Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Open Access

Peer-reviewed

Research Article

Rhodococcus aromaticivorans sp. nov., an o-xylene degrading bacterium, and evidence supporting reclassification of Rhodococcus jostii RHA1

Correction

17 Feb 2026: Muhammad N, Jeon J, Kim E, Kim D, Lee YM (2026) Correction: Rhodococcus aromaticivorans sp. nov., an o-xylene degrading bacterium, and evidence supporting reclassification of Rhodococcus jostii RHA1. PLOS ONE 21(2): e0342933. https://doi.org/10.1371/journal.pone.0342933 View correction

Abstract

A Gram-positive, aerobic, non-motile bacterium with a rod-coccus shape, designated DK17T, was isolated from a crude oil-contaminated soil and identified as a member of the genus Rhodococcus based on 16S rRNA gene analysis, showing highest similarity (99.93%) to Rhodococcus jostii DSM 44719T. However, average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values between strain DK17T and type strains within the genus Rhodococcus were below the species delineation thresholds of 95% and 70%, respectively. In contrast, DK17ᵀ exhibited ANI and dDDH values over 99% and 92%, respectively, with R. jostii RHA1. Comparative genomic analysis revealed that DK17T and RHA1 shared 93.5% of genes, while RHA1 and R. jostii NBRC 16295T shared only 78.6%, indicating a closer relationship between DK17T and RHA1. Both strains possess large genomes (~9.5–9.7 Mb) comprising a linear chromosome and multiple plasmids, and encode multiple dioxygenases and secondary metabolite biosynthetic gene clusters. In vitro assays confirmed o-xylene degradation by both DK17T and R. jostii RHA1, consistent with the presence of the akb gene cluster. Both strains shared C16:0 as a major fatty acid and menaquinone-8 (H2) as the dominant quinone. Based on genomic, phenotypic, and chemotaxonomic data, DK17T (=KCCM 90599T = InaCC B1662T) is proposed as a novel species, Rhodococcus aromaticivorans sp. nov., and R. jostii RHA1 is reclassified as a member of the same species.

Citation: Muhammad N, Jeon J, Kim E, Kim D, Lee YM (2025) Rhodococcus aromaticivorans sp. nov., an o-xylene degrading bacterium, and evidence supporting reclassification of Rhodococcus jostii RHA1. PLoS One 20(12): e0337194. https://doi.org/10.1371/journal.pone.0337194

Editor: Ivan S. Petrushin, Irkutsk State University: Irkutskij gosudarstvennyj universitet, RUSSIAN FEDERATION

Received: June 16, 2025; Accepted: November 4, 2025; Published: December 2, 2025

Copyright: © 2025 Muhammad et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: The 16S rRNA gene sequence of the strain DK17T was submitted to NCBI with the GenBank accession number PQ489412. The raw sequencing reads of DK17T genome have been deposited in the European Nucleotide Archive (ENA) under the accession number PRJEB81518. The genome of the strain DK17T is available in the NCBI with the RefSeq assembly accession number GCF_038447505.2.

Funding: The author(s) received no specific funding for this work.

Competing interests: No authors have competing interests.

1 Introduction

The genus Rhodococcus, belonging to the family Nocardiaceae, order Mycobacteriales, and phylum Actinomycetota, was first proposed in 1891 with Rhodococcus rhodochrous designated as the type species [1,2]. According to LPSN, the genus Rhodococcus currently includes 59 validly published species (https://lpsn.dsmz.de/genus/rhodococcus). However, recent phylogenomic analyses suggest that some of these species may belong to different genera, with several proposed to be reclassified into Rhodococcoides and Prescottella [3,4], indicating that the taxonomy of Rhodococcus is still under revision.

Members of the genus Rhodococcus are Gram-positive and non-motile Actinomycetota that display notable morphological variability depending on growth conditions and are characterized by menaquinone (MK)-8(H2) as their primary isoprenoid quinone [4]. Rhodococcus strains have been isolated from a wide array of environments, including soil, rocks, groundwater, marine sediments, animals, and plants, and are recognized for their extensive physiological versatility [5–11]. This ecological versatility is likely linked to the genomic diversity among Rhodococcus species, with genome sizes ranging from 3.71 to 10.91 Mb (https://www.ncbi.nlm.nih.gov/datasets/genome/?taxon=1827). Such differences in genome size are presumed to contribute to the broad metabolic capabilities of Rhodococcus species, facilitating their adaptation to diverse ecological niches [11].

The metabolic versatility of Rhodococcus species enables them to degrade a wide range of natural and synthetic compounds, including both aliphatic and aromatic hydrocarbons and various environmental pollutants [12]. For example, Rhodococcus sp. RHA1 can metabolize a variety of aromatic compounds, notably polychlorinated biphenyls (PCBs) [13]. R. oxybenzonivorans, isolated from a stream flowing through an industrial complex, can degrade aliphatic and aromatic hydrocarbons, including benzophenone-3, naphthalene, phenanthrene, and hexadecane [14]. Similarly, R. chlorophenolicus thrives in PCB-contaminated sediments, utilizing biphenyl as a carbon source, while R. phenolicus degrades compounds such as phenol, chlorobenzene, and dichlorobenzene [15,16]. The presence of mycolic acids in their cell walls enhances their ability to degrade hydrophobic pollutants, further supporting their role in the breakdown of persistent organic contaminants [12,17]. In particular, R. opacus R7 is well-known for its ability to degrade both naphthalene and o-xylene [18–20]. Recent studies have further demonstrated that Rhodococcus strains are capable of degrading polycyclic aromatic hydrocarbons (PAHs) and dibenzofuran, with biosurfactant production and high cell-surface hydrophobicity enhancing pollutant uptake and bioavailability [21,22]. Collections of alkanotrophic Rhodococcus strains have also revealed broad substrate spectra, including aromatic, N-heterocyclic, and substituted aromatic compounds, highlighting their high efficiency in hydrocarbon-contaminated soil remediation [21,23]. The substantial catabolic diversity observed in several Rhodococcus strains underscores their significant potential for environmental applications, particularly in bioremediation [14,16,24,25].

The large genomes and megaplasmids of Rhodococcus strains confer substantial metabolic versatility, supporting pollutant degradation and the production of secondary metabolites such as antimicrobial agents, bioflocculants, and biosurfactants. The first sequenced Rhodococcus genome, from R. jostii RHA1, spans 9.7 Mbp, including a 7.8 Mbp chromosome and three plasmids, encoding genes for the degradation of diverse compounds [13]. This strain shows extraordinary catabolic diversity including an ability to metabolize halogenated hydrocarbons, ketoaromatic compounds and steroids [13]. R. erythropolis PR4, capable of degrading long-chain alkanes, possesses multiple monooxygenase genes [24,26], while R. opacus R7 is equipped with genes that target a broad range of aliphatic and aromatic hydrocarbons [18,20,27]. The genomic analysis of R. oxybenzonivorans identified 89 monooxygenase and 62 dioxygenase genes, suggesting that this strain has a versatile ability to biodegrade various organic pollutants because the aerobic biodegradation of organic pollutants is generally initiated by oxygenases such as monooxygenase or dioxygenase [14]. These examples highlight the remarkable metabolic potential of Rhodococcus species, underscoring the significance of continued efforts to isolate and investigate novel strains from diverse ecological niches.

Rhodococcus strain DK17T, isolated from crude oil-contaminated soil in Korea, utilizes o-xylene as its sole carbon and energy source, exhibiting a doubling time of 2.4 hours. Although it cannot grow on m- or p-xylenes, DK17T is capable of degrading a variety of alkylbenzenes (e.g., toluene, ethylbenzene, isopropylbenzene), benzene derivatives (e.g., n-propyl- to n-hexylbenzenes, p-toluate, and m- and p-hydroxybenzoates) and phthalate [28–30]. Genome analysis of DK17T revealed the presence of three linear megaplasmids (~399 kb pDK1, ~ 318 kb pDK2, and ~779 kb pDK3), and the akb operon, known to catalyze o-xylene degradation [28], is located on pDK2 [31]. This operon encodes enzymes involved in alkylbenzene catabolism, including two sets of large and small subunits of o-xylene dioxygenase (akbA1a-akbA2a and akbA1b-akbA2b), a ferredoxin (akbA3) and ferredoxin reductase (akbA4), a dehydrogenase (akbB) and a series of meta-cleavage pathway enzymes including meta-cleavage 2,3-dioxygenase (akbC), hydrolase (akbD), hydratase (akbE), and aldolase (akbF) [32]. Functional characterization through recombinant expression in E. coli and gene expression profiling confirmed the conversion of o-xylene to cis-3,4-dihydrodiol by AkbA1A2A3A4, subsequent transformation to 3,4-dimethylcatechol by AkbB, and ring-cleavage by AkbC [33,34], thereby establishing the proposed degradative pathway toward tricarboxylic acid (TCA) cycle intermediates.

