Metabolic reconstruction of sulfur assimilation in the extremophile Acidithiobacillus ferrooxidans based on genome analysis
© Valdés et al; licensee BioMed Central Ltd. 2003
Received: 01 September 2003
Accepted: 15 December 2003
Published: 15 December 2003
Acidithiobacillus ferrooxidans is a gamma-proteobacterium that lives at pH2 and obtains energy by the oxidation of sulfur and iron. It is used in the biomining industry for the recovery of metals and is one of the causative agents of acid mine drainage. Effective tools for the study of its genetics and physiology are not in widespread use and, despite considerable effort, an understanding of its unusual physiology remains at a rudimentary level. Nearly complete genome sequences of A. ferrooxidans are available from two public sources and we have exploited this information to reconstruct aspects of its sulfur metabolism.
Two candidate mechanisms for sulfate uptake from the environment were detected but both belong to large paralogous families of membrane transporters and their identification remains tentative. Prospective genes, pathways and regulatory mechanisms were identified that are likely to be involved in the assimilation of sulfate into cysteine and in the formation of Fe-S centers. Genes and regulatory networks were also uncovered that may link sulfur assimilation with nitrogen fixation, hydrogen utilization and sulfur reduction. Potential pathways were identified for sulfation of extracellular metabolites that may possibly be involved in cellular attachment to pyrite, sulfur and other solid substrates.
A bioinformatic analysis of the genome sequence of A. ferrooxidans has revealed candidate genes, metabolic process and control mechanisms potentially involved in aspects of sulfur metabolism. Metabolic modeling provides an important preliminary step in understanding the unusual physiology of this extremophile especially given the severe difficulties involved in its genetic manipulation and biochemical analysis.
Acidithiobacillus ferrooxidans is a chemolithotrophic, mesophilic, gamma-proteobacterium that lives at pH2. It is found in mine drainage and coal wastes and other acidic environments and is used extensively as part of a consortium of microorganisms for the industrial recovery of metals, such as copper and gold . It can obtain all its energy and electron requirements from the oxidation of various forms of reduced sulfur and ferrous iron. It can also anaerobically reduce sulfur using electrons derived from hydrogen and it can fix nitrogen and carbon dioxide.
The ability of A. ferrooxidans to obtain energy and electrons from sulfur and reduced sulfur compounds has been investigated at the biochemical level. [2–5]. However, less attention has been paid to the mechanisms it uses to take up sulfur (sulfate) from its environment and how it is subsequently assimilated into organic compounds such as the amino acids methionine and cysteine, iron-sulfur centers in electron transfer proteins and various sulfated metabolites . A. ferrooxidans must balance its requirements for sulfate for biosynthesis with its use of sulfur as an energy and electron source. Nothing has been described regarding the regulation of these requirements. Moreover, nothing is known about the possible coordinated regulation of sulfur metabolism and hydrogen utilization and nitrogen fixation that are known from studies in other organisms to impinge upon sulfur metabolism. Furthermore, little is known for any organism about mechanisms for sulfate uptake in acid environments, especially those inhabited by A. ferrooxidans where high concentrations of sulfate ions and potentially competing ions such as molybdenate are frequently found. Although the internal pH of A. ferrooxidans is near neutral, its periplasm and outer membrane are exposed to pH2 and proteins in these locations must fold and function in high concentrations of protons. Questions also arise as to how membrane transport mechanisms function in A. ferrooxidans when confronted by a pH gradient that covers five logs of magnitude, especially considering that in neutrophilic organisms many of these transporters are symporters or antiporters using protons to drive uptake or discharge of metabolites.
Such questions could normally be addressed by a range of genetic and biochemical experiments. Unfortunately, A. ferrooxidans has proved recalcitrant to standard genetic manipulation. There is only one report of transformation in A. ferrooxidans and this may be strain specific and not of general use  and transduction is unknown. Only recently have techniques for conjugation been established and these remain difficult to control and are of low efficiency . Exacerbating the problem is the difficulty of obtaining sufficient cell mass for many biochemical assays. Given these experimental hurdles, we argue that metabolic models derived from bioinformatic analyses offer an especially attractive starting point for unraveling the interesting physiology of A. ferrooxidans. Such studies have already revealed valuable information regarding its amino acid biosynthesis , nitrogen metabolism, metal fluxes and other characteristics .
We have carried out a bioinformatic analysis of the genome sequence of the type strain of A. ferrooxidans ATCC23270 made available by The Institute for Genome Research and Integrated Genomics in an attempt to identify genes and proteins potentially involved in sulfur uptake and assimilation. Using this information, a preliminary model for sulfur assimilation in A. ferrooxidans is presented and possible mechanisms for its biochemical and genetic regulation are discussed. The model serves as a first attempt to understand global sulfur uptake and assimilation in A. ferrooxidans.
Results and Discussion
The results are divided into six sections for convenience of discussion. Section 1 presents the model and lists the genes that constitute the central part of the model. Section 2 describes two possible mechanism for sulfate uptake from the environment. Section 3 describes a potential pathway strongly predicted to be involved in the activation of sulfur and its incorporation into cysteine via adenosine-5'-phosphosulfate (APS). Suggestions are also made as to how this pathway might be regulated. Section 4 identifies a potential pathway for the sulfation of metabolites that might be involved in extra-cellular matrix formation via the PAPS (3'-phosphoadenosine 5'-phosphosulfate) pathway. In Section 5 a proposed connection between cysteine and the formation of Fe-S centers is discussed. We also speculate about the possible role of putative regulatory genes, embedded in predicted regulons for nitrogen fixation and hydrogen oxidation, in sulfur assimilation and sulfur reduction. In Section 6 we have tried to draw together the various postulated regulatory mechanisms in order to paint a more global picture of the regulation of sulfur metabolism in A. ferrooxidans, highlighting aspects of its chemolithoautotrophic physiology.
