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. 2015 Sep;161(Pt 9):1806–1815. doi: 10.1099/mic.0.000130

Quinol oxidase encoded by cyoABCD in Rhizobium etli CFN42 is regulated by ActSR and is crucial for growth at low pH or low iron conditions

Zachary R Lunak 1,†,✉, K Dale Noel 1
PMCID: PMC4635472  PMID: 26297648

Abstract

Rhizobium etli aerobically respires with several terminal oxidases. The quinol oxidase (Cyo) encoded by cyoABCD is needed for efficient adaptation to low oxygen conditions and cyo transcription is upregulated at low oxygen. This study sought to determine how transcription of the cyo operon is regulated. The 5′ sequence upstream of cyo was analysed in silico and revealed putative binding sites for ActR of the ActSR two-component regulatory system. The expression of cyo was decreased in an actSR mutant regardless of the oxygen condition. As ActSR is known to be important for growth under low pH in another rhizobial species, the effect of growth medium pH on cyo expression was tested. As the pH of the media was incrementally decreased, cyo expression gradually increased in the WT, eventually reaching ∼10-fold higher levels at low pH (4.8) compared with neutral pH (7.0) conditions. This upregulation of cyo under decreasing pH conditions was eliminated in the actSR mutant. Both the actSR and cyo mutants had severe growth defects at low pH (4.8). Lastly, the actSR and cyo mutants had severe growth defects when grown in media treated with an iron chelator. Under these conditions, cyo was upregulated in the WT, whereas cyo was not induced in the actSR mutant. Altogether, the results indicated cyo expression is largely dependent on the ActSR two-component system. This study also demonstrated additional physiological roles for Cyo in R. etli CFN42, in which it is the preferred oxidase for growth under acidic and low iron conditions.

Introduction

Bacteria have diverse metabolic capabilities that enable them to cope with the varying environmental conditions. One example is having branched aerobic respiratory chains that terminate at different terminal oxidases, including various cytochrome c oxidases and, alternatively, quinol oxidases. The paths toward these oxidases diverge at quinol. Ubiquinol-cytochrome c oxidoreductase (Fbc), also known as the bc 1 complex, sends electrons from quinol to cytochrome c. Electrons are then transferred from cytochrome c to oxygen via cytochrome c oxidases. Alternatively, electrons can flow directly from quinol to oxygen via quinol oxidases.

The quinol oxidase encoded by cyoABCD is widespread among proteobacteria and firmicutes. This oxidase (Cyo) is often classified as a low-affinity oxidase utilized in high oxygen based on studies in Escherichia coli (García-Horsman et al., 1994; Morris & Schmidt, 2013). Conversely, previous work demonstrated Cyo to be important for growth and adaptation to low oxygen conditions in culture for certain rhizobial species (Surpin & Maier, 1998; Lunak & Noel, 2015). Moreover, in two rhizobial species, cyo genes are upregulated under low oxygen (Trzebiatowski et al., 2001; Bobik et al., 2006; Lunak & Noel, 2015). However, the transcriptional regulatory mechanism of this upregulation is not understood.

Oxygen is a substrate for all the terminal oxidases and is a common factor in how oxidases are regulated (Bueno et al., 2012). Some oxidases are regulated by transcription factors that sense oxygen directly, such as Fnr proteins. Fnr becomes active at low oxygen and binds to a conserved symmetrical motif, TGGAT-N4-ATCAA (Kiley & Beinert, 1998; Körner et al., 2003). Alternatively, oxidases can be regulated by transcription factors that sense oxygen indirectly based on the redox state of certain molecules. The activity of the two-component system RegBA in Rhodobacter capsulatus is regulated in part by the redox state of quinone (Wu & Bauer, 2010). Homologous two-component systems (ActSR in Sinrohizobium meliloti, RegSR in Bradyrhizobium japonicum, PrrBA in Rhodobacter sphaeroides) can complement one another (Emmerich et al., 2000a; Elsen et al., 2004; Wu & Bauer, 2008). However, the DNA binding sites are not as well conserved among organisms compared with the aforementioned Fnr anaerobox. In B. japonicum, the RegA homologue RegR binds to an imperfect repeat NGNGNCN4–6GNNNC (Emmerich et al., 2000b; Lindemann et al., 2007; Torres et al., 2014). In this bacterium, the RegSR two-component system is important for adaptation to low oxygen during processes such as symbiosis with legumes and recently it has been implicated in denitrification (Bauer et al., 1998; Torres et al., 2014). In addition, the RegBA homologue in S. meliloti ActSR is necessary for growth and the induction of genes required for adaptation to low pH conditions (O'Hara et al., 1989; Tiwari et al., 1996).

