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Removal of RNA/DNA hybrids for the maturation of Okazaki fragments on the lagging strand, or due to misincorporation of ribonucleotides by DNA polymerases, is essential for all types of cells. In prokaryotic cells such as Escherichia coli , DNA polymerase 1 and RNase HI are supposed to remove RNA from Okazaki fragments, but many bacteria lack HI-type RNases, such as Bacillus subtilis . Previous work has demonstrated in vitro that four proteins are able to remove RNA from RNA/DNA hybrids, but their actual contribution to DNA replication is unclear. We have studied the dynamics of DNA polymerase A (similar to Pol 1), 5'->3' exonuclease ExoR, and the two endoribonucleases RNase HII and HIII in B. subtilis using single-molecule tracking. We found that all four enzymes show a localization pattern similar to that of replicative DNA helicase. By scoring the distance of tracks to replication forks, we found that all four enzymes are enriched at DNA replication centers. After inducing UV damage, RNase HIII was even more strongly recruited to the replication forks, and PolA showed a more static behavior, indicative of longer binding events, whereas RNase HII and ExoR showed no response. Inhibition of replication by 6(p hydroxyphenylazo)-uracil (HPUra) demonstrated that both RNase HII and RNase HIII are directly involved in the replication. We found that the absence of ExoR increases the likelihood of RNase HIII at the forks, indicating that substrate availability rather than direct protein interactions may be a major driver for the recruitment of RNases to the lagging strands. Thus, B. subtilis replication forks appear to be an intermediate between E. coli type and eukaryotic replication forks and employ a multitude of RNases, rather than any dedicated enzyme for RNA/DNA hybrid removal.

The circumferential motion of MreB filaments plays a key role in cell shape maintenance in many bacteria. It has recently been shown that filament formation of MreB filaments in Bacillus subtilis is influenced by stress conditions. In response to osmotic upshift, MreB molecules were released from filaments, as seen by an increase in freely diffusive molecules, and the peptidoglycan synthesis pattern became less organized, concomitant with slowed-down cell extension. In this study, biotic and abiotic factors were analysed with respect to a possible function in the adaptation of MreB filaments to stress conditions. We show that parallel to MreB, its interactor RodZ becomes more diffusive following osmotic stress, but the remodeling of MreB filaments is not affected by a lack of RodZ. Conversely, mutant strains that prevent efficient potassium influx into cells following osmotic shock show a failure to disassemble MreB filaments, accompanied by less perturbed cell wall extension than is observed in wild type cells. Because potassium ions are known to negatively affect MreB polymerization in vitro, our data indicate that polymer disassembly is directly mediated by the physical consequences of the osmotic stress response. The lack of an early potassium influx response strongly decreases cell survival following stress application, suggesting that the disassembly of MreB filaments may ensure slowed-down cell wall extension to allow for efficient adaptation to new osmotic conditions.

The bacterial cell wall is a meshwork of crosslinked peptidoglycan strands, with a thickness of up to 50 nm in Firmicutes. Little is known about how proteins move through the cell wall to find sites of enzymatic activity. Cell wall synthesis for cell elongation involves the integration of new peptidoglycan strands by integral membrane proteins, as well as the degradation of existing strands by so-called autolysins, soluble proteins that are secreted through the cell membrane. Autolysins comprise different classes of proteases and glucanases and mostly contain cell-wall binding domains in addition to their catalytic domain. We have studied dynamics of Bacillus subtilis autolysins LytC, a major endopeptidase required for lateral cell wall growth, and LytF, a peptidase acting at the newly formed division site in order to achieve separation of daughter cells. We show that both proteins, fused to moxVenus are present as three distinct populations of different diffusion constants. The fastest population is compatible with free diffusion in a crowded liquid environment, that is similar to that of cytosolic enzymes, likely reflecting autolysins diffusing through the periplasm. The medium mobile fraction can be explained by constrained motion through a polymeric substance, indicating mobility of autolysins through the wall similar to that of DNA-binding proteins within the nucleoid. The slow-mobile fraction are most likely autolysins bound to their specific substrate sites. We show that LytF is more static during exponential phase, while LytC appears to be more active during the transition to stationary phase. Both autolysins became more static in backgrounds lacking redundant other autolysins, suggesting stochastic competition for binding sites. On the other hand, lack of inhibitor IseA or autolysin CwlS lead to an altered preference for polar localization of LytF within the cell wall, revealing that inhibitors and autolysins also affect each other's pattern of localization, in addition to their activity.

SecA is a widely conserved ATPase that drives the secretion of proteins across the cell membrane via the SecYEG translocon, while the SRP system is a key player in the insertion of membrane proteins via SecYEG. How SecA gains access to substrate proteins in Bacillus subtilis cells and copes with an increase in substrate availability during biotechnologically desired, high-level expression of secreted proteins is poorly understood. Using single molecule tracking, we found that SecA localization closely mimics that of ribosomes, and its molecule dynamics change similarly to those of ribosomes after inhibition of transcription or translation. These data suggest that B. subtilis SecA associates with signal peptides as they are synthesized at the ribosome, similar to the SRP system. In agreement with this, SecA is a largely mobile cytosolic protein; only a subset is statically associated with the cell membrane, i.e., likely with the Sec translocon. SecA dynamics were considerably different during the late exponential, transition, and stationary growth phases, revealing that single molecule dynamics considerably alter during different genetic programs in cells. During overproduction of a secretory protein, AmyE, SecA showed the strongest changes during the transition phase, i.e., where general protein secretion is high. To investigate whether the overproduction of AmyE also has an influence on other proteins that interact with SecYEG, we analyzed the dynamics of SecDF, YidC, and FtsY with and without AmyE overproduction. SecDF and YidC did not reveal considerable differences in single molecule dynamics during overexpression, while the SRP component FtsY changed markedly in its behavior and became more statically engaged. These findings indicate that the SRP pathway becomes involved in protein secretion upon an overload of proteins carrying a signal sequence. Thus, our data reveal high plasticity of the SecA and SRP systems in dealing with different needs for protein secretion.

Bactofilins have emerged as a widespread family of cytoskeletal proteins with important roles in bacterial morphogenesis, but their precise mode of action is still incompletely understood. In this study, we identify the bactofilin cytoskeleton as a key regulator of cell growth in the stalked budding alphaproteobacterium Hyphomonas neptunium . We show that, in this species, bactofilin polymers localize dynamically to the stalk base and the bud neck, with their absence leading to unconstrained growth of the stalk and bud compartments, indicating a central role in the spatial regulation of cell wall biosynthesis. Database searches reveal that bactofilin genes are often clustered with genes for cell wall hydrolases of the M23 peptidase family, suggesting a functional connection between these two types of proteins. In support of this notion, we find that the H. neptunium M23 peptidase homolog LmdC interacts directly with bactofilin in vitro and is required for proper cell shape in vivo. Complementary studies in the spiral-shaped alphaproteobacterium Rhodospirillum rubrum again reveal a close association of its bactofilin and LmdC homologs, which co-localize at the inner curve of the cell, modulating the degree of cell curvature. Collectively, these findings demonstrate that bactofilins and M23 peptidases form a conserved functional module that promotes local changes in the mode of cell wall biosynthesis, thereby driving cell shape determination in morphologically complex bacteria., Competing Interests: SP, MO, EC, JH, RH, JB, PS, WV, PG, MT No competing interests declared, (© 2023, Pöhl, Osorio-Valeriano, Cserti et al.)

