Growth pattern of the murein sacculus of Escherichia coli

The mechanism by which the murein sacculus of Escherichia coli is being enlarged during growth was investigated by pulse and pulse-chase labeling with [3H]diaminopimelic acid. Changes in the composition of the sacculus during aging were analyzed in detail by high performance liquid chromatography separation of the muropeptide subunits released after complete muramidase digestion. After pulses as short as 10 s, a group of novel phosphorylated muropeptides was detected. The kinetics of their appearance is consistent with these structures being derived from the undecaprenylphosphate-linked growing points of murein. A complex maturation process of murein took place including a rapid decay of pentapeptide side chains and a 10-fold increase in tripeptidyl moieties. In addition, the total degree of cross-linkage increased from 16 to 25%, partly due to a 3-fold increase in the formation of LD-A2pm-A2pm cross-links. In pulse-chase experiments the cross-linkage started to decrease after a maximum at about 35 min of chase. The kinetics in the distribution of the radioactivity among acceptor and donor part in the major cross-bridges Tetra-Tetra and Tetra-Tri differed from each other substantially, indicating that the latter structure is completely cleaved within three generations, whereas only 40% of Tetra-Tetra is cleaved during the same time. Furthermore, the attachment of the lipoprotein to murein was delayed by about one generation. It is proposed that these findings reflect an inside-to-outside growth mechanism of the murein sacculus of E. coli.     

Interactions of Escherichia coli membrane lipoproteins with the murein sacculus.

Bifunctional cross-linking reagents were used to identify cell envelope proteins that interacted with the murein sacculus. This revealed that a number of [3H]leucine-labeled proteins and [3H]palmitate-labeled lipoproteins were reproducibly cross-linked to the sacculus in plasmolyzed cells. The results suggested that most of the cell envelope lipoproteins, and not only the murein lipoprotein, mediate interactions between the murein sacculus and the inner and/or outer membrane of the cell.     

Enzymology of elongation and constriction of the murein sacculus of Escherichia coli

Multiple deletions in murein hydrolases revealed that predominantly amidases are responsible for cleavage of the septum during cell division. Endopeptidases and lytic transglycosylases seem also be involved. In the absence of these enzymes E. coli grows normally but forms chains of adhering cells. Surprisingly, mutants lacking up to eight different murein hydrolases still grow with almost unaffected growth rate. Therefore it is speculated that general enlargement of the murein sacculus may differ from cell division by using transferases rather than the two sets of hydrolytic and synthetic enzymes as seems to be the case for the constriction process. A model is presented that describes growth of the murein of both Gram-positive and -negative bacteria by the activity of murein transferases. It is speculated that enzymes exist that catalyze a transpeptidation of the pre-existing murein onto murein precursors or nascent murein by using the chemical energy present in peptide cross-bridges. Such enzymes would at the same time cleave bonds in the murein net and insert new material into the growing sacculus.     

Evidence for diffuse growth of the cylindrical portion of the Escherichia coli murein sacculus.

High-resolution autoradiography of thin sections of Escherichia coli cells whose murein was pulse-labeled with [3H]diaminopimelic acid after a period of diaminopimelic acid deprivation indicated that elongation of the murein sacculus occurs by a multisite (diffuse) process. Upon chasing, radioactivity in polar murein was stable, whereas radioactivity in cylindrical murein was reduced, indicating that diffuse intercalation of new murein occurred during cell elongation. Elongation and septation were shown to be overlapping processes.     

Phaseolotoxin transport in Escherichia coli and Salmonella typhimurium via the oligopeptide permease.

