The Role of fadD19 and echA19 in Sterol Side Chain Degradation by Mycobacterium smegmatis

Mycobacteria are able to degrade natural sterols and use them as a source of carbon and energy. Several genes which play an important role in cholesterol ring degradation have been described in Mycobacterium smegmatis. However, there are limited data describing the molecular mechanism of the aliphatic side chain degradation by Mycobacterium spp. In this paper, we analyzed the role of the echA19 and fadD19 genes in the degradation process of the side chain of cholesterol and β-sitosterol. We demonstrated that the M. smegmatis fadD19 and echA19 genes are not essential for viability. FadD19 is required in the initial step of the biodegradation of C-24 branched sterol side chains in Mycobacterium smegmatis mc2155, but not those carrying a straight chain like cholesterol. Additionally, we have shown that echA19 is not essential in the degradation of either substrate. This is the first report, to our knowledge, on the molecular characterization of the genes playing an essential role in C-24 branched side chain sterol degradation in M. smegmatis mc2155.


Introduction
Fast-growing mycobacteria (M. smegmatis, M. vaccae, M. phlei, M. fortuitum) have been the subject of biotechnological research for many years. They have been used e.g., in steroid compound biotransformation processes, which provide precursors for the production of steroid drugs [1]. Microbial transformation of steroids is used for the production of novel drugs and also for the synthesis of the key intermediates [2]. In pharmaceutical manufacturing processes the microbiological transformation of steroids are particularly associated with the production of hormones [3]. A great advantage of the bioconversion of steroids is the possibility of modifying a steroid molecule at locations that are typically not available by chemical synthesis. The biotransformation process can be regio-or stereoselective. Moreover, fast growing Mycobacteria are active in the biodegradation of polycyclic aromatic hydrocarbons [4,5], particularly naphthalene, phenantrene, anthracene, fluoranthene, pyrene and benzopyrene [6].
Molecular characterization studies concerning sterol degradation processes could not only be important for the biotechnological production of steroid drugs, but also help widen our knowledge on the pathogenesis of Mycobacterium tuberculosis (Mtb). Recent studies show that cholesterol can play an important role in the host-pathogen interaction [7]. Cholesterol, a component of the host cell membrane, allows mycobacterial survival in the epithelial cells of the alveoli [8]. It was also suggested One of the sterols commonly present in natural environment is β-sitosterol, which belongs to the phytosterols and has a C-24 branched side chain. There are no data available about the molecular mechanisms of β-sitosterol degradation in Mycobacterium.
In this paper we analyzed the role of selected M. smegmatis genes coding for enzymes engaged in the process of degradation of the side chains of cholesterol and β-sitosterol (Scheme 1). We have demonstrated that fadD19 (acyl-CoA-ligase) is required in the initial step of biodegradation of C-24 branched sterols in M. smegmatis. Additionally, we have shown that echA19 (putative enoyl-CoA hydratase) is not essential in the degradation of either substrate (β-sitosterol or cholesterol), but its absence partially inhibits this process.
Molecules 2016, 21,598 3 of 14 One of the sterols commonly present in natural environment is β-sitosterol, which belongs to the phytosterols and has a C-24 branched side chain. There are no data available about the molecular mechanisms of β-sitosterol degradation in Mycobacterium.
In this paper we analyzed the role of selected M. smegmatis genes coding for enzymes engaged in the process of degradation of the side chains of cholesterol and β-sitosterol (Scheme 1). We have demonstrated that fadD19 (acyl-CoA-ligase) is required in the initial step of biodegradation of C-24 branched sterols in M. smegmatis. Additionally, we have shown that echA19 (putative enoyl-CoA hydratase) is not essential in the degradation of either substrate (β-sitosterol or cholesterol), but its absence partially inhibits this process. Scheme 1. Cont. Scheme 1. Cont. Scheme 1. The basic pathways of cholesterol (a) and β-sitosterol (b) side chain degradation containing analyzed metabolites [14,24,25,35,37].

M. smegmatis fadD19 and echA19 Genes are not Essential for Viability
M. smegmatis has an ability to use cholesterol or β-sitosterol as a source of carbon and energy. Culturing of M. smegmatis in the presence of cholesterol results in fast complete degradation of this substrate which was observed as soon as at 24 h ( Figure 1). In the case of β-sitosterol the complete degradation occurred at 96 h ( Figure 2). The two-step recombination protocol of Parish and Stocker [37] was used to obtain the unmarked deletion of M. smegmatis fadD19 and echA19 genes. The successful engineering of mutants confirmed by PCR and Southern blot hybridization (Figures 3 and 4) showed that neither fadD19 nor echa19 is essential for viability of M. smegmatis in rich media.   [14,24,25,35,37].

