AsnB Mediates Amidation of Meso-Diaminopimelic Acid Residues in the Peptidoglycan of Listeria monocytogenes and Affects Bacterial Surface Properties and Host Cell Invasion

A mutant of Listeria monocytogenes ScottA with a transposon in the 5' untranslated region of the asnB gene was identified to be hypersensitive to the antimicrobial t-cinnamaldehyde. Here, we report the functional characterization of AsnB in peptidoglycan (PG) modification and intracellular infection. While AsnB of Listeria is annotated as a glutamine-dependent asparagine synthase, sequence alignment showed that this protein is closely related to a subset of homologs that catalyze the amidation of meso-diaminopimelic acid (mDAP) residues in the peptidoglycan of other bacterial species. Structural analysis of peptidoglycan from an asnB mutant, compared to that of isogenic wild-type (WT) and complemented mutant strains, confirmed that AsnB mediates mDAP amidation in L. monocytogenes. Deficiency in mDAP amidation caused several peptidoglycan- and cell surface-related phenotypes in the asnB mutant, including formation of shorter but thicker cells, susceptibility to lysozyme, loss of flagellation and motility, and a strong reduction in biofilm formation. In addition, the mutant showed reduced invasion of human epithelial JEG-3 and Caco-2 cells. Analysis by immunofluorescence microscopy revealed that asnB inactivation abrogated the proper display at the listerial surface of the invasion protein InlA, which normally gets cross-linked to mDAP via its LPXTG motif. Together, this work shows that AsnB of L. monocytogenes, like several of its homologs in related Gram-positive bacteria, mediates the amidation of mDAP residues in the peptidoglycan and, in this way, affects several cell wall and cell surface-related properties. It also for the first time implicates the amidation of peptidoglycan mDAP residues in cell wall anchoring of InlA and in bacterial virulence.

Download Full-text

Functional characterization of a cellulose synthase, CtCESA1, from the marine red alga Calliarthron tuberculosum (Corallinales)

Journal of Experimental Botany ◽

10.1093/jxb/erab414 ◽

2021 ◽

Author(s):

Jan Xue ◽

Pallinti Purushotham ◽

Justin F Acheson ◽

Ruoya Ho ◽

Jochen Zimmer ◽

...

Keyword(s):

Cell Wall ◽

Bacterial Species ◽

Functional Characterization ◽

Cellulose Synthase ◽

Red Alga ◽

Land Plant ◽

Primary Cell Wall ◽

Vitro Assay ◽

Red Algal

Abstract In land plants and algae, cellulose is important for strengthening cell walls and preventing breakage due to physical forces. Though our understanding of cellulose production by cellulose synthase enzymes (CESAs) has seen significant advances for several land plant and bacterial species, functional characterization of this fundamental protein is absent in red algae. Here we identify CESA gene candidates in the calcifying red alga Calliarthron tuberculosum (Ct) using sequence similarity-based approaches and elucidate their phylogenetic relationship with other CESAs from diverse taxa. One gene candidate, CtCESA1, was closely related to other putative red algal CESAs. To test if CtCESA1 encoded a true cellulose synthase, CtCESA1 protein was expressed and purified from insect and yeast expression systems. CtCESA1 showed glucan synthase activity in glucose tracer assays. CtCESA1 activity was relatively low when compared to plant and bacterial CESA activity. In an in vitro assay, a predicted N-terminal starch-binding domain from CtCESA1 bound red algal floridean starch extracts, representing a unique domain in red algal CESAs not present in CESAs from other lineages. When the CtCESA1 gene was introduced into Arabidopsis thaliana cesa mutants, the red algal CtCESA1 partially rescued the growth defects of the primary cell wall cesa6 mutant, but not cesa3 or secondary cell wall cesa7 mutants. A fluorescently tagged CtCESA1 localized to the plasma membrane in the Arabidopsis cesa6 mutant background. This study presents functional evidence validating the sequence annotation of red algal cellulose synthases. The relatively low activity of CtCESA1, partial complementation in Arabidopsis, and presence of unique protein domains suggest that there are likely functional differences between the algal and land plant CESAs.

Download Full-text

Characterization of O-Acetylation of N-Acetylglucosamine

Journal of Biological Chemistry ◽

10.1074/jbc.m111.241414 ◽

2011 ◽

Vol 286 (27) ◽

pp. 23950-23958 ◽

Cited By ~ 76

Author(s):

Elvis Bernard ◽

Thomas Rolain ◽

Pascal Courtin ◽

Alain Guillot ◽

Philippe Langella ◽

...

