Chemical composition and turnover of peptidoglycan in Neisseria gonorrhoeae.

The peptidoglycan of all four colonial types of a number of strains of Neisseria gonorrhoeae constituted 1 to 2% of the dry weight of the cell. The chemical composition of cell types examined was similar with molar ratios of 1:1:2:1:1 for muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid, respectively. Ninety-six percent of the mass of the peptidoglycan was composed of these compounds. A lipoprotein analogous to that observed in Escherichia coli was not detected. The chain length of the glycan varied from 80 to 110 disaccharide units. The peptide contained equimolar amounts of D- and L-alanine. The rate of turnover of peptidoglycan in strain RD5 was 50% per generation. Turnover proceeded without a lag and followed first-order kinetics.     

Stringent control of peptidoglycan biosynthesis in Escherichia coli K-12.

[3H]Diaminopimelic acid (Dap) was incorporated exclusively into peptidoglycan by Escherichia coli strains auxotrophic for both lysine and Dap. The rate of [3H]Dap incorporation by stringent (rel+) strains was significantly decreased when cells were deprived of required amino acids. The addition of chloramphenicol to amino acid-starved rel+ cultured stimulated both peptidoglycan and ribonucleic acid synthesis. In contrast, a relaxed (relA) derivative incorporated [3H]Dap at comparable rates in the presence or absence of required amino acids. Physiologically significant concentrations of guanosine 5'-diphosphate 3'-diphosphate (ppGpp) inhibited the in vitro synthesis of both carrier lipid-linked intermediate and peptidoglycan catalyzed by a particulate enzyme system. The degree of inhibition was dependent on the concentration of ppGpp in the reaction mixture. Thus, the results of in vivo and in vitro studies indicate that peptidoglycan synthesis is stringently controlled in E. coli.     

Regulation of Bacterial Cell Walls: Turnover of Cell Wall in Staphylococcus aureus

The cell wall of Staphylococcus aureus was shown to undergo turnover during exponential growth. The rate of turnover, about 15% per generation, was identical for both cell wall polymers, peptidoglycan and teichoic acid. Both the old and newly synthesized wall material appeared to undergo turnover at similar rates. The rate of turnover followed first-order kinetics until more than 90% of the original wall was lost. Cell wall turnover was completely blocked under conditions of unbalanced synthesis known to inhibit cellular autolysis, e.g., addition of chloramphenicol. Cell wall turnover was shown to occur in a number of different strains of S. aureus and appears to be widely distributed in this species.     

Evidence for N----O acetyl migration as the mechanism for O acetylation of peptidoglycan in Proteus mirabilis.

O-acetylated peptidoglycan was purified from Proteus mirabilis grown in the presence of specifically radiolabelled glucosamine derivatives, and the migration of the radiolabel was monitored. Mild-base hydrolysis of the isolated peptidoglycan (to release ester-linked acetate) from cells grown in the presence of 40 microM [acetyl-3H]N-acetyl-D-glucosamine resulted in the release of [3H]acetate, as detected by high-pressure liquid chromatography. The inclusion of either acetate, pyruvate, or acetyl phosphate, each at 1 mM final concentration, did not result in a diminution of mild-base-released [3H]acetate levels. No such release of [3H]acetate was observed with peptidoglycan isolated from either Escherichia coli incubated with the same radiolabel or P. mirabilis grown with [1,6-3H]N-acetyl-D-glucosamine or D-[1-14C]glucosamine. These observations support a hypothesis that O acetylation occurs by N----O acetyl transfer within the sacculus. A decrease in [3H]acetate release by mild-base hydrolysis was observed with the peptidoglycan of P. mirabilis cultures incubated in the presence of antagonists of peptidoglycan biosynthesis, penicillin G and D-cycloserine. The absence of free-amino sugars in the peptidoglycan of P. mirabilis but the detection of glucosamine in spent culture broths implies that N----O transacetylation is intimately associated with peptidoglycan turnover.     

Peptidoglycan synthesis and turnover in cell division mutants of Agmenellum.

The synthesis and turnover of peptidoglycan in Agmenellum quadruplicatum was investigated using D-[U-14C]alanine followed by proteolytic digestion. The rate of turnover of alanine in the peptide portion of the peptidoglycan was measured in strain BG-1 and in two division mutants of this strain: one was blocked in cell separation; and the other was a low-temperature, conditional cell division mutant. The peptide portion of peptidoglycan turned over in all three strains tested, but no correlation was observed between septum formation or cell separation and the rate of turnover. Peptidoglycan synthesis was measured during induced division in snake forms of strain SN-29. A stimulation of peptidoglycan synthesis was observed during the period of cross-wall formation, even in the absence of new protein synthesis. Thus in A. quadruplicatum, cross-wall synthesis is accompanied by a stimulation of peptidoglycan synthesis.     

