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Two-component signal transduction systems (TCS) are an important mechanism by which bacteria sense and respond to their environment. Although each two-component system appears to detect and respond to a specific signal(s), it is now evident that they do not always act independently of each other. In this paper we present data indicating regulatory links between the PhoPR two-component system that participates in the cellular response to phosphate limitation, and the essential YycFG two-component system in Bacillus subtilis. We show that the PhoR sensor kinase can activate the YycF response regulator during a phosphate limitation-induced stationary phase, and that this reaction occurs in the presence of the cognate YycG sensor kinase. Phosphorylation of YycF by PhoR also occurs in vitro, albeit at a reduced level. However, the reciprocal cross-phosphorylation does not occur. A second level of interaction between PhoPR and YycFG is indicated by the fact that cells depleted for YycFG have a severely deficient PhoPR-dependent phosphate limitation response and that YycF can bind directly to the promoter of the phoPR operon. YycFG-depleted cells neither activate expression of phoA and phoPR nor repress expression of the essential tagAB and tagDEF operons upon phosphate limitation. This effect is specific to the PhoPR-dependent phosphate limitation response because PhoPR-independent phosphate limitation responses can be initiated in YycFG-depleted cells.

The YycG/YycF two-component system, originally identified in Bacillus subtilis, is very highly conserved and appears to be specific to low G + C Gram-positive bacteria. This system is required for cell viability, although the basis for this and the nature of the YycF regulon remained elusive. Using a combined hybrid regulator/transcriptome approach involving the inducible expression of a PhoP'-'YycF chimerical protein in B. subtilis, we have shown that expression of yocH, which encodes a potential autolysin, is specifically activated by YycF. Gel mobility shift and DNase I footprinting assays were used to show direct binding in vitro of purified YycF to the regulatory regions of yocH as well as ftsAZ, previously reported to be controlled by YycF. Nucleotide sequence analysis and site-directed mutagenesis allowed us to define a potential consensus recognition sequence for the YycF response regulator, composed of two direct repeats: 5'-TGT A/T A A/T/C-N5-TGT A/T A A/T/C-3'. A DNA-motif analysis indicates that there are potentially up to 10 genes within the B. subtilis YycG/YycF regulon, mainly involved in cell wall metabolism and membrane protein synthesis. Among these, YycF was shown to bind directly to the region upstream from the ykvT gene, encoding a potential cell wall hydrolase, and the intergenic region of the tagAB/tagDEF divergon, encoding essential components of teichoic acid biosynthesis. Definition of a potential YycF recognition sequence allowed us to identify likely members of the YycF regulon in other low G + C Gram-positive bacteria, including several pathogens such as Listeria monocytogenes, Staphylococcus aureus and Streptococcus pneumoniae.

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 approximately 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.

Expression of the general stress regulon of Bacillus subtilis is controlled by the alternative transcription factor sigma(B), which is activated when cells encounter growth-limiting energy or environmental stresses. The RsbT serine-threonine kinase is required to convey environmental stress signals to sigma(B), and this kinase activity is magnified in vitro by the RsbR protein, a positive regulator important for full in vivo response to salt or heat stress. Previous genetic analysis suggested that RsbR function is redundant with other unidentified regulators. A search of the translated B. subtilis genome found six paralogous proteins with significant similarity to RsbR: YetI, YezB, YkoB, YojH, YqhA, and YtvA. Their possible regulatory roles were investigated using three different approaches. First, genetic analysis found that null mutations in four of the six paralogous genes have marked effects on the sigma(B) environmental signaling pathway, either singly or in combination. The two exceptions were yetI and yezB, adjacent genes which appear to encode a split paralog. Second, biochemical analysis found that YkoB, YojH, and YqhA are specifically phosphorylated in vitro by the RsbT environmental signaling kinase, as had been previously shown for RsbR, which is phosphorylated on two threonine residues in its C-terminal region. Both residues are conserved in the three phosphorylated paralogs but are absent in the ones that were not substrates of RsbT: YetI and YezB, each of which bears only one of the conserved residues; and YtvA, which lacks both residues and instead possesses an N-terminal PAS domain. Third, analysis in the yeast two-hybrid system suggested that all six paralogs interact with each other and with the RsbR and RsbS environmental regulators. Our data indicate that (i) RsbR, YkoB, YojH, YqhA, and YtvA function in the environmental stress signaling pathway; (ii) YtvA acts as a positive regulator; and (iii) RsbR, YkoB, YojH, and YqhA collectively act as potent negative regulators whose loss increases sigma(B) activity more than 400-fold in unstressed cells.

