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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

A: Broth cultures of E. coli BL21 (DE3) cells, transformed with pETMsRbpA, were grown in LB (with IPTG) to OD600 = 0.3. One set was not induced with IPTG (upper panel) and one set was induced with 1 mM IPTG (lower panel). Serial, ten-fold dilutions were spotted (5 μl) onto LB agar plates supplemented with 100 μg/ml of ampicillin. Two series of plates for each set of broth cultures were made, one supplemented with 1 mM IPTG and the other without IPTG. A gradient of rifampicin was maintained (0 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml and 64 μg/ml). The plates were scanned after 16 h of incubation at 37 °C. None of the tested series (under inducing conditions) showed any increase in MIC values for rifampicin.B: The expression state of MsRbpA in E. coli BL21 (DE3) transformed with pETMsRbpA. The results show the 15% SDS-PAGE expression analyses of MsRbpA for the series of experiments shown in the lower panel of Fig. 3A. Lane 1 = protein marker (kDa); Lane 2 = broth culture induced with 1 mM IPTG; Lanes 3, 5, and 7 = culture from LB agar with 1 mM IPTG; Lanes 4, 6, and 8 = culture from LB Agar with no IPTG.
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f0015: A: Broth cultures of E. coli BL21 (DE3) cells, transformed with pETMsRbpA, were grown in LB (with IPTG) to OD600 = 0.3. One set was not induced with IPTG (upper panel) and one set was induced with 1 mM IPTG (lower panel). Serial, ten-fold dilutions were spotted (5 μl) onto LB agar plates supplemented with 100 μg/ml of ampicillin. Two series of plates for each set of broth cultures were made, one supplemented with 1 mM IPTG and the other without IPTG. A gradient of rifampicin was maintained (0 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml and 64 μg/ml). The plates were scanned after 16 h of incubation at 37 °C. None of the tested series (under inducing conditions) showed any increase in MIC values for rifampicin.B: The expression state of MsRbpA in E. coli BL21 (DE3) transformed with pETMsRbpA. The results show the 15% SDS-PAGE expression analyses of MsRbpA for the series of experiments shown in the lower panel of Fig. 3A. Lane 1 = protein marker (kDa); Lane 2 = broth culture induced with 1 mM IPTG; Lanes 3, 5, and 7 = culture from LB agar with 1 mM IPTG; Lanes 4, 6, and 8 = culture from LB Agar with no IPTG.

Mentions: The expression of MsRbpA in pETMsRbpA is under the control of T7-promoter fused with lac operator, therefore, it can act as a genetic switch to direct the expression of MsRbpA in E. coli BL21 (DE3). For this purpose, we transformed E. coli BL21 (DE3) cells with pETMsRbpA. Subsequently, we grew the transformed E. coli BL21 cells under inducing conditions (1 mM IPTG). The cells from these two sets were plated onto LB agar plates (with 100 μg/ml ampicillin). The plates contained a gradient of rifampicin (0 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml and 64 μg/ml). However, as can be seen from Fig. 3A that overexpression of MsRbpA does not result in an increase in the MIC value of rifampicin for E. coli. At this point of time, it can be questioned as to whether any expression of MsRbpA actually took place in E. coli when the growth was taking place in IPTG. In parallel, it needs to be shown that there was a switch-off in the expression of MsRbpA in the absence of IPTG. Therefore, the growing colonies (shown in Fig. 3A) were picked, lysed and analyzed on a 15% SDS-PAGE. Fig. 3B depicts the results of MsRbpA in a switched-on or switched-off state. Lanes 4, 6, and 8 show the expression of MsRbpA in a switched-off state, while lanes 3, 5 and 7 show its expression in a switched-on state.


Co-evolution of RNA polymerase with RbpA in the phylum Actinobacteria
A: Broth cultures of E. coli BL21 (DE3) cells, transformed with pETMsRbpA, were grown in LB (with IPTG) to OD600 = 0.3. One set was not induced with IPTG (upper panel) and one set was induced with 1 mM IPTG (lower panel). Serial, ten-fold dilutions were spotted (5 μl) onto LB agar plates supplemented with 100 μg/ml of ampicillin. Two series of plates for each set of broth cultures were made, one supplemented with 1 mM IPTG and the other without IPTG. A gradient of rifampicin was maintained (0 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml and 64 μg/ml). The plates were scanned after 16 h of incubation at 37 °C. None of the tested series (under inducing conditions) showed any increase in MIC values for rifampicin.B: The expression state of MsRbpA in E. coli BL21 (DE3) transformed with pETMsRbpA. The results show the 15% SDS-PAGE expression analyses of MsRbpA for the series of experiments shown in the lower panel of Fig. 3A. Lane 1 = protein marker (kDa); Lane 2 = broth culture induced with 1 mM IPTG; Lanes 3, 5, and 7 = culture from LB agar with 1 mM IPTG; Lanes 4, 6, and 8 = culture from LB Agar with no IPTG.
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f0015: A: Broth cultures of E. coli BL21 (DE3) cells, transformed with pETMsRbpA, were grown in LB (with IPTG) to OD600 = 0.3. One set was not induced with IPTG (upper panel) and one set was induced with 1 mM IPTG (lower panel). Serial, ten-fold dilutions were spotted (5 μl) onto LB agar plates supplemented with 100 μg/ml of ampicillin. Two series of plates for each set of broth cultures were made, one supplemented with 1 mM IPTG and the other without IPTG. A gradient of rifampicin was maintained (0 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml and 64 μg/ml). The plates were scanned after 16 h of incubation at 37 °C. None of the tested series (under inducing conditions) showed any increase in MIC values for rifampicin.B: The expression state of MsRbpA in E. coli BL21 (DE3) transformed with pETMsRbpA. The results show the 15% SDS-PAGE expression analyses of MsRbpA for the series of experiments shown in the lower panel of Fig. 3A. Lane 1 = protein marker (kDa); Lane 2 = broth culture induced with 1 mM IPTG; Lanes 3, 5, and 7 = culture from LB agar with 1 mM IPTG; Lanes 4, 6, and 8 = culture from LB Agar with no IPTG.
Mentions: The expression of MsRbpA in pETMsRbpA is under the control of T7-promoter fused with lac operator, therefore, it can act as a genetic switch to direct the expression of MsRbpA in E. coli BL21 (DE3). For this purpose, we transformed E. coli BL21 (DE3) cells with pETMsRbpA. Subsequently, we grew the transformed E. coli BL21 cells under inducing conditions (1 mM IPTG). The cells from these two sets were plated onto LB agar plates (with 100 μg/ml ampicillin). The plates contained a gradient of rifampicin (0 μg/ml, 4 μg/ml, 8 μg/ml, 16 μg/ml, 32 μg/ml and 64 μg/ml). However, as can be seen from Fig. 3A that overexpression of MsRbpA does not result in an increase in the MIC value of rifampicin for E. coli. At this point of time, it can be questioned as to whether any expression of MsRbpA actually took place in E. coli when the growth was taking place in IPTG. In parallel, it needs to be shown that there was a switch-off in the expression of MsRbpA in the absence of IPTG. Therefore, the growing colonies (shown in Fig. 3A) were picked, lysed and analyzed on a 15% SDS-PAGE. Fig. 3B depicts the results of MsRbpA in a switched-on or switched-off state. Lanes 4, 6, and 8 show the expression of MsRbpA in a switched-off state, while lanes 3, 5 and 7 show its expression in a switched-on state.

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