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Serratia marcescens is an opportunistic human pathogen that produces a vibrant red pigment called prodigiosin. Prodigiosin has implications in virulence of S. marcescens and promising clinical applications. We discovered that addition of the virulent flagellotropic bacteriophage χ (Chi) to a culture of S. marcescens stimulates a greater than fivefold overproduction of prodigiosin. Active phage infection is required for the effect, as a χ-resistant strain lacking flagella does not respond to phage presence. Via a reporter fusion assay, we have determined that the addition of a χ-induced S. marcescens cell lysate to an uninfected culture causes a threefold increase in transcription of the pig operon, containing genes essential for pigment biosynthesis. Replacement of the pig promoter with a constitutive promoter abolished the pigmentation increase, indicating that regulatory elements present in the pig promoter likely mediate the phenomenon. We hypothesize that S. marcescens detects the threat of phage-mediated cell death and reacts by producing prodigiosin as a stress response. Our findings are of clinical significance for two main reasons: (i) elucidating complex phage-host interactions is crucial for development of therapeutic phage treatments, and (ii) overproduction of prodigiosin in response to phage could be exploited for its biosynthesis and use as a pharmaceutical. [ABSTRACT FROM AUTHOR]

Sensory adaptation in bacterial chemotaxis is mediated by posttranslational modifications of methyl‐accepting chemotaxis proteins (MCPs). In Escherichia coli, the adaptation proteins CheR and CheB tether to a conserved C‐terminal receptor pentapeptide. Here,we investigated the function of the pentapeptide motif (N/D)WE(E/N)F in Sinorhizobium meliloti chemotaxis. Isothermal titration calorimetry revealed stronger affinity of the pentapeptides to CheR and activated CheB relative to unmodified CheB. Strains with mutations of the conserved tryptophan in one or all four MCP pentapeptides resulted in a significant decrease or loss of chemotaxis to glycine betaine, lysine, and acetate, chemoattractants sensed by pentapeptide‐bearing McpX and pentapeptide‐lacking McpU and McpV, respectively. Importantly, we discovered that the pentapeptide mediates chemotaxis when fused to the C‐terminus of pentapeptide‐lacking chemoreceptors via a flexible linker. We propose that adaptational assistance and a threshold number of available sites enable the efficient docking of adaptation proteins to the chemosensory array. Altogether, these results demonstrate that S. meliloti effectively utilizes a pentapeptide‐dependent adaptation system with a minimal number of tethering units to assist pentapeptide‐lacking chemoreceptors and hypothesize that the higher abundance of CheR and CheB in S. meliloti compared to E. coli allows for ample recruitment of adaptation proteins to the chemosensory array. [ABSTRACT FROM AUTHOR]

The flagellar motor is a powerful macromolecular machine used to propel bacteria through various environments. We determined that flagellar motility of the alpha‐proteobacterium Sinorhizobium meliloti is nearly abolished in the absence of the transcriptional regulator LdtR, known to influence peptidoglycan remodeling and stress response. LdtR does not regulate motility gene transcription. Remarkably, the motility defects of the ΔldtR mutant can be restored by secondary mutations in the motility gene motA or a previously uncharacterized gene in the flagellar regulon, which we named motS. MotS is not essential for S. meliloti motility and may serve an accessory role in flagellar motor function. Structural modeling predicts that MotS comprised an N‐terminal transmembrane segment, a long‐disordered region, and a conserved β‐sandwich domain. The C terminus of MotS is localized in the periplasm. Genetics based substitution of MotA with MotAG12S also restored the ΔldtR motility defect. The MotAG12S variant protein features a local polarity shift at the periphery of the MotAB stator units. We propose that MotS may be required for optimal alignment of stators in wild‐type flagellar motors but becomes detrimental in cells with altered peptidoglycan. Similarly, the polarity shift in stator units composed of MotB/MotAG12S might stabilize its interaction with altered peptidoglycan. [ABSTRACT FROM AUTHOR]

Bacteriophages (phages) are the most abundant biological entities in the biosphere. As viruses that solely infect bacteria, phages have myriad healthcare and agricultural applications including phage therapy and antibacterial treatments in the foodservice industry. Phage therapy has been explored since the turn of the twentieth century but was no longer prioritized following the invention of antibiotics. As we approach a post-antibiotic society, phage therapy research has experienced a significant resurgence for the use of phages against antibiotic-resistant bacteria, a growing concern in modern medicine. Phages are extraordinarily diverse, as are their host receptor targets. Flagellotropic (flagellum-dependent) phages begin their infection cycle by attaching to the flagellum of their motile host, although the later stages of the infection process of most of these phages remain elusive. Flagella are helical appendages required for swimming and swarming motility and are also of great importance for virulence in many pathogenic bacteria of clinical relevance. Not only is bacterial motility itself frequently important for virulence, as it allows pathogenic bacteria to move toward their host and find nutrients more effectively, but flagella can also serve additional functions including mediating bacterial adhesion to surfaces. Flagella are also a potent antigen recognized by the human immune system. Phages utilizing the flagellum for infections are of particular interest due to the unique evolutionary tradeoff they force upon their hosts: by downregulating or abolishing motility to escape infection by a flagellotropic phage, a pathogenic bacterium would also likely attenuate its virulence. This factor may lead to flagellotropic phages becoming especially potent antibacterial agents. This review outlines past, present, and future research of flagellotropic phages, including their molecular mechanisms of infection and potential future applications. [ABSTRACT FROM AUTHOR]