SECTION 1: Proposed preliminary model for sulfur uptake and assimilation
SECTION 2: Possible sulfate uptake mechanisms
Microorganisms obtain sulfur either from inorganic sulfate or from organosulfur compounds such as sulfonates, sulfate esters, or sulfur-containing amino acids. Potential genes with similarity to those for the uptake of methylthioribose, arylsulfonate, sulfur ester, alkene sulfonate or taurine were not detected in the genome of A. ferrooxidans. However, candidate genes potentially encoding uptake of inorganic sulfate were discovered, including a sulfate permease belonging the SulP family of the major facilitator superfamily of transporters (MFS) and a possible sulfate/thiosulfate/molybdenate ATP binding cassette (ABC)-type transporter.
The proposed SulP exhibits eleven predicted transmembrane helices and shows substantial conservation of amino acid sequence and predicted topology of transmembrane regions and extramembrane loops with similar proteins from a variety of neutrophilic bacteria. SulP contains the following motifs that have been identified for sulfate uptake: COG 0659, PFAM 00916 and eMOTIF IPB001902B. It also matched the sulfate uptake Tigerfam 00815, although the score was below the trusted cutoff. SulP is separated by ten base pairs from an upstream gene potentially encoding a carbonic anhydrase gene (cab1). The putative Cab1 of A. ferrooxidans exhibits the Prosite signature (PS00704) of a beta-type, prokaryotic-type carbonic anhydrase. Carbonic anhydrase catalyzes the formation of HCO3 from CO2 + H+. Whereas SulP functions as a H+ symport in most organisms  the juxtaposition of the putative cab1 and sulP in A. ferrooxidans in a potential transcriptional unit might facilitate the function of SulP as a HCO3 - antiporter rather than as a H+ symporter. Supporting this conjecture is experimental evidence for a SulP-like sulfate-HCO3 - antiporter in rat . In addition, linkage of this gene pair is conserved in several organisms and, in the case of Leptospira interrogans, the couple appear as a gene fusion (data not shown), suggesting that the association represents a functional coupling.
An alternative hypothesis is that SulP/Cab1 can function in the reverse direction, exporting sulfate in order to import carbonate for the enhancement of CO2 fixation. Supporting this idea is the finding that carbonic anhydrase activity is associated with CO2 fixation in a number of autotrophic organisms .
The suggested ABC-type membrane uptake mechanism for sulfate/thiosulfate/molybdate uptake in A. ferrooxidans consists of a membrane spanning protein (CysU), that could form a channel for sulfate, and a membrane associated ATP-binding protein (CysA) that could provide energy for the transmembrane transport process. CysU is predicted to contain six transmembrane regions that align with similar regions in similar proteins. The genes cysU and cysA potentially form an operon-like structure (data not shown). Typically, sulfate is delivered to the ABC-uptake pump by a high affinity soluble periplasmic protein . Two candidate genes for this function were detected in A. ferrooxidans. One of them is embedded in a cluster of genes associated with molybdate uptake and is, therefore, more likely to be a molybdate transporter. Both candidates are members of a large paralogous gene family of ABC transporters  and the identification of their functions awaits experimental investigation. However, bioinformatics analysis has pinpointed them as the most likely candidates for sulfate transporters thus enabling a focus for the experimental scientist.
SECTION 3: Proposed pathway for sulfur assimilation into cysteine via APS
Activation of sulfur via APS
After entry into the cell, sulfate is generally made biologically available by activation with ATP in a reaction catalyzed by ATP sulfurylase, yielding APS and PPi. ATP sulfurylase is a heterodimeric enzyme encoded by genes termed cysD and cysN in many organisms . Potential orthologs of cysD and cysN were identified in A. ferrooxidans (Table 1). In addition, experimental evidence demonstrates that sulfate can be incorporated into APS in A. ferrooxidans .
Reduction of APS to sulfite
A potential gene, termed plr (PAPS-like reductase or Cys-H-like), was found in the genome of A. ferrooxidans whose putative protein has weak similarity with PAPS reductase. It also exhibits weak similarity (e -11) to COG0175 (PAPS reductase) and weak similarity to Tigerfam00434 (PAPS reductase) but with a score below the trusted cutoff. An alignment of its hypothetical amino acid sequence with characterized APS and PAPS reductases reveals that it lacks the conserved signature (KRT)ECG(LS)H found in both APS and PAPS reducatses (Figure 2). However, it does have two of the four critical cysteines displayed by APS reductase (highlighted in blue and bold, Figure 2). Plr is located in a gene cluster (which will be described in section 5.3) that contains cysB and this association raises the possibility that the PAPS-like reductase may play a role in sulfur metabolism. However, at present its function remains unknown.
Reduction of sulfite to sulfide
A possible cysG was also detected in A. ferrooxidans (Table 1 – see additional file 1) potentially encoding a uroporphyrin-III C-methyltransferase that has been shown in other organisms to be involved in siroheme formation and is, therefore, involved in the formation of CysJ. In addition to its bioinformatic identification, there is experimental evidence for the presence of a gene encoding this activity in A. ferrooxidans .
Formation of cysteine from O-acetyl-L-serine and sulfide
The final step of sulfur assimilation into cysteine is the synthesis of L-cysteine from O-acetyl-L-serine and sulfide catalyzed by O-acetyl-L-serine (thiol)-lyase encoded by cysM in E. coli . A cysM candidate gene was identified in A. ferrooxidans (Table 1 – see additional file 1). In most organisms O-acetyl-L-serine is synthesized from serine by the activity of serine acetyltransferase encoded by cysE. Two potential cysE genes, cysE1 and cysE2, were detected in A. ferrooxidans embedded in gene clusters predicted to be involved in the formation of Fe-S clusters and in nitrogen fixation. The possible significance of these genetic contexts with respect to the regulation of cysteine biosynthesis is discussed below.
