The many faces of the unusual biofilm activator RemA.

Biofilms can be viewed as tissue-like structures in which microorganisms are organized in a spatial and functional sophisticated manner. Biofilm formation requires the orchestration of a highly integrated network of regulatory proteins to establish cell differentiation and production of a complex extracellular matrix. Here, we discuss the role of the essential Bacillus subtilis biofilm activator RemA. Despite intense research on biofilms, RemA is a largely underappreciated regulatory protein. RemA forms donut-shaped octamers with the potential to assemble into dimeric superstructures. The presumed DNA-binding mode suggests that RemA organizes its target DNA into nucleosome-like structures, which are the basis for its role as transcriptional activator. We discuss how RemA affects gene expression in the context of biofilm formation, and its regulatory interplay with established components of the biofilm regulatory network, such as SinR, SinI, SlrR, and SlrA. We emphasize the additional role of RemA played in nitrogen metabolism and osmotic-stress adjustment.

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

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

TnFLX: a Third-Generation mariner-Based Transposon System for Bacillus subtilis.

Random transposon mutagenesis is a powerful and unbiased genetic approach to answer fundamental biological questions. Here, we introduce an improved mariner-based transposon system with enhanced stability during propagation and versatile applications in mutagenesis. We used a low-copy-number plasmid as a transposon delivery vehicle, which affords a lower frequency of unintended recombination during vector construction and propagation in Escherichia coli We generated a variety of transposons allowing for gene disruption or artificial overexpression, each in combination with one of four different antibiotic resistance markers. In addition, we provide transposons that will report gene/protein expression due to transcriptional or translational coupling. We believe that the TnFLX system will help enhance the flexibility of future transposon modification and application in Bacillus and other organisms.IMPORTANCE The stability of transposase-encoding vectors during cloning and propagation is crucial for the reliable application of transposons. Here, we increased the stability of the mariner delivery vehicle in E. coli Moreover, the TnFLX transposon system will improve the application of forward genetic methods with an increased number of antibiotic resistance markers and the ability to generate unbiased green fluorescent protein (GFP) fusions to report on protein translation and subcellular localization.

Super Resolution Fluorescence Microscopy and Tracking of Bacterial Flotillin (Reggie) Paralogs Provide Evidence for Defined-Sized Protein Microdomains within the Bacterial Membrane but Absence of Clusters Containing Detergent-Resistant Proteins.

Biological membranes have been proposed to contain microdomains of a specific lipid composition, in which distinct groups of proteins are clustered. Flotillin-like proteins are conserved between pro-and eukaryotes, play an important function in several eukaryotic and bacterial cells, and define in vertebrates a type of so-called detergent-resistant microdomains. Using STED microscopy, we show that two bacterial flotillins, FloA and FloT, form defined assemblies with an average diameter of 85 to 110 nm in the model bacterium Bacillus subtilis. Interestingly, flotillin microdomains are of similar size in eukaryotic cells. The soluble domains of FloA form higher order oligomers of up to several hundred kDa in vitro, showing that like eukaryotic flotillins, bacterial assemblies are based in part on their ability to self-oligomerize. However, B. subtilis paralogs show significantly different diffusion rates, and consequently do not colocalize into a common microdomain. Dual colour time lapse experiments of flotillins together with other detergent-resistant proteins in bacteria show that proteins colocalize for no longer than a few hundred milliseconds, and do not move together. Our data reveal that the bacterial membrane contains defined-sized protein domains rather than functional microdomains dependent on flotillins. Based on their distinct dynamics, FloA and FloT confer spatially distinguishable activities, but do not serve as molecular scaffolds.

Chromate Resistance Mechanisms in Leucobacter chromiiresistens.

