OxyR proteins are LysR-type transcriptional regulators, which play an important role in responding to oxidative stress in bacteria. Azospirillum brasilense Sp7 harbours two copies of OxyR. The inactivation of the oxyR1, the gene organized divergently to ahpC in A. brasilense Sp7, led to an increased tolerance to alkyl hydroperoxides, which was corroborated by an increase in alkyl hydroperoxide reductase (AhpC) activity, enhanced expression of ahpC :lacZ fusion and increased synthesis of AhpC protein in the oxyR1::km mutant. The upstream region of ahpC promoter harboured a putative OxyR binding site, T-N11-A. Mutation of T, A or both in the T-N11-Amotif caused derepression of ahpC in A. brasilense suggesting that T-N11-A might be the binding site for a negative regulator. Retardation of the electrophoretic mobility of the T-N11-A motif harbouring oxyR1-ahpC intergenic DNA by recombinant OxyR1, under reducing as well as oxidizing conditions, indicated that OxyR1 acts as a negative regulator of ahpC in A. brasilense. Sequence of the promoter of ahpC, predicted on the basis of transcriptional start site, and an enhanced expression of ahpC:lacZ fusion in chrR2::km mutant background suggested that ahpC promoter was RpoE2 dependent. Thus, this study shows that in A. brasilense Sp7, ahpC expression is regulated negatively by OxyR1 but is regulated positively by RpoE2, an oxidative-stress-responsive sigma factor. It also shows that OxyR1 regulates the expression RpoE1, which is known to play an important role during photooxidative stress in A. brasilense.
AntelmannH.,
EngelmannS.,
SchmidR.,
HeckerM.1996; General and oxidative stress responses in Bacillus subtilis: cloning, expression, and mutation of the alkyl hydroperoxide reductase operon. J Bacteriol 178:6571–6578[PubMed]
ByerlyK. A.,
UrbanowskiM. L.,
StaufferG. V.1991; The metR binding site in the Salmonella typhimurium metH gene: DNA sequence constraints on activation. J Bacteriol 173:3547–3553[PubMed]
ChaeH. Z.,
RobisonK.,
PooleL. B.,
ChurchG.,
StorzG.,
RheeS. G.1994; Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes. Proc Natl Acad Sci U S A 91:7017–7021 [View Article][PubMed]
ChristmanM. F.,
MorganR. W.,
JacobsonF. S.,
AmesB. N.1985; Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium
. Cell 41:753–762 [View Article][PubMed]
DhandayuthapaniS.,
ZhangY.,
MuddM. H.,
DereticV.1996; Oxidative stress response and its role in sensitivity to isoniazid in mycobacteria: characterization and inducibility of ahpC by peroxides in Mycobacterium smegmatis and lack of expression in M. aurum and M. tuberculosis
. J Bacteriol 178:3641–3649[PubMed]
FisherR. F.,
LongS. R.1993; Interactions of NodD at the nod Box: NodD binds to two distinct sites on the same face of the helix and induces a bend in the DNA. J Mol Biol 233:336–348 [View Article][PubMed]
GuptaN.,
KumarS.,
MishraM. N.,
TripathiA. K.2013; A constitutively expressed pair of rpoE2-chrR2 in Azospirillum brasilense Sp7 is required for survival under antibiotic and oxidative stress. Microbiology 159:205–218 [View Article]
HowlettN. G.,
AveryS. V.1997; Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation. Appl Environ Microbiol 63:2971–2976[PubMed]
IyerK. S.,
KleeW. A.1973; Direct spectrophotometric measurement of the rate of reduction of disulfide bonds. The reactivity of the disulfide bonds of bovine -lactalbumin. J Biol Chem 248:707–710[PubMed]
JacobsonF. S.,
MorganR. W.,
ChristmanM. F.,
AmesB. N.1989; An alkyl hydroperoxide reductase from Salmonella typhimurium involved in the defense of DNA against oxidative damage. Purification and properties. J Biol Chem 264:1488–1496[PubMed]
KimY. C.,
MillerC. D.,
AndersonA. J.1999; Transcriptional regulation by iron and role during plant pathogenesis of genes encoding iron- and manganese-superoxide dismutase of Pseudomonas syringae pv. syringae B728a. Physiol Mol Plant Pathol 55:327–339 [View Article]
LeBlancJ. J.,
BrassingaA. K.,
EwannF.,
DavidsonR. J.,
HoffmanP. S.2008; An ortholog of OxyR in Legionella pneumophila is expressed postexponentially and negatively regulates the alkyl hydroperoxide reductase (ahpC2D) operon. J Bacteriol 190:3444–3455 [View Article][PubMed]
MaxonM. E.,
WigboldusJ.,
BrotN.,
WeissbachH.1990; Structure-function studies on Escherichia coli MetR protein, a putative prokaryotic leucine zipper protein. Proc Natl Acad Sci U S A 87:7076–7079 [View Article][PubMed]
MishraM. N.,
ThirunavukkarasuN.,
SharmaI. M.,
JagnnadhamM. V.,
