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Inherent characteristics of gene expression for buffering environmental changes without the corresponding transcriptional regulations

View Article: PubMed Central - PubMed

ABSTRACT

Gene expression patterning is crucial for environmental nutritional responses such as the nitrogen response in Escherichia coli. The nitrogen response is primarily regulated by the expression of glutamine synthetase (GS), which catalyzes the sole reaction of glutamine formation, by cis-logic regulatory circuits. Here, by removing the entire corresponding operator and promoter regions required for the control of GS, we constructed an E. coli strain that enables the detection of the basal GS gene expression, which is expressed from a plain promoter unrelated to the nitrogen response, and measured by co-transcribed GFP expression, an indicator of GS expression. Using strain cultures, we found that the GS expression level was able to shift inversely against the change of the environmental glutamine concentration. As a control experiment, we repeated similar experiments with another strain in which the GS regulatory region remained intact and the GFP gene following the plain promoter was introduced into a different chromosomal site. For this strain, we found that the GFP expression level did not shift in accordance with the environmental glutamine concentration. These results showed that GS expression from the plain promoter exhibited a responsive ability to buffer environmental changes, whereas the GS expression shift did not correlate with the specific characteristics of the plain promoter and GFP expression. This study identifies the inherent characteristics of basal gene expression in response to environmental changes, facilitating a deeper understanding of cellular design principles.

No MeSH data available.


Structure of glnALG operon mutations. (A) The region near the wild-type glnALG operon. (B) The region after modification. (C) The design of three E. coli strains, OSU7, OSU8, and OSU9.
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f1-2_63: Structure of glnALG operon mutations. (A) The region near the wild-type glnALG operon. (B) The region after modification. (C) The design of three E. coli strains, OSU7, OSU8, and OSU9.

Mentions: To assess the performance of basal gene expression, which is expressed without cis-logic gates and topological connectivity, in response to environmental nutritional changes, we constructed two E. coli strains, OSU8 and OSU7. OSU8 was constructed so that the partial region of the glnALG operon, including the entire operator and promoter regions and the glnA gene, was replaced by the reporter cassette sequentially containing the PtetA promoter, the gfpuv5 gene for a GFP variant, and the glnA101 gene for a GS variant (Fig. 1). OSU7 was constructed in the same way as strain OSU8 except for the presence of the introduced gene for GS, glnA100, instead of glnA101 (Fig. 1). Gene expression from the PtetA promoter is constitutive, as its repressor, TetR, is not carried in this strain. As the genes for GFP and GS are co-transcribed from the PtetA promoter, the fluorescence intensity of a cell is expected to indicate the GS expression level. The glnA101 gene has mutations of Asn-219 to Asp and Tyr-397 to His and was obtained from a library generated by the random mutagenesis of a progenitor mutant gene for GS, glnA100, which has a mutation of Tyr-397 to His. Tyr-397 is known to be a target site for the regulation of activity by covalent adenylylation; therefore, GS-Y397H and GS-N219D/Y397H are free from regulation by reversible adenylylation21,22. The enzymatic activity of GS-N219D/Y397H seems to be lower than that of GS-Y397H as the E. coli cells expressing GS-N219D/Y397H grow much more slowly than those expressing GS-Y397H (data not shown). For a control experiment, we constructed an E. coli strain, OSU9, by replacing the intA gene (an unrelated prophage gene with metabolic functions) in a wild-type cell with the cassette sequentially containing the PtetA promoter and the GFP gene (Fig. 1C). In this strain, the glnALG operon is wild-type and, as the genes for GFP and GS are not co-transcribed from the PtetA promoter, the fluorescence intensity of a cell is expected to be independent of the GS expression level.


Inherent characteristics of gene expression for buffering environmental changes without the corresponding transcriptional regulations
Structure of glnALG operon mutations. (A) The region near the wild-type glnALG operon. (B) The region after modification. (C) The design of three E. coli strains, OSU7, OSU8, and OSU9.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036647&req=5

f1-2_63: Structure of glnALG operon mutations. (A) The region near the wild-type glnALG operon. (B) The region after modification. (C) The design of three E. coli strains, OSU7, OSU8, and OSU9.
Mentions: To assess the performance of basal gene expression, which is expressed without cis-logic gates and topological connectivity, in response to environmental nutritional changes, we constructed two E. coli strains, OSU8 and OSU7. OSU8 was constructed so that the partial region of the glnALG operon, including the entire operator and promoter regions and the glnA gene, was replaced by the reporter cassette sequentially containing the PtetA promoter, the gfpuv5 gene for a GFP variant, and the glnA101 gene for a GS variant (Fig. 1). OSU7 was constructed in the same way as strain OSU8 except for the presence of the introduced gene for GS, glnA100, instead of glnA101 (Fig. 1). Gene expression from the PtetA promoter is constitutive, as its repressor, TetR, is not carried in this strain. As the genes for GFP and GS are co-transcribed from the PtetA promoter, the fluorescence intensity of a cell is expected to indicate the GS expression level. The glnA101 gene has mutations of Asn-219 to Asp and Tyr-397 to His and was obtained from a library generated by the random mutagenesis of a progenitor mutant gene for GS, glnA100, which has a mutation of Tyr-397 to His. Tyr-397 is known to be a target site for the regulation of activity by covalent adenylylation; therefore, GS-Y397H and GS-N219D/Y397H are free from regulation by reversible adenylylation21,22. The enzymatic activity of GS-N219D/Y397H seems to be lower than that of GS-Y397H as the E. coli cells expressing GS-N219D/Y397H grow much more slowly than those expressing GS-Y397H (data not shown). For a control experiment, we constructed an E. coli strain, OSU9, by replacing the intA gene (an unrelated prophage gene with metabolic functions) in a wild-type cell with the cassette sequentially containing the PtetA promoter and the GFP gene (Fig. 1C). In this strain, the glnALG operon is wild-type and, as the genes for GFP and GS are not co-transcribed from the PtetA promoter, the fluorescence intensity of a cell is expected to be independent of the GS expression level.

View Article: PubMed Central - PubMed

ABSTRACT

Gene expression patterning is crucial for environmental nutritional responses such as the nitrogen response in Escherichia coli. The nitrogen response is primarily regulated by the expression of glutamine synthetase (GS), which catalyzes the sole reaction of glutamine formation, by cis-logic regulatory circuits. Here, by removing the entire corresponding operator and promoter regions required for the control of GS, we constructed an E. coli strain that enables the detection of the basal GS gene expression, which is expressed from a plain promoter unrelated to the nitrogen response, and measured by co-transcribed GFP expression, an indicator of GS expression. Using strain cultures, we found that the GS expression level was able to shift inversely against the change of the environmental glutamine concentration. As a control experiment, we repeated similar experiments with another strain in which the GS regulatory region remained intact and the GFP gene following the plain promoter was introduced into a different chromosomal site. For this strain, we found that the GFP expression level did not shift in accordance with the environmental glutamine concentration. These results showed that GS expression from the plain promoter exhibited a responsive ability to buffer environmental changes, whereas the GS expression shift did not correlate with the specific characteristics of the plain promoter and GFP expression. This study identifies the inherent characteristics of basal gene expression in response to environmental changes, facilitating a deeper understanding of cellular design principles.

No MeSH data available.