Transitioning to confined spaces impacts bacterial swimming and escape response

Symbiotic bacteria often navigate complex environments before colonizing privileged sites in their host organism. Chemical gradients are known to facilitate directional taxis of these bacteria, guiding them toward their eventual destination. However, less is known about the role of physical features in shaping the path the bacteria take and defining how they traverse a given space. The flagellated marine bacterium Vibrio fischeri, which forms a binary symbiosis with the Hawaiian bobtail squid, Euprymna scolopes, must navigate tight physical confinement during colonization, squeezing through a tissue bottleneck constricting to ～2 μm in width on the way to its eventual home. Using microfluidic in vitro experiments, we discovered that V. fischeri cells alter their behavior upon entry into confined space, straightening their swimming paths and promoting escape from confinement. Using a computational model, we attributed this escape response to two factors: reduced directional fluctuation and a refractory period between reversals. Additional experiments in asymmetric capillary tubes confirmed that V. fischeri quickly escape from confined ends, even when drawn into the ends by chemoattraction. This avoidance was apparent down to a limit of confinement approaching the diameter of the cell itself, resulting in a balance between chemoattraction and evasion of physical confinement. Our findings demonstrate that nontrivial distributions of swimming bacteria can emerge from simple physical gradients in the level of confinement. Tight spaces may serve as an additional, crucial cue for bacteria while they navigate complex environments to enter specific habitats.     

Multiple CheY Homologs Control Swimming Reversals and Transient Pauses in Azospirillum brasilense

Chemotaxis, together with motility, helps bacteria foraging in their habitat. Motile bacteria exhibit a variety of motility patterns, often controlled by chemotaxis, to promote dispersal. Motility in many bacteria is powered by a bidirectional flagellar motor. The flagellar motor has been known to briefly pause during rotation because of incomplete reversals or stator detachment. Transient pauses were previously observed in bacterial strains lacking CheY, and these events could not be explained by incomplete motor reversals or stator detachment. Here, we systematically analyzed swimming trajectories of various chemotaxis mutants of the monotrichous soil bacterium, Azospirillum brasilense. Like other polar flagellated bacterium, the main swimming pattern in A. brasilense is run and reverse. A. brasilense also uses run-pauses and putative run-reverse-flick-like swimming patterns, although these are rare events. A. brasilense mutant derivatives lacking the chemotaxis master histidine kinase, CheA4, or the central response regulator, CheY7, also showed transient pauses. Strikingly, the frequency of transient pauses increased dramatically in the absence of CheY4. Our findings collectively suggest that reversals and pauses are controlled through signaling by distinct CheY homologs, and thus are likely to be functionally important in the lifestyle of this soil organism.     

Roles of CytR,an anti-activator of cyclic-AMP receptor protein (CRP) on flagellar expression and virulence in uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infection (UTI), a common bacterial infectious disease. This bacterium invades the urinary tract cells, where it aggregates, and subsequently forms multicellular colonies termed intracellular bacterial communities (IBCs). The motility of the bacteria plays a key role in the mechanism of virulence in the host bladder. Here, we show that CytR is a modulator of bacterial internalization and aggregation within the bladder epithelial cells sustained by CRP in UPEC. Mutational analyses and gel-shift assays indicated that CytR represses the expression of flhD, thereby encoding a master regulator for flagellar expression that is responsible for bacterial motility when CRP is present, whereas CRP is an activator of flhD expression. Thus, elevated flagellar expression was involved in promoted virulence in the cytR mutant. These combined observations suggest another regulatory layer of flagellar expression and the role of CytR in UPEC virulence.     

Exploring potential applications of a novel extracellular polymeric substance synthesizing bacterium (<Emphasis Type="Italic">Bacillus licheniformis</Emphasis>) isolated from gut contents of earthworm (<Emphasis Type="Italic">Metaphire posthuma</Emphasis>) in environmental remediation

The aim was to isolate, characterize, and explore potentials of gut bacteria from the earthworm (Metaphire posthuma) and imply these bacteria for remediation of Cu(II) and Zn(II). An extracellular polymeric substance (EPS) producing gut bacteria (Bacillus licheniformis strain KX657843) was isolated and identified based on 16S rRNA sequencing and phylogenetic analysis. The strain showed maximum tolerance of 8 and 6 mM for Cu(II) and Zn(II) respectively. It removed 34.5% of Cu(II) and 54.4% of Zn(II) at 25 mg L^?1 after 72 and 96 h incubation respectively. The bacteria possessed a great potential to produce indole acetic acid (38.49 μg mL^?1) at 5 mg mL^?1 l-tryptophan following 12 days incubation. The sterilized seeds of mung beans (Vigna radiata) displayed greater germination and growth under bacterium enriched condition. We observed that the bacterial strain phosphate solubilization ability with a maximum of 204.2 mg L^?1 in absence of Cu(II) and Zn(II). Endowed with biosurfactant property the bacterium exhibited 24% emulsification index. The bacterium offered significant potential of plant growth promotion, Cu(II) and Zn(II) removal, and as such this study is the first report on EPS producing B. licheniformis KX657843 from earthworm which can be applied as powerful tool in remediation programs of Cu(II) and Zn(II) contaminated sites.     

