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.
IMPORTANCE In 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.
INTRODUCTION
Microorganisms living in a fluctuating environment are constantly exposed to a wide variety of environmental stresses, which might be physical, chemical, or biological. The ability of bacteria to sense and respond to the environmental fluctuations is crucial to their survival (1). Bacteria respond to environmental challenges by changing their pattern of gene expression. To enable this, all bacteria harbor multiple sigma factors, which include a primary sigma factor and several alternative sigma factors (2, 3). While the primary sigma factor is responsible for the expression of housekeeping genes, alternative sigma factors regulate the expression of specific set of genes needed to negotiate the environmental fluctuations (4, 5). The majority of sigma factors belong to the σ70 family, and a typical σ70 factor consists of 4 conserved domains (σ1 to σ4) and a nonconserved region (NCR) (6). Heat shock sigma factor (RpoH or σ32) is a member of the σ70 family which lacks σ1 and NCR and recognizes promoters having characteristic −10 and −35 elements. An RpoH protein can be divided into several conserved regions which perform specific roles in the initiation of transcription; e.g., regions 2.1, 2.2, and 3.2 are involved in core binding (7, 8) and regions 2.3, 2.4, 3.1, and 4.2 are involved in strand opening, −10 recognition, DNA binding, and −35 recognition, respectively (6). Region C and the RpoH box are the characteristic regions of RpoH proteins which are absent in other sigma factors. Residues critical for binding to DnaK and FtsH-mediated degradation are located in the highly conserved region 2.1 and region C in the RpoH proteins (71). A stretch of amino acids located between regions 2 and 3 of σ32 is known to participate in the binding of DnaK, which functions as an anti-sigma factor of σ32 (10).
Oxidative stress is one of the most common types of stress faced by aerobic bacteria. During aerobic respiration, movement of electrons from a substrate to the molecular oxygen occurs via various membrane-associated respiratory enzymes. In this process, some of the electrons leak out to produce reactive oxygen species (ROS) such as O2−, H2O2, and ˙OH, which are detrimental to the cell, as they damage biomolecules like DNA, protein, and lipid (11–13). Under normal conditions, cells produce antioxidant enzymes and proteins to detoxify ROS. When the level of ROS generated is higher than the intrinsic capability of cells to detoxify them, oxidative stress results. The levels of ROS in the cell are monitored by two types of transcription regulators. In response to the elevated levels of ROS, DNA binding transcription regulators such as OxyR, SoxR, or OhrR activate oxidative stress-responsive genes by binding to their promoter upstream regions (14). OxyR is a global transcriptional regulator of the LysR family which helps in coping with H2O2-induced oxidative damage by inducing expression of several genes (15, 16). It binds in the upstream region of the target gene promoter, acting as a repressor or activator (17–19). While Escherichia coli contains one copy of OxyR, some bacteria, such as Deinococcus radiodurans and Vibrio cholerae, harbor two paralogs each of OxyR (72). Another class of regulators of oxidative stress includes zinc-binding anti-sigma factors which bind and regulate the activity of their cognate extracytoplasmic function (ECF) sigma factors. They possess a conserved HxxxCxxC zinc-binding motif, which acts as a redox switch to sense peroxide so as to bring a change in their conformation, causing release of the ECF sigma factors they bind (21, 22). The released ECF sigma factor then mediates the expression of genes involved in oxidative stress response (23).
Plant-associated bacteria have to cope with the ROS released by plants in response to bacterial infection and colonization (24, 25). They employ multiple mechanisms for sensing ROS levels and regulating the expression of antioxidant enzymes. Catalase and alkyl hydroperoxide reductase (Ahp) are the two main enzymes in the armory of bacteria which detoxify peroxides such as H2O2, organic peroxides, and peroxynitrite (26). While Ahp is more efficient at scavenging low levels of H2O2, catalases are involved in the detoxification of high levels of H2O2 (27). Escherichia coli produces two types of catalases; one has only catalase activity (KatE; monofunctional catalase), and the other has both catalase and peroxidative activities (KatG; bifunctional catalase). Both catalases contain heme as a prosthetic group. A third type of catalase (pseudocatalase) is also reported for some bacteria which contain Mn instead of heme as a prosthetic group (28). Rhizobia such as Rhizobium etli and Bradyrhizobium japonicum possess only one catalase each (9, 29). Sinorhizobium meliloti, however, produces three catalases: KatA, KatB, and KatC. While KatA and KatC are monofunctional, KatB is bifunctional (30). Among the three catalases of S. meliloti, only KatA is inducible and regulated by OxyR (31).
