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The Transcriptional Repressor, MtrR, of the mtrCDE Efflux Pump Operon of Neisseria gonorrhoeae Can Also Serve as an Activator of "off Target" Gene (glnE) Expression.

Johnson PJ, Shafer WM - Antibiotics (Basel) (2015)

Bottom Line: However, results from a previous transcriptional profiling study suggested that MtrR also represses or activates expression of at least sixty genes outside of the mtr locus.In contrast, evidence for the ability of MtrR to directly activate expression of gonococcal genes has been lacking; herein, we provide such evidence.We now report that MtrR has the ability to directly activate expression of glnE, which encodes the dual functional adenyltransferase/deadenylase enzyme GlnE that modifies GlnA resulting in regulation of its role in glutamine biosynthesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT

MtrR is a well-characterized repressor of the Neisseria gonorrhoeae mtrCDE efflux pump operon. However, results from a previous transcriptional profiling study suggested that MtrR also represses or activates expression of at least sixty genes outside of the mtr locus. Evidence that MtrR can directly repress so-called "off target" genes has previously been reported; in particular, MtrR was shown to directly repress glnA, which encodes glutamine synthetase. In contrast, evidence for the ability of MtrR to directly activate expression of gonococcal genes has been lacking; herein, we provide such evidence. We now report that MtrR has the ability to directly activate expression of glnE, which encodes the dual functional adenyltransferase/deadenylase enzyme GlnE that modifies GlnA resulting in regulation of its role in glutamine biosynthesis. With its capacity to repress expression of glnA, the results presented herein emphasize the diverse and often opposing regulatory properties of MtrR that likely contributes to the overall physiology and metabolism of N. gonorrhoeae.

No MeSH data available.


Related in: MedlinePlus

Identification of the MtrR-binding site in the glnE upstream DNA. (A) The binding specificity of MtrR for the DNA shown in Figure 1 was determined by competitive EMSA; (B) The MtrR-binding sites within this sequence were identified by DNase I protection assays that employed increasing amounts of purified MtrR-MBP (0, 5, 10, and 15 μg) with both sense and anti-sense probes. The protected regions on each probe are identified by the black bars and the two sites on the anti-sense strand are labeled as A′ and B′. Regions containing DNase I hypersensitive sites, which could contain more than one nucleotide, on the sense and antisense strands are denoted by *. The sequencing reactions for each probe are adjacent to the DNase I protection reactions and oriented G, A, T, C.
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Figure 3: Identification of the MtrR-binding site in the glnE upstream DNA. (A) The binding specificity of MtrR for the DNA shown in Figure 1 was determined by competitive EMSA; (B) The MtrR-binding sites within this sequence were identified by DNase I protection assays that employed increasing amounts of purified MtrR-MBP (0, 5, 10, and 15 μg) with both sense and anti-sense probes. The protected regions on each probe are identified by the black bars and the two sites on the anti-sense strand are labeled as A′ and B′. Regions containing DNase I hypersensitive sites, which could contain more than one nucleotide, on the sense and antisense strands are denoted by *. The sequencing reactions for each probe are adjacent to the DNase I protection reactions and oriented G, A, T, C.

Mentions: In order to determine if MtrR activation of glnE is by a direct mechanism, we tested if it can bind to the sequence upstream of glnE (Figure 2). Based on the published MtrR-binding site sequences upstream of mtrCDE [7,10], farR [9], and rpoH [7], we detected four potential MtrR-binding sites on the sense and anti-sense strands in this sequence, which ranged in sequence identity from 67% to 52% for regions on the sense and anti-sense strands, respectively. Using competitive EMSA, we confirmed that the DNA sequence upstream of glnE could bind MtrR (Figure 3A) in a specific manner as such binding was competed by unlabeled specific DNA. The nonspecific DNA failed to show such competition, although at its highest level (100× excess) the electrophoretic mobility of the MtrR∷glnE DNA complex was slightly faster than at lower concentrations of competing DNA; this suggested that some MtrR∷glnE DNA complexes may be weak or non-specific.