All DNA-binding proteins in vivo exist as a population of freely diffusing molecules and of DNA-bound molecules. The molecules bound to DNA can be split into specifically/tightly and nonspecifically bound proteins. Single-molecule tracking (SMT) is a method allowing to visualize protein dynamics in living cells, revealing their behavior in terms of mode of motion, diffusion coefficient/speed, change of dwell times, and unveiling preferred subcellular sites of dwelling. Bleaching-type SMT or fluorescent protein-tagged SMT involves rapid laser-induced bleaching of most fluorophore-labeled molecules. The remaining single fluorescent proteins are then continuously tracked. The trajectories of several fluorescent molecules per cell for a population of cells are analyzed and combined to permit a robust analysis of average behavior of single molecules in live cells, including analyses of protein dynamics in mutant cells or cells exposed to changes in environmental conditions.In this chapter, we describe the preparation of Bacillus subtilis cells, the recording of movies of those cells expressing a monomeric variant of a yellow fluorescent protein (mNeonGreen) fused to a protein of choice, and the subsequent curation of the movie data including the statistical analysis of the protein dynamics. We present a short overview of the analysis program SMTracker 2.0, highlighting its ability to analyze SMT data by non-expert scientists., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

At the transition to stationary phase, a subpopulation of Bacillus subtilis cells can enter the developmental state of competence, where DNA is taken up through the cell envelope, and is processed to single stranded DNA, which is incorporated into the genome if sufficient homology between sequences exists. We show here that the initial step of transport across the cell wall occurs via a true pilus structure, with an average length of about 500 nm, which assembles at various places on the cell surface. Once assembled, the pilus remains at one position and can be retracted in a time frame of seconds. The major pilin, ComGC, was studied at a single molecule level in live cells. ComGC was found in two distinct populations, one that would correspond to ComGC freely diffusing throughout the cell membrane, and one that is relatively stationary, likely reflecting pilus-incorporated molecules. The ratio of 65% diffusing and 35% stationary ComGC molecules changed towards more stationary molecules upon addition of external DNA, while the number of pili in the population did not strongly increase. These findings suggest that the pilus assembles stochastically, but engages more pilin monomers from the membrane fraction in the presence of transport substrate. Our data support a model in which transport of environmental DNA occurs through the entire cell surface by a dynamic pilus, mediating efficient uptake through the cell wall into the periplasm, where DNA diffuses to a cell pole containing the localized transport machinery mediating passage into the cytosol., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Kilb et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: Whereas the translocation of proteins across the cell membrane has been thoroughly investigated, it is still unclear how proteins cross the cell wall in Gram-positive bacteria, which are widely used for industrial applications. We have studied the secretion of α-amylase AmyE within two different Bacillus strains, B. subtilis and B. licheniformis., Results: We show that a C-terminal fusion of AmyE with the fluorescent reporter mCherry is secreted via discrete patches showing very low dynamics. These are visible at many places within the cell wall for many minutes. Expression from a high copy number plasmid was required to be able to see these structures we term "secretion zones". Zones corresponded to visualized AmyE activity on the surface of cells, showing that they release active enzymes. They overlapped with SecA signals but did not frequently co-localize with the secretion ATPase. Single particle tracking showed higher dynamics of SecA and of SecDF, involved in AmyE secretion, at the cell membrane than AmyE. These experiments suggest that SecA initially translocates AmyE molecules through the cell membrane, and then diffuses to a different translocon. Single molecule tracking of SecA suggests the existence of three distinct diffusive states of SecA, which change during AmyE overexpression, but increased AmyE secretion does not appear to overwhelm the system., Conclusions: Because secretion zones were only found during the transition to and within the stationary phase, diffusion rather than passive transport based on cell wall growth from inside to outside may release AmyE and, thus, probably secreted proteins in general. Our findings suggest active transport through the cell membrane and slow, passive transition through the cell wall, at least for overexpressed proteins, in bacteria of the genus Bacillus., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Proteins with a catalytically inactive LytM-type endopeptidase domain are important regulators of cell wall-degrading enzymes in bacteria. Here, we study their representative DipM, a factor promoting cell division in Caulobacter crescentus. We show that the LytM domain of DipM interacts with multiple autolysins, including the soluble lytic transglycosylases SdpA and SdpB, the amidase AmiC and the putative carboxypeptidase CrbA, and stimulates the activities of SdpA and AmiC. Its crystal structure reveals a conserved groove, which is predicted to represent the docking site for autolysins by modeling studies. Mutations in this groove indeed abolish the function of DipM in vivo and its interaction with AmiC and SdpA in vitro. Notably, DipM and its targets SdpA and SdpB stimulate each other's recruitment to midcell, establishing a self-reinforcing cycle that gradually increases autolytic activity as cytokinesis progresses. DipM thus coordinates different peptidoglycan-remodeling pathways to ensure proper cell constriction and daughter cell separation., (© 2023. The Author(s).)

Highly specific interactions between proteins are a fundamental prerequisite for life, but how they evolve remains an unsolved problem. In particular, interactions between initially unrelated proteins require that they evolve matching surfaces. It is unclear whether such surface compatibilities can only be built by selection in small incremental steps, or whether they can also emerge fortuitously. Here, we used molecular phylogenetics, ancestral sequence reconstruction and biophysical characterization of resurrected proteins to retrace the evolution of an allosteric interaction between two proteins that act in the cyanobacterial photoprotection system. We show that this interaction between the orange carotenoid protein (OCP) and its unrelated regulator, the fluorescence recovery protein (FRP), evolved when a precursor of FRP was horizontally acquired by cyanobacteria. FRP's precursors could already interact with and regulate OCP even before these proteins first encountered each other in an ancestral cyanobacterium. The OCP-FRP interaction exploits an ancient dimer interface in OCP, which also predates the recruitment of FRP into the photoprotection system. Together, our work shows how evolution can fashion complex regulatory systems easily out of pre-existing components., (© 2023. The Author(s).)

Background: Early-life exposure to certain environmental bacteria including Acinetobacter lwoffii (AL) has been implicated in protection from chronic inflammatory diseases including asthma later in life. However, the underlying mechanisms at the immune-microbe interface remain largely unknown., Methods: The effects of repeated intranasal AL exposure on local and systemic innate immune responses were investigated in wild-type and Il6 -/- , Il10 -/- , and Il17 -/- mice exposed to ovalbumin-induced allergic airway inflammation. Those investigations were expanded by microbiome analyses. To assess for AL-associated changes in gene expression, the picture arising from animal data was supplemented by in vitro experiments of macrophage and T-cell responses, yielding expression and epigenetic data., Results: The asthma preventive effect of AL was confirmed in the lung. Repeated intranasal AL administration triggered a proinflammatory immune response particularly characterized by elevated levels of IL-6, and consequently, IL-6 induced IL-10 production in CD4 + T-cells. Both IL-6 and IL-10, but not IL-17, were required for asthma protection. AL had a profound impact on the gene regulatory landscape of CD4 + T-cells which could be largely recapitulated by recombinant IL-6. AL administration also induced marked changes in the gastrointestinal microbiome but not in the lung microbiome. By comparing the effects on the microbiota according to mouse genotype and AL-treatment status, we have identified microbial taxa that were associated with either disease protection or activity., Conclusion: These experiments provide a novel mechanism of Acinetobacter lwoffii-induced asthma protection operating through IL-6-mediated epigenetic activation of IL-10 production and with associated effects on the intestinal microbiome., (© 2022 The Authors. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.)

Cation-exchange stationary phases were characterized in different chromatographic modes (HILIC, RPLC, IC) and applied to the separation of non-charged hydrophobic and hydrophilic analytes. The set of columns under investigation included both commercially available cation-exchangers and self-prepared PS/DVB-based columns, the latter consisting of adjustable amounts of carboxylic and sulfonic acid functional groups. The influence of cation-exchange site and polymer substrate on the multimodal properties of cation-exchangers was identified using selectivity parameters, polymer imaging and excess adsorption isotherms. Introducing weakly acidic cation-exchange functional groups to the unmodified PS/DVB-substrate effectively reduced hydrophobic interactions, whilst a low degree of sulfonation (0.09 to 0.27% w/w sulphur) mainly influenced electrostatic interactions. Silica substrate was found to be another important factor for inducing hydrophilic interactions. The presented results demonstrate that cation-exchange resins are suitable for mixed-mode applications and offer versatile selectivity., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