Azospirillum brasilense is a Gram-negative, plant growth-promoting rhizobacterium which is known to promote the growth of nonlegume crops and grasses via nitrogen fixation and phytohormone production (32, 33). While residing in soil or in the rhizosphere it faces a number of abiotic stresses, including change in pH, salinity, temperature, heavy metal, and ROS. During plant root colonization it has to cope with ROS released by roots as a defense response of the host plant. The genome of A. brasilense codes for 1 housekeeping and 22 alternative sigma factors consisting of 10 RpoE, 5 RpoH, 1 RpoN, and 6 FecI sigma factors. Out of the 10 RpoE sigma factors, RpoE1 and RpoE2 are involved in coping with photooxidative as well as oxidative stress (34, 35). Both of these RpoE sigma factors respond to photooxidative or oxidative stress through their cognate anti-sigma factors, which possess redox-active zinc-binding motifs (2). Earlier, we had shown for A. brasilense that carotenoid biosynthesis, which confers protection against photooxidative stress (36–38), is regulated by two cascades of sigma factors, RpoE1→RpoH2 and RpoE2→RpoH1 (39). The genome of A. brasilense also encodes two copies of OxyR regulators, of which one is involved in the negative regulation of AhpC (40). In this study, we have shown that out of the 5 RpoH paralogs encoded in A. brasilense genome, only RpoH1 is responsible for coping with heat stress, while RpoH3 and RpoH5 are involved in the regulation of expression of catalases. Based on this as well as earlier studies, we propose a scheme showing a regulatory network that is involved in the regulation of expression of enzymes involved in oxidative stress response in A. brasilense Sp7.
RESULTS
Bioinformatic analysis of five rpoH paralogs in the A. brasilense genome.A BLASTp search of alphaproteobacterial genomes against E. coli RpoH protein revealed that the genomes in the genus Azospirillum possess more RpoH paralogs than reported so far for any other alphaproteobacterium. Azospirillum strain B510, A. brasilense Sp245, and Azospirillum amazonense harbor 5, 5, and 2 RpoH paralogs, respectively. Magnetospirillum and Rhodospirillum, two genera closely related to Azospirillum, harbor 2 RpoH paralogs each. The five RpoH paralogs, RpoH1, RpoH2, RpoH3, RpoH4, and RpoH5, encoded by the A. brasilense Sp245 genome contain 294, 300, 291, 296 and 299 amino acids, respectively, which show 41%, 36%, 38%, 42%, and 38% identities with E. coli RpoH, respectively.
Comparison of the conserved domains in RpoH paralogs of A. brasilense Sp245.Multiple-sequence alignment of the deduced amino acid sequences of RpoH paralogs in A. brasilense Sp245 showed a high level of similarity in region 2.1 to the RpoH box (particularly regions C and 2.4) and in region 4.2, which are known to be highly conserved among all sigma factors (Fig. 1, middle). Regions 1.2, 2.1, 3.1, 3.2, and 4.1 are relatively less conserved. The 10 amino acid residues of the RpoH boxes of AbRpoH1 and AbRpoH5 were identical (QKKLFFNLRR). RpoH2 (QKSLFFNLRR) and RpoH3 (QKKLFFSLRR) differed from RpoH1 at the third and seventh positions, respectively (underlined). RpoH4 (QKKLFFGLSR) differed from RpoH1 at the seventh as well as ninth position. A close examination of the RpoH boxes of all the five RpoH paralogs of A. brasilense revealed that like all other RpoH factors in alphaproteobacteria, they also contained a lysine (K) at the second position. As observed in other alphaproteobacteria, the third amino acid residue in RpoH paralogs of A. brasilense is a lysine except in case of RpoH2, in which it is a serine. The 4th, 5th, and 6th residues (LFF) are strongly conserved in all the 5 RpoH paralogs.
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.
Complementation of an E. coli rpoH-null mutant with 5 rpoH paralogs of A. brasilense Sp7.In order to check if the five RpoH paralogs of A. brasilense, which show sequence homology with RpoH of E. coli, can complement the heat-sensitive phenotype of an E. coli rpoH-null mutant, we cloned all the 5 rpoH genes in a broad-host-range expression vector, pMMB206, transformed into an E. coli rpoH-null mutant (CAG 9333) and grown with isopropyl-β-d-thiogalactopyranoside (IPTG) at 42°C. Figure 2 shows that an E. coli rpoH-null mutant harboring pMMB206 failed to grow at 42°C. However, the same mutant could grow at 42°C if it expressed any of the 5 rpoH paralogs of A. brasilense (Fig. 2). This indicated that each of the five rpoH paralogs was able to complement an E. coli rpoH-null mutant for heat stress tolerance.
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.
Effects of heat and H2O2 stress on the relative expression of rpoH paralogs.To find the role of 5 RpoH sigma factors of A. brasilense Sp7 in tolerating thermal or oxidative stress (generated by H2O2), we examined the effects of heat (40°C) and H2O2 (1 mM) stress on the expression of the 5 rpoH paralogs in A. brasilense cultures (Fig. 3). Results of the real-time reverse transcription-PCR (RT-PCR) analysis showed that heat stress caused 45-fold induction of rpoH1. However, the expression of other rpoH paralogs was induced only 3- to 7-fold. A. brasilense Sp7 cells treated with 1 mM H2O2 induced the levels of rpoH3 and rpoH5 18- and 58-fold, respectively.
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.
