Hydrometallurgy 94 (2008) 180–184

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Comparative genome analysis of ferrooxidans, A. thiooxidans and A. caldus: Insights into their metabolism and ecophysiology

Jorge Valdés 1, Inti Pedroso, Raquel Quatrini, David S. Holmes ⁎

Center for Bioinformatics and Genome Biology, Life Science Foundation, MIFAB and Andrés Bello University, Santiago, Chile

ARTICLE INFO ABSTRACT

Available online 2 July 2008 Draft genome sequences of Acidthiobacillus thiooxidans and A. caldus have been annotated and compared to the previously annotated genome of A. ferrooxidans. This has allowed the prediction of metabolic and Keywords: regulatory models for each species and has provided a unique opportunity to undertake comparative Acidithiobacillus genomic studies of this group of related . In this paper, the presence or absence of Bioinformatics predicted genes for eleven metabolic processes, electron transfer pathways and other phenotypic Comparative genomics fi Metabolic integration characteristics are reported for the three acidithiobacilli: CO2 xation, the TCA cycle, oxidation, sulfur reduction, iron oxidation, iron assimilation, quorum sensing via the acyl homoserine lactone mechanism, hydrogen oxidation, flagella formation, Che signaling (chemotaxis) and nitrogen fixation. Predicted transcriptional and metabolic interplay between pathways pinpoints possible coordinated responses to environmental signals such as energy source, oxygen and nutrient limitations. The predicted pathway for nitrogen fixation in A. ferrooxidans will be described as an example of such an integrated response. Several responses appear to be especially characteristic of autotrophic microorganisms and may have direct implications for metabolic processes of critical relevance to the understanding of how these microorganisms survive and proliferate in extreme environments, including industrial bioleaching operations. © 2008 Elsevier B.V. All rights reserved.

1. Introduction biologist to focus on key issues suggested by the bioinformatic predictions, thus saving considerable time and resources (Quatrini Acidophilic prokaryotes involved in bioleaching process have been et al., 2007a). the subject of active research from the viewpoints of microbiology, The bioinformatic analysis of the genome sequence of the γ- biophysics, biochemistry and genetics. With the advent of genome proteobacterium A. ferrooxidans has led to the generation of several sequencing and the continuously decreasing costs and time needed to metabolic and regulatory models some of which have been experimen- sequence new genomes, novel opportunities and challenges have tally validated in part, such as sulfur uptake and assimilation (Valdés et arisen for developing integrative metabolic and regulatory models al., 2003), iron uptake and assimilation (Quatrini et al., 2005; Osorio et that couple traditional experimental data sources with information al., 2008-this issue), Fur regulatory gene circuits (Quatrini et al., 2007b), obtained from computationally-derived comparative genome infor- quorum sensing (Rivas et al., 2005; Farah et al., 2005; Rivas et al., 2007), mation. Advanced bioinformatic tools, developed in the last few years, biofilm formation (Barreto et al., 2005), small regulatory RNA gene have allowed relatively rapid ways to generate initial models of prediction (Shmaryahu and Holmes, unpublished results) and carbon metabolic and regulatory features potentially encoded within a metabolism (Barreto et al., 2003; Appia-Ayme et al., 2006). An analysis of genome and to pinpoint key features that can be highlighted for the genome of A. ferrooxidans has also supported and extended earlier subsequent experimental validation. models of iron and sulfur oxidation in this microorganism (Quatrini Chemolithoautotrophic microorganisms in general, and the extreme et al., 2006; Valdés et al., unpublished results). acidophilic ones in particular, are difficult to handle experimentally and Large-scale comparisons of genomes address basic questions, such can be recalcitrant to genetic manipulation. These hurdles can be as the number of functional genes, identification of species-specific compensated for, in part, by the enormous amount of information that genes, distribution of genes among functional families, gene density, can be extracted by careful genome analysis, allowing the experimental preservation of gene order, mechanisms of genome reshuffling, the rate of sequence divergence, etc. (Hardison, 2003). In general, the choice of species that mark evolutionary distances for comparative genomics depends on the aim of the analysis. ⁎ Corresponding author. Tel.: +56 2 239 8969; fax: +56 2 237 2259. In the present study, the genomes of three closely related gram- E-mail addresses: [email protected] (J. Valdés), [email protected] (D.S. Holmes). negative, chemoautotrophic bioleaching microorganisms, A. ferroox- 1 Fax: +56 2 237 2259. idans, A. thiooxidans and A. caldus, were compared, anticipating that