HtrA-type serine proteases participate in folding and degradation of aberrant proteins and in processing and maturation of native proteins. Mutation of the corresponding genes often confers a pleiotropic phenotype that can include temperature sensitivity, sensitivity to osmotic and oxidative stress, and attenuated virulence. There are three HtrA-type serine proteases, YkdA, YvtA, and YycK, encoded in the Bacillus subtilis genome. In this report we show that YkdA and YvtA display many similarities: their expression profiles during the growth cycle in wild-type and mutant backgrounds are very alike, with expression being directed by very similar promoters. Both are induced by temperature upshift and by heterologous amylases at the transition phase of the growth cycle. These characteristics are quite different for YycK, suggesting that it has a cellular function distinct from that of the other two proteases or that it performs the same function but under different conditions. We also show that inactivation of either ykdA or yvtA results in compensating overexpression of the other gene, especially during stress conditions, with a concomitant increase in resistance to heat and hydrogen peroxide stresses. Mutation of both ykdA and yvtA leads to growth defects and to thermosensitivity. The fact that their expression increases dramatically at the transition phase of the growth cycle under certain conditions suggests that the YkdA and YvtA proteases may function in the processing, maturation, or secretion of extracellular enzymes in B. subtilis.

There are three members of the HtrA family of serine proteases, YkdA, YvtA, and YyxA, encoded in the chromosome of Bacillus subtilis. In this study, we report on the promoter structure and regulation of ykdA expression. The ykdA gene is heat inducible, exhibiting a biphasic pattern of expression during a 60-min interval after heat shock. Increased expression after heat shock occurs at the transcriptional level. The heat-shock-inducible promoter has a single mismatch with a SigA-type -10 motif, but does not exhibit similarity to a SigA -35 region. There are six octamer repeats with a consensus TTTTCACA positioned at, and upstream of, the normal position of a -35 region. While repeats V and VI appear dispensable, repeat IV is essential for normal thermoinducible expression. This promoter structure is also found in the control region of yvtA, encoding a second member of this family of proteases. Expression of ykdA is negatively autoregulated both during the growth cycle and during heat shock. Our evidence suggests that YkdA protease activity is not required for this form of regulation. Null mutants of ykdA display increased tolerance to heat and are 80-fold more resistant to 10 mM hydrogen peroxide than wild-type cells. However, ykdA expression is not induced by hydrogen peroxide. These results indicate that the regulon to which YkdA belongs is linked to the oxidative stress response in B. subtilis.

Clp ATPases, which include the ubiquitous HSP100 family, are classified according to their structural features and sequence similarities. During the course of the Bacillus subtilis genome sequencing project, we identified a gene encoding a new member of the HSP100 family. We designated this protein ClpE, as it is the prototype of a novel subfamily among the Clp ATPases, and have identified homologues in several bacteria, including Listeria monocytogenes, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus pneumoniae, Lactobacillus sakei and Clostridium acetobutylicum. A unique feature of these Hsp100-type Clp ATPases is their amino-terminal zinc finger motif. Unlike the other class III genes of B. subtilis (clpC and clpP ), clpE does not appear to be required for stress tolerance. Transcriptional analysis revealed two sigmaA-type promoters, expression from which was shown to be inducible by heat shock and puromycin treatment. Investigation of the regulatory mechanism controlling clpE expression indicates that this gene is controlled by CtsR and is thus a member of the class III heat shock genes of B. subtilis. CtsR negatively regulates clpE expression by binding to the promoter region, in which five CtsR binding sites were identified through DNase I footprinting and sequence analysis.

In this report we present the identification and analysis of two Bacillus subtilis genes, yklA and ykzA, which are homologous to the partially RpoS-controlled osmC gene from Escherichia coli. The yklA gene is expressed at higher levels in minimal medium than in rich medium and is driven by a putative vegetative promoter. Expression of ykzA is not medium dependent but increases dramatically when cells are exposed to stress and starvation. This stress-induced increase in ykzA expression is absolutely dependent on the alternative sigma factor sigmaB, which controls a large stationary-phase and stress regulon. ykzA is therefore another example of a gene common to the RpoS and sigmaB stress regulons of E. coli and B. subtilis, respectively. The composite complex expression pattern of the two B. subtilis genes is very similar to the expression profile of osmC in E. coli.