Chromate is one of the major anthropogenic contaminants on Earth. Leucobacter chromiiresistens is a highly chromate-resistant strain, tolerating chromate concentrations in LB medium of up to 400 mM. In response to chromate stress, L. chromiiresistens forms biofilms, which are held together via extracellular DNA. Inhibition of biofilm formation leads to drastically decreased chromate tolerance. Moreover, chromate is reduced intracellularly to the less-toxic Cr(III). The oxidation status and localization of chromium in cell aggregates were analyzed by energy-dispersive X-ray spectroscopy coupled to scanning transmission electron microscopy and X-ray absorption spectroscopy measurements. Most of the heavy metal is localized as Cr(III) at the cytoplasmic membrane. As a new cellular response to chromate stress, we observed an increased production of the carotenoid lutein. Carotenoid production could increase membrane stability and reduce the concentration of reactive oxygen species. Bioinformatic analysis of the L. chromiiresistens genome revealed several gene clusters that could enable heavy-metal resistance. The extreme chromate tolerance and the unique set of resistance factors suggest the use of L. chromiiresistens as a new model organism to study microbial chromate resistance.IMPORTANCE Chromate is a highly toxic oxyanion. Extensive industrial use and inadequate waste management has caused the toxic pollution of several field sites. Understanding the chromate resistance mechanisms that enable organisms to thrive under these conditions is fundamental to develop (micro)biological strategies and applications aiming at bioremediation of contaminated soils or waters. Potential detoxifying microorganisms are often not sufficient in their resistance characteristics to effectively perform, e.g., chromate reduction or biosorption. In this study, we describe the manifold strategies of L. chromiiresistens to establish an extremely high level of chromate resistance. The multitude of mechanisms conferring it make this organism suitable for consideration as a new model organism to study chromate resistance.

MinD-like ATPase FlhG effects location and number of bacterial flagella during C-ring assembly.

The number and location of flagella, bacterial organelles of locomotion, are species specific and appear in regular patterns that represent one of the earliest taxonomic criteria in microbiology. However, the mechanisms that reproducibly establish these patterns during each round of cell division are poorly understood. FlhG (previously YlxH) is a major determinant for a variety of flagellation patterns. Here, we show that FlhG is a structural homolog of the ATPase MinD, which serves in cell-division site determination. Like MinD, FlhG forms homodimers that are dependent on ATP and lipids. It interacts with a complex of the flagellar C-ring proteins FliM and FliY (also FliN) in the Gram-positive, peritrichous-flagellated Bacillus subtilis and the Gram-negative, polar-flagellated Shewanella putrefaciens. FlhG interacts with FliM/FliY in a nucleotide-independent manner and activates FliM/FliY to assemble with the C-ring protein FliG in vitro. FlhG-driven assembly of the FliM/FliY/FliG complex is strongly enhanced by ATP and lipids. The protein shows a highly dynamic subcellular distribution between cytoplasm and flagellar basal bodies, suggesting that FlhG effects flagellar location and number during assembly of the C-ring. We describe the molecular evolution of a MinD-like ATPase into a flagellation pattern effector and suggest that the underappreciated structural diversity of the C-ring proteins might contribute to the formation of different flagellation patterns.

Bioorthogonal Enzymatic Activation of Caged Compounds.

Engineered cytochrome P450 monooxygenase variants are reported as highly active and selective catalysts for the bioorthogonal uncaging of propargylic and benzylic ether protected substrates, including uncaging in living E. coli. observed selectivity is supported by induced-fit docking and molecular dynamics simulations. This proof-of-principle study points towards the utility of bioorthogonal enzyme/protecting group pairs for applications in the life sciences.

Progress towards bioorthogonal catalysis with organometallic compounds.

The catalysis of bioorthogonal transformations inside living organisms is a formidable challenge--yet bears great potential for future applications in chemical biology and medicinal chemistry. We herein disclose highly active organometallic ruthenium complexes for bioorthogonal catalysis under biologically relevant conditions and inside living cells. The catalysts uncage allyl carbamate protected amines with unprecedented high turnover numbers of up to 270 cycles in the presence of water, air, and millimolar concentrations of thiols. By live-cell imaging of HeLa cells and with the aid of a caged fluorescent probe we could reveal a rapid development of intense fluorescence within the cellular cytoplasm and therefore support the proposed bioorthogonality of the catalysts. In addition, to illustrate the manifold applications of bioorthogonal catalysis, we developed a method for catalytic in-cell activation of a caged anticancer drug, which efficiently induced apoptosis in HeLa cells.

