A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge, Identified in Lactobacillus casei Bacteriophage Endolysins

Regulski, Krzysztof; Courtin, Pascal; Kulakauskas, Saulius; Chapot‐Chartier, Marie‐Pierre

doi:10.1074/jbc.m112.446344

Cited by 27 publications

(24 citation statements)

References 51 publications

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“…PG was then hydrolyzed with mutanolysin, and the resulting soluble muropeptides were reduced and separated by reverse phase HPLC with an Agilent UHPLC 1290 system using an ammonium phosphate buffer and linear methanol gradient as described previously (29). The eluted muropeptides were detected by UV absorbance at 202 nm.…”

Section: Methodsmentioning

confidence: 99%

Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis

Solopova

Formosa-Dague

Courtin

et al. 2016

Journal of Biological Chemistry

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To ensure optimal cell growth and separation and to adapt to environmental parameters, bacteria have to maintain a balance between cell wall (CW) rigidity and flexibility. This can be achieved by a concerted action of peptidoglycan (PG) hydrolases and PG-synthesizing/modifying enzymes. In a search for new regulatory mechanisms responsible for the maintenance of this equilibrium in Lactococcus lactis, we isolated mutants that are resistant to the PG hydrolase lysozyme. We found that 14% of the causative mutations were mapped in the guaA gene, the product of which is involved in purine metabolism. Genetic and transcriptional analyses combined with PG structure determination of the guaA mutant enabled us to reveal the pivotal role of the pyrB gene in the regulation of CW rigidity. Our results indicate that conversion of L-aspartate (L-Asp) to N-carbamoyl-L-aspartate by PyrB may reduce the amount of L-Asp available for PG synthesis and thus cause the appearance of Asp/Asn-less stem peptides in PG. Such stem peptides do not form PG cross-bridges, resulting in a decrease in PG cross-linking and, consequently, reduced PG thickness and rigidity. We hypothesize that the concurrent utilization of L-Asp for pyrimidine and PG synthesis may be part of the regulatory scheme, ensuring CW flexibility during exponential growth and rigidity in stationary phase. The fact that L-Asp availability is dependent on nucleotide metabolism, which is tightly regulated in accordance with the growth rate, provides L. lactis cells the means to ensure optimal CW plasticity without the need to control the expression of PG synthesis genes.Peptidoglycan (PG) 8 is the major component of the Grampositive bacterial cell wall (CW), which envelops the cell as a multilayer sacculus. PG consists of a basic unit made up of N-acetylglucosamine-N-acetylmuramic acid (GlcNAc-MurNAc) disaccharides bound to stem pentapeptides. Disaccharide pentapeptide units are synthesized intracellularly and transported through the cytoplasmic membrane as lipid-disaccharide pentapeptides called lipid II. These blocks are covalently linked to the pre-existing PG polymers by high molecular weight penicillin-binding proteins (PBPs) (1). Class A PBPs contain both transglycosylation and transpeptidation domains, whereas class B PBPs are involved only in transpeptidation. Transglycosylation links the disaccharide pentapeptide to the pre-existing PG chain, whereas transpeptidation connects the stem pentapeptides to neighboring chains, which ensures PG cross-linking through the formation of an interpeptide bridge. Cross-linking in Lactococcus lactis involves the synthesis of an interpeptide bridge made of one D-amino acid (D-Asp or D-Asn), and in this species the PG cross-linking index was estimated to be 35.5% (2). Studies of Staphylococcus aureus have revealed that this PG cross-linking correlates with CW rigidity (3).The basic PG structure is often modified as PG glycan chains can undergo N-deacetylation or O-acetylation, and free carboxyl groups of amino acids of peptide chains ...