The bacterial flagellum is a rotary motor organelle and important virulence factor that propels motile pathogenic bacteria, such as Salmonella enterica, through their surroundings. Bacteriophages, or phages, are viruses that solely infect bacteria. As such, phages have myriad applications in the healthcare field, including phage therapy against antibiotic-resistant bacterial pathogens. Bacteriophage χ (Chi) is a flagellum-dependent (flagellotropic) bacteriophage, which begins its infection cycle by attaching its long tail fiber to the S. enterica flagellar filament as its primary receptor. The interactions between phage and flagellum are poorly understood, as are the reasons that χ only kills certain Salmonella serotypes while others entirely evade phage infection. In this study, we used molecular cloning, targeted mutagenesis, heterologous flagellin expression, and phage-host interaction assays to determine which domains within the flagellar filament protein flagellin mediate this complex interaction. We identified the antigenic N- and C-terminal D2 domains as essential for phage χ binding, with the hypervariable central D3 domain playing a less crucial role. Here, we report that the primary structure of the Salmonella flagellin D2 domains is the major determinant of χ adhesion. The phage susceptibility of a strain is directly tied to these domains. We additionally uncovered important information about flagellar function. The central and most variable domain, D3, is not required for motility in S. Typhimurium 14028s, as it can be deleted or its sequence composition can be significantly altered with minimal impacts on motility. Further knowledge about the complex interactions between flagellotropic phage χ and its primary bacterial receptor may allow genetic engineering of its host range for use as targeted antimicrobial therapy against motile pathogens of the χ-host genera Salmonella, Escherichia, or Serratia. Author summary: Multidrug-resistant (MDR) bacteria kill over one million people per year worldwide. For instance, MDR typhoid fever is a significant concern in developing countries. As we precipitously approach a post-antibiotic society, bacteriophage therapy is a promising solution to the antibiotic crisis. Bacteriophage χ is a virus capable of infecting three species of pathogenic bacteria via their flagella, a significant virulence factor. Flagellum-dependent phages exploit an evolutionary tradeoff: a bacterium suppressing motility to avoid infection by such a phage would likely attenuate its own virulence. This makes flagellum-dependent phages of particular interest for potent therapy against MDR pathogens. In this study, we unveil crucial information about the mechanism of infection of Salmonella enterica by χ, knowledge which has the potential to lead to genetic modification of χ phage's host range and subsequent clinical applications. Similar studies can be conducted in other phage systems to broaden humanity's repertoire of potent therapeutic bacteriophages. [ABSTRACT FROM AUTHOR]

Since the discovery of protein phosphorylation in bacterial nitrogen assimilation and chemotaxis more than 30 years ago, many biochemical techniques for the analysis of two-component signal transduction systems have been developed. Over time the experimental conditions to follow the flow of phosphate groups from histidine kinases to the cognate response regulators in vitro have been fine tuned. Several approaches were applied to circumvent the instability of the phosphorylated form of response regulator proteins to analyze the structures of their activated forms. Recently, a FRET (fluorescence resonance energy transfer) assay was developed to monitor interactions of chemotaxis proteins in vivo. The availability of bacterial genome sequence databases has facilitated the identification of two-component systems and enabled prediction of interacting kinase-response regulators pairs. [Copyright &y& Elsevier]

Bacterial chemoreceptors primarily locate in clusters at the cell pole, where they form large sensory complexes which recruit cytoplasmic components of the signaling pathway. The genome of the soil bacterium Sinorhizobium meliloti encodes seven transmembrane and two soluble chemoreceptors. We have investigated the localization of all nine chemoreceptors in vivo using genome-encoded fusions to a variant of the enhanced green fluorescent protein and to monomeric red fluorescent protein. Six of the transmembrane (McpT to McpX and McpZ) and both soluble (McpY and IcpA) receptors localize to the cell pole. Only McpS, encoded from the symbiotic plasmid pSymA, is evenly distributed in the cell. While the synthesis of all polar localized receptors is confined to exponential growth correlating with the motility phase of cells, McpS is only weakly expressed throughout cell culture growth. Therefore, motile S. meliloti cells form one major chemotaxis cluster that harbors all chemoreceptors except for McpS. Colocalization and deletion analysis demonstrated that formation of polar foci by the majority of receptors is dependent on other chemoreceptors and that receptor clusters are stabilized by the presence of the chemotaxis proteins CheA and CheW. The transmembrane McpV and the soluble IcpA localize to the pole independently of CheA and CheW. However, in mutant strains McpV formed delocalized polar caps that spread throughout the cell membrane while IcpA exhibited increased bipolarity. Immunoblotting of fractionated cells revealed that IcpA, which lacks any hydrophobic domains, nevertheless is associated to the cell membrane. [ABSTRACT FROM AUTHOR]

This past January, in Cuernavaca Mexico, a conglomerate of scientists met to discuss the contemporary view of Bacterial Locomotion and Signal Transduction (BLAST). The BLAST meetings represent a field that has its roots in chemotaxis and the flagellum-based motility but now encompass all types of cellular movement and signalling. The topics varied from the interactions between molecules to the interactions between species. We heard about 3D reconstructions of transmembrane chemoreceptors within cells, new biophysical methods for understanding cellular engines, intricate phosphorelays, elaborate gene networks, new messenger molecules and emerging behaviours within complex populations of cells. At BLAST X we gained an appreciation for the lifestyle choices bacteria make, how they get to where they are going and the molecular mechanisms that underlie their decisions. Herein we review the highlights of the meeting. [ABSTRACT FROM AUTHOR]

fliL is the first gene in a flagellar operon that specifies members of the switch complex and type III export system in Salmonella enterica and Escherichia coli, but no function has been ascribed to this gene thus far. Here we report that a fliL mutant is slightly impaired for swimming but completely defective in swarming in both organisms, and have studied this phenotype further in S. enterica. We have found that on swarm agar, mutant cells release or ‘eject’ their flagellar filaments. The released filaments are attached to the hook and part of the rod structure; we have identified the distal rod protein FlgG but not the proximal rod protein FlgF in these filaments. Rod fracture was not observed if flagellar rotation was prevented by removal of proteins that supply proton flow through the motor. Based on these and other results, we propose that motors experience a higher torque on swarm agar owing to an increased proton motive force, and that FliL allows the rod to withstand the increased torsional stress. The flagella-release phenotype of the S. enterica fliL mutant has a bearing on FliL-dependent flagellar ejection during the swimmer- to stalk-cell transition in the developmental cycle of Caulobacter crescentus. [ABSTRACT FROM AUTHOR]

