scholarly journals The Cpx Envelope Stress Response Modifies Peptidoglycan Cross-Linking via the l,d-Transpeptidase LdtD and the Novel Protein YgaU

2014 ◽  
Vol 197 (3) ◽  
pp. 603-614 ◽  
Author(s):  
Margarita Bernal-Cabas ◽  
Juan Alfonso Ayala ◽  
Tracy L. Raivio
Keyword(s):  
Cell Wall ◽  
Stress Response ◽  
Protein Misfolding ◽  
Diaminopimelic Acid ◽  
Cross Linking ◽  
Content Type ◽  
Lytic Transglycosylase ◽  
Expression Of Genes ◽  
Envelope Stress ◽  
Cross Links

The Cpx envelope stress response mediates a complex adaptation to conditions that cause protein misfolding in the periplasm. A recent microarray study demonstrated that Cpx response activation led to changes in the expression of genes known, or predicted, to be involved in cell wall remodeling. We sought to characterize the changes that the cell wall undergoes during activation of the Cpx pathway inEscherichia coli. Luminescent reporters of gene expression confirmed that LdtD, a putativel,d-transpeptidase; YgaU, a protein of unknown function; and Slt, a lytic transglycosylase, are upregulated in response to Cpx-inducing conditions. Phosphorylated CpxR binds to the upstream regions of these genes, which contain putative CpxR binding sites, suggesting that regulation is direct. We show that the activation of the Cpx response causes an increase in the abundance of diaminopimelic acid (DAP)-DAP cross-links that involves LdtD and YgaU. Altogether, our data indicate that changes in peptidoglycan structure are part of the Cpx-mediated adaptation to envelope stress and indicate a role for the uncharacterized geneygaUin regulating cross-linking.

scholarly journals Characterization of the TyrR Regulon in the Rhizobacterium Enterobacter ludwigii UW5 Reveals Overlap with the CpxR Envelope Stress Response

2020 ◽  
Vol 203 (1) ◽  
Author(s):  
Thomas J. D. Coulson ◽  
René M. Malenfant ◽  
Cheryl L. Patten
Keyword(s):  
Transcription Factor ◽  
Stress Response ◽  
Acid Synthesis ◽  
Plant Interactions ◽  
Amino Acid Synthesis ◽  
Content Type ◽  
E Coli ◽  
Expression Of Genes ◽  
Envelope Stress ◽  
Enterobacter Ludwigii

ABSTRACT The TyrR transcription factor controls the expression of genes for the uptake and biosynthesis of aromatic amino acids in Escherichia coli. In the plant-associated and clinically significant proteobacterium Enterobacter ludwigii UW5, the TyrR orthologue was previously shown to regulate genes that encode enzymes for synthesis of the plant hormone indole-3-acetic acid and for gluconeogenesis, indicating a broader function for the transcription factor. This study aimed to delineate the TyrR regulon of E. ludwigii by comparing the transcriptomes of the wild type and a tyrR deletion strain. In E. ludwigii, TyrR positively or negatively regulates the expression of over 150 genes. TyrR downregulated expression of envelope stress response regulators CpxR and CpxP through interaction with a DNA binding site in the intergenic region between divergently transcribed cpxP and cpxR. Repression of cpxP was alleviated by tyrosine. Methyltransferase gene dmpM, which is possibly involved in antibiotic synthesis, was strongly activated in the presence of tyrosine and phenylalanine by TyrR binding to its promoter region. TyrR also regulated expression of genes for aromatic catabolism and anaerobic respiration. Our findings suggest that the E. ludwigii TyrR regulon has diverged from that of E. coli to include genes for survival in the diverse environments that this bacterium inhabits and illustrate the expansion and plasticity of transcription factor regulons. IMPORTANCE Genome-wide RNA sequencing revealed a broader regulatory role for the TyrR transcription factor in the ecologically versatile bacterium Enterobacter ludwigii beyond that of aromatic amino acid synthesis and transport that constitute the role of the TyrR regulon of E. coli. In E. ludwigii, a plant symbiont and human gut commensal, the TyrR regulon is expanded to include genes that are beneficial for plant interactions and response to stresses. Identification of the genes regulated by TyrR provides insight into the mechanisms by which the bacterium adapts to its environment.

scholarly journals Mycobacteriophage Ms6 LysA: a Peptidoglycan Amidase and a Useful Analytical Tool

