In vivo effects of sporulation kinases on mutant Spo0A proteins in Bacillus subtilis.

The phosphorylated form of the response regulator Spo0A (Spo0A~P) is required for the initiation of sporulation in Bacillus subtilis. Phosphate is transferred to Spo0A from at least four histidine kinases (KinA, KinB, KinC, and KinD) by a phosphotransfer pathway composed of Spo0F and Spo0B. Several mutations in spo0A allow initiation of sporulation in the absence of spo0F and spo0B, but the mechanisms by which these mutations allow bypass of spo0F and spo0B are not fully understood. We measured the ability of KinA, KinB, and KinC to activate sporulation of five spo0A mutants in the absence of Spo0F and Spo0B. We also determined the effect of Spo0E, a Spo0A~P-specific phosphatase, on sporulation of strains containing the spo0A mutations. Our results indicate that several of the mutations relax the specificity of Spo0A, allowing Spo0A to obtain phosphate from a broader group of phosphodonors. In the course of these experiments, we observed medium-dependent effects on the sporulation of different mutants. This led us to identify a small molecule, acetoin, that can stimulate sporulation of some spo0A mutants.

Polymer stability plays an important role in the positional regulation of FtsZ.

We conducted a series of experiments examining the effect of polymer stability on FtsZ localization dynamics in Bacillus subtilis. A loss-of-function mutation in ezrA, a putative polymer-destabilizing factor, suppresses the defects in FtsZ polymer stability associated with minCD overexpression. In addition, a mutation that is predicted to stabilize the FtsZ polymer leads to the formation of polar FtsZ rings. These data support the hypothesis that carefully balanced polymer stability is important for the assembly and localization of FtsZ during the bacterial cell cycle.

Control of a family of phosphatase regulatory genes (phr) by the alternate sigma factor sigma-H of Bacillus subtilis.

A family of 11 phosphatases can help to modulate the activity of response regulator proteins in Bacillus subtilis. Downstream of seven of the rap (phosphatase) genes are phr genes, encoding secreted peptides that function as phosphatase regulators. By using fusions to lacZ and primer extension analysis, we found that six of the seven phr genes are controlled by the alternate sigma factor sigma-H. These results expand the potential of sigma-H to contribute to the output of several response regulators by controlling expression of inhibitors of phosphatases.

Replication initiation proteins regulate a developmental checkpoint in Bacillus subtilis.

We identified a signaling pathway that prevents initiation of sporulation in Bacillus subtilis when replication initiation is impaired. We isolated mutations that allow a replication initiation mutant (dnaA) to sporulate. These mutations affect a small open reading frame, sda, that was overexpressed in replication initiation mutants and appears to be directly regulated by DnaA. Mutations in replication initiation genes inhibit the onset of sporulation by preventing activation of a transcription factor required for sporulation, Spo0A. Deletion of sda restored activation of Spo0A in replication initiation mutants. Overexpression of sda in otherwise wild-type cells inhibited activation of Spo0A and sporulation. Purified Sda inhibited a histidine kinase needed for activation of Spo0A. Our results indicate that control of sda by DnaA establishes a checkpoint that inhibits activation of Spo0A and prevents futile attempts to initiate sporulation.

Effects of replication termination mutants on chromosome partitioning in Bacillus subtilis.

Many circular genomes have replication termination systems, yet disruption of these systems does not cause an obvious defect in growth or viability. We have found that the replication termination system of Bacillus subtilis contributes to accurate chromosome partitioning. Partitioning of the terminus region requires that chromosome dimers, that have formed as a result of RecA-mediated homologous recombination, be resolved to monomers by the site-specific recombinase encoded by ripX. In addition, the chromosome must be cleared from the region of formation of the division septum. This process is facilitated by the spoIIIE gene product which is required for movement of a chromosome out of the way of the division septum during sporulation. We found that deletion of rtp, which encodes the replication termination protein, in combination with mutations in ripX or spoIIIE, led to an increase in production of anucleate cells. This increase in production of anucleate cells depended on recA, indicating that there is probably an increase in chromosome dimer formation in the absence of the replication termination system. Our results also indicate that SpoIIIE probably enhances the function of the RipX recombinase system. We also determined the subcellular location of the replication termination protein and found that it is a good marker for the position of the chromosome terminus.

