Characterization of the Vibrio vulnificus 1-Cys Peroxiredoxin Prx3 and Regulation of Its Expression by the Fe-S Cluster Regulator IscR in Response to Oxidative Stress and Iron Starvation

Lim, Jong Gyu; Bang, Ye Ji; Choi, Sang Ho

doi:10.1074/jbc.m114.611020

Cited by 28 publications

(27 citation statements)

References 53 publications

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“…The protein-DNA binding reactions with purified CRP or SmcR were the same as those with IscR, except that the CRP or SmcR-binding buffer was used as a 1ϫ binding buffer (41,22). Electrophoretic analyses of the DNA-protein complexes were performed, as described previously (32), and visualized as described above for the transcript analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The amplified gbpA, iscR, crp, and smcR ORFs were cloned into pJK1113 under an arabinose-inducible promoter P BAD (32) to create pKK1402, pKK1403, pKK1404, and pKK1405, respectively ( Table 1). The plasmids were transferred into the appropriate mutants by conjugation as described above.…”

Section: Methodsmentioning

confidence: 99%

“…To distinguish these two possibilities, the 390-bp labeled DNA probe encompassing the P gbpA regulatory region (from Ϫ301 to ϩ88) was incubated with increasing amounts of IscR and then subjected to electrophoresis. Because IscR was isolated, purified, and used under aerobic conditions, most of the purified IscR would be in the Fe-S clusterless apo-form (32,47). The addition of IscR resulted in two retarded bands in a concentration-dependent manner, indicating that at least two binding sites for IscR are present in the P gbpA regulatory region (Fig.…”

Section: Iscr Crp and Smcr Function Cooperatively Rather Thanmentioning

confidence: 99%

“…IscR Activates P gbpA by Sensing Reactive Oxygen SpeciesRecently, it has been discovered that IscR senses reactive oxygen species and activates the expression of numerous virulence genes (26,32). This prompted us to examine the effect of oxidative stress on the gbpA expression by measuring the levels of gbpA transcript and VvGbpA protein in the strains grown anaerobically and exposed to a range of H 2 O 2 .…”

Section: Iscr Crp and Smcr Function Cooperatively Rather Thanmentioning

confidence: 99%

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Regulatory Characteristics of Vibrio vulnificus gbpA Gene Encoding a Mucin-binding Protein Essential for Pathogenesis

Jang

Gil

Lim

et al. 2016

Journal of Biological Chemistry

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Binding to mucin is the initial step for enteropathogens to establish pathogenesis. An open reading frame, gbpA, of Vibrio vulnificus was identified and characterized in this study. Compared with wild type, the gbpA mutant was impaired in binding to mucin-agar and the mucin-secreting HT29-methotrexate cells, and the impaired mucin binding was restored by the purified GbpA provided exogenously. The gbpA mutant had attenuated virulence and ability of intestinal colonization in a mouse model, indicating that GbpA is a mucin-binding protein and essential for pathogenesis of V. vulnificus. The gbpA transcription was growth phase-dependent, reaching a maximum during the exponential phase. The Fe-S cluster regulator (IscR) and the cyclic AMP receptor protein (CRP) coactivated, whereas SmcR, a LuxR homologue, repressed gbpA. The cellular levels of IscR, CRP, and SmcR were not significantly affected by one another, indicating that the regulator proteins function cooperatively to regulate gbpA rather than sequentially in a regulatory cascade. The regulatory proteins directly bind upstream of the gbpA promoter P gbpA . DNase I protection assays, together with the deletion analyses of P gbpA , demonstrated that IscR binds to two specific sequences centered at ؊164.5 and ؊106, and CRP and SmcR bind specifically to the sequences centered at ؊68 and ؊45, respectively. Furthermore, gbpA was induced by exposure to H 2 O 2 , and the induction appeared to be mediated by elevated intracellular levels of IscR. Consequently, the combined results indicated that IscR, CRP, and SmcR cooperate for precise regulation of gbpA during the V. vulnificus pathogenesis.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Iscr Crp and Smcr Function Cooperatively Rather Thanmentioning

confidence: 99%

Section: Iscr Crp and Smcr Function Cooperatively Rather Thanmentioning

confidence: 99%

See 2 more Smart Citations

Regulatory Characteristics of Vibrio vulnificus gbpA Gene Encoding a Mucin-binding Protein Essential for Pathogenesis

