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

Abstract

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.

Keywords: M. smegmatis; cholesterol; microbial sterol degradation; sterol side-chain degradation; β-sitosterol.

Scheme 1

The basic pathways of cholesterol (a) and β-sitosterol (b) side chain degradation containing analyzed metabolites [14,24,25,35,37].

Scheme 1

The basic pathways of cholesterol (a) and β-sitosterol (b) side chain degradation containing analyzed metabolites [14,24,25,35,37].

Figure 1

Cholesterol degradation by M. smegmatis based on HPLC analysis. The curves represent the rate of cholesterol degradation in cultures of M. smegmatis mc²155 (×), ΔfadD19 (▲), ΔechA19 (■) in minimal medium. The substrate control (the stability of cholesterol in media) is marked by circles (●). Results are representative of three independent experiments.

Figure 2

β-Sitosterol degradation by M. smegmatis based on HPLC analysis. The curves represent the rate of β-sitosterol degradation in cultures of M. smegmatis mc²155 (stars), ΔfadD19 (▲), ΔechA19 (■), ΔfadD19,attb::PfadD19fadD19 (♦) in minimal medium. The substrate control (the stability of β-sitosterol in media) is marked by circles (●). Results are representative of three independent experiments.

Figure 3

Southern blot-based analysis of mycobacterial mutants generated by directed mutagenesis. (Top) Scheme showing the length of the restriction DNA fragment (2256 bp) and the internal deletion in the mutated gene (706 bp). The chromosomal localization of fadD19 is represented by red arrow, while the internal deletion is marked by black rectangle. (Bottom) Southern blot confirming the internal deletion in the fadD19 gene of M. smegmatis. The lanes represent genomic DNA from: 1, wild-type M. smegmatis; 2–5, double crossover mutants carrying the internally deleted ΔfadD19 gene; 6, single crossover mutant; 7, double crossover mutant carrying the wild-type fadD19 gene.

Figure 4

Southern blot-based analysis of mycobacterial mutants generated by directed mutagenesis. (Top) Scheme showing the length of the restriction DNA fragment (950 bp) and the internal deletion in the mutated gene (404 bp). The chromosomal localization of echA19 is represented by red arrow, while the internal deletion is marked by black rectangle; (Bottom) Southern blot confirming the internal deletion in the echA19 gene of M. smegmatis. The lanes represent genomic DNA from: 1, wild-type M. smegmatis; 2–3, double crossover mutants carrying the internally deleted ΔechA19 gene; 4, single crossover mutant; 5, double crossover mutant carrying the wild-type echA19 gene.

Figure 5

1,4-BNC degradation by M. smegmatis based on HPLC analysis. The curves represent the rate of 1,4-BNC degradation in cultures of M. smegmatis mc²155 (×), ΔfadD19 (▲), ΔfadD19,attb::PfadD19fadD19 (♦) in minimal medium. The substrate control (the stability of β-sitosterol in media) is marked by circles (●). Results are representative of three independent experiments.