Deficiency of Autolytic Activity in and is Associated with a Decreased Permeability of the Wall Free

Abstract

Autolytic-deficient mutants of which grow as chains of non-separated bacilli have been isolated by a procedure involving filtration of mutagenized cultures through glass-sinter filters. The mutants obtained were some 80–90% deficient in both autolysins, -acetylmuramoyl--alanine amidase and endo---acetylglucosaminidase. Treatment of one of these mutants with 5 -LiCl extracted only about 20% of the protein obtained from an equivalent amount of the autolysin-containing bacilli. Similarly, a reduction of 40–60% in LiCl-extractable protein was obtained with autolytic-deficient whether these organisms lacked the autolytic enzyme or were phenotypically deficient by growth in ethanolamine-containing medium. Chromatography on Sephadex G-100 of the protein extracted from and revealed that the major difference between autolytic-deficient and parent organisms was a decrease in proteins of high molecular weight. Smaller differences were observed in a second fraction which contained low molecular weight material and proteins such as the autolysins, whose elution from the column was retarded by interaction with the Sephadex. Further examination of the fractionated protein from by sodium dodecyl sulphate-polyacrylamide gel electrophoresis confirmed that the major difference between the extracts was in the amount of protein present and did not result from marked changes in the size of the extracted proteins. These observations suggest that autolysin deficiency in and results in a change in the porosity (permeability) of the bacterial wall.

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1981-08-01
2024-03-29
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References