Iron may be another important cue for Cyo regulation and utilization. The Fbc pathway to oxygen involves more proteins that contain iron than the Cyo respiratory pathway. Previous work showed cyo to be upregulated in response to low iron conditions in S. meliloti and Pseudomonas aeruginosa (Chao et al., 2005; Kawakami et al., 2010). It is not known how general this regulation is and what the mechanism of regulation might be.

In this study, Rhizobium etli was used as a model organism to determine the major transcription factor involved in regulating cyo. Like other rhizobia, R. etli resides either in the free-soil environment or as a nitrogen-fixing symbiont in root nodules of its leguminous host plant. In both environments, R. etli must cope with varying physiological conditions. R. etli CFN42 is particularly useful for studying Cyo because Cyo is the only quinol oxidase present (Lunak & Noel, 2015).

Use was made of previously isolated mutants whose respiratory paths were constrained to end in cytochrome c oxidases or, conversely, only in quinol oxidases. All the aerobic respiratory chains terminating with cytochrome c oxidases require a functional Fbc. Therefore, an fbc mutant reveals the sufficiency of Cyo function under a given condition because Cyo would be the only viable oxidase option remaining in this mutant. However, a cyo mutant can only respire via cytochrome c oxidases. The inability of a cyo mutant to grow is evidence that the bacterium has evolved to depend on Cyo under the conditions being tested. In the analysis of cyo transcriptional regulation under different conditions, comparisons of an fbc mutant and the WT provide additional physiological insights. If there is no difference between the WT and fbc mutant under a particular condition, this suggests that Fbc is not normally used under that condition. Conversely, a significant increase of cyo transcription in the fbc mutant compared with the WT suggests that Fbc is preferentially utilized under that condition, but the strain can adapt to the loss of Fbc by upregulating cyo.

Methods

Bacterial strains and growth conditions

R. etli strains were derived from CE3, a streptomycin-resistant derivative of the CFN42 WT strain (Noel et al., 1984), whose genome nucleotide sequence has been determined (González et al., 2006). R. etli strains were grown at 30 °C on a rotating shaker in TY liquid medium [0.5 % tryptone (Difco), 0.3 % yeast extract (Difco) and 10 mM CaCl2]. For growth under low pH, strains were grown either in TY buffered with 40 mM MES or in a low pH YGM minimal medium containing 0.4 mM MgSO4, 1.25 mM K2HPO4, 0.11 % sodium glutamate, 0.4 % glucose, 4 mM NH4Cl, 1 mM CaCl2, 0.15 mM FeCl3, 1 μg biotin ml− 1, 1 μg thiamine ml− 1, 1 μg pantothenic acid ml− 1 and 40 mM MES that buffers the pH to ∼4.8. For growth at neutral pH in YGM medium, the pH was adjusted to 7.0 by titration with NaOH. The pH of the medium was tested after growth to ensure the pH was maintained at ± 0.2 pH of the uninoculated medium. For growth under low iron, strains were grown in TY treated with 200 μM 2,2-dipyridl. E. coli strains were grown in LB liquid medium (1.0 % tryptone, 0.5 % yeast extract and 0.5 % NaCl) at 37 °C on a rotating shaker (Sambrook et al., 1989). Agar medium contained 1.5 % Bacto agar (Difco).

