Abstract:
Cyclic di-guanosine-monophosphate (c-di-GMP) has been recognized as a second messenger in bacteria controlling multiple cellular processes such as biofilm formation, motility, and virulence. Proteins containing GGDEF and EAL domains
are engaged in the synthesis and degradation, respectively, of cyclic di-GMP. Some bacteria contain multiple proteins with GGDEF and EAL domains. The genome of Mycobacterium tuberculosis encodes only one protein (Rv1354c) which contains a GGDEF domain. This protein also contains a tandem EAL. The function of this
protein in mycobacteria has not yet been determined. In this study, the orthologue of Rv1354c was investigated in Mycobacterium
smegmatis (MSMEG_2196). The expression of MSMEG_2196 in M. smegmatis was altered by constructing sense and antisense expressing strains. The effect of the altered expression of MSMEG_2196 on M. smegmatis was tested under carbon, oxygen, phosphorous, and nitrogen limited growth conditions. There was no
significant effect on growth in either the antisense or sense expressing strains grown
under nutrient-rich, or carbon-, or oxygen-, or phosphorous limitation conditions. However, a growth effect was observed in the antisense expressing strain when grown under nitrogen-limited conditions. In particular, at mid stationary-phase (1,800 min) the MSMEG_2196 antisense strain had an OD600 value of 0.60, compared to that of the control M. smegmatis/pMind strain (OD600 value of 1.09). These results were further confirmed by the low colony forming units measures observed in MSMEG_2196 antisense strain. Proteomic analysis was carried out on
the MSMEG_2196 antisesne expressing strain grown in the nitrogen-limited condition. Proteins that were differentially expressed were identified by mass spectrometry. A number of the proteins that were down-regulated in the antisense
expressing strain are important in the survival of the bacteria under nitrogen-limited conditions.
This study indicates a role for MSMEG_2196 in growth or survival of mycobacteria under nitrogen-limitations.