2012 ◽  
Vol 79 (3) ◽  
pp. 768-773 ◽  
Author(s):  
Sebabrata Mahapatra ◽  
Charles Piechota ◽  
Filipa Gil ◽  
Yufang Ma ◽  
Hairong Huang ◽  
...  
Keyword(s):  
Cell Wall ◽  
Mycobacterium Tuberculosis ◽  
Recombinant Protein ◽  
Mycobacterium Smegmatis ◽  
Analytical Tool ◽  
Diaminopimelic Acid ◽  
Cross Linking ◽  
Muramic Acid ◽  
Content Type ◽  
Mycobacterial Cell Wall

ABSTRACTSince the peptidoglycan isolated fromMycobacteriumspp. is refractory to commercially available murolytic enzymes, possibly due to the presence of various modifications found on this peptidoglycan, the utility of a mycobacteriophage-derived murolytic enzyme was assessed for an analysis of peptidoglycan from mycobacteria. We cloned, expressed, and purified thelysAgene product, a protein with homology to known peptidoglycan-degrading amidases, from bacteriophage Ms6. The recombinant protein was shown to cleave the bond betweenl-Ala andd-muramic acid of muramyl pentapeptide and to release up to 70% of the diaminopimelic acid present in the isolated mycobacterial cell wall. In contrast to lysozyme, which, in culture, inhibits the growth of bothMycobacterium smegmatisandMycobacterium tuberculosis, LysA had no effect on the growth of either species. However, the enzyme is useful for solubilizing the peptide chains of isolated mycobacterial peptidoglycan for analysis. The data indicate that the stem peptides fromM. smegmatisare heavily amidated, containing few free carboxylic acids, regardless of the cross-linking status.

scholarly journals In VivoStudies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall

2013 ◽  
Vol 57 (9) ◽  
pp. 4470-4480 ◽  
Author(s):  
Min Jung Kwun ◽  
Gabriela Novotna ◽  
Andrew R. Hesketh ◽  
Lionel Hill ◽  
Hee-Jeon Hong
Keyword(s):  
Cell Wall ◽  
Transcriptional Activation ◽  
Streptomyces Coelicolor ◽  
Constitutive Expression ◽  
Transcriptional Response ◽  
Vancomycin Resistance ◽  
Content Type ◽  
Expression Of Genes ◽  
Peptidoglycan Cell Wall

ABSTRACTVanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system inStreptomyces coelicoloras a model, we have undertaken a series ofin vivostudies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with thed-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essentiald-Ala-d-Ala ligase activity by constitutive expression ofvanAencoding a bifunctionald-Ala-d-Ala andd-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containingd-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance ofd-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating ind-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask thed-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting withd-Ala-d-Ala residues, failed to inducevangene expression. Activation of resistance by a vancomycin–d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating ind-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.

scholarly journals Actinomadura geliboluensis sp. nov., isolated from soil

2012 ◽  
Vol 62 (Pt_8) ◽  
pp. 2011-2017 ◽  
Author(s):  
Anil Sazak ◽  
Mustafa Camas ◽  
Cathrin Spröer ◽  
Hans-Peter Klenk ◽  
Nevzat Sahin
Keyword(s):  
Cell Wall ◽  
Type Species ◽  
Diaminopimelic Acid ◽  
Rrna Gene ◽  
Phenotypic Data ◽  
Content Type ◽  
Link Type ◽  
Acid Type ◽  
Gene Sequence Analysis ◽  
Taxonomic Approach

A novel actinobacterium, strain A8036T, isolated from soil, was investigated by using a polyphasic taxonomic approach. The organism formed extensively branched substrate hyphae that generated spiral chains of spores with irregular surfaces. The cell wall contained meso-diaminopimelic acid (type III) and cell-wall sugars were glucose, madurose, mannose and ribose. The predominant menaquinones were MK-9(H6) and MK-9(H4). The phospholipids were diphosphatidylglycerol, phosphatidylinositol and phosphatidylinositol mannosides. The major cellular fatty acids were iso-C16 : 0, C17 : 1 cis9, C16 : 0, C15 : 0 and 10-methyl C17 : 0. Based on 16S rRNA gene sequence analysis, the closest phylogenetic neighbours of strain A8036T were Actinomadura meyerae DSM 44715T (99.23 % similarity), Actinomadura bangladeshensis DSM 45347T (98.9 %) and Actinomadura chokoriensis DSM 45346T (98.3 %). However, DNA–DNA relatedness and phenotypic data demonstrated that strain A8036T could be clearly distinguished from the type strains of all closely related Actinomadura species. Strain A8036T is therefore considered to represent a novel species of the genus Actinomadura , for which the name Actinomadura geliboluensis sp. nov. is proposed. The type strain is A8036T ( = DSM 45508T = KCTC 19868T).

scholarly journals Fusarium head blight resistance in European winter wheat: insights from genome-wide transcriptome analysis

