Localization of translocation complex components in Bacillus subtilis: enrichment of the signal recognition particle receptor at early sporulation septa

We here demonstrate that in Bacillus subtilis, the signal recognition particle receptor, FtsY, transiently localizes to early sporulation septa, whereas three SecYEG translocase-associated membrane proteins (SecDF, SpoIIIJ, and YqjG) are uniformly distributed. These results suggest FtsY delivers secreted proteins to SecYEG at the septum, consistent with initial septal localization of forespore membrane proteins.     

Defining the Region of Bacillus subtilis SpoIIIJ That Is Essential for Its Sporulation-Specific Function

Proteins of the YidC/OxaI/Alb3 family play a crucial role in the insertion, folding, and/or assembly of membrane proteins in prokaryotes and eukaryotes. Bacillus subtilis has two YidC-like proteins, denoted SpoIIIJ and YqjG. SpoIIIJ and YqjG are largely exchangeable in function, but SpoIIIJ has a unique role in sporulation, while YqjG stimulates competence development. To obtain more insight into the regions important for the sporulation specificity of SpoIIIJ, a series of SpoIIIJ/YqjG chimeras was constructed. These chimeras were tested for functionality during vegetative growth and for their ability to complement the sporulation defect of a spoIIIJ deletion strain. The data suggest an important role for the domain comprising transmembrane segment 2 (TMS2) and its flanking loops in sporulation specificity, with lesser contributions to specificity by TMS1 and TMS3.     

Complementary impact of paralogous Oxa1-like proteins of Bacillus subtilis on post-translocational stages in protein secretion

In mitochondria, chloroplasts, and Gram-negative eubacteria, Oxa1p(-like) proteins are critical for the biogenesis of membrane proteins. Here we show that the Gram-positive eubacterium Bacillus subtilis contains two functional Oxa1p orthologues, denoted SpoIIIJ and YqjG. The presence of either SpoIIIJ or YqjG is required for cell viability. Whereas SpoIIIJ is required for sporulation, YqjG is dispensable for this developmental process. The stability of two membrane proteins was found to be mildly affected upon SpoIIIJ limitation in the absence of YqjG. Surprisingly, the topology and stability of other membrane proteins remained unaffected under these conditions. In contrast, SpoIIIJ- and YqjG-limiting conditions resulted in a strong post-translocational defect in the stability of secretory proteins. Together, these data indicate that SpoIIIJ and YqjG of B. subtilis are involved in both membrane protein biogenesis and protein secretion. However, the reduced stability of secretory proteins seems to be the most prominent phenotype of SpoIIIJ/YqjG-depleted B. subtilis cells. In conclusion, our observations show that SpoIIIJ and YqjG have different, but overlapping functions in B. subtilis. Most importantly, it seems that different members of the Oxa1p protein family have acquired at least partly distinct, species-specific, functions that are essential for life.     

Processing of a membrane protein required for cell-to-cell signaling during endospore formation in Bacillus subtilis

Activation of the late prespore-specific RNA polymerase sigma factor sigma(G) during Bacillus subtilis sporulation coincides with completion of the engulfment process, when the prespore becomes a protoplast fully surrounded by the mother cell cytoplasm and separated from it by a double membrane system. Activation of sigma(G) also requires expression of spoIIIJ, coding for a membrane protein translocase of the YidC/Oxa1p/Alb3 family, and of the mother cell-specific spoIIIA operon. Here we present genetic and biochemical evidence indicating that SpoIIIAE, the product of one of the spoIIIA cistrons, and SpoIIIJ interact in the membrane, thereby linking the function of the spoIIIJ and spoIIIA loci in the activation of sigma(G). We also show that SpoIIIAE has a functional Sec-type signal peptide, which is cleaved during sporulation. Furthermore, mutations that reduce or eliminate processing of the SpoIIIAE signal peptide arrest sporulation following engulfment completion and prevent activation of sigma(G). SpoIIIJ-type proteins can function in cooperation with or independently of the Sec system. In one model, SpoIIIJ interacts with SpoIIIAE in the context of the Sec translocon to promote its correct localization and/or topology in the membrane, so that it can signal the activation of sigma(G) following engulfment completion.     

Bacillus subtilis YqjG is required for genetic competence development

Members of the evolutionary conserved Oxa1/Alb3/YidC family have been shown to play an important role in membrane protein insertion, folding and/or assembly. Bacillus subtilis contains two YidC-like proteins, denoted as SpoIIIJ and YqjG. SpoIIIJ and YqjG are largely exchangeable, but SpoIIIJ is essential for spore formation and YqjG cannot complement this activity. To elucidate the role of YqjG, we determined the membrane proteome and functional aspects of B. subtilis cells devoid of SpoIIIJ, YqjG or both. The data show that SpoIIIJ and YqjG have complementary functions in membrane protein insertion and assembly. The reduced levels of F(1)F(O) ATP synthase in cells devoid of both SpoIIIJ and YqjG are due to a defective assembly of the F(1)-domain onto the F(0)-domain. Importantly, for the first time, a specific function is demonstrated for YqjG in genetic competence development.     

