Murein Synthesis Research Articles

Initiation of murein biosynthesis inEscherichia colicells treated with β-lactam antibiotics

The study of the synthesis and accumulation of murein in cells initiating growth in the presence of β-lactam antibiotics indicated: (i) Murein rearrangement contributes substantially to cell enlargement during the first doubling in cell mass; (ii) Penicillin-binding protein 2 has the potential capability to synthesize most, if not all, the new murein required for the cells to double in mass.

Studies on the mode of action of nisin

The interference of the polypeptide antibiotic nisin with membrane function and murein synthesis was studied. The inhibition of the in vivo synthesis of murein in Bacillus subtilis and the resistance of Mycoplasma mycoides to nisin, suggest that the membrane may be excluded as a target. On the other hand a general inhibition of macromolecule syntheses in Micrococcus luteus supports the assumption that the cytoplasmic membrane is a target. In addition, an antagonistic effect on growth inhibition by nisin with both, cytoplasmic membranes and isolated phospholipid components of membranes was demonstrated. The lysis of phospholipid liposomes by nisin and the sensitivity at increased concentration of antibiotic of organisms lacking murein in their cell walls provide further evidence for the cytoplasmic membrane as a target. We therefore assume that the antimicrobial effect of nisin is caused by its interaction with phospholipid components of the cytoplasmic membrane followed by an interference with the membrane function. The sensitivity against nisin is increased in organisms with murein in their cell walls because the inhibition of this polymer causes an additional, inhibitory effect synergistic to the disturbance of the membrane function.

Interaction of FtsA and PBP3 proteins in the Escherichia coli septum.

Mutations in the ftsA gene of Escherichia coli conferred a higher resistance to lysis induced by penicillin or by a combination of cefsulodin and furazlocillin. The ftsA2 allele codes for an FtsA protein which is inactive at 42 degrees C but is able to regain its activity once it is transferred back to 30 degrees C; ftsA2 filaments formed at 42 degrees C in the presence of penicillin divided once the penicillin was removed and the temperature was lowered to 30 degrees C. Potential septation sites in the filaments of wild-type cells treated in the same way remained inactive. The binding of a radioactively labeled derivative of ampicillin to penicillin-binding protein 3 (PBP3) was significantly decreased in strain D-3, containing the mutant allele ftsA3, when the binding assay was performed at the restrictive temperature. A molecular species able to cross-react with an anti-PBP3 serum was nevertheless found to be present in the envelope of D-3 cells. These observations suggested that the FtsA protein, a protein with a structural and regulatory role in septation, and PBP3, a protein enzymatically active in the synthesis of murein for septation, interact with each other.

Molecular cloning and expression of hipA, a gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis.

The hipA gene at 33.8 min on the Escherichia coli chromosome controls the frequency of persistence upon inhibition of murein synthesis; for strains bearing hipA+ the frequency is 10(-6), and for hipA- strains the frequency is 10(-2). hip+ has been cloned by selection for a kanamycin resistance determinant at 33.9 min. hipA+ is dominant over hipA- in both recA+ and recA- backgrounds. The smallest DNA insert which contains hipA+, as determined by the ability of the plasmids to complement hipA- strains, is 1,885 base pairs. Both orientations of hipA+ are obtained when the cloning site of vector is remote from strong promoters; both orientations complement hipA-, and both encode a unique peptide of 50,000 Mr. The probable direction of transcription has been deduced from the pattern of peptides encoded by plasmids from which either end of the insert and adjacent vector sequences have been deleted. This information and the recovery of only one orientation of hipA+ when the cloning site is close to a strong promoter suggest that a high level of expression of the gene is not tolerated by E. coli.

Penicillin binding proteins: role in initiation of murein synthesis in Escherichia coli.

The consequences of the specific inhibition of penicillin binding proteins (Pbps) by beta-lactam antibiotics immediately before resumption of active growth in Escherichia coli suggest that inhibition of murein biosynthesis does not prevent the earlier steps of the initiation of cell growth in mass. The activity of Pbp 2 is apparently critical for the initiation of murein biosynthesis. Provided that Pbp 2 remains active, the other Pbps (1a, 1b, 3, 4, 5, and 6) can be inhibited without any noticeable effect on the initial rate of incorporation of new precursors into macromolecular peptidoglycan. These precursors are, in addition, inserted with a high degree of cross-linkage.

The complete sequence of murein synthesis in ether treated Escherichia coli.

The in vitro synthesis of murein from the precursors UDP-N-acetylglucosamine, L-alanine, D-glutamic acid and meso-diaminopimelic acid was performed with the aid of ether treated Escherichia coli. This synthesis was sensitive to representative inhibitors of early reactions in the cytoplasm as well as of late reactions in the membrane or the cell wall. The sensitivity was higher than in in vitro systems starting with UDP-N-acetylmuramic acid or UDP-N-acetylmuramyl-pentapeptide.