Sinorhizobium meliloti senses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novel S. meliloti chemotaxis protein CheT. While CheT and the Escherichia coli phosphatase CheZ share little sequence homology, CheT is predicted to possess an α‐helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate‐sink response regulator, CheY1~P by enhancing its decay two‐fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a KD of 2.9 μM, which is 25‐fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheT mutant and cheT DXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a KD of 19 μM, and a SEC‐MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation. [ABSTRACT FROM AUTHOR]

The rapid discovery of new and diverse bacteriophages has driven the innovation of approaches aimed at detailing interactions with their bacterial hosts. Previous studies on receptor binding proteins (RBPs) mainly relied on their identification in silico and are based on similarities to well-characterized systems. Thus, novel phage RBPs unlike those currently annotated in genomic and proteomic databases remain largely undiscovered. In this study, we employed a screen to identify RBPs in flagellotropic Agrobacterium phage 7-7-1. Flagellotropic phages utilize bacterial flagella as receptors. The screen identified three candidate RBPs, Gp4, Gp102, and Gp44. Homology modelling predicted that Gp4 is a trimeric, tail associated protein with a central β-barrel, while the structure and function of Gp102 and Gp44 are less obvious. Studies with purified Gp41-247 confirmed its ability to bind and interact with host cells, highlighting the robustness of the RBP screen. We also discovered that Gp41-247 inhibits the growth of host cells in a motility and lipopolysaccharide (LPS) dependent fashion. Hence, our results suggest interactions between Gp41-247, rotating flagellar filaments and host glycans to inhibit host cell growth, which presents an impactful and intriguing focus for future studies. [ABSTRACT FROM AUTHOR]

Looks at the reaction of Escherichia coli, a motile bacterium to a variety of stimuli by modulating the direction of rotation of their flagella. Examination of a binding process in relation to this topic; Methodology used in this experiment; Results from this experiment.

Highlights a study which analyzed the impact of the Escherichia coli's wild type cells, on CheY. Response of flagellated bacteria to positive or negative environmental stimuli; Activity of CheY mutants; Methodology used to conduct the study; Results of the study.

The flagellotropic bacteriophage χ (Chi) infects bacteria via the flagellar filament. Despite years of study, its structural architecture remains partly characterized. Through cryo-EM, we unveil χ′s nearly complete structure, encompassing capsid, neck, tail, and tail tip. While the capsid and tail resemble phage YSD1, the neck and tail tip reveal new proteins and their arrangement. The neck shows a unique conformation of the tail tube protein, forming a socket-like structure for attachment to the neck. The tail tip comprises four proteins, including distal tail protein (DTP), two baseplate hub proteins (BH1P and BH2P), and tail tip assembly protein (TAP) exhibiting minimal organization compared to other siphophages. Deviating from the consensus in other siphophages, DTP in χ forms a trimeric assembly, reducing tail symmetry from 6-fold to 3-fold at the tip. These findings illuminate the previously unexplored structural organization of χ's neck and tail tip. [Display omitted] • Cryo-EM structures of bacteriophage χ's neck and tail tip are solved • Tail tube hexamer adopts a unique conformation at χ's tail-neck junction • Trimeric distal tail protein reduces χ's tail tip symmetry from 6-fold to 3-fold Sonani et al. report the structure of bacteriophage χ using cryo-EM, revealing its capsid, neck, tail, and tail-tip organization. Unique proteins in the neck and tail tip, including a trimeric assembly of distal tail protein, deviate from typical siphophage structures, shedding light on χ's architecture and structural diversity among related bacteriophages. [ABSTRACT FROM AUTHOR]

Large gaps exist in our understanding of how bacteriophages, the most abundant biological entities on Earth, assemble and function. The structure of the "neck" region, where the DNA-filled capsid is connected to the host-recognizing tail remains poorly understood. We describe cryo-EM structures of the neck, the neck-capsid and neck-tail junctions, and capsid of the Agrobacterium phage Milano. The Milano neck 1 protein connects the 12-fold symmetrical neck to a 5-fold vertex of the icosahedral capsid. Comparison of Milano neck 1 homologs leads to four proposed classes, likely evolved from the simplest one in siphophages to more complex ones in myo- and podophages. Milano neck is surrounded by the atypical collar, which covalently crosslinks the tail sheath to neck 1. The Milano capsid is decorated with three types of proteins, a minor capsid protein (mCP) and two linking proteins crosslinking the mCP to the major capsid protein. The extensive network of disulfide bonds within and between neck, collar, capsid and tail provides an exceptional structural stability to Milano. Cryo-EM structures of the neck, neck-capsid and neck-tail junctions, and the capsid of the phage Milano reveal a unique collar structure and an extensive network of disulfide bonds providing structural stability to the phage. [ABSTRACT FROM AUTHOR]

The bacterial flagellum is a complex macromolecular machine that drives bacteria through diverse fluid environments. Although many components of the flagellar motor are conserved across species, the roles of FliL are numerous and species‐specific. Here, we have characterized an additional player required for flagellar motor function in Sinorhizobium meliloti, MotF, which we have identified as a FliL paralog. We performed a comparative analysis of MotF and FliL, identified interaction partners through bacterial two‐hybrid and pull‐down assays, and investigated their roles in motility and motor rotation. Both proteins form homooligomers, and interact with each other, and with the stator proteins MotA and MotB. The ∆motF mutant exhibits normal flagellation but its swimming behavior and flagellar motor activity are severely impaired and erratic. In contrast, the ∆fliL mutant is mostly aflagellate and nonmotile. Amino acid substitutions in cytoplasmic regions of MotA or disruption of the proton channel plug of MotB partially restored motor activity to the ∆motF but not the ∆fliL mutant. Altogether, our findings indicate that both, MotF and FliL, are essential for flagellar motor torque generation in S. meliloti. FliL may serve as a scaffold for stator integration into the motor, and MotF is required for proton channel modulation. [ABSTRACT FROM AUTHOR]

