Bacillus subtilis is both a model organism for basic research and an industrial workhorse, yet there are major gaps in our understanding of the genomic heritage and provenance of many widely used strains. We analyzed 17 legacy strains dating to the early years of B. subtilis genetics. For three-NCIB 3610 T , PY79, and SMY-we performed comparative genome sequencing. For the remainder, we used conventional sequencing to sample genomic regions expected to show sequence heterogeneity. Sequence comparisons showed that 168, its siblings (122, 160, and 166), and the type strains NCIB 3610 and ATCC 6051 are highly similar and are likely descendants of the original Marburg strain, although the 168 lineage shows genetic evidence of early domestication. Strains 23, W23, and W23SR are identical in sequence to each other but only 94.6% identical to the Marburg group in the sequenced regions. Strain 23, the probable W23 parent, likely arose from a contaminant in the mutagenesis experiments that produced 168. The remaining strains are all genomic hybrids, showing one or more "W23 islands" in a 168 genomic backbone. Each traces its origin to transformations of 168 derivatives with DNA from 23 or W23. The common prototrophic lab strain PY79 possesses substantial W23 islands at its trp and sac loci, along with large deletions that have reduced its genome 4.3%. SMY, reputed to be the parent of 168, is actually a 168-W23 hybrid that likely shares a recent ancestor with PY79. These data provide greater insight into the genomic history of these B. subtilis legacy strains.

The rpoS‐encoded sigma(S) subunit of RNA polymerase is a central regulator in a regulatory network that governs the expression of many stationary phase‐induced and osmotically regulated genes in Escherichia coli. sigma(S) is itself induced under these conditions due to an increase in rpoS transcription (only in rich media) and rpoS translation as well as a stabilization of sigma(S) protein which in growing cells is subject to rapid turnover. We demonstrate here that a response regulator, RssB, plays a crucial role in the control of the cellular sigma(S) content. rssB null mutants exhibit nearly constitutively high levels of sigma(S) and are impaired in the post‐transcriptional growth phase‐related and osmotic regulation of sigma(S). Whereas rpoS translational control is not affected, sigma(S) is stable in rssB mutants, indicating that RssB is essential for sigma(S) turnover. RssB contains a unique C‐terminal output domain and is the first known response regulator involved in the control of protein turnover.

The rpoS-encoded cr s subunit of RNA polymerase in Escherichia coil is a global regulatory factor involved in several stress responses. Mainly because of increased rpoS translation and stabilization of cr s, which in nonstressed cells is a highly unstable protein, the cellular ~r s content increases during entry into stationary phase and in response to hyperosmolarity. Here, we identify the hfq-encoded RNA-binding protein HF-I, which has been known previously only as a host factor for the replication of phage Q~ RNA, as an essential factor for rpoS translation. An hfq null mutant exhibits strongly reduced ~r s levels under all conditions tested and is deficient for growth phase-related and osmotic induction of ~r s. Using a combination of gene fusion analysis and pulse-chase experiments, we demonstrate that the hfq mutant is specifically impaired in rpoS translation. We also present evidence that the H-NS protein, which has been shown to affect rpoS translation, acts in the same regulatory pathway as HF-I at a position upstream of HF-I or in conjunction with HF-I. In addition, we show that expression and heat induction of the heat shock ~r factor cr 32 (encoded by rpoH) is not dependent on HF-I, although rpoH and rpoS are both subject to translational regulation probably mediated by changes in mRNA secondary structure. HF-I is the first factor known to be specifically involved in rpoS translation, and this role is the first cellular function to be identified for this abundant ribosome-associated RNA-binding protein in E. coli.