Cloning,sequencing and expression of the flagellin core protein and other genes encoding structural proteins of the Vibrio cholerae flagellum

Vibrio cholerae is a Gram-negative bacterium with a single polar flagellum. Motility is an important virulence factor for this non-invasive pathogen. We cloned and sequenced a locus in V. cholerae V86 (El Tor, Inaba) that contained five different structural genes of the flagellum. The cloned genes and their products were assigned names and functions based on homology with sequences of similar genes and their products from other related bacteria. All of these genes of V. cholerae V86, namely, flgI, J, M, L and flaA, were transcribed in the same direction. These genes respectively encoded the P- and L-ring proteins, the hook-associated proteins 1 and 3 and the flagellin core protein of the flagellum. Our data indicated the presence of more than one flagellar locus in V. cholerae which could provide a means of immunoavoidance during infection. When compared with homologs in other bacteria, the flagellin core protein of V. cholerae exhibited conservation in the N- and C-termini, but had diverged in the central region.     

Confinement Regulates Complex Biochemical Networks: Initiation of Blood Clotting by “Diffusion Acting”

This study shows that environmental confinement strongly affects the activation of nonlinear reaction networks, such as blood coagulation (clotting), by small quantities of activators. Blood coagulation is sensitive to the local concentration of soluble activators, initiating only when the activators surpass a threshold concentration, and therefore is regulated by mass transport phenomena such as flow and diffusion. Here, diffusion was limited by decreasing the size of microfluidic chambers, and it was found that microparticles carrying either the classical stimulus, tissue factor, or a bacterial stimulus, Bacillus cereus, initiated coagulation of human platelet-poor plasma only when confined. A simple analytical argument and numerical model were used to describe the mechanism for this phenomenon: confinement causes diffusible activators to accumulate locally and surpass the threshold concentration. To interpret the results, a dimensionless confinement number, Cn, was used to describe whether a stimulus was confined, and a Damköhler number, Da₂, was used to describe whether a subthreshold stimulus could initiate coagulation. In the context of initiation of coagulation by bacteria, this mechanism can be thought of as “diffusion acting”, which is distinct from “diffusion sensing”. The ability of confinement and diffusion acting to change the outcome of coagulation suggests that confinement should also regulate other biological “on” and “off” processes that are controlled by thresholds.     

The Deep-Sea Bacterium Photobacterium profundum SS9 Utilizes Separate Flagellar Systems for Swimming and Swarming under High-Pressure Conditions

Motility is a critical function needed for nutrient acquisition, biofilm formation, and the avoidance of harmful chemicals and predators. Flagellar motility is one of the most pressure-sensitive cellular processes in mesophilic bacteria; therefore, it is ecologically relevant to determine how deep-sea microbes have adapted their motility systems for functionality at depth. In this study, the motility of the deep-sea piezophilic bacterium Photobacterium profundum SS9 was investigated and compared with that of the related shallow-water piezosensitive strain Photobacterium profundum 3TCK, as well as that of the well-studied piezosensitive bacterium Escherichia coli. The SS9 genome contains two flagellar gene clusters: a polar flagellum gene cluster (PF) and a putative lateral flagellum gene cluster (LF). In-frame deletions were constructed in the two flagellin genes located within the PF cluster (flaA and flaC), the one flagellin gene located within the LF cluster (flaB), a component of a putative sodium-driven flagellar motor (motA2), and a component of a putative proton-driven flagellar motor (motA1). SS9 PF flaA, flaC, and motA2 mutants were defective in motility under all conditions tested. In contrast, the flaB and motA1 mutants were defective only under conditions of high pressure and high viscosity. flaB and motA1 gene expression was strongly induced by elevated pressure plus increased viscosity. Direct swimming velocity measurements were obtained using a high-pressure microscopic chamber, where increases in pressure resulted in a striking decrease in swimming velocity for E. coli and a gradual reduction for 3TCK which proceeded up to 120 MPa, while SS9 increased swimming velocity at 30 MPa and maintained motility up to a maximum pressure of 150 MPa. Our results indicate that P. profundum SS9 possesses two distinct flagellar systems, both of which have acquired dramatic adaptations for optimal functionality under high-pressure conditions.     

