Positive selection of allelic exchange mutants in Mycobacterium bovis BCG

Abstract A resistant mutant with vancomycin MIC of 100 μg/ml was isolated relatively easily through step pressure in the laboratory from a Staphylococcus aureus strain with initial MIC of 1.5 μg/ml for the antibiotic. Upon addition of vancomycin (50 μg/ml) to the growth medium mass increase of the culture and peptidoglycan synthesis continued but cell division (daughter cell separation), cell wall turnover and autolysis were inhibited, resulting in the production of multicellular clumps of bacteria. Parallel with the increase of culture density, the concentration of vancomycin measured both by biological activity and by HPLC gradually declined in the culture medium. Cell division and wall turnover of the culture resumed with the production of cells of normal morphology at the time when the concentration of the drug in the medium decreased below 0.5–1.0 μg/ml. There was no detectable change in the antibiotic concentration in the culture medium during growth of a vancomycin-resistant ( vanA -positive) strain of Enterococcus faecium and an intrinsically vancomycin-resistant strain of Leuconostoc . The vancomycin-resistant staphylococcal mutant gave no signal with the vanA or vanB DNA probes and contained no detectable d-lactate terminating cell wall precursors. The biochemical mechanism and clinical significance of such glycopeptide-resistant mutants remains to be established.     

Reversal of the Vancomycin Inhibition of Peptidoglycan Synthesis by Cell Walls

Addition of cell walls to the peptidoglycan synthetase-acceptor system containing vancomycin (50 μg/ml) prevented the inhibition by the antibiotic. In addition, the inhibition of incorporation of [¹⁴C]muramyl-pentapeptide into peptidoglycan in the presence of vancomycin was reversed by the addition of cell walls to the assay mixture at 60 min. Cell walls previously saturated with vancomycin lost their ability to reverse the inhibition by the antibiotic. The inhibition of peptidoglycan synthesis by ristocetin was partially reversed by the addition of cell walls. The initial stage in peptidoglycan synthesis is catalyzed by phospho-N-acetyl(NAc)muramyl-pentapeptide translocase (uridine 5′-phosphate) according to the reaction: UDP-NAc-muramyl-pentapeptide + acceptor acceptor-phospho-NAc-muramyl-pentapeptide + UMP where acceptor is C₅₅-isoprenoid alcohol phosphate. Vancomycin stimulates the transfer of phospho-NAc-muramyl-pentapeptide to the acceptor, and the addition of cell walls to this assay mixture prevented the stimulation of transfer. In addition to the transfer reaction, the enzyme catalyzes the exchange of [³H]uridine monophosphate (UMP) with UDP-NAc-muramyl-pentapeptide. The exchange reaction is effectively inhibited by vancomycin. For example, 60 μg of vancomycin per ml inhibited the rate of exchange by 50%. Addition of cell walls restored the exchange of UMP with the UMP moiety of UDP-NAc-muramyl-pentapeptide. Thus, cell walls appeared to have a higher affinity for vancomycin than did either the peptidoglycan synthetase-acceptor system or phospho-NAc-muramyl-pentapeptide translocase. These results provide support for the proposal made by Best and Durham that the effective binding of vancomycin to the cell wall could result in the inhibition of transfer of membrane-associated peptidoglycan chains to the growing wall.     

Alterations of cell wall structure and metabolism accompany reduced susceptibility to vancomycin in an isogenic series of clinical isolates of Staphylococcus aureus

