Flagella-mediated Motility Research Articles

The Stand-Alone PilZ-Domain Protein MotL Specifically Regulates the Activity of the Secondary Lateral Flagellar System in Shewanella putrefaciens.

A number of bacterial species control the function of the flagellar motor in response to the levels of the secondary messenger c-di-GMP, which is often mediated by c-di-GMP-binding proteins that act as molecular brakes or clutches to slow the motor rotation. The gammaproteobacterium Shewanella putrefaciens possesses two distinct flagellar systems, the primary single polar flagellum and a secondary system with one to five lateral flagellar filaments. Here, we identified a protein, MotL, which specifically regulates the activity of the lateral, but not the polar, flagellar motors in response to the c-di-GMP levels. MotL only consists of a single PilZ domain binding c-di-GMP, which is crucial for its function. Deletion and overproduction analyses revealed that MotL slows down the lateral flagella at elevated levels of c-di-GMP, and may speed up the lateral flagellar-mediated movement at low c-di-GMP concentrations. In vitro interaction studies hint at an interaction of MotL with the C-ring of the lateral flagellar motors. This study shows a differential c-di-GMP-dependent regulation of the two flagellar systems in a single species, and implicates that PilZ domain-only proteins can also act as molecular regulators to control the flagella-mediated motility in bacteria.

In Vitro Framework to Assess the Anti-Helicobacter pylori Potential of Lactic Acid Bacteria Secretions as Alternatives to Antibiotics

Helicobacter pylori is a prevalent bacterium that can cause gastric ulcers and cancers. Lactic acid bacteria (LAB) ameliorate treatment outcomes against H. pylori, suggesting that they could be a source of bioactive molecules usable as alternatives to current antibiotics for which resistance is mounting. We developed an in vitro framework to compare the anti-H. pylori properties of 25 LAB and their secretions against H. pylori. All studies were done at acidic and neutralized pH, with or without urea to mimic various gastric compartments. Eighteen LAB strains secreted molecules that curtailed the growth of H. pylori and the activity was urea-resistant in five LAB. Several LAB supernatants also reduced the urease activity of H. pylori. Pre-treatment of H. pylori with acidic LAB supernatants abrogated its flagella-mediated motility and decreased its ability to elicit pro-inflammatory IL-8 cytokine from human gastric cells, without reverting the H. pylori-induced repression of other pro-inflammatory cytokines. This study identified the LAB that have the most anti-H. pylori effects, decreasing its viability, its production of virulence factors, its motility and/or its ability to elicit pro-inflammatory IL-8 from gastric cells. Once identified, these molecules can be used as alternatives or complements to current antibiotics to fight H. pylori infections.

Reconstructing ancestral bacteria

Bacterial Phylogeny The origin of the eubacteria and phylogenetic relationships between subgroups have been difficult to resolve. Applying a phylogenetic analysis and recent computational methods to the expanded diversity of bacterial sequences from metagenomic analyses, Coleman et al. infer the root of the eubacterial tree (see the Perspective by Katz). The root was determined without using the Archaea as an outgroup, to avoid the possibility of a false result due to long branch attraction. This method places the eubacterial root in the neighborhood of Fusobacteriota. Using this information, the authors reconstructed the eubacterial ancestor, identifying that this organism likely had a double-membrane cell envelope, flagellum-mediated motility, antiphage defense mechanisms, and diverse metabolic pathways. Science , this issue p. [eabe0511][1]; see also p. [574][2] [1]: /lookup/doi/10.1126/science.abe0511 [2]: /lookup/doi/10.1126/science.abh2814

Characterization of the relationship between polar and lateral flagellar genes in clinical Aeromonas dhakensis: phenotypic, genetic and biochemical analyses

