Proton‐conductivity assay of plugged and unplugged MotA/B proton channel by cytoplasmic pHluorin expressed in <i>Salmonella</i>

Morimoto, Yusuke V.; Che, Yong-Suk; Minamino, Tetsuo; Namba, Keiichi

doi:10.1016/j.febslet.2010.02.051

Cited by 75 publications

(82 citation statements)

References 19 publications

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“…MotA interaction with FliG is essential for switching the flagellar direction from clockwise (CW) movement and stimulating cell tumble to the counterclockwise (CCW) state that promotes the forward movement of the bacteria (39). The effect of deletion of motA and motB, which promotes CCW movement and reduces bacterial chemotaxis, is well known (40)(41)(42) and corroborates our array results indicating reduced chemotaxis for the AIEC LF82 strain (see Fig. S2 in the supplemental material).…”

Section: Discussionsupporting

confidence: 77%

Ribonucleotide Reductase NrdR as a Novel Regulator for Motility and Chemotaxis during Adherent-Invasive Escherichia coli Infection

et al. 2015

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A critical step in the life cycle of all organisms is the duplication of the genetic material during cell division. Ribonucleotide reductases (RNRs) are essential enzymes for this step because they control the de novo production of the deoxyribonucleotides required for DNA synthesis and repair. Enterobacteriaceae have three functional classes of RNRs (Ia, Ib, and III), which are transcribed from separate operons and encoded by the genes nrdAB, nrdHIEF, and nrdDG, respectively. Here, we investigated the role of RNRs in the virulence of adherent-invasive Escherichia coli (AIEC) isolated from Crohn's disease (CD) patients. Interestingly, the LF82 strain of AIEC harbors four different RNRs (two class Ia, one class Ib, and one class III). Although the E. coli RNR enzymes have been extensively characterized both biochemically and enzymatically, little is known about their roles during bacterial infection. We found that RNR expression was modified in AIEC LF82 bacteria during cell infection, suggesting that RNRs play an important role in AIEC virulence. Knockout of the nrdR and nrdD genes, which encode a transcriptional regulator of RNRs and class III anaerobic RNR, respectively, decreased AIEC LF82's ability to colonize the gut mucosa of transgenic mice that express human CEACAM6 (carcinoembryonic antigen-related cell adhesion molecule 6). Microarray experiments demonstrated that NrdR plays an indirect role in AIEC virulence by interfering with bacterial motility and chemotaxis. Thus, the development of drugs targeting RNR classes, in particular NrdR and NrdD, could be a promising new strategy to control gut colonization by AIEC bacteria in CD patients.R ibonucleotide reductase (RNR) is an essential enzyme in all living organisms. It catalyzes the reduction of ribonucleotides (nucleoside triphosphates [NTPs]) to their corresponding 2=-deoxyribonucleotides (deoxynucleoside triphosphates [dNTPs]) and therefore plays an essential role in DNA synthesis and repair. Three RNR classes (classes I, II, and III) exist; these classes exhibit different primary structures, subunit cofactor requirements, and quaternary three-dimensional (3D) structures, but they all are allosterically regulated and share similar catalytic mechanisms (1, 2). Class I RNRs are oxygendependent enzymes that occur in eubacteria, eukaryotes, and some viruses. This class comprises two main subgroups (Ia and Ib). Class Ia RNRs are encoded by an operon containing nrdA and nrdB genes. These genes encode the NrdA subunit, which is catalytically and allosterically regulated, and the NrdB subunit, which possesses radical-generating activity. Class Ib RNRs are encoded by an operon containing the nrdH, nrdI, nrdE, and nrdF genes, which encode the corresponding specific redoxin NrdH, the activating subunit NrdI, the catalytic subunit NrdE, and the radical-generating subunit NrdF. Class III RNRs are present in facultative anaerobic and strict anaerobic microorganisms and use S-adenosylmethionine and iron-sulfur clusters in the NrdG accessory protein to create a stab...