M. smegmatis fadD19 and echA19
Genes are not Essential for Viability M. smegmatis has an ability to use cholesterol or β-sitosterol as a source of carbon and energy. Culturing of M. smegmatis in the presence of cholesterol results in fast complete degradation of this substrate which was observed as soon as at 24 h ( Figure 1). In the case of β-sitosterol the complete degradation occurred at 96 h ( Figure 2). The two-step recombination protocol of Parish and Stocker [37] was used to obtain the unmarked deletion of M. smegmatis fadD19 and echA19 genes. The successful engineering of mutants confirmed by PCR and Southern blot hybridization (Figures 3 and 4) showed that neither fadD19 nor echa19 is essential for viability of M. smegmatis in rich media.

M. smegmatis fadD19 and echA19 Genes are not Essential for Viability
M. smegmatis has an ability to use cholesterol or β-sitosterol as a source of carbon and energy. Culturing of M. smegmatis in the presence of cholesterol results in fast complete degradation of this substrate which was observed as soon as at 24 h ( Figure 1). In the case of β-sitosterol the complete degradation occurred at 96 h ( Figure 2). The two-step recombination protocol of Parish and Stocker [37] was used to obtain the unmarked deletion of M. smegmatis fadD19 and echA19 genes. The successful engineering of mutants confirmed by PCR and Southern blot hybridization (Figures 3 and 4) showed that neither fadD19 nor echa19 is essential for viability of M. smegmatis in rich media.

M. smegmatis fadD19 and echA19 Genes are not Essential for Cholesterol (Side Chain) Degradation
The ability of M. smegmatis ΔfadD19 and ΔechA19 mutants to utilize cholesterol was monitored using the LC-MS/MS technique. The very fast consumption of cholesterol (90% of the substrate was degraded completely at 24 h of culture) was observed for both mutants (Figure 1). M. smegmatis ΔfadD19 and ΔechA19 were capable of cholesterol degradation, as well as cholestenone, AD, ADD production ( Table 1). The first metabolite identified in the degradation process was cholestenone, with the largest amount between 12 h and 24 h of the culture. Cholestenone was more abundant in ΔfadD19 in the 24 h culture, than in the culture of the parent strain.

fadD19 but not echA19 is Required for β-Sitosterol (Side Chain) Degradation
We used β-sitosterol (C-24 branched side chain sterol) as a substrate to analyze the ability of wild type M. smegmatis and its ΔfadD19 and ΔechA19 mutants to degrade side-chain branched sterols. The ability of M. smegmatis ∆fadD19 and ∆echA19 mutants to utilize cholesterol was monitored using the LC-MS/MS technique. The very fast consumption of cholesterol (90% of the substrate was degraded completely at 24 h of culture) was observed for both mutants (Figure 1). M. smegmatis ∆fadD19 and ∆echA19 were capable of cholesterol degradation, as well as cholestenone, AD, ADD production ( Table 1). The first metabolite identified in the degradation process was cholestenone, with the largest amount between 12 h and 24 h of the culture. Cholestenone was more abundant in ∆fadD19 in the 24 h culture, than in the culture of the parent strain.