The flagellotropic phage 7-7-1 specifically adsorbs to Agrobacterium sp. strain H13-3 (formerly Rhizobium lupini H13-3) flagella for efficient host infection. The Agrobacterium sp. H13-3 flagellum is complex and consists of three flagellin proteins: the primary flagellin FIaA, which is essential for motility, and the secondary flagellins FlaB and FIaD, which have minor functions in motility. Using quantitative infectivity assays, we showed that absence of FIaD had no effect on phage infection, while absence of FlaB resulted in a 2.5-fold increase in infectivity. A flaA deletion strain, which produces straight and severely truncated flagella, experienced a significantly reduced infectivity, similar to that ofaflaBflaD strain, which produces a low number of straight flagella. A strain lacking all three flagellin genes is phage resistant. In addition to flagellation, flagellar rotation is required for infection. A strain that is nonmotile due to an in-frame deletion in the gene encoding the motor component MotA is resistant to phage infection. We also generated two strains with point mutations in the moth gene resulting in replacement of the conserved charged residue G1u98, which is important for modulation of rotary speed. A change to the neutral Gln caused the flagellar mo- tor to rotate at a constant high speed, allowing a 2.2-fold-enhanced infectivity. A change to the positively charged Lys caused a jiggly motility phenotype with very slow flagellar rotation, which significantly reduced the efficiency of infection. In conclusion, flagellar number and length, as well as speed of flagellar rotation, are important determinants for infection by phage 7-7-1. [ABSTRACT FROM AUTHOR]

Bacteriophages are the most abundant biological entities in the biosphere. Due to their host specificity and ability to kill bacteria rapidly, bacteriophages have many potential health care applications, including therapy against antibiotic-resistant bacteria. Infection by flagellotropic bacteriophages requires a properly rotating bacterial flagellar filament. The flagella-dependent phage χ (chi) infects serovars of the pathogenic enterobacterium Salmonella enterica. However, cell surface receptors and proteins involved in other stages of χ infection have not been discovered to date. We screened a multigene deletion library of S. enterica serovar Typhimurium by spotting mutants on soft agar plates seeded with bacteriophage χ and monitoring their ability to grow and form a swim ring, a characteristic of bacteriophage-resistant motile mutants. Those multigene deletion regions identified to be important for χ infectivity were further investigated by characterizing the phenotypes of corresponding single-gene deletion mutants. In this way, we identified motile mutants with various degrees of resistance to χ. Deletions in individual genes encoding the AcrABZ-TolC multidrug efflux system drastically reduced infection by bacteriophage χ. Furthermore, an acrABtolC triple deletion strain was fully resistant to χ. Infection was severely reduced but not entirely blocked by the deletion of the gene tig, encoding the molecular chaperone trigger factor. Finally, deletion in genes encoding enzymes involved in the synthesis of the antioxidants glutathione (GSH) and uric acid resulted in reduced infectivity. Our findings begin to elucidate poorly understood processes involved in later stages of flagellotropic bacteriophage infection and inform research aimed at the use of bacteriophages to combat antibiotic-resistant bacterial infections. [ABSTRACT FROM AUTHOR]

Coexistence of bacteriophages, or phages, and their host bacteria plays an important role in maintaining the microbial communities. In natural environments with limited nutrients, motile bacteria can actively migrate towards locations of richer resources. Although phages are not motile themselves, they can infect motile bacterial hosts and spread in space via the hosts. Therefore, in a migrating microbial community coexistence of bacteria and phages implies their co-propagation in space. Here, we combine an experimental approach and mathematical modeling to explore how phages and their motile host bacteria coexist and co-propagate. When lytic phages encountered motile host bacteria in our experimental set up, a sector-shaped lysis zone formed. Our mathematical model indicates that local nutrient depletion and the resulting inhibition of proliferation and motility of bacteria and phages are the key to formation of the observed lysis pattern. The model further reveals the straight radial boundaries in the lysis pattern as a telltale sign for coexistence and co-propagation of bacteria and phages. Emergence of such a pattern, albeit insensitive to extrinsic factors, requires a balance between intrinsic biological properties of phages and bacteria, which likely results from coevolution of phages and bacteria. Author summary: Coexistence of phages and their bacterial hosts is important for maintaining the microbial communities. In a migrating microbial community, coexistence between phages and host bacteria implies that they co-propagate in space. Here we report a novel phage lysis pattern that is indicative of this co-propagation. The corresponding mathematical model we developed highlights a crucial dependence of the lysis pattern and implied phage-bacteria co-propagation on intrinsic properties allowing proliferation and spatial spreading of the microbes. In contrast, extrinsic factors, such as overall nutrient level, do not influence phage-bacteria coexistence and co-propagation. Findings from this work have strong implications for dispersal of phages mediated by motile bacterial communities, which will provide scientific basis for the fast-growing applications of phages. [ABSTRACT FROM AUTHOR]

