1887

Abstract

A simple system has been developed for generating Corynebacterium glutamicum strains containing stable replicative plasmids integrated into the chromosome via homologous recombination. The system is based upon extremely strong incompatibility between two plasmids, which cannot be comaintained even under antibiotic selective pressure. Integration of the resident plasmid that contained the trpD gene of C. glutamicum was achieved by introduction of a second plasmid and subsequent selection for the maintenance of both plasmids. Plasmid integrates positive for both plasmid markers were obtained at a frequency about 10 of the normal transformation frequency with selection for the maintenance of only the second plasmid. Southern analysis revealed that the integration had occurred through a single-crossover homologous recombination between the trpD regions of the host genome and the plasmid. On the basis of the Campbell-type integration, chromosome walking was attempted by using Escherichia coli replication origins that were also present in the integrated plasmid. The chromosomal DNA was digested, ligated, and used to transform E. coli, which enabled recovery of the expected adjacent genomic DNA regions. The plasmid integrate was stably maintained for 30 generations under non-selective culture conditions, suggesting that the integrated sequences carrying a replicon active in the host were maintained as a stable chromosomal insert in C. glutamicum.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-144-7-1863
1998-07-01
2021-07-27
Loading full text...

Full text loading...

/deliver/fulltext/micro/144/7/mic-144-7-1863.html?itemId=/content/journal/micro/10.1099/00221287-144-7-1863&mimeType=html&fmt=ahah

