N-acetylglucosamine-6-phosphate deacetylase (NagA) of Listeria monocytogenes EGD, an essential enzyme for the metabolism and recycling of amino sugars

Abstract

The main aim of our study was to determine the physiological function of NagA enzyme in the Listeria monocytogenes cell. The primary structure of the murein of L. monocytogenes is very similar to that of Escherichia coli, the main differences being amidation of diaminopimelic acid and partial de-N-acetylation of glucosamine residues. NagA is needed for the deacetylation of N-acetyl-glucosamine-6 phosphate to glucosamine-6 phosphate and acetate. Analysis of the L. monocytogenes genome reveals the presence of two proteins with NagA domain, Lmo0956 and Lmo2108, which are cytoplasmic putative proteins. We introduced independent mutations into the structural genes for the two proteins. In-depth characterization of one of these mutants, MN1, deficient in protein Lmo0956 revealed strikingly altered cell morphology, strongly reduced cell wall murein content and decreased sensitivity to cell wall hydrolase, mutanolysin and peptide antibiotic, colistin. The gene products of operon 150, consisting of three genes: lmo0956, lmo0957, and lmo0958, are necessary for the cytosolic steps of the amino-sugar-recycling pathway. The cytoplasmic de-N-acetylase Lmo0956 of L. monocytogenes is required for cell wall peptidoglycan and teichoic acid biosynthesis and is also essential for bacterial cell growth, cell division, and sensitivity to cell wall hydrolases and peptide antibiotics.

Fig. 1

The chemical reaction catalyzed by NagA, a N-acetylglucosamine-6-phosphate de-N-acetylase (N)

Fig. 2

Proposed pathways of recycling and amino-sugar metabolism in L. monocytogenes based on those in B. subtilis, including the proteins predicted to be involved in this process—a schematic representation of L. monocytogenes cytoplasmic proteins potentially involved in amino-sugar metabolism. Abbreviations: Fru fructose, Glu glucose. Gene products: NagA, GlcN-6-P de-N-acetylase; NagB, GlcN-6-P isomerase; GlmS, L-glutamate-d-fructose-6-P amidotransferase; GlmM, phosphoglucomutase; GlmU, UDP-GlcNAc pyrophosphorylase; Pgi, Glc-6-P isomerase; GtaB, UTP-Glc_1-P uridyltransferase; NagP, GamP, phosphotransferases. The symbol (asterisk) indicates genes which are organized in an operon structure (according to Toledo-Arana et al. 2009)

Fig. 3

Model of the protein Lmo0956 generated with the use of Swiss Model server (http://swissmodel.expasy.org). Modeled residue range: (a) 3 to 375 from 377 residues, based on template 2vhlA (2.05 Å), (b) 5 to 363 from 377 residues, based on template 2vhlB (2.05 Å)

Fig. 4

Genetic maps of fragments of L. monocytogenes EGD genome including Lmo0956 (a) and lmo2108 (b) genes. Open reading frames and direction of transcription are indicated with arrows

Fig. 5

Scanning electron micrographs of L. monocytogenes mutant MN1 (a), EGD (b). Bar represents 2 μm

Fig. 6

Transmission electron micrographs of L. monocytogenes EGD (a–d) and mutant MN1 (e–l). Bar represents 100 nm (a, b, d, e, f) and 200 nm (c, g, h, i, j, k, l). At the bacterial wall region; (1) a low inner zone (IWZ) precedes (2) a high-density outer zone (OWZ); then (3) a fibrous layer and (4) the second high-density zone in bacterial wall of the MN1 cells. The plasma membrane resides immediately below the IWZ zone (according to Matias and Beveridge 2006)

Fig. 7

Sensitivity to mutanolysin. Bacteria were incubated with mutanolysin (in final concentration—29 μg/ml) at 37°C for 90 min. Lysis of bacterial cells was measured as a decline in optical density (OD₆₀₀) of the sample over time (at 15-min intervals for 90 min)

Fig. 8

HPLC analysis of the muropeptide composition of L. monocytogenes EGD and nagA mutant strains. HPLC elution patterns of muropeptides after digestion with mutanolysin of murein from L. monocytogenes (black line) and mutant MN1 (gray line). Peaks 1–12 correspond to monomeric muropeptides, 13–22 to dimeric muropeptides, and 23 and over to trimeric muropeptides