Effect of inactivation of rpoH paralogs on the ability of A. brasilense to tolerate heat and H2O2 stress.To check the relative role of RpoH paralogs in the ability of A. brasilense to tolerate heat, we created A. brasilense mutants for each of the 5 rpoH paralogs by inserting a kanamycin resistance gene cassette in each rpoH paralog and placing them in A. brasilense Sp7 by allele replacement. Comparison of the growth of the five rpoH::km mutants at 30°C showed that all of them grew on par with A. brasilense Sp7 except the rpoH5::km mutant, which grew relatively slower than the other 4 rpoH::km mutants (Fig. 4A). At 40°C, however, growth of rpoH1::km was affected most adversely; the growth of the remaining four rpoH::km mutants was affected only marginally (Fig. 4B). The compromised growth of the rpoH1::km mutant was restored to normal by expressing a cloned copy of the rpoH1 gene via an expression vector (Fig. 4C). Since only RpoH1 was found to be important in coping with heat stress, we compared growth of all the rpoH:km mutants under stress caused by H2O2. While growth of the rpoH1::km, rpoH2::km, and rpoH4::km mutants in the presence of 1 mM H2O2 was on par with that of the parent, the rpoH3::km mutant showed a notable reduction in growth rate. The growth of the rpoH5::km mutant, however, was severely retarded (Fig. 4D), indicating that RpoH5 was more important in coping with H2O2-induced oxidative stress than RpoH3. When a plasmid-borne cloned copy of rpoH5 was expressed in the rpoH5::km mutant, growth was restored to a level comparable to that of the parent (Fig. 4E).
(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.
Inactivation of rpoH5 leads to severe reduction in catalase activity.A comparison of the catalase activities present in A. brasilense Sp7 and its 5 rpoH::km mutants was carried out with and without H2O2 treatment by in-gel activity staining (Fig. 5). Without H2O2 treatment, catalase activity in all the 5 rpoH::km mutants was on par with that of the parent. However, upon H2O2 treatment, catalase activity increased severalfold in the parent and all the mutants except the rpoH5::km mutant, in which severe reduction in catalase activity was noticed. The level of activity in the rpoH5::km mutant treated with H2O2 was on par with that in untreated samples, indicating that the inducible catalase activity was absent in the rpoH5::km mutant (Fig. 5). Catalase activity in the rpoH3::km mutant treated with H2O2 was slightly lower than that observed in the parent treated with H2O2.
In-gel assay showing catalase activity of A. brasilense Sp7 and its five rpoH::km mutants with or without 1 mM H2O2.
Effect of inactivation of rpoH paralogs in A. brasilense Sp7 on the expression of katN::lacZ, katAI::lacZ, and katAII::lacZ fusions.In-gel catalase activity in A. brasilense with or without H2O2 treatment indicated that A. brasilense possesses a constitutive as well as an inducible catalase activity. A search of the A. brasilense genome for catalases revealed that its genome encodes three paralogs of catalase. In order to understand which of the 3 catalase paralogs are regulated by RpoH sigma factors, we constructed lacZ fusions with the promoter regions of the three catalase paralogs (katN, katAI, and katAII) and mobilized them into A. brasilense Sp7 as well as in all the 5 rpoH::km mutants. β-Galactosidase assays revealed that katN was not inducible, as treatment with H2O2 did not increase the activity in the parent or the mutants, except the rpoH3::km mutant, which showed substantially compromised promoter activity with or without treatment with H2O2 (Fig. 6B). The katAI promoter activity in the parent as well as in 5 rpoH::km mutants remained unaffected with or without treatment with H2O2 (Fig. 6C). The katAII promoter activity was upregulated by H2O2 treatment in the parent as well as 4 rpoH::km mutants; for the rpoH5::km mutant, the promoter activity with or without H2O2 treatment was reduced drastically (Fig. 6D).
(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.
Effect of overexpression of RpoH paralogs on the expression of katN::lacZ, katAI::lacZ, and katAII::lacZ fusions in an E. coli two-plasmid system.In order to confirm the ability of RpoH sigma factors to activate the promoters of the three paralogs of catalase genes (katN, katAI, and katAII), we used an E. coli DH5α-based two-plasmid system in which each RpoH paralog was overexpressed individually through a broad-host-range expression vector in E. coli DH5α harboring the katN::lacZ, katAI::lacZ, or katAII::lacZ fusion. Figure 7B shows that the expression of katN::lacZ was upregulated severalfold by RpoH3, whereas katAII::lacZ expression was strongly upregulated by RpoH5. katAI::lacZ expression remained unaffected by any of the five RpoH paralogs. These observations further confirmed the ability and specificity of RpoH3 and RpoH5 to activate the promoters of the katN and katAII genes.
(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.