To analyse growth under varying oxygen concentrations, cultures were grown as described previously (Lunak & Noel, 2015). Fully grown cultures were diluted 1 : 200 into 5 ml TY medium, resting in 60 ml serum vials. The serum vials were then capped and the headspace was flushed with nitrogen gas. Using a sterile syringe needle, ambient air (assumed 21 % oxygen) was injected back into the headspace to make it 1 and 0.1 % oxygen. For growth at 21 % oxygen, the vials were covered with aluminium foil. Cultures were then grown at 30 °C on a rotating shaker. To follow growth, 400 μl was removed from the cultures using a sterile syringe needle and the OD600 was measured.

Prediction of ActR binding sites in 5′ upstream of cyo

The genomes of R. etli CFN42, S. meliloti 1021, Rhizobium leguminosarum bv viciae 3841 and B. japonicum USDA 110 have been determined (Finan et al., 2001; Kaneko et al., 2002; González et al., 2006; Young et al., 2006). To identify possible ActR DNA binding sites, various parts of the promoter regions were aligned with known RegR DNA binding sites from B. japonicum (Lindemann et al., 2007; Torres et al., 2014) using clustal w2 software (Thompson et al., 2002).

Materials and techniques for DNA isolation

Genomic DNA for use in cloning was isolated from R. etli strains using a GenElute Bacterial Genomic DNA kit (Sigma). E. coli NEB-5α (Invitrogen) competent cells were transformed (Hanahan, 1983) and plasmids were isolated using a QIAprep Spin Miniprep kit (Qiagen). DNA was recovered from agarose gels using a Gel/PCR DNA Fragments Extraction kit (IBI Scientific) and modified with restriction enzymes purchased from New England BioLabs. Custom primers were synthesized by Eurofins MWG Operon.

Cloning and mutagenesis

The strains used in this study are listed in Table 1. The fbc and cyo mutants were constructed in a previous study (Lunak & Noel, 2015). The cyo mutant (CE574) contains a kanamycin resistance cassette in the SalI site (nt 39 576–39 581 of the R. etli CFN42 genome) of cyoA of the cyoABCD operon. The fbc mutant contains a Tn5 transposon insertion in the fbcF gene of the fbcFBC operon at nt 3 178 288 of the R. etli CFN42 genome (E. Rosado and K. D. Noel, unpublished).

Table 1. Strains and plasmids used in study.

