Limits...
Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae.

Göpel Y, Lüttmann D, Heroven AK, Reichenbach B, Dersch P, Görke B - Nucleic Acids Res. (2010)

Bottom Line: However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription.In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter.Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University, Grisebachstrasse 8, 37077 Göttingen, Germany.

ABSTRACT
Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ(54)-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ(70)-promoters overlap the σ(54)-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ(70)-promoter. Thus, transcription of glmY and glmZ is controlled by σ(54) and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription. In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

Show MeSH

Related in: MedlinePlus

Role of IHF for expression of glmY. (A) Schematic representation of the E. coli glmY promoter region and location of GlrR and putative IHF binding sites. The sequences of the putative IHF binding sites upstream of E. coli glmY and Y. pseudotuberculosis glmZ are shown and the nucleotide exchanges introduced in IHF site 1 of the E. coli glmY promoter are indicated. (B) EMSAs to test binding of purified IHF to the glmY and glmZ promoter regions of E. coli and Y. pseudotuberculosis, respectively. The DNA fragments were obtained by PCR making use of the primer pairs BG377/BG456 and BG700/BG701, respectively. As controls, DNA fragments encompassing the lac promoter were additionally present. (C) Expression of E. coli glmY in ΔihfA and ΔihfB mutants. β-Galactosidase activities of strains carrying the chromosomal E. coli glmY’-lacZ fusion in the context of the wild-type promoter (columns 1–3) or in the context of the mutated σ70-promoter leaving the σ54-promoter as single active promoter (columns 4–6). Genes ihfA or ihfB were deleted as indicated in the legend. The following strains were tested (corresponding to the columns from left to right): Z197, Z395, Z393, Z190, Z394 and Z392. (D) Mutational analysis of the putative IHF site 1 in the E. coli glmY promoter region. β-Galactosidase activities of wild-type and ΔglrR E. coli strains carrying the wild-type or mutated alleles of the E. coli glmY’-lacZ fusion. Mutations were either in the putative IHF-site 1 (columns 3, 4, 7, 8) as indicated in (A) or in the −10 sequence of the glmY promoter (columns 5–8) rendering glmY’-lacZ expression fully dependent on σ54. The following strains were employed (corresponding to the columns from left to right): Z197, Z206, Z370, Z372, Z190, Z196, Z371 and Z373.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3045617&req=5

Figure 6: Role of IHF for expression of glmY. (A) Schematic representation of the E. coli glmY promoter region and location of GlrR and putative IHF binding sites. The sequences of the putative IHF binding sites upstream of E. coli glmY and Y. pseudotuberculosis glmZ are shown and the nucleotide exchanges introduced in IHF site 1 of the E. coli glmY promoter are indicated. (B) EMSAs to test binding of purified IHF to the glmY and glmZ promoter regions of E. coli and Y. pseudotuberculosis, respectively. The DNA fragments were obtained by PCR making use of the primer pairs BG377/BG456 and BG700/BG701, respectively. As controls, DNA fragments encompassing the lac promoter were additionally present. (C) Expression of E. coli glmY in ΔihfA and ΔihfB mutants. β-Galactosidase activities of strains carrying the chromosomal E. coli glmY’-lacZ fusion in the context of the wild-type promoter (columns 1–3) or in the context of the mutated σ70-promoter leaving the σ54-promoter as single active promoter (columns 4–6). Genes ihfA or ihfB were deleted as indicated in the legend. The following strains were tested (corresponding to the columns from left to right): Z197, Z395, Z393, Z190, Z394 and Z392. (D) Mutational analysis of the putative IHF site 1 in the E. coli glmY promoter region. β-Galactosidase activities of wild-type and ΔglrR E. coli strains carrying the wild-type or mutated alleles of the E. coli glmY’-lacZ fusion. Mutations were either in the putative IHF-site 1 (columns 3, 4, 7, 8) as indicated in (A) or in the −10 sequence of the glmY promoter (columns 5–8) rendering glmY’-lacZ expression fully dependent on σ54. The following strains were employed (corresponding to the columns from left to right): Z197, Z206, Z370, Z372, Z190, Z196, Z371 and Z373.

