Sulfite Oxidation in Sinorhizobium Meliloti

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Biochimica et Biophysica Acta 1787 (2009) 1516–1525

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Sulﬁte oxidation in Sinorhizobium meliloti

Jeremy J. Wilson, Ulrike Kappler ⁎

Centre for Metals in Biology, School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Qld 4072, Australia

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Article history: Sulfite-oxidizing enzymes (SOEs) are crucial for the metabolism of many cells and are particularly important Received 26 May 2009 in bacteria oxidizing inorganic or organic sulfur compounds. However, little is known about SOE diversity Received in revised form 16 July 2009 and metabolic roles. Sinorhizobium meliloti contains four candidate genes encoding SOEs of three different Accepted 16 July 2009 types, and in this work we have investigated the role of SOEs in S. meliloti and their possible link to the Available online 24 July 2009 metabolism of the organosulfonate taurine. Low level SOE activity (∼1.4 U/mg) was present under all conditions tested while growth on taurine and thiosulfate induced high activities (5.5–8.8 U/mg) although S. Keywords: fi Sulfite oxidation meliloti cannot metabolize thiosulfate. Protein puri cation showed that although expression of two Metalloenzyme candidate genes matched SOE activity patterns, only a single group 2 SOE, SorT (SMc04049), is responsible Taurine metabolism for this activity. SorT is a heme-free, periplasmic homodimer (78 kDa) that has low homology to other −1 Energy generation bacterial SOEs. SorT has an apparent kcat of 343 s and high affinities for both sulfite (KMapp_pH8 15.5 μM) Gene expression and ferricyanide (KMapp_pH8 3.44 μM), but not cytochrome c, suggesting a need for a high redox potential Enzyme natural electron acceptor. KMapp_sulfite was nearly invariant with pH which is in contrast to all other well characterized SOEs. SorT is part of an operon (SMc04049-04047) also containing a gene for a cytochrome c and an azurin, and these might be the natural electron acceptors for the enzyme. Phylogenetic analysis of SorT-related SOEs and enzymes of taurine degradation indicate that there is no link between the two processes. © 2009 Elsevier B.V. All rights reserved.

1. Introduction unclear is the fate of the sulfonate sulfur, which is initially present as sulfite but is usually recovered as sulfate, indicating the presence of an Bacteria have a major role in both oxidative and reductive reac- as yet incompletely characterized enzymatic oxidation step [4,7,8]. tions of the biological sulfur cycle, and the bacterial sulfur conversions Information on possible links between the occurrence of certain types can be roughly classified into reactions with a role in energy of sulfite-oxidizing enzymes (SOEs) and the presence of organosulfo- metabolism and reactions involved in assimilation of sulfur into cell nate degradation pathways is also scarce. biomass. In soil environments most of the available sulfur occurs as Sulfite can be enzymatically converted to sulfate by molybdenum- organosulfonates or organosulfate esters [1,2]. It is thus not surprising containing enzymes of the sulfite oxidase family [EC1.8.3.1, sulfite that many soil bacteria including plant symbionts such as Sinorhizo- oxidase (SO) and EC1.8.2.1, sulfite dehydrogenase (SDH)] [9,10] and bium meliloti are not only able to utilize these organically bound forms here we have systematically investigated the enzymology of sulfite of sulfur in order to gain access to sulfur for cell biomass, but can use oxidation in S. meliloti grown in the presence of a variety of organic or the carbon skeleton of these compounds as a growth substrate [2–4]. inorganic sulfur compounds. Bacterial sulfite oxidation is generally A particularly abundant organosulfonate that can be used by linked to the respiratory chain and is therefore regarded as part of the many bacteria as a source of cell carbon, sulfur or nitrogen is taurine cell's energy generating systems. The SO family contains three distinct (2-aminoethanesulfonate) [2–4]. A common pathway for taurine groups of enzymes based on the size and structure of the Mo-domain degradation involves taurine deamination by a taurine dehydroge- (Fig. 1) [9], a classification that corresponds well with the conserved nase (TauXY) to sulfoacetaldehyde [5,6] which is then desulfonated domains identified within the SO enzyme family (cd_00321) [11]. by the action of sulfoacetaldehyde acetyltransferase (Xsc), yielding Interestingly, SOE group 2 is the only group that contains sequences of acetyl-phosphate and sulfite. Acetyl-phosphate can either be assimi- eukaryotic origin, while both group 1 and 3 SOEs seem to be exclusive lated into cell biomass via acetyl-CoA or used for substrate level to prokaryotes. phosphorylation with subsequent excretion of acetate [3]. Most steps The first bacterial enzyme of the SO family to be fully characterized of this pathway have been characterized in some form, however, was the SorAB sulfite dehydrogenase from Starkeya novella, a periplasmic group 2 enzyme consisting of a large, Mo-binding subunit (SorA) and a small cytochrome c-binding subunit (SorB) that form an fi ⁎ Corresponding author. Tel.: +61 7 3365 2978; fax: +61 7 3365 4620. integral complex [12,13]. Since then a number of sul te-oxidizing E-mail address: [email protected] (U. Kappler). enzymes from other bacteria have been reported [14–16], but all of

Fig. 1. The sulfite oxidase enzyme family and putative SOEs from S. meliloti: relationship between conserved motifs (COG, cd) and phylogenetic/functional groupings. these enzymes are only distantly related [9]. In addition, other bac- fixation, not the level of reduced carbon compounds present in the terial SO-like enzymes such as the SoxCD sulfur dehydrogenase medium. (group 2) [17,18] and the YedY oxidoreductase (group 1) [19,20] are known that do not oxidize sulfite. 2.2. Growth experiments In this study we have used the α-Proteobacterium S. meliloti as a model organism for elucidating the nature and functions of SOEs in Cultures (10 mL medium in sterile 50 mL tubes) were set up in the metabolism of this bacterium. While S. meliloti is well known for triplicate from precultures grown with the same sulfur or carbon its capacity to act as a nitrogen-fixing symbiont of plants, little is compound and incubated with shaking at 30 °C for 48 h. OD600 known about its ability to grow with inorganic and organic sulfur readings were taken after inoculation, 24 h and 48 h. Cultures were compounds, which is likely, however, to aid the survival of free-living harvested by centrifugation and pellets stored at −20 °C. Growth S. meliloti in soils in the absence of suitable host plants. A complete rates of S. meliloti on taurine, thiosulfate, methanesulfonic acid and operon for taurine catabolism via a taurine dehydrogenase (TauXY) glucose were determined using 250 mL flasks containing 35 mL has been identified in the genome of S. meliloti RM1021 [5]. However, medium. Cultures were inoculated at OD600 0.1 (glucose and taurine) as is the case for other sulfonate-metabolizing bacteria, the nature of or OD600 0.01 (methanesulfonic acid and thiosulfate), OD600 readings the SOE that is part of this process in S. meliloti has not been were taken every hour. Growth characteristics determined here were established, and here we have investigated the molecular details of used in the preparation of cultures for RNA isolation. this process as well as the phylogenetic relationship of SOEs and taurine degradation pathways. 2.3. Biochemical methods

