Insights into Mobile Genetic Elements of the Biocide-Degrading Bacterium Pseudomonas nitroreducens HBP-1

Abstract

The sewage sludge isolate Pseudomonas nitroreducens HBP-1 was the first bacterium known to completely degrade the fungicide 2-hydroxybiphenyl. PacBio and Illumina whole-genome sequencing revealed three circular DNA replicons: a chromosome and two plasmids. Plasmids were shown to code for putative adaptive functions such as heavy metal resistance, but with unclarified ability for self-transfer. About one-tenth of strain HBP-1's chromosomal genes are likely of recent horizontal influx, being part of genomic islands, prophages and integrative and conjugative elements (ICEs). P. nitroreducens carries two large ICEs with different functional specialization, but with homologous core structures to the well-known ICEclc of Pseudomonas knackmussii B13. The variable regions of ICEPni1 (96 kb) code for, among others, heavy metal resistances and formaldehyde detoxification, whereas those of ICEPni2 (171 kb) encodes complete meta-cleavage pathways for catabolism of 2-hydroxybiphenyl and salicylate, a protocatechuate pathway and peripheral enzymes for 4-hydroxybenzoate, ferulate, vanillin and vanillate transformation. Both ICEs transferred at frequencies of 10^-6-10^-8 per P. nitroreducens HBP-1 donor into Pseudomonas putida, where they integrated site specifically into tRNA^Gly-gene targets, as expected. Our study highlights the underlying determinants and mechanisms driving dissemination of adaptive properties allowing bacterial strains to cope with polluted environments.

Keywords: ICEclc; Pseudomonas azelaica; adaptation; aromatic compounds; evolution; heavy metal; integrative and conjugative elements.

Figure 1

Circular maps of replicons of the P. nitroreducens HBP-1 genome. (A). Chromosome with predictions of the main genomic island (GIPni1), prophages (P) and ICEs (ICEPni1 and ICEPni2). The outermost circles show the location of predicted ORFs on the top strand (light blue) and bottom strand (red), followed by IslandViewer and PHASTER predictions showing regions of genome plasticity (dark blue). The inner circles represent BlastN comparisons with genomes of other Pseudomonas species (alternating green and purple circles), from the outside to the inside: Pseudomonas sp. AK6U (Acc. No., NZ_CP025229.1), Pseudomonas knackmussii B13 (NZ_HG322950.1), Pseudomonas putida KT2440 (NC_002947.4), Pseudomonas aeruginosa PAO1 (NC_002516.2), Pseudomonas citronellolis SJTE-3 (NZ_CP015878.1), Pseudomonas oryzihabitans USDA-ARS-USMARC-56511 (NZ_CP013987.1), Pseudomonas pseudoalcaligenes CECT 5344 (NZ_HG916826.1), Pseudomonas resinovorans NBRC 106553 (NC_021499.1), Pseudomonas stutzeri 28a24 (NZ_CP007441.1), and Pseudomonas veronii 1YdBTEX2 (LT599583.1). The two innermost circles depict the GC plot (penultimate circle), and the GC skew (most central circle). Numbers around the black circle indicate the size in base pairs. (B). Plasmid pPniHBP1_1 with the indication of heavy metal resistance loci (HMR), active partition system (parAB or PRTRC), initiation replication protein (rep), relaxase (rel), helicase (hel). Organization is the same as that described for the map of the chromosome except for the BlastN comparisons, which were performed with (from the outside to the inside) Pseudomonas putida KT2440 (NC_002947.4), Pseudomonas aeruginosa genomic island PAGI-5 (EF611301.1), Pseudomonas aeruginosa strain PA298 plasmid pBM908 (CP040126.1), and Pseudomonas aeruginosa strain T2101 plasmid pBT2101 (CP039991.1). (C). Plasmid pPniHBP1_2 with the same indications as for pPniHBP1_1, and other features of interest (umuCD, ndpA). Organization is the same as that described for the map of the chromosome except for the BlastN comparisons, which were performed with (from the outside to the inside). Pseudomonas sp. SCB32 chromosome (CP045118.1), Pseudomonas aeruginosa strain AR_0356 plasmid (pAR0356, CP027167.1).

Figure 2

Comparison of ICEPni1 and ICEPni2 from P. nitroreducens, and ICEclc from P. knackmussi B13 (A). Linear map of ICEPni1, ICEPni2, and ICEclc with indication of the location of predicted ORFs on the top strand (light blue boxes) and bottom strand (red boxes), the integrase-encoding gene (int), the attachment sites (att, black boxes), and variable regions (VR). The core region of ICEclc was framed with dashed black rectangles. The integrase gene of ICEclc on the rightmost end of the element was used as a reference point for the alignments. Comparisons were performed using BlastN and are displayed by colored areas linking related regions in the same (red) and inverted (purple) orientation. The intensity of the colored area reflects the percentage of nucleotide identity (minimum 65%) between the sequences. (B). Schematic representation (drawn to scale) of the genetic organization of ICEclc, ICEPni1 and ICEPni2 regulation loci. Genes are represented by arrowed boxes, and color-coded according to bioinformatic prediction or experimental demonstration of their function: purple, integration/excision; orange, active partition; light to dark blue, transcriptional activators; black, toxic effect; pink, single-stranded DNA protection; light yellow, tRNA; gray, unknown function. Promoters are represented by angled arrows pointing towards the transcription orientation. Numbers under genes (x/y) indicate the percentage of amino acid identity (x) and the coverage (y) of corresponding gene product in ICEclc.