FtsK and SpoIIIE, coordinators of chromosome segregation and envelope remodeling in bacteria.

The translocation of DNA during bacterial cytokinesis is mediated by the SpoIIIE/FtsK family of proteins. These proteins ensure efficient chromosome segregation into sister cells by ATP-driven translocation of DNA and they control chromosome dimer resolution. How FtsK/SpoIIIE mediate chromosome translocation during cytokinesis in Gram-positive and Gram-negative organisms has been the subject of debate. Studies on FtsK in Escherichia coli, and recent work on SpoIIIE in Bacillus subtilis, have identified interactions between each translocase and the division machinery, supporting the idea that SpoIIIE and FtsK coordinate the final steps of cytokinesis with completion of chromosome segregation. Here we summarize and discuss the view that SpoIIIE and FtsK play similar roles in coordinating cytokinesis with chromosome segregation, during growth and differentiation.

Chromosome Segregation and Peptidoglycan Remodeling Are Coordinated at a Highly Stabilized Septal Pore to Maintain Bacterial Spore Development.

Asymmetric division, a hallmark of endospore development, generates two cells, a larger mother cell and a smaller forespore. Approximately 75% of the forespore chromosome must be translocated across the division septum into the forespore by the DNA translocase SpoIIIE. Asymmetric division also triggers cell-specific transcription, which initiates septal peptidoglycan remodeling involving synthetic and hydrolytic enzymes. How these processes are coordinated has remained a mystery. Using Bacillus subtilis, we identified factors that revealed the link between chromosome translocation and peptidoglycan remodeling. In cells lacking these factors, the asymmetric septum retracts, resulting in forespore cytoplasmic leakage and loss of DNA translocation. Importantly, these phenotypes depend on septal peptidoglycan hydrolysis. Our data support a model in which SpoIIIE is anchored at the edge of a septal pore, stabilized by newly synthesized peptidoglycan and protein-protein interactions across the septum. Together, these factors ensure coordination between chromosome translocation and septal peptidoglycan remodeling to maintain spore development.

Shaping an Endospore: Architectural Transformations During Bacillus subtilis Sporulation.

Endospore formation in Bacillus subtilis provides an ideal model system for studying development in bacteria. Sporulation studies have contributed a wealth of information about the mechanisms of cell-specific gene expression, chromosome dynamics, protein localization, and membrane remodeling, while helping to dispel the early view that bacteria lack internal organization and interesting cell biological phenomena. In this review, we focus on the architectural transformations that lead to a profound reorganization of the cellular landscape during sporulation, from two cells that lie side by side to the endospore, the unique cell within a cell structure that is a hallmark of sporulation in B. subtilis and other spore-forming Firmicutes. We discuss new insights into the mechanisms that drive morphogenesis, with special emphasis on polar septation, chromosome translocation, and the phagocytosis-like process of engulfment, and also the key experimental advances that have proven valuable in revealing the inner workings of bacterial cells.

Milestones in Bacillus subtilis sporulation research.

Endospore formation has been a rich field of research for more than a century, and has benefited from the powerful genetic tools available in Bacillus subtilis. In this review, we highlight foundational discoveries that shaped the sporulation field, from its origins to the present day, tracing a chronology that spans more than one hundred eighty years. We detail how cell-specific gene expression has been harnessed to investigate the existence and function of intercellular proteinaceous channels in sporulating cells, and we illustrate the rapid progress in our understanding of the cell biology of sporulation in recent years using the process of chromosome translocation as a storyline. Finally, we sketch general aspects of sporulation that remain largely unexplored, and that we envision will be fruitful areas of future research.

DNA Organization and Superesolved Segregation.

With single-molecule localization microscopy (SMLM) it is possible to reveal the internal composition, architecture, and dynamics of molecular machines and large cellular complexes. SMLM remains technically challenging, and frequently its implementation requires tailored experimental conditions that depend on the complexity of the subcellular structure of interest. Here, we describe two simple, robust, and high-throughput protocols to study molecular motors and machineries responsible for chromosome transport and organization in bacteria using 2D- and 3D-SMLM.

Mechanochemical coupling and bi-phasic force-velocity dependence in the ultra-fast ring ATPase SpoIIIE.