Keyword(s):

Cell Wall ◽

Structural Characterization ◽

Bacterial Species ◽

Muramic Acid ◽

Gram Positive Bacteria ◽

Mutant Strains ◽

Encoding Genes ◽

Hydrolysis Of ◽

Cell Wall Hydrolysis

Peptidoglycan (PG) N-acetyl muramic acid (MurNAc) O-acetylation is widely spread in Gram-positive bacteria and is generally associated with resistance against lysozyme and endogenous autolysins. We report here the presence of O-acetylation on N-acetylglucosamine (GlcNAc) in Lactobacillus plantarum PG. This modification of glycan strands was never described in bacteria. Fine structural characterization of acetylated muropeptides released from L. plantarum PG demonstrated that both MurNAc and GlcNAc are O-acetylated in this species. These two PG post-modifications rely on two dedicated O-acetyltransferase encoding genes, named oatA and oatB, respectively. By analyzing the resistance to cell wall hydrolysis of mutant strains, we showed that GlcNAc O-acetylation inhibits N-acetylglucosaminidase Acm2, the major L. plantarum autolysin. In this bacterial species, inactivation of oatA, encoding MurNAc O-acetyltransferase, resulted in marked sensitivity to lysozyme. Moreover, MurNAc over-O-acetylation was shown to activate autolysis through the putative N-acetylmuramoyl-l-alanine amidase LytH enzyme. Our data indicate that in L. plantarum, two different O-acetyltransferases play original and antagonistic roles in the modulation of the activity of endogenous autolysins.

Download Full-text

The Sortase SrtA of Listeria monocytogenes Is Involved in Processing of Internalin and in Virulence

Infection and Immunity ◽

10.1128/iai.70.3.1382-1390.2002 ◽

2002 ◽

Vol 70 (3) ◽

pp. 1382-1390 ◽

Cited By ~ 87

Author(s):

Caroline Garandeau ◽

Hélène Réglier-Poupet ◽

Iharilalao Dubail ◽

Jean-Luc Beretti ◽

Patrick Berche ◽

...

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Protein A ◽

Knockout Mutant ◽

Reporter Protein ◽

Gram Positive ◽

Bacterial Surface ◽

Gram Positive Bacteria ◽

Covalently Linked

ABSTRACT Listeria monocytogenes is an intracellular gram-positive human pathogen that invades eucaryotic cells. Among the surface-exposed proteins playing a role in this invasive process, internalin belongs to the family of LPXTG proteins, which are known to be covalently linked to the bacterial cell wall in gram-positive bacteria. Recently, it has been shown in Staphylococcus aureus that the covalent anchoring of protein A, a typical LPXTG protein, is due to a cysteine protease, named sortase, required for bacterial virulence. Here, we identified in silico from the genome of L. monocytogenes a gene, designated srtA, encoding a sortase homologue. The role of this previously unknown sortase was studied by constructing a sortase knockout mutant. Internalin was used as a reporter protein to study the effects of the srtA mutation on cell wall anchoring of this LPXTG protein in L. monocytogenes. We show that the srtA mutant (i) is affected in the display of internalin at the bacterial surface, (ii) is significantly less invasive in vitro, and (iii) is attenuated in its virulence in the mouse. These results demonstrate that srtA of L. monocytogenes acts as a sortase and plays a role in the pathogenicity.

Download Full-text

Discovery of fibrillar adhesins across bacterial species

BMC Genomics ◽

10.1186/s12864-021-07586-2 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Vivian Monzon ◽

Aleix Lafita ◽

Alex Bateman

Keyword(s):

