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Tn5 transposition in Escherichia coli is repressed by Hfq and activated by over-expression of the small non-coding RNA SgrS.

Ross JA, Trussler RS, Black MD, McLellan CR, Haniford DB - Mob DNA (2014)

Bottom Line: As Hfq does not typically function directly in transcription, we searched for a transcription factor that also down-regulated IS50 transposase transcription and is itself under Hfq control.We show that Crp (cyclic AMP receptor protein) fits these criteria as: (1) disruption of the crp gene led to an increase in IS50 transposase expression and the magnitude of this increase was comparable to that observed for an hfq disruption; and (2) Crp expression decreased in hfq (-) .Preliminary results support the possibility that this regulation is mediated through Crp.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Western Ontario, London, ONN6A 5C1 Canada.

ABSTRACT

Background: Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base pairing of mRNAs with trans-encoded sRNAs. It was previously shown that Hfq down-regulates Tn10 transposition by inhibiting IS10 transposase expression at the post-transcriptional level. This provided the first example of Hfq playing a role in DNA transposition and led us to ask if a related transposon, Tn5, is similarly regulated.

Results: We show that Hfq strongly suppresses Tn5 transposition in Escherichia coli by inhibiting IS50 transposase expression. However, in contrast to the situation for Tn10, Hfq primarily inhibits IS50 transposase transcription. As Hfq does not typically function directly in transcription, we searched for a transcription factor that also down-regulated IS50 transposase transcription and is itself under Hfq control. We show that Crp (cyclic AMP receptor protein) fits these criteria as: (1) disruption of the crp gene led to an increase in IS50 transposase expression and the magnitude of this increase was comparable to that observed for an hfq disruption; and (2) Crp expression decreased in hfq (-) . We also demonstrate that IS50 transposase expression and Tn5 transposition are induced by over-expression of the sRNA SgrS and link this response to glucose limitation.

Conclusions: Tn5 transposition is negatively regulated by Hfq primarily through inhibition of IS50 transposase transcription. Preliminary results support the possibility that this regulation is mediated through Crp. We also provide evidence that glucose limitation activates IS50 transposase transcription and transposition.

No MeSH data available.


Related in: MedlinePlus

Frequencies ofTn5transposition inhfq−versuswtstrains ofE. coli. (A)Tn5 transposition from the chromosome of DBH179 and derivatives (hfq− and dam−) was measured by the conjugal ‘mating out’ assay as described in Methods. For purposes of trans-complementation, strains contained an empty vector or a low-copy plasmid encoding either wild type hfqWT or mutant forms of hfq (K56A or Y25A) expressed from the hfq P3 promoter. The data was compiled from four independent experiments, each with at least three isolates of each strain. The average transposition frequency was 8.33 × 10−5 events per mL of mating mix for the wt strain (no ‘hfq plasmid’) and for purposes of comparison this value was set at 1 and all other values normalized to this. The illustration shows the structure of an Hfq hexamer with RNA (gold) bound either to the proximal or distal face [9]. The Y25A mutation inhibits RNA binding to the distal face and the K56A mutation inhibits RNA binding to the proximal face. Adapted from Nature Reviews: Microbiology [9] with permission from Macmillan Publishers. (B)Tn5 transposition from the chromosome of DBH261 and derivatives (hfq− and dam−) was measured as in (A). The data shown is from one experiment with five independent isolates of each strain. The average transposition frequency for the wt strain was 2.57 × 10−6 events per mL of mating mix. In (A) and (B) the error bars indicate standard error of the mean.
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Fig2: Frequencies ofTn5transposition inhfq−versuswtstrains ofE. coli. (A)Tn5 transposition from the chromosome of DBH179 and derivatives (hfq− and dam−) was measured by the conjugal ‘mating out’ assay as described in Methods. For purposes of trans-complementation, strains contained an empty vector or a low-copy plasmid encoding either wild type hfqWT or mutant forms of hfq (K56A or Y25A) expressed from the hfq P3 promoter. The data was compiled from four independent experiments, each with at least three isolates of each strain. The average transposition frequency was 8.33 × 10−5 events per mL of mating mix for the wt strain (no ‘hfq plasmid’) and for purposes of comparison this value was set at 1 and all other values normalized to this. The illustration shows the structure of an Hfq hexamer with RNA (gold) bound either to the proximal or distal face [9]. The Y25A mutation inhibits RNA binding to the distal face and the K56A mutation inhibits RNA binding to the proximal face. Adapted from Nature Reviews: Microbiology [9] with permission from Macmillan Publishers. (B)Tn5 transposition from the chromosome of DBH261 and derivatives (hfq− and dam−) was measured as in (A). The data shown is from one experiment with five independent isolates of each strain. The average transposition frequency for the wt strain was 2.57 × 10−6 events per mL of mating mix. In (A) and (B) the error bars indicate standard error of the mean.

Mentions: We asked if Hfq regulates Tn5 transposition in E. coli by measuring the frequency of Tn5 transposition under conditions of hfq deficiency using the ‘mating out’ assay. In this assay, an F+ donor strain harboring a chromosomal copy of Tn5 was mated to an F− recipient strain and the mating efficiency and number of transposition events were measured by plating mating mixes on the appropriate selective media (see Methods). We show in Figure 2A that in one donor strain background (DBH179) Tn5 transposition increased by close to 75-fold under conditions of hfq deficiency. Note that we did not have a defective copy of Tn5 to act as a negative control in this experiment. In lieu of this, we carried out physical mapping on a sampling of colonies present on ‘hop’ plates to ensure that bona fide transposition events were being measured in both wt and hfq− strains (Additional file 1).Figure 2


Tn5 transposition in Escherichia coli is repressed by Hfq and activated by over-expression of the small non-coding RNA SgrS.

