Requirements for Efficient Thiosulfate Oxidation in Bradyrhizobium

G C A T T A C G G C A T genes

Article Requirements for Efﬁcient Thiosulfate Oxidation in Bradyrhizobium diazoefﬁciens

Sachiko Masuda 1,*, Hauke Hennecke 2 and Hans-Martin Fischer 2

1 RIKEN Center for Sustainable Resource Science, Yokohama 230-0045, Japan 2 ETH Zurich, Institute of Microbiology, Vladimir-Prelog-Weg 4, CH-8093 Zurich, Switzerland [email protected] (H.H.); ﬁ[email protected] (H.-M.F.) * Correspondence: [email protected]; Tel.: +81-45-503-9574

Received: 8 November 2017; Accepted: 12 December 2017; Published: 15 December 2017

Abstract: One of the many disparate lifestyles of Bradyrhizobium diazoefficiens is chemolithotrophic growth with thiosulfate as an electron donor for respiration. The employed carbon source may be CO2 (autotrophy) or an organic compound such as succinate (mixotrophy). Here, we discovered three new facets of this capacity: (i) When thiosulfate and succinate were consumed concomitantly in conditions of mixotrophy, even a high molar excess of succinate did not exert efficient catabolite repression over the use of thiosulfate. (ii) Using appropriate cytochrome mutants, we found that electrons derived from thiosulfate during chemolithoautotrophic growth are preferentially channeled via cytochrome c550 to the aa3-type heme-copper cytochrome oxidase. (iii) Three genetic regulators were identified to act at least partially in the expression control of genes for chemolithoautotrophic thiosulfate oxidation: RegR and CbbR as activators, and SoxR as a repressor.

Keywords: Bradyrhizobium diazoefﬁciens; thiosulfate; chemolithoautotroph; regulation; cytochrome

1. Introduction The α-proteobacterium Bradyrhizobium diazoefficiens, a facultative nitrogen fixer and root-nodule symbiont of soybean, is not only a versatile heterotroph but may also entertain a chemolithotrophic lifestyle in the non-symbiotic state. Hydrogen [1], carbon monoxide [2], and thiosulfate [3] have been reported to be the inorganic substrates that serve as electron donors for energy metabolism. In a previous study, Masuda et al. [3] demonstrated chemolithotrophic growth of B. diazoefficiens with 2− thiosulfate as a reductant for oxic respiration according to the following equation: SSO3 + H2O + 2 2− + O2 → 2 SO4 + 2 H . The electrons derived from the oxidation of thiosulfate may also be used for the reduction of carbon dioxide as the sole carbon source (chemolithoautotrophy; [4]). CO2 can be replaced by an organic carbon source such as succinate, a growth phenotype that has been termed mixotrophy [3]. An inspection of the B. diazoefficiens genome sequence [3,5] has revealed the existence of sulfur oxidation genes that typically occur in α-proteobacteria (soxTRSVWXYZABCD; Figure1A) and code for proteins of the so-called Sox complex [6–8]. Therein, the SoxYZ proteins play a key role by binding thiosulfate covalently to a cysteine thiolate and forming a disulfide bridge in a reaction that − 2− − − releases the first couple of electrons: [SoxYZ]-S + SSO3 → [SoxYZ]-S-S-SO3 + 2 e [8]. Curiously, the B. diazoefficiens genome harbors two soxY homologs (soxY1, soxY2; 3) of which, only soxY1 of sox locus I (Figure1A) is essential for thiosulfate oxidation [3]. Following up on a previous publication [3], the present study aimed at addressing three unsolved aspects related to the physiology, biochemistry, and genetic regulation of thiosulfate oxidation. First, we wished to shed more light on the physiology of mixotrophic growth. To which extent are succinate and thiosulfate consumed concomitantly? Is succinate solely used as a carbon source or does it

Genes 2017, 8, 390; doi:10.3390/genes8120390 www.mdpi.com/journal/genes Genes 2017, 8, 390 2 of 12 also replace thiosulfate as an energy source? Second, we wished to better understand the path electrons take from thiosulfate to oxygen as the terminal electron acceptor. The most prominent oxygen reductase during aerobic growth of B. diazoefficiens is the aa3-type cytochrome oxidase encoded by the coxBAFC genes [9]. Therefore, using appropriate B. diazoefficiens cytochrome mutants, we examined the possible involvement of a soluble c-type cytochrome and of aa3-type cytochrome oxidase in thiosulfate-dependent respiration. Third, we wished to investigate the genetic control of sox gene expression in B. diazoefficiens. Several indications as to which regulatory proteins might be involved came from studies with other bacteria. For example, negative control of sox gene expression had been discovered in Paracoccus pantotrophus GB17 and Pseudaminobacter salicylatoxidans KCT001 in which SoxR was shown to act as a repressor of sox genes in the absence of thiosulfate [10,11]. How sox gene induction occurs in the presence of thiosulfate is not known. The sox locus I of B. diazoefficiens (Figure1A) harbors a soxR-homologous gene, which prompted us to explore its possible role in the regulation of thiosulfate oxidation. Another candidate regulator was the CbbR protein. Being an activator of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes cbbLS and of genes for Calvin-Benson-Bassham-cycle enzymes in many autotrophic bacteria [12–14], CbbR might be a regulator also for chemolithoautotrophic growth of B. diazoefficiens with thiosulfate and CO2. A cbbR-like gene is present in the cbb cluster of the B. diazoefficiens genome ([4,5] Figure1B). Finally, we were interested to know whether the two-component regulatory system RegSR [15,16] is involved in thiosulfate-dependent growth. The B. diazoefficiens RegS/RegR sensor/regulator system is a member of the highly conserved RegB/RegA family which is present in a wide range of bacteria and plays a fundamental role in the transcription of redox-regulated genes for numerous energy-generating and energy-utilizing processes such as photosynthesis, CO2 fixation, nitrogen fixation, hydrogen utilization, aerobic and anaerobic respiration, denitrification, and electron transport [17]. Therefore, thiosulfate oxidation might also be a target for control by RegSR in B. diazoefficiens.