Visualization of mismatch repair in bacterial cells.

We determined the localizations of mismatch repair proteins in living Bacillus subtilis cells. MutS-GFP colocalized with the chromosome in all cells and formed foci in a subset of cells. MutL-GFP formed foci in a subset of cells, and its localization was MutS dependent. The introduction of mismatches by growth in 2-aminopurine caused a replication-dependent increase in the number of cells with MutS and MutL foci. Approximately half of the MutS foci colocalized with DNA polymerase foci. We conclude that MutS is associated with the entire chromosome, poised to detect mismatches. After detection, it appears that mismatch repair foci assemble at mismatches as they emerge from the DNA polymerase and are then carried away from the replisome by continuing replication.

Control of sporulation gene expression in Bacillus subtilis by the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB).

Two chromosome partitioning proteins, Soj (ParA) and Spo0J (ParB), regulate the initiation of sporulation in Bacillus subtilis. In a spo0J null mutant, sporulation is inhibited by the action of Soj. Soj negatively regulates expression of several sporulation genes by binding to the promoter regions and inhibiting transcription. All of the genes known to be inhibited by Soj are also activated by the phosphorylated form of the transcription factor Spo0A (Spo0A approximately P). We found that, in a spo0J null mutant, Soj affected sporulation, in part, by decreasing the level of Spo0A protein. Soj negatively regulated transcription of spo0A and associated with the spo0A promoter region in vivo. Expression of spo0A from a heterologous promoter in a spo0J null mutant restored Spo0A levels and partly bypassed the sporulation and gene expression defects. Soj did not appear to significantly affect phosphorylation of Spo0A. Thus, in the absence of Spo0J, Soj inhibits sporulation and sporulation gene expression by inhibiting accumulation of the activator protein Spo0A and by acting downstream of Spo0A to inhibit gene expression directly.

Control of initiation of sporulation by replication initiation genes in Bacillus subtilis.

Initiation of spore formation in Bacillus subtilis appears to depend on initiation of DNA replication. This regulation was first identified using a temperature-sensitive mutation in dnaB. We found that mutations in the replication initiation genes dnaA and dnaD also inhibit sporulation, indicating that inhibition of sporulation is triggered by general defects in the function of replication initiation proteins.

Movement of replicating DNA through a stationary replisome.

We found that DNA is replicated at a central stationary polymerase, and each replicated region moves away from the replisome. In Bacillus subtilis, DNA polymerase is predominantly located at or near midcell. When replication was blocked in a specific chromosomal region, that region was centrally located with DNA polymerase. Upon release of the block, each copy of the duplicated region was located toward opposite cell poles, away from the central replisome. In a roughly synchronous population of cells, a region of chromosome between origin and terminus moved to the replisome prior to duplication. Thus, the polymerase at the replication forks is stationary, and the template is pulled in and released outward during duplication. We propose that B. subtilis, and probably many bacteria, harness energy released during nucleotide condensation by a stationary replisome to facilitate chromosome partitioning.

Synthetic lethal phenotypes caused by mutations affecting chromosome partitioning in Bacillus subtilis.

We investigated the genetic interactions between mutations affecting chromosome structure and partitioning in Bacillus subtilis. Loss-of-function mutations in spoIIIE (encoding a putative DNA translocase) and smc (involved in chromosome structure and partitioning) caused a synthetic lethal phenotype. We constructed a conditional mutation in smc and found that many of the spoIIIE smc double-mutant cells had a chromosome bisected by a division septum. The growth defect of the double mutant was exacerbated by a null mutation in the chromosome partitioning gene spo0J. These results suggest that mutants defective in nucleoid structure are unable to move chromosomes out of the way of the invaginating septum and that SpoIIIE is involved in repositioning these bisected chromosomes during vegetative growth.

