scholarly journals Insertional Inactivation of Genes Encoding Components of the Sodium-Type Flagellar Motor and Switch ofVibrio parahaemolyticus

2000 ◽  
Vol 182 (4) ◽  
pp. 1035-1045 ◽  
Author(s):  
Blaise R. Boles ◽  
Linda L. McCarter
Keyword(s):  
Motor Function ◽  
Vibrio Parahaemolyticus ◽  
Common Mechanism ◽  
Flagellar Motor ◽  
E Coli ◽  
Insertional Inactivation ◽  
Torque Generation ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Type Motor

ABSTRACT Vibrio parahaemolyticus possesses two types of flagella, polar and lateral, powered by distinct energy sources, which are derived from the sodium and proton motive forces, respectively. Although proton-powered flagella in Escherichia coli andSalmonella enterica serovar Typhimurium have been extensively studied, the mechanism of torque generation is still not understood. Molecular knowledge of the structure of the sodium-driven motor is only now being developed. In this work, we identify the switch components, FliG, FliM, and FliN, of the sodium-type motor. This brings the total number of genes identified as pertinent to polar motor function to seven. Both FliM and FliN possess charged domains not found in proton-type homologs; however, they can interact with the proton-type motor of E. coli to a limited extent. Residues known to be critical for torque generation in the proton-type motor are conserved in the sodium-type motor, suggesting a common mechanism for energy transfer at the rotor-stator interface regardless of the driving force powering rotation. Mutants representing a complete panel of insertionally inactivated switch and motor genes were constructed. All of these mutants were defective in sodium-driven swimming motility. Alkaline phosphatase could be fused to the C termini of MotB and MotY without abolishing motility, whereas deletion of the unusual, highly charged C-terminal domain of FliM disrupted motor function. All of the mutants retained proton-driven, lateral motility over surfaces. Thus, although central chemotaxis genes are shared by the polar and lateral systems, genes encoding the switch components, as well as the motor genes, are distinct for each motility system.

scholarly journals Effect of the MotA(M206I) Mutation on Torque Generation and Stator Assembly in theSalmonellaH+-Driven Flagellar Motor

2019 ◽  
Vol 201 (6) ◽  
Author(s):  
Yuya Suzuki ◽  
Yusuke V. Morimoto ◽  
Kodai Oono ◽  
Fumio Hayashi ◽  
Kenji Oosawa ◽  
...  
Keyword(s):  
Ion Flux ◽  
Motive Force ◽  
Flagellar Motor ◽  
Wild Type ◽  
Torque Generation ◽  
Unit Speed ◽  
Bacterial Flagellar Motor ◽  
Ph Dependent ◽  
Type Motor ◽  
External Ph

ABSTRACTThe bacterial flagellar motor is composed of a rotor and a dozen stators and converts the ion flux through the stator into torque. Each stator unit alternates in its attachment to and detachment from the rotor even during rotation. In some species, stator assembly depends on the input energy, but it remains unclear how an electrochemical potential across the membrane (e.g., proton motive force [PMF]) or ion flux is involved in stator assembly dynamics. Here, we focused on pH dependence of a slow motile MotA(M206I) mutant ofSalmonella. The MotA(M206I) motor produces torque comparable to that of the wild-type motor near stall, but its rotation rate is considerably decreased as the external load is reduced. Rotation assays of flagella labeled with 1-μm beads showed that the rotation rate of the MotA(M206I) motor is increased by lowering the external pH whereas that of the wild-type motor is not. Measurements of the speed produced by a single stator unit using 1-μm beads showed that the unit speed of the MotA(M206I) is about 60% of that of the wild-type and that a decrease in external pH did not affect the MotA(M206I) unit speed. Analysis of the subcellular stator localization revealed that the number of functional stators is restored by lowering the external pH. The pH-dependent improvement of stator assembly was observed even when the PMF was collapsed and proton transfer was inhibited. These results suggest that MotA-Met206 is responsible for not only load-dependent energy coupling between the proton influx and rotation but also pH-dependent stator assembly.IMPORTANCEThe bacterial flagellar motor is a rotary nanomachine driven by the electrochemical transmembrane potential (ion motive force). About 10 stators (MotA/MotB complexes) are docked around a rotor, and the stator recruitment depends on the load, ion motive force, and coupling ion flux. The MotA(M206I) mutation slows motor rotation and decreases the number of docked stators inSalmonella. We show that lowering the external pH improves the assembly of the mutant stators. Neither the collapse of the ion motive force nor a mutation mimicking the proton-binding state inhibited stator localization to the motor. These results suggest that MotA-Met206 is involved in torque generation and proton translocation and that stator assembly is stabilized by protonation of the stator.

