CTP regulates membrane-binding activity of the nucleoid occlusion protein Noc.

ATP- and GTP-dependent molecular switches are extensively used to control functions of proteins in a wide range of biological processes. However, CTP switches are rarely reported. Here, we report that a nucleoid occlusion protein Noc is a CTPase enzyme whose membrane-binding activity is directly regulated by a CTP switch. In Bacillus subtilis, Noc nucleates on 16 bp NBS sites before associating with neighboring non-specific DNA to form large membrane-associated nucleoprotein complexes to physically occlude assembly of the cell division machinery. By in vitro reconstitution, we show that (1) CTP is required for Noc to form the NBS-dependent nucleoprotein complex, and (2) CTP binding, but not hydrolysis, switches Noc to a membrane-active state. Overall, we suggest that CTP couples membrane-binding activity of Noc to nucleoprotein complex formation to ensure productive recruitment of DNA to the bacterial cell membrane for nucleoid occlusion activity.

RefZ and Noc Act Synthetically to Prevent Aberrant Divisions during Bacillus subtilis Sporulation.

During sporulation, Bacillus subtilis undergoes an atypical cell division that requires overriding mechanisms that protect chromosomes from damage and ensure inheritance by daughter cells. Instead of assembling between segregated chromosomes at midcell, the FtsZ-ring coalesces polarly, directing division over one chromosome. The DNA-binding protein RefZ facilitates the timely assembly of polar Z-rings and partially defines the region of chromosome initially captured in the forespore. RefZ binds to motifs (RBMs) located proximal to the origin of replication (oriC). Although refZ and the RBMs are conserved across the Bacillus genus, a refZ deletion mutant sporulates with wild-type efficiency, so the functional significance of RefZ during sporulation remains unclear. To further investigate RefZ function, we performed a candidate-based screen for synthetic sporulation defects by combining ΔrefZ with deletions of genes previously implicated in FtsZ regulation and/or chromosome capture. Combining ΔrefZ with deletions of ezrA, sepF, parA, or minD did not detectably affect sporulation. In contrast, a ΔrefZ Δnoc mutant exhibited a sporulation defect, revealing a genetic interaction between RefZ and Noc. Using reporters of sporulation progression, we determined the ΔrefZ Δnoc mutant exhibited sporulation delays after Spo0A activation but prior to late sporulation, with a subset of cells failing to divide polarly or activate the first forespore-specific sigma factor, SigF. The ΔrefZ Δnoc mutant also exhibited extensive dysregulation of cell division, producing cells with extra, misplaced, or otherwise aberrant septa. Our results reveal a previously unknown epistatic relationship that suggests refZ and noc contribute synthetically to regulating cell division and supporting spore development. IMPORTANCE The DNA-binding protein RefZ and its binding sites (RBMs) are conserved in sequence and location on the chromosome across the Bacillus genus and contribute to the timing of polar FtsZ-ring assembly during sporulation. Only a small number of noncoding and nonregulatory DNA motifs are known to be conserved in chromosomal position in bacteria, suggesting there is strong selective pressure for their maintenance; however, a refZ deletion mutant sporulates efficiently, providing no clues as to their functional significance. Here, we find that in the absence of the nucleoid occlusion factor Noc, deletion of refZ results in a sporulation defect characterized by developmental delays and aberrant divisions.

Bacterial Cell Division: Nonmodels Poised to Take the Spotlight.

The last three decades have witnessed an explosion of discoveries about the mechanistic details of binary fission in model bacteria such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus. This was made possible not only by advances in microscopy that helped answer questions about cell biology but also by clever genetic manipulations that directly and easily tested specific hypotheses. More recently, research using understudied organisms, or nonmodel systems, has revealed several alternate mechanistic strategies that bacteria use to divide and propagate. In this review, we highlight new findings and compare these strategies to cell division mechanisms elucidated in model organisms.

The Division Defect of a Bacillus subtilis minD noc Double Mutant Can Be Suppressed by Spx-Dependent and Spx-Independent Mechanisms.

