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Substrate Preferences Establish the Order of Cell Wall Assembly in Staphylococcus aureus.

J Am Chem Soc. 2018 02 21;140(7):2442-2445

Authors: Schaefer K, Owens TW, Kahne D, Walker S

Abstract
The Gram-positive bacterial cell wall is a large supramolecular structure and its assembly requires coordination of complex biosynthetic pathways. In the step that merges the two major biosynthetic pathways in Staphylococcus aureus cell wall assembly, conserved protein ligases attach wall teichoic acids to peptidoglycan, but the order of biosynthetic events is a longstanding question. Here, we use a chemical approach to define which of the possible peptidoglycan intermediates are substrates for wall-teichoic acid ligases, thereby establishing the order of cell wall assembly. We have developed a strategy to make defined glycan chain-length polymers of either un-cross-linked or cross-linked peptidoglycan, and we find that wall teichoic acid ligases cannot transfer wall teichoic acid precursors to the cross-linked substrates. A 1.9 Å crystal structure of a LytR-CpsA-Psr (LCP) family ligase in complex with a wall teichoic acid precursor defines the location of the peptidoglycan binding site as a long, narrow groove, and suggests that the basis for selectivity is steric exclusion of cross-linked peptidoglycan. Consistent with this hypothesis, we have found that chitin oligomers are good substrates for transfer, showing that LCPs do not discriminate cross-linked from un-cross-linked peptidoglycan substrates by recognizing features of the un-cross-linked stem peptide. We conclude that wall teichoic acids are coupled to un-cross-linked peptidoglycan chains at an early stage of peptidoglycan synthesis and may create marks that define the proper spacing of subsequent cross-links.

PMID: 29402087 [PubMed - indexed for MEDLINE]

Abstract

Abstract

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Dual regulation of activity and intracellular localization of the PASTA kinase PrkC during Bacillus subtilis growth.

Sci Rep. 2018 Jan 26;8(1):1660

Authors: Pompeo F, Byrne D, Mengin-Lecreulx D, Galinier A

Abstract
The activity of the PrkC protein kinase is regulated in a sophisticated manner in Bacillus subtilis cells. In spores, in the presence of muropeptides, PrkC stimulates dormancy exit. The extracellular region containing PASTA domains binds peptidoglycan fragments to probably enhance the intracellular kinase activity. During exponential growth, the cell division protein GpsB interacts with the intracellular domain of PrkC to stimulate its activity. In this paper, we have reinvestigated the regulation of PrkC during exponential and stationary phases. We observed that, during exponential growth, neither its septal localization nor its activity are influenced by the addition of peptidoglycan fragments or by the deletion of one or all PASTA domains. However, Dynamic Light Scattering experiments suggest that peptidoglycan fragments bind specifically to PrkC and induce its oligomerization. In addition, during stationary phase, PrkC appeared evenly distributed in the cell wall and the deletion of one or all PASTA domains led to a non-activated kinase. We conclude that PrkC activation is not as straightforward as previously suggested and that regulation of its kinase activity via the PASTA domains and peptidoglycan fragments binding occurs when PrkC is not concentrated to the bacterial septum, but all over the cell wall in non-dividing bacillus cells.

PMID: 29374241 [PubMed - in process]

Octapeptins are colistin-like lipopeptide antibiotics with activity against multi- and extensively drug-resistant (MDR and XDR) Gram-negative bacteria. We describe the design, synthesis, and early preclinical evaluation of a novel series of octapeptins with superior activity and pharmacokinetics.

Much like animals and to a degree humans, bacteria enjoy a good fight. They stab, shove and poison each other in pursuit of the best territory. While this much is clear, little is known about the tactics and strategy that bacteria use during their miniature wargames.

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Peptidoglycan compositional analysis of Enterococcus faecalis biofilm by stable isotope labeling by amino acids in bacterial culture.