Flagellar-mediated motility is a crucial virulence factor in many bacterial species. A dual flagellar system has been described in aeromonads; however, there is no flagella-related study in the emergent human pathogen Aeromonas dhakensis. Using 46 clinical A. dhakensis, phenotypic motility, genotypic characteristics (flagellar genes and sequence types), biochemical properties and their relationship were investigated in this study. All 46 strains showed swimming motility at 30 °C in 0.3% Bacto agar and carried the most prevalent 6 polar flagellar genes cheA, flgE, flgG, flgH, flgL, and flgN. On the contrary, only 18 strains (39%) demonstrated swarming motility on 0.5% Eiken agar at 30 °C and they harbored 11 lateral flagellar genes lafB, lafK, lafS, lafT, lafU, flgCL, flgGL, flgNL, fliEL, fliFL, and fliGL. No association was found between biochemical properties and motility phenotypes. Interestingly, a significant association between swarming and strains isolated from pus was observed (p = 0.0171). Three strains 187, 277, and 289 isolated from pus belonged to novel sequence types (ST522 and ST524) exhibited fast swimming and swarming profiles, and they harbored > 90% of the flagellar genes tested. Our findings provide a fundamental understanding of flagellar-mediated motility in A. dhakensis.

The enteric pathogen Clostridium difficile chemotaxes towards mucin glycans during the early colonization phase of infection

BackgroundInfection with the pathogen Clostridium difficile is a major cause of antibiotic‐induced diarrhea and colitis. In the United States, C. difficile infects millions of patients each year and results in healthcare associated costs of over 1 billion dollars. Despite the significant burden of C. difficile infection, the exact mechanisms of C. difficile host colonization remain unknown. It is well known that antibiotic use sets the stage for infection and that toxin production induces symptoms. However, how C. difficile identifies and colonizes its ecological niche are unclear. Similar to other pathogens, flagellum‐mediated motility and the ability to interact with the intestinal mucus layer are likely critical factors for C. difficile colonization. We hypothesized that C. difficile would recognize mucin glycans present on human MUC2 and would chemotax towards these oligosaccharides. We also speculated that MUC2 would influence C. difficile gene expression and promote colonization.Methods & ResultsTo address these gaps in knowledge we fluorescently tagged C. difficile R20291 with CFDA‐SE and measured chemotaxis using a standard capillary assay. We observed that C. difficile was able to chemotax towards purified human MUC2 from a variety of sources, including human goblet cell lines HT29‐MTX, LS174T, T84 as well as human stool. We also found that C. difficile could recognize oligosaccharides present on MUC2 glycans, such as fucose, galactose, mannose, GalNac, GluNAc, and neurominic acid. The greatest accumulation of C. difficile was observed with mannose. To confirm these findings, we live‐imaged C. difficile chemotaxis in IBIDI chemotaxis slides and found that the presence of MUC2 or mannose increased the velocity and distance traveled by C. difficile compared to buffer medium alone. Examination of C. difficile genomes from several ribotypes revealed that C. difficile lacks the glycosyl hydrolases (GH) required for O‐linked mucin glycan degradation, although it did contain GH family 38, which cleaves mannose residues. To assess whether C. difficile could use mucin oligosaccharides as a primary carbon source, we grew C. difficile in a fully defined minimal medium (CDMM) in the absence of glucose supplemented with individual glycan oligosaccharides or MUC2. Consistent with our GH analysis, C. difficile was unable to grow on intact MUC2 alone. However, C. difficile was able to utilize all mucin oligosaccharides for growth. Further analysis of the C. difficile genome confirmed the transporters required for uptake of these sugars are present in all species. Finally, RNAseq demonstrated shifts in gene expression in C. difficile R20291 in response to human MUC2.ConclusionsThese data indicate that C. difficile recognizes and utilizes mucin‐derived oligosaccharides. We also demonstrate that C. difficile does not possess the enzymatic capacity to degrade these glycans on its own. Therefore, it must require the presence of other mucin‐degrading microbes for growth, pointing to the role of the microbial community in supporting C. difficile colonization.

Small RNAs OmrA and OmrB promote class III flagellar gene expression by inhibiting the synthesis of anti-Sigma factor FlgM

