Genetic and morphologic variations during L-form conversion in Mycobacterium tuberculosis

Abstract

The loss of cell wall in bacteria may result in uncoordinated propagation and occurrence of highly pleomorphic forms with new biologic characteristics, known as L-forms. The ability of Mycobacterium tuberculosis to survive and reproduce as L-forms is assumed to be an adaptive reaction towards unfavourable factors. In this respect, stress induced L-form conversion could provide an interesting opportunity to study morphologic and genetic changes in mycobacteria. Stressful starvation followed by passages in semisolid medium was used for induction and selective isolation of M. tuberculosis Lforms from three strains (S-18, S-43 and CRBIP7.10). Colonial growth and morphologic ultrastructure, as well as genetic changes in 16S rRNA gene and mycobacteria specific direct repeats (DRs) were evaluated during L-form conversion. Electron microscopy showed exhibition of specific ultrastructure of cell wall deficient bacteria, which were consistent with the formation of typical "fried eggs" shaped Lform colonies. Based on sequence analysis of 16S rRNA gene fragment, we found variable sites in Lform of strain S-18. Spoligotyping analysis demonstrated appearance of genetic variations in DRs locus of all L-forms. In conclusion, we suggested that appearance of genetic changes in M. tuberculosis could be driven and accelerated by L-form transformation. Both morphologic and genetic variations found in L-forms of M. tuberculosis, can be regarded as a manifestation of adaptive plasticity and capability of mycobacteria to survive under unfavorable environmental factors.