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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.


Overexpression of AsrC increased rpoE mRNA and protein.RpoE mRNA assessed by Northern hybridization (A) and qRT-PCR (B). Total RNAs were isolated from WT+pBAD and WT+pBAD-asrC strains grown to OD600 0.4 at 0, 20, 40, and 60 min after addition of L-arabinose (0.2% final concentration). Levels of 5S rRNA were the internal reference. (C) Western blot of RpoE protein relative to DnaK in WT+pBAD and WT+pBAD-asrC strains. Representative blots are shown. (D) Quantitation of RpoE protein levels based on Western blots. Experiments were repeated three times and error bars indicate standard deviations. ∗∗P < 0.01 compared with control group.
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Figure 6: Overexpression of AsrC increased rpoE mRNA and protein.RpoE mRNA assessed by Northern hybridization (A) and qRT-PCR (B). Total RNAs were isolated from WT+pBAD and WT+pBAD-asrC strains grown to OD600 0.4 at 0, 20, 40, and 60 min after addition of L-arabinose (0.2% final concentration). Levels of 5S rRNA were the internal reference. (C) Western blot of RpoE protein relative to DnaK in WT+pBAD and WT+pBAD-asrC strains. Representative blots are shown. (D) Quantitation of RpoE protein levels based on Western blots. Experiments were repeated three times and error bars indicate standard deviations. ∗∗P < 0.01 compared with control group.

Mentions: The last gene of the rpoE operon is rseC. RseC positively modulates the transcriptional activity of σE (Missiakas et al., 1997). Thus, we investigated if AsrC overexpression influenced the expression of rpoE. Northern hybridization and qRT-PCR both showed that overexpression of AsrC increased rpoE mRNA, but the WT+pBAD control strain displayed no significant differences in rpoE mRNA level after L-arabinose induction for 60 min (Figures 6A,B). The level of rpoE mRNA increased about threefold after 60 min of L-arabinose induction in the WT+pBAD-asrC strain (Figure 6B). RpoE protein increased time-dependently after AsrC overexpression was induced (Figure 6C). Western blot quantification showed that RpoE protein was significantly elevated after L-arabinose induction for 2 h and reached a maximum (5.3-fold) after induction for 4 h (Figure 6D). Expression of rpoE was also detected in ΔPasrC+pBAD-lepA strain and WT+pBAD control strain. qRT-PCR results showed that mRNA level of rpoE significantly decreased in ΔPasrC+pBAD-lepA strain compared to WT+pBAD control strain (Supplementary Figure S2). These results indicated that expression of RpoE was positively affected by AsrC.


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)

Overexpression of AsrC increased rpoE mRNA and protein.RpoE mRNA assessed by Northern hybridization (A) and qRT-PCR (B). Total RNAs were isolated from WT+pBAD and WT+pBAD-asrC strains grown to OD600 0.4 at 0, 20, 40, and 60 min after addition of L-arabinose (0.2% final concentration). Levels of 5S rRNA were the internal reference. (C) Western blot of RpoE protein relative to DnaK in WT+pBAD and WT+pBAD-asrC strains. Representative blots are shown. (D) Quantitation of RpoE protein levels based on Western blots. Experiments were repeated three times and error bars indicate standard deviations. ∗∗P < 0.01 compared with control group.
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Figure 6: Overexpression of AsrC increased rpoE mRNA and protein.RpoE mRNA assessed by Northern hybridization (A) and qRT-PCR (B). Total RNAs were isolated from WT+pBAD and WT+pBAD-asrC strains grown to OD600 0.4 at 0, 20, 40, and 60 min after addition of L-arabinose (0.2% final concentration). Levels of 5S rRNA were the internal reference. (C) Western blot of RpoE protein relative to DnaK in WT+pBAD and WT+pBAD-asrC strains. Representative blots are shown. (D) Quantitation of RpoE protein levels based on Western blots. Experiments were repeated three times and error bars indicate standard deviations. ∗∗P < 0.01 compared with control group.
Mentions: The last gene of the rpoE operon is rseC. RseC positively modulates the transcriptional activity of σE (Missiakas et al., 1997). Thus, we investigated if AsrC overexpression influenced the expression of rpoE. Northern hybridization and qRT-PCR both showed that overexpression of AsrC increased rpoE mRNA, but the WT+pBAD control strain displayed no significant differences in rpoE mRNA level after L-arabinose induction for 60 min (Figures 6A,B). The level of rpoE mRNA increased about threefold after 60 min of L-arabinose induction in the WT+pBAD-asrC strain (Figure 6B). RpoE protein increased time-dependently after AsrC overexpression was induced (Figure 6C). Western blot quantification showed that RpoE protein was significantly elevated after L-arabinose induction for 2 h and reached a maximum (5.3-fold) after induction for 4 h (Figure 6D). Expression of rpoE was also detected in ΔPasrC+pBAD-lepA strain and WT+pBAD control strain. qRT-PCR results showed that mRNA level of rpoE significantly decreased in ΔPasrC+pBAD-lepA strain compared to WT+pBAD control strain (Supplementary Figure S2). These results indicated that expression of RpoE was positively affected by AsrC.

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.