Analysis of HubP-dependent cell pole protein targeting in Vibrio cholerae uncovers novel motility regulators

In rod-shaped bacteria, the emergence and maintenance of long-axis cell polarity is involved in key cellular processes such as cell cycle, division, environmental sensing and flagellar motility among others. Many bacteria achieve cell pole differentiation through the use of polar landmark proteins acting as scaffolds for the recruitment of functional macromolecular assemblies. In Vibrio cholerae a large membrane-tethered protein, HubP, specifically interacts with proteins involved in chromosome segregation, chemotaxis and flagellar biosynthesis. Here we used comparative proteomics, genetic and imaging approaches to identify additional HubP partners and demonstrate that at least six more proteins are subject to HubP-dependent polar localization. These include a cell-wall remodeling enzyme (DacB), a likely chemotaxis sensory protein (HlyB), two presumably cytosolic proteins of unknown function (VC1210 and VC1380) and two membrane-bound proteins, named here MotV and MotW, that exhibit distinct effects on chemotactic motility. We show that while both ΔmotW and ΔmotV mutants retain monotrichous flagellation, they present significant to severe motility defects when grown in soft agar. Video-tracking experiments further reveal that ΔmotV cells can swim in liquid environments but are unable to tumble or penetrate a semisolid matrix, whereas a motW deletion affects both tumbling frequency and swimming speed. Motility suppressors and gene co-occurrence analyses reveal co-evolutionary linkages between MotV, a subset of non-canonical CheV proteins and flagellar C-ring components FliG and FliM, whereas MotW regulatory inputs appear to intersect with specific c-di-GMP signaling pathways. Together, these results reveal an ever more versatile role for the landmark cell pole organizer HubP and identify novel mechanisms of motility regulation.

Metformin Alters the Chemotaxis and Flagellar Motility of Escherichia coli

Metformin is a biguanide molecule that is widely prescribed to treat type 2 diabetes and metabolic syndrome. Although it is known that metformin promotes the lifespan by altering intestinal microorganism metabolism, how metformin influences and alters the physiological behavior of microorganisms remains unclear. Here we studied the effect of metformin on the behavior alterations of the model organism Escherichia coli (E. coli), including changes in chemotaxis and flagellar motility that plays an important role in bacterial life. It was found that metformin was sensed as a repellent to E. coli by tsr chemoreceptors. Moreover, we investigated the chemotactic response of E. coli cultured with metformin to two typical attractants, glucose and α-methyl-DL-aspartate (MeAsp), finding that metformin prolonged the chemotactic recovery time to the attractants, followed by the recovery time increasing with the concentration of stimulus. Metformin also inhibited the flagellar motility of E. coli including the flagellar motor rotation and cell swimming. The inhibition was due to the reduction of torque generated by the flagellar motor. Our discovery that metformin alters the behavior of chemotaxis and flagellar motility of E. coli could provide potential implications for the effect of metformin on other microorganisms.

Bacterial lifestyle switch in response to algal metabolites

Unicellular algae, termed phytoplankton, greatly impact the marine environment by serving as the basis of marine food webs and by playing central roles in biogeochemical cycling of elements. The interactions between phytoplankton and heterotrophic bacteria affect the fitness of both partners. It is becoming increasingly recognized that metabolic exchange determines the nature of such interactions, but the underlying molecular mechanisms remain underexplored. Here, we investigated the molecular and metabolic basis for the bacterial lifestyle switch, from coexistence to pathogenicity, in Sulfitobacter D7 during its interaction with Emiliania huxleyi, a cosmopolitan bloom-forming phytoplankter. To unravel the bacterial lifestyle switch, we profiled bacterial transcriptomes in response to infochemicals derived from algae in exponential and stationary growth, which induced the Sulfitobacter D7 coexistence and pathogenicity lifestyles, respectively. We found that algal dimethylsulfoniopropionate (DMSP) was a pivotal signaling molecule that mediated the transition between the lifestyles. However, the coexisting and pathogenic lifestyles were evident only in the presence of additional algal metabolites. In the pathogenic mode, Sulfitobacter D7 upregulated flagellar motility and many transport systems, presumably to maximize assimilation of E. huxleyi-derived metabolites released by algal cells upon cell death. Specifically, we discovered that algae-produced benzoate promoted the growth of Sulfitobacter D7, and negated the DMSP-inducing lifestyle switch to pathogenicity, demonstrating that benzoate is important for maintaining the coexistence of algae and bacteria. We propose that bacteria can sense the physiological status of the algal host through changes in the metabolic composition, which will determine the bacterial lifestyle during the interactions.

