To Feed or to Stick? Genomic Analysis Offers Clues for the Role of a Molecular Machine in Endospore Formers.

Sporulation in Firmicutes starts with the formation of two adjacent cells and proceeds with the engulfment of the smaller one, the forespore, by the larger one, the mother cell. This critical step involves a core set of conserved genes, some transcribed in the forespore, such as spoIIQ, and others transcribed in the mother cell, such as the eight-gene spoIIIA operon. A model has been proposed in which the SpoIIIA and the SpoIIQ proteins form a channel connecting the mother cell and the forespore, playing the role of a secretion apparatus allowing the mother cell to nurture the fully engulfed forespore. Exploration of the genomes of Caryophanaceae and Erysipelotrichales has provided informations that are not fully congruent with data from Bacillaceae or Clostridia. The differences observed are correlated with specific physiological features, and alternate, not mutually exclusive views of the function of the SpoIIIA-SpoIIQ complex are presented.

CsfG, a sporulation-specific, small non-coding RNA highly conserved in endospore formers.

Endospore formation is a characteristic shared by some Bacilli and Clostridia that involves the creation of two cell types, the forespore and the mother cell. Hundreds of protein-encoding genes have been shown to be transcribed in a cell-specific fashion during this developmental process in Bacillus subtilis. We have used a phylogenetic profiling procedure to identify clusters of B. subtilis coding and non-coding sequences that co-occur in other endospore formers. One such cluster shows a strong bias for sporulation-related genes (42 % among 156 genes) and is enriched in potential non-coding RNAs. We have studied one RNA candidate, encoded in the ylbG-ylbH interval. In vivo analysis using a transcriptional fusion to the Escherichia coli lacZ gene demonstrates that this region of the chromosome contains a gene, csfG, encoding a 147-nucleotide RNA that is transcribed only during sporulation, specifically in the forespore. csfG is present in many endospore formers, mostly Bacilli and some Clostridia, whereas it is absent from bacteria that do not produce endospores. All CsfG RNAs contain a strongly conserved, pyrimidine-rich, central motif that overlaps a potential stem-loop structure. The remarkable conservation of this sequence in widely divergent bacteria suggests that it plays a conserved physiological role, presumably by interacting with an unidentified target in the forespore, where it contributes to the acquisition of the spore properties.

Do mycobacteria produce endospores?

The genus Mycobacterium, which is a member of the high G+C group of Gram-positive bacteria, includes important pathogens, such as M. tuberculosis and M. leprae. A recent publication in PNAS reported that M. marinum and M. bovis bacillus Calmette-Guérin produce a type of spore known as an endospore, which had been observed only in the low G+C group of Gram-positive bacteria. Evidence was presented that the spores were similar to endospores in ultrastructure, in heat resistance and in the presence of dipicolinic acid. Here, we report that the genomes of Mycobacterium species and those of other high G+C Gram-positive bacteria lack orthologs of many, if not all, highly conserved genes diagnostic of endospore formation in the genomes of low G+C Gram-positive bacteria. We also failed to detect the presence of endospores by light microscopy or by testing for heat-resistant colony-forming units in aged cultures of M. marinum. Finally, we failed to recover heat-resistant colony-forming units from frogs chronically infected with M. marinum. We conclude that it is unlikely that Mycobacterium is capable of endospore formation.

Genetic dissection of an inhibitor of the sporulation sigma factor sigma(G).

Sporulation in Bacillus subtilis is controlled by a cascade of four sigma factors that are held into inactive form until the proper stage of development. The Gin protein, encoded by csfB, is able to strongly inhibit the activity of one of these factors, sigma(G), in vivo. The csfB gene is present in a large number of endospore formers, but the various Gin orthologues show little conservation, in striking contrast to their sigma(G) counterparts. We have carried out a mutagenesis analysis of the Gin protein in order to understand its inhibitory properties. By measuring sigma(G) inhibition in the presence of Gin in vivo, assessing Gin ability to bind sigma(G) in a yeast two-hybrid assay, and quantifying Gin-sigma(G) interaction in B. subtilis, we have identified specific residues that play an essential role in binding sigma(G) or in preventing sigma(G) transcriptional activity. Two cysteine pairs, conserved in all Gin orthologues, are essential for Gin activity. Mutations in the first pair are partially complemented by mutations in the second pair, suggesting that Gin exists in oligomeric form, at least as a dimer. Dimerisation is consistent with our in vitro analysis of a purified Gin recombinant protein, which shows that Gin contains 0.5 zinc atom per monomer. Altogether, these results indicate that the conserved cysteines play a structural role, whereas another less conserved region of the protein is involved in interacting with sigma(G). Interestingly, some mutants have kept most of their ability to bind sigma(G) but are completely unable to inhibit sigma(G) transcriptional activity, raising the possibility that Gin might act by a mechanism more complex than just sequestration of sigma(G).