Bacterial strain or plasmid Description, genotype or phenotype Reference or source
R. etli strains
 CE3 WT strain, str-1 Noel et al. (1984)
 CE3/pZL73 CE3 carrying pZL73, TcR This study
 CE119 CE3 derivative; str-1 fbcF : : Tn5 Lunak & Noel (2015)
 CE119/pZL73 CE119 carrying pZL73; TcR This study
 CE574 CE3 derivative; str-1 cyoA : : Km Lunak & Noel (2015)
 CE574/pZL34 CE574 carrying pZL34, TcR Lunak & Noel (2015)
 CE605 CE3 derivative; str-1 actS : : Km : : actR This study
 CE605/pZL73 CE605 carrying pZL73, TcR This study
 CE605/pZL51 CE605 carrying pZL51, TcR This study
E. coli strains
 NEB-5α Competent strain used for cloning New England Biolabs
 MT616 pro thi endA hsdR supE44 recA-J6 pRK2013Km : : Tn9 Finan et al. (1986)
Plasmids
 pBSL86 nptII gene cassette; KmR Alexeyev (1995)
 pCR2.1 AprKmr, TA cloning vector for PCR products Invitrogen
 pEX18Tc Suicide plasmid; TcR oriT sacB Hoang et al. (1998)
 pFAJ1708 Expression vector with nptII promoter Dombrecht et al. (2001)
 pMP220 Transcriptional lacZ fusion vector, TcR Spaink et al. (1987)
 pZL34 1.3 kb BamHI/PstI fragment with cyoA in pFAJ1708 Lunak & Noel (2015)
 pZL48 Km cassette replacing 1.7 kb of the actSR operon, KmR This study
 pZL51 3.1 kb EcoRI/HndIII fragment with actSR in pFAJ1708 This study
 pZL73 pMP220-derived, 475 bp KpnI/XbaI fragment upstream  of cyoA fused with lacZ This study
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The actSR mutant (CE605) was made by deleting 1148 bp of actS and additionally 511 bp of actR. To mutate actSR, the initial 181 bp of actSR together with 566 bp of 5′ upstream sequence (nt 59 093–58 347 of the R. etli CFN42 genome) was amplified by PCR using ActSup-EcoRI and ActSup-XbaI primers (listed in Table S1, available in the online Supplementary Material). The PCR product was then ligated into the TA cloning vector pCR2.1 (Invitrogen) to create pZL45. In addition, the last 65 bp of actR together with 586 bp of 3′ downstream sequence (nt 56 614–55 964 of the R. etli CFN42 genome) was amplified by PCR using ActRdwn-XbaI and ActRdwn-HndIII primers (Table S1) and ligated into pCR2.1 to create pZL46. pZL45 was then digested with EcoRI/XbaI to release the 747 bp fragment and pZL46 was digested with XbaI/HndIII to release the 651 bp fragment. Both of these fragments were ligated into pEX18Tc (Hoang et al., 1998) to create pZL47. pZL47 was then digested with XbaI and a kanamycin resistance cassette (Alexeyev, 1995) was inserted into this site to create plasmid pZL48. pZL48 was transferred into R. etli CE3 by using the plasmid-mobilizer strain MT616 on TY agar plates (Finan et al., 1986; Glazebrook & Walker, 1991).

CE3 transconjugants containing pZL48 were selected and purified as described previously (Ojeda et al., 2010). Double-crossover recombinants were screened on TY agar plates supplemented with 1 μg tetracycline ml− 1. Of the recombinants that were sensitive to 1 μg tetracycline ml− 1 and resistant to 8 % sucrose on TY agar, it was then verified by PCR that the colonies contained only the mutant allele and that the WT allele was absent.

Complementation of actSR and cyo

The entire actSR operon including the flanking sequence (nt 59 093–55 964 of the R. etli CFN42 genome) was amplified using the primers listed in Table S1. The DNA PCR product was originally inserted into the TA cloning vector pCR2.1. The plasmid was then digested with XbaI/BamHI to release the 3.1 kb fragment containing actSR, which then was ligated to pFAJ1708 (Dombrecht et al., 2001). The resulting plasmid pZL51 was transferred into CE605 (actSR mutant) by triparental mating. Strains containing pZL51 were selected for tetracycline resistance (5 μg ml− 1). A similar approach was used to complement the cyo mutant CE574 and has been described previously (Lunak & Noel, 2015).

lacZ fusion and β-galactosidase measurements

To generate a cyoA : : lacZ transcriptional fusion, the promoter region of cyoA (nt 40 020–40 490 of the R. etli CFN42 genome) was amplified from R. etli CE3 genomic DNA by PCR using the primers listed in Table S1. The PCR product was then inserted into the TA cloning vector pCR2.1 to create plasmid pZL67. The 485 bp fragment was released from pZL67 using XbaI/KpnI and ligated into the pMP220 plasmid (Spaink et al., 1987) at the KpnI/XbaI restriction sites to create pZL73. pZL73 was transferred into CE3 using the MT616 plasmid-mobilizer strain. The empty vector pMP220 was introduced into CE3 as a negative control. Under different oxygen (1 or 21 %) and pH (4.8–7.5) conditions, 1 ml culture was withdrawn and washed with cold Z-buffer. The β-galactosidase assay was performed and Miller units were calculated as described previously (Sambrook et al., 1989).

