The recent revision of the Acidithiobacillia class using genomic taxonomy methods has shown that, in addition to the existence of previously unrecognized genera and species, some species of the class harbor levels of divergence that are congruent with ongoing differentiation processes. In this study, we have performed a subspecies-level analysis of sequenced strains of Acidithiobacillus ferrooxidans to prove the existence of distinct sublineages and identify the discriminant genomic/genetic characteristics linked to these sublineages, and to shed light on the processes driving such differentiation. Differences in the genomic relatedness metrics, levels of synteny, gene content, and both integrated and episomal mobile genetic elements (MGE) repertoires support the existence of two subspecies-level taxa within A. ferrooxidans. While sublineage 2A harbors a small plasmid related to pTF5, this episomal MGE is absent in sublineage 2B strains. Likewise, clear differences in the occurrence, coverage and conservation of integrated MGEs are apparent between sublineages. Differential MGE-associated gene cargo pertained to the functional categories of energy metabolism, ion transport, cell surface modification, and defense mechanisms. Inferred functional differences have the potential to impact long-term adaptive processes and may underpin the basis of the subspecies-level differentiation uncovered within A. ferrooxidans. Genome resequencing of iron- and sulfur-adapted cultures of a selected 2A sublineage strain (CCM 4253) showed that both episomal and large integrated MGEs are conserved over twenty generations in either growth condition. In turn, active insertion sequences profoundly impact short-term adaptive processes. The ISAfe1 element was found to be highly active in sublineage 2A strain CCM 4253. Phenotypic mutations caused by the transposition of ISAfe1 into the pstC2 encoding phosphate-transport system permease protein were detected in sulfur-adapted cultures and shown to impair growth on ferrous iron upon the switch of electron donor. The phenotypic manifestation of the △pstC2 mutation, such as a loss of the ability to oxidize ferrous iron, is likely related to the inability of the mutant to secure the phosphorous availability for electron transport-linked phosphorylation coupled to iron oxidation. Depletion of the transpositional △pstC2 mutation occurred concomitantly with a shortening of the iron-oxidation lag phase at later transfers on a ferrous iron-containing medium. Therefore, the pstII operon appears to play an essential role in A. ferrooxidans when cells oxidize ferrous iron. Results highlight the influence of insertion sequences and both integrated and episomal mobile genetic elements in the short- and long-term adaptive processes of A. ferrooxidans strains under changing growth conditions.

Centro Científico y Tecnológico de Excelencia Ciencia and Vida Santiago Chile

Centro de Genómica y Bioinformática Facultad de Ciencias Universidad Mayor Camino La Piramide 5750 8580000 Huechuraba Santiago Chile

College of Natural Sciences Bangor University Bangor LL57 2UW UK

Department of Biochemistry Faculty of Science Masaryk University 61137 Brno Czech Republic

Facultad de Ciencias Biológicas Universidad Nacional Mayor de San Marcos Lima Peru

Facultad de Ingeniería Arquitectura y Diseño Universidad San Sebastián Santiago Chile

Facultad de Medicina y Ciencia Universidad San Sebastián 7510157 Providencia Santiago Chile

Faculty of Health and Life Sciences Coventry University Coventry CV1 5FB UK

Fundación Ciencia and Vida Avenida Del Valle Norte 725 8580702 Huechuraba Santiago Chile

Natural History Museum London UK

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Moya-Beltrán A, et al. Genomic evolution of the class Acidithiobacillia: Deep-branching Proteobacteria living in extreme acidic conditions. ISME J. 2021;15:3221–3238. doi: 10.1038/s41396-021-00995-x. PubMed DOI PMC

Nuñez H, et al. Molecular systematics of the genus Acidithiobacillus: Insights into the phylogenetic structure and diversification of the taxon. Front. Microbiol. 2017 doi: 10.3389/fmicb.2017.00030. PubMed DOI PMC

Wu W, et al. Discovery of a new subgroup of sulfur dioxygenases and characterization of sulfur dioxygenases in the sulfur metabolic network of Acidithiobacillus caldus. PLoS ONE. 2017;12:e0183668. doi: 10.1371/journal.pone.0183668. PubMed DOI PMC