An iron-regulated gene required for utilization of aerobactin as an exogenous siderophore in Vibrio parahaemolyticus

Microbiology ◽  
2003 ◽  
Vol 149 (5) ◽  
pp. 1217-1225 ◽  
Author(s):  
Tatsuya Funahashi ◽  
Tomotaka Tanabe ◽  
Hiroaki Aso ◽  
Hiroshi Nakao ◽  
Yoshio Fujii ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Vibrio Parahaemolyticus ◽  
Outer Membrane Receptor ◽  
Gene Encoding ◽  
E Coli ◽  
Iron Deficient ◽  
Insertional Inactivation ◽  
Titration Assay ◽  
Binding Sequence

A previous investigation using the Fur titration assay system showed that Vibrio parahaemolyticus possesses a gene encoding a protein homologous to IutA, the outer-membrane receptor for ferric aerobactin in Escherichia coli. In this study, a 5·6 kb DNA region from the V. parahaemolyticus WP1 genome was cloned and two entire genes, iutA and alcD homologues, were identified which are absent from Vibrio cholerae genomic sequences. The V. parahaemolyticus IutA and AlcD proteins share 43 % identity with the Escherichia coli IutA protein and 24 % identity with the Bordetella bronchiseptica AlcD protein of unknown function, respectively. Primer extension analysis revealed that the iutA gene is transcribed in response to low-iron availability from a putative promoter overlapped with a sequence resembling a consensus E. coli Fur-binding sequence. In agreement with the above finding, V. parahaemolyticus effectively utilized exogenously supplied aerobactin for growth under iron-limiting conditions. Moreover, insertional inactivation of iutA impaired growth in the presence of aerobactin and incapacitated the outer-membrane fraction from iron-deficient cells for binding 55Fe-labelled aerobactin. These results indicate that the V. parahaemolyticus iutA homologue encodes an outer-membrane protein which functions as the receptor for ferric aerobactin. Southern blot analysis revealed that the iutA homologues are widely distributed in clinical and environmental isolates of V. parahaemolyticus. However, additional genes required for ferric aerobactin transport across the inner membrane remain to be clarified.

scholarly journals Suppressor Analysis of the MotB(D33E) Mutation To Probe Bacterial Flagellar Motor Dynamics Coupled with Proton Translocation

2008 ◽  
Vol 190 (20) ◽  
pp. 6660-6667 ◽  
Author(s):  
Yong-Suk Che ◽  
Shuichi Nakamura ◽  
Seiji Kojima ◽  
Nobunori Kami-ike ◽  
Keiichi Namba ◽  
...  
Keyword(s):  
High Speed ◽  
Proton Translocation ◽  
Energy Coupling ◽  
Flagellar Motor ◽  
Transmembrane Helices ◽  
Wild Type ◽  
Torque Generation ◽  
Serovar Typhimurium ◽  
Bacterial Flagellar Motor ◽  
Protein Instability

ABSTRACT MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow with motor rotation. Asp-33 of Salmonella enterica serovar Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, the mechanism of energy coupling remains unknown. Here, we carried out genetic and motility analysis of a slowly motile motB(D33E) mutant and its pseudorevertants. We first confirmed that the poor motility of the motB(D33E) mutant is due to neither protein instability, mislocalization, nor impaired interaction with MotA. We isolated 17 pseudorevertants and identified the suppressor mutations in the transmembrane helices TM2 and TM3 of MotA and in TM and the periplasmic domain of MotB. The stall torque produced by the motB(D33E) mutant motor was about half of the wild-type level, while those for the pseudorevertants were recovered nearly to the wild-type levels. However, the high-speed rotations of the motors under low-load conditions were still significantly impaired, suggesting that the rate of proton translocation is still severely limited at high speed. These results suggest that the second-site mutations recover a torque generation step involving stator-rotor interactions coupled with protonation/deprotonation of Glu-33 but not maximum proton conductivity.