2. Materials and Methods

2.1. Bacterial Strains, Media, and Growth Conditions Bacterial strains and plasmids used in this study are listed in Table1. Strain 110 spc4 was used as the B. diazoefficiens wild type [18]. It is a spectinomycin-resistant derivative of USDA110 that, until recently, carried the species name B. japonicum [19]. B. diazoefficiens strains were pre-cultivated in peptone salts-yeast extract medium [18] supplemented with 0.1% L-arabinose. Precultures were washed twice with minimal medium lacking thiosulfate and succinate as described previously [3]. Then, fresh cultures were inoculated, and the strains were cultivated aerobically at 28 ◦C in minimal medium with sodium thiosulfate for chemolithoautotrophic growth, with sodium thiosulfate plus sodium succinate for mixotrophic growth, or with sodium succinate for heterotrophic growth, as described previously [3]. The concentrations of these ingredients will be mentioned in the context of the respective growth experiments. When appropriate, media for B. diazoefficiens cultivation contained antibiotics at the following concentrations (µg mL−1): spectinomycin, 100; streptomycin, 50; gentamycin, 100; kanamycin, 100; and tetracycline, 50 (solid medium) and 25 (liquid medium). Escherichia coli was grown in Luria-Bertani medium [20] containing the following concentrations of antibiotics for plasmid selection (µg mL−1): ampicillin, 200; kanamycin, 30; and tetracycline, 10. Genes 2017, 8, 390 3 of 12

Table 1. Bacterial strains and plasmids used in this study.

Strain or Plasmid Relevant Genotype or Phenotype Reference or Source Strains Bradyrhizobium diazoefﬁciens 110spc4 Spr wild type [18] 6806 Spr Kmr soxR::aphII (same orientation) This study 6807 Spr Kmr soxR::aphII (opposite orientation) This study 2494 Spr Kmr cbbR::aphII This study 2426 Spr Smr regR::Ω [15] cox132 Spr Kmr coxA::Tn5 [21] 3447 Spr Gmr cycA::gen [22] C3505 Spr Smr cycB::Ω [23] C3524 Spr Smr Kmr cycB::Ω cycC::aphII [22] 3448 Spr Smr Kmr Gmr cycA::gen cycB::Ω cycC::aphII [22] Escherichia coli − − − BL21 (DE3) E. coli BF dcm ompT hsdSB (rB mB ) gal λ(DE3) [24] supE44 ∆lacU169 (Φ80lacZ∆M15) hsdR17 recA1 DH5α BRL a gyrA96 thi-1 relA1 Smr Spr hsdR (RP4-2 kan::Tn7 tet::Mu; integrated in S17-1 [25] the chromosome) Plasmids pBSL14 Apr Kmr [26] pBSL86 Apr Kmr [26] pSUP202pol4 Tcr (pSUP202) oriT of RP4 [27] pUC18 Apr cloning vector [28] pBluescript II SK+ Apr cloning vector Stratagene b Apr (pBluescript II SK+) genomic 3,182-bp pRJ2492 This study NotI-BamHI fragment spanning cbbR-cbbF’ Tcr (pSUP202pol4) cbbR-cbbF’, 2,353-bp EcoRI pRJ2493 This study fragment of pRJ2492 Tcr Kmr (pSUP202pol4) ∆cbbR::aphII (same pRJ2494 orientation), 1,211-bp Acc65I-SalI fragment of This study pBSL14 inserted into Acc65I-XhoI-digested pRJ2493 Apr (pUC18) overlapped-extension PCR-generated pRJ6802 in frame deletion of soxR on 1.6-kb EcoRI-HindIII This study fragment cloned into pUC18 Apr Kmr (pRJ6802) ∆soxR::aphII, 1,260-bp KpnI pRJ6803 fragment from pBSL86 inserted into KpnI site of This study EcoRI-HindIII fragment (same orientation as soxR) Apr Kmr (pRJ6802) ∆soxR::aphII, 1,260-bp KpnI fragment from pBSL86 inserted into KpnI site of pRJ6804 This study EcoRI-HindIII fragment (opposite orientation to soxR) Kmr Tcr (pSUP202pol4) ∆soxR::aphII, 2,894-bp pRJ6806 EcoRI/HindIII fragment from pRJ6803 inserted into This study SmaI site (aphII and soxR in same orientation) Kmr Tcr (pSUP202pol4) ∆soxR::aphII, 2,894-bp pRJ6807 EcoRI/HindIII fragment from pRJ6804 inserted into This study SmaI site (aphII and soxR in opposite orientation) Spr, spectinomycin resistant; Kmr, kanamycin resistant; Smr, streptomycin resistant; Gmr, gentamicin resistant; Apr, ampicillin resistant; Tcr, tetracycline resistant. a Gaithersburg MD, USA; b La Jolla CA, USA. Genes 2017, 8, 390 4 of 12