During growth, bacteria increase in size and divide. Division is initiated by the formation of the Z-ring, a ring-like cytoskeletal structure formed by treadmilling protofilaments of the tubulin homolog FtsZ. FtsZ localization is thought to be controlled by the Min and Noc systems, and here we explore why cell division fails at high temperature when the Min and Noc systems are simultaneously mutated. Microfluidic analysis of a minD noc double mutant indicated that FtsZ formed proto-Z-rings at periodic interchromosome locations but that the rings failed to mature and become functional. Extragenic suppressor analysis indicated that a variety of mutations restored high temperature growth to the minD noc double mutant, and while many were likely pleiotropic, others implicated the proteolysis of the transcription factor Spx. Further analysis indicated that a Spx-dependent pathway activated the expression of ZapA, a protein that primarily compensates for the absence of Noc. In addition, an Spx-independent pathway reduced the length of the cytokinetic period, perhaps by increasing divisome activity. Finally, we provide evidence of an as-yet-unidentified protein that is activated by Spx and governs the frequency of polar division and minicell formation. IMPORTANCE Bacteria must properly position the location of the cell division machinery in order to grow, divide, and ensure each daughter cell receives one copy of the chromosome. In Bacillus subtilis, cell division site selection depends on the Min and Noc systems, and while neither is individually essential, cells fail to grow at high temperature when both are mutated. Here, we show that cell division fails in the absence of Min and Noc, due not to a defect in FtsZ localization but rather to a failure in the maturation of the cell division machinery. Suppressor mutations that restored growth were selected, and while some activated the expression of ZapA via the Spx stress response pathway, others appeared to directly enhance divisome activity.

Frequency modulation of the Min-protein oscillator by nucleoid-associated factors in Escherichia coli.

In E. coli, the Min-protein oscillator, together with the nucleoid occlusion (NO), destabilizes the Z-ring assembly away from the midcell to ensure faithful septation. These two inhibitory pathways are thought to be working independently for division site placement. Even though the Min-protein oscillator has been displayed by synthetic minimal systems, it is unclear the interplays of Min proteins and compartment geometry are sufficient to bolster oscillation stability in vivo. By probing if NO plays a role in the Min oscillation, we study the oscillation frequency in the anucleate and nucleoid-perturbed cells. Surprisingly, we found that the oscillation periods of the Min-protein oscillators were seriously deviated in the anucleate and nucleoid-perturbed cells, but the oscillation frequency either went up in the anucleate or down in the nucleoid-perturbed cells. Intriguingly, enhanced stability and reduced frequency were observed in the cells expressing the NO factor SlmA higher than the native level. Our results reveal an unanticipated role of the nucleoid in modulating the frequency and stability of Min-protein system. SlmA is indicated to facilitate such modulations, potentially via directly interacting with the Min-protein system. A fresh perspective is suggested that frequency modulation of Min-protein oscillator is mediated via the act of nucleoid-associated factors.

A DNA-Binding Protein Tunes Septum Placement during Bacillus subtilis Sporulation.

Bacillus subtilis is a bacterium capable of differentiating into a spore form more resistant to environmental stress. Early in sporulation, each cell possesses two copies of a circular chromosome. A polar FtsZ ring (Z ring) directs septation over one of the chromosomes, generating two cell compartments. The smaller "forespore" compartment initially contains only 25 to 30% of one chromosome, and this transient genetic asymmetry is required for differentiation. Timely assembly of polar Z rings and precise capture of the chromosome in the forespore both require the DNA-binding protein RefZ. To mediate its role in chromosome capture, RefZ must bind to specific DNA motifs (RBMs) that localize near the poles at the time of septation. Cells artificially induced to express RefZ during vegetative growth cannot assemble Z rings, an effect that also requires DNA binding. We hypothesized that RefZ-RBM complexes mediate precise chromosome capture by modulating FtsZ function. To investigate, we isolated 10 RefZ loss-of-function (rLOF) variants unable to inhibit cell division yet still capable of binding RBMs. Sporulating cells expressing the rLOF variants in place of wild-type RefZ phenocopied a ΔrefZ mutant, suggesting that RefZ acts through an FtsZ-dependent mechanism. The crystal structure of RefZ was solved, and wild-type RefZ and the rLOF variants were further characterized. Our data suggest that RefZ's oligomerization state and specificity for the RBMs are critical determinants influencing RefZ's ability to affect FtsZ dynamics. We propose that RBM-bound RefZ complexes function as a developmentally regulated nucleoid occlusion system for fine-tuning the position of the septum relative to the chromosome during sporulation.IMPORTANCE The bacterial nucleoid forms a large, highly organized structure. Thus, in addition to storing the genetic code, the nucleoid harbors positional information that can be leveraged by DNA-binding proteins to spatially constrain cellular activities. During B. subtilis sporulation, the nucleoid undergoes reorganization, and the cell division protein FtsZ assembles polarly to direct septation over one chromosome. The TetR family protein RefZ binds DNA motifs (RBMs) localized near the poles at the time of division and is required for both timely FtsZ assembly and precise capture of DNA in the future spore compartment. Our data suggest that RefZ exploits nucleoid organization by associating with polarly localized RBMs to modulate the positioning of FtsZ relative to the chromosome during sporulation.

