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

Blocking Chromosome Translocation during Sporulation of Bacillus subtilis Can Result in Prespore-Specific Activation of σ(G) That Is Independent of σ(E) and of Engulfment

Formation of spores by Bacillus subtilis is characterized by cell compartment-specific gene expression directed by four RNA polymerase σ factors, which are activated in the order σ(F)-σ(E)-σ(G)-σ(K). Of these, σ(G) becomes active in the prespore upon completion of engulfment of the prespore by the mother cell. Transcription of the gene encoding σ(G), spoIIIG, is directed in the prespore by RNA polymerase containing σ(F) but also requires the activity of σ(E) in the mother cell. When first formed, σ(G) is not active. Its activation requires expression of additional σ(E)-directed genes, including the genes required for completion of engulfment. Here we report conditions in which σ(G) becomes active in the prespore in the absence of σ(E) activity and of completion of engulfment. The conditions are (i) having an spoIIIE mutation, so that only the origin-proximal 30% of the chromosome is translocated into the prespore, and (ii) placing spoIIIG in an origin-proximal location on the chromosome. The main function of the σ(E)-directed regulation appears to be to coordinate σ(G) activation with the completion of engulfment, not to control the level of σ(G) activity. It seems plausible that the role of σ(E) in σ(G) activation is to reverse some inhibitory signal (or signals) in the engulfed prespore, a signal that is not present in the spoIIIE mutant background. It is not clear what the direct activator of σ(G) in the prespore is. Competition for core RNA polymerase between σ(F) and σ(G) is unlikely to be of major importance

Contrasting Effects of σ(E) on Compartmentalization of σ(F) Activity during Sporulation of Bacillus subtilis

Spore formation by Bacillus subtilis is a primitive form of development. In response to nutrient starvation and high cell density, B. subtilis divides asymmetrically, resulting in two cells with different sizes and cell fates. Immediately after division, the transcription factor σ(F) becomes active in the smaller prespore, which is followed by the activation of σ(E) in the larger mother cell. In this report, we examine the role of the mother cell-specific transcription factor σ(E) in maintaining the compartmentalization of gene expression during development. We have studied a strain with a deletion of the spoIIIE gene, encoding a DNA translocase, that exhibits uncompartmentalized σ(F) activity. We have determined that the deletion of spoIIIE alone does not substantially impact compartmentalization, but in the spoIIIE mutant, the expression of putative peptidoglycan hydrolases under the control of σ(E) in the mother cell destroys the integrity of the septum. As a consequence, small proteins can cross the septum, thereby abolishing compartmentalization. In addition, we have found that in a mutant with partially impaired control of σ(F), the activation of σ(E) in the mother cell is important to prevent the activation of σ(F) in this compartment. Therefore, the activity of σ(E) can either maintain or abolish the compartmentalization of σ(F), depending upon the genetic makeup of the strain. We conclude that σ(E) activity must be carefully regulated in order to maintain compartmentalization of gene expression during development

Expression of the σF-Directed csfB Locus Prevents Premature Appearance of σG Activity during Sporulation of Bacillus subtilis▿

During sporulation, σG becomes active in the prespore upon the completion of engulfment. We show that the inactivation of the σF-directed csfB locus resulted in premature activation of σG. CsfB exerted control distinct from but overlapping with that exerted by LonA to prevent inappropriate σG activation. The artificial induction of csfB severely compromised spore formation

Control of the Expression and Compartmentalization of σ(G) Activity during Sporulation of Bacillus subtilis by Regulators of σ(F) and σ(E)

During formation of spores by Bacillus subtilis the RNA polymerase factor σ(G) ordinarily becomes active during spore formation exclusively in the prespore upon completion of engulfment of the prespore by the mother cell. Formation and activation of σ(G) ordinarily requires prior activity of σ(F) in the prespore and σ(E) in the mother cell. Here we report that in spoIIA mutants lacking both σ(F) and the anti-sigma factor SpoIIAB and in which σ(E) is not active, σ(G) nevertheless becomes active. Further, its activity is largely confined to the mother cell. Thus, there is a switch in the location of σ(G) activity from prespore to mother cell. Factors contributing to the mother cell location are inferred to be read-through of spoIIIG, the structural gene for σ(G), from the upstream spoIIG locus and the absence of SpoIIAB, which can act in the mother cell as an anti-sigma factor to σ(G). When the spoIIIG locus was moved away from spoIIG to the distal amyE locus, σ(G) became active earlier in sporulation in spoIIA deletion mutants, and the sporulation septum was not formed, suggesting that premature σ(G) activation can block septum formation. We report a previously unrecognized control in which SpoIIGA can prevent the appearance of σ(G) activity, and pro-σ(E) (but not σ(E)) can counteract this effect of SpoIIGA. We find that in strains lacking σ(F) and SpoIIAB and engineered to produce active σ(E) in the mother cell without the need for SpoIIGA, σ(G) also becomes active in the mother cell

Loss of Compartmentalization of σE Activity Need Not Prevent Formation of Spores by Bacillus subtilis▿ †

Compartmentalization of the activities of RNA polymerase sigma factors is a hallmark of formation of spores by Bacillus subtilis. It is initiated soon after the asymmetrically located sporulation division takes place with the activation of σF in the smaller cell, the prespore. σF then directs a signal via the membrane protease SpoIIGA to activate σE in the larger mother cell by processing of pro-σE. Here, we show that σE can be activated in the prespore with little effect on sporulation efficiency, implying that complete compartmentalization of σE activity is not essential for spore formation. σE activity in the prespore can be obtained by inducing transcription in the prespore of spoIIGA or of sigE*, which encodes a constitutively active form of σE, but not of spoIIGB, which encodes pro-σE. We infer that σE compartmentalization is partially attributed to a competition between the compartments for the activation signaling protein SpoIIR. Normally, SpoIIGA is predominantly located in the mother cell and as a consequence confines σE activation to it. In addition, we find that CsfB, previously shown to inhibit σG, is independently inhibiting σE activity in the prespore. CsfB thus appears to serve a gatekeeper function in blocking the action of two sigma factors in the prespore: it prevents σG from becoming active before completion of engulfment and helps prevent σE from becoming active at all

Bex, the Bacillus subtilis Homolog of the Essential Escherichia coli GTPase Era, Is Required for Normal Cell Division and Spore Formation

The Bacillus subtilis bex gene complemented the defect in an Escherichia coli era mutant. The Bex protein showed 39% identity and 67% similarity to the E. coli Era GTPase. In contrast to era, bex was not essential in all strains. bex mutant cells were elongated and filled with diffuse nucleoid material. They grew slowly and exhibited severely impaired spore formation