Four genes identified within the late operon of PBSX show characteristics expected of a host cell lysis system; they are xepA, encoding an exported protein; xhlA, encoding a putative membrane-associated protein; xhlB, encoding a putative holin; and xlyA, encoding a putative endolysin. In this work, we have assessed the contribution of each gene to host cell lysis by expressing the four genes in different combinations under the control of their natural promoter located on the chromosome of Bacillus subtilis 168. The results show that xepA is unlikely to be involved in host cell lysis. Expression of both xhlA and xhlB is necessary to effect host cell lysis of B. subtilis. Expression of xhlB (encoding the putative holin) together with xlyA (encoding the endolysin) cannot effect cell lysis, indicating that the PBSX lysis system differs from those identified in the phages of gram-negative bacteria. Since host cell lysis can be achieved when xlyA is inactivated, it is probable that PBSX encodes a second endolysin activity which also uses XhlA and XhlB for export from the cell. The chromosome-based expression system developed in this study to investigate the functions of the PBSX lysis genes should be a valuable tool for the analysis of other host cell lysis systems and for expression and functional analysis of other lethal gene products in gram-positive bacteria.

Bacillus subtilis is the best-characterized member of the Gram-positive bacteria. Its genome of 4,214,810 base pairs comprises 4,100 protein-coding genes. Of these protein-coding genes, 53% are represented once, while a quarter of the genome corresponds to several gene families that have been greatly expanded by gene duplication, the largest family containing 77 putative ATP-binding transport proteins. In addition, a large proportion of the genetic capacity is devoted to the utilization of a variety of carbon sources, including many plant-derived molecules. The identification of five signal peptidase genes, as well as several genes for components of the secretion apparatus, is important given the capacity of Bacillus strains to secrete large amounts of industrially important enzymes. Many of the genes are involved in the synthesis of secondary metabolites, including antibiotics, that are more typically associated with Streptomyces species. The genome contains at least ten prophages or remnants of prophages, indicating that bacteriophage infection has played an important evolutionary role in horizontal gene transfer, in particular in the propagation of bacterial pathogenesis.

The transition state regulator AbrB functions as an activator, a repressor, and a preventer of gene expression in Bacillus subtilis. In this paper, we show that expression of abrB is growth phase dependent. Accumulation of abrB transcript is restricted to a short period spanning the transition between the lag and exponential phases of the growth cycle. The level of abrB transcript then falls sharply, and transcript cannot be detected at the mid-exponential period of the growth cycle. The level of AbrB protein is also maximal during early exponential growth but decreases gradually throughout the remainder of the growth cycle. The abrupt reduction of abrB transcript level during the early period of the growth cycle is effected by the phosphorylated form of the response regulator Spo0p3and to a lesser extent by negative autoregulation. The growth cycle-dependent expression of abrB is very similar to that observed for fis in Escherichia coli and in Salmonella typhimurium. Although AbrB and Fis are not homologous proteins, they display extensive similarity in terms of size, DNA binding characteristics, growth cycle-dependent patterns of expression, and their control over the expression of a varied group of operons. We hypothesize therefore that AbrB, like Fis, is a nucleoid binding protein.

PBSX and skin are two unusual genetic elements resident on the Bacillus subtilis chromosome. PBSX is a phage-like element located at approximately 100 degrees which is induced by the SOS response and results in cell lysis with the release of phage-like particles. The phage particles contain bacterial chromosomal DNA and kill sensitive bacteria without injecting DNA. The skin element is located at approximately 230 degrees on the chromosome and is positioned within the sigK open reading frame (ORF). It is excised at a particular stage of sporulation, leading to reconstitution of the complete sigK gene. In this paper, we show that there are phage-like operons present in the skin element which are highly homologous to the region of PBSX comprising part of the control region and the late operon. These operons are similar in terms of their gene organization, the percentage identity of the products of homologous ORFs and the positioning and strengths of ribosome-binding sites for each ORF. Although this high degree of conservation suggests that the phange-like operons in skin can be expressed, expression of the late operon was not detected during exponential growth, during sporulation or after induction of the SOS response. However two non-phage-like operons in the skin element are expressed and have distinct expression profiles that are dependent on the growth and developmental status of the cell.

The members of the bacterial genus Bacillus are important organisms for both research and industrial purposes, and a major international effort is under way to sequence the complete genome of Bacillus subtilis, the type species for this genus. In this article the organization of the project is summarized; the strategies employed for cloning, sequencing and data handling; the progress to date, and the likely benefits which will accrue to basic research and to the biotechnology industry upon completion of the sequence.

PBSX is a phage-like bacteriocin (phibacin) of Bacillus subtilis 168. Bacteria carrying the PBSX genome are induced by DNA-damaging agents to lyse and produce PBSX particles. The particles cannot propagate the PBSX genome. The particles produced by this suicidal response kill strains nonlysogenic for PBSX. A 5.2-kb region which controls the induction of PBSX has been sequenced. The genes identified include the previously identified repressor gene xre and a positive control factor gene, pcf. Pcf is similar to known sigma factors and acts at the late promoter PL, which has been located distal to pcf. The first two genes expressed from the late promoter show homology to genes encoding the subunits of phage terminases.