A series of isogenic methicillin-resistant Staphylococcus aureus isolates recovered from a bacteremic patient were shown to acquire gradually increasing levels of resistance to vancomycin during chemotherapy with the drug (K. Sieradzki, T. Leski, L. Borio, J. Dick, and A. Tomasz, J. Clin. Microbiol. 41:1687-1693, 2003). We compared properties of the earliest (parental) vancomycin-susceptible isolate, JH1 (MIC, 1 microg/ml), to two late (progeny) isolates, JH9 and JH14 (vancomycin MIC, 8 microg/ml). The resistant isolates produced abnormally thick cell walls and poorly separated cells when grown in antibiotic-free medium. Chemical analysis of the resistant isolates showed decreased cross-linkage of the peptidoglycan and drastically reduced levels of PBP4 as determined by the fluorographic assay. Resistant isolates showed reduced rates of cell wall turnover and autolysis. In vitro hydrolysis of resistant cell walls by autolytic extracts prepared from either susceptible or resistant strains was also slow, and this abnormality could be traced to a quantitative (or qualitative) change in the wall teichoic acid component of resistant isolates. Some change in the structure and/or metabolism of teichoic acids appears to be an important component of the mechanism of decreased susceptibility to vancomycin in S. aureus.     

Relative affinity of vancomycin and ristocetin for cell walls and uridine diphosphate-N-acetylmuramyl pentapeptide

A determination of the relative affinity of vancomycin and ristocetin for isolated cell walls and for a peptidoglycan precursor was made. These antibiotics had previously been shown to adsorb to cell walls and to complex with peptides containing a d-alanyl-d-alanine C-terminus. By using (14)C-uridine diphosphate (UDP)-N-acetylmuramyl pentapeptide, it was shown that the complex which is formed between this peptidoglycan precursor and either vancomycin or ristocetin does not preclude adsorption of the antibiotics to cell walls of Micrococcus lysodeikticus. Complex formation between ristocetin and UDP-N-acetylmuramyl pentapeptide was assured by differential absorption spectra. However, when the complex was mixed with cell walls, the antibiotic was sedimented with the walls, and the radioactivity remained in the supernatant solution. This indication that ristocetin and vancomycin have a greater affinity for walls than for UDP-N-acetylmuramyl pentapeptide and that the complex per se does not bind to cell walls suggests that adsorption of these antibiotics to cell walls is probably responsible for the inhibition of peptidoglycan synthesis. This proposal is strengthened by the observation that complexed antibiotic is no less inhibitory for growth of Bacillus subtilis than free vancomycin or ristocetin.     

Peptidoglycan synthesis in Bacillus licheniformis. The inhibition of cross-linking by benzylpenicillin and cephaloridine in vivo accompanied by the formation of soluble peptidoglycan.

The synthesis of peptidoglycan by an autolysin-deficient beta-lactamase-negative mutant of Bacillus licheniformis was studied in vivo in the absence of protein synthesis. Benzylpenicillin and cephaloridine inhibited the formation of cross-bridges between newly synthesized peptidoglycan and the pre-existing cell wall. This inhibition, detected by measurement of the incorporation of N-acetyl[14C]glucosamine into the glycan fraction of the cell wall, was reversed by treatment with beta-lactamase and washing. Inhibition of D-alanine carboxypeptidase by benzylpenicillin was not reversed under similar conditions. Cells in which the initial penicillin inhibition of transpeptidation had been reversed showed an increased sensitivity to a subsequent addition of the antibiotic. Chemical analysis of peptidoglycan synthesized after reversal of penicillin inhibition revealed the presence of excess of alanine resulting from the continued inhibition of D-alanine carboxypeptidase. When the cell walls were digested to yield muropeptides so that the degree of cross-linking could be measured, the product after reversal of penicillin inhibition contained fewer cross-links than did the control preparation. Cultures treated with benzylpenicillin and cephaloridine continued to synthesize uncross-linked soluble peptidoglycan, which accumulated in the medium. This soluble material was all newly synthesized peptidoglycan and did not result from autolysis of the bacteria. The average chain lengths of the glycan synthesized in vivo and released as soluble peptidoglycan in the presence of both benzylpenicillin and cephaloridine were similar to those found previously in this organism.     

