Cloning and Expression of FixA and FixB Genes and Their Role in the Formation of Dipicolinic Acid in Mycobacterium marinum CCUG 20988

Dipicolinic acid (DPA) comprising 5-15% of the spore dry weight is important for spore stability and resistance properties. In some Gram-positive bacteria such as Clostridium perfringens, an electron transfer flavoprotein, EtfA, acts in an incorporative way in DPA formation. The aim of this study was to explore if the fixA and fixB genes which are electron transfer flavoproteins could incorporate in DPA formation in Mycobacterium marinum CCUG 20988. In this descriptive-analytic study, the fixA and fixB genes were PCR-amplified and ligated between the Nde I and Hind III sites of pET19b vector with ampicillin resistance gene in frame with an N-terminal T7-promotor and a C-terminal of 6-histidin tag and transformed into BL21-Codon Plus-RIL Escherichia coli competent cells. For over expression of fixA and fixB genes, SDS-polyacrylamide gel electrophoresis was used. Also the direct interaction between dihydrodipicolinate acid (DHDPA) synthase and fixA and fixB proteins for DPA formation was investigated. Detectable DPA formation has been identified upon isopropyl-β-D-thiogalactopyranoside (IPTG) induction using fixA and fixB genes together. The amount of dipicolinic was about 32.8 and 49.2 µg/mL acids in LB and M9 media, respectively. It can be concluded that both fixA and fixB genes as electron transfer flavoproteins may incorporate in dipicolinic acid formation in M. marinum. We assume that if sporulation turns out to be a common mechanism used by mycobacteria in response to environmental conditions, it might be one of the means by which it attains dormancy within the host.