Cell Surface ◽

Bacterial Species ◽

Domain Architecture ◽

Surface Proteins ◽

Bacterial Surface ◽

Bacterial Phyla ◽

Gram Positive Bacteria ◽

Invasion Mechanisms ◽

C Terminus ◽

Wide Range

Abstract Background Fibrillar adhesins are long multidomain proteins that form filamentous structures at the cell surface of bacteria. They are an important yet understudied class of proteins composed of adhesive and stalk domains that mediate interactions of bacteria with their environment. This study aims to characterize fibrillar adhesins in a wide range of bacterial phyla and to identify new fibrillar adhesin-like proteins to improve our understanding of host-bacteria interactions. Results Through careful literature and computational searches, we identified 82 stalk and 27 adhesive domain families in fibrillar adhesins. Based on the presence of these domains in the UniProt Reference Proteomes database, we identified and analysed 3,542 fibrillar adhesin-like proteins across species of the most common bacterial phyla. We further enumerate the adhesive and stalk domain combinations found in nature and demonstrate that fibrillar adhesins have complex and variable domain architectures, which differ across species. By analysing the domain architecture of fibrillar adhesins, we show that in Gram positive bacteria, adhesive domains are mostly positioned at the N-terminus and cell surface anchors at the C-terminus of the protein, while their positions are more variable in Gram negative bacteria. We provide an open repository of fibrillar adhesin-like proteins and domains to enable further studies of this class of bacterial surface proteins. Conclusion This study provides a domain-based characterization of fibrillar adhesins and demonstrates that they are widely found in species across the main bacterial phyla. We have discovered numerous novel fibrillar adhesins and improved our understanding of pathogenic adhesion and invasion mechanisms.

Download Full-text

GtcA is required for LTA glycosylation in Listeria monocytogenes serovar 1/2a and Bacillus subtilis

10.1101/2019.12.12.873851 ◽

2019 ◽

Cited By ~ 2

Author(s):

Jeanine Rismondo ◽

Talal F. M. Haddad ◽

Yang Shen ◽

Martin J. Loessner ◽

Angelika Gründling

Keyword(s):

Cell Wall ◽

Bacillus Subtilis ◽

Listeria Monocytogenes ◽

Teichoic Acid ◽

Lipoteichoic Acid ◽

Wall Teichoic Acid ◽

Gram Positive Bacteria ◽

Cell Wall Polymers ◽

Galactose Residue

ABSTRACTThe cell wall polymers wall teichoic acid (WTA) and lipoteichoic acid (LTA) are often modified with glycosyl and D-alanine residues. Recent studies have shown that a three-component glycosylation system is used for the modification of LTA in several Gram-positive bacteria including Bacillus subtilis and Listeria monocytogenes. In the L. monocytogenes 1/2a strain 10403S, the cytoplasmic glycosyltransferase GtlA is thought to use UDP-galactose to produce the C55-P-galactose lipid intermediate, which is transported across the membrane by an unknown flippase. Next, the galactose residue is transferred onto the LTA backbone on the outside of the cell by the glycosyltransferase GtlB. Here we show that GtcA is necessary for the glycosylation of LTA in L. monocytogenes 10403S and B. subtilis 168 and we hypothesize that these proteins act as C55-P-sugar flippases. With this we revealed that GtcA is involved in the glycosylation of both teichoic acid polymers in L. monocytogenes 10403S, namely WTA with N-acetylglucosamine and LTA with galactose residues. These findings indicate that the L. monocytogenes GtcA protein can act on different C55-P-sugar intermediates. Further characterization of GtcA in L. monocytogenes led to the identification of residues essential for its overall function as well as residues, which predominately impact WTA or LTA glycosylation.GRAPHICAL ABSTRACT

Download Full-text

Identification and Characterization of a Peptidoglycan Hydrolase, MurA, of Listeria monocytogenes, a Muramidase Needed for Cell Separation

Journal of Bacteriology ◽

10.1128/jb.185.23.6801-6808.2003 ◽

2003 ◽

Vol 185 (23) ◽

pp. 6801-6808 ◽

Cited By ~ 56

Author(s):

Shannon A. Carroll ◽

Torsten Hain ◽

Ulrike Technow ◽

Ayub Darji ◽

Philippos Pashalidis ◽

...

