Some Properties of Two Autolytic-Defective Mutants of Streptococcus faecalis ATCC 9790

Staphylococcus aureus, which usually forms grape-like clusters, has the ability to form regularly arranged cell packets. These regular cell packets are formed when the activity of its separation enzyme(s) is lost either by treatment with detergents, such as sodium dodecyl sulfate or Trition X-100, or by mutation of the cells. These cell packets consisted of 8 to 64 spherical cells that have a three-dimensional arrangement. Some irregularity in the arragement of cells in packets, however, can be observed by scanning electron microscopy. It is concluded that S. aureus fundametally divides along three definitely oriented planes that are located at right angles to each other. After cell division, the cells usually become translocated due to the action of a separation enzyme(s) to form grape-like clusters.

Section: Discussionsupporting

confidence: 59%

Formation of regular packets of Staphylococcus aureus cells

Koyama¹,

Yamada²,

Matsuhashi³

1977

“…These strains usually grow as long chains of cells in the presence or absence of novobiocin, and it was concluded that this failure to undergo normal cell separation is a consistent consequence of novobiocin resistance in this organism. Mutants exhibiting failure to complete normal cell separation have been described in Micrococcus lysodeikticus (33), B. licheniformis NCTC 6346 (15), B. subtilis (13), Streptococcus faecalis (27,29), and Staphylococcus aureus H (6). Studies on the mutants confirmed earlier suggestions (11,24) that autolytic enzymes are required for cell separation in these gram-positive organisms and that the chain morphology occurs as a direct result of a deficiency in autolytic activity.…”

mentioning

confidence: 53%

Role of teichuronic acid in Bacillus licheniformis: defective autolysis due to deficiency of teichuronic acid in a novobiocin-resistant mutant

Robson¹,

Baddiley²

1977

nov-12, a novobiocin-resistant mutant of Bacillus licheniformis ATCC 9945, grows as long chains of cells, a characteristic of autolytic-deficient (Lyt-) mutants. Isolated walls from nov-12 autolyzed at a rate equal to 5% of that displayed by wild-type walls, thus confirming the Lyt- phenotype. Protein-free nov-12 walls displayed marked resistance to, and also failure to bind, added autolysin solubilized from wild-type walls. Comparison of isolated cell walls revealed a deficiency in teichuronic acid in the mutant. Lesser differences were observed in walls of this strain, including a reduction in galactose, an increase in the proportion of peptidoglycan, and small quantitative differences in peptidoglycan composition though the proportions of protein and teichoic acid were similar in walls of both strains. Autolytic sensitivity was studied in walls in which protein, teichoic acid, and teichuronic acid were removed successively by selective extraction procedures. Autolysis of wild-type walls was unaffected by removal or protein or teichoic acid, but teichuronic acid removal rendered wild-type walls as insensitive to autolysis as mutant walls had been throughout. Therefore, in this mutant, deficiency in teichuronic acid alone leads to the Lyt- phenotype and, hence, activity and binding of autolysin(s) are dependent upon teichuronic acid but not teichoic acid. Also, the potential rate of autolysis of cell walls in this organism was correlated with the proportion of teichuronic acid in the wall. The possible significance of these findings with respect to control of autolysis and cell separation is discussed.

“…N-Acetylmuramyl(MurNAc)-L-alanine amidase, which hydrolyzes the amide linkage between glycan and peptide in peptidoglycan, is one of the major cell wall lytic enzymes in grampositive bacteria and also is responsible for postfissional separation of cells (2,5,10). On the other hand, the enzyme with a similar name in gram-negative bacteria does not hydrolyze cell wall peptidoglycan but only splits the MurNAc-L-alanine linkage of smaller molecules that do not contain N-acetylglucosamine (13).…”

mentioning

confidence: 99%

Mutation of the N-acetylmuramyl-L-alanine amidase gene of Escherichia coli K-12

Tomioka¹,

Nikaido²,

Miyakawa³

et al. 1983

Mutants of Escherichia coli with very low N-acetylmuramyl-L-alanine amidase activity were isolated. The gene amiA responsible for most of this enzyme activity was mapped at 51 min on the E. coli chromosome, with the most plausible gene order assumed to be amiA pts(H or I) purC. The mutant phenotype was recessive and physiologically undiscemible. N-Acetylmuramyl(MurNAc)-L-alanine amidase, which hydrolyzes the amide linkage between glycan and peptide in peptidoglycan, is one of the major cell wall lytic enzymes in grampositive bacteria and also is responsible for postfissional separation of cells (2, 5, 10). On the other hand, the enzyme with a similar name in gram-negative bacteria does not hydrolyze cell wall peptidoglycan but only splits the MurNAc-L-alanine linkage of smaller molecules that do not contain N-acetylglucosamine (13). Therefore, functions of the enzyme in gram-negative bacteria are unknown. Recently, we isolated a mutant of Escherichia coli that lacks almost all the activity of this enzyme. The mutant cell was viable and showed no distinct phenotypical changes. The gene amiA, which is responsible for the enzyme mutation, was mapped, and a double mutant involving the envA mutation, which is supposed to function in regulating the level of the amidase activity, was constructed.Characterization of the MurNAc-L-alanine amidase activity that became defective by the ami4 mutation. Enzyme assay was carried out with MurNAc-tripeptide (L-Ala-D-Glu-meso-[1,7-14C]diaminopemilic acid (A2pm) as substrate.The reaction mixture for the amidase assay contained, in a final volume of 30 RI, 50 mM Tris-hydrochloride buffer (pH 7.5), 67 pmol of labeled MurNAc-tripeptide (specific activity, 50 Ci/mol) and crude cell extract (supernatant of 6,000 x g [10 min] centrifugation) containing 30 to 70 ,ug of protein. The mixture was incubated at 30°C for various times, and the reaction was terminated by boiling the mixture for 90 s. The radioactive product and substrate were separated by paper electrophoresis in 0.1 M formic acid (11 V/cm, 2 h), and the radioactivities on the paper were counted in a liquid scintillation spect Present address: Department of Fermentation Industry, University of Hiroshima, Hiroshima, Japan. trometer in toluene-2,5-diphenyloxazole-1,4bis-(5-phenyloxazolyl)benzene (1 liter:4 g:100 mg). The amidase reaction obeyed first-order kinetics until about 40% of the substrate was degraded. The amidase activity was calculated from the amounts of disappearance of substrate and appearance of the product after calibration on the basis of a first-order reaction. Evidence that the amidase activity in the mutant was not reduced by the presence of an inhibitor was obtained in an experiment with a mixture of sonic cell extracts of the parent and the mutant; the amidase activity in the amiA strain (parent) was neither inhibited nor stimulated by the presence of twice as much (as protein) of the amiA mutant cell extract.A single product was detected after incubation with cell extracts of amiA+ strains. The product tripep...