Consensus nomenclature for dyneins and associated assembly factors

Dyneins are highly complex, multicomponent, microtubule-based molecular motors. These enzymes are responsible for numerous motile behaviors in cytoplasm, mediate retrograde intraflagellar transport (IFT), and power ciliary and flagellar motility. Variants in multiple genes encoding dyneins, outer dynein arm (ODA) docking complex subunits, and cytoplasmic factors involved in axonemal dynein preassembly (DNAAFs) are associated with human ciliopathies and are of clinical interest. Therefore, clear communication within this field is particularly important. Standardizing gene nomenclature, and basing it on orthology where possible, facilitates discussion and genetic comparison across species. Here, we discuss how the human gene nomenclature for dyneins, ODA docking complex subunits, and DNAAFs has been updated to be more functionally informative and consistent with that of the unicellular green alga Chlamydomonas reinhardtii, a key model organism for studying dyneins and ciliary function. We also detail additional nomenclature updates for vertebrate-specific genes that encode dynein chains and other proteins involved in dynein complex assembly.

In planta transcriptomics reveals conflicts between pattern-triggered immunity and the AlgU sigma factor regulon

In previous work, we determined the transcriptomic impacts of flg22 pre-induced Pattern Triggered Immunity (PTI) in Arabidopsis thaliana on the pathogen Pseudomonas syringae pv. tomato DC3000 (Pto). During PTI exposure we observed expression patterns in Pto reminiscent of those previously observed in a Pto algU mutant. AlgU is a conserved extracytoplasmic function sigma factor which has been observed to regulate over 950 genes in Pto in vitro. We sought to identify the AlgU regulon in planta.and which PTI-regulated genes overlapped with AlgU-regulated genes. In this study, we analyzed transcriptomic data from RNA-sequencing to identify the AlgU in planta regulon and its relationship with PTI. Our results showed that approximately 224 genes are induced by AlgU, while another 154 genes are downregulated by AlgU in Arabidopsis during early infection. Both stress response and virulence-associated genes were induced by AlgU, while the flagellar motility genes are downregulated by AlgU. Under the pre-induced PTI condition, more than half of these AlgU-regulated genes have lost induction/suppression in contrast to naive plants, and almost all function groups regulated by AlgU were affected by PTI.

The Campylobacter jejuni Response Regulator and Cyclic-Di-GMP Binding CbrR Is a Novel Regulator of Flagellar Motility

A leading cause of bacterial gastroenteritis, Campylobacter jejuni is also associated with broad sequelae, including extragastrointestinal conditions such as reactive arthritis and Guillain-Barré Syndrome (GBS). CbrR is a C. jejuni response regulator that is annotated as a diguanylate cyclase (DGC), an enzyme that catalyzes the synthesis of c-di-GMP, a universal bacterial second messenger, from GTP. In C. jejuni DRH212, we constructed an unmarked deletion mutant, cbrR−, and complemented mutant, cbrR+. Motility assays indicated a hyper-motile phenotype associated with cbrR−, whereas motility was deficient in cbrR+. The overexpression of CbrR in cbrR+ was accompanied by a reduction in expression of FlaA, the major flagellin. Biofilm assays and scanning electron microscopy demonstrated similarities between DRH212 and cbrR−; however, cbrR+ was unable to form significant biofilms. Transmission electron microscopy showed similar cell morphology between the three strains; however, cbrR+ cells lacked flagella. Differential radial capillary action of ligand assays (DRaCALA) showed that CbrR binds GTP and c-di-GMP. Liquid chromatography tandem mass spectrometry detected low levels of c-di-GMP in C. jejuni and in E. coli expressing CbrR. CbrR is therefore a negative regulator of FlaA expression and motility, a critical virulence factor in C. jejuni pathogenesis.

Distinct within-host bacterial populations ensure function, colonization and transmission in leaf symbiosis