The csa-15 locus of Bacillus subtilis corresponds to an operon encoding proteins which display features characteristic of the ABC group of transporters. Sequence analysis reveals a very high level of identity to the ribose transport operon of Escherichia coli. This hypothesis is supported by the observation that strains carrying mutagenic insertions in this operon are unable to grow on ribose as sole carbon source. Expression of this operon is directed by a single SigA-type promoter which is negatively regulated by Spo0A during the late-exponential/transition state of the growth cycle. Expression is also subject to catabolite repression and this mode of regulation is dominant to control of expression by Spo0A.

The citrulline biosynthetic operon argC-F located at 100 degrees on the Bacillus subtilis chromosome contains seven open reading frames which encode all the enzymes required for the biosynthesis of citrulline. The operon is transcribed as a single transcription unit. The second cistron of the operon is homologous to ArgJ (ornithine acetyltransferase) from Bacillus stearothermophilus and Neisseria gonorrhoeae, suggesting that the acetylation of glutamate and the deacetylation of acetylornithine are carried out by a single enzyme in a cyclical pathway. The argF gene is an orthologue of argF from Pseudomonas aeruginosa and a paralogue of arcB from P. aeruginosa and argF/argI from Escherichia coli.

A method is described here that can be used to identify operons whose expression is controlled by any particular regulator protein. This method was used to identify operons whose expression is negatively regulated by Spo0A in Bacillus subtilis. Twenty-eight strains were identified, each of which contains an operon-lacZ transcriptional fusion, negatively regulated, either directly or indirectly, by Spo0A. In one of these strains (CSA8), the lacZ gene is fused to the argC-F operon positioned at 100 degrees on the B. subtilis chromosome. The regulated expression of this operon by Spo0A-P is mediated indirectly through the transition state regulator AbrB and is manifest only during growth on solid medium. In a second strain (CSA15), the lacZ gene is fused to an operon encoding a transport system which displays features characteristic of the ABC group of transporters, and which has a very high level of identity to the ribose transport system from Escherichia coli. Expression of the ribose transport operon is directed by a single SigA-type promoter. Transcription from this promoter is repressed by the phosphorylated form of Spo0A during the late-exponential/transition phase of the growth cycle and this control is not mediated through the transition-state regulator, AbrB.

The features which contribute to the activity of the single-stranded origin of the Bacillus plasmid pBAA1 were investigated. This origin is contained on a DNA fragment greater than 116 but less than 191 bases in size. There is the potential to form three stem-loop structures within this fragment. Comparison of the sequence of this origin from pBAA1 with the sequence of a homologous fragment from the Bacillus thuringiensis plasmid pGI2 indicates that both the structure and the relative positioning of the predicted stem-loops are important for origin activity. Deletion analysis suggests that it is the structure of stem-loop III which is important, because it can be replaced by a nonrelated dyad element without significant loss of origin activity. Three sequence motifs are conserved between the origins from pBAA1 and pGI2. Mutation of motif 1 leads to attenuation of single-stranded origin activity. A second motif (motif 3) shares significant homology with a group of single-strand initiation (ssi) sites found on plasmids isolated from Escherichia coli, suggesting that it also contributes to single-stranded origin activity. Our results also indicate that RNA polymerase is utilized to synthesize the RNA primer at the pBAA1 single-stranded origin and that this origin can function in both Bacillus subtilis and Staphylococcus aureus.

The pattern and extent of DNA sequence variability at the rplX locus (encoding ribosomal protein L24) has been investigated in nine strains of Bacillus subtilis. Overall, there is a very low level of nucleotide diversity, even at silent sites, which is probably due to selection among synonymous codons. By analogy with Escherichia coli, there may also be some effect of the relative proximity of rplX to the chromosomal origin of replication. The small number of nucleotide substitutions are non-randomly distributed: all of the synonymous changes are in valine codons. From the sequence differences the strains can be divided into two groups, which are not coincident with their previous classification; this observation is consistent with recombination among strains.

The results obtained during the first year of the project involving the sequencing of the Bacillus subtilis genome are presented. Different gene libraries using a yeast artificial chromosome vector and bacteriophage vectors, lambda FixII and phi 105J124, have been constructed. A total of 300 kbp have been cloned using the lambda FixII vector, 68 kbp of which have been fully sequenced. Several open reading frames showing homologies with genes of other organisms were found. Two genes, previously unknown in this organism, have been identified.

