce plasmid pGZRS-19 into A. succinogenes by electroporation, and since pGZRS-19 is stably maintained in A. succinogenes, we tested it as an expression vector in A. succinogenes. In a first step, we subcloned the pckA promoter region into pGZRS-19, yielding plasmid pLGZ901. Because we did not know which of the two putative promoter regions was the pckA promoter, we subcloned a fragment that encompassed both putative promoters (FIG. 1). This fragment also contained the pckA putative ribosome-binding site. The A. succinogenes pckA and E. coli lacZα open reading frames were then cloned downstream of the pckA promoter and ribosome binding site in pLGZ901, yielding plasmids pLGZ902 and pLGZ903, respectively. Electroporation of A. succinogenes with pLGZ901 and pLGZ903 gave transformation efficiencies similar to those obtained with pGZRS-19 (Table 4).
β-Galatosidase activity was followed in cultures of A. succinogenes 130Z comprising pLGZ903, grown in medium A and TS broth (FIG. 2). β-Galactosidase activity was up to 150 units per mg-protein after only 2 hours growth. It then leveled off between 200 and 250 units per mg-protein after 8 hours. No difference in activity was observed between the cultures grown in glucose-based and TS media. Similar β-galactosidase activity levels were observed in A. succinogenes FZ6 comprising pLGZ903, grown in the same conditions. These results indicate that the pckA promoter-ribosome binding site cassette allows the expression of a foreign protein at high levels in A. succinogenes. These results were verified by testing the activity of PEPCK in A. succinogenes strains 130Z and FZ6 comprising plasmid pLZG902. As seen in Table 5, 130Z and FZ6 comprising pLZG902 showed twice as much PEPCK activity as the strains devoid of plasmid.







TABLE 5







PEPCK activity in A. succinogenes strains grown in


medium A











PEPCK activity



Strain
(nmole/min · mg protein)







130Z
 732 ± 26



130Z(pLZ902)
1470 ± 34



FZ6
 851 ± 19



FZ6(pLZ902)
1591 ± 27










EXAMPLE 7
Expression of TetR and CmR in A. succinogenes. We need several markers to conveniently engineer A. succinogenes. One can be used to label a gene deletion, while another one can be used to select for the maintenance of a recombinant gene on a plasmid replicon. To determine whether, once expressed, CmR and TetR could be used as selective markers in A. succinogenes, the Tn9 CmR and Tn10 TetR genes were cloned into pLGZ920 under control of the A. succinogenes pckA promoter, yielding plasmids pLGZ921 and pLGZ922, respectively. After electroporation, A. succinogenes 130Z cells harboring pLGZ921 and pLGZ922 were spread on TS-agar (30 g/l TS, 10 g/l glucose) plates containing variable amounts of tetracycline and chloramphenicol (FIG. 9). Strains 130Z(pLGZ921) plated on chloramphenicol plates and 130Z(pLGZ922) plated on tetracycline plates were used as the negative controls. As seen in FIG. 9, tetracycline-sensitive 130Z(pLGZ922) strain grew on plates containing up to 1.0 μg/ml tetracycline, but showed no growth after one week on plates containing higher tetracycline concentrations. In contrast, strain 130Z(pLGZ921) grew on plates containing up to 5.0 μg/ml tetracycline. Chloramphenicol-sensitive 130Z(pLGZ921) strain grew on plates containing up to 0.4 μg/ml tetracycline, but not on plates containing higher chloramphenicol concentrations. Strain 130Z(pLGZ922) grew on plates containing up to 5.0 μg/ml chloramphenicol. These results indicate that, once under control of the A. succinogenes pckA promoter, the Tn10 TetR and Tn9 CmR genes are expressed and functional in A. succinogenes. These results also suggest that these two genes expressed under control of the pckA promoter can be used as selective markers in media containing the corresponding antibiotic at 1.5 to 4.0 μg/ml.
While the present invention is described herein with reference to illustrated embodiments, it should be understood that the invention is not limited hereto. Those having ordinary skill in the art and access to the teachings herein will recognize additional modifications and embodiments within the scope thereof. Therefore, the present invention is limited only by the claims attached herein.

## Claims

1. A method for producing a recombinant Actinobacillus succinogenes comprising:
(a) providing a plasmid comprising at least one marker gene which confers resistance to an antibiotic comprising a Tn3 AmpR gene, or an ampicillin, tetracycline or chloramphenicol resistance gene operably linked to a first promoter comprising one or more pairs of −35 and −10 promoter elements of an Actinobacillus succinogenes pckA gene; an origin of replication comprising at least a 1.5 kB PstI-HaeII fragment of a pGZRS-1 replicon so as to be functional in Actinobacillus succinogenes; a second promoter comprising one or more pairs of −35 and −10 promoter elements of the Actinobacillus succinogenes pckA gene; and a cloning site for a nucleic acid downstream of the second promoter;
(b) transforming Actinobacillus succinogenes with the plasmid; and
(c) selecting in the presence of the antibiotic the recombinant Actinobacillus succinogenes from non-transformed Actinobacillus succinogenes.

2. The method of claim 1, wherein the transformation is electroporation.

3. The method of claim 1 wherein the recombinant Actinobacillus succinogenes is selected from non-transformed Actinobacillus succinogenes by culturing in the presence of the antibiotic.

4. The method of claim 1 wherein the second promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 21 from between about nucleotide 25 and nucleotide 255.

5. The method of claim 1 wherein the second promoter provides a ribosome binding site comprising the nucleotide sequence AGGTG.

6. The method of claim 1 wherein the plasmid further includes a ColE1 origin of replication.

7. The method of claim 1 wherein the plasmid is pLGZ901 or pLGZ920.