Localization of FtsZ in Helicobacter pylori and consequences for cell division.

Of the various kinds of cell division, the most common mode is binary fission, the division of a cell into two morphologically identical daughter cells. However, in the case of asymmetric cell division, Caulobacter crescentus produces two morphologically and functionally distinct cell types. Here, we have studied cell cycle progression of the human pathogen Helicobacter pylori using a functional green fluorescent protein (GFP) fusion of FtsZ protein and membrane staining. In small cells, representing newly divided cells, FtsZ localizes to a single cell pole. During the cell cycle, spiral intermediates are formed until an FtsZ ring is positioned with very little precision, such that central as well as acentral rings can be observed. Daughter cells showed considerably different sizes, suggesting that H. pylori divides asymmetrically. Fluorescence recovery after photobleaching (FRAP) analyses demonstrate that the H. pylori FtsZ ring is about as dynamic as that of Escherichia coli but that polar assemblies show less turnover. Strikingly, our results demonstrate that H. pylori cell division follows a different route from that in E. coli and Bacillus subtilis. It is also different from that in C. crescentus, where cytokinesis regulation proteins like MipZ play a role. Therefore, this report provides the first cell-biological analysis of FtsZ dynamics in the human pathogen H. pylori and even in epsilonproteobacteria to our knowledge. In addition, analysis of the filament architecture of H. pylori and E. coli FtsZ filaments in the heterologous system of Drosophila melanogaster S2 Schneider cells revealed that both have different filamentation properties in vivo, suggesting a unique intrinsic characteristic of each protein.

Structural and functional analysis of the cerato-platanin-like protein Cpl1 suggests diverging functions in smut fungi.

Plant-pathogenic fungi are causative agents of the majority of plant diseases and can lead to severe crop loss in infected populations. Fungal colonization is achieved by combining different strategies, such as avoiding and counteracting the plant immune system and manipulating the host metabolome. Of major importance are virulence factors secreted by fungi, which fulfil diverse functions to support the infection process. Most of these proteins are highly specialized, with structural and biochemical information often absent. Here, we present the atomic structures of the cerato-platanin-like protein Cpl1 from Ustilago maydis and its homologue Uvi2 from Ustilago hordei. Both proteins adopt a double-Ψß-barrel architecture reminiscent of cerato-platanin proteins, a class so far not described in smut fungi. Our structure-function analysis shows that Cpl1 binds to soluble chitin fragments via two extended grooves at the dimer interface of the two monomer molecules. This carbohydrate-binding mode has not been observed previously and expands the repertoire of chitin-binding proteins. Cpl1 localizes to the cell wall of U. maydis and might synergize with cell wall-degrading and decorating proteins during maize infection. The architecture of Cpl1 harbouring four surface-exposed loop regions supports the idea that it might play a role in the spatial coordination of these proteins. While deletion of cpl1 has only mild effects on the virulence of U. maydis, a recent study showed that deletion of uvi2 strongly impairs U. hordei virulence. Our structural comparison between Cpl1 and Uvi2 reveals sequence variations in the loop regions that might explain a diverging function.

Synthetic motility and cell shape defects associated with deletions of flotillin/reggie paralogs in Bacillus subtilis and interplay of these proteins with NfeD proteins.