Despite the advancements in genome-based taxonomy, the precise classification of industrially significant Rhodococcus strains, including DK17T, remains unresolved. Recent studies suggest that certain strains, such as DK17T and RHA1, may belong to a distinct group within the genus, referred to as Cluster C or subgenus Anisorhodococcus, indicating ongoing challenges in defining species boundaries [3,35,36]. To address this, we conducted a comparative analysis of physiological and genomic characteristics of DK17T strain in relation to closely related Rhodococcus species, with the aim of clarifying its taxonomic position and evaluating its biotechnological potential in environmental applications and secondary metabolite production.

2 Materials and methods

2.1 Isolation and culturing conditions

Soil samples were collected in 1999 from an open access area within the Yeocheon Industrial Complex (Yeosu, South Korea). At the time of the sampling, the site was publicly accessible and no specific permits were required. Strain DK17 was isolated from o-xylene-degrading enrichment cultures using minimal salts basal (MSB) medium [37], with o-xylene supplied in the vapor phase and incubation at 30°C with shaking [28]. A 5 mL aliquot of the enriched culture, free of soil particles, was transferred to fresh MSB supplemented with o-xylene, followed by streaking onto MSB with 20 mM glucose (MSB + Glu) or Reasoner’s 2A (R2A) agar. The strain that grew well on both media was designated DK17T. Pure colonies were preserved in 20% glycerol at −80°C. Strain DK17T was deposited to the Korea Culture Center of Microorganisms (KCCM) and the Indonesian Culture Collection (InaCC).

2.2 Sequencing of 16S rRNA gene and phylogenetic analysis

Genomic DNA from strain DK17T, grown on MSB + Glu, was extracted by using the genomic DNA preparation reagent (PrepMan Ultra Sample Preparation Reagent, Thermo Fisher Scientific). The 16S rRNA gene was amplified using the universal primers, 27F and 1492R [38]. PCR products were purified using a PCR purification kit and sequenced using primers, 785F and 907R [38]. The 16S rRNA gene sequences were assembled using Vector NTI software and searched in the EzBioCloud database for matching sequences [39]. The sequences downloaded from EzBioCloud were aligned and trimmed using BioEdit software. Three types of phylogenetic trees were constructed utilizing the molecular evolutionary genetics analysis (MEGA X) software [40]: neighbor-joining (NJ) [41], maximum likelihood algorithms (ML) [42] and the maximum parsimony (MP) method [43]. All the phylogenetic trees were evaluated using 1,000 bootstrap iterations, with Corynebacterium diphtheriae NCTC 11397T (X84248) as the outgroup.

2.3 Genome sequencing, assembly, and annotation

The complete genome sequence of strain DK17T was sequenced using a combination of Oxford Nanopore Technology (ONT) and Illumina NovaSeq, as previously described by Jeon et al. (2025) [31]. Raw reads were quality-filtered using NanoFilt (v2.6.0) with parameters of -q 10 and -l 5000 for Nanopore and Trimmomatic (v0.21.0) for Illumina [44,45]. Hybrid assembly was performed using Unicycler (v0.4.8-beta) [46], and genome completeness and contamination were assessed using CheckM (v1.2.2) [47]. Annotation was conducted with the NCBI Prokaryotic Genome Annotation Pipeline [48]. Functional analyses including detection of biosynthetic gene clusters (BGCs), were performed using RAST [49] and antiSMASH (v7.0) [50] with default parameters.

2.4 Genome-based relatedness and functional features comparison

For phylogenomic analysis, multiple sequence alignments of the concatenated 120 ubiquitous single-copy proteins were performed using GTDB-Tk [51]. Two datasets were analyzed: one comprising thirty-two available type strain genomes within the genus Rhodococcus along with DK17T, R. jostii RHA1 and R. opacus R7, and the other including six additional genomes designated as R. jostii (R. jostii NPDC059932, R. jostii NPDC059950, R. jostii NPDC127600, R. jostii IEGM 60, R. jostii DSM 44719T, and R. jostii CCM 4760). Phylogenomic trees were reconstructed based on 1,000 sets of sequence replications using the maximum likelihood algorithm by MEGA X [40,42,52]. To assess genome-based similarity among 35 available Rhodococcus genomes, pairwise genome-based relatedness was estimated by calculating average nucleotide identity (ANI) using EzBioCloud (www.ezbiocloud.net/tools/ani) [39]. Digital DNA–DNA hybridization (dDDH) among genomes that showed > 94% ANI values with DK17T, including R. jositi RHA1, R. opacus ATCC 51881T, R. opacus R7, and R. wratislaviensis NBRC 100605T, as well as the type strain R. jostii NBRC 16295T (genome-sequenced strain, equivalent to the type strain DSM 44719T) was calculated using the genome-to-genome distance calculator (GGDC) (version 3.0, DSMZ; https://ggdc.dsmz.de/ggdc.php#) [39,53]. Core gene sharing analysis and comparative assessment of the genomic basis for o-xylene degradation and synthesis of secondary metabolites were performed using genomes that showed >94% ANI values with DK17T, including R. jostii RHA1, R. opacus ATCC 51881T, R. opacus R7, and R. wratislaviensis NBRC 100605T, as well as the type strain R. jostii NBRC 16295T. Comparative genome analysis was performed by sequence clustering using CD-HIT (v4.8.1) [54] with a 70% sequence identity threshold to investigate core genes between genomes. UpSet plot was visualized using UpSetR package (v1.4.0) in R [55].

2.5 Physiological and biochemical characterization

To determine physiological and biochemical characteristics, strains DK17T, R. jostii RHA1, and R. opacus KCTC 9811T (=ATCC 51881T) were cultivated on R2A agar. Gram reaction was performed using Gram-stain kit (Sigma). The morphology of the cells was examined using transmission electron microscopy (TEM; CM200, Philips). For TEM analysis, the cells of strains DK17T and R. jostii RHA1 were negatively stained with 2.0% uranyl acetate on a carbon-coated copper grid [56]. Motility of strains DK17T, R. jostii RHA1, and R. opacus KCTC 9811T was determined by the observation of growth after inoculation in the R2A liquid medium with 0.4% agar. The optimal temperature for growth of strains DK17T, R. jostii RHA1, and R. opacus KCTC 9811T was determined by culturing strains on R2A at different temperatures (0, 4, 10, 15, 20, 25, 30, and 37°C). NaCl tolerance tests for all three strains were conducted on R2A agar supplemented with varying concentration of NaCl (0, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, and 9% w/v) [57]. Growth on agar plate was evaluated based on visible colony formation after incubation.

The optimal pH for growth for all three strains was determined in R2A liquid medium. The pH was adjusted using the following buffering systems; Na2HPO4-buffered citric acid, pH 4.0–5.0; MES, pH 5.5–6.0; MOPS, pH 6.5–7.0; AMPD, pH 8.0–9.5; CAPS, pH 10.0. The growth of each culture was assessed by measuring optical density at 600 nm (EnVision plate reader, PerkinElmer) [56]. The growth under anaerobic culture condition was determined using a jar containing an AnaeroPak (Mitsubishi Gas Chemical). Biochemical characterization for strains DK17T, R. jostii RHA1, and R. opacus KCTC 9811T were determined. Catalase activity was tested using 3% (v/v) hydrogen peroxide and oxidase activity was assessed with 1% (w/v) tetramethyl-p-phenylenediamine dihydrochloride [58,59]. The ability to hydrolyze casein, chitin, hypoxanthine, starch, xanthine, and Tweens 20, 40, 60, and 80 was evaluated on R2A agar [60]. Biochemical characteristics and enzymatic activities were determined using API ZYM and API 20NE following the manufacturer’s instructions (bioMérieux).