Lysine represses transcription of the Escherichia coli dapB gene by preventing its activation by the ArgP activator.

The Escherichia coli dapB gene encodes one of the enzymes of the biosynthetic pathway leading to lysine and its immediate precursor, diaminopimelate. Expression of dapB is repressed by lysine, but no trans-acting regulator has been identified so far. Our analysis of the dapB regulatory region shows that sequences located in the -81/-118 interval upstream of the transcription start site are essential for full expression of dapB, as well as for lysine repression. Screening a genomic library for a gene that could alleviate lysine repression when present in multicopy led to the recovery of argP, a gene encoding an activating protein of the LysR-type family, known to use lysine as an effector. An argP null mutation strongly decreases dapB transcription that becomes insensitive to lysine. Purified His(6)-tagged ArgP protein binds with an apparent K(d) of 35 nM to the dapB promoter in a gel retardation assay, provided that sequences up to -103 are present. In the presence of L-lysine and L-arginine, the binding of ArgP to dapB is partly relieved. These results fit with a model in which ArgP contributes to enhanced transcription of dapB when lysine becomes limiting.

How the early sporulation sigma factor sigmaF delays the switch to late development in Bacillus subtilis.

Sporulation in Bacillus subtilis is a primitive differentiation process involving two cell types, the forespore and the mother cell. Each cell implements two successive transcription programmes controlled by specific sigma factors. We report that activity of sigma(G), the late forespore sigma factor, is kept in check by Gin, the product of csfB, a gene controlled by sigma(F), the early forespore sigma factor. Gin abolishes sigma(G) transcriptional activity when sigma(G) is artificially synthesized during growth, but has no effect on sigma(F). Gin interacts strongly with sigma(G) but not with sigma(F) in a yeast two-hybrid experiment. The absence of Gin allows sigma(G) to be active during sporulation independently of the mother-cell development to which it is normally coupled. Premature sigma(G) activity leads to the formation of slow-germinating spores, and complete deregulation of sigma(G) synthesis is lethal when combined with gin inactivation. Gin allows sigma(F) to delay the switch to the late forespore transcription programme by preventing sigma(G) to take over before the cell has reached a critical stage of development. A similar strategy, following a completely unrelated route, is used by the mother cell.

To kill but not be killed: a delicate balance.

Before launching a missile, it is necessary to design an efficient safety net for self-protection. In this issue of Cell, Ellermeier et al. (2006) describe the mechanism underlying a biological safety net for the soil bacterium Bacillus subtilis. This bacterium protects itself from a toxic protein it secretes by upregulating an immunity protein, which it does by sequestering a transcriptional repressor at the plasma membrane.

The sigmaE regulon and the identification of additional sporulation genes in Bacillus subtilis.

We report the identification and characterization on a genome-wide basis of genes under the control of the developmental transcription factor sigma(E) in Bacillus subtilis. The sigma(E) factor governs gene expression in the larger of the two cellular compartments (the mother cell) created by polar division during the developmental process of sporulation. Using transcriptional profiling and bioinformatics we show that 253 genes (organized in 157 operons) appear to be controlled by sigma(E). Among these, 181 genes (organized in 121 operons) had not been previously described as members of this regulon. Promoters for many of the newly identified genes were located by transcription start site mapping. To assess the role of these genes in sporulation, we created null mutations in 98 of the newly identified genes and operons. Of the resulting mutants, 12 (in prkA, ybaN, yhbH, ykvV, ylbJ, ypjB, yqfC, yqfD, ytrH, ytrI, ytvI and yunB) exhibited defects in spore formation. In addition, subcellular localization studies were carried out using in-frame fusions of several of the genes to the coding sequence for GFP. A majority of the fusion proteins localized either to the membrane surrounding the developing spore or to specific layers of the spore coat, although some fusions showed a uniform distribution in the mother cell cytoplasm. Finally, we used comparative genomics to determine that 46 of the sigma(E)-controlled genes in B.subtilis were present in all of the Gram-positive endospore-forming bacteria whose genome has been sequenced, but absent from the genome of the closely related but not endospore-forming bacterium Listeria monocytogenes, thereby defining a core of conserved sporulation genes of probable common ancestral origin. Our findings set the stage for a comprehensive understanding of the contribution of a cell-specific transcription factor to development and morphogenesis.