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. 1994 Oct;176(20):6312–6323. doi: 10.1128/jb.176.20.6312-6323.1994

Characterization of mutants of Caulobacter crescentus defective in surface attachment of the paracrystalline surface layer.

S G Walker 1, D N Karunaratne 1, N Ravenscroft 1, J Smit 1
PMCID: PMC196973  PMID: 7929003

Abstract

Strains of Caulobacter crescentus express a paracrystalline surface layer (S-layer) consisting of the protein RsaA. Mutants of C. crescentus NA1000 and CB2, isolated for their ability to grow in the absence of calcium ions, uniformly no longer had the S-layer attached to the cell surface. However, RsaA was still produced, and when colonies grown on calcium-sufficient medium were examined, large two-dimensional arrays of S-layer were found intermixed with the cells. Such arrays were not found in calcium-deficient medium even when high levels of magnesium ions were provided. The arrays could be disrupted with divalent ion chelators and more readily with the calcium-selective ethylene glycol-bis (beta-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA). Thus, the outer membrane surface was not needed as a template for self-assembly, but calcium likely was. The cell surface and S-layer gene of assembly-defective mutants of NA1000 were examined to determine the basis of the S-layer surface attachment defect. Mutants had no detectable alteration in the rough lipopolysaccharide (LPS) or a characterized capsular polysaccharide, but another polysaccharide molecule was greatly reduced or absent in all calcium-independent mutants. The molecule was shown to be a smooth LPS with a core sugar and fatty acid complement identical to those of the rough LPS and an O polysaccharide of homogeneous length, tentatively considered to be composed of 4,6-dideoxy-4-amino hexose, 3,6-dideoxy-3-amino hexose, and glycerol in equal proportions. This molecule (termed SLPS) was detectable by surface labeling with a specific antiserum only when the S-layer was not present. The rsaA genes from three calcium-independent mutants were cloned and expressed in an S-layer-negative, SLPS-positive strain. A normal S-layer was produced, ruling out defects in rsaA in these cases. It is proposed that SLPS is required for S-layer surface attachment, possibly via calcium bridging. The data support the possibility that calcium binding is required to prevent an otherwise lethal effect of SLPS. If true, mutations that eliminate the O polysaccharide of SLPS eliminate the lethal effects of calcium-deprived SLPS, at the expense of S-layer attachment.