2. Materials and methods The activity of sulfite-oxidizing enzymes was routinely determined using 20 mM Tris–Cl buffer pH 8.0, 1 mM ferricyanide, 2 mM 2.1. Bacterial strains, media and growth conditions freshly prepared sodium sulfite and varying amounts of cell extracts. Assays were performed at 25 °C by monitoring the absorbance change − 1 −1 S. meliloti strain 1021 [21] was routinely grown aerobically at 30 °C at 420 nm (ɛferricyanide =1.09 mM cm ). One unit of activity using liquid or solid TYS medium [22] containing 25 μg/mL Strep- corresponds to the amount of enzyme required to oxidize 1 μmol of tomycin. Sulfur compound utilization was assessed using a modified sulfite per minute. For easy comparison with data reported by others, version of DSMZ (German type culture collection) medium 69 activities are also reported in mkat/kg where applicable. Cytochrome containing twice the original concentration of phosphate buffer. The c based assays for SOEs [23] were used where specified. Kinetic medium was prepared as a basal salt medium without the addition of parameters of SorT were determined using the ferricyanide assay and thiosulfate, and different carbon or sulfur sources (methanol, ethanol, 20 mM Tris-acetate buffers. Kinetic parameters were derived by direct sodium thiosulfate, potassium tetrathionate, taurine, methanesulfo- non-linear fitting of the data to the Michaelis–Menten equation using nic acid, ethanesulfonic acid, dimethylsulfoxide, sodium fumarate, SigmaPlot 9 (SYSTAT Software). The pH dependence of activity was sodium hydrogen carbonate) were added at a final concentration of determined in 50 mM buffers (bis-Tris, Tris, glycine, CAPS) between 20 mM, glucose was used at a final concentration of 10 mM. Solutions pH 6 and 12. The activity of malate dehydrogenase was determined as of volatile or instable compounds were prepared immediately prior to in [24]. Assays for sulfite oxidase activity were carried out using a use. Where necessary, substrate stock solutions were adjusted to near Hansatech Oxygen electrode essentially as in [25]. neutral pH before use. Cultures containing inorganic sulfur com- Small scale cell-free extracts for use in enzyme assays were pounds as primary electron donor were grown under autotrophic prepared from cell pellets of 10 mL cultures using BugBuster conditions in the absence of added carbon other than the 0.3 g/L yeast Mastermix™ (Novagen) according to the manufacturer's instructions. extract contained in the medium base. Cultures supplemented with Cellular fractionation was performed on cultures in late exponential sodium carbonate are equivalent to basal salt controls as addition of growth phase using the osmotic shock method [26]. After removal of sodium carbonate only alters the level of carbon dioxide available for the periplasmic fraction, cell pellets were resuspended in 20 mM Tris– 1518 J.J. Wilson, U. Kappler / Biochimica et Biophysica Acta 1787 (2009) 1516–1525

Cl pH 8, passed three times through a French Pressure cell (Aminco, free RNA samples were used for RT-PCR and quantitative PCR. RNA 12000 psi) followed by separation of the membrane and soluble concentrations were determined using the Quant-it RNA kit (Invitro- protein fractions by ultracentrifugation (90 min, 4 °C, 145000 ×g)ina gen). cDNA was synthesized from 500 μg RNA using the Superscript III Beckman L8-80 ultracentrifuge. Membrane fractions were resus- reverse transcriptase (Invitrogen) and the RNAsin RNAse Inhibitor pended in 20 mM Tris–Cl pH 8 using a handheld glass homogenizer. (Promega). Diluted cDNA (1:10 to 1:100000) was used as the Cellular fractionation by osmotic shock uses very mild conditions, and template for real-time and standard PCR. Quantitative real-time PCR as no enzymatic digestion of the cell wall is involved, release of used PCR products of 200 bp and the SYBR green Mastermix (Applied periplasmic proteins is often incomplete. However, the osmotic shock Biosystems). Real-time PCR reactions were set up using an epMotion method avoids accidental release of cytoplasmic proteins due to cell robot (Eppendorf) and performed on an Applied Biosystems 7900T lysis during isolation of periplasmic proteins using spheroplasts and cycler in 384-well plates. Primer concentrations were optimized for was chosen for this reason. each target gene. Data analysis was carried out as in [31]. Protein gel electrophoresis used the method of [27]. Protein determinations were performed using the BCA-1 kit (Sigma Aldrich) 2.6. Bioinformatic analysis of the S. meliloti RM1021 genome or the 2DQuant kit (GE Healthcare Biosciences). Mass fingerprints of proteins separated on SDS-PAGE gels were prepared as in [28] and A number of known genes involved in the metabolism of sulfur analyzed using a VoyagerSTR MALDI-Tof mass spectrometer (Applied compounds were translated into amino acid sequences and used in Biosystems). Electrospray mass spectrometry was performed on a BLAST_P searches [32] of the S. meliloti RM1021 genome and the Q-Star mass spectrometer (Applied Biosystems) essentially as in [28]. megaplasmids pSymA and pSymB. These included the tau operon from Molecular mass determination of native, purified proteins by MALLS Paracoccus pantotrophus (taurine metabolism, genes tauR, tauA, tauB, (Multi Angle Laser Light Scattering) was carried out as in [29] using a tauC, tauX, tauY, xsc, tauZ, acc. no AY498615.3), the SO family-related miniDAWN Tristar laser light scattering photometer and Optilab DSP genes yedY (Escherichia coli, acc. no NC_000913.2, proteins NP_416480 interferometric refractometer (both Wyatt technology) equipped and NP_416481), sorA and soxC (both from St. novella,acc.no with a Superdex 75 HR (10/30) gel filtration column and using 20 mM AF139113 and AF154565), the DMSO reductase genes dorA (Rhodo- Tris–Cl pH 7.8, 150 mM NaCl as the buffer. A sample volume of 50 μLof bacter capsulatus, acc. no U49506) and dmsABC (E. coli, acc. no. an approximately 200 μM protein solution was used per injection, J03412) as well as the sox gene cluster from St. novella (genes soxC, experimental errors are reported as standard deviations of the mole- soxB, soxD, soxY, soxZ, soxA , soxX, acc. no AF154565). Candidate genes cular mass estimate. Mo-content of protein samples was determined identified in the S. meliloti genome were analyzed using the programs by ICP-MS at the ENTOX centre at the University of Queensland. ProtParam [33], TMHMM [34,35], DAS [36], TMPRed [37], TatP [38] and Signal P [39,40] available through the Expasy homepage (www. 2.4. Purification of the S. meliloti sulfite-oxidizing enzyme expasy.org; accessed May 2009) and the Vector NTi Advance 10 program suite (Invitrogen). 35–40 g (wet weight) of S. meliloti cell material was resuspended in Phylogenetic analyses were performed on sequence homologues 20 mM Tris–Cl pH 8.8 and passed three times through a French Press of the Xsc (SMb21530), TauY (SMb21529) and SorT (SMc04049) (see above). Cell debris was removed by centrifugation (30000 ×g, proteins from S. meliloti. Homologous sequences were aligned using 30 min, 4 °C), and the resultant crude extract applied to a DEAE- AlignX (Vector NTi Advance11, Invitrogen). The MEGA4.0 software Sepharose column (2.6×24.5 cm, GE Healthcare Biosciences) equili- package [41] was used for phylogenetic and bootstrap analyses. brated in 20 mM Tris–Cl pH 8.8. Proteins were eluted using a linear Evolutionary distances were computed using the Poisson correction gradient from 0 to 300 mM NaCl and fractions testing positive for SDH method and are in the units of the number of amino acid substitutions activity pooled. Ammonium sulfate (15% w/v) was added to the pool per site. All positions containing gaps and missing data were which was then applied to a Phenyl-Sepharose FF column (1.6×20 cm, eliminated from the datasets. GE Healthcare Biosciences) equilibrated in 20 mM Tris–Cl pH 7.8, 15% (w/v) ammonium sulfate. SDH activity eluted in the flow-through and 3. Results was concentrated (AmiconUltra 30 kDa MWCO, Millipore) before being applied to a Superdex 75 (16/60, GE Healthcare Biosciences) gel 3.1. Identification of genes encoding putative sulfite-oxidizing enzymes filtration column (running buffer: 20 mM Tris–Cl pH 7.8, 150 mM in the S. meliloti genome NaCl). Where further purification was required, pooled samples were desalted by dialysis against 20 mM Tris–Cl pH 8.8 and applied to Using the sorA and soxC genes from St. novella and the yedY gene a MonoQ column (5/5, GE Healthcare Biosciences) (gradient: from E. coli [19] as the search models, four candidate genes that 0–250 mM NaCl). Protein purity and enrichment were monitored by encode proteins related to the molybdenum subunit of enzymes of the SDS-PAGE and SDH activity assays throughout the purification. SO family (Table 1, Fig. 2) were identified in S. meliloti [9].No homologues of dissimilatory adenylylphosphosulfate reductases were 2.5. Molecular biological methods found, and therefore, indirect oxidation of sulfite [10] was not considered any further in this context. All four putative S. meliloti Standard methods were used throughout [26]. All oligonucleotide SOEs belong to the COG2041 group (sulfite oxidase and related primers were from Invitrogen. Genomic DNA was isolated using the enzymes), and to the conserved domain cd_00321 ‘SO_family_Moco’ DNAZOL reagent (Invitrogen), standard PCR reactions used the GoTaq [11]. Cd_00321 is a superfamily that contains 4 major subcategories green Mastermix (Promega) according to the manufacturer's instruc- (Fig. 1), cd_02107 to cd_02110 (YedY-like Moco; bact_SO_family_ tions. RNA was isolated from S. meliloti cultures grown to mid- Moco; arch_bact_SO_family_Moco and SO_family Moco_dimer), with exponential phase and preserved using the Bacteria Protect Reagent the latter, cd_02110, containing another four subgroups, cd_02111 to (Qiagen) using either the Qiagen RNeasy Kit or the Illustra RNAspin cd_02114 that are based on different, well characterized pro- and Mini kit (GE Healthcare Biosciences) according to the manufacturer's eukaryotic SOEs (Fig. 1). In the classification of SOEs based on the instructions. DNA contamination of RNA samples was assessed by PCR structure of the catalytic Mo-binding domain [9] cd_02107 and using generic primers 27F and 1492R [30] that target the bacterial 16S cd_02108 form group 1 ‘enzymes from pathogenic bacteria’, cd_02110 rRNA gene. If a PCR product was detectable after 34 cycles of corresponds to group 2 ‘sulfite-oxidizing enzymes and plant nitrate amplification, the RNA sample was subjected to further DNAse treat- reductases’ and cd_02109 is equivalent to group 3 ‘enzymes from ment and PCR-testing until no PCR product was detectable. Only DNA- archaea and soil bacteria’ (Fig. 1). J.J. Wilson, U. Kappler / Biochimica et Biophysica Acta 1787 (2009) 1516–1525 1519

