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PubMed1972

Regulation of the bacterial cell wall: analysis of a mutant of Bacillus subtilis defective in biosynthesis of teichoic acid.

Bacillus subtilis 168ts-200B is a temperature-sensitive mutant of B. subtilis 168 which grows as rods at 30 C but as irregular spheres at 45 C. Growth at the nonpermissive temperature resulted in a deficiency of teichoic acid in the cell wall. A decrease in teichoic acid synthesis coupled with the rapid turnover of this polymer led to a progressive loss until less than 20% of the level found in wild-type rods remained in spheres. Extracts of cells grown at 45 C contained amounts of the enzymes involved in the biosynthesis and glucosylation of teichoic acids that were equal to or greater than those found in normal rods. Cell walls of the spheres were deficient also in the endogenous autolytic enzyme (N-acyl muramyl-l-alanine amidase). Genetic analysis of the mutant by PBS1-mediated transduction and deoxyribonucleic acid-mediated transformation demonstrated that the lesion responsible for these effects (tag-1) is tightly linked to the genes which regulate the glucosylation of teichoic acid in the mid-portion of the chromosome of B. subtilis.

Alanine↗

PubMed1972

Autolysis of microbial cells: salt activation of autolytic enzymes in a mutant of Staphylococcus aureus.

The effect of various salts on the autolysis of cell wall of a ribitol teichoic acid-deficient mutant of Staphylococcus aureus H (strain 52A5 carrying tar-1) was compared with the parent strain. In the presence of high concentrations of certain salts such as 1.0 m NaCl, the mutant undergoes autolysis with the release of osmotically sensitive spheroplasts. The parent strain is not affected by these conditions. The stimulation of lysis is related to an activation of N-acylmuramyl-l-alanine amidase.

Amino Acids↗

PubMed1973

Effect of phosphate limitation on the morphology and wall composition of Bacillus licheniformis and its phosphoglucomutase-deficient mutants.

Two very poorly lytic mutants of Bacillus licheniformis 6346 that had no teichuronic acid or glucose in their walls were phosphoglucomutase deficient. The walls of the mutants were less autolytic, and the lesion in the phosphoglucomutase gene and the formation of lytic amidase seemed to be interrelated. When phosphoglucomutase was regained or the effects of the deficiency were circumvented by the presence of galactose in the medium, the lytic enzyme was partially regained. When subjected to growth limitation by the supply of inorganic phosphate, the mutants ceased to make teichoic acid, and their walls contained a greatly increased proportion of mucopeptide. Under these conditions they formed irregular spheres which changed back to rods when inorganic phosphate was supplied. Both wall and protein synthesis were necessary for the changes in morphology. An intermediate crescent-shaped cell was formed in the change from sphere to a rod. The possible relationship of this morphological change to the distribution of biosynthetic sites is discussed.

Amidohydrolases↗

PubMed1974

Specificity of the autolysin of Streptococcus (Diplococcus) pneumoniae.

A Streptococcus (Diplococcus) pneumoniae autolysin, partially purified from cellular autolysates, was optimally active at pH 7.0 and was stimulated by monovalent cations. Addition of autolysin to walls resulted in the appearance of only N-terminal l-alanine, whereas no glycosidase activity was observed. Walls which had been solubilized by autolysin were separated by gel filtration into a low-molecular-weight peptide containing amino acids in the same ratios found in intact walls and a high molecular fraction containing the amino acid-deficient peptidoglycan backbone. Thus, the major activity is an N-acetylmuramyl-l-alanine amidase. In addition, walls undergoing spontaneous lysis revealed no glycosidase activity but showed an increase in only N-terminal alanine. Autolysin, which was bound to walls in saline, was almost completely removed when walls were washed in distilled water, and all of the activity was recovered in the water wash fluid.

Alanine↗

PubMed1974

Cell wall turnover at the hemispherical caps of Bacillus subtilis.

Cell walls made by Bacillus subtilis bacteria grown in D(2)O medium have buoyant densities in CsCl which are different from walls made by cells grown in H(2)O medium. Cell wall turnover was studied by measuring the change in wall buoyant density after a B. subtilis culture was shifted from growth in D(2)O medium to aeration in H(2)O medium. Walls from the hemispherical caps were isolated after preferential digestion of wall from the cylindrical regions using the B. subtilis autolytic amidase. The walls from the polar regions were found to turn over extensively.

