scholarly journals The FlgN chaperone activates the Na+-driven engine of the Salmonella flagellar protein export apparatus

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Tohru Minamino ◽  
Miki Kinoshita ◽  
Yusuke V. Morimoto ◽  
Keiichi Namba
Keyword(s):  
Conformational Change ◽  
Environmental Conditions ◽  
Proton Motive Force ◽  
Protein Export ◽  
Motive Force ◽  
Structural Proteins ◽  
Wild Type ◽  
Flagellar Protein

AbstractThe bacterial flagellar protein export machinery consists of a transmembrane export gate complex and a cytoplasmic ATPase complex. The gate complex has two intrinsic and distinct H+-driven and Na+-driven engines to drive the export of flagellar structural proteins. Salmonella wild-type cells preferentially use the H+-driven engine under a variety of environmental conditions. To address how the Na+-driven engine is activated, we analyzed the fliJ(Δ13–24) fliH(Δ96–97) mutant and found that the interaction of the FlgN chaperone with FlhA activates the Na+-driven engine when the ATPase complex becomes non-functional. A similar activation can be observed with either of two single-residue substitutions in FlhA. Thus, it is likely that the FlgN-FlhA interaction generates a conformational change in FlhA that allows it to function as a Na+ channel. We propose that this type of activation would be useful for flagellar construction under conditions in which the proton motive force is severely restricted.

scholarly journals Chemical or genetic alteration of proton motive force results in loss of virulence of Burkholderia glumae , the cause of rice bacterial panicle blight.

2021 ◽  
Author(s):  
Asif Iqbal ◽  
Pradip R. Panta ◽  
John Ontoy ◽  
Jobelle Bruno ◽  
Jong Hyun Ham ◽  
...  
Keyword(s):  
Sodium Bicarbonate ◽  
Wild Type Strain ◽  
Proton Motive Force ◽  
Membrane Transporters ◽  
Motive Force ◽  
Wild Type ◽  
Bacterial Genomes ◽  
Burkholderia Glumae ◽  
Sheath Rot ◽  
Panicle Blight

Rice is an important source of food for more than half the world’s population. Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae . B. glumae synthesizes toxoflavin, an essential virulence factor, that is required for symptoms of the disease. The products of the tox operons, ToxABCDE and ToxFGHI, are responsible for the synthesis and the proton motive force (PMF)-dependent secretion of toxoflavin, respectively. The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Our previous work has demonstrated that absence of certain DedA family members results in pleiotropic effects, impacting multiple pathways that are energized by PMF. We have demonstrated that a member of the DedA family from Burkholderia thailandensis , named DbcA, is required for the extreme polymyxin resistance observed in this organism. B. glumae encodes a homolog of DbcA with 73% amino acid identity to Burkholderia thailandensis DbcA. Here, we created and characterized a B. glumae Δ dbcA strain. In addition to polymyxin sensitivity, B. glumae Δ dbcA is compromised for virulence in several BPB infection models and secretes only low amounts of toxoflavin (∼15% of wild type levels). Changes in membrane potential in B. glumae Δ dbcA were reproduced in the wild type strain by the addition of sub-inhibitory concentrations of sodium bicarbonate, previously demonstrated to cause disruption of PMF. Sodium bicarbonate inhibited B. glumae virulence in rice suggesting a possible non-toxic chemical intervention for bacterial panicle blight. IMPORTANCE Bacterial panicle blight (BPB) is a disease of rice characterized by grain discoloration or sheath rot caused mainly by Burkholderia glumae . The DedA family is a highly conserved membrane protein family found in most bacterial genomes that likely function as membrane transporters. Here, we constructed a B. glumae mutant with a deletion in a DedA family member named dbcA and report a loss of virulence in models of BPB. Physiological analysis of the mutant shows that the proton motive force is disrupted, leading to reduction of secretion of the essential virulence factor toxoflavin. The mutant phenotypes are reproduced in the virulent wild type strain without an effect on growth using sodium bicarbonate, a nontoxic buffer that has been reported to disrupt the PMF. The results presented here suggest that bicarbonate may be an effective antivirulence agent capable of controlling BPB without imposing an undue burden on the environment.

scholarly journals High-Resolution pH Imaging of Living Bacterial Cells To Detect Local pH Differences

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Yusuke V. Morimoto ◽  
Nobunori Kami-ike ◽  
Tomoko Miyata ◽  
Akihiro Kawamoto ◽  
Takayuki Kato ◽  
...  
Keyword(s):  
High Resolution ◽  
Atp Hydrolysis ◽  
Imaging System ◽  
Proton Motive Force ◽  
Energy Transduction ◽  
Protein Export ◽  
Motive Force ◽  
Bacterial Flagellum ◽  
Ph Imaging ◽  
Local Ph