Keyword(s):

Cell Wall ◽

Listeria Monocytogenes ◽

Cell Separation ◽

Bacterial Species ◽

Surface Protein ◽

Wild Type ◽

Terminal Domain ◽

Cell Wall Hydrolase ◽

Characteristic Features ◽

Type Gene

ABSTRACT A novel cell wall hydrolase encoded by the murA gene of Listeria monocytogenes is reported here. Mature MurA is a 66-kDa cell surface protein that is recognized by the well-characterized L. monocytogenes-specific monoclonal antibody EM-7G1. MurA displays two characteristic features: (i) an N-terminal domain with homology to muramidases from several gram-positive bacterial species and (ii) four copies of a cell wall-anchoring LysM repeat motif present within its C-terminal domain. Purified recombinant MurA produced in Escherichia coli was confirmed to be an authentic cell wall hydrolase with lytic properties toward cell wall preparations of Micrococcus lysodeikticus. An isogenic mutant with a deletion of murA that lacked the 66-kDa cell wall hydrolase grew as long chains during exponential growth. Complementation of the mutant strain by chromosomal reintegration of the wild-type gene restored expression of this murein hydrolase activity and cell separation levels to those of the wild-type strain. Studies reported herein suggest that the MurA protein is involved in generalized autolysis of L. monocytogenes.

Download Full-text

Biochemical characterization of a cortexless mutant of a variant of Bacillus cereus

Canadian Journal of Microbiology ◽

10.1139/m76-147 ◽

1976 ◽

Vol 22 (7) ◽

pp. 1007-1012 ◽

Cited By ~ 2

Author(s):

Susanne M. Pearce

Keyword(s):

Cell Wall ◽

Fatty Acid Composition ◽

Fatty Acid ◽

Bacillus Cereus ◽

Biochemical Characterization ◽

Diaminopimelic Acid ◽

Peptidoglycan Synthesis ◽

Biochemical Lesion ◽

Do So

Previous studies on this cortexless mutant of Bacillus cereus var. alesti indicated that the forespore membrane was the site of the biochemical lesion. This hypothesis is supported by the results presented here: fatty acid composition of sporulating cells of the mutant is altered, while in vegetative cells it is comparable to the parent; soluble precursors of peptidoglycan synthesis are accumulated in the mutant, at the time of cortex formation; homogenates of the mutant prepared at the time of cortex formation are unable to incorporate tritiated diaminopimelic acid into peptidoglycan, while homogenates of cells forming germ cell wall do so to an extent comparable to that of the parent; lipid-linked intermediates are formed by the mutant as in the parent. Apparently the mutant is unable either to transfer disaccharide penta-peptide units from the carrier lipid to the growing peptidoglycan acceptor, or to transport lipid-linked intermediates across the forespore membrane.

Download Full-text

In silico and functional characterization of the promoter of a Eucalyptussecondary cell wall associated cellulose synthase gene (EgCesA1)

BMC Proceedings ◽

10.1186/1753-6561-5-s7-p107 ◽

2011 ◽

Vol 5 (S7) ◽

Cited By ~ 1

Author(s):

Nicky Creux ◽

Minique De Castro ◽

Martin Ranik ◽

Antanas Spokevicius ◽

Gerd Bossinger ◽

...

Keyword(s):

Cell Wall ◽

In Silico ◽

Functional Characterization ◽

Cellulose Synthase ◽

Cellulose Synthase Gene

Download Full-text

Identification and Functional Characterization of the CVOMT and EOMT Genes Promoters from Ocimum Basilicum L

10.21203/rs.3.rs-642688/v1 ◽

2021 ◽

Author(s):

Fatemeh Khakdan ◽

Zahra Shirazi ◽

Mojtaba Ranjbar

Keyword(s):

Water Deficit ◽

Transcriptional Control ◽

Functional Characterization ◽

Pcr Analysis ◽

Water Deficit Stress ◽

Specific Expression ◽

Stress Dependent ◽

First Time ◽

Dependent Mechanism

Abstract Methyl chavicol and methyl eugenol are important phenylpropanoid compounds previously purified from basil. These compounds are significantly enhanced by the water deficit stress-dependent mechanism. Here, for the first time, pObCVOMT and pObEOMT promoters were extracted by the genome walking method. They were then cloned into the upstream of the β-glucuronidase (GUS) reporter gene to identify the pattern of GUS water deficit stress-specific expression. Histochemical GUS assays showed in transgenic tobacco lines bearing the GUS gene driven by pObCVOMT and pObEOMT promoters, GUS was strongly expressed under water deficit stress. qRT-PCR analysis of pObCVOMT and pObEOMT transgenic plants confirmed the histochemical assays, indicating that the GUS expression is also significantly induced and up-regulated by increasing density of water deficit stress. This indicates these promoters are able to drive inducible expression. The cis-acting elements analysis showed that the pObCVOMT and pObEOMT promoters contained dehydration or water deficit-related transcriptional control elements.

Download Full-text