A Non-Poissonian Flagellar Motor Switch Increases Bacterial Chemotactic Potential

We investigate bacterial chemotactic strategies using run-tumble and run-reverse-flick motility patterns. The former is typically observed in enteric bacteria such as Escherichia coli and Salmonella and the latter was recently observed in the marine bacteria Vibrio alginolyticus and is possibly exhibited by other polar flagellated species. It is shown that although the three-step motility pattern helps the bacterium to localize near hot spots, an exploitative behavior, its exploratory potential in short times can be significantly enhanced by employing a non-Poissonian regulation scheme for its flagellar motor switches.     

Colonization of Lutzomyia verrucarum and Lutzomyia longipalpis Sand Flies (Diptera: Psychodidae) by Bartonella bacilliformis,the Etiologic Agent of Carrión’s Disease

Bartonella bacilliformis is a pathogenic bacterium transmitted to humans presumably by bites of phlebotomine sand flies, infection with which results in a bi-phasic syndrome termed Carrión’s disease. After constructing a low-passage GFP-labeled strain of B. bacilliformis, we artificially infected Lutzomyia verrucarum and L. longipalpis populations, and subsequently monitored colonization of sand flies by fluorescence microscopy. Initially, colonization of the two fly species was indistinguishable, with bacteria exhibiting a high degree of motility, yet still confined to the abdominal midgut. After 48h, B. bacilliformis transitioned from bacillus-shape to a non-motile, small coccoid form and appeared to be digested along with the blood meal in both fly species. Differences in colonization patterns became evident at 72h when B. bacilliformis was observed at relatively high density outside the peritrophic membrane in the lumen of the midgut in L. verrucarum, but colonization of L. longipalpis was limited to the blood meal within the intra-peritrophic space of the abdominal midgut, and the majority of bacteria were digested along with the blood meal by day 7. The viability of B. bacilliformis in L. longipalpis was assessed by artificially infecting, homogenizing, and plating for determination of colony-forming units in individual flies over a 13-d time course. Bacteria remained viable at relatively high density for approximately seven days, suggesting that L. longipalpis could potentially serve as a vector. The capacity of L. longipalpis to transmit viable B. bacilliformis from infected to uninfected meals was analyzed via interrupted feeds. No viable bacteria were retrieved from uninfected blood meals in these experiments. This study provides significant information toward understanding colonization of sand flies by B. bacilliformis and also demonstrates the utility of L. longipalpis as a user-friendly, live-vector model system for studying this severely neglected tropical disease.     

Coarse-grained molecular dynamics simulations of a rotating bacterial flagellum

Many types of bacteria propel themselves using elongated structures known as flagella. The bacterial flagellar filament is a relatively simple and well-studied macromolecular assembly, which assumes different helical shapes when rotated in different directions. This polymorphism enables a bacterium to switch between running and tumbling modes; however, the mechanism governing the filament polymorphism is not completely understood. Here we report a study of the bacterial flagellar filament using numerical simulations that employ a novel coarse-grained molecular dynamics method. The simulations reveal the dynamics of a half-micrometer-long flagellum segment on a timescale of tens of microseconds. Depending on the rotation direction, specific modes of filament coiling and arrangement of monomers are observed, in qualitative agreement with experimental observations of flagellar polymorphism. We find that solvent-protein interactions are likely to contribute to the polymorphic helical shapes of the filament.     

Agrobacterium tumefaciens Site Attachment as a Necessary Prerequisite for Crown Gall Tumor Formation on Potato Discs

The infectivity of Agrobacterium tumefaciens strain B6 was inhibited about 50% when these bacteria were inoculated on potato discs with equal viable cell counts of a weakly virulent strain of A. tumefaciens (B-48) or autoclaved strains of B6 or B-48. Inhibition by B-48 or autoclaved B6 could still be obtained when these cells were added up to a maximum of 10 minutes after the addition of viable B6. Maximum inhibition occurred when these cells were added 10 minutes prior to the addition of B6. There was no inhibition observed when equal cell counts of B6 were added along with a Gram-positive bacterium or yeast cell, while inhibition was observed when these B6 cells were added simultaneously with other Gram-negative cells. These results suggest that a physical, specific bacterial attachment that occurs within 10 minutes is necessary for tumor formation on potato discs.     

