The SMC Condensin Complex Is Required for Origin Segregation in Bacillus subtilis

SummarySMC condensin complexes play a central role in organizing and compacting chromosomes in all domains of life [1, 2]. In the bacterium Bacillus subtilis, cells lacking SMC are viable only during slow growth and display decondensed chromosomes, suggesting that SMC complexes function throughout the genome [3, 4]. Here, we show that rapid inactivation of SMC or its partner protein ScpB during fast growth leads to a failure to resolve newly replicated origins and a complete block to chromosome segregation. Importantly, the loss of origin segregation is not due to an inability to unlink precatenated sister chromosomes by Topoisomerase IV. In support of the idea that ParB-mediated recruitment of SMC complexes to the origin is important for their segregation, cells with reduced levels of SMC that lack ParB are severely impaired in origin resolution. Finally, we demonstrate that origin segregation is a task shared by the condensin complex and the parABS partitioning system. We propose that origin-localized SMC constrains adjacent DNA segments along their lengths, drawing replicated origins in on themselves and away from each other. This SMC-mediated lengthwise condensation, bolstered by the parABS system, drives origin segregation

Bacillus Subtilis SMC Complexes Juxtapose Chromosome Arms as They Travel from Origin to Terminus

Structural maintenance of chromosomes (SMC) complexes play critical roles in chromosome dynamics in virtually all organisms, but how they function remains poorly understood. In the bacterium Bacillus subtilis, SMC-condensin complexes are topologically loaded at centromeric sites adjacent to the replication origin. Here we provide evidence that these ring-shaped assemblies tether the left and right chromosome arms together while traveling from the origin to the terminus (>2 megabases) at rates >50 kilobases per minute. Condensin movement scales linearly with time, providing evidence for an active transport mechanism. These data support a model in which SMC complexes function by processively enlarging DNA loops. Loop formation followed by processive enlargement provides a mechanism by which condensin complexes compact and resolve sister chromatids in mitosis and by which cohesin generates topologically associating domains during interphase. Keywords: SMC; ParB; condensin; cohesion; loop extrusion; TADNational Institutes of Health (U.S.) (Grant GM082899

Novel Secretion Apparatus Maintains Spore Integrity and Developmental Gene Expression in Bacillus subtilis

Sporulation in Bacillus subtilis involves two cells that follow separate but coordinately regulated developmental programs. Late in sporulation, the developing spore (the forespore) resides within a mother cell. The regulation of the forespore transcription factor σG that acts at this stage has remained enigmatic. σG activity requires eight mother-cell proteins encoded in the spoIIIA operon and the forespore protein SpoIIQ. Several of the SpoIIIA proteins share similarity with components of specialized secretion systems. One of them resembles a secretion ATPase and we demonstrate that the ATPase motifs are required for σG activity. We further show that the SpoIIIA proteins and SpoIIQ reside in a multimeric complex that spans the two membranes surrounding the forespore. Finally, we have discovered that these proteins are all required to maintain forespore integrity. In their absence, the forespore develops large invaginations and collapses. Importantly, maintenance of forespore integrity does not require σG. These results support a model in which the SpoIIIA-SpoIIQ proteins form a novel secretion apparatus that allows the mother cell to nurture the forespore, thereby maintaining forespore physiology and σG activity during spore maturation

Bypass of a protein roadblock by a replicative DNA helicase

Replicative DNA helicases generally unwind DNA as a single hexamer that encircles and translocates along one strand of the duplex while excluding the complementary strand (“steric exclusion”). In contrast, large T antigen (T-ag), the replicative DNA helicase of the Simian Virus 40 (SV40), is reported to function as a pair of stacked hexamers that pumps double-stranded DNA through its central channel while laterally extruding single-stranded DNA. Here, we use single-molecule and ensemble assays to show that T-ag assembled on the SV40 origin unwinds DNA efficiently as a single hexamer that translocates on single-stranded DNA in the 3′ to 5′ direction. Unexpectedly, T-ag unwinds DNA past a DNA-protein crosslink on the translocation strand, suggesting that the T-ag ring can open to bypass bulky adducts. Together, our data underscore the profound conservation among replicative helicase mechanisms while revealing a new level of plasticity in their interactions with DNA damage

