Novel mutations that alter the regulation of sporulation in Bacillus subtilis. Evidence that phosphorylation of regulatory protein SpoOA controls the initiation of sporulation

Sporulation in Bacillus subtilis is a complex developmental process that occurs in response to nutrient deprivation. To identify components of the mechanism that allows cells to monitor their nutritional status and to understand how this sensory information is transduced into a signal to activate specific sporulation genes, we have isolated mutants that are able to sporulate efficiently under nutritional conditions that strongly inhibit sporulation in wild-type bacteria, a phenotype we refer to as Coi (control of initiation). Four coi mutations were found to be within the coding sequence of spoOA, a gene in which null mutations prevent the initiation of sporulation and a gene whose product shares a domain of homology with phosphorylation-activated proteins that play signal transduction roles in bacteria. All four of the spoOA mutations were within this conserved domain and in close proximity to the presumptive phosphoacceptor site. The wild-type and one of the mutant SpoOA proteins were purified and shown to be competent to accept phosphoryl groups from a phosphohistidine within a bacterial signal transduction kinase (CheA). The mutant SpoOA protein exhibited enhanced phosphoacceptor activity compared with the wild-type. This property of the mutant protein, together with additional genetic information, supports a model for regulation of sporulation initiation by control of the phosphorylation level of SpoOA.     

Sensor domains encoded in Bacillus anthracis virulence plasmids prevent sporulation by hijacking a sporulation sensor histidine kinase

Anthrax toxin and capsule, determinants for successful infection by Bacillus anthracis, are encoded on the virulence plasmids pXO1 and pXO2, respectively. Each of these plasmids also encodes proteins that are highly homologous to the signal sensor domain of a chromosomally encoded major sporulation sensor histidine kinase (BA2291) in this organism. B. anthracis Sterne overexpressing the plasmid pXO2-61-encoded signal sensor domain exhibited a significant decrease in sporulation that was suppressed by the deletion of the BA2291 gene. Expression of the sensor domains from the pXO1-118 and pXO2-61 genes in Bacillus subtilis strains carrying the B. anthracis sporulation sensor kinase BA2291 gene resulted in BA2291-dependent inhibition of sporulation. These results indicate that sporulation sensor kinase BA2291 is converted from an activator to an inhibitor of sporulation in its native host by the virulence plasmid-encoded signal sensor domains. We speculate that activation of these signal sensor domains contributes to the initiation of B. anthracis sporulation in the bloodstream of its infected host, a salient characteristic in the virulence of this organism, and provides an additional role for the virulence plasmids in anthrax pathogenesis.     

Relation between inhibition of Bacilli sporulation and synthesis of lytic enzymes.

In two strains of Bacillus, the synthesis of two specific lytic enzymes was studied concomitantly with an inhibition of the sporulation: LD-carboxypeptidase synthesis was unaffected whereas γ-D-glutamyl-(L)meso-diaminopimelyl endopeptidase synthesis was shown to be closely related to sporulation. The endopeptidase production is totaly inhibited when netropsin inhibits sporulation in B. sphaericus and is low in B. subtilis Thy^?A when sporulation is inhibited by thymidine starvation. This enzyme seems directly connected with the sporulation sequence.     

Purification and preliminary crystallographic studies on the sporulation response regulatory phosphotransferase protein,spo0B,from Bacillus subtilis

The phosphotransferase protein Spo0B, a component of the sporulation signal transduction system in Bacillus subtilis was expressed from the Escherichia coli strain BL21DE3. It was purified, crystallized, and 2.25 Å data measured using the synchrotron source at the Stanford Linear Accelerator Center. The search for heavy atom derivatives is in progress. © 1997 Wiley-Liss Inc.     

Repeated triggering of sporulation in Bacillus subtilis selects against a protein that affects the timing of cell division

Bacillus subtilis sporulation is a last-resort phenotypical adaptation in response to starvation. The regulatory network underlying this developmental pathway has been studied extensively. However, how sporulation initiation is concerted in relation to the environmental nutrient availability is poorly understood. In a fed-batch fermentation set-up, in which sporulation of ultraviolet (UV)-mutagenized B. subtilis is repeatedly triggered by periods of starvation, fitter strains with mutated tagE evolved. These mutants display altered timing of phenotypical differentiation. The substrate for the wall teichoic acid (WTA)-modifying enzyme TagE, UDP-glucose, has recently been shown to be an intracellular proxy for nutrient availability, and influences the timing of cell division. Here we suggest that UDP-glucose also influences timing of cellular differentiation.     

