A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. This allows, on X-Gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, greatly facilitating clone identification during mutagenesis. The plasmid used Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct with...

Bacteria elongation involves moving synthetic complexes around the cell wall.

Summary The Hsp100/Clp ATPases constitute a family of closely related proteins which some members function solely as chaperones whereas others additionally can associate with the unrelated ClpP peptidase forming Clp proteolytic complex. We have investigated role four in versatile pathogen, Staphylococcus aureus . Previously, we showed that ClpX is required for expression major virulence factors and S. , but not survival during heat shock. In present study, inactivated clpC clpB clpL and,...

A model system for investigating how developmental regulatory networks determine cell fate is spore formation in Bacillus subtilis . The master regulator sporulation Spo0A, which activated by phosphorylation via a phosphorelay that subject to three positive feedback loops. ultimate decision sporulate is, however, stochastic only portion of the population sporulates even under optimal conditions. It was previously assumed activation Spo0A and hence entry into bistable switch mediated one or...

ABSTRACT In vitro mariner transposon mutagenesis of Streptococcus pneumoniae chromosomal DNA was used to isolate regulatory mutants affecting expression the comCDE operon, encoding peptide quorum-sensing two-component signal transduction system controlling competence development. A insertion leading increased comC found lie directly upstream from S. clpP gene, proteolytic subunit Clp ATP-dependent protease, whose in Bacillus subtilis is controlled by CtsR repressor. order examine clp gene...

Multiple regulatory mechanisms for coping with stress co-exist in low G+C Gram-positive bacteria. Among these, the HrcA and CtsR repressors control distinct regulons model organism, Bacillus subtilis. We recently identified an orthologue of regulator response major pathogen, Staphylococcus aureus. Sequence analysis S. aureus genome revealed presence potential operator sites not only upstream from genes encoding subunits Clp ATP-dependent protease, as B. subtilis, but also, unexpectedly,...

The cytoskeleton occupies a central role in cellular immunity by promoting bacterial sensing and antibacterial functions. Septins are cytoskeletal proteins implicated various processes, including cell division. also assemble into cage-like structures that entrap cytosolic Shigella, yet how septins recognize bacteria is poorly understood. Here, we discover recruited to regions of micron-scale membrane curvature upon invasion division variety species. Cardiolipin, curvature-specific...

Abstract How cells control their shape and size is a long-standing question in cell biology. Many rod-shaped bacteria elongate sidewalls by the action of wall synthesizing machineries that are associated to actin-like MreB cortical patches. However, little known about how elongation regulated enable varied growth rates sizes. Here we use total internal reflection fluorescence microscopy single-particle tracking visualize isoforms, as proxy for synthesis, Bacillus subtilis Escherichia coli...

ABSTRACT Clp-HSP100 ATPases are a widespread family of ubiquitous proteins that occur in both prokaryotes and eukaryotes play important roles the folding newly synthesized refolding aggregated proteins. They have also been shown to participate virulence several pathogens, including Listeria monocytogenes . Here, we describe member L. harbors all characteristics ClpB subclass, which is absent closely related gram-positive model organism, Bacillus subtilis. Transcriptional analysis clpB...

The AbrB protein of the spore-forming bacterium Bacillus subtilis is a repressor numerous genes that are switched on during transition from exponential to stationary phase growth. gene for under negative control master regulator entry into sporulation, Spo0A-P. It has generally been assumed derepression achieved by Spo0A-P-mediated repression abrB followed rapid degradation protein. Here, we report levels do decrease phase, but this not entire basis which AbrB-controlled derepressed....

ABSTRACT The response regulator Spo0A governs multiple developmental processes in Bacillus subtilis , including most conspicuously sporulation. is activated by phosphorylation via a multicomponent phosphorelay. Previous work has shown that the protein not rate limiting for Rather, present at high levels growing cells, rapidly rising to yet higher under sporulation-inducing conditions, suggesting synthesis of subject just-in-time control mechanism. Transcription spo0A governed promoter...

Summary The conversion of a growing cell into an endospore in Bacillus subtilis involves phagocytic‐like process which the developing spore (the forespore) is wholly engulfed by adjacent mother cell. A prerequisite for engulfment removal peptidoglycan from septum that separates forespore cell, depends on autolysin SpoIID and two proteins unknown function, SpoIIM SpoIIP. Here we present evidence SpoIIP also autolysin, it acts tandem with SpoIID, all three are complex each other. We further...