Flotillin/reggie proteins are membrane-associated proteins present in all kinds of cells and belong to the family of proteins carrying the SPFH (stomatin, prohibitin, flotillin, and HflK/HflC) domain. In addition to this domain of unknown function, flotillin proteins are characterized by the flotillin domain, which is rich in heptad repeats. Bacterial flotillin orthologs have recently been shown to be part of lipid rafts, like their eukaryotic counterparts, and to be involved in signaling events. Double deletions of floT and the gene encoding the second flotillin-like protein in Bacillus subtilis, floA, show strong synthetic defects in cell morphology, motility, and transformation efficiency. The lack of FloT resulted in a marked defect in motility. Using total internal reflection fluorescence (TIRF) microscopy, we show that both proteins localize in characteristic focal structures within the cell membrane, which move in a highly dynamic and random manner but localize independently of each other. Thus, flotillin paralogs act in a spatially distinct manner. Flotillin domains in both FloA and FloT are essential for focal assemblies and for the proper function of flotillins. Both flotillin genes are situated next to genes encoding NfeD proteins. FloT dramatically affects the localization of NfeD2: FloT apparently recruits NfeD2 into the focal assemblies, documenting a close interaction between flotillins and NfeDs in bacteria. In contrast, the localization of NfeD1b is not affected by FloA, FloT, or NfeD2. FloA does not show a spatial connection with the upstream-encoded NfeD1b (YqeZ). Our work establishes that bacterial flotillin-like proteins have overlapping functions in a variety of membrane-associated processes and that flotillin domain-mediated assembly and NfeD proteins play important roles in setting up the flotillin raft-like structures in vivo.

The deletion of bacterial dynamin and flotillin genes results in pleiotrophic effects on cell division, cell growth and in cell shape maintenance.

BACKGROUND: In eukaryotic cells, dynamin and flotillin are involved in processes such as endocytosis and lipid raft formation, respectively. Dynamin is a GTPase that exerts motor-like activity during the pinching off of vesicles, while flotillins are coiled coil rich membrane proteins with no known enzymatic activity. Bacteria also possess orthologs of both classes of proteins, but their function has been unclear. RESULTS: We show that deletion of the single dynA or floT genes lead to no phenotype or a mild defect in septum formation in the case of the dynA gene, while dynA floT double mutant cells were highly elongated and irregularly shaped, although the MreB cytoskeleton appeared to be normal. DynA colocalizes with FtsZ, and the dynA deletion strain shows aberrant FtsZ rings in a subpopulation of cells. The mild division defect of the dynA deletion is exacerbated by an additional deletion in ezrA, which affects FtsZ ring formation, and also by the deletion of a late division gene (divIB), indicating that DynA affects several steps in cell division. DynA and mreB deletions generated a synthetic defect in cell shape maintenance, showing that MreB and DynA play non-epistatic functions in cell shape maintenance. TIRF microscopy revealed that FloT forms many dynamic membrane assemblies that frequently colocalize with the division septum. The deletion of dynA did not change the pattern of localization of FloT, and vice versa, showing that the two proteins play non redundant roles in a variety of cellular processes. Expression of dynamin or flotillin T in eukaryotic S2 cells revealed that both proteins assemble at the cell membrane. While FloT formed patch structures, DynA built up tubulated structures extending away from the cells. CONCLUSIONS: Bacillus subtilis dynamin ortholog DynA plays a role during cell division and in cell shape maintenance. It shows a genetic link with flotillin T, with both proteins playing non-redundant functions at the cell membrane, where they assemble even in the absence of any bacterial cofactor.

TnFLXopen: Markerless Transposons for Functional Fluorescent Fusion Proteins and Protein Interaction Prediction.