References

  1. Aiba S., Tsunekawa H., Imanaka T. 1982; New approach to tryptophan production by Escherichia coli: genetic manipulation of composite plasmids in vitro.. Appl Environ Microbiol 43:289–297
    [Google Scholar]
  2. Archer J.A.G, Solow-Cordero D.E., Sinskey A.J. 1991; A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by l-threonine.. Gene 107:53–59
    [Google Scholar]
  3. Fedorova N.D., Highlander S.K. 1997; Generation of targeted nonpolar gene insertions and operon fusions in Pasteurella haemolytica and creation of a strain that produces and secretes inactive leukotoxin.. Infect Immun 65:2593–2598
    [Google Scholar]
  4. Gutterson N.I., Koshland D.E. 1983; Replacement and amplification of bacterial genes with sequences altered in vitro.. Proc Natl Acad Sci USA 804894–4898
    [Google Scholar]
  5. Ikeda M., Katsumata R. 1992; Metabolic engineering to produce tyrosine or phenylalanine in a tryptophan-producing Corynebacterium glutamicum strain.. Appl Environ Microbiol 58:781–785
    [Google Scholar]
  6. Ikeda M., Ozaki A., Katsumata R. 1993; Phenylalanine production by metabolically engineered Corynebacterium glutamicum with the pheA gene of Escherichia coli.. Appl Microbiol Biotechnol 39:318–323
    [Google Scholar]
  7. Ikeda M., Nakanishi K., Kino K., Katsumata R. 1994; Fermentative production of tryptophan by a stable recombinant strain of Corynebacterium glutamicum with a modified serinebiosynthetic pathway.. Biosci Biotechnol Biochem 58:674–678
    [Google Scholar]
  8. Katsumata R., Ikeda M. 1993; Hyperproduction of tryptophan in Corynebacterium glutamicum by pathway engineering.. Bio/ Technology 11:921–925
    [Google Scholar]
  9. Katsumata R., Ozaki A., Oka T., Furuya A. 1984; Protoplast transformation of glutamate-producing bacteria with plasmid DNA.. J Bacterial 159:306–311
    [Google Scholar]
  10. Katsumata R., Mizukami T., Ozaki A., Kikuchi Y., Kino K., Oka T., Furuya A. 1987; Gene cloning in glutamic acid bacteria: the system and its applications.. In Proceedings of the 4th European Congress on Biotechnology, Amsterdam 4 pp. 767–776 Edited by Neijssel O. M. and others Amsterdam: Elsevier;
    [Google Scholar]
  11. Kinoshita S., Nakayama K. 1978; Amino acids.. In Primary Products of Metabolism pp. 209–261 Edited by Rose A. H. London, New York & San Francisco: Academic Press;
    [Google Scholar]
  12. Leenhouts K.J., Kok J., Venema G. 1989; Campbell-like integration of heterologous plasmid DNA into the chromosome of Lactococcus lactis subsp. lactis.. Appl Environ Microbiol 55:394–400
    [Google Scholar]
  13. Leenhouts K.J., Kok J., Venema G. 1990; Stability of integrated plasmids in the chromosome of Lactococcus lactis.. Appl Environ Microbiol 56:2726–2735
    [Google Scholar]
  14. Novick R.P. 1987; Plasmid incompatibility.. Microbiol Rev 51:381–395
    [Google Scholar]
  15. Ozaki A., Katsumata R., Oka T., Furuya A. 1984; Functional expression of the genes of Escherichia coli in gram-positive Corynebacterium glutamicum.. Mol Gen Genet 196:175–178
    [Google Scholar]
  16. Ozaki A., Katsumata R., Oka T., Furuya A. 1985; Cloning of the gene concerned in phenylalanine biosynthesis in Corynebacterium glutamicum and its application to breeding of a phenylalanine producing strain.. Agric Biol Chern 49:2925–2930
    [Google Scholar]
  17. Reyes O., Guyonvarch A., Bonamy C, Salti V., David F., Leblon G. 1991; ‘Integron’-bearing vectors: a method suitable for stable chromosomal integration in highly restrictive corynebacteria.. Gene 107:61–68
    [Google Scholar]
  18. Rigby P.W.J., Dieckman M., Rhodes C., Berg P. 1977; Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I.. J Mol Biol 113:237–251
    [Google Scholar]
  19. Saito H., Miura K. 1963; Preparation of transforming deoxyribonucleic acid by phenol treatment.. Biochim Biophys Acta 72:619–629
    [Google Scholar]
  20. Sambrook J., Fritsch E.F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn.. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  21. Santamaria R., Gil J.A., Mesas J.M., Martin J.F. 1984; Characterization of an endogenous plasmid and development of cloning vectors and a transformation system in Brevibacterium lactofermentum.. J Gen Microbiol 130:2237–2246
    [Google Scholar]
  22. Schwarzer A., Pühler A. 1991; Manipulation of Corynebacterium glutamicum by gene disruption and replacement.. Bio/Technology 9:84–87
    [Google Scholar]
  23. Simon R., Priefer U., Pühler A. 1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria.. Bio/Technology 1:784–790
    [Google Scholar]
  24. Smith M.D., Flickinger J.L, Lineberger D. W., Schmidt B. 1986; Protoplast transformation in coryneform bacteria and introduction of an α-amylase gene from Bacillus amyloliquefaciens into Brevibacterium lactofermentum.. Appl Environ Microbiol 51:636–639
    [Google Scholar]
  25. Sugiura M., Suzuki S., Kisumi M. 1987; Improvement of histidine-producing strains of Serratia marcescens by cloning of a mutant allele of the histidine operon on a mini-F plasmid vector.. Agric Biol Chem 51:371–377
    [Google Scholar]
  26. Vertés A.A., Hatakeyama K., Inui M., Kobayashi M., Kurusu Y., Yukawa H. 1993; Replacement recombination in Coryneform bacteria: high efficiency integration requirement for non-meth-ylated plasmid DNA.. Biosci Biotechnol Biochem 57:2036–2038
    [Google Scholar]
  27. Yoshihama M., Higashiro K., Rao E.A., Akedo M., Shanabruch W.G., Follettie M.T., Walker G.C., Sinskey A.J. 1985; Cloning vector system for Corynebacterium glutamicum.. J Bacteriol 162:591–597
    [Google Scholar]
  28. Young M., Ehrlich S.D. 1989; Stability of reiterated sequences in the Bacillus subtilis chromosome.. J Bacteriol 171:2653–2656
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-144-7-1863
Loading
/content/journal/micro/10.1099/00221287-144-7-1863
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error