scholarly journals Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

2016 ◽  
Vol 198 (21) ◽  
pp. 2925-2935 ◽  
Author(s):  
Heng Zhao ◽  
Yingjie Sun ◽  
Jason M. Peters ◽  
Carol A. Gross ◽  
Ethan C. Garner ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Transcriptional Repression ◽  
Cell Envelope ◽  
Turgor Pressure ◽  
Teichoic Acid ◽  
Lethal Gene ◽  
Genetic Redundancy ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Lipid Ii

ABSTRACTThe integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. InBacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C55lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimizedclusteredregularlyinterspacedshortpalindromicrepeat (CRISPR) system with catalytically inactive (“dead”)CRISPR-associated protein9(dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate thatB. subtilisrequires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the σM-dependent cell envelope stress response, includingbcrC, which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of theB. subtilisUPP-Pase enzymes, and provide further evidence linking the σMregulon to cell envelope homeostasis pathways.IMPORTANCEThe emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

scholarly journals Genome-Wide Analysis of Phosphorylated PhoP Binding to Chromosomal DNA Reveals Several Novel Features of the PhoPR-Mediated Phosphate Limitation Response in Bacillus subtilis

2015 ◽  
Vol 197 (8) ◽  
pp. 1492-1506 ◽  
Author(s):  
Letal I. Salzberg ◽  
Eric Botella ◽  
Karsten Hokamp ◽  
Haike Antelmann ◽  
Sandra Maaß ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Response Regulator ◽  
Teichoic Acid ◽  
Phosphate Limitation ◽  
Chromosomal Dna ◽  
Cell Wall Metabolism ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Two Component

ABSTRACTThe PhoPR two-component signal transduction system controls one of three responses activated byBacillus subtilisto adapt to phosphate-limiting conditions (PHO response). The response involves the production of enzymes and transporters that scavenge for phosphate in the environment and assimilate it into the cell. However, inB. subtilisand some otherFirmicutesbacteria, cell wall metabolism is also part of the PHO response due to the high phosphate content of the teichoic acids attached either to peptidoglycan (wall teichoic acid) or to the cytoplasmic membrane (lipoteichoic acid). Prompted by our observation that the phosphorylated WalR (WalR∼P) response regulator binds to more chromosomal loci than are revealed by transcriptome analysis, we established the PhoP∼P bindome in phosphate-limited cells. Here, we show that PhoP∼P binds to the chromosome at 25 loci: 12 are within the promoters of previously identified PhoPR regulon genes, while 13 are newly identified. We extend the role of PhoPR in cell wall metabolism showing that PhoP∼P binds to the promoters of four cell wall-associated operons (ggaAB,yqgS,wapA, anddacA), although none show PhoPR-dependent expression under the conditions of this study. We also show that positive autoregulation ofphoPRexpression and full induction of the PHO response upon phosphate limitation require PhoP∼P binding to the 3′ end of thephoPRoperon.IMPORTANCEThe PhoPR two-component system controls one of three responses mounted byB. subtilisto adapt to phosphate limitation (PHO response). Here, establishment of the phosphorylated PhoP (PhoP∼P) bindome enhances our understanding of the PHO response in two important ways. First, PhoPR plays a more extensive role in adaptation to phosphate-limiting conditions than was deduced from transcriptome analyses. Among 13 newly identified binding sites, 4 are cell wall associated (ggaAB,yqgS,wapA, anddacA), revealing that PhoPR has an extended involvement in cell wall metabolism. Second, amplification of the PHO response must occur by a novel mechanism since positive autoregulation ofphoPRexpression requires PhoP∼P binding to the 3′ end of the operon.

scholarly journals Structure and Mechanism of LcpA, a Phosphotransferase That Mediates Glycosylation of a Gram-Positive Bacterial Cell Wall-Anchored Protein

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Sara D. Siegel ◽  
Brendan R. Amer ◽  
Chenggang Wu ◽  
Michael R. Sawaya ◽  
Jason E. Gosschalk ◽  
...  
Keyword(s):  
Cell Wall ◽  
Disulfide Bond ◽  
Cell Envelope ◽  
Teichoic Acid ◽  
Surface Proteins ◽  
Wall Teichoic Acid ◽  
Gram Positive ◽  
Content Type ◽  
X Ray Crystallography ◽  
Gram Positive Bacteria