Genetic organization and regulation of the cys operon
An additional facet of cys operon regulation is that transcriptional activation via CysB requires the presence of the coinducer N-acetylserine. In E. coli and S. typhimurium it has been shown that a reduction in the availability of sulfide leads to the accumulation of O-acetyl-L-serine that spontaneously isomerizes to N-acetyl-L-serine. This latter compound binds to CysB inducing a conformational change that permits the protein to interact with its regulatory sites, upregulating the cys operon and restoring sulfide levels [22, 25, 26]. O-acetyl-L-serine is synthesized via the acetylation of serine by the enzyme serine acetyltranferase that is the product of cysE. It is speculated that the hypothetical CysE1and CysE2, previoiusly mentioned in the formation of cysteine, are also responsible for the formation of N-acetyl-L-serine and therefore play an important role in the regulation of the cys operon.
A second mechanism regulating cys operon activity has been proposed . Excess cysteine is toxic because it promotes oxidative DNA damage by promoting the Fenton reaction  and the synthesis of cysteine can be down-regulated to prevent excess buildup via feedback inhibition on serine acetyltransferase (CysE) by cysteine. This, in turn, limits the production of O-acetyl-L-serine and, since this compound is one of the precursors of cysteine, its diminution causes a concomitant decrease in the synthesis of cysteine. A reduction of CysE activity is also predicted to reduce CysB activity that, in turn, will downregulate the expression of the cys operon.
The notable conservation of the genes and their organization in the proposed cys operon, taken together with the conservation of both the predicted CysB transcriptional activator and CysE co-activator, strongly suggests that A. ferrooxidans can regulate the cys operon in a manner comparable to that described for other organisms. This speculation should provoke future bioinformatic and experimental searches for CysB regulatory binding sites in appropriate regions of the A. ferrooxidans genome. Given this conservation and the fact that the pH of the cytoplasm of A. ferrooxidans is near neutral suggests that the bioinformatic predictions may be open to experimental validation by complementation of well characterized mutants in other organisms such as E. coli (21, 29).
SECTION 4: Proposed sulfation of metabolites via the PAPS pathway
The hypothetical CysNC1 and CysNC2 of A. ferrooxidans exhibit several motifs and key conserved amino acids characteristic of the fused ATP sulfurylase/kinases of other organisms (Figure 6), suggesting that they may carry out a role in PAPS biosynthesis similar to that determined in these organisms . It has also been demonstrated that a segment of A. ferrooxidans DNA, prepared from a randomly cloned genomic library, complemented an E. coli mutant deficient in CysC activity  providing experimental support for the bioinformatic identification of a possible gene encoding ATP sulfurylase/kinase activity.
Fused cysN/cysC genes have been described in Rhizobium sp. where the fusion is termed nodQ . In R. meliloti, NodP (similar to the A. ferrooxidans CysD) and NodQ (similar to the A. ferrooxidans CysNC) are required for the synthesis of PAPS which provides activated sulfur for the modification of the reducing end of Nod, a factor essential for the formation of root nodules . In addition, PAPS is used in S. meliloti for the sulfation of cell surface polysaccharides . There are two copies of nodPQ in R. meliloti, which are functionally redundant in the sense that both copies have to be inactivated to impair nodule formation in alfalfa . PAPS also serves as a substrate in Mycobacteria for the biosynthesis of sulfolipids that may function as virulence factors . In general, sulfated molecules are directed outside the cell and act as modulators of cell-cell communication and cell adhesion and PAPS serves as a universal intermediate in their biosynthesis. Given the juxtaposition of cysNC1 with a gene potentially encoding a fibronectin-like binding protein that may be involved in cell adhesion and the juxtaposition of cysNC2 with a cluster of genes potentially encoding functions involved in capsular biosynthesis (Figure 6), we speculate that A. ferrooxidans also utilizes PAPS as a donor for the sulfation of extra-cellular proteins which may be involved in adhesion of the organism to substrates such as sulfur and pyrite . Genes potentially encoding the SecA translocase system and the PapCD chaperone-usher system were detected in the genome of A. ferrooxidans (data not shown), suggesting that the organism has the capacity to export and assemble adhesive structures.
An alternative, but not necessarily mutually exclusive, role for the function of the fused ATP sulfurylase/kinase protein has been suggested from studies of the kinetic constants of the A. aeolicus enzyme. This enzyme appears to be optimized, not for the formation of PAPS, but in the reverse direction generating ATP and sulfate. Consequently, in addition to its postulated role in the formation of PAPS, it may function in the opposite direction as a route to recycle PPi produced by biosynthetic reactions and for the formation of sulfate in the absence of an inorganic source of sulfate . Therefore, APS appears to lie at a critical metabolic branch point  channeling sulfur to cysteine or to PAPS, where PAPS can serves as a substrate for sulfation of other metabolites, or, in the reverse direction for recycling PPi and sulfate. It will be important in the future to investigate these predictions and to determine the regulatory mechanisms that control the flux of sulfur to the two pathways.
Consistent with the contention that A. ferrooxidans is capable of PAPS formation is the presence of a candidate cysQ, associated with the cysD2NC2 gene cluster, but separated from it by a gene potentially encoding a transposase (Figure 6). CysQ encodes a 3'(2'),5'-bisphosphate nucleotidase that removes the 3'-phosphate from APS to regenerate AMP, helping to drive the APS-PAPS cycle and diminishing the toxicity of PAPS . We speculate that the hypothetical CysQ performs a similar function in A. ferrooxidans.
The candidate CysQ of A. ferrooxidans exhibits the two prosite motifs (PDOC00547) characteristic of the enzyme as shown in the following two segments of the predicted amino acid sequence where the prosite motifs are underlined: WGFFWLVDPLDGTKEFIR KNGEYT and LGPTMEWDTAAAQIIATESGC RVEA the prosite patterns are: [FWV]-x(0,1)-[LIVM]-D-P-[LIVM]-D-[SG]-[ST]-x(2)-[FYA]-x-[HKRNSTY] where the first D and the T bind a metal ion and [WYV]-D-x-[AC]-[GSA]-[GSAPV]-x-[LIVFACP]-[LIVM]-[LIVAC]-x(3)-[GH]-[GA], (the final [GA] is replaced by a [C] in the A. ferrooxidans sequence).