TripathiA. K.2008; Mutation in a gene encoding anti-sigma factor in A. brasilense confers tolerance to elevated temperature, antibacterial peptide and PEG-200 via carotenoid synthesis. FEMS Microbiol Lett 287:221–229 [View Article][PubMed]
MishraM. N.,
KumarS.,
GuptaN.,
KaurS.,
GuptaA.,
TripathiA. K.2011; An extracytoplasmic function sigma factor cotranscribed with its cognate anti-sigma factor confers tolerance to NaCl, ethanol and methylene blue in Azospirillum brasilense Sp7. Microbiology 157:988–999 [View Article][PubMed]
MongkolsukS.,
LoprasertS.,
WhangsukW.,
FuangthongM.,
AtichartpongkunS.1997; Characterization of transcription organization and analysis of unique expression patterns of an alkyl hydroperoxide reductase C gene (ahpC) and the peroxide regulator operon ahpF-oxyR-orfX from Xanthomonas campestris pv. phaseoli
. J Bacteriol 179:3950–3955[PubMed]
NamT. W.,
ZiegelhofferE. C.,
LemkeR. A.,
DonohueT. J.2013; Proteins needed to activate a transcriptional response to the reactive oxygen species singlet oxygen. MBio 4:e00541-12 [View Article][PubMed]
NurI.,
YuvalL. S.,
OkonY.,
HenisY.1981; Carotenoid composition & function in nitrogen fixing bacteria of the genus Azospirillum
. J Gen Microbiol 122:27–32
NurI.,
OkonY.,
HenisY.1982; Effect of dissolved oxygen tension on production of carotenoids, poly-β-hydroxybutyrate, succinate oxidase and superoxide dismutase by Azospirillum brasilenseCd grown in continuous culture. J Gen Microbiol 128:2937–2943 [View Article]
Pagán-RamosE.,
SongJ.,
McFaloneM.,
MuddM. H.,
DereticV.1998; Oxidative stress response and characterization of the oxyR-ahpC and furA-katG loci in Mycobacterium marinum
. J Bacteriol 180:4856–4864[PubMed]
ParsekM. R.,
YeR. W.,
PunP.,
ChakrabartyA. M.1994; Critical nucleotides in the interaction of a LysR-type regulator with its target promoter region. catBC promoter activation by CatR. J Biol Chem 269:11279–11284[PubMed]
RochaE. R.,
SmithC. J.1999; Role of the alkyl hydroperoxide reductase (ahpCF) gene in oxidative stress defense of the obligate anaerobe Bacteroides fragilis
. J Bacteriol 181:5701–5710[PubMed]
SchellM. A.,
PoserE. F.1989; Demonstration, characterization, and mutational analysis of NahR protein binding to nah and sal promoters. J Bacteriol 171:837–846[PubMed]
SchellM. A.,
BrownP. H.,
RajuS.1990; Use of saturation mutagenesis to localize probable functional domains in the NahR protein, a LysR-type transcription activator. J Biol Chem 265:3844–3850[PubMed]
SimonR.,
PrieferU.,
PuehlerA.1983; A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology 1:784–791 [View Article]
StorzG.,
JacobsonF. S.,
TartagliaL. A.,
MorganR. W.,
SilveiraL. A.,
AmesB. N.1989; An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp
. J Bacteriol 171:2049–2055[PubMed]
StothardP.2000; The sequence manipulation suite: JavaScript programs for analyzing and formatting protein and DNA sequences. Biotechniques 28:1102–1104[PubMed]
TartagliaL. A.,
StorzG.,
AmesB. N.1989; Identification and molecular analysis of oxyR-regulated promoters important for the bacterial adaptation to oxidative stress. J Mol Biol 210:709–719 [View Article][PubMed]
TatsuzawaH.,
MaruyamaT.,
MisawaN.,
FujimoriK.,
NakanoM.2000; Quenching of singlet oxygen by carotenoids produced in Escherichia coli - attenuation of singlet oxygen-mediated bacterial killing by carotenoids. FEBS Lett 484:280–284 [View Article][PubMed]
TeramotoH.,
InuiM.,
YukawaH.2013; OxyR acts as a transcriptional repressor of hydrogen peroxide-inducible antioxidant genes in Corynebacterium glutamicum R. FEBS J 280:3298–3312 [View Article][PubMed]
ThirunavukkarasuN.,
MishraM. N.,
SpaepenS.,
VanderleydenJ.,
GrossC. A.,
TripathiA. K.2008; An extra-cytoplasmic function sigma factor and anti-sigma factor control carotenoid biosynthesis in Azospirillum brasilense
. Microbiology 154:2096–2105 [View Article][PubMed]
ToledanoM. B.,
KullikI.,
TrinhF.,
BairdP. T.,
SchneiderT. D.,
StorzG.1994; Redox-dependent shift of OxyR-DNA contacts along an extended DNA-binding site: a mechanism for differential promoter selection. Cell 78:897–909 [View Article][PubMed]
VanstockemM.,
MichielsK.,
VanderleydenJ.,
Van GoolA. P.1987; Transposon mutagenesis of Azospirillum brasilense and Azospirillum lipoferum: physical analysis of Tn5 and Tn5-Mob insertion mutants. Appl Environ Microbiol 53:410–415[PubMed]
WasimM.,
BibleA. N.,
XieZ.,
AlexandreG.2009; Alkyl hydroperoxide reductase has a role in oxidative stress resistance and in modulating changes in cell-surface properties in Azospirillum brasilense Sp245. Microbiology 155:1192–1202 [View Article][PubMed]