ABSTRACT Bacteria can move by a variety of mechanisms, the best understood being flagella-mediated motility. Flagellar genes are organized in a three-tiered cascade allowing for temporally regulated expression that involves both transcriptional and post-transcriptional control. The class I operon encodes the master regulator FlhDC that drives class II gene transcription. Class II genes include fliA and flgM, which encode the Sigma factor σ28, required for class III transcription, and the anti-Sigma factor FlgM, which inhibits σ28 activity, respectively. The flhDC mRNA is regulated by several small regulatory RNAs (sRNAs). Two of these, the sequence-related OmrA and OmrB RNAs, inhibit FlhD synthesis. Here, we report on a second layer of sRNA-mediated control downstream of FhlDC in the flagella pathway. By mutational analysis, we confirm that a predicted interaction between the conserved 5ʹ seed sequences of OmrA/B and the early coding sequence in flgM mRNA reduces FlgM expression. Regulation is dependent on the global RNA-binding protein Hfq. In vitro experiments support a canonical mechanism: binding of OmrA/B prevents ribosome loading and decreases FlgM protein synthesis. Simultaneous inhibition of both FlhD and FlgM synthesis by OmrA/B complicated an assessment of how regulation of FlgM alone impacts class III gene transcription. Using a combinatorial mutation strategy, we were able to uncouple these two targets and demonstrate that OmrA/B-dependent inhibition of FlgM synthesis liberates σ28 to ultimately promote higher expression of the class III flagellin gene fliC.

Chemotaxis and cyclic‐di‐GMP signalling control surface attachment of Escherichia coli

Attachment to surfaces is an important early step during bacterial infection and during formation of submerged biofilms. Although flagella-mediated motility is known to be important for attachment of Escherichia coli and other bacteria, implications of motility regulation by cellular signalling remain to be understood. Here, we show that motility largely promotes attachment of E. coli, including that mediated by type 1 fimbriae, by allowing cells to reach, get hydrodynamically trapped at and explore the surface. Inactivation or inhibition of the chemotaxis signalling pathway improves attachment by suppressing cell reorientations and thereby increasing surface residence times. The attachment is further enhanced by deletion of genes encoding the cyclic diguanosine monophosphate (c-di-GMP)-dependent flagellar brake YcgR or the diguanylate cyclase DgcE. Such increased attachment in absence of c-di-GMP signalling is in contrast to its commonly accepted function as a positive regulator of the sessile state. It is apparently due to the increased swimming speed of E. coli in absence of YcgR-mediated motor control, which strengthens adhesion mediated by the type 1 fimbriae. Thus, both signalling networks that regulate motility of E. coli also control its engagement with both biotic and abiotic surfaces, which has likely implications for infection and biofilm formation.

Sensing and Approaching Toxic Arsenate by Shewanella putrefaciens CN-32.

Although arsenic at a high concentration imposes strong selective pressure on microbes, various microbes have been found to grow in As-rich environments. So far, little is known about how microbes can sense and move toward arsenate in the environment, and the underlying molecular mechanisms have not been revealed. Here, we report the chemotaxis response toward arsenate (As(V)) by Shewanella putrefaciens CN-32, a model dissimilatory metal-reducing bacterium (DMRB), and elucidate the mechanisms. We find that S. putrefaciens CN-32 exhibits a chemotactic behavior toward As(V) and diverse electron acceptors. To sense As(V), S. putrefaciens CN-32 requires functional arsenate respiratory reductase but does not depend on its metal-reducing-like respiratory pathway. We observe that such a sense is governed by an energy taxis mechanism and mediated by several methyl-accepting chemotaxis proteins (MCPs), rather than a specific MCP. Moreover, we reveal that the chemotactic signal transduction pathway is conserved in Shewanella, and histidine kinase and flagella-mediated motility are essential for taxis toward As(V). This work reverses the conventional view about arsenic as a chemotactic inhibitor to microbes by revealing the positive chemotaxis of Shewanella to As(V).

Crystal structure of the flagellar cap protein FliD from Bdellovibrio bacteriovorus

Bdellovibrio bacteriovorus is a predator bacterial species of the Deltaproteobacteria class that requires flagellum-mediated motility to initiate the parasitization of other gram-negative bacteria. The flagellum is capped by FliD, which polymerizes flagellin into a flagellar filament. FliD has been reported to function as a species-specific oligomer, such as a tetramer, a pentamer, or a hexamer, in members of the Gammaproteobacteria class. However, the oligomeric state and structural features of FliD from bacterial species outside the Gammaproteobacteria class are unknown. Based on structural and biochemical analyses, we report here that B. bacteriovorus FliD (bbFliD) forms a tetramer. bbFliD tetramerizes in a circular head-to-tail arrangement by inserting the D2 domain of one subunit into the concave surface of the second subunit generated between the D2 and D3 domains as observed in Serratia marcescens FliD. However, bbFliD adopts a more compact and flat oligomeric structure, which exhibits a more extended tetramerization interface flanked by two additional surfaces due to different intersubunit and interdomain organizations as well as an elongated loop. In conclusion, FliD from B. bacteriovorus, which belongs to the Deltaproteobacteria class, also produces a tetramer similar to FliD from Gammaproteobacterial species but adopts a unique species-specific oligomeric structure.