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Maria Buerstmayr ◽  
Christian Wagner ◽  
Tetyana Nosenko ◽  
Jimmy Omony ◽  
Barbara Steiner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Wall ◽  
Stress Response ◽  
Secondary Cell Wall ◽  
Fungal Colonization ◽  
Head Blight ◽  
Resistance Level ◽  
Expression Of Genes ◽  
European Winter Wheat ◽  
Fhb Resistance

Abstract Background Fusarium head blight (FHB) is a devastating disease of wheat worldwide. Resistance to FHB is quantitatively controlled by the combined effects of many small to medium effect QTL. Flowering traits, especially the extent of extruded anthers, are strongly associated with FHB resistance. Results To characterize the genetic basis of FHB resistance, we generated and analyzed phenotypic and gene expression data on the response to Fusarium graminearum (Fg) infection in 96 European winter wheat genotypes, including several lines containing introgressions from the highly resistant Asian cultivar Sumai3. The 96 lines represented a broad range in FHB resistance and were assigned to sub-groups based on their phenotypic FHB severity score. Comparative analyses were conducted to connect sub-group-specific expression profiles in response to Fg infection with FHB resistance level. Collectively, over 12,300 wheat genes were Fusarium responsive. The core set of genes induced in response to Fg was common across different resistance groups, indicating that the activation of basal defense response mechanisms was largely independent of the resistance level of the wheat line. Fg-induced genes tended to have higher expression levels in more susceptible genotypes. Compared to the more susceptible non-Sumai3 lines, the Sumai3-derivatives demonstrated higher constitutive expression of genes associated with cell wall and plant-type secondary cell wall biogenesis and higher constitutive and Fg-induced expression of genes involved in terpene metabolism. Gene expression analysis of the FHB QTL Qfhs.ifa-5A identified a constitutively expressed gene encoding a stress response NST1-like protein (TraesCS5A01G211300LC) as a candidate gene for FHB resistance. NST1 genes are key regulators of secondary cell wall biosynthesis in anther endothecium cells. Whether the stress response NST1-like gene affects anther extrusion, thereby affecting FHB resistance, needs further investigation. Conclusion Induced and preexisting cell wall components and terpene metabolites contribute to resistance and limit fungal colonization early on. In contrast, excessive gene expression directs plant defense response towards programmed cell death which favors necrotrophic growth of the Fg pathogen and could thus lead to increased fungal colonization.

scholarly journals Site-Directed Cross-Linking Identifies the Stator-Rotor Interaction Surfaces in a Hybrid Bacterial Flagellar Motor

2021 ◽  
Vol 203 (9) ◽  
Author(s):  
Hiroyuki Terashima ◽  
Seiji Kojima ◽  
Michio Homma
Keyword(s):  
Escherichia Coli ◽  
Cross Linking ◽  
Physical Interaction ◽  
Flagellar Motor ◽  
Intact Cells ◽  
Content Type ◽  
Bacterial Flagellum ◽  
Cross Links ◽  
Bacterial Flagellar Motor ◽  
Rotary Motor

ABSTRACT The bacterial flagellum is the motility organelle powered by a rotary motor. The rotor and stator elements of the motor are located in the cytoplasmic membrane and cytoplasm. The stator units assemble around the rotor, and an ion flux (typically H+ or Na+) conducted through a channel of the stator induces conformational changes that generate rotor torque. Electrostatic interactions between the stator protein PomA in Vibrio (MotA in Escherichia coli) and the rotor protein FliG have been shown by genetic analyses but have not been demonstrated biochemically. Here, we used site-directed photo-cross-linking and disulfide cross-linking to provide direct evidence for the interaction. We introduced a UV-reactive amino acid, p-benzoyl-l-phenylalanine (pBPA), into the cytoplasmic region of PomA or the C-terminal region of FliG in intact cells. After UV irradiation, pBPA inserted at a number of positions in PomA and formed a cross-link with FliG. PomA residue K89 gave the highest yield of cross-links, suggesting that it is the PomA residue nearest to FliG. UV-induced cross-linking stopped motor rotation, and the isolated hook-basal body contained the cross-linked products. pBPA inserted to replace residue R281 or D288 in FliG formed cross-links with the Escherichia coli stator protein, MotA. A cysteine residue introduced in place of PomA K89 formed disulfide cross-links with cysteine inserted in place of FliG residues R281 and D288 and some other flanking positions. These results provide the first demonstration of direct physical interaction between specific residues in FliG and PomA/MotA. IMPORTANCE The bacterial flagellum is a unique organelle that functions as a rotary motor. The interaction between the stator and rotor is indispensable for stator assembly into the motor and the generation of motor torque. However, the interface of the stator-rotor interaction has only been defined by mutational analysis. Here, we detected the stator-rotor interaction using site-directed photo-cross-linking and disulfide cross-linking approaches. We identified several residues in the PomA stator, especially K89, that are in close proximity to the rotor. Moreover, we identified several pairs of stator and rotor residues that interact. This study directly demonstrates the nature of the stator-rotor interaction and suggests how stator units assemble around the rotor and generate torque in the bacterial flagellar motor.

