Limits...
Co-evolution of RNA polymerase with RbpA in the phylum Actinobacteria

View Article: PubMed Central - PubMed

ABSTRACT

The role of RbpA in the backdrop of M. smegmatis showed that it rescues mycobacterial RNA polymerase from rifampicin-mediated inhibition (Dey et al., 2010; Dey et al., 2011). Paget and co-workers (Paget et al., 2001; Newell et al., 2006) have revealed that RbpA homologs occur exclusively in actinobacteria. Newell et al. (2006) showed that MtbRbpA, when complemented in a ∆rbpA mutant of S. coelicolor, showed a low recovery of MIC (from 0.75 to 2 μg/ml) as compared to complementation by native RbpA of S. coelicolor (MIC increases from 0.75 to 11 μg/ml). Our studies on MsRbpA show that it is a differential marker for M. smegmatis RNA polymerase as compared to E. coli RNA polymerase at IC50 levels of rifampicin. A recent sequence-based analysis by Lane and Darst (2010) has shown that RNA polymerases from Proteobacteria and Actinobacteria have had a divergent evolution. E. coli is a representative of Proteobacteria and M. smegmatis is an Actinobacterium. RbpA has an exclusive occurrence in Actinobacteria. Since protein–protein interactions might not be conserved across different species, therefore, the probable reason for the indifference of MsRbpA toward E. coli RNA polymerase could be the lineage-specific differences between actinobacterial and proteobacterial RNA polymerases. These observations led us to ask the question as to whether the evolution of RbpA in Actinobacteria followed the same route as that of RNA polymerase subunits from actinobacterial species. We show that the exclusivity of RbpA in Actinobacteria and the unique evolution of RNA polymerase in this phylum share a co-evolutionary link. We have addressed this issue by a blending of experimental and bioinformatics based approaches. They comprise of induction of bacterial cultures coupled to rifampicin-tolerance, transcription assays and statistical comparison of phylogenetic trees for different pairs of proteins in actinobacteria.

No MeSH data available.


Related in: MedlinePlus

MB 7H9 broth cultures of Jmc2155 and JRmc2155 cells were grown in presence of rifampicin (0, 10, 20, 40 and 80 μg/ml; shown in white) under inducing conditions of 2% acetamide. The surviving cells were pelleted, resuspended in 5 μl of LB and patched onto LB agar plates supplemented with 25 μg/ml of kanamycin and 2% glucose. The plates were scanned after 24 h of incubation at 37 °C. The M. smegmatis strain overexpressing MsRbpA (JRmc2155) showed increase in MIC value for rifampicin as compared to the strain housing the vector backbone (Jmc2155) only.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5121209&req=5

f0010: MB 7H9 broth cultures of Jmc2155 and JRmc2155 cells were grown in presence of rifampicin (0, 10, 20, 40 and 80 μg/ml; shown in white) under inducing conditions of 2% acetamide. The surviving cells were pelleted, resuspended in 5 μl of LB and patched onto LB agar plates supplemented with 25 μg/ml of kanamycin and 2% glucose. The plates were scanned after 24 h of incubation at 37 °C. The M. smegmatis strain overexpressing MsRbpA (JRmc2155) showed increase in MIC value for rifampicin as compared to the strain housing the vector backbone (Jmc2155) only.

Mentions: We chose the acetamidase promoter in order to have sufficient overexpression to allow expression of detectable amount of protein from mycobacterial cells. We cloned MsRbpA under acetamidase promoter in pJAM2 and electroporated the construct pJAM2MsRbpA into competent mc2155 cells. As a control, only pJAM2 vector was also electroporated into competent mc2155 cells (Dey et al., 2010). The vector pJAM2 has a kanamycin resistance marker. The resulting strains Jmc2155 (carrying pJAM2) and JRmc2155 (carrying pJAM2MsRbpA) were screened for the overexpression of MsRbpA in the presence of acetamide. The strains were then tested at different levels of rifampicin. The strain overexpressing MsRbpA, JRmc2155, in the inducing conditions of 2% acetamide grew at rifampicin concentrations of 20 μg/ml, 40 μg/ml and 80 μg/ml, while the strain carrying pJAM2, Jmc2155, was incapable of growing at these concentrations (Fig. 2). Thus, we found that overexpression of MsRbpA leads to increase in the rifampicin tolerance level in an otherwise rifampicin-sensitive strain carrying the vector alone.