Nucleoid occlusion protein Noc recruits DNA to the bacterial cell membrane.

To proliferate efficiently, cells must co-ordinate division with chromosome segregation. In Bacillus subtilis, the nucleoid occlusion protein Noc binds to specific DNA sequences (NBSs) scattered around the chromosome and helps to protect genomic integrity by coupling the initiation of division to the progression of chromosome replication and segregation. However, how it inhibits division has remained unclear. Here, we demonstrate that Noc associates with the cell membrane via an N-terminal amphipathic helix, which is necessary for function. Importantly, the membrane-binding affinity of this helix is weak and requires the assembly of nucleoprotein complexes, thus establishing a mechanism for DNA-dependent activation of Noc. Furthermore, division inhibition by Noc requires recruitment of NBS DNA to the cell membrane and is dependent on its ability to bind DNA and membrane simultaneously. Indeed, Noc production in a heterologous system is sufficient for recruitment of chromosomal DNA to the membrane. Our results suggest a simple model in which the formation of large membrane-associated nucleoprotein complexes physically occludes assembly of the division machinery.

Structures of the nucleoid occlusion protein SlmA bound to DNA and the C-terminal domain of the cytoskeletal protein FtsZ.

Cell division in most prokaryotes is mediated by FtsZ, which polymerizes to create the cytokinetic Z ring. Multiple FtsZ-binding proteins regulate FtsZ polymerization to ensure the proper spatiotemporal formation of the Z ring at the division site. The DNA-binding protein SlmA binds to FtsZ and prevents Z-ring formation through the nucleoid in a process called "nucleoid occlusion" (NO). As do most FtsZ-accessory proteins, SlmA interacts with the conserved C-terminal domain (CTD) that is connected to the FtsZ core by a long, flexible linker. However, SlmA is distinct from other regulatory factors in that it must be DNA-bound to interact with the FtsZ CTD. Few structures of FtsZ regulator-CTD complexes are available, but all reveal the CTD bound as a helix. To deduce the molecular basis for the unique SlmA-DNA-FtsZ CTD regulatory interaction and provide insight into FtsZ-regulator protein complex formation, we determined structures of Escherichia coli, Vibrio cholera, and Klebsiella pneumonia SlmA-DNA-FtsZ CTD ternary complexes. Strikingly, the FtsZ CTD does not interact with SlmA as a helix but binds as an extended conformation in a narrow, surface-exposed pocket formed only in the DNA-bound state of SlmA and located at the junction between the DNA-binding and C-terminal dimer domains. Binding studies are consistent with the structure and underscore key interactions in complex formation. Combined, these data reveal the molecular basis for the SlmA-DNA-FtsZ interaction with implications for SlmA's NO function and underscore the ability of the FtsZ CTD to adopt a wide range of conformations, explaining its ability to bind diverse regulatory proteins.

