Limits...
Regulation of gene expression in restriction-modification system Eco29kI.

Nagornykh M, Zakharova M, Protsenko A, Bogdanova E, Solonin AS, Severinov K - Nucleic Acids Res. (2011)

Bottom Line: We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts.The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation.Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA.

ABSTRACT
The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

Show MeSH

Related in: MedlinePlus

Production of M.Eco29kI from internal promoter Meth_P. (A) Horizontal lines show the overnight growth of E. coli Z85 strain harboring a plasmid carrying the entire Eco29kI R-M system (p29) or a mutant plasmid with inactivated eco29kIR promoters (p29-ResPmut) on LB plates. Cells were spotted with indicated dilutions of λvir phage lysate. Phages collected from infections of cells carrying the mutant plasmid (second row) were used in infections shown at the bottom of the panel (this phage is labeled λvir-Eco29mod). The phenotypes of cells with respect to Eco29kI Res and Met activities are indicated at the right. (B) Determining the relative abundance of bicistronic and monocistronic Eco29kI RNA by qRT–PCR. Reverse transcription reactions were carried out with a primer annealing at the end of the eco29kIM gene using RNA samples purified from cells harboring indicated plasmids and qRT–PCR was performed using eco29kIM specific primers. qRT–PCR reactions run with bla-specific primers were used for normalization. (C) Primer extension analysis carried out with RNA purified from cells harboring a plasmid with entire Eco29kI system (p29) or cells harboring the same plasmid with inactive eco29kIR promoters (p29-ResPmut) using a primer designed to reveal transcripts initiated from Meth_P. Reverse transcription reaction products that correspond to the bicistronic mRNA degradation intermediates are marked with asterisks.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3113576&req=5

Figure 4: Production of M.Eco29kI from internal promoter Meth_P. (A) Horizontal lines show the overnight growth of E. coli Z85 strain harboring a plasmid carrying the entire Eco29kI R-M system (p29) or a mutant plasmid with inactivated eco29kIR promoters (p29-ResPmut) on LB plates. Cells were spotted with indicated dilutions of λvir phage lysate. Phages collected from infections of cells carrying the mutant plasmid (second row) were used in infections shown at the bottom of the panel (this phage is labeled λvir-Eco29mod). The phenotypes of cells with respect to Eco29kI Res and Met activities are indicated at the right. (B) Determining the relative abundance of bicistronic and monocistronic Eco29kI RNA by qRT–PCR. Reverse transcription reactions were carried out with a primer annealing at the end of the eco29kIM gene using RNA samples purified from cells harboring indicated plasmids and qRT–PCR was performed using eco29kIM specific primers. qRT–PCR reactions run with bla-specific primers were used for normalization. (C) Primer extension analysis carried out with RNA purified from cells harboring a plasmid with entire Eco29kI system (p29) or cells harboring the same plasmid with inactive eco29kIR promoters (p29-ResPmut) using a primer designed to reveal transcripts initiated from Meth_P. Reverse transcription reaction products that correspond to the bicistronic mRNA degradation intermediates are marked with asterisks.

Mentions: To show that eco29kIR promoter-independent eco29kIM transcription is biologically relevant, we determined the ability of cells harboring a plasmid expressing the complete Eco29kI system or derivative plasmids to restrict and/or modify the λvir phage (Figure 4A). As expected, the infection of cells harboring the entire Eco29kI system by unmodified phage was inefficient. Unmodified phage effectively infected cells carrying a plasmid with mutated eco29kIR promoters, also as expected. Importantly, phage progeny from the latter infections was not restricted by cells harboring complete R-M system, indicating that sufficient amounts of methylase are synthesized even in the absence of transcription from Res_P1/Res_P2.Figure 4.


Regulation of gene expression in restriction-modification system Eco29kI.

