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.

Analysis of teichoic acid biosynthesis regulation reveals that the extracytoplasmic function sigma factor σ M is induced by phosphate depletion in Bacillus subtilis W23

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 3041-3049 ◽  
Author(s):  
Kathrin Minnig ◽  
Vladimir Lazarevic ◽  
Blazenka Soldo ◽  
Catherine Mauël
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Teichoic Acid ◽  
Regulatory System ◽  
Phosphate Starvation ◽  
Phosphate Limitation ◽  
Wall Structure ◽  
Wall Teichoic Acid ◽  
Activity Data ◽  
Two Component

The expression of the Bacillus subtilis W23 tar genes specifying the biosynthesis of the major wall teichoic acid, the poly(ribitol phosphate), was studied under phosphate limitation using lacZ reporter fusions. Three different regulation patterns can be deduced from these β-galactosidase activity data: (i) tarD and tarL gene expression is downregulated under phosphate starvation; (ii) tarA and, to a minor extent, tarB expression after an initial decrease unexpectedly increases; and (iii) tarO is not influenced by phosphate concentration. To dissect the tarA regulatory pattern, its two promoters were analysed under phosphate limitation: The P tarA -ext promoter is repressed under phosphate starvation by the PhoPR two-component system, whereas, under the same conditions, the P tarA -int promoter is upregulated by the action of an extracytoplasmic function (ECF) σ factor, σ M. In contrast to strain 168, σ M is activated in strain W23 in phosphate-depleted conditions, a phenomenon indirectly dependent on PhoPR, the two-component regulatory system responsible for the adaptation to phosphate starvation. These results provide further evidence for the role of σ M in cell-wall stress response, and suggest that impairment of cell-wall structure is the signal activating this ECF σ factor.

scholarly journals Cell envelope gene expression in phosphate-limited Bacillus subtilis cells

Microbiology ◽  
2011 ◽  
Vol 157 (9) ◽  
pp. 2470-2484 ◽  
Author(s):  
Eric Botella ◽  
Sebastian Hübner ◽  
Karsten Hokamp ◽  
Annette Hansen ◽  
Paola Bisicchia ◽  
...  
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Cell Envelope ◽  
Expression Profiles ◽  
Phosphate Limitation ◽  
Envelope Gene ◽  
Two Component System ◽  
Cell Wall Metabolism ◽  
Component System ◽  
Two Component

The high phosphate content of Bacillus subtilis cell walls dictates that cell wall metabolism is an important feature of the PhoPR-mediated phosphate limitation response. Here we report the expression profiles of cell-envelope-associated and PhoPR regulon genes, determined by live cell array and transcriptome analysis, in exponentially growing and phosphate-limited B. subtilis cells. Control by the WalRK two-component system confers a unique expression profile and high level of promoter activity on the genes of its regulon with yocH and cwlO expression differing both qualitatively and quantitatively from all other autolysin-encoding genes examined. The activity of the PhoPR two-component system is restricted to the phosphate-limited state, being rapidly induced in response to the cognate stimulus, and can be sustained for an extended phosphate limitation period. Constituent promoters of the PhoPR regulon show heterogeneous induction profiles and very high promoter activities. Phosphate-limited cells also show elevated expression of the actin-like protein MreBH and reduced expression of the WapA cell wall protein and WprA cell wall protease indicating that cell wall metabolism in this state is distinct from that of exponentially growing and stationary-phase cells. The PhoPR response is very rapidly deactivated upon removal of the phosphate limitation stimulus with concomitant increased expression of cell wall metabolic genes. Moreover expression of genes encoding enzymes involved in sulphur metabolism is significantly altered in the phosphate-limited state with distinct perturbations being observed in wild-type 168 and AH024 (ΔphoPR) cells.

