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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

Transposase-lacZexpression assays in cells over-expressing sRNAs. (A) Transposase expression from an IS50 translational fusion (TLF) (see Figure 3A) present on a low-copy plasmid (pDH798) was measured in the presence of a compatible plasmid expressing one of the indicated sRNAs from the inducible pLlacO promoter in DBH33. Cells were grown in M9 glucose and 0.1 mM IPTG was added to subcultures to induce sRNA expression. Transposase expression was measured 4 hours after IPTG addition. Expression levels were normalized to the strain with the vector only control. (B) The impact of different growth media on SgrS-induced up-regulation of transposase expression was evaluated using a single-copy TCF fusion (see Figure 3A) present in the chromosome of DBH265. Note that the sgrS1 allele of SgrS contains a two-nucleotide mutation that inhibits its ability to down-regulate expression of the glucose transporter encoded by ptsG. Subcultures were grown in either M9 glucose, M9 glucose + glycerol, or Luria broth (LB), as indicated. β-galactosidase activity was measured approximately 4 to 6 hours after subcultures were started. In (A) and (B) mean and standard error values of duplicate experiments, each of which included at least three replicates, are shown. (C) Northern blot of RNA isolated from cells in (B). RNA was extracted from cells immediately before starting the Miller assay and visualized by Northern blotting with 32P-labeled RNA probes complementary to either SgrS or the 5S rRNA (internal control).
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Fig8: Transposase-lacZexpression assays in cells over-expressing sRNAs. (A) Transposase expression from an IS50 translational fusion (TLF) (see Figure 3A) present on a low-copy plasmid (pDH798) was measured in the presence of a compatible plasmid expressing one of the indicated sRNAs from the inducible pLlacO promoter in DBH33. Cells were grown in M9 glucose and 0.1 mM IPTG was added to subcultures to induce sRNA expression. Transposase expression was measured 4 hours after IPTG addition. Expression levels were normalized to the strain with the vector only control. (B) The impact of different growth media on SgrS-induced up-regulation of transposase expression was evaluated using a single-copy TCF fusion (see Figure 3A) present in the chromosome of DBH265. Note that the sgrS1 allele of SgrS contains a two-nucleotide mutation that inhibits its ability to down-regulate expression of the glucose transporter encoded by ptsG. Subcultures were grown in either M9 glucose, M9 glucose + glycerol, or Luria broth (LB), as indicated. β-galactosidase activity was measured approximately 4 to 6 hours after subcultures were started. In (A) and (B) mean and standard error values of duplicate experiments, each of which included at least three replicates, are shown. (C) Northern blot of RNA isolated from cells in (B). RNA was extracted from cells immediately before starting the Miller assay and visualized by Northern blotting with 32P-labeled RNA probes complementary to either SgrS or the 5S rRNA (internal control).

Mentions: Over-expression of sRNAs can alter Hfq-regulated networks by limiting the availability of Hfq [21,22]. Given our findings that Tn5 transposition and transposase gene expression are affected by hfq status, we asked if IS50 transposase expression might be sensitive to Hfq-titration. Towards this end, we measured transposase expression from the TLF under conditions where a single sRNA was over-expressed from an IPTG inducible promoter (pLlacO) in DBH33, which is lacIq. Our initial screen included four different Hfq-dependent sRNAs, including RybB, RyeB, MicC and SgrS, all of which are expected to tightly bind Hfq in vivo; apparent equilibrium dissociation constants of approximately 3.3 nM and < 20 nM have been measured for MicC and SgrS, respectively [23-25]. Cells were grown in M9 glucose and sRNA expression was induced for 4 hours in exponential phase. We show in Figure 8A that only one of the sRNAs tested, SgrS, had a significant impact on transposase expression. Induction of SgrS increased transposase expression just over three-fold. Given the comparable Hfq binding affinities of the sRNAs tested, it seemed unlikely that SgrS expression was increasing transposase expression through an Hfq-titration mechanism.Figure 8


