Identification and characterization of the dif Site from Bacillus subtilis

Bacteria with circular chromosomes have evolved systems that ensure multimeric chromosomes, formed by homologous recombination between sister chromosomes during DNA replication, are resolved to monomers prior to cell division. The chromosome dimer resolution process in Escherichia coli is mediated by two tyrosine family site-specific recombinases, XerC and XerD, and requires septal localization of the division protein FtsK. The Xer recombinases act near the terminus of chromosome replication at a site known as dif (Ecdif). In Bacillus subtilis the RipX and CodV site-specific recombinases have been implicated in an analogous reaction. We present here genetic and biochemical evidence that a 28-bp sequence of DNA (Bsdif), lying 6° counterclockwise from the B. subtilis terminus of replication (172°), is the site at which RipX and CodV catalyze site-specific recombination reactions required for normal chromosome partitioning. Bsdif in vivo recombination did not require the B. subtilis FtsK homologues, SpoIIIE and YtpT. We also show that the presence or absence of the B. subtilis SPβ-bacteriophage, and in particular its yopP gene product, appears to strongly modulate the extent of the partitioning defects seen in codV strains and, to a lesser extent, those seen in ripX and dif strains

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

Inverse Conformational Selection in Lipid–Protein Binding

Interest in lipid interactions with proteins and other biomolecules is emerging not only in fundamental biochemistry but also in the field of nanobiotechnology where lipids are commonly used, for example, in carriers of mRNA vaccines. The outward-facing components of cellular membranes and lipid nanoparticles, the lipid headgroups, regulate membrane interactions with approaching substances, such as proteins, drugs, RNA, or viruses. Because lipid headgroup conformational ensembles have not been experimentally determined in physiologically relevant conditions, an essential question about their interactions with other biomolecules remains unanswered: Do headgroups exchange between a few rigid structures, or fluctuate freely across a practically continuous spectrum of conformations? Here, we combine solid-state NMR experiments and molecular dynamics simulations from the NMRlipids Project to resolve the conformational ensembles of headgroups of four key lipid types in various biologically relevant conditions. We find that lipid headgroups sample a wide range of overlapping conformations in both neutral and charged cellular membranes, and that differences in the headgroup chemistry manifest only in probability distributions of conformations. Furthermore, the analysis of 894 protein-bound lipid structures from the Protein Data Bank suggests that lipids can bind to proteins in a wide range of conformations, which are not limited by the headgroup chemistry. We propose that lipids can select a suitable headgroup conformation from the wide range available to them to fit the various binding sites in proteins. The proposed inverse conformational selection model will extend also to lipid binding to targets other than proteins, such as drugs, RNA, and viruses

Novel spoIIE Mutation That Causes Uncompartmentalized σF Activation in Bacillus subtilis

During sporulation, Bacillus subtilis undergoes an asymmetric division that results in two cells with different fates, the larger mother cell and the smaller forespore. The protein phosphatase SpoIIE, which is required for activation of the forespore-specific transcription factor σF, is also required for optimal efficiency and timing of asymmetric division. We performed a genetic screen for spoIIE mutants that were impaired in sporulation but not σF activity and isolated a strain with the mutation spoIIEV697A. The mutant exhibited a 10- to 40-fold reduction in sporulation and a sixfold reduction in asymmetric division compared to the parent. Transcription of the σF-dependent spoIIQ promoter was increased more than 10-fold and was no longer confined to the forespore. The excessive σF activity persisted even when asymmetric division was prevented. Disruption of spoIIGB did not restore asymmetric division to the spoIIEV697A mutant, indicating that the deficiency is not a consequence of predivisional activation of the mother cell-specific transcription factor σE. Deletion of the gene encoding σF (spoIIAC) restored asymmetric division; however, a mutation that dramatically reduced the number of promoters responsive to σF, spoIIAC561 (spoIIACV233 M), failed to do so. This result suggests that the block is due to expression of one of the small subset of σF-dependent genes expressed in this background or to unregulated interaction of σF with some other factor. Our results indicate that regulation of SpoIIE plays a critical role in coupling asymmetric division to σF activation in order to ensure proper spatial and temporal expression of forespore-specific genes

Compartmentalization of Gene Expression during Bacillus subtilis Spore Formation

Gene expression in members of the family Bacillaceae becomes compartmentalized after the distinctive, asymmetrically located sporulation division. It involves complete compartmentalization of the activities of sporulation-specific sigma factors, σ(F) in the prespore and then σ(E) in the mother cell, and then later, following engulfment, σ(G) in the prespore and then σ(K) in the mother cell. The coupling of the activation of σ(F) to septation and σ(G) to engulfment is clear; the mechanisms are not. The σ factors provide the bare framework of compartment-specific gene expression. Within each σ regulon are several temporal classes of genes, and for key regulators, timing is critical. There are also complex intercompartmental regulatory signals. The determinants for σ(F) regulation are assembled before septation, but activation follows septation. Reversal of the anti-σ(F) activity of SpoIIAB is critical. Only the origin-proximal 30% of a chromosome is present in the prespore when first formed; it takes ≈15 min for the rest to be transferred. This transient genetic asymmetry is important for prespore-specific σ(F) activation. Activation of σ(E) requires σ(F) activity and occurs by cleavage of a prosequence. It must occur rapidly to prevent the formation of a second septum. σ(G) is formed only in the prespore. SpoIIAB can block σ(G) activity, but SpoIIAB control does not explain why σ(G) is activated only after engulfment. There is mother cell-specific excision of an insertion element in sigK and σ(E)-directed transcription of sigK, which encodes pro-σ(K). Activation requires removal of the prosequence following a σ(G)-directed signal from the prespore

Compartmentalization of Gene Expression during Sporulation of Bacillus subtilis Is Compromised in Mutants Blocked at Stage III of Sporulation

Mutations in the spoIIIA and spoIIIJ loci disrupt the compartmentalization of gene expression during sporulation of Bacillus subtilis. The breakdown in compartmentalization is not the cause of their being blocked in spore formation. Rather, it appears to be a consequence of the engulfed prespore's being unstable

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