Patrick J. Piggot

Compartmentalization of Gene Expression during Bacillus subtilis Spore Formation

SUMMARY Gene expression in members of the family Bacillaceae becomes compartmentalized after the distinctive, asymmetrically located sporulation division. It involves complete compartmentalization of the activities of sporulation-specific sigma factors, σF in the prespore and then σE in the mother cell, and then later, following engulfment, σG in the prespore and then σK in the mother cell. The coupling of the activation of σF to septation and σG to engulfment is clear; the mechanisms are not. The σ factors provide the bare framework of compartment-specific gene expression. Within each σ regulon are several temporal classes of genes, and for key regulators, timing is critical. There are also complex intercompartmental regulatory signals. The determinants for σF regulation are assembled before septation, but activation follows septation. Reversal of the anti-σF activity of SpoIIAB is critical. Only the origin-proximal 30% of a chromosome is present in the prespore when first formed; it takes ≈15 min for the rest to be transferred. This transient genetic asymmetry is important for prespore-specific σF activation. Activation of σE requires σF activity and occurs by cleavage of a prosequence. It must occur rapidly to prevent the formation of a second septum. σG is formed only in the prespore. SpoIIAB can blockσ G activity, but SpoIIAB control does not explain why σG is activated only after engulfment. There is mother cell-specific excision of an insertion element in sigK and σE-directed transcription of sigK, which encodes pro-σK. Activation requires removal of the prosequence following aσ G-directed signal from the prespore.

Control of cell shape and elongation by the rodA gene in Bacillus subtilis.

The Escherichia coli rodA and ftsW genes and the spoVE gene of Bacillus subtilis encode membrane proteins that control peptidoglycan synthesis during cellular elongation, division and sporulation respectively. While rodA and ftsW are essential genes in E. coli, the B. subtilis spoVE gene is dispensable for growth and is only required for the synthesis of the spore cortex peptidoglycan. In this work, we report on the characterization of a B. subtilis gene, designated rodA, encoding a homologue of E. coli RodA. We found that the growth of a B. subtilis strain carrying a fusion of rodA to the IPTG‐inducible Pspac promoter is inducer dependent. Limiting concentrations of inducer caused the formation of spherical cells, which eventually lysed. An increase in the level of IPTG induced a sphere‐to‐short rod transition that re‐established viability. Higher levels of inducer restored normal cell length. Staining of the septal or polar cap peptidoglycan by a fluorescent lectin was unaffected during growth of the mutant under restrictive conditions. Our results suggest that rodA functions in maintaining the rod shape of the cell and that this function is essential for viability. In addition, RodA has an irreplaceable role in the extension of the lateral walls of the cell. Electron microscopy observations support these conclusions. The ultrastructural analysis further suggests that the growth arrest that accompanies loss of the rod shape is caused by the cells inability to construct a division septum capable of spanning the enlarged cell. RodA is similar over its entire length to members of a large protein family (SEDS, for shape, elongation, division and sporulation). Members of the SEDS family are probably present in all eubacteria that synthesize peptidoglycan as part of their cell envelope.

Partial penetrance facilitates developmental evolution in bacteria

Development normally occurs similarly in all individuals within an isogenic population, but mutations often affect the fates of individual organisms differently. This phenomenon, known as partial penetrance, has been observed in diverse developmental systems. However, it remains unclear how the underlying genetic network specifies the set of possible alternative fates and how the relative frequencies of these fates evolve. Here we identify a stochastic cell fate determination process that operates in Bacillus subtilis sporulation mutants and show how it allows genetic control of the penetrance of multiple fates. Mutations in an intercompartmental signalling process generate a set of discrete alternative fates not observed in wild-type cells, including rare formation of two viable ‘twin’ spores, rather than one within a single cell. By genetically modulating chromosome replication and septation, we can systematically tune the penetrance of each mutant fate. Furthermore, signalling and replication perturbations synergize to significantly increase the penetrance of twin sporulation. These results suggest a potential pathway for developmental evolution between monosporulation and twin sporulation through states of intermediate twin penetrance. Furthermore, time-lapse microscopy of twin sporulation in wild-type Clostridium oceanicum shows a strong resemblance to twin sporulation in these B. subtilis mutants. Together the results suggest that noise can facilitate developmental evolution by enabling the initial expression of discrete morphological traits at low penetrance, and allowing their stabilization by gradual adjustment of genetic parameters.

Construction of gusA transcriptional fusion vectors for Bacillus subtilis and their utilization for studies of spore formation

A series of gusA transcriptional fusion vectors is described for Bacillus subtilis (Bs). The series includes a vector for use with the amyE system of Shimotsu and Henner [Gene 43 (1986) 85-94], an integrative vector and vectors that provide gusA or gusA neo cassettes. The gusA fusions are compatible with lacZ fusion vectors that are widely used with Bs, and gusA and lacZ fusions are expressed at similar levels. beta-Glucuronidase (beta Glu) and beta-galactosidase (beta Gal) do not exhibit any cross-reactivity, there is very little endogenous beta Glu activity in Bs, and there is no indication of mutation to high-level expression. We have use strains containing both gusA and lacZ fusions to compare the times of expression of different genes during sporulation.

TRANSCRIPTION OF THE BACILLUS SUBTILIS SPOIIA LOCUS

The spoIIA operon encodes three genes, including the structural gene for a sporulation-induced sigma factor sigma F. We used deletion analysis of spoIIA-lacZ fusions to define the location of the spoIIA promoter. We found that sigma H-RNA polymerase transcribes spoIIA accurately in vitro and propose that sigma H directs transcription of spoIIA during sporulation.

