To estimate the minimal gene set required to sustain bacterial life in nutritious conditions, we carried out a systematic inactivation of Bacillus subtilis genes. Among Ϸ4,100 genes of the organism, only 192 were shown to be indispensable by this or previous work. Another 79 genes were predicted to be essential. The vast majority of essential genes were categorized in relatively few domains of cell metabolism, with about half involved in information processing, one-fifth involved in the synthesis of cell envelope and the determination of cell shape and division, and one-tenth related to cell energetics. Only 4% of essential genes encode unknown functions. Most essential genes are present throughout a wide range of Bacteria, and almost 70% can also be found in Archaea and Eucarya. However, essential genes related to cell envelope, shape, division, and respiration tend to be lost from bacteria with small genomes. Unexpectedly, most genes involved in the Embden-Meyerhof-Parnas pathway are essential. Identification of unknown and unexpected essential genes opens research avenues to better understanding of processes that sustain bacterial life.

SummaryThe induction of stress proteins is an important component of the adaptional network of a non-growing cell of Bacillus subtilis. A diverse range of stresses such as heat shock, salt stress, ethanol, starvation for oxygen or nutrients etc. induce the same set of proteins, called general stress proteins. Although the adaptive functions of these proteins are largely unknown, they are proposed to provide general and rather non-specific protection of the cell under these adverse conditions. In addition to these non-specific general stress proteins, all extracellular signals induce a set of specific stress proteins that may confer specific protection against a particular stress factor. In B. subtilis at least three different classes of heat-inducible genes can be defined by their common regulatory characteristics: Class I genes, as exemplified by the dnaK and groE operons, are most efficiently induced by heat stress. Their expression involves a

By using an internal part of the dnaK gene from BaciUlus megaterium as a probe, a 5.2-kb Hindlll fragment of chromosomal DNA of BaciUlus subtilis was cloned. Downstream sequences were isolated by in vivo chromosome walking. Sequencing of 5,085 bp revealed four open reading frames in the order orf9-grpEdnaK-dnaJ. orJ39 encodes a 39-kDa polypeptide of unknown biological function with no noticeable homology to any other protein within the data bases. Alignment of the GrpE protein with those of three other bacterial species revealed a low overall homology, but a higher homology restricted to two regions which might be involved in interactions with other proteins. Alignment of the DnaK protein with six bacterial DnaK polypeptides revealed that a contiguous region of 24 amino acids is absent from the DnaK proteins of all known gram-positive species. Primer extension studies revealed three potential transcription start sites, two preceding orJ39 (Si and S2) and a third one in front of grpE (S3). S2 and S3 were activated at a high temperature. Northern (RNA) analysis led to the detection of three mRNA species of 4.9, 2.6, and 1.5 kb. RNA dot blot experiments revealed an at-least-fivefold increase in the amount of specific mRNA from 0 to 5 min postinduction and then a rapid decrease. A transcriptional fusion between dnaK and the amyL reporter gene exhibited a slight increase in a-amylase activity after heat induction. A 9-bp inverted repeat was detected in front of the coding region of orJ39. This inverted repeat is present in a number of other heat shock operons in other microorganisms ranging from cyanobacteria to mycobacteria. The biological property of this inverted repeat as a putative key element in the induction of heat shock genes is discussed. The dnaK locus was mapped at about 2230 on the B. subtilis genetic map.The heat shock response is a homeostatic mechanism that enables cells to survive a variety of environmental stresses. It is characterized by the increased synthesis of a group of evolutionarily conserved proteins, heat shock proteins (HSPs), and is a universal feature of both prokaryotic and eukaryotic cells (35). When Eschertichia coli cells are shifted to a high temperature, the synthesis of a set of about 20 HSPs transiently increases and then declines rapidly to steady-state levels characteristic of the new ambient temperature (44).The highly conserved HSPs perform similar functions in all organisms. One of these functions is the well-established regulation of protein-protein interactions by the chaperonins (69). One of the most abundant HSPs, HSP70, is highly conserved in evolution. It is found in such diverse organisms as E. coli, Saccharomyces cerevisiae, Drosophila melanogaster, and Homo sapiens (14, 35).The dnaK gene of E. coli was originally discovered because mutations in it blocked bacteriophage lambda DNA replication at all temperatures (22,23). Subsequently, the dnaK gene product was shown to be essential for E. coli viability at high and low temperatures (22,30,46,51,52), and genetic evid...