An autoregulatory circuit affecting peptide signaling in Bacillus subtilis.

The competence and sporulation factor (CSF) of Bacillus subtilis is an extracellular pentapeptide produced from the product of phrC. CSF has at least three activities: (i) at low concentrations, it stimulates expression of genes activated by the transcription factor ComA; at higher concentrations, it (ii) inhibits expression of those same genes and (iii) stimulates sporulation. Because the activities of CSF are concentration dependent, we measured the amount of extracellular CSF produced by cells. We found that by mid-exponential phase, CSF accumulated to concentrations (1 to 5 nM) that stimulate ComA-dependent gene expression. Upon entry into stationary phase, CSF reached 50 to 100 nM, concentrations that stimulate sporulation and inhibit ComA-dependent gene expression. Transcription of phrC was found to be controlled by two promoters: P1, which precedes rapC, the gene upstream of phrC; and P2, which directs transcription of phrC only. Both RapC and CSF were found to be part of autoregulatory loops that affect transcription from P1, which we show is activated by ComA approximately P. RapC negatively regulates its own expression, presumably due to its ability to inhibit accumulation of ComA approximately P. CSF positively regulates its own expression, presumably due to its ability to inhibit RapC activity. Transcription from P2, which is controlled by the alternate sigma factor sigma(H), increased as cells entered stationary phase, contributing to the increase in extracellular CSF at this time. In addition to controlling transcription of phrC, sigmaH appears to control expression of at least one other gene required for production of CSF.

Identification and characterization of a negative regulator of FtsZ ring formation in Bacillus subtilis.

During the bacterial cell cycle, the tubulin-like cell-division protein FtsZ polymerizes into a ring structure that establishes the location of the nascent division site. We have identified a regulator of FtsZ ring formation in Bacillus subtilis. This protein, EzrA, modulates the frequency and position of FtsZ ring formation. The loss of ezrA resulted in cells with multiple FtsZ rings located at polar as well as medial sites. Moreover, the critical concentration of FtsZ required for ring formation was lower in ezrA null mutants than in wild-type cells. EzrA was associated with the cell membrane and also colocalized with FtsZ to the nascent septal site. We propose that EzrA interacts either with FtsZ or with one of its binding partners to promote depolymerization.

Control of development by altered localization of a transcription factor in B. subtilis.

In B. subtilis, the chromosome partitioning proteins Soj (ParA) and Spo0J (ParB) regulate the initiation of sporulation. Soj is a negative regulator of sporulation gene expression, and Spo0J antagonizes Soj function. Using fusions of Soj to green fluorescent protein, we found that Soj localized near the cell poles and upon entry into stationary phase oscillated from pole to pole. In the absence of Spo0J, Soj was associated predominantly with DNA. By in vivo cross-linking and immunoprecipitation, we found that Soj physically associates with developmentally regulated promoters, and this association increased in the absence of Spo0J. These results show that Soj switches localization and function depending on the chromosome partitioning protein Spo0J. We further show that mutations in the Soj ATPase domain disrupt localization and function and render Soj insensitive to regulation by Spo0J.

Localization of bacterial DNA polymerase: evidence for a factory model of replication.

Two general models have been proposed for DNA replication. In one model, DNA polymerase moves along the DNA (like a train on a track); in the other model, the polymerase is stationary (like a factory), and DNA is pulled through. To distinguish between these models, we visualized DNA polymerase of the bacterium Bacillus subtilis in living cells by the creation of a fusion protein containing the catalytic subunit (PolC) and green fluorescent protein (GFP). PolC-GFP was localized at discrete intracellular positions, predominantly at or near midcell, rather than being distributed randomly. These results suggest that the polymerase is anchored in place and thus support the model in which the DNA template moves through the polymerase.