scholarly journals A Suppressor Mutation That Creates a Faster and More Robust σE Envelope Stress Response

2016 ◽  
Vol 198 (17) ◽  
pp. 2345-2351 ◽  
Author(s):  
Anna Konovalova ◽  
Jaclyn A. Schwalm ◽  
Thomas J. Silhavy
Keyword(s):  
Stress Response ◽  
Stress Responses ◽  
Novel Mutation ◽  
Signal Transduction Pathway ◽  
Normal Growth ◽  
Internal Stresses ◽  
Content Type ◽  
Envelope Stress ◽  
Genetic Perturbations ◽  
Periplasmic Stress

ABSTRACTThe σE envelope stress response is an essential signal transduction pathway which detects and removes mistargeted outer membrane (OM) β-barrel proteins (OMPs) in the periplasm ofEscherichia coli. It relies on σE, an alternative sigma factor encoded by therpoEgene. Here we report a novel mutation, a nucleotide change of C to A in the third base of the second codon, which increases levels of σE (rpoE_S2R). TherpoE_S2Rmutation does not lead to the induction of the stress response during normal growth but instead changes the dynamics of induction upon periplasmic stress, resulting in a faster and more robust response. This allows cells to adapt faster to the periplasmic stress, avoiding lethal accumulation of unfolded OMPs in the periplasm caused by severe defects in the OMP assembly pathway.IMPORTANCESurvival of bacteria under conditions of external or internal stresses depends on timely induction of stress response signaling pathways to regulate expression of appropriate genes that function to maintain cellular homeostasis. Previous studies have shown that strong preinduction of envelope stress responses can allow bacteria to survive a number of lethal genetic perturbations. In our paper, we describe a unique mutation that enhances kinetics of the σE envelope stress response pathway rather than preinducing the response. This allows bacteria to quickly adapt to sudden and severe periplasmic stress.

scholarly journals Two DD-Carboxypeptidases fromMycobacterium smegmatisAffect Cell Surface Properties through Regulation of Peptidoglycan Cross-Linking and Glycopeptidolipids

2018 ◽  
Vol 200 (14) ◽  
Author(s):  
Satya Deo Pandey ◽  
Shilpa Pal ◽  
Ganesh Kumar N ◽  
Ankita Bansal ◽  
Sathi Mallick ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Cross Linking ◽  
Surface Lipid ◽  
Cross Link ◽  
Content Type ◽  
Murine Macrophages ◽  
Cross Links ◽  
Link Formation

ABSTRACTDuring the peptidoglycan (PG) maturation of mycobacteria, the glycan strands are interlinked by both 3-3 (between twomeso-diaminopimelic acids [meso-DAPs]) and 4-3 cross-links (betweend-Ala andmeso-DAP), though there is a predominance (60 to 80%) of 3-3 cross-links. Thedd-carboxypeptidases (dd-CPases) act on pentapeptides to generate tetrapeptides that are used byld-transpeptidases as substrates to form 3-3 cross-links. Therefore,dd-CPases play a crucial role in mycobacterial PG cross-link formation. However, the physiology ofdd-CPases in mycobacteria is relatively unexplored. In this study, we deleted twodd-CPase genes,msmeg_2433andmsmeg_2432, both individually and in combination, fromMycobacterium smegmatismc2155. Though the singledd-CPase gene deletions had no significant impact on the mycobacterial physiology, many interesting functional alterations were observed in the double-deletion mutant,viz., a predominance in PG cross-link formation was shifted from 3-3 cross-links to 4-3, cell surface glycopeptidolipid (GPL) expression was reduced, and susceptibility to β-lactams and antitubercular agents was enhanced. Moreover, the survival rate of the double mutant within murine macrophages was higher than that of the parent. Interestingly, the complementation with any one of thedd-CPase genes could restore the wild-type phenotype. In a nutshell, we infer that the altered ratio of 4-3 to 3-3 PG cross-links might have influenced the expression of surface GPLs, colony morphology, biofilm formation, drug susceptibility, and subsistence of the cells within macrophages.IMPORTANCEThe glycan strands in mycobacterial peptidoglycan (PG) are interlinked by both 3-3 and 4-3 cross-links. Thedd-CPases generate tetrapeptides by acting on the pentapeptides, andld-transpeptidases use tetrapeptides as substrates to form 3-3 cross-links. In this study, we showed that simultaneous deletions of twodd-CPases alter the nature of PG cross-linking from 3-3 cross-links to 4-3 cross-links. The deletions subsequently decrease the expression of glycopeptidolipids (significant surface lipid present in many nontuberculous mycobacteria, includingMycobacterium smegmatis) and affect other physiological parameters, like cell morphology, growth rate, biofilm formation, antibiotic susceptibility, and survival within murine macrophages. Thus, unraveling the physiology ofdd-CPases might help us design antimycobacterial therapeutics in the future.