A Bacillus subtilis morphogene cluster that includes spoVE is homologous to the mra region of Escherichia coli

It is known that there is a strong similarity in amino acid sequence between the products of the Escherichia coli morphogenes ftsW (mra region at 2 min) and rodA (mrd region at 14 min) and the Bacillus subtilis SpoVE protein which is required for spore cortex formation. We show here that the predicted amino acid sequences coded for by the genes flanking spoVE are homologous to the products of the E coli genes murD and murG, which flank ftsW, and are involved in peptidoglycan biosynthesis. During vegetative growth and early stationary phase spoVE is cotranscribed with murD and murG in the form of very long polycistronic messages originating upstream of murD. However, this transcriptional activity is shut-off soon (approximately 1 h) after the cells enter stationary phase, and spoVE is then transcribed at two times during sporulation from its own promoter(s). Insertional in vitro mutagenesis of the region revealed that although murD and murG are essential for normal vegetative growth, spoVE is only required for sporulation: spoVE null mutants display a sporulation stage V phenotype indistinguishable by light microscopy from the phenotype conferred by the spoVE85 and spoVE153 alleles that originally defined the locus.

Persistence of Streptococcus mutans in Stationary-Phase Batch Cultures and Biofilms

ABSTRACT Streptococcus mutans is a member of oral plaque biofilms and is considered the major etiological agent of dental caries. We have characterized the survival of S. mutans strain UA159 in both batch cultures and biofilms. Bacteria grown in batch cultures in a chemically defined medium, FMC, containing an excess of glucose or sucrose caused the pH to decrease to 4.0 at the entry into stationary phase, and they survived for about 3 days. Survival was extended up to 11 days when the medium contained a limiting concentration of glucose or sucrose that was depleted by the time the bacteria reached stationary phase. Sugar-limited cultures maintained a pH of 7.0 throughout stationary phase. Their survival was shortened to 3 days by the addition of exogenous lactic acid at the entry into stationary phase. Sugar starvation did not lead to comparable survival in biofilms. Although the pH remained at 7.0, bacteria could no longer be cultured from biofilms 4 days after the imposition of glucose or sucrose starvation; BacLight staining results did not agree with survival results based on culturability. In both batch cultures and biofilms, survival could be extended by the addition of 0.5% mucin to the medium. Batch survival increased to an average of 26 (±8) days, and an average of 2.7 × 105 CFU per chamber were still present in biofilms that were starved of sucrose for 12 days.

Identification of different sites of expression for spo loci by transformation of Bacillus subtilis.

Asporogenous mutants of Bacillus subtilis were rendered capable of forming heat-resistant spores by transformation with wild-type (spo+) DNA at, or near, the start of sporulation. For several mutants up to about 50% of the colonies derived from heat-resistant spores, formed as a result of the transformation, remained genetically asporogenous (spo). This was thought to indicate that the genome of the mother cell, but not that of the forespore, was transformed to spo+ and that correct expression of the spo locus in the mother cell was sufficient for spore formation. At the end of the process the mother cell was destroyed, leaving a mature heat-resistant spore that was genetically asporogenous. It is concluded that the loci spoIIID, spIVA, spoVB and spoVE are expressed in the mother cell. For one mutant more than 99% of the colonies derived from heat-resistant spores were genetically spo+. It is concluded that the locus involved, spoVA, had to be expressed in the forespore. Thus different sporulation-specific loci are expressed in the mother cell and in the forespore. The loci expressed in the mother cell are expressed in one cell type so that another cell type, the forespore, can develop into a heat-resistant spore. Other unselected donor markers could be introduced into the recipient during transformation provided high concentrations of DNA were used. The frequency of congression was the same for spo survivors as for spo+ survivors. This implies that there was no correlation between the DNA strand into which the selected spo+ and the unselected donor markers integrated.

Role of Intracellular Polysaccharide in Persistence of Streptococcus mutans

Intracellular polysaccharide (IPS) is accumulated by Streptococcus mutans when the bacteria are grown in excess sugar and can contribute toward the cariogenicity of S. mutans. Here we show that inactivation of the glgA gene (SMU1536), encoding a putative glycogen synthase, prevented accumulation of IPS. IPS is important for the persistence of S. mutans grown in batch culture with excess glucose and then starved of glucose. The IPS was largely used up within 1 day of glucose starvation, and yet survival of the parental strain was extended by at least 15 days beyond that of a glgA mutant; potentially, some feature of IPS metabolism distinct from providing nutrients is important for persistence. IPS was not needed for persistence when sucrose was the carbon source or when mucin was present.

Growth Phase Variation in Cell and Nucleoid Morphology in a Bacillus subtilis recA Mutant

The major role of RecA is thought to be in helping repair and restart stalled replication forks. During exponential growth, Bacillus subtilis recA cells exhibited few microscopically observable nucleoid defects. However, the efficiency of plating was about 12% of that of the parent strain. A substantial and additive defect in viability was also seen for addB and recF mutants, suggesting a role for the corresponding recombination paths during normal growth. Upon entry into stationary phase, a subpopulation (approximately 15%) of abnormally long cells and nucleoids developed in B. subtilis recA mutants. In addition, recA mutants showed a delay in, and a diminished capacity for, effecting prespore nucleoid condensation.