The Program of Gene Transcription for a Single Differentiating Cell Type During Sporulation in \u3cem\u3eBacillus subtilis\u3c/em\u3e

Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: σE, σK, GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The σE factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the σE regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of σK. Next, σK activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by σK while switching on a final set of 36 genes. Evidence is presented that repression and activation contribute to proper morphogenesis. The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins. The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription. Similar regulatory circuits could be a common feature of other systems of cellular differentiation

The Program of Gene Transcription for a Single Differentiating Cell Type during Sporulation in Bacillus subtilis

Asymmetric division during sporulation by Bacillus subtilis generates a mother cell that undergoes a 5-h program of differentiation. The program is governed by a hierarchical cascade consisting of the transcription factors: σ(E), σ(K), GerE, GerR, and SpoIIID. The program consists of the activation and repression of 383 genes. The σ(E) factor turns on 262 genes, including those for GerR and SpoIIID. These DNA-binding proteins downregulate almost half of the genes in the σ(E) regulon. In addition, SpoIIID turns on ten genes, including genes involved in the appearance of σ(K) (.) Next, σ(K) activates 75 additional genes, including that for GerE. This DNA-binding protein, in turn, represses half of the genes that had been activated by σ(K) while switching on a final set of 36 genes. Evidence is presented that repression and activation contribute to proper morphogenesis. The program of gene expression is driven forward by its hierarchical organization and by the repressive effects of the DNA-binding proteins. The logic of the program is that of a linked series of feed-forward loops, which generate successive pulses of gene transcription. Similar regulatory circuits could be a common feature of other systems of cellular differentiation

Reconstitution of Peptidoglycan Cross-Linking Leads to Improved Fluorescent Probes of Cell Wall Synthesis

The peptidoglycan precursor, Lipid II, produced in the model Gram-positive bacterium Bacillus subtilis differs from Lipid II found in Gram-negative bacteria such as Escherichia coli by a single amidation on the peptide side chain. How this difference affects the cross-linking activity of penicillin-binding proteins (PBPs) that assemble peptidoglycan in cells has not been investigated because B. subtilis Lipid II was not previously available. Here we report the synthesis of B. subtilis Lipid II and its use by purified B. subtilis PBP1 and E. coli PBP1A. While enzymes from both organisms assembled B. subtilis Lipid II into glycan strands, only the B. subtilis enzyme cross-linked the strands. Furthermore, B. subtilis PBP1 catalyzed the exchange of both d-amino acids and d-amino carboxamides into nascent peptidoglycan, but the E. coli enzyme only exchanged d-amino acids. We exploited these observations to design a fluorescent d-amino carboxamide probe to label B. subtilis PG in vivo and found that this probe labels the cell wall dramatically better than existing reagents

A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis

How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely suppresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development

An experimentally supported model of the Bacillus subtilis global transcriptional regulatory network

Organisms from all domains of life use gene regulation networks to control cell growth, identity, function, and responses to environmental challenges. Although accurate global regulatory models would provide critical evolutionary and functional insights, they remain incomplete, even for the best studied organisms. Efforts to build comprehensive networks are confounded by challenges including network scale, degree of connectivity, complexity of organism–environment interactions, and difficulty of estimating the activity of regulatory factors. Taking advantage of the large number of known regulatory interactions in Bacillus subtilis and two transcriptomics datasets (including one with 38 separate experiments collected specifically for this study), we use a new combination of network component analysis and model selection to simultaneously estimate transcription factor activities and learn a substantially expanded transcriptional regulatory network for this bacterium. In total, we predict 2,258 novel regulatory interactions and recall 74% of the previously known interactions. We obtained experimental support for 391 (out of 635 evaluated) novel regulatory edges (62% accuracy), thus significantly increasing our understanding of various cell processes, such as spore formation