Multi-subunit ring-shaped ATPases are molecular motors that harness chemical free energy to perform vital mechanical tasks such as polypeptide translocation, DNA unwinding, and chromosome segregation. Previously we reported the intersubunit coordination and stepping behavior of the hexameric ring-shaped ATPase SpoIIIE (Liu et al., 2015). Here we use optical tweezers to characterize the motor's mechanochemistry. Analysis of the motor response to external force at various nucleotide concentrations identifies phosphate release as the likely force-generating step. Analysis of SpoIIIE pausing indicates that pauses are off-pathway events. Characterization of SpoIIIE slipping behavior reveals that individual motor subunits engage DNA upon ATP binding. Furthermore, we find that SpoIIIE's velocity exhibits an intriguing bi-phasic dependence on force. We hypothesize that this behavior is an adaptation of ultra-fast motors tasked with translocating DNA from which they must also remove DNA-bound protein roadblocks. Based on these results, we formulate a comprehensive mechanochemical model for SpoIIIE.

Single-Molecule Tracking of DNA Translocases in Bacillus subtilis Reveals Strikingly Different Dynamics of SftA, SpoIIIE, and FtsA.

Like many bacteria, Bacillus subtilis possesses two DNA translocases that affect chromosome segregation at different steps. Prior to septum closure, nonsegregated DNA is moved into opposite cell halves by SftA, while septum-entrapped DNA is rescued by SpoIIIE. We have used single-molecule fluorescence microscopy and tracking (SMT) experiments to describe the dynamics of the two different DNA translocases, the cell division protein FtsA and the glycolytic enzyme phosphofructokinase (PfkA), in real time. SMT revealed that about 30% of SftA molecules move through the cytosol, while a fraction of 70% is septum bound and static. In contrast, only 35% of FtsA molecules are static at midcell, while SpoIIIE molecules diffuse within the membrane and show no enrichment at the septum. Several lines of evidence suggest that FtsA plays a role in septal recruitment of SftA: an ftsA deletion results in a significant reduction in septal SftA recruitment and a decrease in the average dwell time of SftA molecules. FtsA can recruit SftA to the membrane in a heterologous eukaryotic system, suggesting that SftA may be partially recruited via FtsA. Therefore, SftA is a component of the division machinery, while SpoIIIE is not, and it is otherwise a freely diffusive cytosolic enzyme in vivo Our developed SMT script is a powerful technique to determine if low-abundance proteins are membrane bound or cytosolic, to detect differences in populations of complex-bound and unbound/diffusive proteins, and to visualize the subcellular localization of slow- and fast-moving molecules in live cells.IMPORTANCE DNA translocases couple the late events of chromosome segregation to cell division and thereby play an important role in the bacterial cell cycle. The proteins fall into one of two categories, integral membrane translocases or nonintegral translocases. We show that the membrane-bound translocase SpoIIIE moves slowly throughout the cell membrane in B. subtilis and does not show a clear association with the division septum, in agreement with the idea that it binds membrane-bound DNA, which can occur through cell division across nonsegregated chromosomes. In contrast, SftA behaves like a soluble protein and is recruited to the division septum as a component of the division machinery. We show that FtsA contributes to the recruitment of SftA, revealing a dual role of FtsA at the division machinery, but it is not the only factor that binds SftA. Our work represents a detailed in vivo study of DNA translocases at the single-molecule level.

Chromosome Translocation Inflates Bacillus Forespores and Impacts Cellular Morphology.

The means by which the physicochemical properties of different cellular components together determine bacterial cell shape remain poorly understood. Here, we investigate a programmed cell-shape change during Bacillus subtilis sporulation, when a rod-shaped vegetative cell is transformed to an ovoid spore. Asymmetric cell division generates a bigger mother cell and a smaller, hemispherical forespore. The septum traps the forespore chromosome, which is translocated to the forespore by SpoIIIE. Simultaneously, forespore size increases as it is reshaped into an ovoid. Using genetics, timelapse microscopy, cryo-electron tomography, and mathematical modeling, we demonstrate that forespore growth relies on membrane synthesis and SpoIIIE-mediated chromosome translocation, but not on peptidoglycan or protein synthesis. Our data suggest that the hydrated nucleoid swells and inflates the forespore, displacing ribosomes to the cell periphery, stretching septal peptidoglycan, and reshaping the forespore. Our results illustrate how simple biophysical interactions between core cellular components contribute to cellular morphology.

Isolation of a Membrane Protein Complex for Type VII Secretion in Staphylococcus aureus.