Fosfomycin inactivates its target enzyme in Escherichia coli cells carrying a fosfomycin resistance plasmid.

The target enzyme for fosfomycin antibiotic action, enolpyruvyl transferase, was synthesized in similar amounts and with similar kinetic and inhibition constants for phosphoenolpyruvate and fosfomycin, respectively, by an Escherichia coli strain containing the fosfomycin resistance plasmid pOU900 and by plasmid-free strains. Neither phosphoenolpyruvate level nor murein synthesis was altered by pOU900.

HipA, a newly recognized gene of Escherichia coli K-12 that affects frequency of persistence after inhibition of murein synthesis.

Except for a small fraction of persisters, 10(-6) to 10(-5), Escherichia coli K-12 is killed by prolonged inhibition of murein synthesis. The progeny of persisters are neither more resistant to inhibition of murein synthesis nor more likely to persist than normal cells. Mutants have been isolated in which a larger fraction, 10(-2), persists. The persistent response of the mutants, Hip (high persistence), is to inhibition of murein synthesis at early or late steps by antibiotics (phosphomycin, cycloserine, and ampicillin) or by metabolic block (starvation for diaminopimelic acid). Killing of the parent strain by each of the four inhibitors has two phases: The first is rapid and lasts about 30 min; the second is slower, but still substantial, and lasts 3 to 4 h. The first phase also occurs in the Hip mutants, but then viability of the mutants remains constant after about 30 min. Neither tolerance, resistance, impaired growth, nor reversion of spheroplasts accounts for high-frequency persistence. Two of the mutations map at 33.8 min in a region containing few other recognized functions. This position and the phenotypes define hipA as a newly recognized gene. Transposons Tn5 and Tn10 have been inserted close to hipA making it possible to explore the molecular genetics of persistence, a long recognized but poorly understood phenomenon.

Volume growth, murein synthesis, and murein cross-linkage during the division cycle of Escherichia coli PA3092.

Cells of Escherichia coli PA3092 were synchronized by centrifugal elutriation. The synchronously growing cells were double labeled with -3H or DL-[meso-2,6-14C]diaminopimelic acid (DAP) at different times. Cells incorporated [3H]DAP at a continuously increasing rate during their cycle, with a maximum occurring at about 30 min before division for trichloroacetic acid-precipitated cells (whole cells) and about 10 min before division for sodium dodecyl sulfate-treated cells (sacculi). This was in good agreement with the observed kinetics of volume growth under these conditions. Furazlocillin, which preferentially interacts with penicillin-binding protein 3, modified the pattern of incorporation of [3H]DAP. Electron microscopy indicated that furazlocillin did not inhibit the initiation of division but rather its completion. In addition, we measured the cross-linking of the murein inserted at different times during synchronous growth. The highest percentages were found to occur around division. At this same time, the cross-linking of old peptidoglycan was found to be decreased.

Murein synthesis and beta-lactam antibiotic susceptibility during rod-to-sphere transition in a pbpA(Ts) mutant of Escherichia coli.

The conditional morphology mutant of Escherichia coli SP45 grows as a rod at 30 degrees C and assumes a spherical shape after 90 min of incubation at 42 degrees C. The rod-to-sphere morphological transition has been found to be associated with the disappearance of penicillin-binding protein 2 (PBP-2), the progressive reduction (as much as 50%) of murein synthesis, as measured both in intact cells and ether-permeabilized bacteria, and alterations in the structure of the cell envelope, including detachment of the outer membrane from the underlying structures. The detachment was initially localized at the poles of the cells and then spread over the entire surface. Shape transition was also linked to increased susceptibility to beta-lactam antibiotics which preferentially bound to PBP-1A (cephalothin, cephaloridine) or to PBP-3 (furazlocillin, piperacillin). Treatment with beta-lactams possessing a high affinity for PBP-1A, although inducing a low degree of peptidoglycan synthesis inhibition (5 to 10%), was associated with a marked loss of cell viability and massive lysis. On the other hand, the simultaneous absence of PBP-2 and inhibition of PBP-3 causes a significant reduction of peptidoglycan synthesis, yet only slightly affected cell viability. Whereas PBP-1A inhibition during shape transition had no effect on morphology, addition of antibiotics binding to PBP-3 30 min after the temperature shift-up caused formation of elongated cells with a centrally located bulge, not observed in similarly treated cells grown at 30 degrees C. Inhibition of PBP-3 in round cells 90 min after temperature shift caused formation of giant cells, indicating complete loss of elongation ability. The different effects of the simultaneous inhibition of two PBPs, combining mutational loss with specific binding in vivo of another PBP by beta-lactams, provide new insight into the role of these proteins and the killing mechanisms of this class of antibiotics.