Effect of inactivation of the two oxyR paralogs on growth and catalase activity in A. brasilense Sp7.Previously, we have shown that A. brasilense Sp7 genome encodes two copies of the OxyR transcriptional regulator. The gene encoding OxyR1, was oriented divergently to ahpC gene and shown to negatively regulate the expression of ahpC (40). The gene encoding OxyR2, however, is oriented divergently to the katAII gene (AZOBR_31180). In this study, we constructed an oxyR2::km mutant in A. brasilense Sp7 by inserting a kanamycin resistance gene cassette in oxyR2 and mobilizing the gene into A. brasilense Sp7 genome by allele replacement as described earlier (40). Comparison of the growth of the two oxyR1::km mutants with that of their parent showed that growth of the oxyR1::km mutant was on par with that of the parent. The growth of the oxyR2::km mutant, however, was drastically compromised (Fig. 8A). Functional complementation with the cloned oxyR2 gene revealed that expression of OxyR2 restored the growth of the oxyR2::km mutant (see Fig. S2 in the supplemental material at http://www.cimap.res.in/ENGLISH/images/Director/Rai_et_al_2018_AEM_Suppl_Figures_S1_S2.pdf). When we carried out the in-gel catalase assay, we observed that the catalase activities of the two mutants and the parent were comparable in the absence of H2O2. In the presence of H2O2, however, the catalase activities were induced severalfold in A. brasilense Sp7 as well as the oxyR1::km mutant. Such an induction of catalase activity, however, was absent in the case of the oxyR2::km mutant (Fig. 8C). We also compared peroxide levels in A. brasilense Sp7 and its two oxyR::km mutants, which showed that the oxyR2::km mutant accumulated considerably higher levels of peroxide, which might have adversely affected its growth (Fig. 8B). This also indicated that OxyR2 acts as a positive regulator of the expression of its divergently transcribed gene, katAII.
(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.
Expression of katN::lacZ, katAI::lacZ, and katAII::lacZ fusions in A. brasilense Sp7 and its oxyR1::km and oxyR2::km mutants.To study the role of OxyR paralogs in regulating the expression of three catalase paralogs, we mobilized katN::lacZ, katAI::lacZ, and katAII::lacZ fusions in A. brasilense Sp7 and its oxyR1::km and oxyR2::km mutants. β-Galactosidase assays with or without H2O2 treatment showed that the expression of katN::lacZ and katAI::lacZ was not affected by the inactivation of any of the two OxyRs (Fig. 9A and B). However, the expression of katAII::lacZ was severely affected in the oxyR2::km mutant (Fig. 9C). This observation further confirmed that out of the three catalase paralogs in A. brasilense Sp7, expression of only katAII was regulated by OxyR2.
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.
Identification of cis-acting regulatory elements of the inducible catalase gene regulated by RpoH5.Since katAII was the catalase gene that was induced by H2O2 and regulated by RpoH5 as well as OxyR2, we determined the transcription start site (TSS) of katAII by 5′ rapid amplification of cDNA ends (RACE) to predict the −10 and −35 promoter elements. The TSS of katAII was located 46 nucleotides upstream of the initiation codon AUG, and GGATTT and TTGGAT were predicted as −10 and −35 elements, respectively (Fig. 10A). The −35 element of the katAII gene of A. brasilense Sp7 was identical to the −35 element of the previously characterized katA gene of Pseudomonas aeruginosa. To further confirm whether the predicted promoter elements were correct, the −35 element TTGGAT was replaced with AAGGAT by site-directed mutagenesis. The native promoter (with TTGGAT as the −35 element) and the mutant promoter (with AAGGAT as the −35 element) were transcriptionally fused with the promoterless lacZ reporter in a broad-host-range vector and mobilized into A. brasilense Sp7. A β-galactosidase assay revealed that there was severe reduction in the promoter activity from the mutated promoter compared to the native promoter, indicating that the predicted −35 element was required for the promoter activity (Fig. 10B). Examination of the upstream region of the katAII promoter revealed presence of a TN11A motif which is characteristic of an OxyR binding site. The TN11A motif, however, was absent in the region upstream of the initiation codons of katN and katAI. We also created two deletions, one (P2) carrying deletion of one arm of the TN11A motif and the second (P3) carrying deletion of both the arms of the TN11A motif. Fusion of both these deletions with lacZ showed drastically reduced β-galactosidase activity in comparison to that shown by the native katAII promoter upstream region (Fig. 10C). This further indicated that the predicted TN11A motif was important for the activation of the katAII gene.
(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.
DISCUSSION
Although the occurrence of two copies of genes encoding RpoH sigma factor is a characteristic feature of alphaproteobacteria, A. brasilense possesses 5 paralogs of RpoH, the maximum reported for any bacterium so far. While all members of gammaproteobacteria, including E. coli, harbor a single copy of the rpoH gene, most alphaproteobacteria possess two RpoH paralogs. Bacteria possessing two RpoH paralogs include rhizobia such as R. etli, S. meliloti, Magnetospirillum loti, and Rhizobium leguminosarum and nonrhizobial species such as Rhodobacter sphaeroides, Brucella melitensis, Rhodospirillum rubrum, and Bartonella quintana. Bradyrhizobium japonicum is the only alphaproteobacterium which was shown to harbor more than two copies of rpoH (41): RpoH1 was involved in the regulation of the heat stress response, while RpoH2 was essential for the synthesis of cellular proteins under normal growth conditions (41, 42). In R. sphaeroides also, RpoH1 was involved in the regulation of the heat stress response, while RpoH2 was the major player in 1O2 stress response (43). In R. etli, however, RpoH1 was involved in responding to heat shock as well as oxidative stress, but RpoH2 was involved in osmotolerance (44).