2.2. Construction of soxR and cbbR Deletion Mutants Plasmids for mutant construction and the resulting mutants are listed in Table1. The B. diazoefficiens mutantsGenes 2017, were 8, 390 generated by marker replacement mutagenesis [29]. For deletion of soxR, upstream4 of and 12 downstream flanking regions of the soxR gene were amplified and cloned into pUC18, yielding pRJ6802. TheinsertedaphII ingene either encoding of two kanamycinorientations resistance at a KpnIwas site cutin-between out from the pBSL86 up- and and downstream inserted in eitherregions, of tworesulting orientations in pRJ6803 at a and KpnI pRJ6804. site in-between The mutated the up-soxR and DNA downstream constructs regions,were excised resulting and cloned in pRJ6803 into andthe suicide pRJ6804. plasmid The mutated pSUP202pol4,soxR DNA yielding constructs pRJ6806 were and excisedpRJ6807. and Mobilization cloned into of the these suicide plasmids plasmid into pSUP202pol4,B. diazoefficiens yielding110spc4 pRJ6806was carried and pRJ6807.out via E. Mobilization coli S17-1 and of thesefollowed plasmids by screening into B. diazoefficiens for double 110recombinationspc4 was carried events, out resulting via E. in coli mutantS17-1 strains and followed 6806 (∆soxR by screening, same orientation for double of the recombination aphII gene) events,and 6807 resulting (∆soxR in, opposite mutant strainsorientation). 6806 ( ∆ThesoxR precise, same orientationdeletion ends of the withaphII referencegene) and to 6807the genome (∆soxR, oppositepositions orientation).[5] are depicted The precisein Figure deletion 1A. A ends simila withr mutagenesis reference to the strategy genome was positions applied [5 ]to are generate depicted a inpartial Figure cbbR1A. deletion A similar mutant. mutagenesis To this end, strategy a genomic was applied 3,182-bp to generateNotI-BamHI a partial DNAcbbR fragmentdeletion comprising mutant. TocbbR this and end, cbbF’ a genomic was cloned 3,182-bp in plasmid NotI-BamHI pRJ2492 DNA from fragment which it comprising was excisedcbbR as andan EcoRIcbbF’ wasfragment cloned and in plasmidinserted pRJ2492in vector from pSUP202pol4 which it was to yield excised pRJ2493. as an EcoRI A 290-bp fragment cbbR-internal and inserted Acc65I-XhoI in vector fragment pSUP202pol4 was toreplaced yield pRJ2493.by the kanamycin A 290-bp resistancecbbR-internal cassette Acc65I-XhoI present on fragment a 1,211-bp was Acc65I-SalI replaced byfragment the kanamycin isolated resistancefrom pBSL14. cassette The resulting present on plasmid a 1,211-bp pRJ2494 Acc65I-SalI was used fragment for marker isolated replacement from pBSL14. mutagenesis The resulting to yield plasmidthe cbbR pRJ2494mutant strain was used 2494. for The marker precise replacement deletion ends mutagenesis with reference to yield to the the genomecbbR mutant positions strain [5] 2494. are Theshown precise in Figure deletion 1B. ends with reference to the genome positions [5] are shown in Figure1B.

Figure 1. Genetic mapsmaps ofofthe thesox soxand andcbb cbbgene gene clusters clusters in inB. B. diazoefficiens diazoefficiens.(A. )(A Genes) Genes of soxof soxlocus locus I in I the in wildthe wild type, type, and and the genotypethe genotype of soxR of soxRdeletion deletion mutants mutants 6806 (6806∆soxR (∆,soxR same, same orientation orientation of the ofaphII the gene)aphII andgene) 6807 and (∆ 6807soxR ,(∆aphIIsoxRin, aphII opposite in opposite orientation). orientation). The ∆soxR Thedeletion ∆soxR endsdeletion are definedends are by defined the nucleotide by the positionsnucleotide in positions the B. diazoefficiens in the B. diazoefficiensgenome. (B) genome. Genes of ( theB) Genescbb cluster of the in cbb the cluster wild type, in the and wild the type, genotype and ofthecbbR genotypemutant of 2494.cbbR mutant The ∆cbbR 2494.deletion The ∆cbbR ends deletion are defined ends by are the defined nucleotide by the positions nucleotide in the positions genome. in the genome. 2.3. Expression of sox and cbb Genes 2.3. Expression of sox and cbb Genes B. diazoefficiens strains were grown chemolithoautotrophically, mixotrophically, or heterotrophically in 100B. mLdiazoefficiens of minimal mediumstrains (seewere above) grown in 500-mL chemolithoautotr Erlenmeyerophically, flasks. Total mixotrophically, RNA was extracted or fromheterotrophically chemolithoautotrophic in 100 mL of cultures minimal of medium the wild (see type above) andof in mutants500-mL Erlenmeyer 6806 and 6807 flasks. at 6 Total days RNA after inoculation,was extracted and from of chemolithoautotrophic mutants 2494 and 2429 cultures at 18 days of the after wild inoculation. type and of Total mutants RNA 6806 was and extracted 6807 at from6 days mixotrophically after inoculation, and and heterotrophically of mutants 2494 grown and 2429 cultures at 18 afterdays 30 after h of inoculation. growth. Cell Total harvest, RNA RNA was extraction,extracted from and cDNAmixotrophically synthesis were and doneheterotrophica as describedlly previouslygrown cultures [16]. The after expression 30 h of ofgrowth.sox and Cellcbb harvest, RNA extraction, and cDNA synthesis were done as described previously [16]. The expression of sox and cbb genes was analyzed by quantitative reverse transcription-PCR (qRT-PCR) with techniques described previously [16], using suitable primers based on the respective sox and cbb gene sequences. The precise primer sequences are available from authors on request. For normalization we used the sigA gene which, based on our previous microarray data, was found to be unchanged under many different growth conditions (for example, see [16]).

Genes 2017, 8, 390 5 of 12 genes was analyzed by quantitative reverse transcription-PCR (qRT-PCR) with techniques described previously [16], using suitable primers based on the respective sox and cbb gene sequences. The precise primer sequences are available from authors on request. For normalization we used the sigA gene which, based on our previous microarray data, was found to be unchanged under many different growth conditions (for example, see [16]).

