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The novel cis-encoded antisense RNA AsrC positively regulates the expression of rpoE-rseABC operon and thus enhances the motility of Salmonella enterica serovar typhi.

Zhang Q, Zhang Y, Zhang X, Zhan L, Zhao X, Xu S, Sheng X, Huang X - Front Microbiol (2015)

Bottom Line: We found that AsrC increased the levels of rseC mRNA and protein.The expression of rpoE was also increased in S. typhi after overexpression of AsrC, which was dependent on rseC.Thus, we propose that AsrC increased RseC level and indirectly activating RpoE which can initiate fliA expression and promote the motility of S. typhi.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Jiangsu University - School of Medicine Zhenjiang, China ; Danyang People's Hospital of Jiangsu Province Danyang, China.

ABSTRACT
Bacterial non-coding RNAs are essential in many cellular processes, including response to environmental stress, and virulence. Deep sequencing analysis of the Salmonella enterica serovar typhi (S. typhi) transcriptome revealed a novel antisense RNA transcribed in cis on the strand complementary to rseC, an activator gene of sigma factor RpoE. In this study, expression of this antisense RNA was confirmed in S. typhi by Northern hybridization. Rapid amplification of cDNA ends and sequence analysis identified an 893 bp sequence from the antisense RNA coding region that covered all of the rseC coding region in the reverse direction of transcription. This sequence of RNA was named as AsrC. After overexpression of AsrC with recombinantant plasmid in S. typhi, the bacterial motility was increased obviously. To explore the mechanism of AsrC function, regulation of rseC and rpoE expression by AsrC was investigated. We found that AsrC increased the levels of rseC mRNA and protein. The expression of rpoE was also increased in S. typhi after overexpression of AsrC, which was dependent on rseC. Thus, we propose that AsrC increased RseC level and indirectly activating RpoE which can initiate fliA expression and promote the motility of S. typhi.

No MeSH data available.


Related in: MedlinePlus

Expression of rpoE mRNA and protein in rseC mutant strain after overexpression of AsrC. (A) qRT-PCR for rpoE mRNA levels in ΔrseC+pBAD and ΔrseC+pBAD-asrC strains. Total RNA was isolated from ΔrseC+pBAD and ΔrseC+pBAD-asrC strains grown to OD600 0.4 at 0 min, 20 min, 40 min, and 60 min after addition of L-arabinose (0.2% final concentration). Levels of 5S rRNA were the internal reference. (B) Western blot of RpoE protein relative to DnaK in ΔrseC+pBAD and ΔrseC+pBAD-asrC strains. (C) Quantitation of RpoE protein based on Western blots. Proteins were isolated from ΔrseC+pBAD and ΔrseC+pBAD-asrC strains grown to OD600 0.4 at 0 h, 0.5 h, 1 h, 2 h, 4 h and 6 h after addition of L-arabinose (0.2% final concentration). Experiments were repeated three times and error bars indicate standard deviations.
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Figure 7: Expression of rpoE mRNA and protein in rseC mutant strain after overexpression of AsrC. (A) qRT-PCR for rpoE mRNA levels in ΔrseC+pBAD and ΔrseC+pBAD-asrC strains. Total RNA was isolated from ΔrseC+pBAD and ΔrseC+pBAD-asrC strains grown to OD600 0.4 at 0 min, 20 min, 40 min, and 60 min after addition of L-arabinose (0.2% final concentration). Levels of 5S rRNA were the internal reference. (B) Western blot of RpoE protein relative to DnaK in ΔrseC+pBAD and ΔrseC+pBAD-asrC strains. (C) Quantitation of RpoE protein based on Western blots. Proteins were isolated from ΔrseC+pBAD and ΔrseC+pBAD-asrC strains grown to OD600 0.4 at 0 h, 0.5 h, 1 h, 2 h, 4 h and 6 h after addition of L-arabinose (0.2% final concentration). Experiments were repeated three times and error bars indicate standard deviations.

Mentions: The results above indicated that AsrC overexpression increased rseC and rpoE mRNA and protein. RseC positively modulate σE (RpoE) activity (Missiakas et al., 1997), suggesting that AsrC might elevate rpoE mRNA and protein through increased transcription and translation of rseC. We investigated rpoE mRNA and protein in an rseC mutant containing a pBAD-asrC plasmid (ΔrseC+pBAD-asrC) or pBAD control plasmid (ΔrseC+pBAD). No significant expression difference in mRNA or protein was observed between the ΔrseC+pBAD-asrC strain and the ΔrseC+pBAD control strain (Figures 7A–C). These results indicated that increased rpoE mRNA and protein induced by AsrC overexpression was rseC dependent, primarily through increasing rseC transcription and translation.


