Acquisition of Type I methyltransferase via horizontal gene transfer increases the drug resistance of Aeromonas veronii

Abstract

Aeromonas veronii is an opportunistic pathogen that affects both fish and mammals, including humans, leading to bacteraemia, sepsis, meningitis and even death. The increasing virulence and drug resistance of A. veronii are of significant concern and pose a severe risk to public safety. The Type I restriction-modification (RM) system, which functions as a bacterial defence mechanism, can influence gene expression through DNA methylation. However, little research has been conducted to explore its origin, evolutionary path, and relationship to virulence and drug resistance in A. veronii. In this study, we analysed the pan-genome of 233 A. veronii strains, and the results indicated that it was 'open', meaning that A. veronii has acquired additional genes from other species. This suggested that A. veronii had the potential to adapt and evolve rapidly, which might have contributed to its drug resistance. One Type I methyltransferase (MTase) and two complete Type I RM systems were identified, namely AveC4I, AveC4II and AveC4III in A. veronii strain C4, respectively. Notably, AveC4I was exclusive to A. veronii C4. Phylogenetic analysis revealed that AveC4I was derived from horizontal gene transfer from Thiocystis violascens and exchanged genes with the human pathogen Comamonas kerstersii. Single molecule real-time sequencing was applied to identify the motif methylated by AveC4I, which was unique and not recognized by any reported MTases in the REBASE database. We also annotated the functions and pathways of the genes containing the motif, revealing that AveC4I may control drug resistance in A. veronii C4. Our findings provide new insight on the mechanisms underlying drug resistance in pathogenic bacteria. By identifying the specific genes and pathways affected by AveC4I, this study may aid in the development of new therapeutic approaches to combat A. veronii infections.

Keywords: Aeromonas veronii; Type I restriction-modification system; drug resistance; horizontal gene transfer; methylome.

Fig. 1.

Pan-genome structure of 20 Aeromonas veronii strains. (a) Comparative genomic map of 20 A. veronii strains. The blue bar represents the number of gene families in each strain. The green bar on the genomic map denotes the count of either unique genes (glaucous dots) present in individual strains, or accessory genes (glaucous dots connected by the glaucous line) found in at least two strains. The flower plot represents the number of core genes and the remaining genes (defined as variable genes) in each strain. (b) Gene accumulation curves for the pan-genome and core genome of 20 A. veronii strains. (c) Distribution of Clusters of Orthologous Groups (COG) functional annotations of 20 A. veronii strains. [C] Energy production and conversion, [D] Cell cycle control, cell division, chromosome partitioning, [E] Amino acid transport and metabolism, [F] Nucleotide transport and metabolism, [G] Carbohydrate transport and metabolism, [H] Coenzyme transport and metabolism, [I] Lipid transport and metabolism, [J] Translation, ribosomal structure and biogenesis, [K] Transcription, [L] Replication, recombination and repair, [M] Cell wall/membrane/envelope biogenesis, [N] Cell motility, [O] Posttranslational modification, protein turnover, chaperones, [P] Inorganic ion transport and metabolism, [Q] Secondary metabolite biosynthesis, transport and catabolism, [R] General function prediction only, [S] Function unknown, [T] Signal transduction mechanisms, [U] Intracellular trafficking, secretion and vesicular transport, [V] Defence mechanisms, [Y] Nuclear structure.

Fig. 2.

Distribution of hsd genes in 20 Aeromonas veronii strains. (a) Phylogenetic analysis of 20 A . veronii strains. The phylogenetic tree based on 2728 single-copy core genes of the 20 strains is divided into five clades. Gene gain (light blue) and loss (dark blue) are represented by pie charts. Geographical location and isolation conditions are represented by heat maps. The gene cluster consists of homologous genes of eight hsd genes in A. veronii C4. Bootstrap values from 1000 replicates are shown. (b) Gene clusters of AveC4I, AveC4II and AveC4III in A. veronii C4.

Fig. 3.

Comparison between phylogenetic trees. (a) The phylogenetic tree of HsdM1 was reconstructed based on 109 homologous HsdM1 (left), and the phylogenetic tree of the 107 species containing homologous HsdM1 was reconstructed based on 21 single-copy core genes (right). (b) The phylogenetic tree of HsdS1 was reconstructed based on 59 homologous HsdS1 (left), and the phylogenetic tree of the 58 species containing homologous HsdS1 was reconstructed based on 26 single-copy core genes (right). In (a) and (b) dots of various colours symbolize the species belonging to different classes. Different background colours highlight the species that come from diverse sources or have different characteristics. Lines connect the same species. A. veronii C4 is marked with a red arrowhead.

Fig. 4.

Methylome analysis of AveC4I. (a) Distribution of the motif methylated by AveC4I in A. veronii C4. The motif is enriched in the region (2.9–3.0 Mb), which includes ONR73_14145 (Type I secretion C-terminal target domain-containing protein), ONR73_14150 (T1SS 143 repeat domain-containing protein) and ONR73_14155 (Ca²⁺-binding protein). (b) Scatter plot of KEGG analysis of the genes with the motif. The ‘Bacterial chemotaxis’ pathway showed the most significant enrichment (hypergeometric test, P<0.01).