Amidohydrolases↗

PubMed1974

Autolysis in Staphylococcus aureus: preferential release of old cell walls.

The kinetics of release of old versus new cell wall in two strains of Staphylococcus aureus were studied during autolysis. In both strains the autolytic enzyme is an amidase. Cells were double labeled with (3)H and (14)C, and the distribution of radioactivity in the cell walls was monitored during autolysis. In all cases the rate of release of steady-state lable from peptidoglycan was significantly higher than that of pulse label. Identical results were obtained with whole cells or isolated cell walls. The results suggest that in S. aureus the old cell wall is preferentially released during autolysis.

Amidohydrolases↗

PubMed1975

Proteinase C (carboxypeptidase Y) mutant of yeast.

A mutant of yeast lacking proteinase C (carboxypeptidase Y) activity has been found by using a histochemical stain to screen mutagenized colonies. This defect segregates 2:2 in meiotic tetrads. Cell extracts lacked the esterolytic, amidase, and proteolytic activities associated with proteinase C. The absence of proteinase C does not affect mitotic growth and has no obvious effect on the formation of viable ascospores or meiotic segregation. The mutant grows on peptides known to be cleaved by proteinase C in vitro. This finding is consistent with the idea that other enzymes exist in vivo with overlapping substrate specificities.

Anilides↗

PubMed1976

Mechanism of autolysis of Neisseria gonorrhoeae.

The major autolysin(s) of Neisseria gonorrhoeae was solubilized from envelopes by extraction with 2% Triton X-100 containing 0.5 M NaCl. Neither Triton X-100 nor NaCl alone could effectively release the autolysin(s). The major autolysin is N-acetylmuramyl-L-alanine amidase (EC 3.5.1.28). The pH optimum for this reaction was broad, ranging from 5.5 to 8.5. Optimal hydrolysis of peptidoglycan occurred in 2% Triton X-100 in 0.1 M KCl. Attempts to purify the autolysin were unsuccessful. A rapid assay for enzyme activity was developed using radioactive cell walls as a substrate ([3H]diaminopimelic acid).

Amidohydrolases↗

PubMed1976

Autolytic enzyme-deficient mutants of Bacillus subtilis 168.

Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. These mutants grow at normal rates as very long chains of unseparated cells. The length of the chains is directly related to the growth rates. They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology. Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter. Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6. Likewise, Microcococcus luteus walls at pH 5.6 and B. subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts. Thus, the activity of both autolytic enzymes in the mutants is depressed. The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation. The mutants did not produce more protease or amylase than did the wild type. They sporulate and the spores germinate normally. The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type. The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.

Acetylglucosaminidase↗

PubMed1977

Characterization of autolysins from Mycobacterium smegmatis.

This study demonstrates, for the first time, the autolytic enzymes associated with mycobacterial cell walls. Based on the release of radioactivity and ninhydrin-reactive material from isolated cell walls, it was shown that maximum activity occurs during the late log phase of growth and at a buffer pH of about 8.0. Chemical analyses of autolytic digests of isolated cell walls indicated that at least three autolysins are active under the conditions used. These are N-glycolylmuramic acid-L-alanine amidase, an aminopeptidase that releases L-alanine, and an endopeptidase that solubilizes and L-alanyl-D-glutamic acid dippetide. No other endopeptidase, carboxypeptidase, or glycosidase activity was detected.

Alanine↗

PubMed1979

Local anesthetics block induction of the Pseudomonas alk regulon.

The local anesthetics procaine and piperocaine blocked induction of the plasmid-determined enzymatic activities involved in the metabolism of n-alkanes in Pseudomonas putida. Procaine reversibly inhibited existing alkane hydroxylase activity. Induction of a soluble aliphatic amidase activity was not affected. These results support the hypothesis that induction of the plasmid-determined alkane metabolic system in P. putida involves a membrane component(s).

Alkanes↗

PubMed1980

Bacillus cereus autolytic endoglucosaminidase active on cell wall peptidoglycan with N-unsubstituted glucosamine residues.