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 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.

scholarly journals Control of teichoic acid and teichuronic acid biosynthesis in chemostat cultures of Bacillus subtilis var. niger

1969 ◽  
Vol 111 (1) ◽  
pp. 1-5 ◽  
Author(s):  
D C Ellwood ◽  
D. W. Tempest
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Teichoic Acid ◽  
Extracellular Fluid ◽  
Acid Synthesis ◽  
Growth Cycle ◽  
Wall Material ◽  
Wall Teichoic Acid ◽  
Anionic Polymers ◽  
Teichuronic Acid

1. Quantitative determination of the anionic polymers present in the walls of Bacillus subtilis var. niger organisms undergoing transition, in a chemostat culture, from either Mg2+-limitation to PO43−-limitation or K+-limitation to PO43−-limitation showed that teichuronic acid synthesis started immediately the culture became PO43−-limited and proceeded at a rate substantially faster than the rate of biomass synthesis. 2. Simultaneously, the cell-wall teichoic acid content diminished at a rate greater than that due to dilution by newly synthesized wall material, and fragments of teichoic acid and mucopeptide accumulated in the culture extracellular fluid. 3. Equally rapid reverse changes occurred when a PO43−-limited B. subtilis var. niger culture was returned to being Mg2+-limited. 4. It is concluded that in this organism both teichoic acid and teichuronic acid syntheses are expressions of a single genotype, and a mechanism for the control of synthesis of both polymers is suggested. 5. These results are discussed with reference to the constantly changing environmental conditions that obtain in a batch culture and the variation in bacterial cell-wall composition that is reported to occur throughout the growth cycle.

scholarly journals Analysis of Bacillus subtilis tagAB andtagDEF Expression during Phosphate Starvation Identifies a Repressor Role for PhoP∼P

1998 ◽  
Vol 180 (3) ◽  
pp. 753-758 ◽  
Author(s):  
Wei Liu ◽  
Stephen Eder ◽  
F. Marion Hulett
Keyword(s):  
Cell Wall ◽  
Bacillus Subtilis ◽  
Teichoic Acid ◽  
Phosphate Starvation ◽  
Acid Synthesis ◽  
Pho Regulon ◽  
Wall Teichoic Acid ◽  
Negative Role ◽  
First Time ◽  
Starvation Conditions

ABSTRACT The tagAB and tagDEF operons, which are adjacent and divergently transcribed, encode genes responsible for cell wall teichoic acid synthesis in Bacillus subtilis. TheBacillus data presented here suggest that PhoP and PhoR are required for direct repression of transcription of the two operons under phosphate starvation conditions but have no regulatory role under phosphate-replete conditions. These data identify for the first time that PhoP∼P has a negative role in Pho regulon gene regulation.

scholarly journals Wall Teichoic Acids Restrict Access of Bacteriophage Endolysin Ply118, Ply511, and PlyP40 Cell Wall Binding Domains to the Listeria monocytogenes Peptidoglycan

2012 ◽  
Vol 194 (23) ◽  
pp. 6498-6506 ◽  
Author(s):  
Marcel R. Eugster ◽  
Martin J. Loessner
Keyword(s):  
Cell Wall ◽  
Listeria Monocytogenes ◽  
Teichoic Acid ◽  
Single Copy ◽  
Wall Teichoic Acid ◽  
Content Type ◽  
Binding Domains ◽  
Cell Wall Binding ◽  
Wall Teichoic Acids

ABSTRACTThe C-terminal cell wall binding domains (CBDs) of phage endolysins direct the enzymes to their binding ligands on the bacterial cell wall with high affinity and specificity. TheListeria monocytogenesPly118, Ply511, and PlyP40 endolysins feature related CBDs which recognize the directly cross-linked peptidoglycan backbone structure ofListeria. However, decoration with fluorescently labeled CBDs primarily occurs at the poles and septal regions of the rod-shaped cells. To elucidate the potential role of secondary cell wall-associated carbohydrates such as the abundant wall teichoic acid (WTA) on this phenomenon, we investigated CBD binding usingL. monocytogenesserovar 1/2 and 4 cells deficient in WTA. Mutants were obtained by deletion of two redundanttagOhomologues, whose products catalyze synthesis of the WTA linkage unit. While inactivation of eithertagO1(EGDelmo0959) ortagO2(EGDelmo2519) alone did not affect WTA content, removal of both alleles following conditional complementation yielded WTA-deficientListeriacells. Substitution oftagOfrom an isopropyl-β-d-thiogalactopyranoside-inducible single-copy integration vector restored the original phenotype. Although WTA-deficient cells are viable, they featured severe growth inhibition and an unusual coccoid morphology. In contrast to CBDs from otherListeriaphage endolysins which directly utilize WTA as binding ligand, the data presented here show that WTAs are not required for attachment of CBD118, CBD511, and CBDP40. Instead, lack of the cell wall polymers enables unrestricted spatial access of CBDs to the cell wall surface, indicating that the abundant WTA can negatively regulate sidewall localization of the cell wall binding domains.

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