Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1982 Sep;151(3):1543–1552. doi: 10.1128/jb.151.3.1543-1552.1982

Isolation and analysis of sacculi from Streptococcus sanguis.

V M Reusch Jr
PMCID: PMC220436  PMID: 7107558

Abstract

Sacculi were prepared from Streptococcus sanguis 34 by exhaustive extraction of bacteria with hot 1% sodium dodecyl sulfate-0.5% 2-mercaptoethanol. Lyophilized residue was dissociated by brief sonication to single bodies closely resembling streptococci in phase-contrast microscopic density, staining properties, and morphology. Electron micrographs revealed bodies that contained variable amounts of cellular contents and were bounded by intact cell walls. Chemical analyses of sacculi demonstrated the presence of peptidoglycan, carbohydrate, protein, and phosphate. The hexose content of sacculi varied 10-fold depending upon the composition of the growth medium. When sacculi were subjected to treatment with 5 M LiCl, 8 M urea, 40% phenol (25 degrees C), or dimethyl sulfoxide most of the nitrogen and carbohydrate present was recovered in the insoluble fraction. These data suggest that sacculi contain the cell wall fraction of the extracted bacteria and that most of the carbohydrates and proteins of sacculi are firmly bound to the insoluble fraction, which contains the peptidoglycan matrix.