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Selected References

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

  1. Anast Nick, Smit John. Isolation and Characterization of Marine Caulobacters and Assessment of Their Potential for Genetic Experimentation. Appl Environ Microbiol. 1988 Mar;54(3):809–817. doi: 10.1128/aem.54.3.809-817.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bagdasarian M., Lurz R., Rückert B., Franklin F. C., Bagdasarian M. M., Frey J., Timmis K. N. Specific-purpose plasmid cloning vectors. II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for gene cloning in Pseudomonas. Gene. 1981 Dec;16(1-3):237–247. doi: 10.1016/0378-1119(81)90080-9. [DOI] [PubMed] [Google Scholar]
  3. Barany F. Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering. Gene. 1985;37(1-3):111–123. doi: 10.1016/0378-1119(85)90263-x. [DOI] [PubMed] [Google Scholar]
  4. Belland R. J., Trust T. J. Synthesis, export, and assembly of Aeromonas salmonicida A-layer analyzed by transposon mutagenesis. J Bacteriol. 1985 Sep;163(3):877–881. doi: 10.1128/jb.163.3.877-881.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Beveridge T. J., Murray R. G. Dependence of the superficial layers of Spirillum putridiconchylium on Ca2+ or Sr2+. Can J Microbiol. 1976 Sep;22(9):1233–1244. doi: 10.1139/m76-183. [DOI] [PubMed] [Google Scholar]
  6. Bingle W. H., Kurtz H. D., Jr, Smit J. An "all-purpose" cellulase reporter for gene fusion studies and application to the paracrystalline surface (S)-layer protein of Caulobacter crescentus. Can J Microbiol. 1993 Jan;39(1):70–80. doi: 10.1139/m93-010. [DOI] [PubMed] [Google Scholar]
  7. Brent R., Ptashne M. The lexA gene product represses its own promoter. Proc Natl Acad Sci U S A. 1980 Apr;77(4):1932–1936. doi: 10.1073/pnas.77.4.1932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. COHEN-BAZIRE G., SISTROM W. R., STANIER R. Y. Kinetic studies of pigment synthesis by non-sulfur purple bacteria. J Cell Physiol. 1957 Feb;49(1):25–68. doi: 10.1002/jcp.1030490104. [DOI] [PubMed] [Google Scholar]
  9. Caroff M., Bundle D. R., Perry M. B. Structure of the O-chain of the phenol-phase soluble cellular lipopolysaccharide of Yersinia enterocolitica serotype O:9. Eur J Biochem. 1984 Feb 15;139(1):195–200. doi: 10.1111/j.1432-1033.1984.tb07994.x. [DOI] [PubMed] [Google Scholar]
  10. Chart H., Shaw D. H., Ishiguro E. E., Trust T. J. Structural and immunochemical homogeneity of Aeromonas salmonicida lipopolysaccharide. J Bacteriol. 1984 Apr;158(1):16–22. doi: 10.1128/jb.158.1.16-22.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Darveau R. P., Hancock R. E. Procedure for isolation of bacterial lipopolysaccharides from both smooth and rough Pseudomonas aeruginosa and Salmonella typhimurium strains. J Bacteriol. 1983 Aug;155(2):831–838. doi: 10.1128/jb.155.2.831-838.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dooley J. S., Lallier R., Shaw D. H., Trust T. J. Electrophoretic and immunochemical analyses of the lipopolysaccharides from various strains of Aeromonas hydrophila. J Bacteriol. 1985 Oct;164(1):263–269. doi: 10.1128/jb.164.1.263-269.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Dooley J. S., Trust T. J. Surface protein composition of Aeromonas hydrophila strains virulent for fish: identification of a surface array protein. J Bacteriol. 1988 Feb;170(2):499–506. doi: 10.1128/jb.170.2.499-506.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Edwards P., Smit J. A transducing bacteriophage for Caulobacter crescentus uses the paracrystalline surface layer protein as a receptor. J Bacteriol. 1991 Sep;173(17):5568–5572. doi: 10.1128/jb.173.17.5568-5572.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Garduño R. A., Phipps B. M., Baumeister W., Kay W. W. Novel structural patterns in divalent cation-depleted surface layers of Aeromonas salmonicida. J Struct Biol. 1992 Nov-Dec;109(3):184–195. doi: 10.1016/1047-8477(92)90030-e. [DOI] [PubMed] [Google Scholar]
  16. Gilchrist A., Fisher J. A., Smit J. Nucleotide sequence analysis of the gene encoding the Caulobacter crescentus paracrystalline surface layer protein. Can J Microbiol. 1992 Mar;38(3):193–202. doi: 10.1139/m92-033. [DOI] [PubMed] [Google Scholar]
  17. Gilchrist A., Smit J. Transformation of freshwater and marine caulobacters by electroporation. J Bacteriol. 1991 Jan;173(2):921–925. doi: 10.1128/jb.173.2.921-925.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Godchaux W., 3rd, Gorski L., Leadbetter E. R. Outer membrane polysaccharide deficiency in two nongliding mutants of Cytophaga johnsonae. J Bacteriol. 1990 Mar;172(3):1250–1255. doi: 10.1128/jb.172.3.1250-1255.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hitchcock P. J. Aberrant migration of lipopolysaccharide in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Eur J Biochem. 1983 Jul 1;133(3):685–688. doi: 10.1111/j.1432-1033.1983.tb07517.x. [DOI] [PubMed] [Google Scholar]
  20. Hitchcock P. J., Brown T. M. Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels. J Bacteriol. 1983 Apr;154(1):269–277. doi: 10.1128/jb.154.1.269-277.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kabir S. Composition and immunochemical properties of the cell surface proteins of Vibrio cholerae. J Gen Microbiol. 1986 Aug;132(8):2235–2242. doi: 10.1099/00221287-132-8-2235. [DOI] [PubMed] [Google Scholar]