Evidence for involvement of penicillin-binding protein 3 in murein synthesis during septation but not during cell elongation.

Furazlocillin (1 microgram/ml) and piperacillin (5 microgram/ml) bound specifically to penicillin-binding protein 3 (PBP-3) and not to the other major PBPs in intact Escherichia coli cells. The effect of this specific binding to PBP-3 on murein synthesis of elongating and synchronously septating cells was investigated in two thermosensitive division mutants, E. coli BUG6 and E. coli JE10730, the latter possessing a thermolabile PBP-3. Synchronous cell division was induced by shifting the cultures from the nonpermissive temperature (42 degrees C) to 30 degrees C. Both [14C]diaminopimelic acid incorporation into murein of intact cells and [14C]N-acetylglucosamine incorporation into murein of cells permeabilized with ether was inhibited by an average of 42% in septating cells. In filaments growing at the nonpermissive temperature, we detected no inhibition and, frequently, a 10 to 15% stimulation of murein synthesis. The two drugs, at concentrations used in the above experiments, bound exclusively to PBP-3 both in elongating and septating intact cells and in ether-treated cells. These results support the hypothesis that PBP-3 activity is exclusively required for septal murein synthesis.

Alterations of the cytoplasmic and outer membranes ofEscherichia coli infected with bacteriophage ϕX174

InEscherichia coli C infected with bacteriophage ϕX174, the cytoplasmic and outer membranes of the host bacterium exhibit various alterations in their protein composition as revealed by sodium dodecyl sulfate gel electrophoresis of purified membranes. These alterations result mainly from the action of the lysis gene product of the phage. One effect of the changes occurring in the membranes results in different rates of release of wild-type phage and its lysis-negative mutant from glycine spheroplasts. The activity of phospho-MurNAc-pentapeptide translocase, an enzyme involved in murein synthesis and located in the cytoplasmic membrane, is unimpaired by these alterations.

Effects of furazlocillin, a beta-lactam antibiotic which binds selectively to penicillin-binding protein 3, on Escherichia coli mutants deficient in other penicillin-binding proteins

Furazlocillin binds selectively to penicillin-binding protein 3 (PBP-3), prevents septation of Escherichia coli, and allows the cells to form long filaments without lysis. The effect of furazlocillin on the morphology, autolysis, and murein synthesis of E. coli mutants deficient in either PBP-1A, PBP-1Bs, or PBP-2 was studied. The results reveal that PBP-1A and PBP-1Bs functions are not equivalent since furazlocillin affects the morphology, autolysis, and murein synthesis of PBP1A- mutants quite differently from that of PBP-1Bs mutants. Different "PBP-2-" mutants were found to respond to furazlocillin in dramatically different ways: strain LS-1 cells formed elongated rods with a central bulge which eventually lysed, whereas SP6 cells formed stable "barbells" in which the two daughter cells were well separated but remained connected by a thick central region.

Murein biosynthesis in ether permeabilized Escherichia coli starting from early peptidoglycan precursors

ETB, ether treated bacteria, from E. coli and other Gram-negative strains, contain in a cell-free system all enzymes necessary for murein biosynthesis. Starting with a variety of combinations of peptidoglycan precursors, high yields of sodium dodecylsulfate (SDS, 4%) insoluble murein or murein like material were synthesized. The amount of newly synthesized SDS insoluble material (NSM) was dependent upon the growing phase at which cells had been harvested for preparation of ETB. This data may provide some insight into the regulation of peptidoglycan biosynthesis. Starting from early peptidoglycan precursors, the cell-free synthesis of NSM was inhibited by specific inhibitors of murein synthesis, such as D-cycloserine, D-fluoroalanine, 2-amino-ethylphosphonate, analogues of D-alanyl-D-alanine and beta-lactam antibiotics at appropriate concentrations. Some D-alanyl-D-alanine analogues and 4-chlorodiaminopimelic acid were incorporated into NSM in place of their corresponding natural substrates.

Inhibition of peptidoglycan synthesis in ether-permeabilized Escherichia coli cells by structural analogs of D-alanyl-D-alanine.

Several analogs of D-alanyl-D-alanine have been proved to be competitive inhibitors of murein (cross-linked peptidoglycan) synthesis in either-permeabilized cells of Escherichia coli. Some analogs, distinguished from D-alanyl-D-alanine only by minor structural deviations, were incorporated into a murein-like sodium dodecyl sulfate-insoluble material in place of the natural substrate. These analogs therefore could be designated as competitive substrates of the cross-linked end product of murein synthesis. In contrast, others, even those containing bulky residues at the methyl group of the amino-terminal D-alanine, exhibited inhibition of murein synthesis. The last-mentioned group of analogs also inhibited a blank value which seems to be a characteristic feature of this system without added D-alanyl-D-alanine. From this data, a steady-state concentration of D-alanyl-D-alanine or D-alanine or both in growing cells of E. coli could be calculated as approximately 0.4 x 10(-3) M.