2.4. Analysis of Thiosulfate, Succinate and Fumarate Thiosulfate concentration was measured spectrophotometrically as previously described [3]. Extracellular succinate and fumarate concentrations were measured with a high-performance liquid chromatograph (Waters Alliance model 2690, Milford MA, USA) at 210 nm [30]. Two ml of cell cultures were harvested, ﬁltered, and samples for HPLC analysis were taken from the supernatant. Sodium tartrate (10 mM) was added to the cell-free supernatant as an internal standard [30].

3. Results and Discussion

3.1. Concurrent Consumption of Thiosulfate and Succinate during Mixotrophic Growth The performance of B. diazoefficiens growth in mixotrophic condition was explored. While the initial concentration of thiosulfate (4 mM) was kept constant, the initial concentration of succinate in the medium was varied between 2 mM and 20 mM (Figure2; Table S1). Succinate at 2, 4, and 8 mM in the medium appeared to have a beneficial effect on growth because growth yields increased, and succinate was consumed completely without substantial accumulation of its immediate oxidation product, fumarate, in the medium. Surprisingly, thiosulfate was also consumed entirely in these cultures. Obviously, thiosulfate and succinate were consumed concomitantly if mixotrophic cultures contained 8 mM succinate or less. Even in the presence of higher succinate concentrations (12, 16, 20 mM) almost 50% of the available thiosulfate was still consumed while the remainder was detectable in the medium (Figure2). Unphysiologically high succinate concentrations (16 and 20 mM) slightly impaired the final cell density, and neither succinate nor its oxidation product, fumarate was completely degraded by, or incorporated into, cells, which was indicated by the massive accumulation of unused fumarate in the medium. The most remarkable aspect of these results is that succinate exerts no or only partial catabolite repression over the use of thiosulfate. We interpret this to mean that succinate did either not or only partly replace thiosulfate as a source of reducing equivalents and hence, energy, but rather served predominantly as a carbon source. An alternative interpretation for the apparently obligate consumption of up to 50% thiosulfate in the presence of saturating amounts of succinate could be that thiosulfate was used as a sulfur source. This possibility appeared unlikely to us because the growth medium always contained a non-limiting amount of Mg-sulfate (3.25 mM) as the standard source of sulfur. Genes 2017, 8, 390 6 of 12 Genes 2017, 8, 390 6 of 12

Figure 2.2. Mixotrophic growth of the B. diazoefﬁciensdiazoefficiens wildwild type.type. The growth media contained 4 mM thiosulfate and and the the amounts amounts of of succin succinateate indicated indicated above above each each pair pair of panels of panels (from (from 2 to 220 to mM). 20 mM). Cell growth was measured as optical density (OD) at A600, which is shown in the upper panels together Cell growth was measured as optical density (OD) at A600, which is shown in the upper panels together with thiosulfatethiosulfate consumption. consumption. Succinate Succinate consumption consumption and and fumarate fumarate production production are shown are shown in the in lower the oflower panels. of panels. Two independent Two independent experiments experiments were performed, were performed, and one and of themone of is them shown. is shown.

3.2. Chemolithoautotrophic Growth Partly Depends on Cytochrome c550 and the aa3-Type Cytochrome Oxidase 3.2. Chemolithoautotrophic Growth Partly Depends on Cytochrome c550 and the aa3-Type Cytochrome Oxidase Being a a strictly strictly oxic oxic process, process, thiosulfate thiosulfate oxidatio oxidationn was was expected expected to deliver to deliver electrons electrons to the to most the mostprominent prominent terminal terminal oxygen oxygen reductase reductase present present in aerobically in aerobically grown grownB. diazoefficiensB. diazoefficiens cells, i.e.,cells, the aa i.e.,3- type cytochrome oxidase [9]. To test this inference, a cytochrome aa3 subunit-I mutant (coxA::Tn5, the aa3-type cytochrome oxidase [9]. To test this inference, a cytochrome aa3 subunit-I mutant (straincoxA::Tn cox132)5, strain completely cox132) completely defective defectivein the inactivity the activity of this of thisoxidase oxidase was was examined examined for chemolithoautotrophic growth with thiosulfate and CO2 and compared with the wild type. While all chemolithoautotrophic growth with thiosulfate and CO2 and compared with the wild type. While all strains grewgrew substantially substantially slower slower under under chemolithoautotrophic chemolithoautotrophic conditions conditions compared withcompared mixotrophic with conditions,mixotrophic growth conditions, of the growthcoxA mutant of the was coxA particularly mutant was impaired, particularly and thiosulfate impaired, consumption and thiosulfate was stronglyconsumption delayed was instrongly comparison delayed with in the comparison wild type wi (Figureth the3 ;wild Table type S1). (Figure This suggested 3; Table S1). that This the suggested that the aa3-type cytochrome oxidase plays an important, though not essential role in aa3-type cytochrome oxidase plays an important, though not essential role in thiosulfate oxidation. Sincethiosulfate oxidases oxidation. of this Since type oxidases receive electronsof this type directly receive from electrons reduced directly cytochrome from reducedc, we cytochrome also tested whichc, we also of the tested soluble whichc-type of the cytochromes soluble c-type previously cytochromes identified previously in B.diazoefficiens identified in[ B.21 –diazoefficiens23] might fulfill [21– 23] might fulfill such a task. Cytochromes c552 and c555, encoded by cycB and cycC, respectively, could such a task. Cytochromes c552 and c555, encoded by cycB and cycC, respectively, could be excluded be excluded because a cycB deletion mutant (strain C3505) and a cycB+C double deletion mutant (strain C3524) were proficient in chemolithoautotrophic growth and thiosulfate consumption just like the wild type (Figure 3). By contrast, a cytochrome c550 deletion mutant (ΔcycA, strain 3447) and a