ABSTRACT The widely conserved LytR-CpsA-Psr (LCP) family of enzymes in Gram-positive bacteria is known to attach glycopolymers, including wall teichoic acid, to the cell envelope. However, it is undetermined if these enzymes are capable of catalyzing glycan attachment to surface proteins. In the actinobacterium Actinomyces oris, an LCP homolog here named LcpA is genetically linked to GspA, a glycoprotein that is covalently attached to the bacterial peptidoglycan by the housekeeping sortase SrtA. Here we show by X-ray crystallography that LcpA adopts an α-β-α structural fold, akin to the conserved LCP domain, which harbors characteristic catalytic arginine residues. Consistently, alanine substitution for these residues, R149 and R266, abrogates GspA glycosylation, leading to accumulation of an intermediate form termed GspALMM, which is also observed in the lcpA mutant. Unlike other LCP proteins characterized to date, LcpA contains a stabilizing disulfide bond, mutations of which severely affect LcpA stability. In line with the established role of disulfide bond formation in oxidative protein folding in A. oris, deletion of vkor, coding for the thiol-disulfide oxidoreductase VKOR, also significantly reduces LcpA stability. Biochemical studies demonstrated that the recombinant LcpA enzyme possesses pyrophosphatase activity, enabling hydrolysis of diphosphate bonds. Furthermore, this recombinant enzyme, which weakly interacts with GspA in solution, catalyzes phosphotransfer to GspALMM. Altogether, the findings support that A. oris LcpA is an archetypal LCP enzyme that glycosylates a cell wall-anchored protein, a process that may be conserved in Actinobacteria, given the conservation of LcpA and GspA in these high-GC-content organisms. IMPORTANCE In Gram-positive bacteria, the conserved LCP family enzymes studied to date are known to attach glycopolymers, including wall teichoic acid, to the cell envelope. It is unknown if these enzymes catalyze glycosylation of surface proteins. We show here in the actinobacterium Actinomyces oris by X-ray crystallography and biochemical analyses that A. oris LcpA is an LCP homolog, possessing pyrophosphatase and phosphotransferase activities known to belong to LCP enzymes that require conserved catalytic Arg residues, while harboring a unique disulfide bond critical for protein stability. Importantly, LcpA mediates glycosylation of the surface protein GspA via phosphotransferase activity. Our studies provide the first experimental evidence of an archetypal LCP enzyme that promotes glycosylation of a cell wall-anchored protein in Gram-positive bacteria.

scholarly journals Exposure ofStaphylococcus aureusto Targocil Blocks Translocation of the Major Autolysin Atl across the Membrane, Resulting in a Significant Decrease in Autolysis

2018 ◽  
Vol 62 (7) ◽  
pp. e00323-18 ◽  
Author(s):  
Kiran B. Tiwari ◽  
Craig Gatto ◽  
Suzanne Walker ◽  
Brian J. Wilkinson
Keyword(s):  
Transcriptional Repression ◽  
Cell Walls ◽  
Teichoic Acid ◽  
Pcr Analysis ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Autolytic Activity ◽  
Zymographic Analysis ◽  
Resistant Strains ◽  
Wall Components

ABSTRACTPeptidoglycan (PG) and wall teichoic acid (WTA) are the major staphylococcal cell wall components, and WTA biosynthesis has recently been explored for drug development. Targocil is a novel agent that targets the TarG subunit of the WTA translocase (TarGH) that transports WTA across the membrane to the wall. Previously we showed that targocil treatment of a methicillin-susceptibleStaphylococcus aureusstrain led to a rapid shut down of cellular autolysis. Targocil II, which targets the TarH subunit of TarGH, also resulted in a drastic decrease in autolysis. Here, we address the mechanism of targocil-mediated decreased autolysis. The mechanism is WTA dependent since targocil treatment decreased autolysis in methicillin-resistant strains but not in a WTA-deficient mutant. Similar to cellular autolysis, autolysin-retaining crude cell walls isolated from targocil-treated cells had vastly decreased autolytic activity compared to those from untreated cells. Purified cell walls from control and targocil-treated cells, which lack autolytic activity, were similarly susceptible to lysozyme and lysostaphin and had similar O-acetyl contents, indicating that targocil treatment did not grossly alter PG structure and chemistry. Purified cell walls from targocil-treated cells were highly susceptible to autolysin extracts, supporting the notion that targocil treatment led to decreased autolysin in the crude cell walls. Quantitative real-time PCR analysis revealed that the decrease in autolysis in the targocil-exposed cells was not due to transcriptional repression of the autolysin genesatl,lytM,lytN, andsle1. Zymographic analysis of peptidoglycan hydrolase profiles showed a deficiency of cell surface autolysins in targocil-treated cells but higher activity in cell membrane fractions. Here, we propose that the untranslocated WTA molecules in the targocil-exposed cells sequester Atl at the membrane, resulting in significantly decreased autolysis.

scholarly journals BrpA Is Involved in Regulation of Cell Envelope Stress Responses in Streptococcus mutans

2012 ◽  
Vol 78 (8) ◽  
pp. 2914-2922 ◽  
Author(s):  
J. P. Bitoun ◽  
S. Liao ◽  
X. Yao ◽  
S.-J. Ahn ◽  
R. Isoda ◽  
...  
Keyword(s):  
Streptococcus Mutans ◽  
Stress Responses ◽  
Antimicrobial Agents ◽  
Galleria Mellonella ◽  
Cell Envelope ◽  
Luciferase Reporter ◽  
Luciferase Reporter Gene ◽  
Content Type ◽  
Lipid Ii ◽  
The Impact