Phaseolotoxin [(N delta-phosphosulfamyl)ornithylalanylhomoarginine], a phytotoxic tripeptide produced by Pseudomonas syringae pv. phaseolicola that inhibits ornithine carbamoyltransferase, is transported into Escherichia coli and Salmonella typhimurium via the oligopeptide transport system (Opp). Mutants defective in oligopeptide permease (Opp-) were resistant to phaseolotoxin. Spontaneous phaseolotoxin-resistant mutants (Toxr) lacked the Opp function as evidenced by their cross-resistance to triornithine and failure to utilize glycylhistidylglycine as a source of histidine. Growth inhibition by phaseolotoxin was prevented by peptides known to be transported via the Opp system and by treatment of the toxin with L-aminopeptidase. In both E. coli and S. typhimurium, Toxr mutations were cotransducible with trp, suggesting that the opp locus occupies similar positions in genetic maps of these bacteria.     

Regulation of peptide transport in Escherichia coli: induction of the trp-linked operon encoding the oligopeptide permease.

Growth of Escherichia coli in medium containing leucine results in increased entry of exogenously supplied tripeptides into the bacterial cell. This leucine-mediated elevation of peptide transport required expression of the trp-linked opp operon and was accompanied by increased sensitivity to toxic tripeptides, by an enhanced capacity to utilize nutritional peptides, and by an increase in both the velocity and apparent steady-state level of L-[U-14C]alanyl-L-alanyl-L-alanine accumulation for E. coli grown in leucine-containing medium relative to these parameters of peptide transport measured with bacteria grown in media lacking leucine. Direct measurement of opp operon expression by pulse-labeling experiments demonstrated that growth of E. coli in the presence of leucine resulted in increased synthesis of the oppA-encoded periplasmic binding protein.     

The N-acetyl-D-glucosamine kinase of Escherichia coli and its role in murein recycling

N-acetyl-D-glucosamine (GlcNAc) is a major component of bacterial cell wall murein and the lipopolysaccharide of the outer membrane. During growth, over 60% of the murein of the side wall is degraded, and the major products, GlcNAc-anhydro-N-acetylmuramyl peptides, are efficiently imported into the cytoplasm and cleaved to release GlcNAc, anhydro-N-acetylmuramic acid, murein tripeptide (L-Ala-D-Glu-meso-diaminopimelic acid), and D-alanine. Like murein tripeptide, GlcNAc is readily recycled, and this process was thought to involve phosphorylation, since GlcNAc-6-phosphate (GlcNAc-6-P) is efficiently used to synthesize murein or lipopolysaccharide or can be metabolized by glycolysis. Since the gene for GlcNAc kinase had not been identified, in this work we purified GlcNAc kinase (NagK) from Escherichia coli cell extracts and identified the gene by determining the N-terminal sequence of the purified kinase. A nagK deletion mutant lacked phosphorylated GlcNAc in its cytoplasm, and the cell extract of the mutant did not phosphorylate GlcNAc, indicating that NagK is the only GlcNAc kinase expressed in E. coli. Unexpectedly, GlcNAc did not accumulate in a nagK nagEBACD mutant, though both GlcNAc and GlcNAc-6-P accumulate in the nagEBACD mutant, suggesting the existence of an alternative pathway (presumably repressed by GlcNAc-6-P) that reutilizes GlcNAc without the involvement of NagK.     

The lactose permease of Escherichia coli: evidence in favor of a dimer

Lactose permease from Escherichia coli T 206 was purified in octyl-beta-D-glucopyranoside (octyl-glucoside) according to Newman et al. [J. Biol. Chem. (1981) 256, 11804-11808]. In this detergent the protein has a very high tendency to aggregate nonspecifically. Therefore, exchange of octyl-glucoside was performed for another nonionic detergent, dodecyl octaethylene glycol monoether (C12E8), in which the protein is more stable. The amounts of bound C12E8 and phospholipids were measured using radioactive detergent and gas chromatography, respectively, and were found to be respectively 0.2 and 0.15 g/g protein. Analytical ultracentrifugation (sedimentation velocity and sedimentation equilibrium) and gel filtration (conventional and high performance liquid chromatography) experiments indicated that in this detergent the lactose permease existed mainly as a dimer. This result is at variance with the monomeric state of the protein reported by Wright et al. [FEBS Lett. (1983) 162, 11-15] in another nonionic detergent (dodecyl-o-beta-maltoside). We discuss the possible reason for this discrepancy and suggest that the dimeric state of association may well reflect the situation that prevails in the membrane.     