The dnaK and groESL operons of Bacillus subtilis are preceded by a potential r43 promoter sequence (recognized by the vegetative cf factor) and by an inverted repeat (IR) consisting of 9 bp separated by a 9-bp spacer. Since this IR has been found in many bacterial species, we suspected that it might be involved in heat shock regulation. In order to test this hypothesis, three different mutational alterations of three bases were introduced within the IR preceding the dnaK operon. These mutations were crossed into the chromosome of B. subtilis, and expression of the dnaK and of the unlinked groESL operons was studied. The dnaK operon exhibited increased expression at low temperature and a reduction in the stimulation after temperature upshift. Furthermore, these mutations reduced expression of the groESL operon at low temperature by 50% but did not interfere with stimulation after heat shock. These experiments show that the IR acts as a negative cis element of the dnaK operon. This conclusion was strengthened by the observation that the IR reduced expression of two different transcriptional fusions significantly after its insertion between the promoter and the reporter gene. Since this IR has been described in many bacterial species as preceding only genes of the dnaK and groESL operons, both encoding molecular chaperones (39 cases are documented so far), we designated this heat shock element CIRCE (controlling IR of chaperone expression). Furthermore, we suggest that this novel mechanism is more widespread among eubacteria than the regulation mechanism described for Escherichia coli and has a more ancient origin.A temperature upshift from 30°C to 42°C transiently induces the heat shock genes in Escherichia coli by activating transcription from promoters specifically recognized by RNA polymerase containing u32 (5, 6) encoded by the rpoH gene (14,25). Enhanced transcription from heat shock promoters is caused by a transient increase in the cellular level of &32 (7, 21) as a result of increased synthesis and stabilization (20). The intracellular concentration of &32 increases 15-to 20-fold within 5 min after temperature upshift and then declines to a new steady-state level. This regulation pathway serves as a paradigm for the eubacteria.Recently, we started analysis of the regulation of heat shock response in Bacillus subtilis. When transcriptional fusions between various E. coli heat shock promoters fused to two different reporter genes were analyzed with B. subtilis, no temperature-dependent induction could be measured (23). We concluded that, in contrast to vegetative promoters, E. coli heat shock promoters are not recognized in B. subtilis. Transcriptional analysis of the dnaK and groESL operons of B. subtilis revealed in both cases a DNA sequence resembling the canonical sequence for vegetative promoters preceding the potential transcriptional start sites, which were activated after temperature upshift (18,24). A close inspection of the DNA sequences around the transcription start sites led to the detection of ...

When confronted with a stress factor, bacteria react with a specific stress response, a genetically encoded programme resulting in the transiently enhanced expression of a subset of genes. One of these stress factors is a sudden increase in the external pH. As a first step to understand the response of Bacillus subtilis cells towards an alkali shock at the transcriptional level, we attempted to identify alkali‐inducible genes using the DNA macroarray technique. To define the appropriate challenging conditions, we used the ydjF gene, the orthologue of the Escherichia coli pspA, as a model gene for an alkali‐inducible gene. Hybridization of 33P‐labelled cDNA to a DNA macroarray revealed induction of more than 80 genes by a sudden increase in the external pH value from 6.3 to 8.9. It was discovered that a large subset of these genes belong to the recently described σW regulon, which was confirmed by the analysis of a sigW knockout. A comparison of B. subtilis wild type with the congenic sigW knockout also led to the discovery of new members of the σW regulon. In addition, we found several genes clearly not belonging to that regulon. This analysis represents the first report of an extracellular stimulus inducing the σW regulon.