Biochemistry. 2018 Jan 25;:

Authors: Chang JD, Wallace AG, Foster EE, Kim SJ

Abstract
Peptidoglycan (PG) is a major component of the cell wall in Enterococcus faecalis. Accurate analysis of PG composition provides crucial insights into bacteria's cellular functions and responses to external stimuli, but this analysis remains challenging due to various chemical modifications to PG-repeat subunits. We characterized changes to the PG composition in E. faecalis grown as planktonic bacteria and biofilm by developing "Stable Isotope Labeling by Amino Acids in Bacterial Culture" (SILAB) optimized for bacterial cultures with incomplete amino acid labeling. This comparative analysis by mass spectrometry was carried out by labeling E. faecalis in biofilm with heavy-Lys (L-[13C6, 2D9, 15N2]Lys) and planktonic bacteria with natural abundance L-Lys, then mixing the equal amount of bacteria from each condition and carrying out cell-wall isolation and mutanolysin digestion necessary for liquid chromatography-mass spectrometry. An analytical method was developed to determine muropeptide abundances using correction factors to compensate for incomplete heavy-Lys isotopic enrichment (98.33% ± 0.05%) and incorporation (83.23% ± 1.16%). SILAB analysis of 47 pairs of PG fragment ions from isolated cell walls of planktonic and biofilm samples were selected for analysis. We found that the PG in biofilm showed increased PG cross-linking, increased N-deacetylation of GlcNAc, decreased O-acetylation of MurNAc, and increased stem modifications by D,D- and L,D-carboxypeptidases.

PMID: 29368511 [PubMed - as supplied by publisher]

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Stress-Induced Reorganization of the Mycobacterial Membrane Domain.

MBio. 2018 Jan 23;9(1):

Authors: Hayashi JM, Richardson K, Melzer ES, Sandler SJ, Aldridge BB, Siegrist MS, Morita YS

Abstract
Cell elongation occurs primarily at the mycobacterial cell poles, but the molecular mechanisms governing this spatial regulation remain elusive. We recently reported the presence of an intracellular membrane domain (IMD) that was spatially segregated from the conventional plasma membrane in Mycobacterium smegmatis The IMD is enriched in the polar region of actively elongating cells and houses many essential enzymes involved in envelope biosynthesis, suggesting its role in spatially restricted elongation at the cell poles. Here, we examined reorganization of the IMD when the cells are no longer elongating. To monitor the IMD, we used a previously established reporter strain expressing fluorescent IMD markers and grew it to the stationary growth phase or exposed the cells to nutrient starvation. In both cases, the IMD was delocalized from the cell pole and distributed along the sidewall. Importantly, the IMD could still be isolated biochemically by density gradient fractionation, indicating its maintenance as a membrane domain. Chemical and genetic inhibition of peptidoglycan biosynthesis led to the delocalization of the IMD, suggesting the suppression of peptidoglycan biosynthesis as a trigger of spatial IMD rearrangement. Starved cells with a delocalized IMD can resume growth upon nutrient repletion, and polar enrichment of the IMD coincides with the initiation of cell elongation. These data reveal that the IMD is a membrane domain with the unprecedented capability of subcellular repositioning in response to the physiological conditions of the mycobacterial cell.IMPORTANCE Mycobacteria include medically important species, such as the human tuberculosis pathogen Mycobacterium tuberculosis The highly impermeable cell envelope is a hallmark of these microbes, and its biosynthesis is a proven chemotherapeutic target. Despite the accumulating knowledge regarding the biosynthesis of individual envelope components, the regulatory mechanisms behind the coordinated synthesis of the complex cell envelope remain elusive. We previously reported the presence of a metabolically active membrane domain enriched in the elongating poles of actively growing mycobacteria. However, the spatiotemporal dynamics of the membrane domain in response to stress have not been examined. Here, we show that the membrane domain is spatially reorganized when growth is inhibited in the stationary growth phase, under nutrient starvation, or in response to perturbation of peptidoglycan biosynthesis. Our results suggest that mycobacteria have a mechanism to spatiotemporally coordinate the membrane domain in response to metabolic needs under different growth conditions.

PMID: 29362232 [PubMed - in process]

PBP4 activity and its overexpression are necessary for PBP4-mediated high-level β-lactam resistance.

