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Glycine-rich RNA binding protein of Oryza sativa inhibits growth of M15 E. coli cells.

Singh U, Deb D, Singh A, Grover A - BMC Res Notes (2011)

Bottom Line: Removal of the inducer, IPTG, resulted in re-growth of the cells, indicating that effect of the foreign proteins was of reversible nature.Expression of eukaryotic, stress-associated OsGR-RBP4 protein in prokaryotic E. coli M15 cells proves injurious to the growth of the bacterial cells.E. coli genome does not appear to encode for any protein that has significant homology to OsGR-RBP4 protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India. anil.anilgrover@gmail.com.

ABSTRACT

Background: Plant glycine-rich RNA binding proteins have been implicated to have roles in diverse abiotic stresses.

Findings: E. coli M15 cells transformed with full-length rice glycine-rich RNA binding protein4 (OsGR-RBP4), truncated rice glycine-rich RNA binding protein4 (OsGR-RBP4ΔC) and rice FK506 binding protein (OsFKBP20) were analyzed for growth profiles using both broth and solid media. Expression of OsGR-RBP4 and OsGR-RBP4ΔC proteins caused specific, inhibitory effect on growth of recombinant M15 E. coli cells. The bacterial inhibition was shown to be time and incubation temperature dependent. Removal of the inducer, IPTG, resulted in re-growth of the cells, indicating that effect of the foreign proteins was of reversible nature. Although noted at different levels of dilution factors, addition of purified Os-GR-RBP4 and OsGR-RBP4ΔC showed a similar inhibitory effect as seen with expression inside the bacterial cells.

Conclusions: Expression of eukaryotic, stress-associated OsGR-RBP4 protein in prokaryotic E. coli M15 cells proves injurious to the growth of the bacterial cells. E. coli genome does not appear to encode for any protein that has significant homology to OsGR-RBP4 protein. Therefore, the mechanism of inhibition appears to be due to some illegitimate interactions of the OsGR-RBP4 with possibly the RNA species of the trans-host bacterial cells. The detailed mechanism underlying this inhibition remains to be worked out.

No MeSH data available.


Related in: MedlinePlus

Dynamics of E. coli before and after induction with IPTG. A. Growth profile of E. coli M15 cells. Panels a, b, c and d show growth profiles of pQE30/M15, OsGR-RBP4/M15, OsGR-RBP4ΔC/M15 andOsFKBP0/M15 E. coli cell types, respectively. Purple line denotes un-induced and blue line denotes induced E. coli M15 cells. B. a. Plate culture assay of OsGR-RBP4/M15, OsGR- RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells (incubation at 37°C). Cells were harvested at various time points after IPTG induction as shown. Serial dilutions were made and equal number of cells was spotted on LB agar plates with antibiotic selection. Plates were photographed after 16 h of incubation. +IPTG refers to induction by IPTG for the time periods indicated at the top. b. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells (incubation temperature of the plates = 20°C). Cells were harvested at various time points after IPTG induction as shown. c. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells, (incubation temperature of the plates = 45°C).
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Figure 2: Dynamics of E. coli before and after induction with IPTG. A. Growth profile of E. coli M15 cells. Panels a, b, c and d show growth profiles of pQE30/M15, OsGR-RBP4/M15, OsGR-RBP4ΔC/M15 andOsFKBP0/M15 E. coli cell types, respectively. Purple line denotes un-induced and blue line denotes induced E. coli M15 cells. B. a. Plate culture assay of OsGR-RBP4/M15, OsGR- RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells (incubation at 37°C). Cells were harvested at various time points after IPTG induction as shown. Serial dilutions were made and equal number of cells was spotted on LB agar plates with antibiotic selection. Plates were photographed after 16 h of incubation. +IPTG refers to induction by IPTG for the time periods indicated at the top. b. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells (incubation temperature of the plates = 20°C). Cells were harvested at various time points after IPTG induction as shown. c. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells, (incubation temperature of the plates = 45°C).

Mentions: Next, growth profiles of OsGR-RBP4/M15, OsGR-RBP4ΔC/M15, OsFKBP20/M15 and pQE30/M15 cells were monitored spectrophotometrically both for the IPTG un-induced and the induced cultures. The overnight grown primary cultures were used to set the secondary cultures beginning with 0.5 O.D. O.D. values were subsequently recorded after every 30 min interval. Growth curves were then plotted for the O.D. values against time intervals. From Figure 2A, it is clear that the growth rates of all four cells types (OsGR-RBP4/M15, OsGR-RBP4ΔC/M15, OsFKBP20/M15 and pQE30/M15) were nearly comparable under IPTG un-induced conditions (shown by blue line color). Strikingly, OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells types after IPTG induction failed to grow at all on secondary culturing (shown by pink line color). There was no effect of IPTG addition to pQE30/M15 cell type. While OsFKBP20/M15 cells did grow upon IPTG induction, its growth was slightly less than pQE30/M15 cell type.