The Transcriptional Repressor, MtrR, of the mtrCDE Efflux Pump Operon of Neisseria gonorrhoeae Can Also Serve as an Activator of "off Target" Gene (glnE) Expression.

Johnson PJ, Shafer WM - Antibiotics (Basel) (2015)

Identification of the MtrR-binding site in the glnE upstream DNA. (A) The binding specificity of MtrR for the DNA shown in Figure 1 was determined by competitive EMSA; (B) The MtrR-binding sites within this sequence were identified by DNase I protection assays that employed increasing amounts of purified MtrR-MBP (0, 5, 10, and 15 μg) with both sense and anti-sense probes. The protected regions on each probe are identified by the black bars and the two sites on the anti-sense strand are labeled as A′ and B′. Regions containing DNase I hypersensitive sites, which could contain more than one nucleotide, on the sense and antisense strands are denoted by *. The sequencing reactions for each probe are adjacent to the DNase I protection reactions and oriented G, A, T, C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4464784&req=5

Figure 3: Identification of the MtrR-binding site in the glnE upstream DNA. (A) The binding specificity of MtrR for the DNA shown in Figure 1 was determined by competitive EMSA; (B) The MtrR-binding sites within this sequence were identified by DNase I protection assays that employed increasing amounts of purified MtrR-MBP (0, 5, 10, and 15 μg) with both sense and anti-sense probes. The protected regions on each probe are identified by the black bars and the two sites on the anti-sense strand are labeled as A′ and B′. Regions containing DNase I hypersensitive sites, which could contain more than one nucleotide, on the sense and antisense strands are denoted by *. The sequencing reactions for each probe are adjacent to the DNase I protection reactions and oriented G, A, T, C.
Mentions: In order to determine if MtrR activation of glnE is by a direct mechanism, we tested if it can bind to the sequence upstream of glnE (Figure 2). Based on the published MtrR-binding site sequences upstream of mtrCDE [7,10], farR [9], and rpoH [7], we detected four potential MtrR-binding sites on the sense and anti-sense strands in this sequence, which ranged in sequence identity from 67% to 52% for regions on the sense and anti-sense strands, respectively. Using competitive EMSA, we confirmed that the DNA sequence upstream of glnE could bind MtrR (Figure 3A) in a specific manner as such binding was competed by unlabeled specific DNA. The nonspecific DNA failed to show such competition, although at its highest level (100× excess) the electrophoretic mobility of the MtrR∷glnE DNA complex was slightly faster than at lower concentrations of competing DNA; this suggested that some MtrR∷glnE DNA complexes may be weak or non-specific.

Bottom Line: However, results from a previous transcriptional profiling study suggested that MtrR also represses or activates expression of at least sixty genes outside of the mtr locus.In contrast, evidence for the ability of MtrR to directly activate expression of gonococcal genes has been lacking; herein, we provide such evidence.We now report that MtrR has the ability to directly activate expression of glnE, which encodes the dual functional adenyltransferase/deadenylase enzyme GlnE that modifies GlnA resulting in regulation of its role in glutamine biosynthesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, Emory University School of Medicine, Atlanta, GA 30322, USA.

ABSTRACT

MtrR is a well-characterized repressor of the Neisseria gonorrhoeae mtrCDE efflux pump operon. However, results from a previous transcriptional profiling study suggested that MtrR also represses or activates expression of at least sixty genes outside of the mtr locus. Evidence that MtrR can directly repress so-called "off target" genes has previously been reported; in particular, MtrR was shown to directly repress glnA, which encodes glutamine synthetase. In contrast, evidence for the ability of MtrR to directly activate expression of gonococcal genes has been lacking; herein, we provide such evidence. We now report that MtrR has the ability to directly activate expression of glnE, which encodes the dual functional adenyltransferase/deadenylase enzyme GlnE that modifies GlnA resulting in regulation of its role in glutamine biosynthesis. With its capacity to repress expression of glnA, the results presented herein emphasize the diverse and often opposing regulatory properties of MtrR that likely contributes to the overall physiology and metabolism of N. gonorrhoeae.

No MeSH data available.


Related in: MedlinePlus