Heat shock response is a universal phenomenon that enables living cells to overcome several environmental stresses, including heat stress, which causes proteins to unfold and aggregate (45, 46). E. coli possesses only one RpoH sigma factor, which drives the expression of more than 100 genes, including heat shock proteins (47). However, inactivation of rpoH in E. coli also leads to defects in coping with oxidative stress (48). In Salmonella enterica serovar Typhimurium also, inactivation of rpoH leads to a sensitivity 10 times higher for H2O2 (49). This is because ROS generated during oxidative stress often cause misfolding of proteins and enzymes (50). The presence of multiple RpoH sigma factors in alphaproteobacteria, therefore, is an evolutionary adaptation to meet a biological need in the free-living or plant-associated aerobic bacteria, which are frequently exposed to oxidative stress. Exposure of cells to one type of stress can confer protection against other stresses. For example, bacteria challenged with elevated osmolarity acquire increased tolerance to elevated temperature and ROS (7, 27, 51–53). The level of oxidized glutathione also increases in bacteria after heat shock treatment (54). This is also the reason for the observed cross-protection provided by one type of stress by exposure to mild levels of other types of stresses (55, 56). In Rhodobacter sphaeroides, RpoH2 is shown to be involved in controlling the oxidative stress defense system (43) Thus, having two sets of RpoH regulons, one committed for coping with thermal stress and the other taking care of the oxidative stress, is a very useful adaptation, particularly for the plant-associated bacteria, which encounter ROS burst while colonizing the plant (25).
Duplication and divergence of rpoH in A. brasilense and other bacteria seem to have generated multiple copies, of which only one remains committed for the heat stress. The other RpoH paralogs, however, seem to have specialized in coping with different types of stresses such as oxidative and photooxidative stress, which also leads to misfolding of proteins. The process of duplication and divergence for the evolution of multiple RpoH paralogs runs the risk of cross talk in the form of leaky expression of genes of a different RpoH regulon (57, 58). For the leakproof and tightly regulated expression of each regulon, promoters of the target genes of each regulon need to differentiate distinctly. At the same time, the amino acid residues of regions 2.4, 3.0, and 4.2 of the RpoH, which are involved in the recognition of the −10, extended −10, and −35 promoter elements of the heat shock genes (14), also need to change accordingly so as to recognize their target promoters and to insulate each regulon from the other (57, 58).
Individual RpoH family members from different bacteria can completely, or partially, complement the temperature sensitivity of the E. coli rpoH mutant, indicating that they are functionally similar to σ32. RpoH1 and RpoH2 sigma factors of R. sphaeroides and Rhizobium meliloti complement the growth defect of the temperature-sensitive E. coli σ32 mutant (44, 59, 60). In A. brasilense Sp7 also, all the 5 RpoH paralogs individually complemented the heat-sensitive phenotype of the E. coli mutant, indicating that all of them are functional RpoH proteins. While creating rpoH::km mutants individually for each rpoH paralog in A. brasilense, we anticipated that it might not be possible to isolate a heat-sensitive rpoH::km mutant, as other copies of rpoH paralogs might complement for the inactivated copy of rpoH. However, interestingly, the growth of rpoH1::km mutant was adversely affected at 40°C. However, the growth of the other 4 rpoH::km mutants was affected only marginally at this temperature. To understand the reason behind the failure of the rpoH1::km mutant to grow at 40°C (although it harbors 4 other functional copies of the rpoH gene), we carried out real-time RT-PCR to study the inducibility of expression of the 5 rpoH paralogs at 40°C. The results showed that heat stress induced the expression of only rpoH1 and not of the remaining 4 rpoH paralogs (Fig. 3), which might be the reason for the failure of rpoH1::km mutant to grow at 40°C.
Since only RpoH1 was found to be responsible for coping with heat stress in A. brasilense Sp7, we investigated the role of the other 4 RpoH paralogs in coping with other stresses; our findings showed different levels of expression of the 5 rpoH paralogs under different abiotic stress conditions. Since oxidative and photooxidative stresses often lead to the denaturation of proteins, it was hypothesized that other paralogs might be involved in coping with stresses that cause protein denaturation. We have previously shown an involvement of RpoH2 in tolerating photooxidative stress in A. brasilense (36). In this study, the inability of rpoH3::km and rpoH5::km mutants to grow at 1 mM H2O2 and severalfold induction of rpoH3 and rpoH5 transcripts by 1 mM H2O2 indicated that both RpoH3 and RpoH5 were involved in coping with H2O2 stress. The observation that the catalase activity of rpoH5::km mutant, even after treatment with H2O2, was on par with that of the untreated mutant, indicated that inactivation of rpoH5 had led to the loss of inducible catalase activity in the rpoH5::km mutant. The in-gel catalase activity clearly showed that the treatment of A. brasilense Sp7 with H2O2 leads to a substantially higher level of induction, indicating the important role played by RpoH5 in regulating the expression of inducible catalase activity.
The A. brasilense Sp245 genome encodes 3 paralogs of catalases: KatN, KatAI, and KatAII. Expression studies with katN::lacZ, katAI::lacZ, and katAII::lacZ transcriptional fusions in A. brasilense and its 5 rpoH::km mutants clearly revealed that katAII was inducible and regulated by RpoH5. katN was regulated by RpoH3 but not inducible. We also reconfirmed these observations using a two-plasmid system in E. coli, in which each RpoH sigma factor was expressed via an inducible PtacUV5 promoter (39) to check their ability to activate the expression of the katN::lacZ, katAI::lacZ, and katAII::lacZ fusions. This study also confirmed that the katN::lacZ and katAII::lacZ fusions were activated by RpoH3 and RpoH5 sigma factors, respectively.