Conjugal transfer of beta-lactamase-producing plasmids of Neisseria gonorrhoeae to Neisseria meningitidis

Twenty clinical isolates of beta-lactamase-producing Neisseria gonorrhoeae from Japanese sources were studied to define their ability to serve as donors for their plasmids in conjugation with Neisseria meningitidis. These twenty strains of N. gonorrhoeae harbored the 4.5-megadalton (Mdal) beta-lactamase-producing plasmids and the 24.5-Mdal conjugative plasmids. We found that only three of twenty N. gonorrhoeae strains showed a detectable conjugation frequency (greater than 10(-5)) with N. meningitidis as the recipient although all strains were capable of mobilizing beta-lactamase-producing plasmids to N. gonorrhoeae and to Escherichia coli. The 4.5-Mdal beta-lactamase-producing plasmid was maintained in N. meningitidis, but the large 24.5-Mdal conjugative plasmid has not been found in N. meningitidis transconjugants.     

In vitro metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal protozoa and bacteria.

Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid [( 3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. These radiolabeled bacterial mutants were incubated alone (control) and with mixed ruminal bacteria or protozoa, and the metabolic processes, rates, and patterns of radiolabeled products released from them were studied. Control incubations revealed an inherent difference between the two substrates; gram-positive supernatants consistently contained 5% radioactivity, whereas even at 0 h, those from the gram-negative mutant released 22%. Incubations with ruminal microorganisms showed that the two mutants were metabolized differently and that protozoa were the major effectors of their metabolism. Protozoa exhibited differential rates of engulfment (150 B. megaterium GW1 and 4,290 E. coli W7-M5 organisms per protozoan per h), and they extensively degraded [3H]DAP-labeled B. megaterium GW1 at rates up to nine times greater than those of ruminal bacteria. By contrast, [3H]DAP-labeled E. coli W7-M5 degradation by either ruminal bacteria or ruminal protozoa was more limited. These fundamental differences in the metabolism of the two mutants, especially by ruminal protozoa, were reflected in the patterns and rates of radiolabeled metabolites produced; many were rapidly released from [3H]DAP-labeled B. megaterium GW1, whereas few were slowly released from [3H]DAP-labeled E. coli W7-M5. Most radiolabeled products derived from [3H]DAP-labeled B. megaterium GW1 were peptides of bacterial peptidoglycan origin. The ruminal metabolism of DAP-containing gram-positive and gram-negative bacteria, even with the same peptidoglycan chemotype, is thus likely to be profoundly different.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro metabolism of 2,2'-diaminopimelic acid from gram-positive and gram-negative bacterial cells by ruminal protozoa and bacteria

Bacillus megaterium GW1 and Escherichia coli W7-M5 were specifically radiolabeled with 2,2'-diamino[G-3H]pimelic acid [( 3H]DAP) as models of gram-positive and gram-negative bacteria, respectively. These radiolabeled bacterial mutants were incubated alone (control) and with mixed ruminal bacteria or protozoa, and the metabolic processes, rates, and patterns of radiolabeled products released from them were studied. Control incubations revealed an inherent difference between the two substrates; gram-positive supernatants consistently contained 5% radioactivity, whereas even at 0 h, those from the gram-negative mutant released 22%. Incubations with ruminal microorganisms showed that the two mutants were metabolized differently and that protozoa were the major effectors of their metabolism. Protozoa exhibited differential rates of engulfment (150 B. megaterium GW1 and 4,290 E. coli W7-M5 organisms per protozoan per h), and they extensively degraded [3H]DAP-labeled B. megaterium GW1 at rates up to nine times greater than those of ruminal bacteria. By contrast, [3H]DAP-labeled E. coli W7-M5 degradation by either ruminal bacteria or ruminal protozoa was more limited. These fundamental differences in the metabolism of the two mutants, especially by ruminal protozoa, were reflected in the patterns and rates of radiolabeled metabolites produced; many were rapidly released from [3H]DAP-labeled B. megaterium GW1, whereas few were slowly released from [3H]DAP-labeled E. coli W7-M5. Most radiolabeled products derived from [3H]DAP-labeled B. megaterium GW1 were peptides of bacterial peptidoglycan origin. The ruminal metabolism of DAP-containing gram-positive and gram-negative bacteria, even with the same peptidoglycan chemotype, is thus likely to be profoundly different.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aerobactin utilization by Neisseria gonorrhoeae and cloning of a genomic DNA fragment that complements Escherichia coli fhuB mutations.

