Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Feb;179(3):677–683. doi: 10.1128/jb.179.3.677-683.1997

Secretion of nuclease across the outer membrane of Serratia marcescens and its energy requirements.

Y Suh 1, M J Benedik 1
PMCID: PMC178747  PMID: 9006020

Abstract

Extracellular secretion of Serratia marcescens nuclease occurs as a two-step process via a periplasmic intermediate. Unlike other extracellular proteins secreted by gram-negative bacteria by the general secretory pathway, nuclease accumulates in the periplasm in its active form for an unusually long time before its export into the growth medium. The energy requirements for extracellular secretion of nuclease from the periplasm were investigated. Our results suggest that the second step of secretion across the outer membrane is dependent upon the external pH; acidic pH effectively but reversibly blocks extracellular secretion. However, electrochemical proton gradient, and possibly ATP hydrolysis, are not required for this step. We suggest that nuclease uses a novel mechanism for the second step of secretion in S. marcescens.

Full Text

The Full Text of this article is available as a PDF (294.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akrim M., Bally M., Ball G., Tommassen J., Teerink H., Filloux A., Lazdunski A. Xcp-mediated protein secretion in Pseudomonas aeruginosa: identification of two additional genes and evidence for regulation of xcp gene expression. Mol Microbiol. 1993 Oct;10(2):431–443. doi: 10.1111/j.1365-2958.1993.tb02674.x. [DOI] [PubMed] [Google Scholar]
  2. Arkowitz R. A., Wickner W. SecD and SecF are required for the proton electrochemical gradient stimulation of preprotein translocation. EMBO J. 1994 Feb 15;13(4):954–963. doi: 10.1002/j.1460-2075.1994.tb06340.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Ball T. K., Suh Y., Benedik M. J. Disulfide bonds are required for Serratia marcescens nuclease activity. Nucleic Acids Res. 1992 Oct 11;20(19):4971–4974. doi: 10.1093/nar/20.19.4971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Ball T. K., Wasmuth C. R., Braunagel S. C., Benedik M. J. Expression of Serratia marcescens extracellular proteins requires recA. J Bacteriol. 1990 Jan;172(1):342–349. doi: 10.1128/jb.172.1.342-349.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bardwell J. C., McGovern K., Beckwith J. Identification of a protein required for disulfide bond formation in vivo. Cell. 1991 Nov 1;67(3):581–589. doi: 10.1016/0092-8674(91)90532-4. [DOI] [PubMed] [Google Scholar]
  6. Bieker-Brady K., Silhavy T. J. Suppressor analysis suggests a multistep, cyclic mechanism for protein secretion in Escherichia coli. EMBO J. 1992 Sep;11(9):3165–3174. doi: 10.1002/j.1460-2075.1992.tb05393.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bortoli-German I., Brun E., Py B., Chippaux M., Barras F. Periplasmic disulphide bond formation is essential for cellulase secretion by the plant pathogen Erwinia chrysanthemi. Mol Microbiol. 1994 Feb;11(3):545–553. doi: 10.1111/j.1365-2958.1994.tb00335.x. [DOI] [PubMed] [Google Scholar]
  8. Chen Y. C., Shipley G. L., Ball T. K., Benedik M. J. Regulatory mutants and transcriptional control of the Serratia marcescens extracellular nuclease gene. Mol Microbiol. 1992 Mar;6(5):643–651. doi: 10.1111/j.1365-2958.1992.tb01512.x. [DOI] [PubMed] [Google Scholar]
  9. Chen Y. C., Suh Y., Riise E., Kartman B., Jin S., Benedik M. J. Inhibition of Serratia marcescens nuclease secretion by a truncated nuclease peptide. Gene. 1996 Jun 12;172(1):9–16. doi: 10.1016/0378-1119(96)00187-4. [DOI] [PubMed] [Google Scholar]
