Diversity and Horizontal Transfer of Antarctic Pseudomonas spp. Plasmids

Abstract

Pseudomonas spp. are widely distributed in various environments around the world. They are also common in the Antarctic regions. To date, almost 200 plasmids of Pseudomonas spp. have been sequenced, but only 12 of them were isolated from psychrotolerant strains. In this study, 15 novel plasmids of cold-active Pseudomonas spp. originating from the King George Island (Antarctica) were characterized using a combined, structural and functional approach, including thorough genomic analyses, functional analyses of selected genetic modules, and identification of active transposable elements localized within the plasmids and comparative genomics. The analyses performed in this study increased the understanding of the horizontal transfer of plasmids found within Pseudomonas populations inhabiting Antarctic soils. It was shown that the majority of the studied plasmids are narrow-host-range replicons, whose transfer across taxonomic boundaries may be limited. Moreover, structural and functional analyses enabled identification and characterization of various accessory genetic modules, including genes encoding major pilin protein (PilA), that enhance biofilm formation, as well as active transposable elements. Furthermore, comparative genomic analyses revealed that the studied plasmids of Antarctic Pseudomonas spp. are unique, as they are highly dissimilar to the other known plasmids of Pseudomonas spp.

Keywords: Antarctica; Pseudomonas; biofilm; horizontal gene transfer; plasmid; transposable element.

Figure 1

Plasmid occurrence and possible directions of the horizontal transfer of plasmids between the selected Pseudomonas strains isolated from the Antarctic soil. (a) The phylogenetic tree of plasmid-carrying Antarctic Pseudomonas spp. constructed based on 16S rDNA sequences. The unrooted tree was constructed using the maximum-likelihood algorithm (with the Tamura-Nei model), and statistical support for internal nodes was determined by 1000 bootstrap replicates. Values of >50% are shown. Pseduomonas putida ATCC 12633 was used as an out-group. (b) The UPGMA dendrogram was constructed based on results of analysis of heavy-metal resistance of Antarctic Pseudomonas spp. Circles and triangles on both trees indicate particular plasmids (identified in this study, plus pA3J1, characterized previously [26]). (c,d) The schemes showing the distribution of: (c) pA3J1, pA4J1, pA7J1 and pA29J1 plasmids, and (d) pA16J1, pA22BJ1 and pA22BJ2 plasmids. Plasmids with the same names but occurring in various strains are identical (100% of nucleotide sequence identity). Arrows indicate the possible directions of horizontal transfer of the analyzed plasmids. A dotted line indicates (potential) horizontal transfer of plasmid along with its possible point mutations.

Figure 2

Linear maps showing the genetic structure and organization of the circular plasmids of the Antarctic Pseudomonas spp. Arrows indicate genes and their transcriptional orientation. The predicted genes/genetic modules: CAP—capsule polysaccharide export system; HSP—heat shock protein; MOB—mobilization to conjugal transfer; PAR—partitioning; PIL—pilus assembly; REP—replication; RM—restriction–modification system; SUG—type I polysaccharide secretion; TA—toxin–antitoxin; TE—transposable element; TER—tellurium resistance are indicated.

Figure 3

(a) Attachment of E. coli DH5α harboring the plasmids pBBR1MCS-2, pBBR1-Pil6 (with PIL module of pA6H3), pBBR1-Pil46 (with the PIL module of pA46H2) and pBBR1-Pil62 (with the PIL module of pA62H1) to the polystyrene surface of microtitration plates, assessed by crystal violet staining. Error bars represent standard deviations. *—mean statistical significance p < 0.0001, compared to the adherence of E. coli DH5α harboring plasmids pBBR1MCS-2 (negative control). (b) Biofilm structure of E. coli DH5α harboring plasmids pBBR1MCS-2, pBBR1-Pil6 (with the PIL module of pA6H3), pBBR1-Pil46 (with the PIL module of pA46H2) and pBBR1-Pil62 (with the PIL module of pA62H1) after 24 h of cultivation, visualized by scanning confocal laser microscopy. Scale bars for biofilm thickness are presented. Horizontal optical sections were recorded at depths of 5 and 10 µm from the bottom of the dish.

Figure 4

Nucleotide sequence-based comparison of the plasmids identified in this study (plus pA3J1, that was identified previously and originates from the same environment) with 192 Pseudomonas spp. plasmids retrieved from the GenBank (NCBI) database. Whole-plasmid-genome similarity analysis was performed using Circoletto, with 1 × 10⁻⁵⁰ as the threshold. Plasmid sequences are represented as ideograms on the innermost ring. The size of the ideograms is proportional to the size of the plasmids; however, the plasmids identified in this study and pA3J1 were enlarged five times for a better visibility. The start and end positions of each plasmid genome are represented as green and orange blocks within the ideograms. The outer rings indicate the psychrotolerance of the host strain and name or accession number of the plasmid, respectively. The ribbon colors reflect the percentage identity of particular genomic regions.

Figure 5

Protein-based similarity network of Pseudomonas spp. plasmids. The network was constructed based on the comparison of the predicted proteins encoded by each plasmid compared all-against-all using BLASTp with e-value of 1 × 10⁻¹⁰, query coverage of HSP and sequence identity of at least 90% thresholds. Each node (circle) represents a single plasmid. Its size corresponds to the size of the plasmid and its color reflects psychrotolerance characteristics of the host strain. The color intensity of the node reflects the presence (intense color) or absence (bleached color) of at least one protein homologous to a protein encoded in at least one plasmid identified in this study. The number of homologous proteins encoded in two compared plasmids is reflected by the thickness of the edge (link) connecting each pair of nodes. For the clarity of the network, only the names of plasmids identified in this study (plus pA3J1) were shown, while the other plasmids are represented by numbers that correspond with the list of plasmids presented in the Supplementary Table S7.