Mentions: The sequence alignment analyses detected two additional sequence motifs with similarity to the binding site of the global transcriptional regulator IHF. These sequence elements were detectable in all species, except for the glmZ promoters of Escherichia, Shigella and Klebsiella (Supplementary Figures S3 and S4), which according to all evidence are transcribed from single σ70-promoters. This suggested a role of these sites for activities of the σ54-promoters upstream of glmY and glmZ (Figure 6A). Therefore, we tested whether IHF is able to bind to the promoter fragments of E. coli glmY and Y. pseudotuberculosis glmZ. Both DNA fragments were bound by IHF protein (Figure 6B). The lacZ promoter fragments, which served as internal controls, were also bound, but at higher protein concentrations. The lacZ promoter is not known to contain any IHF site indicating unspecific binding. To confirm this conclusion, we repeated the experiments using a DNA fragment covering the ptsG promoter from Bacillus subtilis as internal control. B. subtilis does not possess IHF. Once more, efficient binding of the glmY and glmZ promoters could be observed, while the ptsG promoter was only bound at higher protein concentrations (Supplementary Figure S9). Hence, binding of IHF to the lacZ and ptsG promoters is unspecific, which is in line with previous data reporting that IHF binds DNA with lower affinity also in sequence-independent manner (30).Figure 6.


Common and divergent features in transcriptional control of the homologous small RNAs GlmY and GlmZ in Enterobacteriaceae.

Göpel Y, Lüttmann D, Heroven AK, Reichenbach B, Dersch P, Görke B - Nucleic Acids Res. (2010)

Role of IHF for expression of glmY. (A) Schematic representation of the E. coli glmY promoter region and location of GlrR and putative IHF binding sites. The sequences of the putative IHF binding sites upstream of E. coli glmY and Y. pseudotuberculosis glmZ are shown and the nucleotide exchanges introduced in IHF site 1 of the E. coli glmY promoter are indicated. (B) EMSAs to test binding of purified IHF to the glmY and glmZ promoter regions of E. coli and Y. pseudotuberculosis, respectively. The DNA fragments were obtained by PCR making use of the primer pairs BG377/BG456 and BG700/BG701, respectively. As controls, DNA fragments encompassing the lac promoter were additionally present. (C) Expression of E. coli glmY in ΔihfA and ΔihfB mutants. β-Galactosidase activities of strains carrying the chromosomal E. coli glmY’-lacZ fusion in the context of the wild-type promoter (columns 1–3) or in the context of the mutated σ70-promoter leaving the σ54-promoter as single active promoter (columns 4–6). Genes ihfA or ihfB were deleted as indicated in the legend. The following strains were tested (corresponding to the columns from left to right): Z197, Z395, Z393, Z190, Z394 and Z392. (D) Mutational analysis of the putative IHF site 1 in the E. coli glmY promoter region. β-Galactosidase activities of wild-type and ΔglrR E. coli strains carrying the wild-type or mutated alleles of the E. coli glmY’-lacZ fusion. Mutations were either in the putative IHF-site 1 (columns 3, 4, 7, 8) as indicated in (A) or in the −10 sequence of the glmY promoter (columns 5–8) rendering glmY’-lacZ expression fully dependent on σ54. The following strains were employed (corresponding to the columns from left to right): Z197, Z206, Z370, Z372, Z190, Z196, Z371 and Z373.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045617&req=5