Amouric A, Brochier-Armanet C, Johnson DB, Bonnefoy V, Hallberg KB. Phylogenetic and genetic variation among Fe(II)-oxidizing acidithiobacilli supports the view that these comprise multiple species with different ferrous iron oxidation pathways. Microbiology (N Y) 2011;157:111–122. PubMed

Hallberg KB, González-Toril E, Johnson DB. Acidithiobacillus ferrivorans, sp. nov.; facultatively anaerobic, psychrotolerant iron-, and sulfur-oxidizing acidophiles isolated from metal mine-impacted environments. Extremophiles. 2010;14:9–19. doi: 10.1007/s00792-009-0282-y. PubMed DOI

Hedrich S, Johnson DB. Acidithiobacillus ferridurans sp. nov., an acidophilic iron-, sulfur- and hydrogen-metabolizing chemolithotrophic gammaproteobacterium. Int. J. Syst. Evolut. Microbiol. 2013;63:4018–4025. doi: 10.1099/ijs.0.049759-0. PubMed DOI

Falagán C, Barrie Johnson D. Acidithiobacillus ferriphilus sp. nov., a facultatively anaerobic iron- and sulfur-metabolizing extreme acidophile. Int. J. Syst. Evolut. Microbiol. 2016;66:206–211. doi: 10.1099/ijsem.0.000698. PubMed DOI PMC

Norris PR, et al. Acidithiobacillus ferrianus sp. nov.: An ancestral extremely acidophilic and facultatively anaerobic chemolithoautotroph. Extremophiles. 2020;24(2):329–337. doi: 10.1007/s00792-020-01157-1. PubMed DOI PMC

Temple KL, Colmer AR. The autotrophic oxidation of iron by a new bacterium, Thiobacillus ferrooxidans. J. Bacteriol. 1951;62:605–611. doi: 10.1128/jb.62.5.605-611.1951. PubMed DOI PMC

Johnson DB. Biomining—Biotechnologies for extracting and recovering metals from ores and waste materials. Curr. Opin. Biotechnol. 2014;30:24–31. doi: 10.1016/j.copbio.2014.04.008. PubMed DOI

Quatrini R, Johnson DB. Acidithiobacillus ferrooxidans. Trends Microbiol. 2019;27:282–283. doi: 10.1016/j.tim.2018.11.009. PubMed DOI

Zhang X, et al. Comparative genomics unravels metabolic differences at the species and/or strain level and extremely acidic environmental adaptation of ten bacteria belonging to the genus Acidithiobacillus. Syst. Appl. Microbiol. 2016;39:493–502. doi: 10.1016/j.syapm.2016.08.007. PubMed DOI

Chen P, et al. Draft genome sequence of extremely acidophilic bacterium Acidithiobacillus ferrooxidans DLC-5 isolated from acid mine drainage in Northeast China. Genom. Data. 2015;6:267–268. doi: 10.1016/j.gdata.2015.10.018. PubMed DOI PMC

Latorre M, et al. The bioleaching potential of a bacterial consortium. Bioresour. Technol. 2016;218:659–666. doi: 10.1016/j.biortech.2016.07.012. PubMed DOI

Yan L, et al. Draft genome sequence of Acidithiobacillus ferrooxidans YQH-1. Genom. Data. 2015;6:269–270. doi: 10.1016/j.gdata.2015.10.009. PubMed DOI PMC

Ulloa R, et al. Domestication of local microbial consortia for efficient recovery of gold through top-down selection in airlift bioreactors. Front. Microbiol. 2019;10:60. doi: 10.3389/fmicb.2019.00060. PubMed DOI PMC

Valdés J, et al. Acidithiobacillus ferrooxidans metabolism: From genome sequence to industrial applications. BMC Genom. 2008;9:597. doi: 10.1186/1471-2164-9-597. PubMed DOI PMC

Koonin EV. Horizontal gene transfer: Essentiality and evolvability in prokaryotes, and roles in evolutionary transitions. F1000Res. 2016;5:F1000 Faculty Rev-1805. doi: 10.12688/f1000research.8737.1. PubMed DOI PMC