Reverse transcription quantitative PCR (RT-qPCR)

Cultures were pelleted, immediately frozen in dry ice and stored at − 80 °C. When ready for testing, cells were thawed on ice and RNA was extracted using a NucleoSpin RNA II kit (Macherey Nagel). The RNA concentration was measured by a NanoDrop spectrophotometer and 1 μg RNA was converted into cDNA using an EasyScript cDNA synthesis kit (MidSci) with the specific reverse primer for the gene of interest. As a negative control, water was added instead of the reverse transcriptase. cDNA products were quantified by real-time PCR using EvaGreen qPCR Mastermix (MidSci), gene-specific primers and a Bio-Rad iCycler. For analysing cyo expression, primers were designed to detect a 118 bp fragment in cyoB (Table S1). Samples were initially denatured at 95 °C for 10 min followed by a 40-cycle amplification protocol (95 °C for 15 s, 60 °C for 60 s). After the PCR, a melt curve analysis was performed to ensure only one amplification product was present. The expression of the 16S RNA gene was analysed using the same approach. Results for cyoB expression were normalized to the expression of the 16S RNA gene.

Results

cyo 5′ upstream sequence and prediction of regulatory elements

The genome nucleotide sequence of R. etli CFN42 has been determined (González et al., 2006). The 5′ upstream DNA of the cyo operon is depicted in Fig. 1. The promoter region contained three putative ActR DNA binding sites that aligned with the consensus RegR DNA binding site in B. japonicum (Figs. 1 and S1). Two of the possible ActR binding sites were in succession (nt 40 164–40 130 of the R. etli CFN42 genome sequence). The other potential ActR DNA binding site and a potential FNR anaerobox were further upstream (nt 40 387–40 421). Potential ActR DNA binding sites were also present in the 5′ regions upstream of cyo in S. meliloti, R. leguminosarum and B. japonicum (Fig. S1).

Fig. 1. The 5′ upstream promoter region of cyo. The sequence contains 5′ DNA upstream of the cyoA ORF (nt 40 430–39 982 of R. etli CFN42 genome). Putative ActR DNA binding sites are underlined. A putative Crp-Fnr anaerobox is dash-underlined. Possible translation start sites are highlighted in black.

Fig. 1.

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Mutagenesis of actSR and its effect on cyo expression under low oxygen

To test whether ActSR regulates cyo expression, mutant strain (CE605) was constructed by replacing the actSR operon with a kanamycin resistance cassette. The expression of cyo was analysed in this actSR mutant under various oxygen conditions using RT-qPCR and a cyo : : lacZ fusion (Fig. 2a, b). As seen previously (Lunak & Noel, 2015), the WT significantly upregulated cyo at 1 % compared with 21 % oxygen concentration. In the actSR mutant, cyo transcript levels were approximately eightfold lower compared with WT levels under low oxygen (1 %). Under high oxygen conditions, the expression of cyo was decreased in the actSR mutant (around twofold) compared with the WT levels. The expression of cyo was restored to WT levels after transferring the WT copy of actSR, pZL51, into the actSR background. In addition, cyo promoter activity in an fbc mutant was assessed by use of the cyo : : lacZ fusion carried on a plasmid in mutant cells grown under 1 and 21 % oxygen. Under both low and high oxygen conditions, the fbc mutant exhibited higher cyo promoter activity than the WT (Fig. 2b).

Fig. 2. cyo expression under 1 and 21 % oxygen. Strains were grown under either 21 or 1 % oxygen conditions. Protein or RNA was extracted from cells in exponential phase. (a) RT-qPCR of cyoB. RNA was extracted and converted into cDNA using the reverse gene-specific primer. As described in Methods, cDNA was then quantified by qPCR. The amount of cyoB cDNA was then normalized to the amount of 16S RNA cDNA from the original RNA sample. Mean ± sd values were calculated from three separate qPCR assays. (b) β-Galactosidase activity of the strains carrying the transcriptional fusion plasmid pZL73 (cyoA : : lacZ). Mean ± sd values were calculated from three or more separate lacZ assays from two different cultures.

Fig. 2.