Jang

Gil

Lim

et al. 2016

Journal of Biological Chemistry

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“…For quantitative real-time PCR, cDNA was synthesized using the iScript TM cDNA synthesis kit (Bio-Rad), and real-time PCR amplification of the cDNA was performed using the Chromo 4 real-time PCR detection system (Bio-Rad) with pairs of primers listed in Table 2. Relative expression levels of the specific transcripts were calculated using the 16S rRNA expression level as the internal reference for normalization (24).…”

Section: Elisa-thementioning

confidence: 99%

OxyR2 Functions as a Three-state Redox Switch to Tightly Regulate Production of Prx2, a Peroxiredoxin of Vibrio vulnificus

Bang

Lee

Kim

et al. 2016

Journal of Biological Chemistry

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The bacterial transcriptional regulator OxyR is known to function as a two-state redox switch. OxyR senses cellular levels of H 2 O 2 via a "sensing cysteine" that switches from the reduced to a disulfide state upon H 2 O 2 exposure, inducing the expression of antioxidant genes. The reduced and disulfide states of OxyR, respectively, bind to extended and compact regions of DNA, where the reduced state blocks and the oxidized state allows transcription and further induces target gene expression by interacting with RNA polymerase. Vibrio vulnificus OxyR2 senses H 2 O 2 with high sensitivity and induces the gene encoding the antioxidant Prx2. In this study, we used mass spectrometry to identify a third redox state of OxyR2, in which the sensing cysteine was overoxidized to S-sulfonated cysteine (Cys-SO 3 H) by high H 2 O 2 in vitro and in vivo, where the modification deterred the transcription of prx2. The DNA binding preferences of OxyR2 5CA -C206D, which mimics overoxidized OxyR2, suggested that overoxidized OxyR2 binds to the extended DNA site, masking the ؊35 region of the prx2 promoter. These combined results demonstrate that OxyR2 functions as a three-state redox switch to tightly regulate the expression of prx2, preventing futile production of Prx2 in cells exposed to high levels of H 2 O 2 sufficient to inactivate Prx2. We further provide evidence that another OxyR homolog, OxyR1, displays similar three-state behavior, inviting further exploration of this phenomenon as a potentially general regulatory mechanism.

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The Iron–sulfur Cluster Biosynthesis Regulator IscR Contributes to Iron Homeostasis and Resistance to Oxidants inPseudomonas Aeruginosa

Romsang

Dubbs

Mongkolsuk

2016

Stress and Environmental Regulation of Gene Expression and Adaptation in Bacteria

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Characterization of the Vibrio vulnificus 1-Cys Peroxiredoxin Prx3 and Regulation of Its Expression by the Fe-S Cluster Regulator IscR in Response to Oxidative Stress and Iron Starvation

Cited by 28 publications

References 53 publications

Regulatory Characteristics of Vibrio vulnificus gbpA Gene Encoding a Mucin-binding Protein Essential for Pathogenesis

Regulatory Characteristics of Vibrio vulnificus gbpA Gene Encoding a Mucin-binding Protein Essential for Pathogenesis

OxyR2 Functions as a Three-state Redox Switch to Tightly Regulate Production of Prx2, a Peroxiredoxin of Vibrio vulnificus

The Iron–sulfur Cluster Biosynthesis Regulator IscR Contributes to Iron Homeostasis and Resistance to Oxidants inPseudomonas Aeruginosa

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