RecA plays a central role in DNA repair and is a main actor involved in homologous recombination (HR). In vivo , RecA forms filamentous structures termed "threads," which are essential for HR, but whose nature is still ill defined. We show that RecA from Bacillus subtilis having lower ATP binding activity can still form nucleoprotein filaments in vitro , features lower dsDNA binding activity, but still retains most of wild type RecA activity in vivo . Contrarily, loss of ATPase activity strongly reduced formation of nucleoprotein filaments in vitro , and effectivity to repair double strand breaks (DSBs) in vivo. In the presence of wild type RecA protein, additionally expressed RecA with lowered ATPbinding activity only moderately affected RecA dynamics, while loss of ATPase activity leads to a large reduction of the formation of threads, as well as of their dynamic changes observed in a seconds-scale. Single molecule tracking of RecA revealed incorporation of freely diffusing and nonspecifically DNA-bound molecules into threads upon induction of a single DSB. This change of dynamics was highly perturbed in the absence of ATPase activity, revealing that filamentous forms of RecA as well as their dynamics depend on ATPase activity. Based on the idea that ATPase activity of RecA is most important for DNA strand exchange activity, our data suggest that extension and retraction of threads due is to many local strand invasion events during the search for sequences homologous to the induced DNA break site. IMPORTANCE Single-strand (ss) DNA binding ATPase RecA is the central recombinase in homologous recombination, and therefore essential for DNA repair pathways involving DNA strand exchange reactions. In several bacterial, RecA forms filamentous structures along the long axis of cells after induction of double strand breaks (DSBs) in the chromosome. These striking assemblies likely reflect RecA/ssDNA nucleoprotein filaments, which can extend and remodel within a time frame of few minutes. We show that ATPase activity of RecA is pivotal for these dynamic rearrangements, which include recruitment of freely diffusing molecules into low-mobile molecules within filaments. Our data suggest that ssDNA binding- and unbinding reactions are at the heart of RecA dynamics that power the dynamics of subcellular filamentous assemblies, leading to strand exchange reactions over a distance of several micrometers.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Stoll, Zegarra, Bange and Graumann.)

Bacterial Type IV CRISPR-Cas systems are thought to rely on multi-subunit ribonucleoprotein complexes to interfere with mobile genetic elements, but the substrate requirements and potential DNA nuclease activities for many systems within this type are uncharacterized. Here we show that the native Pseudomonas oleovorans Type IV-A CRISPR-Cas system targets DNA in a PAM-dependent manner and elicits interference without showing DNA nuclease activity. We found that the first crRNA of P. oleovorans contains a perfect match in the host gene coding for the Type IV pilus biogenesis protein PilN. Deletion of the native Type IV CRISPR array resulted in upregulation of pilN operon transcription in the absence of genome cleavage, indicating that Type IV-A CRISPR-Cas systems can function in host gene regulation. These systems resemble CRISPR interference (CRISPRi) methodology but represent a natural CRISPRi-like system that is found in many Pseudomonas and Klebsiella species and allows for gene silencing using engineered crRNAs., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Bacterial gene expression depends on the efficient functioning of global transcriptional networks, however their interconnectivity and orchestration rely mainly on the action of individual DNA binding proteins called transcription factors (TFs). TFs interact not only with their specific target sites, but also with secondary (off-target) sites, and vary in their promiscuity. It is not clear yet what mechanisms govern the interactions with secondary sites, and how such rewiring affects the overall regulatory network, but this could clearly constrain horizontal gene transfer. Here, we show the molecular mechanism of one such off-target interaction between two unrelated TFs in Escherichia coli: the C regulatory protein of a Type II restriction-modification system, and the RacR repressor of a defective prophage. We reveal that the C protein interferes with RacR repressor expression, resulting in derepression of the toxic YdaT protein. These results also provide novel insights into regulation of the racR-ydaST operon. We mapped the C regulator interaction to a specific off-target site, and also visualized C protein dynamics, revealing intriguing differences in single molecule dynamics in different genetic contexts. Our results demonstrate an apparent example of horizontal gene transfer leading to adventitious TF cross-talk with negative effects on the recipient's viability. More broadly, this study represents an experimentally-accessible model of a regulatory constraint on horizontal gene transfer., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Signaling of two-component systems by phosphoryl transfer requires interaction of the sensor kinase with the response regulator. Interaction of the C4-dicarboxylate-responsive and membrane-integral sensor kinase DcuS with the response regulator DcuR was studied. In vitro , the cytoplasmic part of DcuS (PAS C -Kin) was employed. Stable complexes were formed, when either DcuS or DcuR were phosphorylated ( K d 22 ± 11 and 28 ± 7 nM, respectively). The unphosphorylated proteins produced a more labile complex ( K d 1380 ± 395 nM). Bacterial two-hybrid studies confirm interaction of DcuR with DcuS (and PAS C -Kin) in vivo . The absolute contents of DcuR (197-979 pmol mg -1 protein) in the bacteria exceeded those of DcuS by more than 1 order of magnitude. According to the K d values, DcuS exists in complex, with phosphorylated but also unphosphorylated DcuR. In live cell imaging, the predominantly freely diffusing DcuR becomes markedly less mobile after phosphorylation and activation of DcuS by fumarate. Portions of the low mobility fraction accumulated at the cell poles, the preferred location of DcuS, and other portions within the cell, representing phosphorylated DcuR bound to promoters. In the model, acitvation of DcuS increases the affinity toward DcuR, leading to DcuS-P × DcuR formation and phosphorylation of DcuR. The complex is stable enough for phosphate-transfer, but labile enough to allow exchange between DcuR from the cytosol and DcuR-P of the complex. Released DcuR-P diffuses to target promoters and binds. Uncomplexed DcuR-P in the cytosol binds to nonactivated DcuS and becomes dephosphorylated. The lower affinity between DcuR and DcuS avoids blocking of DcuS and allows rapid exchange of DcuR. IMPORTANCE Complex formation of membrane-bound sensor kinases with the response regulators represents an inherent step of signaling from the membrane to the promoters on the DNA. In the C4-dicarboxylate-sensing DcuS-DcuR two-component system, complex formation is strengthened by activation (phosphorylation) in vitro and in vivo , with trapping of the response regulator DcuR at the membrane. Single-molecule tracking of DcuR in the bacterial cell demonstrates two populations of DcuR with decreased mobility in the bacteria after activation: one at the membrane, but a second in the cytosol, likely representing DNA-bound DcuR. The data suggest a model with binding of DcuR to DcuS-P for phosphorylation, and of DcuR-P to DcuS for dephosphorylation, allowing rapid adaptation of the DcuR phosphorylation state. DcuR-P is released and transferred to DNA by 3D diffusion.

Objective: Bactofilins can assemble into polymeric structures and play important roles in cell shape maintenance, chromosome segregation and motility. Bacillus subtilis bactofilins BacE and BacF were shown to be important for swimming motility in strain PY79, and single gene deletions were reported to be lethal, in contrast to a double bacEF deletion., Results: Extending this work, we show that motility defects vary between different B. subtilis strains, with strain 168 showing no defect in motility, and 3610 showing delayed induction of swimming. Generation of single gene deletions in PY79 was possible by transferring corresponding deletions from 168. In the natural isolate 3610, gene deletions also showed a negative effect on biofilm formation, revealing an additional function for BacE and BacF. A spontaneous arising suppressor mutation in PY79 was mapped to the flhO gene, a constituent of the flagellum, which obtained an 18 amino acid extension at its C-terminus. Our findings show that bactofilin gene deletions lead to different motility phenotypes dependent on the strain background, and affect biofilm formation in the natural isolate 3610. Our data reinforce the idea of a connection between bactofilins and motion via the flagellum, and suggest that they operate in a switch like manner., (© 2022. The Author(s).)

In bacteria, the monopolar localization of enzymes and protein complexes can result in a bimodal distribution of enzyme activity between the dividing cells and heterogeneity of cellular behaviors. In Shewanella putrefaciens, the multidomain hybrid diguanylate cyclase/phosphodiesterase PdeB, which degrades the secondary messenger c-di-GMP, is located at the flagellated cell pole. Here, we show that direct interaction between the inactive diguanylate cyclase (GGDEF) domain of PdeB and the FimV domain of the polar landmark protein HubP is crucial for full function of PdeB as a phosphodiesterase. Thus, the GGDEF domain serves as a spatially controlled on-switch that effectively restricts PdeBs activity to the flagellated cell pole. PdeB regulates abundance and activity of at least two crucial surface-interaction factors, the BpfA surface-adhesion protein and the MSHA type IV pilus. The heterogeneity in c-di-GMP concentrations, generated by differences in abundance and timing of polar appearance of PdeB, orchestrates the population behavior with respect to cell-surface interaction and environmental spreading., (© 2022. The Author(s).)