Characterization of the parB-Like yyaA Gene of Bacillus subtilis

We have characterized the yyaA gene of Bacillus subtilis, located near the origin of chromosome replication (oriC). Its protein product is similar to the Spo0J protein, which belongs to the ParB family of chromosome- and plasmid-partitioning proteins. Insertional inactivation of the yyaA gene had no apparent effect on chromosome organization and partitioning during vegetative growth or sporulation. Subcellular localization of YyaA by immunofluorescence microscopy indicated that it colocalizes with the nucleoid, and gel retardation studies confirmed that YyaA binds relatively nonspecifically to DNA. Overexpression of yyaA caused a sporulation defect characterized by the formation of multiple septa within the cell. This phenotype indicates that YyaA may have a regulatory role at the onset of sporulation.     

The putative DNA translocase SpoIIIE is required for sporulation of the symmetrically dividing coccal species Sporosarcina ureae

The spoIIIE gene of Sporosarcina ureae encodes a 780-residue protein, showing 58% identity to the SpoIIIE protein of Bacillus subtilis, which is thought to be a DNA translocase. Expression of the S. ureae spoIIIE gene is able to restore sporulation in a B. subtilis spoIIIE mutant. Inactivation of the S. ureae spoIIIE gene blocks sporulation of S. ureae at stage III. Within the limits of detection, the sporulation division in S. ureae shows the same symmetry, or near symmetry, as the vegetative division (in contrast to the highly asymmetric location of the sporulation division for B. subtilis), and so it is inferred that SpoIIIE facilitates chromosome partitioning during sporulation, even when the division is not grossly asymmetric. It is suggested that chromosome partitioning lags behind division during sporulation but not during vegetative growth.     

Expression of a Bacillus megaterium sporulation-specific gene during sporulation of Bacillus subtilis

The gene for the Bacillus megaterium spore C protein, a sporulation-specific gene, has been transferred into Bacillus subtilis. The B. megaterium gene was expressed little, if at all, during log-phase and early-stationary-phase growth, but was expressed during sporulation with the same kinetics as and at a level similar to that of the analogous B. subtilis genes. This finding is most consistent with the regulation of this class of genes by a mechanism of positive control.     

The program of protein synthesis during sporulation in Bacillus subtilis

The program of protein synthesis was examined during sporulation in Bacillus subtilis as an index of the control of gene expression. At various stages of growth and spore formation, cells of B. subtilis were pulse-labeled with ³⁵S-methionine. Protein was extracted from the radioactively labeled bacteria and then subjected to high resolution one-dimensional and two-dimensional slab gel electrophoresis. We report that sporulating cells restricted or “turned off” the synthesis of certain polypeptides characteristic of the vegetative phase of growth. In certain cases, this “turn off” was prevented in a mutant (SpoOa-5NA) blocked at the first stage of spore formation. Sporulating bacteria also elaborated new polypeptide species that could not be detected in vegetatively growing cells or in cells of the asporogenous mutant SpoOa-5NA in sporulation medium. The synthesis of these sporulation-specific proteins was “turned on” in a temporally defined sequence throughout the period of spore formation. Spore coat protein, for example, was first synthesized at 4 hr after the onset of sporulation, the time at which refractile prespores appeared. Certain sporulation-specific polypeptides including the coat protein were among the most actively produced polypeptides in sporulating cells.     

A novel pathway of intercellular signalling in Bacillus subtilis involves a protein with similarity to a component of type III secretion channels

During spore formation in Bacillus subtilis, sigma(E)-directed gene expression in the mother-cell compartment of the sporangium triggers the activation of sigma(G) in the forespore by a pathway of intercellular signalling that is composed of multiple proteins of unknown function. Here, we confirm that the vegetative protein SpoIIIJ, the forespore protein SpoIIQ and eight membrane proteins (SpoIIIAA through SpoIIIAH) produced in the mother cell under the control of sigma(E) are ordinarily required for intercellular signalling. In contrast, an anti-sigma(G) factor previously implicated in the pathway is shown to be dispensable. We also present evidence suggesting that SpoIIIJ is a membrane protein translocase that facilitates the insertion of SpoIIIAE into the membrane. In addition, we report the isolation of a mutation that partially bypasses the requirement for SpoIIIJ and for SpoIIIAA through SpoIIIAG, but not for SpoIIIAH or SpoIIQ, in the activation of sigma(G). We therefore propose that under certain genetic conditions, SpoIIIAH and SpoIIQ can constitute a minimal pathway for the activation of sigma(G). Finally, based on the similarity of SpoIIIAH to a component of type III secretion systems, we speculate that signalling is mediated by a channel that links the mother cell to the forespore.     

Recent progress in Bacillus subtilis sporulation

The Gram-positive bacterium Bacillus subtilis can initiate the process of sporulation under conditions of nutrient limitation. Here, we review some of the last 5?years of work in this area, with a particular focus on the decision to initiate sporulation, DNA translocation, cell-cell communication, protein localization and spore morphogenesis. The progress we describe has implications not only just for the study of sporulation but also for other biological systems where homologs of sporulation-specific proteins are involved in vegetative growth.     

