Evidence for function overlapping of <scp>CymA</scp> and the cytochrome <scp>bc</scp>1 complex in the <scp>S</scp>hewanella oneidensis nitrate and nitrite respiration

Fu, Huihui; Jin, Miao; Ju, Lin; Mao, Yinting; Gao, Haichun

doi:10.1111/1462-2920.12457

Cited by 90 publications

(139 citation statements)

References 69 publications

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“…The ␤-galactosidase activity assay was performed to determine gene expression using the integrative lacZ reporter pHGEI01 (34). In brief, the sequence of ϳ400 bp upstream from the gene of interest was amplified and placed in front of the full-length E. coli lacZ gene.…”

Section: Methodsmentioning

confidence: 99%

Unraveling the Mechanism for the Viability Deficiency of Shewanella oneidensis oxyR Null Mutant

et al. 2015

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Oxidative stresses triggered by reactive oxygen species (ROS) that damage various cellular components are unavoidable for virtually all living organisms. In defense, microorganisms have evolved sophisticated mechanisms to sense, respond to, and battle against ROS. Shewanella oneidensis, an important research model for applied and environmental microbes, employs OxyR to mediate the response to H 2 O 2 by derepressing the production of the major H 2 O 2 scavenger KatB as a major means toward these goals. Surprisingly, despite enhanced H 2 O 2 degradation, the oxyR mutant carries a viability deficiency phenotype (plating defect), which can be suppressed by the addition of exogenous iron species. Experiments showed that the defect was not due to iron starvation. Rather, multiple lines of evidence suggested that H 2 O 2 generated abiotically in lysogeny broth (LB) is responsible for the defect by quickly killing mutant cells. We then showed that the iron species suppressed the plating defect by two distinct mechanisms, either as an H 2 O 2 scavenger without involving living cells or as an environmental cue to stimulate an OxyR-independent response to help cells cope with oxidative stress. Based on the suppression of the plating defect by overproduction of H 2 O 2 scavengers in vivo, we propose that cellular components that are vulnerable to H 2 O 2 and responsible for the defect may reside outside the cytoplasm. IMPORTANCEIn bacteria, OxyR is the major regulator controlling the cellular response to H 2 O 2 . The loss of OxyR results in reduced viability in many species, but the underlying mechanism is unknown. We showed in S. oneidensis that this defect was due to H 2 O 2 generated abiotically in LB. We then showed that this defect could be corrected by the addition of Fe 2؉ or catalase to the LB or increased intracellular production of catalase. Further analyses revealed that Fe 2؉ was able not only to decompose H 2 O 2 directly but also to stimulate the activity of OxyR-independent H 2 O 2 -scavenging enzymes. Our data indicate that iron species play a previously underappreciated role in protecting cells from H 2 O 2 in environments.

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Section: Methodsmentioning

confidence: 99%

Unraveling the Mechanism for the Viability Deficiency of Shewanella oneidensis oxyR Null Mutant

et al. 2015

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“…For the integrative lacZ reporter system, fragments indicated in the text or figure legends were cloned into the reporter vector pHGEI01 to generate transcriptional fusions (25). The resultant vectors were then verified by sequencing and transferred into relevant strains by conjugation.…”

Section: Methodsmentioning

confidence: 99%

“…To eliminate the antibiotic marker, helper plasmid pBBR-Cre was transferred into the strains carrying a correctly integrated construct (26). Midlog phase cultures were harvested, aliquoted, and subjected to ␤-galactosidase activity assays as described before (25).…”

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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“…␤-Galactosidase assays were performed to determine gene expression with the integrative lacZ reporter plasmid pHGEI01 (32). In brief, the sequence of ϳ400 bp upstream of a gene of interest was amplified and placed immediately upstream of the full-length E. coli lacZ gene.…”

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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Evidence for function overlapping of CymA and the cytochrome bc₁ complex in the Shewanella oneidensis nitrate and nitrite respiration

Cited by 90 publications

References 69 publications

Unraveling the Mechanism for the Viability Deficiency of Shewanella oneidensis oxyR Null Mutant

Unraveling the Mechanism for the Viability Deficiency of Shewanella oneidensis oxyR Null Mutant

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

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

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Evidence for function overlapping of CymA and the cytochrome bc1 complex in the Shewanella oneidensis nitrate and nitrite respiration

Cited by 90 publications

References 69 publications

Unraveling the Mechanism for the Viability Deficiency of Shewanella oneidensis oxyR Null Mutant

Unraveling the Mechanism for the Viability Deficiency of Shewanella oneidensis oxyR Null Mutant

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

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

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Evidence for function overlapping of CymA and the cytochrome bc₁ complex in the Shewanella oneidensis nitrate and nitrite respiration