scholarly journals The Complex Flagellar Torque Generator of Pseudomonas aeruginosa

2004 ◽  
Vol 186 (19) ◽  
pp. 6341-6350 ◽  
Author(s):  
Timothy B. Doyle ◽  
Andrew C. Hawkins ◽  
Linda L. McCarter
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Membrane Proteins ◽  
Motor Function ◽  
Swimming Speed ◽  
Flagellar Motor ◽  
Loss Of Function ◽  
Wild Type ◽  
Aqueous Environments ◽  
Genes Encoding ◽  
Rotary Motors

ABSTRACT Flagella act as semirigid helical propellers that are powered by reversible rotary motors. Two membrane proteins, MotA and MotB, function as a complex that acts as the stator and generates the torque that drives rotation. The genome sequence of Pseudomonas aeruginosa PAO1 contains dual sets of motA and motB genes, PA1460-PA1461 (motAB) and PA4954-PA4953 (motCD), as well as another gene, motY (PA3526), which is known to be required for motor function in some bacteria. Here, we show that these five genes contribute to motility. Loss of function of either motAB-like locus was dispensable for translocation in aqueous environments. However, swimming could be entirely eliminated by introduction of combinations of mutations in the two motAB-encoding regions. Mutation of both genes encoding the MotA homologs or MotB homologs was sufficient to abolish motility. Mutants carrying double mutations in nonequivalent genes (i.e., motA motD or motB motC) retained motility, indicating that noncognate components can function together. motY appears to be required for motAB function. The combination of motY and motCD mutations rendered the cells nonmotile. Loss of function of motAB, motY, or motAB motY produced similar phenotypes; although the swimming speed was only reduced to ∼85% of the wild-type speed, translocation in semisolid motility agar and swarming on the surface of solidified agar were severely impeded. Thus, the flagellar motor of P. aeruginosa represents a more complex configuration than the configuration that has been studied in other bacteria, and it enables efficient movement under different circumstances.

scholarly journals Functional Analysis of Genes in the rfbLocus of Leptospira borgpetersenii Serovar Hardjo Subtype Hardjobovis

2000 ◽  
Vol 68 (7) ◽  
pp. 3793-3798 ◽  
Author(s):  
Dieter M. Bulach ◽  
Thareerat Kalambaheti ◽  
Alejandro de la Peña-Moctezuma ◽  
Ben Adler
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Open Reading Frames ◽  
New Host ◽  
E Coli ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Leptospira Borgpetersenii ◽  
Nucleotide Sugars

ABSTRACT Lipopolysaccharide (LPS) is a key antigen in immunity to leptospirosis. Its biosynthesis requires enzymes for the biosynthesis and polymerization of nucleotide sugars and the transport through and attachment to the bacterial membrane. The genes encoding these functions are commonly clustered into loci; for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis, this locus, named rfb, spans 36.7 kb and contains 31 open reading frames, of which 28 have been assigned putative functions on the basis of sequence similarity. Characterization of the function of these genes is hindered by the fact that it is not possible to construct isogenic mutant strains in Leptospira. We used two approaches to circumvent this problem. The first was to clone the entire locus into a heterologous host system and determine if a “recombinant” LPS or polysaccharide was synthesized in the new host. The second approach used putative functions to identify mutants in other bacterial species whose mutations might be complemented by genes on the leptospiralrfb locus. This approach was used to investigate the function of three genes in the leptospiral rfb locus and demonstrated function for orfH10, which complemented awbpM strain of Pseudomonas aeruginosa, andorfH13, which complemented an rfbW strain ofVibrio cholerae. However, despite the similarity of OrfH11 to WecC, a wecC strain of E. coli was not complemented by orfH11. The predicted protein encoded byorfH8 is similar to GalE from a number of organisms. ASalmonella enterica serovar Typhimurium strain producing no GalE was used as a background in which orfH8 produced detectable GalE enzyme activity.