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It is known that cyo mutants have growth defects at 0.1 and 1 % oxygen. The above results suggest that actSR mutants should have the same defects. Therefore, growth of the actSR mutant was analysed under various oxygen concentrations in TY medium. The actSR mutant failed to grow under 0.1 % oxygen and had a severe growth defect under 1.0 % oxygen (Fig. 3a, b). Addition of the WT actSR operon (pZL51) restored growth similar to that of the WT. Under high oxygen conditions (21 %), the mutant had a slight growth defect (Fig. 3c).

Fig. 3. Growth curves of the actSR mutant under (a) 0.1, (b) 1.0 and (c) 21 % oxygen. Strains were initially grown in TY liquid under a gas phase with 21 % oxygen. At full growth they were subcultured 1 : 200 into 5 ml TY liquid in 60 ml serum vials. As described in Methods, nitrogen and air were added to the headspaces in the vials above the liquid to give the indicated concentrations of oxygen. Growth was followed by measuring the OD600. Bars, sd from at least three separate cultures.

Fig. 3.

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cyo expression under varying pH conditions

Given ActSR's role as a global regulator in acidic conditions (O'Hara et al., 1989; Tiwari et al., 1996), cyo promoter activity was measured under various pH conditions. As the pH in the medium was incrementally lowered from 7.5 to 4.8, cyo promoter activity began to increase at pH < 6.5 (Fig. 4a). cyo expression was investigated more closely at pH 4.8 versus 7.0 using RT-qPCR in addition to the cyo : : lacZ fusion. Transcription activity was ∼10-fold higher at pH 4.8 compared with pH 7.0 in the WT (Fig. 4b, c). In the actSR mutant, cyo transcript levels and promoter activity were significantly lower compared with WT levels under any pH condition. cyo expression was significantly increased after transferring the WT copy of the actSR operon pZL51 into the actSR background, but was still lower compared with WT under low pH (Fig. 4c). In the fbc mutant, the promoter activity was significantly higher compared with the WT when cells were grown at neutral pH (Fig. 4b). However, the promoter activities were similar between the WT and fbc mutant under low pH conditions.

Fig. 4. cyo expression under varying pH conditions. (a) WT cells, carrying pZL73 (cyo : : lacZ), were harvested from exponentially growing cultures at different pH (4.8–7.5) in YGM media and the β-galactosidase assay was performed. Specific activity is given in Miller units. Mean ± sd values were calculated from three or more separate lacZ assays from two different cultures. (b) Strains, carrying pZL73 (cyo : : lacZ), were grown in YGM buffered with MES at pH 4.8 or 7.0. Cells were harvested in exponential phase and the β-galactosidase activity (Miller units) was determined. Mean ± sd values were calculated from three or more separate assays from two different cultures. (c) Strains were grown in YGM media buffered with MES at pH 4.8 or 7.0. RNA was extracted from exponential phase, and cyoB transcript levels were determined and normalized to the 16S RNA levels. Mean ± sd values were calculated from three separate qPCR assays.

Fig. 4.

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Growth under low pH

To determine if Cyo and ActSR had a significant physiological role at low pH, the growth of the mutants was analysed at pH 4.8 and 7.0 in YGM media. Both the cyo and actSR mutants had significantly prolonged lag phases when grown under low pH (Fig. 5a). Conversely, the fbc mutant grew comparably to the WT. The cyo and actSR mutants also had growth defects in TY media adjusted to pH 4.8 (Fig. 5b). The growth defects were alleviated after transferring the WT copies of cyoA (pZL34) and actSR (pZL51) into their respective mutant backgrounds (Fig. 5b, S2a). When grown under neutral pH, the cyo mutant grew similarly to the WT, whereas both the actSR and fbc mutants had slight growth defects (Fig. 5c).

