Proteins encoded by plasmid DNA are specifically labeled in uv-irradiated cells of Escherichia coli carrying recA and uvrA mutations because extensive degradation of the chromosome DNA occurs concurrently with amplification of plasmid DNA.

Simple method for identification of plasmid-coded proteins. [Escherichia coli, uv radiation]

Peptidoglycan molecular requirements allowing detection by Nod1 and Nod2.

The biosynthesis of bacterial cell wall peptidoglycan is a complex process that involves enzyme reactions that take place in the cytoplasm (synthesis of the nucleotide precursors) and on the inner side (synthesis of lipid-linked intermediates) and outer side (polymerization reactions) of the cytoplasmic membrane. This review deals with the cytoplasmic steps of peptidoglycan biosynthesis, which can be divided into four sets of reactions that lead to the syntheses of (1) UDP-N-acetylglucosamine from fructose 6-phosphate, (2) UDP-N-acetylmuramic acid from UDP-N-acetylglucosamine, (3) UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid and (4) D-glutamic acid and dipeptide D-alanyl-D-alanine. Recent data concerning the different enzymes involved are presented. Moreover, special attention is given to (1) the chemical and enzymatic synthesis of the nucleotide precursor substrates that are not commercially available and (2) the search for specific inhibitors that could act as antibacterial compounds.

Cytoplasmic steps of peptidoglycan biosynthesis.

Vancomycin resistance in Enterococcus faecium BM4 147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alaninemalanine ligase of altered substrate specificity (Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-20211, while the sequence of VanH is related to those of a-keto acid dehydrogenases (Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)). We report purification of VanH to homogeneity, characterization as a D-specific a-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2- hydroxybutyrate (K, = 0.60 mM). The VanA product ~-alanyl-~-2-hydroxybutyrate could then be in- corporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D- Ala-D- Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-~-alanyl-~-2-hydroxybutyrate (& > 73 mM) was > 1 000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (& = 54 pM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.

Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA.

Summary: The phenomenon of peptidoglycan recycling is reviewed. Gram-negative bacteria such as Escherichia coli break down and reuse over 60% of the peptidoglycan of their side wall each generation. Recycling of newly made peptidoglycan during septum synthesis occurs at an even faster rate. Nine enzymes, one permease, and one periplasmic binding protein in E. coli that appear to have as their sole function the recovery of degradation products from peptidoglycan, thereby making them available for the cell to resynthesize more peptidoglycan or to use as an energy source, have been identified. It is shown that all of the amino acids and amino sugars of peptidoglycan are recycled. The discovery and properties of the individual proteins and the pathways involved are presented. In addition, the possible role of various peptidoglycan degradation products in the induction of β-lactamase is discussed.

How Bacteria Consume Their Own Exoskeletons (Turnover and Recycling of Cell Wall Peptidoglycan)

The cellular pool levels of most of the cytoplasmic precursors of peptidoglycan synthesis were determined for normally growing cells of Escherichia coli K-12. In particular, a convenient method for analyzing the uridine nucleotide precursor contents was developed by associating gel filtration and reverse-phase high-pressure liquid chromatography techniques. The enzymatic parameters of the four synthetases which catalyze the stepwise addition of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine to uridine diphosphate-N-acetylmuramic acid were determined. It was noteworthy that the pool levels of L-alanine, D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine were much higher than the Km values determined for these substrates, whereas the molar concentrations of the uridine nucleotide precursors were lower than or about the same order of magnitude as the corresponding Km values. Taking into consideration the data obtained, an attempt was made to compare the in vitro activities of the D-glutamic acid, meso-diaminopimelic acid, and D-alanyl-D-alanine adding enzymes with their in vivo functioning, expressed by the amounts of peptidoglycan synthesized. The results also suggested that these adding activities were not in excess in the cell under normal growth conditions, but their amounts appeared adjusted to the requirements of peptidoglycan synthesis. Under the different in vitro conditions considered, only low levels of L-alanine adding activity were observed.

Cytoplasmic steps of peptidoglycan synthesis in Escherichia coli.

The 2-min region of the Escherichia coli genome contains a large cluster of genes from pbpB to envA that code for proteins involved in peptidoglycan biosynthesis and cell division. From pLC26-6 of the collection of Clarke and Carbon (L. Clarke and J. Carbon, Cell 9:91-99, 1976) plasmids carrying different fragments from the 8-kilobase-pair region downstream of pbpB were constructed and analyzed for their ability to direct protein synthesis in maxicells, to complement various thermosensitive mutations, and to overproduce enzymatic activities. We report the localization of the previously unidentified murD gene coding for the D-glutamic acid-adding enzyme within this region. Our data show that the genes are in the order pbpB-murE-murF-X-murD-Y-murG, where X and Y represent chromosomal fragments from 1 to 1.5 kilobase pairs, possibly coding for unknown proteins. Furthermore, the murE and murF genes, encoding the meso-diaminopimelic acid and D-alanyl-D-alanine-adding enzymes, respectively, may be translationally coupled when transcription is initiated upstream of murE, within the preceding structural gene pbpB coding for penicillin-binding protein 3.

Organization of the murE-murG region of Escherichia coli: identification of the murD gene encoding the D-glutamic-acid-adding enzyme.

The d-glutamate-adding and d-alanyl-d-alanine-adding enzymes from Escherichia coli were partially purified by fast protein liquid chromatography on an anion exchanger. Their relative molecular masses, determined by gel filtration on Superose 12, were 54000 ± 2000 and 51000 ± 2000, respectively. In order to investigate the specificity of these ligases, several compounds derived from their respective nucleotide substrates were prepared. In the case of the d-Glu-adding enzyme, DDP-MurNAc-l-Ala (DDP = dihydrouridine 5′-diphosphate) and P1-MurNAc-l-Ala were substrates of the reaction. In the case of the d-Ala-d-Ala-adding enzyme, only DDP-MurNAc-l-Ala-d-Glu(-meso-A2pm) was a subsrate; P1-MurNAc-l-Ala-d-Glu(-meso-A2pm) was neither a substrate nor an inhibitor. Concerning the amino acid site of the d-Glu-adding enzyme, even closely related analogues of d-glutamate hardly inhibited the reaction.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1987.tb13560.x

Partial purification and specificity studies of the D-glutamate-adding and D-alanyl-D-alanine-adding enzymes from Escherichia coli K12.

Nucleotide sequence of the murF gene encoding the UDP-MurNAc-pentapeptide synthetase of Escherichia coli

/pdf/revised-interpretation-of-the-sequence-containing-the-mure-13czj19vse.pdf

Revised interpretation of the sequence containing the murE gene encoding the UDP-N-acetylmuramyl-tripeptide synthetase of Escherichia coli

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