ABSTRACTProtons are utilized for various biological activities such as energy transduction and cell signaling. For construction of the bacterial flagellum, a type III export apparatus utilizes ATP and proton motive force to drive flagellar protein export, but the energy transduction mechanism remains unclear. Here, we have developed a high-resolution pH imaging system to measure local pH differences within livingSalmonella entericacells, especially in close proximity to the cytoplasmic membrane and the export apparatus. The local pH near the membrane was ca. 0.2 pH unit higher than the bulk cytoplasmic pH. However, the local pH near the export apparatus was ca. 0.1 pH unit lower than that near the membrane. This drop of local pH depended on the activities of both transmembrane export components and FliI ATPase. We propose that the export apparatus acts as an H+/protein antiporter to couple ATP hydrolysis with H+flow to drive protein export.IMPORTANCEThe flagellar type III export apparatus is required for construction of the bacterial flagellum beyond the cellular membranes. The export apparatus consists of a transmembrane export gate and a cytoplasmic ATPase complex. The export apparatus utilizes ATP and proton motive force as the energy source for efficient and rapid protein export during flagellar assembly, but it remains unknown how. In this study, we have developed anin vivopH imaging system with high spatial and pH resolutions with a pH indicator probe to measure local pH near the export apparatus. We provide direct evidence suggesting that ATP hydrolysis by the ATPase complex and the following rapid protein translocation by the export gate are both linked to efficient proton translocation through the gate.

scholarly journals Yersinia enterocolitica Type III Secretion Depends on the Proton Motive Force but Not on the Flagellar Motor Components MotA and MotB

2004 ◽  
Vol 72 (7) ◽  
pp. 4004-4009 ◽  
Author(s):  
Gottfried Wilharm ◽  
Verena Lehmann ◽  
Kristina Krauss ◽  
Beatrix Lehnert ◽  
Susanna Richter ◽  
...  
Keyword(s):  
Yersinia Enterocolitica ◽  
Type Iii Secretion ◽  
Proton Motive Force ◽  
Motive Force ◽  
Host Cells ◽  
Infection Model ◽  
Flagellar Motor ◽  
Wild Type ◽  
Secretion Systems ◽  
Type Iii

ABSTRACT The flagellum is believed to be the common ancestor of all type III secretion systems (TTSSs). In Yersinia enterocolitica, expression of the flagellar TTSS and the Ysc (Yop secretion) TTSS are inversely regulated. We therefore hypothesized that the Ysc TTSS may adopt flagellar motor components in order to use the pathogenicity-related translocon in a drill-like manner. As a prerequisite for this hypothesis, we first tested a requirement for the proton motive force by both systems using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP). Motility as well as type III-dependent secretion of Yop proteins was inhibited by CCCP. We deleted motAB, which resulted in an immotile phenotype. This mutant, however, secreted amounts of Yops to the supernatant comparable to those of the wild type. Translocation of Yops into host cells was also not affected by the motAB deletion. Virulence of the mutant was comparable to that of the wild type in the mouse oral infection model. Thus, the hypothesis that the Ysc TTSS might adopt flagellar motor components was not confirmed. The finding that, in addition to consumption of ATP, Ysc TTSS requires the proton motive force is discussed.

scholarly journals Limitations to photosynthesis by proton motive force-induced photosystem II photodamage

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Geoffry A Davis ◽  
Atsuko Kanazawa ◽  
Mark Aurel Schöttler ◽  
Kaori Kohzuma ◽  
John E Froehlich ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Proton Motive Force ◽  
Motive Force ◽  
Limiting Factor ◽  
Wild Type ◽  
Recombination Rates ◽  
Atp Production ◽  
Photosynthetic Regulation ◽  
Light Capture

The thylakoid proton motive force (pmf) generated during photosynthesis is the essential driving force for ATP production; it is also a central regulator of light capture and electron transfer. We investigated the effects of elevated pmf on photosynthesis in a library of Arabidopsis thaliana mutants with altered rates of thylakoid lumen proton efflux, leading to a range of steady-state pmf extents. We observed the expected pmf-dependent alterations in photosynthetic regulation, but also strong effects on the rate of photosystem II (PSII) photodamage. Detailed analyses indicate this effect is related to an elevated electric field (Δψ) component of the pmf, rather than lumen acidification, which in vivo increased PSII charge recombination rates, producing singlet oxygen and subsequent photodamage. The effects are seen even in wild type plants, especially under fluctuating illumination, suggesting that Δψ-induced photodamage represents a previously unrecognized limiting factor for plant productivity under dynamic environmental conditions seen in the field.