SECTION 5: Formation of iron-sulfur centers and the proposed connection of sulfur metabolism with nitrogen fixation, hydrogen oxidation and sulfur reduction
Proposed isc operon for the formation of Fe-S centers
Cysteine serves as the main source of sulfur for the biosynthesis of Fe-S centers. Proteins with Fe-S centers are key catalysts in metabolism and are central for many processes that involve electron transfer. Fe-S proteins are also involved in sensing and transcriptional control . One route for Fe-S cluster biogenesis is exemplified by a protein complex encoded by the iscSUA operon involved in the formation nitrogenase and other proteins containing Fe-S centers . In this system, iscS (also known as nifS) encodes a cysteine desulfurase that removes an active sulfur from cysteine and icsU and iscA encode proteins that are thought to serve as a scaffold for Fe-S center assembly. Also, frequently forming part of the operon are hscA and hscB that encode chaperones thought to aid in Fe-S center assembly, and fdx encoding a ferredoxin. The negative regulator iscR is generally found upstream of the operon. IscR has been shown to contain a 2Fe-2S center that appears to be required for IscR to bind to its regulatory site and repress transcription. A model has been proposed in which IscR can sense the level of Fe-S center formation and adjust the expression of the isc operon in accordance with requirements for Fe-S center formation .
Additional copies of iscS in a proposed nitrogen fixing regulon
Proposed functional relationship of cysB1 and cysB2 with hoxA and tcrr
Hydrogen is an obligatory by-product of nitrogen fixation in a reaction that consumes about 27% of the electron flux. Some organisms export the hydrogen, while others have genes that encode hydrogen utilization functions permitting them to recover usable energy from the hydrogen, thereby increasing their metabolic efficiency . In addition, a number of organisms can oxidize hydrogen derived from the environment. Hydrogen oxidation can proceed aerobically in which electrons withdrawn from hydrogen reduce oxygen to water via a series of cytochromes resulting in the generation of a proton motif force for energy transduction. Hydrogen oxidation can also occur anaerobically in which the electrons from hydrogen are fed into the APS pathway via a protein complex encoded by the hmc operon (high molecular weight cytochrome) resulting in the reduction of sulfur to sulfide . The HMC complex includes a protein with an iron-sulfur center of the "bacterial-type" 4Fe-4S ferredoxins. A predicted gene cluster potentially encoding an HMC complex was detected in the A. ferrooxidans genome (data not shown) and we propose that A. ferrooxidans has the capacity to feed electrons from hydrogen oxidation into the sulfur reduction pathway.
Since A. ferrooxidans fixes nitrogen  it presumably generates hydrogen in the process. Various strains of A. ferrooxidans have also been shown to oxidize externally supplied hydrogen both aerobically and anaerobically using, in the latter case, Fe+3 or S0 as electron acceptors [49, 50] Unexpectedly, hydrogen did not support the growth of the strain of A. ferrooxidans used in the genome sequencing studies (type strain ATCC23270)  despite the predicted presence of the required hydrogen utilization genes. One possible explanation is that the predicted hydrogen utilization genes are used only for hydrogen oxidation during nitrogen fixation and cannot support growth using an exogenous hydrogen source. Alternatively, the right physiological conditions might not have been used in the experimental design to provoke the activity of the genes. The strong bioinformatic predictions for the presence and conservation of two potential gene clusters predicted to be involved in hydrogen utilization suggest that further experimental work is warranted to reexplore the issue of hydrogen utilization in the type strain of A. ferrooxidans.
The significance of the proposed transcriptional coupling between the predicted cysB and hoxA genes requires investigation. We speculate that simultaneous transcriptional activation of cysB and hoxA promotes the concomitant expression of the hox1 regulon (and possible also the hox2 regulon) and the cys operon. Activation of the cys operon will, in turn, provide additional enzymatic activity to absorb the electron flux delivered by the oxidation of hydrogen. It will also provide increased amounts of cysteine to serve as a source of sulfur for the formation of Fe-S centers used by proteins involved in nitrogen fixation and hydrogen oxidation. In other organisms CysB and HoxA are autorepressors and their predicted juxtaposition in the A. ferrooxidans genome raises additional questions regarding their possible autoregulation and their role in global regulatory control of hydrogen utilization and sulfur metabolism.
A second copy of the cys operon regulator cysB (cysB2) is found adjacent to a gene termed tcrr (Fig. 9B). Tcrr exhibits the CheY-like receiver and DNA binding domains of the AtoC (COG2204.1) family of two component response regulators but it lacks the AAA-like ATPase domain characteristic of this family. It has weak similarity to HoxA (expectancy in Blast = 10 -8)We speculate that the co-transcription of tcrr and cysB2 could simultaneously activate the unknown gene targets of the tcrrA transcriptional regulator and also the cys regulon via the activity of cysB2. Tcrr is predicted to be the receiver partner of a two component transcriptional regulatory system but the activator of this partnership is not known. A consideration that may shed light on the significance of the juxtaposition of the cysB2-tcrr couple is that a potential gene, cobS, was detected that may lie within the same transcriptional unit as cysB2-tcrr (Figure 9B). CobS is predicted to be involved in cobalmin biosynthesis which is required, inter alia, for the conversion of homocysteine to methionine. This impinges on sulfur metabolism because cysteine serves as the source of sulfur for methioine biosynthesis . Furthermore, downstream from cobS, but separated from it by three open reading frames whose predicted proteins have no matches in GenBank, is the putative cysH-like gene plr that was described earlier. Our bioinformatic analysis underscores the need to evaluate the expression and function of plr and the predicted orphan genes since these may encode sulfur related functions peculiar to A. ferrooxidans and, therefore, merit special attention. The best hit of plr is to a phage protein (Table 1 – [see additional file 1]) suggesting that this region may have entered the A. ferrooxidans genome by lateral gene transfer.
