Analysis of the biodegradative and adaptive potential of the novel polychlorinated biphenyl degrader Rhodococcus sp. WAY2 revealed by its complete genome sequence

The complete genome sequence of Rhodococcus sp. WAY2 (WAY2) consists of a circular chromosome, three linear replicons and a small circular plasmid. The linear replicons contain typical actinobacterial invertron-type telomeres with the central CGTXCGC motif. Comparative phylogenetic analysis of the 16S rRNA gene along with phylogenomic analysis based on the genome-to-genome blast distance phylogeny (GBDP) algorithm and digital DNA–DNA hybridization (dDDH) with other Rhodococcus type strains resulted in a clear differentiation of WAY2, which is likely a new species. The genome of WAY2 contains five distinct clusters of bph, etb and nah genes, putatively involved in the degradation of several aromatic compounds. These clusters are distributed throughout the linear plasmids. The high sequence homology of the ring-hydroxylating subunits of these systems with other known enzymes has allowed us to model the range of aromatic substrates they could degrade. Further functional characterization revealed that WAY2 was able to grow with biphenyl, naphthalene and xylene as sole carbon and energy sources, and could oxidize multiple aromatic compounds, including ethylbenzene, phenanthrene, dibenzofuran and toluene. In addition, WAY2 was able to co-metabolize 23 polychlorinated biphenyl congeners, consistent with the five different ring-hydroxylating systems encoded by its genome. WAY2 could also use n-alkanes of various chain-lengths as a sole carbon source, probably due to the presence of alkB and ladA gene copies, which are only found in its chromosome. These results show that WAY2 has a potential to be used for the biodegradation of multiple organic compounds.


Methods
To test the topology of the pRWAY01, pRWAY02 and pRWAY03 replicons, different sets of primers were designed at least at 800 bp from each replicon end to avoid telomeric sequences. A reverse primer in each of the left ends of the three replicons (1L3, 2L3 and 3L3), was designed in order to amplify with its respective forward primer in the replicons right ends (1R1, 2R1, 3R1) only in case of circular topology. Additionally, forward primers in the left ends (1L2, 2L1 and 3L2) and reverse primers in the right ends (1R3, 2R2 and 3R3), were designed to be combined with the ones previously described as positive controls. The sequences of the primers, Tm, positions in each replicon and combinations used can be seen below and also a scheme of the priming sites. For the small circular plasmid pRWAY04, two sets of primers (4F1-4R3 and 4F2-4R4) were designed to amplify ~10 Kbps of overlapping fragments that covers the total plasmid length (14.8 Kbps). Melting temperature of the primers, absence of dimerization and harping formation and lack of secondary priming sites were assessed using the OligoAnalyzer tool available at https://eu.idtdna.com/calc/analyzer. For pRWAY01, pRWAY02 and pRWAY03, PCR reactions were carried out in a total volume of 25 L containing 2.5 L of 10x PCB buffer MgCl2 free, 1 L MgCl2 50 mM, 0.5 L of DMSO (dimethyl sulfoxide) at 10% (v/v), 0.5 L dNTP mix 10 mM (2.5 mM each), 1 L of each primer at 10 M, 1 L of Taq DNA polymerase 1 U/L (Biotools) and 1 L of Rhodococcus sp. WAY2 genomic DNA at a 30-50 ng/L. The cycling conditions consisted in a first denaturation step at 95 ºC for 5 min followed by 27 cycles of amplification (1 min denaturation at 95 ºC, 45 s of primer annealing at 60 ºC and an elongation step at 72 ºC for 1.5 min) followed by a final elongation step at 72 ºC for 7 min. For pRWAY04, PCR reactions were carried out in a total volume of 25 L containing 12.5 L of Master Mix Q5' High Fidelity 2x (New England BioLabs), 1.25 L of each primer at 10 M, 0.5 L of DMSO at 10% (v/v) and 9.5 L of WAY2 genomic DNA at a 30-50 ng/uL. The cycling conditions consisted inn a first denaturation step at 98 ºC for 3 min followed by 35 cycles of amplification (10 s denaturation at 98 ºC, 30 s of primer annealing at 71 ºC and an elongation step at 72 ºC for 7 min) followed by a final elongation step at 72 ºC for 3 min. PCR products were electrophoretically separated in 0.8% (w/v) agarose gels and dyed with Gel Red.

Results
All the primer combinations to test the linear topology of the pRWAY01, pRWAY02 and pRWAY03 plasmids resulted in positive amplification of the controls (image), with amplicon sizes congruent with the theoretically expected, and negative amplification with the primer combinations designed to amplify only in case of circular topology (image: 1L3-1R1, 2L3-2R1 and 3L3-3R1, lanes 3, 6 and 9). These results validate the linear topology of the pRWAY01, pRWAY02 and pRWAY03 replicons of Rhodococcus sp. WAY2, as predicted by its genome sequencing. The two PCRs of the pRWAY04 circular small plasmid resulted in amplicon sizes of ~10 Kbps, congruent with the theoretically expected and validate the circular topology of this plasmid.
(A) Schematic representation of the primers designed to test the topology of the pRWAY01, pRWAY02 and pRWAY03 replicons of Rhodococcus sp. WAY2. Red arrows indicate the combination of primers which will result in positive amplification in case of circular topology. (B) Schematic representation of the primers designed to test the topology of the small pRWAY04 replicon. (C) PCR results in agarose gels at 0.8% (w/v). Black typing lanes show positive control amplicons (arrows), while red typing lanes (3, 6 and 9) show no amplification, which correspond with a linear topology of the pRWAY01, pRWAY02 and pRWAY03 replicons. Lanes 10 and 11 show the PCR products of the pRWAY04 small replicon, congruent with a circular topology.
Primers designed to test the topology of the Rhodococcus sp. WAY2 extrachromosomal replicons. Supplementary File S3. Whole genome-based taxonomic analysis of Rhodococcus sp. WAY2 using the Type (Strain) Genome Server (TYGS).

Forward (F) / Reverse (R) Sequence (5' -3') Tm (C)
GBDP tree based in whole-genome sequences of the 10 closest type strain genomes to R. sp. WAY2 (red typing) inferred with FastME from GBDP distances. Tree was rooted at midpoint. Pseudo-bootstrap support values are shown below branches and were calculated over 100 replicates, with an average branch support of 91.5%.
GBDP tree based in 16S rRNA gene sequences of the 10 closest type strain to R. sp. WAY2 (red typing) inferred with FastME from GBDP distances. Tree was rooted at midpoint. Pseudo-bootstrap support values are shown below branches and were calculated over 100 replicates, with an average branch support of 62.5%.
Pairwise comparisons of Rhodococcus sp. WAY2 against the 10 closest type strain genomes.