fadD19 but not echA19 is Required for β-Sitosterol (Side Chain) Degradation
We used β-sitosterol (C-24 branched side chain sterol) as a substrate to analyze the ability of wild type M. smegmatis and its ∆fadD19 and ∆echA19 mutants to degrade side-chain branched sterols. We found that mutant ∆fadD19 is unable to use β-sitosterol as the only source of carbon and energy, suggesting the fundamental role of FadD19 in β-sitosterol degradation by M. smegmatis (Figure 2, Table 2). Moreover, we observed that ∆echA19 inactivation delayed the β-sitosterol degradation by M. smegmatis ∆echA19 (Figure 2, Table 2), which was associated with a slower initial growth phase (24 h) (unpublished data). We also tested the ability of the wild type strain and its mutants to utilize 1,4-BNC (3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid), a steroid with a partially preserved aliphatic chain ( Figure 5). It was observed that both wild type and ∆fadD19 strains were able to degrade 1,4-BNC. We found that mutant ∆fadD19 is unable to use β-sitosterol as the only source of carbon and energy, suggesting the fundamental role of FadD19 in β-sitosterol degradation by M. smegmatis (Figure 2, Table 2). Moreover, we observed that ΔechA19 inactivation delayed the β-sitosterol degradation by M. smegmatis ΔechA19 (Figure 2, Table 2), which was associated with a slower initial growth phase (24 h) (unpublished data). We also tested the ability of the wild type strain and its mutants to utilize 1,4-BNC (3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid), a steroid with a partially preserved aliphatic chain ( Figure 5). It was observed that both wild type and ΔfadD19 strains were able to degrade 1,4-BNC. To confirm the essential role of FadD19 in the early step of β-sitosterol degradation, a complemented mutant was constructed carrying an intact fadD19 under control of its natural putative promoter (M. smegmatis ∆fadD19; attB::PfadD19fadD19). The complemented strain, but not the ∆fadD19 mutant, was able to degrade β-sitosterol with kinetics similar to that of wild type M. smegmatis (Figure 2). The complementation confirmed that the inhibition of β-sitosterol degradation by M. smegmatis ∆fadD19 was directly due to the absence of an intact fad gene, not to a polar effect.
In the case of the ΔechA19 mutant we observed a delay in the degradation of β-sitosterol compared to a wild type strain (Figure 2, Table 2). Analysis of the growth kinetics (unpublished data) on minimal medium supplemented with β-sitosterol confirms that inactivation of echA19 results in To confirm the essential role of FadD19 in the early step of β-sitosterol degradation, a complemented mutant was constructed carrying an intact fadD19 under control of its natural putative promoter (M. smegmatis ∆fadD19; attB::P fadD19 fadD19). The complemented strain, but not the ∆fadD19 mutant, was able to degrade β-sitosterol with kinetics similar to that of wild type M. smegmatis (Figure 2). The complementation confirmed that the inhibition of β-sitosterol degradation by M. smegmatis ∆fadD19 was directly due to the absence of an intact fad gene, not to a polar effect.
In the case of the ∆echA19 mutant we observed a delay in the degradation of β-sitosterol compared to a wild type strain (Figure 2, Table 2). Analysis of the growth kinetics (unpublished data) on minimal medium supplemented with β-sitosterol confirms that inactivation of echA19 results in slower consumption of this substrate.

Discussion
In this study we demonstrated for the first time an important role of fadD19 in the initial step of β-sitosterol degradation by M. smegmatis. Moreover, this is the first report on the molecular characterization of the gene playing an essential role in C 24-branched chain sterol degradation in M. smegmatis. In addition, we observed that echA19 inactivation delayed the process of β-sitosterol degradation by M. smegmatis ∆echA19. This effect is specific to β-sitosterol, because the addition of acetate or propionate abolishes this effect between the wild type strain M. smegmatis mc 2 155 and the ∆echA19 mutant (unpublished data). The literature data concerning EchA19 is very limited, so the role of EchA19 in sterol side-chain degradation will be further examined.
Fast growing mycobacteria, including M. smegmatis, are able to degrade sterols carrying straight or branched side chains. Here, we found that FadD19 in M. smegmatis is essential to utilize branched side chain sterols, but not those carrying a straight chain, like cholesterol. Using 1,4-BNC as a substrate, we found that FadD19 acts in the early step of β-sitosterol degradation. On the other hand, EchA19 appeared not to be essential for degrading either β-sitosterol or cholesterol. Similar observations were made by Wilbrink et al. [26] with Rhodococcus rhodochrous as a model strain. The mutant defective in the synthesis of FadD19 was not able to utilize sitosterol and campesterol but degraded cholesterol [26]. The essentiality for degradation of steroid substrates carrying branched but not straight side chains was also shown in the case of aldol-lyases Ltp3 and Ltp4 [24].
The knowledge of genes involved in sterol side-chain degradation is very limited in contrast to enzymes catalyzing the sterol ring degradation. However, a catabolic gene cluster was identified in M. smegmatis and R. jostii, as encoding the enzymes which are probably responsible for the β-oxidation process [22,25]. The β-oxidation process requires several enzymes, such as enoyl-CoA hydratase (EchA), acyl-CoA dehydrogenase (FadE), 3-hydroxy-acyl-CoA-dehydrogenase (FadB) and 3-keto-acyl-CoA thiolase (FadA) [38]. Steroid-CoA ligase (fadD19), which plays a key role in C-24 branched sterol degradation, has been identified by Wilbrink et al. [26]. A second gene which is analyzed in this study is echA19 (putative enoyl-CoA hydratase). EchA5 was described as non-essential for M. tuberculosis growth in vitro and in vivo [39]. Yang et al. showed that MaoC-like enoyl-CoA hydratase, ChsH1-ChsH2, catalyzes the hydratation of 3-oxo-4,17-pregnadiene-20-carboxyl-CoA to 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA in cholesterol side chain degradation [40]. Moreover, Casabon et al. characterized acyl-CoA synthetases involved in steroid side-chain degradation in M. tuberculosis and R. jostii RHA1 [41]. In the genome of pathogenic mycobacteria, Mtb, orthologs of analyzed genes fadD19 and echA19 can be identified with an 82.7% and 81.6% identity at the amino acids level, respectively. FadD19 was predicted to be specifically required in Mtb for growth on cholesterol [17]. However, the specific inactivation of both genes, ∆(echA19-fadD19), in Mtb did not affect the process of cholesterol degradation (Brzostek et al., unpublished data). There are no published data about the utilization of β-sitosterol by Mtb, however, a number of different sterols are available in the host.
The degradation of the sterol aliphatic side-chain is still not well understood. However, it is generally accepted that the side-chain of sterols is shortened by β-oxidation reactions. Some genes encoding putative β-oxidation enzymes in the cholesterol regulons of Mtb and Rhodococcus rhodochrous DSM43269 were identified. FadA5 catalyzes the thiolysis of acetoacetyl-CoA in vitro yielding androsterone metabolites [e.g., 4-androstenedione (AD) and 1,4-androstenedione (ADD)] [23].
Thiolase activity appeared to be required for growth on cholesterol and for Mtb virulence, especially during the late stage of mouse infection. Cytochrome P450 monooxygenase (Cyp125) was identified as a selective sterol side chain degradation enzyme in the Rhodococcus rhodochrous RG32 strain. The orthologue of cyp 125 in M. tuberculosis (rv3545c) was suggested to be associated with the process of pathogenesis [42].Understanding of the process of sterol degradation by mycobacteria is important not only from the point of view of Mtb pathogenesis but can also have practical implications in the production of steroid drugs (AD, ADD). These compounds might be synthesized using non-pathogenic mycobacterial strains [2,43].