Genes 2017, 8, 390 7 of 12 because a cycB deletion mutant (strain C3505) and a cycB+C double deletion mutant (strain C3524) wereGenes proficient2017, 8, 390 in chemolithoautotrophic growth and thiosulfate consumption just like the wild7 type of 12 (Figure3). By contrast, a cytochrome c550 deletion mutant (∆cycA, strain 3447) and a cycA+B+C triplecycA+ mutantB+C triple (strain mutant 3448) (strain displayed 3448) impaired displayed chemolithoautotrophic impaired chemolithoautotrophic growth and growth delayed and thiosulfate delayed consumptionthiosulfate consumption in a similar in way a similar as observed way as with observed the coxA withmutant the coxA (Figure mutant3). It(Figure appears 3). probable,It appears therefore,probable, thattherefore, electrons that derived electrons from thiosulfatederived from oxidation thiosulfate are transferred oxidation through are transferred cytochrome throughc550 to thecytochromeaa3-type cytochromec550 to the aa oxidase3-type cytochrome for the reduction oxidase of for O2 the. Notably, reduction however, of O2. cycANotably,and however,coxA mutants cycA retainedand coxA partial mutants chemolithoautotrophic retained partial chemolithoautotrophic activity with thiosulfate, activity which with suggests thiosulfate, that alternative which suggests routes ofthat electron alternative transfer routes might of existelectron in these transf mutants.er might Notably, exist inB. these diazoefficiens mutants.possesses Notably, twoB. diazoefficiens additional cytochromepossesses two oxidases additional and four cytochrome additional ubiquinoloxidases and oxidases four that,additional collectively, ubiquinol might beoxidases responsible that, forcollectively, the residual might chemolithotrophic be responsible activityfor the residual [9,31]. Whether chemolithotrophic one of these activity six oxidases [9,31]. plays Whether an equally one of prominentthese six oxidases role as theplaysaa3 an-type equally oxidase prominent in thiosulfate role as oxidation the aa3-type is difficult oxidase toin predict,thiosulfate as weoxidation have no is knowledgedifficult to aboutpredict, the biochemicalas we have componentsno knowledge (quinols, about cytochromes) the biochemical to which components the enzymatic (quinols, Sox complexcytochromes) of B. diazoefficiensto which thedelivers enzymatic its electronsSox complex first. of B. diazoefficiens delivers its electrons first.

FigureFigure 3.3. ContributionContribution ofof cytochromescytochromes toto chemolithoautotrophicchemolithoautotrophic growthgrowth ofof B.B. diazoefﬁciensdiazoefficiens withwith

thiosulfatethiosulfate andand CO CO22.. The The analyzed analyzed strains strains were were the wild the wild type typeand the and cytochrome the cytochrome mutants mutants cox132 cox132(lacking (lacking subunit subunitI of aa3-type I of cytochromeaa3-type cytochrome oxidase), 3447 oxidase), (lacking 3447 cytochrome (lacking c550 cytochrome), C3505 (lackingc550), C3505cytochrome (lacking c552 cytochrome), C3524 (lackingc552), C3524 cytochromes (lacking c cytochromes552 and c555), cand552 and 3448c555 (lacking), and 3448all three (lacking c-type all threecytochromes).c-type cytochromes). The starting The startingmedia contained media contained 4 mM 4 mMthiosulfate. thiosulfate. The The panels panels show show thiosulfatethiosulfate consumptionconsumption andand growthgrowth measuredmeasured asas colony-formingcolony-forming unitsunits (CFU).(CFU). CFUCFU countscounts werewere assessedassessed byby takingtaking samplessamples fromfrom thethe culturescultures andand plating plating out out dilutions dilutions on on agar agar plates plates with with peptone-salts-yeast peptone-salts-yeast extractextract mediummedium [18[18].]. Shown are the meanmean valuesvalues andand standardstandard deviationdeviation derivedderived fromfrom threethree independentindependent cultures. cultures.

3.3.3.3. InvolvementInvolvement ofof CbbRCbbR andand RegRRegR inin thethe RegulationRegulationof ofChemolithotrophic Chemolithotrophic Thiosulfate Thiosulfate Oxidation Oxidation ThreeThree regulatoryregulatory proteinsproteins (SoxR,(SoxR, CbbR,CbbR, RegR)RegR) werewere thoughtthought toto bebe candidatescandidates forfor possiblypossibly controllingcontrolling chemolithotrophicchemolithotrophic thiosulfate oxidation (s (seeee Introduction). Introduction). To To test test their their involvement involvement in inthis this phenotype, phenotype, we we constructed constructed soxRsoxR andand cbbRcbbR deletion mutants andand analyzedanalyzed chemolithotrophicchemolithotrophic thiosulfate oxidation in comparison with the wild type. The ΔsoxR mutants were strains 6806 and 6807, and the ΔcbbR mutant was strain 2494 (Figure 1; Table 1). We included in this study a regR knock-out mutant (strain 2426) that was available from earlier work [15]. The two ∆soxR strains showed a similar growth behavior as the wild type (shown in Figure 4 for strain 6806). Given the fact that SoxR is a repressor in other thiosulfate-oxidizing bacteria [10,11], the lack of a mutant phenotype