Differential Stabilities of Phosphorylated Response Regulator Domains Reflect Functional Roles of the Yeast Osmoregulatory SLN1 and SSK1 Proteins

Osmoregulation in Saccharomyces cerevisiae involves a multistep phosphorelay system requiring three proteins, SLN1, YPD1, and SSK1, that are related to bacterial two-component signaling proteins, in particular, those involved in regulating sporulation in Bacillus subtilis and anaerobic respiration in Escherichia coli. The SLN1-YPD1-SSK1 phosphorelay regulates a downstream mitogen-activated protein kinase cascade which ultimately controls the concentration of glycerol within the cell under hyperosmotic stress conditions. The C-terminal response regulator domains of SLN1 and SSK1 and full-length YPD1 have been overexpressed and purified from E. coli. A heterologous system consisting of acetyl phosphate, the bacterial chemotaxis response regulator CheY, and YPD1 has been developed as an efficient means of phosphorylating SLN1 and SSK1 in vitro. The homologous regulatory domains of SLN1 and SSK1 exhibit remarkably different phosphorylated half-lives, a finding that provides insight into the distinct roles that these phosphorylation-dependent regulatory domains play in the yeast osmosensory signal transduction pathway.     

The relationship of serine protease activity to RNA polymerase modification and sporulation in Bacillus subtilis

The isolation and properties of a single site temperature sensitive protease mutant of Bacillus subtilis are described. Numerous criteria suggest that the mutation resides in the structural gene coding for a basic serine protease. The mutation has been mapped between aroD and lys-1 on the Bacillus subtilis chromosome. This protease exists as an intracellular and extracellular enzyme. The mutant cells are temperature sensitive for sporulation, antibiotic production, and the sporulation-specific alteration in DNA-dependent RNA polymerase β subunit. Several types of evidence indicate a direct involvement of this enzyme in a limited proteolytic cleavage of vegetative RNA polymerase β subunit, which produces the lower molecular weight β subunit found in sporulating cells. The derangement in this process is sufficient to account for the stoppage of sporulation at stage 0 when the mutant cells are grown at the non-permissive temperature.     

Cell cycle and sporulation in Bacillus subtilis

Recent work on cell division and chromosome orientation and partitioning in Bacillus subtilis has provided insights into cell cycle regulation during growth and development. The cell cycle is an integral part of development and entrance into sporulation is modulated by signals that transmit the status of DNA integrity, chromosome replication and segregation. In addition, B. subtilis modifies cell division and DNA segregation to establish cell-type-specific gene expression during sporulation.     

Insight into the sporulation phosphorelay: Crystal structure of the sensor domain of Bacillus subtilis histidine kinase,KinD

The Bacillus subtilis KinD signal‐transducing histidine kinase is a part of the sporulation phosphorelay known to regulate important developmental decisions such as sporulation and biofilm formation. We have determined crystal structures of the extracytoplasmic sensing domain of KinD, which was copurified and crystallized with a pyruvate ligand. The structure of a ligand‐binding site mutant was also determined; it was copurified and crystallized with an acetate ligand. The structure of the KinD extracytoplasmic segment is similar to that of several other sensing domains of signal transduction proteins and is composed of tandem Per‐Arnt‐Sim (PAS)‐like domains. The KinD ligand‐binding site is located on the membrane distal PAS‐like domain and appears to be highly selective; a single mutation, R131A, abolishes pyruvate binding and the mutant binds acetate instead. Differential scanning fluorimetry, using a variety of monocarboxylic and dicarboxylic acids, identified pyruvate, propionate, and butyrate but not lactate, acetate, or malate as KinD ligands. A recent report found that malate induces biofilm formation in a KinD‐dependent manner. It was suggested that malate might induce a metabolic shift and increased secretion of the KinD ligand of unknown identity. The structure and binding assays now suggests that this ligand is pyruvate and/or other small monocarboxylic acids. In summary, this study gives a first insight into the identity of a molecular ligand for one of the five phosphorelay kinases of B. subtilis.     

Septation and chromosome segregation during sporulation in Bacillus subtilis

The early stages of sporulation in Bacillus subtilis incorporate a modified, highly asymmetric cell division. It is now clear that most, if not all, of the components of the vegetative division machinery are used also for asymmetric division. However, the machinery for chromosome segregation may differ significantly between vegetative growth and sporulation. Several interesting checkpoint mechanisms couple cell cycle events to gene expression early in sporulation. This review summarises important advances in the understanding of chromosome segregation and cell division at the onset of sporulation in B.subtilis in the past three years.     