scholarly journals 4-Chlorobenzoate Uptake in Comamonas sp. Strain DJ-12 Is Mediated by a Tripartite ATP-Independent Periplasmic Transporter

2006 ◽  
Vol 188 (24) ◽  
pp. 8407-8412 ◽  
Author(s):  
Jong-Chan Chae ◽  
Gerben J. Zylstra
Keyword(s):  
Growth Rate ◽  
Gene Cluster ◽  
Type Strain ◽  
Wild Type Strain ◽  
Proton Motive Force ◽  
Significant Inhibition ◽  
Motive Force ◽  
Wild Type ◽  
Knockout Mutant

ABSTRACT The fcb gene cluster involved in the hydrolytic dehalogenation of 4-chlorobenzoate is organized in the order fcbB-fcbA-fcbT1-fcbT2-fcbT3-fcbC in Comamonas sp. strain DJ-12. The genes are operonic and inducible with 4-chloro-, 4-iodo-, and 4-bromobenzoate. The fcbT1, fcbT2, and fcbT3 genes encode a transporter in the secondary TRAP (tripartite ATP-independent periplasmic) family. An fcbT1T2T3 knockout mutant shows a much slower growth rate on 4-chlorobenzoate compared to the wild type. 4-Chlorobenzoate is transported into the wild-type strain five times faster than into the fcbT1T2T3 knockout mutant. Transport of 4-chlorobenzoate shows significant inhibition by 4-bromo-, 4-iodo-, and 4-fluorobenzoate and mild inhibition by 3-chlorobenzoate, 2-chlorobenzoate, 4-hydroxybenzoate, 3-hydroxybenzoate, and benzoate. Uptake of 4-chlorobenzoate is significantly inhibited by ionophores which collapse the proton motive force.

scholarly journals FliK-Driven Conformational Rearrangements of FlhA and FlhB Are Required for Export Switching of the Flagellar Protein Export Apparatus

2019 ◽  
Vol 202 (3) ◽  
Author(s):  
Tohru Minamino ◽  
Yumi Inoue ◽  
Miki Kinoshita ◽  
Keiichi Namba
Keyword(s):  
Substrate Specificity ◽  
Conformational Change ◽  
Ring Structure ◽  
Protein Export ◽  
Structural Remodeling ◽  
Type Iii ◽  
Type Protein ◽  
Flagellar Assembly ◽  
Flagellar Protein ◽  
Conformational Rearrangements

ABSTRACT FlhA and FlhB are transmembrane proteins of the flagellar type III protein export apparatus, and their C-terminal cytoplasmic domains (FlhAC and FlhBC) coordinate flagellar protein export with assembly. FlhBC undergoes autocleavage between Asn-269 and Pro-270 in a well-conserved NPTH loop located between FlhBCN and FlhBCC polypeptides and interacts with the C-terminal domain of the FliK ruler when the length of the hook has reached about 55 nm in Salmonella. As a result, the flagellar protein export apparatus switches its substrate specificity, thereby terminating hook assembly and initiating filament assembly. The mechanism of export switching remains unclear. Here, we report the role of FlhBC cleavage in the switching mechanism. Photo-cross-linking experiments revealed that the flhB(N269A) and flhB(P270A) mutations did not affect the binding affinity of FlhBC for FliK. Genetic analysis of the flhB(P270A) mutant revealed that the P270A mutation affects a FliK-dependent conformational change of FlhBC, thereby inhibiting the substrate specificity switching. The flhA(A489E) mutation in FlhAC suppressed the flhB(P270A) mutation, suggesting that an interaction between FlhBC and FlhAC is critical for the export switching. We propose that the interaction between FliKC and a cleaved form of FlhBC promotes a conformational change in FlhBC responsible for the termination of hook-type protein export and a structural remodeling of the FlhAC ring responsible for the initiation of filament-type protein export. IMPORTANCE The flagellar type III protein export apparatus coordinates protein export with assembly, which allows the flagellum to be efficiently built at the cell surface. Hook completion is an important morphological checkpoint for the sequential flagellar assembly process. The protein export apparatus switches its substrate specificity from the hook protein to the filament protein upon hook completion. FliK, FlhB, and FlhA are involved in the export-switching process, but the mechanism remains a mystery. By analyzing a slow-cleaving flhB(P270A) mutant, we provide evidence that an interaction between FliK and FlhB induces conformational rearrangements in FlhB, followed by a structural remodeling of the FlhA ring structure that terminates hook assembly and initiates filament formation.