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Section: Methodsmentioning

confidence: 99%

Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis

Solopova

Formosa-Dague

Courtin

et al. 2016

Journal of Biological Chemistry

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“…Like bacterial lysins, CLEs usually contain a binding domain with binding sites for a particular type of PG structure, and a catalytic domain for cleaving PG with high efficiency. It has long been believed that the specificity of these enzymes resides in the binding domain (Donovan et al, ; Regulski et al, ). However, the catalytic domains of several lytic enzymes also retain specificity (Heffron et al, ; Li et al, ; Mayer et al, ).…”

Section: Resultsmentioning

confidence: 99%

Characterization of the activity of the spore cortex lytic enzyme CwlJ1

Grover

Paskaleva

et al. 2015

Biotech & Bioengineering

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The germination enzyme CwlJ1 plays an important role in degrading the cortex during the germination of Bacillus anthracis spores. However, the specific function and catalytic activity of CwlJ1 remain elusive. Here we report for the first time a detailed in vitro mechanistic study of CwlJ1 expressed in Escherichia coli and its activity against the spore cortical fragments of B. anthracis when added exogenously. CwlJ1 was active on both decoated spores and spore cortical fragments. Through liquid chromatography-mass spectrometry analysis of the digested cortical fragments, we determined that CwlJ1 was a thermostable N-acetylmuramoyl-L-alanine amidase. CwlJ1 mainly recognized large segments of glycan chains in the cortex instead of the minimal structural unit tetrasaccharide, with specificity for muramic acid-δ-lactam-containing glycan chains and preference for the tetrapeptide side chain. Unlike most amidases, CwlJ1 did not appear to contain a divalent cation, as it retained its activity in the presence of EDTA. This study shines some light on the mechanism of spore germination, and provides increased insight into the development of sporicidal enzyme systems for decontamination of B. anthracis and other related bacteria.

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“…Interestingly, we found that a few A. baumannii isolates were resistant to endolysin from the same species of bacteriophage that targets this bacterium (i.e., we found that PlyAB1 did not effectively lyse 6 of the 212 additional clinical MDRAB isolates that we tested). The reason for this may relate to potential differences in the peptidoglycan of PlyAB1-sensitive A. baumannii strains and that of PlyAB1-resistant strains [ 25 ],[ 26 ].…”

Section: Discussionmentioning

confidence: 99%

Antibacterial properties of Acinetobacter baumanniiphage Abp1 endolysin (PlyAB1)

et al. 2014

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BackgroundAcinetobacter baumannii has emerged as one of the most important hospital-acquired pathogens in the world, because of its resistance to almost all available antibiotic drugs. Endolysins from phages are attracting increasing interest as potential antimicrobial agents, especially for drug-resistant bacteria. We previously isolated and characterized Abp1, a virulent phage targeting the multidrug-resistant A. baumannii strain, AB1.MethodsTo evaluate the antimicrobial potential of endolysin from the Abp1 phage, the endolysin gene plyAB1 was cloned and over-expressed in Escherichia coli, and the lytic activity of the recombinant protein (PlyAB1) was tested by turbidity assessment and bacteria counting assays.ResultsPlyAB1 exhibits a marked lytic activity against A. baumannii AB1, as shown by a decrease in the number of live bacteria following treatment with the enzyme. Moreover, PlyAB1 displayed a highly specific lytic effect against all of the 48 hospital-derived pandrug-resistant A. baumannii isolates that were tested. These isolates were shown to belong to different ST clones by multilocus sequence typing.ConclusionsThe results presented here show that PlyAB1 has potential as an antibiotic against drug-resistant A. baumannii.Electronic supplementary materialThe online version of this article (doi:10.1186/s12879-014-0681-2) contains supplementary material, which is available to authorized users.

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A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge, Identified in Lactobacillus casei Bacteriophage Endolysins

Cited by 27 publications

References 51 publications

Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis

Regulation of Cell Wall Plasticity by Nucleotide Metabolism in Lactococcus lactis

Characterization of the activity of the spore cortex lytic enzyme CwlJ1

Antibacterial properties of Acinetobacter baumanniiphage Abp1 endolysin (PlyAB1)

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