Identification of genes involved in triacylglycerol metabolism of Rhodococcus opacus PD630

ALVAREZ AF,; ALVAREZ, H. M.; KALSCHEUER R,; STEINBÜCHEL A,

Badajoz, España

Congreso; International Conference on Environmental, Industrial and Applied Microbiology (BioMicroWorld-2005); 2005

Rhodococcus opacus PD630 accumulates large amounts of triacylglycerols (TAGs) and minor amounts of wax esters as storage compounds. This characteristic is restricted to a few genera of bacteria such as Mycobacterium, Nocardia, Streptomyces, Rhodococcus and Acinetobacter. Fatty acids in acylglycerols in cells of R. opacus PD630 account for up to 76 % of the cellular dry weight with gluconate as carbon source [1]. Thus, R. opacus PD630 is considered as an oleaginous model bacterium.   The central region of a putative diacylglycerol acyltransfrase (dgat) gene was amplified using degenerate primers designed based on consensus sequences from multiple alignments of a wax ester synthase/diacylglycerol acyltransferase (wax/dgat) of Acinetobacter calcoaceticus and 8 homologous genes of Mycobaterium smegmatis mc2155. This 828-bp fragment (RhoE) was used for the identification of a 4-kbp DNA fragment from R. opacus PD630 through dot-blot from a partial BamHI genomic library. The 4-kbp DNA fragment was sequenced and a 1.4-kbp ORF was identified. Its deduced amino acid sequence showed 25 to 30 % identity to WS/DGAT from A. calcoaceticus and related proteins from different species of Mycobacterium. The sequence showed also common conservated motives with related proteins, including its putative active site HHXLXDGXS [2]. Southern blot experiments with total DNA from R. opacus PD630 showed only one hybridization signal which is with fragment RhoE. Therefore, in case that strain PD630 has more genes with the same function, like Mycobacterium smegmatis mc2155 and other Mycobacterium species, they share low similarity in their nucleotide sequence.   Furthermore, an other ORF was identified upstream of the putative dgat gene, which showed similarity with diverse esterases and lipases of related bacteria, such as Nocardia and Mycobacterium spp. Both identified genes, which may be involved in the TAG metabolism in R. opacus PD630, may have an independent transcription regulation, since they possess different transcription directions in the chromosome.   We are now cloning the putative dgat gene of strain PD630 into an expression vector in E. coli, in order to study the enzymatic activity of the protein and to accomplish its functional and molecular characterization. In addition, we are producing a knock-out mutant of R. opacus PD630 for this gene for studying its role in lipid metabolism and in the physiology of this TAG-accumulating bacterium.   Acknowledgements A.F. Alvarez is indebted to the Deutscher Akademischer Austauschdienst (DAAD) for the award of a doctoral scholarship.   References [1] Alvarez HM, Mayer F, Fabritius D, Steinbüchel A. Arch Microbiol 165(1996), p. 337-386   [2] Kalscheuer R. and Steinbüchel A. J Biol Chem 278(2003), p. 8075-8082