Metabolic turnover of mRNA is fundamental to the control gene expression in all organisms, notably fast-adapting prokaryotes. In many bacteria, RNase Y initiates global decay via an endonucleolytic cleavage, as shown Gram-positive model organism Bacillus subtilis This enzyme tethered inner cell membrane, a pseudocompartmentalization coherent with its task initiating cleavage/maturation mRNAs that are translated at periphery. Here, we used total internal reflection fluorescence microscopy...

Summary MreB proteins play a major role during morphogenesis of rod‐shaped bacteria by organizing biosynthesis the peptidoglycan cell wall. However, mechanisms underlying this process are not well understood. In B acillus subtilis , membrane‐associated polymers have been shown to be associated elongation‐specific complexes containing transmembrane morphogenetic factors and extracellular wall assembly proteins. We now found that an early intracellular step synthesis is also . show previously...

During bacterial exponential growth, the morphogenetic actin-like MreB proteins form membrane-associated assemblies that move processively following trajectories perpendicular to long axis of cell. Such structures are thought scaffold and restrict movement peptidoglycan synthesizing machineries, thereby coordinating sidewall elongation. In Bacillus subtilis, this function is performed by redundant action three isoforms, namely MreB, Mbl MreBH. mreB mbl highly transcribed from vegetative...

The actin-like MreB protein is a key player of the machinery controlling elongation and maintenance cell shape most rod-shaped bacteria. This known to be highly dynamic, moving along short axis cells, presumably reflecting movement wall synthetic machineries during enzymatic assembly peptidoglycan mesh. ability proteins form polymers not debated, but their structure, length, conditions establishment have remained unclear subject conflicting reports. Here we analyze various strains...

Nucleotide sequence analysis of the Streptococcus salivarius clpP locus revealed potential binding sites for both CtsR and HrcA repressors. Dual regulation by was demonstrated using Bacillus subtilis as a heterologous host, shown to bind directly promoter sequence. This is first example gene under control HrcA.

Transcription termination factor Rho is known for its ubiquitous role in suppression of pervasive, mostly antisense, transcription. In the model Gram-positive bacterium Bacillus subtilis , de-repression pervasive transcription by inactivation rho revealed regulation post-exponential differentiation programs. To identify other aspects regulatory during adaptation to starvation, we have constructed a B . strain (Rho + ) that expresses at relatively stable high level order compensate decrease...

In vivo, bacterial actin MreB assembles into dynamic membrane-associated filamentous structures that exhibit circumferential motion around the cell. Current knowledge of biochemical and polymerization properties in vitro remains limited is mostly based on proteins from Gram-negative species. this study, we report first observation organized protofilaments by electron microscopy 3D-structure a Gram-positive bacterium. We show Geobacillus stearothermophilus forms straight pairs lipid surfaces...

ABSTRACT Pathogenic Shigella bacteria are a paradigm to address key issues of cell and infection biology. Polar localisation the autotransporter protein IcsA is essential for actin tail formation, which necessary bacterium travel from cell-to-cell; yet how proteins targeted bacterial pole poorly understood. The homologue MreB has been extensively studied in broth culture using model organisms including Escherichia coli, Bacillus subtilis Caulobacter crescentus, but never visualised...

B. subtilis adapts to changing environments by reprogramming its genetic expression through a variety of transcriptional regulators from the global transition state that allow complete resetting cell expression, stress specific controlling only limited number key genes required for optimal adaptation. Among them, MarR-type are known respond stresses including antibiotics or oxidative stress, and control catabolic virulence gene expression. Here we report characterization ydcFGH operon...

How cells control their shape and size is a fundamental question of biology. In most bacteria, cell imposed by the peptidoglycan (PG) polymeric meshwork that surrounds cell. Thus, bacterial morphogenesis results from coordinated action proteins assembling degrading PG shell. Remarkably, during steady-state growth, bacteria maintain defined along generations, suggesting error-proof mechanisms tightly process. rod-shaped model for Gram-positive bacterium Bacillus subtilis, average length...