In nature, bacteria reside in biofilms- multicellular differentiated communities held together by an extracellular matrix. This work identified a novel subpopulation-mineral-forming cells-that is essential for biofilm formation in Bacillus subtilis biofilms. This subpopulation contains an intracellular calcium-accumulating niche, in which the formation of a calcium carbonate mineral is initiated. As the biofilm colony develops, this mineral grows in a controlled manner, forming a functional macrostructure that serves the entire community. Consistently, biofilm development is prevented by the inhibition of calcium uptake. Our results provide a clear demonstration of the orchestrated production of calcite exoskeleton, critical to morphogenesis in simple prokaryotes., Competing Interests: The authors declare no competing interests., (© 2022 The Authors.)

Flavins are ubiquitous molecules in life as they serve as important enzyme cofactors. In the Gram-positive, soil-dwelling bacterium Bacillus subtilis , four well-characterized gene products (the enzymes RibDG, RibE, RibAB, and RibH) catalyze the biosynthesis of riboflavin (RF) from guanosine-triphosphate (GTP) and ribulose-5-phosphate (R5P). The corresponding genes form an operon together with the gene ribT ( ribDG-E-AB-H-T ), wherein the function of this terminal gene remained enigmatic. RibT has been structurally characterized as a GCN5-like acetyltransferase (GNAT), however, with unidentified target molecules. Bacterial two-hybrid system revealed interactions between RibT, RibH, and RibE, forming the heavy RF synthase complex. Applying single particle tracking (SPT), we found that confined (sub)diffusion of RibT is largely dependent on interacting RibE and, to a lesser degree, on interacting RibH. By induced expression of otherwise low-expressed ribT from an ectopic locus, we observed a decrease in the subpopulation considered to represent capsids of the heavy RF synthase and an increase in the subpopulation thought to represent pentamers of RibH, pointing to a putative role for RibT in capsid disassembly. Complementarily, either deletion of ribT or mutation of a key residue from RibH (K29) suspected to be the substrate of RibT for acetylation leads to increased levels of subpopulations considered as capsids of RibH-mVenus (RibH-mV) in comparison to wild-type (wt)-like cells. Thus, we provide evidence for an indirect involvement of RibT in RF biosynthesis by a putative capsid disassembling mechanism considered to involve acetylation of RibH residue K29 at the three-fold symmetry axis of 60-mer capsids., Competing Interests: DAOR was employed by BioNTech Manufacturing Marburg GmbH. CH was employed by BioSpringBiotechnolgie GmbH. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Rotter, Heger, Kühm, Schmidt, Schäfer, Heimerl, Mack and Graumann.)

DNA helicases of the RecD2 family are ubiquitous. Bacillus subtilis RecD2 in association with the single-stranded binding protein SsbA may contribute to replication fork progression, but its detailed action remains unknown. In this work, we explore the role of RecD2 during DNA replication and its interaction with the RecA recombinase. RecD2 inhibits replication restart, but this effect is not observed in the absence of SsbA. RecD2 slightly affects replication elongation. RecA inhibits leading and lagging strand synthesis, and RecD2, which physically interacts with RecA, counteracts this negative effect. In vivo results show that recD2 inactivation promotes RecA-ssDNA accumulation at low mitomycin C levels, and that RecA threads persist for a longer time after induction of DNA damage. In vitro, RecD2 modulates RecA-mediated DNA strand-exchange and catalyzes branch migration. These findings contribute to our understanding of how RecD2 may contribute to overcome a replicative stress, removing RecA from the ssDNA and, thus, it may act as a negative modulator of RecA filament growth., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In competent Gram-negative and Gram-positive bacteria, double-stranded DNA is taken up through the outer cell membrane and/or the cell wall and is bound by ComEA, which in Bacillus subtilis is a membrane protein. DNA is converted to single-stranded DNA and transported through the cell membrane via ComEC. We show that in Bacillus subtilis, the C terminus of ComEC, thought to act as a nuclease, is important not only for DNA uptake, as judged from a loss of transformability, but also for the localization of ComEC to the cell pole and its mobility within the cell membrane. Using single-molecule tracking, we show that only 13% of ComEC molecules are statically localized at the pole, while 87% move throughout the cell membrane. These experiments suggest that recruitment of ComEC to the cell pole is mediated by a diffusion/capture mechanism. Mutation of a conserved aspartate residue in the C terminus, likely affecting metal binding, strongly impairs transformation efficiency, suggesting that this periplasmic domain of ComEC could indeed serve a catalytic function as a nuclease. By tracking fluorescently labeled DNA, we show that taken-up DNA has a similar mobility as a protein, in spite of being a large polymer. DNA dynamics are similar within the periplasm as those of ComEA, suggesting that most taken-up molecules are bound to ComEA. We show that DNA can be highly mobile within the periplasm, indicating that this subcellular space can act as reservoir for taken-up DNA, before its entry into the cytosol. IMPORTANCE Bacteria can take up DNA from the environment and incorporate it into their chromosome, which is termed "natural competence" that can result in the uptake of novel genetic information. We show that fluorescently labeled DNA moves within the periplasm of competent Bacillus subtilis cells, with similar dynamics as DNA receptor ComEA. This finding indicates that DNA can accumulate in the periplasm, likely bound by ComEA, and thus can be stored before uptake at the cell pole, via integral membrane DNA permease ComEC. Assembly of ComEC at the cell pole likely occurs by a diffusion-capture mechanism. DNA uptake into cells thus takes a detour through the entire periplasm and involves a high degree of free diffusion along and within the cell membrane.

Bacteria are dependent on rapid alterations in gene expression. A prerequisite for rapid adaptations is efficient RNA turnover, with endonuclease RNase Y playing a crucial role in mRNA stability as well as in maturation. In Bacillus subtilis , RNase Y in turn interacts with the so-called "Y-complex" consisting of three proteins, which play important functions in sporulation, natural transformation and biofilm formation. It is thought that the Y-complex acts as an accessory factor in RNase Y regulation but might also have independent functions. Using single-molecule tracking, we show that all three Y-complex proteins exhibit three distinct mobilities, including movement through the cytosol and confined motion, predominantly at membrane-proximal sites but also within the cell center. A transcriptional arrest leads to a strong change in localization and dynamics of YmcA, YlbF and YaaT, supporting their involvement in global RNA degradation. However, Y-complex proteins show distinguishable protein dynamics, and the deletion of yaaT or ylbF shows a minor effect on the dynamics of YmcA. Cell fractionation reveals that YaaT displays a mixture of membrane association and presence in the cytosol, while YlbF and YmcA do not show direct membrane attachment. Taken together, our experiments reveal membrane-associated and membrane-independent activities of Y-complex proteins and a dynamic interplay between them with indirect membrane association of YmcA and YlbF via YaaT.

In order to survive, bacterial cells rely on precise spatiotemporal organization and coordination of essential processes such as cell growth, chromosome segregation, and cell division. Given the general lack of organelles, most bacteria are forced to depend on alternative localization mechanisms, such as, for example, geometrical cues. DivIVA proteins are widely distributed in mainly Gram-positive bacteria and were shown to bind the membrane, typically in regions of strong negative curvature, such as the cell poles and division septa. Here, they have been shown to be involved in a multitude of processes: from apical cell growth and chromosome segregation in actinobacteria to sporulation and inhibition of division re-initiation in firmicutes. Structural analyses revealed that DivIVA proteins can form oligomeric assemblies that constitute a scaffold for recruitment of other proteins. However, it remained unclear whether interaction with partner proteins influences DivIVA dynamics. Using structured illumination microscopy (SIM), single-particle tracking (SPT) microscopy, and fluorescent recovery after photobleaching (FRAP) experiments, we show that DivIVA from Corynebacterium glutamicum is mobilized by its binding partner ParB. In contrast, we show that the interaction between Bacillus subtilis DivIVA and its partner protein MinJ reduces DivIVA mobility. Furthermore, we show that the loss of the rod-shape leads to an increase in DivIVA dynamics in both organisms. Taken together, our study reveals the modulation of the polar scaffold protein by protein interactors and cell morphology. We reason that this leads to a very simple, yet robust way for actinobacteria to maintain polar growth and their rod-shape. In B. subtilis , however, the DivIVA protein is tailored towards a more dynamic function that allows quick relocalization from poles to septa upon division.

