Cloning of an Internal Fragment of pimA Gene Coding Glycosyl-transferase of Corynebacterium glutamicum

摘要

Background and Objectives: Mycolyl-transferases are a clan of proteins that are especially present in the CMN genera ( Corynebacterium , Mycobacterium and Nocardia ), mycolyl-transferases are responsible for cell wall components synthesis. The mycobacteria and corynebacteria envelopes share some gross structural features and similar cell wall architecture. The aim of the present work is to identify C. glutamicum genes encoding for glycosyl-transferases enzymes activity. Materials and Methods: In silico search for “glycosyl transferases” that were common both to M. tuberculosis and Corynebacterium difteriae revealed the presence of PimA-like sequences in the actinomycete Streptomyces coelicolor and in the extremophile archeons Pyrococcus horikoshii , Aeropyrum pernix and Pyrococcus abyssi . Results: Highly conserved regions obtained permitted the design of mixtures of oligonucleotides pairs intended to PCR amplification of a pim A gene fragment. The integration of the internal pim A gene fragment at the bacterial genome was done by a single homologous recombination event at the identical wild-type pim A gene of C. glutamicum Or2262. The pim A gene (belonging to the locus pgs A- htr B- pim A) and encoding for glycosyl-transferase enzyme activity of the species C. glutamicum ATCC13032 and C. glutamicum sp. 2262 (reclassified as C. glutamicum Or2262) was successfully cloned. Conclusion: A comparison of the transformability of C. glutamicum Or2262 and f. C. glutamicum ATCC13032 RES167 revealed 18.0 times difference in the ratio of transformability, which suggested that it is attributed to the difference in the efficiency of plasmid-host recombination rather than the efficiency of diffusion of the plasmid through the bacterial envelope.