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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

Motility assay of the wild type, ΔPasrC, WT+pBAD and WT+pBAD-asrC strains. (A) AsrC-promoter mutant strain with decreased motility. Overexpression of AsrC increased motility on swim-agar plates. (B) Motility ring diameters of S. typhi strains. Bacterial were spotted onto LB plates with 0.3% agar and incubated at 37°C for 8 h. The image is representative of three independent experiments. (C) mRNA of flagellar genes (flhD, fliA, and fliD) determined by qRT-PCR. RNA was extracted from three independent cultures for each strain grown to OD600 0.4. Levels of 5S rRNA were the internal reference. ∗∗P < 0.01 compared with control group.
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Figure 4: Motility assay of the wild type, ΔPasrC, WT+pBAD and WT+pBAD-asrC strains. (A) AsrC-promoter mutant strain with decreased motility. Overexpression of AsrC increased motility on swim-agar plates. (B) Motility ring diameters of S. typhi strains. Bacterial were spotted onto LB plates with 0.3% agar and incubated at 37°C for 8 h. The image is representative of three independent experiments. (C) mRNA of flagellar genes (flhD, fliA, and fliD) determined by qRT-PCR. RNA was extracted from three independent cultures for each strain grown to OD600 0.4. Levels of 5S rRNA were the internal reference. ∗∗P < 0.01 compared with control group.

Mentions: To investigate the function of the AsrC, the asrC-promoter mutant stain (ΔPasrC) of S. typhi was prepared. qRT-PCR results showed that asrC had almost no expression in ΔPasrC strain (Supplementary Figure S1). Overexpression of AsrC was performed in S. typhi which was transformed by a recombinant plasmid pBAD-asrC and induced with L-arabinose. The motility of S. typhi influence by the AsrC was assessed by using motility-swim agar plates. The motility of ΔPasrC was significantly decreased compared to the wild-type strain (WT). After overexpression of AsrC, S. typhi (WT+pBAD-asrC) had significantly increased motility compared to the control strain (WT+pBAD; Figures 4A,B). Transcript levels of flagellar genes flhD, fliA, and fliD were compared among WT, ΔPasrC, WT+pBAD, and WT+pBAD-asrC strains using qRT-PCR. Expression of flhD, fliA, and fliD in the mutant stain ΔPasrC was decreased compared to the WT strain and increased in the WT+pBAD-asrC strain compared to the empty-plasmid control (Figure 4C). LepA complementary strain and control strain were constructed by transferring the recombinant plasmid pBAD-lepA and the empty vector pBAD/gIII into the ΔPasrC mutant strain. Expression of flagella-related genes of the two strains was observed to be identical (data not shown). These results suggested that AsrC can up-regulate S. typhi motility.


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)

Motility assay of the wild type, ΔPasrC, WT+pBAD and WT+pBAD-asrC strains. (A) AsrC-promoter mutant strain with decreased motility. Overexpression of AsrC increased motility on swim-agar plates. (B) Motility ring diameters of S. typhi strains. Bacterial were spotted onto LB plates with 0.3% agar and incubated at 37°C for 8 h. The image is representative of three independent experiments. (C) mRNA of flagellar genes (flhD, fliA, and fliD) determined by qRT-PCR. RNA was extracted from three independent cultures for each strain grown to OD600 0.4. Levels of 5S rRNA were the internal reference. ∗∗P < 0.01 compared with control group.
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Related In: Results  -  Collection

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Figure 4: Motility assay of the wild type, ΔPasrC, WT+pBAD and WT+pBAD-asrC strains. (A) AsrC-promoter mutant strain with decreased motility. Overexpression of AsrC increased motility on swim-agar plates. (B) Motility ring diameters of S. typhi strains. Bacterial were spotted onto LB plates with 0.3% agar and incubated at 37°C for 8 h. The image is representative of three independent experiments. (C) mRNA of flagellar genes (flhD, fliA, and fliD) determined by qRT-PCR. RNA was extracted from three independent cultures for each strain grown to OD600 0.4. Levels of 5S rRNA were the internal reference. ∗∗P < 0.01 compared with control group.
Mentions: To investigate the function of the AsrC, the asrC-promoter mutant stain (ΔPasrC) of S. typhi was prepared. qRT-PCR results showed that asrC had almost no expression in ΔPasrC strain (Supplementary Figure S1). Overexpression of AsrC was performed in S. typhi which was transformed by a recombinant plasmid pBAD-asrC and induced with L-arabinose. The motility of S. typhi influence by the AsrC was assessed by using motility-swim agar plates. The motility of ΔPasrC was significantly decreased compared to the wild-type strain (WT). After overexpression of AsrC, S. typhi (WT+pBAD-asrC) had significantly increased motility compared to the control strain (WT+pBAD; Figures 4A,B). Transcript levels of flagellar genes flhD, fliA, and fliD were compared among WT, ΔPasrC, WT+pBAD, and WT+pBAD-asrC strains using qRT-PCR. Expression of flhD, fliA, and fliD in the mutant stain ΔPasrC was decreased compared to the WT strain and increased in the WT+pBAD-asrC strain compared to the empty-plasmid control (Figure 4C). LepA complementary strain and control strain were constructed by transferring the recombinant plasmid pBAD-lepA and the empty vector pBAD/gIII into the ΔPasrC mutant strain. Expression of flagella-related genes of the two strains was observed to be identical (data not shown). These results suggested that AsrC can up-regulate S. typhi motility.

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