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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

Assays for E. coli viability. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells after the washing off the IPTG. Other details same as in Figure 2B.
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Figure 3: Assays for E. coli viability. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells after the washing off the IPTG. Other details same as in Figure 2B.

Mentions: Next we tested the effect of temperature on bacterial growth. Secondary culture (0.5 O.D. of the overnight grown primary culture used to set the secondary culture) was set up for all the four transformed E. coli M15 cell types. After 3 h, total culture volume was divided into two parts: one part was induced by IPTG and other was left un-induced. After every 30 min interval, cells from the un-induced and induced cultures were serially diluted and spotted on LB agar plates with carbenicillin and kanamycin. Three sets of plates were prepared and one set of each of these was incubated for 16 h at 20°C, 37°C and 45°C in separate incubators. At 20°C (Figure 2B. b) and 37°C (Figure 2B. a), all the four cells types showed comparable growth profile in the sets made without induction by IPTG (Figure 2B. b). In sets prepared with IPTG induced cell types, OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells showed lower growth profiles than OsFKBP20/M15 and pQE30/M15 cell types (Figure 2B. c). At both the temperatures of plate incubation, this effect was more pronounced after at least 60 min of IPTG induction. At 45°C (Figure 2B.c), increased lethality to OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells as against OsFKBP20/M15 and pQE30/M15 cells was apparent even at 30 min of IPTG induction. In this stress regime, it was further noted that OsFKBP20M15 and pQE30/M15 cells were able to grow gradually with time following induction by IPTG. However, OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells failed to overcome the lethality effects following IPTG induction (Figure 3).


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)

Assays for E. coli viability. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells after the washing off the IPTG. Other details same as in Figure 2B.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: Assays for E. coli viability. Plate assay of OsGR-RBP4/M15, OsGR-RBP4PΔC/M15, OsFKBP20/M15 and pQE30/M15 E. coli cells after the washing off the IPTG. Other details same as in Figure 2B.
Mentions: Next we tested the effect of temperature on bacterial growth. Secondary culture (0.5 O.D. of the overnight grown primary culture used to set the secondary culture) was set up for all the four transformed E. coli M15 cell types. After 3 h, total culture volume was divided into two parts: one part was induced by IPTG and other was left un-induced. After every 30 min interval, cells from the un-induced and induced cultures were serially diluted and spotted on LB agar plates with carbenicillin and kanamycin. Three sets of plates were prepared and one set of each of these was incubated for 16 h at 20°C, 37°C and 45°C in separate incubators. At 20°C (Figure 2B. b) and 37°C (Figure 2B. a), all the four cells types showed comparable growth profile in the sets made without induction by IPTG (Figure 2B. b). In sets prepared with IPTG induced cell types, OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells showed lower growth profiles than OsFKBP20/M15 and pQE30/M15 cell types (Figure 2B. c). At both the temperatures of plate incubation, this effect was more pronounced after at least 60 min of IPTG induction. At 45°C (Figure 2B.c), increased lethality to OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells as against OsFKBP20/M15 and pQE30/M15 cells was apparent even at 30 min of IPTG induction. In this stress regime, it was further noted that OsFKBP20M15 and pQE30/M15 cells were able to grow gradually with time following induction by IPTG. However, OsGR-RBP4/M15 and OsGR-RBP4ΔC/M15 cells failed to overcome the lethality effects following IPTG induction (Figure 3).

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