It has long become clear that in spite of generally lacking internal membrane systems, bacteria contain well-structured subcellular structures of usually filamentous proteins, and a preferred 3D arrangement of their chromosome(s). Some of these systems are set up by so-called cytoskeletal elements, or by polar landmark proteins, but the mechanism of specific localization is still unclear in most cases. Intriguingly, apart from such spatially organizing systems, the bacterial cytoplasm has unusual properties in terms of the diffusion of molecules, which varies between different sites within the cell. In many bacteria, chromosomes are compacted into centrally located nucleoids, being orderly folded as opposed to consisting of random coils of DNA. In these bacteria, there is a separation of transcription and translation, such that transcription by RNA polymerase occurs on the nucleoids, and translation takes place mostly at the cell poles and directly underneath the cell membrane, because 70S ribosomes accumulate at sites surrounding the nucleoids. Interestingly, accumulation of ribosomes appears to slow down diffusion of enzymes, noticeable for larger enzyme complexes, while nucleoids provide areas of confined motion for DNA-binding proteins, yet acceleration zones for non-DNA-binding proteins. Crowded regions at the cell poles set up zones of higher concentration of the translation machinery, shortening diffusion distances for rate-limiting translation factor/ribosome interactions, and of metabolic enzymes, possibly speeding up pathways containing low concentrations of metabolites. Thus, heterogeneous diffusion adds another layer of subcellular organization on top of cytoskeletal elements., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Dynamin-like proteins are membrane-associated GTPases, conserved in bacteria and in eukaryotes, that can mediate nucleotide-driven membrane deformation or membrane fusion reactions. Bacillus subtilis' DynA has been shown to play an important role in protecting cells against chemicals that induce membrane leakage, and to form an increased number of membrane-associated structures after induction of membrane stress. We have studied the dynamics of DynA at a single molecule level in real time, to investigate how assembly of stress-induced structures is accompanied by changes in molecule dynamics. We show that DynA molecule displacements are best described by the existence of three distinct populations, a static mode, a low-mobility, and a fast-mobile state. Thus, DynA is most likely freely diffusive within the cytosol, moves along the cell membrane with a low mobility, and arrests at division sites or at stress-induced lesions at the membrane. In response to stress-inducing membrane leakage, but not to general stress, DynA molecules become slightly more static, but largely retain their mobility, suggesting that only few molecules are involved in the repair of membrane lesions, while most molecules remain in a dynamic mode scanning for lesions. Our data suggest that even moderate changes in single molecule dynamics can lead to visible changes in protein localization patterns., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Messenger RNA molecules have been localized to different positions in cells and have been followed by time-lapse microscopy. We have used MS2-mVenus-labeled mRNA and single-particle tracking to obtain information on the dynamics of single-mRNA molecules in real time. Using single-molecule tracking, we show that several mRNA molecules visualized via two MS2-binding sites and MS2-mVenus expressed in Bacillus subtilis cells show free diffusion through the entire cell and constrained motion predominantly close to the cell membrane and at the polar regions of the cells. Because constrained motion of mRNAs likely reflects molecules complexed with ribosomes, our data support the idea that translation occurs at sites surrounding the nucleoids. Squared displacement analyses show the existence of at least two distinct populations of molecules with different diffusion constants or possibly of three populations, for example, freely mobile mRNAs, mRNAs in transition complexes, or in complex with polysomes. Diffusion constants between differently sized mRNAs did not differ dramatically and were much lower than that of cytosolic proteins. These data agree with the large size of mRNA molecules and suggest that, within the viscous cytoplasm, size variations do not translate into mobility differences. However, at observed diffusion constants, mRNA molecules would be able to reach all positions within cells in a frame of seconds. We did not observe strong differences in the location of confined motion for mRNAs encoding mostly soluble or membrane proteins, indicating that there is no strong bias for localization of membrane protein-encoding transcripts for the cell membrane., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sattler and Graumann.)

Single-molecule (particle) tracking is a powerful method to study dynamic processes in cells at highest possible spatial and temporal resolution. We have developed SMTracker, a graphical user interface for automatic quantifying, visualizing and managing of data. Version 2.0 determines distributions of positional displacements in x- and y-direction using multi-state diffusion models, discriminates between Brownian, sub- or superdiffusive behaviour, and locates slow or fast diffusing populations in a standardized cell. Using SMTracker, we show that the Bacillus subtilis RNA degradosome consists of a highly dynamic complex of RNase Y and binding partners. We found similar changes in molecule dynamics for RNase Y, CshA, PNPase and enolase, but not for phosphofructokinase, RNase J1 and J2, to inhibition of transcription. However, the absence of PfkA or of RNase J2 affected molecule dynamics of RNase Y-mVenus, indicating that these two proteins are indeed part of the degradosome. Molecule counting suggests that RNase Y is present as a dimer in cells, at an average copy number of about 500, of which 46% are present in a slow-diffusive state and thus likely engaged within degradosomes. Thus, RNase Y, CshA, PNPase and enolase likely play central roles, and RNase J1, J2 and PfkA more peripheral roles, in degradosome architecture., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Knowledge on the localization and mobility of enzymes inside bacterial cells is scarce, but important for understanding spatial regulation of metabolism. The four central enzymes (Rib enzymes) of the riboflavin (RF) biosynthesis pathway in the Gram positive model bacterium Bacillus subtilis have been studied extensively in vitro, especially the heavy RF synthase, a large protein complex with a capsid structure formed by RibH and an encapsulated RibE homotrimer, which mediates substrate-channeling. However, little is known about the behavior and mobility of these enzymes in vivo., Results: We have investigated the localization and diffusion of the Rib enzymes in the cytoplasm of B. subtilis. By characterizing the diffusion of Rib enzymes in live cells using single particle tracking (SPT) we provide evidence for confined diffusion at the cell poles and otherwise Brownian motion. A majority of RibH particles showed clear nucleoid occlusion and a high degree of confined motion, which is largely abolished after treatment with Rifampicin, revealing that confinement is dependent on active transcription. Contrarily, RibE is mostly diffusive within the cell, showing only 14% encapsulation by RibH nanocompartments. By localizing different diffusive populations within single cells, we find that fast diffusion occurs mostly across the nucleoids located in the cell centers, while the slower, confined subdiffusion occurs at the crowded cell poles., Conclusions: Our results provide evidence for locally different motion of active enzymes within the bacterial cytoplasm, setting up metabolic compartmentalization mostly at the poles of cells., (© 2021. The Author(s).)

We have studied the localization and dynamics of bacterial Ffh, part of the SRP complex, its receptor FtsY, and of ribosomes in the Gamma-proteobacterium Shewanella putrefaciens. Using structured illumination microscopy, we show that ribosomes show a pronounced accumulation at the cell poles, whereas SRP and FtsY are distributed at distinct sites along the cell membrane, but they are not accumulated at the poles. Single molecule dynamics can be explained by assuming that all three proteins/complexes move as three distinguishable mobility fractions: a low mobility/static fraction may be engaged in translation, medium-fast diffusing fractions may be transition states, and high mobility populations likely represent freely diffusing molecules/complexes. Diffusion constants suggest that SRP and FtsY move together with slow-mobile ribosomes. Inhibition of transcription leads to loss of static molecules and reduction of medium-mobile fractions, in favor of freely diffusing subunits, while inhibition of translation appears to stall the medium mobile fractions. Depletion of FtsY leads to aggregation of Ffh, but not to loss of the medium mobile fraction, indicating that Ffh/SRP can bind to ribosomes independently from FtsY. Heat maps visualizing the three distinct diffusive populations show that while static molecules are mostly clustered at the cell membrane, diffusive molecules are localized throughout the cytosol. The medium fast populations show an intermediate pattern of preferential localization, suggesting that SRP/FtsY/ribosome transition states may form within the cytosol to finally find a translocon., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mayer, Schwan, Oviedo-Bocanegra, Bange, Thormann and Graumann.)