scholarly journals Separation and enrichment of sodium-motile bacteria using cost-effective microfluidics

2020 ◽  
Author(s):  
Jyoti P Gurung ◽  
Moein Navvab Kashani ◽  
Sanaz Agarwal ◽  
Murat Gel ◽  
Matthew AB Baker
Keyword(s):  
Low Cost ◽  
Cost Effective ◽  
Flagellar Motor ◽  
Term Selection ◽  
E Coli ◽  
Fold Enrichment ◽  
Torque Generation ◽  
Motile Species ◽  
Motile Bacteria

AbstractMany motile bacteria are propelled by the rotation of flagellar filaments. This rotation is driven by a membrane protein known as the stator-complex, which drives the rotor of the bacterial flagellar motor. Torque generation is powered in most cases by proton transit through the stator complex, with the next most common ionic power source being sodium. Synthetic chimeric stators which combine sodium- and proton-powered stators have enabled the interrogation of sodium-stators in species that are typically proton-powered, such as the sodium powered PomA-PotB stator complex in E. coli. Much is known about the signalling cascades that respond to attractant and govern switching bias as an end-product of chemotaxis, however less is known about how energetics and chemotaxis interact to affect the colonisation of environmental niches where ion concentrations and compositions may vary. Here we designed a fluidics system at low cost for rapid prototyping to separate motile and non-motile populations of bacteria. We measure separation efficiencies at varying ionic concentrations and confirm using fluorescence that our device can deliver eight-fold enrichment of the motile proportion of a mixed population of motile and non-motile species. Furthermore, our results show that we can select bacteria from reservoirs where sodium is not initially present. Overall, this technique can be used to implement long-term selection from liquid culture for directed evolution approaches to investigate the adaptation of motility in bacterial ecosystems.

scholarly journals Bifidobacterium mongoliense genome seems particularly adapted to milk oligosaccharides digestion leading to production of antivirulent metabolites

2020 ◽  
Author(s):  
Pauline Bondue ◽  
Christian Milani ◽  
Emilie Arnould ◽  
Marco Ventura ◽  
Georges Daube ◽  
...  
Keyword(s):  
Bovine Milk ◽  
Transport Systems ◽  
Exact Nature ◽  
High Concentration ◽  
Milk Oligosaccharides ◽  
E Coli ◽  
A Genome ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Intestinal Pathogens

Abstract Human milk oligosaccharides (HMO) could promote the growth of bifidobacteria, improving young children’s health. In addition, fermentation of carbohydrates by bifidobacteria can result in the production of metabolites presenting an antivirulent activity against intestinal pathogens. Bovine milk oligosaccharides (BMO), structurally similar to HMO, are found at high concentration in cow whey. This is particularly observed for 3’-sialyllactose (3’SL). This study focused on enzymes and transport systems involved in HMO/BMO metabolism contained in B. crudilactis and B. mongoliense genomes. The ability of B. mongoliense to grow in media supplemented with whey or 3’SL was assessed. Next, the effects of cell-free spent media (CFSM) were tested against the virulence expression of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Due to the presence of genes encoding β-galactosidases, β-hexosaminidases, α-sialidases and α-fucosidases, B. mongoliense presents a genome more sophisticated and more adapted to the digestion of BMO/HMO than B. crudilactis . In addition, HMO/BMO digestion involves genes encoding oligosaccharide transport systems found in B. mongoliense but not in B. crudilactis . B. mongoliense seemed able to grow on media supplemented with whey or 3’SL as main source of carbon (8.3±1.0 and 6.7±0.3 log cfu/mL, respectively). CFSM obtained from whey resulted in a significant under-expression of ler , fliC , luxS , stx1 and qseA genes (-2.2, -5.3, -2.4, -2.5 and -4.8, respectively; P<0.05) of E. coli O157:H7. CFSM from 3’SL resulted in a significant up-regulation of luxS (2.0; P<0.05) gene and a down-regulation of fliC (-5.0; P<0.05) gene. CFSM obtained from whey resulted in significant up-regulations of sopD and hil genes (2.9 and 3.5, respectively; P<0.05) of S. Typhimurium, while CFSM obtained from 3’SL fermentation down-regulated hil and sopD genes (-2.7 and -4.2, respectively; P<0.05). From enzymes and transporters highlighted in the genome of B. mongoliense and its potential ability to metabolise 3’SL and whey, B. mongoliense seems well able to digest HMO/BMO. The exact nature of the metabolites contained in CFSM has to be identified still. These results suggest that BMO associated with B. mongoliense could be an interesting synbiotic formulation to maintain or restore intestinal health of young children.

