Structural basis for the peptidoglycan editing activity of YfiH

Bacterial cells are encased in peptidoglycan (PG), a polymer of disaccharide N-acetyl-glucosamine (GlcNAc) and N-acetyl-muramic acid (MurNAc) cross-linked by peptide stems. PG is synthesized in the cytoplasm as UDP-MurNAc-peptide precursors, of which the amino-acid composition of the peptide is unique, with L-Ala added at the first position in most bacteria but L-Ser or Gly in some bacteria. YfiH is a PG-editing factor whose absence causes misincorporation of L-Ser instead of L-Ala into peptide stems; but its mechanistic function is unknown. Here we report the crystal structures of substrate-bound and product-bound YfiH, showing that YfiH is a cytoplasmic amidase that controls the incorporation of the correct amino acid to the nucleotide precursors by preferentially cleaving the nucleotide precursor byproduct UDP-MurNAc-L-Ser. This work reveals an editing mechanism in the cytoplasmic steps of peptidoglycan biosynthesis.

A muramidase from Acremonium alcalophilum hydrolyse peptidoglycan found in the gastrointestinal tract of broiler chickens

This study evaluates peptidoglycan hydrolysis by a microbial muramidase from the fungus Acremonium alcalophilum in vitro and in the gastrointestinal tract of broiler chickens. Peptidoglycan used for in vitro studies was derived from 5 gram-positive chicken gut isolate type strains. In vitro peptidoglycan hydrolysis was studied by three approaches: a) helium ion microscopy to identify visual phenotypes of hydrolysis b) reducing end assay to quantify solubilization of peptidoglycan fragments and c) mass spectroscopy to estimate relative abundances of soluble substrates and reaction products. Visual effects of peptidoglycan hydrolysis could be observed by helium ion microscopy and the increase in abundance of soluble peptidoglycan due to hydrolysis was quantified by a reducing end assay. Mass spectroscopy confirmed the release of hydrolysis products and identified muropeptides from the five different peptidoglycan sources. Peptidoglycan hydrolysis in chicken crop, jejunum and caecum samples was measured by quantifying the total and soluble muramic acid content. A significant increase in the proportion of the soluble muramic acid was observed in all three segments upon inclusion of the microbial muramidase in the diet.

A new method to measure amino sugar isomers and amino acids in soil extracts and soil hydrolysates based on AccQ.Tag-chemistry and reversed phase ultra-high-performance liquid chromatography

<p>Soil microbial necromass represents a significant proportion (>50%) of soil organic matter (SOM). Microbial necromass consists mainly of particulate organic residues from fragmented cells walls and other slow turnover cytoplasmic components of dead fungi and bacteria. Some of the key components of microbial cell walls, such as peptides and amino sugar polymers, can remain and accumulate in the soil over prolonged times. Amino sugars have been used as biomarkers to quantify the contribution of microbial necromass to stabilized SOM. The different amino sugars present in polymeric form in soils can be released by acid hydrolysis and allow the estimation of the contribution of both fungal and bacterial necromass to the SOM pool. Among the amino sugars, hexosamine isomers (glucosamine, galactosamine, mannosamine) and muramic acid (the ether of lactic acid and glucosamine) are the most abundant ones. Muramic acid is specific to bacterial peptidoglycan while glucosamine is an abundant cell wall component of both, fungal chitin and bacterial peptidoglycan.</p><p>There are several chromatographic methods to measure free and bound amino sugars and amino acids in soil extracts and soil hydrolysates, but none of them allow the combined determination of amino sugar biomarkers and amino acids simultaneously in a single assay for rapid analysis. This is important as a large fraction of soil necromass N (>50%) consists of non-amino sugar-N, such as proteins and nucleic acids. In this study we therefore adopt a method based on 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ.Tag) derivatization of amino compounds and optimized chromatographic (reversed phase) separation to simultaneously measure amino sugars (isomers) and amino acids in soil extracts and soil hydrolysates using ultra-high-performance liquid chromatography coupled to fluorescence or UV detection.</p><p>The use of this method allows for fast, robust and highly sensitive quantification of amino acids and amino sugars in environmental samples at sub-micromolar levels. This approach will help to improve our understanding of soil microbial necromass dynamics and their inherent effect on soil C and N sequestration. The AccQ.Tag chemistry also allows compound detection by electrospray ionization (ESI)-mass spectrometry, enabling isotope (<sup>13</sup>C, <sup>15</sup>N) tracing applications.</p>

Disruption ofmplActivates β-Lactamase Production inStenotrophomonas maltophiliaandPseudomonas aeruginosaClinical Isolates

ABSTRACTThe hyperproduction of chromosomally encoded β-lactamases is a key method of acquired resistance to ceftazidime, aztreonam, and, when seen in backgrounds having reduced envelope permeability, carbapenems. Here, we show that the loss of Mpl, a UDP-muramic acid/peptide ligase, is a common and previously overlooked cause of chromosomally encoded β-lactamase hyperproduction in clinical isolates ofStenotrophomonas maltophiliaandPseudomonas aeruginosa, important pathogens notorious for their β-lactam-resistant phenotypes.