An autolytic glycosidase from a lysozyme-resistant strain of Bacillus cereus capable of cleaving the glycosidic linkages of N-unsubstituted glucosamine in the cell wall peptidoglycan was studied. This glycosidase activity, together with N-acetylmuramyl-L-alanine amidase activity, was found in an autolytic enzyme preparation obtained from the 20,000 x g precipitate fraction by means of autolysis followed by ammonium sulfate fractionation. The major saccharide fragments resulting from digestion of the untreated, non-N-acetylated, cell wall peptidoglycan of B. cereus with the autolytic enzyme preparation were identified as N-acetylmuramyl-glucosamine and its dimer. The peptidoglycan N-acetylated with acetic anhydride could also be digested with the same enzyme preparation, giving N-acetylmuramyl-N-acetylglucosamine and its dimer as the major saccharide fragments.

Bacillus cereus↗

PubMed1983

Cell wall-DNA association in Bacillus subtilis.

Autolysis of cell walls of Bacillus subtilis 168 resulted in solubilization of wall-associated DNA. Most of the DNA was solubilized only in the later stages of autolysis. Solubilization of up to 70% of the wall by autolysins resulted in only 25 to 30% solubilization of wall-associated DNA. When the wall fragments remaining after 70% autolysis were analyzed by electron microscopy, it was observed that the preparations were highly enriched for completed septa, or poles. Partial autolysis at pH 5.2 or pH 8.6, both of which reflect hydrogen ion levels that permit either N-acetylglucosaminidase or N-acetylmuramyl-L-alanine amidase, but not both, to act, gave rise to enrichment of cell poles. When walls were incubated with subtilisin, DNase, or RNase, release of DNA (or DNA fragments) was accelerated. Density gradient centrifugation patterns of lysates of cells pulse-labeled with N-[3H]acetylglucosamine and then chased revealed that a small, but significant, proportion of the radioactivity sedimented to a density position equivalent to that of DNA-membrane complexes. Because the pulse-chase sequence enriched for radioactivity in cell poles, the results suggest that at least some molecules from polar cell walls have an affinity for DNA-membrane complexes. We suggest that DNA binds strongly, possibly via a DNA-membrane complex, to cell poles of B. subtilis. The results provide support for a view offered previously (Koch et al., FEMS Microbiol. Lett. 12:201-208, 1981) that some special structure in or very near the poles of gram-positive bacilli is involved in the segregation of DNA during cell division.

Bacillus subtilis↗

PubMed1985

Generation of purpura-producing principle from pneumococcal cell walls.

The in vitro kinetics of muramic acid-alanine bond hydrolysis and pneumococcal purpura-producing principle generation by incubation of Streptococcus pneumoniae cell wall preparations with the bacterial autolysin N-acetylmuramyl-L-alanine amidase were similar. The generated purpura-producing principle preparation had a weight-average molecular weight of ca. 2.6 X 10(7) and possessed the glycan and teichoic acid constituents of the pneumococcal cell wall. The results support the idea that the pneumococcal purpura-producing principle is a high-molecular-weight, glycan-teichoic acid fragment released by hydrolysis of the muramic acid-alanine bonds in the bacterial cell wall.

Amino Acids↗

PubMed1985

Peptidoglycan cross-linking and teichoic acid attachment in Streptococcus pneumoniae.

Autolysin-defective pneumococci continue to synthesize both peptidoglycan and teichoic acid polymers (Fischer and Tomasz, J. Bacteriol. 157:507-513, 1984). Most of these peptidoglycan polymers are released into the surrounding medium, and a smaller portion becomes attached to the preexisting cell wall. We report here studies on the degree of cross-linking, teichoic acid substitution, and chemical composition of these peptidoglycan polymers and compare them with normal cell walls. peptidoglycan chains released from the penicillin-treated pneumococci contained no attached teichoic acids. The released peptidoglycan was hydrolyzed by M1 muramidase; over 90% of this material adsorbed to vancomycin-Sepharose and behaved like disaccharide-peptide monomers during chromatography, indicating that the released peptidoglycan contained un-cross-linked stem peptides, most of which carried the carboxy-terminal D-alanyl-D-alanine. The N-terminal residue of the released peptidoglycan was alanine, with only a minor contribution from lysine. In addition to the usual stem peptide components of pneumococcal cell walls (alanine, lysine, and glutamic acid), chemical analysis revealed the presence of significant amounts of serine, aspartate, and glycine and a high amount of alanine and glutamate as well. We suggest that these latter amino acids and the excess alanine and glutamate are present as interpeptide bridges. Heterogeneity of these was suggested by the observation that digestion of the released peptidoglycan with the pneumococcal murein hydrolase (amidase) produced peptides that were resolved by ion-exchange chromatography into two distinct peaks; the more highly mobile of these was enriched with glycine and aspartate. The peptidoglycan chains that became attached to the preexisting cell wall in the presence of penicillin contained fewer peptide cross-links and proportionally fewer attached teichoic acids than did their normal counterparts. The normal cell wall was heavily cross-linked, and the cross-linked peptides were distributed equally between the teichoic acid-linked and teichoic acid-free fragments.