Figure 6: Role of IHF for expression of glmY. (A) Schematic representation of the E. coli glmY promoter region and location of GlrR and putative IHF binding sites. The sequences of the putative IHF binding sites upstream of E. coli glmY and Y. pseudotuberculosis glmZ are shown and the nucleotide exchanges introduced in IHF site 1 of the E. coli glmY promoter are indicated. (B) EMSAs to test binding of purified IHF to the glmY and glmZ promoter regions of E. coli and Y. pseudotuberculosis, respectively. The DNA fragments were obtained by PCR making use of the primer pairs BG377/BG456 and BG700/BG701, respectively. As controls, DNA fragments encompassing the lac promoter were additionally present. (C) Expression of E. coli glmY in ΔihfA and ΔihfB mutants. β-Galactosidase activities of strains carrying the chromosomal E. coli glmY’-lacZ fusion in the context of the wild-type promoter (columns 1–3) or in the context of the mutated σ70-promoter leaving the σ54-promoter as single active promoter (columns 4–6). Genes ihfA or ihfB were deleted as indicated in the legend. The following strains were tested (corresponding to the columns from left to right): Z197, Z395, Z393, Z190, Z394 and Z392. (D) Mutational analysis of the putative IHF site 1 in the E. coli glmY promoter region. β-Galactosidase activities of wild-type and ΔglrR E. coli strains carrying the wild-type or mutated alleles of the E. coli glmY’-lacZ fusion. Mutations were either in the putative IHF-site 1 (columns 3, 4, 7, 8) as indicated in (A) or in the −10 sequence of the glmY promoter (columns 5–8) rendering glmY’-lacZ expression fully dependent on σ54. The following strains were employed (corresponding to the columns from left to right): Z197, Z206, Z370, Z372, Z190, Z196, Z371 and Z373.
Mentions: The sequence alignment analyses detected two additional sequence motifs with similarity to the binding site of the global transcriptional regulator IHF. These sequence elements were detectable in all species, except for the glmZ promoters of Escherichia, Shigella and Klebsiella (Supplementary Figures S3 and S4), which according to all evidence are transcribed from single σ70-promoters. This suggested a role of these sites for activities of the σ54-promoters upstream of glmY and glmZ (Figure 6A). Therefore, we tested whether IHF is able to bind to the promoter fragments of E. coli glmY and Y. pseudotuberculosis glmZ. Both DNA fragments were bound by IHF protein (Figure 6B). The lacZ promoter fragments, which served as internal controls, were also bound, but at higher protein concentrations. The lacZ promoter is not known to contain any IHF site indicating unspecific binding. To confirm this conclusion, we repeated the experiments using a DNA fragment covering the ptsG promoter from Bacillus subtilis as internal control. B. subtilis does not possess IHF. Once more, efficient binding of the glmY and glmZ promoters could be observed, while the ptsG promoter was only bound at higher protein concentrations (Supplementary Figure S9). Hence, binding of IHF to the lacZ and ptsG promoters is unspecific, which is in line with previous data reporting that IHF binds DNA with lower affinity also in sequence-independent manner (30).Figure 6.

Bottom Line: However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription.In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter.Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

View Article: PubMed Central - PubMed

Affiliation: Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August-University, Grisebachstrasse 8, 37077 Göttingen, Germany.

ABSTRACT
Small RNAs GlmY and GlmZ compose a cascade that feedback-regulates synthesis of enzyme GlmS in Enterobacteriaceae. Here, we analyzed the transcriptional regulation of glmY/glmZ from Yersinia pseudotuberculosis, Salmonella typhimurium and Escherichia coli, as representatives for other enterobacterial species, which exhibit similar promoter architectures. The GlmY and GlmZ sRNAs of Y. pseudotuberculosis are transcribed from σ(54)-promoters that require activation by the response regulator GlrR through binding to three conserved sites located upstream of the promoters. This also applies to glmY/glmZ of S. typhimurium and glmY of E. coli, but as a difference additional σ(70)-promoters overlap the σ(54)-promoters and initiate transcription at the same site. In contrast, E. coli glmZ is transcribed from a single σ(70)-promoter. Thus, transcription of glmY and glmZ is controlled by σ(54) and the two-component system GlrR/GlrK (QseF/QseE) in Y. pseudotuberculosis and presumably in many other Enterobacteria. However, in a subset of species such as E. coli this relationship is partially lost in favor of σ(70)-dependent transcription. In addition, we show that activity of the σ(54)-promoter of E. coli glmY requires binding of the integration host factor to sites upstream of the promoter. Finally, evidence is provided that phosphorylation of GlrR increases its activity and thereby sRNA expression.

Show MeSH
Related in: MedlinePlus