scholarly journals The Cell Envelope Stress Response of Bacillus subtilis towards Laspartomycin C

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 729
Author(s):  
Angelika Diehl ◽  
Thomas M. Wood ◽  
Susanne Gebhard ◽  
Nathaniel I. Martin ◽  
Georg Fritz
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Stress Response ◽  
Cell Envelope ◽  
Resistance Mechanisms ◽  
Detailed Knowledge ◽  
Knock Out ◽  
Lipid Ii ◽  
Envelope Stress ◽  
The Impact

Cell wall antibiotics are important tools in our fight against Gram-positive pathogens, but many strains become increasingly resistant against existing drugs. Laspartomycin C is a novel antibiotic that targets undecaprenyl phosphate (UP), a key intermediate in the lipid II cycle of cell wall biosynthesis. While laspartomycin C has been thoroughly examined biochemically, detailed knowledge about potential resistance mechanisms in bacteria is lacking. Here, we use reporter strains to monitor the activity of central resistance modules in the Bacillus subtilis cell envelope stress response network during laspartomycin C attack and determine the impact on the resistance of these modules using knock-out strains. In contrast to the closely related UP-binding antibiotic friulimicin B, which only activates ECF σ factor-controlled stress response modules, we find that laspartomycin C additionally triggers activation of stress response systems reacting to membrane perturbation and blockage of other lipid II cycle intermediates. Interestingly, none of the studied resistance genes conferred any kind of protection against laspartomycin C. While this appears promising for therapeutic use of laspartomycin C, it raises concerns that existing cell envelope stress response networks may already be poised for spontaneous development of resistance during prolonged or repeated exposure to this new antibiotic.

scholarly journals Role of the msaABCR Operon in Cell Wall Biosynthesis, Autolysis, Integrity, and Antibiotic Resistance in Staphylococcus aureus

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Bibek G C ◽  
Gyan S. Sahukhal ◽  
Mohamed O. Elasri
Keyword(s):  
Staphylococcus Aureus ◽  
Cell Wall ◽  
Antibiotic Resistance ◽  
Teichoic Acid ◽  
Cross Linking ◽  
Wall Defect ◽  
Content Type ◽  
Mrsa Strains ◽  
Mutant Cells

ABSTRACT Staphylococcus aureus is an important human pathogen in both community and health care settings. One of the challenges with S. aureus as a pathogen is its acquisition of antibiotic resistance. Previously, we showed that deletion of the msaABCR operon reduces cell wall thickness, resulting in decreased resistance to vancomycin in vancomycin-intermediate S. aureus (VISA). In this study, we investigated the nature of the cell wall defect in the msaABCR operon mutant in the Mu50 (VISA) and USA300 LAC methicillin-resistant Staphylococcus aureus (MRSA) strains. Results showed that msaABCR mutant cells had decreased cross-linking in both strains. This defect is typically due to increased murein hydrolase activity and/or nonspecific processing of murein hydrolases mediated by increased protease activity in mutant cells. The defect was enhanced by a decrease in teichoic acid content in the msaABCR mutant. Therefore, we propose that deletion of the msaABCR operon results in decreased peptidoglycan cross-linking, leading to increased susceptibility toward cell wall-targeting antibiotics, such as β-lactams and vancomycin. Moreover, we also observed significantly downregulated transcription of early cell wall-synthesizing genes, supporting the finding that msaABCR mutant cells have decreased peptidoglycan synthesis. More specifically, the msaABCR mutant in the USA300 LAC strain (MRSA) showed significantly reduced expression of the murA gene, whereas the msaABCR mutant in the Mu50 strain (VISA) showed significantly reduced expression of glmU, murA, and murD. Thus, we conclude that the msaABCR operon controls the balance between cell wall synthesis and cell wall hydrolysis, which is required for maintaining a robust cell wall and acquiring resistance to cell wall-targeting antibiotics, such as vancomycin and the β-lactams.

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