Abstract
Extracellular secretion of the Serratia marcescens nuclease occurs in a two-step process: (i) rapidly to the periplasm via a signal sequence-dependent pathway and then (ii) slowly to the extracellular growth medium without cell lysis. There are two major isoforms of the nuclease in the culture supernatant of S. marcescens. We have isolated, purified, and determined the sequences of both isoforms. The first isoform, the mature nuclease (Sm2), is the result of signal sequence processing. The second isoform (Sm1) has three additional amino acids missing from the N terminus of the mature nuclease. Sm1 starts to appear extracellularly only during prolonged growth of a culture (16 to 48 h), probably because of cell lysis. However, pulse-chase experiments show that it is made early with Sm2 but is not secreted efficiently.
Full Text
The Full Text of this article is available as a PDF (655.7 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- 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]
- Andersson H., von Heijne G. A 30-residue-long "export initiation domain" adjacent to the signal sequence is critical for protein translocation across the inner membrane of Escherichia coli. Proc Natl Acad Sci U S A. 1991 Nov 1;88(21):9751–9754. doi: 10.1073/pnas.88.21.9751. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ball T. K., Saurugger P. N., Benedik M. J. The extracellular nuclease gene of Serratia marcescens and its secretion from Escherichia coli. Gene. 1987;57(2-3):183–192. doi: 10.1016/0378-1119(87)90121-1. [DOI] [PubMed] [Google Scholar]
- 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]
- Bally M., Filloux A., Akrim M., Ball G., Lazdunski A., Tommassen J. Protein secretion in Pseudomonas aeruginosa: characterization of seven xcp genes and processing of secretory apparatus components by prepilin peptidase. Mol Microbiol. 1992 May;6(9):1121–1131. doi: 10.1111/j.1365-2958.1992.tb01550.x. [DOI] [PubMed] [Google Scholar]
- Biedermann K., Jepsen P. K., Riise E., Svendsen I. Purification and characterization of a Serratia marcescens nuclease produced by Escherichia coli. Carlsberg Res Commun. 1989;54(1):17–27. doi: 10.1007/BF02910469. [DOI] [PubMed] [Google Scholar]
- Bromke B. J., Hammel J. M. Regulation of extracellular protease formation by Serratia marcescens. Can J Microbiol. 1979 Jan;25(1):47–52. doi: 10.1139/m79-008. [DOI] [PubMed] [Google Scholar]
- Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Devine J. H., Shadel G. S., Baldwin T. O. Identification of the operator of the lux regulon from the Vibrio fischeri strain ATCC7744. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5688–5692. doi: 10.1073/pnas.86.15.5688. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eaves G. N., Jeffries C. D. ISOLATION AND PROPERTIES OF AN EXOCELLULAR NUCLEASE OF SERRATIA MARCESCENS. J Bacteriol. 1963 Feb;85(2):273–278. doi: 10.1128/jb.85.2.273-278.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Filimonova M. N., Dement'ev A. A., Leshchinskaia I. B., Bakulina G. Iu, Shliapnikov S. V. Vydelenie i kharakteristika izoform vnekletochnoi nuklezy Serratia marcescens. Biokhimiia. 1991 Mar;56(3):508–520. [PubMed] [Google Scholar]
- Filimonova M. N., Krause K. L., Benedik M. J. Kinetic studies of the Serratia marcescens extracellular nuclease isoforms. Biochem Mol Biol Int. 1994 Aug;33(6):1229–1236. [PubMed] [Google Scholar]
- Grimont P. A., Grimont F. The genus Serratia. Annu Rev Microbiol. 1978;32:221–248. doi: 10.1146/annurev.mi.32.100178.001253. [DOI] [PubMed] [Google Scholar]
- He S. Y., Schoedel C., Chatterjee A. K., Collmer A. Extracellular secretion of pectate lyase by the Erwinia chrysanthemi out pathway is dependent upon Sec-mediated export across the inner membrane. J Bacteriol. 1991 Jul;173(14):4310–4317. doi: 10.1128/jb.173.14.4310-4317.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Howard S. P., Buckley J. T. Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila. J Bacteriol. 1985 Mar;161(3):1118–1124. doi: 10.1128/jb.161.3.1118-1124.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Kunkel T. A., Roberts J. D., Zakour R. A. Rapid and efficient site-specific mutagenesis without phenotypic selection. Methods Enzymol. 1987;154:367–382. doi: 10.1016/0076-6879(87)54085-x. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Laforet G. A., Kendall D. A. Functional limits of conformation, hydrophobicity, and steric constraints in prokaryotic signal peptide cleavage regions. Wild type transport by a simple polymeric signal sequence. J Biol Chem. 1991 Jan 15;266(2):1326–1334. [PubMed] [Google Scholar]