Bacterial Strains and Culture Conditions
The following bacterial strains were used: Escherichia coli Top 10 (Invitrogen, Carlsbad, CA, USA), Mycobacterium smegmatis mc 2 155 (Snapper et al., [44]). E. coli strains were grown in LB medium. For growth on solid medium, 1% (w/v) Difco agar (Becton, Franklin Lakes, NJ, USA) was added. The mycobacterial strains were cultured in Middlebrook 7H9 broth or 7H10 agar plates supplemented with albumin-dextrose-sodium chloride and/or kanamycin (25 µg¨mL´1 NB medium (100 mL) was inoculated with M. smegmatis and incubated overnight at 37˝C with shaking at 140 r.p.m. min´1. An overnight culture of M. smegmatis was harvested by centrifugation for 10 min at 5000 r.p.m. The cells were resuspended in 10 mL minimal medium and a sample (about 2-4 mL) was transferred to 96-98 mL fresh minimal medium in a 1 L flask. The optical density of bacteria was 0.1. Finally, cholesterol or β-sitosterol were added to the medium at a final concentration of 100 mg/L. The cultures were incubated on a shaker (140 r.p.m.) at 37˝C.

Plasmid Constructions
To perform unmarked deletion in fadD19 and echA19 genes of Mycobacterium smegmatis, suicidal recombination delivery vectors were constructed. In the first step the 5' ends of the genes (fadD19 or echA19) and upstream regions were amplified using primers MsfadD19Gr1 (CCAGACGCCGGACGGATCCCAGGC) and MsfadD19Gr2 (CGCCTGTGTGGATACCAGCCATTG) for fadD19, and MsechA19Gr1 (GCTGCAGATCGGTGATGACGCGGTTGG) and MsechA19Gr2 (CAAGCTTGACGATGAGGGTGTGTCCGC) for echA19, and cloned into the KpnI/BamHI and PstI/HindIII sites of p2NIL to create pNW03 and pNW08, respectively. Subsequently, the 3' ends of the genes (fadD19 or echA19) and downstream regions were amplified using primers MsfadD19Gr3 (CGGGATCCCCCGACGAGGTGTGGCAGG) and MsfadD19Gr4 (GCAAGCTTCCATCCAGACG CCGGACG) for fadD19, and MsechA19Gr3 (CAAGCTTTGGACAAGGCGCTGGAGATCG) and MsechA19Gr4 (CGGTACCTCACATTCGGGAACGGTGGGAC) for echA19, and cloned into the BamHI/HindIII sites of pNW03 and HindIII/KpnI sites of pNW08. The ligated 5' and 3' fragments of the fadD19 and echA19 genes in the resulting vectors were out of frame. The resultant vectors were named pNW04 and pNW09, respectively. Finally, PacI marker gene cassette from pGOAL17 carrying lacZ and sacB genes was cloned into the PacI site of pNW04 and pNW09 to create pNW05 and pNW010, respectively. The plasmids used in this work are listed in Table 3.

