Genetic and Molecular Characterization of Flagellar Assembly in Shewanella oneidensis

Wu, Lin; Wang, Jixuan; Tang, Peng; Chen, Haijiang; Gao, Haichun

doi:10.1371/journal.pone.0021479

Cited by 93 publications

(209 citation statements)

References 82 publications

(147 reference statements)

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“…Widespread presence of FlgP and other C. jejuni diskcomplex proteins (i.e., FlgQ, PflA, and PflB) across the e-proteobacteria, together with previous observations of flagellar motors incorporating disk complexes (18,29), confirm that other e-proteobacteria such as Helicobacter and Wolinella genera also assemble C. jejuni-type homologous disk complexes. FlgP also is widespread among many γ-proteobacterial genera including Vibrio, Shewanella, and Rhodobacter, but in these genera components of the Vibrio disk complex MotX, MotY, FlgO, and FlgT are encoded, as is consistent with assembly of Vibrio-type diskcomplex homologs (45,46). Intriguingly, however, the widespread FlgP basal disks do not interact directly with the stator complexes in either the Vibrio or e-proteobacterial disk-complex variants.…”

Section: Discussionmentioning

confidence: 95%

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Beeby

Ribardo

Brennan

et al. 2016

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

185

262

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Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, from Campylobacter and Vibrio species. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes.

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Section: Discussionmentioning

confidence: 95%

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Beeby

Ribardo

Brennan

et al. 2016

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

185

262

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“…All mutants from previous studies were successfully complemented by a copy of the corresponding gene on plasmids pHG101 and pHG102 (Table 1) (24,25). In this study, these complementation vectors were used with similar results.…”

Section: Methodsmentioning

confidence: 99%

“…The resulting vector was maintained in E. coli WM3064 and transferred to S. oneidensis after verification by sequencing. Log-phase cells (OD 600 , ϳ0.2) were harvested by centrifugation, washed with PBS, suspended in lysis buffer (0.25 M Tris HCl, 0.5% Triton X-100, pH 7.5) for 30 min, and assayed with ortho-nitrophenyl-␤-D-galactopyranoside as described previously (24). Activity was determined by monitoring color development at 420 nm with a Synergy 2 Multi-Detection microplate reader (M200 Pro; Tecan) and reported in Miller units.…”

Section: Methodsmentioning

confidence: 99%

A Matter of Timing: Contrasting Effects of Hydrogen Sulfide on Oxidative Stress Response in Shewanella oneidensis

Wan

et al. 2015

J Bacteriol

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Hydrogen sulfide (H 2 S), well known for its toxic properties, has recently become a research focus in bacteria, in part because it has been found to prevent oxidative stress caused by treatment with some antibiotics. H 2 S has the ability to scavenge reactive oxygen species (ROS), thus preventing oxidative stress, but it is also toxic, leading to conflicting reports of its effects in different organisms. Here, with Shewanella oneidensis as a model, we report that the effects of H 2 S on the response to oxidative stress are time dependent. When added simultaneously with H 2 O 2 , H 2 S promoted H 2 O 2 toxicity by inactivating catalase, KatB, a hemecontaining enzyme involved in H 2 O 2 degradation. Such an inhibitory effect may apply to other heme-containing proteins, such as cytochrome cbb 3 oxidase. When H 2 O 2 was supplied 20 min or later after the addition of H 2 S, the oxidative-stress-responding regulator OxyR was activated, resulting in increased resistance to H 2 O 2 . The activation of OxyR was likely triggered by the influx of iron, a response to lowered intracellular iron due to the iron-sequestering property of H 2 S. Given that Shewanella bacteria thrive in redox-stratified environments that have abundant sulfur and iron species, our results imply that H 2 S is more important for bacterial survival in such environmental niches than previously believed. IMPORTANCE Previous studies have demonstrated that H 2 S is either detrimental or beneficial to bacterial cells. While it can act as a growth-inhibiting molecule by damaging DNA and denaturing proteins, it helps cells to combat oxidative stress. Here we report that H 2 S indeed has these contrasting biological functions and that its effects are time dependent. Immediately after H 2 S treatment, there is growth inhibition due to damage of heme-containing proteins, at least to catalase and cytochrome c oxidase. In contrast, when added a certain time later, H 2 S confers an enhanced ability to combat oxidative stress by activating the H 2 O 2 -responding regulator OxyR. Our data reconcile conflicting observations about the functions of H 2 S.