High level oxacillin and vancomycin resistance and altered cell wall composition in Staphylococcus aureus carrying the staphylococcal mecA and the enterococcal vanA gene complex

Recently, for the first time in the history of this bacterial species, methicillin-resistant Staphylococcus aureus (MRSA) carrying the enterococcal vanA gene complex and expressing high level resistance to vancomycin was identified in clinical specimens (CDC (2002) MMWR 51, 565-567). The purpose of our studies was to understand how vanA is expressed in the heterologous background of S. aureus and how it interacts with the mecA-based resistance mechanism, which is also present in these strains and is targeted on cell wall biosynthesis. The vanA-containing staphylococcal plasmid was transferred from the clinical vancomycin-resistant S. aureus (VRSA) strain HIP11714 (CDC (2002) MMWR 51, 565-567) to the methicillin-resistant S. aureus (MRSA) strain COL for which extensive genetic and biochemical information is available on staphylococcal cell wall biochemistry and drug resistance mechanisms. The transconjugant named COLVA showed high and homogeneous resistance to both oxacillin and vancomycin. COLVA grown in vancomycin-containing medium produced an abnormal peptidoglycan: all pentapeptides were replaced by tetrapeptides, and the peptidoglycan contained at least 22 novel muropeptide species that frequently showed a deficit or complete absence of pentaglycine branches. The UDP-MurNAc-pentapeptide, the major component of the cell wall precursor pool in vancomycin-sensitive cells was replaced by UDP-MurNAc-depsipeptide and UDP-MurNAc-tetrapeptide. Transposon inactivation of the beta-lactam resistance gene mecA caused complete loss of beta-lactam resistance but had no effect on the expression of vancomycin resistance. The two major antibiotic resistance mechanisms encoded by mecA and vanA residing in the same S. aureus appear to use different sets of enzymes for the assembly of cell walls.     

Gradual alterations in cell wall structure and metabolism in vancomycin-resistant mutants of Staphylococcus aureus

In five vancomycin-resistant laboratory step mutants selected from the highly and homogeneously methicillin-resistant Staphylococcus aureus strain COL (MIC of methicillin, 800 microg/ml; MIC of vancomycin, 1.5 microg/ml), the gradually increasing levels of resistance to vancomycin were accompanied by parallel decreases in the levels of methicillin resistance and abnormalities in cell wall metabolism. The latter included a gradual reduction in the proportion of highly cross-linked muropeptide species in peptidoglycan, down-regulation of the production of penicillin-binding protein 2A (PBP2A) and PBP4, and hypersensitivity to beta-lactam antibiotics each with a relatively selective affinity for the various staphylococcal PBPs; the PBP2-specific inhibitor ceftizoxime was particularly effective.     

Vancomycin-resistant Staphylococcus aureus

The evolution and molecular mechanisms of vancomycin resistance in Staphylococcus aureus were reviewed. Case reports and research studies on biochemestry, electron microscopy and molecular biology of Staphylococcus aureus were selected from Medline database and summarized in the following review. After almost 40 years of successful treatment of S. aureus with vancomycin, several cases of clinical failures have been reported (since 1997). S. aureus strains have appeared with intermediate susceptibility (MIC 8-16 microg/ml), as well as strains with heterogeneous resistance (global MIC < or =4 microg/ml), but with subpopulations of intermediate susceptibility. In these cases, resistance is mediated by cell wall thickening with reduced cross linking. This traps the antibiotic before it reaches its major target, the murein monomers in the cell membrane. In 2002, a total vancomycin resistant strain (MIC > or =32 microg/ml) was reported with vanA genes from Enterococcus spp. These genes induce the change of D-Ala-D-Ala terminus for D-Ala-D-lactate in the cell wall precursors, leading to loss of affinity for glycopeptides. Vancomycin resistance in S. aureus has appeared; it is mediated by cell wall modifications that trap the antibiotic before it reaches its action site. In strains with total resistance, Enterococcus spp. genes have been acquired that lead to modification of the glycopeptide target.     

