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PubMed1987

Relation of the heat resistance of bacterial spores to chemical composition and structure. II. Relation to cortex and structure.

The relation between the amount of cortex, measured as total hexosamine, as diaminopimelic acid and as muramic lactam, and the heat resistance of spores of five different strains of Bacillus stearothermophilus was studied. Electron micrographs of thin sections of the spores were made to relate the structure of the spores to chemical and thermal characteristics. It was found that the amount of the cortex was significantly related to heat resistance of the spores. Strains with more electron-dense and better organized cortices were found to express higher heat resistance.

Amino Acids, Diamino↗
PubMed2004

Production of muramic delta-lactam in Bacillus subtilis spore peptidoglycan.

Bacterial spore heat resistance is primarily dependent upon dehydration of the spore cytoplasm, a state that is maintained by the spore peptidoglycan wall, the spore cortex. A peptidoglycan structural modification found uniquely in spores is the formation of muramic delta-lactam. Production of muramic delta-lactam in Bacillus subtilis requires removal of a peptide side chain from the N-acetylmuramic acid residue by a cwlD-encoded muramoyl-L-Alanine amidase. Expression of cwlD takes place in both the mother cell and forespore compartments of sporulating cells, though expression is expected to be required only in the mother cell, from which cortex synthesis derives. Expression of cwlD in the forespore is in a bicistronic message with the upstream gene ybaK. We show that ybaK plays no apparent role in spore peptidoglycan synthesis and that expression of cwlD in the forespore plays no significant role in spore peptidoglycan formation. Peptide cleavage by CwlD is apparently followed by deacetylation of muramic acid and lactam ring formation. The product of pdaA (yfjS), which encodes a putative deacetylase, has recently been shown to also be required for muramic delta-lactam formation. Expression of CwlD in Escherichia coli results in muramoyl L-Alanine amidase activity but no muramic delta-lactam formation. Expression of PdaA alone in E. coli had no effect on E. coli peptidoglycan structure, whereas expression of CwlD and PdaA together resulted in the formation of muramic delta-lactam. CwlD and PdaA are necessary and sufficient for muramic delta-lactam production, and no other B. subtilis gene product is required. PdaA probably carries out both deacetylation and lactam ring formation and requires the product of CwlD activity as a substrate.

Amidohydrolases↗
PubMed1976

Relationship between cortex content and properties of Bacillus sphaericus spores.

The muramic lactam content of spores of Bacillus sphaericus mutants defective in meso-diaminopimelic acid synthesis increases almost linearly with an increase of meso-diaminopimelic acid concentration in the medium. Since muramic lactam content is a measure of cortex content, the amount of cortex in spores of the mutants can be easily varied by changing the meso-diaminopimelic acid concentration in the medium. Characteristic properties were tested in spores containing different amounts of cortex. Critical amounts of cortex were associated with different spore properties. Refractility and dipicolinic acid accumulation in the spores both required about 20% of the maximum cortex content (although refractility is independent of dipicolinic acid content). For xylene octanol resistance, about 25% of the maximum cortex content was required.

Bacillus↗
PubMed1981

[Characteristics of the active and inactive variants of Actinomyces sp. 26-115, a producer of actinomycin C].

Two natural variants, i.e. No. 1 and No. 2, not producing actinomycin were isolated from cultures of the actinomycin C-producing organism Actinomyces sp. 26-115. Variant No. 1 differed from the active variant by the growth dynamics and colony morphology. Variant No. 2 was close to the active variant by the growth dynamics. It was shown with electron microscopy that the cells of variant No. 1 differed from those of the active variant in the number and form of the mycelial septa, more even and compact structure of the cell walls and higher sensitivity to actinomycin. Still, they were more stable to the effect of lysozyme and ultrasound. The cell walls of the inactive variant No. 1 gradually lost teichoic acid during development, while the loss of peptidoglycan was observed only on transfer to the stationary phase. The cell walls of the active variant lost teichoic acid and peptidoglycan at the same time on transfer to the stationary phase. Peptidoglycans of both variants contained diaminopimelic acid (the configuration of which was not determined) and glycine (1:1) as differentiating amino acids. The two adjacent tetrapeptides were joined with one glycine radical. The peptidoglycan peptide chains of both variants contained muramic, glutamic and diaminopimelic acids and alanine (1:1:1:2). The peptidoglycans of the inactive variant No. 1 contained in addition valine and isoleucine. However, it is hardly probable that they are contained by the peptidoglycan peptide chains.