The genome of Ensifer alkalisoli YIC4027 provides insights for host specificity and environmental adaptations

BackgroundEnsifer alkalisoli YIC4027, a recently characterized nitrogen-fixing bacterium of the genus Ensifer, has been isolated from root nodules of the host plant Sesbania cannabina. This plant is widely used as green manure and for soil remediation. E. alkalisoli YIC4027 can grow in saline-alkaline soils and is a narrow-host-range strain that establishes a symbiotic relationship with S. cannabina. The complete genome of this strain was sequenced to better understand the genetic basis of host specificity and adaptation to saline-alkaline soils.ResultsE. alkalisoli YIC4027 was found to possess a 6.1-Mb genome consisting of three circular replicons: one chromosome (3.7 Mb), a chromid (1.9 Mb) and a plasmid (0.46 Mb). Genome comparisons showed that strain YIC4027 is phylogenetically related to broad-host-range Ensifer fredii strains. Synteny analysis revealed a strong collinearity between chromosomes of E. alkalisoli YIC4027 and those of the E. fredii NGR234 (3.9 Mb), HH103 (4.3 Mb) and USDA257 (6.48 Mb) strains. Notable differences were found for genes required for biosynthesis of nodulation factors and protein secretion systems, suggesting a role of these genes in host-specific nodulation. In addition, the genome analysis led to the identification of YIC4027 genes that are presumably related to adaptation to saline-alkaline soils, rhizosphere colonization and nodulation competitiveness. Analysis of chemotaxis cluster genes and nodulation tests with constructed che gene mutants indicated a role of chemotaxis and flagella-mediated motility in the symbiotic association between YIC4027 and S. cannabina.ConclusionsThis study provides a basis for a better understanding of host specific nodulation and of adaptation to a saline-alkaline rhizosphere. This information offers the perspective to prepare optimal E. alkalisoli inocula for agriculture use and soil remediation.

Sulfur Assimilation Alters Flagellar Function and Modulates the Gene Expression Landscape of Serratia marcescens.

Sulfur is an essential nutrient that contributes to cellular redox homeostasis, transcriptional regulation, and translation initiation when incorporated into different biomolecules. Transport and reduction of extracellular sulfate followed by cysteine biosynthesis is a major pathway of bacterial sulfur assimilation. For the opportunistic pathogen Serratia marcescens, function of the cysteine biosynthesis pathway is required for extracellular phospholipase activity and flagellum-mediated surface motility, but little else is known about the influence of sulfur assimilation on the physiology of this organism. In this work, it was determined that an S. marcescens cysteine auxotroph fails to differentiate into hyperflagellated and elongated swarmer cells and that cysteine, but not other organic sulfur molecules, restores swarming motility to these bacteria. The S. marcescens cysteine auxotroph further exhibits reduced transcription of phospholipase, hemolysin, and flagellin genes, each of which is subject to transcriptional control by the flagellar regulatory system. Based on these data and the central role of cysteine in sulfur assimilation, it was reasoned that environmental sulfur availability may contribute to the regulation of these functions in S. marcescens Indeed, bacteria that are starved for sulfate exhibit substantially reduced transcription of the genes for hemolysin, phospholipase, and the FlhD flagellar master regulator. A global transcriptomic analysis further defined a large set of S. marcescens genes that are responsive to extracellular sulfate availability, including genes that encode membrane transport, nutrient utilization, and metabolism functions. Finally, sulfate availability was demonstrated to alter S. marcescens cytolytic activity, suggesting that sulfate assimilation may impact the virulence of this organism.IMPORTANCE Serratia marcescens is a versatile bacterial species that inhabits diverse environmental niches and is capable of pathogenic interactions with host organisms ranging from insects to humans. This report demonstrates for the first time the extensive impacts that environmental sulfate availability and cysteine biosynthesis have on the transcriptome of S. marcescens The finding that greater than 1,000 S. marcescens genes are differentially expressed depending on sulfate availability suggests that sulfur abundance is a crucial factor that controls the physiology of this organism. Furthermore, the high relative expression levels for the putative virulence factors flagella, phospholipase, and hemolysin in the presence of sulfate suggests that a sulfur-rich host environment could contribute to the transcription of these genes during infection.