SECTION 6. Proposed integration of the regulation of sulfur assimilation with nitrogen fixation, hydrogen oxidation and sulfur reduction
At the heart of the proposed regulatory network is the pathway from sulfate to cysteine via APS, catalyzed by the cys operon cysJIHDN under the control of the positive transcriptional regulator CysB. In the event that the cell requires activation of the nitrogen fixing (nif) operon, the need for sulfur for the Fe-S centers of nitrogenase could be met by activation of the isc cluster in which iscS2 and iscS3 encoding cysteine desulfurase are embedded (Figure 8). The withdrawal of sulfur by cysteine desulfurase will lead to the depletion of cysteine. However, activation of the nif and isc operons is predicted to activate the cysE genes embedded in these operons resulting in the production of N-acetyl-L-serine that, in turn, will activate the cys operon via CysB helping to restore cysteine levels. Nitrogen fixation takes place anaerobically or microaerophilically, an important point that we will return to in the next paragraph.
The hydrogen byproduct of nitrogen fixation is predicted to activate one or both of the hydrogen regulons of A. ferrooxidans via hoxA. Hydrogen utilization requires proteins with Fe-S centers calling again for products of the isc operon. In addition, since the cell is under anaerobic conditions, the electrons from the oxidation of hydrogen will be fed into the APS pathway thereby reducing sulfate to some form of reduced sulfur, such as polysulfate, or polythionate which could be stored for later use as an energy and electron source when aerobic oxidation is restored. Electrons may also be used to reduce sulfate to sulfide. The concomitant increase of electron flux in the sulfur reduction pathway probably requires greater activity of the cys operon genes to provide the additional enzymatic activity to absorb the flux.
Associated with hoxA, the proposed transcriptional regulator of the hox1, is one of the two copies of cysB. Increased CysB activity, coupled with the proposed up-regulated activity of CysE, resulting from the aforementioned activation of the nif regulon, is predicted to increase levels of cys operon expression that, in turn, will help to cope with the increased electron flux from hydrogen oxidation. In addition, transcriptional activation of tcrr could lead to increased expression of both cobS, involved in cobalmin biosynthesis and thus methionine formation, and also of the putative gene plr with weak similarity to cysH.
The presence of redundant copies of both cysB and cysE could reflect the need for alternative routes to activate the cys operon depending on different regulatory signals. This possibility merits further investigation. The model of sulfur regulation also highlights other nodes of potential regulation that deserve further exploration. For example, what are the mechanisms that dictate the flow of activated sulfur to cysteine or to sulfated metabolites at the proposed APS decision point? What regulates the expression of cysQ that is predicted to convert PAPS to APS? Absent from the model, at the present time, are the pathway connections and regulatory mechanisms that could incorporate sulfur oxidation, iron oxidation and iron homeostasis into the description of sulfur assimilation. However, their integration into the model awaits a more adequate description of these processes in A. ferrooxidans.
We posit that the preliminary metabolic model for sulfur uptake and assimilation in A. ferrooxidans presented here has permitted the capture of potentially biologically valid relations that have now been highlighted for experimental validation. By deconvoluting aspects of the metabolic pathway and control mechanisms involved in sulfur metabolism and how these might be integrated with other metabolic processes such as nitrogen fixation, hydrogen utilization and sulfur reduction, we hope to have provided a glimpse of the autotrophic physiology that makes A. ferrooxidans of particular interest. It is also hoped that the work of the experimental scientist has been facilitated by this study, enabling a focus on the possible and likely, pinpointing areas of particular interest and reducing the labor in search of less likely pathways and mechanisms.
The proposed metabolic model provides an important preliminary step in understanding global sulfur metabolism in A. ferrooxidans especially given the extreme difficulties involved in its genetic manipulation and biochemical analysis. It identifies some of the easier and more conspicuous aspects of the regulation of sulfur assimilation. Enfolded within the suggested model are much more complex and subtle mechanisms of regulation whose understanding will require additional bioinformatic analysis and experimental investigation.
Known metabolic pathways of sulfur assimilation were obtained from the MetaCyc database , KEGG  and ERGO . Amino acid sequences derived from genes identified as being involved in sulfur assimilation were used as query sequences to search the partial genome sequence of A. ferrooxidans ATCC 23270 in the TIGR (preliminary sequence data was obtained from The Institute for Genomic Research through the website at http://www.tigr.org) and ERGO  data bases using TblastN and BlastP , respectively, with default parameters. When a prospective candidate gene was identified its predicted amino acid sequence was used to formulate a BlastP search of the non-redundant data base at NCBI. Only bidirectional best hits were accepted as evidence for putative orthologs. Candidate genes and their translated proteins were further characterized employing the following bioinformatic tools available in the web: primary structure similarity relations ClustalW , secondary structure predictions PSI-PRED  and SSpred , transmembrane predictions TMHMM , motif predictions Prosite  and PRINTS , domain predictions ProDom , Pfam  and Tigrfam , prediction of protein localization sites Psort . Information regarding the genetic organization of genes in organisms other than A. ferrooxidans were obtained from Integrated Genomics .
Work supported by Fondecyt No. 1010623. We thank the Institute of Genome Research (TIGR) and Integrated Genomics, Inc. (IG) for the use of their partial sequence of the A. ferrooxidans genome. Sequencing of A. ferrooxidans was accomplished with support from the USA Dept. of Energy (DOE).