0304-386X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2008.05.039 J. Valdés et al. / Hydrometallurgy 94 (2008) 180–184 181 such a study would help to consolidate the prediction and identifica- tion of genes and conserved gene clusters involved in various predicted metabolic models and that the identification of conserved intergenic regions between the three species would facilitate the prediction of small regulatory RNA genes and transcriptional regulatory regions. It was also hoped that it would allow the identification of genes involved in iron oxidation specificto A. ferrooxidans and might, in the long term, suggest the genetic basis of other potential differences between the three species such as the ability of A. caldus to grow at 45–55 °C compared to the mesophilic temperatures of the other two species (Hallberg and Lindstrom, 1994). Most importantly, it was hoped that a comparative genomics study could identify novel genetic differences between the three species, suggest models of the evolution of their respective genomes and allows the prediction of mutual interactions between the three acidithiobacilli that might occur in their natural environment and in operations (ecophysiology).

2. Methods

The complete genome sequence of Acidithiobacillus ferrooxidans ATCC 23270 was obtained from the Institute for Genomic Research database (TIGR, Valdés et al., unpublished results). Draft genome sequences of Fig. 1. Schematic representation of the bioinformatics pipeline used in this study. A. thiooxidans ATCC 19377 and A. caldus ATCC 51756 were obtained from Abbreviations: ACT, Artemis comparison tool; NR-DB, Non-redundant database; MG- the Center for Bioinformatics and Genome Biology (CBGB), Santiago, DB, microbial genomes database. Additional information can be found in the Methods. Chile. Candidate protein coding genes were identified in genome sequences using Glimmer (Salzberg et al., 1998), Critica (Badger and 3.2. Whole genome comparison Olsen,1999)andBlastX(Altschul et al.,1997). The 5′ and 3′regions of each ORF were inspected to define initiation codons using homologies, To identify genomic regions with significant similarity, predicted position of ribosomal binding sites, and transcriptional terminators. protein comparisons were performed between the A. ferrooxidans The following bioinformatic programs were used to further complete genome sequence and the two draft genomes sequences of characterize candidate genes and their predicted protein products: A. caldus and A. thiooxidans. Fig. 2 shows a similarity map that BlastP and PsiBlast (Altschul et al., 1997), the suite of protein facilitates the identification of regions in the A. ferrooxidans genome comparison and classification programs available in InterproScan that have no counterparts in the other two genomes. This first step is (Mulder and Apweiler, 2007). Model metabolic pathways were required for a rapid identification of potential functional modules reconstructed using PRIAM (Claudel-Renard et al., 2003)and present in the model organism A. ferrooxidans and their respective compared to those obtained from BIOCYC (Caspi et al., 2008), KEGG locations in the genome sequence. Most of the exclusive regions of the (Kanehisa et al., 2008) and ERGO (Overbeek et al., 2003). Amino acid A. ferrooxidans genome are characterized by abnormal GC content and sequences derived for the genes identified in the genome sequence of GC skew signatures. Fig. 2 has four highlighted regions that A. ferrooxidans ATCC 23270 and draft genome sequences for A. caldus correspond to examples of conserved/non-conserved genomic loci: ATCC 51756 and A. thiooxidans ATCC 19377 were annotated, analyzed (A) pet-I and rus operons involved in iron oxidation in A. ferrooxidans. and compared to known metabolic models using perl scripts These loci are absent in the other two acidithiobacilli. They are located developed in our laboratory. The annotated genomes were displayed close to, and on either side of, the origin of replication; a position which in the interactive format of Artemis (Rutherford et al., 2000). provides duplicated copies early in the DNA replication cycle. It is not The genomes were compared at the amino acid level using tblastx known whether this location is important to augment the copy number − 5 (Altschul et al., 1997) using an e-value of 