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Rhodococcus bacterial strains are characterized by wide metabolic versatility and extraordinary resistance to environmental stresses. The high versatility and adaptability of Rhodococcus strains is partly related with large and complex genomes (up to 10.1 Mbp) including high genetic redundancy and the presence of several circular and linear (mega)plasmids, which harbour peculiar catabolic and biosynthetic genes. Within Rhodococcus genus, R. opacus strain PD630 is considered a model oleaginous strain for its ability to produce and accumulate lipids (mostly triacyglycerols, TAGs) using different carbon sources, including low-cost and renewable resources such as lignocellulose. Notably, under specific growth conditions, this strain is capable of accumulating up to 80% of its cellular dry weight in TAGs; that is a rare feature in the prokaryotic and eukaryotic kingdoms. Multi-omic approaches have been applied to obtain system-level information about metabolic and regulatory pathways involved in these biosynthetic processes. Novel molecular tools for genome editing (CRISPR/Cas9 and recombineering) have been recently developed highlighting the possible utilization of R. opacus PD630 as synthetic biology platform for lipids production.

In this paper comparative genome and phenotype microarray analyses of Rhodococcus sp. BCP1 and Rhodococcus opacus R7 were performed. Rhodococcus sp. BCP1 was selected for its ability to grow on short-chain n-alkanes and R. opacus R7 was isolated for its ability to grow on naphthalene and on o-xylene. Results of genome comparison, including BCP1, R7, along with other Rhodococcus reference strains, showed that at least 30% of the genome of each strain presented unique sequences and only 50% of the predicted proteome was shared. To associate genomic features with metabolic capabilities of BCP1 and R7 strains, hundreds of different growth conditions were tested through Phenotype Microarray, by using Biolog plates and plates manually prepared with additional xenobiotic compounds. Around one-third of the surveyed carbon sources was utilized by both strains although R7 generally showed higher metabolic activity values compared to BCP1. Moreover, R7 showed broader range of nitrogen and sulphur sources. Phenotype Microarray data were combined with genomic analysis to genetically support the metabolic features of the two strains. The genome analysis allowed to identify some gene clusters involved in the metabolism of the main tested xenobiotic compounds. Results show that R7 contains multiple genes for the degradation of a large set of aromatic and PAHs compounds, while a lower variability in terms of genes predicted to be involved in aromatic degradation was found in BCP1. This genetic feature can be related to the strong genetic pressure exerted by the two different environment from which the two strains were isolated. According to this, in the BCP1 genome the smo gene cluster involved in the short-chain n-alkanes degradation, is included in one of the unique regions and it is not conserved in the Rhodococcus strains compared in this work. Data obtained underline the great potential of these two Rhodococcus spp. strains for biodegradation and environmental decontamination processes.

Rhodococcus opacus PD630 is a biotechnologically important bacterium with metabolic capability for bioremediation, metal recovery, and storage of triacylglycerols. Genome editing by homologous recombination in R. opacus is hampered by a very low combined frequency of DNA transfer and recombination. To improve recombination in the species, a conjugative, conditional suicide plasmid based on the replicon derived from the Corynebacterium glutamicum plasmid pGA1 was constructed and evaluated in R. opacus . The replication of this plasmid is controlled by a dual inducible and repressible promoter system originally developed for Mycobacterium spp. Next, we demonstrated that a derivative of this plasmid containing sacB as a counterselection marker and homologous regions of R. opacus could be used for homologous recombination, and that the problem of obtaining recombinants had been solved. Like for other Corynebacteriales , the cell wall of Rhodococcus spp. contains mycolic acids which form a hydrophobic and impermeable outer layer. Mycolic acids are essential for Mycobacterium smegmatis , but not for Corynebacterium glutamicum , and the new vector was used to study if mycolic acid is essential for R. opacus . We found that accD3 that is necessary for mycolic acid synthesis could only be deleted from the chromosome in strains containing a plasmid-encoded copy of accD3. This indicates that mycolic acid is important for R. opacus viability. The conditional suicide vector should be useful for homologous recombination or for delivering gene products like recombinases or Cas proteins and gRNA to Rhodococcus and related genera, while the approach should be applicable for any plasmid needing a plasmid-encoded protein for replication. Key points • Improved vector for homologous recombination in R. opacus . • Mycolic acid is important for survival of R. opacus like it is for Mycobacterium . • Similar conditional suicide plasmids may be constructed for other bacteria . Graphical abstract

