Cell wall associated poly-L-glutamine (PLG) layer synthesis is directly linked to glutamine synthetase (GS) encoded by glnA1 in tuberculosis causing mycobacteria. Avirulent Mycobacterium smegmatis (M. smegmatis) despite of having a glnA1 homolog lacks cell wall associated PLG layer. In the present study, we complemented a Delta glnA1 mutant of Mycobacterium bovis (lack PLG in cell wall) with M. smegmatis glnA1 cloned under M. bovis glnA1 promoter. PLC synthesis was restored in the cell wall of complemented strain. The complemented strain also showed increased resistance to physical stresses such as. lysozyme, SDS and increased survival in THP-1 macrophages in comparison to the knockout. Further, in beta-galactosidase reporter assay M. smegmatis glnA1 promoter showed ten times less activity as compared to M. bovis glnA1 promoter. GACT(-8-11) -> TGAC mutations in the M. smegrnatis glnA1 promoter restored its activity by 60% as compared to the activity of promoter of M. bovis. This mutation also showed increased GS expression and produced cell wall associated PLC in M. smegmatis. The results of this study demonstrate that glnA1 promoter of M. smegmatis accounts for low expression level of GS and apparently responsible for absence of cell wall associated PLG layer. (C) 2015 Elsevier Inc. All rights reserved.