Fluoroquinolone resistance ofSerratia marcescens: sucrose, salicylate, temperature, and pH induction of phenotypic resistance

2007 ◽  
Vol 53 (11) ◽  
pp. 1239-1245 ◽  
Author(s):  
Sanela Begic ◽  
Elizabeth A. Worobec
Keyword(s):  
Nalidixic Acid ◽  
Environmental Conditions ◽  
Efflux Pump ◽  
Proton Motive Force ◽  
Motive Force ◽  
Natural Resistance ◽  
Fluoroquinolone Resistance ◽  
Efflux Pump Inhibitor ◽  
Phenotypic Resistance ◽  
Acid Accumulation

Serratia marcescens is a nosocomial agent with a natural resistance to a broad spectrum of antibiotics, making the treatment of its infections very challenging. This study examines the influence of salicylate, sucrose, temperature, and pH variability on membrane permeability and susceptibility of S. marcescens to norfloxacin (hydrophilic fluoroquinolone) and nalidixic acid (hydrophobic quinolone). Resistance of wild-type S. marcescens UOC-67 (ATCC 13880) to norfloxacin and nalidixic acid was assessed by minimal inhibitory concentration (MIC) assays after growth in the presence of various concentrations of sucrose and salicylate and different temperatures and pH values. Norfloxacin and nalidixic acid accumulation was determined in the absence and presence of (i) carbonyl cyanide m-chlorophenylhydrazone (CCCP), a proton-motive-force collapser, and (ii) Phe-Arg β-naphthylamide (PAβN), an efflux pump inhibitor. Accumulation of norfloxacin decreased when S. marcescens was grown in high concentrations of salicylate (8 mmol/L) and sucrose (10% m/v), at high temperature (42 °C), and at pH 6, and it was restored in the presence of CCCP because of the collapse of proton-gradient-dependent efflux in S. marcescens. Although nalidixic acid accumulation was observed, it was not affected by salicylate, sucrose, pH, or temperature changes. In the absence of PAβN, and either in the presence or absence of CCCP, a plateau was reached in the nalidixic acid accumulation for all environmental conditions. With the addition of 20 mg/L PAβN nalidixic acid accumulation is restored for all environmental conditions, suggesting that this quinolone is recognized by a yet to be identified S. marcescens pump that does not use proton motive force as its energy source.

scholarly journals Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression

Microbiology ◽  
2009 ◽  
Vol 155 (4) ◽  
pp. 1170-1180 ◽  
Author(s):  
Todd G. Smith ◽  
Lara Pereira ◽  
Timothy R. Hoover
Keyword(s):  
Helicobacter Pylori ◽  
Substrate Specificity ◽  
Reporter Genes ◽  
Inhibitory Effect ◽  
Protein Export ◽  
Flagellar Gene ◽  
Wild Type ◽  
Flagellar Assembly ◽  
Flagellar Protein ◽  
H Pylori

Regulation of the Helicobacter pylori flagellar gene cascade involves the transcription factors σ 54 (RpoN), employed for expression of genes required midway through flagellar assembly, and σ 28 (FliA), required for expression of late genes. Previous studies revealed that mutations in genes encoding components of the flagellar protein export apparatus block expression of the H. pylori RpoN and FliA regulons. FlhB is a membrane-bound component of the export apparatus that possesses a large cytoplasmic domain (FlhBC). The hook length control protein FliK interacts with FlhBC to modulate the substrate specificity of the export apparatus. FlhBC undergoes autocleavage as part of the switch in substrate specificity. Consistent with previous reports, deletion of flhB in H. pylori interfered with expression of RpoN-dependent reporter genes, while deletion of fliK stimulated expression of these reporter genes. In the ΔflhB mutant, disrupting fliK did not restore expression of RpoN-dependent reporter genes, suggesting that the inhibitory effect of the ΔflhB mutation is not due to the inability to export FliK. Amino acid substitutions (N265A and P266G) at the putative autocleavage site of H. pylori FlhB prevented processing of FlhB and export of filament-type substrates. The FlhB variants supported wild-type expression of RpoN- and FliA-dependent reporter genes. In the strain producing FlhBN265A, expression of RpoN- and FliA-dependent reporter genes was inhibited when fliK was disrupted. In contrast, expression of these reporter genes was unaffected or slightly stimulated when fliK was disrupted in the strain producing FlhBP266G. H. pylori HP1575 (FlhX) shares homology with the C-terminal portion of FlhBC (FlhBCC) and can substitute for FlhBCC in flagellar assembly. Disrupting flhX inhibited expression of a flaB reporter gene in the wild-type but not in the ΔfliK mutant or strains producing FlhB variants, suggesting a role for FlhX or FlhBCC in normal expression of the RpoN regulon. Taken together, these data indicate that the mechanism by which the flagellar protein export apparatus exerts control over the H. pylori RpoN regulon is complex and involves more than simply switching substrate specificity of the flagellar protein export apparatus.

Sign in / Sign up

Export Citation Format

Share Document