E. coli Cell Cycle Machinery.

Cytokinesis in E. coli is organized by a cytoskeletal element designated the Z ring. The Z ring is formed at midcell by the coalescence of FtsZ filaments tethered to the membrane by interaction of FtsZ's conserved C-terminal peptide (CCTP) with two membrane-associated proteins, FtsA and ZipA. Although interaction between an FtsZ monomer and either of these proteins is of low affinity, high affinity is achieved through avidity - polymerization linked CCTPs interacting with the membrane tethers. The placement of the Z ring at midcell is ensured by antagonists of FtsZ polymerization that are positioned within the cell and target FtsZ filaments through the CCTP. The placement of the ring is reinforced by a protein network that extends from the terminus (Ter) region of the chromosome to the Z ring. Once the Z ring is established, additional proteins are recruited through interaction with FtsA, to form the divisome. The assembled divisome is then activated by FtsN to carry out septal peptidoglycan synthesis, with a dynamic Z ring serving as a guide for septum formation. As the septum forms, the cell wall is split by spatially regulated hydrolases and the outer membrane invaginates in step with the aid of a transenvelope complex to yield progeny cells.

Bacterial Nucleoid Occlusion: Multiple Mechanisms for Preventing Chromosome Bisection During Cell Division.

In most bacteria cell division is driven by the prokaryotic tubulin homolog, FtsZ, which forms the cytokinetic Z ring. Cell survival demands both the spatial and temporal accuracy of this process to ensure that equal progeny are produced with intact genomes. While mechanisms preventing septum formation at the cell poles have been known for decades, the means by which the bacterial nucleoid is spared from bisection during cell division, called nucleoid exclusion (NO), have only recently been deduced. The NO theory was originally posited decades ago based on the key observation that the cell division machinery appeared to be inhibited from forming near the bacterial nucleoid. However, what might drive the NO process was unclear. Within the last 10 years specific proteins have been identified as important mediators of NO. Arguably the best studied NO mechanisms are those employed by the Escherichia coli SlmA and Bacillus subtilis Noc proteins. Both proteins bind specific DNA sequences within selected chromosomal regions to act as timing devices. However, Noc and SlmA contain completely different structural folds and utilize distinct NO mechanisms. Recent studies have identified additional processes and factors that participate in preventing nucleoid septation during cell division. These combined data show multiple levels of redundancy as well as a striking diversity of mechanisms have evolved to protect cells against catastrophic bisection of the nucleoid. Here we discuss these recent findings with particular emphasis on what is known about the molecular underpinnings of specific NO machinery and processes.

Single-molecule dynamics suggest that ribosomes assemble at sites of translation in Bacillus subtilis.

Eukaryotic cells transcribe ribosomal RNA and largely assemble ribosomes in a structure called the nucleolus, where chromosomal regions containing rRNA operons are clustered. In bacteria, many rRNA operons cluster close to the origin regions that are positioned on the outer borders of nucleoids, close to polar areas, where translating 70S ribosomes are located. Because outer regions of the nucleoids contain the highest accumulation of RNA polymerase, it has been hypothesized that bacteria contain "nucleolus-like" structures. However, ribosome subunits freely diffuse through the entire cells, and could thus be assembled and matured throughout the non-compartmentalized cell. By tracking single molecules of two GTPases that play an essential role in ribosomal folding and processing in Bacillus subtilis, we show that this process takes place at sites of translation, i.e., predominantly at the cell poles. Induction of the stringent response led to a change in the population of GTPases assumed to be active in maturation, but did not abolish nucleoid occlusion of ribosomes or of GTPases. Our findings strongly support the idea of the conceptualization of nucleolus-like structures in bacteria, i.e., rRNA synthesis, ribosomal protein synthesis and subunit assembly occurring in close proximity at the cell poles, facilitating the efficiency of ribosome maturation even under conditions of transient nutrient deprivation.

Noc Corrals Migration of FtsZ Protofilaments during Cytokinesis in Bacillus subtilis.