Amino Acids↗

PubMed1989

Isolation, purification, and properties of Penicillium charlesii alkaline protease.

A serine protease with a pH optimum from 7 to 9 and activity over the range of pH 3 to 10 was isolated and purified from culture filtrates of Penicillium charlesii 16 days after inoculation. The enzyme was purified by the following sequence of procedures: (i) gel permeation chromatography through Sephacryl S-200, (ii) DEAE-Sepharose anion-exchange chromatography, and (iii) fast protein liquid chromatography (FPLC) over Superose 12. Anion-exchange chromatography separated the protease activity into a major activity (protease PII, 82%) and two minor activities (proteases PI and PIII, 10 and 8%, respectively, of the total activity). Protease PII has a molecular mass of 44 kilodaltons. Purified preparations of this enzyme are susceptible to autodegradation. FPLC of heat-treated PII gave one major species (PIIa), whereas untreated enzyme resulted in three species (PIIb, PIIc, and PIId). PIIb and PIIc also catalyzed the hydrolysis of protein (hide powder azure). PIIb and PIIc were in the molecular mass range of 10 to 20 kilodaltons. Protease PII is completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The protease has primary substrate specificity for phenylalanyl or arginyl amino acyl residues attached to amines. The enzyme has amidase, but no esterase activity toward similar synthetic substrates such as occurs with trypsinlike microbial serine proteases. The addition of PMSF (final concentration, 10(-4) M) to 1- and 2-day-old cultures of P. charlesii inhibited the production of extracellular peptidophosphogalactomannan (pPGM) by 41 and 34%, respectively, and inhibited the alkaline protease activity by 85%. These results suggest that the production and release of pPGM may be affected by alkaline protease.

Chromatography, High Pressure Liquid↗

PubMed1989

13N isotope studies of glutamine assimilation pathways in Neurospora crassa.

L-[amide-13N]glutamine in Neurospora crassa is metabolized to [13N]glutamate by glutamate synthase and to [13N]ammonium by the glutamine transaminase-omega-amidase pathway. The [13N]ammonium released is assimilated by glutamate dehydrogenase and glutamine synthetase, confirming the operation of a glutamine cycle. Most of the nitrogen is retained during cycling between glutamate and glutamine.

Ammonia↗

PubMed1990

Purification and characterization of a protease from Clostridium botulinum type A that nicks single-chain type A botulinum neurotoxin into the di-chain form.

A protease that nicks the approximately 150-kilodalton (kDa) single-chain type A botulinum neurotoxin into the approximately 150-kDa di-chain form in vitro was isolated from Clostridium botulinum type A (Hall strain) cultures. The di-chain neurotoxin generated in vitro is composed of an approximately 50-kDa light chain and an approximately 100-kDa heavy chain which are disulfide linked and is indistinguishable from the di-chain neurotoxin that forms in vivo and is routinely isolated (M.L. Dekleva and B.R. DasGupta, Biochem. Biophys. Res. Commun. 162:767-772, 1989). This enzyme was purified greater than 1,000-fold by ammonium sulfate precipitation, QAE-Sephadex Q-50, Sephadex G-100, and CM-Sephadex C-50 chromatography steps with the synthetic substrate N-benzoyl-DL-arginine-p-nitroanilide. The approximately 62-kDa amidase (protease) is a complex of 15.5- and 48-kDa polypeptides (determined by polyacrylamide gel electrophoresis) that could not be separated without sodium dodecyl sulfate. The enzyme has an isoelectric point of pH 5.73, a pH optimum of 6.2 to 6.4, an absolute requirement for a thiol-reducing agent as well as a divalent metallic cation (probably Ca2+) for activity, and a temperature optimum of 70 degrees C. Tests with several synthetic substrates indicated the high specificity of the enzyme for arginyl amide bonds.

Botulinum Toxins↗