Different responses to Spo0A-mediated suppression of the related Bacillus subtilis phages Nf and φ29

The φ29 family of phages is divided in three groups. Members of groups 1 and 2 infect the spore-forming bacterium Bacillus subtilis . Previous studies showed that group 1 phage φ29 adapts its infection strategy to the physiological state of the host. Thus, the lytic cycle of φ29 is suppressed when cells are infected during the early stages of sporulation and the infecting genome becomes trapped into the spore. A major element of this adaptive strategy is a very sensitive response to the host-encoded Spo0A protein, the key regulator for sporulation activation, which is directly responsible for suppression of φ29 development. Here we analysed if this adaptation is conserved in phage Nf belonging to group 2. The results obtained show that although Nf also possesses the alternative infection strategy, it is clearly less sensitive to Spo0A-mediated suppression than φ29. Sequence determination of the Nf genome revealed striking differences in the number of Spo0A binding site sequences. The results provide evidence that the life style of two highly related phages is distinctly tuned by differences in binding sites for a host-encoded regulatory protein, being a good example of how viruses have evolved to optimally exploit features of their host.     

Genetic analysis of spo0A and spo0C mutants of Bacillus subtilis with a phi 105 prophage merodiploid system.

An 8.0-kilobase chromosomal fragment of Bacillus subtilis which contained an intact spo0A gene was recloned onto temperate phage phi 105 from the rho 11dspo0A+-1 transducing phage. A specialized transducing phage, phi 105-dspo0A+-1, was constructed and used to transduce the spo0A12 mutant strain 1S9. A Spo+ transductant which was a single lysogen of the phi 105dspo0A+-1 transducing phage was isolated. From competent cells of this Spo+ transductant was isolated a Spo- (Spo0A) strain which was immune to phi 105. It was used to prepare a lysate of the phi 105dspo0A12 phage. Transduction of the spo0C9V recE4 strain with the phi 105dspo0A12 and phi 105dspo0A+-1 phages was carried out. The phi 105dspo0A+-1 phage gave rise to a large number of heat-resistant cells, but the phi 105dspo0A12 phage formed no heat-resistant cells. These results indicate that the spo0A12 and spo0C9V mutant genes do not complement each other in the ability to sporulate and that the spo0C9V mutation is located within the spo0A gene. Although the spo0C9V strain was completely asporogenous, the spo0C9V/spo0C9V diploid strain produced heat-resistant cells at a frequency of ca. 10(-3) in the sporulation medium. This result indicates that two copies of the spo0C9V mutant gene partially restore the ability of these cells to sporulate.     

Behavior of a temperate bacteriophage in differentiating cells of Bacillus subtilis.

During the first 6 hr of sporulation, infection of Bacillus subtilis by by phi105 wild type or the clear-plaque mutant phi105 c30 was nonproductive, but phage DNA was trapped inside developing spores. After infection with either wild-type or mutant phage at early times of sporulation (T1-T3), phage DNA entered the developing spores in a heat-stable form, which may represent integration of the phage DNA into the host chromosome. Phage DNA in carrier spores produced by infection at later times (T4-T6) was much more heat sensitive. Spore preparations containing either phi105 wild type or phi105 c30 carrier spores gave rise to a spontaneous burst of phage during outgrowth, although the fraction of carried wild-type phage that chose lysis over lysogeny at germination has not been determined. Heat induction of the thermoinducible lysogen 3610 (phi105 cts23) was also abortive during sporulation. Furthermore, induction neither prevented eventual spore formation nor resulted in the conversion of prophage DNA to the carrier state; during outgrowth, the previously induced lysogenic spores remained stable lysogens. However, if the sporulating lysogenic cells were plated immediately after induction, they did not form colonies at high efficiency, as though transfer to fresh medium allowed sufficient phage expression to kill the host.     

The bacterial chromosome segregation protein Spo0J spreads along DNA from parS nucleation sites

Regulation of chromosome inheritance is essential to ensure proper transmission of genetic information. To accomplish accurate genome segregation, cells organize their chromosomes and actively separate them prior to cytokinesis. In Bacillus subtilis the Spo0J protein is required for accurate chromosome segregation and it regulates the developmental switch from vegetative growth to sporulation. Spo0J is a DNA-binding protein that recognizes at least eight identified parS sites located near the origin of replication. As judged by fluorescence microscopy, Spo0J forms discrete foci associated with the oriC region of the chromosome throughout the cell cycle. In an attempt to determine the mechanisms utilized by Spo0J to facilitate productive chromosome segregation, we have investigated the DNA binding activity of Spo0J. In vivo we find Spo0J associates with several kilobases of DNA flanking its specific binding sites (parS) through a parS-dependent nucleation event that promotes lateral spreading of Spo0J along the chromosome. Using purified components we find that Spo0J has the ability to coat non-specific DNA substrates. These 'Spo0J domains' provide large structures near oriC that could potentially demark, organize or localize the origin region of the chromosome.     