Bacteria that divide by binary fission form FtsZ rings at the geometric midpoint of the cell between the bulk of the replicated nucleoids. In Bacillus subtilis, the DNA- and membrane-binding Noc protein is thought to participate in nucleoid occlusion by preventing FtsZ rings from forming over the chromosome. To explore the role of Noc, we used time-lapse fluorescence microscopy to monitor FtsZ and the nucleoid of cells growing in microfluidic channels. Our data show that Noc does not prevent de novo FtsZ ring formation over the chromosome nor does Noc control cell division site selection. Instead, Noc corrals FtsZ at the cytokinetic ring and reduces migration of protofilaments over the chromosome to the future site of cell division. Moreover, we show that FtsZ protofilaments travel due to a local reduction in ZapA association, and the diffuse FtsZ rings observed in the Noc mutant can be suppressed by ZapA overexpression. Thus, Noc sterically hinders FtsZ migration away from the Z-ring during cytokinesis and retains FtsZ at the postdivisional polar site for full disassembly by the Min system.IMPORTANCE In bacteria, a condensed structure of FtsZ (Z-ring) recruits cell division machinery at the midcell, and Z-ring formation is discouraged over the chromosome by a poorly understood phenomenon called nucleoid occlusion. In B. subtilis, nucleoid occlusion has been reported to be mediated, at least in part, by the DNA-membrane bridging protein, Noc. Using time-lapse fluorescence microscopy of cells growing in microchannels, we show that Noc neither protects the chromosome from proximal Z-ring formation nor determines the future site of cell division. Rather, Noc plays a corralling role by preventing protofilaments from leaving a Z-ring undergoing cytokinesis and traveling over the nucleoid.

How similar or dissimilar cells are produced by bacterial cell division?

DNA replication, segregation and cell division are vital processes and require an interplay of multiple proteins. These processes are highly conserved across bacteria yet similar or dissimilar progeny are produced after cell division. This review describes the bacterial cell division in considerable detail. This includes studies on model microorganisms which produce similar progeny such as Escherichia coli and Vibrio cholerae, and dissimilar progeny such as sporulating Bacillus subtilis, Actinobacteria, Caulobacter crescentus etc. The mechanism of symmetric and asymmetric cell division and its regulation has also been discussed.

The Nucleoid Occlusion Protein SlmA Binds to Lipid Membranes.

Protection of the chromosome from scission by the division machinery during cytokinesis is critical for bacterial survival and fitness. This is achieved by nucleoid occlusion, which, in conjunction with other mechanisms, ensures formation of the division ring at midcell. In Escherichia coli, this mechanism is mediated by SlmA, a specific DNA binding protein that antagonizes assembly of the central division protein FtsZ into a productive ring in the vicinity of the chromosome. Here, we provide evidence supporting direct interaction of SlmA with lipid membranes, tuned by its binding partners FtsZ and SlmA binding sites (SBS) on chromosomal DNA. Reconstructions in minimal membrane systems that mimic cellular environments show that SlmA binds to lipid-coated microbeads or locates at the edge of microfluidic-generated microdroplets, inside which the protein is encapsulated. DNA fragments containing SBS sequences do not seem to be recruited to the membrane by SlmA but instead compete with SlmA's ability to bind lipids. The interaction of SlmA with FtsZ modulates this behavior, ultimately triggering membrane localization of the SBS sequences alongside the two proteins. The ability of SlmA to bind lipids uncovered in this work extends the interaction network of this multivalent regulator beyond its well-known protein and nucleic acid recognition, which may have implications in the overall spatiotemporal control of division ring assembly.IMPORTANCE Successful bacterial proliferation relies on the spatial and temporal precision of cytokinesis and its regulation by systems that protect the integrity of the nucleoid. In Escherichia coli, one of these protectors is SlmA protein, which binds to specific DNA sites around the nucleoid and helps to shield the nucleoid from inappropriate bisection by the cell division septum. Here, we discovered that SlmA not only interacts with the nucleoid and septum-associated cell division proteins but also binds directly to cytomimetic lipid membranes, adding a novel putative mechanism for regulating the local activity of these cell division proteins. We find that interaction between SlmA and lipid membranes is regulated by SlmA's DNA binding sites and protein binding partners as well as chemical conditions, suggesting that the SlmA-membrane interactions are important for fine-tuning the regulation of nucleoid integrity during cytokinesis.

Diversification of DNA-Binding Specificity by Permissive and Specificity-Switching Mutations in the ParB/Noc Protein Family.