Genes 2017, 8, 390 8 of 12 thiosulfate oxidation in comparison with the wild type. The ∆soxR mutants were strains 6806 and 6807, and the ∆cbbR mutant was strain 2494 (Figure1; Table1). We included in this study a regR knock-out mutant (strain 2426) that was available from earlier work [15]. The two ∆soxR strains showed a Genessimilar 2017 growth, 8, 390 behavior as the wild type (shown in Figure4 for strain 6806). Given the fact8 that of 12 SoxR is a repressor in other thiosulfate-oxidizing bacteria [10,11], the lack of a mutant phenotype in B.in diazoefficiensB. diazoefficiens∆soxR ΔsoxRstrains strains did did not not come come as a as surprise. a surprise. This This made made further further analyses analyses necessary necessary in order in orderto support to support a negative a negative regulatory regula functiontory function of SoxR of alsoSoxR in alsoB. diazoefficiens in B. diazoefficiens(see below). (see below). CbbR CbbR and RegR and RegRare positive are positive regulators regulators [12–16]. [12 Both−16]. proteins Both proteins appeared appeared to be involved to be involved in efficient in thiosulfate-dependent efficient thiosulfate- dependentchemolithoautotrophy chemolithoautotrophy of B. diazoefficiens of B. diazoefficiens, indicated, by indicated the increased by the generationincreased generation time and decreased time and decreasedfinal cell density, final cell and density, a diminished and a diminished thiosulfate thiosulfate oxidation rateoxidation of the ratecbbR ofand the regRcbbR mutantsand regR (Figure mutants4; Table(Figure S1). 4; Table Since S1). the Since mutations the mutations affected affected these properties these properties only partly, only partly we conclude, we conclude that CbbRthat CbbR and andRegR RegR are important, are important, but not but essential not essential activators activa involvedtors involved in chemolithotrophic in chemolithotrophic thiosulfate thiosulfate oxidation oxidationin B. diazoefficiens in B. diazoefficiens, which suggests, which thesuggests existence the existence of other, soof other, far unknown so far unknown regulators regulators in this process. in this Whileprocess. the While importance the importance of CbbR of might CbbR be might explained be ex byplained its likely by its role likely as an role activator as an activator of CO2 fixation of CO2 fixationgenes, the genes, role the of RegR role of remains RegR remains unknown. unknown. Gene expression Gene expression studies studies were therefore were therefore designed designed to help toclarify help theseclarify issues. these issues.

Figure 4. Contribution of transcription transcription regulato regulatorsrs to chemolithoautotrophic chemolithoautotrophic growth of B. diazoefficiens diazoefﬁciens with thiosulfate and CO2. The analyzed strains were the wild type and the regulatory mutants 6806 with thiosulfate and CO2. The analyzed strains were the wild type and the regulatory mutants 6806 (lacking the SoxR repressor), 2494 (lacking the CbbR activator), and 2426 (lacking the RegR response regulator of of the the two-component two-component RegSR RegSR system). system). The The starting starting media media contained contained 4 mM 4 mMthiosulfate. thiosulfate. The panelsThe panels show show thiosulfate thiosulfate consumption consumption and growth and growth measured measured as colony-forming as colony-forming units (CFU). units Shown (CFU). areShown the mean are the values mean and values standard and standard deviatio deviationn derived derived from three from independent three independent cultures. cultures.

3.4. Expression of Cbb and Sox Genes during Chemolithoautotrophic Growth To support the physiologic observations with genetic data, the expression of the Calvin-cycle genes cbbL and cbbF and the thiosulfate oxidation gene soxY1 was examined. Using qRT-PCR, gene genes cbbL and cbbF and the thiosulfate oxidation gene soxY1 was examined. Using qRT-PCR, expressiongene expression in cells in cellsgrown grown chemolithoautotrophically chemolithoautotrophically was was compared compared with with that that in in heterotrophically heterotrophically grown cells.cells. TheThe strains strains used used for for this this experiment experiment were were the wildthe wild type type and theand regulatory the regulatory mutants mutants∆soxR, Δ∆soxRcbbR, andΔcbbR∆regR and. Δ TheregR results. The results are displayed are displayed in Figure in Figure5. A significant 5. A significant decrease decrease in expression in expression was wasobserved observed only only for the forcbbF the andcbbFccbL andgenes ccbL genes in chemolithoautotrophically in chemolithoautotrophically grown grown cells of cells the cbbR of themutant cbbR (strainmutant 2494).(strain This 2494). confirms This confir the rolems ofthe CbbR role asof anCbbR activator as an foractivator chemolithoautotrophy, for chemolithoautotrophy, especially especially CO2 fixation. By contrast, the growth defect caused by the regR mutation in strain 2426 (see CO2 fixation. By contrast, the growth defect caused by the regR mutation in strain 2426 (see Figure4) Figure 4) was not reflected in a decreased expression of the investigated soxY1 and cbbL and cbbF was not reflected in a decreased expression of the investigated soxY1 and cbbL and cbbF genes (Figuregenes (Figure5). The chemolithoautotrophic5). The chemolithoautotrophic growth defect growth of adefectregR mutantof a regR must mutant therefore must have therefore other, sohave far other,unknown so far causes. unknown The only causes. effect The seen only in theeffectsoxR seenmutant in the (strain soxR 6806)mutant was (strain an apparent 6806) was de-repression an apparent of de-repression of soxY1 expression in cells grown heterotrophically (Figure 5, and leftmost portion of soxY1 expression in cells grown heterotrophically (Figure5, and leftmost portion of Figure6). This was Figurethe first 6). indication This was in theB. diazoefficiensfirst indicationthat in SoxR B. diazoefficiens might be a negativethat SoxR regulator might be which, a negative when regulator mutated, which,leads to when an enhanced mutated, expression leads to an of enhanced genes that expres are normallysion of genes repressed that inare the normally wild type repressed in the absence in the wildof thiosulfate. type in the absence of thiosulfate.