  10. Cline K., Ettinger W. F., Theg S. M. Protein-specific energy requirements for protein transport across or into thylakoid membranes. Two lumenal proteins are transported in the absence of ATP. J Biol Chem. 1992 Feb 5;267(4):2688–2696. [PubMed] [Google Scholar]
  11. Douwe de Boer A., Weisbeek P. J. Chloroplast protein topogenesis: import, sorting and assembly. Biochim Biophys Acta. 1991 Nov 13;1071(3):221–253. doi: 10.1016/0304-4157(91)90015-o. [DOI] [PubMed] [Google Scholar]
  12. Driessen A. J. Bacterial protein translocation: kinetic and thermodynamic role of ATP and the protonmotive force. Trends Biochem Sci. 1992 Jun;17(6):219–223. doi: 10.1016/0968-0004(92)90381-i. [DOI] [PubMed] [Google Scholar]
  13. EAVES G. N., JEFFRIES C. D. EFFECT OF PH ON THE FORMATION OF EXOCELLULAR NUCLEASE IN AGING BROTH CULTURES OF SERRATIA MARCESCENS. J Bacteriol. 1963 Jun;85:1194–1196. doi: 10.1128/jb.85.6.1194-1196.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geller B. L. Energy requirements for protein translocation across the Escherichia coli inner membrane. Mol Microbiol. 1991 Sep;5(9):2093–2098. doi: 10.1111/j.1365-2958.1991.tb02138.x. [DOI] [PubMed] [Google Scholar]
  15. Glick B. S., Beasley E. M., Schatz G. Protein sorting in mitochondria. Trends Biochem Sci. 1992 Nov;17(11):453–459. doi: 10.1016/0968-0004(92)90487-t. [DOI] [PubMed] [Google Scholar]
  16. Henry R., Kapazoglou A., McCaffery M., Cline K. Differences between lumen targeting domains of chloroplast transit peptides determine pathway specificity for thylakoid transport. J Biol Chem. 1994 Apr 8;269(14):10189–10192. [PubMed] [Google Scholar]
  17. Hirst T. R., Holmgren J. Transient entry of enterotoxin subunits into the periplasm occurs during their secretion from Vibrio cholerae. J Bacteriol. 1987 Mar;169(3):1037–1045. doi: 10.1128/jb.169.3.1037-1045.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Howard S. P., Critch J., Bedi A. Isolation and analysis of eight exe genes and their involvement in extracellular protein secretion and outer membrane assembly in Aeromonas hydrophila. J Bacteriol. 1993 Oct;175(20):6695–6703. doi: 10.1128/jb.175.20.6695-6703.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hultgren S. J., Jacob-Dubuisson F., Jones C. H., Bränden C. I. PapD and superfamily of periplasmic immunoglobulin-like pilus chaperones. Adv Protein Chem. 1993;44:99–123. doi: 10.1016/s0065-3233(08)60565-3. [DOI] [PubMed] [Google Scholar]
  20. Jacob-Dubuisson F., Striker R., Hultgren S. J. Chaperone-assisted self-assembly of pili independent of cellular energy. J Biol Chem. 1994 Apr 29;269(17):12447–12455. [PubMed] [Google Scholar]
  21. Jiang B., Howard S. P. The Aeromonas hydrophila exeE gene, required both for protein secretion and normal outer membrane biogenesis, is a member of a general secretion pathway. Mol Microbiol. 1992 May;6(10):1351–1361. doi: 10.1111/j.1365-2958.1992.tb00856.x. [DOI] [PubMed] [Google Scholar]
  22. Jin S., Chen Y., Christie G. E., Benedik M. J. Regulation of the Serratia marcescens extracellular nuclease: positive control by a homolog of P2 Ogr encoded by a cryptic prophage. J Mol Biol. 1996 Feb 23;256(2):264–278. doi: 10.1006/jmbi.1996.0084. [DOI] [PubMed] [Google Scholar]
  23. Joly J. C., Wickner W. The SecA and SecY subunits of translocase are the nearest neighbors of a translocating preprotein, shielding it from phospholipids. EMBO J. 1993 Jan;12(1):255–263. doi: 10.1002/j.1460-2075.1993.tb05651.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kamitani S., Akiyama Y., Ito K. Identification and characterization of an Escherichia coli gene required for the formation of correctly folded alkaline phosphatase, a periplasmic enzyme. EMBO J. 1992 Jan;11(1):57–62. doi: 10.1002/j.1460-2075.1992.tb05027.