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Section: Discussionsupporting

confidence: 77%

Ribonucleotide Reductase NrdR as a Novel Regulator for Motility and Chemotaxis during Adherent-Invasive Escherichia coli Infection

et al. 2015

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“…A plasmid, pNSK9-D33N, which encodes MotA and MotB(D33N) on pTrc99A (Pharmacia) was transformed into both cells to reduce the number of functional stators. To carry out resurrection experiments, we used a plasmid, pYC20, which encodes MotA and MotB on pBAD24 (24). DNA manipulation was carried out as previously described (38).…”

Section: Methodsmentioning

confidence: 99%

Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

Nakamura

Kami-ike

Yokota

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The bacterial flagellar motor can rotate in both counterclockwise (CCW) and clockwise (CW) directions. It has been shown that the sodium ion-driven chimeric flagellar motor rotates with 26 steps per revolution, which corresponds to the number of FliG subunits that form part of the rotor ring, but the size of the backward step is smaller than the forward one. Here we report that the protondriven flagellar motor of Salmonella also rotates with 26 steps per revolution but symmetrical in both CCW and CW directions with occasional smaller backward steps in both directions. Occasional shift in the stepping positions is also observed, suggesting the frequent exchange of stators in one of the 11-12 possible anchoring positions around the rotor. These observations indicate that the elementary process of torque generation by the cyclic association/ dissociation of the stator with every FliG subunit along the circumference of the rotor is symmetric in CCW and CW rotation even though the structure of FliG is highly asymmetric and suggests a 180°rotation of a FliG domain for the rotor-stator interaction to reverse the direction of rotation.FliG | MotAB stator complex | Salmonella | stepping rotation | switch

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“…A conserved proline residue of MotA, Pro-173 and Pro-222, are thought to be involved in conformational changes of the stator complex that couple proton influx with torque generation (Braun et al, 1999;Nakamura et al, 2009b). The absolutely conserved and functionally critical aspartic acid residue, Asp-33 of MotB is Rotation rate (Hz) Rotation rate (Hz) located near the cytoplasmic end of its TM and is postulated to be a proton-binding site (Sharp et al, 1995;Togashi et al, 1997;Zhou et al, 1998b;Che et al, 2008;Morimoto et al, 2010a). MotB exists as a dimer in the stator complex and these aspartic acid residues are positioned on the surface of the MotB-TM dimer facing MotA-TMs, suggesting that the stator complex is likely to have two proton-conducting pathways (Fig.…”

Section: Arrangement Of Transmembrane Segments Of the Mota/b Complexmentioning

confidence: 99%

“…Therefore, it has been suggested that the proton conductivity of the MotA/B complex is suppressed prior to stator assembly around a motor. An in-frame deletion of residues 51-70 or 52-71 within the periplasmic domain of E. coli MotB or Salmonella MotB, respectively, a region, which is highly conserved among the MotB orthologs, causes considerable proton leakage, thereby arresting cell growth (Hosking et al, 2006;Morimoto et al, 2010a). This suggests that the deleted region of MotB acts as a plug segment that inserts into a proton channel within the cytoplasmic membrane to prevent from premature proton translocation through the channel before the association with a motor and that upon stator assembly in the motor, the plug leaves the channel, allowing the stator to conduct protons (Fig.…”

Section: Motb C Controls the Proton Channel Activity During Stator Asmentioning

confidence: 99%

“…A high-resolution observation of flagellar motor rotation has revealed a fine stepping motion with 26 steps per revolution, which corresponds to that of FliG subunits in the Cring (Sowa et al, 2005;Nakamura et al, 2010). The proton conductivity of the MotA/B proton channel complex is proposed to be suppressed by a plug segment of MotB when the MotA/B complex is not assembled around the basal body (Hosking et al, 2006;Morimoto et al, 2010a). Although a low-resolution structure of the entire basal body containing the stator complexes has been visualized in situ by electron cryotomography (Fig.…”

Section: Introductionmentioning

confidence: 99%

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Assembly and Activation of the MotA/B Proton Channel Complex of the Proton-Driven Flagellar Motor of Salmonella enterica

Morimoto¹,

Minamino²

2012

Salmonella - Distribution, Adaptation, Control Measures and Molecular Technologies

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Proton‐conductivity assay of plugged and unplugged MotA/B proton channel by cytoplasmic pHluorin expressed in Salmonella

Cited by 75 publications

References 19 publications

Ribonucleotide Reductase NrdR as a Novel Regulator for Motility and Chemotaxis during Adherent-Invasive Escherichia coli Infection

Ribonucleotide Reductase NrdR as a Novel Regulator for Motility and Chemotaxis during Adherent-Invasive Escherichia coli Infection

Evidence for symmetry in the elementary process of bidirectional torque generation by the bacterial flagellar motor

Assembly and Activation of the MotA/B Proton Channel Complex of the Proton-Driven Flagellar Motor of Salmonella enterica

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