Genes organized divergently to the genes encoding transcriptional regulators are most often regulated by the divergently organized regulator(s) (61). Since katAII in A. brasilense was also organized divergently to the gene encoding OxyR2, we investigated the role of OxyR2 in regulating the H2O2 stress response. Previously, we had shown that oxyR1, which is divergently organized to ahpC in A. brasilense Sp7, was involved in a negative regulation of ahpC expression (40). In this study, we showed that inactivation of oxyR2, even without H2O2 treatment, led to a drastic reduction in the growth of A. brasilense. Since the inducible catalase activity was absent in the oxyR2::km mutant, it might have resulted in an accumulation of ROS leading to a severely compromised growth. These observations suggested that OxyR2 is required for the expression of katAII. Expression of the katN::lacZ, katAI::lacZ, and katAII::lacZ fusions in A. brasilense Sp7 and its oxyR1::km and oxyR2::km mutants further confirmed that OxyR2 was required for katAII expression.
Since KatAII was the most important enzyme involved in coping with H2O2 stress, we also analyzed the katAII upstream region to show that the predicted −35 element of the katAII promoter was identical to that found upstream of the katAII promoter of P. aeruginosa (17). Similarly, we detected a TN11A motif, a potential OxyR binding site, truncation or deletion of which led to the loss of katAII promoter activity in A. brasilense. These studies revealed probable binding sites of RpoH5 and OxyR2 in the upstream region of the katAII gene in A. brasilense Sp7. However, whether RpoH5 and OxyR2 actually bind to these putative binding motifs would need additional biochemical evidence. Positive regulation of the target genes of OxyR is carried out by the binding of OxyR to the operator elements which precede promoter element, and of sigma factor to the promoter. Binding sites of the sigma factor RpoH5 and the transcriptional regulator OxyR lie in juxtaposition encompassing a 65-bp sequence. Binding of the tetrameric OxyR to their operator elements is weak and degenerate so that under oxidizing or reducing conditions, it can bind or detach easily from the surface (62). This is why the operator sequence always deviates from the consensus so that OxyR can bind loosely to the operator sequence (62, 63). Thus, under oxidizing conditions, RpoH5 probably binds to the identified promoter element (TTGGAT and GGATTT) and OxyR2 to the TN11A motif preceding the promoter. Simultaneous and juxtaposed binding of RpoH5 and OxyR2 might facilitate interaction of the α subunit of RpoH5 with the C terminus of the OxyR protein, which is necessary for the maximal expression of KatAII (64).
A. brasilense is one of the most rhizocompetent bacteria and colonizes the roots of a large number of crops and grasses (33). In order to combat the ROS released by plants in response to microbial infection, root-colonizing bacteria need to be armed with a robust and fine-tuned mechanism of sensing ROS and inducing expression of antioxidant enzymes and proteins. Our present and previous studies show that the A. brasilense genome encodes 2 paralogs of OxyR, 2 RpoE sigma factors whose activity is controlled by their cognate redox-sensitive, zinc-binding anti-sigma factors, 5 paralogs of RpoH, 3 paralogs of catalases, and 1 alkyl hydroperoxide reductase (34, 37, 39, 40). Based on these studies, we propose a scheme showing a regulatory network that is involved in the regulation of expression of enzymes involved in oxidative stress response in A. brasilense (Fig. 11). While OxyR1 negatively regulates the expression of alkyl hydroperoxide reductase (62), OxyR2 positively regulated expression of the inducible catalase. While alkyl hydroperoxide reductase is known to detoxify alkyl hydroperoxides as well as low levels of H2O2, inducible catalase (KatAII) deals with the enhanced levels of extracellular H2O2. The two zinc-binding anti-sigma factors (ChrR1 and ChrR2) of A. brasilense, which regulate the activity of their cognate extracytoplasmic sigma factors (RpoE1 and RpoE2), are sensitive to H2O2 (34) and participate in the photooxidative and oxidative stress response. RpoE1 sigma factor regulates the expression of RpoH2 and RpoH5, whereas RpoE2 regulates the expression of RpoH1 and RpoH4 (39). While the RpoE1→RpoH2 cascade is involved in carotenoid biosynthesis as well as in photooxidative stress response (39), this study shows that the RpoE1→RpoH5 cascade together with OxyR2 regulates the expression of KatAII, which is the most potent and important enzyme for coping with enhanced levels of H2O2. We do not yet know how the expression of RpoH3 is regulated. This study clearly elucidates the role of RpoH paralogs (RpoH3 and RpoH5) as well as a transcriptional regulator (OxyR2) in the regulation of catalase expression in bacteria. The presence of multiple copies of redox-responsive sigma factors such as RpoE, RpoH, OxyR, and catalases might provide A. brasilense a robust mechanism to protect itself against diverse types of stresses, including oxidative stress prevalent in soil and rhizosphere.