- Li P., Beckwith J., Inouye H. Alteration of the amino terminus of the mature sequence of a periplasmic protein can severely affect protein export in Escherichia coli. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7685–7689. doi: 10.1073/pnas.85.20.7685. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lory S., Tai P. C., Davis B. D. Mechanism of protein excretion by gram-negative bacteria: Pseudomonas aeruginosa exotoxin A. J Bacteriol. 1983 Nov;156(2):695–702. doi: 10.1128/jb.156.2.695-702.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lu H. M., Mizushima S., Lory S. A periplasmic intermediate in the extracellular secretion pathway of Pseudomonas aeruginosa exotoxin A. J Bacteriol. 1993 Nov;175(22):7463–7467. doi: 10.1128/jb.175.22.7463-7467.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Lyerly D. M., Kreger A. S. Importance of serratia protease in the pathogenesis of experimental Serratia marcescens pneumonia. Infect Immun. 1983 Apr;40(1):113–119. doi: 10.1128/iai.40.1.113-119.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Miller M. D., Benedik M. J., Sullivan M. C., Shipley N. S., Krause K. L. Crystallization and preliminary crystallographic analysis of a novel nuclease from Serratia marcescens. J Mol Biol. 1991 Nov 5;222(1):27–30. doi: 10.1016/0022-2836(91)90734-n. [DOI] [PubMed] [Google Scholar]
- Miller M. D., Tanner J., Alpaugh M., Benedik M. J., Krause K. L. 2.1 A structure of Serratia endonuclease suggests a mechanism for binding to double-stranded DNA. Nat Struct Biol. 1994 Jul;1(7):461–468. doi: 10.1038/nsb0794-461. [DOI] [PubMed] [Google Scholar]
- Nakahama K., Yoshimura K., Marumoto R., Kikuchi M., Lee I. S., Hase T., Matsubara H. Cloning and sequencing of Serratia protease gene. Nucleic Acids Res. 1986 Jul 25;14(14):5843–5855. doi: 10.1093/nar/14.14.5843. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Palzkill T., Le Q. Q., Wong A., Botstein D. Selection of functional signal peptide cleavage sites from a library of random sequences. J Bacteriol. 1994 Feb;176(3):563–568. doi: 10.1128/jb.176.3.563-568.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Puziss J. W., Strobel S. M., Bassford P. J., Jr Export of maltose-binding protein species with altered charge distribution surrounding the signal peptide hydrophobic core in Escherichia coli cells harboring prl suppressor mutations. J Bacteriol. 1992 Jan;174(1):92–101. doi: 10.1128/jb.174.1.92-101.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 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]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shen L. M., Lee J. I., Cheng S. Y., Jutte H., Kuhn A., Dalbey R. E. Use of site-directed mutagenesis to define the limits of sequence variation tolerated for processing of the M13 procoat protein by the Escherichia coli leader peptidase. Biochemistry. 1991 Dec 24;30(51):11775–11781. doi: 10.1021/bi00115a006. [DOI] [PubMed] [Google Scholar]
- Sokalski S. J., Jewell M. A., Asmus-Shillington A. C., Mulcahy J., Segreti J. An outbreak of Serratia marcescens in 14 adult cardiac surgical patients associated with 12-lead electrocardiogram bulbs. Arch Intern Med. 1992 Apr;152(4):841–844. [PubMed] [Google Scholar]
- Tommassen J., Filloux A., Bally M., Murgier M., Lazdunski A. Protein secretion in Pseudomonas aeruginosa. FEMS Microbiol Rev. 1992 Sep;9(1):73–90. doi: 10.1016/0378-1097(92)90336-m. [DOI] [PubMed] [Google Scholar]
- Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
- Wilfert J. N., Barrett F. F., Ewing W. H., Finland M., Kass E. H. Serratia marcescens: biochemical, serological, and epidemiological characteristics and antibiotic susceptibility of strains isolated at Boston City Hospital. Appl Microbiol. 1970 Feb;19(2):345–352. doi: 10.1128/am.19.2.345-352.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wilkowske C. J., Washington J. A., 2nd, Martin W. J., Ritts R. E., Jr Serratia marcescens. Biochemical characteristics, antibiotic susceptibility patterns, and clinical significance. JAMA. 1970 Dec 21;214(12):2157–2162. doi: 10.1001/jama.214.12.2157. [DOI] [PubMed] [Google Scholar]
- 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]
- Yamane K., Mizushima S. Introduction of basic amino acid residues after the signal peptide inhibits protein translocation across the cytoplasmic membrane of Escherichia coli. Relation to the orientation of membrane proteins. J Biol Chem. 1988 Dec 25;263(36):19690–19696. [PubMed] [Google Scholar]
- Yanagida N., Uozumi T., Beppu T. Specific excretion of Serratia marcescens protease through the outer membrane of Escherichia coli. J Bacteriol. 1986 Jun;166(3):937–944. doi: 10.1128/jb.166.3.937-944.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- von Heijne G. Net N-C charge imbalance may be important for signal sequence function in bacteria. J Mol Biol. 1986 Nov 20;192(2):287–290. doi: 10.1016/0022-2836(86)90365-7. [DOI] [PubMed] [Google Scholar]