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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

Overexpression of AsrC increased rseC mRNA and protein expression levels. mRNA levels of resC were assessed by Northern hybridization (A) and qRT-PCR (B). Total RNA from wild-type (WT+pBAD) and overexpression strain (WT+pBAD-asrC) 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 profile of RseC protein relative to DnaK in WT+pBAD and WT+pBAD-asrC strains. Representative blots are shown. (D) Quantitation of RseC protein based on Western blots. Proteins were isolated from WT+pBAD and WT+pBAD-asrC strains grown to OD600 0.4 at 0, 0.5, 1, 2, 4, and 6 h after addition of L-arabinose (0.2% final concentration). Experiments were repeated three times and error bars indicate standard deviations. ∗∗P < 0.01 compared with control group.
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Figure 5: Overexpression of AsrC increased rseC mRNA and protein expression levels. mRNA levels of resC were assessed by Northern hybridization (A) and qRT-PCR (B). Total RNA from wild-type (WT+pBAD) and overexpression strain (WT+pBAD-asrC) 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 profile of RseC protein relative to DnaK in WT+pBAD and WT+pBAD-asrC strains. Representative blots are shown. (D) Quantitation of RseC protein based on Western blots. Proteins were isolated from WT+pBAD and WT+pBAD-asrC strains grown to OD600 0.4 at 0, 0.5, 1, 2, 4, and 6 h after addition of L-arabinose (0.2% final concentration). Experiments were repeated three times and error bars indicate standard deviations. ∗∗P < 0.01 compared with control group.

Mentions: AsrC is transcribed from the DNA strand opposite the rseC gene and therefore has perfect complementarity with rseC mRNA. To investigate the effect of asrC expression on resC levels, WT+pBAD and WT+pBAD-asrC strains were grown to exponential phase (OD600 0.4) and treated with L-arabinose for 0, 20, 40, or 60 min. Changes in mRNA abundance were monitored by Northern hybridization. The mRNA level of rseC increased time-dependently when AsrC was overexpressed, but no additional increase was seen in the WT+pBAD strain (Figure 5A). These results were confirmed by qRT-PCR. The mRNA level of rseC was significantly higher when AsrC was overexpressed compared to the WT+pBAD strain (Figure 5B). The effect of AsrC overexpression on the protein level of RseC was analyzed. Western blots showed an increase in RseC protein levels in the WT+pBAD-asrC strain compared to the WT+pBAD strain during induction of asrC expression for the first 4 h with recovery to basal level at 6 h. No significant expression difference was observed in the WT+pBAD control strain (Figures 5C,D). The mRNA level of rseC was also detected in ΔPasrC lepA complementary (ΔPasrC+pBAD-lepA) strain and WT+pBAD control strain. qRT-PCR results showed that rseC expression significantly decreased in ΔPasrC+pBAD-lepA strain compared to WT+pBAD control strain (Supplementary Figure S2). These observations suggested that AsrC might be vital for in increasing rseC mRNA and protein levels.


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 rseC mRNA and protein expression levels. mRNA levels of resC were assessed by Northern hybridization (A) and qRT-PCR (B). Total RNA from wild-type (WT+pBAD) and overexpression strain (WT+pBAD-asrC) 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 profile of RseC protein relative to DnaK in WT+pBAD and WT+pBAD-asrC strains. Representative blots are shown. (D) Quantitation of RseC protein based on Western blots. Proteins were isolated from WT+pBAD and WT+pBAD-asrC strains grown to OD600 0.4 at 0, 0.5, 1, 2, 4, and 6 h after addition of L-arabinose (0.2% final concentration). Experiments were repeated three times and error bars indicate standard deviations. ∗∗P < 0.01 compared with control group.
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Figure 5: Overexpression of AsrC increased rseC mRNA and protein expression levels. mRNA levels of resC were assessed by Northern hybridization (A) and qRT-PCR (B). Total RNA from wild-type (WT+pBAD) and overexpression strain (WT+pBAD-asrC) 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 profile of RseC protein relative to DnaK in WT+pBAD and WT+pBAD-asrC strains. Representative blots are shown. (D) Quantitation of RseC protein based on Western blots. Proteins were isolated from WT+pBAD and WT+pBAD-asrC strains grown to OD600 0.4 at 0, 0.5, 1, 2, 4, and 6 h after addition of L-arabinose (0.2% final concentration). Experiments were repeated three times and error bars indicate standard deviations. ∗∗P < 0.01 compared with control group.
Mentions: AsrC is transcribed from the DNA strand opposite the rseC gene and therefore has perfect complementarity with rseC mRNA. To investigate the effect of asrC expression on resC levels, WT+pBAD and WT+pBAD-asrC strains were grown to exponential phase (OD600 0.4) and treated with L-arabinose for 0, 20, 40, or 60 min. Changes in mRNA abundance were monitored by Northern hybridization. The mRNA level of rseC increased time-dependently when AsrC was overexpressed, but no additional increase was seen in the WT+pBAD strain (Figure 5A). These results were confirmed by qRT-PCR. The mRNA level of rseC was significantly higher when AsrC was overexpressed compared to the WT+pBAD strain (Figure 5B). The effect of AsrC overexpression on the protein level of RseC was analyzed. Western blots showed an increase in RseC protein levels in the WT+pBAD-asrC strain compared to the WT+pBAD strain during induction of asrC expression for the first 4 h with recovery to basal level at 6 h. No significant expression difference was observed in the WT+pBAD control strain (Figures 5C,D). The mRNA level of rseC was also detected in ΔPasrC lepA complementary (ΔPasrC+pBAD-lepA) strain and WT+pBAD control strain. qRT-PCR results showed that rseC expression significantly decreased in ΔPasrC+pBAD-lepA strain compared to WT+pBAD control strain (Supplementary Figure S2). These observations suggested that AsrC might be vital for in increasing rseC mRNA and protein levels.

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