Approximately 47% of the genes of the Gram-positive bacterium Bacillus subtilis belong to paralogous gene families. The present studies were aimed at the functional analysis of the sip gene family of B. subtilis, consisting of five chromosomal genes, denoted sipS, sipT, sipU, sipV, and sipW. All five sip genes specify type I signal peptidases (SPases), which are actively involved in the processing of secretory preproteins. Interestingly, strains lacking as many as four of these SPases could be obtained. As shown with a temperature-sensitive SipS variant, only cells lacking both SipS and SipT were not viable, which may be caused by jamming of the secretion machinery with secretory preproteins. Thus, SipS and SipT are of major importance for protein secretion. This conclusion is underscored by the observation that only the transcription of the sipS and sipT genes is temporally controlled via the DegS-DegU regulatory system, in concert with the transcription of most genes for secretory preproteins. Notably, the newly identified SPase SipW is highly similar to SPases from archaea and the ER membrane of eukaryotes, suggesting that these enzymes form a subfamily of the type I SPases, which is conserved in the three domains of life.

Whereas in Escherichia coli only one heat shock regulon is transiently induced by mild heat stress, for Bacillus subtilis three classes of heat shock genes regulated by different mechanisms have been described. Regulation of class I heat shock genes (dnaK and groE operons) involves an inverted repeat (CIRCE element) which most probably serves as an operator for a repressor. Here, we report on the analyses of an hrcA null mutant (⌬hrcA), in which hrcA, the first gene of the dnaK operon, was deleted from the B. subtilis chromosome. This strain was perfectly viable at low and high temperatures. Transcriptional analysis of the deletion mutant revealed a high level of constitutive expression of both the dnaK and groE operons even at a low temperature. A further increase in the amount of groE transcript was observed after temperature upshift, suggesting a second induction mechanism for this operon. Overproduction of HrcA protein from a second copy of hrcA derived from a plasmid (phrcA ؉ ) in B. subtilis wild-type and ⌬hrcA strains prevented heat shock induction of the dnaK and groE operons at the level of transcription almost completely and strongly reduced the amounts of mRNA at a low temperature as well. Whereas the wild-type strain needed 4 h to resume growth after temperature upshift, the ⌬hrcA strain stopped growth only for about 1 h. Overproduction of HrcA protein prior to a heat shock almost completely prevented growth at a high temperature. These data clearly demonstrate that the hrcA product serves as a negative regulator of class I heat shock genes.The heat shock response is an important homeostatic mechanism that enables cells from animals, plants, and bacteria to survive a variety of environmental stresses (21,22). It is characterized by the transiently increased synthesis of a number of proteins, which are called heat shock proteins (HSPs). The strong evolutionary conservation of the heat shock response argues that this response is beneficial for many kinds of cells. HSPs have essential roles in the synthesis, transport, and folding of proteins and are often referred to as molecular chaperones (9). In prokaryotes, the major HSPs are encoded by single genes expressed constitutively at all temperatures. Following a temperature upshift, the rates of expression of these genes abruptly accelerate. After about 8 min, the rates of synthesis of the HSPs are turned down. In Escherichia coli, the heat shock response is positively regulated by the alternate sigma factor 32 and is negatively regulated by the products of the heat shock genes dnaK, dnaJ, and grpE (for recent reviews, see references 5 and 39).In contrast to E. coli, Bacillus subtilis contains three classes of heat shock genes which are turned on by mild heat stress (12). Class I heat shock genes, as exemplified by the dnaK and the groE operons, are expressed from the vegetative promoter P A (6), and their expression involves a cis-active inverted repeat called CIRCE (41). We suggested that class I heat shock genes are negatively regulated by a repressor...

SummaryThe Bacillus subtilis s s s s W regulon is induced by different stresses such as alkaline shock, salt shock, phage infection and certain antibiotics that affect cell wall biosynthesis.