Essential Role of σ Factor RpoF in Flagellar Biosynthesis and Flagella-Mediated Motility of Acidithiobacillus caldus.

Acidithiobacillaceae, an important family of acidophilic and chemoautotrophic sulfur or iron oxidizers, participate in geobiochemical circulation of the elements and drive the release of heavy metals in mining associated habitats. Because of their environmental adaptability and energy metabolic systems, Acidithiobacillus spp. have become the dominant bacteria used in bioleaching for heavy metal recovery. Flagella-driven motility is associated with bacterial chemotaxis and bacterial responses to environmental stimuli. However, little is known about how the flagellum of Acidithiobacillus spp. is regulated and how the flagellum affects the growth of these chemoautotrophic bacteria. In this study, we analyzed the flagellar gene clusters in Acidithiobacillus strains and uncovered the close relationship between flagella and the sulfur-oxidizing systems (Sox system). The σ28 gene (rpoF) knockout and overexpression strains of Acidithiobacillus caldus were constructed. Scanning electron microscopy shows that A. caldus ΔrpoF cells lacked flagella, indicating the essential role of RpoF in regulating flagella synthesis in these chemoautotrophic bacteria. Motility analysis suggests that the deletion of rpoF resulted in the reduction of swarming capability, while this capability was enhanced in the rpoF overexpression strain. Both static cultivation and low concentration of energy substrates (elemental sulfur or tetrathionate) led to weak growth of A. caldus ΔrpoF cells. The deletion of rpoF promoted bacterial attachment to the surface of elemental sulfur in static cultivation. The absence of RpoF caused an obvious change in transcription profile, including genes in flagellar cluster and those involved in biofilm formation. These results provide an understanding on the regulation of flagellar hierarchy and the flagellar function in these sulfur or iron oxidizers.

In vitro efficacy of phytotherapeutics suggested for prevention and therapy of urinary tract infections.

To analyse the therapeutic efficacy of various phytotherapeutics and their antimicrobial compounds with regard to strain specificity and dose dependence. A representative strain collection of 40 uropathogenic bacteria isolated from complicated and uncomplicated urinary tract infection was subjected to various virulence assays (bacterial growth, mannose-sensitive agglutination, and motility) to determine the therapeutic impact of various compounds with antimicrobial activity. We tested proanthocyanidins (PAC), D-mannose, rosemary extract (Canephron®), and isothiocyanates (Angocin®). D-mannose efficiently blocked the adhesive properties of all type 1 fimbriae-positive isolates in low concentration (0.2%), but showed no bacteriostatic effect. PAC also actively blocked agglutination, but the concentration varied considerably among isolates. Escherichia coli required the highest concentration (10%), while Enterobacter cloacae responded to low concentrations (0.1%). Allyl isothiocyanates not only impaired agglutination in all tested isolates, but also had a dramatic impact on flagella-mediated motility in Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis (p < 0.001). The administration of rosemary extracts revealed a strong bacteriostatic effect in growth assays. All tested strains were strongly inhibited by the addition of 10μg/ml or 1μg/ml of purified rosemary extractions with the exception of Serratia marcescens. Morganella morganii responded only to 10μg/ml. Phytotherapeutics and small-molecular compounds like mannosides have the potential to become an integral part in a multi-modal treatment concept for the treatment and prevention of urinary tract infections. Their efficiency can be optimised when strain specificities and therapeutic concentrations are taken into account.

Vibrio Flagellar Synthesis.

Vibrio spp. are highly motile Gram-negative bacteria, ubiquitously found in aquatic environments. Some Vibrios are responsible for disease and morbidity of marine invertebrates and humans, while others are studied for their symbiotic interactions. Vibrio spp. are motile due to synthesis of flagella that rotate and propel the bacteria. Many Vibrio spp. synthesize monotrichous polar flagella (e.g., V. cholerae, V. alginolyticus); however, some synthesize peritrichous or lophotrichous flagella. Flagellar-mediated motility is intimately connected to biological and cellular processes such as chemotaxis, biofilm formation, colonization, and virulence of Vibrio spp. This review focuses on the polar flagellum and its regulation in regard to Vibrio virulence and environmental persistence.

