Prevalence of the gene lsr2 among the genus Mycobacterium and an investigation into changes in biofilm formation when inactivating lsr2 regulated genes in Mycobacterium smegmatis

Abstract

Mycobacterium smegmatis is a non-pathogenic model organism commonly used to study the human pathogen Mycobacterium tuberculosis. M. smegmatis has a rough colony morphology and is capable of forming biofilms, however, when the DNA-regulating protein Lsr2 is inactivated the bacteria forms a smooth colony and loses biofilm formation. This study investigates the phenotypic effect on colony morphology and biofilm formation when Lsr2-regulated genes are inactivated. A mutant strain of M. smegmatis with defective lsr2 (M. smegmatis Δlsr2) was used as a parent strain for the study and the gene for mycocerosic acid synthase (mas) was selected for inactivation. Genes downstream of mas from MSMEG_4727 to MSMEG_4733 and MSMEG_4741were also selected for inactivation. Genes were inactivated using homologous recombination via "suicide vector" plasmids lacking a Mycobacterial origin of replication and carrying kanamycin resistance and PCR fragments of the target genes. M. smegmatis Δlsr2 was transformed by electroporation and transformants were plated and screened for kanamycin resistance. The mutant strain lacking functional lsr2 and mas (M. smegmatis Δlsr2Δmas) regained the ability to form biofilms and exhibited colony morphology similar to wild-type M. smegmatis. Inactivation of genes MSMEG_4727 to MSMEG_4733 produced similar results. Inactivation of MSMEG_4741 caused no change in the Δlsr2 mutant phenotype. These results indicate that mas and genes downstream are involved with the biofilm formation and colony morphology change associated with the M. smegmatis Δlsr2 mutant strain.