Ligand-induced conformational changes in the lactose permease of Escherichia coli: evidence for two binding sites.

By using a lactose permease mutant containing a single Cys residue in place of Val 331 (helix X), conformational changes induced by ligand binding were studied. With right-side-out membrane vesicles containing Val 331-->Cys permease, lactose transport is inactivated by either N-ethylmaleimide (NEM) or 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM). Remarkably, beta,D-galactopyranosyl 1-thio-beta,D-galactopyranoside (TDG) enhances the rate of inactivation by CPM, a hydrophobic sulfhydryl reagent, whereas NEM inactivation is attenuated by the ligand. Val 331-->Cys permease was then purified and studied in dodecyl-beta,D-maltoside by site-directed fluorescence spectroscopy. The reactivity of Val 331-->Cys permease with 2-(4'-maleimidylanilino)-naphthalene-6-sulfonic acid (MIANS) is not changed over a low range of TDG concentrations (< 0.8 mM), but the fluorescence of the MIANS-labeled protein is quenched in a saturable manner (apparent Kd approximately equal to 0.12 mM) without a change in emission maximum. In contrast, over a higher range of TDG concentrations (1-10 mM), the reactivity of Val 331-->Cys permease with MIANS is enhanced and the emission maximum of MIANS-labeled permease is blue shifted by 3-7 nm. Furthermore, the fluorescence of MIANS-labeled Val 331 -->Cys permease is quenched by both acrylamide and iodide, but the former is considerably more effective. A low concentration of TDG (0.2 mM) does not alter quenching by either compound, whereas a higher concentration of ligand (10 mM) decreases the quenching constant for iodide by about 50% and for acrylamide by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acetate metabolism in Escherichia coli strains lacking phosphoenolpyruvate: carbohydrate phosphotransferase system; evidence of carbon recycling strategies and futile cycles

The ptsHIcrr operon was deleted from Escherichia coli wild-type JM101 to generate strain PB11 (PTS(-)). In a mutant derived from PB11 that partially recovered its growth capacity on glucose by an adaptive evolution process (PB12, PTS(-)Glc(+)), part of the phosphoenolpyruvate not used in glucose transport has been utilized for the synthesis of aromatic compounds. In this report, it is shown that on acetate as a carbon source, PB11 displayed a specific growth rate (mu) higher than PB12 (0.21 and 0.13 h(-1), respectively) while JM101 had a mu of 0.28 h(-1). To understand these growth differences on acetate, we compared the expression profiles of central metabolic genes by RT-PCR analysis. Obtained data revealed that some gluconeogenic genes were downregulated in both PTS(-) strains as compared to JM101, while most glycolytic genes were upregulated in PB12 in contrast to PB11 and JM101. Furthermore, inactivation of gluconeogenic genes, like ppsA, sfcA, and maeB,and poxB gene that codes for pyruvate oxidase, has differential impacts in the acetate metabolism of these strains. Results indicate that growth differences on acetate in the PTS(-) derivatives are due to potential carbon recycling strategies, mainly in PB11, and futile carbon cycles, especially in PB12.     

Turnover of murein in a diaminopimelic acid dependent mutant ofEscherichia coli

Escherichia coli 173-25, whose cell wall was labelled with¹⁴C-diaminopimelic acid, was found to lose about 15% radioactivity during growth in a fresh medium, two thirds or more being lost during the first two generations. Degradation products of the cell wall were mostly of low-molecular type. About 5% of the cells lyzed as a result of transfer associated with filtration, washing and resuspension of the bacterial population in a diaminopimelic acid (DAP) deficient medium. The degradation was very low during the first 20 min. The amount of wall material released from the cells increased between 20–30 min and a sudden decrease of viability of the population was observed. The degradation of murein triggered by starvation for DAP continued when supplementing the deficient medium with DAP and when growth was resumed. About one-half of the cell wall material released into the medium under these conditions was macromolecular. However, lysis of the cells and release of proteins into the medium were rapidly interrupted after DAP was added to the starving culture and the differential rate of synthesis of the cell wall increased. Turnover of murein was not associated with protein turnover.     