ABSTRACTPrevious studies have shown that BrpA plays a major role in acid and oxidative stress tolerance and biofilm formation byStreptococcus mutans. Mutant strains lacking BrpA also display increased autolysis and decreased viability, suggesting a role for BrpA in cell envelope integrity. In this study, we examined the impact of BrpA deficiency on cell envelope stresses induced by envelope-active antimicrobials. Compared to the wild-type strain UA159, the BrpA-deficient mutant (TW14D) was significantly more susceptible to antimicrobial agents, especially lipid II inhibitors. Several genes involved in peptidoglycan synthesis were identified by DNA microarray analysis as downregulated in TW14D. Luciferase reporter gene fusion assays also revealed that expression ofbrpAis regulated in response to environmental conditions and stresses induced by exposure to subinhibitory concentrations of cell envelope antimicrobials. In aGalleria mellonella(wax worm) model, BrpA deficiency was shown to diminish the virulence ofS. mutansOMZ175, which, unlikeS. mutansUA159, efficiently kills the worms. Collectively, these results suggest that BrpA plays a role in the regulation of cell envelope integrity and that deficiency of BrpA adversely affects the fitness and diminishes the virulence of OMZ175, a highly invasive strain ofS. mutans.

scholarly journals Localization and Interactions of Teichoic Acid Synthetic Enzymes in Bacillus subtilis

2007 ◽  
Vol 190 (5) ◽  
pp. 1812-1821 ◽  
Author(s):  
Alex Formstone ◽  
Rut Carballido-López ◽  
Philippe Noirot ◽  
Jeffery Errington ◽  
Dirk-Jan Scheffers
Keyword(s):  
Bacillus Subtilis ◽  
Cell Shape ◽  
Spatial Organization ◽  
Structural Integrity ◽  
Fluorescent Protein ◽  
Teichoic Acid ◽  
Cytoskeletal Proteins ◽  
Thick Wall ◽  
Wall Teichoic Acid ◽  
Critical Function

ABSTRACT The thick wall of gram-positive bacteria is a polymer meshwork composed predominantly of peptidoglycan (PG) and teichoic acids, both of which have a critical function in maintenance of the structural integrity and the shape of the cell. In Bacillus subtilis 168 the major teichoic acid is covalently coupled to PG and is known as wall teichoic acid (WTA). Recently, PG insertion/degradation over the lateral wall has been shown to occur in a helical pattern. However, the spatial organization of WTA assembly and its relationship with cell shape and PG assembly are largely unknown. We have characterized the localization of green fluorescent protein fusions to proteins involved in several steps of WTA synthesis in B. subtilis: TagB, -F, -G, -H, and -O. All of these localized similarly to the inner side of the cytoplasmic membrane, in a pattern strikingly similar to that displayed by probes of nascent PG. Helix-like localization patterns are often attributable to the morphogenic cytoskeletal proteins of the MreB family. However, localization of the Tag proteins did not appear to be substantially affected by single disruption of any of the three MreB homologues of B. subtilis. Bacterial and yeast two-hybrid experiments revealed a complex network of interactions involving TagA, -B, -E, -F, -G, -H, and -O and the cell shape determinants MreC and MreD (encoded by the mreBCD operon and presumably involved in the spatial organization of PG synthesis). Taken together, our results suggest that, in B. subtilis at least, the synthesis and export of WTA precursors are mediated by a large multienzyme complex that may be associated with the PG-synthesizing machinery.

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 LytR-CpsA-Psr Enzymes as Determinants of Bacillus anthracis Secondary Cell Wall Polysaccharide Assembly

2014 ◽  
Vol 197 (2) ◽  
pp. 343-353 ◽  
Author(s):  
Megan Liszewski Zilla ◽  
Yvonne G. Y. Chan ◽  
Justin Mark Lunderberg ◽  
Olaf Schneewind ◽  
Dominique Missiakas
Keyword(s):  
Cell Wall ◽  
Bacillus Anthracis ◽  
Chain Length ◽  
Secondary Cell Wall ◽  
Teichoic Acid ◽  
Cell Wall Polysaccharide ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Associated Proteins ◽  
Layer Assembly

Bacillus anthracis, the causative agent of anthrax, replicates as chains of vegetative cells by regulating the separation of septal peptidoglycan. Surface (S)-layer proteins and associated proteins (BSLs) function as chain length determinants and bind to the secondary cell wall polysaccharide (SCWP). In this study, we identified theB. anthracislcpDmutant, which displays increased chain length and S-layer assembly defects due to diminished SCWP attachment to peptidoglycan. In contrast, theB. anthracislcpB3variant displayed reduced cell size and chain length, which could be attributed to increased deposition of BSLs. In other bacteria, LytR-CpsA-Psr (LCP) proteins attach wall teichoic acid (WTA) and polysaccharide capsule to peptidoglycan.B. anthracisdoes not synthesize these polymers, yet its genome encodes six LCP homologues, which, when expressed inS. aureus, promote WTA attachment. We propose a model wherebyB. anthracisLCPs promote attachment of SCWP precursors to discrete locations in the peptidoglycan, enabling BSL assembly and regulated separation of septal peptidoglycan.

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