Fluorescence microscopy of cells expressing proteins translationally linked to a fluorophore can be a powerful tool to investigate protein localization dynamics in vivo. One major obstacle to reliably analyze biologically relevant localization is the construction of a fusion protein that is both fluorescent and functional. Here, we develop a strategy to construct fluorescent fusions at theoretically any location in the protein by using TnFLXopen random transposon mutagenesis to randomly insert a gene encoding a fluorescent protein. Moreover, insertions within a target gene are enriched by an inducible gene-trap strategy and selection by fluorescence activated cell sorting. Using this approach, we isolate a variety of fluorescent fusions to FtsZ that exhibit ring-like localization and a fusion to the flagellar stator protein that both is functional for supporting motility and localizes as fluorescent puncta. Finally, we further modify TnFLXopen to insert the coding sequence for the C-terminal half of mVenus for use in bimolecular fluorescence complementation (BiFC) and the in vivo detection of protein-protein interaction candidates. As proof-of-concept, the DivIVA polar scaffolding protein was fused to the N terminus of mVenus, the C terminus of mVenus was delivered by transposition, and a combination of fluorescence activated cell sorter (FACS) sorting and whole-genome sequencing identified the known self-interaction of DivIVA as well as other possible candidate interactors. We suggest that the FACS selection is a viable alternative to antibiotic selection in transposon mutagenesis that can generate new fluorescent tools for in vivo protein characterization. IMPORTANCE Transposon mutagenesis is a powerful tool for random mutagenesis, as insertion of a transposon and accompanying antibiotic resistance cassette often disrupt gene function. Here, we present a series of transposons with fluorescent protein genes which, when integrated in frame, may be selected with a fluorescence activated cell sorter (FACS). An open reading frame runs continuously through the transposon such that fluorescent protein fusions may be inserted theoretically anywhere in the primary sequence and potentially preserve function of the target protein. Finally, the transposons were further modified to randomly insert a partial fluorescent protein compatible with bimolecular fluorescence complementation (BiFC) to identify protein interaction candidates.

A novel system of cytoskeletal elements in the human pathogen Helicobacter pylori.

Pathogenicity of the human pathogen Helicobacter pylori relies upon its capacity to adapt to a hostile environment and to escape from the host response. Therefore, cell shape, motility, and pH homeostasis of these bacteria are specifically adapted to the gastric mucus. We have found that the helical shape of H. pylori depends on coiled coil rich proteins (Ccrp), which form extended filamentous structures in vitro and in vivo, and are differentially required for the maintenance of cell morphology. We have developed an in vivo localization system for this pathogen. Consistent with a cytoskeleton-like structure, Ccrp proteins localized in a regular punctuate and static pattern within H. pylori cells. Ccrp genes show a high degree of sequence variation, which could be the reason for the morphological diversity between H. pylori strains. In contrast to other bacteria, the actin-like MreB protein is dispensable for viability in H. pylori, and does not affect cell shape, but cell length and chromosome segregation. In addition, mreB mutant cells displayed significantly reduced urease activity, and thus compromise a major pathogenicity factor of H. pylori. Our findings reveal that Ccrp proteins, but not MreB, affect cell morphology, while both cytoskeletal components affect the development of pathogenicity factors and/or cell cycle progression.

The MocR/GabR Ectoine and Hydroxyectoine Catabolism Regulator EnuR: Inducer and DNA Binding.

The compatible solutes ectoine and 5-hydroxyectoine are widely synthesized by bacteria as osmostress protectants. These nitrogen-rich tetrahydropyrimidines can also be exploited as nutrients by microorganisms. Many ectoine/5-hydroxyectoine catabolic gene clusters are associated with a regulatory gene (enuR: ectoine nutrient utilization regulator) encoding a repressor protein belonging to the MocR/GabR sub-family of GntR-type transcription factors. Focusing on EnuR from the marine bacterium Ruegeria pomeroyi, we show that the dimerization of EnuR is mediated by its aminotransferase domain. This domain can fold independently from its amino-terminal DNA reading head and can incorporate pyridoxal-5'-phosphate (PLP) as cofactor. The covalent attachment of PLP to residue Lys302 of EnuR was proven by mass-spectrometry. PLP interacts with system-specific, ectoine and 5-hydroxyectoine-derived inducers: alpha-acetyldiaminobutyric acid (alpha-ADABA), and hydroxy-alpha-acetyldiaminobutyric acid (hydroxy-alpha-ADABA), respectively. These inducers are generated in cells actively growing with ectoines as sole carbon and nitrogen sources, by the EutD hydrolase and targeted metabolic analysis allowed their detection. EnuR binds these effector molecules with affinities in the low micro-molar range. Studies addressing the evolutionary conservation of EnuR, modelling of the EnuR structure, and docking experiments with the inducers provide an initial view into the cofactor and effector binding cavity. In this cavity, the two high-affinity inducers for EnuR, alpha-ADABA and hydroxy-alpha-ADABA, are positioned such that their respective primary nitrogen group can chemically interact with PLP. Purified EnuR bound with micro-molar affinity to a 48 base pair DNA fragment containing the sigma-70 type substrate-inducible promoter for the ectoine/5-hydroxyectoine importer and catabolic gene cluster. Consistent with the function of EnuR as a repressor, the core elements of the promoter overlap with two predicted EnuR operators. Our data lend themselves to a straightforward regulatory model for the initial encounter of EnuR-possessing ectoine/5-hydroxyectoine consumers with environmental ectoines and for the situation when the external supply of these compounds has been exhausted by catabolism.