Table 1 Properties of SDH-related genes identiﬁed in the S. meliloti genome.

Gene name SMc01281 SMb20584 SMc04049 SMa02103 CDD domainsa cd02107, COG2041 cd02109, COG2041 cd02110, COG2041 cd02110, COG2041 SO family groupb Group 1A Group 3 Group 2 Group 2 Location in S. meliloti genome Chromosome pSYMb Chromosome pSYMa Cellular location Periplasmic Cytoplasmic Periplasmic Cytoplasmic No. of aa 313 239 399 468 MW (kDa) 34.69 27.38 42.47 51.13 pI 6.78 5.65 5.88 5.55 Transmembrane domains No No No No Signal peptide Yes (TatP), 49.9% (Signal P) No Yes No SP cleavage site res. 46–47, AAA-LE res. 31–32, AEA-KE MW-processed protein 29.82 39.42 pI processed protein 5.43 5.7

a Based on [11]. b Based on [9].

Of the four putative SOEs present in S. meliloti, two are group 2 pounds (glucose, formate, methanol, ethanol, hydrogencarbonate) as SOEs (cd_02110 SO_family_Moco_dimer) while one each belongs to well as inorganic (thiosulfate, tetrathionate) and organic sulfur the group 1 (cd_02107) and group 3 (cd_02109) SO family proteins compounds (methanesulfonic acid (MSA), ethanesulfonic acid, taurine, (Table 1, Fig. 1). Although the group 2 SOEs (cd_02110) contain four dimethylsulfoxide). Significant biomass production was observed only well established subgroups, neither of the group 2 S. meliloti SOEs was when glucose or taurine were present in the growth medium (final strongly associated any of these subgroups (Fig. 1), indicating that OD600 of ∼2.4 and ∼1.2, respectively), while formate supported some further differentiation of cd_02110 may be necessary in the future. growth (final OD600 ∼0.46) (Table 2). All other substrates led to final The S. meliloti group 1 SOE SMc01281 is related to YedY from E. coli OD600 values of 0.2–0.22. As S. meliloti can grow to an OD600 of ∼0.2 on (59% identity/69% conserved amino acids). This periplasmically the base medium without added carbon sources it is likely that this level located protein is encoded by the chromosomally located yedY gene of growth is supported by the small amount of yeast extract (0.3 g/L) (SMc01281) which, like the majority of yedY genes [9], is found in present in the basal medium. It is thus unclear whether any of the conjunction with a gene (yedZ) encoding a heme b-binding protein additional substrates tested actually contributed to the growth of S. with 6 transmembrane helices (Fig. 2, Table 1). The two group 2 SOE- meliloti. like proteins (cd_02110) are encoded by genes SMa02103 and Cell extracts prepared for all of the above growth conditions SMc04049. The SMa02103 gene is located on megaplasmid pSymA (Table 2) contained mostly low levels of SOE activity (between 0.5 and encodes a cytoplasmic protein (Fig. 2), while the chromosomally and 1.9 U/mg, average 1.4±0.4 U/mg), while high levels of activity located SMc04049 gene encodes a periplasmic protein. The fourth were only detected in cell extracts of cultures grown on taurine (5.54± SOE-candidate gene, SMb20584, is located on megaplasmid pSYMb 0.42 U/mg) or thiosulfate (8.8±0.63 U/mg). This is a surprising finding and encodes a cytoplasmic group 3 SOE with no characterized as S. meliloti lacks a pathway for thiosulfate utilization. Cell extracts from relatives to date. MSA grown cultures had a slightly enhanced SOE activity of 2.43± 0.13 U/mg (Table 2). Thus, the substrates taurine, thiosulfate, MSA and 3.2. When does S. meliloti require sulfite-oxidizing enzymes? glucose were chosen for further investigation as they are representative of the three different types of growth substrates used in this study, and To establish conditions where sulfite oxidation is required by S. distinct SOE-like enzymes might be associated with different prevailing meliloti, cells were grown in the presence of a number of carbon com- growth modes.

