B. subtilis Sec and Srp Systems Show Dynamic Adaptations to Different Conditions of Protein Secretion.

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.

Dynamics of cell wall-binding proteins at a single molecule level: B. subtilis autolysins show different kinds of motion.

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.

A dynamic bactofilin cytoskeleton cooperates with an M23 endopeptidase to control bacterial morphogenesis.

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.

Uptake of environmental DNA in Bacillus subtilis occurs all over the cell surface through a dynamic pilus structure.

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.

Protein secretion zones during overexpression of amylase within the Gram-positive cell wall.

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.

DipM controls multiple autolysins and mediates a regulatory feedback loop promoting cell constriction in Caulobacter crescentus.

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.

Fortuitously compatible protein surfaces primed allosteric control in cyanobacterial photoprotection.

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.

Factors affecting mixed-mode retention properties of cation-exchange stationary phases.

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.

Intranasal administration of Acinetobacter lwoffii in a murine model of asthma induces IL-6-mediated protection associated with cecal microbiota changes.

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.

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

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

ATPase Activity of Bacillus subtilis RecA Affects the Dynamic Formation of RecA Filaments at DNA Double Strand Breaks.

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.

Molecular basis for lethal cross-talk between two unrelated bacterial transcription factors - the regulatory protein of a restriction-modification system and the repressor of a defective prophage.

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.

Heterogeneity of Subcellular Diffusion in Bacteria Based on Spatial Segregation of Ribosomes and Nucleoids.

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.

Characterization of the self-targeting Type IV CRISPR interference system in Pseudomonas oleovorans.

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.

Tight Complex Formation of the Fumarate Sensing DcuS-DcuR Two-Component System at the Membrane and Target Promoter Search by Free DcuR Diffusion.

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 (PASC-Kin) was employed. Stable complexes were formed, when either DcuS or DcuR were phosphorylated (Kd 22 ± 11 and 28 ± 7 nM, respectively). The unphosphorylated proteins produced a more labile complex (Kd 1380 ± 395 nM). Bacterial two-hybrid studies confirm interaction of DcuR with DcuS (and PASC-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 Kd 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.

The roles of intracellular and extracellular calcium in Bacillus subtilis biofilms.

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.

The Acetyltransferase RibT From Bacillus subtilis Affects in vivo Dynamics of the Multimeric Heavy Riboflavin Synthase Complex.

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.

Strain-dependent motility defects and suppression by a flhO mutation for B. subtilis bactofilins.

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.

GGDEF domain as spatial on-switch for a phosphodiesterase by interaction with landmark protein HubP.

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.

Y-Complex Proteins Show RNA-Dependent Binding Events at the Cell Membrane and Distinct Single-Molecule Dynamics.

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.