2.6 Chemotaxonomic characterization

To determine the cellular fatty acid profile, cells from strains DK17T, R. jostii RHA1, R. opacus KCTC 9811T were collected from R2A agar after incubation at 30°C for two days. The fatty acids were extracted following the MIDI protocol (Sherlock Microbial Identification System version 6.0) and analyzed using a gas chromatography (GC) system at KCCM (Seoul, Korea) [61]. Quinones were extracted as described by Collins and Jones and analyzed by high-performance liquid chromatography (HPLC) at KCCM [62].

2.7 In vitro assay for o-xylene degradation

For the degradation of o-xylene, freshly prepared cultures of DK17T, R. jostii RHA1, and R. opacus KCTC 9811T, were transferred to MSB agar plates, with o-xylene supplied as the sole carbon and energy source. o-Xylene in liquid form was added to a glass bulb with a cotton stopper and directly supplied to the strains as a vaporized gas in an airtight container [28]. As a control, the strains were also transferred to MSB agar without o-xylene. The plates were incubated at 30°C for three days, and after incubation, growth on the MSB media was observed and compared between the presence and absence of o-xylene.

3 Results and discussion

3.1 16S rRNA gene-based phylogeny

The phylogenetic analysis using 16S rRNA gene sequence confirmed that strain DK17T is affiliated to the genus Rhodococcus, showing the highest sequence similarities with R. jostii DSM 44719T (99.93%), R. koreensis DSM 44489T (99.36%), R. oxybenzonivorans S2-17T (99.21%), and R. percolatus MBS1T (99.06%). The 16S rRNA gene similarity values between strain DK17T and closely related strains exceeded the cutoff values of species discrimination (98.7%) [63]. Phylogenetic trees based on 16S rRNA gene sequences showed a monophyletic clustering of the strain DK17T with reference strains R. jostii DSM 44719T, R. jostii RHA1, and R. koreensis DSM 44498T (Figure S1 in S1 File).

3.2 Genome-based relationship and comparison of general genomic features

The CheckM analysis indicated a value of 99.59% completeness and 2.01% contamination, demonstrating the high quality of DK17T genome (Table S1 in S2 File). The 16S rRNA gene sequences of strain DK17T (1,430 bp), determined by direct sequencing, were identical to those retrieved from its genome sequences. A genome-based phylogenetic tree demonstrated a robust clustering of strain DK17T and R. jostii RHA1 within the genus Rhodococcus, supporting their close evolutionary relationship while R. jostii NBRC 16295T was more distantly related to RHA1 (Fig 1). In addition, four genomes deposited under the name R. jostii (R. jostii NPDC059932, R. jostii NPDC059950, R. jostii NPDC127600, and R. jostii IEGM 60) also clustered with DK17T and R. jostii RHA1 (Figure S2 in S1 File), suggesting potential misidentification of these strains. To further confirm the taxonomic position and novelty of strain DK17T, ANI and dDDH values were calculated against the closely related strains forming a monophyletic clade in the phylogenomic tree. The ANI and dDDH values between strain DK17T and closely related strains in the phylogenomic tree, except for R. jostii RHA1, were below the standard cut-off values for speciation, with ANI values under 95–96% and dDDH values below 70% (Fig 1, Table S2 in S2 Table) [53,64]. This suggests that DK17T can be considered a novel species. In contrast, the ANI and dDDH values between strain DK17T and R. jostii RHA1 were 99% and 92%, respectively, supporting their classification as the same species. In addition, four genomes deposited under the name R. jostii (R. jostii NPDC059932, NPDC059950, NPDC127600, and IEGM 60) showed >95% ANI values with DK17T, whereas their ANI values with the R. jostii type strain genomes were approximately 90% (Figure S2 in S1 File). Previously, Rhodococcus strain RHA1 was classified as a bona fide member of the species R. jostii based on a combination of phenotypic characteristics and DNA-DNA relatedness, which was manually determined to exceed 87%, well above the 70% threshold for species delineation [65]. However, this study re-evaluates the classification of Rhodococcus strain RHA1 using a comprehensive genome-based approach, providing a more precise and robust basis that refines prior classifications. Our analysis showed that the ANI values between R. jostii RHA1 and the R. jostii type strains, R. jostii DSM 44719T and R. jostii NBRC 16295T were 90% indicating that R. jostii RHA1 is distinct from the established R. jostii type strains. Consistent with recent analyses of intrageneric structure within Rhodococcus, DK17T and RHA1 clearly fall within Cluster C, which corresponds to the proposed subgenus Anisorhodococcus [35,36]. This cluster is phylogenetically distinct from the one containing the authentic R. jostii type strain, further supporting that RHA1 and DK17T should not be classified as R. jostii. Furthermore, our analysis showed that R. opacus R7 and R. wratislaviensis NBRC 100605T belong to the same species, with a pairwise ANI of 98.7% and a dDDH value of 90% (Fig 1, Table S2 in S2 Table).

thumbnail
Download:
Fig 1. Maximum likelihood phylogenomic tree inferred from concatenated alignments of 120 single-copy amino acid sequences in Genome Taxonomy Database (GTDB).

Genomic sequences were obtained from the NCBI RefSeq database under the corresponding assembly accession number. Bootstrap values are represented by the nodes as filled circle (>90%) and empty circle (>50%). Corynebacterium diphtheria NCTC11397T (GCF_001457455) was used as an outgroup. Scale bar, 0.05 substitutions per amino acid position. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) were calculated for the following genomes: 1. DK17T, 2. R. jostii RHA1, 3. R. opacus ATCC 51881T, 4. R. opacus R7, 5. R. wratislaviensis NBRC100605T, 6. R. koreensis DSM 44498T, 7. R. pseudokoreensis R79T, 8. R. jostii NBRC 16295T.

https://doi.org/10.1371/journal.pone.0337194.g001

The total genome size of strain DK17T is 9.5 Mb, with a GC content of 67.1% (Fig 1, Table S1 in S2 File). The genome sizes of Rhodococcus strains that formed a monophyletic clade with DK17T in the phylogenomic tree ranged from 8.1 to 10.4 Mb, which is larger than those in other clades (3.7–7.9 Mb) (Fig 1). Notably, our phylogenomic analysis shows that RHA1 is well separated from the R. jostii type strain, consistent with the observations reported by Sangal et al. (2019) [35]. The GC contents of strains forming a monophyletic clade with DK17T ranged from 66.7 to 67.4%, which is consistent within genus Rhodococcus (61.7–73.1%) (Fig 1). The genome of strain DK17T comprises one linear chromosome, three linear mega-plasmids, and two circular plasmids (Table S1 in S2 File). Rhodococcus jostii RHA1 and R. opacus R7 possess three and five linear plasmids, respectively (Table S1 in S2 File). In contrast, plasmids in R. opacus ATCC 51881T, R. wratislaviensis NBRC 100605T, and R. jostii NBRC 16295T were not fully determined, partially due to the low sequencing depths.

Identification of core and unique genes among strain DK17T and five Rhodococcus strains, R. jostii RHA1, R. opacus R7, R. opacus ATCC 51881T, R. wratislaviensis NBRC 100605T, and R. jostii NBRC 16295T, revealed that all strains share 4,880 core genes, with 759 unique genes identified in strain DK17T (Fig 2). Notably, DK17T shared 93.4–93.5% of its genes with R. jostii RHA1 while only 79.9–80.0% with R. jostii NBRC 16295T. In addition, the proportion of shared genes between R. jostii RHA1 and R. jostii NBRC 16295T is 78.6–78.8%. This suggests that R. jostii RHA1 and R. jostii NBRC 16295T are distinct taxa, while DK17T and RHA1 are more closely related.

thumbnail
Download:
Fig 2. Comparative genome analysis of DK17T with closely related Rhodococcus strains.