The effect of nisin on murein synthesis.

Nisin inhibits murein synthesis with concomitant accumulation of undecaprenyl-pyrophospho-MurHAc(pentapeptide) (lipid intermediate I). This inhibition is caused by the formation of a complex between the antibiotic and lipid intermediate I. Undecaprenyl-pyrophospho-MurNAc(pentapeptide)-GlcNAc (lipid intermediate II) also forms a complex with nisin. However, when murein synthesis is inhibited by nisin, this latter complex is not formed since lipid intermediate II is not longer synthesized.

On the process of cellular division in Escherichia coli: isolation and characterization of penicillin-binding proteins 1a, 1b, and 3.

Multiple mutants of Escherichia coli defective in penicillin-binding proteins (PBPs) were constructed, and into these strains Co1E1 plasmids carrying the genes for PBP-1a, -1b, or -3 were introduced. From these plasmid-carrying strains, PBP-1a and -1b were purified by ampicillin-Sepharose affinity chromatography and PBP-3 by cephalexin-Sepharose affinity chromatography. Improved purification was achieved by differential elution with NH2OH. Purified PBP-1b synthesized murein when added to the membrane fraction of a PBP-1b-defective mutant, which by itself failed to support murein synthesis in vitro. The PBP-1b preparation was able to synthesize murein from the lipid intermediate extracted with chloroform/methanol but was unable to utilize UDP-linked precursors for murein synthesis. Murein synthesis was inhibited by vancomysin, ristocetin, moenomycin, and enduracidin, but not by beta-lactam antibiotics. The synthesized murein was shown to contain crosslinked muropeptides. Their crosslinking was abolished by action of beta-lactam antibiotics. The PBP-1a and -3 preparations showed substantially no activity for murein synthesis in the same reaction system. None of the three PBPs showed D-alanine carboxypeptidase activity with UDP-N-acetylmuramoyl-pentapeptide as substrate or endopeptidase activity with bis(disaccharide-peptide) as substrate.

The sensitivity of the pseudomurein-containing genus Methanobacterium to inhibitors of murein synthesis

The sensitivity to inhibitors of various steps of murein synthesis was studied with six strains of methanogenic bacteria. Four of the strains belong to the genus Methanobacterium, which contains pseudomurein in its cell walls. This polymer-as well as murein-is not present in the two control organisms, Methanosarcina barkeri and Methanospirillum hungatii, which were found to be resistant to all inhibitors of murein synthesis. The four strains of Methanobacterium were resistant to the antibiotics fosfomycin, D-cycloserine, vancomycin, penicillin G and cephalosporin C, all of which inhibit the synthesis or function of the peptide subunits of murein. On the other hand, the four strains were susceptible to bacitracin, nisin, gardimycin and enduracidin. It is therefore assumed that the biosynthesis of murein and pseudomurein, respectively, may have some reactions of the so-called lipid cycle and the polymerization of the heteroglycan strands in common.

Murein biosynthesis during a synchromous cell cycle of Escherichia coli B

The last stages of murein biosynthesis were studied in relation to the division cycle of Escherichia coli in cells synchronized by amino acid starvation (Ron et al., J. Bacteriol. 123:374--376, 1975). Murein synthesis and the activities of the D-alanine carboxypeptidase and transpeptidase were found to vary significantly during the cell cycle. Maximal synthesis and transpeptidation were observed immediately after cell division, whereas maximal D-alanine carboxypeptidase activity was detected before cell division. These results are in agreement with our earlier findings that before cell division there is a stage of increased hydrolysis of the C-terminal D-alanine moiety of newly synthesized murein strands.

Effect of benzylpenicillin on the synthesis and structure of the cell envelope of Neisseria gonorrhoeae.

The effect of benzylpenicillin on the synthesis and morphology of the cell envelope of Neisseria gonorrhoeae was examined. Penicillin immediately stopped murein synthesis; it also enhanced the rate of turnover of glucosamine, but not diaminopimelic acid, in the murein. In addition, penicillin greatly increased the shedding of lipid and lipopolysaccharide into the medium. In the electron microscope, protrusions of the cell membrane were evident, as well as apparent holes in the murein cell wall. All of these changes occurred while active synthesis was taking place, before the lysis of the cells. Lysis could be prevented by growing the cells at low pH and high concentrations of Mg2+; however, the effects of penicillin on murein synthesis and turnover and on the release of lipid were not affected.