Disruption of fadD19 and echA19 Genes
The protocol of Parish and Stocker [29] was used to disrupt fadD19 or echA19 genes at their native loci on the chromosome. Plasmid DNA (pNW05, pNW10) was treated with NaOH (2 mM) and integrated into the M. smegmatis chromosome by homologous recombination. The resulting single crossover (SCO) recombinant mutant colonies were blue, Km R and sensitive to sucrose. The site of recombination was confirmed by PCR and Southern blot hybridization. The SCO strains were further processed to select DCO (double-crossover) mutants that were white, Km S and resistant to sucrose. PCR and Southern blot hybridization were used to distinguish between the wild-type and DCO mutants. The probes were generated by PCR with primers binding to the 5 1 and 3 1 of each gene and DNA of pNW04 and pNW09 as templates, and labeled with a nonradioactive primer extension system (DIG-labeling system, Amersham, Uppsala, Sweden).

Complementation Constructs
The complementation vector was engineered by amplifying the fad gene with its own promoter region (345 bp) using M. smegmatis chromosomal DNA as a template. The PCR product was initially cloned into pJet1.2, verified by sequencing and subsequently re-cloned into the pMV306Km integrative vector using XbaI and HindIII.

Preparation of Cholesterol
The substrate was dissolved in a solution of 96% ethanol-Triton WR1339 (1:1) at 80˝C.

Preparation of β-Sitosterol
β-Sitosterol (15 g) and Tween 80 (2 g) were suspended in distilled water (50 mL). The suspension was homogenized at 94˝C for 20 min. Further the speed of homogenizer was reduced to 1000 rpm/min and gradually water was added to the mixture to a final volume of 200 mL. The suspension was then homogenized for 10 min. reaching up to 2000/min. In the final step the microscopic control of the substrate was performed by measuring the β-sitosterol crystal size. The resulting sample of β-sitosterol was in the shape of needles with a length of 2-4 µm and 1-2 µm in width. The steroid compound was sterilized by boiling three times in 1 h at 24 h intervals. β-Sitosterol was stored at 4˝C.

HPLC MS/MS Analysis of Sterol Degradation
Sterol degradation analysis was performed using an Agilent 1200 HPLC (Santa Clara CA, USA) system and a 3200 QTRAP mass spectrometer (AB Sciex, Framingham, MA, USA) with an atmospheric pressure chemical ionization APCI source. Chromatographic separation was performed using an XDB-C18 column (2.1 mmˆ50 mmˆ1.8 µm; Agilent Technologies). The mobile phase consisted of 2 mM ammonium formate and 0.2% formic acid in water (A) and 2 mM ammonium formate and 0.2% formic acid in methanol (B). The column temperature was maintained at 40˝C and the flow rate was 0.5 mL¨min´1. The ion source settings were as follows: curtain gas: 25 (CUR), IS: 5500 V, nebulizer gas: 50 (GS1), drying gas: 50 (GS2) and temperature of 600˝C. Data analysis was performed with the Analyst™ v1.5.2 software (AB Sciex). Tandem mass spectrometry for the quantitation of sterols degradation courses was made using positive ionization in the multiple reaction monitoring (MRM) mode. The quantitative analysis of sterols and its degradation products was based on standard curves with a linearity range from 1 ng¨mL´1 to 10 ng¨mL´1 (correlation coefficient R 2 = 0.951). All the experimental data represent the means of at least three independent experiments.

Conclusions
The obtained results allows us to conclude that the FadD19 plays an important role in the initial step of biodegradation of C-24 branched sterol side chains in Mycobacterium smegmatis mc 2 155. The extension of our knowledge in the area of sterol degradation could have significant implications for the production of steroid drugs. Additionally, a detailed explanation of the molecular mechanism of sterol degradation could help to explain M. tuberculosis pathogenesis.