J Antimicrob Chemother. 2018 Jan 19;:

Authors: Basuino L, Jousselin A, Alexander JAN, Strynadka NCJ, Pinho MG, Chambers HF, Chatterjee SS

Abstract
Background: PBP4 is typically considered unimportant for conferring high-level β-lactam resistance in Staphylococcus aureus. Mutations in PBP4 have been associated with β-lactam non-susceptibility among natural strains of S. aureus. We have previously shown that PBP4 can mediate high-level β-lactam resistance in laboratory-generated strains passaged in β-lactam antibiotics. Mutations in the pbp4 promoter that up-regulate its expression and missense mutations that surround PBP4's active site were detected in high frequencies among passaged strains, suggesting PBP4 plays a key role in resistance. How these mutations participate in PBP4's ability to provide high-level β-lactam resistance is unknown.
Objectives: To determine whether enzymatic activity of PBP4 is required for high-level β-lactam resistance and to investigate how the pbp4-associated mutations provide β-lactam resistance.
Methods: The catalytic activity of PBP4 was disabled through introduction of a serine to alanine point mutation in its active site (Ser-75→Ala) in a representative and well-studied passaged strain, CRB. pbp4 promoter and missense mutations detected in CRB were reconstituted in a WT strain individually and in combination. β-Lactam resistance of the resultant strains was evaluated by population analysis. Bacterial peptidoglycan composition of the pbp4 mutants was evaluated with and without antibiotic treatment using LC.
Results: PBP4 inactivation imparted complete β-lactam susceptibility of CRB. Reconstitution of PBP4 missense mutations alone did not impart β-lactam resistance, but did so in synergism with pbp4 promoter mutation. A similar synergistic interaction of pbp4 mutations was observed in enhanced peptidoglycan cross-linking upon antibiotic treatment.
Conclusions: PBP4's activity and overexpression both contribute to high-level β-lactam resistance.

PMID: 29360990 [PubMed - as supplied by publisher]

Allostery, Recognition of Nascent Peptidoglycan and Crosslinking of the Cell Wall by the Essential Penicillin-Binding Protein 2x of Streptococcus pneumoniae.

ACS Chem Biol. 2018 Jan 22;:

Authors: Bernardo-Garcia N, Mahasenan KV, Batuecas MT, Lee M, Hesek D, Petráčková D, Doubravová L, Branny P, Mobashery S, Hermoso JA

Abstract
Transpeptidases, members of the penicillin-binding protein (PBP) families, catalyze crosslinking of the bacterial cell wall. This transformation is critical for the survival of bacteria and it is the target of inhibition by -lactam antibiotics. We report herein our structural insights into catalysis by the essential PBP2x of Streptococcus pneumoniae by disclosing a total of four X-ray structures, two computational models based on the crystal structures and molecular-dynamics simulations. The X-ray structures are for the apo PBP2x, the enzyme modified covalently in the active site by oxacillin (a penicillin antibiotic), the enzyme modified by oxacillin in the presence of a synthetic tetrasaccharide surrogate for the cell-wall peptidoglycan and a non-covalent complex of cefepime (a cephalosporin antibiotic) bound to the active site. A pre-requisite for catalysis by transpeptidases, including PBP2x, is the molecular recognition of nascent peptidoglycan strands, which harbor pentapeptide stems. We disclose that the recognition of nascent peptidoglycan by PBP2x takes place by complexation of one pentapeptide stem at an allosteric site located in the PASTA domains of this enzyme. This binding predisposes the third pentapeptide stem in the same nascent peptidoglycan strand to penetration into the active site for the turnover events. The complexation of the two pentapeptide stems in the same peptidoglycan strand is a recognition motif for the nascent peptidoglycan, critical for the cell-wall crosslinking reaction.

PMID: 29357220 [PubMed - as supplied by publisher]

Abstract

Related Articles

Discovery of genes required for lipoteichoic acid glycosylation predicts two distinct mechanism for wall teichoic acid glycosylation.

J Biol Chem. 2018 Jan 17;:

