Isolation of putative KshA and KshB genes from A. simplex USA-18
To clone KshA genes from A. simplex USA-18, the amino acid sequences of several bacterial Rieske [2Fe-2S] terminal oxygenases, including those isolated from R. jostii RHA1 (YP_704482), M. smegmatis (YP_890151), Burkholderia cenocepacia J2315 (YP_002234232), Ralstonia eutropha JMP134 (YP_295786) and Comamonas testosteroni KF-1 (WP_003057373), were aligned. Consensus motifs such as (R/T)(Y/F)(A/P)RGW and CP(F/Y)H(G/D)W were chosen for the design of sense degenerate primers (primer 1 and 3, respectively, Table 1), while the (N/I)(C/M)H(Y/V/T)P(I/V) motif was used for the design of an antisense degenerate primer (primer 2, Table 1). PCR using primer 1 and 2 gave rise to a 728-bp DNA (S1A2), while primer 3 and 2 produced a 532-bp DNA (S3A2). These two DNA fragments share 72.3% identity within the overlapped region, suggested that A. simplex USA-18 contains at least two potential KshA genes. The upstream and downstream regions of S1A2 were obtained by inverse PCR and DNA walking, and thus an S1A2-containing open reading frame (ORF) of 1155 nucleotides (accession number KJ598876) was identified. Searching databases using BLASTn algorithms revealed that the ORF exhibits 99.7% identity to a putative iron-sulfur binding oxidoreductase gene (REQ_45190) of R. equi 103S [11]. Similarly, the nucleotide sequences flanking S3A2 were determined and an ORF of 1161 nucleotides (accession number KJ598877) was identified. It shares 99.7% identity with another iron-sulfur binding oxidoreductase gene (REQ_06790) of R. equi 103S [11]. To clone KshB gene, the amino acid sequences of the reductase subunit of 3-ketosteroid 9α-hydroxylases from M. smegmatis (WP_003894254), Pseudovibrio sp. (WP_008550016), R. erythropolis (AAL96830), and R. jostii RHA1 (YP_705768) were aligned. Accordingly, the conserved GSGITP and PYSC(R/Q/K)(E/S)G motifs were chosen to design the sense and antisense degenerate primers, respectively (primer 4 and 5, Table 1). PCR using this pair of primer generated a 530-bp fragment (S4A5). A putative ORF of 1185 nucleotides (accession number KJ598878) was subsequently identified after the flanking regions of S4A5 were obtained by DNA walking. The 1185-bp ORF was found to have an identical nucleotide sequence to REQ_36320 of R. equi 103S that presumably encoding the reductase component of 3-ketosteroid 9α-hydroxylase [11].
Reclassification of A. simplex USA-18 as R. equi USA-18
The great resemblance of the genes cloned in this study and the cholesterol oxidase gene cloned previously [22] to those of R. equi 103S raised a suspicion of whether the taxonomic classification of A. simplex USA-18 had been properly determined. The gene encoding for 16S ribosomal RNA was amplified from A. simplex USA-18 by PCR using the universal primer 8 F and U1492R [25]. Blastn showed that the gene, with the accession number KJ598875, is highly similar, with identities over 99%, to the 16S ribosomal RNA genes isolated from a variety of R. equi strains. However, the identities between the gene and those from Arthrobacter strains are about 91-92%, suggesting that A. simplex USA-18 is phylogenetically closer to R. equi than to A. simplex.
To confirm the 16S ribosomal RNA sequence-based suggestion, the metabolic profile of A. simplex USA-18 was checked using the Biolog Identification System, in which the ability of the bacterium to metabolize 71 carbon sources and sensitivity to 23 chemicals were analyzed. The profile identifies the test strain USA18 as Rhodococcus equi, with similarity index between 0.774 and 0.789. Accordingly, A. simplex USA-18 was renamed R. equi USA-18 hereafter.
Deletion of the REQ_36320 ortholog in R. equi USA-18
Searching protein homologs of the REQ_45190 product within R. equi 103S using Blastp algorithm found another six potential Rieske [2Fe-2S] terminal oxygenases, which are products of REQ_06790, REQ_08980, REQ_15470, REQ_40110, REQ_42740, and REQ_43730. The amino acid sequence identities between the REQ_45190 product and each of the homologs are 61.6, 57.3, 91.7, 63.4, 70.7, and 67.8%, respectively. None of their enzyme activities has been characterized. As to REQ_36320, no significant homolog was found. Considering the possible redundancy of KshA genes in R. equi USA-18, we chose the REQ_36320 ortholog as the target for the activity disruption of 3-ketosteroid hydroxylase. Plasmid pUC-ΔΒ-Kan was constructed as described in materials and methods for the PCR-targeted gene disruption. The NdeI-linearized pUC-ΔB-Kan was introduced into R. equi USA-18 competent cells by electroporation. The colonies that survived on kanamycin-containing agar medium were further examined to assure the occurrence of a double crossover event in the flanking regions of the REQ_36320 ortholog between chromosome and the introduced DNA by PCR. PCR amplification using primer 10 and 12 would generate a predetermined ~2.8-kb DNA product if the REQ_36320 ortholog in chromosome had been replaced by Kan (Figure 2). PCR using primer 13 and 12 would otherwise generate another ~2.8-kb DNA fragment if the chromosome of R. equi USA-18 remained unchanged. Generation of the predefined PCR product in response to primer 10 and 12 but not to primer 13 and 12 suggests that the REQ_36320 ortholog had been replaced with Kan in the chromosome of the two transformants (USA-18ΔB2 and USA-18ΔB8). PCR using REQ_36320 specific primers (primer 13 and 14) confirmed the absence of the gene in these two transformants.
