Influence of mobile genetic elements and insertion sequences in long- and short-term adaptive processes of Acidithiobacillus ferrooxidans strains

Abstract

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.

Pangenome-level analysis of nucleoid-associated proteins in the Acidithiobacillia class: insights into their functional roles in mobile genetic elements biology

“…Yet, apart from KfrA, the only NAP families detected in plasmids of the class, were HU, IHF_A and IHF_B, all of which are well known for their role in DNA bending. In this regard, the presence of IHF-binding sites in the ori region of several A. ferrooxidans pTF5-like plasmids is noteworthy ( Chakravarty et al, 1995 ; Moya-Beltrán et al, 2023 ). These findings suggest that plasmid-encoded NAPs of the acidithiobacilli may participate in NAP induced DNA-bending within the ori and/or oriT regions to facilitate replication and/or transfer processes, as is the case for IHF proteins during E. coli pSC101 plasmid replication ( Biek and Cohen, 1989 ) and IncFV plasmid pED208 DNA-transfer initiation ( Di Laurenzio et al, 1995 ).…”

“… # Species names as proposed in Moya-Beltrán et al (2021) ; ICE names as proposed in Bustamante et al (2012) , Acuña et al (2013) , and Moya-Beltrán et al (2023) . …”

“…The class pertains to the phylum Proteobacteria ( Williams and Kelly, 2013 ) and contains a single order and two families, the Acidithiobacillaceae with 10 validly described species ( Boden and Hutt, 2019a ), and the more ancestral Thermithiobacillaceae with only two acknowledged species ( Boden and Hutt, 2019b ). Extensive genomic comparisons of sequenced members of the class have revealed important lacunae in our comprehension of the phylogenetic space occupied by the class ( Nuñez et al, 2017 ) and expanded its genomic contours to an increasing total of 19 lineages ( Moya-Beltrán et al, 2021 , 2023 ). These linages rank at different taxonomic levels, including three novel genera named as ‘ Fervidacidithiobacillus ’ (ex.…”

“…MGE-encoded candidate NAPs identified in Acidithiobacillia class genomes.Species names as proposed in Moya-Beltrán et al(2021); ICE names as proposed inBustamante et al (2012),Acuña et al (2013), andMoya-Beltrán et al (2023).…”

Acidithiobacillia class members originating at sites within the Pacific Ring of Fire and other tectonically active locations and description of the novel genus ‘Igneacidithiobacillus’

“…Phenotypic and genotypic variability of its members are well acknowledged and have motivated several instances of revision of the taxonomy of the group ( Johnson and Quatrini, 2020 ). Currently, the class comprises ten validated species ( Boden and Hutt, 2019 ) and eight recently acknowledged novel species ( Moya-Beltrán et al, 2021 ), in addition to several subspecies-level lineages ( Nuñez et al, 2017 ; Moya-Beltrán et al, 2023 ). These studies have significantly expanded the genomic contours of the class and highlighted important lacunae in our comprehension of the phylogenetic space occupied by acidithiobacilli-like bacteria.…”