Mechanism of intrinsic resistance to vancomycin in Clostridium innocuum NCIB 10674

We have studied the basis for intrinsic resistance to low levels of vancomycin in Clostridium innocuum NCIB 10674 (MIC = 8 microg/ml). Analysis by high-pressure liquid chromatography (HPLC) and mass spectrometry of peptidoglycan nucleotide precursors pools revealed the presence of two types of UDP-MurNac-pentapeptide precursors constitutively produced, an UDP-MurNAc-pentapeptide with a serine at the C terminus which represented 93% of the pool and an UDP-MurNAc-pentapeptide with an alanine at the C terminus which represented the rest of the pool. C. innocuum cell wall muropeptides containing pentapeptide[Ser], either dialanine substituted on the epsilon amino group of lysine or not, were identified and represented about 10% of the monomers while only 1% of pentapeptide[D-Ala] monomers were found. The sequence of a 2,465-bp chromosomal fragment from C. innocuum was determined and revealed the presence of ddl(c. innocuum) and C. innocuum racemase genes putatively encoding homologues of D-Ala:D-X ligases and amino acid racemases, respectively. Analysis of the pool of precursors of Enterococcus faecalis JH2-2, containing cloned ddl(c. innocuum) and C. innocuum racemase genes showed in addition to the UDP-MurNAc-pentapeptide[D-Ala], the presence of an UDP-MurNAc-pentapeptide[D-Ser] precursor. However, the expression of low-level resistance to vancomycin was observed only when both genes were cloned in E. faecalis JH2-2 together with the vanXYc gene from Enterococcus gallinarum BM4174 which encodes a d,d-peptidase which eliminates preferentially the high affinity vancomycin UDP-MurNAc-pentapeptide [D-Ala] precursors produced by the host. We conclude that resistance to vancomycin in C. innocuum NCIB 10674 was related to the presence of the two chromosomal ddl(c. innocuum) and C. innocuum racemase genes allowing the synthesis of a peptidoglycan precursor terminating in serine with low affinity for vancomycin.     

The synthesis of peptidoglycan in an autolysin-deficient mutant of Bacillus licheniformis N.C.T.C. 6346 and the effect of β-lactam antibiotics, bacitracin and vancomycin

The synthesis of peptidoglycan by cell-free membrane and membrane+wall preparations from an autolysin-deficient, beta-lactamase-negative mutant of Bacillus licheniformis N.C.T.C. 6346 was studied. The membrane preparation synthesized un-cross-linked polymer, the formation of which was not inhibited by beta-lactam antibiotics. Release of d-alanine by the action of d-alanine carboxypeptidase was inhibited variably according to the antibiotic. This inhibition was reversed by neutral hydroxylamine but not by the action of beta-lactamases or by washing. Bacitracin inhibited peptidoglycan synthesis, but not the d-alanine carboxypeptidase. Examination of peptidoglycan synthesized in the presence of excess of bacitracin showed that synthesis was not restricted to the addition of one disaccharide-pentapeptide unit at each synthetic site, an average of 2-3 disaccharide-pentapeptide units being added. Peptidoglycan synthesis was three- to four-fold more sensitive to vancomycin than was the release of d-alanine by the action of the carboxypeptidase. Incorporation of newly synthesized peptidoglycan into pre-existing cell wall was studied in membrane+wall preparations. This incorporation was catalysed by a benzylpenicillin- and cephaloridine-sensitive transpeptidase. The concentrations of these antibiotics giving 50% inhibition of incorporation were almost identical with those required to inhibit growth of the bacillus. Inhibition of the transpeptidase was reversed by treatment with beta-lactamase or by washing.     