Harnessing ß-Lactam Antibiotics for Illumination of the Activity of Penicillin-Binding Proteins in Bacillus subtilis.

Selective chemical probes enable individual investigation of penicillin-binding proteins (PBPs) and provide critical information about their enzymatic activity with spatial and temporal resolution. To identify scaffolds for novel probes to study peptidoglycan biosynthesis in Bacillus subtilis, we evaluated the PBP inhibition profiles of 21 ß-lactam antibiotics from different structural subclasses using a fluorescence-based assay. Most compounds readily labeled PBP1, PBP2a, PBP2b, or PBP4. Almost all penicillin scaffolds were coselective for all or combinations of PBP2a, 2b, and 4. Cephalosporins, on the other hand, possessed the lowest IC50 values for PBP1 alone or along with PBP4 (ceftriaxone, cefoxitin) and 2b (cefotaxime) or 2a, 2b, and 4 (cephalothin). Overall, five selective inhibitors for PBP1 (aztreonam, faropenem, piperacillin, cefuroxime, and cefsulodin), one selective inhibitor for PBP5 (6-aminopenicillanic acid), and various coselective inhibitors for other PBPs in B. subtilis were discovered. Surprisingly, carbapenems strongly inhibited PBP3, formerly shown to have low affinity for ß-lactams and speculated to be involved in ß-lactam resistance in B. subtilis. To investigate the specific roles of PBP3, we developed activity-based probes based on the meropenem core and utilized them to monitor the activity of PBP3 in living cells. We showed that PBP3 activity localizes as patches in single cells and concentrates as a ring at the septum and the division site during the cell growth cycle. Our activity-based approach enabled spatial resolution of the transpeptidation activity of individual PBPs in this model microorganism, which was not possible with previous chemical and biological approaches.

Role for Cell-Surface Collagen of Streptococcus pyogenes in Infections.

Group A Streptococcus (GAS) displays cell-surface proteins that resemble human collagen. We find that a fluorophore-labeled collagen mimetic peptide (CMP) labels GAS cells but not Escherichia coli or Bacillus subtilis cells, which lack such proteins. The CMP likely engages in a heterotrimeric helix with endogenous collagen, as the nonnatural d enantiomer of the CMP does not label GAS cells. To identify a molecular target, we used reverse genetics to "knock-in" the GAS genes that encode two proteins with collagen-like domains, Scl1 and Scl2, into B. subtilis. The fluorescent CMP labels the cells of these B. subtilis strains. Moreover, these strains bind tightly to a surface of mammalian collagen. These data are consistent with streptococcal collagen forming triple helices with damaged collagen in a wound bed and thus have implications for microbial virulence.

The Min System Disassembles FtsZ Foci and Inhibits Polar Peptidoglycan Remodeling in Bacillus subtilis.