The α-proteobacterium Sinorhizobium meliloti can live freely in the soil or engage in a symbiosis with its legume host. S. meliloti facilitates nitrogen fixation in root nodules, thus providing pivotal, utilizable nitrogen to the host. The organism has eight chemoreceptors, namely McpT to McpZ and IcpA that facilitate chemotaxis. McpX is the first known bacterial sensor of quaternary ammonium compounds (QACs) such as choline and betaines. Because QACs are exuded at chemotaxis-relevant concentrations by germinating alfalfa seeds, McpX has been proposed to contribute to host-specific chemotaxis. We have determined the crystal structure of the McpX periplasmic region (McpXPR) in complex with the proline betaine at 2.7 Å resolution. In the crystal, the protein forms a symmetric dimer with one proline betaine molecule bound to each monomer of McpXPR within membrane-distal CACHE module. The ligand is bound through cation-finteractions with four aromatic amino acid residues. Mutational analysis in conjunction with binding studies revealed that a conserved aspartate residue is pivotal for ligand binding. We discovered that, in a striking example of convergent evolution, the ligand-binding site of McpXPR resembles that of a group of structurally unrelated betaine-binding proteins including ProX and OpuAC. Through this comparison and docking studies, we rationalized the specificity of McpXPR for this specific group of ligands. Collectively, our structural, biochemical, and molecular docking data have revealed the molecular determinants in McpX that are crucial for its rare ligand specificity for QACs. [ABSTRACT FROM AUTHOR]

Bacteria, including strains of Salmonella , have been researched and applied as therapeutic cancer agents for centuries. Salmonella are particularly of interest due to their facultative anaerobic nature, facilitating colonization of differentially oxygenated tumor regions. Additionally, Salmonella can be manipulated with relative ease, resulting in the ability to attenuate the pathogen or engineer vectors for drug delivery. It was recently discovered that the anti-cancer Salmonella enterica serovar Typhimurium strain VNP20009 is lacking in chemotactic ability, due to a non-synonymous single nucleotide polymorphism in cheY . Replacing the mutated copy of cheY with the wild-type sequence restored chemotaxis to 70% of the parental strain. We aimed to investigate further if chemotaxis of VNP20009 can be optimized. By restoring the gene msbB in VNP20009 cheY + , which confers attenuation by lipid A modification, we observed a 9% increase in swimming speed, 13% increase in swim plate performance, 19% increase in microfluidic device partitioning towards the attractant at the optimum concentration gradient, and mitigation of a non-motile cell subpopulation. We conclude that chemotaxis can be enhanced further but at the cost of changing one defining characteristic of VNP20009. A less compromised strain might be needed to employ for investigating bacterial chemotaxis in tumor interactions. [ABSTRACT FROM AUTHOR]

The bacterium Sinorhizobium meliloti is attracted to seed exudates of its host plant alfalfa ( Medicago sativa). Since quaternary ammonium compounds (QACs) are exuded by germinating seeds, we assayed chemotaxis of S. meliloti towards betonicine, choline, glycine betaine, stachydrine and trigonelline. The wild type displayed a positive response to all QACs. Using LC-MS, we determined that each germinating alfalfa seed exuded QACs in the nanogram range. Compared to the closely related nonhost species, spotted medic ( Medicago arabica), unique profiles were released. Further assessments of single chemoreceptor deletion strains revealed that an mcpX deletion strain displayed little to no response to these compounds. Differential scanning fluorimetry showed interaction of the isolated periplasmic region of McpX (McpXPR and McpX34-306) with QACs. Isothermal titration calorimetry experiments revealed tight binding to McpXPR with dissociation constants ( Kd) in the nanomolar range for choline and glycine betaine, micromolar Kd for stachydrine and trigonelline and a Kd in the millimolar range for betonicine. Our discovery of S. meliloti chemotaxis to plant-derived QACs adds another role to this group of compounds, which are known to serve as nutrient sources, osmoprotectants and cell-to-cell signalling molecules. This is the first report of a chemoreceptor that mediates QACs taxis through direct binding. [ABSTRACT FROM AUTHOR]

The supercoiling of bacterial and archaeal flagellar filaments is required for motility. Archaeal flagellar filaments have no homology to their bacterial counterparts and are instead homologs of bacterial type IV pili. How these prokaryotic flagellar filaments, each composed of thousands of copies of identical subunits, can form stable supercoils under torsional stress is a fascinating puzzle for which structural insights have been elusive. Advances in cryoelectron microscopy (cryo-EM) make it now possible to directly visualize the basis for supercoiling, and here, we show the atomic structures of supercoiled bacterial and archaeal flagellar filaments. For the bacterial flagellar filament, we identify 11 distinct protofilament conformations with three broad classes of inter-protomer interface. For the archaeal flagellar filament, 10 protofilaments form a supercoil geometry supported by 10 distinct conformations, with one inter-protomer discontinuity creating a seam inside of the curve. Our results suggest that convergent evolution has yielded stable superhelical geometries that enable microbial locomotion. [Display omitted] • Cryo-EM can resolve the atomic structures of supercoiled flagellar filaments • Supercoiled atomic structures show 11 states in bacteria and 10 states in archaea • Distinctly different structures have evolved to form similar supercoils Although lacking homology between their structural components, archaeal and bacterial flagellar filaments achieve the supercoiling that powers locomotion through analogous mechanisms. [ABSTRACT FROM AUTHOR]

The role of chemotaxis and motility in Salmonella enterica serovar Typhimurium tumor colonization remains unclear. We determined through swim plate assays that the well-established anticancer agent S. Typhimurium VNP20009 is deficient in chemotaxis, and that this phenotype is suppressible. Through genome sequencing, we revealed that VNP20009 and four selected suppressor mutants had a single nucleotide polymorphism (SNP) in cheY causing a mutation in the conserved proline residue at position 110. CheY is the response regulator that interacts with the flagellar motor-switch complex and modulates rotational bias. The four suppressor mutants additionally carried non-synonymous SNPs in fliM encoding a flagellar switch protein. The CheY-P110S mutation in VNP20009 likely rendered the protein unable to interact with FliM, a phenotype that could be suppressed by mutations in FliM. We replaced the mutated cheY in VNP20009 with the wild-type copy and chemotaxis was partially restored. The swim ring of the rescued strain, VNP20009 cheY + , was 46% the size of the parental strain 14028 swim ring. When tested in capillary assays, VNP20009 cheY + was 69% efficient in chemotaxis towards the attractant aspartate as compared to 14028. Potential reasons for the lack of complete restoration and implications for bacterial tumor colonization will be discussed. [ABSTRACT FROM AUTHOR]