Background Bacteria belonging to the Rhodococcus genus play an important role in the degradation of many contaminants, including methylbenzenes. These bacteria, widely distributed in the environment, are known to be a powerhouse of numerous degradation functions, due to their ability to metabolize a wide range of organic molecules including aliphatic, aromatic, polycyclic aromatic compounds (PAHs), phenols, and nitriles. In accordance with their immense catabolic diversity, Rhodococcus spp. possess large and complex genomes, which contain a multiplicity of catabolic genes, a high genetic redundancy of biosynthetic pathways and a sophisticated regulatory network. The present study aimed to identify genes involved in the o -xylene degradation in R. opacus strain R7 through a genome-based approach. Results Using genome-based analysis we identified all the sequences in the R7 genome annotated as dioxygenases or monooxygenases/hydroxylases and clustered them into two different trees. The akb , phe and prm sequences were selected as genes encoding respectively for dioxygenases, phenol hydroxylases and monooxygenases and their putative involvement in o -xylene oxidation was evaluated. The involvement of the akb genes in o -xylene oxidation was demonstrated by RT-PCR/qPCR experiments after growth on o -xylene and by the selection of the R7–50 leaky mutant. Although the akb genes are specifically activated for o -xylene degradation, metabolic intermediates of the pathway suggested potential alternative oxidation steps, possibly through monooxygenation. This led us to further investigate the role of the prm and the phe genes. Results showed that these genes were transcribed in a constitutive manner, and that the activity of the Prm monooxygenase was able to transform o -xylene slowly in intermediates as 3,4-dimethylphenol and 2-methylbenzylalcohol. Moreover, the expression level of phe genes, homologous to the phe genes of Rhodococcus spp. 1CP and UPV-1 with a 90% identity, could explain their role in the further oxidation of o -xylene and R7 growth on dimethylphenols. Conclusions These results suggest that R7 strain is able to degrade o -xylene by the Akb dioxygenase system leading to the production of the corresponding dihydrodiol. Likewise, the redundancy of sequences encoding for several monooxygenases/phenol hydroxylases, supports the involvement of other oxygenases converging in the o -xylene degradation pathway in R7 strain.

Rhodococcus opacus PD630 is an oleaginous bacterium able to accumulate large amounts of triacylglycerols (TAG) in different carbon sources. The last reaction for TAG biosynthesis is catalyzed by the bifunctional wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) enzymes encoded by atf genes. R. opacus PD630 possesses at least 17 putative atf homologous genes in its genome, but only atf 1 and atf 2 exhibited a significant DGAT activity when expressed in E. coli , as revealed in a previous study. The contribution of atf 1 gene to TAG accumulation by strain PD630 has been demonstrated previously, although additional Atfs may also contribute to lipid accumulation, since the atf 1-disrupted mutant is still able to produce significant amounts of TAG (Alvarez et al., Microbiology 154:2327–2335, 2008 ). In this study, we investigated the in vivo role of atf 2 gene in TAG accumulation by R. opacus PD630 by using different genetic strategies. The atf 2-disrupted mutant exhibited a decrease in TAG accumulation (up to 25–30 %, w/w) and an approximately tenfold increase in glycogen formation in comparison with the wild-type strain. Surprisingly, in contrast to single mutants, a double mutant generated by the disruption of atf 1 and atf 2 genes only showed a very low effect in TAG and in glycogen accumulation under lipid storage conditions. Overexpression of atf 1 and atf 2 genes in strain PD630 promoted an increase of approximately 10 % (w/w) in TAG accumulation, while heterologous expression of atf 2 gene in Mycobacterium smegmatis caused an increase in TAG accumulation during cultivation in nitrogen-rich media. This study demonstrated that, in addition to atf 1 gene, atf 2 is actively involved in TAG accumulation by the oleaginous R. opacus PD630.

Bacteria make a huge contribution to the purification of the environment from toxic stable pollutants of anthropogenic and natural origin due to the diversity of their enzyme systems. For example, the ability to decompose 3-chlorobenzoate (3CBA) by the four representative genera of Actinobacteria, such as Rhodococcus, Gordonia, Microbacterium, and Arthrobacter, was studied. In most cases, the formation of 4-chlorocatechol as the only key intermediate during the decomposition of 3CBA was observed. However, Rhodococcus opacus strain 1CP was an exception, whose cells decomposed 3CBA via both 3-chloro- and 4-chlorocatechol. The enzyme 3-Chlorobenzoate 1,2-dioxygenase (3CBDO) induced during the growth of these bacteria in the presence of 3CBA differed significantly in substrate specificity from the benzoate dioxygenases induced upon growth in the presence of benzoate. The R. opacus 6a strain was found to contain genes encoding chlorocatechol 1,2-dioxygenase, chloromuconate cycloisomerase, and dienelactone hydrolase, whose nucleotide sequence was 100% consistent with the sequences of the corresponding genes encoding the enzymes of the modified 4-chlorocatechol ortho-cleavage pathway of the strain R. opacus 1CP. However, the gene encoding chloromuconolactone dehalogenase (clcF) was not found in the representatives of the actinomycete genera, including Gordonia and Arthrobacter. A linear mega-plasmid carrying 3-chlorocatechol degradation genes remained stable after maintaining the R. opacus 1CP strain on an agar-rich medium for 25 years. In general, a similar plasmid was absent in actinobacteria of other genera, as well as in closely related species of R. opacus 6a.

