Functional Characterization of a Catabolic Plasmid from Polychlorinated- Biphenyl-Degrading Rhodococcus sp. Strain RHA1

Abstract

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative "genomic islands" (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.Rhodococcus is a widely occurring genus of aerobic, nonmotile soil bacteria that are closely related to three other genera of GC-rich actinomycetes: Gordonia, Nocardia, and Mycobacterium. Rhodococci degrade an extraordinarily wide variety of organic substrates and thus play an important role in the global C cycle. The unusual armamentarium of enzymatic activities involved in these processes has been exploited in applications ranging from commodity chemical production to the desulfurization of fossil fuels (6). Consequently, the metabolic capabilities of rhodococci are of interest to the pharmaceutical, environmental, chemical, and energy sectors.Rhodococcus sp. strain RHA1 is characterized by its exceptional ability to transform polychlorinated biphenyls (PCBs) (53), a particularly widespread and persistent class of environmental pollutants. It is generally thought that in aerobic bacteria, PCBs are cometabolized by the bph pathway, which is responsible for the aerobic degradation of biphenyl (23). The upper bph pathway consists of four enzymatic activities that together transform biphenyl to benzoate and 2-hydroxypenta-2,4-dienoate. For each of these four steps, RHA1 appears to possess multiple isozymes, which may help explain the strain's superior PCB-transforming capabilities. Thus, the strain contains at least three bph-type ring-hydroxylating di...

Catabolism of Benzoate and Phthalate in Rhodococcus sp. Strain RHA1: Redundancies and Convergence

“…The reductase, encoded by fpr254A1, is predicted to harbor a 2Fe-2S cluster and flavin adenine dinucleotide/NAD domains. This is the second putative class V P450 system (66) found in RHA1 to date (84). The N-terminal 22 amino acids of the cyp254A1-encoded oxygenase shares 85% sequence identity with N-terminal peptide of the cytochrome P450 2EP that catalyzes dealkylation of 2-ethoxyphenol (27), strongly suggesting that these enzymes have similar substrate specificities.…”

Phenylacetate Catabolism in Rhodococcus sp. Strain RHA1: a Central Pathway for Degradation of Aromatic Compounds

“…(7) and modified for Rhodococcus strain RHA1 (16). Fosmid clones containing the target genes were selected from an RHA1 library (22) and used to transform E. coli BW25113 containing the -Red recombination plasmid pKD46 (Amp r ). Gene replacement cassettes consisting of Apr r oriT flanked by sequences from both sides of the target gene were amplified by PCR and introduced into E. coli BW25113 (pKD46) containing the appropriate fosmid.…”

TraA is required for megaplasmid conjugation in Rhodococcus erythropolis AN12

“…The sequences and gene annotations of Wve rhodococcal megaplasmidsp33701 from Rhodococcus equi ATCC33701 (Takai et al, 2000), pBD2 from Rhodococcus erythropolis BD2 (Stecker et al, 2003), pRHL3 from Rhodococcus sp. RHA1 (Warren et al, 2004), as well as pREC1 and pREL1 from R. erythropolis PR4 (Sekine et al, 2006)-were recently completed. Analysis of pREC1 (Genbank Accession No.…”