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).
MATERIALS AND METHODS
Plasmids, bacterial strains, and growth conditions.Azospirillum brasilense Sp7 was used as a model organism, E. coli DH5α as a host for cloning and expression, and E. coli S17.1 for conjugative mobilization. While E. coli strains were grown on Luria-Bertani (LB) medium at 37°C, A. brasilense was grown on LB medium or MMAB minimal medium (67) at 30°C. Liquid cultures were grown with shaking at 200 rpm. Table 1 lists the strains and plasmids used in this study.
Bacterial strains and plasmids used
Chemical compounds and antibiotics.Wherever necessary, ampicillin (100 μg ml−1), tetracycline (10 μg ml−1), kanamycin (100 μg ml−1), chloramphenicol (40 μg ml−1), 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (X-Gal; 40 μg ml−1), or IPTG (1 mM) was added to the growth medium. H2O2 used in this study was purchased from Merck, Germany. Enzymes used for DNA manipulation and cloning were from New England BioLabs (NEB; UK), and those for RACE were from Thermo Scientific (USA).
Gene organization, sequence alignments, and phylogenetic analysis of RpoH paralogs.Deduced amino acid sequences of RpoH homologs were retrieved from the NCBI database (http://www.ncbi.nlm.nih.gov/) and used for clustal-W alignment at EBI server (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Similarity and identity were checked by Sequence Manipulation Suite.
Effect of heat and H2O2 on relative expression of rpoH paralogs in A. brasilense Sp7.To check the effects of elevated temperature (37°C) and 1 mM H2O2 on the relative expression of the 5 rpoH paralogs, the transcript levels (mRNA) of each rpoH paralog were analyzed by real-time RT-PCR. The A. brasilense Sp7 culture was grown an 30°C up to an optical density at 600 nm (OD600) of 0.4 and then shifted for growth at 37°C as well as in medium containing 1 mM H2O2 for 1 h (up to mid-log phase). The whole experiment was set in triplicate with controls. Cultures were harvested and cell pellets used for RNA extraction by the TRIzol method. To avoid DNA contamination in RNA, RNA extract was treated with DNase I (NEB) for 1 h at 37°C and heat inactivated using EDTA at 65°C for 10 min. RNA integrity was confirmed by denaturing agarose gel electrophoresis. DNA-free RNA was quantified in a NanoDrop (ND-1000). The cDNA was synthesized by using 2 μg of RNA according to the manufacturer's instructions (Fermentas). PCR with Taq polymerase and the same set of RT primers was used to check the DNA contamination for each RNA sample by PCR (ABI) using housekeeping gene rpoD. Expression of each rpoH paralog was quantified by real-time PCR using SYBR green I (Roche) in a Light Cycler 480 II instrument. Quantitative PCR (qPCR) was carried out according to the manufacturer's instructions (Roche). The protocol used was as follows. The real-time PCR mixture contained 5 μl of 2× Light Cycler 480 SYBR green I, 0.5 μM each primer, and 1 μl (2 to 5 ng) of cDNA. The cycling conditions included an initial incubation step at 95°C for 5 min, followed by 45 cycles of amplification for 10 s at 95°C, 10 s at 62°C (single acquisition), and 12 s at 72°C. The final cooling step was at 40°C for 30 s. The housekeeping gene (rpoD) was set as a calibrator.
Inactivation of rpoH1, rpoH3, rpoH4, rpoH5, and oxyR2 in A. brasilense Sp7.Insertional mutants of rpoH1, rpoH3, rpoH4, rpoH5, and the transcriptional regulator oxyR2 were constructed by inserting a kanamycin resistance gene cassette in the middle of the open reading frame (ORF) of each gene and placed in the genome by allele replacement (39) (see Fig. S1 in the supplemental material available at http://www.cimap.res.in/ENGLISH/images/Director/Rai_et_al_2018_AEM_Suppl_Figures_S1_S2.pdf). Primers along with restriction sites used are given in Table 2.
Primers used in this studya
Growth curve.For plotting growth curves of A. brasilense Sp7 and its mutants, bacterial colonies were freshly streaked on MMAB plates and incubated overnight. The next day, a single colony was picked up, inoculated in 40 ml of MMAB in a 150-ml flask, and allowed to grow up to an OD of 0.4. A. brasilense Sp7 and its rpoH::km mutants were exposed to 1 mM H2O2. Optical density of the cultures was recorded at 600 nm at every 4 h up to 24 h in a UV-visible spectrophotometer (Thermo Scientific, USA). Similarly, to check the role of different paralogs of rpoH in heat stress, overnight grown cultures of the rpoH::km mutants were diluted 100-fold in MMAB and incubated at 30°C and 40°C. Growth was recorded in triplicate at two independent times and plotted using GraphPad Prism.