Mannose-Binding Lectin Inhibits the Motility of Pathogenic Salmonella by Affecting the Driving Forces of Motility and the Chemotactic Response

Mannose-binding lectin (MBL) is a key pattern recognition molecule in the lectin pathway of the complement system, an important component of innate immunity. MBL functions as an opsonin which enhances the sequential immune process such as phagocytosis. We here report an inhibitory effect of MBL on the motility of pathogenic bacteria, which occurs by affecting the energy source required for motility and the signaling pathway of chemotaxis. When Salmonella cells were treated with a physiological concentration of MBL, their motile fraction and free-swimming speed decreased. Rotation assays of a single flagellum showed that the flagellar rotation rate was significantly reduced by the addition of MBL. Measurements of the intracellular pH and membrane potential revealed that MBL affected a driving force for the Salmonella flagellum, the electrochemical potential difference of protons. We also found that MBL treatment increased the reversal frequency of Salmonella flagellar rotation, which interfered with the relative positive chemotaxis toward an attractive substrate. We thus propose that the motility inhibition effect of MBL may be secondarily involved in the attack against pathogens, potentially facilitating the primary role of MBL in the complement system.     

FlgM Is Secreted by the Flagellar Export Apparatus in Bacillus subtilis

The bacterial flagellum is assembled from over 20 structural components, and flagellar gene regulation is morphogenetically coupled to the assembly state by control of the anti-sigma factor FlgM. In the Gram-negative bacterium Salmonella enterica, FlgM inhibits late-class flagellar gene expression until the hook-basal body structural intermediate is completed and FlgM is inhibited by secretion from the cytoplasm. Here we demonstrate that FlgM is also secreted in the Gram-positive bacterium Bacillus subtilis and is degraded extracellularly by the proteases Epr and WprA. We further demonstrate that, like in S. enterica, the structural genes required for the flagellar hook-basal body are required for robust activation of σ^D-dependent gene expression and efficient secretion of FlgM. Finally, we determine that FlgM secretion is strongly enhanced by, but does not strictly require, hook-basal body completion and instead demands a minimal subset of flagellar proteins that includes the FliF/FliG basal body proteins, the flagellar type III export apparatus components FliO, FliP, FliQ, FliR, FlhA, and FlhB, and the substrate specificity switch regulator FliK.     

Identification and Characterization of a Novel Flagellum-Dependent Salmonella-Infecting Bacteriophage,iEPS5

A novel flagellatropic phage of Salmonella enterica serovar Typhimurium, called iEPS5, was isolated and characterized. iEPS5 has an icosahedral head and a long noncontractile tail with a tail fiber. Genome sequencing revealed a double-stranded DNA of 59,254 bp having 73 open reading frames (ORFs). To identify the receptor for iEPS5, Tn5 transposon insertion mutants of S. Typhimurium SL1344 that were resistant to the phage were isolated. All of the phage-resistant mutants were found to have mutations in genes involved in flagellar formation, suggesting that the flagellum is the adsorption target of this phage. Analysis of phage infection using the ΔmotA mutant, which is flagellated but nonmotile, demonstrated the requirement of flagellar rotation for iEPS5 infection. Further analysis of phage infection using the ΔcheY mutant revealed that iEPS5 could infect host bacteria only when the flagellum is rotating counterclockwise (CCW). These results suggested that the CCW-rotating flagellar filament is essential for phage adsorption and required for successful infection by iEPS5. In contrast to the well-studied flagellatropic phage Chi, iEPS5 cannot infect the ΔfliK mutant that makes a polyhook without a flagellar filament, suggesting that these two flagellatropic phages utilize different infection mechanisms. Here, we present evidence that iEPS5 injects its DNA into the flagellar filament for infection by assessing DNA transfer from SYBR gold-labeled iEPS5 to the host bacteria.     

An Element of Determinism in a Stochastic Flagellar Motor Switch

Marine bacterium Vibrio alginolyticus uses a single polar flagellum to navigate in an aqueous environment. Similar to Escherichia coli cells, the polar flagellar motor has two states; when the motor is counter-clockwise, the cell swims forward and when the motor is clockwise, the cell swims backward. V. alginolyticus also incorporates a direction randomization step at the start of the forward swimming interval by flicking its flagellum. To gain an understanding on how the polar flagellar motor switch is regulated, distributions of the forward Δ_f and backward Δ_b intervals are investigated herein. We found that the steady-state probability density functions, P(Δ_f) and P(Δ_b), of freely swimming bacteria are strongly peaked at a finite time, suggesting that the motor switch is not Poissonian. The short-time inhibition is sufficiently strong and long lasting, i.e., several hundred milliseconds for both intervals, which is readily observed and characterized. Treating motor reversal dynamics as a first-passage problem, which results from conformation fluctuations of the motor switch, we calculated P(Δ_f) and P(Δ_b) and found good agreement with the measurements.     