(a) UpSet plot represents the core, unique, and accessory genes between Rhodococcus genomes. (b) The proportion of shared genes.

https://doi.org/10.1371/journal.pone.0337194.g002

3.3 Physiological and biochemical characterization

Strain DK17T was Gram-positive, strictly aerobic, and non-motile. TEM images revealed that DK17T exhibited a rod–coccus morphology, with cell length of 1.1–1.7 μm and a diameter ranging from 0.8–0.9 μm. R. jostii RHA1 displayed a similar morphology, with cell lengths of 0.8–1.2 μm and diameters of 0.6–0.9 μm. The growth temperature range for DK17T was 10–30°C (optimum 25–30°C). DK17T grew within a pH range of 5.5–8.0 (optimum 7.0) and in the NaCl concentration of 0–8% (w/v) (optimum 0–6%) (Table 1). The growth temperature and NaCl tolerance of strain DK17T are similar to those of R. jostii RHA1 and R. opacus KCTC 9811T. However, the pH tolerance range for R. jostii RHA1 and R. opacus KCTC 9811T (5.0–9.0) is slightly broader than that of strain DK17T. The detailed physiological characteristics of strain DK17T and closely related Rhodococcus strains are summarized in (Table 1).

thumbnail
Download:
Table 1. Differential physiological properties of the strain DK17T and phylogenetically closely related Rhodococcus species. Strains: 1, DK17T; 2, R. jostii RHA1 [65]; 3, R. opacus KCTC 9811T [91]; 4, R. wratislaviensis N805T [5]; 5, R. jostii IFO 16295T [92].

https://doi.org/10.1371/journal.pone.0337194.t001

Strain DK17T along with R. jostii RHA1 and R. opacus KCTC 9811T was catalase positive and oxidase negative. The strain DK17T hydrolyzed the casein, hypoxanthine, Tween 20, 40, and 60. Interestingly, R. jostii RHA1 can hydrolyze casein, hypoxanthine, and Tween 20, whereas R. opacus KCTC 9811T is not capable of hydrolyzing the macromolecules. In API ZYM test, all three Rhodococcus strains, DK17T, R. jostii RHA1, and R. opacus KCTC 9811T showed nearly similar results. The strain DK17T was positive for the activities of acid phosphatase, cysteine arylamidase, α-gulcosidase, lipase C14, leucine arylamidase, naphthol-AS-BI-phosphohydrolase, trypsin, and valine arylamidase while weak positive for α-chymotrypsin. In API 20 NE, the strain DK17T was positive for the reduction of nitrates to nitrites, for the hydrolysis of esculin ferric citrate, and production of urease enzyme. However, the strain R. opacus KCTC 9811T was only weakly positive for urease activity. Most of the physiological and biochemical characteristics determined in this study were similar between DK17T and R. jostii RHA1. However, hydrolysis of Tween 40 and 60, which was positive in DK17T, was negative in RHA1 (Table 1).

3.4 Chemotaxonomic characterization

The fatty acid profile for strain DK17T, R. jostii RHA1, and R. opacus KCTC 9811T were determined. The predominant fatty acids found in strain DK17T, R. jostii RHA1 and other closely related strains, R. opacus KCTC 9811T, R. wratislaviensis NCTC 13229T, and R. jostii IFO 16295T were C16:0 and C17:0, ranging from 22.0–33.0% and 7.0–16.5% (Table 2). In DK17T and R. jostii RHA1, C17:1 ω8c and summed features 3 were also predominant, ranging from 15.6–17.0% and 14.0–14.6%, respectively (Table 2). Additionally, only R. jostii RHA1 and R. opacus KCTC 9811T possessed C18:1 ω9c at proportions of 14.2% and 11.0%, respectively. Notably, C15:0 was not detected in strains DK17ᵀ and R. jostii RHA1, which may serve as a distinguishing feature from other Rhodococcus species where this fatty acid is typically more abundant. Strain DK17T possesses menaquinone 8 (MK-8) as its respiratory quinone, consistent with R. jostii RHA1 [65].

thumbnail
Download:
Table 2. Cellular fatty acid composition of the strain DK17T and phylogenetically closely related Rhodococcus species. Strains: 1, DK17T; 2, R. jostii RHA1; 3, R. opacus KCTC 9811T; 4, R. wratislaviensis N805T; 5, R. jostii IFO 16295T. Data for DK17T, R. jostii RHA1, and R. opacus KCTC 9811Tare from the present study, while data for the other strains are from previously published sources4-5.

https://doi.org/10.1371/journal.pone.0337194.t002

3.5 Comparative analysis of genome functions

3.5.1 Aromatic compound metabolism.

The RAST analysis showed that strain DK17T carried 9,548 coding sequences (CDSs) and 61 RNAs and R. jostii RHA1 exhibited 9,680 CDSs and 66 RNAs. In all six genomes used for comparison, the largest number of annotated genes were assigned to amino acids and derivatives (631–778), carbohydrate metabolism (527–778), and fatty acids, lipids, and isoprenoid synthesis (300–503) (Table S3 in S2 File). Notably, all the six Rhodococcus strains exhibited a high number of genes related to the metabolism of aromatic compounds, with a total range of 138–177 genes identified. These genes are involved in the metabolism of various aromatic compounds such as biphenyl, benzoate, quinic acid, salicylate, gentisate, catechol, protocatechuate, and β-ketoadipate.

Strain DK17ᵀ and R. jostii RHA1 harbor a comparable repertoire of genes involved in the degradation of aromatic compounds, although DK17ᵀ had slightly fewer CDSs than R. jostii RHA1. Both strains contain genes for aromatic monooxygenases, dioxygenases, and the β-ketoadipate pathway, which are consistent with their experimentally observed ability to degrade a broad range of aromatic compounds. Among these compounds, benzoate, a simple aromatic carboxylate that is both a widespread environmental pollutant and utilizable growth substrate is metabolized by diverse microorganisms. For example, Pseudomonas putida KT2440 degrades benzoate through the catechol intermediates [66], while Acinetobacter baylyi and Aspergillus nidulans utilize quinate via the quinate/shikimate pathway [67,68]. In Rhodococcus species, benzoate is commonly degraded under aerobic conditions via benzoate 1,2-dioxygenase or benzoate monooxygenase. The benzoate-degrading ability of strain DK17ᵀ was also demonstrated in pure culture experiments [69]. These degradation processes play important roles in soil bioremediation by contributing to pollutant detoxification in terrestrial and aquatic environments [70,71]. Furthermore, the capacity to metabolize benzoate is often associated with microbial strains that can degrade structurally complex molecules, including steroids. For instance, cholesterol degradation in Rhodococcus begins with oxidation to cholestenone via extracellular cholesterol oxidase [72] and efficient conversion has been demonstrated in Rhodococcus erythropolis using alkylated cyclodextrin [73]. A cholesterol oxidase gene (choG) was identified in Rhodococcus sp. strain CECT3014 [74]. However, this gene was not detected from strain DK17T and R. jostii RHA1, despite the confirmation of initial ring oxidation in R. jostii RHA1. These observations highlight the metabolic flexibility of Rhodococcus and their potential in bioremediation and steroid bioconversion.

Beyond biodegradation, recent studies have highlighted the ecological roles of Rhodococcus species in interactions with plants [75,76]. While pathogenic species such as Rhodococcus equi and Rhodococcus fascians cause foal pneumonia and leafy gall disease in plants [77,78], other strains exhibit traits associated with nitrogen metabolism and plant growth promotion. For example, R. jostii RHA1 encodes nitrogen lipid regulator (NlpR), suggesting regulatory capacity for nitrogen metabolism [79]. In addition, Rhodococcus erythropolis KB1 has been shown to synergize with Medicago sativa to enhance petroleum hydrocarbon degradation (up to 95%) while simultaneously promoting plant growth and nutrient enrichment [80], and Rhodococcus qingshengii RL1, a plant-associated strain isolated from Eruca sativa, carries genes enabling nitrogen fixation, phytohormone and siderophore production, and root colonization, highlighting its role in stress tolerance and plant–microbe interactions [81]. Although DK17ᵀ has not been directly evaluated for plant growth-promoting effects, its genome harbors genes encoding inorganic phosphatase (ppx and ppa) and alkaline phosphatase (phoD), which may support phosphate solubilization. These features, together with predicted capabilities for secondary metabolite production, provide a foundation for future studies exploring the potential of DK17ᵀ in plant–microbe interactions. Validating these metabolic traits experimentally, including biodegradation, nutrient cycling, and symbiotic interactions, will be crucial to uncovering the putative ecological functions of this strain.

3.5.2 Biosynthetic gene clusters and secondary metabolite potential.

Analysis of biosynthetic gene clusters (BGCs) involved in the synthesis of secondary metabolites using antiSMASH revealed that the genomes of the strains used for comparison contained 18–23 BGCs (Fig 3). These BGCs belong to various groups, including non-ribosomal peptide synthases (NRPSs), polyketides, beta-lactones, and terpenes and they encode compounds that act as antibiotics, anticancer agents, antioxidants, and siderophores.

thumbnail
Download:
Fig 3. Biosynthetic gene clusters (BGCs) in Rhodococcus strains.