10 as cutoff to identify of the iron oxidation genes to provide increased transcriptional activity; syntenic regions. Syntenic regions were displayed and analyzed using B) nitrogen fixation gene cluster in A. ferrooxidans absent in the other the Artemis comparison tool (ACT) (Carver et al., 2005)and two acidithiobacilli; (C) large region present in A. ferrooxidans that is Genomeviz (Ghai and Chakraborty, 2007). absent in the other two acidithiobacilli. This region is rich in predicted hypothetical genes and potential mobile sequences including predicted 3. Results and discussion plasmid and phage elements suggesting that this is a region acquired in A. ferrooxidans after its divergence from the last common ancestor of the 3.1. Comparative genomics pipeline three acidithiobacilli. However, this hypothesis requires substantiation by phylogenic analysis. It also remains to be investigated if the A bioinformatic pipeline was developed to undertake the genetic hypothetical genes in this region provide A. ferrooxidans with species- and metabolic comparison of the three acidothiobacilli (Fig. 1). The specific properties; (D) a region that is highly conserved between the pipeline is composed of two initial bioinformatics steps that cooperate three acidithiobacilli that is mainly composed of genes predicted to to generate the searchable Structural Query Language (SQL) server encode rRNAs, ribosomal proteins and other genes related to protein termed the “Acidthiobacillus database” that is at the core of the synthesis functions. analysis. These steps are i) open reading frame (ORF) prediction using Glimmer and Critica where predicted ORFs are then compared to 3.3. Function predictions annotated genes deposited in NR-DB and MG-DB using InterproScan, PRIAM and Blast in order to assign potential function and ii) BlastX of A comparative analysis of the genomes of A. ferrooxidans and the the genomes against the NR-DB to improve the annotation developed other acidithiobacilli revealed fundamental similarities and differ- in (i). The SQL database was then mined and results visualized using ences that reflect their specific metabolic capacities. Table 1 lists a four tools: Artemis, ACT, GenomeViz and KEGG maps. number of predicted functions for each of the three acidithiobacilli. All 182 J. Valdés et al. / Hydrometallurgy 94 (2008) 180–184

Fig. 2. Whole genome comparison of the A. ferrooxidans genome and the two draft genomes of A. caldus and A. thiooxidans. First circle, examples of conserved/non-conserved regions in the three acidithiobacilli. A–D, information discussed in the text; second outer circle (green), A. caldus blast hits mapped against the A. ferrooxidans genome; third circle (orange), A. thiooxidans blast hits mapped against the A. ferrooxidans genome; fourth and fifth circles, A. ferrooxidans protein coding genes; sixth circle, A. ferrooxidans GC content; seventh circle, A. ferrooxidans GC skew. The predicted origin of replication is located at “12 o'clock”.

three have genes for the Calvin-Benson scheme for CO2 fixation and differences point to distinctive functional roles in iron management lack the gene for α-keto glutarate dehydrogenase suggesting that they between the three acidithiobacilli and the future study of these have an incomplete TCA cycle, as has been found in all obligate distinctions will contribute to our understanding of the ecophysiology chemoautotrophs to date. However, they differ in the genes predicted and general survival strategies in acidic and iron loaded environments. to be involved in sulfur oxidation. A. thiooxidans and A. caldus have a predicted suite of essential soxABXYZ genes with similarity to the well studied SOX (sulfur oxidases) system (Friedrich et al., 2001). In Table 1 contrast, A. ferrooxidans lacks genes encoding the SOX system but has Presence (Yes) or absence (No = not detected) of predicted genes for several metabolic genes with similarity to those encoding SQR (sulfide quinone and phenotypic characteristics of A. ferrooxidans, A. caldus and A. thiooxidans based on oxidoreductases), TQR (thiosulfate quinone oxidoreducatase) and bioinformatics genome comparisons TetH (tetrathionate hydrolase), suggesting that it has evolved a Microorganism A. ferrooxidans A. thiooxidans A. caldus different mechanism(s) for sulfur oxidation. Predicted genes for:

