Wolfgang Schumann

Essential Bacillus subtilis genes

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 ≈4,100 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.

Heat-shock and general stress response in Bacillus subtilis.

The 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 σA‐dependent promoter, an inverted repeat (called the CIRCE element) highly conserved among eubacteria, and probably a repressor interacting with the CIRCE element. The majority of general stress genes (class II, more than 40) are induced at σB‐dependent promoters by different growth‐inhibiting conditions. The activation of σB by stress or starvation is the crucial event in the induction of this large stress regulon. Only a few genes, including lonclpCclpP, and ftsH, can respond to different stress factors independently of σB or CIRCE (class III). Stress induction of these genes occurs at promoters presumably recognized by σA and probably involves additional regulatory elements which remain to be defined.

The GroE chaperonin machine is a major modulator of the CIRCE heat shock regulon of Bacillus subtilis

Class I heat‐inducible genes in Bacillus subtilis consist of the heptacistronic dnaK and the bicistronic groE operon and form the CIRCE regulon. Both operons are negatively regulated at the level of transcription by the HrcA repressor interacting with its operator, the CIRCE element. Here, we demonstrate that the DnaK chaperone machine is not involved in the regulation of HrcA and that the GroE chaperonin exerts a negative effect in the post‐transcriptional control of HrcA. When expression of the groE operon was turned off, the dnaK operon was significantly activated and large amounts of apparently inactive HrcA repressor were produced. Overproduction of GroEL, on the other hand, resulted in decreased expression of the dnaK operon. Introduction of the hrcA gene and its operator into Escherichia coli was sufficient to elicit a transient heat shock response, indicating that no additional Bacillus‐specific gene(s) was needed. As in B.subtilis, the groEL gene of E.coli negatively influenced the activity of HrcA. HrcA could be overproduced in E.coli, but formed inclusion bodies which could be dissolved in 8 M urea. Upon removal of urea, HrcA had a strong tendency to aggregate, but aggregation could be suppressed significantly by the addition of GroEL. Purified HrcA repressor was able specifically to retard a DNA fragment containing the CIRCE element, and the amount of retarded DNA was increased significantly in the presence of GroEL. These results suggest that the GroE chaperonin machine modulates the activity of the HrcA repressor and therefore point to a novel function of GroE as a modulator of the heat shock response.

Alkaline shock induces the Bacillus subtilisσW regulon

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.

The Bacillus subtilis heat shock stimulon

Abstract All organisms respond to a sudden increase in temperature by the so-called heat shock response. This response results in the induction of a subset of genes, designated heat shock genes coding for heat shock proteins, which allow the cell to cope with the stress regimen. Research carried out during the last 10 years with eubacteria has revealed that the heat shock genes of a given species fall into different classes (regulons), where each class is regulated by a different transcriptional regulator, which could be an alternative sigma factor, a transcriptional activator, or a transcriptional repressor. All regulons of a single species constitute the heat shock stimulon. In Bacillus subtilis, more than 200 genes representing over 7% of the transcriptionally active genes are induced at least 3-fold in response to a heat shock. This response becomes apparent within the first minute after exposure to heat stress, is transient, and is coordinated by at least 5 transcriptional regulator proteins, including 2 repressors, an alternate sigma-factor, and a 2-component signal transduction system. A detailed analysis of the regulation of all known heat shock genes has shown that they belong to at least 6 regulons that together comprise the B subtilis heat shock stimulon. Potential thermosensors are discussed in this article.

The ftsH gene of Bacillus subtilis is involved in major cellular processes such as sporulation, stress adaptation and secretion

The ftsH gene of Bacillus subtilis has been identified as a general stress gene which is transiently induced after thermal or osmotic upshift. The FtsH protein exhibits 70.1% homology to FtsH of Escherichia coli which constitutes an essential ATP‐ and Zn2+‐dependent protease anchored in the cytoplasmic membrane via two N‐terminal transmembrane domains. This paper describes the isolation and functional characterization of an ftsH null mutant which was obtained by integration of a cat‐cassette near the 5′ end of ftsH, thereby preventing the synthesis of FtsH protein. In contrast to the situation in E. coli, ftsH is dispensable in B. subtilis but results in a pleiotropic phenotype. While the mutant cells grew mostly as large filaments under physiological conditions, they turned out to be extremely sensitive to heat and salt stress. Although ftsH is necessary for adaptation to heat, it is not involved in the regulation of the heat‐shock response. The induction profiles of representative genes of the CIRCE and sigma‐B regulon and class III heat‐shock genes lon and clpC were identical in the wild type and the ftsH null mutant. Furthermore, the ftsH knockout strain was unable to sporulate, and this failure was probably due to the absence of Spo0A protein which is essential for entry into the sporulation programme. In addition, secretion of bulk exoproteins was severely impaired in the ftsH null mutant after entry into stationary phase. The α‐amylase and subtilisin activity in the supernatant was specifically tested. Whereas the activity of α‐amylase increased after entry into stationary phase in both the wild type and the ftsH mutant strain, that of subtilisin encoded by aprE was prevented at the level of transcription in the mutant. Most of these results can be explained by the failure to synthesize appropriate amounts of Spo0A protein in the ftsH null mutant and point to ftsH as a developmental checkpoint