Characterization of bacterial innate and engineered cooperative behavior, regulated through chemical signaling in a process known as quorum sensing, is critical to development of a myriad of bacteria-enabled systems including biohybrid drug delivery systems and biohybrid mobile sensor networks. Here, we demonstrate, for the first time, that microfluidic diffusive mixers can be used for spatiotemporally high-throughput characterization of bacterial quorum-sensing response. Using this batch characterization method, the quorum-sensing response in Escherichia coli MG 1655, transformed with a truncated lux operon from Vibriofischcri, in the presence of 1-100 nM exogenous acyl-homoserine lactone - molecules has been quantified. This method provides a rapid and facile tool for high-throughput characterization of the quorumsensing response of genetically modified bacteria in the presence of a wide concentration range of signaling molecules with a precision of °0.5 nM. Furthermore, the quorum-sensing response of BacteriaBots has been characterized to determine if the results obtained from a large bacterial population can serve as a robust predictive tool for the small bacterial population attached to each BacteriaBot. [ABSTRACT FROM AUTHOR]

Synthetic biology aims to design de novo biological systems and reengineer existing ones. These efforts have mostly focused on transcriptional circuits, with reengineering of signaling circuits hampered by limited understanding of their systems dynamics and experimental challenges. Bacterial two-component signaling systems offer a rich diversity of sensory systems that are built around a core phosphotransfer reaction between histidine kinases and their output response regulator proteins, and thus are a good target for reengineering through synthetic biology. Here, we explore the signal-response relationship arising from a specific motif found in two-component signaling. In this motif, a single histidine kinase (HK) phosphotransfers reversibly to two separate output response regulator (RR) proteins. We show that, under the experimentally observed parameters from bacteria and yeast, this motif not only allows rapid signal termination, whereby one of the RRs acts as a phosphate sink towards the other RR (i.e. the output RR), but also implements a sigmoidal signal-response relationship. We identify two mathematical conditions on system parameters that are necessary for sigmoidal signal-response relationships and define key parameters that control threshold levels and sensitivity of the signal-response curve. We confirm these findings experimentally, by in vitro reconstitution of the one HK-two RR motif found in the Sinorhizobium meliloti chemotaxis pathway and measuring the resulting signal-response curve. We find that the level of sigmoidality in this system can be experimentally controlled by the presence of the sink RR, and also through an auxiliary protein that is shown to bind to the HK (yielding Hill coefficients of above 7). These findings show that the one HK-two RR motif allows bacteria and yeast to implement tunable switch-like signal processing and provides an ideal basis for developing threshold devices for synthetic biology applications. [ABSTRACT FROM AUTHOR]

Bacterial chemotaxis is an important attribute that aids in establishing symbiosis between rhizobia and their legume hosts. Plant roots and seeds exude a spectrum of molecules into the soil to attract their bacterial symbionts. The alfalfa symbiont Sinorhizobium meliloti possesses eight chemoreceptors to sense its environment and mediate chemotaxis toward its host. The methyl accepting chemotaxis protein McpU is one of the more abundant S. meliloti chemoreceptors and an important sensor for the potent attractant proline. We established a dominant role of McpU in sensing molecules exuded by alfalfa seeds. Mass spectrometry analysis determined that a single germinating seed exudes 3.72 nmol of proline, producing a millimolar concentration near the seed surface which can be detected by the chemosensory system of S. meliloti. Complementation analysis of the mcp 17 deletion strain verified McpU as the key proline sensor. A structure-based homology search identified tandem Cache (calcium channels and chemotaxis receptors) domains in the periplasmic region of McpU. Conserved residues Asp-155 and Asp-182 of the N-termi-nal Cache domain were determined to be important for proline sensing by evaluating mutant strains in capillary and swim plate assays. Differential scanning fluorimetry revealed interaction of the isolated periplasmic region of McpU (McpU40-284) with proline and the importance of Asp-182 in this interaction. Using isothermal titration calorimetry, we determined that proline binds with a Kd (dissociation constant) of 104 μM to McpU40-284) while binding was abolished when Asp-182 was substituted by Glu. Our results show that McpU is mediating chemotaxis toward host plants by direct proline sensing. [ABSTRACT FROM AUTHOR]

Type IVb pili in enteropathogenic bacteria function as a host colonization factor by mediating tight adherence to host cells, but their role in bacterium-plant symbiosis is currently unknown. The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains two clusters encoding proteins for type IVb pili of the Flp (fimbrial low-molecular-weight protein) subfamily. To establish the role of Flp pili in the symbiotic interaction of S. meliloti and its host, Medicago sativa, we deleted pilA1, which encodes the putative pilin subunit in the chromosomal flp-1 cluster and conducted competitive nodulation assays. The pilA1 deletion strain formed 27% fewer nodules than the wild type. Transmission electron microscopy revealed the presence of bundle-forming pili protruding from the polar and lateral region of S. meliloti wild-type cells. The putative pilus assembly ATPase CpaE1 fused to mCherry showed a predominantly unilateral localization. Transcriptional reporter gene assays demonstrated that expression of pilA1 peaks in early stationary phase and is repressed by the quorum-sensing regulator ExpR, which also controls production of exopolysaccharides and motility. Binding of acyl homoserine lactone-activated ExpR to the pilA1 promoter was confirmed with electrophoretic mobility shift assays. A 17-bp consensus sequence for ExpR binding was identified within the 28-bp protected region by DNase I footprinting analyses. Our results show that Flp pili are important for efficient symbiosis of S. meliloti with its plant host. The temporal inverse regulation of exopolysaccharides and pili by ExpR enables S. meliloti to achieve a coordinated expression of cellular processes during early stages of host interaction. [ABSTRACT FROM AUTHOR]