Specific interactions between proteins and DNA are essential to many biological processes. Yet, it remains unclear how the diversification in DNA-binding specificity was brought about, and the mutational paths that led to changes in specificity are unknown. Using a pair of evolutionarily related DNA-binding proteins, each with a different DNA preference (ParB [Partitioning Protein B] and Noc [Nucleoid Occlusion Factor], which both play roles in bacterial chromosome maintenance), we show that specificity is encoded by a set of four residues at the protein-DNA interface. Combining X-ray crystallography and deep mutational scanning of the interface, we suggest that permissive mutations must be introduced before specificity-switching mutations to reprogram specificity and that mutational paths to new specificity do not necessarily involve dual-specificity intermediates. Overall, our results provide insight into the possible evolutionary history of ParB and Noc and, in a broader context, might be useful for understanding the evolution of other classes of DNA-binding proteins.

Asymmetric cell division during Bacillus subtilis sporulation.

Bacillus subtilis is a rod-shaped bacterium which divides precisely at mid-cell during vegetative growth. Unlike Escherichia coli, another model organism used for studying cell division, B. subtilis can also divide asymmetrically during sporulation, the simplest cell differentiation process. The asymmetrically positioned sporulation septum serves as a morphological foundation for establishing differential gene expression in the smaller forespore and larger mother cell. Both vegetative and sporulation septation events are fine-tuned with cell cycle, and placement of both septa are highly precise. We understand in some detail how this is achieved during vegetative growth but have limited information about how the asymmetric septation site is determined during sporulation.

Spatial coordination between chromosomes and cell division proteins in Escherichia coli.

To successfully propagate, cells need to coordinate chromosomal replication and segregation with cell division to prevent formation of DNA-less cells and cells with damaged DNA. Here, we review molecular systems in Escherichia coli that are known to be involved in positioning the divisome and chromosome relative to each other. Interestingly, this well-studied micro-organism has several partially redundant mechanisms to achieve this task; none of which are essential. Some of these systems determine the localization of the divisome relative to chromosomes such as SlmA-dependent nucleoid occlusion, some localize the chromosome relative to the divisome such as DNA translocation by FtsK, and some are likely to act on both systems such as the Min system and newly described Ter linkage. Moreover, there is evidence that E. coli harbors other divisome-chromosome coordination systems in addition to those known. The review also discusses the minimal requirements of coordination between chromosomes and cell division proteins needed for cell viability. Arguments are presented that cells can propagate without any dedicated coordination between their chromosomes and cell division machinery at the expense of lowered fitness.

Localization of aggregating proteins in bacteria depends on the rate of addition.

Many proteins are observed to localize to specific subcellular regions within bacteria. Recent experiments have shown that proteins that have self-interactions that lead them to aggregate tend to localize to the poles. Theoretical modeling of the localization of aggregating protein within bacterial cell geometries shows that aggregates can spontaneously localize to the pole due to nucleoid occlusion. The resulting polar localization, whether it be to a single pole or to both was shown to depend on the rate of protein addition. Motivated by these predictions we selected a set of genes from Escherichia coli, whose protein products have been reported to localize when tagged with green fluorescent protein (GFP), and explored the dynamics of their localization. We induced protein expression from each gene at different rates and found that in all cases unipolar patterning is favored at low rates of expression whereas bipolar is favored at higher rates of expression. Our findings are consistent with the predictions of the model, suggesting that localization may be due to aggregation plus nucleoid occlusion. When we expressed GFP by itself under the same conditions, no localization was observed. These experiments highlight the potential importance of protein aggregation, nucleoid occlusion and rate of protein expression in driving polar localization of functional proteins in bacteria.

Division site positioning in bacteria: one size does not fit all.

Spatial regulation of cell division in bacteria has been a focus of research for decades. It has been well studied in two model rod-shaped organisms, Escherichia coli and Bacillus subtilis, with the general belief that division site positioning occurs as a result of the combination of two negative regulatory systems, Min and nucleoid occlusion. These systems influence division by preventing the cytokinetic Z ring from forming anywhere other than midcell. However, evidence is accumulating for the existence of additional mechanisms that are involved in controlling Z ring positioning both in these organisms and in several other bacteria. In some cases the decision of where to divide is solved by variations on a common evolutionary theme, and in others completely different proteins and mechanisms are involved. Here we review the different ways bacteria solve the problem of finding the right place to divide. It appears that a one-size-fits-all model does not apply, and that individual species have adapted a division-site positioning mechanism that best suits their lifestyle, environmental niche and mode of growth to ensure equal partitioning of DNA for survival of the next generation.