Fig. 2. S. meliloti operons encoding proteins related to known sulﬁte-oxidizing enzymes. Panel A: SMc01281 gene region encoding a YedY related SOE group 1A protein. Panel B: SMc04049 gene region (SOE group 2). Panel C: SMa02103 gene region (SOE group 2). Panel D: SMb20584 gene region (SOE group 3). Colour coding for genes shown: light grey: Molybdenum subunits, grey: heme-containing proteins, dark grey: pseudoazurin-related proteins, hatched: repeat regions, open arrows: unrelated genes present in the gene region. 1520 J.J. Wilson, U. Kappler / Biochimica et Biophysica Acta 1787 (2009) 1516–1525

Table 2 Growth characteristics and sulﬁte-oxidizing enzyme activity observed for S. meliloti grown with different carbon or sulfur sources.

Condition Average U/mg SDH activity OD600 after 48 h Methanol (20 mM) 1.31±0.08 0.203 Ethanol (20 mM) 1.89±0.19 0.241 Glucose (10 mM) 1.12±0.06 2.41 Potassium tetrathionate (10 mM) 0.52±0.04 0.205 Sodium thiosulfate (20 mM) 8.85±0.63 0.22 Taurine (20 mM) 5.54±0.42 1.15 MSA (20 mM) 2.43±0.13 0.201 ESA (20 mM) 1.45±0.09 0.199 DMSO (20 mM) 1.72±0.15 0.206 Sodium carbonate (20 mM) 1.74±0.11 0.209 Sodium formate (20 mM) 1.57±0.07 0.464

Enzyme activity averages shown are the mean of at least 14 determinations carried out on crude extracts from three separate cultures for each condition. Errors are given as 95% conﬁdence intervals. Shown in bold and italics: growth substrates chosen for further study. ESA = ethanesulfonic acid, MSA = methanesulfonic acid, DMSO = dimethylsulfoxide.

The cellular location of SOE activity was determined for the four model substrates: in all cases, the fractionation by osmotic shock was successful as indicated by the absence of the cytoplasmic marker enzyme, malate dehydrogenase, in the periplasmic protein fraction (data not shown). Periplasmic fractions contained signiﬁcant amounts of SOE activity (1.67–2.04 U/mg for taurine and thiosulfate), indicating a periplasmic location of the responsible enzyme in all four cases and thus involvement of either SMc04049 (SorT) or the YedY-like SMc01281.

3.3. Expression of SOE genes in S. meliloti

Expression of the genes encoding the four putative S. meliloti SOEs was determined, initially using two-step RT-PCR (Fig. 3A). As a control the xsc gene encoding a sulfoacetaldehyde acetyltransferase that is Fig. 3. Expression of SOE-like genes from S. meliloti in cells grown in the presence of specific to taurine metabolism was used, and the control gene was glucose (G), thiosulfate (TS), taurine (T) or methanesulfonic acid (MSA). Panel A: Reverse Transcriptase PCR. RNA was isolated from culture in early to mid-exponential only expressed in cells that had been grown on taurine (Fig. 3A). phase, for glucose (G) and taurine (T) additional RNA samples (G2, T2) were isolated Similar results were obtained for the tauY gene that encodes a subunit from cells in mid to late exponential growth phase. The latter samples were only used in of taurine dehydrogenase (data not shown). In contrast, three of the RT-PCR not qPCR experiments, N = no template control. The cDNA was diluted 1:100 four SDH-like genes were expressed in varying amounts under all for RT-PCR except for SMb20584 where a dilution of 1:10 was used. Panel B: Average normalized gene expression derived from quantitative real-time PCR experiments conditions tested, while no expression of the gene encoding the carried out using the SYBR green technology. Gene expression was normalized relative cytoplasmic group 2 SMa02103 protein was detected in any of the to the expression of the 16S rRNA gene in each sample. Experimental errors are given as samples, ruling out its involvement in sulfite oxidation (Fig. 3A). relative standard deviation. Expression of the SMb20584 gene encoding the cytoplasmic group 3 SOE-like protein was very low throughout, confirming that the two Co-transcription with neighboring genes was tested for the periplasmic enzymes SMc01281 (YedY-like, group 1) and SMc04049 SMc04049/sorT and SMc01281/yedY genes that are found in conserved (SorT, group 2) were most likely causing the observed SDH activity. genetic environments. As shown in Fig. 4, the putative SMc01281/yedY Quantification of gene expression by real-time PCR revealed that and SMc01282/yedZ genes form a transcriptional unit, while the SMc04049/sorT gene (group 2 SOE) had the highest expression SMc04049/sorT is co-transcribed with both the cytochrome c encoding levels under all conditions tested as well as an expression pattern SMc04048 gene and the azu2 gene that encodes a pseudoazurin (Figs. 1 matching the observed SOE activity and was thus likely to be the and 3). This suggests that the two most highly expressed SOE-like major S. meliloti SOE (Fig. 3B). In fact, during growth on taurine and proteins in S. meliloti are either multisubunit proteins or require thiosulfate expression of this gene was similar to that of the xsc gene accessory proteins for function. that is induced to high levels in the presence of taurine and is central to taurine catabolism. The SMc01281 gene encoding the second 3.4. Purification and characterization of the sulfite-oxidizing enzyme periplasmic SOE (YedY-like, group 1) also showed increased expres- from taurine-grown S. meliloti cells sion in the presence of taurine and thiosulfate, the two growth conditions associated with the highest observed SOE activity in S. Purification of the S. meliloti SOE was undertaken using cells grown meliloti cell extracts. However, expression of SMc01281 on thiosulfate on taurine. Throughout the purification SOE activity was always and taurine reached only 7% and 8.8% of the transcript levels detected associated with a single protein peak, making the involvement of more for the SMc04049/sorT gene under the respective growth conditions. than one enzyme unlikely. As reported for the SOEs from Comamonas/ In contrast, the SMb20584 gene was expressed at low levels (0.7% and Delftia acidovorans and Cupriavidus necator [7,8], several unknown 1.7% of SMc04049/sorT expression on thiosulfate and taurine), with proteins tended to co-purify with SMc04049 (SorT). Using peptide the highest expression for this gene being observed in the presence of mass fingerprints the major co-purifying proteins were identified as a glucose or thiosulfate. This differing expression pattern and the putative dipeptide binding periplasmic protein (SMc02634), a cytoplasmic location of the encoded protein indicate that the group 3 putative oligopeptide transporter protein (SMb21196), a probable protein encoded by SMb20584 is not likely to be involved in sulfite fructose-bisphosphate aldolase class I protein (SMc03983) and a oxidation in S. meliloti. putative uncharacterized protein (SMc02156). No functional link J.J. Wilson, U. Kappler / Biochimica et Biophysica Acta 1787 (2009) 1516–1525 1521