  1. Andrews P. 1970; Estimation of molecular size and molecular weights of biological compounds by gel filtration. Methods of Biochemical Analysis 18:1–53
    [Google Scholar]
  2. Bott K. F., Wilson G. A. 1968; Metabolic and nutritional factors influencing the development of competence for transfection of Bacillus subtilis. Bacteriological Reviews 32:370–378
    [Google Scholar]
  3. Brown W. C. 1973; Rapid methods for extracting autolysins from Bacillus subtilis. Applied Microbiology 25:295–300
    [Google Scholar]
  4. Brown W. C., Wilson C. R., Lukehart S., Young F. E., Shiflett M. A. 1976; Analysis of autolysins in temperature-sensitive mutants of Bacillus subtilis. Journal of Bacteriology 125:166–173
    [Google Scholar]
  5. Fairbanks G., Steck T. L., Wallach D. H. F. 1971; Electrophoretic analysis of the major polypeptides of the human erythrocyte membrane. Biochemistry 10:2606–2617
    [Google Scholar]
  6. Fan D. P., Beckman M. M. 1971; Mutant of Bacillus subtilis demonstrating requirement of lysis for growth. Journal of Bacteriology 105:629–636
    [Google Scholar]
  7. Fan D. P., Beckman M. M. 1972; New centrifugation technique for isolating enzymes from large cell structures: isolation and characterisation of two Bacillus subtilis autolysins. Journal of Bacteriology 109:1258–1265
    [Google Scholar]
  8. Fan D. P., Beckman M. M. 1973a; Structural differences between walls from hemispherical caps and partial septa of Bacillus subtilis. Journal of Bacteriology 114:790–797
    [Google Scholar]
  9. Fan D. P., Beckman M. M. 1973b; Micrococcus lysodeikticus bacterial walls as a substrate specific for the autolytic glycosidase of Bacillus subtilis. Journal of Bacteriology 114:804–813
    [Google Scholar]
  10. Fein J. E. 1979; Possible involvement of bacterial autolytic enzymes in flagellar morphogenesis. Journal of Bacteriology 137:933–946
    [Google Scholar]
  11. Fein J. E., Rogers H. J. 1976; Autolytic enzyme defective mutants of Bacillus subtilis 168. Journal of Bacteriology 127:1427–1442
    [Google Scholar]
  12. Fernandez-Sousa J.-M., Perez-Castells R., Rodriguez R. 1978; A simple, one-step chromatographic procedure for the purification of lysozyme. Biochimica et biophysica acta 523:430–434
    [Google Scholar]
  13. Forsberg C. W., Rogers H. J. 1971; Autolytic enzymes in growth of bacteria. Nature; London: 229272–273
    [Google Scholar]
  14. Gerhardt P., Judge J. A. 1964; Porosity of isolated cell walls of Saccharomyces cerevisiae and Bacillus megaterium. Journal of Bacteriology 87:945–951
    [Google Scholar]
  15. Ghosh B. K., Wouters J. T. M., Lampen J. O. 1971; Distribution of the sites of alkaline phosphatase(s) in vegetative cells of Bacillus subtilis. Journal of Bacteriology 108:928–937
    [Google Scholar]
  16. Giles K. W., Myers A. 1965; An improved diphenylamine method for the estimation of deoxyribonucleic acid. Nature; London: 20693
    [Google Scholar]
  17. Glenn A. R., Mandelstam J. 1971; Sporulation in Bacillus subtilis 168. Comparison of alkaline phosphatase from sporulating and vegetative cells. Biochemical Journal 123:129–138
    [Google Scholar]
  18. Hakenbeck R., Waks S., Tomasz A. 1978; Characterisation of cell wall polymers secreted into the growth medium of lysis-defective pneumococci during treatment with penicillin and other inhibitors of cell wall synthesis. Antimicrobial Agents and Chemotherapy 13:302–311
    [Google Scholar]
  19. Halvorson H., Ellias L. 1958; The purification and properties of an α-glucosidase of Saccharomyces italicus Y1225. Biochimica et biophysica acta 30:28–40
    [Google Scholar]
  20. Herbold D. R., Glaser L. 1975; Bacillus subtilis N-acetylmuramic acid l-alanine amidase. Journal of Biological Chemistry 250:1676–1682
    [Google Scholar]
  21. Höltje J.-V., Tomasz A. 1976; Purification of the pneumococcal N-acetylmuramyl-l-alanine amidase to biochemical homogeneity. Journal of Biological Chemistry 251:4199–4207
    [Google Scholar]
  22. Hranueli D., Piggot P. J., Mandelstam J. 1974; Statistical estimate of the total number of operons specific for Bacillus subtilis sporulation. Journal of Bacteriology 119:684–690
    [Google Scholar]
  23. Hughes R. C., Thurman P. F., Stokes E. 1975; Estimates of the porosity of Bacillus licheniformis and Bacillus subtilis cell walls. Zeitschrift für Immunitätsforschung 149:126–135
    [Google Scholar]
  24. Larner J., Gillespie R. E. 1956; Gastrointestinal digestion of starch. II. Properties of the intestinal carbohydrase. Journal of Biological Chemistry 223:709–726
    [Google Scholar]
  25. Marmur J. 1961; A procedure for the isolation of deoxyribonucleic acid from micro-organisms. Journal of Molecular Biology 3:208–218
    [Google Scholar]
  26. Pollock M. R. 1961; The measurement of the liberation of penicillinase from Bacillus subtilis. Journal of General Microbiology 26:239–253
    [Google Scholar]
  27. Prasad C., Freese E. 1974; Cell lysis of Bacillus subtilis caused by intracellular accumulation of glucose-1-phosphate. Journal of Bacteriology 118:1111–1122
    [Google Scholar]
  28. Razin S., Argaman M. 1963; Lysis of mycoplasma, bacterial protoplasts, spheroplasts and l-forms by various agents. Journal of General Microbiology 30:155–172
    [Google Scholar]
  29. Robson R. L., Baddiley J. 1977; Role of teichuronic acid in Bacillus licheniformis: defective autolysis due to deficiency in teichuronic acid in a novobiocin-resistant mutant. Journal of Bacteriology 129:1051–1058
    [Google Scholar]
  30. Rogers H. J. 1979; The function of bacterial autolysins. In Microbial Polysaccharides and Polysaccharases pp. 237–268 Berkeley R. C. W., Gooday G. W., Ellwood D. C. Edited by London & New York: Academic Press.;
    [Google Scholar]
  31. Scherrer R., Gerhardt P. 1971; Molecular sieving by the Bacillus megaterium cell wall and protoplast. Journal of Bacteriology 107:718–735
    [Google Scholar]
  32. Spizizen J. 1958; Transformation of biochemically deficient strains of Bacillus subtilis by deoxyribonucleate. Proceedings of the National Academy of Sciences of the United States of America 44:1072–1078
    [Google Scholar]
  33. Taku A., Gardner H. L., Fan D. P. 1975; Reconstitution of cell wall synthesis in toluene- and LiCl-treated Bacillus megaterium by addition of a soluble protein extract. Journal of Biological Chemistry 250:3375–3380
    [Google Scholar]
  34. Taylor C., Rogers H. J., Ward J. B. 1980; The autolytic endo-β-N-acetylglucosaminidase of Bacillus subtilis: purification and properties. Society for General Microbiology Quarterly 7:73–74
    [Google Scholar]
  35. Ujita S., Kimura K. 1975; Studies of glucose metabolism in Bacillus subtilis: 1. Purification of glucose-6-phosphate dehydrogenase from the vegetative cell and its properties in comparison with the spore enzyme. Journal of Biochemistry 77:197–206
    [Google Scholar]
  36. Waks S., Tomasz A. 1978; Secretion of cell wall polymers into the growth medium of lysis-defective pneumococci during treatment with penicillin and other inhibitors of cell wall synthesis. Antimicrobial Agents and Chemotherapy 13:293–301
    [Google Scholar]
  37. Whitaker J. R. 1963; Determination of molecular weights of proteins by gel filtration on Sephadex. Analytical Chemistry 35:1950–1953
    [Google Scholar]
  38. Wood D. A. W., Tristram H. 1970; Localisation in the cell and extraction of alkaline phosphatase from Bacillus subtilis. Journal of Bacteriology 104:1045–1051
    [Google Scholar]
  39. Yamane K., Yamaguchi K., Marou B. 1973; Purification and properties of a cross-reacting material related to α-amylase and biochemical comparison with the parent a-amylase. Biochimica et biophysica acta 295:323–340
    [Google Scholar]
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