An uncharacterised protein mediates motility, biofilm formation, and host colonisation in adherent invasive Escherichia coli

Adherent Invasive Escherichia coli (AIEC) is a non-diarrhoeagenic intestinal E. coli patho type with a putativea etiological role in Crohn’s Disease (CD). AIEC pathogenes is ischaracterised by adhesion to, invasion of, and replication within intestinal epithelial cells and macrophages, and biofilm formation. Using a heterologous expression screen, we identified a gene in the AIEC LF82 genome encoding a protein which self-assembles into filaments in HeLa cells, which we hypothesised was a novel pilin or biofilm matrix component, designated bcmA (biofilm coupled to motility in AIEC). Using a crystal violet-based assay, we found LF82ΔbcmA have defective biofilm formation, which can be fully complemented by episomal bcmA expression. Microscopic analysis of LF82 biofilms demonstrated LF82ΔbcmA form patchy, sparse biofilms, and revealed an intracellular localisation for GFP-tagged bcmA, suggesting the protein is not a surface-exposed adhesin or biofilm matrix component. We therefore assessed the role of bcmA in flagellar-mediated motility and found that – despite displaying wild-type flagellar morphologies – LF82ΔbcmA have profound swimming and swarming defects. Work in a Caenorhabdit is elegans infection model suggests bcmA is not required for full virulence; however, preliminary evidence suggests LF82ΔbcmA have defective C. elegans gut colonisation. Taken together, our data demonstrates roles for bcmA in AIEC host colonisation via an undefined role in motility. bcmA is highly conserved among pathogenic gammaproteobacteria, including Salmonella Typhi, Shigella boydii and Klebsiella pneumoniae. This suggests bcmA may not only be significant for AIEC pathogenesis, but may also represent an important virulence determinant in several major gammaproteobacterial pathogens.

Bacillus subtilis Biofilms: a Matter of Individual Choice.

Bacillus subtilis has the capacity to choose between two mutually exclusive lifestyles: biofilm formation and flagellum-mediated swimming motility. Interestingly, this choice is made at the individual cell level, with bacterial cells in a population expressing genes required for biofilm formation or genes required for swimming motility but not both. A bistable switch controls the biofilm-versus-swimming decision, resulting in an evolutionarily favorable strategy known as "bet hedging" that ensures that subpopulations of bacteria continue to grow as conditions change and/or become unfavorable. In a recent issue of mBio, J. Kampf and colleagues (mBio 9:e01464-18, 2018, https://doi.org/10.1128/mBio.01464-18) reported the use of a combination of genetics and microfluidics to reveal that the interplay that occurs between the SinR and YmdB proteins underlies the B. subtilis choice between biofilm formation and swimming motility. Their report suggests that B. subtilis experiences selective pressure to form biofilms while maintaining reserve cell subpopulations with the capacity to swim away.

MrpJ Directly Regulates Proteus mirabilis Virulence Factors, Including Fimbriae and Type VI Secretion, during Urinary Tract Infection

Proteus mirabilis is a leading cause of catheter-associated urinary tract infections (CAUTIs) and urolithiasis. The transcriptional regulator MrpJ inversely modulates two critical aspects of P. mirabilis UTI progression: fimbria-mediated attachment and flagellum-mediated motility. Transcriptome data indicated a network of virulence-associated genes under MrpJ's control. Here, we identify the direct gene regulon of MrpJ and its contribution to P. mirabilis pathogenesis, leading to the discovery of novel virulence targets. Chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) was used for the first time in a CAUTI pathogen to probe for in vivo direct targets of MrpJ. Selected MrpJ-regulated genes were mutated and assessed for their contribution to UTI using a mouse model. ChIP-seq revealed a palindromic MrpJ binding sequence and 78 MrpJ-bound regions, including binding sites upstream of genes involved in motility, fimbriae, and a type VI secretion system (T6SS). A combinatorial mutation approach established the contribution of three fimbriae (fim8A, fim14A, and pmpA) to UTI and a new pathogenic role for the T6SS in UTI progression. In conclusion, this study (i) establishes the direct gene regulon and an MrpJ consensus binding site and (ii) led to the discovery of new virulence genes in P. mirabilis UTI, which could be targeted for therapeutic intervention of CAUTI.