The genome of the symbiotic soil bacterium Sinorhizobium meliloti contains eight genes coding for methyl-accepting chemotaxis proteins (MCPs) McpS to McpZ and one gene coding for a transducer-like protein, IcpA. Seven of the MCPs are localized in the cytoplasmic membrane via two membrane-spanning regions, whereas McpY and IcpA lack such hydrophobic regions. The periplasmic regions of McpU, McpV, and McpX contain the small-ligand-binding domain Cache. In addition, McpU possesses the ligand-binding domain TarH. By probing gene expression with lacZ fusions, we have identified mcpU and mcpX as being highly expressed. Deletion of any one of the receptor genes caused impairments in the chemotactic response toward most organic acids, amino acids, and sugars in a swarm plate assay. The data imply that chemoreceptor proteins in S. meliloti can sense more than one class of carbon source and suggest that many or all receptors work as an ensemble. Tactic responses were virtually eliminated for a strain lacking all nine receptor genes. Capillary assays revealed three important sensors for the strong attractant proline: McpU, McpX, and McpY. Receptor deletions variously affected free-swimming speed and attractant-induced chemokinesis. Noticeably, cells lacking mcpU were swimming 9% slower than the wild-type control. We infer that McpU inhibits the kinase activity of CheA in the absence of an attractant. Cells lacking one of the two soluble receptors were impaired in chemokinetic proficiency by more than 50%. We propose that the internal sensors, IcpA and the PAS domain containing McpY, monitor the metabolic state of S. meliloti. [ABSTRACT FROM AUTHOR]

Chemoreceptors enable the legume symbiont Sinorhizobium meliloti to detect and respond to specific chemicals released from their host plant alfalfa, which allows the establishment of a nitrogen-fixing symbiosis. The periplasmic region (PR) of transmembrane chemoreceptors act as the sensory input module for chemotaxis systems via binding of specific ligands, either directly or indirectly. S. meliloti has six transmembrane and two cytosolic chemoreceptors. However, the function of only three of the transmembrane receptors have been characterized so far, with McpU, McpV, and McpX serving as general amino acid, short-chain carboxylate, and quaternary ammonium compound sensors, respectively. In the present study, we analyzed the S. meliloti chemoreceptor McpT. High-throughput differential scanning fluorimetry assays, using Biolog phenotype microarray plates, identified 15 potential ligands for McpTPR, with the majority classified as mono-, di-, and tricarboxylates. S. meliloti exhibited positive chemotaxis toward seven selected carboxylates, namely, a-ketobutyrate, citrate, glyoxylate, malate, malonate, oxalate, and succinate. These carboxylates were detected in seed exudates of the alfalfa host. Deletion of mcpT resulted in a significant decrease of chemotaxis to all carboxylates except for citrate. Isothermal titration calorimetry revealed that McpTPR bound preferentially to the monocarboxylate glyoxylate and with lower affinity to the dicarboxylates malate, malonate, and oxalate. However, no direct binding was detected for the remaining three carboxylates that elicited an McpT-dependent chemotaxis response. Taken together, these results demonstrate that McpT is a broad-range carboxylate chemoreceptor that mediates chemotactic response via direct ligand binding and an indirect mechanism that needs to be identified. [ABSTRACT FROM AUTHOR]

Highlights: [•] The genome sequence of S. meliloti RU11/001, a model organism for flagellum structure, motility and chemotaxis is reported. [•] The S. meliloti RU11/001 genome consists of a chromosome, the megaplasmids pSymA and pSymB and three accessory plasmids. [•] The RU11/001 genome harbors the accessory plasmid pSmeRU11a that is missing in the related strain S. meliloti SM11. [•] The S. meliloti RU11/001 motility regulon comprises genes required for flagellum assembly, motility and chemotaxis. [ABSTRACT FROM AUTHOR]

Chemotaxis and motility are important traits that support bacterial survival in various ecological niches and in pathogenic and symbiotic host interaction. Chemotactic stimuli are sensed by chemoreceptors or methyl-accepting chemotaxis proteins (MCPs), which direct the swimming behavior of the bacterial cell. In this study, we present evidence that the cellular abundance of chemoreceptors in the plant symbiont Sinorhizobium meliloti can be altered by the addition of several to as few as one amino acid residues and by including common epitope tags such as 3×FLAG and 6×His at their C termini. To further dissect this phenomenon and its underlying molecular mechanism, we focused on a detailed analysis of the amino acid sensor McpU. Controlled proteolysis is important for the maintenance of an appropriate stoichiometry of chemoreceptors and between chemoreceptors and chemotactic signaling proteins, which is essential for an optimal chemotactic response. We hypothesized that enhanced stability is due to interference with protease binding, thus affecting proteolytic efficacy. Location of the protease recognition site was defined through McpU stability measurements in a series of deletion and amino acid substitution mutants. Deletions in the putative protease recognition site had similar effects on McpU abundance, as did extensions at the C terminus. Our results provide evidence that the programmed proteolysis of chemotaxis proteins in S. meliloti is cell cycle regulated. This posttranslational control, together with regulatory pathways on the transcriptional level, limits the chemotaxis machinery to the early exponential growth phase. Our study identified parallels to cell cycle-dependent processes during asymmetric cell division in Caulobacter crescentus. [ABSTRACT FROM AUTHOR]