Analysis of murein and murein precursors during antibiotic-induced lysis of Escherichia coli.

Lysis of Escherichia coli induced by either D-cycloserine, moenomycin, or penicillin G was monitored by studying murein metabolism. The levels of the soluble murein precursor UDP-N-acetylmuramyl-L-alanyl-D-glutamyl-m-diaminopimelyl-D-alanyl- D-alanine (UDP-MurNAc-pentapeptide) and the carrier-linked MurNAc-(pentapeptide)-pyrophosphoryl-undecaprenol as well as N-acetylglucosamine-beta-1,4-MurNAc-(pentapeptide)-pyrophosphoryl- undecaprenol varied in a specific way. In the presence of penicillin, which is known to interfere with the cross-linking of murein, the concentration of the lipid-linked precursors unexpectedly decreased before the onset of lysis, although the level of UDP-MurNAc-pentapeptide remained normal. In the case of moenomycin, which specifically blocks the formation of the murein polysaccharide strands, the lipid-linked precursors as well as UDP-MurNAc-pentapeptide accumulated as was expected. D-Cycloserine, which inhibits the biosynthesis of UDP-MurNAc-pentapeptide, consequently caused a decrease in all three precursors. The muropeptide composition of the murein showed general changes such as an increase in the unusual DL-cross bridge between two neighboring meso-diaminopimelic acid residues and, as a result of uncontrolled DL- and DD-carboxypeptidase activity, an increase in tripeptidyl and a decrease in tetrapeptidyl and pentapeptidyl moieties. The average length of the glycan strands decreased. When the glycan strands were fractionated according to length, a dramatic increase in the amount of single disaccharide units was observed not only in the presence of penicillin but also in the presence of moenomycin. This result is explained by the action of an exo-muramidase, such as the lytic transglycosylases present in E. coli. It is proposed that antibiotic-induced bacteriolysis is the result of a zipperlike splitting of the murein net by exo-muramidases locally restricted to the equatorial zone of the cell.     

Identification of a dedicated recycling pathway for anhydro-N-acetylmuramic acid and N-acetylglucosamine derived from Escherichia coli cell wall murein

Turnover and recycling of the cell wall murein represent a major metabolic pathway of Escherichia coli. It is known that E. coli efficiently reuses, i.e., recycles, its murein tripeptide, L-alanyl-gamma-D-glutamyl-meso-diaminopimelate, to form new murein. However, the question of whether the cells also recycle the amino sugar moieties of cell wall murein has remained unanswered. It is demonstrated here that E. coli recycles the N-acetylglucosamine present in cell wall murein degradation products for de novo murein and lipopolysaccharide synthesis. Furthermore, E. coli also recycles the anhydro-N-acetylmuramic acid moiety by first converting it into N-acetylglucosamine. Based on the results obtained by studying mutants unable to recycle amino sugars, the pathway for recycling is revealed.     

Arrangement of glycan chains in the sacculus of Escherichia coli.

A novel of Escherichia coli endopeptidase was used for a selective partial hydrolysis of the peptide bridges which interlink the glycan chains in E. coli sacculi. The loosening of the murein network revealed, in the electron microscope, a preferential orientation of the glycan chains, more or less perpendicular to the length axis of the cell. Control incubations with E. coli transglycosylase or egg-white lysozyme did not leave ordered structures behind.     

Novel type of murein transglycosylase in Escherichia coli.

