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. 1995 Dec;177(23):6874–6880. doi: 10.1128/jb.177.23.6874-6880.1995

Characterization of a chimeric proU operon in a subtilin-producing mutant of Bacillus subtilis 168.

Y Lin 1, J N Hansen 1
PMCID: PMC177556  PMID: 7592481

Abstract

The ability to respond to osmotic stress by osmoregulation is common to virtually all living cells. Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium can achieve osmotolerance by import of osmoprotectants such as proline and glycine betaine by an import system encoded in an operon called proU with genes for proteins ProV, ProW, and ProX. In this report, we describe the discovery of a proU-type locus in the gram-positive bacterium Bacillus subtilis. It contains four open reading frames (ProV, ProW, ProX, and ProZ) with homology to the gram-negative ProU proteins, with the B. subtilis ProV, ProW, and ProX proteins having sequence homologies of 35, 29, and 17%, respectively, to the E. coli proteins. The B. subtilis ProZ protein is similar to the ProW protein but is smaller and, accordingly, may fulfill a novel role in osmoprotection. The B. subtilis proU locus was discovered while exploring the chromosomal sequence upstream from the spa operon in B. subtilis LH45, which is a subtilin-producing mutant of B. subtilis 168. B. subtilis LH45 had been previously constructed by transformation of strain 168 with linear DNA from B. subtilis ATCC 6633 (W. Liu and J. N. Hansen, J. Bacteriol. 173:7387-7390, 1991). Hybridization experiments showed that LH45 resulted from recombination in a region of homology in the proV gene, so that the proU locus in LH45 is a chimera between strains 168 and 6633. Despite being a chimera, this proU locus was fully functional in its ability to confer osmotolerance when glycine betaine was available in the medium. Conversely, a mutant (LH45 deltaproU) in which most of the proU locus had been deleted grew poorly at high osmolarity in the presence of glycine betaine. We conclude that the proU-like locus in B. subtilis LH45 is a gram-positive counterpart of the proU locus in gram-negative bacteria and probably evolved prior to the evolutionary split of prokaryotes into gram-positive and gram-negative forms.