Restriction-modification (R-M) systems represent a first line of defense against invasive DNAs, such as bacteriophage DNAs, and are widespread among bacteria and archaea. By acquiring a Type II R-M system via horizontal gene transfer, the new hosts generally become more resistant to phage infection, through the action of a restriction endonuclease (REase), which cleaves DNA at or near specific sequences. A modification methyltransferase (MTase) serves to protect the host genome against its cognate REase activity. The production of R-M system components upon entering a new host cell must be finely tuned to confer protective methylation before the REase acts, to avoid host genome damage. Some type II R-M systems rely on a third component, the controller (C) protein, which is a transcription factor that regulates the production of REase and/or MTase. Previous studies have suggested C protein effects on the dynamics of expression of an R-M system during its establishment in a new host cell. Here, we directly examine these effects. By fluorescently labelling REase and MTase, we demonstrate that lack of a C protein reduces the delay of REase production, to the point of being simultaneous with, or even preceding, production of the MTase. Single molecule tracking suggests that a REase and a MTase employ different strategies for their target search within host cells, with the MTase spending much more time diffusing in proximity to the nucleoid than does the REase. This difference may partially ameliorate the toxic effects of premature REase expression., (© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The search for novel drugs that efficiently eliminate prokaryotic pathogens is one of the most urgent health topics of our time. Robust evaluation methods for monitoring the antibiotic stress response in prokaryotes are therefore necessary for developing respective screening strategies. Besides advantages of common in vitro  techniques, there is a growing demand for in vivo  information based on imaging techniques that allow to screen antibiotic candidates in a dynamic manner. Gathering information from imaging data in a reproducible manner, robust data processing and analysis workflows demand advanced (semi-)automation and data management to increase reproducibility. Here we demonstrate a versatile and robust semi-automated image acquisition, processing and analysis workflow to investigate bacterial cell morphology in a quantitative manner. The presented workflow, A.D.I.C.T, covers aspects of experimental setup deployment, data acquisition and handling, image processing (e.g. ROI management, data transformation into binary images, background subtraction, filtering, projections) as well as statistical evaluation of the cellular stress response (e.g. shape measurement distributions, cell shape modeling, probability density evaluation of fluorescence imaging micrographs) towards antibiotic-induced stress, obtained from time-course experiments. The imaging workflow is based on regular brightfield images combined with live-cell imaging data gathered from bacteria, in our case from recombinant Shewanella  cells, which are processed as binary images. The model organism expresses target proteins relevant for membrane-biogenesis that are functionally fused to respective fluorescent proteins. Data processing and analysis are based on customized scripts using ImageJ2/FIJI, Celltool and R packages that can be easily reproduced and adapted by users. Summing up, our approach aims at supporting life-scientists to establish their own imaging-pipeline in order to exploit their data as versatile as possible and in a reproducible manner., Competing Interests: No competing interests were disclosed., (Copyright: © 2022 Mayer B et al.)

The precise spatiotemporal control of cell proliferation is key to the morphogenesis of epithelial tissues. Epithelial cell divisions lead to tissue crowding and local changes in force distribution, which in turn suppress the rate of cell divisions. However, the molecular mechanisms underlying this mechanical feedback are largely unclear. Here, we identify a critical requirement of B-plexin transmembrane receptors in the response to crowding-induced mechanical forces during embryonic skin development. Epidermal stem cells lacking B-plexins fail to sense mechanical compression, resulting in disinhibition of the transcriptional coactivator YAP, hyperproliferation, and tissue overgrowth. Mechanistically, we show that B-plexins mediate mechanoresponses to crowding through stabilization of adhesive cell junctions and lowering of cortical stiffness. Finally, we provide evidence that the B-plexin-dependent mechanochemical feedback is also pathophysiologically relevant to limit tumor growth in basal cell carcinoma, the most common type of skin cancer. Our data define a central role of B-plexins in mechanosensation to couple cell density and cell division in development and disease.

Bacillus subtilis DynA is a member of the dynamin superfamily, involved in membrane remodeling processes. DynA was shown to catalyze full membrane fusion and it plays a role in membrane surveillance against antibiotics. We show here that DynA also provides a novel resistance mechanism against phage infection. Cells lacking DynA are efficiently lysed after phage infection and virus replication. DynA does not prevent phage infection and replication in individual cells, but significantly delays host cell lysis, thereby slowing down the release of phage progeny from the host cells. During the process, DynA forms large, almost immobile clusters on the cell membrane that seem to support membrane integrity. Single-molecule tracking revealed a shift of freely diffusive molecules within the cytosol toward extended, confined motion at the cell membrane following phage induction. Thus, the bacterial dynamins are the first anti-phage system reported to delay host cell lysis and the last line of defense of a multilayered antiviral defense. DynA is therefore providing protective effects on the population, but not on single cell level. IMPORTANCE Bacteria have to cope with myriads of phages in their natural environments. Consequently, they have evolved sophisticated systems to prevent phage infection or epidemic spreading of the infection in the population. We show here that a bacterial dynamin-like protein is involved in phage resistance. The Bacillus subtilis DynA protein delays lysis of infected bacteria and reduces spreading of the phage particles. Thus, the dynamin mediated protection is not at the level of the individual cell, but on the population level. The bacterial DynA is the last line of defense to reduce the deleterious effect of a phage infection in a bacterial community. Interestingly, dynamin-like proteins such as Mx proteins are also involved in antiviral activities in Eukaryotes. Thus, the interaction of dynamin-like proteins and viruses seem to be an evolutionary ancient process.

Replication forks must respond to changes in nutrient conditions, especially in bacterial cells. By investigating the single-molecule dynamics of replicative helicase DnaC, DNA primase DnaG, and lagging-strand polymerase DnaE in the model bacterium Bacillus subtilis , we show that proteins react differently to stress conditions in response to transient replication blocks due to DNA damage, to inhibition of the replicative polymerase, or to downshift of serine availability. DnaG appears to be recruited to the forks by a diffusion and capture mechanism, becomes more statically associated after the arrest of polymerase, but binds less frequently after fork blocks due to DNA damage or to nutritional downshift. These results indicate that binding of the alarmone (p)ppGpp due to stringent response prevents DnaG from binding to forks rather than blocking bound primase. Dissimilar behavior of DnaG and DnaE suggests that both proteins are recruited independently to the forks rather than jointly. Turnover of all three proteins was increased during replication block after nutritional downshift, different from the situation due to DNA damage or polymerase inhibition, showing high plasticity of forks in response to different stress conditions. Forks persisted during all stress conditions, apparently ensuring rapid return to replication extension. IMPORTANCE All cells need to adjust DNA replication, which is achieved by a well-orchestrated multiprotein complex, in response to changes in physiological and environmental conditions. For replication forks, it is extremely challenging to meet with conditions where amino acids are rapidly depleted from cells, called the stringent response, to deal with the inhibition of one of the centrally involved proteins or with DNA modifications that arrest the progression of forks. By tracking helicase (DnaC), primase (DnaG), and polymerase (DnaE), central proteins of Bacillus subtilis replication forks, at a single molecule level in real time, we found that interactions of the three proteins with replication forks change in different manners under different stress conditions, revealing an intriguing plasticity of replication forks in dealing with replication obstacles. We have devised a new tool to determine rates of exchange between static movement (binding to a much larger complex) and free diffusion, showing that during stringent response, all proteins have highly increased exchange rates, slowing down overall replication, while inactivation of polymerase or replication roadblocks leaves forks largely intact, allowing rapid restart once obstacles are removed., (Copyright © 2021 Hernández-Tamayo et al.)

In Rhizobiales bacteria, such as Sinorhizobium meliloti, cell elongation takes place only at new cell poles, generated by cell division. Here, we show that the role of the FtsN-like protein RgsS in S. meliloti extends beyond cell division. RgsS contains a conserved SPOR domain known to bind amidase-processed peptidoglycan. This part of RgsS and peptidoglycan amidase AmiC are crucial for reliable selection of the new cell pole as cell elongation zone. Absence of these components increases mobility of RgsS molecules, as well as abnormal RgsS accumulation and positioning of the growth zone at the old cell pole in about one third of the cells. These cells with inverted growth polarity are able to complete the cell cycle but show partially impaired chromosome segregation. We propose that amidase-processed peptidoglycan provides a landmark for RgsS to generate cell polarity in unipolarly growing Rhizobiales.