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Koronakis V., Hughes C., Koronakis E. Energetically distinct early and late stages of HlyB/HlyD-dependent secretion across both Escherichia coli membranes. EMBO J. 1991 Nov;10(11):3263–3272. doi: 10.1002/j.1460-2075.1991.tb04890.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lill R., Dowhan W., Wickner W. The ATPase activity of SecA is regulated by acidic phospholipids, SecY, and the leader and mature domains of precursor proteins. Cell. 1990 Jan 26;60(2):271–280. doi: 10.1016/0092-8674(90)90742-w. [DOI] [PubMed] [Google Scholar]
  27. Lindeberg M., Collmer A. Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi: sequence comparison with secretion genes from other gram-negative bacteria. J Bacteriol. 1992 Nov;174(22):7385–7397. doi: 10.1128/jb.174.22.7385-7397.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liu J., Walsh C. T. Peptidyl-prolyl cis-trans-isomerase from Escherichia coli: a periplasmic homolog of cyclophilin that is not inhibited by cyclosporin A. Proc Natl Acad Sci U S A. 1990 Jun;87(11):4028–4032. doi: 10.1073/pnas.87.11.4028. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Matsuyama S., Fujita Y., Mizushima S. SecD is involved in the release of translocated secretory proteins from the cytoplasmic membrane of Escherichia coli. EMBO J. 1993 Jan;12(1):265–270. doi: 10.1002/j.1460-2075.1993.tb05652.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Miyazaki H., Yanagida N., Horinouchi S., Beppu T. Characterization of the precursor of Serratia marcescens serine protease and COOH-terminal processing of the precursor during its excretion through the outer membrane of Escherichia coli. J Bacteriol. 1989 Dec;171(12):6566–6572. doi: 10.1128/jb.171.12.6566-6572.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Neidhardt F. C., Bloch P. L., Smith D. F. Culture medium for enterobacteria. J Bacteriol. 1974 Sep;119(3):736–747. doi: 10.1128/jb.119.3.736-747.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Neupert W., Hartl F. U., Craig E. A., Pfanner N. How do polypeptides cross the mitochondrial membranes? Cell. 1990 Nov 2;63(3):447–450. doi: 10.1016/0092-8674(90)90437-j. [DOI] [PubMed] [Google Scholar]
  33. Peek J. A., Taylor R. K. Characterization of a periplasmic thiol:disulfide interchange protein required for the functional maturation of secreted virulence factors of Vibrio cholerae. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):6210–6214. doi: 10.1073/pnas.89.13.6210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Pohlner J., Halter R., Beyreuther K., Meyer T. F. Gene structure and extracellular secretion of Neisseria gonorrhoeae IgA protease. 1987 Jan 29-Feb 4Nature. 325(6103):458–462. doi: 10.1038/325458a0. [DOI] [PubMed] [Google Scholar]
  35. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pugsley A. P. Translocation of a folded protein across the outer membrane in Escherichia coli. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):12058–12062. doi: 10.1073/pnas.89.24.12058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Randall L. L., Hardy S. J. Correlation of competence for export with lack of tertiary structure of the mature species: a study in vivo of maltose-binding protein in E. coli. Cell. 1986 Sep 12;46(6):921–928. doi: 10.1016/0092-8674(86)90074-7. [DOI] [PubMed] [Google Scholar]
  38. Reeves P. J., Whitcombe D., Wharam S., Gibson M., Allison G., Bunce N., Barallon R., Douglas P., Mulholland V., Stevens S. Molecular cloning and characterization of 13 out genes from Erwinia carotovora subspecies carotovora: genes encoding members of a general secretion pathway (GSP) widespread in gram-negative bacteria. Mol Microbiol. 1993 May;8(3):443–456. doi: 10.1111/j.1365-2958.1993.tb01589.x. [DOI] [PubMed] [Google Scholar]