Brevibacterium divaricatum: Influence of nutritional conditions on the wall composition and release of uncross-linked peptidoglycan chains into medium

Purified cell walls, originating from penicillin-treated (3 g/ml, 1 h) and-untreated Brevibacterium divaricatum cells grown on complex (CM) and glucose minimal medium with (MM) or without (Ca-free MM) calcium carbonate, were isolated by two procedures. Electron micrographs and chemical analysis revealed no differences between identically isolated walls with respect to the presence or absence of either penicillin or calcium carbonate in the glucose growth medium. On the contrary, the appearance and peptidoglycan content of the walls was greatly dependent on the procedure used for their isolation and the walls isolated from the cells grown on complex medium contained more materials other than peptidoglycan. It was shown that the presence of calcium carbonate in the glucose minimal medium was essential for accumulation of large amounts of peptidoglycan chains into the medium. Penicillin-induced interruption of cell wall synthesis was prerequisite for manifestation of the calcium carbonate stimulating effect.Abbreviations CM complex medium - MM chemically defined minimal medium based on glucose and containing calcium carbonate - Ca-free MM MM modified only by the omission of calcium carbonate - ET-walls Enzyme treated walls - FPR-walls French press-ruptured walls     

Inactivated pbp4 in highly glycopeptide-resistant laboratory mutants of Staphylococcus aureus.

Both vancomycin- and teicoplanin-resistant laboratory mutants of Staphylococcus aureus produce peptidoglycans of altered composition in which the proportion of highly cross-linked muropeptide species is drastically reduced with a parallel increase in the representation of muropeptide monomers and dimers (Sieradzki, K., and Tomasz, A. (1997) J. Bacteriol. 179, 2557-2566; and Sieradzki, K. , and Tomasz, A. (1998) Microb. Drug Resist. 4, 159-168). We now report that the distorted peptidoglycan composition is related to defects in penicillin-binding protein 4 (PBP4); no PBP4 was detectable by the fluorographic assay in membrane preparations from the mutants, and comparison of the sequence of pbp4 amplified from the mutants indicated disruption of the gene by two types of abnormalities, a 17-amino acid long duplication starting at position 305 of the pbp4 gene was detected in the vancomycin-resistant mutant, and a stop codon was found to be introduced into the pbp4 KTG motif at position 261 in the mutant selected for teicoplanin resistance. Additional common patterns of disturbances in the peptidoglycan metabolism of the mutants are indicated by the increased sensitivity of mutant cell walls to the M1 muramidase and decreased sensitivity to lysostaphin, which is a reversal of the susceptibility pattern of the parental cell walls. Furthermore, the results of high performance liquid chromatography analysis of lysostaphin digests of peptidoglycan suggest an increase in the average chain length of the glycan strands in the peptidoglycan of the glycopeptide-resistant mutants. The increased molar proportion of muropeptide monomers in the cell wall of the glycopeptide-resistant mutants should provide binding sites for the "capture" of vancomycin and teicoplanin molecules, which may be part of the mechanism of glycopeptide resistance in S. aureus.     

Growth of Streptococcus mutans protoplasts is not inhibited by penicillin.

A method is described in which cells of Streptococcus mutans BHT can be converted to spherical, osmotically fragile protoplasts. Exponential-phase cells were suspended in a solution containing 0.5 M melezitose, and their cell walls were hydrolyzed with mutanolysin (M-1 enzyme). When the resultant protoplasts were incubated in a chemically defined growth medium containing 0.5 M NH4Cl, the protoplast suspensions increased in turbidity, protein, ribonucleic acid, and deoxyribonucleic acid in a balanced fashion. In the presence of benzylpenicillin (5 microgram/ml), balanced growth of protoplasts was indistinguishable from untreated controls. This absence of inhibition of protoplast growth in the presence of benzylpenicillin was apparently not due to inactivation of the antibiotic. When exponential-phase cells of S. mutans BHT were first exposed to 5 microgram of benzyl-penicillin per ml for 1 h and then converted to protoplasts, these protoplasts were also able to grow in chemically defined, osmotically stabilized medium. The ability of wall-free protoplasts to grow and to synthesize ribonucleic acid and protein in the presence of a relatively high concentration of benzylpenicillin contrasts with the previously reported rapid inhibition of ribonucleic acid and protein synthesis in intact streptococci. These data suggest that this secondary inhibition of ribonucleic acid and protein synthesis in whole cells is due to factors involved with the continued assembly of an intact, insoluble cell wall rather than with earlier stages of peptidoglycan synthesis.     