NRPS, non-ribosomal peptides synthase; NAPAA, non-alpha-poly amino acid; T1PKS, type I polyketide synthase.

https://doi.org/10.1371/journal.pone.0337194.g003

Among the BGCs, NRPSs represent biosynthetic capacity for a structurally diverse class of secondary metabolites known for their wide range of biological activities and pharmacological properties [82,83]. Strain DK17T harbors NRPSs BGCs predicted to produce compounds such as atratumycin, galtamycin, indigoidine, oxalomycin B, rimosamide, and legonindolizidine A6 (Fig 3). These antibiotics represent an important group of biocontrol agents that protect plants from bacterial and fungal pathogens, thereby underscoring the significance of these strains as valuable biotechnological resources [82,84,85]. In particular, BGCs for atratumycin, fluvirucin B1, and auroramucin were found only in DK17T and R. jostii RHA1 while other genomes did not harbor these BGCs. Interestingly, among the Rhodococcus strains, only DK17T contains BGCs associated with fluvirucin B2, petrichorin, auroramycin, legonindolizidine A6, and indigoidine, all of which exhibit antimicrobial activity. R. jostii RHA1 carries a BGC predicted to encode biosynthesis of lavendiol, which functions as an antimicrobial and antioxidant compound [86]. In contrast, another group of NRPSs, including those associated with thermochelin and rhodochelin, function as siderophores that enable bacteria to sequester iron from the environment, thereby facilitating their growth under challenging conditions were found in all genomes compared [87].

The second major class of BGCs encodes the biosynthetic capacity for polyketides, which are synthesized by polyketide synthases, multi-domain enzymes responsible for producing a diverse range of secondary metabolites [88]. These BGCs encode compounds such as tetrocarcin A and lobosamide, which function as antibiotics and growth inhibitors. Furthermore, all genomes contained BGCs for isorenieratene, which are characterized by their five-carbon hydrocarbon skeleton and function as antioxidants and photoprotective pigments [89]. The results underscore the valuable contributions of DK17T and related Rhodococcus strains to environmental and biotechnological applications through the production of significant metabolites.

3.6 o-Xylene degradation gene analysis and in vitro assay

The genus Rhodococcus is well-known for its ability to degrade aromatic compounds, with various strains effectively breaking down substances such as pyridine, biphenyl, and oxybenzone [14–16]. Dioxygenase plays a key role in this process by incorporating both atoms of molecular oxygen into their substrates for the degradation of aromatic compounds [90]. Strain DK17T encodes a total of 76 dioxygenases or dioxygenase components, 15 of which are related to aromatic dioxygenase. Among them, four dioxygenase components (akbA1a, akbA1b, akbA2a, and akbA2b encoding aromatic ring-oxidizing dioxygenase) and one dioxygenase (akbC encoding aromatic ring-cleavage dioxygenase) were included in the akb gene cluster, indicating a role in the o-xylene degradation pathway. A genomic investigation of the akb gene cluster, an o-xylene degradation gene cluster, in six Rhodococcus species (DK17T, R. jostii RHA1, R. jostii NBRC 16295T, R. opacus ATCC 51881T, R. opacus R7, and R. wratislaviensis NBRC 100605T) revealed that DK17T, R. jostii RHA1, and R. opacus R7 harbored akb cluster-related genes in their genomes, while the other species did not (Fig 4). Furthermore, the akb cluster-related genes in pDK2 of DK17T share an average amino acid sequence identity of 99.9% with those in plasmid pRLH2 of R. jostii RHA1. This finding suggests that the ability to degrade o-xylene is strain-specific within the genus Rhodococcus.

thumbnail
Download:
Fig 4. Comparison of the genetic organization of the akb gene cluster.

The plot was visualized using the syntenyPlotteR package (v1.0.0) in R (v4.3.3). Putative genes are colored by dark gray and pseudogenes are shown in a dashed arrow shape. akbS and akbT, two-component regulatory genes; akbA1a-akbA2a and akbA1b-akbA2b, o-xylene dioxygenase—two sets of large and small oxygenase subunits; akbA3, a gene for ferredoxin component; akbA4, a gene for ferredoxin reductase component; akbB, cis-dihydrodiol dehydrogenase; akbC, meta-cleavage 2,3-dioxygenase; akbD, hydrolase; akbE, hydratase; akbF, aldolase.

https://doi.org/10.1371/journal.pone.0337194.g004

In vitro capacity for o-xylene degradation of strains DK17T, R. jostii RHA1, and R. opacus KCTC 9811T was performed to confirm the involvement of akb cluster-related genes in the o-xylene degradation. The strain DK17T and R. jostii RHA1 showed optimum growth on MSB agar plates in the presence of o-xylene vapor while very weak growth on MSB in the absence of o-xylene. The strain R. opacus KCTC 9811T showed a very weak growth on MSB supplemented with o-xylene vapor. These observations are consistent with the presence of a complete akb gene cluster in DK17ᵀ and R. jostii RHA1, and its absence in R. opacus KCTC 9811ᵀ. Overall, our analysis highlights the important contribution of strain DK17T in the degradation of various alkylbenzenes including o-xylene, which is crucial for bioremediation, carbon source utilization, and toxicity reduction in contaminated environments.

4 Conclusion

4.1 Description of Rhodococcus aromaticivorans sp. nov.

Rhodococcus aromaticivorans (a.ro.ma.ti.ci’vo.rans. L. masc. adj. aromaticus, aromatic, fragrant; L. pres. part. vorans, devouring; N.L. masc. part. adj. aromaticivorans, devouring aromatic compounds).

Cells are Gram-positive, aerobic, non-motile, catalase-positive, and exhibit a cocci-rod shape, measuring 1.1–1.7 μm in length and 0.8–0.9 μm in diameter. Colonies on R2A agar are mucoid after 48 h incubation at 30°C. The strain can grow at temperatures ranging from 10–30°C (optimum, 30°C), pH 5.5–8.0 (optimum, pH 7.0) and with 0–8% NaCl (w/v; optimum, 0–6%).

Cells are positive for the hydrolysis of casein and Tween 20 while negative for Tween 80, starch, and chitin. In the API ZYM kit, the strain was positive for the enzymatic activities of acid phosphatase, cystine arylamidase, lipase (C14), leucine arylamidase, α-glucosidase, β-glucosidase, naphthol-AS-BI-phosphohydrolase, trypsin, and valine arylamidase. In the API 20NE assay, the strain was positive for nitrate reduction, urease production, and hydrolysis of esculin ferric citrate. The predominant fatty acids found in strain DK17T were C16:0, C17:1 ω8c, and summed features 3. The respiratory quinone is MK-8.

The genomic DNA G + C content of the type strain is 67.09%. The type strain, DK17T (=KCCM 90599T = InaCC B1662T), was isolated from crude oil-contaminated site in Yeocheon, Republic of Korea. The 16S rRNA gene sequence of the strain DK17T was submitted to NCBI with the GenBank accession number PQ489412. The raw sequencing reads of DK17T genome have been deposited in the European Nucleotide Archive (ENA) under the accession number PRJEB81518. The genome of the strain DK17T is available in the NCBI with the RefSeq assembly accession number GCF_038447505.2.

4.2 Reclassification of Rhodococcus jostii RHA1 as Rhodococcus aromaticivorans

Our comprehensive genome-based and physiological analyses support the reclassification of R. jostii RHA1, distinguishing it from the established R. jostii. Phylogenomic analysis revealed a strong evolutionary relationship between DK17T and RHA1, with ANI (99%) and dDDH (92%) values well above species delineation thresholds, while RHA1 exhibited substantial genetic divergence from R. jostii DSM 44719T and NBRC 16295T. Comparative genomic analysis further reinforced this distinction, as DK17T and RHA1 shared a significantly higher proportion of genes with each other than with R. jostii type strains. Physiological and biochemical characteristics, including unique substrate utilization patterns and fatty acid profiles, also supported this classification. Furthermore, the akb cluster-related genes in plasmid pDK2 of DK17T share an average amino acid sequence identity of 99.9% with those in plasmid pRLH2 of R. jostii RHA1, highlighting a close genetic relationship between these two strains in their capacity for o-xylene degradation. These findings provide strong evidence that RHA1 is more closely related to DK17T than to R. jostii type strain, necessitating its taxonomic reassignment and refining the classification of these industrially significant Rhodococcus strains.