Co-evolution of RNA polymerase with RbpA in the phylum Actinobacteria
MB 7H9 broth cultures of Jmc2155 and JRmc2155 cells were grown in presence of rifampicin (0, 10, 20, 40 and 80 μg/ml; shown in white) under inducing conditions of 2% acetamide. The surviving cells were pelleted, resuspended in 5 μl of LB and patched onto LB agar plates supplemented with 25 μg/ml of kanamycin and 2% glucose. The plates were scanned after 24 h of incubation at 37 °C. The M. smegmatis strain overexpressing MsRbpA (JRmc2155) showed increase in MIC value for rifampicin as compared to the strain housing the vector backbone (Jmc2155) only.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121209&req=5

f0010: MB 7H9 broth cultures of Jmc2155 and JRmc2155 cells were grown in presence of rifampicin (0, 10, 20, 40 and 80 μg/ml; shown in white) under inducing conditions of 2% acetamide. The surviving cells were pelleted, resuspended in 5 μl of LB and patched onto LB agar plates supplemented with 25 μg/ml of kanamycin and 2% glucose. The plates were scanned after 24 h of incubation at 37 °C. The M. smegmatis strain overexpressing MsRbpA (JRmc2155) showed increase in MIC value for rifampicin as compared to the strain housing the vector backbone (Jmc2155) only.
Mentions: We chose the acetamidase promoter in order to have sufficient overexpression to allow expression of detectable amount of protein from mycobacterial cells. We cloned MsRbpA under acetamidase promoter in pJAM2 and electroporated the construct pJAM2MsRbpA into competent mc2155 cells. As a control, only pJAM2 vector was also electroporated into competent mc2155 cells (Dey et al., 2010). The vector pJAM2 has a kanamycin resistance marker. The resulting strains Jmc2155 (carrying pJAM2) and JRmc2155 (carrying pJAM2MsRbpA) were screened for the overexpression of MsRbpA in the presence of acetamide. The strains were then tested at different levels of rifampicin. The strain overexpressing MsRbpA, JRmc2155, in the inducing conditions of 2% acetamide grew at rifampicin concentrations of 20 μg/ml, 40 μg/ml and 80 μg/ml, while the strain carrying pJAM2, Jmc2155, was incapable of growing at these concentrations (Fig. 2). Thus, we found that overexpression of MsRbpA leads to increase in the rifampicin tolerance level in an otherwise rifampicin-sensitive strain carrying the vector alone.

View Article: PubMed Central - PubMed

ABSTRACT

The role of RbpA in the backdrop of M. smegmatis showed that it rescues mycobacterial RNA polymerase from rifampicin-mediated inhibition (Dey et al., 2010; Dey et al., 2011). Paget and co-workers (Paget et al., 2001; Newell et al., 2006) have revealed that RbpA homologs occur exclusively in actinobacteria. Newell et al. (2006) showed that MtbRbpA, when complemented in a ∆rbpA mutant of S. coelicolor, showed a low recovery of MIC (from 0.75 to 2 μg/ml) as compared to complementation by native RbpA of S. coelicolor (MIC increases from 0.75 to 11 μg/ml). Our studies on MsRbpA show that it is a differential marker for M. smegmatis RNA polymerase as compared to E. coli RNA polymerase at IC50 levels of rifampicin. A recent sequence-based analysis by Lane and Darst (2010) has shown that RNA polymerases from Proteobacteria and Actinobacteria have had a divergent evolution. E. coli is a representative of Proteobacteria and M. smegmatis is an Actinobacterium. RbpA has an exclusive occurrence in Actinobacteria. Since protein–protein interactions might not be conserved across different species, therefore, the probable reason for the indifference of MsRbpA toward E. coli RNA polymerase could be the lineage-specific differences between actinobacterial and proteobacterial RNA polymerases. These observations led us to ask the question as to whether the evolution of RbpA in Actinobacteria followed the same route as that of RNA polymerase subunits from actinobacterial species. We show that the exclusivity of RbpA in Actinobacteria and the unique evolution of RNA polymerase in this phylum share a co-evolutionary link. We have addressed this issue by a blending of experimental and bioinformatics based approaches. They comprise of induction of bacterial cultures coupled to rifampicin-tolerance, transcription assays and statistical comparison of phylogenetic trees for different pairs of proteins in actinobacteria.

No MeSH data available.


Related in: MedlinePlus