Aerobactin, a dihydroxamate siderophore produced by many strains of enteric bacteria, stimulated the growth of Neisseria gonorrhoeae FA19 and F62 in iron-limiting medium. However, gonococci did not produce detectable amounts of aerobactin in the Escherichia coli LG1522 aerobactin bioassay. We probed gonococcal genomic DNA with the cloned E. coli aerobactin biosynthesis (iucABCD), aerobactin receptor (iutA), and hydroxamate utilization (fhuCDB) genes. Hybridization was detected with fhuB sequences but not with the other genes under conditions which will detect 70% or greater homology. Similar results were obtained with 21 additional strains of gonococci by colony filter hybridization. A library of DNA from N. gonorrhoeae FA19 was constructed in the phasmid vector lambda SE4, and a clone was isolated that complemented the fhuB mutation in derivatives of E. coli BU736 and BN3307. These results suggest that fhuB is a conserved gene and may play a fundamental role in iron acquisition by N. gonorrhoeae.     

Kinetics of uptake and incorporation of meso-diaminopimelic acid in different Escherichia coli strains.

The rate at which the peptidoglycan precursor meso-diaminopimelic acid (DAP) is incorporated into the cell wall of Escherichia coli cells was determined by pulse-label experiments. For different E. coli strains, the incorporation rate was compared with the rate of uptake of DAP into the cell. With E. coli W7, a dap lys mutant generally used in this kind of studies, steady-state incorporation was reached only after about 0.75 of the doubling time. This lag period can be ascribed to the presence of a large internal DAP pool in the cells. An E. coli K-12 lysA strain was constructed which could be grown without DAP in its medium. Consequently, due to the higher specific activity of the added [3H]DAP, faster incorporation and higher levels of radioactivity in the peptidoglycan layer were observed in the K-12 lysA strain than in the W7 strain. In addition, uptake and incorporation were faster in steady state (within about 0.2 of the doubling time), indicating a smaller DAP pool. The lag period could be further diminished and the incorporation rate could be increased by feedback inhibition of the biosynthetic pathway to DAP with threonine and methionine. These results make MC4100 lysA a suitable strain for studies on peptidoglycan synthesis. To explain our observations, we suggest the existence of an expandable pool of DAP in E. coli which varies with the DAP concentration in the growth medium. With 2 microgram of DAP per ml, the size of the pool is severalfold the amount of DAP contained in the cell wall. This pool can be partly washed out of the cells. Grown without DAP, MC4100 lysA still has a small pool caused by endogenous synthesis, which accounts for the fact that steady-state [3H]DAP incorporation in the lysA strain still shows a lag period.     

Turnover and recycling of the murein sacculus in oligopeptide permease-negative strains of Escherichia coli: indirect evidence for an alternative permease system and for a monolayered sacculus.

Turnover of murein in oligopeptide permease-negative Escherichia coli cells appeared to be minimal or nonexistent. In one strain in which it was possible to measure turnover during the first generation of chase, it was found that the rate of turnover was constant throughout a chase of three generations. This result suggests that an "inside-to-outside" mode of growth of the sacculus does not occur in E. coli. Turnover, though minimal, was significantly higher from cells labeled uniformly than from cells labeled only in the lateral wall, suggesting that a significant portion of the observed turnover is related to cell separation. Actually, turnover only appeared to be minimal in opp mutant strains. Tripeptides were being released by turnover at a rate of about 50% per generation and then were efficiently recycled. This suggests that in addition to opp, a low-affinity uptake system for tripeptide derived from the sacculus may exist.     

Site of Inhibition of Peptidoglycan Biosynthesis During the Stringent Response in Escherichia coli

The site of inhibition of peptidoglycan synthesis during the stringent response in Escherichia coli was determined in strains which were auxotrophic for both lysine and diaminopimelic acid (DAP). Cells were labeled with [(3)H]DAP for 30 to 60 min in the presence and absence of required amino acids, and the cellular distribution of [(3)H]DAP was determined. In both stringent (rel(+)) and relaxed (relA) strains, amino acid deprivation did not inhibit the incorporation of [(3)H]DAP into the nucleotide precursor and lipid intermediate fractions. The amount of [(3)H]DAP incorporated into the peptidoglycan fraction by the amino acid-deprived relA strain was over 70% of the amount incorporated in the presence of required amino acids. In contrast, the amounts of labeled peptidoglycan in amino acid-deprived rel(+) strains were only 20 to 44% of the amounts synthesized in the presence of amino acids. These results indicate that a late step in peptidoglycan synthesis is inhibited during the stringent response. The components of the lipid intermediate fraction synthesized by rel(+) strains in the presence and absence of required amino acids were quantitated. Amino acid deprivation did not inhibit the synthesis of either the monosaccharide-pentapeptide or the disaccharide-pentapeptide derivatives of the lipid intermediate. Thus, the reaction which is most likely inhibited during the stringent response is the terminal one involving the incorporation of the disaccharide-pentapeptide into peptidoglycan.     