Glycine-rich RNA binding protein of Oryza sativa inhibits growth of M15 E. coli cells.

Singh U, Deb D, Singh A, Grover A - BMC Res Notes (2011)

Dynamics of E. coli before and after induction with IPTG. A. Growth profile of E. coli M15 cells. Panels a, b, c and d show growth profiles of pQE30/M15, OsGR-RBP4/M15, OsGR-RBP4ΔC/M15 andOsFKBP0/M15 E. coli cell types, respectively. Purple line denotes un-induced and blue line denotes induced E. coli M15 cells. B. a. Plate culture assay of OsGR-RBP4/M15, OsGR- RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells (incubation at 37°C). Cells were harvested at various time points after IPTG induction as shown. Serial dilutions were made and equal number of cells was spotted on LB agar plates with antibiotic selection. Plates were photographed after 16 h of incubation. +IPTG refers to induction by IPTG for the time periods indicated at the top. b. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells (incubation temperature of the plates = 20°C). Cells were harvested at various time points after IPTG induction as shown. c. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells, (incubation temperature of the plates = 45°C).
© Copyright Policy - open-access
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Figure 2: Dynamics of E. coli before and after induction with IPTG. A. Growth profile of E. coli M15 cells. Panels a, b, c and d show growth profiles of pQE30/M15, OsGR-RBP4/M15, OsGR-RBP4ΔC/M15 andOsFKBP0/M15 E. coli cell types, respectively. Purple line denotes un-induced and blue line denotes induced E. coli M15 cells. B. a. Plate culture assay of OsGR-RBP4/M15, OsGR- RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells (incubation at 37°C). Cells were harvested at various time points after IPTG induction as shown. Serial dilutions were made and equal number of cells was spotted on LB agar plates with antibiotic selection. Plates were photographed after 16 h of incubation. +IPTG refers to induction by IPTG for the time periods indicated at the top. b. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells (incubation temperature of the plates = 20°C). Cells were harvested at various time points after IPTG induction as shown. c. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells, (incubation temperature of the plates = 45°C).
Mentions: Next, growth profiles of OsGR-RBP4/M15, OsGR-RBP4ΔC/M15, OsFKBP20/M15 and pQE30/M15 cells were monitored spectrophotometrically both for the IPTG un-induced and the induced cultures. The overnight grown primary cultures were used to set the secondary cultures beginning with 0.5 O.D. O.D. values were subsequently recorded after every 30 min interval. Growth curves were then plotted for the O.D. values against time intervals. From Figure 2A, it is clear that the growth rates of all four cells types (OsGR-RBP4/M15, OsGR-RBP4ΔC/M15, OsFKBP20/M15 and pQE30/M15) were nearly comparable under IPTG un-induced conditions (shown by blue line color). Strikingly, OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells types after IPTG induction failed to grow at all on secondary culturing (shown by pink line color). There was no effect of IPTG addition to pQE30/M15 cell type. While OsFKBP20/M15 cells did grow upon IPTG induction, its growth was slightly less than pQE30/M15 cell type.

Bottom Line: Removal of the inducer, IPTG, resulted in re-growth of the cells, indicating that effect of the foreign proteins was of reversible nature.Expression of eukaryotic, stress-associated OsGR-RBP4 protein in prokaryotic E. coli M15 cells proves injurious to the growth of the bacterial cells.E. coli genome does not appear to encode for any protein that has significant homology to OsGR-RBP4 protein.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Plant Molecular Biology, University of Delhi South Campus, New Delhi, India. anil.anilgrover@gmail.com.

ABSTRACT

Background: Plant glycine-rich RNA binding proteins have been implicated to have roles in diverse abiotic stresses.

Findings: E. coli M15 cells transformed with full-length rice glycine-rich RNA binding protein4 (OsGR-RBP4), truncated rice glycine-rich RNA binding protein4 (OsGR-RBP4ΔC) and rice FK506 binding protein (OsFKBP20) were analyzed for growth profiles using both broth and solid media. Expression of OsGR-RBP4 and OsGR-RBP4ΔC proteins caused specific, inhibitory effect on growth of recombinant M15 E. coli cells. The bacterial inhibition was shown to be time and incubation temperature dependent. Removal of the inducer, IPTG, resulted in re-growth of the cells, indicating that effect of the foreign proteins was of reversible nature. Although noted at different levels of dilution factors, addition of purified Os-GR-RBP4 and OsGR-RBP4ΔC showed a similar inhibitory effect as seen with expression inside the bacterial cells.

Conclusions: Expression of eukaryotic, stress-associated OsGR-RBP4 protein in prokaryotic E. coli M15 cells proves injurious to the growth of the bacterial cells. E. coli genome does not appear to encode for any protein that has significant homology to OsGR-RBP4 protein. Therefore, the mechanism of inhibition appears to be due to some illegitimate interactions of the OsGR-RBP4 with possibly the RNA species of the trans-host bacterial cells. The detailed mechanism underlying this inhibition remains to be worked out.

No MeSH data available.


Related in: MedlinePlus