Beard S, Ossandon FJ, Rawlings DE, Quatrini R. The flexible genome of acidophilic prokaryotes. Curr. Issues Mol. Biol. 2020;40:231–266. PubMed

Orellana LH, Jerez CA. A genomic island provides Acidithiobacillus ferrooxidans ATCC 53993 additional copper resistance: A possible competitive advantage. Appl. Microbiol. Biotechnol. 2011;92:761–767. doi: 10.1007/s00253-011-3494-x. PubMed DOI

Kotze AA, Tuffin IM, Deane SM, Rawlings DE. Cloning and characterization of the chromosomal arsenic resistance genes from Acidithiobacillus caldus and enhanced arsenic resistance on conjugal transfer of ars genes located on transposon TnAtcArs. Microbiology (Reading, England) 2006;152(Pt 12):3551–3560. doi: 10.1099/mic.0.29247-0. PubMed DOI

Flores-Ríos R, et al. The type IV secretion system of ICEAfe1: Formation of a conjugative pilus in Acidithiobacillus ferrooxidans. Front. Microbiol. 2019;10:30. doi: 10.3389/fmicb.2019.00030. PubMed DOI PMC

Bustamante P, et al. ICE Afe 1, an actively excising genetic element from the biomining bacterium Acidithiobacillus ferrooxidans. J. Mol. Microbiol. Biotechnol. 2012;22:399–407. PubMed

Bustamante P, Tello M, Orellana O. Toxin-antitoxin systems in the mobile genome of Acidithiobacillus ferrooxidans. PLoS ONE. 2014;9:e112226. doi: 10.1371/journal.pone.0112226. PubMed DOI PMC

Castillo A, et al. A DNA segment encoding the anticodon stem/loop of tRNA determines the specific recombination of integrative-conjugative elements in Acidithiobacillus species. RNA Biol. 2018;15:492–499. doi: 10.1080/15476286.2017.1408765. PubMed DOI PMC

Levicán G, et al. A 300 kpb genome segment, including a complete set of tRNA genes, is dispensable for Acidithiobacillus Ferrooxidans. Adv. Mater. Res. 2009;71–73:187–190. doi: 10.4028/www.scientific.net/AMR.71-73.187. DOI

Alamos P, et al. Functionality of tRNAs encoded in a mobile genetic element from an acidophilic bacterium. RNA Biol. 2018;15:518–527. doi: 10.1080/15476286.2017.1349049. PubMed DOI PMC

Moya-Beltrán A, et al. Evolution of type IV CRISPR-cas systems: Insights from CRISPR loci in integrative conjugative elements of Acidithiobacillia. CRISPR J. 2021;4:656–672. doi: 10.1089/crispr.2021.0051. PubMed DOI PMC

Mamani S, et al. Insights into the quorum sensing regulon of the acidophilic Acidithiobacillus ferrooxidans revealed by transcriptomic in the presence of an acyl homoserine lactone superagonist analog. Front. Microbiol. 2016;7:1365. doi: 10.3389/fmicb.2016.01365. PubMed DOI PMC

Rivas M, Seeger M, Holmes DS, Jedlicki E. A lux-like quorum sensing system in the extreme acidophile Acidithiobacillus ferrooxidans. Biol. Res. 2005;38:283–297. doi: 10.4067/S0716-97602005000200018. PubMed DOI

Barreto M, Jedlicki E, Holmes DS. Identification of a gene cluster for the formation of extracellular polysaccharide precursors in the chemolithoautotroph Acidithiobacillus ferrooxidans. Appl. Environ. Microbiol. 2005;71:2902–2909. doi: 10.1128/AEM.71.6.2902-2909.2005. PubMed DOI PMC

Valdés J, et al. Comparative genomics begins to unravel the ecophysiology of bioleaching. Hydrometallurgy. 2010;104:471–476. doi: 10.1016/j.hydromet.2010.03.028. DOI

