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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

Design of constructs, confirmation of transcription and translation of rice proteins in E. coli. a. Linear maps of the recombinant constructs employed in this study. b. RT-PCR analysis of IPTG un-induced and induced samples of transformed E. coli M15 cells. Upper panel shows RT-PCR depicting expression of the respective genes. Lower panel shows EtBr stained gel depicting comparable staining of the ribosomal RNA species, 2 μg of total RNA was loaded in each lane. c. Expression of the foreign proteins in the transformed E. coli cells. Total protein from the IPTG un-induced and induced cells were separately analyzed. Induced proteins are indicated by arrows. Pre-stained markers are shown on the left side of the panel. UI and I refer to IPTG un-induced and induced samples, respectively.
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Figure 1: Design of constructs, confirmation of transcription and translation of rice proteins in E. coli. a. Linear maps of the recombinant constructs employed in this study. b. RT-PCR analysis of IPTG un-induced and induced samples of transformed E. coli M15 cells. Upper panel shows RT-PCR depicting expression of the respective genes. Lower panel shows EtBr stained gel depicting comparable staining of the ribosomal RNA species, 2 μg of total RNA was loaded in each lane. c. Expression of the foreign proteins in the transformed E. coli cells. Total protein from the IPTG un-induced and induced cells were separately analyzed. Induced proteins are indicated by arrows. Pre-stained markers are shown on the left side of the panel. UI and I refer to IPTG un-induced and induced samples, respectively.

Mentions: This study aimed at the analysis of the OsGR-RBP4 expression in E. coli M15 cells. To accomplish this, OsGR-RBP4 cloned in pQE30 vector was used. We also used OsGR-RBP4ΔC (truncated version of the Osgr-rbp4 with intact RRM but lacking the glycine rich domain sequences) cloned in pQE30. In addition, we used OsFKBP20 cDNA cloned in pQE30 as control (Figure 1a). As a plasmid control, pQE30 was also transformed in E. coli M15 cells. Four types of recombinant E. coli cells thus analyzed were as per the following constitution: OsGR-RBP4 in M15 cells (OsGR-RBP4/M15), OsGR-RBP4ΔC/M15, OsFKBP20/M15 and pQE30/M15. Selection of the transformed bacterial cells on antibiotic provided first line of evidence that the cells were transformed. Further, expression of the foreign inserts in the E. coli M15 host cells were examined at the transcript level. RNAs isolated from OsGR-RBP4/M15, OsGR-RBP4ΔC/M15 and OsFKBP20/M15 cells were used for cDNA preparation. cDNAs were used for RT-PCR reactions and the products of these reactions were analyzed on 1% agarose gel. As shown in Figure 1b, clear expression was noted for OsGR-RBP4, OsGR-RBP4ΔC and OsFKBP20 RNAs in RT-PCR reaction from the IPTG-induced cells. It is noteworthy that low level expression was also apparent for the IPTG un-induced samples of various transformants (particularly for the OsGR-RBP4ΔC/pQE30 lane). Subsequently, expression of the foreign protein was examined in the E. coli M15 host cells. Total proteins isolated from OsGR-RBP4/M15, OsGR-RBP4ΔC/M15 and OsFKBP20/M15 cells were loaded on 12% SDS-gel. As shown in Figure 1c, the expressed foreign proteins were clearly noted in the IPTG induced samples (shown by arrows). No such protein was detected in E. coli M15 cells transformed with pQE30 plasmid backbone.


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)

Design of constructs, confirmation of transcription and translation of rice proteins in E. coli. a. Linear maps of the recombinant constructs employed in this study. b. RT-PCR analysis of IPTG un-induced and induced samples of transformed E. coli M15 cells. Upper panel shows RT-PCR depicting expression of the respective genes. Lower panel shows EtBr stained gel depicting comparable staining of the ribosomal RNA species, 2 μg of total RNA was loaded in each lane. c. Expression of the foreign proteins in the transformed E. coli cells. Total protein from the IPTG un-induced and induced cells were separately analyzed. Induced proteins are indicated by arrows. Pre-stained markers are shown on the left side of the panel. UI and I refer to IPTG un-induced and induced samples, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Design of constructs, confirmation of transcription and translation of rice proteins in E. coli. a. Linear maps of the recombinant constructs employed in this study. b. RT-PCR analysis of IPTG un-induced and induced samples of transformed E. coli M15 cells. Upper panel shows RT-PCR depicting expression of the respective genes. Lower panel shows EtBr stained gel depicting comparable staining of the ribosomal RNA species, 2 μg of total RNA was loaded in each lane. c. Expression of the foreign proteins in the transformed E. coli cells. Total protein from the IPTG un-induced and induced cells were separately analyzed. Induced proteins are indicated by arrows. Pre-stained markers are shown on the left side of the panel. UI and I refer to IPTG un-induced and induced samples, respectively.
Mentions: This study aimed at the analysis of the OsGR-RBP4 expression in E. coli M15 cells. To accomplish this, OsGR-RBP4 cloned in pQE30 vector was used. We also used OsGR-RBP4ΔC (truncated version of the Osgr-rbp4 with intact RRM but lacking the glycine rich domain sequences) cloned in pQE30. In addition, we used OsFKBP20 cDNA cloned in pQE30 as control (Figure 1a). As a plasmid control, pQE30 was also transformed in E. coli M15 cells. Four types of recombinant E. coli cells thus analyzed were as per the following constitution: OsGR-RBP4 in M15 cells (OsGR-RBP4/M15), OsGR-RBP4ΔC/M15, OsFKBP20/M15 and pQE30/M15. Selection of the transformed bacterial cells on antibiotic provided first line of evidence that the cells were transformed. Further, expression of the foreign inserts in the E. coli M15 host cells were examined at the transcript level. RNAs isolated from OsGR-RBP4/M15, OsGR-RBP4ΔC/M15 and OsFKBP20/M15 cells were used for cDNA preparation. cDNAs were used for RT-PCR reactions and the products of these reactions were analyzed on 1% agarose gel. As shown in Figure 1b, clear expression was noted for OsGR-RBP4, OsGR-RBP4ΔC and OsFKBP20 RNAs in RT-PCR reaction from the IPTG-induced cells. It is noteworthy that low level expression was also apparent for the IPTG un-induced samples of various transformants (particularly for the OsGR-RBP4ΔC/pQE30 lane). Subsequently, expression of the foreign protein was examined in the E. coli M15 host cells. Total proteins isolated from OsGR-RBP4/M15, OsGR-RBP4ΔC/M15 and OsFKBP20/M15 cells were loaded on 12% SDS-gel. As shown in Figure 1c, the expressed foreign proteins were clearly noted in the IPTG induced samples (shown by arrows). No such protein was detected in E. coli M15 cells transformed with pQE30 plasmid backbone.

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