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.     

Formation of cell wall polymers by reverting protoplasts of Bacillus licheniformis.

The biosynthesis of peptidoglycan and teichoic acid by reverting protoplasts of Bacillus licheniformis 6346 His-, in cubated at 35 C on medium containing 2.5% agar, is detectable after 40 min. The amount of N-acetyl-[1-14C]glucosamine incorporated into peptidoglycan and teichoic acid on continued incubation doubles at the same rate as the incorporation of [3H]tryptophan into protein. At the early stages of reversion the average glycan chain length, measured by the ratio of free reducing groups of muramic acid and glucosamine to total muramic acid present, is very short. As reversion proceeds, the average chain length increases to a value similar to the found in the wall of the parent bacillus. The extent of cross-linkage found in the peptide side chains of the peptidoglycan also increases as reversion proceeds. At the completion of reversion the wall material synthesized has similar characteristics to those of the walls of the parent bacilli, containing peptidoglycan and teichoic and teichuronic acids in about the same proportions. Soluble peptidoglycan can be isolated from the reversion medium, amounting to 30% of the total formed after 3 h of incubation and 8% after 12 h. This amount was reduced by the presence in the medium of the walls of an autolysin-deficient mutant; they were not formed at all by reverting protoplasts of the autolysin-deficient mutant itself. Analysis of the soluble material provided additional evidence for their being autolytic products rather than small unchanged molecules. When protoplasts were incubated on medium containing only 0.8% agar, 53 to 67% of the peptidoglycan formed after 3 h of incubation was soluble, and 21% after 12 h. Fibers that appeared to be sheared from the protoplasts at intermediate stages of reversion on medium containing 2.5% agar were similar in composition to the bacillary walls.     

Evidence for the synthesis of soluble peptidoglycan fragments by protoplasts of Streptococcus faecalis.

Growing protoplasts of Streptococcus faecalis 9790 were found to synthesize and excrete soluble peptidoglycan fragments. The presence of soluble peptidoglycan derivatives in culture supernatants was determined by (i) incorporation of three different radioactively labeled precursors (L-lysine, D-alanine, and acetate) into products which, after hen egg-white lysozyme hydrolysis, had the same KD values on gel filtration as muramidase hydrolysis products of isolated walls; (ii) inhibition of net synthesis of these products by cycloserine and vancomycin; and (iii) identification of disaccharide-peptide monomer using the beta-elimination reaction, gel filtration, and high-voltage paper electrophoresis. Under the conditions of these experiments the presence of newly synthesized, acid-precipitable (macromolecular) peptidoglycan was not detected. The predominance of monomer (70 to 80%) in lysozyme digests of peptidoglycan synthesized by protoplasts was in sharp contrast to digest of walls from intact streptococci which contain mostly peptide cross-linked products. Biosynthesis and release of relatively uncross-linked, soluble peptidoglycan fragments by protoplasts was related to the absence of suitable, preexisting acceptor wall.     

Study of the composition of cell wall of group A Streptococcus after hydrolysis using muramidase from Streptomyces levoris

The aim of the experiment was to study the lysis products of cell walls of group A streptococci resulting from exposure to N-acetylmuramidase. It was shown that for isolating surface proteins free of polysaccharide and peptidoglycan fragments it was necessary to treat the streptococcal cell walls with endo-beta-N-acetylmuramidase for no more than 30 minutes. Prolonged hydrolysis with muramidase led to the presence of polysaccharide and the peptidoglycan fragments in the protein fractions, intracellular wall proteins covalently bound to the peptidoglycan fragments and polysaccharide being also released.     