Supporting information

S1 File. Additional figures related to the identification of strain DK17T. Figure S1. Neighbor-Joining tree based on 16S rRNA gene sequences showing the phylogenetic relationship of DK17T and closely related species in the genus Rhodococcus.

Bootstrap values (>70%) in the order of NJ/ML/MP are shown at the branch points based on 1,000 replications. An asterisk (*) indicates bootstrap values below 70% in the order of NJ/ML/MP. GenBank accession numbers are shown in parentheses. Bar, 0.01 substitutions per nucleotide position. Corynebacterium diphtheriae NCTC 11397T (X84248) was used as an outgroup. Figure S2. Maximum likelihood phylogenomic tree inferred from concatenated alignments of 120 single-copy amino acid sequences in Genome Taxonomy Database (GTDB) (a) and Average nucleotide identity (b). Genomic sequences were obtained from the NCBI RefSeq database under the corresponding assembly accession number. Genomes highlighted in the grey box represent non-type strains deposited under the name Rhodococcus jostti whereas those in the dark grey box are type strains. Bootstrap values (>70%) are indicated on the nodes. Corynebacterium diphtheria NCTC11397T (GCF_001457455) was used as an outgroup. Scale bar, 0.05 substitutions per amino acid position.

https://doi.org/10.1371/journal.pone.0337194.s001

(PPTX)

S2 File. Additional tables related to DK17T genome. Table S1. Summary of genome statistics. Table S3. Overview of the RAST subsystem and the numbers of genes involved in each metabolism of DK17T and the closest reference strains within the genus Rhodococcus. Strains: 1, DK17T; 2, R. jostii RHA1; 3, R. opacus R7: 4, R. opacus ATCC 51881T: 5, R. wratislaviensis NCTC13229T; 6, R jostii NBRC 16295T

https://doi.org/10.1371/journal.pone.0337194.s002

(DOCX)

S2 Table. Additional table for DK17T genome comparison. Table S2. Average nucleotide identity among Rhodococcus genomes.

https://doi.org/10.1371/journal.pone.0337194.s003

(XLSX)

Acknowledgments

We thank Professor Aharon Oren for his expert advice on the Latin etymology of the species name.