The defective prophage of Bacillus subtilis 168, PBSX, is a chromosomally based element which encodes a non-infectious phage-like particle with bactericidal activity. PBSX is induced by agents which elicit the SOS response. In a PBSX thermoinducible strain which carries the xhi1479 mutation, PBSX is induced by raising the growth temperature from 37 degrees C to 48 degrees C. A 1.2-kb fragment has been cloned which complements the xhi1479 mutation. The nucleotide sequence of this fragment contains an open reading frame (ORF) which encodes a protein of 113 amino acids (aa). This aa sequence resembles that of other bacteriophage repressors and suggests that the N-terminal region forms a helix-turn-helix motif, typical of the DNA-binding domain of many bacterial regulatory proteins. The ORF is preceded by four 15-bp direct repeats, each of which contains an internal palindromic sequence, and by sequences resembling a SigA-dependent promoter. The nt sequence of an equivalent fragment from the PBSX thermoinducible strain has also been determined. There are three aa differences within the ORF compared to the wild type, one of which lies within the helix-turn-helix segment. This ORF encodes a repressor protein of PBSX.

PBSX, a defective Bacillus subtilis prophage, maps to the metA-metC region of the chromosome. DNA (33 kilobases) from this region of the chromosome was cloned and analyzed by insertional mutagenesis with the integrating plasmid pWD3. This plasmid had a promoterless alpha-amylase gene (amyL) that provided information on the direction and level of transcription at the site of integration. Transcription under the control of the PBSX repressor proceeded in the direction metA to metC over a distance of at least 18 kilobases. Electrophoretic analysis of proteins produced by different integrant strains upon PBSX induction and by fragments subcloned in Escherichia coli allowed the identification of early and late regions of the prophage. A set of contiguous fragments directing mutagenic integration suggested that the minimum size of an operon that encodes phage structural proteins is 19 kilobases. The adaptation of PBSX transcriptional and replicational functions to a chromosomally based, thermoinducible expression system is discussed.

Pretreatment of Bacillus subtilis with low concentrations of hydrogen peroxide protected the cells against the lethal effects of higher levels of oxidative stress. During the period of adaptation, eight proteins were induced, as detected by one-dimensional gel electrophoresis. Four of these proteins were the same size as four of the proteins induced by the temperature upshift. The range of proteins synthesized in response to an elevation in temperature depended both on the starting (lower) temperature and on the temperature to which the cells were shifted. Both catalase and superoxide dismutase were present at high levels in B. subtilis, but neither was induced by oxidative stress or temperature upshift. In fact, catalase activity was reduced after the temperature upshift.

A cryptic plasmid, pBAA1, was identified in an industrial Bacillus strain. The plasmid is 6.8 kilobases in size and is present in cells at a copy number of approximately 5 per chromosome equivalent. The plasmid has been maintained under industrial fermentation conditions without apparent selective pressure and so is assumed to be partition proficient. The minimal replicon was localized to a 1.4-kilobase fragment which also contains the functions required for copy number control. The very low level of segregational instability of the minimal replicon suggests that it also contains functions involved in plasmid maintenance. Comparison with other plasmids indicates that pBAA1 belongs to the group of small gram-positive plasmids which replicate by a rolling cycle-type mechanism. A sequence was identified which is required for the efficient conversion of the single plus strand to the double-stranded form during plasmid replication. Deletion of this sequence resulted in a low level of segregational plasmid instability.

The sensitivity of Bacillus subtilis to hydrogen peroxide (oxidative stress) was found to vary with the position of the culture in the growth cycle. The most dramatic change occurred at the stationary phase, when the cells became totally resistant to 10 mM H2O2, in contrast to the loss of 3 to 4 log units of viability when treated at the early log phase. Two of the eight proteins induced by a protective concentration of H2O2 (50 muM) were also induced (in the absence of oxidative stress) on entry into the late log phase of growth. The response of five isogenic spo0 mutants (spo0B, spo0E, spo0F, spo0H, and spo0J) to oxidative stress was identical to that of the wild-type parental strain. In an isogenic spo0A strain, mid-log-phase cells were 100-fold less sensitive to 10 mM H2O2 than was the wild type. Pretreatment with 50 microM H2O2 induced little further protection, suggesting that the response is constitutive in this strain. By comparison of proteins induced by 50 microM H2O2 in the wild-type, spo0A, spo0H, and spo0J strains, four proteins were identified that may be essential for protection against lethal concentrations of H2O2. The presence of multiple copies of the spo0H gene in a spo0A background converted the stress phenotype of the spo0A mutant to that of the wild type but left the sporulation phenotype unaltered.