Actinomyces↗
PubMed1976

Conditional spore cortex-less mutants of Bacillus sphaericus 9602.

Lysine-requiring mutants of Bacillus sphaericus 9602 were isolated and classified into three groups by their mutation site in the pathway of lysine biosynthesis. The Group I mutant lacks meso-alpha, epsilon-diaminopimelic acid (meso-Dap) decarboxylase activity, but Group II and III mutants have a normal level of Dap decarboxylase activity. A Group II mutant makes dipicolinic acid from an intermediate in the lysine pathway, but Group III mutants do not. In the absence of meso-Dap in the culture, muramic lactam content in the spore cortex of Group II and III mutants is very low, compared to the wild type content. Addition of meso-Dap to the culture causes an increase of muramic lactam content. Since meso-Dap is detectable only in the spore cortex of B. sphaericus 9602, almost all of the muramic lactam in the spore is probably also located in the cortex. Group I mutants grown in the presence of L-lysine sporulate normally. Group II and III mutants produce oval and nonrefractile spores under the same conditions but the addition of meso-Dap to the culture results in the production of round and refractile spores. Thus, the presence of cortex in the spore is essential to give the round and refractile spores in B. sphaericus. The presence of cortex is also required for the accumulation of dipicolinic acid in the sporulating cells. Furthermore, 1-octanol resistance of the spore depends only on the presence of cortex but both cortex and dipicolinic acid are required for heat resistance of the spore.

Bacillus↗
PubMed2002

A polysaccharide deacetylase gene (pdaA) is required for germination and for production of muramic delta-lactam residues in the spore cortex of Bacillus subtilis.

The predicted amino acid sequence of Bacillus subtilis yfjS (renamed pdaA) exhibits high similarity to those of several polysaccharide deacetylases. Beta-galactosidase fusion experiments and results of Northern hybridization with sporulation sigma mutants indicated that the pdaA gene is transcribed by E(sigma)(G) RNA polymerase. pdaA-deficient spores were bright by phase-contrast microscopy, and the spores were induced to germination on the addition of L-alanine. Germination-associated spore darkening, a slow and partial decrease in absorbance, and slightly lower dipicolinic acid release compared with that by the wild-type strain were observed. In particular, the release of hexosamine-containing materials was lacking in the pdaA mutant. Muropeptide analysis indicated that the pdaA-deficient spores completely lacked muramic delta-lactam. A pdaA-gfp fusion protein constructed in strain 168 and pdaA-deficient strains indicated that the protein is localized in B. subtilis spores. The biosynthetic pathway of muramic delta-lactam is discussed.

Amidohydrolases↗
PubMed1996

Fermentation of carbohydrate in rat ileal excreta is enhanced with cecal inocula compared with fecal inocula.

The differential fermentative capacities of microflora from two regions of the large bowel and how fermentation was altered by prior exposure of the microflora to the substrate to be fermented were studied using an in vitro fermentation system. Bacterial inocula were prepared from cecal contents and feces from three groups of rats fed purified diets containing 100 g/kg dietary fiber from canned peas or psyllium seed husk, or a nonpurified diet containing 170 g/kg dietary fiber. The substrate for all fermentations was ileal excreta from colectomized rats fed a purified diet containing 100 g/kg dietary fiber from canned peas. Anaerobic fermentations were conducted for 0, 3, 6, 12, 24, 48 and 96 h. Sugars of the unfermented polysaccharides were measured by gas chromatography following acid hydrolysis; disappearance was the measure of fermentation. Independent of inoculum source, > 90% of the starch and arabinose and 75% of the uronic acids, but < 30% of non-starch glucose (the measure of cellulose), were fermented by 24 h. Cecal inocula fermented arabinose and uronic acids more quickly (P < 0.05) and fermented more (P < 0.05) non-starch glucose than fecal inocula. Inocula adapted to psyllium seed husk fermented starch faster (P < 0.05) and non-starch glucose, arabinose and uronic acids more slowly (P < 0.05) than inocula adapted to peas or nonpurified diet. Bacterial efficiency of carbohydrate utilization, the increase in muramic acid/mole carbohydrate fermented, was greatest (P < 0.05) with cecal inocula adapted to peas. We conclude that using cecal microflora as the inoculum source provides a more accurate index of fermentation during transit through the large bowel and that noncellulosic and storage polysaccharides of the plant cell wall are utilized before cellulose.

