Abstract
The Helicobacter pylori pss gene, coding for phosphatidylserine synthase (PSS), was cloned and sequenced in this study. A polypeptide of 237 amino acids was deduced from the PSS sequence. H. pylori PSS exhibits significant amino acid sequence identity with the PSS proteins found in the archaebacterium Methanococcus jannaschii, the gram-positive bacterium Bacillus subtilis, and the yeast Saccharomyces cerevisiae but none with its Escherichia coli counterpart. Expression of the putative pss gene in maxicells gave rise to a product of approximately 26 kDa, which is in agreement with the predicted molecular mass of 26,617 Da. A manganese-dependent PSS activity was found in the membrane fractions of the E. coli cells overexpressing the H. pylori pss gene product. This result indicates that this enzyme is a membrane-bound protein, a conclusion which is supported by the fact that the PSS protein contains several local hydrophobic segments which could form transmembrane helices. The pss gene was inactivated with a chloramphenicol acetyltransferase cassette on the plasmid. However, an isogenic pss gene-disrupted mutant of H. pylori UA802 could not be obtained, suggesting that this enzyme plays an essential role in the growth of this organism.
Full Text
The Full Text of this article is available as a PDF (320.3 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Bae-Lee M. S., Carman G. M. Phosphatidylserine synthesis in Saccharomyces cerevisiae. Purification and characterization of membrane-associated phosphatidylserine synthase. J Biol Chem. 1984 Sep 10;259(17):10857–10862. [PubMed] [Google Scholar]
- Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bult C. J., White O., Olsen G. J., Zhou L., Fleischmann R. D., Sutton G. G., Blake J. A., FitzGerald L. M., Clayton R. A., Gocayne J. D. Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science. 1996 Aug 23;273(5278):1058–1073. doi: 10.1126/science.273.5278.1058. [DOI] [PubMed] [Google Scholar]
- DeChavigny A., Heacock P. N., Dowhan W. Sequence and inactivation of the pss gene of Escherichia coli. Phosphatidylethanolamine may not be essential for cell viability. J Biol Chem. 1991 Mar 15;266(8):5323–5332. [PubMed] [Google Scholar]
- Dutt A., Dowhan W. Characterization of a membrane-associated cytidine diphosphate-diacylglycerol-dependent phosphatidylserine synthase in bacilli. J Bacteriol. 1981 Aug;147(2):535–542. doi: 10.1128/jb.147.2.535-542.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ezaki T., Takeuchi N., Liu S. L., Kai A., Yamamoto H., Yabuuchi E. Small-scale DNA preparation for rapid genetic identification of Campylobacter species without radioisotope. Microbiol Immunol. 1988;32(2):141–150. doi: 10.1111/j.1348-0421.1988.tb01373.x. [DOI] [PubMed] [Google Scholar]
- Ge Z., Hiratsuka K., Taylor D. E. Nucleotide sequence and mutational analysis indicate that two Helicobacter pylori genes encode a P-type ATPase and a cation-binding protein associated with copper transport. Mol Microbiol. 1995 Jan;15(1):97–106. doi: 10.1111/j.1365-2958.1995.tb02224.x. [DOI] [PubMed] [Google Scholar]
- Ge Z., Taylor D. E. Helicobacter pylori genes hpcopA and hpcopP constitute a cop operon involved in copper export. FEMS Microbiol Lett. 1996 Dec 1;145(2):181–188. doi: 10.1111/j.1574-6968.1996.tb08575.x. [DOI] [PubMed] [Google Scholar]
- Ge Z., Taylor D. E. Sequencing, expression, and genetic characterization of the Helicobacter pylori ftsH gene encoding a protein homologous to members of a novel putative ATPase family. J Bacteriol. 1996 Nov;178(21):6151–6157. doi: 10.1128/jb.178.21.6151-6157.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Gopalakrishnan A. S., Chen Y. C., Temkin M., Dowhan W. Structure and expression of the gene locus encoding the phosphatidylglycerophosphate synthase of Escherichia coli. J Biol Chem. 1986 Jan 25;261(3):1329–1338. [PubMed] [Google Scholar]
- Hamamatsu S., Shibuya I., Takagi M., Ohta A. Loss of phosphatidylserine synthesis results in aberrant solute sequestration and vacuolar morphology in Saccharomyces cerevisiae. FEBS Lett. 1994 Jul 4;348(1):33–36. doi: 10.1016/0014-5793(94)00576-1. [DOI] [PubMed] [Google Scholar]