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Selected References

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  1. Banerjee S., Hansen J. N. Structure and expression of a gene encoding the precursor of subtilin, a small protein antibiotic. J Biol Chem. 1988 Jul 5;263(19):9508–9514. [PubMed] [Google Scholar]
  2. Barr G. C., Ni Bhriain N., Dorman C. J. Identification of two new genetically active regions associated with the osmZ locus of Escherichia coli: role in regulation of proU expression and mutagenic effect of cya, the structural gene for adenylate cyclase. J Bacteriol. 1992 Feb;174(3):998–1006. doi: 10.1128/jb.174.3.998-1006.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bernardini M. L., Fontaine A., Sansonetti P. J. The two-component regulatory system ompR-envZ controls the virulence of Shigella flexneri. J Bacteriol. 1990 Nov;172(11):6274–6281. doi: 10.1128/jb.172.11.6274-6281.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berry A., DeVault J. D., Chakrabarty A. M. High osmolarity is a signal for enhanced algD transcription in mucoid and nonmucoid Pseudomonas aeruginosa strains. J Bacteriol. 1989 May;171(5):2312–2317. doi: 10.1128/jb.171.5.2312-2317.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Boch J., Kempf B., Bremer E. Osmoregulation in Bacillus subtilis: synthesis of the osmoprotectant glycine betaine from exogenously provided choline. J Bacteriol. 1994 Sep;176(17):5364–5371. doi: 10.1128/jb.176.17.5364-5371.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cairney J., Booth I. R., Higgins C. F. Osmoregulation of gene expression in Salmonella typhimurium: proU encodes an osmotically induced betaine transport system. J Bacteriol. 1985 Dec;164(3):1224–1232. doi: 10.1128/jb.164.3.1224-1232.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cangelosi G. A., Martinetti G., Nester E. W. Osmosensitivity phenotypes of Agrobacterium tumefaciens mutants that lack periplasmic beta-1,2-glucan. J Bacteriol. 1990 Apr;172(4):2172–2174. doi: 10.1128/jb.172.4.2172-2174.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Chambers S. T., Kunin C. M. Isolation of glycine betaine and proline betaine from human urine. Assessment of their role as osmoprotective agents for bacteria and the kidney. J Clin Invest. 1987 Mar;79(3):731–737. doi: 10.1172/JCI112878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chung Y. J., Hansen J. N. Determination of the sequence of spaE and identification of a promoter in the subtilin (spa) operon in Bacillus subtilis. J Bacteriol. 1992 Oct;174(20):6699–6702. doi: 10.1128/jb.174.20.6699-6702.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chung Y. J., Steen M. T., Hansen J. N. The subtilin gene of Bacillus subtilis ATCC 6633 is encoded in an operon that contains a homolog of the hemolysin B transport protein. J Bacteriol. 1992 Feb;174(4):1417–1422. doi: 10.1128/jb.174.4.1417-1422.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Csonka L. N., Hanson A. D. Prokaryotic osmoregulation: genetics and physiology. Annu Rev Microbiol. 1991;45:569–606. doi: 10.1146/annurev.mi.45.100191.003033. [DOI] [PubMed] [Google Scholar]
  12. Csonka L. N., Ikeda T. P., Fletcher S. A., Kustu S. The accumulation of glutamate is necessary for optimal growth of Salmonella typhimurium in media of high osmolality but not induction of the proU operon. J Bacteriol. 1994 Oct;176(20):6324–6333. doi: 10.1128/jb.176.20.6324-6333.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Csonka L. N. Physiological and genetic responses of bacteria to osmotic stress. Microbiol Rev. 1989 Mar;53(1):121–147. doi: 10.1128/mr.53.1.121-147.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Dattananda C. S., Gowrishankar J. Osmoregulation in Escherichia coli: complementation analysis and gene-protein relationships in the proU locus. J Bacteriol. 1989 Apr;171(4):1915–1922. doi: 10.1128/jb.171.4.1915-1922.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Dattananda C. S., Rajkumari K., Gowrishankar J. Multiple mechanisms contribute to osmotic inducibility of proU operon expression in Escherichia coli: demonstration of two osmoresponsive promoters and of a negative regulatory element within the first structural gene. J Bacteriol. 1991 Dec;173(23):7481–7490. doi: 10.1128/jb.173.23.7481-7490.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Doige C. A., Ames G. F. ATP-dependent transport systems in bacteria and humans: relevance to cystic fibrosis and multidrug resistance. Annu Rev Microbiol. 1993;47:291–319. doi: 10.1146/annurev.mi.47.100193.001451. [DOI] [PubMed] [Google Scholar]
  17. Dorman C. J., Chatfield S., Higgins C. F., Hayward C., Dougan G. Characterization of porin and ompR mutants of a virulent strain of Salmonella typhimurium: ompR mutants are attenuated in vivo. Infect Immun. 1989 Jul;57(7):2136–2140. doi: 10.1128/iai.57.7.2136-2140.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dorman C. J., Ni Bhriain N., Higgins C. F. DNA supercoiling and environmental regulation of virulence gene expression in Shigella flexneri. Nature. 1990 Apr 19;344(6268):789–792. doi: 10.1038/344789a0. [DOI] [PubMed] [Google Scholar]
  19. Druger-Liotta J., Prange V. J., Overdier D. G., Csonka L. N. Selection of mutations that alter the osmotic control of transcription of the Salmonella typhimurium proU operon. J Bacteriol. 1987 Jun;169(6):2449–2459. doi: 10.1128/jb.169.6.2449-2459.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Dylan T., Helinski D. R., Ditta G. S. Hypoosmotic adaptation in Rhizobium meliloti requires beta-(1----2)-glucan. J Bacteriol. 1990 Mar;172(3):1400–1408. doi: 10.1128/jb.172.3.1400-1408.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Faatz E., Middendorf A., Bremer E. Cloned structural genes for the osmotically regulated binding-protein-dependent glycine betaine transport system (ProU) of Escherichia coli K-12. Mol Microbiol. 1988 Mar;2(2):265–279. doi: 10.1111/j.1365-2958.1988.tb00028.x. [DOI] [PubMed] [Google Scholar]