Developmentally-Regulated Excision of the SPβ Prophage Reconstitutes a Gene Required for Spore Envelope Maturation in Bacillus subtilis

Temperate phages infect bacteria by injecting their DNA into bacterial cells, where it becomes incorporated into the host genome as a prophage. In the genome of Bacillus subtilis 168, an active prophage, SPβ, is inserted into a polysaccharide synthesis gene, spsM. Here, we show that a rearrangement occurs during sporulation to reconstitute a functional composite spsM gene by precise excision of SPβ from the chromosome. SPβ excision requires a putative site-specific recombinase, SprA, and an accessory protein, SprB. A minimized SPβ, where all the SPβ genes were deleted, except sprA and sprB, retained the SPβ excision activity during sporulation, demonstrating that sprA and sprB are necessary and sufficient for the excision. While expression of sprA was observed during vegetative growth, sprB was induced during sporulation and upon mitomycin C treatment, which triggers the phage lytic cycle. We also demonstrated that overexpression of sprB (but not of sprA) resulted in SPβ prophage excision without triggering the lytic cycle. These results suggest that sprB is the factor that controls the timing of phage excision. Furthermore, we provide evidence that spsM is essential for the addition of polysaccharides to the spore envelope. The presence of polysaccharides on the spore surface renders the spore hydrophilic in water. This property may be beneficial in allowing spores to disperse in natural environments via water flow. A similar rearrangement occurs in Bacillus amyloliquefaciens FZB42, where a SPβ-like element is excised during sporulation to reconstitute a polysaccharide synthesis gene, suggesting that this type of gene rearrangement is common in spore-forming bacteria because it can be spread by phage infection.     

Assembly of phage phi 29 genome with viral protein p6 into a compact complex.

The formation of a multimeric nucleoprotein complex by the phage phi 29 dsDNA binding protein p6 at the phi 29 DNA replication origins, leads to activation of viral DNA replication. In the present study, we have analysed protein p6-DNA complexes formed in vitro along the 19.3 kb phi 29 genome by electron microscopy and micrococcal nuclease digestion, and estimated binding parameters. Under conditions that greatly favour protein-DNA interaction, the saturated phi 29 DNA-protein p6 complex appears as a rigid, rod-like, homogeneous structure. Complex formation was analysed also by a psoralen crosslinking procedure that did not disrupt complexes. The whole phi 29 genome appears, under saturating conditions, as an irregularly spaced array of complexes approximately 200-300 bp long; however, the size of these complexes varies from approximately 2 kb to 130 bp. The minimal size of the complexes, confirmed by micrococcal nuclease digestion, probably reflects a structural requirement for stability. The values obtained for the affinity constant (K(eff) approximately 10(5) M-1) and the cooperativity parameter (omega approximately 100) indicate that the complex is highly dynamic. These results, together with the high abundance of protein p6 in infected cells, lead us to propose that protein p6-DNA complexes could have, at least at some stages, during infection, a structural role in the organization of the phi 29 genome into a nucleoid-type, compact nucleoprotein complex.     

The FtsEX ABC transporter directs cellular differentiation in Bacillus subtilis

A fundamental challenge in developmental biology is to elucidate the regulatory events that trigger cellular differentiation. Sporulation in the Gram-positive bacterium Bacillus subtilis serves as a simple experimental model system to address this challenge. The hallmark of sporulation is the formation of an asymmetrically positioned septum that divides the cell into unequally sized progeny. Here we describe the role of an ABC transporter, comprising the FtsE and FtsX proteins, in the initiation of spore formation. We discovered that in the absence of this transporter, entry into sporulation is delayed and an atypical symmetric septum is formed instead of a polar one. We show that this phenotype can be suppressed by artificially activating the master regulator of sporulation, Spo0A, or by activating the histidine kinases that function upstream of Spo0A. Our data indicate that the FtsEX transporter is one of the top components in the hierarchy of factors required to initiate sporulation, and thus it is essential for establishing proper temporal activation of the process.     

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.     