As predicted from experimental evidence, A. caldus and CO2 fixation Calvin-Benson Calvin-Benson Calvin-Benson A. thiooxidans lack the genes that permit A. ferrooxidans to oxidize TCA cycle Incomplete Incomplete Incomplete ferrous iron. In A. ferrooxidans the genes coding for the iron oxidation S oxidation SQR system SOX system SOX system S reduction Yes No No system are organized in two transcriptional units, the petI (petC-1, Fe(II) oxidation Yes No No petB-1, petA-1, sdrA-1 and cycA-1) and rus (cyc2, cyc1, hyp, coxB, coxA No. of Fe(III) uptake OMRs 11 8 6 coxC, coxD and rus) operons (Holmes and Bonnefoy, 2006 and Quorum sensing by the AHL system Yes No No references therein) that were not found by similarity searches. Hydrogen oxidation Yes NI Yes Regarding iron assimilation and homeostasis, A. ferrooxidans has Flagella formation No Yes Yes Che signaling No Yes Yes more predicted outer membrane receptors (OMRs) for Fe(III) side- Nitrogen fixation Yes No No rophores than either of the other two acidithiobacilli (Table 1). There SQR = sulfide quinone oxidoreductase; SOX = sulfur oxidase; APS = adenosine-5′- are also differences in the number of TonB and ABC transporters phosphosulfate; PAPS = 3′ phosphoadenosine-5′-phosphosulfate; OMRs = outer involved in Fe(III) siderophore uptake between the three microorgan- membrane Fe(III) siderophore receptors; AHL = acyl homoserine lactone; NI = no isms as well as Fe(II) uptake systems (data not shown). These information. J. Valdés et al. / Hydrometallurgy 94 (2008) 180–184 183

Fig. 3. Predicted metabolic and regulatory interplay between nitrogen and carbon metabolism in A. ferrooxidans. Proposed connections to sulfur, hydrogen and energy are not shown for simplicity.

For example, the significantly larger number of Fe(III) siderophore over NifA) and post-translational level (by the reversible modification OMRs found in A. ferrooxidans might help to explain its greater of the nitrogenase enzyme by the Drag/DraT proteins) and ammonia sensitivity to Fe(III) rich environments. availability in many bacteria (Dixon and Kahn, 2004). Some strains of A. ferrooxidans exhibit chemotaxis to thiosulfate Nitrogen metabolism has a critical signal transduction component, (Chakraborty and Roy, 1992) and have single coiled flagellum and pili the P-II protein, that is encoded by the genes glnK and glnB in bacteria (DiSpirito et al., 1982; Gonzalez and Cotoras, 1987; Kelly and Wood, and archaea. These genes are predicted in the A. ferrooxidans genome. 2000) that can mediate the attachment to sulfur (Ohmura et al., 1996). The active form of P-II is a trimer that can inhibit the activity of NtrB However, the genome of the type strain of A. ferrooxidans lacks genes and represses activity of the ammonia transporter encoded by amtB for both flagella formation and Che two-component signaling (Dixon and Kahn, 2004). NtrB is a positive regulator that, via NifA, transduction. This makes it unlikely that it can carry out chemotaxis, activates the operon used to fix nitrogen (N2-ase in Fig. 3). NifL is a at least by the well-studied pathways and suggests that the species negative regulator of the N2-ase operon that transduces the signal A. ferrooxidans has a diverse phenotype. The other two acidithiobacilli generated by O2 concentration. Hence, in the presence of P-II and/or contain genes for both properties. A word of caution is needed to point oxygen, nitrogen cannot be fixed. However, in the presence of ATP and out that all three sequenced genomes come from strains grown in alpha-ketoglutarate P-II is inactivated (Ninfa and Jiang, 2005). Thus laboratory conditions prior to being deposited in culture banks, when energy is abundant and there is not enough reduced nitrogen in meaning that original properties of the environmental founders could the form of ammonia and glutamine, information can be channeled have been lost during passage in the laboratory. This is unlikely in the from the CO2 fixation machinery via the TCA cycle and energy status case of the Che genes because they are typically distributed via ATP synthesis to inactivate P-II and allow N2 fixation in the absence throughout the genome, meaning that multiple excision events of O2. would be necessary for their elimination. The flagella genes, on the However, in leaner times P-II is not inactivated and so the energy other hand, are often located in one large gene cluster (about 30 that could have been used for N2 fixation can be used by other genes) and so could be lost by one excision event. metabolic processes. A. ferrooxidans exhibits candidate genes for the