Effect of minCD on FtsZ ring position and polar septation in Bacillus subtilis.

We examined the pattern of FtsZ localization in a Bacillus subtilis minCD mutant. When grown in minimal medium, the majority (approximately 89%) of the minCD mutant cells with an FtsZ ring had a single, medially positioned FtsZ ring. These results indicate that genes in addition to minCD function to restrict the number and position of FtsZ rings. When grown in rich medium, greater than 50% of the minCD mutant cells had multiple FtsZ rings, indicating significant differences in regulation of FtsZ ring formation based on growth medium.

Chromosome arrangement within a bacterium.

BACKGROUND: The contour length of the circular chromosome of bacteria is greater than a millimeter but must be accommodated within a cell that is only a few micrometers in length. Bacteria do not have nucleosomes and little is known about the arrangement of the chromosome inside a prokaryotic cell. RESULTS: We have investigated the arrangement of chromosomal DNA within the bacterium Bacillus subtilis by using fluorescence microscopy to visualize two sites on the chromosome simultaneously in the same cell. Indirect immunofluorescence with antibodies against the chromosome partition protein Spo0J were used to visualize the replication origin region of the chromosome. Green fluorescent protein fused to the lactose operon repressor Lacl was used to decorate tandem copies of the lactose operon operator lacO. A cassette of tandem operators was separately inserted into the chromosome near the origin (359 degrees), near the replication terminus (181 degrees), or at two points in between (90 degrees and 270 degrees). The results show that the layout of the chromosome is dynamic but is principally arranged with the origin and terminus maximally apart and the quarter points of the chromosome in between. CONCLUSIONS: The use of cytological methods to visualize two chromosomal sites in the same cell has provided a glimpse of the arrangement of a bacterial chromosome. We conclude that, to a first approximation, the folding of the bacterial chromosome is consistent with, and may preserve, the linear order of genes on the DNA.

The ins and outs of peptide signaling.

Many bacteria communicate by secreting and responding to extracellular peptides (pheromones). Some peptide pheromones act via receptors on the cell surface, which are often membrane-bound histidine protein kinases. Other peptide pheromones are transported into the cell by an oligopeptide permease and interact with intracellular receptors to modulate gene expression.

Characterization of a prokaryotic SMC protein involved in chromosome partitioning.

smc of Bacillus subtilis encodes a homolog of eukaryotic SMC proteins involved in chromosome condensation, pairing, and partitioning. A null mutation in B. subtilis smc caused a temperature-sensitive-lethal phenotype in rich medium. Under permissive conditions, the mutant had abnormal nucleoids, approximately 10% of the cells were anucleate, and assembly of foci of the chromosome partitioning protein Spo0J was altered. In combination with a null mutation in spo0J, the smc mutation caused a synthetic phenotype; cell growth was slower and approximately 25% of the cells were anucleate. Our results demonstrate that the B. subtilis Smc protein, like its eukaryotic counterpart, plays an important role in chromosome structure and partitioning.

Identification and characterization of a bacterial chromosome partitioning site.

We have identified a DNA site involved in chromosome partitioning in B. subtilis. This site was identified in vivo as the binding site for the chromosome partitioning protein Spo0J, a member of the ParB family of partitioning proteins. Spo0J is a site-specific DNA-binding protein that recognizes a 16 bp sequence found in spo0J. Allowing two mismatches, this sequence occurs ten times in the entire B. subtilis chromosome, all in the origin-proximal approximately 20%. Eight of the ten sequences are bound to Spo0J in vivo. The presence of a site on an otherwise unstable plasmid stabilized the plasmid in a Spo0J-dependent manner, demonstrating that this site, called parS, can function as a partitioning site. This site and Spo0J are conserved in a wide range of bacterial species.