Heterologous expression of Bacillus intermedius gene of glutamyl endopeptidase in Bacillus subtilis strains defective in regulatory proteins

Expression of the gene of glutamyl endopeptidase from Bacillus intermedius (gseBi) cloned on the plasmid pV has been studied in Bacillus subtilis recombinant strains with mutations of the regulatory proteins involved in sporogenesis and spore germination. It has been established that inactivation of the regulatory protein Spo0A involved in sporulation initiation resulted in a decrease in the expression of the gseBi gene by 65% on average. A mutation in the gene of the sensor histidine kinase kinA had no effect on the biosynthesis of the enzyme. Inactivation of Ger proteins regulating bacterial spore germination resulted in a 1.5-5-fold decrease in glutamyl endopeptidase activity. It has been concluded that expression of the B. intermedius glutamyl endopeptidase gene from plasmid pV in recombinant cells of B. subtilis is under impaired control by the regulatory system of Spo0F/Spo0A phosphorelay, which participates in sporulation initiation. The regulatory Ger proteins responsible for spore germination also affect expression of the gene of this enzyme.     

Dynamic relocalization of phage phi 29 DNA during replication and the role of the viral protein p16.7

We have examined the localization of DNA replication of the Bacillus subtilis phage phi 29 by immunofluorescence. To determine where phage replication was localized within infected cells, we examined the distribution of phage replication proteins and the sites of incorporation of nucleotide analogues into phage DNA. On initiation of replication, the phage DNA localized to a single focus within the cell, nearly always towards one end of the host cell nucleoid. At later stages of the infection cycle, phage replication was found to have redistributed to multiple sites around the periphery of the nucleoid, just under the cell membrane. Towards the end of the cycle, phage DNA was once again redistributed to become located within the bulk of the nucleoid. Efficient redistribution of replicating phage DNA from the initial replication site to various sites surrounding the nucleoid was found to be dependent on the phage protein p16.7.     

Roles of phosphoinositides and of Spo14p (phospholipase D)-generated phosphatidic acid during yeast sporulation

During yeast sporulation, internal membrane synthesis ensures that each haploid nucleus is packaged into a spore. Prospore membrane formation requires Spo14p, a phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2]-stimulated phospholipase D (PLD), which hydrolyzes phosphatidylcholine (PtdCho) to phosphatidic acid (PtdOH) and choline. We found that both meiosis and spore formation also require the phosphatidylinositol (PtdIns)/PtdCho transport protein Sec14p. Specific ablation of the PtdIns transport activity of Sec14p was sufficient to impair spore formation but not meiosis. Overexpression of Pik1p, a PtdIns 4-kinase, suppressed the sec14-1 meiosis and spore formation defects; conversely, pik1-ts diploids failed to undergo meiosis and spore formation. The PtdIns(4)P 5-kinase, Mss4p, also is essential for spore formation. Use of phosphoinositide-specific GFP-PH domain reporters confirmed that PtdIns(4,5)P2 is enriched in prospore membranes. sec14, pik1, and mss4 mutants displayed decreased Spo14p PLD activity, whereas absence of Spo14p did not affect phosphoinositide levels in vivo, suggesting that formation of PtdIns(4,5)P2 is important for Spo14p activity. Spo14p-generated PtdOH appears to have an essential role in sporulation, because treatment of cells with 1-butanol, which supports Spo14p-catalyzed PtdCho breakdown but leads to production of Cho and Ptd-butanol, blocks spore formation at concentrations where the inert isomer, 2-butanol, has little effect. Thus, rather than a role for PtdOH in stimulating PtdIns(4,5)P2 formation, our findings indicate that during sporulation, Spo14p-mediated PtdOH production functions downstream of Sec14p-, Pik1p-, and Mss4p-dependent PtdIns(4,5)P2 synthesis.     

A Secreted Factor Coordinates Environmental Quality with Bacillus Development

Entry into sporulation is governed by the master regulator Spo0A. Spo0A accumulates in its active form, Spo0A-P, as cells enter stationary phase. Prior reports have shown that the acute induction of constitutively active Spo0A during exponential growth does not result in sporulation. However, a subsequent study also found that a gradual increase in Spo0A-P, mediated through artificial expression of the kinase, KinA, during exponential growth, is sufficient to trigger sporulation. We report here that sporulation via KinA induction depends on the presence of an extracellular factor or factors (FacX) that only accumulates to active levels during post-exponential growth. FacX is retained by dialysis with a cutoff smaller than 500 Dalton, can be concentrated, and is susceptible to proteinase K digestion, similar to described quorum-sensing peptides shown to be involved in promoting sporulation. However, unlike previously characterized peptides, FacX activity does not require the Opp or App oligopeptide transporter systems. In addition, FacX activity does not depend on SigH, Spo0A, or ComX. Importantly, we find that in the presence of FacX, B. subtilis can be induced to sporulate following the artificial induction of constitutively active Spo0A. These results indicate that there is no formal requirement for gradual Spo0A-P accumulation and instead support the idea that sporulation requires both sufficient levels of active Spo0A and at least one other signal or condition.