Fig. 4. Co-transcription of SOE-related genes in S. meliloti. Black bars indicate the position and expected sizes of the PCR products generated. Panel A: yedYZ genes (SMc01281 and 01282), panel B: SMc04047-04049 genes. Lanes: R = cDNA template generated from RNA isolated from cells grown with taurine, N = no template control, P = positive control with genomic DNA as template. Amplification conditions were the same for all samples. between these proteins and SMc04049 (SorT) is apparent. The amount (data not shown). The molybdenum content of the purified enzyme of co-purifying proteins could be reduced by changes in the was 59% as determined by ICP-MS. purification parameters. The final enzyme preparation contained a protein with an apparent molecular mass of ∼40 kDa (Fig. 5, top, 3.5. Characterization of SMc04049 (SorT) inset). The preparation was essentially free of other proteins, although minute amounts of a contaminating cytochrome c were present in The SMc04049 (SorT) protein eluted from a calibrated Superdex75 some fractions. The purified protein was tryptically digested and the (16/60) gelfiltration column with an apparent molecular mass of resulting mass fingerprint clearly identified it as SMc04049 (SorT) ∼68 kDa which is larger than the mass of a monomer, but too small for a dimer. In the case of the SorAB sulfite dehydrogenase a similar discrepancy had been due to the presence of an additional subunit with unusual electrophoretic properties [13,42]. As SorT appeared to consist of a single, ∼40 kDa subunit, we used a combination of electrospray mass spectrometry and MALLS to determine the molecular masses of the subunit and the native protein. The mature SMc04049 (SorT) protein has a predicted molecular mass of 39.42 kDa, and using electrospray mass spectrometry a subunit molecular mass of 39.424± 0.005 kDa was determined for SorT. In contrast, MALLS analysis of the size of the native protein resulted in a mass of 78.04±0.8 kDa (peak polydispersity: 1.001), clearly showing that the native protein is a

homodimer (α2) without additional subunits. The purified SorT (SMc04049) SOE was kinetically characterized using the three established electron acceptors for SOEs, ferricyanide, cytochrome c (horse heart) and oxygen. SorT activity with cytochrome c was only ∼13% (65±6 U/mg or 1081±106 mkat/kg) of the activity observed with ferricyanide (522±17 U/mg or 8701±284 mkat/kg). No oxygen dependent sulfite oxidation was detected although S. meliloti cells show respiratory activity with sulfite (Voreck and Kappler, unpublished observation), indicating that the reaction catalysed by SorT is linked to the respiratory chain. All further characterization was therefore carried out using ferricyanide as the electron acceptor. At pH

8.0 in 20 mM Tris-acetate buffer, an apparent KMsulfite of 15.5±2.0 μM was determined (Fig. 5, top), the corresponding apparent KMferricyanide −1 was 3.4±0.8 μM and the apparent kcat was 343±11 s (Table 3). The pH dependence of SorT activity was determined between pH 6 and pH 12. The data could be fitted to a bell-shaped curve with apparent pKa values of pH 5.5 and pH 11.1 and maximal sulfite-oxidizing activity between pH 8 and 9 (Fig. 5, bottom).

Table 3 Variation of catalytic parameters of the SorT (SMc04049) sulfite dehydrogenase with pH. Fig. 5. Kinetic data for purified SorT (SMc04049) S. meliloti sulfite dehydrogenase. Top: fi fi Direct non-linear t of data for KMapp_sulfite at pH 8; inset: SDS-PAGE of puri ed S. pH 6 7 8 8.8 9.6 meliloti SorT (SMc04049), left lane: molecular mass standard, right lane: purified SorT KMapp_sulfite (μM) 22.1±2.7 10.6±1.3 15.5±1.9 10.8±1.2 7.9±1.2 (SMc04049) (1.2 μg). Bottom: pH dependence of sulfite-oxidizing activity fitted to a − k (s 1) 309±8 341±9 343±11 385±9 387±11 bell-shaped curve. cat 1522 J.J. Wilson, U. Kappler / Biochimica et Biophysica Acta 1787 (2009) 1516–1525

Apparent KMsulfite and kcat values of SorT were also determined at pH also supported by the position of the two enzymes in question within 6, 7, 8.8 and 9.6 (Table 3). Both parameters were remarkably insensitive the cd_02110 SOE group: SorA from St. novella is a representative of to pH, with apparent KMsulfite values varying between 22.1±2.7 μMat subgroup cd_02114 ‘SorA-Moco_dimer’, while the S. meliloti SorT pH 6 and 7.9±1.2 μM at pH 9.6. Turnover numbers increased slightly at enzyme does not fall into any of the four existing subcategories of higher pH values with values varying between 309±8 and 387±11 s−1 cd_02110 (Fig. 1). at pH 6 and pH 9.6 respectively. This behaviour of the catalytic Unlike the well studied SOE from St. novella, SorT is a homodimeric parameters is clearly different from similar data reported for the enzyme containing only a molybdenum redox centre, a structure that vertebrate sulfite oxidases or the bacterial SorAB sulfite dehydrogenase is reminiscent of that of the plant sulfite oxidase (PSO), however, SorT

[43,44], where both apparent KMsulfite and kcat values are strongly pH is not a sulfite oxidase as it cannot transfer electrons to molecular dependent (Fig. S1). oxygen. Heme groups, that are a typical feature of vertebrate sulfite Unlike many of the well characterized SOs and SDHs [13,44,45] oxidases and some bacterial sulfite deydrogenases [43,44], are absent SorT was found to be relatively insensitive to inhibition by changes in from SorT. As for most bacterial SOEs [7,8,14,15], the natural electron ionic strength, with 200 mM Tris-acetate buffer causing ∼20% acceptor for SorT is unknown at present and all these enzymes show inhibition of activity, while sodium chloride at 40 mM and 200 mM much larger activities with the artificial electron acceptor ferricyanide caused 11% and 32% inhibition, respectively. Sodium sulfate was a than with mitochondrial cytochrome c which is preferred by the slightly more potent inhibitor of activity with addition of 10 mM and heme-containing bacterial SOEs [13,16] found in St. novella and 50 mM causing 9% and 23% inhibition of activity. In contrast, the Campylobacter jejuni. The high SorT activities observed with ferricy- presence of 15 mM sodium sulfate causes 50% inhibition of enzyme anide may reflect a need for an electron acceptor with a higher redox activity in the St. novella SorAB sulfite dehydrogenase [43]. potential than that of the vertebrate cytochrome and/or a differing primary structure that enables more efficient interactions between 4. Discussion SorT and the acceptor protein. Small bacterial electron transfer proteins with high redox potentials include copper proteins such as This is the first study of sulfite-oxidizing enzymes in S. meliloti and azurin [47], ferredoxins and certain cytochromes c [48], and we have the relationship of these enzymes to pathways for the degradation of shown here that genes encoding both a cytochrome c and an azurin sulfur compounds. S. meliloti contains four genes that encode SOEs are co-transcribed with the sorT gene. The need for a different elec- and these include representatives of all three known groups of sulfite- tron acceptor may also be related to the absence of a heme domain/ oxidizing enzymes, making S. meliloti an ideal model organism for subunit in SorT as these domains are thought to mediate electron studying bacterial sulfite oxidation. The ability of S. meliloti to oxidize transfer to cytochrome c in SorAB and the vertebrate SOs [12]. sulfur compounds has also not been investigated in detail so far, In addition to its unusual structural features, the catalytic mecha- although a complete operon for the degradation of taurine had been nism of SorT also shows novel features that differ from those of all SOEs previously identified in the S. meliloti genome [5]. Here we have that have been studied in detail so far. SorT has high affinities for its shown that while taurine is readily metabolized neither related sulfo- substrate, sulfite, and fast turnover rates, however, while in vertebrate nates such as methane- and ethanesulfonic acid nor a number of other SOs and bacterial SorAB SOEs KMapp_sulfite is clearly pH dependent organic and inorganic sulfur compounds support growth. with very strong increases seen above pH 8 (Fig. S1) [43,44],inSorT