Shuttle mutagenesis of Neisseria gonorrhoeae: pilin null mutations lower DNA transformation competence.

The method of shuttle mutagenesis has been extended to Neisseria gonorrhoeae. We have constructed a defective mini-Tn3 derivative that encodes chloramphenicol resistance in both N. gonorrhoeae and Escherichia coli and selected for mutations in the chloramphenicol resistance gene that express higher levels of antibiotic resistance in N. gonorrhoeae. Isogenic N. gonorrhoeae strains that differ only in pilin expression were constructed and used to test the effect of pilin null mutations on DNA transformation competence.     

Transfer of a gonococcal beta-lactamase plasmid to conjugation-deficient Neisseria cinerea strains by transformation

We have previously shown that some strains of Neisseria cinerea can serve as recipients in conjugation (Con+) with Neisseria gonorrhoeae while others cannot (Con-). To determine if a replication defect contributes to the inability of certain strains of N. cinerea to serve as recipients in conjugation, we attempted to introduce a naturally occurring gonococcal beta-lactamase plasmid into N. cinerea by transformation. Various methods were employed, and all proved unsuccessful. Since specific sequences are required for DNA uptake in transformation of N. gonorrhoeae, we constructed a number of hybrid plasmids containing N. cinerea chromosomal DNA inserted into the N. gonorrhoeae/Escherichia coli beta-lactamase shuttle vector, pLES2. When nine randomly selected plasmids with inserts were used to transform an N. cinerea strain which did not accept the gonococcal beta-lactamase plasmid by conjugation, transformants were observed with four of the hybrid plasmids. The presence of one of the hybrid plasmids, pCAG9, in transformants was confirmed by agarose gel electrophoresis, Southern hybridization, and beta-lactamase production. When an N. gonorrhoeae donor strain containing pCAG9 was used in conjugation with several N. cinerea strains, only those strains that were previously shown to act as recipients could accept and maintain pCAG9. The ability of pCAG9 and the other three hybrid plasmids to transform Con- strains demonstrates that the beta-lactamase plasmid can replicate in Con- strains, and, therefore, the Con- phenotype is due to a block in some other stage of the conjugation process.     

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.     

O-acetylation of peptidoglycan in Neisseria gonorrhoeae. Investigation of lipid-linked intermediates and glycan chains newly incorporated into the cell wall

Radioactive labelling of the amino sugars in gonococcal peptidoglycan was followed by treatment with Chalaropsis muramidase and TLC separation of the products. Even after very brief periods of labelling (0.5 min) the peptidoglycan was already cross-linked to some 80% of the final value and little change occurred within 2 min. The remaining cross-linking was achieved only over a period of about one generation time. Streptomycete endopeptidase was used to show the extent to which new chains were cross-linked to old. Even at the earliest times many cross-linked units contained new material in both moieties and by 3 min there was little distinction in relative labelling, indicating that in Neisseria gonorrhoeae most newly synthesized glycan chains are cross-linked to other new chains rather than to pre-existing peptidoglycan. A model is proposed in which newly polymerized monomer units are predestined either towards dimer formation with other new chains, which are then rapidly O-acetylated and not further cross-linked, or towards the formation of trimers and higher oligomers, the latter being a slower process. Although significant O-acetylation of peptidoglycan was detectable even at the earliest times, efforts to detect O-acetylated lipid intermediates were unsuccessful. The chief lipid intermediate found was apparently the disaccharide-peptide unit linked to undecaprenol.     

Extracellular proteases modify cell wall turnover in Bacillus subtilis.

The rate of turnover of peptidoglycan in exponentially growing cultures of Bacillus subtilis was observed to be sensitive to extracellular protease. In protease-deficient mutants the rates of cell wall turnover were greater than that of wild-type strain 168, whereas hyperprotease-producing strains exhibited decreased rates of peptidoglycan turnover. The rate of peptidogylcan turnover in a protease-deficient strain was decreased when the mutant was grown in the presence of a hyperprotease-producing strain. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to cultures of hyperprotease-producing strains increased their rates of cell wall turnover. Isolated cell walls of all protease mutants contained autolysin levels equal to or greater than that of wild-type strain 168. The presence of filaments, or cells with incomplete septa, was observed in hyperprotease-producing strains or when a protease-deficient strain was grown in the presence of subtilisin. The results suggest that the turnover of cell walls in B. subtilis may be regulated by extracellular proteases.