Ross JA, Trussler RS, Black MD, McLellan CR, Haniford DB - Mob DNA (2014)

Frequencies ofTn5transposition inhfq−versuswtstrains ofE. coli. (A)Tn5 transposition from the chromosome of DBH179 and derivatives (hfq− and dam−) was measured by the conjugal ‘mating out’ assay as described in Methods. For purposes of trans-complementation, strains contained an empty vector or a low-copy plasmid encoding either wild type hfqWT or mutant forms of hfq (K56A or Y25A) expressed from the hfq P3 promoter. The data was compiled from four independent experiments, each with at least three isolates of each strain. The average transposition frequency was 8.33 × 10−5 events per mL of mating mix for the wt strain (no ‘hfq plasmid’) and for purposes of comparison this value was set at 1 and all other values normalized to this. The illustration shows the structure of an Hfq hexamer with RNA (gold) bound either to the proximal or distal face [9]. The Y25A mutation inhibits RNA binding to the distal face and the K56A mutation inhibits RNA binding to the proximal face. Adapted from Nature Reviews: Microbiology [9] with permission from Macmillan Publishers. (B)Tn5 transposition from the chromosome of DBH261 and derivatives (hfq− and dam−) was measured as in (A). The data shown is from one experiment with five independent isolates of each strain. The average transposition frequency for the wt strain was 2.57 × 10−6 events per mL of mating mix. In (A) and (B) the error bars indicate standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
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getmorefigures.php?uid=PMC4265352&req=5

Fig2: Frequencies ofTn5transposition inhfq−versuswtstrains ofE. coli. (A)Tn5 transposition from the chromosome of DBH179 and derivatives (hfq− and dam−) was measured by the conjugal ‘mating out’ assay as described in Methods. For purposes of trans-complementation, strains contained an empty vector or a low-copy plasmid encoding either wild type hfqWT or mutant forms of hfq (K56A or Y25A) expressed from the hfq P3 promoter. The data was compiled from four independent experiments, each with at least three isolates of each strain. The average transposition frequency was 8.33 × 10−5 events per mL of mating mix for the wt strain (no ‘hfq plasmid’) and for purposes of comparison this value was set at 1 and all other values normalized to this. The illustration shows the structure of an Hfq hexamer with RNA (gold) bound either to the proximal or distal face [9]. The Y25A mutation inhibits RNA binding to the distal face and the K56A mutation inhibits RNA binding to the proximal face. Adapted from Nature Reviews: Microbiology [9] with permission from Macmillan Publishers. (B)Tn5 transposition from the chromosome of DBH261 and derivatives (hfq− and dam−) was measured as in (A). The data shown is from one experiment with five independent isolates of each strain. The average transposition frequency for the wt strain was 2.57 × 10−6 events per mL of mating mix. In (A) and (B) the error bars indicate standard error of the mean.
Mentions: We asked if Hfq regulates Tn5 transposition in E. coli by measuring the frequency of Tn5 transposition under conditions of hfq deficiency using the ‘mating out’ assay. In this assay, an F+ donor strain harboring a chromosomal copy of Tn5 was mated to an F− recipient strain and the mating efficiency and number of transposition events were measured by plating mating mixes on the appropriate selective media (see Methods). We show in Figure 2A that in one donor strain background (DBH179) Tn5 transposition increased by close to 75-fold under conditions of hfq deficiency. Note that we did not have a defective copy of Tn5 to act as a negative control in this experiment. In lieu of this, we carried out physical mapping on a sampling of colonies present on ‘hop’ plates to ensure that bona fide transposition events were being measured in both wt and hfq− strains (Additional file 1).Figure 2

Bottom Line: As Hfq does not typically function directly in transcription, we searched for a transcription factor that also down-regulated IS50 transposase transcription and is itself under Hfq control.We show that Crp (cyclic AMP receptor protein) fits these criteria as: (1) disruption of the crp gene led to an increase in IS50 transposase expression and the magnitude of this increase was comparable to that observed for an hfq disruption; and (2) Crp expression decreased in hfq (-) .Preliminary results support the possibility that this regulation is mediated through Crp.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Western Ontario, London, ONN6A 5C1 Canada.

ABSTRACT

Background: Hfq functions in post-transcriptional gene regulation in a wide range of bacteria, usually by promoting base pairing of mRNAs with trans-encoded sRNAs. It was previously shown that Hfq down-regulates Tn10 transposition by inhibiting IS10 transposase expression at the post-transcriptional level. This provided the first example of Hfq playing a role in DNA transposition and led us to ask if a related transposon, Tn5, is similarly regulated.

Results: We show that Hfq strongly suppresses Tn5 transposition in Escherichia coli by inhibiting IS50 transposase expression. However, in contrast to the situation for Tn10, Hfq primarily inhibits IS50 transposase transcription. As Hfq does not typically function directly in transcription, we searched for a transcription factor that also down-regulated IS50 transposase transcription and is itself under Hfq control. We show that Crp (cyclic AMP receptor protein) fits these criteria as: (1) disruption of the crp gene led to an increase in IS50 transposase expression and the magnitude of this increase was comparable to that observed for an hfq disruption; and (2) Crp expression decreased in hfq (-) . We also demonstrate that IS50 transposase expression and Tn5 transposition are induced by over-expression of the sRNA SgrS and link this response to glucose limitation.

Conclusions: Tn5 transposition is negatively regulated by Hfq primarily through inhibition of IS50 transposase transcription. Preliminary results support the possibility that this regulation is mediated through Crp. We also provide evidence that glucose limitation activates IS50 transposase transcription and transposition.

No MeSH data available.


Related in: MedlinePlus