Nagornykh M, Zakharova M, Protsenko A, Bogdanova E, Solonin AS, Severinov K - Nucleic Acids Res. (2011)

Production of M.Eco29kI from internal promoter Meth_P. (A) Horizontal lines show the overnight growth of E. coli Z85 strain harboring a plasmid carrying the entire Eco29kI R-M system (p29) or a mutant plasmid with inactivated eco29kIR promoters (p29-ResPmut) on LB plates. Cells were spotted with indicated dilutions of λvir phage lysate. Phages collected from infections of cells carrying the mutant plasmid (second row) were used in infections shown at the bottom of the panel (this phage is labeled λvir-Eco29mod). The phenotypes of cells with respect to Eco29kI Res and Met activities are indicated at the right. (B) Determining the relative abundance of bicistronic and monocistronic Eco29kI RNA by qRT–PCR. Reverse transcription reactions were carried out with a primer annealing at the end of the eco29kIM gene using RNA samples purified from cells harboring indicated plasmids and qRT–PCR was performed using eco29kIM specific primers. qRT–PCR reactions run with bla-specific primers were used for normalization. (C) Primer extension analysis carried out with RNA purified from cells harboring a plasmid with entire Eco29kI system (p29) or cells harboring the same plasmid with inactive eco29kIR promoters (p29-ResPmut) using a primer designed to reveal transcripts initiated from Meth_P. Reverse transcription reaction products that correspond to the bicistronic mRNA degradation intermediates are marked with asterisks.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3113576&req=5

Figure 4: Production of M.Eco29kI from internal promoter Meth_P. (A) Horizontal lines show the overnight growth of E. coli Z85 strain harboring a plasmid carrying the entire Eco29kI R-M system (p29) or a mutant plasmid with inactivated eco29kIR promoters (p29-ResPmut) on LB plates. Cells were spotted with indicated dilutions of λvir phage lysate. Phages collected from infections of cells carrying the mutant plasmid (second row) were used in infections shown at the bottom of the panel (this phage is labeled λvir-Eco29mod). The phenotypes of cells with respect to Eco29kI Res and Met activities are indicated at the right. (B) Determining the relative abundance of bicistronic and monocistronic Eco29kI RNA by qRT–PCR. Reverse transcription reactions were carried out with a primer annealing at the end of the eco29kIM gene using RNA samples purified from cells harboring indicated plasmids and qRT–PCR was performed using eco29kIM specific primers. qRT–PCR reactions run with bla-specific primers were used for normalization. (C) Primer extension analysis carried out with RNA purified from cells harboring a plasmid with entire Eco29kI system (p29) or cells harboring the same plasmid with inactive eco29kIR promoters (p29-ResPmut) using a primer designed to reveal transcripts initiated from Meth_P. Reverse transcription reaction products that correspond to the bicistronic mRNA degradation intermediates are marked with asterisks.
Mentions: To show that eco29kIR promoter-independent eco29kIM transcription is biologically relevant, we determined the ability of cells harboring a plasmid expressing the complete Eco29kI system or derivative plasmids to restrict and/or modify the λvir phage (Figure 4A). As expected, the infection of cells harboring the entire Eco29kI system by unmodified phage was inefficient. Unmodified phage effectively infected cells carrying a plasmid with mutated eco29kIR promoters, also as expected. Importantly, phage progeny from the latter infections was not restricted by cells harboring complete R-M system, indicating that sufficient amounts of methylase are synthesized even in the absence of transcription from Res_P1/Res_P2.Figure 4.

Bottom Line: We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts.The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation.Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

View Article: PubMed Central - PubMed

Affiliation: Waksman Institute of Microbiology, 190 Frelinghuysen Road, Piscataway, NJ 08854, USA.

ABSTRACT
The Eco29kI restriction-modification (R-M) system consists of two partially overlapping genes, eco29kIR, encoding a restriction endonuclease and eco29kIM, encoding methyltransferase. The two genes are thought to form an operon with the eco29kIR gene preceding the eco29kIM gene. Such an organization is expected to complicate establishment of plasmids containing this R-M system in naive hosts, since common logic dictates that methyltransferase should be synthesized first to protect the DNA from cleavage by the endonuclease. Here, we characterize the Eco29kI gene transcription. We show that a separate promoter located within the eco29kIR gene is sufficient to synthesize enough methyltransferase to completely modify host DNA. We further show that transcription from two intragenic antisense promoters strongly decreases the levels of eco29kIR gene transcripts. The antisense transcripts act by preventing translation initiation from the bicistronic eco29kIR-eco29kIM mRNA and causing its degradation. Both eco29kIM and antisense promoters are necessary for Eco29kI genes establishment and/or stable maintenance, indicating that they jointly contribute to coordinated expression of Eco29kI genes.

Show MeSH
Related in: MedlinePlus