Abstract
Mycobacterium tuberculosis, the etiological agent of tuberculosis, is surrounded by a complex envelope of unusually low permeability which contributes to antibiotic resistance and evasion of the host defence mechanisms. A distinguishing feature of mycobacteria is their high content of lipids, many of which are unique to this genus. Acyl-CoA carboxylases catalyse the first committed step of lipid synthesis. Mycobacterial acyl-CoA carboxylases consist of two subunits, only one of which is biotinylated. The gene for the biotin containing subunit AccA1 from M. tuberculosis has been isolated and sequenced at the University of Surrey. To define the physiological role of mycobacterial acyl-CoA carboxylases, a combination of molecular biology approaches was used to cause a reduction in the activity of the enzyme. The following strategies were adopted: i) Inhibition of acyl-CoA carboxylase by depletion of biotin both extracellularly and intracellularly. Extracellular depletion of biotin was achieved by adding avidin to the culture medium used to grow the mycobacterial strains. Intracellular depletion of biotin was attempted through the expression by a plasmid of an additional biotinylated protein, enzymatically inactive to capture most of the biotin present in the cell. ii) Reduction of acyl-CoA carboxylase activity by producing an inactive hybrid enzyme containing a non-functional AccA1 subunit. A mutation affecting biotinylation was introduced by site directed mutagenesis at the 3’ end of the accA1 gene from M. tuberculosis. The mutated gene was expressed by a plasmid and controlled by the use of promoters of different strength. iii) Diminution of the production of acyl-CoA carboxylase through interference with the expression of the biotinylated subunit of the enzyme by an antisense approach. A fragment of the 5’ end of the accA1 gene containing the Shine-Dalgarno sequence and the translational start site was cloned in an opposite direction with respect to the promoter. The amount of anti-sense mRNA produced was controlled by the use of promoters of different strength. A reduction in the activity of mycobacterial acyl-CoA carboxylase would be expected to impair lipid synthesis, and ultimately cause a decrease in the amount of lipids that constitute the cell wall. The lack of such effects may be ascribed to the presence of multiple acyl-CoA carboxylases, detected by the genome sequencing but not apparent at the time this work was performed. An understanding of the biochemistry and molecular genetics of the cell wall lipids would provide a rational basis for seeking drugs targeted at the production of such specific compounds. Moreover, the mycobacterial cell wall is also important in pathology and further knowledge of the complex nature of its biosynthesis would enable to identify critical genes whose inactivation could lead to attenuation.