The second messenger cyclic di-GMP regulates a variety of processes in bacteria, many of which are centered around the decision whether to adopt a sessile or a motile life style. Regulatory circuits include pathogenicity, biofilm formation, and motility in a wide variety of bacteria, and play a key role in cell cycle progression in Caulobacter crescentus. Interestingly, multiple, seemingly independent c-di-GMP pathways have been found in several species, where deletions of individual c-di-GMP synthetases (DGCs) or hydrolases (PDEs) have resulted in distinct phenotypes that would not be expected based on a freely diffusible second messenger. Several recent studies have shown that individual signaling nodes exist, and additionally, that protein/protein interactions between DGCs, PDEs and c-di-GMP receptors play an important role in signaling specificity. Additionally, subcellular clustering has been shown to be employed by bacteria to likely generate local signaling of second messenger, and/or to increase signaling specificity. This review highlights recent findings that reveal how bacteria employ spatial cues to increase the versatility of second messenger signaling.

Objective: Structural maintenance of chromosomes (SMC) proteins are key players in chromosome dynamics in all types of organisms. The so-called condensin subfamily is essential for chromosome condensation in eukaryotic cells, as is the bacterial SMC complex (called MukBEF in Escherichia coli). We expressed the Bacillus subtilis Smc protein and its two complex partners ScpA and ScpB in E. coli cells, and monitored effects on chromosome compaction by DNA staining of live cells using epifluorescence microscopy., Data Description: We show that expression of BsSmc leads to strong chromosome compaction, while expression of ScpAB does not show any effect. Chromosome compaction by Smc was also found for mutant versions lacking ATP binding or ability for head engagement, and was counteracted by concomitant expression of ScpAB. Our findings show that the SMC complex can act as autonomous condensation system in a heterologous bacterial host system, for which neither ATP binding nor ATP hydrolysis are required. Our investigation suggests that the negative effect on compaction activity of Smc exerted by ScpAB in vivo does not involve an effect on ATPase activity, but more likely a stabilization of the engagement of head domains, which in turn may affect ATPase activity.

Background: MreB is a bacterial ortholog of actin and forms mobile filaments underneath the cell membrane, perpendicular to the long axis of the cell, which play a crucial role for cell shape maintenance. We wished to visualize Bacillus subtilis MreB in vitro and therefore established a protocol to obtain monomeric protein, which could be polymerized on a planar membrane system, or associated with large membrane vesicles., Results: Using a planar membrane system and electron microscopy, we show that Bacillus subtilis MreB forms bundles of filaments, which can branch and fuse, with an average width of 70 nm. Fluorescence microscopy of non-polymerized YFP-MreB, CFP-Mbl and mCherry-MreBH proteins showed uniform binding to the membrane, suggesting that 2D diffusion along the membrane could facilitate filament formation. After addition of divalent magnesium and calcium ions, all three proteins formed highly disordered sheets of filaments that could split up or merge, such that at high protein concentration, MreB and its paralogs generated a network of filaments extending away from the membrane. Filament formation was positively affected by divalent ions and negatively by monovalent ions. YFP-MreB or CFP-Mbl also formed filaments between two adjacent membranes, which frequently has a curved appearance. New MreB, Mbl or MreBH monomers could add to the lateral side of preexisting filaments, and MreB paralogs co-polymerized, indicating direct lateral interaction between MreB paralogs., Conclusions: Our data show that B. subtilis MreB paralogs do not easily form ordered filaments in vitro, possibly due to extensive lateral contacts, but can co-polymerise. Monomeric MreB, Mbl and MreBH uniformly bind to a membrane, and form irregular and frequently split up filamentous structures, facilitated by the addition of divalent ions, and counteracted by monovalent ions, suggesting that intracellular potassium levels may be one important factor to counteract extensive filament formation and filament splitting in vivo.

Objective: The bacterial cell cycle comprises initiation of replication and ensuing elongation, concomitant chromosome segregation (in some organisms with a delay termed cohesion), and finally cell division. By quantifying the number of origin and terminus regions in exponentially growing Bacillus subtilis cells, and after induction of DNA damage, we aimed at determining cell cycle parameters at different growth rates at a single cell level., Results: B. subtilis cells are mostly mero-oligoploid during fast growth and diploid during slow growth. However, we found that the number of replication origins and of termini is highly heterogeneous within the cell population at two different growth rates, and that even at slow growth, a majority of cells attempts to maintain more than a single chromosome at all times of the cell cycle. Heterogeneity of chromosome copy numbers may reflect different subpopulations having diverging growth rates even during exponential growth conditions. Cells continued to initiate replication and segregate chromosomes after induction of DNA damage, as judged by an increase in origin numbers per cell, showing that replication and segregation are relatively robust against cell cycle perturbation.

Although DNA-compacting proteins have been extensively characterized in vitro , knowledge of their DNA binding dynamics in vivo is greatly lacking. We have employed single-molecule tracking to characterize the motion of the three major chromosome compaction factors in Bacillus subtilis , Smc ( s tructural m aintenance of c hromosomes) proteins, topoisomerase DNA gyrase, and histone-like protein HBsu. We show that these three proteins display strikingly different patterns of interaction with DNA; while Smc displays two mobility fractions, one static and one moving through the chromosome in a constrained manner, gyrase operates as a single slow-mobility fraction, suggesting that all gyrase molecules are catalytically actively engaged in DNA binding. Conversely, bacterial histone-like protein HBsu moves through the nucleoid as a larger, slow-mobility fraction and a smaller, high-mobility fraction, with both fractions having relatively short dwell times. Turnover within the SMC complex that makes up the static fraction is shown to be important for its function in chromosome compaction. Our report reveals that chromosome compaction in bacteria can occur via fast, transient interactions in vivo , avoiding clashes with RNA and DNA polymerases. IMPORTANCE All types of cells need to compact their chromosomes containing their genomic information several-thousand-fold in order to fit into the cell. In eukaryotes, histones achieve a major degree of compaction and bind very tightly to DNA such that they need to be actively removed to allow access of polymerases to the DNA. Bacteria have evolved a basic, highly dynamic system of DNA compaction, accommodating rapid adaptability to changes in environmental conditions. We show that the Bacillus subtilis histone-like protein HBsu exchanges on DNA on a millisecond scale and moves through the entire nucleoid containing the genome as a slow-mobility fraction and a dynamic fraction, both having short dwell times. Thus, HBsu achieves compaction via short and transient DNA binding, thereby allowing rapid access of DNA replication or transcription factors to DNA. Topoisomerase gyrase and B. subtilis Smc show different interactions with DNA in vivo , displaying continuous loading or unloading from DNA, or using two fractions, one moving through the genome and one statically bound on a time scale of minutes, respectively, revealing three different modes of DNA compaction in vivo ., (Copyright © 2020 Schibany et al.)

The movement of filamentous, actin-like MreB and of enzymes synthesizing the bacterial cell wall has been proposed to be highly coordinated. We have investigated the motion of MreB and of RodA and PbpH cell wall synthesis enzymes at 500 ms and at 20 ms time scales, allowing us to compare the motion of entire MreB filaments as well as of single molecules with that of the two synthesis proteins. While all three proteins formed assemblies that move with very similar trajectory orientation and with similar velocities, their trajectory lengths differed considerably, with PbpH showing shortest and MreB longest trajectories. These experiments suggest different on/off rates for RodA and PbpH at the putative peptidoglycan-extending machinery (PGEM), and during interaction with MreB filaments. Single molecule tracking revealed distinct slow-moving and freely diffusing populations of PbpH and RodA, indicating that they change between free diffusion and slow motion, indicating a dynamic interaction with the PGEM complex. Dynamics of MreB molecules and the orientation and speed of filaments changed markedly after induction of salt stress, while there was little change for RodA and PbpH single molecule dynamics. During the stress adaptation phase, cells continued to grow and extended the cell wall, while MreB formed fewer and more static filaments. Our results show that cell wall synthesis during stress adaptation occurs in a mode involving adaptation of MreB dynamics, and indicate that Bacillus subtilis cell wall extension involves an interplay of enzymes with distinct binding kinetics to sites of active synthesis., (Copyright © 2020 Dersch, Mehl, Stuckenschneider, Mayer, Roth, Rohrbach and Graumann.)

