Catalase Expression in Azospirillum brasilense Sp7 Is Regulated by a Network Consisting of OxyR and Two RpoH Paralogs and Including an RpoE1→RpoH5 Regulatory Cascade

Abstract

The genome of Azospirillum brasilense encodes five RpoH sigma factors: two OxyR transcription regulators and three catalases. The aim of this study was to understand the role they play during oxidative stress and their regulatory interconnection. Out of the 5 paralogs of RpoH present in A. brasilense, inactivation of only rpoH1 renders A. brasilense heat sensitive. While transcript levels of rpoH1 were elevated by heat stress, those of rpoH3 and rpoH5 were upregulated by H₂O₂ Catalase activity was upregulated in A. brasilense and its rpoH::km mutants in response to H₂O₂ except in the case of the rpoH5::km mutant, suggesting a role for RpoH5 in regulating inducible catalase. Transcriptional analysis of the katN, katAI, and katAII genes revealed that the expression of katN and katAII was severely compromised in the rpoH3::km and rpoH5::km mutants, respectively. Regulation of katN and katAII by RpoH3 and RpoH5, respectively, was further confirmed in an Escherichia coli two-plasmid system. Regulation of katAII by OxyR2 was evident by a drastic reduction in growth, KatAII activity, and katAII::lacZ expression in an oxyR2::km mutant. This study reports the involvement of RpoH3 and RpoH5 sigma factors in regulating oxidative stress response in alphaproteobacteria. We also report the regulation of an inducible catalase by a cascade of alternative sigma factors and an OxyR. Out of the three catalases in A. brasilense, those corresponding to katN and katAII are regulated by RpoH3 and RpoH5, respectively. The expression of katAII is regulated by a cascade of RpoE1→RpoH5 and OxyR2.IMPORTANCEIn silico analysis of the A. brasilense genome showed the presence of multiple paralogs of genes involved in oxidative stress response, which included 2 OxyR transcription regulators and 3 catalases. So far, Deinococcus radiodurans and Vibrio cholerae are known to harbor two paralogs of OxyR, and Sinorhizobium meliloti harbors three catalases. We do not yet know how the expression of multiple catalases is regulated in any bacterium. Here we show the role of multiple RpoH sigma factors and OxyR in regulating the expression of multiple catalases in A. brasilense Sp7. Our work gives a glimpse of systems biology of A. brasilense used for responding to oxidative stress.

Keywords: cascade; catalase; paralogs; sigma factor RpoH; transcriptional regulator OxyR.

FIG 1

Multiple-sequence alignment of amino acid sequences of the 5 RpoH paralogs in Azospirillum brasilense with RpoH of E. coli K-12. The sequences below bidirectional arrows indicate conserved regions in RpoH. The unique RpoH box is present within the region C in all the 5 RpoH sequences.

FIG 2

Plate assay showing effect of overexpression of five rpoH paralogs (H1 to H5) of A. brasilense on the heat-sensitive phenotype of the rpoH-null mutant of E. coli CAG 9333. Vector pMMB206 was used as a negative control.

FIG 3

Relative expression of rpoH paralogs determined by quantitative RT-PCR by using threshold cycle values obtained from RNA samples of treated (1 mM H₂O₂ and 40°C temperature) and untreated A. brasilense Sp7. For normalization, mRNA levels for rpoD (housekeeping sigma factor gene) were used as an internal standard.

FIG 4

(A) Comparison of the growth of A. brasilense Sp7 and its five rpoH::km mutants at 30°C. (B) Comparison of the growth of A. brasilense Sp7 and its five rpoH::km mutants at 40°C. (C) Growth curve showing ability of the cloned copy of the rpoH1 gene to complement the rpoH1::km mutant at 40°C. (D) Comparison of the growth of A. brasilense Sp7 and its five rpoH::km mutants treated with 1 mM H₂O₂. (E) Growth curve showing the ability of the cloned copy of the rpoH5 gene to complement the rpoH5::km mutant in the presence of 1 mM H₂O₂. Each point of the curve shows the mean of triplicates of three independent experiments, and error bars show SD at each point of the growth.

FIG 5

In-gel assay showing catalase activity of A. brasilense Sp7 and its five rpoH::km mutants with or without 1 mM H₂O₂.

FIG 6

(A) Genetic organization of the three paralogs of catalase genes: katAII, katAI, and katN. (B to D) Comparison of β-galactosidase activities of katN::lacZ (B), katAI::lacZ (C), and katAII::lacZ (D) in A. brasilense Sp7 and its five rpoH::km mutants in the absence or presence of 1 mM H₂O₂.

FIG 7

(A) Scheme of a two-plasmid system showing design for activation of the katN::lacZ, katAI::lacZ, and katAII::lacZ fusions by each of the five paralogs of the rpoH gene in E. coli. (B) β-Galactosidase assay showing the ability of the 5 RpoH paralogs to activate the katN::lacZ, katAI::lacZ, and katAII::lacZ fusions in E. coli.

FIG 8

(A) Comparison of the growth of A. brasilense Sp7 and its oxyR1::km and oxyR2::km mutants in MMAB. (B) Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assay showing relative accumulation of ROS in A. brasilense Sp7 and its two oxyR::km mutants. (C) In-gel catalase assay of A. brasilense Sp7 and its oxyR1::km and oxyR2::km mutants with or without 1 mM H₂O₂.

FIG 9

Comparison of β-galactosidase activity from the katN::lacZ (A), katAI::lacZ (B), and katAII::lacZ (C) fusions in A. brasilense Sp7 and its oxyR1::km and oxyR2::km mutants in the presence or absence of 1 mM H₂O₂.

FIG 10

(A) Genetic organization of oxyR2-katAII. For determination of the transcription start site (boldface G) of katAII, we conducted 5′ RACE to predict the −10 (GGATAA) and −35 (TTGGAT) elements of the promoters. (B) β-Galactosidase activity from the promoter of the katAII gene with native −35 element and with a mutant −35 element. (C) Effect of deletion of the promoter upstream region of katAII on the β-galactosidase activity from the katAII::lacZ fusion. P1 encompasses 186 nucleotides downstream TSS and 61 nucleotides upstream of the TSS. P2 encompasses 186 nucleotides downstream TSS and 52 nucleotides upstream of the TSS. P3 encompasses 186 nucleotides downstream TSS and 42 nucleotides upstream of the TSS. Conserved T and A nucleotides with a space of 11 nucleotides are underlined and are shown in bold.

FIG 11

Model showing cascades and network involved in regulating the peroxide stress response in A. brasilense. Two paralogs of rpoE, five paralogs of rpoH, and two paralogs of oxyR regulate the expression of three paralogs of catalase and one alkyl hydroperoxide reductase. RpoE1 regulates the expression of RpoH2 and RpoH5, and RpoE2 regulates the expression of RpoH1 and RpoH4 (39). Expression of katAII is positively regulated by RpoH5 as well as OxyR2. At the bottom is an enlarged image showing OxyR2 binding to the operator region of katAII through the conserved TN₁₁A motif and RpoH5 binding; the −35 and −10 regions are identified. RpoH3 positively regulates the expression of katN. We have previously shown that OxyR1 is involved in the regulation of the ahpC gene (40).