The final uncharacterized step in the degradation of organosulfo- KMapp_sulfite is nearly invariant with pH. In fact, in contrast to the other nates is the conversion of sulfite, which is highly reactive and therefore SOEs the highest substrate affinities were observed at pH values above 8. cytotoxic [10,46], to sulfate and our work has unambiguously identified Similarly, kcat_app was very insensitive to pH changes, with only a small the group 2 SOE SorT (SMc04049) as the enzyme responsible for SOE increase seen towards the high pH range (Table 3). In vertebrate SOs and activity observed in S. meliloti grown in the presence of thiosulfate and the SorAB SOE kcat_app is clearly pH dependent and shows either one or the alkanesulfonate taurine (Table 2, Fig. 3A). two pKa values between pH 6 and 10. For vertebrate SOs and SorAB We also detected expression of the genes encoding two other SDHs it has been suggested that the higher substrate affinity at low pH is SOEs, the group 3 enzyme SMb20584 and a group 1, YedY-like SOE indicative of a higher affinity of the enzyme for the protonated form of − (SMc01281). While expression levels for the former were low and its the substrate, HSO3 [43,49,50]. It is then possible that the differing possible function is unknown, expression of the yedY-related gene behaviour of KMapp_sulfite seen in SorT could indicate that this enzyme − 2− SMc01281 was high during growth of S. meliloti on taurine and works equally well with either HSO3 or SO3 . There is some possibility thiosulfate, indicating a possible role for this enzyme in S. meliloti that the use of an artificial electron acceptor such as ferricyanide, could sulfur metabolism although the nature of this role is unclear and does have some influence on the pH dependence of kcat_app [43,44]. However, not appear to involve sulfite oxidation. To the best of our knowledge the altered behaviour of KMapp_sulfite is highly unlikely to be due to the this is the first time that growth conditions under which a YedY-like use of ferricyanide as an electron acceptor in SorT steady-state assays: It SOE is expressed have been identified for any bacterium, thereby has been shown for several other SOEs including those found in opening up new possibilities for future studies of this class of SOE. vertebrates that the strong increase in KMapp_sulfite above pH 8 is a The S. meliloti SorT SOE belongs, like all other pro- and eukaryotic property of the Mo centre alone and it has been seen in steady-state SOEs studied to date, to the group 2 SOEs (cd_02110) within the assays, and is also apparent when only the reductive half reaction of the sulfite oxidase enzyme family, however, it is not closely related to any enzyme is studied, i.e. when no electron acceptor is present [43,49,50]. of the bacterial SOEs studied to date. SorT shares 59% sequence The altered properties of the SorT catalytic parameters are an exciting identity and 76% sequence similarity with the largely uncharacterized finding and make SorT a prime candidate for elucidating how changes in enzyme from Delftia acidovorans [7,8], while sequence identities with the redox properties of the Mo centre and in the active site environment the other bacterial SOEs that have been studied in varying levels alter the kinetics of sulfite oxidation. of detail range between 27 and 35% (sequence similarity values: Is there a link between the pathway for taurine oxidation and the 44–50%) (Table S1). In view of the low similarity of the SorT sequence presence of a particular type of SOE? Sulfite-oxidizing enzymes appear to other bacterial SOEs and the obvious structural and kinetic to be associated with the metabolism of taurine in many bacteria [4], differences discussed below we have decided to refer to this enzyme and we have therefore investigated whether SorT represents an SOE- as SorT rather than naming it ‘SorA’ as has been done in some cases type that is generally associated with taurine degradation pathways [7]. The designation ‘SorA’ was coined for the heterodimeric, heme- (represented by the TauY and Xsc proteins from S. meliloti). In both containing SOE from St. novella which has only 32% sequence identity cases sequences originating from α-andβ-Proteobacteria form to SorT and also has a clearly differing structure. This classification is distinct but related clusters (Fig. S2), however, while there is a good J.J. Wilson, U. Kappler / Biochimica et Biophysica Acta 1787 (2009) 1516–1525 1523 correlation between the presence of TauY and the presence of Xsc, chromes with between 63 and 90% amino acid similarity to the Xsc-like sequences exist in many organisms that do not appear to cytochrome encoded by the SMc04048 gene (Fig. 2), i.e. these enzymes contain a TauY-like taurine dehydrogenase. For both TauY and Xsc have an operon structure that on the surface resembles that of the from S. meliloti the most closely related sequences were from Ochro- heme-containing, heterodimeric SorAB SOE from St. novella [9,12,13]. bacterium, Roseobacter and Rhodobacter species. However, despite these similarities, neither SorT nor any of the other However, there was no obvious correlation between the occur- characterized SOEs contain a heme group as an integral part of the rence of TauY/Xsc-like sequences and SorT-related sequences. SorT enzyme. This proves that it is not possible at present to predict the groups with sequences from both α- and β-Proteobacteria such as structure of bacterial SOEs based on an inspection of the genetic Sulfitobacter, Acidovorax, Comamonas and Delftia acidovorans. SOE- environment of SOE-encoding genes alone. As there is no direct link related sequences from Roseobacter and Rhodobacter species that between taurine metabolism and the occurrence of a particular type of contain sequences closely related to TauY and Xsc from S. meliloti, fall SOEs, the question as to what the main function of bacterial SOEs in into a different clade that also contains sequences from Nitrobacter cellular metabolism is remains to be answered. As a result of our (Fig. 6). analyses it seems possible that contrary to earlier assumptions [4,7,51], The majority (N90%) of the SOE-encoding genes in this group, SOEs are not linked to particular metabolic pathways but may be including the SOEs from D. acidovorans, C. necator or T. thermophilus induced in response to exposure of the bacteria to sulfite. This is [7,15] are found upstream of genes encoding soluble c-type cyto- supported by the expression pattern of sorT that differed noticeably

