SummaryDuring carbon-starvation-induced entry into stationary phase, Escherichia coli cells exhibit a variety of physiological and morphological changes that ensure survival during periods of prolonged starvation. Induction of 30-50 proteins of mostly unknown function has been shown under these conditions. In an attempt to identify C-starvation-regulated genes we isolated and characterized chromosomal C-starvation-induced csi::/acZ fusions using the x.p/acMu syste-;". One operon fusion (csi2::/acZ) has been studied in detail. csi2::/acZ was induced during transition from exponential to stationary phase and was negatively regulated by cAMP. It was mapped at 59 min on the E. coli chromosome and conferred a pleiotropic phenotype. As demonstrated by two-dimensional gel electrophoresis, cells carrying csi2::/acZ did not synthesize at least 16 proteins present in an isogenic csi2+ strain. Cells containing csi2::/acZ or csi2::Tn 10 did not produce glycogen, did not develop thermotolerance and H20 2 resistance, and did not induce a stationary-phase-specific acidic phosphatase (AppA) as well as another csi fusion (csi5::/acZ). Moreover, they died off much more rapidly than wild-type cells during prolonged starvation. We conclude that csi2::/acZ defines a regulatory gene of central importance for stationary phase E. coli cells. These results and the cloning of the wild-type gene corresponding to csi2 demonstrated that the csi2 locus is allelic with the previously identified regulatory genes katF and appR. The katF sequence indicated that its gene product is a novel sigma factor supposed to regulate expression of catalase HPII and exonuclease III (Mulvey and Loewen, 1989). We suggest that this novel sigma subunit of RNA polymerase defined by csi21 katF/appR is a central early regulator of a large starvation/stationary phase regulon in E. coli and propose 'rpoS' (' us,) as appropriate designations.

The second vegetative sigma factor crs (encoded by the rpoS gene) is the master regulator in a complex regulatory network that governs the expression of many stationary phase-induced and osmotically regulated genes in Escherichia coil Using a combination of gene-fusion technology and quantitative immunoblot, pulse-labeling, and immunoprecipitation analyses, we demonstrate here that rpoS/~r s expression is not only transcriptionally controlled, but is also extensively regulated at the levels of translation and protein stability. rpoS transcription is inversely correlated with growth rate and and is negatively controlled by cAMP-CRP. In complex medium rpoS transcription is stimulated during entry into stationary phase, whereas in minimal media, it is not significantly induced, rpoS translation is stimulated during transition into stationary phase as well as by an increase in medium osmolarity. A model involving mRNA secondary structure is suggested for this novel type of post-transcriptional growth phase-dependent and osmotic regulation. Furthermore, ~r s is a highly unstable protein in exponentially growing cells (with a half-life of 1.4 min), that is stabilized at the onset of starvation. When cells are grown in minimal glucose medium, translational induction and ~r s stabilization occur in a temporal order with the former being stimulated already in late exponential phase and the latter taking place at the onset of starvation. Although crs does not control its own transcription, it is apparently indirectly involved in a negative feedback control that operates on the post-transcriptional level. Our analysis also indicates that at least five different signals [cAMP, a growth rate-related signal (ppGpp?I, a cell density signal, an osmotic signal, and a starvation signal] are involved in the control of all these processes that regulate rpoS/~r s expression.

The rpoS (katF) gene of Escherichia coli encodes a putative sigma factor (ajS) required for the expression of a variety of stationary phase-induced genes, for the development of stationary-phase stress resistance, and for long-term starvation survival (R. Lange and R. Hengge-Aronis, Mol. Microbiol. 5:49-59, 1991). Here we show that the genes otsA, otsB, treA, and osmB, previously known to be osmotically regulated, are also induced during transition into stationary phase in a rs-dependent manner. otsA and otsB, which encode trehalose-6-phosphate synthase and trehalose-6-phosphate phosphatase, respectively, are involved in os-dependent stationary-phase thermotolerance. Neither os nor trehalose, however, is required for the development of adaptive thermotolerance in growing cells, which might be controlled by cJE.

The novel sigma factor (as) encoded by rpoS (katF) is required for induction of many growth phase-regulated genes and expression of a variety of stationary-phase phenotypes in Escherichia coli. Here we demonstrate that wild-type cells exhibit spherical morphology in stationary phase, whereas rpoS mutant cells remain rod shaped and are generally larger. Size reduction of E. coli cells along the growth curve is a continuous and at least biphasic process, the second phase of which is absent in rpoS-deficient cells and correlates with induction of the morphogene bol in wild-type cells. Stationary-phase induction of boUl is dependent on as. The "gearbox," a characteristic sequence motif present in the cS-dependent growth phase-and growth rate-regulated bolAp1 promoter, is not recognized by cs, since stationary-phase induction of the mcbA promoter, which also contains a gearbox, does not require rs, and other crs-controlled promoters do not contain gearboxes. However, good homology to the potential -35 and -10 consensus sequences for crs regulation is found in the bolAp, promoter.

We have identified in the Streptococcus pneumoniae genome sequence a two-component system (TCS13, Blp [bacteriocin-like peptide]) which is closely related to quorum-sensing systems regulating cell density-dependent phenotypes such as the development of genetic competence or the production of antimicrobial peptides in lactic acid bacteria. In this study we present evidence that TCS13 is a peptide-sensing system that controls a regulon including genes encoding Blps. Downstream of the Blp TCS (BlpH R) we identified open reading frames (blpAB) that have the potential to encode an ABC transporter that is homologous to the ComA/B export system for the competence-stimulating peptide ComC. The putative translation product of blpC, a small gene located downstream of blpAB, has a leader peptide with a Gly-Gly motif. This leader peptide is typical of precursors processed by this family of transporters. Microarray-based expression profiling showed that a synthetic oligopeptide corresponding to the processed form of BlpC (BlpC*) induces a distinct set of 16 genes. The changes in the expression profile elicited by synthetic BlpC* depend on BlpH since insertional inactivation of its corresponding gene abolishes differential gene induction. Comparison of the promoter regions of the blp genes disclosed a conserved sequence element formed by two imperfect direct repeats upstream of extended ؊10 promoter elements. We propose that BlpH is the sensor for BlpC* and the conserved sequence element is a recognition sequence for the BlpR response regulator.Signaling mechanisms controlling multicellular behavior of bacteria have attracted much attention in current research. In gram-negative bacteria, homoserine-lactone-based communication systems are prominent. Research in this area led to the term "quorum sensing" for phenomena that are controlled by cell density (12). In gram-positive bacteria, quorum sensing is accomplished by signaling systems that depend on the secretion and sensing of small peptides (11,19). At least two different mechanisms for sensing the presence of pheromone-like peptides are known (21). The first involves import of the peptide and interaction with an intracellular factor (22); the second involves binding to the extracellular portion of a membranebound histidine kinase. This leads to the autophosphorylation of the kinase and subsequent activation, e.g., phosphorylation of a cognate response regulator that mediates changes in gene expression. Quorum-sensing systems regulate a plethora of cellular functions. In Staphylococcus aureus, the AgrC-AgrAsystem is involved in the density-dependent regulation of virulence (18). In Lactobacillus strains, the production of bacteriocins is dependent on peptide-regulated two-component systems (TCS) (4, 10). In Streptococcus pneumoniae, the development of genetic competence (the natural ability to take up DNA) has been shown to be regulated by the comC-DE system (29).The com system of S. pneumoniae was the first quorumsensing system for which a biological function was defined. ...