- Rawlings DE: Heavy metal mining using microbes. Annu Rev Microbiol. 2002, 56: 65-91. 10.1146/annurev.micro.56.012302.161052.View ArticlePubMedGoogle Scholar
- Rohwerder T, Sand W: The sulfane sulfur of persulfides is the actual substrate of the sulfur-oxidizing enzymes from Acidithiobacillus and Acidiphilium spp. Microbiology. 2003, 149: 1699-1710. 10.1099/mic.0.26212-0.View ArticlePubMedGoogle Scholar
- Ramirez P, Toledo H, Guiliani N, Jerez CA: An exported rhodanese-like protein is induced during growth of Acidithiobacillus ferrooxidans in metal sulfides and different sulfur compounds. Appl Environ Microbiol. 2002, 68: 1837-1845. 10.1128/AEM.68.4.1837-1845.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Masau RJ, Oh JK, Suzuki I: Mechanism of oxidation of inorganic sulfur compounds by thiosulfate-grown Thiobacillus thiooxidans. Can J Microbiol. 2001, 47: 348-358. 10.1139/cjm-47-4-348.View ArticlePubMedGoogle Scholar
- Sugio T, Hirose T, Ye LZ, Tano T: Purification and some properties of sulfite:ferric ion oxidoreductase from Thiobacillus ferrooxidans. J Bacteriol. 1992, 174: 4189-4192.PubMed CentralPubMedGoogle Scholar
- Tuovinen OH, Kelley BC, Nicholas DJ: The uptake and assimilation of sulphate by Thiobacillus ferrooxidans. Arch Microbiol. 1975, 105: 123-127.View ArticlePubMedGoogle Scholar
- Kusano T, Sugawara K, Inoue C, Takeshima T, Numata M, Shiratori T: Electrotransformation of Thiobacillus ferrooxidans with plasmids containing a mer determinant. J Bacteriol. 1992, 174: 6617-6623.PubMed CentralPubMedGoogle Scholar
- Liu Z, Guiliani N, Appia-Ayme C, Borne F, Ratouchniak J, Bonnefoy V: Construction and characterization of a recA mutant of Thiobacillus ferrooxidans by marker exchange mutagenesis. J Bacteriol. 2000, 182: 2269-2276. 10.1128/JB.182.8.2269-2276.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Selkov E, Overbeek R, Kogan Y, Chu L, Vonstein V, Holmes D, Silver S, Haselkorn R, Fonstein M: Functional analysis of gapped microbial genomes: amino acid metabolism of Thiobacillus ferrooxidans. Proc Natl Acad Sci USA. 2000, 97: 3509-3514. 10.1073/pnas.97.7.3509.PubMed CentralView ArticlePubMedGoogle Scholar
- Barreto M, Quatrini R, Bueno S, Arraigada S, Valdes J, Silver S, Jedlicki E, Holmes DS: Aspects of the predicted physiology of Acidithiobacillus ferrooxidans deduced from an analysis of its partial genome sequence. Hydrometallurgy. 2003, 71: 97-105. 10.1016/S0304-386X(03)00145-2.View ArticleGoogle Scholar
- Tripp BC, Smith K, Ferry JG: Carbonic anhydrase: new insights for an ancient enzyme. J Biol Chem. 2001, 52: 48615-48618. 10.1074/jbc.R100045200.View ArticleGoogle Scholar
- Bissig M, Hagenbuch B, Stieger B, Koller T, Meier PJ: Functional expression cloning of the canalicular sulfate transport system of rat hepatocytes. J Biol Chem. 1994, 269: 3017-3021.PubMedGoogle Scholar
- Jacobson BL, He JJ, Vermersch PS, Lemon DD, Quiocho FA: Engineered interdomain disulfide in the periplasmic receptor for sulfate transport reduces flexibility. Site-directed mutagenesis and ligand-binding studies. J Biol Chem. 1991, 266: 5220-5225.PubMedGoogle Scholar
- Holland B, Blight MA: ABC-ATPases, adaptable energy generators fuelling transmembrane movement of a variety of molecules in organisms from bacteria to humans. J Mol Biol. 1999, 293: 381-399. 10.1006/jmbi.1999.2993.View ArticlePubMedGoogle Scholar
- Leyh TS, Taylor JC, Markham GD: The sulfate activation locus of Escherichia coli K12: cloning, genetic, and enzymatic characterization. J Biol Chem. 1988, 263: 2409-2416.PubMedGoogle Scholar
- Berendt U, Haverkamp T, Prior A, Schwenn JD: Reaction mechanism of thioredoxin: 3'-phospho-adenylylsulfate reductase investigated by site-directed mutagenesis. Eur J Biochem. 1995, 233: 347-356.View ArticlePubMedGoogle Scholar
- Kopriva S, Buchert T, Fritz G, Suter M, Benda R, Schunemann V, Koprivova A, Schurmann P, Trautwein AX, Kroneck PM, Brunold C: The presence of an iron-sulfur cluster in adenosine 5'-phosphosulfate reductase separates organisms utilizing adenosine 5'-phosphosulfate and phosphoadenosine 5'-phosphosulfate for sulfate assimilation. J Biol Chem. 2002, 277: 21786-21791. 10.1074/jbc.M202152200.View ArticlePubMedGoogle Scholar
- Kredich NM: Biosynthesis of Cysteine. In: Escherichia coli and Salmonella. Edited by: Neidhardt FC. 1996, ASM Press, Washington, DC, 1: 514-527. 2Google Scholar
- Nakayama M, Akashi T, Hase T: Plant sulfite reductase: molecular structure, catalytic function and interaction with ferredoxin. J Inorg Biochem. 2000, 82: 27-32. 10.1016/S0162-0134(00)00138-0.View ArticlePubMedGoogle Scholar
- Crane BR, Siegel LM, Getzoff ED: Sulfite reductase structure at 1.6 Å: evolution and catalysis for reduction of inorganic anions. Science. 1995, 270: 59-67.View ArticlePubMedGoogle Scholar
- Sugio T, Suzuki H, Tanaka T, Matsugi S, Tanaka K, Kanao T, Tano T: A DNA region that complements on Escherichia coli cysG mutation in Thiobacillus ferrooxidans. Biosci Biotechnol Biochem. 1995, 59: 728-730.View ArticlePubMedGoogle Scholar
