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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

Affect of affinity purified proteins on bacterial growth. a. Purification of OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins by affinity column chromatography. Arrows indicate the presence of partially purified proteins in respective panels. Pre-stained markers are shown on the extreme left panel. b. Effect of purified OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins on the survival of E. coli M15 cells under in vitro conditions. Photograph shows serial dilutions made after 8 h and spotted on LB agar plates with antibiotic selection.
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Figure 4: Affect of affinity purified proteins on bacterial growth. a. Purification of OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins by affinity column chromatography. Arrows indicate the presence of partially purified proteins in respective panels. Pre-stained markers are shown on the extreme left panel. b. Effect of purified OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins on the survival of E. coli M15 cells under in vitro conditions. Photograph shows serial dilutions made after 8 h and spotted on LB agar plates with antibiotic selection.

Mentions: Further, growth profiles of E. coli M15 cells were monitored in response to addition of purified OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins. The respective proteins were partially purified from the corresponding cultures using affinity column chromatography (respective proteins shown by arrows in Figure 4a). Equal amounts of proteins (120 μg) were added directly into the E. coli M15 cultures. It may be noted here that because of the variations in the molecular weights of the purified OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins, the number of individual protein molecules in 120 μg of protein may vary. After ~8 h of growth, cultures were spotted on LB agar plates with selection (kan). Exogenous addition of the OsGR-RBP4 and OsGR-RBP4PΔC proteins showed a distinct toxic effect on the growth of E. coli M15 cells. Out of these two, OsGR-RBP4PΔC appeared more toxic to cells than OsGR-RBP4. This again may be possible because of higher ability of OsGR-RBP4PΔC protein to enter into E. coli owing to its lower molecular weight. On the other hand, exogenous addition of OsFKBP20 protein didn't result in growth inhibition of bacterial cells (Figure 4b).


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)

Affect of affinity purified proteins on bacterial growth. a. Purification of OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins by affinity column chromatography. Arrows indicate the presence of partially purified proteins in respective panels. Pre-stained markers are shown on the extreme left panel. b. Effect of purified OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins on the survival of E. coli M15 cells under in vitro conditions. Photograph shows serial dilutions made after 8 h and spotted on LB agar plates with antibiotic selection.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040685&req=5

Figure 4: Affect of affinity purified proteins on bacterial growth. a. Purification of OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins by affinity column chromatography. Arrows indicate the presence of partially purified proteins in respective panels. Pre-stained markers are shown on the extreme left panel. b. Effect of purified OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins on the survival of E. coli M15 cells under in vitro conditions. Photograph shows serial dilutions made after 8 h and spotted on LB agar plates with antibiotic selection.
Mentions: Further, growth profiles of E. coli M15 cells were monitored in response to addition of purified OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins. The respective proteins were partially purified from the corresponding cultures using affinity column chromatography (respective proteins shown by arrows in Figure 4a). Equal amounts of proteins (120 μg) were added directly into the E. coli M15 cultures. It may be noted here that because of the variations in the molecular weights of the purified OsGR-RBP4, OsGR-RBP4PΔC and OsFKBP20 proteins, the number of individual protein molecules in 120 μg of protein may vary. After ~8 h of growth, cultures were spotted on LB agar plates with selection (kan). Exogenous addition of the OsGR-RBP4 and OsGR-RBP4PΔC proteins showed a distinct toxic effect on the growth of E. coli M15 cells. Out of these two, OsGR-RBP4PΔC appeared more toxic to cells than OsGR-RBP4. This again may be possible because of higher ability of OsGR-RBP4PΔC protein to enter into E. coli owing to its lower molecular weight. On the other hand, exogenous addition of OsFKBP20 protein didn't result in growth inhibition of bacterial cells (Figure 4b).

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