Moya-Beltrán A, et al. Nucleotide Second messenger-based signaling in extreme acidophiles of the Acidithiobacillus species complex: Partition between the core and variable gene complements. Front. Microbiol. 2019 doi: 10.3389/fmicb.2019.00381. PubMed DOI PMC

Holmes DS, et al. ISAfe1, an ISL3 family insertion sequence from Acidithiobacillus ferrooxidans ATCC 19859. J. Bacteriol. 2001;183(14):4323–4329. doi: 10.1128/JB.183.14.4323-4329.2001. PubMed DOI PMC

Yates JR, Holmes DS. Two families of repeated DNA sequences in Thiobacillus ferrooxidans. J. Bacteriol. 1987;169:1861–1870. doi: 10.1128/jb.169.5.1861-1870.1987. PubMed DOI PMC

Schrader JA, Holmes DS. Phenotypic switching of Thiobacillus ferrooxidans. J. Bacteriol. 1988;170:3915–3923. doi: 10.1128/jb.170.9.3915-3923.1988. PubMed DOI PMC

Cabrejos M-E, et al. IST1 insertional inactivation of the resB gene: Implications for phenotypic switching in Thiobacillus ferrooxidans. FEMS Microbiol. Lett. 1999;175:223–229. doi: 10.1111/j.1574-6968.1999.tb13624.x. PubMed DOI

Bonnefoy V, Grail BM, Johnson DB. Salt stress-induced loss of iron oxidoreduction activities and reacquisition of that phenotype depend on rus operon transcription in Acidithiobacillus ferridurans. Appl. Environ. Microbiol. 2018;84(7):e02795-17. doi: 10.1128/AEM.02795-17. PubMed DOI PMC

Tamames J, Puente-Sánchez F. SqueezeMeta, A highly portable, fully automatic metagenomic analysis pipeline. Front. Microbiol. 2018;9:3349. doi: 10.3389/fmicb.2018.03349. PubMed DOI PMC

González C, et al. Genetic variability of psychrotolerant Acidithiobacillus ferrivorans revealed by (meta)genomic analysis. Res. Microbiol. 2014;165:726–734. doi: 10.1016/j.resmic.2014.08.005. PubMed DOI

Chen J, Liu Y, Diep P, Mahadevan R. Genomic analysis of a newly isolated Acidithiobacillus ferridurans JAGS strain reveals its adaptation to acid mine drainage. Minerals. 2021;11:74. doi: 10.3390/min11010074. DOI

Dominy CN, Coram NJ, Rawlings DE. Sequence analysis of plasmid pTF5, a 19.8-kb geographically widespread member of the Thiobacillus ferrooxidan spTFI91-like plasmid family. Plasmid. 1998;40:50–57. doi: 10.1006/plas.1998.1344. PubMed DOI

Chakravarty L, et al. Characterization of the pTFI91-family replicon of Thiobacillus ferrooxidans plasmids. Can. J. Microbiol. 2011;41:354–365. doi: 10.1139/m95-048. PubMed DOI

Hsieh YJ, Wanner BL. Global regulation by the seven-component Pi signaling system. Curr. Opin. Microbiol. 2010;13:198–203. doi: 10.1016/j.mib.2010.01.014. PubMed DOI PMC

Landesman J, Duncan DW, Walden CC. Oxidation of inorganic sulfur compounds by washed cell suspensions of Thiobacillus ferrooxidans. Can. J. Microbiol. 2011;12:957–964. doi: 10.1139/m66-129. PubMed DOI

Yarzábal A, Duquesne K, Bonnefoy V. Rusticyanin gene expression of Acidithiobacillus ferrooxidans ATCC 33020 in sulfur- and in ferrous iron media. Hydrometallurgy. 2003;71:107–114. doi: 10.1016/S0304-386X(03)00146-4. DOI

Yarzábal A, Appia-Ayme C, Ratouchniak J, Bonnefoy V. Regulation of the expression of the Acidithiobacillus ferrooxidans rus operon encoding two cytochromes c, a cytochrome oxidase and rusticyanin. Microbiology. 2004;150:2113–2123. doi: 10.1099/mic.0.26966-0. PubMed DOI