In-gel catalase activity.To compare catalase activities of A. brasilense Sp7 and its mutants, overnight-grown cultures were diluted 100-fold and allowed to grow up to mid-log phase, after which half of the culture was treated with 1 mM H2O2 and grown for additional 1 h. Crude lysates were prepared from the treated and untreated cultures, and 100 μg of protein was loaded for examining catalase activity. Protein was estimated using the Bradford assay (20). Briefly, each sample was loaded onto a native gel (7%) and resolved by electrophoresis at 4°C. The gel was washed three times with distilled water for 10 min each at room temperature, followed by treatment with 0.003% (vol/vol) H2O2 for 10 min. The gel was then rinsed twice with water for 5 min. Equal volumes of 2% ferric chloride and 2% potassium ferricyanide were poured on top of the gel for staining. After the appearance of the bands showing catalase activity, the stain was poured off and rinsed 3 or 4 times with water, and the image was captured in an Alpha imager.
Construction of lacZ fusions with the promoters of katN, katAI, and katAII.To understand regulation of the promoters of genes encoding three catalases by five paralogs of RpoH and two paralogs of OxyR in A. brasilense, ∼450-bp upstream regions (relative to initiation codon ATG) of catalase katN (AZOBR_140171), katAI (AZOBR_20017), and katAII (AZOBR_p440183) genes were amplified using primer pairs having restriction sites XbaI and HindIII (listed in Table 2). These amplified PCR products were digested with restriction enzymes XbaI and HindIII and cloned into a similarly digested vector, pCZ750. Once clones were confirmed through restriction digestion, colony PCR, and sequencing, recombinant plasmids were isolated and transformed into E. coli S17.1, which was then used as a donor strain for conjugative mobilization of recombinant plasmids into A. brasilense Sp7 and its 5 rpoH::km mutants (rpoH1::km, rpoH2::km, rpoH3::km, rpoH4::km, and rpoH5::km) and 2 oxyR::km mutants (oxyR1::km and oxyR2::km). Exconjugants were selected on chloramphenicol plate for A. brasilense Sp7 and on chloramphenicol and kanamycin plates for rpoH::km and oxyR::km mutants.
Two-plasmid system.To ascertain the regulation of promoters of katN, katAI, and katAII by the five rpoH paralogs, we used an E. coli DH5α based two-plasmid assay system (9) in which each of the 5 rpoH paralogs was cloned downstream of the an IPTG-inducible promoter in pMMB206 and cotransformed individually with the katN::lacZ, katAI::lacZ, and katAII::lacZ fusions in the vector pCZ750.
β-Galactosidase assay.A. brasilense Sp7 and its mutants were grown overnight in 3 ml of MMAB medium, and on the next day, cultures were diluted 100-fold into 40 ml of MMAB and allowed to grow up to an OD600 of 0.4. Cultures were then divided equally into two flasks. One of the flasks was treated with 1 mM H2O2 and allowed to grow for additional 1 h. Cultures (2 ml) were pelleted from each sample in triplicate, and the β-galactosidase assay was carried out (65).
Determination of transcription start site by 5′ RACE.In order to determine the transcriptional start site (TSS) of the gene katAII, A. brasilense cultures were grown up to an OD of 0.4 (mid-log phase), treated with 1 mM H2O2, and grown for additional 1 h followed by the isolation of total RNA. To remove DNA contamination, isolated RNA was treated with DNase. To ensure elimination of DNA in the samples, isolated RNA was used as the template for the amplification of the katAII gene using Taq polymerase. The absence of any amplification indicated that the isolated RNA was DNA free. To determine the TSS, three different rounds of PCR were carried out. In the first round, cDNA was synthesized using a single gene-specific primer (GSP3 [Table 2]) with reverse transcriptase (Thermo Scientific, USA). Formation of cDNA was confirmed by obtaining a PCR product of a specific size using primer pair katAII F/GSP3 (Table 2) with Taq polymerase. After the confirmation of cDNA synthesis, poly(A) tailing was carried out with terminal transferase (Thermo Scientific, USA). Once the poly(A) tail was added at the 3′ end, a second round of PCR was carried out using primer pair GSP2/oligo(dT) anchor primer (Table 2). To further specify, a third round of PCR was carried out using primer pair GSP1/anchor primer (Table 2). A specific size of PCR product was obtained from the third round of PCR, which was cloned into pGEM-T Easy vector and transformed into the competent cells of E. coli XL-1 Blue. The next day, some of the randomly picked clones were processed for plasmid DNA isolation, followed by sequencing (Sci Genome, India) to map the TSS.
Site-directed mutagenesis.To validate the essentiality of the −35 element predicted on the basis of the TSS, we replaced the native hexameric sequence (TTGGAT) with the mutated hexameric sequence (AAGGAT), where underlined sequence was changed using site-directed mutagenesis, accomplished by using cloned promoter of katAII in plasmid pCZ750 as a template. PCR cycling conditions used were as described earlier (9). Primers used for site-directed mutagenesis are given in Table 2.
ACKNOWLEDGMENTS
This work was supported by Department of Biotechnology, Government of India. S.S. and A.K.R. were supported by fellowships from DBT and ICMR, respectively.
We appreciate the support received from the coordinator of the School of Biotechnology, Banaras Hindu University.
We declare no conflicts of interest.
FOOTNOTES
- Received 22 July 2018.
- Accepted 1 September 2018.
- Accepted manuscript posted online 14 September 2018.
- Copyright © 2018 American Society for Microbiology.