The ESAT6-like secretion system (ESS) of Staphylococcus aureus promotes effector protein transport across the bacterial envelope. Genes in the ESS cluster are required for S. aureus establishment of persistent abscess lesions and the modulation of immune responses during bloodstream infections. However, the biochemical functions of most of the ESS gene products, specifically the identity of secretion machine components, are unknown. Earlier work demonstrated that deletion of essB, which encodes a membrane protein, abolishes S. aureus ESS secretion. Loss-of-function mutations truncating the essB gene product cause dominant-negative phenotypes on ESS secretion, suggesting that EssB is a central component of the secretion machinery. To test this prediction, we purified native and affinity-tagged EssB from staphylococcal membranes via dodecyl-maltoside extraction, identifying a complex assembled from five proteins, EsaA, EssA, EssB, EssD, and EsxA. All five proteins are essential for secretion, as knockout mutations in the corresponding genes abolish ESS transport. Biochemical and bacterial two-hybrid analyses revealed a direct interaction between EssB and EsaA that, by engaging a mobile machine component, EsxA, may also recruit EssA and EssD.IMPORTANCE Type VII secretion systems support the lifestyle of Gram-positive bacteria, including important human pathogens such as Bacillus anthracis, Mycobacterium tuberculosis, and Staphylococcus aureus Genes encoding SpoIIIE-FtsK-like ATPases and WXG100-secreted products are conserved features of type VII secretion pathways; however, most of the genes in T7SS clusters are not conserved between different bacterial species. Here, we isolate a complex of proteins from the membranes of S. aureus that appears to represent the core secretion machinery, designated ESS. These results suggest that three membrane proteins, EsaA, EssB, and EssA, form a secretion complex that associates with EssC, the SpoIIIE-FtsK-like ATPase, and with EsxA, a mobile machine component and member of the WXG100 protein family.

Two-subunit DNA escort mechanism and inactive subunit bypass in an ultra-fast ring ATPase.

SpoIIIE is a homo-hexameric dsDNA translocase responsible for completing chromosome segregation in Bacillus subtilis. Here, we use a single-molecule approach to monitor SpoIIIE translocation when challenged with neutral-backbone DNA and non-hydrolyzable ATP analogs. We show that SpoIIIE makes multiple essential contacts with phosphates on the 5'→3' strand in the direction of translocation. Using DNA constructs with two neutral-backbone segments separated by a single charged base pair, we deduce that SpoIIIE's step size is 2 bp. Finally, experiments with non-hydrolyzable ATP analogs suggest that SpoIIIE can operate with non-consecutive inactive subunits. We propose a two-subunit escort translocation mechanism that is strict enough to enable SpoIIIE to track one DNA strand, yet sufficiently compliant to permit the motor to bypass inactive subunits without arrest. We speculate that such a flexible mechanism arose for motors that, like SpoIIIE, constitute functional bottlenecks where the inactivation of even a single motor can be lethal for the cell.

The conjugative DNA-transfer apparatus of Streptomyces.

Conjugation is a major route of horizontal gene transfer, an important driving force in the evolution of bacterial genomes. Since antibiotic producing streptomycetes represent a natural reservoir of antibiotic resistance genes, the Streptomyces conjugation system might have a particular role in the dissemination of the resistance genes. Streptomycetes transfer DNA in a unique process, clearly distinguished from the well-known DNA-transfer by type IV secretion systems. A single plasmid-encoded DNA-translocase, TraB, transfers a double-stranded DNA-molecule to the recipient. Elucidation of the structure, pore forming ability and DNA binding characteristics of TraB indicated that the TraB conjugation system is derived from an FtsK-like ancestor protein suggesting that Streptomyces adapted the FtsK/SpoIIIE chromosome segregation system to transfer DNA between two distinct Streptomyces cells. Following the primary transfer, a multi-protein DNA-translocation apparatus consisting of TraB and several Spd-proteins spreads the newly transferred DNA to the neighbouring mycelial compartments resulting in the rapid colonization of the recipient mycelium by the donor DNA.

Polymorphisms of FtsK/SpoIIIE protein in Mycobacterium tuberculosis complex strains may affect both protein function and host immune reaction.

FtsK/SpoIIIE family ATPases are conserved throughout bacteria and are involved in the translocation of DNA and proteins through membrane-spanning pores. Through comparison of 26 whole-genome-sequenced M. tuberculosis complex (MTBC) strains downloaded from NCBI website, we found that FtsK/SpoIIIE protein presented polymorphisms. One M. bovis strain and three BCG strains even showed the two T cell epitopes missing. Then we chose 159 clinical M. tuberculosis isolates from China, amplified gene encoding FtsK/SpoIIIE protein (Rv3871) and compared the sequences. The results showed that there are polymorphisms existed in FtsK/SpoIIIE protein among MTBC, which may affect both protein function and host immune reaction. In addition, position 1497 could be used as a good phylogenetic marker for Beijing strains.