Kucera J, et al. Ferrous iron oxidation by sulfur-oxidizing Acidithiobacillus ferrooxidans and analysis of the process at the levels of transcription and protein synthesis. Antonie Van Leeuwenhoek. 2013;103:905–919. doi: 10.1007/s10482-012-9872-2. PubMed DOI

Johnson DB. Selective solid media for isolating and enumerating acidophilic bacteria. J. Microbiol. Methods. 1995;23:205–218. doi: 10.1016/0167-7012(95)00015-D. DOI

Mandl M, Nováková O. An ultraviolet spectrophotometric method for the determination of oxidation of iron sulphide minerals by bacteria. Biotechnol. Tech. 1993;7:573–574. doi: 10.1007/BF00156331. DOI

Nieto PA, Covarrubias PC, Jedlicki E, Holmes DS, Quatrini R. Selection and evaluation of reference genes for improved interrogation of microbial transcriptomes: Case study with the extremophile Acidithiobacillus ferrooxidans. BMC Mol. Biol. 2009;10:63. doi: 10.1186/1471-2199-10-63. PubMed DOI PMC

Bolger AM, Lohse M, Usadel B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30:2114–2120. doi: 10.1093/bioinformatics/btu170. PubMed DOI PMC

Bankevich A, et al. SPAdes: A new genome assembly algorithm and its applications to single-cell sequencing. J. Comput. Biol. 2012;19:455–477. doi: 10.1089/cmb.2012.0021. PubMed DOI PMC

Darling ACE, Mau B, Blattner FR, Perna NT. Mauve: Multiple alignment of conserved genomic sequence with rearrangements. Genome Res. 2004;14:1394–1403. doi: 10.1101/gr.2289704. PubMed DOI PMC

Tatusova T, et al. NCBI prokaryotic genome annotation pipeline. Nucleic Acids Res. 2016;44:6614–6624. doi: 10.1093/nar/gkw569. PubMed DOI PMC

Raes J, Korbel JO, Lercher MJ, von Mering C, Bork P. Prediction of effective genome size in metagenomic samples. Genome Biol. 2007;8:R10. doi: 10.1186/gb-2007-8-1-r10. PubMed DOI PMC

Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM. BUSCO update: Novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol. Biol. Evol. 2021;38:4647–4654. doi: 10.1093/molbev/msab199. PubMed DOI PMC

Aziz RK, et al. The RAST Server: Rapid annotations using subsystems technology. BMC Genom. 2008;9:75. doi: 10.1186/1471-2164-9-75. PubMed DOI PMC

Drillon G, Carbone A, Fischer G. SynChro: A fast and easy tool to reconstruct and visualize synteny blocks along eukaryotic chromosomes. PLoS ONE. 2014;9:e92621. doi: 10.1371/journal.pone.0092621. PubMed DOI PMC

Siguier P, Perochon J, Lestrade L, Mahillon J, Chandler M. ISfinder: The reference centre for bacterial insertion sequences. Nucleic Acids Res. 2006;34(Database issue):D32–D36. doi: 10.1093/nar/gkj014. PubMed DOI PMC

Xie Z, Tang H. ISEScan: Automated identification of insertion sequence elements in prokaryotic genomes. Bioinformatics. 2017;33:3340–3347. doi: 10.1093/bioinformatics/btx433. PubMed DOI

Riadi G, Medina-Moenne C, Holmes DS. TnpPred: A web service for the robust prediction of prokaryotic transposases. Comp. Funct. Genom. 2012;2012:1–5. doi: 10.1155/2012/678761. PubMed DOI PMC

Hsiao W, Wan I, Jones SJ, Brinkman FSL. IslandPath: Aiding detection of genomic islands in prokaryotes. Bioinformatics. 2003;19:418–420. doi: 10.1093/bioinformatics/btg004. PubMed DOI

Vernikos GS, Parkhill J. Interpolated variable order motifs for identification of horizontally acquired DNA: Revisiting the Salmonella pathogenicity islands. Bioinformatics. 2006;22:2196–2203. doi: 10.1093/bioinformatics/btl369. PubMed DOI