Rotational-echo double resonance characterization of vancomycin binding sites in Staphylococcus aureus

Solid-state NMR experiments with stable isotope-labeled Staphylococcus aureus have provided insight into the structure of the peptidoglycan binding site of a potent fluorobiphenyl derivative of chloroeremomycin (Eli Lilly LY329332). Rotational-echo double resonance (REDOR) NMR provided internuclear distances from the 19F of this glycopeptide antibiotic to natural-abundance 31P and to specific 13C and 15N labels biosynthetically incorporated into the bacteria from labeled alanine, glycine, or lysine in the growth medium. Results from experiments with intact late log phase bacteria and cell walls indicated homogeneous drug-peptidoglycan binding. Drug dimers were not detected in situ, and the hydrophobic fluorobiphenyl group of LY329332 did not insert into the bilayer membrane. A model of the binding site consistent with the REDOR results positions the vancomycin cleft around an un-cross-linked D-Ala-D-Ala peptide stem with the fluorobiphenyl moiety of the antibiotic near the base of a second, proximate stem in a locally ordered peptidoglycan matrix.     

Morphologic mutant of Escherichia coli K12 having disorders in morphology, division and cell wall biosynthesis]

A study was made of the properties of a spherical mutant obtained from the E. coli K12 HfrC strain under the effect of N-nitroso-N-methyl-urea. The growth of the mutant of full value media was characterized by a marked reduction of the cell division at the rest phase, but exponential growth phase failed to differ from the growth of the parental strain. Electron microscopic study of surface structures of the mutant cells which grew under physiological conditions permitted to distinguish two types: the first type had a typical structure of the cell wall characteristic of Gram negative microbes; the second type was framed by a bicontour membrane without any distinct structure. The presence of these two types of cells was also confirmed by their different sensitivity to the ionic detergents. On the basis of chemical analysis of peptidoglycan of the cell wall (which was markedly decreased in amount in the mutant cells), and also of the unsually high accumlation of the UDP-precursors of peptidoglycan under conditions of penicillin action it is supposed that normal regulation of metabolism of the cell walls was deranged. Mutation designated by 11rA symbol was plotted by phase PI transduction alongside of strA gene.     

Reassembly in vitro of hexagonal surface arrays in a protein-producing bacterium, Bacillus brevis 47.

Bacillus brevis 47 had two protein layers (the outer and middle walls) and a peptidoglycan layer (the inner wall) and contained two major proteins with approximate molecular weights of 130,000 and 150,000 in the cell wall. Both the total and Triton-insoluble envelopes revealed a hexagonal lattice array with a lattice constant of 14.5 nm. The proteins of 130,000 and 150,000 molecular weight isolated from the Triton-insoluble envelopes were serologically different from each other and assembled in vitro on the peptidoglycan layer. A mixture of 130,000- and 150,000-molecular-weight proteins led to the formation of a five-layered cell wall structure, two layers on each side of the peptidoglycan layer, which resembled closely the Triton-insoluble envelopes. A three-layered cell wall structure, one layer on each side of the peptidoglycan layer, was reconstituted when only the 150,000-molecular-weight protein was used. Both five- and three-layered cell walls reconstituted in vitro also contained hexagonally arranged arrays with the same lattice constant as that of the total and Triton-insoluble envelopes. A mutant, strain 47-57, which was isolated as a phage-resistant colony, had a two-layered cell wall consisting of the middle and inner wall layers and contained only 150,000-molecular-weight protein as the major cell wall protein. The cell envelopes of the mutant revealed the hexagonal arrays with the same lattice constant as that of the wild-type cell envelopes. We conclude that the outer and middle wall layers consist of proteins with approximate molecular weights of 130,000 and 150,000, respectively. Furthermore, the 150,000-molecular-weight protein formed the hexagonal arrays in the middle wall layer.