Regulation of Flagellum Biosynthesis in Response to Cell Envelope Stress in Salmonella enterica Serovar Typhimurium.

ABSTRACTFlagellum-driven motility of Salmonella enterica serovar Typhimurium facilitates host colonization. However, the large extracellular flagellum is also a prime target for the immune system. As consequence, expression of flagella is bistable within a population of Salmonella, resulting in flagellated and nonflagellated subpopulations. This allows the bacteria to maximize fitness in hostile environments. The degenerate EAL domain protein RflP (formerly YdiV) is responsible for the bistable expression of flagella by directing the flagellar master regulatory complex FlhD4C2 with respect to proteolytic degradation. Information concerning the environmental cues controlling expression of rflP and thus about the bistable flagellar biosynthesis remains ambiguous. Here, we demonstrated that RflP responds to cell envelope stress and alterations of outer membrane integrity. Lipopolysaccharide (LPS) truncation mutants of Salmonella Typhimurium exhibited increasing motility defects due to downregulation of flagellar gene expression. Transposon mutagenesis and genetic profiling revealed that σ24 (RpoE) and Rcs phosphorelay-dependent cell envelope stress response systems sense modifications of the lipopolysaccaride, low pH, and activity of the complement system. This subsequently results in activation of RflP expression and degradation of FlhD4C2 via ClpXP. We speculate that the presence of diverse hostile environments inside the host might result in cell envelope damage and would thus trigger the repression of resource-costly and immunogenic flagellum biosynthesis via activation of the cell envelope stress response.

A dual role for proline iminopeptidase in the regulation of bacterial motility and host immunity.

During plant-pathogen interactions, pathogenic bacteria have evolved multiple strategies to cope with the sophisticated defence systems of host plants. Proline iminopeptidase (PIP) is essential to Xanthomonas campestris pv. campestris (Xcc) virulence, and is conserved in many plant-associated bacteria, but its pathogenic mechanism remains unclear. In this study, we found that disruption of pip in Xcc enhanced its flagella-mediated bacterial motility by decreasing intracellular bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) levels, whereas overexpression of pip in Xcc restricted its bacterial motility by elevating c-di-GMP levels. We also found that PIP is a type III secretion system-dependent effector capable of eliciting a hypersensitive response in non-host, but not host plants. When we transformed pip into the host plant Arabidopsis, higher bacterial titres were observed in pip-overexpressing plants relative to wild-type plants after Xcc inoculation. The repressive function of PIP on plant immunity was dependent on PIP's enzymatic activity and acted through interference with the salicylic acid (SA) biosynthetic and regulatory genes. Thus, PIP simultaneously regulates two distinct regulatory networks during plant-microbe interactions, i.e. it affects intracellular c-di-GMP levels to coordinate bacterial behaviour, such as motility, and functions as a type III effector translocated into plant cells to suppress plant immunity. Both processes provide bacteria with the regulatory potential to rapidly adapt to complex environments, to utilize limited resources for growth and survival in a cost-efficient manner and to improve the chances of bacterial survival by helping pathogens to inhabit the internal tissues of host plants.

Fungal networks shape dynamics of bacterial dispersal and community assembly in cheese rind microbiomes

Most studies of bacterial motility have examined small-scale (micrometer–centimeter) cell dispersal in monocultures. However, bacteria live in multispecies communities, where interactions with other microbes may inhibit or facilitate dispersal. Here, we demonstrate that motile bacteria in cheese rind microbiomes use physical networks created by filamentous fungi for dispersal, and that these interactions can shape microbial community structure. Serratia proteamaculans and other motile cheese rind bacteria disperse on fungal networks by swimming in the liquid layers formed on fungal hyphae. RNA-sequencing, transposon mutagenesis, and comparative genomics identify potential genetic mechanisms, including flagella-mediated motility, that control bacterial dispersal on hyphae. By manipulating fungal networks in experimental communities, we demonstrate that fungal-mediated bacterial dispersal can shift cheese rind microbiome composition by promoting the growth of motile over non-motile community members. Our single-cell to whole-community systems approach highlights the interactive dynamics of bacterial motility in multispecies microbiomes.