Genes 2017, 8, 390 9 of 12 Genes 2017, 8, 390 9 of 12

FigureFigure 5. ExpressionExpression of of the the soxYsoxY1,1 ,cbbFcbbF, ,and and cbbLcbbL genesgenes in in the the B.B. diazoefficiens diazoefﬁciens wildwild type type (WT) (WT) and and in inregulatory regulatory mutants mutants ∆soxR∆soxR (6806),(6806), ∆cbbR∆cbbR (2494)(2494) and and∆regR∆regR (2426).(2426). Expression Expression was measured was measured by qRT- by qRT-PCRPCR in cells in cellsgrown grown heterotrophically heterotrophically (with (with4 mM 4succinate) mM succinate) and chemolitho and chemolithoautotrophicallyautotrophically (with 4 (withmM thiosulfate). 4 mM thiosulfate). The expression The expression of each of gene each geneunde underr each eachcondition condition had hadbeen been normalized normalized to that to that of ofthe the housekeeping housekeeping sigma sigma factor factor gene gene sigAsigA. The. The column column heights heights and and error error bars bars represent represent the means the means and andstandard standard deviations deviations for three for three cultures cultures wher wheree each each culture culture was was measured measured in triplicate. in triplicate.

3.5.3.5. Expression of the Sox GenesGenes andand EvidenceEvidence forfor SoxRSoxR asas aa NegativeNegative RegulatorRegulator

TheThe expression studies studies in in the the soxRsoxR mutantmutant background background were were expanded expanded to include to include not only not soxY only1 soxYbut also1 but additional also additional genes genesof the of sox the genesox genecluster. cluster. Based Based on studies on studies with withP. pantotrophusP. pantotrophus GB17GB17 and andP.salicylatoxidansP. salicylatoxidans KCT001KCT001 [10,11], [10 the,11], soxSR the soxSR and soxVWand soxVW genes genesare divergently are divergently transcribed transcribed from SoxR- from SoxR-regulatedregulated promoters promoters located located between between soxS andsoxS soxVand. GenesoxV expression. Gene expression was monitored was monitored with qRT-PCR with qRT-PCRunder mixotrophic under mixotrophic and heterotrophic and heterotrophic growth conditions growth conditionsin the wild type in the and wild in the type soxR and mutant in the soxR6806 mutant(Table 2, 6806 Figure (Table 6).2 The, Figure soxSR,6). ThesoxV,soxSR soxW, andsoxV soxY, soxW1 genesand soxYwere1 expressedgenes were most expressed strongly most in stronglythe wild intype the grown wild type under grown mixotrophic under mixotrophic condition where condition thiosu wherelfate was thiosulfate present. was In heterotrophic present. In heterotrophic conditions, conditions,i.e., in the absence i.e., in of the thiosulfate, absence of the thiosulfate, soxY1 gene the is poorlysoxY1 geneexpressed is poorly in the expressed wild type. in The the other wild genes type. Thealso othershowed genes a decreased also showed expression a decreased in heterotrop expressionhically in heterotrophically grown cells, albeit grown less cells,pronounced albeit less as pronouncedcompared with as comparedsoxY1. In the with soxRsoxY mutant1. In the strainsoxR 6806,mutant however, strain the 6806, soxS however,, soxV, soxW the soxS and, soxYsoxV1, genessoxW andshowedsoxY an1 genes enhanced showed expression an enhanced in heterotrophicall expression iny heterotrophically grown cells as compared grown cells with as respective compared wild- with respectivetype cells. wild-typeThis is especially cells. This true is for especially the soxY1 true gene for (leftmost the soxY portion1 gene (leftmostof Figure portion6). These of results Figure are6). Thesefully in results line with are fully the purported in line with role the of purported SoxR as rolea repressor of SoxR of as sox a repressor genes in ofthesox absencegenesin of the thiosulfate absence of[10,11]. thiosulfate The data [10, 11in]. Table The data 2 gives in Table an esti2 givesmate an of estimate the factors of the of de-repression factors of de-repression of sox genes of sox in thegenes wild in thetype wild when type thiosulfate when thiosulfate is present is presentduring mixo duringtrophic mixotrophic growth. growth. Again, the Again, de-repression the de-repression of soxY of1 standssoxY1 standsout most out prominently. most prominently. Biochemical Biochemical transcription transcription studies studies in vitroin with vitro purifiedwith puriﬁed components components will now will nowbe necessary be necessary to understand to understand the mechanism the mechanism of So ofxR-mediated SoxR-mediated negative negative control control in the in the absence absence of ofthiosulfate. thiosulfate.

TableTable 2.2.Ratio Ratio of of relative relative expression expression of soxof soxgenes genes in B. in diazoefficiens B. diazoefficienswild typewild andtypesoxR andmutant soxR mutant 6806 grown 6806 a undergrown mixotrophic under mixotrophic and heterotrophic and heterotrophic conditions conditions. a.

Genes Genes Wild WildType Type 6806 6806 (∆ soxR(ΔsoxR) ) soxR 16 ± 8 --- soxR 16 ± 8 — soxS 50 ± 17 1.2 ± 0.5 soxS 50 ± 17 1.2 ± 0.5 soxV 50 ± 26 0.7 ± 0.3 soxV 50 ± 26 0.7 ± 0.3 soxW soxW 90 ± 4590 ± 45 0.8 0.8± 0.7± 0.7 soxY1 4500 ± 1500 1.4 ± 0.4 soxY1 4500 ± 1500 1.4 ± 0.4 a a Expression of of each each gene gene under under heterotrophic heterotrophic andmixotrophic and mixotrophic growth growth condition condition was normalized was normalized to that of the to housekeepingthat of the housekeeping sigma factor gene sigmasigA .factor Numbers gene reﬂect sigA the. Numbers relative expression reflect the level relative in cells expression grown under level mixotrophic in cells conditiongrown under divided mixotrophic by the expression condition levels in divided cells grown by under the heterotrophicexpression conditions.levels in Thecells means grown± standard under deviations of three biological replicates are shown. heterotrophic conditions. The means ± standard deviations of three biological replicates are shown.