Spatiotemporal distribution of bacteriochlorophylls in the meromictic Lake Suigetsu, Japan

The spatiotemporal distribution of chlorophyll pigments (chloropigments) in the water column of a meromictic lake, Lake Suigetsu (Fukui, Japan), was investigated. Water samples were collected from the central basin of Lake Suigetsu bimonthly between May 2008 and March 2010 at appropriate depths, including the oxic surface, oxic–anoxic interface, and anoxic bottom layers. Chlorophyll a, related to cyanobacteria and eukaryotic phytoplankton, was detected throughout the water column during the years of the study, whereas bacteriochlorophyll e, related to brown-colored green sulfur bacteria, was detected in the anoxic layers below the chemocline at a maximum concentration of 825 μg L^?1. The concentration of bacteriochlorophyll e was generally maximal at or just below the chemocline of the lake. The cellular content of bacteriochlorophyll e was estimated to be low in the upper part of the chemocline and tended to increase with increasing water depth. Bacteriochlorophyll a, which was presumably related to purple sulfur bacteria, was only detected at the chemocline during summer and autumn at concentrations of 5.4–16.3 μg L^?1. Our analysis of the chloropigment distribution for the two years of the study suggested that brown-colored green sulfur bacteria are the predominant phototroph in the anoxic layers of Lake Suigetsu, and that these play a significant role in the carbon and sulfur cycling of the lake, especially from spring to summer.     

Cellular Architecture of Treponema pallidum: Novel Flagellum, Periplasmic Cone, and Cell Envelope as Revealed by Cryo Electron Tomography

High-resolution cryo electron tomography (cryo-ET) was utilized to visualize Treponema pallidum, the causative agent of syphilis, at the molecular level. Three-dimensional (3D) reconstructions from 304 infectious organisms revealed unprecedented cellular structures of this unusual member of the spirochetal family. High-resolution cryo-ET reconstructions provided detailed structures of the cell envelope, which is significantly different from that of Gram-negative bacteria. The 4-nm lipid bilayer of both outer membrane and cytoplasmic membrane resolved in 3D reconstructions, providing an important marker for interpreting membrane-associated structures. Abundant lipoproteins cover the outer leaflet of the cytoplasmic membrane, in contrast to the rare outer membrane proteins visible by scanning probe microscopy. High-resolution cryo-ET images also provided the first observation of T. pallidum chemoreceptor arrays, as well as structural details of the periplasmically located cone-shaped structure at both ends of the bacterium. Furthermore, 3D subvolume averages of periplasmic flagellar motors and flagellar filaments from living organisms revealed the novel flagellar architectures that may facilitate their rotation within the confining periplasmic space. Our findings provide the most detailed structural understanding of periplasmic flagella and the surrounding cell envelope, which enable this enigmatic bacterium to efficiently penetrate tissue and to escape host immune responses.     

Enrichment and physiological characterization of an anaerobic ammonium-oxidizing bacterium ‘Candidatus Brocadia sapporoensis’

We successfully enriched a novel anaerobic ammonium-oxidizing (anammox) bacterium affiliated with the genus ‘Candidatus Brocadia’ with high purity (>90%) in a membrane bioreactor (MBR). The enriched bacterium was distantly related to the hitherto characterized ‘Ca. Brocadia fulgida’ and ‘Ca. Brocadia sinica’ with 96% and 93% of 16S ribosomal RNA gene sequence identity, respectively. The bacterium exhibited the common structural features of anammox bacteria and produced hydrazine in the presence of hydroxylamine under anoxic conditions. The temperature range of anammox activity was 20–45 °C with a maximum activity at 37 °C. The maximum specific growth rate (μ_max) was 0.0082 h^?1 at 37 °C, corresponding to a doubling time of 3.5 days. The half-saturation constant (K_S) for nitrite was 5 ± 2.5 μM. The anammox activity was inhibited by nitrite (IC₅₀ = 11.6 mM) but not by formate and acetate. The major respiratory quinone was identified to be menaquinone-7 (MK-7). The enriched anammox bacterium shared nearly half of genes with ‘Ca. Brocadia sinica’ and ‘Ca. Brocadia fulgida’. The enriched bacterium showed all known physiological characteristics of anammox bacteria and can be distinguished from the close relatives by its 16S rRNA gene sequence. Therefore, we proposed the name ‘Ca. Brocadia sapporoensis’ sp. nov.     

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.