- Lochowska A, Iwanicka-Nowicka R, Plochocka D, Hryniewicz MM: Functional dissection of the LysR-type CysB transcriptional regulator. Regions important for DNA binding, inducer response, oligomerization, and positive control. J Biol Chem. 2001, 276: 2098-2107. 10.1074/jbc.M007192200.View ArticlePubMedGoogle Scholar
- Ostrowski J, Kredich NM: Negative autoregulation of cysB in Salmonella typhimurium: in vitro interactions of CysB protein with the cysB promoter. J Bacteriol. 1991, 173: 2212-2218.PubMed CentralPubMedGoogle Scholar
- Hryniewicz MM, Kredich NM: Hydroxyl radical footprints and half-site arrangements of binding sites for the CysB transcriptional activator of Salmonella typhimurium. J Bacteriol. 1995, 177: 2343-2353.PubMed CentralPubMedGoogle Scholar
- Hryniewicz MM, Kredich NM: The cysP promoter of Salmonella typhimurium : characterization of two binding sites for CysB protein, studies of in vivo transcription initiation, and demonstration of the anti-inducer effects of thiosulfate. J Bacteriol. 1991, 173: 5876-5886.PubMed CentralPubMedGoogle Scholar
- Monroe RS, Ostrowski J, Hryniewicz MM, Kredich NM: In vitro interactions of CysB protein with the cysK and cysJIH promoter regions of Salmonella typhimurium. J Bacteriol. 1990, 172: 6919-6929.PubMed CentralPubMedGoogle Scholar
- Park S, Imlay JA: High levels of intracellular cysteine promote oxidative DNA damage by driving the fenton reaction. J Bacteriol. 2003, 185: 1942-1950. 10.1128/JB.185.6.1942-1950.2003.PubMed CentralView ArticlePubMedGoogle Scholar
- Schwedock JS, Liu C, Leyh TS, Long SR: Rhizobium meliloti NodP and NodQ form a multifunctional sulfate-activating complex requiring GTP for activity. J Bacteriol. 1994, 176: 7055-7064.PubMed CentralPubMedGoogle Scholar
- Fry IJ, Garcia E: Cloning and caracterization of Thiobacillus ferrooxidans genes involved in sulfur assimilation. In Biohydrometallurgy. Edited by: Salley J, McCready RGL, Wichlacz PL. 1989, 171-185.Google Scholar
- Laeremans T, Coolsaet N, Verreth C, Snoeck C, Hellings N, Vanderleyden J, Martinez-Romero E: Functional redundancy of genes for sulphate activation enzymes in Rhizobium sp. BR816. Microbiology. 1997, 143: 3933-3942.View ArticlePubMedGoogle Scholar
- Abola AP, Willits MG, Wang RC, Long SR: Reduction of adenosine-5'-phosphosulfate instead of 3'-phosphoadenosine-5'-phosphosulfate in cysteine biosynthesis by Rhizobium meliloti and other members of the family Rhizobiaceae. J Bacteriol. 1999, 181: 5280-5287.PubMed CentralPubMedGoogle Scholar
- Keating DH, Willits MG, Long SR: A Sinorhizobium meliloti lipopolysaccharide mutant altered in cell surface sulfation. J Bacteriol. 2002, 184: 6681-6689. 10.1128/JB.184.23.6681-6689.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Schwedock JS, Long SR: Rhizobium meliloti genes involved in sulfate activation: the two copies of nodPQ and a new locus, saa. Genetics. 1992, 132: 899-909.PubMed CentralPubMedGoogle Scholar
- Williams SJ, Senaratne RH, Mougous JD, Riley LW, Bertozzi CR: 5'-adenosinephosphosulfate lies at a metabolic branch point in mycobacteria. J Biol Chem. 2002, 277: 32606-3215. 10.1074/jbc.M204613200.View ArticlePubMedGoogle Scholar
- Gehrke T, Hallmann R, Kinzler K, Sand W: The EPS of Acidithiobacillus ferrooxidans a model for structure-function relationships of attached bacteria and their physiology. Water Sci Technol. 2001, 43: 159-167.PubMedGoogle Scholar
- Hanna E, MacRae IJ, Medina DC, Fisher AJ, Segel IH: ATP sulfurylase from the hyperthermophilic chemolithotroph Aquifex aeolicus. Arch Biochem Biophys. 2002, 406: 275-288. 10.1016/S0003-9861(02)00428-9.View ArticlePubMedGoogle Scholar
- Neuwald AF, Krishnan BR, Brikun I, Kulakauskas S, Suziedelis K, Tomcsanyi T, Leyh TS, Berg DE: cysQ, a gene needed for cysteine synthesis in Escherichia coli K-12 only during aerobic growth. J Bacteriol. 1992, 174: 415-425.PubMed CentralPubMedGoogle Scholar
- Kiley PJ, Beinert H: The role of Fe-S proteins in sensing and regulation in bacteria. Current Opinion Microbiol. 2003, 6: 181-185. 10.1016/S1369-5274(03)00039-0.View ArticleGoogle Scholar
- Yuvaniyama P, Agar JN, Cash VL, Johnson MK, Dean DR: NifS-directed assembly of a transient [2Fe-2S] cluster within the NifU protein. Proc Natl Acad Sci. 2000, 97 : 599-604. 10.1073/pnas.97.2.599.PubMed CentralView ArticlePubMedGoogle Scholar
- Frazzon J, Dean DR: Feedback regulation of iron-sulfur cluster biosynthesis. Proc Nal Acad Sci. 2001, 98: 14751-14753. 10.1073/pnas.011579098.View ArticleGoogle Scholar
- Persson BC, Jager G, Gustafsson C: The spoU gene of Escherichia coli, the fourth gene of the spoT operon, is essential for tRNA (Gm18) 2'-O-methyltransferase activity. Nucleic Acids Res. 1997, 25: 4093-4097. 10.1093/nar/25.20.4093.PubMed CentralView ArticlePubMedGoogle Scholar