Only A. ferrooxidans has genes for homoserine lactone-based above mentioned activities and can thus balance the fixation of N2 and quorum sensing and these been experimentally demonstrated to importation of NH4 according to metabolic circumstances and oxygen occur through the classic LuxIR system (Rivas et al., 2005; Farah et al., concentration, whereas A. thiooxidans and A. caldus lack the N2-ase 2005). A. ferrooxidans also has a potential quorum sensing system operon and must rely on A. ferrooxidans for fixed nitrogen, presumably + based on act gene cluster activity; alternatively this system may be supplied as NH4. involved in cell membrane formation and the two possibilities remain to be explored experimentally (Rivas et al., 2007). 4. Conclusions A. ferrooxidans is the only one of the three to have predicted nitrogenase genes and so is the only one to be able to fix atmospheric Comparative genomics provides a rich source of information for nitrogen as experimentally demonstrated for A. ferrooxidans by improving gene annotations, assigning gene functions, identifying Mackintosh (Mackintosh, 1978). This means that, in a consortium of potential regulatory regions and for building metabolic models. It also the three bacteria and in the absence of fixed nitrogen such as provides insight into gene and genome evolution. Comparison of the ammonia, A. ferrooxidans would have to support the entire commu- closely related A. ferrooxidans, A. thiooxidans and A. caldus genomes nity's requirements for nitrogen. This prediction remains to be tested has identified sets of genes that could be responsible for differences in but could potentially have important consequences in bioleaching iron and sulfur oxidation in these organisms. It also suggests that fixed operations and natural communities where sources exogenous fixed nitrogen can only be supplied by A. ferrooxidans, suggesting that it is nitrogen might be limiting. the major contributor of nitrogen in a consortium of the three bacteria such as might be found in a bioleaching heap. Surprisingly, the 3.4. Interplay between assimilatory pathways in acidithiobacilli using genome of the type strain of A. ferrooxidans does not encode flagella or nitrogen fixation in a. ferrooxidans as an example the che signaling genes associated with chemotaxis whereas the other two acidithiobacilli have these capacities. In this study we have identified several regulatory components that provide potential for crosstalk between assimilatory and anabolic Acknowledgments pathways. Most of the assimilatory processes detected have home- ostatically controlled systems to detect the bioavailability of a specific Work supported by Fondecyt 1050063, Fondecyt 11060164, DI- element. Nitrogen metabolism is mainly regulated by oxygen UNAB 34-06, DI-UNAB 15-06/I and a Microsoft Sponsored Research concentration at the transcriptional (by the repressor activity of NifL Award. 184 J. Valdés et al. / Hydrometallurgy 94 (2008) 180–184

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