Fig. 5. Growth curves at low and neutral pH. Strains were grown aerobically in (a) YGM media buffered at pH 4.8, (b) TY media buffered at pH 4.8 (c) or YGM media buffered at pH 7.0. Growth was followed by measuring the OD600. After growth, the pH was measured to ensure the pH of the culture was maintained at ± 0.2 pH of the uninoculated cultures. Bars, sd from at least three separate experiments.

Fig. 5.

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When streaked on low pH YGM plates (pH 4.8), the actSR mutant did not start to form colonies until 7 days after streaking (Fig. S3), whereas the WT consistently formed isolated colonies 2–3 days after streaking on low pH plates. Isolated colonies were consistently observed in the cyo mutant 4 days after streaking. After transferring the WT copies of cyoA (pZL34) and actSR (pZL51) into their respective mutant backgrounds, the onset of isolated colonies was similar to that of the WT (2–3 days after streaking).

Growth analysis and cyo expression under low iron

To test the importance of Cyo under low iron conditions, growth and cyo expression were analysed in cells grown in TY medium treated with an iron chelator, 2,2-dipyridyl. Both the cyo and actSR mutants had a severe growth defect under this condition (Fig. 6a). However, growth of the fbc mutant was comparable to that of the WT. Addition of the WT copies (pZL34 and pZL51) to their respective mutant backgrounds alleviated the observed growth defects (Fig. S2b). In the WT, the expression of cyo was approximately fivefold higher in this low iron medium (Fig. 6b). Conversely, cyo expression was not induced under low iron concentrations in the actSR mutant. In the fbc mutant, cyo expression was similar to the WT under the low iron condition.

Fig. 6. Growth and cyo expression under low iron. (a) Strains were grown aerobically in TY treated with 200 μM 2,2-dipyridyl. Growth was followed by measuring the OD600. Bars, sd from at least three separate cultures. (b) Strains carrying pZL73 were grown in 2,2-dipyridyl-treated and untreated TY media. β-Galactosidase activities were determined from cells in exponential phase. Mean ± sd values were calculated from three or more separate lacZ assays from two different cultures.

Fig. 6.

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Discussion

Potential ActR DNA binding sites are present in the DNA 5′ to cyoA in strains of R. etli, R. leguminosarum, S. meliloti and B. japonicum. This suggests the existence of common mechanisms for regulating cyo in these species. The focus of this study was to investigate the possible role of ActSR and the genetic results imply that this two-component system is a key regulator of cyo expression in R. etli CFN42.

First, an actSR mutant was constructed and its cyo expression was tested under varying oxygen concentrations. The actSR mutation abrogated the strong upregulation in cyo expression observed in the WT under low oxygen conditions. This result correlates with a recent microarray study in B. japonicum, where cyo genes were significantly decreased under anoxic conditions in a mutant defective in the ActSR homologue RegSR (Torres et al., 2014). In addition, this result may explain how cyo is upregulated under low oxygen in S. meliloti, where it had been shown that cyo is upregulated independently of FixJ (Bobik et al., 2006).

The actSR mutant had a significant growth defect under low oxygen (0.1 and 1.0 %) concentrations. This is consistent with findings in B. japonicum where it is considered a global regulator for adaptation to low oxygen conditions (Bauer et al., 1998; Torres et al., 2014).

The ActSR two-component system is an important global regulator under acidic conditions in S. meliloti (O'Hara et al., 1989; Tiwari et al., 1996). Therefore, it was predicted that cyo might be upregulated under low pH. The expression of cyo remained at a basal level under neutral pH but, as pH in the growth medium was lowered below 6.5, cyo expression rose in inverse proportion to the pH drop. Increased cyo expression in response to acidic conditions has been observed in a microarray study in S. meliloti (Hellweg et al., 2009). Similar to the response to varying oxygen concentrations, deletion of actSR eliminated the increased cyo expression under low pH.