Sterol catabolism in REQ_36320 knockout mutant
R. equi USA-18 and R. equi USA-18ΔB8 were cultivated in medium that was supplemented with 0.2% (w/v) cholesterol or phytosterol and 0.2% Tween 20 or Tween 80. The broth, harvested at 4 and 6 days post-inoculation, was extracted with ethyl acetate and the compounds in the extract were analyzed by TLC (Figure 3). The parental strain USA-18 did not produce discernible metabolic intermediate of sterols. However, the knockout strain gave rise to a prominent spot having the same migration distance as ADD on TLC plate. Other minor substances in the extract of R. equi USA-18ΔB8 were noticed, particularly in the prolonged culture that contained Tween 80.
To further identify the compounds in the spot that had the same migration rate as ADD on TLC, the culture broth was harvested at 5 and 8 days after inoculation, extracted with ethyl acetate, and analyzed with an analytical C18 reverse-phase HPLC column. Two peaks were found in the elution profile (Figure 4). The first peak has a retention time of ~5.3 min, approximately same as ADD standard, and its magnitude increased with the culture time. The second peak, with a retention time of ~6.5 min, remained relatively constant during the culture course. The chemical nature of the substance with the 5.3-min retention time was determined with UHPLC-MS/MS (Figure 5). This substance has a molecular weight of 284 Daltons, consistent with that of ADD. In addition, it generated a fragmentation pattern exactly identical to that of ADD. These data confirm that the substance, with the 5.3-min retention time, accumulated in the culture broth of R. equi USA-18ΔB8 is ADD. The molecular weight of the substance with the retention time of 6.5 min was also determined by mass spectrometry (data not shown). Its molecular weight was determined to be 342 Daltons. Presumably, this substance is Δ1,4-BNC, a precursor of ADD, according to its molecular weight and the fact that it was produced only when sterols were included in the culture medium.
Accumulation of Δ1,4-BNC in the broth suggests a catabolic pathway of sterol to ADD via Δ1,4-BNC (Figure 1). In other words, ADD not necessarily descended from AD after a dehydrogenation reaction. To know whether R. equi USA-18ΔB8 was capable of converting AD to ADD, the cells were cultivated in glycerol minimal medium supplemented with 0.5% (w/v) AD and 0.2% Tween 20 at 37°C for 5 days. TLC analysis indicated that most AD in the medium had been converted into ADD (Figure 6). Taken together, sterols are catabolized to ADD in R. equi USA-18 via at least two routes, namely from Δ1,4-BNC to ADD and from Δ4-BNC, AD to ADD.
Time course of ADD production
To assess the capability of R. equi USA-18ΔB8 for ADD production, the cells were cultivated in shaken flasks in glycerol minimal medium supplemented with 0.2% (w/v) cholesterol and 0.5% (v/v) Tween 20 at 28°C. An aliquot of the broth was withdrawn at daily intervals and the cell density and ADD within were determined. The cell density reached a plateau at about day 4, while the ADD concentration continuously increased to 0.58 mg/ml, equivalent to 40% molar yield, at day 7 (Figure 7).
Restricted growth of R. equi USA-18 in macrophages
Virulent strains of R. equi are recognized as facultative intracellular pathogens that cause severe pyogranulomatous bronchopneumonia in young foals. The pathogenic strains also cause an opportunistic infection in humans, particularly in HIV-infected and immunosuppressed patients [26]. Virulent plasmids, with the size up to 100 kb, are required for the virulent strains to survive within macrophages and for virulence in the susceptible hosts [27]. In addition, the sterol catabolic pathway is important for pathogenesis of R. equi[28]. R. equi is also a common soil-dwelling microorganism thriving on plant and animal sterols. R. equi USA-18 was originally isolated from soil [19]. No virulent plasmid was found in this strain. To determine whether R. equi USA-18 is of virulence, the growth of the strain within human macrophages was assayed as described in materials and methods. The cell number, cfu/ml, of E. coli Top10F' within macrophages continuously decreased during the incubation period, only 2.5% left at 96 h. As with the decrease of E. coli in macrophages, the viable counts of R. equi USA-18 and R. equi USA-18ΔB8 also decreased with time; however, they dropped to an undetectable level at 96 h. (Figure 8). Inability to persistently grow in macrophages suggests that R. equi USA-18ΔB8 has an application potential in industry.