Chemotaxis systems enable microbes to sense their immediate environment, moving toward beneficial stimuli and away from those that are harmful. In an effort to better understand the chemotaxis system of Sinorhizobium meliloti, a symbiont of the legume alfalfa, the cellular stoichiometries of all ten chemotaxis proteins in S. meliloti were determined. A combination of quantitative immunoblot and mass spectrometry revealed that the protein stoichiometries in S. meliloti varied greatly from those in Escherichia coli and Bacillus subtilis. To compare protein ratios to other systems, values were normalized to the central kinase CheA. All S. meliloti chemotaxis proteins exhibited increased ratios to various degrees. The 10-fold higher molar ratio of adaptor proteins CheW1 and CheW2 to CheA might result in the formation of rings in the chemotaxis array that consist of only CheW instead of CheA and CheW in a 1:1 ratio. We hypothesize that the higher ratio of CheA to the main response regulator CheY2 is a consequence of the speed-variable motor in S. meliloti, instead of a switch-type motor. Similarly, proteins involved in signal termination are far more abundant in S. meliloti, which utilizes a phosphate sink mechanism based on CheA retrophosphorylation to inactivate the motor response regulator versus CheZ-catalyzed dephosphorylation as in E. coli and B. subtilis. Finally, the abundance of CheB and CheR, which regulate chemoreceptor methylation, was increased compared to CheA, indicative of variations in the adaptation system of S. meliloti. Collectively, these results mark significant differences in the composition of bacterial chemotaxis systems. [ABSTRACT FROM AUTHOR]

Sinorhizobium meliloti is a soil-dwelling endosymbiont of alfalfa that has eight chemoreceptors to sense environmental stimuli during its free-living state. The functions of two receptors have been characterized, with McpU and McpX serving as general amino acid and quaternary ammonium compound sensors, respectively. Both receptors use a dual Cache (calcium channels and chemotaxis receptors) domain for ligand binding. We identified that the ligand-binding periplasmic region (PR) of McpV contains a single Cache domain. Homology modeling revealed that McpVPR is structurally similar to a sensor domain of a chemoreceptor with unknown function from Anaeromyxobacter dehalogenans, which crystallized with acetate in its binding pocket. We therefore assayed McpV for carboxylate binding and S. meliloti for carboxylate sensing. Differential scanning fluorimetry identified 10 potential ligands for McpVPR. Nine of these are monocarboxylates with chain lengths between two and four carbons. We selected seven compounds for capillary assay analysis, which established positive chemotaxis of the S. meliloti wild type, with concentrations of peak attraction at 1 mM for acetate, propionate, pyruvate, and glycolate, and at 100 mM for formate and acetoacetate. Deletion of mcpV or mutation of residues essential for ligand coordination abolished positive chemotaxis to carboxylates. Using microcalorimetry, we determined that dissociation constants of the seven ligands with McpVPR were in the micromolar range. An McpVPR variant with a mutation in the ligand coordination site displayed no binding to isobutyrate or propionate. Of all the carboxylates tested as attractants, only glycolate was detected in alfalfa seed exudates. This work examines the relevance of carboxylates and their sensor to the rhizobium-legume interaction. [ABSTRACT FROM AUTHOR]

Bacteriophage 7-7-1, a member of the family Myoviridae, infects the soil bacterium Agrobacterium sp. strain H13-3. Infection requires attachment to actively rotating bacterial flagellar filaments, with flagellar number, length, and rotation speed being important determinants for infection efficiency. To identify the secondary receptor(s) on the cell surface, we isolated motile, phage-resistant Agrobacterium sp. H13-3 transposon mutants. Transposon insertion sites were pinpointed using arbitrary primed PCR and bioinformatics analyses. Three genes were recognized, whose corresponding proteins had the following computationally predicted functions: AGROH133_07337, a glycosyltransferase; AGROH133_13050, a UDP-glucose 4-epimerase; and AGROH133_08824, an integral cytoplasmic membrane protein. The first two gene products are part of the lipopolysaccharide (LPS) synthesis pathway, while the last is predicted to be a relatively small (13.4-kDa) cytosolic membrane protein with up to four transmembrane helices. The phenotypes of the transposon mutants were verified by complementation and site-directed mutagenesis. Additional characterization of motile, phage-resistant mutants is also described. Given these findings, we propose a model for Agrobacterium sp. H13-3 infection by bacteriophage 7-7-1 where the phage initially attaches to the flagellar filament and is propelled down toward the cell surface by clockwise flagellar rotation. The phage then attaches to and degrades the LPS to reach the outer membrane and ejects its DNA into the host using its syringe-like contractile tail. We hypothesize that the integral membrane protein plays an important role in events following viral DNA ejection or in LPS processing and/or deployment. The proposed two-step attachment mechanism may be conserved among other flagellotropic phages infecting Gram-negative bacteria. [ABSTRACT FROM AUTHOR]

The chemosensory system in Sinorhizobium meliloti has several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA in S. meliloti. Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell in S. meliloti. The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen in Bacillus subtilis (23:1) and about 10 times higher than that in Escherichia coli (3.4:1). Different from E. coli, the high-abundance receptors in S. meliloti are lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptional lacZ fusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system in S. meliloti deviates significantly from the established enterobacterial paradigm but shares some similarities with B. subtilis. [ABSTRACT FROM AUTHOR]

A mutagenized and genetically modified derivative of Salmonella enterica serovar Typhimurium 14028S, VNP20009 (ATCC 202165), is attenuated in host virulence and accumulates preferentially in tumors. Here, we report the complete genome of this anticancer therapy agent consisting of one chromosome and one virulence plasmid. The major genetic features that distinguish VNP20009 from its parental strain are: an engineered msbB deletion, a 3′-extension in pykA , a Tn10-caused 16.6-kbp inversion leading to the disruption of purM , a 108-kbp Suwwan deletion resulting in the loss of 128 genes, and 50 non-synonymous SNPs. [ABSTRACT FROM AUTHOR]