Plasmids of Psychrotolerant Polaromonas spp. Isolated From Arctic and Antarctic Glaciers - Diversity and Role in Adaptation to Polar Environments

Abstract

Cold-active bacteria of the genus Polaromonas (class Betaproteobacteria) are important components of glacial microbiomes. In this study, extrachromosomal replicons of 26 psychrotolerant Polaromonas strains, isolated from Arctic and Antarctic glaciers, were identified, sequenced, and characterized. The plasmidome of these strains consists of 13 replicons, ranging in size from 3,378 to 101,077 bp. In silico sequence analyses identified the conserved backbones of these plasmids, composed of genes required for plasmid replication, stable maintenance, and conjugal transfer. Host range analysis revealed that all of the identified plasmids are narrow-host-range replicons, only able to replicate in bacteria of closely related genera (Polaromonas and Variovorax) of the Comamonadaceae family. Special attention was paid to the identification of plasmid auxiliary genetic information, which may contribute to the adaptation of bacteria to environmental conditions occurring in glaciers. Detailed analysis revealed the presence of genes encoding proteins potentially involved in (i) protection against reactive oxygen species, ultraviolet radiation, and low temperatures; (ii) transport and metabolism of organic compounds; (iii) transport of metal ions; and (iv) resistance to heavy metals. Some of the plasmids also carry genes required for the molecular assembly of iron-sulfur [Fe-S] clusters. Functional analysis of the predicted heavy metal resistance determinants demonstrated that their activity varies, depending on the host strain. This study provides the first molecular insight into the mobile DNA of Polaromonas spp. inhabiting polar glaciers. It has generated valuable data on the structure and properties of a pool of plasmids and highlighted their role in the biology of psychrotolerant Polaromonas strains and their adaptation to the environmental conditions of Arctic and Antarctic glaciers.

Keywords: Antarctica; Arctic; Polaromonas; bacterial adaptation; glacier; heavy metal resistance; iron–sulfur [Fe-S] cluster; plasmid.

FIGURE 1

Phylogenetic tree for 16S rDNA sequences of Polaromonas spp. The tree was constructed by applying the neighbor-joining algorithm with Kimura corrected distances. Statistical support for the internal nodes was determined by 1000 bootstrap replicates and values of ≥50% are shown. The 16S rDNA sequence of Comamonas phosphati WYH 22-41 was used as an outgroup. GenBank accession numbers of the 16S rDNA sequences used for the phylogenetic analysis are given in square brackets. The scale bar represents 0.01 substitutions per nucleotide position. S.I and S.II – clusters of Antarctic strains (originating from King George Island), N.I and N.II – clusters of Arctic strains (originating from Spitsbergen Island). Strains analyzed in this study are in bold text. The source of each isolate is indicated in round brackets (I – ice, C – cryoconite material). The presence of a particular module within a given plasmid is shown by a colored dot. The following modules are indicated: AM, amino acid metabolism; AT, amino acid transport; CM, carbohydrate metabolism; CoM, coenzyme metabolism; E, energy production and metabolism; [Fe-S], [Fe-S] cluster assembly; HMR, heavy metal resistance; IM, ion metabolism; IT, ion transport; LM, lipid metabolism; MOB, conjugal transfer system; NM, nucleotide metabolism; OM, metabolism of other compounds; OS, oxidative stress response; PAR, partitioning system; POT, polyamine transport system; REP, replication system; TA, toxin–antitoxin system; UVR, UV radiation response.

FIGURE 2

Linear maps showing the genetic structure of the circular Polaromonas plasmids encoding replication initiation proteins of the TrfA and Rep3 families. Arrows indicate genes and their transcriptional orientation. Predicted genetic modules are indicated by colored boxes: AM, amino acid metabolism; AT, amino acid transport; CM, carbohydrate metabolism; CoM, coenzyme metabolism; E, energy production and metabolism; [Fe-S], [Fe-S] cluster assembly; HMR, heavy metal resistance; IM, ion metabolism; IT, ion transport; LM, lipid metabolism; MOB, mobilization for conjugal transfer; NM, nucleotide metabolism; OM, metabolism of other compounds; OS, oxidative stress response; PAR, partitioning system; POT, polyamine transport system; REP, replication system; TA, toxin–antitoxin system; UVR, UV radiation response. Orphan genes encoding predicted ParAs and antitoxins of TA systems are not shown as modules. The linear maps of plasmids pE3SP1, pE5SP1, pE10SP1, pH6NP1, and pW11NP2 were divided for clarity.

FIGURE 3

Linear maps showing the genetic structure of the circular Polaromonas plasmids encoding replication initiation proteins of the RepA_C, RPA, and RepL families, and unknown family plasmid pW11NP1. Arrows indicate genes and their transcriptional orientation. Predicted genetic modules are indicated by colored boxes: CM, carbohydrate metabolism; HMR, heavy metal resistance; IT, ion transport; MOB, conjugal transfer system; PAR, partitioning system; REP, replication system; TA, toxin–antitoxin system. Orphan genes encoding predicted ParAs and antitoxins of TA systems are not shown as modules.

FIGURE 4

Comparative analysis of genetic modules involved in [Fe-S] cluster assembly. The coordinates of the gene clusters identified in Polaromonas plasmids, as well as the chromosomes of Rhodoferax ferrireducens T118 (GenBank: CP000267) and Polaromonas sp. CJ666 (GenBank: CP000316) are shown in parentheses. The gray-shaded areas connect genes encoding proteins sharing ≥70% (dark gray) or 40–70% (light gray) aa identity, respectively.

FIGURE 5

The influence of predicted CDF, MER, and ZNT resistance modules on the heavy metal tolerance of Agrobacterium tumefaciens LBA288, Escherichia coli DH5α, Pseudomonas aeruginosa PAO1161, and V. paradoxus EPS. The resistance phenotypes of the transformed strains were tested at both 15 and 37°C. CDF-H6N – cadmium, cobalt, copper, manganese, nickel, and/or zinc resistance module of plasmid pH6NP1; MER-H6N – mercury resistance module of plasmid pH6NP1; ZNT-E3S and ZNT-E10S – P-type heavy metal-transporting ATPase of plasmids pE3SP1 and pE10SP, respectively.

FIGURE 6

Plasmid-encoded features contributing to adaptation of cold-active bacteria to polar environments. The presence of particular feature within Polaromonas plasmids is indicated by black dot.