1887

Abstract

To examine the possible physiological significance of Mrp, a multi-subunit cation/proton antiporter from , a chromosomal deletion Δ of was constructed and characterized. The resulting mutant showed a consistent early growth defect in LB broth that became more evident at elevated pH of the growth medium and increasing Na or K loads. After 24 h incubation, these differences disappeared likely due to the concerted effort of other cation pumps in the mutant. Phenotype MicroArray analyses revealed an unexpected systematic defect in nitrogen utilization in the Δ mutant that was complemented by using the operon on an arabinose-inducible expression vector. Deletion of the operon also led to hypermotility, observable on LB and M9 semi-solid agar. Surprisingly, Δ mutation resulted in wild-type biofilm formation in M9 despite a growth defect but the reverse was true in LB. Furthermore, the Δ strain exhibited higher susceptibility to amphiphilic anions. These pleiotropic phenotypes of the Δ mutant demonstrate how the chemiosmotic activity of Mrp contributes to the survival potential of despite the presence of an extended battery of cation/proton antiporters of varying ion selectivity and pH profile operating in the same membrane.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.000384
2016-12-21
2020-04-04
Loading full text...

Full text loading...

/deliver/fulltext/micro/162/12/2147.html?itemId=/content/journal/micro/10.1099/mic.0.000384&mimeType=html&fmt=ahah