scholarly journals Curli Loci of Shigella spp

2000 ◽  
Vol 68 (6) ◽  
pp. 3780-3783 ◽  
Author(s):  
Harry Sakellaris ◽  
Nerissa K. Hannink ◽  
Kumar Rajakumar ◽  
Dieter Bulach ◽  
Meredith Hunt ◽  
...  
Keyword(s):  
Selective Pressure ◽  
Shigella Flexneri ◽  
Pcr Analysis ◽  
Environmental Isolates ◽  
E Coli ◽  
Wide Range ◽  
Serovar Typhimurium ◽  
Genes Encoding ◽  
Shigella Spp ◽  
Pathoadaptive Mutations

ABSTRACT An unstable chromosomal element encoding multiple antibiotic resistance in Shigella flexneri serotype 2a was found to include sequences homologous to the csg genes encoding curli in Escherichia coli and Salmonella enterica serovar Typhimurium. As curli have been implicated in the virulence of serovar Typhimurium, we investigated thecsg loci in all four species of Shigella. DNA sequencing and PCR analysis showed that the csg loci of a wide range of Shigella strains, of diverse serotypes and different geographical distributions, were almost universally disrupted by deletions or insertions, indicating the existence of a strong selective pressure against the expression of curli. Strains of enteroinvasive E. coli (EIEC), which share virulence traits with Shigella spp. and cause similar diseases in humans, also possessed insertions or deletions in the csg locus or were otherwise unable to produce curli. Since the production of curli is a widespread trait in environmental isolates of E. coli, our results suggest that genetic lesions that abolish curli production in the closely related genus Shigella and in EIEC are pathoadaptive mutations.

scholarly journals Coupling Ion Specificity of the Flagellar Stator Proteins MotA1/MotB1 of Paenibacillus sp. TCA20

Biomolecules ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1078
Author(s):  
Sakura Onoe ◽  
Myu Yoshida ◽  
Naoya Terahara ◽  
Yoshiyuki Sowa
Keyword(s):  
Cytoplasmic Membrane ◽  
Divalent Cations ◽  
Monovalent Cation ◽  
Flagellar Motor ◽  
Ion Specificity ◽  
E Coli ◽  
Paenibacillus Sp ◽  
Complex Functions ◽  
Torque Generation ◽  
Monovalent Ion

The bacterial flagellar motor is a reversible rotary molecular nanomachine, which couples ion flux across the cytoplasmic membrane to torque generation. It comprises a rotor and multiple stator complexes, and each stator complex functions as an ion channel and determines the ion specificity of the motor. Although coupling ions for the motor rotation were presumed to be only monovalent cations, such as H+ and Na+, the stator complex MotA1/MotB1 of Paenibacillus sp. TCA20 (MotA1TCA/MotB1TCA) was reported to use divalent cations as coupling ions, such as Ca2+ and Mg2+. In this study, we initially aimed to measure the motor torque generated by MotA1TCA/MotB1TCA under the control of divalent cation motive force; however, we identified that the coupling ion of MotA1TCAMotB1TCA is very likely to be a monovalent ion. We engineered a series of functional chimeric stator proteins between MotB1TCA and Escherichia coli MotB. E. coli ΔmotAB cells expressing MotA1TCA and the chimeric MotB presented significant motility in the absence of divalent cations. Moreover, we confirmed that MotA1TCA/MotB1TCA in Bacillus subtilis ΔmotABΔmotPS cells generates torque without divalent cations. Based on two independent experimental results, we conclude that the MotA1TCA/MotB1TCA complex directly converts the energy released from monovalent cation flux to motor rotation.

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