Hereditary symbioses have the potential to drive transgenerational effects, yet the mechanisms responsible for transmission of heritable plant symbionts are still poorly understood. The leaf symbiosis between Dioscorea sansibarensis and the bacterium Orrella dioscoreae offers an appealing model system to study how heritable bacteria are transmitted to the next generation. Here, we demonstrate that inoculation of apical buds with a bacterial suspension is sufficient to colonize newly-formed leaves and propagules, and to ensure transmission to the next plant generation. Flagellar motility is not required for movement inside the plant, but is important for the colonization of new hosts. Further, stringent tissue-specific regulation of putative symbiotic functions highlight the presence of two distinct subpopulations of bacteria in the leaf gland and at the shoot meristem. We propose that bacteria in the leaf gland dedicate resources to symbiotic functions, while dividing bacteria in the shoot tip ensure successful colonization of meristematic tissue, glands and propagules. Compartmentalization of intra-host populations, together with tissue-specific regulation may serve as a robust mechanism for the maintenance of mutualism in leaf symbiosis.ImportanceSeveral plant species form associations with bacteria in their leaves, called leaf symbiosis. These associations are highly specific, but the mechanisms responsible for symbiont transmission are poorly understood. Using the association between the yam species Dioscorea sansibarensis and Orrella dioscoreae as a model leaf symbiosis, we provide experimental evidence that bacteria are transmitted vertically and distributed to specific leaf structures via association with shoot meristems. Flagellar motility is required for initial infection, but does not contribute to spread within host tissue. We also provide evidence that bacterial subpopulations at the meristem or in the symbiotic leaf gland differentially express key symbiotic genes. We argue that this separation of functional symbiont populations, coupled to tight control over bacterial infection and transmission, explain the evolutionary robustness of leaf symbiosis. These findings may provide insights into how plants may recruit and maintain beneficial symbionts at the leaf surface.

Using Experimentally Calibrated Regularized Stokeslets to Assess Bacterial Flagellar Motility Near a Surface

The presence of a nearby boundary is likely to be important in the life cycle and evolution of motile flagellate bacteria. This has led many authors to employ numerical simulations to model near-surface bacterial motion and compute hydrodynamic boundary effects. A common choice has been the method of images for regularized Stokeslets (MIRS); however, the method requires discretization sizes and regularization parameters that are not specified by any theory. To determine appropriate regularization parameters for given discretization choices in MIRS, we conducted dynamically similar macroscopic experiments and fit the simulations to the data. In the experiments, we measured the torque on cylinders and helices of different wavelengths as they rotated in a viscous fluid at various distances to a boundary. We found that differences between experiments and optimized simulations were less than 5% when using surface discretizations for cylinders and centerline discretizations for helices. Having determined optimal regularization parameters, we used MIRS to simulate an idealized free-swimming bacterium constructed of a cylindrical cell body and a helical flagellum moving near a boundary. We assessed the swimming performance of many bacterial morphologies by computing swimming speed, motor rotation rate, Purcell’s propulsive efficiency, energy cost per swimming distance, and a new metabolic energy cost defined to be the energy cost per body mass per swimming distance. All five measures predicted that the optimal flagellar wavelength is eight times the helical radius independently of body size and surface proximity. Although the measures disagreed on the optimal body size, they all predicted that body size is an important factor in the energy cost of bacterial motility near and far from a surface.

Non-motile subpopulations of Pseudomonas aeruginosa repress flagellar motility in motile cells through a type IV pili- and Pel-dependent mechanism

The downregulation of P. aeruginosa flagellar motility is a key event in biofilm formation, host-colonization, and the formation of microbial communities, but the external factors that repress motility are not well understood. Here, we report that under swarming conditions, motility can be repressed by cells that are non-motile due to the absence of a flagellum or flagellar rotation. Non-motile cells, due to mutations that prevent either flagellum biosynthesis or rotation, present at 5% of the total population suppressed swarming of wild-type cells under the conditions tested in this study. Non-motile cells required functional type IV pili and the ability to produce the Pel exopolysaccharide to suppress swarming by the motile wild type. In contrast, motile cells required only type IV pili, but not Pel production, in order for swarming to be repressed by non-motile cells. We hypothesize that interactions between motile and non-motile cells may enhance the formation of sessile communities including those involving multiple genotypes, phenotypically-diverse cells, and perhaps other species.

Reduced Infection Efficiency of Phage NCTC 12673 on Non-Motile Campylobacter jejuni Strains Is Related to Oxidative Stress

Campylobacter jejuni is a Gram-negative foodborne pathogen that causes diarrheal disease and is associated with severe post-infectious sequelae. Bacteriophages (phages) are a possible means of reducing Campylobacter colonization in poultry to prevent downstream human infections. However, the factors influencing phage-host interactions must be better understood before this strategy can be predictably employed. Most studies have focused on Campylobacter phage binding to the host surface, with all phages classified as either capsule- or flagella-specific. Here we describe the characterization of a C. jejuni phage that requires functional flagellar glycosylation and motor genes for infection, without needing the flagella for adsorption to the cell surface. Through phage infectivity studies of targeted C. jejuni mutants, transcriptomic analysis of phage-resistant mutants, and genotypic and phenotypic analysis of a spontaneous phage variant capable of simultaneously overcoming flagellar gene dependence and sensitivity to oxidative stress, we have uncovered a link between oxidative stress, flagellar motility, and phage infectivity. Taken together, our results underscore the importance of understanding phage-host interactions beyond the cell surface and point to host oxidative stress state as an important and underappreciated consideration for future phage-host interaction studies.