Authors: Rismondo J, Percy MG, Gründling A

Abstract
The bacterial cell wall is an important and highly complex structure that is essential for bacterial growth because it protects bacteria from cell lysis and environmental insults. A typical Gram-positive bacterial cell wall is composed of peptidoglycan and the secondary cell wall polymers, wall teichoic acid (WTA) and lipoteichoic acid (LTA). In many Gram-positive bacteria, LTA is a polyglycerol-phosphate chain that is decorated with D-alanine and sugar residues. However, the function of and proteins responsible for the glycosylation of LTA are either unknown or not well-characterized. Here, using bioinformatics, genetic, and NMR spectroscopy approaches, we found that the Bacillus subtilis csbB and yfhO genes are essential for LTA glycosylation. Interestingly, the Listeria monocytogenes gene lmo1079, which encodes a YfhO ortholog, was not required for LTA glycosylation, but instead was essential for WTA glycosylation. LTA is polymerized on the outside of the cell and hence can only be glycosylated extracellularly. Based on the similarity of the genes coding for YfhO orthologs that are required in B. subtilis for LTA glycosylation or in L. monocytogenes for WTA glycosylation, we hypothesize that WTA glycosylation might also occur extracellularly in Listeria species. Finally, we discovered that in L. monocytogenes lmo0626 (gtlB) was required for LTA glycosylation, indicating that the encoded protein has a similar function to YfhO, even though the proteins are not homologous. Together, our results enable us to propose an updated model for LTA glycosylation and also indicate that glycosylation of WTA might occur through two different mechanisms in Gram-positive bacteria.

PMID: 29343515 [PubMed - as supplied by publisher]

Abstract

The human skin microbiome

The human skin microbiome, Published online: 15 January 2018; doi:10.1038/nrmicro.2017.157

Our skin is home to millions of bacteria, fungi and viruses that comprise the skin microbiota. In this Review, Byrd and colleagues discuss recent insights into skin microbial communities, including their composition in health and disease, dynamics between species and interactions with the immune system.

It's official—there are some 42 million protein molecules in a simple cell, revealed a team of researchers led by Grant Brown, a biochemistry professor in the University of Toronto's Donnelly Centre for Cellular and Biomolecular Research. Analyzing data from almost two dozen large studies of protein abundance in yeast cells, the team was able to produce for the first time reliable estimates for the number of molecules for each protein, as revealed in a study published this week in the journal Cell Systems.

Summary

Peptidoglycan recognition by the innate immune system

Peptidoglycan recognition by the innate immune system, Published online: 02 January 2018; doi:10.1038/nri.2017.136

Peptidoglycan is an important structural component of bacterial cell walls, and mammalian cells express a number of distinct pattern-recognition receptors that detect peptidoglycan fragments. Here, the authors discuss new insights into the role of peptidoglycan recognition in inflammation, metabolism and disease.

In most bacteria, the essential targets of β-lactam antibiotics are the d,d-transpeptidases that catalyze the last step of peptidoglycan polymerization by forming 4->3 cross-links. The peptidoglycan of Clostridium difficile is unusual since it mainly contains 3->3 cross-links generated by l,d-transpeptidases. To gain insight into the characteristics of C. difficile peptidoglycan cross-linking enzymes, we purified the three putative C. difficile l,d-transpeptidase paralogues Ldt_Cd1, Ldt_Cd2, and Ldt_Cd3, which were previously identified by sequence analysis. The catalytic activities of the three proteins were assayed with a disaccharide-tetrapeptide purified from the C. difficile cell wall. Ldt_Cd2 and Ldt_Cd3 catalyzed the formation of 3->3 cross-links (l,d-transpeptidase activity), the hydrolysis of the C-terminal d-Ala residue of the disaccharide-tetrapeptide substrate (l,d-carboxypeptidase activity), and the exchange of the C-terminal d-Ala for d-Met. Ldt_Cd1 displayed only l,d-carboxypeptidase activity. Mass spectrometry analyses indicated that Ldt_Cd1 and Ldt_Cd2 were acylated by β-lactams belonging to the carbapenem (imipenem, meropenem, and ertapenem), cephalosporin (ceftriaxone), and penicillin (ampicillin) classes. Acylation of Ldt_Cd3 by these β-lactams was not detected. The acylation efficacy of Ldt_Cd1 and Ldt_Cd2 was higher for the carbapenems (480 to 6,600 M^–1 s^–1) than for ampicillin and ceftriaxone (3.9 to 82 M^–1 s^–1). In contrast, the efficacy of the hydrolysis of β-lactams by Ldt_Cd1 and Ldt_Cd2 was higher for ampicillin and ceftriaxone than for imipenem. These observations indicate that Ldt_Cd1 and Ldt_Cd2 are inactivated only by β-lactams of the carbapenem class due to a combination of rapid acylation and the stability of the resulting covalent adducts.