References

  1. 1. Zopf W. Über ausscheidung von fettfarbstoffen (lipochromen) seitens gewisser spaltpilze. Ber Dtsch Bot Ges. 1891;9(1):22–8.
  2. 2. Val-Calvo J, Vázquez-Boland JA. Mycobacteriales taxonomy using network analysis-aided, context-uniform phylogenomic approach for non-subjective genus demarcation. mBio. 2023;14(5):e0220723. pmid:37796005
  3. 3. Sangal V, Goodfellow M, Jones AL, Sutcliffe IC. A stable home for an equine pathogen: valid publication of the binomial Prescottella equi gen. nov., comb. nov., and reclassification of four rhodococcal species into the genus Prescottella. Int J Syst Evol Microbiol. 2022;72(9). pmid:36107761
  4. 4. Jones A, Goodfellow M. Genus IV Rhodococcus (Zopf 1891) emend. Goodfellow, Alderson and Chun 1998a. Bergey’s Manual of Systematic Bacteriology. 2012;5:437–63.
  5. 5. Kaparullina EN, Trotsenko YuA, Doronina NV. Characterization of Rhodococcus wratislaviensis, a New Gram-Positive Facultative Methylotroph, and Properties of Its C1 Metabolism. Microbiology. 2019;88(1):46–53.
  6. 6. Chaudhary DK, Kim J. Rhodococcus olei sp. nov., with the ability to degrade petroleum oil, isolated from oil-contaminated soil. Int J Syst Evol Microbiol. 2018;68(5):1749–56. pmid:29620494
  7. 7. Kämpfer P, Dott W, Martin K, Glaeser SP. Rhodococcus defluvii sp. nov., isolated from wastewater of a bioreactor and formal proposal to reclassify [Corynebacterium hoagii] and Rhodococcus equi as Rhodococcus hoagii comb. nov. Int J Syst Evol Microbiol. 2014;64(Pt 3):755–61. pmid:24198057
  8. 8. Lee SD, Kim IS, Kim Y-J, Joung Y. Rhodococcus cavernicola sp. nov., isolated from a cave, and Rhodococcus degradans is a later heterosynonym of Rhodococcus qingshengii. Int J Syst Evol Microbiol. 2020;70(7):4409–15. pmid:32538737
  9. 9. Ramaprasad EVV, Mahidhara G, Sasikala C, Ramana CV. Rhodococcus electrodiphilus sp. nov., a marine electro active actinobacterium isolated from coral reef. Int J Syst Evol Microbiol. 2018;68(8):2644–9. pmid:29957174
  10. 10. Zhang D, Su Z-Y, Li L, Tang W-Z. Rhodococcus spongiicola sp. nov. and Rhodococcus xishaensis sp. nov., from marine sponges. Int J Syst Evol Microbiol. 2021;71(7):10.1099/ijsem.0.004863. pmid:34296990
  11. 11. Cappelletti M, Presentato A, Piacenza E, Firrincieli A, Turner RJ, Zannoni D. Biotechnology of Rhodococcus for the production of valuable compounds. Appl Microbiol Biotechnol. 2020;104(20):8567–94. pmid:32918579
  12. 12. Martínková L, Uhnáková B, Pátek M, Nesvera J, Kren V. Biodegradation potential of the genus Rhodococcus. Environ Int. 2009;35(1):162–77. pmid:18789530
  13. 13. McLeod MP, Warren RL, Hsiao WWL, Araki N, Myhre M, Fernandes C, et al. The complete genome of Rhodococcus sp. RHA1 provides insights into a catabolic powerhouse. Proc Natl Acad Sci U S A. 2006;103(42):15582–7. pmid:17030794
  14. 14. Baek JH, Baek W, Jeong SE, Lee SC, Jin HM, Jeon CO. Rhodococcus oxybenzonivorans sp. nov., a benzophenone-3-degrading bacterium, isolated from stream sediment. Int J Syst Evol Microbiol. 2022;72(6):10.1099/ijsem.0.005433. pmid:35704462
  15. 15. Su X, Liu Y, Hashmi MZ, Hu J, Ding L, Wu M, et al. Rhodococcus biphenylivorans sp. nov., a polychlorinated biphenyl-degrading bacterium. Antonie Van Leeuwenhoek. 2015;107(1):55–63. pmid:25315102
  16. 16. Rehfuss M, Urban J. Rhodococcus phenolicus sp. nov., a novel bioprocessor isolated actinomycete with the ability to degrade chlorobenzene, dichlorobenzene and phenol as sole carbon sources. Syst Appl Microbiol. 2005;28(8):695–701. pmid:16261859
  17. 17. Ceniceros A, Dijkhuizen L, Petrusma M, Medema MH. Genome-based exploration of the specialized metabolic capacities of the genus Rhodococcus. BMC Genomics. 2017;18(1):593. pmid:28793878
  18. 18. Di Canito A, Zampolli J, Orro A, D’Ursi P, Milanesi L, Sello G, et al. Genome-based analysis for the identification of genes involved in o-xylene degradation in Rhodococcus opacus R7. BMC Genomics. 2018;19(1):587. pmid:30081830
  19. 19. Di Gennaro P, Rescalli E, Galli E, Sello G, Bestetti G. Characterization of Rhodococcus opacus R7, a strain able to degrade naphthalene and o-xylene isolated from a polycyclic aromatic hydrocarbon-contaminated soil. Res Microbiol. 2001;152(7):641–51. pmid:11605984
  20. 20. Orro A, Cappelletti M, D’Ursi P, Milanesi L, Di Canito A, Zampolli J, et al. Genome and Phenotype Microarray Analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7: Genetic Determinants and Metabolic Abilities with Environmental Relevance. PLoS One. 2015;10(10):e0139467. pmid:26426997
  21. 21. Ma J, Zhuang Y, Wang Y, Zhu N, Wang T, Xiao H, et al. Update on new trend and progress of the mechanism of polycyclic aromatic hydrocarbon biodegradation by Rhodococcus, based on the new understanding of relevant theories: a review. Environ Sci Pollut Res Int. 2023;30(41):93345–62. pmid:37548784
  22. 22. Wu YA, Wang X, Zhao WH, Wang XD, Yang ZK, Li L. Roles of n-hexadecane in the degradation of dibenzofuran by a biosurfactant-producing bacterium Rhodococcus sp. J Clean Produc. 2024;434:140500.
  23. 23. Krivoruchko A, Kuyukina M, Peshkur T, Cunningham CJ, Ivshina I. Rhodococcus Strains from the Specialized Collection of Alkanotrophs for Biodegradation of Aromatic Compounds. Molecules. 2023;28(5):2393. pmid:36903638
  24. 24. Laczi K, Kis Á, Horváth B, Maróti G, Hegedüs B, Perei K, et al. Metabolic responses of Rhodococcus erythropolis PR4 grown on diesel oil and various hydrocarbons. Appl Microbiol Biotechnol. 2015;99(22):9745–59. pmid:26346267
  25. 25. Xu J-L, He J, Wang Z-C, Wang K, Li W-J, Tang S-K, et al. Rhodococcus qingshengii sp. nov., a carbendazim-degrading bacterium. Int J Syst Evol Microbiol. 2007;57(Pt 12):2754–7. pmid:18048720
  26. 26. Sekine M, Tanikawa S, Omata S, Saito M, Fujisawa T, Tsukatani N, et al. Sequence analysis of three plasmids harboured in Rhodococcus erythropolis strain PR4. Environ Microbiol. 2006;8(2):334–46. pmid:16423019
  27. 27. Zampolli J, Collina E, Lasagni M, Di Gennaro P. Biodegradation of variable-chain-length n-alkanes in Rhodococcus opacus R7 and the involvement of an alkane hydroxylase system in the metabolism. AMB Express. 2014;4:73. pmid:25401074
  28. 28. Kim D, Kim Y-S, Kim S-K, Kim SW, Zylstra GJ, Kim YM, et al. Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp. strain DK17. Appl Environ Microbiol. 2002;68(7):3270–8. pmid:12089003
  29. 29. Choi KY, Kim D, Sul WJ, Chae J-C, Zylstra GJ, Kim YM, et al. Molecular and biochemical analysis of phthalate and terephthalate degradation by Rhodococcus sp. strain DK17. FEMS Microbiol Lett. 2005;252(2):207–13. pmid:16181748
  30. 30. Kim D. Functional genomic studies on alkylbenzene degradation by Rhodococcus sp. strain DK17. Yonsei University; 2003.
    • 31. Jeon J, Kim D, Kim E, Lee YM. Refining the genome of alkylbenzene-degrading Rhodococcus sp. DK17 and comparative analysis with genomes of its deletion mutants. Microbiol Resour Announc. 2025;14(4):e01134-24. pmid:39998182
    • 32. Kim D, Choi KY, Yoo M, Zylstra GJ, Kim E. Biotechnological Potential of Rhodococcus Biodegradative Pathways. J Microbiol Biotechnol. 2018;28(7):1037–51. pmid:29913546
    • 33. Kim D, Chae J-C, Zylstra GJ, Kim Y-S, Kim S-K, Nam MH, et al. Identification of a novel dioxygenase involved in metabolism of o-xylene, toluene, and ethylbenzene by Rhodococcus sp. strain DK17. Appl Environ Microbiol. 2004;70(12):7086–92. pmid:15574904
    • 34. Kim D, Chae J-C, Jang JY, Zylstra GJ, Kim YM, Kang BS, et al. Functional characterization and molecular modeling of methylcatechol 2,3-dioxygenase from o-xylene-degrading Rhodococcus sp. strain DK17. Biochem Biophys Res Commun. 2005;326(4):880–6. pmid:15607751
    • 35. Sangal V, Goodfellow M, Jones AL, Seviour RJ, Sutcliffe IC. Refined systematics of the genus Rhodococcus based on whole genome analyses. In: Alvarez HM, editor. Biology of Rhodococcus. Cham: Springer International Publishing; 2019. p. 1–21.
      • 36. Val-Calvo J, Scortti M, Vázquez-Boland JA. Leveraging the subgenus category to address genus over-splitting exemplified with Prescottella and other recently proposed Mycobacteriales genera. bioRxiv. 2025;2025.04.27.650868.
      • 37. Stanier RY, Palleroni NJ, Doudoroff M. The aerobic pseudomonads: a taxonomic study. J Gen Microbiol. 1966;43(2):159–271. pmid:5963505
      • 38. Lane D. 16S/23S rRNA sequencing. Nucleic acid techniques in bacterial systematics. Chichester: Wiley; 1991. p. 125–75.
        • 39. Yoon S-H, Ha S-M, Kwon S, Lim J, Kim Y, Seo H, et al. Introducing EzBioCloud: a taxonomically united database of 16S rRNA gene sequences and whole-genome assemblies. Int J Syst Evol Microbiol. 2017;67(5):1613–7. pmid:28005526
        • 40. Kumar S, Stecher G, Li M, Knyaz C, Tamura K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018;35(6):1547–9. pmid:29722887
        • 41. Zhang W, Sun Z. Random local neighbor joining: a new method for reconstructing phylogenetic trees. Mol Phylogenet Evol. 2008;47(1):117–28. pmid:18343690
        • 42. Felsenstein J. Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol. 1981;17(6):368–76. pmid:7288891
        • 43. Fitch WM. Toward Defining the Course of Evolution: Minimum Change for a Specific Tree Topology. Systematic Biol. 1971;20(4):406–16.
        • 44. De Coster W, D’Hert S, Schultz DT, Cruts M, Van Broeckhoven C. NanoPack: visualizing and processing long-read sequencing data. Bioinformatics. 2018;34(15):2666–9. pmid:29547981
        • 45. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114–20. pmid:24695404