The purification and properties of a novel type of murein transglycosylase from Escherichia coli are described. The purified enzyme appears as a single band on sodium dodecyl sulfate-polyacrylamide gels and has an apparent molecular weight of approximately 65,000 as estimated by gel filtration and gel electrophoresis. It degrades pure murein sacculi from E. coli almost completely into low-molecular-weight products. The two prominent muropeptide fragments in the digest are the disaccharide-tripeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid and the corresponding disaccharide-tetrapeptide N-acetylglucosamine-N-acetylmuramic acid-L-alanine-D-iso-glutamic acid-meso-diaminopimelic acid-D-alanine. The unique feature of these compounds is that the disaccharide has no reducing end group and that the muramic acid residue possesses an internal 1 leads to 6 anhydro linkage. The new lytic enzyme is designated as a murein: murein transglycosylase. Its possible role in the rearrangement of murein during cell growth and division is discussed.     

Absence of oligomeric murein intermediates in Escherichia coli.

The intermediates in the biosynthetic pathway of murein were examined in two strains of Escherichia coli to determine whether they synthesized oligomeric precursors in vivo. No oligomeric precursors could be detected; the only intermediates found were the previously described UDP-N-acetylmuramyl peptides, and the two lipid-linked compounds, N-acetylglucosamyl-N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol and N-acetylmuramyl-(pentapeptide)-pyrophosphoryl-undecaprenol. It was concluded that lipid-linked monomers are directly incorporated into the murein sacculus in vivo and do not pass through an oligomeric stage.     

Interactions of membrane lipoproteins with the murein sacculus of Escherichia coli as shown by chemical crosslinking studies of intact cells

Proteins that were closely associated with murein in intact cells of Escherichia coli were studied by treating [3H]leucine and [3H]palmitate-labeled cells with the chemical crosslinking reagent dithiobis(succinimidylpropionate). Murein was purified and crosslinked peptides were released from the murein by treatment with beta-mercaptoethanol. Nine murein-associated [3H]leucine-labeled peptides were identified. Five of the nine peptides were lipoproteins, based on labeling with [3H]palmitate, protease sensitivity and gel electrophoretic correspondence to membrane lipoproteins present in uncrosslinked cell envelope preparations. The results suggest that these membrane lipoproteins may play a significant role in the structural integration of the murein and membrane layers of the cell envelope.     

Genetic characterization of an Escherichia coli mutant altered in the structure of murein lipoprotein.

Mutants defective in the structure, biosynthesis, and assembly of murein lipoprotein have been isolated. One of these mutants has been shown to synthesize a structurally altered lipoprotein. The biochemical features of the mutant lipoprotein (lipid deficiency, dimer formation, and a reduced, bound form of lipoprotein) could be attributed to a single mutation (or closely linked mutations) located at 36.4 min of the Escherichia coli map. We propose that this mutant is altered in the structural gene for murein lipoprotein (mlpA). Biochemical studies carried out with a heterogenote, mlpA/F'mlpA+, revealed the biochemical codominance of the wild-type and mutant genes.     

Multiplicity of oligopeptide transport systems in Escherichia coli.

The ability of Escherichia coli K-12 4212 to utilize a variety of oligopeptides as sources of required amino acids was examined. Triornithine-resistant mutants of this strain were oligopeptide permease deficient (Opp-) as judged by their inability to utilize (Lys)3 and (Lys)4 as sources of lysine and their resistance to the toxic tripeptide (Val)3. These same mutants were able to grow when Met-Met-Met, Met-Gly-Met, Met-Gly-Gly, Gly-Met-Gly, Gly-Gly-Met, Gly-Met-Met, Met-Met-Gly, or Leu-Leu-Leu were supplied in place of the requisite amino acid. The system mediating the uptake of these peptides, herein designated Opr I, was not able to transport N-alpha-acetylated peptides, nor the tetrapeptides Met-Gly-Met-Met, Met-Met-Gly-Met, or Met-Met-Met-Gly. Competition experiments indicated that trimethionine and trileucine enter E. coli K-12 via either Opp or Opr I. Analogous results were found using the methionine, leucine-requiring auxotroph E. coli B163. It appears that more than one oligopeptide transport system exists in E. coli and that the system mediating peptide uptake is complex.