  39. Robinson C., Cai D., Hulford A., Brock I. W., Michl D., Hazell L., Schmidt I., Herrmann R. G., Klösgen R. B. The presequence of a chimeric construct dictates which of two mechanisms are utilized for translocation across the thylakoid membrane: evidence for the existence of two distinct translocation systems. EMBO J. 1994 Jan 15;13(2):279–285. doi: 10.1002/j.1460-2075.1994.tb06260.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Salmond G. P., Reeves P. J. Membrane traffic wardens and protein secretion in gram-negative bacteria. Trends Biochem Sci. 1993 Jan;18(1):7–12. doi: 10.1016/0968-0004(93)90080-7. [DOI] [PubMed] [Google Scholar]
  41. Schiebel E., Driessen A. J., Hartl F. U., Wickner W. Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase. Cell. 1991 Mar 8;64(5):927–939. doi: 10.1016/0092-8674(91)90317-r. [DOI] [PubMed] [Google Scholar]
  42. Segui-Real B., Kispal G., Lill R., Neupert W. Functional independence of the protein translocation machineries in mitochondrial outer and inner membranes: passage of preproteins through the intermembrane space. EMBO J. 1993 May;12(5):2211–2218. doi: 10.1002/j.1460-2075.1993.tb05869.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stock J. B., Rauch B., Roseman S. Periplasmic space in Salmonella typhimurium and Escherichia coli. J Biol Chem. 1977 Nov 10;252(21):7850–7861. [PubMed] [Google Scholar]
  44. Stuart R. A., Cyr D. M., Craig E. A., Neupert W. Mitochondrial molecular chaperones: their role in protein translocation. Trends Biochem Sci. 1994 Feb;19(2):87–92. doi: 10.1016/0968-0004(94)90041-8. [DOI] [PubMed] [Google Scholar]
  45. Suh Y., Alpaugh M., Krause K. L., Benedik M. J. Differential secretion of isoforms of Serratia marcescens extracellular nuclease. Appl Environ Microbiol. 1995 Nov;61(11):4083–4088. doi: 10.1128/aem.61.11.4083-4088.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Suh Y., Jin S., Ball T. K., Benedik M. J. Two-step secretion of the Serratia marcescens extracellular nuclease. J Bacteriol. 1996 Jul;178(13):3771–3778. doi: 10.1128/jb.178.13.3771-3778.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tommassen J., de Vrije T., de Cock H., Bosch D., de Kruijff B. Involvement of membrane lipids in protein export in Escherichia coli. J Cell Sci Suppl. 1989;11:73–83. doi: 10.1242/jcs.1989.supplement_11.6. [DOI] [PubMed] [Google Scholar]
  48. Wandersman C., Létoffé S. Involvement of lipopolysaccharide in the secretion of Escherichia coli alpha-haemolysin and Erwinia chrysanthemi proteases. Mol Microbiol. 1993 Jan;7(1):141–150. doi: 10.1111/j.1365-2958.1993.tb01105.x. [DOI] [PubMed] [Google Scholar]
  49. Wandersman C. Secretion across the bacterial outer membrane. Trends Genet. 1992 Sep;8(9):317–322. doi: 10.1016/0168-9525(92)90264-5. [DOI] [PubMed] [Google Scholar]
  50. Winkler U., Timmis K. Pleiotropic mutations in Serratia marcescens which increase the synthesis of certain exocellular proteins and the rate of spontaneous prophage induction. Mol Gen Genet. 1973 Aug 17;124(3):197–206. doi: 10.1007/BF00293091. [DOI] [PubMed] [Google Scholar]
  51. Wong K. R., Buckley J. T. Proton motive force involved in protein transport across the outer membrane of Aeromonas salmonicida. Science. 1989 Nov 3;246(4930):654–656. doi: 10.1126/science.2814486. [DOI] [PubMed] [Google Scholar]
  52. Yu J., Webb H., Hirst T. R. A homologue of the Escherichia coli DsbA protein involved in disulphide bond formation is required for enterotoxin biogenesis in Vibrio cholerae. Mol Microbiol. 1992 Jul;6(14):1949–1958. doi: 10.1111/j.1365-2958.1992.tb01368.x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES