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csrR, a Paralog and Direct Target of CsrA, Promotes Legionella pneumophila Resilience in Water.

Abbott ZD, Yakhnin H, Babitzke P, Swanson MS - MBio (2015)

Bottom Line: A potent regulator of this pathogen's intracellular life cycle is CsrA, a protein widely distributed among bacterial species that is understood quite well.Our finding that every sequenced L. pneumophila strain carries several csrA paralogs-including two common to all isolates--indicates that the legionellae exploit CsrA regulatory switches for multiple purposes.Our discovery that one paralog, CsrR, is a target of CsrA that enhances survival in water is an important step toward understanding colonization of the engineered environment by pathogenic L. pneumophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

No MeSH data available.


Related in: MedlinePlus

CsrA binds tightly and specifically to csrR RNA. (A) 5′-end-labeled csrR RNA (0.1 nM) was incubated with CsrA protein at the indicated concentration (nM), and the complexes were separated by gel electrophoresis. Positions of bound (marked B) and free (marked F) RNA species are shown. The binding constant (Kd) was calculated from the binding curve shown at right. (B) Labeled csrR RNA (0.1 nM) was incubated with CsrA protein in the absence or presence of unlabeled specific (csrR) or nonspecific (E. coliphoB) competitor RNA at the concentrations indicated. (C) 5′-End-labeled csrR RNA was treated with RNase T1 in the presence of CsrA protein at the concentration indicated. Also shown are partial alkaline hydrolysis (column OH) and RNase T1 digestion (column T1) ladders, as well as a control sample that lacked RNase T1 treatment (column C). Labels indicate residues for which CsrA protein reduced RNase T1 cleavage (BS1 to BS3), the csrR Shine-Dalgarno (SD) sequence, and the translation initiation codon (Met). Numbering is with respect to the start site of csrR transcription (+1).
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fig4: CsrA binds tightly and specifically to csrR RNA. (A) 5′-end-labeled csrR RNA (0.1 nM) was incubated with CsrA protein at the indicated concentration (nM), and the complexes were separated by gel electrophoresis. Positions of bound (marked B) and free (marked F) RNA species are shown. The binding constant (Kd) was calculated from the binding curve shown at right. (B) Labeled csrR RNA (0.1 nM) was incubated with CsrA protein in the absence or presence of unlabeled specific (csrR) or nonspecific (E. coliphoB) competitor RNA at the concentrations indicated. (C) 5′-End-labeled csrR RNA was treated with RNase T1 in the presence of CsrA protein at the concentration indicated. Also shown are partial alkaline hydrolysis (column OH) and RNase T1 digestion (column T1) ladders, as well as a control sample that lacked RNase T1 treatment (column C). Labels indicate residues for which CsrA protein reduced RNase T1 cleavage (BS1 to BS3), the csrR Shine-Dalgarno (SD) sequence, and the translation initiation codon (Met). Numbering is with respect to the start site of csrR transcription (+1).

Mentions: To test directly whether CsrA protein binds to csrR mRNA, we performed an in vitro assay. Purified CsrA protein was incubated with 5‚Ä≤-end-labeled csrR RNA, and then binding was analyzed by gel electromobility shift assay. As predicted by the in silico analysis, CsrA protein bound tightly to csrR RNA, with a calculated dissociation constant of 32 ¬Ī 9¬†nM (Fig.¬†4A). This physical interaction was specific, since incubation with an excess of unlabeled csrR RNA effectively competed with CsrA protein binding to labeled csrR RNA (Fig.¬†4B). In contrast, incubation with even higher concentrations of a heterologous competitor RNA, E.¬†coliphoB, did not inhibit CsrA binding to csrR. Therefore, CsrA protein binds csrR RNA tightly and specifically in vitro.


csrR, a Paralog and Direct Target of CsrA, Promotes Legionella pneumophila Resilience in Water.

Abbott ZD, Yakhnin H, Babitzke P, Swanson MS - MBio (2015)

CsrA binds tightly and specifically to csrR RNA. (A) 5′-end-labeled csrR RNA (0.1 nM) was incubated with CsrA protein at the indicated concentration (nM), and the complexes were separated by gel electrophoresis. Positions of bound (marked B) and free (marked F) RNA species are shown. The binding constant (Kd) was calculated from the binding curve shown at right. (B) Labeled csrR RNA (0.1 nM) was incubated with CsrA protein in the absence or presence of unlabeled specific (csrR) or nonspecific (E. coliphoB) competitor RNA at the concentrations indicated. (C) 5′-End-labeled csrR RNA was treated with RNase T1 in the presence of CsrA protein at the concentration indicated. Also shown are partial alkaline hydrolysis (column OH) and RNase T1 digestion (column T1) ladders, as well as a control sample that lacked RNase T1 treatment (column C). Labels indicate residues for which CsrA protein reduced RNase T1 cleavage (BS1 to BS3), the csrR Shine-Dalgarno (SD) sequence, and the translation initiation codon (Met). Numbering is with respect to the start site of csrR transcription (+1).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4471563&req=5

fig4: CsrA binds tightly and specifically to csrR RNA. (A) 5′-end-labeled csrR RNA (0.1 nM) was incubated with CsrA protein at the indicated concentration (nM), and the complexes were separated by gel electrophoresis. Positions of bound (marked B) and free (marked F) RNA species are shown. The binding constant (Kd) was calculated from the binding curve shown at right. (B) Labeled csrR RNA (0.1 nM) was incubated with CsrA protein in the absence or presence of unlabeled specific (csrR) or nonspecific (E. coliphoB) competitor RNA at the concentrations indicated. (C) 5′-End-labeled csrR RNA was treated with RNase T1 in the presence of CsrA protein at the concentration indicated. Also shown are partial alkaline hydrolysis (column OH) and RNase T1 digestion (column T1) ladders, as well as a control sample that lacked RNase T1 treatment (column C). Labels indicate residues for which CsrA protein reduced RNase T1 cleavage (BS1 to BS3), the csrR Shine-Dalgarno (SD) sequence, and the translation initiation codon (Met). Numbering is with respect to the start site of csrR transcription (+1).
Mentions: To test directly whether CsrA protein binds to csrR mRNA, we performed an in vitro assay. Purified CsrA protein was incubated with 5‚Ä≤-end-labeled csrR RNA, and then binding was analyzed by gel electromobility shift assay. As predicted by the in silico analysis, CsrA protein bound tightly to csrR RNA, with a calculated dissociation constant of 32 ¬Ī 9¬†nM (Fig.¬†4A). This physical interaction was specific, since incubation with an excess of unlabeled csrR RNA effectively competed with CsrA protein binding to labeled csrR RNA (Fig.¬†4B). In contrast, incubation with even higher concentrations of a heterologous competitor RNA, E.¬†coliphoB, did not inhibit CsrA binding to csrR. Therefore, CsrA protein binds csrR RNA tightly and specifically in vitro.

Bottom Line: A potent regulator of this pathogen's intracellular life cycle is CsrA, a protein widely distributed among bacterial species that is understood quite well.Our finding that every sequenced L. pneumophila strain carries several csrA paralogs-including two common to all isolates--indicates that the legionellae exploit CsrA regulatory switches for multiple purposes.Our discovery that one paralog, CsrR, is a target of CsrA that enhances survival in water is an important step toward understanding colonization of the engineered environment by pathogenic L. pneumophila.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan, USA.

No MeSH data available.


Related in: MedlinePlus