Animals↗
PubMed1975

Autolysis of Neisseria gonorrhoeae.

Physiological conditions that would provide maximal rates of autolysis of Neisseria gonorrhoeae were examined. Autolysis was found to occur over a broad pH range with the optimum at pH 9.0 IN 0.05 M tris(hydroxymethyl)amino-methane-maleate buffer. The temperature optimum was found to be 40 C. Potassium ions greatly stimulated autolysis at a concentration of 0.01 M. Exposure of growing N. gonorrhoeae cells to penicillin, vancomycin, or D-cycloserine influenced the susceptibility to the autolysis, whereas chloramphenicol afforded some protection against autolysis. The primary structure of the peptidoglycan is composed of muramic acid/glutamic acid/alanine/diaminopimelic acid/glucosamine in approximate molar ratios of 1:1:2:1:1, respectively. Exogenous radioactive diaminopimelic acid, D-glucosamine, and D-alanine were incorporated into peptidoglycan. During autolysis these radioactive fragments were released from cells.

Alanine↗
PubMed1996

Methotrexate therapy for ectopic pregnancy in adolescents.

STUDY OBJECTIVE: To determine the suitability and efficacy of methotrexate (MTX) therapy for adolescent patients with ectopic pregnancy. MATERIALS: Patients with ectopic pregnancy were treated with methotrexate, 50 mg/m2 intramuscularly. Follow-up determinations of quantitative beta hCG levels were done on days 1, 4, and 7. They were then seen weekly until beta hCG levels were less than 15 mIU/ml. PARTICIPANTS: Adolescents with ectopic pregnancy (55 patients) received MTX therapy and comprised the study group. They were compared with 186 adults similarly treated. The groups were compared for treatment outcome and for compliance with the rigid protocol requirements. MAIN OUTCOME MEASURES: Treatment success was defined as a patient who required no surgical intervention and whose beta hCG levels declined to < 15 mIU/ml. RESULTS: 85% of patients required no surgical intervention following MTX injection. Surgical intervention rates were slightly higher and success rates slightly lower for adolescent patients, but these differences were not statistically significant (p = .71). Although adult patients were more likely to have a perfect compliance rating, comparison of all compliance categories showed no significant differences (p = .17). CONCLUSIONS: MTX therapy is satisfactory for adolescent patients. It is effective, and adolescent patients are able to comply with the requirements of a rigid protocol.

Adolescent↗
PubMed1996

Biochemical evidence for the formation of a covalent acyl-phosphate linkage between UDP-N-acetylmuramate and ATP in the Escherichia coli UDP-N-acetylmuramate:L-alanine ligase-catalyzed reaction.

In the peptidoglycan biosynthesis pathway in Escherichia coli, UDP-N-acetylmuramate:L-alanine ligase (MurC) catalyzes the formation of UDP-N-acetylmuramyl-L-alanine. A peptide bond is formed in this reaction and an ATP molecule is hydrolyzed concomitantly to produce ADP and orthophosphate. A biochemical approach was devised to elucidate the role of ATP in this reaction. A fusion construct pMAL::murC was prepared and the maltose binding protein--UDP-N-acetylmuramyl:L-alanine ligase fusion protein was overproduced in E. coli/pMal::murC upon isopropyl beta-thiogalactoside induction. The fusion protein was purified to > or = 90% homogeneity by a single-step affinity chromatography. Subsequently, the ligase was released from the maltose binding protein by proteolytic cleavage and was purified to > or = 95% homogeneity by an ion-exchange chromatographic step. The kinetic parameters of the regenerated ligase are comparable to those of the purified native enzyme. This ligase was used to investigate the role that ATP plays in the formation of UDP-N-acetylmuramyl-L-alanine. UDP-N-acetyl[18O]muramate (with 18O located at the carboxylate function only) was prepared by a combination of chemical and enzymatic processes and was used as the substrate of the ligase to probe the reaction mechanism. All reaction products were purified and subjected to liquid chromatographic-mass spectrometric analysis. A single [18O]oxygen was transferred from UDP-N-acetyl[18O]muramate to the orthophosphate produced in the reaction. No [18O]oxygen was detected in the adenosine nucleotides recovered from the reaction. These results strongly suggest that this ligase-catalyzed peptide formation proceeds through an activated acyl-phosphate linkage during the reaction process. ATP therefore assists in the process of the peptide bond formation by donating its gamma-phosphoryl group to activate the carboxyl group of UDP-N-acetylmuramic acid.