References

  1. Asai Y., Yakushi T., Kawagishi I., Homma M.. 2003; Ion-coupling determinants of Na+-driven and H+-driven flagellar motors. J Mol Biol327:453–463 [CrossRef][PubMed]
    [Google Scholar]
  2. Bochner B. R., Gadzinski P., Panomitros E.. 2001; Phenotype microarrays for high-throughput phenotypic testing and assay of gene function. Genome Res11:1246–1255 [CrossRef][PubMed]
    [Google Scholar]
  3. Chatterjee A., Chaudhuri S., Saha G., Gupta S., Chowdhury R.. 2004; Effect of bile on the cell surface permeability barrier and efflux system of Vibrio cholerae. J Bacteriol186:6809–6814 [CrossRef][PubMed]
    [Google Scholar]
  4. Dibrov P., Rimon A., Dzioba J., Winogrodzki A., Shalitin Y., Padan E.. 2005; 2-Aminoperimidine, a specific inhibitor of bacterial NhaA Na(+)/H(+) antiporters. FEBS Lett579:373–378 [CrossRef][PubMed]
    [Google Scholar]
  5. Donnenberg M. S., Kaper J. B.. 1991; Construction of an eae deletion mutant of enteropathogenic Escherichia coli by using a positive-selection suicide vector. Infect Immun 59:4310–4317[PubMed]
    [Google Scholar]
  6. Dzioba J., Ostroumov E., Winogrodzki A., Dibrov P.. 2002; Cloning, functional expression in Escherichia coli and primary characterization of a new Na+/H+ antiporter, NhaD, of Vibrio cholerae. Mol Cell Biochem229:119–124[PubMed][CrossRef]
    [Google Scholar]
  7. Dzioba-Winogrodzki J., Winogrodzki O., Krulwich T. A., Boin M. A., Häse C. C., Dibrov P.. 2009; The Vibrio cholerae Mrp system: cation/proton antiport properties and enhancement of bile salt resistance in a heterologous host. J Mol Microbiol Biotechnol16:176–186 [CrossRef][PubMed]
    [Google Scholar]
  8. Habibian R., Dzioba J., Barrett J., Galperin M. Y., Loewen P. C., Dibrov P.. 2005; Functional analysis of conserved polar residues in Vc-NhaD, Na+/H+ antiporter of Vibrio cholerae. J Biol Chem280:39637–39643 [CrossRef][PubMed]
    [Google Scholar]
  9. Hamamoto T., Hashimoto M., Hino M., Kitada M., Seto Y., Kudo T., Horikoshi K.. 1994; Characterization of a gene responsible for the Na+/H+ antiporter system of alkalophilic Bacillus species strain C-125. Mol Microbiol14:939–946 [CrossRef][PubMed]
    [Google Scholar]
  10. Häse C. C., Barquera B.. 2001; Role of sodium bioenergetics in Vibrio cholerae. Biochim Biophys Acta1505:169–178 [CrossRef][PubMed]
    [Google Scholar]
  11. Häse C. C., Mekalanos J. J.. 1999; Effects of changes in membrane sodium flux on virulence gene expression in Vibrio cholerae. Proc Natl Acad Sci U S A96:3183–3187 [CrossRef][PubMed]
    [Google Scholar]
  12. Häse C. C., Fedorova N. D., Galperin M. Y., Dibrov P. A.. 2001; Sodium ion cycle in bacterial pathogens: evidence from cross-genome comparisons. Microbiol Mol Biol Rev65:353–370 Table of contents [CrossRef][PubMed]
    [Google Scholar]
  13. Heithoff D. M., Mahan M. J.. 2004; Vibrio cholerae biofilms: stuck between a rock and a hard place. J Bacteriol186:4835–4837 [CrossRef][PubMed]
    [Google Scholar]
  14. Herz K., Vimont S., Padan E., Berche P.. 2003; Roles of NhaA, NhaB, and NhaD Na+/H+ antiporters in survival of Vibrio cholerae in a saline environment. J Bacteriol185:1236–1244 [CrossRef][PubMed]
    [Google Scholar]
  15. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R.. 1989; Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene77:51–59 [CrossRef][PubMed]
    [Google Scholar]
  16. Ito M., Guffanti A. A., Oudega B., Krulwich T. A.. 1999; Mrp, a multigene, multifunctional locus in Bacillus subtilis with roles in resistance to cholate and to Na+ and in pH homeostasis. J Bacteriol181:2394–2402[PubMed]
    [Google Scholar]
  17. Ito M., Guffanti A. A., Wang W., Krulwich T. A.. 2000; Effects of nonpolar mutations in each of the seven Bacillus subtilis mrp genes suggest complex interactions among the gene products in support of Na(+) and alkali but not cholate resistance. J Bacteriol182:5663–5670 [CrossRef][PubMed]
    [Google Scholar]
  18. Kojima S., Yamamoto K., Kawagishi I., Homma M.. 1999; The polar flagellar motor of Vibrio cholerae is driven by an Na+ motive force. J Bacteriol181:1927–1930[PubMed]
    [Google Scholar]
  19. Kosono S., Morotomi S., Kitada M., Kudo T.. 1999; Analyses of a Bacillus subtilis homologue of the Na+/H+ antiporter gene which is important for pH homeostasis of alkaliphilic Bacillus sp. C-125. Biochim Biophys Acta1409:171–175 [CrossRef][PubMed]
    [Google Scholar]
  20. Kosono S., Ohashi Y., Kawamura F., Kitada M., Kudo T.. 2000; Function of a principal Na(+)/H(+) antiporter, ShaA, is required for initiation of sporulation in Bacillus subtilis. J Bacteriol182:898–904 [CrossRef][PubMed]