        • 46. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: Resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6):e1005595. pmid:28594827
        • 47. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res. 2015;25(7):1043–55. pmid:25977477
        • 48. Li W, O’Neill KR, Haft DH, DiCuccio M, Chetvernin V, Badretdin A, et al. RefSeq: expanding the Prokaryotic Genome Annotation Pipeline reach with protein family model curation. Nucleic Acids Res. 2021;49(D1):D1020–8. pmid:33270901
        • 49. Aziz RK, Bartels D, Best AA, DeJongh M, Disz T, Edwards RA, et al. The RAST Server: rapid annotations using subsystems technology. BMC Genomics. 2008;9:75. pmid:18261238
        • 50. Blin K, Shaw S, Augustijn HE, Reitz ZL, Biermann F, Alanjary M, et al. antiSMASH 7.0: new and improved predictions for detection, regulation, chemical structures and visualisation. Nucleic Acids Res. 2023;51(W1):W46–50. pmid:37140036
        • 51. Chaumeil PA, Mussig AJ, Hugenholtz P, Parks DH. GTDB-Tk: a toolkit to classify genomes with the genome taxonomy database. Bioinformatics. 2019;36(6):1925–7. pmid:31730192
        • 52. Parks DH, Chuvochina M, Waite DW, Rinke C, Skarshewski A, Chaumeil P-A, et al. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol. 2018;36(10):996–1004. pmid:30148503
        • 53. Meier-Kolthoff JP, Auch AF, Klenk H-P, Göker M. Genome sequence-based species delimitation with confidence intervals and improved distance functions. BMC Bioinformatics. 2013;14:60. pmid:23432962
        • 54. Li W, Godzik A. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics. 2006;22(13):1658–9. pmid:16731699
        • 55. Lex A, Gehlenborg N, Strobelt H, Vuillemot R, Pfister H. UpSet: Visualization of Intersecting Sets. IEEE Trans Vis Comput Graph. 2014;20(12):1983–92. pmid:26356912
        • 56. Noh H-J, Shin SC, Park Y, Choi A, Baek K, Hong SG, et al. Lichenicola cladoniae gen. nov., sp. nov., a member of the family Acetobacteraceae isolated from an Antarctic lichen. Int J Syst Evol Microbiol. 2020;70(11):5918–25. pmid:33034550
        • 57. Huq MA, Rahman MS, Rahman MM. Aquincola agrisoli sp. nov., isolated from rhizospheric soil of eggplant and in silico genome mining for the prediction of biosynthetic gene clusters. Int J Syst Evol Microbiol. 2024;74(4):006355. pmid:38683662
        • 58. Reiner K. Catalase test protocol. Am Soc Microbiol. 2010;1(1):1–9.
        • 59. Shields P, Cathcart L. Oxidase test protocol. Am Soc Microbiol. 2010;4:1–9.
        • 60. Park Y, Noh H-J, Hwang CY, Shin SC, Hong SG, Jin YK, et al. Hymenobacter siberiensis sp. nov., isolated from a marine sediment of the East Siberian Sea and Hymenobacter psoromatis sp. nov., isolated from an Antarctic lichen. Int J Syst Evol Microbiol. 2022;72(3):10.1099/ijsem.0.005290. pmid:35348450
        • 61. Sasser M. Bacterial identification by gas chromatographic analysis of fatty acids methyl esters (GC-FAME). Newark (NY): Microbial ID; 2006.
          • 62. Collins MD, Jones D. Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implication. Microbiol Rev. 1981;45(2):316–54. pmid:7022156
          • 63. Yarza P, Yilmaz P, Pruesse E, Glöckner FO, Ludwig W, Schleifer K-H, et al. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat Rev Microbiol. 2014;12(9):635–45. pmid:25118885
          • 64. Yoon S-H, Ha S-M, Lim J, Kwon S, Chun J. A large-scale evaluation of algorithms to calculate average nucleotide identity. Antonie Van Leeuwenhoek. 2017;110(10):1281–6. pmid:28204908
          • 65. Jones AL, Davies J, Fukuda M, Brown R, Lim J, Goodfellow M. Rhodococcus jostii: a home for Rhodococcus strain RHA1. Antonie Van Leeuwenhoek. 2013;104(3):435–40. pmid:23851715
          • 66. Sudarsan S, Blank LM, Dietrich A, Vielhauer O, Takors R, Schmid A, et al. Dynamics of benzoate metabolism in Pseudomonas putida KT2440. Metab Eng Commun. 2016;3:97–110. pmid:29468117
          • 67. Stuani L, Lechaplais C, Salminen AV, Ségurens B, Durot M, Castelli V, et al. Novel metabolic features in Acinetobacter baylyi ADP1 revealed by a multiomics approach. Metabolomics. 2014;10(6):1223–38. pmid:25374488
          • 68. Wheeler KA, Lamb HK, Hawkins AR. Control of metabolic flux through the quinate pathway in Aspergillus nidulans. Biochem J. 1996;315 ( Pt 1)(Pt 1):195–205. pmid:8670107
          • 69. Choi KY, Zylstra GJ, Kim E. Benzoate catabolite repression of the phthalate degradation pathway in Rhodococcus sp. strain DK17. Appl Environ Microbiol. 2007;73(4):1370–4. pmid:17158614
          • 70. Gerginova M, Spankulova G, Paunova-Krasteva T, Peneva N, Stoitsova S, Alexieva Z. Effects of Aromatic Compounds Degradation on Bacterial Cell Morphology. Fermentation. 2023;9(11):957.
          • 71. Haritash AK, Kaushik CP. Biodegradation aspects of polycyclic aromatic hydrocarbons (PAHs): a review. J Hazard Mater. 2009;169(1–3):1–15. pmid:19442441
          • 72. García JL, Uhía I, Galán B. Catabolism and biotechnological applications of cholesterol degrading bacteria. Microb Biotechnol. 2012;5(6):679–99. pmid:22309478
          • 73. Jadoun J, Bar R. Microbial transformations in a cyclodextrin medium. Part 3. Cholesterol oxidation by Rhodococcus erythropolis. Appl Microbiol Biotechnol. 1993;40(2–3).
          • 74. Fernández de Las Heras L, Mascaraque V, García Fernández E, Navarro-Llorens JM, Perera J, Drzyzga O. ChoG is the main inducible extracellular cholesterol oxidase of Rhodococcus sp. strain CECT3014. Microbiol Res. 2011;166(5):403–18. pmid:20630728
          • 75. Li P, Tian Y, Yang K, Tian M, Zhu Y, Chen X, et al. Mechanism of microbial action of the inoculated nitrogen-fixing bacterium for growth promotion and yield enhancement in rice (Oryza sativa L.). Adv Biotechnol (Singap). 2024;2(4):32. pmid:39883349
          • 76. Suyal DC, Joshi D, Kumar S, Soni R, Goel R. Differential protein profiling of soil diazotroph Rhodococcus qingshengii S10107 towards low-temperature and nitrogen deficiency. Sci Rep. 2019;9(1):20378. pmid:31889061
          • 77. Dhaouadi S, A. H M, Rhouma A. The plant pathogen Rhodococcus fascians. History, disease symptomatology, host range, pathogenesis and plant–pathogen interaction. Ann Appl Biol. 2020;177(1):4–15.
          • 78. Suzuki Y, Takahashi K, Takase F, Sawada N, Nakao S, Toda A, et al. Serological epidemiological surveillance for vapN-harboring Rhodococcus equi infection in goats. Comp Immunol Microbiol Infect Dis. 2020;73:101540. pmid:32911379
          • 79. Hernández MA, Lara J, Gago G, Gramajo H, Alvarez HM. The pleiotropic transcriptional regulator NlpR contributes to the modulation of nitrogen metabolism, lipogenesis and triacylglycerol accumulation in oleaginous rhodococci. Mol Microbiol. 2017;103(2):366–85. pmid:27786393
          • 80. Zhu N, Sun S, Guo X, Luo W, Zhuang Y, Lei T, et al. Integration of physiology, genomics and microbiomics analyses reveal the biodegradation mechanism of petroleum hydrocarbons by Medicago sativa L. and growth-promoting bacterium Rhodococcus erythropolis KB1. Bioresour Technol. 2025;415:131659. pmid:39426428
          • 81. Kuhl T, Chowdhury SP, Uhl J, Rothballer M. Genome-Based Characterization of Plant-Associated Rhodococcus qingshengii RL1 Reveals Stress Tolerance and Plant-Microbe Interaction Traits. Front Microbiol. 2021;12:708605. pmid:34489897
          • 82. Amsaveni R, Sureshkumar M, Aravinth A, Reshma Mary J, Vivekanandhan G. Production of Non-Ribosomal Peptide Synthetase (NRPS)-Dependent Siderophore by Aeromonas Isolates. Iran Biomed J. 2016;20(4):235–40. pmid:27155016
          • 83. Süssmuth RD, Mainz A. Nonribosomal Peptide Synthesis-Principles and Prospects. Angew Chem Int Ed Engl. 2017;56(14):3770–821. pmid:28323366
          • 84. Felnagle EA, Jackson EE, Chan YA, Podevels AM, Berti AD, McMahon MD, et al. Nonribosomal peptide synthetases involved in the production of medically relevant natural products. Mol Pharm. 2008;5(2):191–211. pmid:18217713
          • 85. Iacovelli R, Bovenberg RAL, Driessen AJM. Nonribosomal peptide synthetases and their biotechnological potential in Penicillium rubens. J Ind Microbiol Biotechnol. 2021;48(7–8):kuab045. pmid:34279620
          • 86. Pait IGU, Kitani S, Roslan FW, Ulanova D, Arai M, Ikeda H, et al. Discovery of a new diol-containing polyketide by heterologous expression of a silent biosynthetic gene cluster from Streptomyces lavendulae FRI-5. J Ind Microbiol Biotechnol. 2018;45(2):77–87. pmid:29255990
          • 87. Bosello M, Robbel L, Linne U, Xie X, Marahiel MA. Biosynthesis of the siderophore rhodochelin requires the coordinated expression of three independent gene clusters in Rhodococcus jostii RHA1. J Am Chem Soc. 2011;133(12):4587–95. pmid:21381663
          • 88. Hochmuth T, Piel J. Polyketide synthases of bacterial symbionts in sponges--evolution-based applications in natural products research. Phytochemistry. 2009;70(15–16):1841–9. pmid:19443000
          • 89. Helfrich EJN, Lin G-M, Voigt CA, Clardy J. Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering. Beilstein J Org Chem. 2019;15:2889–906. pmid:31839835
          • 90. Bugg TDH. Dioxygenase enzymes: catalytic mechanisms and chemical models. Tetrahedron. 2003;59(36):7075–101.
          • 91. Klatte S, Kroppenstedt RM, Rainey FA. Rhodococcus opacus sp.nov., An Unusual Nutritionally Versatile Rhodococcus-species. Syst Appl Microbiol. 1994;17(3):355–60.
          • 92. Takeuchi M, Hatano K, Sedlácek I, Pácová Z. Rhodococcus jostii sp. nov., isolated from a medieval grave. Int J Syst Evol Microbiol. 2002;52(Pt 2):409–13. pmid:11931149