Fig. 6. Phylogenetic relationship of S. meliloti SorT to other bacterial sulfite-oxidizing enzymes. The tree was generated using the neighbor-joining algorithm integrated into MEGA4.0 [41], bootstrap values (500 replicates) above 50% are shown. SorT and other bacterial SOEs that have been described in the literature are shown in bold. 1524 J.J. Wilson, U. Kappler / Biochimica et Biophysica Acta 1787 (2009) 1516–1525 from those of xsc and tauY which encode key enzymes of taurine Escherichia coli sulfite oxidase homologue reveals the role of a conserved active site cysteine in assembly and function, Biochemistry 44 (2005) 10339–10348. degradation. Clearly further work is needed to uncover the factors [20] L. Loschi, S.J. Brokx, T.L. Hills, G. Zhang, M.G. Bertero, A.L. Lovering, J.H. Weiner, governing the formation of bacterial SOEs and the functional differences N.C.J. Strynadka, Structural and biochemical identification of a novel bacterial inherent in their structure in order to understand how these enzyme are oxidoreductase, J. Biol. Chem. 279 (2004) 50391–50400. [21] F. Galibert, T.M. Finan, S.R. Long, A. Puhler, P. Abola, F. Ampe, F. Barloy-Hubler, M.J. integrated into cellular metabolism. Barnett, A. Becker, P. Boistard, G. Bothe, M. Boutry, L. Bowser, J. Buhrmester, E. Cadieu, D. Capela, P. Chain, A. Cowie, R.W. Davis, S. Dreano, N.A. Federspiel, R.F. Acknowledgements Fisher, S. Gloux, T. Godrie, A. Goffeau, B. Golding, J. Gouzy, M. Gurjal, I. Hernandez- Lucas, A. Hong, L. Huizar, R.W. Hyman, T. Jones, D. Kahn, M.L. Kahn, S. Kalman, D.H. Keating, E. Kiss, C. Komp, V. Lalaure, D. Masuy, C. Palm, M.C. Peck, T.M. Pohl, D. This work was supported by the Australian Research Council by an Portetelle, B. Purnelle, U. Ramsperger, R. Surzycki, P. Thebault, M. Vandenbol, F.J. Australian Research Fellowship (ARF) and an ARC discovery project Vorholter, S. Weidner, D.H. Wells, K. Wong, K.C. Yeh, J. Batut, The composite (DP0878525) to UK and by the University of Queensland. We would genome of the legume symbiont Sinorhizobium meliloti, Science 293 (2001) 668–672. like to thank Prof. G. King and Dr. S. Rowland for making their MALLS [22] J.E. Beringer, R factor transfer in Rhizobium leguminosarum, J. Gen. Microbiol. 84 analysis facility available for use in this project and for assisting with (1974) 188–198. the analysis of the resulting data. We thank Dr. C.J. Jones for critically [23] U. Kappler, S. Bailey, C.J. Feng, M.J. Honeychurch, G.R. Hanson, P.V. Bernhardt, G. Tollin, J.H. Enemark, Kinetic and structural evidence for the importance of Tyr236 for reviewing this manuscript. the integrity of the Mo active site in a bacterial sulfite dehydrogenase, Biochemistry 45 (2006) 9696–9705. Appendix A. Supplementary data [24] J.P. Markwell, J. Lascelles, Membrane-bound, pyridine nucleotide-independent L-lactate dehydrogenase of Rhodopseudomonas sphaeroides, J. Bact. 133 (1978) 593–600. Supplementary data associated with this article can be found, in [25] R. Hansch, C. Lang, E. Riebeseel, R. Lindigkeit, A. Gessler, H. Rennenberg, R.R. Mendel, the online version, at doi:10.1016/j.bbabio.2009.07.005. Plant sulfite oxidase as novel producer of hydrogen peroxide — combination of enzyme catalysis with a subsequent non-enzymatic reaction step, J. Biol. Chem. 281 (2006) 6884–6888. References [26] F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, K. Struhl, in: K. Janssen (Ed.), Current Protocols in Molecular Biology, vol. 1, John Wiley and [1] M.A. Kertesz, P. Mirleau, The role of soil microbes in plant sulphur nutrition, J. Exp. Sons Inc., Hoboken, NJ, 2005. Bot. 55 (2004) 1939–1945. [27] U.K. Laemmli, Cleavage of structural protein during the assembly of the head of [2] M.A. Kertesz, Riding the sulfur cycle — metabolism of sulfonates and sulfate esters bacteriophage T4, Nature 227 (1970) 680–685. in Gram-negative bacteria, FEMS Microbiol. Rev. 24 (1999) 135–175. [28] U. Kappler, K.F. Aguey-Zinsou, G.R. Hanson, P.V. Bernhardt, A.G. McEwan, [3] A.M. Cook, K. Denger, T.H.M. Smits, Dissimilation of C3-sulfonates Arch, Microbiol. Cytochrome c551 from Starkeya novella: characterization, spectroscopic proper- 185 (2006) 83–90. ties, and phylogeny of a diheme protein of the SoxAX family, J. Biol. Chem. 279 [4]A.M.Cook,K.Denger,S.S.Oja,P.Saransaari,Metabolismoftaurinein (2004) 6252–6260. microorganisms — a primer in molecular biodiversity? Advances in Experimental [29] S.L. Rowland, W.F. Burkholder, K.A. Cunningham, M.W. Maciejewski, A.D. Medicine and Biology, Springer, Berlin, 2006, pp. 3–13. Grossman, G.F. King, Structure and mechanism of action of Sda, an inhibitor of [5] C. Bruggeman, K. Denger, A.M. Cook, J. Ruff, Enzymes and genes of taurine and the histidine kinases that regulate initiation of sporulation in Bacillus subtilis, Mol. isethionate dissimilation in Paracoccus denitrificans, Microbiology 150 (2004) Cell 13 (2004) 689–701. 805–816. [30] D.J. Lane, E. Stackebrandt, M. Goodfellow, 16S–23S rRNA sequencing, Nucleic [6] J. Ruff, K. Denger, A.M. Cook, Sulphoacetaldehyde acetyltransferase yields acetyl Acid Techniques in Bacterial Systematics, John Wiley and Sons, Chichester, 1991, phosphate: purification from Alcaligenes defragrans and gene clusters in taurine pp. 115–175. degradation, Biochem. J. 369 (2003) 275–285. [31] U. Kappler, L.I. Sly, A.G. McEwan, Respiratory gene clusters of Metallosphaera [7] K. Denger, S. Weinitschke, T.H.M. Smits, D. Schleheck, A.M. Cook, Bacterial sulfite sedula — differential expression and transcriptional organization, Microbiology dehydrogenases in organotrophic metabolism: separation and identification in 151 (2005) 35–43. Cupriavidus necator H16 and in Delftia acidovorans SPH-1, Microbiology 154 [32] S.F. Altschul, T.L. Madden, A.A. SchÑffer, J. Zhang, Z. Zhang, W. Miller, D.J. Lipman, (2008) 256–263. Gapped BLAST and PSI-BLAST: a new generation of protein database search [8] W. Reichenbecher, D.P. Kelly, J.C. Murrell, Desulfonation of propanesulfonic acid programs, Nucleic Acids Res. 25 (1997) 3389–3402. by Comamonas acidovorans strain P53: evidence for an alkanesulfonate sulfona- [33] E. Gasteiger, C. Hoogland, A. Gattiker, S. Duvaud, M.R. Wilkins, R.D. Appel, A. tase and an atypical sulfite dehydrogenase, Arch. Microbiol. 