    [Google Scholar]
  21. Kosono S., Haga K., Tomizawa R., Kajiyama Y., Hatano K., Takeda S., Wakai Y., Hino M., Kudo T.. 2005; Characterization of a multigene-encoded sodium/hydrogen antiporter (sha) from Pseudomonas aeruginosa: its involvement in pathogenesis. J Bacteriol187:5242–5248 [CrossRef][PubMed]
    [Google Scholar]
  22. Krulwich T. A., Hicks D. B., Ito M.. 2009; Cation/proton antiporter complements of bacteria: why so large and diverse?. Mol Microbiol74:257–260 [CrossRef]
    [Google Scholar]
  23. Lauriano C. M., Ghosh C., Correa N. E., Klose K. E.. 2004; The sodium-driven flagellar motor controls exopolysaccharide expression in Vibrio cholerae. J Bacteriol186:4864–4874 [CrossRef][PubMed]
    [Google Scholar]
  24. Magariyama Y., Sugiyama S., Muramoto K., Maekawa Y., Kawagishi I., Imae Y., Kudo S.. 1994; Very fast flagellar rotation. Nature371:752 [CrossRef][PubMed]
    [Google Scholar]
  25. Metcalf W. W., Jiang W., Daniels L. L., Kim S. K., Haldimann A., Wanner B. L.. 1996; Conditionally replicative and conjugative plasmids carrying lacZ alpha for cloning, mutagenesis, and allele replacement in bacteria. Plasmid35:1–13 [CrossRef][PubMed]
    [Google Scholar]
  26. Miller C. J., Drasar B. S., Feachem R. G.. 1984; Response of toxigenic Vibrio cholerae 01 to physico-chemical stresses in aquatic environments. J Hygiene93:475–495 [CrossRef]
    [Google Scholar]
  27. Minato Y., Fassio S. R., Reddekopp R. L., Häse C. C.. 2014; Inhibition of the sodium-translocating NADH-ubiquinone oxidoreductase [Na+-NQR] decreases cholera toxin production in Vibrio cholerae O1 at the late exponential growth phase. Microb Pathog66:36–39 [CrossRef][PubMed]
    [Google Scholar]
  28. Prouty M. G., Correa N. E., Klose K. E.. 2001; The novel sigma54- and sigma28-dependent flagellar gene transcription hierarchy of Vibrio cholerae. Mol Microbiol39:1595–1609 [CrossRef][PubMed]
    [Google Scholar]
  29. Putnoky P., Kereszt A., Nakamura T., Endre G., Grosskopf E., Kiss P., Kondorosi A.. 1998; The pha gene cluster of Rhizobium meliloti involved in pH adaptation and symbiosis encodes a novel type of K+ efflux system. Mol Microbiol 28:1091–1101 [CrossRef][PubMed]
    [Google Scholar]
  30. Quinn M. J., Resch C. T., Sun J., Lind E. J., Dibrov P., Häse C. C.. 2012; NhaP1 is a K+(Na+)/H+ antiporter required for growth and internal pH homeostasis of Vibrio cholerae at low extracellular pH. Microbiology158:1094–1105 [CrossRef][PubMed]
    [Google Scholar]
  31. Resch C. T., Winogrodzki J. L., Patterson C. T., Lind E. J., Quinn M. J., Dibrov P., Häse C. C.. 2010; The putative Na+/H+ antiporter of Vibrio cholerae, Vc-NhaP2, mediates the specific K+/H+ exchange in vivo. Biochemistry49:2520–2528 [CrossRef][PubMed]
    [Google Scholar]
  32. Singleton F. L., Attwell R., Jangi S., Colwell R. R.. 1982a; Effects of temperature and salinity on Vibrio cholerae growth. Appl Environ Microbiol44:1047–1058
    [Google Scholar]
  33. Singleton F. L., Attwell R. W., Jangi M. S., Colwell R. R.. 1982b; Influence of salinity and organic nutrient concentration on survival and growth of Vibrio cholerae in aquatic microcosms. Appl Environ Microbiol43:1080–1085
    [Google Scholar]
  34. Swartz T. H., Ikewada S., Ishikawa O., Ito M., Krulwich T. A.. 2005; The Mrp system: a giant among monovalent cation/proton antiporters?. Extremophiles9:345–354 [CrossRef][PubMed]
    [Google Scholar]
  35. Watnick P. I., Kolter R.. 1999; Steps in the development of a Vibrio cholerae El Tor biofilm. Mol Microbiol34:586–595 [CrossRef][PubMed]
    [Google Scholar]
  36. Watnick P. I., Fullner K. J., Kolter R.. 1999; A role for the mannose-sensitive hemagglutinin in biofilm formation by Vibrio cholerae El Tor. J Bacteriol181:3606–3609[PubMed]
    [Google Scholar]
  37. Wiens E. J., Winogrodzki J. L., Resch C. T., Orriss G. L., Stetefeld J., Dibrov P.. 2014; The C-terminal cytoplasmic portion of the NhaP2 cation-proton antiporter from Vibrio cholerae affects its activity and substrate affinity. Mol Cell Biochem389:51–58 [CrossRef][PubMed]
    [Google Scholar]
  38. Zhou L., Lei X. H., Bochner B. R., Wanner B. L.. 2003; Phenotype microarray analysis of Escherichia coli K-12 mutants with deletions of all two-component systems. J Bacteriol185:4956–4972 [CrossRef][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.000384
Loading
/content/journal/micro/10.1099/mic.0.000384
Loading

Data & Media loading...

Supplements

Supplementary File 1

PDF

Most cited this month

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