Cell division in bacteria is carried out by about a dozen proteins which assemble at midcell and form a complex known as the divisome. To study the dynamics and temporal hierarchy of divisome assembly in Bacillus subtilis, we have examined the in vivo localization pattern of a set of division proteins fused to green fluorescent protein in germinating spores and vegetative cells. Using time series and time-lapse microscopy, we show that the FtsZ ring assembles early and concomitantly with FtsA, ZapA, and EzrA. After a time delay of at least 20% of the cell cycle, a second set of division proteins, including GpsB, FtsL, DivIB, FtsW, Pbp2B, and DivIVA, are recruited to midcell. Together, our data provide in vivo evidence for two-step assembly of the divisome. Interestingly, overproduction of FtsZ advances the temporal assembly of EzrA but not of DivIVA, suggesting that a signal different from that of FtsZ polymerization drives the assembly of late divisome proteins. Microarray analysis shows that FtsZ depletion or overexpression does not significantly alter the transcription of division genes, supporting the hypothesis that cell division in B. subtilis is mainly regulated at the posttranscriptional level.

Bacteria can become dormant or form spores when they are starved for nutrients. Here, we find that non-sporulating Bacillus subtilis cells can survive deep starvation conditions for many months. During this period, cells adopt an almost coccoid shape and become tolerant to antibiotics. Unexpectedly, these cells appear to be metabolically active and show a transcriptome profile very different from that of stationary phase cells. We show that these starved cells are not dormant but are growing and dividing, albeit with a doubling time close to 4 days. Very low nutrient levels, comparable to 10,000-fold diluted lysogeny broth (LB), are sufficient to sustain this growth. This extreme slow growth, which we propose to call ‘oligotrophic growth state’, provides an alternative strategy for B. subtilis to endure nutrient depletion and environmental stresses. Further work is warranted to test whether this state can be found in other bacterial species to survive deep starvation conditions.

The transcription error rate estimated from mistakes in end product RNAs is 10−3–10−5. We analyzed the fidelity of nascent RNAs from all actively transcribing elongation complexes (ECs) in Escherichia coli and Saccharomyces cerevisiae and found that 1–3% of all ECs in wild-type cells, and 5–7% of all ECs in cells lacking proofreading factors are, in fact, misincorporated complexes. With the exception of a number of sequence-dependent hotspots, most misincorporations are distributed relatively randomly. Misincorporation at hotspots does not appear to be stimulated by pausing. Since misincorporation leads to a strong pause of transcription due to backtracking, our findings indicate that misincorporation could be a major source of transcriptional pausing and lead to conflicts with other RNA polymerases and replication in bacteria and eukaryotes. This observation implies that physical resolution of misincorporated complexes may be the main function of the proofreading factors Gre and TFIIS. Although misincorporation mechanisms between bacteria and eukaryotes appear to be conserved, the results suggest the existence of a bacteria-specific mechanism(s) for reducing misincorporation in protein-coding regions. The links between transcription fidelity, human disease, and phenotypic variability in genetically-identical cells can be explained by the accumulation of misincorporated complexes, rather than mistakes in mature RNA.

Transcription by RNA polymerase may be interrupted by pauses caused by backtracking or misincorporation that can be resolved by the conserved bacterial Gre-factors. However, the consequences of such pausing in the living cell remain obscure. Here, we developed molecular biology and transcriptome sequencing tools in the human pathogen Streptococcus pneumoniae and provide evidence that transcription elongation is rate-limiting on highly expressed genes. Our results suggest that transcription elongation may be a highly regulated step of gene expression in S. pneumoniae. Regulation is accomplished via long-living elongation pauses and their resolution by elongation factor GreA. Interestingly, mathematical modeling indicates that long-living pauses cause queuing of RNA polymerases, which results in ‘transcription traffic jams’ on the gene and thus blocks its expression. Together, our results suggest that long-living pauses and RNA polymerase queues caused by them are a major problem on highly expressed genes and are detrimental for cell viability. The major and possibly sole function of GreA in S. pneumoniae is to prevent formation of backtracked elongation complexes.

