A shuttle vector designated pMAD was constructed for quickly generating gene inactivation mutants in naturally nontransformable gram-positive bacteria. This vector allows, on X-Gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) plates, a quick colorimetric blue-white discrimination of bacteria which have lost the plasmid, greatly facilitating clone identification during mutagenesis. The plasmid was used in Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus to efficiently construct mutants with or without an associated antibiotic resistance gene.

The peptidoglycan cell wall and the actin-like MreB cytoskeleton are major determinants of cell shape in rod-shaped bacteria. The prevailing model postulates that helical, membrane-associated MreB filaments organize elongation-specific peptidoglycan-synthesizing complexes along sidewalls. We used total internal reflection fluorescence microscopy to visualize the dynamic relation between MreB isoforms and cell wall synthesis in live Bacillus subtilis cells. During exponential growth, MreB proteins did not form helical structures. Instead, together with other morphogenetic factors, they assembled into discrete patches that moved processively along peripheral tracks perpendicular to the cell axis. Patch motility was largely powered by cell wall synthesis, and MreB polymers restricted diffusion of patch components in the membrane and oriented patch motion.

SummaryThe Hsp100/Clp ATPases constitute a family of closely related proteins of which some members function solely as chaperones whereas others additionally can associate with the unrelated ClpP peptidase forming a Clp proteolytic complex. We have investigated the role of four Clp ATPases in the versatile pathogen, Staphylococcus aureus . Previously, we showed that ClpX is required for expression of major virulence factors and for virulence of S. aureus , but not for survival during heat shock. In the present study, we have inactivated clpC , clpB and clpL and, while none of these mutations affected toxin production, both ClpC and ClpB and to a minor extent ClpL were required for intracellular multiplication within bovine mammary epithelial cells. These defects were paralleled by an inability of the clpC mutant to grow at high temperature and of the clpB mutant to induce thermotolerance indicating that the protective functions of these proteins are required both at high temperature and during infection. By primer extension analysis and footprint studies, we show that expression of clpC and clpB is controlled by the negative heatshock regulator, CtsR, and that ClpC is required for its repressor activity. Thus, ClpC is a likely sensor of stress encountered during both environmental stress and infection. In addition to virulence factor production the ability to form biofilms is of importance to S. aureus as a nosocomial pathogen. Interestingly, biofilm formation was reduced in the absence of ClpX or ClpC whereas it was enhanced in the absence of ClpP. Thus, our data show that Clp proteolytic complexes and the Clp ATPases control several key processes of importance to the success of S. aureus as a pathogen.

A model system for investigating how developmental regulatory networks determine cell fate is spore formation in Bacillus subtilis. The master regulator for sporulation is Spo0A, which is activated by phosphorylation via a phosphorelay that is subject to three positive feedback loops. The ultimate decision to sporulate is, however, stochastic in that only a portion of the population sporulates even under optimal conditions. It was previously assumed that activation of Spo0A and hence entry into sporulation is subject to a bistable switch mediated by one or more feedback loops. Here we reinvestigate the basis for bimodality in sporulation. We show that none of the feedback loops is rate limiting for the synthesis and phosphorylation of Spo0A. Instead, the loops ensure a just-in-time supply of relay components for rising levels of phosphorylated Spo0A, with phosphate flux through the relay being limiting for Spo0A activation and sporulation. In addition, genes under Spo0A control did not exhibit a bimodal pattern of expression as expected for a bistable switch. In contrast, we observed a highly heterogeneous pattern of Spo0A activation that increased in a nonlinear manner with time. We present a computational model for the nonlinear increase and propose that the phosphorelay is a noise generator and that only cells that attain a threshold level of phosphorylated Spo0A sporulate.A challenge in developmental biology is to understand how cells in an apparently homogeneous population adopt different fates. An attractive organism in which to address this challenge is Bacillus subtilis, which can adopt a variety of alternative fates depending on growth conditions (1-3). In some cases, cell population heterogeneity is generated stochastically. That is, fluctuations in gene expression due to noise can be amplified by feedback loops to lock cells in alternative stable states, resulting in a bimodal distribution of cell types. This is exemplified by genetic competence in which a positive feedback loop acting as a bistable switch creates such a distribution (2, 4). We use bimodal to mean systems that exhibit two discrete states and bistable to specify a class of bimodal systems in which nonlinear reinforcement stabilizes the alternative states. Here we are concerned with bimodality in the capacity of B. subtilis to sporulate.The master regulator for entry into sporulation, Spo0A (0A), accumulates gradually over the first 90 min of sporulation (5) (Fig. S1.) and is only active in its phosphorylated form (0A∼P) (6). Some genes under its control, such as those involved in biofilm formation and cannibalism, have strong binding sites for 0A∼P and are switched ON at low levels of 0A∼P. Other genes, such as those for spore formation, have weak binding sites and are only activated when 0A∼P accumulates to high levels (5, 7). The accumulation of 0A∼P is governed by a regulatory network built around a four-component cascade in which the relay protein Spo0F (0F) is phosphorylated by KinA and other kinases (6,8). 0F∼P, in turn, transfer...