  22. Kido N., Ohta M., Kato N. Detection of lipopolysaccharides by ethidium bromide staining after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J Bacteriol. 1990 Feb;172(2):1145–1147. doi: 10.1128/jb.172.2.1145-1147.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kokka R. P., Lindquist D., Abbott S. L., Janda J. M. Structural and pathogenic properties of Aeromonas schubertii. Infect Immun. 1992 May;60(5):2075–2082. doi: 10.1128/iai.60.5.2075-2082.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Loenen W. A., Brammar W. J. A bacteriophage lambda vector for cloning large DNA fragments made with several restriction enzymes. Gene. 1980 Aug;10(3):249–259. doi: 10.1016/0378-1119(80)90054-2. [DOI] [PubMed] [Google Scholar]
  25. Mandatori R., Penner J. L. Structural and antigenic properties of Campylobacter coli lipopolysaccharides. Infect Immun. 1989 Nov;57(11):3506–3511. doi: 10.1128/iai.57.11.3506-3511.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Merker R. I., Smit J. Characterization of the adhesive holdfast of marine and freshwater caulobacters. Appl Environ Microbiol. 1988 Aug;54(8):2078–2085. doi: 10.1128/aem.54.8.2078-2085.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Merril C. R., Goldman D., Sedman S. A., Ebert M. H. Ultrasensitive stain for proteins in polyacrylamide gels shows regional variation in cerebrospinal fluid proteins. Science. 1981 Mar 27;211(4489):1437–1438. doi: 10.1126/science.6162199. [DOI] [PubMed] [Google Scholar]
  28. Messner P., Sleytr U. B. Crystalline bacterial cell-surface layers. Adv Microb Physiol. 1992;33:213–275. doi: 10.1016/s0065-2911(08)60218-0. [DOI] [PubMed] [Google Scholar]
  29. POINDEXTER J. S. BIOLOGICAL PROPERTIES AND CLASSIFICATION OF THE CAULOBACTER GROUP. Bacteriol Rev. 1964 Sep;28:231–295. doi: 10.1128/br.28.3.231-295.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Poindexter J. S. Selection for nonbuoyant morphological mutants of Caulobacter crescentus. J Bacteriol. 1978 Sep;135(3):1141–1145. doi: 10.1128/jb.135.3.1141-1145.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Preston M. A., Penner J. L. Structural and antigenic properties of lipopolysaccharides from serotype reference strains of Campylobacter jejuni. Infect Immun. 1987 Aug;55(8):1806–1812. doi: 10.1128/iai.55.8.1806-1812.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Quigley N. B., Reeves P. R. Chloramphenicol resistance cloning vector based on pUC9. Plasmid. 1987 Jan;17(1):54–57. doi: 10.1016/0147-619x(87)90008-4. [DOI] [PubMed] [Google Scholar]
  33. Ravenscroft N., Walker S. G., Dutton G. G., Smit J. Identification, isolation, and structural studies of extracellular polysaccharides produced by Caulobacter crescentus. J Bacteriol. 1991 Sep;173(18):5677–5684. doi: 10.1128/jb.173.18.5677-5684.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ravenscroft N., Walker S. G., Dutton G. G., Smith J. Identification, isolation, and structural studies of the outer membrane lipopolysaccharide of Caulobacter crescentus. J Bacteriol. 1992 Dec;174(23):7595–7605. doi: 10.1128/jb.174.23.7595-7605.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Remaut E., Stanssens P., Fiers W. Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda. Gene. 1981 Oct;15(1):81–93. doi: 10.1016/0378-1119(81)90106-2. [DOI] [PubMed] [Google Scholar]
  36. Smit J., Agabian N. Cell surface patterning and morphogenesis: biogenesis of a periodic surface array during Caulobacter development. J Cell Biol. 1982 Oct;95(1):41–49. doi: 10.1083/jcb.95.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Smit J., Agabian N. Cloning of the major protein of the Caulobacter crescentus periodic surface layer: detection and characterization of the cloned peptide by protein expression assays. J Bacteriol. 1984 Dec;160(3):1137–1145. doi: 10.1128/jb.160.3.1137-1145.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Smit J., Engelhardt H., Volker S., Smith S. H., Baumeister W. The S-layer of Caulobacter crescentus: three-dimensional image reconstruction and structure analysis by electron microscopy. J Bacteriol. 1992 Oct;174(20):6527–6538. doi: 10.1128/jb.174.20.6527-6538.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Smit J., Grano D. A., Glaeser R. M., Agabian N. Periodic surface array in Caulobacter crescentus: fine structure and chemical analysis. J Bacteriol. 1981 Jun;146(3):1135–1150. doi: 10.1128/jb.146.3.1135-1150.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Tsai C. M., Frasch C. E. A sensitive silver stain for detecting lipopolysaccharides in polyacrylamide gels. Anal Biochem. 1982 Jan 1;119(1):115–119. doi: 10.1016/0003-2697(82)90673-x. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Walker S. G., Smith S. H., Smit J. Isolation and comparison of the paracrystalline surface layer proteins of freshwater caulobacters. J Bacteriol. 1992 Mar;174(6):1783–1792. doi: 10.1128/jb.174.6.1783-1792.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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