- Hikiji T., Miura K., Kiyono K., Shibuya I., Ohta A. Disruption of the CHO1 gene encoding phosphatidylserine synthase in Saccharomyces cerevisiae. J Biochem. 1988 Dec;104(6):894–900. doi: 10.1093/oxfordjournals.jbchem.a122579. [DOI] [PubMed] [Google Scholar]
- Hirabayashi T., Larson T. J., Dowhan W. Membrane-associated phosphatidylglycerophosphate synthetase from Escherichia coli: purification by substrate affinity chromatography on cytidine 5'-diphospho-1,2-diacyl-sn-glycerol sepharose. Biochemistry. 1976 Nov 30;15(24):5205–5211. doi: 10.1021/bi00669a002. [DOI] [PubMed] [Google Scholar]
- Janitor M., Jarosch E., Schweyen R. J., Subík J. Molecular characterization of the PEL1 gene encoding a putative phosphatidylserine synthase. Yeast. 1995 Oct;11(13):1223–1231. doi: 10.1002/yea.320111302. [DOI] [PubMed] [Google Scholar]
- Janitor M., Obernauerová M., Kohlwein S. D., Subík J. The pel1 mutant of Saccharomyces cerevisiae is deficient in cardiolipin and does not survive the disruption of the CHO1 gene encoding phosphatidylserine synthase. FEMS Microbiol Lett. 1996 Jun 15;140(1):43–47. doi: 10.1111/j.1574-6968.1996.tb08312.x. [DOI] [PubMed] [Google Scholar]
- Jiang Q., Hiratsuka K., Taylor D. E. Variability of gene order in different Helicobacter pylori strains contributes to genome diversity. Mol Microbiol. 1996 May;20(4):833–842. doi: 10.1111/j.1365-2958.1996.tb02521.x. [DOI] [PubMed] [Google Scholar]
- KANFER J., KENNEDY E. P. METABOLISM AND FUNCTION OF BACTERIAL LIPIDS. II. BIOSYNTHESIS OF PHOSPHOLIPIDS IN ESCHERICHIA COLI. J Biol Chem. 1964 Jun;239:1720–1726. [PubMed] [Google Scholar]
- 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]
- Larson T. J., Dowhan W. Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol. Biochemistry. 1976 Nov 30;15(24):5212–5218. doi: 10.1021/bi00669a003. [DOI] [PubMed] [Google Scholar]
- Larson T. J., Hirabayshi T., Dowhan W. Phosphatidylglycerol biosynthesis in Bacillus licheniformis Resolution of membrane-bound enzymes by affinity chromatography on cytidinediphospho-sn-1,2-diacylglycerol Sepharose. Biochemistry. 1976 Mar 9;15(5):974–979. doi: 10.1021/bi00650a005. [DOI] [PubMed] [Google Scholar]
- Letts V. A., Klig L. S., Bae-Lee M., Carman G. M., Henry S. A. Isolation of the yeast structural gene for the membrane-associated enzyme phosphatidylserine synthase. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7279–7283. doi: 10.1073/pnas.80.23.7279. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Louie K., Chen Y. C., Dowhan W. Substrate-induced membrane association of phosphatidylserine synthase from Escherichia coli. J Bacteriol. 1986 Mar;165(3):805–812. doi: 10.1128/jb.165.3.805-812.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Louie K., Dowhan W. Investigations on the association of phosphatidylserine synthase with the ribosomal component from Escherichia coli. J Biol Chem. 1980 Feb 10;255(3):1124–1127. [PubMed] [Google Scholar]
- Marshall B. J., Warren J. R. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984 Jun 16;1(8390):1311–1315. doi: 10.1016/s0140-6736(84)91816-6. [DOI] [PubMed] [Google Scholar]
- Nikawa J., Kodaki T., Yamashita S. Primary structure and disruption of the phosphatidylinositol synthase gene of Saccharomyces cerevisiae. J Biol Chem. 1987 Apr 5;262(10):4876–4881. [PubMed] [Google Scholar]
- Nikawa J., Tsukagoshi Y., Kodaki T., Yamashita S. Nucleotide sequence and characterization of the yeast PSS gene encoding phosphatidylserine synthase. Eur J Biochem. 1987 Aug 17;167(1):7–12. doi: 10.1111/j.1432-1033.1987.tb13297.x. [DOI] [PubMed] [Google Scholar]
- Nishijima S., Asami Y., Uetake N., Yamagoe S., Ohta A., Shibuya I. Disruption of the Escherichia coli cls gene responsible for cardiolipin synthesis. J Bacteriol. 1988 Feb;170(2):775–780. doi: 10.1128/jb.170.2.775-780.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ohta A., Shibuya I. Membrane phospholipid synthesis and phenotypic correlation of an Escherichia coli pss mutant. J Bacteriol. 1977 Nov;132(2):434–443. doi: 10.1128/jb.132.2.434-443.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Okada M., Matsuzaki H., Shibuya I., Matsumoto K. Cloning, sequencing, and expression in Escherichia coli of the Bacillus subtilis gene for phosphatidylserine synthase. J Bacteriol. 1994 Dec;176(24):7456–7461. doi: 10.1128/jb.176.24.7456-7461.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Parsonnet J., Friedman G. D., Vandersteen D. P., Chang Y., Vogelman J. H., Orentreich N., Sibley R. K. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med. 1991 Oct 17;325(16):1127–1131. doi: 10.1056/NEJM199110173251603. [DOI] [PubMed] [Google Scholar]
