Comparative genome analysis of Acidithiobacillus 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 ABSTRACT ARTICLE INFO Available online 2 July 2008 Keywords: Acidithiobacillus Bioinformatics Comparative genomics Metabolic integration 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 regulatory models for each species and has provided a unique opportunity to undertake comparative genomic studies of this group of related bioleaching bacteria. In this paper, the presence or absence of predicted genes for eleven metabolic processes, electron transfer pathways and other phenotypic characteristics are reported for the three acidithiobacilli: CO 2 fixation, the TCA cycle, sulfur 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 Acidophilic prokaryotes involved in bioleaching process have been the subject of active research from the viewpoints of microbiology, biophysics, biochemistry and genetics. With the advent of genome sequencing and the continuously decreasing costs and time needed to sequence new genomes, novel opportunities and challenges have arisen for developing integrative metabolic and regulatory models that couple traditional experimental data sources with information obtained from computationally-derived comparative genome infor- mation. Advanced bioinformatic tools, developed in the last few years, have allowed relatively rapid ways to generate initial models of metabolic and regulatory features potentially encoded within a genome and to pinpoint key features that can be highlighted for subsequent experimental validation. Chemolithoautotrophic microorganisms in general, and the extreme acidophilic ones in particular, are difficult to handle experimentally and can be recalcitrant to genetic manipulation. These hurdles can be compensated for, in part, by the enormous amount of information that can be extracted by careful genome analysis, allowing the experimental biologist to focus on key issues suggested by the bioinformatic predictions, thus saving considerable time and resources (Quatrini et al., 2007a). The bioinformatic analysis of the genome sequence of the γ- proteobacterium A. ferrooxidans has led to the generation of several metabolic and regulatory models some of which have been experimen- tally validated in part, such as sulfur uptake and assimilation (Valdés et al., 2003), iron uptake and assimilation (Quatrini et al., 2005; Osorio et al., 2008-this issue), Fur regulatory gene circuits (Quatrini et al., 2007b), quorum sensing (Rivas et al., 2005; Farah et al., 2005; Rivas et al., 2007), biofilm formation (Barreto et al., 2005), small regulatory RNA gene prediction (Shmaryahu and Holmes, unpublished results) and carbon metabolism (Barreto et al., 2003; Appia-Ayme et al., 2006). An analysis of the genome of A. ferrooxidans has also supported and extended earlier models of iron and sulfur oxidation in this microorganism (Quatrini et al., 2006; Valdés et al., unpublished results). Large-scale comparisons of genomes address basic questions, such as the number of functional genes, identification of species-specific genes, distribution of genes among functional families, gene density, 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. In the present study, the genomes of three closely related gram- negative, chemoautotrophic bioleaching microorganisms, A. ferroox- idans, A. thiooxidans and A. caldus, were compared, anticipating that Hydrometallurgy 94 (2008) 180–184 ⁎ Corresponding author. Tel.: +56 2 239 8969; fax: +56 2 237 2259. E-mail addresses: jorge.valdes@gmail.com (J. Valdés), dsholmes2000@yahoo.com (D.S. Holmes). 1 Fax: +56 2 237 2259. 0304-386X/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.hydromet.2008.05.039 Contents lists available at ScienceDirect Hydrometallurgy journal homepage: www.elsevier.com/locate/hydromet