Adenosine Triphosphate↗
PubMed2000

Comparison of the UDP-N-acetylmuramate:L-alanine ligase enzymes from Mycobacterium tuberculosis and Mycobacterium leprae.

In the peptidoglycan of Mycobacterium leprae, L-alanine of the side chain is replaced by glycine. When expressed in Escherichia coli, MurC (UDP-N-acetyl-muramate:L-alanine ligase) of M. leprae showed K(m) and V(max) for L-alanine and glycine similar to those of Mycobacterium tuberculosis MurC, suggesting that another explanation should be sought for the presence of glycine.

Alanine↗
PubMed2006

Subcellular localization of a germiantion-specific cortex-lytic enzyme, SleB, of Bacilli during sporulation.

The subcellular localization of a germination-specific cortex-lytic enzyme, SleB, of Bacillus subtilis during sporulation was observed by using fusions of N-terminal region of SleB to the green fluorescent protein (GFP). A fusion with a putative peptidoglycan-binding motif (SleB1-108-GFP) formed a fluorescent ring around the forespore of the wild type strain, as expected from the known location of the intact SleB in the dormant spore. SleB1-108-GFP formed a similar fluorescent ring around the forespore of the gerE mutant which has a severe defect in the coat structure, and of the cwlD mutant which lacks a muramic delta-lactam unique to the spore peptidoglycan (cortex), whereas the fusion could not attach to the spore of the cwlDgerE mutant. By contrast, a fusion without the motif (SleB1-45-GFP) could not be recruited around the forespore of the gerE mutant though it appeared to be accumulated on the outside of the spore of the wild type strain. Since SleB was shown to degrade only the cortex with muramic delta-lactam, these results suggested that a proper localization of SleB requires a strict interaction between the motif of the enzyme and the delta-lactam structure of the cortex, not the formation of normal coat layer.

Amidohydrolases↗
PubMed1998

Peptidoglycan structural dynamics during germination of Bacillus subtilis 168 endospores.

Peptidoglycan structural dynamics during endospore germination of Bacillus subtilis 168 have been examined by muropeptide analysis. The first germination-associated peptidoglycan structural changes are detected within 3 min after the addition of the specific germinant L-alanine. We detected in the spore-associated material new muropeptides which, although they have slightly longer retention times by reversed-phase (RP)-high-pressure liquid chromatography (HPLC) than related ones in dormant spores, show the same amino acid composition and molecular mass. Two-dimensional nuclear magnetic resonance (NMR) analysis shows that the chemical changes to the muropeptides on germination are minor and are probably limited to stereochemical inversion. These new muropeptides account for almost 26% of the total muropeptides in spore-associated material after 2 h of germination. The exudate of germinated spores of B. subtilis 168 contains novel muropeptides in addition to those present in spore-associated material. Exudate-specific muropeptides have longer retention times, have no reducing termini, and exhibit a molecular mass 20 Da lower than those of related reduced muropeptides. These new products are anhydro-muropeptides which are generated by a lytic transglycosylase, the first to be identified in a gram-positive bacterium. There is also evidence for the activity of a glucosaminidase during the germination process. Quantification of muropeptides in spore-associated material indicates that there is a heterogeneous distribution of muropeptides in spore peptidoglycan. The spore-specific residue, muramic delta-lactam, is proposed to be a major substrate specificity determinant of germination-specific lytic enzymes, allowing cortex hydrolysis without any effect on the primordial cell wall.

Bacillus subtilis↗