Genome-wide comparative analysis of clinical and environmental strains of the opportunistic pathogen Paracoccus yeei (Alphaproteobacteria)

Abstract

Introduction: Paracoccus yeei is the first species in the genus Paracoccus to be implicated in opportunistic infections in humans. As a result, P. yeei strains provide a valuable model for exploring how bacteria shift from a saprophytic to a pathogenic lifestyle, as well as for investigating the role of horizontally transferred DNA in this transition. In order to gain deeper insights into the unique characteristics of this bacterium and the molecular mechanisms underlying its opportunistic behavior, a comparative physiological and genomic analysis of P. yeei strains was performed.

Results: Complete genomic sequences of 7 P. yeei isolates (both clinical and environmental) were obtained and analyzed. All genomes have a multipartite structure comprising numerous extrachromosomal replicons (59 different ECRs in total), including large chromids of the DnaA-like and RepB families. Within the mobile part of the P. yeei genomes (ECRs and transposable elements, TEs), a novel non-autonomous MITE-type element was identified. Detailed genus-wide comparative genomic analysis permitted the identification of P. yeei-specific genes, including several putative virulence determinants. One of these, the URE gene cluster, determines the ureolytic activity of P. yeei strains-a unique feature among Paracoccus spp. This activity is induced by the inclusion of urea in the growth medium and is dependent on the presence of an intact nikR regulatory gene, which presumably regulates expression of nickel (urease cofactor) transporter genes.

Discussion: This in-depth comparative analysis provides a detailed insight into the structure, composition and properties of P. yeei genomes. Several predicted virulence determinants (including URE gene clusters) were identified within ECRs, indicating an important role for the flexible genome in determining the opportunistic properties of this bacterium.

Keywords: Paracoccus yeei; chromid; evolution of pathogenic bacteria; multipartite genome; non-autonomous transposable element; opportunistic pathogen; transposable element; urease.

Figure 1

Distribution of ECRs in P. yeei genomes. (A) Phylogenetic tree of P. yeei constructed using core gene nucleotide sequences. Three other members of the genus Paracoccus—P. aminophilus JCM 7686, P. aminovorans JCM 7685, and P. denitrificans PD1222—as well as Roseobacter denitrificans Och 114 (as an outgroup) were included in the analysis. Bar represents 0.1 nucleotide substitutions per position. (B) Summary of data for ECRs of the analyzed Paracoccus spp. strains. (C) Distribution of different REP module types among ECRs of Paracoccus spp.

Figure 2

ECRs identified in the genomes of P. yeei strains. (A) Genetic organization of the replication (REP) and partitioning modules of the ECRs. The box indicates the location of sequence motifs specific to each type of replication initiation protein. (B) Size range of P. yeei replicons carrying a given REP module type (yellow). The blue color indicates replicons present in the genomes of P. aminophilus JCM 7686, P. aminovorans JCM 7685, and P. denitrificans PD1222.

Figure 3

Comparative genomic analysis of three P. yeei strains, CCUG 17731, LM20, and CCUG 46822, illustrating the two chromosome structural variants identified in this species.

Figure 4

Distribution of genes encoding proteins belonging to different COG functional categories within (A) the chromosomes and (B) ECRs of the analyzed P. yeei strains (*environmental isolates). Each colored segment indicates the relative contribution of a functional category as a percentage of total COGs. Each ring represents a different strain of P. yeei. COG functional categories: C, energy production and conversion; 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; P, inorganic ion transport and metabolism; Q, secondary metabolite biosynthesis, transport and catabolism; D, cell cycle control, cell division, chromosome partitioning; M, cell wall/membrane/envelope biogenesis; N, cell motility; O, posttranslational modification, protein turnover, chaperones; T, signal transduction mechanisms; U, intracellular trafficking, secretion and vesicular transport; V, defense mechanisms; W, extracellular structures; Z, cytoskeleton; J, translation, ribosomal structure and biogenesis; K, transcription; L, replication, recombination and repair; X, mobilome, i.e., prophages, transposons; R, general function, prediction only; S, function unknown, COG not assigned.

Figure 5

Distribution of insertion sequences in P. yeei genomes (*environmental isolates).

Figure 6

Non-autonomous transposable element MITEPye1 identified in P. yeei strains. (A) Alignment of terminal inverted repeat nucleotide sequences (IRL, left IR; IRR, right IR) of complete MITEPye1 elements identified in P. yeei genomes and ISPye18. Identical residues are indicated by gray shading. Nucleotide sequences of direct repeats (DRs) generated by MITEPye1 elements during transposition are indicated by underlining. (B) RNA secondary structures predicted by in silico folding using Mfold software for MITEPye1. The minimum folding energy (ΔG −47.93) of the predicted secondary structures was calculated by Mfold.

Figure 7

Genetic organization of urease gene clusters (URE) located in P. yeei ECRs—URE type 1 (present in all DnaA-like chromids) and URE type 2 (present in two RepABC plasmids of P. yeei CCUG 13493 and CCUG 32052). Shaded areas connect homologous DNA regions.