In vitro mariner transposon mutagenesis of Streptococcus pneumoniae chromosomal DNA was used to isolate regulatory mutants affecting expression of the comCDE operon, encoding the peptide quorum-sensing twocomponent signal transduction system controlling competence development. A transposon insertion leading to increased comC expression was found to lie directly upstream from the S. pneumoniae clpP gene, encoding the proteolytic subunit of the Clp ATP-dependent protease, whose expression in Bacillus subtilis is controlled by the CtsR repressor. In order to examine clp gene regulation in S. pneumoniae, a detailed analysis of the complete genome sequence was performed, indicating that there are five likely CtsR-binding sites located upstream from the clpE, clpP, and clpL genes and the ctsR-clpC and groESL operons. The S. pneumoniae ctsR gene was cloned under the control of an inducible promoter and used to demonstrate regulation of the S. pneumoniae clpP and clpE genes and the clpC and groESL operons by using B. subtilis as a heterologous host. The CtsR protein of S. pneumoniae was purified and shown to bind specifically to the clpP, clpC, clpE, and groESL regulatory regions. S. pneumoniae ⌬ctsR, ⌬clpP, ⌬clpC, and ⌬clpE mutants were constructed by gene deletion/replacement. ClpP was shown to act as a negative regulator, preventing competence gene expression under inappropriate conditions. Phenotypic analyses also indicated that ClpP and ClpE are both required for thermotolerance. Contrary to a previous report, we found that ClpC does not play a major role in competence development, autolysis, pneumolysin production, or growth at high temperature of S. pneumoniae.

SummaryThe conversion of a growing cell into an endospore in Bacillus subtilis involves a phagocytic-like process in which the developing spore (the forespore) is wholly engulfed by the adjacent mother cell. A prerequisite for engulfment is the removal of peptidoglycan from the septum that separates the forespore from the mother cell, a process that depends on the autolysin SpoIID and two proteins of unknown function, SpoIIM and SpoIIP. Here we present evidence that SpoIIP is also an autolysin, that it acts in tandem with SpoIID, and that all three proteins are in a complex with each other. We further show that the members of the complex exhibit a hierarchical relationship in which SpoIIM is responsible for localization to the septal membrane, SpoIIP localizes to the septal membrane by interacting with SpoIIM, and SpoIID, in turn, localizes by interacting with SpoIIP. Finally, we show that localization of SpoIIM depends on a fourth protein SpoIIB, raising the possibility that the complex contains an additional component and creating an overall hierarchy of the form: SpoIIB,SpoIIM, SpoIIP,SpoIID.

SummaryMultiple 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 in the model organism, Bacillus subtilis . We recently identified an orthologue of the CtsR regulator of stress response in the major pathogen, Staphylococcus aureus . Sequence analysis of the S . aureus genome revealed the presence of potential CtsR operator sites not only upstream from genes encoding subunits of the Clp ATP-dependent protease, as in B . subtilis , but also, unexpectedly, within the promoter regions of the dnaK and groESL operons known to be specifically controlled by HrcA. The tandem arrangement of the CtsR and HrcA operators suggests a novel mode of dual heat shock regulation by these two repressors. The S . aureus ctsR and hrcA genes were cloned under the control of the P xylA xylose-inducible promoter and used to demonstrate dual regulation of the dnaK and groESL operons by both CtsR and HrcA, using B . subtilis as a heterologous host. Direct binding by both repressors was shown in vitro by gel mobility shift and DNase I footprinting experiments using purified S .

SummaryThe cytoskeleton occupies a central role in cellular immunity by promoting bacterial sensing and antibacterial functions. Septins are cytoskeletal proteins implicated in various cellular processes, including cell division. Septins also assemble into cage-like structures that entrap cytosolic Shigella, yet how septins recognize bacteria is poorly understood. Here, we discover that septins are recruited to regions of micron-scale membrane curvature upon invasion and division by a variety of bacterial species. Cardiolipin, a curvature-specific phospholipid, promotes septin recruitment to highly curved membranes of Shigella, and bacterial mutants lacking cardiolipin exhibit less septin cage entrapment. Chemically inhibiting cell separation to prolong membrane curvature or reducing Shigella cell growth respectively increases and decreases septin cage formation. Once formed, septin cages inhibit Shigella cell division upon recruitment of autophagic and lysosomal machinery. Thus, recognition of dividing bacterial cells by the septin cytoskeleton is a powerful mechanism to restrict the proliferation of intracellular bacterial pathogens.