Pleiotropic mutations affecting sporulation conditions and the syntheses of extracellular enzymes in Bacillus subtilis 168

Pleiotropic mutations of the chromosome of Bacillus subtilis 168 affecting simultaneously the levels of extracellular levansucrase and proteolytic activities are described. These mutations have been mapped at the sacU locus identified by PBS 1 mediated transduction. Several pleotropic hyperproducers and pleiotropic hypoproducers of these extracellular enzymatic activities, genotypically designated sacU^h and sacU⁻ respectively, have been isolated. sacU^h mutants are capable of sporulation in rich media or in mineral media containing amino acids in the presence of an excess of glucose in both cases; under these conditions the sporulation of the wild type strain 168 is inhibited. One pleiotropic mutation conferring hyperproduction of levansucrase and proteolytic activities was mapped at the sacQ locus distant from sacU.The sacU and sacQ mutants may be affected in a not yet identified regulation mechanism which controls simultaneously the production of several extracellular enzymatic activities and the sporulation conditions of B. subtilis 168.     

Template specificity changes of DNA-dependent RNA polymerase in B. subtilis during sporulation

Previous work has indicated that loss of ability of DNA dependent RNA polymerase, from stationary phase cultures of $\text{B. subtilis}$, to transcribe phage øe DNA was a $\text{sine qua non}$ for sporulation. To ascertain if this change in template specificity was sporulation-specific, we repeated these experiments using a defined sporulation medium. The changes observed previously did not occur in the defined medium although sporulation was normal. The ability of the enzyme to transcribe other DNA templates was also examined. Similar studies were carried out using a polymerase from a rifamycin-resistant, sporulation conditional mutant. The significance of these findings with regard to the regulation of sporulation in $\text{B. subtilis}$ is discussed.     

Single-gene regulated non-spore-forming Bacillus subtilis: Construction,transcriptome responses,and applications for producing enzymes and surfactin

Bacillus subtilis, a spore-forming industrial bacterium, is widely used for production of enzymes and valuable chemicals. The spore-formation, however, always results in remarkably reduced cell-density, thereby reducing product yield. Here, we constructed different non-spore-forming B. subtilis mutants via single-gene regulation. During the three spore-forming stages: signal sensing, transduction, and sporulation, we found that deleting only a single gene of sporulation, i.e. spo0A, spoIIIE, and spoIVB, can completely block the spore generation. Interestingly, the engineered non-sporulating mutants exhibited physiological heterogeneity and distinct synthetic capabilities. The spo0A-null spore-free mutant displayed remarkably high enzyme production capacity, such as 194% enhance amylase production. However, the spoIVB-null non-spore-forming mutant was especially efficient in producing secondary metabolites, such as surfactin; its flask titer increased significantly to 16.7 g/L, with the overexpression and Leu addition strategy. Our results offer a new strategy for re-modeling B. subtilis to further improve its fermentation efficiency and application.     

Genomic determinants of sporulation in Bacilli and Clostridia: towards the minimal set of sporulation‐specific genes

Three classes of low‐G+C Gram‐positive bacteria (Firmicutes), Bacilli, Clostridia and Negativicutes, include numerous members that are capable of producing heat‐resistant endospores. Spore‐forming firmicutes include many environmentally important organisms, such as insect pathogens and cellulose‐degrading industrial strains, as well as human pathogens responsible for such diseases as anthrax, botulism, gas gangrene and tetanus. In the best‐studied model organism Bacillus subtilis, sporulation involves over 500 genes, many of which are conserved among other bacilli and clostridia. This work aimed to define the genomic requirements for sporulation through an analysis of the presence of sporulation genes in various firmicutes, including those with smaller genomes than B. subtilis. Cultivable spore‐formers were found to have genomes larger than 2300 kb and encompass over 2150 protein‐coding genes of which 60 are orthologues of genes that are apparently essential for sporulation in B. subtilis. Clostridial spore‐formers lack, among others, spoIIB, sda, spoVID and safA genes and have non‐orthologous displacements of spoIIQ and spoIVFA, suggesting substantial differences between bacilli and clostridia in the engulfment and spore coat formation steps. Many B. subtilis sporulation genes, particularly those encoding small acid‐soluble spore proteins and spore coat proteins, were found only in the family Bacillaceae, or even in a subset of Bacillus spp. Phylogenetic profiles of sporulation genes, compiled in this work, confirm the presence of a common sporulation gene core, but also illuminate the diversity of the sporulation processes within various lineages. These profiles should help further experimental studies of uncharacterized widespread sporulation genes, which would ultimately allow delineation of the minimal set(s) of sporulation‐specific genes in Bacilli and Clostridia.