Vasant K. Chary

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.

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 percent identity and 67 percent 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.

Control of the Expression and Compartmentalization of σG Activity during Sporulation of Bacillus subtilis by Regulators of σF and σE

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

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.

Blocking Chromosome Translocation during Sporulation of Bacillus subtilis Can Result in Prespore-Specific Activation of σG That Is Independent of σE and of Engulfment

Formation of spores by Bacillus subtilis is characterized by cell compartment-specific gene expression directed by four RNA polymerase sigma factors, which are activated in the order sigma(F)-sigma(E)-sigma(G)-sigma(K). Of these, sigma(G) becomes active in the prespore upon completion of engulfment of the prespore by the mother cell. Transcription of the gene encoding sigma(G), spoIIIG, is directed in the prespore by RNA polymerase containing sigma(F) but also requires the activity of sigma(E) in the mother cell. When first formed, sigma(G) is not active. Its activation requires expression of additional sigma(E)-directed genes, including the genes required for completion of engulfment. Here we report conditions in which sigma(G) becomes active in the prespore in the absence of sigma(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 sigma(E)-directed regulation appears to be to coordinate sigma(G) activation with the completion of engulfment, not to control the level of sigma(G) activity. It seems plausible that the role of sigma(E) in sigma(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 sigma(G) in the prespore is. Competition for core RNA polymerase between sigma(F) and sigma(G) is unlikely to be of major importance.

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.

Postdivisional Synthesis of the Sporosarcina ureae DNA Translocase SpoIIIE either in the Mother Cell or in the Prespore Enables Bacillus subtilis To Translocate DNA from the Mother Cell to the Prespore

The differentiation of vegetative cells of Bacillus subtilis into spores involves asymmetric cell division, which precedes complete chromosome partitioning. The DNA translocase SpoIIIE is required to translocate the origin distal 70% of the chromosome from the larger mother cell into the smaller prespore, the two cells that result from the division. We have tested the effect of altering the time and location of SpoIIIE synthesis on spore formation. We have expressed the spoIIIE homologue from Sporosarcina ureae in B. subtilis under the control of different promoters. Expression from either a weak mother cell-specific (sigma(E)) promoter or a weak prespore-specific (sigma(F)) promoter partly complemented the sporulation defect of a spoIIIE36 mutant; however, expression from a strong prespore-specific (sigma(F)) promoter did not. DNA translocation from the mother cell to the prespore was assayed using spoIIQ-lacZ inserted at thrC; transcription of spoIIQ occurs only in the prespore. Translocation of thrC::spoIIQ-lacZ into the prespore occurred efficiently when spoIIIE(Su) was expressed from the weak sigma(E)- or sigma(F)-controlled promoters but not when it was expressed from the strong sigma(F)-controlled promoter. It is speculated that the mechanism directing SpoIIIE insertion into the septum in the correct orientation may accommodate slow postseptational, prespore-specific SpoIIIE synthesis but may be swamped by strong prespore-specific synthesis.

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 σ(F) in the smaller cell, the prespore. σ(F) then directs a signal via the membrane protease SpoIIGA to activate σ(E) in the larger mother cell by processing of pro-σ(E). Here, we show that σ(E) can be activated in the prespore with little effect on sporulation efficiency, implying that complete compartmentalization of σ(E) activity is not essential for spore formation. σ(E) 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 σ(E), but not of spoIIGB, which encodes pro-σ(E). We infer that σ(E) 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 σ(E) activation to it. In addition, we find that CsfB, previously shown to inhibit σ(G), is independently inhibiting σ(E) 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 σ(G) from becoming active before completion of engulfment and helps prevent σ(E) from becoming active at all.

Contrasting Effects of σE on Compartmentalization of σF 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 sigmaF becomes active in the smaller prespore, which is followed by the activation of sigmaE in the larger mother cell. In this report, we examine the role of the mother cell-specific transcription factor sigmaE 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 sigmaF 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 sigmaE 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 sigmaF, the activation of sigmaE in the mother cell is important to prevent the activation of sigmaF in this compartment. Therefore, the activity of sigmaE can either maintain or abolish the compartmentalization of sigmaF, depending upon the genetic makeup of the strain. We conclude that sigmaE activity must be carefully regulated in order to maintain compartmentalization of gene expression during development.

Contrasting Effects of ¿ E on Compartmentalization of ¿ 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 sigmaF becomes active in the smaller prespore, which is followed by the activation of sigmaE in the larger mother cell. In this report, we examine the role of the mother cell-specific transcription factor sigmaE 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 sigmaF 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 sigmaE 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 sigmaF, the activation of sigmaE in the mother cell is important to prevent the activation of sigmaF in this compartment. Therefore, the activity of sigmaE can either maintain or abolish the compartmentalization of sigmaF, depending upon the genetic makeup of the strain. We conclude that sigmaE activity must be carefully regulated in order to maintain compartmentalization of gene expression during development.