The novel cis-encoded antisense RNA AsrC positively regulates the expression of rpoE-rseABC operon and thus enhances the motility of Salmonella enterica serovar typhi.

Zhang Q, Zhang Y, Zhang X, Zhan L, Zhao X, Xu S, Sheng X, Huang X - Front Microbiol (2015)

Expression of rpoE mRNA and protein in rseC mutant strain after overexpression of AsrC. (A) qRT-PCR for rpoE mRNA levels in ΔrseC+pBAD and ΔrseC+pBAD-asrC strains. Total RNA was isolated from ΔrseC+pBAD and ΔrseC+pBAD-asrC strains grown to OD600 0.4 at 0 min, 20 min, 40 min, and 60 min after addition of L-arabinose (0.2% final concentration). Levels of 5S rRNA were the internal reference. (B) Western blot of RpoE protein relative to DnaK in ΔrseC+pBAD and ΔrseC+pBAD-asrC strains. (C) Quantitation of RpoE protein based on Western blots. Proteins were isolated from ΔrseC+pBAD and ΔrseC+pBAD-asrC strains grown to OD600 0.4 at 0 h, 0.5 h, 1 h, 2 h, 4 h and 6 h after addition of L-arabinose (0.2% final concentration). Experiments were repeated three times and error bars indicate standard deviations.
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Related In: Results  -  Collection

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Show All Figures
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Figure 7: Expression of rpoE mRNA and protein in rseC mutant strain after overexpression of AsrC. (A) qRT-PCR for rpoE mRNA levels in ΔrseC+pBAD and ΔrseC+pBAD-asrC strains. Total RNA was isolated from ΔrseC+pBAD and ΔrseC+pBAD-asrC strains grown to OD600 0.4 at 0 min, 20 min, 40 min, and 60 min after addition of L-arabinose (0.2% final concentration). Levels of 5S rRNA were the internal reference. (B) Western blot of RpoE protein relative to DnaK in ΔrseC+pBAD and ΔrseC+pBAD-asrC strains. (C) Quantitation of RpoE protein based on Western blots. Proteins were isolated from ΔrseC+pBAD and ΔrseC+pBAD-asrC strains grown to OD600 0.4 at 0 h, 0.5 h, 1 h, 2 h, 4 h and 6 h after addition of L-arabinose (0.2% final concentration). Experiments were repeated three times and error bars indicate standard deviations.
Mentions: The results above indicated that AsrC overexpression increased rseC and rpoE mRNA and protein. RseC positively modulate σE (RpoE) activity (Missiakas et al., 1997), suggesting that AsrC might elevate rpoE mRNA and protein through increased transcription and translation of rseC. We investigated rpoE mRNA and protein in an rseC mutant containing a pBAD-asrC plasmid (ΔrseC+pBAD-asrC) or pBAD control plasmid (ΔrseC+pBAD). No significant expression difference in mRNA or protein was observed between the ΔrseC+pBAD-asrC strain and the ΔrseC+pBAD control strain (Figures 7A–C). These results indicated that increased rpoE mRNA and protein induced by AsrC overexpression was rseC dependent, primarily through increasing rseC transcription and translation.

Bottom Line: We found that AsrC increased the levels of rseC mRNA and protein.The expression of rpoE was also increased in S. typhi after overexpression of AsrC, which was dependent on rseC.Thus, we propose that AsrC increased RseC level and indirectly activating RpoE which can initiate fliA expression and promote the motility of S. typhi.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Jiangsu University - School of Medicine Zhenjiang, China ; Danyang People's Hospital of Jiangsu Province Danyang, China.

ABSTRACT
Bacterial non-coding RNAs are essential in many cellular processes, including response to environmental stress, and virulence. Deep sequencing analysis of the Salmonella enterica serovar typhi (S. typhi) transcriptome revealed a novel antisense RNA transcribed in cis on the strand complementary to rseC, an activator gene of sigma factor RpoE. In this study, expression of this antisense RNA was confirmed in S. typhi by Northern hybridization. Rapid amplification of cDNA ends and sequence analysis identified an 893 bp sequence from the antisense RNA coding region that covered all of the rseC coding region in the reverse direction of transcription. This sequence of RNA was named as AsrC. After overexpression of AsrC with recombinantant plasmid in S. typhi, the bacterial motility was increased obviously. To explore the mechanism of AsrC function, regulation of rseC and rpoE expression by AsrC was investigated. We found that AsrC increased the levels of rseC mRNA and protein. The expression of rpoE was also increased in S. typhi after overexpression of AsrC, which was dependent on rseC. Thus, we propose that AsrC increased RseC level and indirectly activating RpoE which can initiate fliA expression and promote the motility of S. typhi.

No MeSH data available.


Related in: MedlinePlus