A microfluidic system coupled with fluorescence microscopy is a powerful approach for quantitative analysis of bacterial growth. Here, we measure parameters of growth and dynamic localization of the cell division initiation protein FtsZ in Bacillus subtilis Consistent with previous reports, we found that after division, FtsZ rings remain at the cell poles, and polar FtsZ ring disassembly coincides with rapid Z-ring accumulation at the midcell. In cells mutated for minD, however, the polar FtsZ rings persist indefinitely, suggesting that the primary function of the Min system is in Z-ring disassembly. The inability to recycle FtsZ monomers in the minD mutant results in the simultaneous maintenance of multiple Z-rings that are restricted by competition for newly synthesized FtsZ. Although the parameters of FtsZ dynamics change in the minD mutant, the overall cell division time remains the same, albeit with elongated cells necessary to accumulate a critical threshold amount of FtsZ for promoting medial division. Finally, the minD mutant characteristically produces minicells composed of polar peptidoglycan shown to be inert for remodeling in the wild type. Polar peptidoglycan, however, loses its inert character in the minD mutant, suggesting that the Min system not only is important for recycling FtsZ but also may have a secondary role in the spatiotemporal regulation of peptidoglycan remodeling.IMPORTANCE Many bacteria grow and divide by binary fission in which a mother cell divides into two identical daughter cells. To produce two equally sized daughters, the division machinery, guided by FtsZ, must dynamically localize to the midcell each cell cycle. Here, we quantitatively analyzed FtsZ dynamics during growth and found that the Min system of Bacillus subtilis is essential to disassemble FtsZ rings after division. Moreover, a failure to efficiently recycle FtsZ results in an increase in cell size. Finally, we show that the Min system has an additional role in inhibiting cell wall turnover and contributes to the "inert" property of cell walls at the poles.

Genetic links between bacterial dynamin and flotillin proteins.

Dynamin is a membrane-associated GTPase that confers motor-like functions in membrane dynamics, such as endocytosis, in eukaryotic cells. Flotillin (reggie) proteins are also a widely conserved class of membrane proteins, associated with the formation of protein assemblies within the membrane, and with endocytotic processes. Bacterial dynamin has been shown to bind to membranes in vitro and to mediate membrane fusion. Bacillus subtilis DynA localizes to the cell division septum, and it was recently shown that it indeed plays a role in cell division. Interestingly, dynamin shows a genetic interaction with flotillin proteins in this prokaryotic model organism and the absence of both proteins results in a cell division and cell shape defect. Here, we show that in addition to the morphological phenotypes, a dynamin/flotillin double deletion strain shows a synthetic defect in cell motility, much stronger than that of flotillin single mutant cells. While the contribution of altered cell shape and slower growth of the double deletion strain on motility cannot be clearly assessed, our data emphasize the fact that dynamin and flotillin proteins play tightly connected functions in a wide range of aspects in membrane processes in bacteria.

Polar localization of a tripartite complex of the two-component system DcuS/DcuR and the transporter DctA in Escherichia coli depends on the sensor kinase DcuS.

The C4-dicarboxylate responsive sensor kinase DcuS of the DcuS/DcuR two-component system of E. coli is membrane-bound and reveals a polar localization. DcuS uses the C4-dicarboxylate transporter DctA as a co-regulator forming DctA/DcuS sensor units. Here it is shown by fluorescence microscopy with fusion proteins that DcuS has a dynamic and preferential polar localization, even at very low expression levels. Single assemblies of DcuS had high mobility in fast time lapse acquisitions, and fast recovery in FRAP experiments, excluding polar accumulation due to aggregation. DctA and DcuR fused to derivatives of the YFP protein are dispersed in the membrane or in the cytosol, respectively, when expressed without DcuS, but co-localize with DcuS when co-expressed at appropriate levels. Thus, DcuS is required for location of DctA and DcuR at the poles and formation of tripartite DctA/DcuS/DcuR sensor/regulator complexes. Vice versa, DctA, DcuR and the alternative succinate transporter DauA were not essential for polar localization of DcuS, suggesting that the polar trapping occurs by DcuS. Cardiolipin, the high curvature at the cell poles, and the cytoskeletal protein MreB were not required for polar localization. In contrast, polar localization of DcuS required the presence of the cytoplasmic PAS(C) and the kinase domains of DcuS.