Genes 2017, 8, 390 10 of 12 Genes 2017, 8, 390 10 of 12

FigureFigure 6. 6. ExpressionExpression of the soxSsoxS,, soxVsoxV,, soxWsoxW,, and andsoxY soxY1 1genes genes in in the theB. B. diazoefﬁciens diazoefficienswild wild type type and and in thein thesoxR soxRmutant mutant 6806. 6806. Expression Expression was was measured measured in cells in cell growns grown under under heterotrophic heterotrophic (4 mM (4 mM succinate) succinate) and andmixotrophic mixotrophic conditions conditions (4 mM (4 mM succinate succinate plus plus 4 mM 4 thiosulfate).mM thiosulfate). The expressionThe expression of each of geneeach undergene undereach condition each condition had been had normalized been normalized to that to of that the housekeepingof the housekeeping sigma factorsigma gene factorsigA gene. The sigA column. The columnheights heights and error and bars error represent bars represent the means the and means standard and deviationsstandard deviations of three cultures of three where cultures each culturewhere eachwas measuredculture was in measured triplicate. in triplicate.

InIn conclusion, conclusion, our our study study contributes contributes to to a a bette betterr understanding understanding of of the the complex complex and and versatile versatile physiologyphysiology of of B.B. diazoefficeins. diazoefficeins. While While studying studying mixotrophic mixotrophic and and ch chemolithoautotrophicemolithoautotrophic growth growth of of B.diazoefficiensB. diazoefficiens wildwild type type and and a aseries series of of defined defined mutants, mutants, we we have have identified identified two two members members of of the the electron transfer pathway from thiosulfate to oxygen, cytochrome CycA and aa3-type cytochrome electron transfer pathway from thiosulfate to oxygen, cytochrome CycA and aa3-type cytochrome oxidaseoxidase CoxBAFC. CoxBAFC. Moreover, Moreover, we we have have demonstrated demonstrated forfor thethe first first time time the involvement the involvement of CbbR of CbbR and RegRand RegR in autotrophic in autotrophic growth growth of B. of diazoefficiensB. diazoefficiens, which, which expands expands the the functional functional relevance relevance of of these these regulatoryregulatory proteinsproteins inin complex complex genetic genetic networks networks present present in this in bacterium. this bacterium. At this stage,At this the stage, molecular the molecularsignals which signals are which sensed are and sensed transduced and transduced by CbbR by and CbbR RegR and remain RegR remain to be identified. to be identified. The same The sameapplies applies to the to previously the previously uncharacterized uncharacterized SoxR protein SoxR protein for which for wewhich demonstrated we demonstrated a role in a negative role in negativeregulation regulation of sox genes of sox in genes heterotrophically in heterotrophically grown cells grown of B. cells diazoefficiens of B. diazoefficiens. . Supplementary Materials: The following are available online at www.mdpi.com/2073-4425/8/12/390/s1. Supplementary Materials: The following are available online at www.mdpi.com/2073-4425/8/12/390/s1. Table S1: Generation time of all strains used in this study grown under the indicated conditions. Table S1: Generation time of all strains used in this study grown under the indicated conditions. Acknowledgments:Acknowledgments: WeWe are are grateful grateful to to Karin Karin Schneider Schneider for for help help with with the the measurement measurement of of succinate succinate and and fumarate fumarate concentrations.concentrations. M.S. acknowledgesacknowledges the the receipt receipt of of a fellowshipa fellowship from from the Japanthe Japan Society Society for the for Promotion the Promotion of Science. of Science. Author Contributions: S.M. performed all experiments. All authors designed the experiment and wrote the Authormain manuscript. Contributions: S.M. performed all experiments. All authors designed the experiment and wrote the mainConflicts manuscript. of Interest: The authors declare no conflict of interest. Conflicts of Interest: The authors declare no conflict of interest. References References 1. Hanus, F.J.; Maier, R.J.; Evans, H.J. Autotrophic growth of H2-uptake-positive strains of Rhizobium japonicum in an atmosphere supplied with hydrogen gas. Proc. Natl. Acad. Sci. USA 1979, 76, 1788–1792. [CrossRef] 1. Hanus, F.J.; Maier, R.J.; Evans, H.J. Autotrophic growth of H2-uptake-positive strains of Rhizobium japonicum[PubMed ]in an atmosphere supplied with hydrogen gas. Proc. Natl. Acad. Sci. USA 1979, 76, 1788–1792. 2.2. Lorite,Lorite, M.J.; M.J.; Tachil, Tachil, J.; J.; Sanjuán, Sanjuán, J.; J.; Meyer, Meyer, O.; O.; Bedmar, Bedmar, E.J. E.J. Carbon Carbon monoxide monoxide dehydrogenase dehydrogenase activity activity in in BradyrhizobiumBradyrhizobium japonicum japonicum.. Appl.Appl. Environ. Environ. Microbiol. Microbiol. 20002000, ,6666, ,1871–1876. 1871–1876. [CrossRef][PubMed] 3.3. Masuda,Masuda, S.; S.; Eda, Eda, S.; S.; Ikeda, Ikeda, S.; S.; Mitsui, H.; Minamisa Minamisawa,wa, K. Thiosulfate-depend Thiosulfate-dependentent chemolithoautotrophic chemolithoautotrophic growthgrowth of of BradyrhizobiumBradyrhizobium japonicum japonicum.. Appl.Appl. Environ. Environ. Microbiol. Microbiol. 20102010, ,7676, ,2402–2409. 2402–2409. [CrossRef][PubMed] 4. Masuda, S.; Eda, S.; Sugawara, C.; Mitsui, H.; Minamisawa, K. The cbbL gene is required for thiosulfate- dependent autotrophic growth of Bradyrhizobium japonicum. Microbes Environ. 2010, 25, 220–223.

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