Microorganisms capable of decomposing hydrophobic substrates in cold climates are of considerable interest both in terms of studying adaptive reactions to low temperatures and in terms of their application in biotechnologies for cleaning up oil spills in a crude-oil polluted soil. The aim of this work was to investigate the genome of Rhodococcus opacus S8 and explore behavior traits of this strain grown in the presence of hexadecane. The genome size of strain S8 is 8.78 Mb, of which the chromosome size is 7.75 Mb. The S8 strain contains 2 circular plasmids of 135 kb and 105 kb and a linear plasmid with a size of 788 kb. The analysis of the genome revealed the presence of genes responsible for the degradation of alkanes and synthesis of biosurfactants. The peculiarities of morphology of microbial cells when interacting with a hydrophobic substrate were revealed. An adaptive mechanism responsible in the absence of oxygen for maintaining the process of degradation of hexadecane is discussed. The data obtained show that the strain S8 has great potential to be used in biotechnologies.

We screened bacteria that use E2 as its sole source of carbon and energy for growth and identified them as Rhodococcus, and we named them DSSKP-R-001. For a better understanding of the metabolic potential of the strain, whole genome sequencing of Rhodococcus DSSKP-R-001 and annotation of the functional genes were performed. The genomic sketches included a predicted protein-coding gene of approximately 5.4 Mbp with G + C content of 68.72% and 5180. The genome of Rhodococcus strain DSSKP-R-001 consists of three replicons: one chromosome and two plasmids of 5.2, 0.09, and 0.09, respectively. The results showed that there were ten steroid-degrading enzymes distributed in the whole genome of the strain. The existence and expression of estradiol-degrading enzymes were verified by PCR and RTPCR. Finally, comparative genomics was used to compare multiple strains of Rhodococcus. It was found that Rhodococcus DSSKP-R-001 had the highest similarity to Rhodococcus sp. P14 and there were 2070 core genes shared with Rhodococcus sp. P14, Rhodococcus jostii RHA1, Rhodococcus opacus B4, and Rhodococcus equi 103S, showing evolutionary homology. In summary, this study provides a comprehensive understanding of the role of Rhodococcus DSSKP-R-001 in estradiol-efficient degradation of these assays for Rhodococcus. DSSKP-R-001 in bioremediation and evolution within Rhodococcus has important meaning.

Lignocellulosic biomass is currently underutilized, but it offers promise as a resource for the generation of commercial end-products, such as biofuels, detergents, and other oleochemicals. Rhodococcus opacus PD630 is an oleaginous, Gram-positive bacterium with an exceptional ability to utilize recalcitrant aromatic lignin breakdown products to produce lipid molecules such as triacylglycerols (TAGs), which are an important biofuel precursor. Lipid carbon storage molecules accumulate only under growth-limiting low nitrogen conditions, representing a significant challenge toward using bacterial biorefineries for fuel precursor production. In this work, we screened overexpression of 27 native transcriptional regulators for their abilities to improve lipid accumulation under nitrogen-rich conditions, resulting in three strains that accumulate increased lipids, unconstrained by nitrogen availability when grown in phenol or glucose. Transcriptomic analyses revealed that the best strain (#13) enhanced FA production via activation of the β-ketoadipate pathway. Gene deletion experiments confirm that lipid accumulation in nitrogen-replete conditions requires reprogramming of phenylalanine metabolism. By generating mutants decoupling carbon storage from low nitrogen environments, we move closer toward optimizing R. opacus for efficient bioproduction on lignocellulosic biomass.

The 165-kb megaplasmid pAO1 of Arthrobacter nicotinovorans carries two large gene clusters, one involved in nicotine catabolism ( nic -gene cluster) and one in carbohydrate utilization ( ch -gene cluster). Here, we propose that both gene clusters were acquired by A . nicotinovorans by horizontal gene transfer mediated by pAO1. Protein–protein blast search showed that none of the published Arthrobacter genomes contains nic -genes, but Rhodococcus opacus carries on its chromosome a nic -gene cluster highly similar to that of pAO1. Analysis of the nic -genes in the two species suggested a recombination event between their nic -gene clusters. Apparently, there was a gene exchange between pAO1, or a precursor plasmid, and a nic -gene cluster of an as yet unidentified Arthrobacter specie or other soil bacterium, possibly related to Rhodococcus, leading to the transfer by pAO1 of this catabolic trait to A. nicotinovorans . Analysis of the pAO1 ch -gene cluster revealed a virtually identical counterpart on the chromosome of Arthrobacter phenanthrenivorans . Moreover, the sequence analysis of the genes flanking the ch -gene cluster suggested that it was acquired by pAO1 by Xer-related site directed recombination and transferred via the plasmid to A. nicotinovorans . The G+C content, the level of sequence identity, gene co-linearity of nic - and ch -gene clusters as well as the signs of recombination events clearly supports the notion of pAO1 and its precursor plasmids as vehicles in HGT among Gram + soil bacteria.