[87] Peptidoglycan transpeptidase in Bacillus megaterium

257

236

A series of mutants defective in penicillinbinding components (or proteins) (PBCs) was isolated from a collection of thermosensitive mutants of Escherichia coli. The mutants included mutations for each PBC (1 through 4) and a mutation in the activity of D-alanine carboxypeptidase la (PBC-5/6). PBC-1 was resolved into two components, PBC-la and PBC-lb, which were the products of different genes referred to as ponA and ponB, respectively.No mutation examined in ponA or ponB-, by itself, was associated with a thermosensitive defect in growth. The mutants having ponB-were hypersensitive to cephalosporins, which showed unusually high affinity to PBC-la. Murein synthetic activity decreased markedly in cell-free preparations from the ponB mutant. The double mutation ponA-ponB-was found to be lethal. A mutant having ponAts and ponB-revealed thermosensitivity of growth and Iysed at the restrictive temperature. It is concluded that PBC-1a and PBC-1b could share a biochemical reaction necessary for cell elongation so that the function of either one may bypass the loss of the function of the other.With the series of mutants, the chromosomal locus of the gene for each PBC was mapped: ponA (PBC-la) 73.5 min, ponB

Section: Resultssupporting

confidence: 71%

Section: Resultsmentioning

confidence: 99%

On the process of cellular division in Escherichia coli: a series of mutants of E. coli altered in the penicillin-binding proteins.

Suzuki¹,

Nishimura²,

Hirota³

1978

257

236

“…Previous work showed a correlation between spore MAL content and degree of heat resistance (28), but in that case MAL content was probably a measure of total peptidoglycan content (29). Our results indicate that the observed correlation was certainly between total spore peptidoglycan content and heat resistance.…”

Section: Discussionsupporting

confidence: 69%

Muramic lactam in peptidoglycan of Bacillus subtilis spores is required for spore outgrowth but not for spore dehydration or heat resistance

Popham

Helin²,

Costello³

et al. 1996

204

345

Bacterial endospores derive much of their longevity and resistance properties from the relative dehydration of their protoplasts. The spore cortex, a peptidoglycan structure surrounding the protoplasm, maintains, and is postulated to have a role in attaining, protoplast dehydration. A structural modification unique to the spore cortex is the removal of all or part of the peptide side chains from the majority of the muramic acid residues and the conversion of 50% of the muramic acid to muramic lactam. A mutation in the cwlD gene of Bacillus subtilis, predicted to encode a muramoyl-L-alanine amidase, results in the production of spores containing no muramic lactam. These spores have normally dehydrated protoplasts but are unable to complete the germination͞outgrowth process to produce viable cells. Addition of germinants resulted in the triggering of germination with loss of spore refractility and the release of dipicolinic acid but no degradation of cortex peptidoglycan. Germination in the presence of lysozyme allowed the cwlD spores to produce viable cells and showed that they have normal heat resistance properties. These results (i) suggest that a mechanical activity of the cortex peptidoglycan is not required for the generation of protoplast dehydration but rather that it simply serves as a static structure to maintain dehydration, (ii) demonstrate that degradation of cortex peptidoglycan is not required for spore solute release or partial spore core rehydration during germination, (iii) indicate that muramic lactam is a major specificity determinant of germination lytic enzymes, and (iv) suggest the mechanism by which the spore cortex is degraded during germination while the germ cell wall is left intact.The ability of endospores produced by Gram-positive eubacteria (i.e., Bacillus and Clostridium) to survive harsh physical and chemical treatments and to remain in a dormant state for long periods is important in the food preservation industry and represents an interesting biological problem. The protoplast of the spore is relatively dehydrated (even when suspended in H 2 O) in comparison to that of a vegetative cell, resulting in metabolic dormancy, and this dehydration is responsible in large part for the heat and hydrogen peroxide resistance properties of the spore (1-3). The mechanisms by which this dehydration is achieved and maintained are not clear and remain an outstanding question in the field. Maintenance of protoplast dehydration is clearly dependent on the integrity of a surrounding peptidoglycan wall and the unique structure of this peptidoglycan has been postulated to confer on it a role in attaining protoplast dehydration (4, 5). The spore peptidoglycan is comprised of two contiguous structures, an inner layer called the germ cell wall and a thicker outer layer called the cortex. The germ cell wall appears to have a structure similar to that of vegetative cell wall peptidoglycan (6) and serves as the initial cell wall during spore outgrowth. The vegetative peptidoglycan of Bacillus subti...

“…In some cases it may be that organisms are killed by a mechanism that does not involve covalent binding of penicillin. The transpeptidase of Bacillus megaterium is competitively rather than irreversibly inhibited by penicillins (13). Moreover, both E. coli and Streptomyces strains contain or excrete soluble D-alanine carboxypeptidases that are reversibly inhibited by penicillins (14,15).…”

mentioning

confidence: 99%

Isolation by Covalent Affinity Chromatography of the Penicillin-Binding Components from Membranes of Bacillus subtilis

Blumberg¹,

Strominger²

1972

Self Cite

An affinity chromatography technique was developed to isolate the five penicillin-binding components present in Bacillus subtilis membranes. The proteins were solubilized by the detergent Nonidet P-40, bound covalently to penicillin-substituted Sepharose, and subsequently eluted from the matrix with neutral hydroxylamine, which cleaves the penicilloyl-enzyme bond. Penicillin binding-component V, the D-alanine carboxypeptidase, makes up 1% of the total membrane protein. A modification of the above procedure enabled this enzyme to be obtained from the membrane in pure form in a single step with 50% overall recovery of enzymatic activity.Penicillin kills bacteria by preventing the last stage in cellwall biosynthesis, the cross-linking of nascent cell wall by the transpeptidase (1-3). In Escherichia coli, and probably other microorganisms, the transpeptidase is irreversibly inactivated by penicillin (4-6). It has been hypothesized that this inactivation is due to penicilloylation (2). Many strains of bacteria have been shown to bind radioactive penicillin. Moreover, the sensitivity of the organisms to penicillin correlates roughly with the rate at which penicillin binds to the cells (7-12). In some cases it may be that organisms are killed by a mechanism that does not involve covalent binding of penicillin. The transpeptidase of Bacillus megaterium is competitively rather than irreversibly inhibited by penicillins (13). Moreover, both E. coli and Streptomyces strains contain or excrete soluble D-alanine carboxypeptidases that are reversibly inhibited by penicillins (14, 15). The Streptomyces carboxypeptidase catalyzes a transpeptidation reaction using synthetic substrates (16), but it has not yet been shown that this extracellular enzyme is the physiological transpeptidase. In any case, the analysis of penicillin-binding components offers one promising approach to the study of cell-wall biosynthesis and the mode of action of penicillin.It has now become clear that there exists in a bacterial membrane not one protein that reacts irreversibly with penicillins, but several. Two lines of experimentation first suggested the occurrence of multiple penicillin-binding components. The D-alanine carboxypeptidase of B. subtilis, which is irreversibly inactivated by penicillins (17), could be inhibited in growing cells by 6-aminopenicillanic acid without killing the cells. In contrast, the cells were killed by cephalothin at concentrations that caused no inhibition of the carboxypeptidase (18). This result implied the presence of at least two penicillin-binding components, the cephalothinresistant carboxypeptidase and the cephalothin-sensitive penicillin killing site. In fact, isoelectric focusing of B. subtilis Abbreviation: SDS, sodium dodecyl sulfate. 3751 membranes to which radioactive penicillin had been bound revealed multiple peaks of radioactivity. Prior treatment with cephalothin abolished some but not all of the bands (19,20