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)

Transposase-lacZexpression assays in cells over-expressing sRNAs. (A) Transposase expression from an IS50 translational fusion (TLF) (see Figure 3A) present on a low-copy plasmid (pDH798) was measured in the presence of a compatible plasmid expressing one of the indicated sRNAs from the inducible pLlacO promoter in DBH33. Cells were grown in M9 glucose and 0.1 mM IPTG was added to subcultures to induce sRNA expression. Transposase expression was measured 4 hours after IPTG addition. Expression levels were normalized to the strain with the vector only control. (B) The impact of different growth media on SgrS-induced up-regulation of transposase expression was evaluated using a single-copy TCF fusion (see Figure 3A) present in the chromosome of DBH265. Note that the sgrS1 allele of SgrS contains a two-nucleotide mutation that inhibits its ability to down-regulate expression of the glucose transporter encoded by ptsG. Subcultures were grown in either M9 glucose, M9 glucose + glycerol, or Luria broth (LB), as indicated. β-galactosidase activity was measured approximately 4 to 6 hours after subcultures were started. In (A) and (B) mean and standard error values of duplicate experiments, each of which included at least three replicates, are shown. (C) Northern blot of RNA isolated from cells in (B). RNA was extracted from cells immediately before starting the Miller assay and visualized by Northern blotting with 32P-labeled RNA probes complementary to either SgrS or the 5S rRNA (internal control).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Fig8: Transposase-lacZexpression assays in cells over-expressing sRNAs. (A) Transposase expression from an IS50 translational fusion (TLF) (see Figure 3A) present on a low-copy plasmid (pDH798) was measured in the presence of a compatible plasmid expressing one of the indicated sRNAs from the inducible pLlacO promoter in DBH33. Cells were grown in M9 glucose and 0.1 mM IPTG was added to subcultures to induce sRNA expression. Transposase expression was measured 4 hours after IPTG addition. Expression levels were normalized to the strain with the vector only control. (B) The impact of different growth media on SgrS-induced up-regulation of transposase expression was evaluated using a single-copy TCF fusion (see Figure 3A) present in the chromosome of DBH265. Note that the sgrS1 allele of SgrS contains a two-nucleotide mutation that inhibits its ability to down-regulate expression of the glucose transporter encoded by ptsG. Subcultures were grown in either M9 glucose, M9 glucose + glycerol, or Luria broth (LB), as indicated. β-galactosidase activity was measured approximately 4 to 6 hours after subcultures were started. In (A) and (B) mean and standard error values of duplicate experiments, each of which included at least three replicates, are shown. (C) Northern blot of RNA isolated from cells in (B). RNA was extracted from cells immediately before starting the Miller assay and visualized by Northern blotting with 32P-labeled RNA probes complementary to either SgrS or the 5S rRNA (internal control).
Mentions: Over-expression of sRNAs can alter Hfq-regulated networks by limiting the availability of Hfq [21,22]. Given our findings that Tn5 transposition and transposase gene expression are affected by hfq status, we asked if IS50 transposase expression might be sensitive to Hfq-titration. Towards this end, we measured transposase expression from the TLF under conditions where a single sRNA was over-expressed from an IPTG inducible promoter (pLlacO) in DBH33, which is lacIq. Our initial screen included four different Hfq-dependent sRNAs, including RybB, RyeB, MicC and SgrS, all of which are expected to tightly bind Hfq in vivo; apparent equilibrium dissociation constants of approximately 3.3 nM and < 20 nM have been measured for MicC and SgrS, respectively [23-25]. Cells were grown in M9 glucose and sRNA expression was induced for 4 hours in exponential phase. We show in Figure 8A that only one of the sRNAs tested, SgrS, had a significant impact on transposase expression. Induction of SgrS increased transposase expression just over three-fold. Given the comparable Hfq binding affinities of the sRNAs tested, it seemed unlikely that SgrS expression was increasing transposase expression through an Hfq-titration mechanism.Figure 8

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