- Iciek M, Wodek L: Biosynthesis and biological properties of compounds containing highly reactive, reduced sulfane sulfur. Pol J Pharmacol. 2001, 53: 215-225.PubMedGoogle Scholar
- Huang TC, Lin RF, Chu MK, Chen HM: Organization and expression of nitrogen-fixation genes in the aerobic nitrogen-fixing unicellular cyanobacterium Synechococcus sp. strain RF-1. Microbiology. 1999, 145: 743-753.View ArticlePubMedGoogle Scholar
- Van Soom C, Verreth C, MJ Vanderleyden J: Identification of a potential transcriptional regulator of hydrogenase activity in free-living Bradyrhizobium japonicum strains. Mol Gen Genet. 1993, 239: 235-240.View ArticlePubMedGoogle Scholar
- Durmowicz MC, Maier RJ: Roles of HoxX and HoxA inbiosynthesis of hydrogenase in Bradyrhizobium japonicum. J Bacteriol. 179: 3676-3682.Google Scholar
- Lenz O, Bernhard M, Buhrke T, Schwartz E, Friedrich B: The hydrogen-sensing apparatus in Ralstonia eutropha. J Mol Microbiol Biotechnol. 2002, 4: 255-262.PubMedGoogle Scholar
- Rossi M, Pollock WB, Reij MW, Keon RG, Fu R, Voordouw G: The HMC operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex. J Bacteriol. 1993, 175: 4699-4711.PubMed CentralPubMedGoogle Scholar
- Mackintosh ME: Nitrogen fixation in Thiobacillus ferrooxidans. J Gen Microbiol. 1978, 105: 215-218.View ArticleGoogle Scholar
- Drobner E, Huber H, Stetter KO: Thiobacillus ferrooxidans, a facultative hydrogen oxidizer. Appl Environ Microbiol,. 1990, 56: 2922-2923.Google Scholar
- Ohmura N, Sasaki K, Matsumoto N, Saiki H: Anaerobic respiration using Fe(3+), S(0), and H(2) in the chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans. J Bacteriol. 2002, 184: 2081-2087. 10.1128/JB.184.8.2081-2087.2002.PubMed CentralView ArticlePubMedGoogle Scholar
- Roth JR, Lawrence JG, Bobik TA: Cobalmin (Coenzyme B12): Synthesis and Biological Significance. Annu Rev Microbiol. 1996, 50: 137-181. 10.1146/annurev.micro.50.1.137.View ArticlePubMedGoogle Scholar
- Karp PD, Riley M, Paley SM, Pellegrini-Toole A: The MetaCyc database. Nucleic Acids Res. 2002, 30: 59-61. 10.1093/nar/30.1.59.PubMed CentralView ArticlePubMedGoogle Scholar
- Kanehisa M, Goto S, Kawashima S, Nakaya A: The KEGG databases at GenomeNet. Nucleic Acids Res. 2002, 30: 42-46. 10.1093/nar/30.1.42.PubMed CentralView ArticlePubMedGoogle Scholar
- Overbeek R, Larsen N, Walunas T, D'Souza M, Pusch G, Selkov EJr, Liolios K, Joukov V, Kaznadzey D, Anderson I, Bhattacharyya A, Burd H, Gardner W, Hanke P, Kapatral V, Mikhailova N, Vasieva O, Osterman A, Vonstein V, Fonstein M, Ivanova N, Kyrpides N: The ERGO genome analysis and discovery system. Nucleic Acids Res. 2003, 31: 164-171. 10.1093/nar/gkg148.PubMed CentralView ArticlePubMedGoogle Scholar
- Altschul SF, Madden TL, Scha!er AA, Zhang J, Zheng Z, Miller W, Lipman DJ: Gapped BLAST and PSIBLAST – A new generation of protein database search programs. Nucleic Acids Res. 1997, 25: 3389-3402. 10.1093/nar/25.17.3389.PubMed CentralView ArticlePubMedGoogle Scholar
- Higgins D, Thompson J, Gibson T, Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994, 22: 4673-4680.PubMed CentralView ArticlePubMedGoogle Scholar
- Jones DT: Protein secondary structure prediction based on position-specific scoring matrices. J Mol Biol. 1999, 292: 195-202. 10.1006/jmbi.1999.3091.View ArticlePubMedGoogle Scholar
- Pollastri G, Przybylski D, Rost B, Baldi P: Improving the prediction of protein secondary structure in three and eight classes using recurrent neural networks and profiles. Proteins. 2002, 47: 228-235. 10.1002/prot.10082.View ArticlePubMedGoogle Scholar
- Krogh A, Larsson B, vonHeijne G, Sonnhammer ELL: Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes. J Mol Biol. 2001, 305: 567-580. 10.1006/jmbi.2000.4315.View ArticlePubMedGoogle Scholar
- Hofmann K, Bucher P, Falquet L, Bairoch A: The PROSITE database, its status in 1999. Nucleic Acids Res. 1999, 27: 215-219. 10.1093/nar/27.1.215.PubMed CentralView ArticlePubMedGoogle Scholar
- Attwood TK, Beck ME, Bleasby AJ, Parry-Smith DJ: PRINTS – A database of protein motif fingerprints. Nucleic Acids Res. 1994, 22: 3590-3596.PubMed CentralPubMedGoogle Scholar
- Servant F, Bru C, Carrère S, Courcelle E, Gouzy J, Peyruc D, Kahn D: ProDom: Automated clustering of homologous domains. Briefings in Bioinformatics. 2002, 3: 246-251.View ArticlePubMedGoogle Scholar
- Bateman A, Birney E, Cerruti L, Durbin R, Etwiller L, Eddy SR, Griffiths-Jones S, Howe KL, Marshall M, Sonnhammer EL: The Pfam protein families database. Nucleic Acids Res. 2002, 30: 276-280. 10.1093/nar/30.1.276.PubMed CentralView ArticlePubMedGoogle Scholar
- Haft DH, Selengut JD, White O: The TIGRFAMs database of protein families. Nucleic Acids Res. 2003, 31: 371-373. 10.1093/nar/gkg128.PubMed CentralView ArticlePubMedGoogle Scholar
- Nakai K, Horton P: PSORT: a program for detecting sorting signals in proteins and predicting their subcellular localization. Trends Biochem Sci. 1999, 24: 34-36. 10.1016/S0968-0004(98)01336-X.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL.