Through 16S rRNA research, it was found that ZJ belongs to the genus Ferroplasma (Supplementary Figure S1). To investigate environmental adaptation mechanisms within Ferroplasma, we conducted a comparative genomic analysis of all isolated whole-genome sequenced strains. The examined strains exhibited conserved genomic characteristics, as evidenced by their comparable GC content and genome architecture (Table 1). Notably, all four strains maintained narrow GC content ranges between 36.50 and 36.95%, with genome sizes averaging 1.85 Mbp. The predicted protein-coding gene counts demonstrated moderate variation across strains, ranging from 1,842 to 2,084 genes. These conserved genomic parameters suggest evolutionary stability in fundamental genomic organization among Ferroplasma species, while the observed gene content variations may reflect niche-specific adaptations.

The ANI serves as a robust genomic metric for evaluating phylogenetic relatedness between microbial strains, with established thresholds defining species boundaries (de Albuquerque and Haag, 2023). Conventionally, ANI values exceeding 95% are considered indicative of strains belonging to the same recognized microbial species (Luis et al., 2023). Our analysis revealed observed ANI values between the newly isolated strains ZJ and F. acidiphilum exceeding this benchmark (Figure 2A), demonstrating their close phylogenetic affiliation. Notably, the calculated ANI between F. acidiphilum DSM 28986 and strain ZJ reached 100% genomic identity, strongly supporting their classification as conspecific strains according to current taxonomic standards (Viver et al., 2024). This genomic identity threshold corroborates their taxonomic assignment within the same species despite potential phenotypic variations.

Based on comprehensive phylogenetic analysis of complete genome sequences performed in this study, we propose that strain ZJ should be taxonomically classified within F. acidiphilum species, as evidenced by its consistent clustering with reference strains in the phylogenetic reconstruction (Figure 2B) (Hu et al., 2024).

As illustrated in Figure 3, pangenome analysis conducted on the four Ferroplasma strains delineated three distinct genomic components: the core genome, accessory genome, and unique genome (Horesh et al., 2021). Subsequent COG functional annotation revealed characteristic distribution patterns of COG categories across these genomic compartments. While the current analysis suggests an open pan-genome state, this configuration may transition to a closed state with expanded sampling. Notably, Ferroplasma exhibits evolutionary flexibility through HGT mechanisms, enabling interspecies acquisition of MGEs that contribute to its adaptive genomic repertoire (Vernikos et al., 2015). This metabolic plasticity is particularly significant given the organism’s extreme acidophilic habitat requirements.

The core genome of Ferroplasma comprises 1,498 CDSs, with F. acidiphilum ZJ harbored 276 accessory genes among studied species. These accessory genomic elements, known to regulate genetic traits associated with environmental adaptability, stress resistance, and virulence (Medini et al., 2005), contribute significantly to intraspecies diversity. Compared with the genomes from the Ferroplasma, F. acidiphilum ZJ harbored the largest accessory genes (276 genes) apart from other genomes, implying a relatively high degree of genomic diversity. Functional annotation of accessory genes (Figure 4) reveals three predominant categories: [M] cell wall/membrane/envelope biogenesis, [V] defense mechanisms, and [Q] secondary metabolites biosynthesis, transport, and catabolism. This distribution may suggest evolutionary specialization through structural adaptations (particularly in cellular envelope components) and sophisticated defense systems to counter extreme pH stress. Notably, SMs appear to play pivotal roles in Ferroplasma’s unique environmental adaptation strategies. The observed genomic diversity and functional specialization collectively provide a competitive advantage for niche colonization in harsh ecosystems, demonstrating a robust evolutionary framework for extremophile survival.

A comprehensive analysis of IS elements across Ferroplasma genomes revealed nine distinct IS families comprising 195 total copies, as detailed in Supplementary Table S1. Comparative genomic profiling demonstrated significant interspecies variation, with reference strains F. acidarmanus fer1 (76 copies) and F. acidiphilum Y (70 copies) harboring substantially higher IS loads compared to F. acidiphilum DSM 28986 (27 copies) and F. acidiphilum ZJ (22 copies). Six predominant families (IS256, IS200/IS6, IS5, IS4, ISH3, and IS481) collectively accounted for >85% of IS elements across the four genomes. Core IS families (IS4, ISH3, IS200/IS6, IS256, IS481, IS5, and IS110) were conserved in all strains, while strain-specific distributions emerged for IS1634 (exclusive to F. acidiphilum Y and F. acidarmanus fer1) and ISNCY (unique to F. acidarmanus fer1). Notably, IS4 demonstrated universal distribution and particular functional relevance, potentially serving as an evolutionary toolkit through HGT to enhance microbial adaptation in extreme environments, as evidenced by its prevalence in extremophiles and pathogenic bacteria (Lysnyansky et al., 2009; Touchon and Rocha, 2007). Mechanistic studies suggest IS4 may modulate salinity tolerance modulation and genomic plasticity in response to environmental stressors (Bonnefoy et al., 2018).

Previous studies proved that the insertion and deletion of IS4 in specific sequences affects the ability of extreme acidophilies in the AMD environment, to utilize iron (Bonnefoy et al., 2018). Functional characterization of IS4 through adjacent gene analysis revealed distinct genomic insertion patterns (Figure 5). Unlike other IS families, IS4 frequently co-occurred with FOG (Functional Orthologous Group) transposase derivatives, suggesting potential archaeal-specific transposition mechanisms. Comparative mapping identified preferential IS4 insertion near critical metabolic loci, including: (1) heavy metal transporter genes (e.g., CzcD transporters for cobalt/zinc/cadmium detoxification) (Hassan et al., 2013); (2) membrane biosynthesis components (Radical SAM proteins and aldo/keto reductase for QS molecule synthesis in F. acidiphilum Y and F. acidarmanus fer1); and (3) structural elements supporting biofilm maintenance (cellulose synthase catalytic subunits in F. acidiphilum ZJ and DSM 28986 genomes) (Römling and Galperin, 2015). These strategic insertion patterns indicated that IS4-mediated genomic restructuring enhances environmental adaptability through several mechanisms: improving heavy metal transport, optimizing conversion rates, reinforcing cell membrane integrity, and augmenting specialized metabolic functions. The distribution pattern of IS4 and its association with gene neighborhoods offer mechanistic insights into how MGEs influence the functional genomic evolution of extreme acidophilic archaea and enhance their environmental remediation capabilities.

Five GIs ranging from 11.5 to 51.0 kb were identified in the F. acidiphilum ZJ genome (Supplementary Figure S2), harboring 159 predicted genes. Comparative analysis revealed interspecies variability in GI content: F. acidiphilum DSM 28986 contained the highest gene count (210 genes) distributed across 8 GIs, while F. acidarmanus fer1 exhibited the most extensive GI architecture with 11 GIs encompassing 176 genes. F. acidiphilum Y displayed intermediate characteristics, with 173 genes organized into 7 GIs (Supplementary Table S2). Subsequent functional annotation highlighted a predominance of MGE-associated genes, including 18 coding for transposases, integrases, and recombinases. This finding aligns with the established role of GIs in facilitating HGT through transformation, conjugation, and transduction mechanisms (Juhas et al., 2009). Additionally, genes encoding transferases, hydrolases, and transporter proteins were identified. Notably, a CRISPR-associated Cas1 protein was detected within F. acidiphilum ZJ’s GI, suggesting potential CRISPR-mediated defense against MGEs or phage infection. However, no complete CRISPR arrays were identified in the genome. The analysis further predicted five restriction-modification system subunits within GIs, implicating these regions in restriction-based defense systems. Of particular ecological relevance, heavy metal resistance genes were systematically identified across all Ferroplasma strains. This included the conserved cobalt/zinc/cadmium resistance protein CzcD, a cation-transporting transmembrane protein previously characterized in acidophilic archaea (Deppenmeier et al., 2002). Heavy metal-binding proteins encoded in the Ferroplasma archaea genome are crucial for mitigating heavy metal toxicity. These proteins safeguard cellular structure and function by actively exporting heavy metal ions, preventing their accumulation to toxic levels. Toxin-Antitoxin systems are widely present in archaea and typically consist of two genes: one encodes a toxin and the other encodes an antitoxin. In addition to this, the prevalence of efflux pumps, transporters, and heavy metal resistance factors in GIs underscores their role in adapting to extreme environments and suggests potential applications in environmental remediation.

The prediction of secondary metabolite synthesis gene clusters in three F. acidiphilum strains (ZJ, DSM 28986, and Y) revealed their ability to produce two types of exopolysaccharides: lipopolysaccharide and capsular polysaccharide. Different from other strains, F. acidarmanus fer1 can only synthesize lipopolysaccharide and lacks the ability to synthesize capsular polysaccharide. The absence of a cell wall in Ferroplasma likely increases its reliance on EPS for protection. These EPS, including capsular polysaccharides and lipopolysaccharides, can serve as effective biosorbents for the removal of heavy metals from the environment (Song et al., 2020). The negatively charged functional groups (e.g., carboxyl, sulfate, phosphate) present in lipopolysaccharide and capsular polysaccharide facilitate electrostatic adsorption or complexation with heavy metal ions (Pb²⁺, Cu²⁺, Cd²⁺, etc.) (Ghimire and McCarthy, 2018). Additionally, the formation of biofilms by polysaccharides: lipopolysaccharide and capsular polysaccharide enhances the tolerance of microorganisms to heavy metal stress. However, the structural diversity of polysaccharides: lipopolysaccharide and capsular polysaccharide among different microorganisms results in varying adsorption capacities for heavy metals (Gao et al., 2023). The high molecular weight and negative charge of EPS enable it to adsorb and immobilize heavy metal ions. The presence of numerous metal-binding proteins within MGEs suggests that Ferroplasma may secrete these proteins to work in conjunction with EPS, thereby enhancing its capacity to capture heavy metals.

As illustrated in Figure 7, the genomes of F. acidiphilum ZJ and F. acidiphilum DSM 28986 contain an auxiliary BGC responsible for synthesizing falcarindiol, a characteristic acetylenic lipid compound (Jin et al., 2012). Notably, falcarindiol demonstrates QS-modulatory activity, with experimental evidence showing downregulation of QS-associated genes (rhlA, rhlI, pqsA, and rhlR) in Pseudomonas aeruginosa (Zhao et al., 2021). This suggests falcarindiol may predominantly target the Rhl-QS system and the PQS system in acidophiles. The Rhl-QS system, extensively conserved in Acidithiobacillus species, contains a putative LuxI/R homolog pair that facilitates the production of the C₄-HSL signaling molecule. While the PQS system remains undocumented in acidophiles, genomic analyses reveal widespread distribution of PQS biosynthetic genes phnA and phnB among AMD-associated acidophiles (Huang et al., 2022).

Multiple sequence alignment of PhnA and PhnB homologs in Ferroplasma (Supplementary Figure S3) identified critical conserved residues within their active sites: PhnA contains signature residues G₄₉, E₅₅, G₅₈, I₆₄, F₆₆, K₆₈, L₇₃, and P₁₂₀, while PhnB features I₇₃, Y₇₄, E₁₁₈, G₁₂₁, I₁₂₇, R₁₂₉, K₁₃₁, and L₁₃₆. Notably, the genomic region associated with falcarindiol biosynthesis encodes an “Aldo/keto reductase family oxidoreductase” (Pfam domain), suggesting this enzyme family may play a pivotal role in falcarindiol synthesis, potentially coupled with IS-mediated genetic transfer. Combined with evidence of intrageneric HGT, these findings imply that falcarindiol biosynthesis could exert broad ecological impacts on environmental acidophiles, potentially enhancing the adaptive capacity and ecological competitiveness of Ferroplasma archaea through QS modulation.

The archaeon Ferroplasma employs specialized MGEs and BGCs as key environmental adaptation mechanisms, as illustrated in Figure 8. Our findings imply that MGEs may mediate HGT, thereby affecting QS regulatory networks, although this link requires further substantiation (e.g., GC content anomalies or phylogenetic incongruence). Ferroplasma engages in the redox cycles of metals like iron and sulfur via transporters and invertases, facilitating environmental remediation. Similar to other acidophilic bacteria, its QS system may enhance EPS secretion in response to certain levels of organic carbon or metal ions by regulating EPS synthesis gene transcription (Luo et al., 2024). This mechanism aids biofilm formation, shielding microorganisms from extreme environmental harm, and boosts remediation efficiency through EPS’s heavy metal ion complexation. Thus, EPS synthesis genes are pivotal in Ferroplasma’s environmental remediation. Furthermore, QS may optimize microbial metabolic division of labor, addressing resource competition in complex environments by modulating iron oxidation gene expression (Huang et al., 2022). Additionally, its unique membrane lipids, such as calditol-linked glycerol dialkyl glycerol tetraethers, ensure cell stability in highly acidic conditions, supporting sustained remediation efforts. The study revealed substantial genetic diversity and secondary metabolite production capabilities within Ferroplasma genomes, highlighting their biotechnological potential for targeted metabolite discovery through systematic genomic exploration.