The Bacillus subtilisσW anti‐sigma factor RsiW is degraded by intramembrane proteolysis through YluC

The Bacillus subtilisσW regulon is induced by different stresses such as alkaline shock, salt shock, phage infection and certain antibiotics that affect cell wall biosynthesis. The activity of the alternative, extracytoplasmic function (ECF) sigma factor σW is modulated by a specific anti‐sigma factor (RsiW or YbbM) encoded by the rsiW (ybbM) gene located immediately downstream of sigW. The RsiW membrane topology was determined, and a specific reporter system for RsiW function was constructed. Experiments using the yeast two‐hybrid system suggested a direct interaction of σW with the cytoplasmic part of RsiW. Analysis of truncated forms of the RsiW protein revealed that σW induction by alkaline shock is dependent on both the transmembrane and the extracytoplasmic domain of RsiW. Western blot and pulse–chase experiments demonstrated degradation of RsiW after an alkaline shock. A B. subtilis mutant strain deleted for the Escherichia coli yaeL orthologue yluC, encoding a transmembrane protease, was defective in inducing a σW‐controlled promoter after alkaline shock and accumulated a membrane‐bound truncated form of RsiW, suggesting that the activity of σW is controlled by the proteolysis of RsiW by at least two different proteolytic steps.

SsrA-Mediated Tagging in Bacillus subtilis

A general mechanism in bacteria to rescue stalled ribosomes involves a stable RNA encoded by the ssrA gene. This RNA, termed tmRNA, encodes a proteolytic peptide tag which is cotranslationally added to truncated polypeptides, thereby targeting them for rapid proteolysis. To study this ssrA-mediated mechanism in Bacillus subtilis, a bipartite detection system was constructed that was composed of the HrcA transcriptional repressor and the bgaB reporter gene coding for a heat-stable beta-galactosidase fused to an HrcA-controlled promoter. After the predicted proteolysis tag was fused to HrcA, the reporter beta-galactosidase was expressed constitutively at a high level due to the instability of the tagged HrcA. Replacement of the two C-terminal alanine residues of the tag by aspartate rendered the repressor stable. Replacement of the hrcA stop codon by a transcriptional terminator sequence rendered the protein unstable; this was caused by trans translational addition of the proteolytic tag. Inactivating the B. subtilis ssrA or smpB (yvaI) gene prevented the trans translational tagging reaction. Various protease-deficient strains of B. subtilis were tested for proteolysis of tagged HrcA. HrcA remained stable only in clpX or clpP knockouts, which suggests that this ATP-dependent protease is primarily responsible for the degradation of SsrA-tagged proteins in B. subtilis.

Development of a New Integration Site within the Bacillus subtilis Chromosome and Construction of Compatible Expression Cassettes

The Bacillus subtilis lacA gene, coding for beta-galactosidase, has been explored as a new site able to accept DNA sequences from nonreplicating delivery vectors. Two such delivery expression vectors have been constructed and shown to be useful in obtaining regulated expression from the chromosomal location. In another experiment, it was shown that the integration of a regulatory gene at the lacA locus was able to control the expression of a transcriptional fusion at the amyE locus. These experiments demonstrate that both integration sites can be used simultaneously to obtain regulated expression of desired genes.

Construction and Application of Epitope- and Green Fluorescent Protein-Tagging Integration Vectors for Bacillus subtilis

ABSTRACT Here we describe the construction and application of six new tagging vectors allowing the fusion of two different types of tagging sequences, epitope and localization tags, to any Bacillus subtilis protein. These vectors are based on the backbone of pMUTIN2 and replace the lacZ gene with tagging sequences. Fusion of the tagging sequences occurs by PCR amplification of the 3′ terminal part of the gene of interest (about 300 bp), insertion into the tagging vector in such a way that a fusion protein will be synthesized upon integration of the whole vector via homologous recombination with the chromosomal gene. Three of these tagging sequences (FLAG, hemagglutinin, and c-Myc) allow the covalent addition of a short epitope tag and thereby detection of the fusion proteins in immunoblots, while three other tags (green fluorescent protein+, yellow fluorescent protein, and cyan fluorescent protein) are helpful in assigning proteins within one of the compartments of the cell. The versatility of these vectors was demonstrated by fusing these tags to the cytoplasmically located HtpG and the inner membrane protein FtsH.