Tu Q, Ding D. Detecting pathogenicity islands and anomalous gene clusters by iterative discriminant analysis. FEMS Microbiol. Lett. 2003;221:269–275. doi: 10.1016/S0378-1097(03)00204-0. PubMed DOI

Fouts DE. Phage_Finder: Automated identification and classification of prophage regions in complete bacterial genome sequences. Nucleic Acids Res. 2006;34:5839–5851. doi: 10.1093/nar/gkl732. PubMed DOI PMC

Akhter S, Aziz RK, Edwards RA. PhiSpy: A novel algorithm for finding prophages in bacterial genomes that combines similarity- and composition-based strategies. Nucleic Acids Res. 2012;40(16):e126. doi: 10.1093/nar/gks406. PubMed DOI PMC

Guglielmini J, et al. Key components of the eight classes of type IV secretion systems involved in bacterial conjugation or protein secretion. Nucleic Acids Res. 2014;42:5715–5727. doi: 10.1093/nar/gku194. PubMed DOI PMC

Martínez-García PM, Ramos C, Rodríguez-Palenzuela P. T346Hunter: A novel web-based tool for the prediction of type III, type IV and type VI secretion systems in bacterial genomes. PLoS ONE. 2015;10(4):e0119317. doi: 10.1371/journal.pone.0119317. PubMed DOI PMC

Souza RC, et al. AtlasT4SS: A curated database for type IV secretion systems. BMC Microbiol. 2012;12:172. doi: 10.1186/1471-2180-12-172. PubMed DOI PMC

Lowe TM, Eddy SR. tRNAscan-SE: A program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25:955. doi: 10.1093/nar/25.5.955. PubMed DOI PMC

Laslett D, Canback B. ARAGORN, a program to detect tRNA genes and tmRNA genes in nucleotide sequences. Nucleic Acids Res. 2004;32:11–16. doi: 10.1093/nar/gkh152. PubMed DOI PMC

Solovyev V, Salamov A. Automatic annotation of microbial genomes and metagenomic sequences. In: Li RW, editor. Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies. Nova Science Publishers; 2011. pp. 61–78.

Gilchrist CLM, Chooi YH. clinker & clustermap.js: Automatic generation of gene cluster comparison figures. Bioinformatics. 2021;37:2473–2475. doi: 10.1093/bioinformatics/btab007. PubMed DOI

Krzywinski M, et al. Circos: An information aesthetic for comparative genomics. Genome Res. 2009;19:1639–1645. doi: 10.1101/gr.092759.109. PubMed DOI PMC

Pritchard L, Glover RH, Humphris S, Elphinstone JG, Toth IK. Genomics and taxonomy in diagnostics for food security: Soft-rotting enterobacterial plant pathogens. Anal. Methods. 2015;8:12–24. doi: 10.1039/C5AY02550H. DOI

Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinform. 2013;14:60. doi: 10.1186/1471-2105-14-60. PubMed DOI PMC

Meier-Kolthoff JP, Klenk HP, Göker M. Taxonomic use of DNA G+C content and DNA-DNA hybridization in the genomic age. Int. J. Syst. Evol. Microbiol. 2014;64:352–356. doi: 10.1099/ijs.0.056994-0. PubMed DOI

Richter M, Rosselló-Móra R. Shifting the genomic gold standard for the prokaryotic species definition. Proc. Natl. Acad. Sci. U.S.A. 2009;106:19126–19131. doi: 10.1073/pnas.0906412106. PubMed DOI PMC

Contreras-Moreira B, Vinuesa P. GET_HOMOLOGUES, a versatile software package for scalable and robust microbial pangenome analysis. Appl. Environ. Microbiol. 2013;79:7696–7701. doi: 10.1128/AEM.02411-13. PubMed DOI PMC

Tatusov RL, et al. The COG database: An updated version includes eukaryotes. BMC Bioinform. 2003;4:41. doi: 10.1186/1471-2105-4-41. PubMed DOI PMC

Kanehisa M, Goto S. KEGG: Kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28:27–30. doi: 10.1093/nar/28.1.27. PubMed DOI PMC

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