TheS subunit of RNA polymerase (encoded by the rpoS gene) is the master regulator in a complex regulatory network that controls stationary-phase induction and osmotic regulation of many genes in Escherichia coli. Here we demonstrate that the histone-like protein H-NS is also a component of this network, in which it functions as a global inhibitor of gene expression during the exponential phase of growth. On two-dimensional gels, at least 22 S -controlled proteins show increased expression in an hns mutant. H-NS also inhibits the expression of S itself by a mechanism that acts at the posttranscriptional level. Our results indicate that relief of repression by H-NS plays a role in stationary-phase induction as well as in hyperosmotic induction of rpoS translation. Whereas certain S -dependent genes (e.g., osmY) are only indirectly regulated by H-NS via its role in the control of S expression, others are also H-NS-regulated in a S -independent manner. (For this latter class of genes, rpoS hns double mutants show higher levels of expression than mutants deficient in rpoS alone.) In addition, we demonstrate that the slow-growth phenotype of hns mutants is suppressed in hns rpoS double mutants and that many second-site suppressor mutants that spontaneously arise from hns strains carry lesions that affect the expression of S . TheS subunit of RNA polymerase acts as a master regulator in a regulatory network that controls the expression of numerous stationary-phase-induced and osmotically regulated genes in Escherichia coli (11,12,15). rpoS, the structural gene for S , is itself induced during entry into the stationary phase (22,23,25,30,34,39) and in response to an increase in medium osmolarity (23). Under both conditions, control of the cellular S level is largely posttranscriptional, involving stimulation of translation (23, 30) as well as changes in S stability (23,43). So far, more than 30 genes or operons that are under direct or indirect control of S have been identified. Within this large S regulon, differential regulation has been observed for subsets of genes, for instance in response to anaerobiosis (3, 5, 6) or oxidative stress (1, 26). In addition, during entry into the stationary phase, the induction of various S -dependent genes follows different kinetics. These observations indicate that additional factors besides S participate in the fine regulation of these genes. The cyclic AMP (cAMP)-cAMP receptor protein complex (13,20,21,29,47), integration host factor (1, 20), and Lrp (20) have been identified as modulating factors in the control of various S -dependent genes. The data presently available indicate that various combinations of regulatory factors that can act either positively or negatively are used for the control of various stationary-phaseinducible genes. This regulatory strategy results in a high degree of specific fine modulation of S -controlled genes with respect to the time of induction during entry into the stationary phase and in response to additional environmental parameters.In the prese...

The s subunit of RNA polymerase (encoded by the rpoS gene) is a master regulator in a complex regulatory network that governs the expression of many stationary-phase-induced and osmotically regulated genes in Escherichia coli. rpoS expression is itself osmotically regulated by a mechanism that operates at the posttranscriptional level. Cells growing at high osmolarity already exhibit increased levels of s during the exponential phase of growth. Osmotic induction of rpoS can be triggered by addition of NaCl or sucrose and is alleviated by glycine betaine. Stimulation of rpoS translation and a change in the half-life of s from 3 to 50 min both contribute to osmotic induction. Experiments with lacZ fusions inserted at different positions within the rpoS gene indicate that an element required for s degradation is encoded between nucleotides 379 and 742 of the rpoS coding sequence.Like most single-cell organisms, bacteria must be able to cope with extreme fluctuations in the composition and physical parameters of their environments. One of these parameters is the osmolarity of the surrounding medium. When Escherichia coli cells experience a shift to high osmolarity, influx of potassium ions and synthesis of glutamate are strongly stimulated. This rapid response is followed by uptake from the medium and/or synthesis of compatible solutes and osmoprotectants like glycine betaine, proline, or trehalose (for a review, see reference 5). In parallel, the induction of numerous proteins can be observed by two-dimensional O'Farrell gel electrophoresis (4, 14). Several corresponding genes have been identified, for instance, by isolating hyperosmotically inducible lacZ or phoA gene fusions (1,3,6,(8)(9)(10)35).With respect to the regulatory mechanisms involved, two systems have been studied in detail. One is the proU operon, which encodes a glycine betaine uptake system (for a recent review, see reference 24), whereas the other is the ompF/ompC porin system, which is controlled by a typical two-component regulatory system consisting of the membrane-bound sensory histidine kinase EnvZ and the response regulatory OmpR (15,17,26,27). However, the regulatory mechanisms involved do not seem to play a general role in the osmotic regulation of many genes, and efforts to identify a global osmotic regulator have failed.By contrast, several other hyperosmotically induced genes (otsBA, treA, osmB, osmY, and bolA) are under the control of s , a sigma subunit of RNA polymerase in E. coli that is encoded by the rpoS gene (13,14,19,34). This seems to implicate s as a global regulator in the osmotic control of gene expression. In fact, this would be a second global regulatory role for s , which is usually regarded as a stationary-phasespecific sigma factor since the genes mentioned above, as well as many other s -dependent genes, are induced during entry into stationary phase (for recent reviews, see references 11, 12, and 23). Besides being growth phase regulated at the levels of transcription, translation, and s protein stability, rpoS has a...