Full text

PDF
1543

Images in this article

1545Image
on p.1545
1548Image
on p.1548
1549Image
on p.1549
1549Image
on p.1549

Selected References

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

  1. AVIGAD G., AMARAL D., ASENSIO C., HORECKER B. L. The D-galactose oxidase of Polyporus circinatus. J Biol Chem. 1962 Sep;237:2736–2743. [PubMed] [Google Scholar]
  2. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  3. Becker R., Helenius A., Simons K. Solubilization of the Semliki Forest virus membrane with sodium dodecyl sulfate. Biochemistry. 1975 May 6;14(9):1835–1841. doi: 10.1021/bi00680a005. [DOI] [PubMed] [Google Scholar]
  4. Berg S. P., Nesbitt D. M. Chromium oxalate: a new spin label broadening agent for use with thylakoids. Biochim Biophys Acta. 1979 Dec 6;548(3):608–615. doi: 10.1016/0005-2728(79)90068-9. [DOI] [PubMed] [Google Scholar]
  5. Boothby D., Daneo-Moore L., Shockman G. D. A rapid, guantitative, and selective estimation of radioactively labeled peptidoglycan in gram-positive bacteria. Anal Biochem. 1971 Dec;44(2):645–653. doi: 10.1016/0003-2697(71)90255-7. [DOI] [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  7. Conover M. J., Thompson J. S., Shockman G. D. Autolytic enzyme of Streptococcus faecalis: release of soluble enzyme from cell walls. Biochem Biophys Res Commun. 1966 Jun 13;23(5):713–719. doi: 10.1016/0006-291x(66)90459-1. [DOI] [PubMed] [Google Scholar]
  8. Doyle R. J., Streips U. N., Fan V. S., Brown W. C., Mobley H., Mansfield J. M. Cell wall protein in Bacillus subtilis. J Bacteriol. 1977 Jan;129(1):547–549. doi: 10.1128/jb.129.1.547-549.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Engelman D. M., Terry T. M., Morowitz H. J. Characterization of the plasma membrane of Mycoplasma laidlawii. I. Sodium dodecyl sulfate solubilization. Biochim Biophys Acta. 1967 Jul 3;135(3):381–390. doi: 10.1016/0005-2736(67)90028-4. [DOI] [PubMed] [Google Scholar]
  10. Evans M. J., Lingrel J. B. Hemoglobin messenger ribonucleic acid. Distribution of the 9S ribonucleic acid in polysomes of different sizes. Biochemistry. 1969 Mar;8(3):829–831. doi: 10.1021/bi00831a010. [DOI] [PubMed] [Google Scholar]
  11. Fein J. E., Rogers H. J. Autolytic enzyme-deficient mutants of Bacillus subtilis 168. J Bacteriol. 1976 Sep;127(3):1427–1442. doi: 10.1128/jb.127.3.1427-1442.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Helenius A., Simons K. Removal of lipids from human plasma low-density lipoprotein by detergents. Biochemistry. 1971 Jun 22;10(13):2542–2547. doi: 10.1021/bi00789a019. [DOI] [PubMed] [Google Scholar]
  13. Holden K. G., Yim N. C., Griggs L. J., Weisbach J. A. Gel electrophoresis of mucous glycoproteins. II. Effect of physical deaggregation and disulfide-bond cleavage. Biochemistry. 1971 Aug 3;10(16):3110–3113. doi: 10.1021/bi00792a020. [DOI] [PubMed] [Google Scholar]
  14. Kline B. C., Helinski D. R. F 1 sex factor of Escherichia coli. Size and purification in the form of a strand-specific relaxation complex of supercoiled deoxyribonucleic acid and protein. Biochemistry. 1971 Dec 21;10(26):4975–4980. doi: 10.1021/bi00802a022. [DOI] [PubMed] [Google Scholar]
  15. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  16. Lacks S. A., Springhorn S. S. Renaturation of enzymes after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. J Biol Chem. 1980 Aug 10;255(15):7467–7473. [PubMed] [Google Scholar]
  17. 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]
  18. McIntire F. C., Vatter A. E., Baros J., Arnold J. Mechanism of coaggregation between Actinomyces viscosus T14V and Streptococcus sanguis 34. Infect Immun. 1978 Sep;21(3):978–988. doi: 10.1128/iai.21.3.978-988.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Nachman R. L., Ferris B. Human platelet membrane protein. Biochemistry. 1970 Jan 20;9(2):200–205. doi: 10.1021/bi00804a002. [DOI] [PubMed] [Google Scholar]
  20. Neuhaus F. C., Tobin C. E., Ahlgren J. A. Membrane-wall interrelationship in Gaffkya homari: sulfhydryl sensitivity and heat lability of nascent peptidoglycan incorporation into walls. J Bacteriol. 1980 Jul;143(1):112–119. doi: 10.1128/jb.143.1.112-119.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. PARK J. T., HANCOCK R. A fractionation procedure for studies of the synthesis of cell-wall mucopeptide and of other polymers in cells of Staphylococcus aureus. J Gen Microbiol. 1960 Feb;22:249–258. doi: 10.1099/00221287-22-1-249. [DOI] [PubMed] [Google Scholar]
  22. PERKINS H. R. THE ACTION OF HOT FORMAMIDE ON BACTERIAL CELL WALLS. Biochem J. 1965 Jun;95:876–882. doi: 10.1042/bj0950876. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. ROSEN H. A modified ninhydrin colorimetric analysis for amino acids. Arch Biochem Biophys. 1957 Mar;67(1):10–15. doi: 10.1016/0003-9861(57)90241-2. [DOI] [PubMed] [Google Scholar]
  24. Ray A., Reynolds J. A., Polet H., Steinhardt J. Binding of large organic anions and neutral molecules by native bovine serum albumin. Biochemistry. 1966 Aug;5(8):2606–2616. doi: 10.1021/bi00872a019. [DOI] [PubMed] [Google Scholar]
  25. Reusch V. M., Panos C. Defective synthesis of lipid intermediates for peptidoglycan formation in a stabilized L-form of Streptococcus pyogenes. J Bacteriol. 1976 Apr;126(1):300–311. doi: 10.1128/jb.126.1.300-311.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Reynolds J. A., Tanford C. Binding of dodecyl sulfate to proteins at high binding ratios. Possible implications for the state of proteins in biological membranes. Proc Natl Acad Sci U S A. 1970 Jul;66(3):1002–1007. doi: 10.1073/pnas.66.3.1002. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Reynolds J. A., Tanford C. The gross conformation of protein-sodium dodecyl sulfate complexes. J Biol Chem. 1970 Oct 10;245(19):5161–5165. [PubMed] [Google Scholar]
  28. Rosan B. Relationship of the cell wall composition of group H streptococci and Streptococcus sanguis to their serological properties. Infect Immun. 1976 Apr;13(4):1144–1153. doi: 10.1128/iai.13.4.1144-1153.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rosenberg S. A., Guidotti G. The protein of human erythrocyte membranes. I. Preparation, solubilization, and partial characterization. J Biol Chem. 1968 Apr 25;243(8):1985–1992. [PubMed] [Google Scholar]
  30. SCHIFFMAN G., KABAT E. A., THOMPSON W. IMMUNOCHEMICAL STUDIES ON BLOOD GROUPS. XXX. CLEAVAGE OF A, B, AND H BLOOD-GROUP SUBSTANCES BY ALKALI. Biochemistry. 1964 Jan;3:113–120. doi: 10.1021/bi00889a018. [DOI] [PubMed] [Google Scholar]
  31. Schleifer K. H., Kandler O. Peptidoglycan types of bacterial cell walls and their taxonomic implications. Bacteriol Rev. 1972 Dec;36(4):407–477. doi: 10.1128/br.36.4.407-477.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Shirey T., Huang R. C. Use of sodium dodecyl sulfate, alone, to separate chromatin proteins from deoxyribonucleoprotein of Arbacia punctulata sperm chromatin. Biochemistry. 1969 Oct;8(10):4138–4148. doi: 10.1021/bi00838a035. [DOI] [PubMed] [Google Scholar]
  33. Shockman G. D., Thompson J. S., Conover M. J. The autolytic enzyme system of Streptococcus faecalis. II. Partial characterization of the autolysin and its substrate. Biochemistry. 1967 Apr;6(4):1054–1065. doi: 10.1021/bi00856a014. [DOI] [PubMed] [Google Scholar]
  34. TREVELYAN W. E., PROCTER D. P., HARRISON J. S. Detection of sugars on paper chromatograms. Nature. 1950 Sep 9;166(4219):444–445. doi: 10.1038/166444b0. [DOI] [PubMed] [Google Scholar]
  35. Tsien H. C., Shockman G. D., Higgins M. L. Structural arrangement of polymers within the wall of Streptococcus faecalis. J Bacteriol. 1978 Jan;133(1):372–386. doi: 10.1128/jb.133.1.372-386.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Ward J. B. Teichoic and teichuronic acids: biosynthesis, assembly, and location. Microbiol Rev. 1981 Jun;45(2):211–243. doi: 10.1128/mr.45.2.211-243.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  38. White P. J., Gilvarg C. A teichuronic acid containing rhamnose from cell walls of Bacillus megaterium. Biochemistry. 1977 May 31;16(11):2428–2435. doi: 10.1021/bi00630a018. [DOI] [PubMed] [Google Scholar]

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

RESOURCES