  22. Galán J. E., Curtiss R., 3rd Expression of Salmonella typhimurium genes required for invasion is regulated by changes in DNA supercoiling. Infect Immun. 1990 Jun;58(6):1879–1885. doi: 10.1128/iai.58.6.1879-1885.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Gouesbet G., Jebbar M., Talibart R., Bernard T., Blanco C. Pipecolic acid is an osmoprotectant for Escherichia coli taken up by the general osmoporters ProU and ProP. Microbiology. 1994 Sep;140(Pt 9):2415–2422. doi: 10.1099/13500872-140-9-2415. [DOI] [PubMed] [Google Scholar]
  24. Gowrishankar J., Jayashree P., Rajkumari K. Molecular cloning of an osmoregulatory locus in Escherichia coli: increased proU gene dosage results in enhanced osmotolerance. J Bacteriol. 1986 Dec;168(3):1197–1204. doi: 10.1128/jb.168.3.1197-1204.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Gowrishankar J. Nucleotide sequence of the osmoregulatory proU operon of Escherichia coli. J Bacteriol. 1989 Apr;171(4):1923–1931. doi: 10.1128/jb.171.4.1923-1931.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Graham J. E., Wilkinson B. J. Staphylococcus aureus osmoregulation: roles for choline, glycine betaine, proline, and taurine. J Bacteriol. 1992 Apr;174(8):2711–2716. doi: 10.1128/jb.174.8.2711-2716.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gutowski-Eckel Z., Klein C., Siegers K., Bohm K., Hammelmann M., Entian K. D. Growth phase-dependent regulation and membrane localization of SpaB, a protein involved in biosynthesis of the lantibiotic subtilin. Appl Environ Microbiol. 1994 Jan;60(1):1–11. doi: 10.1128/aem.60.1.1-11.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Higgins C. F., Sutherland L., Cairney J., Booth I. R. The osmotically regulated proU locus of Salmonella typhimurium encodes a periplasmic betaine-binding protein. J Gen Microbiol. 1987 Feb;133(2):305–310. doi: 10.1099/00221287-133-2-305. [DOI] [PubMed] [Google Scholar]
  29. Klein C., Entian K. D. Genes involved in self-protection against the lantibiotic subtilin produced by Bacillus subtilis ATCC 6633. Appl Environ Microbiol. 1994 Aug;60(8):2793–2801. doi: 10.1128/aem.60.8.2793-2801.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Klein C., Kaletta C., Entian K. D. Biosynthesis of the lantibiotic subtilin is regulated by a histidine kinase/response regulator system. Appl Environ Microbiol. 1993 Jan;59(1):296–303. doi: 10.1128/aem.59.1.296-303.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Klein C., Kaletta C., Schnell N., Entian K. D. Analysis of genes involved in biosynthesis of the lantibiotic subtilin. Appl Environ Microbiol. 1992 Jan;58(1):132–142. doi: 10.1128/aem.58.1.132-142.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  33. Landfald B., Strøm A. R. Choline-glycine betaine pathway confers a high level of osmotic tolerance in Escherichia coli. J Bacteriol. 1986 Mar;165(3):849–855. doi: 10.1128/jb.165.3.849-855.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Liu W., Hansen J. N. Conversion of Bacillus subtilis 168 to a subtilin producer by competence transformation. J Bacteriol. 1991 Nov;173(22):7387–7390. doi: 10.1128/jb.173.22.7387-7390.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Liu W., Hansen J. N. Enhancement of the chemical and antimicrobial properties of subtilin by site-directed mutagenesis. J Biol Chem. 1992 Dec 15;267(35):25078–25085. [PubMed] [Google Scholar]
  36. Lucht J. M., Bremer E. Adaptation of Escherichia coli to high osmolarity environments: osmoregulation of the high-affinity glycine betaine transport system proU. FEMS Microbiol Rev. 1994 May;14(1):3–20. doi: 10.1111/j.1574-6976.1994.tb00067.x. [DOI] [PubMed] [Google Scholar]
  37. May G., Faatz E., Lucht J. M., Haardt M., Bolliger M., Bremer E. Characterization of the osmoregulated Escherichia coli proU promoter and identification of ProV as a membrane-associated protein. Mol Microbiol. 1989 Nov;3(11):1521–1531. doi: 10.1111/j.1365-2958.1989.tb00138.x. [DOI] [PubMed] [Google Scholar]
  38. Mellies J., Brems R., Villarejo M. The Escherichia coli proU promoter element and its contribution to osmotically signaled transcription activation. J Bacteriol. 1994 Jun;176(12):3638–3645. doi: 10.1128/jb.176.12.3638-3645.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Mohana Rao J. K., Argos P. A conformational preference parameter to predict helices in integral membrane proteins. Biochim Biophys Acta. 1986 Jan 30;869(2):197–214. doi: 10.1016/0167-4838(86)90295-5. [DOI] [PubMed] [Google Scholar]
  40. Overdier D. G., Olson E. R., Erickson B. D., Ederer M. M., Csonka L. N. Nucleotide sequence of the transcriptional control region of the osmotically regulated proU operon of Salmonella typhimurium and identification of the 5' endpoint of the proU mRNA. J Bacteriol. 1989 Sep;171(9):4694–4706. doi: 10.1128/jb.171.9.4694-4706.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. SCOTT W. J. Water relations of Staphylococcus aureus at 30 degrees C. Aust J Biol Sci. 1953 Nov;6(4):549–564. [PubMed] [Google Scholar]
  42. Stirling D. A., Hulton C. S., Waddell L., Park S. F., Stewart G. S., Booth I. R., Higgins C. F. Molecular characterization of the proU loci of Salmonella typhimurium and Escherichia coli encoding osmoregulated glycine betaine transport systems. Mol Microbiol. 1989 Aug;3(8):1025–1038. doi: 10.1111/j.1365-2958.1989.tb00253.x. [DOI] [PubMed] [Google Scholar]
  43. Yancey P. H., Clark M. E., Hand S. C., Bowlus R. D., Somero G. N. Living with water stress: evolution of osmolyte systems. Science. 1982 Sep 24;217(4566):1214–1222. doi: 10.1126/science.7112124. [DOI] [PubMed] [Google Scholar]
  44. del Castillo I., Gómez J. M., Moreno F. mprA, an Escherichia coli gene that reduces growth-phase-dependent synthesis of microcins B17 and C7 and blocks osmoinduction of proU when cloned on a high-copy-number plasmid. J Bacteriol. 1990 Jan;172(1):437–445. doi: 10.1128/jb.172.1.437-445.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

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