Mutagenesis and mapping of the gene for a sporulation-specific penicillin-binding protein in Bacillus subtilis.

Penicillin-binding protein (PBP) 5* is produced by Bacillus subtilis only during sporulation and is believed to be required for synthesis of the peptidoglycan-like cortex layer of the spore. The structural gene (dacB) for PBP 5* was insertionally mutagenized by integration of a plasmid bearing an internal fragment of the gene, and the phenotype of the null mutant was characterized. The mutant had no apparent vegetative growth or germination defect, but it produced extremely heat-sensitive spores. This property is consistent with a defect in the amount or assembly of the cortex and supports the hypothesis that PBP 5* is required for synthesis of this structure. Analysis of the progeny after spontaneous excision of the integrated plasmid led to the conclusion that expression of the dacB gene was required only in the mother cell compartment during sporulation, which is also consistent with a role for PBP 5* in cortex synthesis and with its location in the outer forespore membrane. Genetic mapping located dacB midway between aroC (206 degrees) and lys (210 degrees) on the B. subtilis chromosome. This is a region where there are no other known spo, ger, or PBP genes. In related studies, we found that a null mutant of dacA, the structural gene for vegetative PBP 5, produced normal heat-resistant spores, which suggests that this PBP is not essential for cortex synthesis. In addition, a candidate for another sporulation-specific PBP was revealed on gels at approximately the same position as PBP 5*. The two PBPs could be distinguished by immunoassays.     

Requirement for the cell division protein DivIB in polar cell division and engulfment during sporulation in Bacillus subtilis

During spore formation in Bacillus subtilis, cell division occurs at the cell pole and is believed to require essentially the same division machinery as vegetative division. Intriguingly, although the cell division protein DivIB is not required for vegetative division at low temperatures, it is essential for efficient sporulation under these conditions. We show here that at low temperatures in the absence of DivIB, formation of the polar septum during sporulation is delayed and less efficient. Furthermore, the polar septa that are complete are abnormally thick, containing more peptidoglycan than a normal polar septum. These results show that DivIB is specifically required for the efficient and correct formation of a polar septum. This suggests that DivIB is required for the modification of sporulation septal peptidoglycan, raising the possibility that DivIB either regulates hydrolysis of polar septal peptidoglycan or is a hydrolase itself. We also show that, despite the significant number of completed polar septa that form in this mutant, it is unable to undergo engulfment. Instead, hydrolysis of the peptidoglycan within the polar septum, which occurs during the early stages of engulfment, is incomplete, producing a similar phenotype to that of mutants defective in the production of sporulation-specific septal peptidoglycan hydrolases. We propose a role for DivIB in sporulation-specific peptidoglycan remodelling or its regulation during polar septation and engulfment.     

SmeA, a small membrane protein with multiple functions in Streptomyces sporulation including targeting of a SpoIIIE/FtsK-like protein to cell division septa

Sporulation in aerial hyphae of Streptomyces coelicolor involves profound changes in regulation of fundamental morphogenetic and cell cycle processes to convert the filamentous and multinucleoid cells to small unigenomic spores. Here, a novel sporulation locus consisting of smeA (encoding a small putative membrane protein) and sffA (encoding a SpoIIIE/FtsK-family protein) is characterized. Deletion of smeA-sffA gave rise to pleiotropic effects on spore maturation, and influenced the segregation of chromosomes and placement of septa during sporulation. Both smeA and sffA were expressed specifically in apical cells of sporogenic aerial hyphae simultaneously with or slightly after Z-ring assembly. The presence of smeA-like genes in streptomycete chromosomes, plasmids and transposons, often paired with a gene for a SpoIIIE/FtsK- or Tra-like protein, indicates that SmeA and SffA functions might be related to DNA transfer. During spore development SffA accumulated specifically at sporulation septa where it colocalized with FtsK. However, sffA did not show redundancy with ftsK, and SffA function appeared distinct from the DNA translocase activity displayed by FtsK during closure of sporulation septa. The septal localization of SffA was dependent on SmeA, suggesting that SmeA may act as an assembly factor for SffA and possibly other proteins required during spore maturation.     

The Streptomyces coelicolor ssgB gene is required for early stages of sporulation

ssgB was identified as a novel early sporulation gene in Streptomyces coelicolor. An ssgB deletion mutant failed to sporulate, over-produced actinorhodin, and its colonies were significantly larger than those of the parental strain, suggesting an important role for the ssgB gene product in the process of growth cessation prior to sporulation-specific cell division. This places ssgB temporally before the paralogous sporulation gene ssgA. Analysis of ssgB mutant hyphae by electron microscopy and by confocal fluorescence microscopy showed that it was defective in the initiation of sporulation, as no sporulation septa could be identified, and DNA segregation had not yet been initiated in the mutant.