- Parsonnet J., Hansen S., Rodriguez L., Gelb A. B., Warnke R. A., Jellum E., Orentreich N., Vogelman J. H., Friedman G. D. Helicobacter pylori infection and gastric lymphoma. N Engl J Med. 1994 May 5;330(18):1267–1271. doi: 10.1056/NEJM199405053301803. [DOI] [PubMed] [Google Scholar]
- Raetz C. R., Carman G. M., Dowhan W., Jiang R. T., Waszkuc W., Loffredo W., Tsai M. D. Phospholipids chiral at phosphorus. Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast. Biochemistry. 1987 Jun 30;26(13):4022–4027. doi: 10.1021/bi00387a042. [DOI] [PubMed] [Google Scholar]
- Raetz C. R., Kantor G. D., Nishijima M., Newman K. F. Cardiolipin accumulation in the inner and outer membranes of Escherichia coli mutants defective in phosphatidylserine synthetase. J Bacteriol. 1979 Aug;139(2):544–551. doi: 10.1128/jb.139.2.544-551.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Raetz C. R., Kennedy E. P. The association of phosphatidylserine synthetase with ribosomes in extracts of Escherichia coli. J Biol Chem. 1972 Apr 10;247(7):2008–2014. [PubMed] [Google Scholar]
- Sancar A., Hack A. M., Rupp W. D. Simple method for identification of plasmid-coded proteins. J Bacteriol. 1979 Jan;137(1):692–693. doi: 10.1128/jb.137.1.692-693.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shi W., Bogdanov M., Dowhan W., Zusman D. R. The pss and psd genes are required for motility and chemotaxis in Escherichia coli. J Bacteriol. 1993 Dec;175(23):7711–7714. doi: 10.1128/jb.175.23.7711-7714.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shibuya I., Miyazaki C., Ohta A. Alteration of phospholipid composition by combined defects in phosphatidylserine and cardiolipin synthases and physiological consequences in Escherichia coli. J Bacteriol. 1985 Mar;161(3):1086–1092. doi: 10.1128/jb.161.3.1086-1092.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shine J., Dalgarno L. The 3'-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci U S A. 1974 Apr;71(4):1342–1346. doi: 10.1073/pnas.71.4.1342. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Turner R. J., Weiner J. H. Evaluation of transmembrane helix prediction methods using the recently defined NMR structures of the coat proteins from bacteriophages M13 and Pf1. Biochim Biophys Acta. 1993 Sep 3;1202(1):161–168. doi: 10.1016/0167-4838(93)90078-6. [DOI] [PubMed] [Google Scholar]
- Usui M., Sembongi H., Matsuzaki H., Matsumoto K., Shibuya I. Primary structures of the wild-type and mutant alleles encoding the phosphatidylglycerophosphate synthase of Escherichia coli. J Bacteriol. 1994 Jun;176(11):3389–3392. doi: 10.1128/jb.176.11.3389-3392.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wang Y., Roos K. P., Taylor D. E. Transformation of Helicobacter pylori by chromosomal metronidazole resistance and by a plasmid with a selectable chloramphenicol resistance marker. J Gen Microbiol. 1993 Oct;139(10):2485–2493. doi: 10.1099/00221287-139-10-2485. [DOI] [PubMed] [Google Scholar]
- Wang Y., Taylor D. E. Chloramphenicol resistance in Campylobacter coli: nucleotide sequence, expression, and cloning vector construction. Gene. 1990 Sep 28;94(1):23–28. doi: 10.1016/0378-1119(90)90463-2. [DOI] [PubMed] [Google Scholar]
- Whelan K. F., Sherburne R. K., Taylor D. E. Characterization of a region of the IncHI2 plasmid R478 which protects Escherichia coli from toxic effects specified by components of the tellurite, phage, and colicin resistance cluster. J Bacteriol. 1997 Jan;179(1):63–71. doi: 10.1128/jb.179.1.63-71.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- von Heijne G. Membrane protein structure prediction. Hydrophobicity analysis and the positive-inside rule. J Mol Biol. 1992 May 20;225(2):487–494. doi: 10.1016/0022-2836(92)90934-c. [DOI] [PubMed] [Google Scholar]