e Bacterial cell division is a highly coordinated process that begins with the polymerization of the tubulin-like protein FtsZ at midcell. FtsZ polymerization is regulated by a set of conserved cell division proteins, including ZapA. However, a zapA mutation does not result in a clear phenotype in Bacillus subtilis. In this study, we used a synthetic-lethal screen to find genes that become essential when ZapA is mutated. Three transposon insertions were found in yvcL. The deletion of yvcL in a wild-type background had only a mild effect on growth, but a yvcL zapA double mutant is very filamentous and sick. This filamentation is caused by a strong reduction in FtsZ-ring assembly, suggesting that YvcL is involved in an early stage of cell division. YvcL is 25% identical and 50% similar to the Streptomyces coelicolor transcription factor WhiA, which induces ftsZ and is required for septation of aerial hyphae during sporulation. Using green fluorescent protein fusions, we show that YvcL localizes at the nucleoid. Surprisingly, transcriptome analyses in combination with a ChIP-on-chip assay gave no indication that YvcL functions as a transcription factor. To gain more insight into the function of YvcL, we searched for suppressors of the filamentous phenotype of a yvcL zapA double mutant. Transposon insertions in gtaB and pgcA restored normal cell division of the double mutant. The corresponding proteins have been implicated in the metabolic sensing of cell division. We conclude that YvcL (WhiA) is involved in cell division in B. subtilis through an as-yet-unknown mechanism.

Gene expression can be highly heterogeneous in isogenic cell populations. An extreme type of heterogeneity is the so-called bistable or bimodal expression, whereby a cell can differentiate into two alternative expression states. Stochastic fluctuations of protein levels, also referred to as noise, provide the necessary source of heterogeneity that must be amplified by specific genetic circuits in order to obtain a bimodal response. A classical model of bimodal differentiation is the activation of genetic competence in Bacillus subtilis. The competence transcription factor ComK activates transcription of its own gene, and an intricate regulatory network controls the switch to competence and ensures its reversibility. However, it is noise in ComK expression that determines which cells activate the ComK autostimulatory loop and become competent for genetic transformation. Despite its important role in bimodal gene expression, noise remains difficult to investigate due to its inherent stochastic nature. We adapted an artificial autostimulatory loop that bypasses all known ComK regulators to screen for possible factors that affect noise. This led to the identification of a novel protein Kre (YkyB) that controls the bimodal regulation of ComK. Interestingly, Kre appears to modulate the induction of ComK by affecting the stability of comK mRNA. The protein influences the expression of many genes, however, Kre is only found in bacteria that contain a ComK homologue and, importantly, kre expression itself is downregulated by ComK. The evolutionary significance of this new feedback loop for the reduction of transcriptional noise in comK expression is discussed. Our findings show the importance of mRNA stability in bimodal regulation, a factor that requires more attention when studying and modelling this non-deterministic developmental mechanism.

HighlightsThe Trigger Loop is one of the major determinants of transcription fidelity.Intrinsic proofreading occurs via transcript-assisted cleavage.Factor-assisted proofreading takes place via exchange of RNAP active centres.Misincorporation is a major source of transcription pausing.Another role of fidelity is the prevention of conflicts with other cellular processes.

Five essential proteins are known to assemble at the division site of Bacillus subtilis. However, the recruitment of the FtsW homolog is still unclear. Here, we take advantage of spore germination to facilitate the depletion of essential proteins and to study the divisome assembly in the absence of previous division events. We show that, unlike what has been shown for the Escherichia coli divisome, the assembly of FtsW is interdependent with the localization of PBP 2B and FtsL, which are key components of the membrane bound division complex. Interestingly, the Z-ring appeared to disassemble upon prolonged depletion of late division proteins. Nevertheless, we could restore Z-ring formation and constriction by re-inducing FtsW, which suggests that the stability of the Z-ring is stimulated by the assembly of a functional division complex.