172 (1999) 387–392. Bairoch, J.M. Walker, Protein identification and analysis tools on the ExPASy [9] U. Kappler, C.G. Friedrich, C. Dahl, Bacterial sulfite dehydrogenases — enzymes Server, The Proteomics Protocols Handbook, Humana Press, 2005, pp. 571–607. for chemolithtrophs only?, Microbial Sulfur Metabolism, Springer, Berlin, 2008, [34] A. Krogh, B. Larsson, G. VonHeijne, E.L.L. Sonnhammer, Predicting transmembrane pp. 151–169. protein topology with a hidden Markov model: application to complete genomes, [10] U. Kappler, C. Dahl, Enzymology and molecular biology of prokaryotic sulfite J. Mol. Biol. 305 (2001) 567–580. oxidation, FEMS Microbiol. Lett. 203 (2001) 1–9. [35] S. Moller, M.D.R. Croning, R. Apweiler, Evaluation of methods for the prediction of [11] A. Marchler-Bauer, J.B. Anderson, P.F. Cherukuri, C. DeWeese-Scott, L.Y. Geer, M. membrane spanning regions, Bioinformatics 17 (2001) 633–646. Gwadz, S. He, D.I. Hurwitz, J.D. Jackson, Z. Ke, C. Lanczycki, C.A. Liebert, C. Liu, F. Lu, [36] M. Cserzo, E. Wallin, I. Simon, G. VonHeijne, A. Elofsson, Prediction of transmem- G.H. Marchler, M. Mullokandov, B.A. Shoemaker, V. Simonyan, J.S. Song, P.A. brane alpha-helices in prokaryotic membrane proteins: the Dense Alignment Thiessen, R.A. Yamashita, J.J. Yin, D. Zhang, S.H. Bryant, CDD: a Conserved Domain Surface method, Prot. Eng. 10 (1997) 673–676. Database for protein classification, Nucleic Acids Res. 33 (2005) D192–196. [37] K. Hofmann, W. Stoffel, TMbase — a database of membrane spanning proteins [12] U. Kappler, S. Bailey, Molecular basis of intramolecular electron transfer in sulfite- segments, Biol. Chem. 374 (1993) 166. oxidizing enzymes is revealed by high resolution structure of a heterodimeric [38] J.D. Bendtsen, H. Nielsen, D. Widdick, T. Palmer, S. Brunak, Prediction of twin- complex of the catalytic molybdopterin subunit and a c-type cytochrome subunit, arginine signal peptides, BMC Bioinformatics 6 (2005) 167–176. J. Biol. Chem. 280 (2005) 24999–25007. [39] O. Emanuelsson, S. Brunak, G. von Heijne, H. Nielsen, Locating proteins in the cell [13] U. Kappler, B. Bennett, J. Rethmeier, G. Schwarz, R. Deutzmann, A.G. McEwan, C. using TargetP, SignalP and related tools, Nat. Protocols 2 (2007) 953–971. Dahl, Sulfite: cytochrome c oxidoreductase from Thiobacillus novellus — purification, [40] H. Nielsen, S. Brunak, G. VonHeijne, Machine learning approaches to the predic- characterization and molecular biology of a heterodimeric member of the sulfite tion of signal peptides and other protein sorting signals, Prot. Eng. 12 (1999) 3–9. oxidase family, J. Biol. Chem. 275 (2000) 13202–13212. [41] K. Tamura, J. Dudley, M. Nei, S. Kumar, MEGA4: Molecular Evolutionary Genetics [14] G. D'Errico, A. Di Salle, F. La Cara, M. Rossi, R. Cannio, Identification and Analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24 (2007) 1596–1599. characterization of a novel bacterial sulfite oxidase with no heme binding domain [42] F. Toghrol, W.M. Southerland, Purifi cation of Thiobacillus novellus sulfite oxidase. from Deinococcus radiodurans, J. Bact. 188 (2006) 694–701. Evidence for the presence of heme and molybdenum, J. Biol. Chem. 258 (1983) [15] A. Di Salle, G. D'Errico, F. La Cara, R. Cannio, M. Rossi, A novel thermostable sulfite 6762–6766. oxidase from Thermus thermophilus: characterization of the enzyme, gene cloning [43] S. Bailey, T. Rapson, K. Winters-Johnson, A.V. Astashkin, J.H. Enemark, U. Kappler, and expression in Escherichia coli, Extremophiles 10 (2006) 587–598. Molecular basis for enzymatic sulfite oxidation — how three conserved active site [16] J.D. Myers, D.J. Kelly, A sulphite respiration system in the chemoheterotrophic residues shape enzyme activity, J. Biol. Chem. 284 (2009) 2053–2063. human pathogen Campylobacter jejuni, Microbiology 151 (2005) 233–242. [44] M.S. Brody, R. Hille, The kinetic behavior of chicken liver sulfite oxidase, Biochemistry [17] F. Bardischewsky, A. Quentmeier, D. Rother, P. Hellwig, S. Kostka, C.G. Friedrich, 38 (1999) 6668–6677. Sulfur dehydrogenase of Paracoccus pantotrophus: the heme-2 domain of the molyb- [45] C.J. Feng, R.V. Kedia, J.T. Hazzard, J.K. Hurley, G. Tollin, J.H. Enemark, Effect of solution doprotein cytochrome c complex is dispensable for catalytic activity, Biochemistry viscosity on intramolecular electron transfer in sulfite oxidase, Biochemistry 41 44 (2005) 7024–7034. (2002) 5816–5821. [18] A. Quentmeier, R. Kraft, S. Kostka, R. Klockenkamper, C.G. Friedrich, Character- [46] H. Niknahad, P.J. O'Brien, Mechanism of sulfite cytotoxicity in isolated rat ization of a new type of sulfite dehydrogenase from Paracoccus pantotrophus hepatocytes, Chem. Biol. Interact. 174 (2008) 147–154. GB17, Arch. Microbiol. 173 (2000) 117–125. [47] M. Kukimoto, M. Nishiyama, T. Ohnuki, S. Turley, E.T. Adman, S. Horinouchi, T. [19] S.J. Brokx, R.A. Rothery, G.J. Zhang, D.P. Ng, J.H. Weiner, Characterization of an Beppu, Identification of interaction site of pseudoazurin with its redox partner, J.J. Wilson, U. Kappler / Biochimica et Biophysica Acta 1787 (2009) 1516–1525 1525

copper-containing nitrite reductase from Alcaligenes faecalis S-6, Prot. Eng. dehydrogenase: mechanistic insights and contrasts with related Mo enzymes, 8 (1995) 153–158. Biochim. Biophys. Acta-Bioenerg. 1777 (2008) 1319–1325. [48] C.A. McDevitt, P. Hugenholtz, G.R. Hanson, A.G. McEwan, Molecular analysis of [50] H.L. Wilson, K.V. Rajagopalan, The role of tyrosine 343 in substrate binding and dimethylsulfide dehydrogenase from Rhodovulum sulfidophilum; its place in the catalysis by human sulfite oxidase, J. Biol. Chem. 279 (2004) 15105–15113. DMSO reductase family of microbial molybdopterin-containing enzymes, Mol. [51] U. Kappler, C.G. Friedrich, H.G. Truper, C. Dahl, Evidence for two pathways of Microbiol. 44 (2002) 1576–1587. thiosulfate oxidation in Starkeya novella (formerly Thiobacillus novellus), Arch. [49] T.D. Rapson, U. Kappler, P.V. Bernhardt, Direct catalytic electrochemistry of sulfite Microbiol. 175 (2001) 102–111.