Although several proteins have been identified that facilitate chromosome segregation in bacteria, no clear analogue of the mitotic machinery in eukaryotic cells has been identified. In order to investigate if recognizable patterns of segregation exist during the cell cycle, we tracked the segregation of duplicated origin regions in Bacillus subtilis for 60 min in the fastest practically achievable resolution, achieving 10-s intervals. We found that while separation occurred in random patterns, often including backwards movement, overall, segregation of loci near the origins of replication was linear for the entire cell cycle. Thus, the process of partitioning can be best described as directed motion. Simulations with entropy-driven separation of polymers synthesized by two polymerases show sudden bursts of movement and segregation patterns compatible with the observed in vivo patterns, showing that for Bacillus , segregation patterns can be modeled based on entropic forces. To test if obstacles for replication forks lead to an alteration of the partitioning pattern, we challenged cells with chemicals inducing DNA damage or blocking of topoisomerase activity. Both treatments led to a moderate slowing down of separation, but linear segregation was retained, showing that chromosome segregation is highly robust against cell cycle perturbation. IMPORTANCE We have followed the segregation of origin regions on the Bacillus subtilis chromosome in the fastest practically achievable temporal manner, for a large fraction of the cell cycle. We show that segregation occurred in highly variable patterns but overall in an almost linear manner throughout the cell cycle. Segregation was slowed down, but not arrested, by treatment of cells that led to transient blocks in DNA replication, showing that segregation is highly robust against cell cycle perturbation. Computer simulations based on entropy-driven separation of newly synthesized DNA polymers can recapitulate sudden bursts of movement and segregation patterns compatible with the observed in vivo patterns, indicating that for Bacillus , segregation patterns may include entropic forces helping to separate chromosomes during the cell cycle., (Copyright © 2020 El Najjar et al.)

Bacillus subtilis can import DNA from the environment by an uptake machinery that localizes to a single cell pole. We investigated the roles of ComEB and of the ATPase ComGA during the state of competence. We show that ComEB plays an important role during competence, possibly because it is necessary for the recruitment of GomGA to the cell pole. ComEB localizes to the cell poles even upon expression during exponential phase, indicating that it can serve as polar marker. ComEB is also a deoxycytidylate monophosphate (dCMP) deaminase, for the function of which a conserved cysteine residue is important. However, cysteine-mutant ComEB is still capable of natural transformation, while a comEB deletion strain is highly impaired in competence, indicating that ComEB confers two independent functions. Single-molecule tracking (SMT) reveals that both proteins exchange at the cell poles between bound and unbound in a time scale of a few milliseconds, but turnover of ComGA increases during DNA uptake, whereas the mobility of ComEB is not affected. Our data reveal a highly dynamic role of ComGA during DNA uptake and an unusual role for ComEB as a mediator of polar localization, localizing by diffusion-capture on an extremely rapid time scale and functioning as a moonlighting enzyme., (© 2020 John Wiley & Sons Ltd.)

Background: Fluorescence microscopy is a powerful tool in cell biology, especially for the study of dynamic processes. Intensive irradiation of bacteria with UV, blue and violet light has been shown to be able to kill cells, but very little information is available on the effect of blue or violet light during live-cell imaging., Results: We show here that in the model bacterium Bacillus subtilis chromosome segregation and cell growth are rapidly halted by standard violet (405 nm) and blue light (CFP) (445-457 nm) excitation, whereas they are largely unaffected by green light (YFP). The stress sigma factor σ B and the blue-light receptor YtvA are not involved in growth arrest. Using synchronized B. subtilis cells, we show that the use of blue light for fluorescence microscopy likely induces non-specific toxic effects, rather than a specific cell cycle arrest. Escherichia coli and Caulobacter crescentus cells also stop to grow after 15 one-second exposures to blue light (CFP), but continue growth when imaged under similar conditions in the YFP channel. In the case of E. coli, YFP excitation slows growth relative to white light excitation, whereas CFP excitation leads to cell death in a majority of cells. Thus, even mild violet/blue light excitation interferes with bacterial growth. Analyzing the dose-dependent effects of violet light in B. subtilis, we show that short exposures to low-intensity violet light allow for continued cell growth, while longer exposures do not., Conclusions: Our experiments show that care must be taken in the design of live-cell imaging experiments in that violet or blue excitation effects must be closely controlled during and after imaging. Violet excitation during sptPALM or other imaging studies involving photoactivation has a threshold, below which little effects can be seen, but above which a sharp transition into cell death occurs. YFP imaging proves to be better suited for time-lapse studies, especially when cell cycle or cell growth parameters are to be examined.

Bacillus subtilis contains two known cyclic di-GMP (c-di-GMP)-dependent receptors, YdaK and DgrA, as well as three diguanylate cyclases (DGCs): soluble DgcP and membrane-integral DgcK and DgcW. DgrA regulates motility, while YdaK is responsible for the formation of a putative exopolysaccharide, dependent on the activity of DgcK. Using single-molecule tracking, we show that a majority of DgcK molecules are statically positioned in the cell membrane but significantly less so in the absence of YdaK but more so upon overproduction of YdaK. The soluble domains of DgcK and of YdaK show a direct interaction in vitro , which depends on an intact I-site within the degenerated GGDEF domain of YdaK. These experiments suggest a direct handover of a second messenger at a single subcellular site. Interestingly, all three DGC proteins contribute toward downregulation of motility via the PilZ protein DgrA. Deletion of dgrA also affects the mobility of DgcK within the membrane and also that of DgcP, which arrests less often at the membrane in the absence of DgrA. Both, DgcK and DgcP interact with DgrA in vitro , showing that divergent as well as convergent direct connections exist between cyclases and their effector proteins. Automated determination of molecule numbers in live cells revealed that DgcK and DgcP are present at very low copy numbers of 6 or 25 per cell, respectively, such that for DgcK, a part of the cell population does not contain any DgcK molecule, rendering signaling via c-di-GMP extremely efficient. IMPORTANCE Second messengers are free to diffuse through the cells and to activate all responsive elements. Cyclic di-GMP (c-di-GMP) signaling plays an important role in the determination of the life style transition between motility and sessility/biofilm formation but involves numerous distinct synthetases (diguanylate cyclases [DGCs]) or receptor pathways that appear to act in an independent manner. Using Bacillus subtilis as a model organism, we show that for two c-di-GMP pathways, DGCs and receptor molecules operate via direct interactions, where a synthesized dinucleotide appears to be directly used for the protein-protein interaction. We show that very few DGC molecules exist within cells; in the case of exopolysaccharide (EPS) formation via membrane protein DgcK, the DGC molecules act at a single site, setting up a single signaling pool within the cell membrane. Using single-molecule tracking, we show that the soluble DGC DgcP arrests at the cell membrane, interacting with its receptor, DgrA, which slows down motility. DgrA also directly binds to DgcK, showing that divergent as well as convergent modules exist in B. subtilis Thus, local-pool signal transduction operates extremely efficiently and specifically., (Copyright © 2020 Kunz et al.)

Ubiquitous RarA AAA + ATPases play crucial roles in the cellular response to blocked replication forks in pro- and eukaryotes. Here, we provide evidence that absence of RarA reduced the viability of Δ recA , Δ recO , and recF 15 cells during unperturbed growth. The rarA gene was epistatic to recO and recF genes in response to H 2 O 2 - or MMS-induced DNA damage. Conversely, the inactivation of rarA partially suppressed the HR defect of mutants lacking end-resection (Δ addAB , Δ recJ , Δ recQ , Δ recS ) or branch migration (Δ ruvAB , Δ recG , Δ radA ) activity. RarA contributes to RecA thread formation, that are thought to be the active forms of RecA during homology search. The absence of RarA reduced RecA accumulation, and the formation of visible RecA threads in vivo upon DNA damage. When Δ rarA was combined with mutations in genuine RecA accessory genes, RecA accumulation was further reduced in Δ rarA Δ recU and Δ rarA Δ recX double mutant cells, and was blocked in Δ rarA recF 15 cells. These results suggest that RarA contributes to the assembly of RecA nucleoprotein filaments onto single-stranded DNA, and possibly antagonizes RecA filament disassembly., (Copyright © 2020 Romero, Serrano, Hernández-Tamayo, Carrasco, Cárdenas, Ayora, Graumann and Alonso.)