An fbc mutant was included in this study, because theory and all evidence to date indicate Cyo is the only viable respiratory option in an R. etli CFN42 mutant lacking Fbc. Based on this premise, it reveals the capability of Cyo to function under varying conditions. For instance, this mutant was able to grow under either low or neutral pH conditions, implying that Cyo provides a useful function under a wide range of pH conditions. Moreover, Cyo seems to function at a wide range of iron concentrations, given that the fbc mutant can grow at both low and high iron concentrations. In conditions where cyo is marginally expressed in the WT, such as high oxygen or neutral pH, the fbc mutant had approximately threefold higher levels of cyo expression compared with the WT. This indicates the WT prefers the Fbc pathway under these conditions, but the mutant is still able to respond to the lack of Fbc activity by upregulating cyo and presumably increasing its utilization. However, under conditions where cyo is upregulated in the WT (low oxygen, low pH and low iron) the differences in cyo expression were minimal between the fbc mutant and the WT. These results further support that Cyo is the preferable oxidase under these conditions.

How ActSR becomes active has been characterized in vitro in the homologous RegBA system in Rhodobacter capsulatus. One study indicated autophosphorylation activity of the ActS homologue RegB can be influenced by the redox state of quinone (Wu & Bauer, 2010). Although both quinone (oxidized) and quinol (reduced) can bind to RegB with equal affinity, only quinone will promote an inactive conformation. Thus, a higher quinol : quinone ratio in the cell will lead to a more active RegBA. This may explain how fbc mutation leads to upregulation of cyo under both aerobic and neutral pH conditions. In this mutant, the Fbc pathway is non-functional, which would presumably cause a build-up of quinol, causing the quinol : quinone ratio to increase. The increase of quinol would further activate ActSR and consequently increase cyo expression. It is predicted that the increased cyo expression observed in the fbc mutant is fully attributable to ActSR as the actSR/fbc double mutant was unattainable in aerobic conditions.

The quinol : quinone ratio may also be affected at low pH. The premise is based on several studies in another alphaproteobacterium, Rhodobacter sphaeroides, where Fbc activity was optimum at pH 8.0 and dropped to a relatively low level at pH < 6.0 (Crofts et al., 1999; Guergova-Kuras et al., 2000; Lhee et al., 2010; Zhou et al., 2012). If these in vitro conditions are relevant to changes the pH of the growth medium, an acidic pH would cause lower Fbc activity leading to a higher quinol : quinone ratio and, consequently, greater ActSR activity and higher levels of cyo expression.

Previous work has shown cyo to be upregulated in response to low iron conditions in S. meliloti and P. aeruginosa (Chao et al., 2005; Kawakami et al., 2010). This study indicates cyo is not only upregulated in R. etli, but also necessary for efficient growth under low iron conditions as the cyo mutant had a severe growth defect. The results indicate ActSR is necessary for cyo induction under low iron similar to the other conditions tested. To our knowledge, ActSR has yet to be linked to low iron conditions. Its requirement may be that it is necessary for any high level of expression of cyo. Common iron regulatory elements (RirA, Irr, Fur) were not identified in the 5′ promoter region of cyo (Rudolph et al., 2006). One rationale for regulation by ActSR starts with the observation that cytochrome c 1 levels are vastly decreased in B. japonicum under low iron conditions (Gao & O'Brian, 2005). Theoretically, this would cause a build-up of quinol as proposed for the fbc mutant described above and consequently cause an increase of ActSR activity leading to higher cyo levels.

In summary, this study indicates that the ActSR two-component system is necessary for the transcriptional activation of cyo under varying physiological conditions. Knowing the involvement of this regulator led to the discovery of other physiological roles for the Cyo quinol oxidase, as the cyo mutant also had severe growth defects under low pH and iron conditions. Given that cyo was also significantly upregulated in these conditions, the implication is that Cyo is the preferred oxidase at low pH and low iron in R. etli CFN42.

Acknowledgements

Funds from the National Institutes of Health (1 R15 GM087699-01A1) helped support this research.

Supplementary Data

Supplementary Data

Click here for additional data file. (927.2KB, pdf)

Abbreviations:

RT-qPCR

reverse transcription quantitative PCR

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Supplementary Data

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