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“…Plasmid pHG102, which carries the constitutively active S. oneidensis arcA promoter, was used in genetic complementation of fabB, fabF1, and fabF2 mutants (18,19). After being verified by sequencing, the vectors were introduced into the relevant mutants for phenotypic assays.…”

Section: Methodsmentioning

confidence: 99%

“…Promoterless plasmid pHG101, present in ϳ5 copies in S. oneidensis, was used to assay the effects of tandem repeat (TR) copy numbers on mutation rates (19,26). DNA fragments of interest were generated by PCR with primers given in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

Suppression of fabB Mutation by fabF1 Is Mediated by Transcription Read-through in Shewanella oneidensis

Quanfei

et al. 2016

J Bacteriol

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As type II fatty acid synthesis is essential for the growth of Escherichia coli, its many components are regarded as potential targets for novel antibacterial drugs. Among them, ␤-ketoacyl-acyl carrier protein (ACP) synthase (KAS) FabB is the exclusive factor for elongation of the cis-3-decenoyl-ACP (cis-3-C 10 -ACP). In our previous study, we presented evidence to suggest that this may not be the case in Shewanella oneidensis, an emerging model gammaproteobacterium renowned for its respiratory versatility. Here, we identified FabF1, another KAS, as a functional replacement for FabB in S. oneidensis. In fabB ؉ or desA ؉ (encoding a desaturase) cells, which are capable of making unsaturated fatty acids (UFA), FabF1 is barely produced. However, UFA auxotroph mutants devoid of both fabB and desA genes can be spontaneously converted to suppressor strains, which no longer require exogenous UFAs for growth. Suppression is caused by a TGTTTT deletion in the region upstream of the fabF1 gene, resulting in enhanced FabF1 production. We further demonstrated that the deletion leads to transcription read-through of the terminator for acpP, an acyl carrier protein gene immediately upstream of fabF1. There are multiple tandem repeats in the region covering the terminator, and the TGTTTT deletion, as well as others, compromises the terminator efficacy. In addition, FabF2 also shows an ability to complement the FabB loss, albeit substantially less effectively than FabF1. IMPORTANCEIt has been firmly established that FabB for UFA synthesis via type II fatty acid synthesis in FabA-containing bacteria such as E. coli is essential. However, S. oneidensis appears to be an exception. In this bacterium, FabF1, when sufficiently expressed, is able to fully complement the FabB loss. Importantly, such a capability can be obtained by spontaneous mutations, which lead to transcription read-through. Therefore, our data, by identifying the functional overlap between FabB and FabFs, provide new insights into the current understanding of KAS and help reveal novel ways to block UFA synthesis for therapeutic purposes. The de novo fatty acid synthesis (FAS) pathway, namely, type II, is the predominant, if not exclusive, route for endogenous production of fatty acids (1). The FAS pathway, the current knowledge of which derives mainly from the model organism Escherichia coli, is highly conserved in bacteria. Central to the pathway are reactions catalyzed by ␤-ketoacyl-acyl carrier protein (ACP) synthases (KAS), including FabH, FabB, and FabF. FabH is responsible for the condensation of an acyl coenzyme A (acylCoA) unit and a malonyl-acyl carrier protein (malonyl-ACP) unit, but cannot work with acetyl-ACP, the substrate of FabB and FabF; as a consequence, FabH could not function as a replacement for FabB or FabF (1). While FabB and FabF are exchangeable in elongation of saturated intermediates, each catalyzes a reaction with the unsaturated branch that the other cannot or at least much less effectively (2) (Fig. 1). The key reaction catalyze...

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Genetic and Molecular Characterization of Flagellar Assembly in Shewanella oneidensis

Cited by 93 publications

References 82 publications

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold

A Matter of Timing: Contrasting Effects of Hydrogen Sulfide on Oxidative Stress Response in Shewanella oneidensis

Suppression of fabB Mutation by fabF1 Is Mediated by Transcription Read-through in Shewanella oneidensis

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