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Transcriptional and posttranscriptional regulation of Bacillus sp. CDB3 arsenic-resistance operon ars1.

Yu X, Zheng W, Bhat S, Aquilina JA, Zhang R - PeerJ (2015)

Bottom Line: Compared to other ars gene clusters, regulation of the Bacillus sp.CDB3 ars1 operon is more complex.It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, University of Wollongong , Wollongong, NSW , Australia.

ABSTRACT
Bacillus sp. CDB3 possesses a novel eight-gene ars cluster (ars1, arsRYCDATorf7orf8) with some unusual features in regard to expression regulation. This study demonstrated that the cluster is a single operon but can also produce a short three-gene arsRYC transcript. A hairpin structure formed by internal inverted repeats between arsC and arsD was shown to diminish the expression of the full operon, thereby probably acting as a transcription attenuator. A degradation product of the arsRYC transcript was also identified. Electrophoretic mobility shift analysis demonstrated that ArsR interacts with the ars1 promoter forming a protein-DNA complex that could be impaired by arsenite. However, no interaction was detected between ArsD and the ars1 promoter, suggesting that the CDB3 ArsD protein may not play a regulatory role. Compared to other ars gene clusters, regulation of the Bacillus sp. CDB3 ars1 operon is more complex. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons.

No MeSH data available.


mRNA secondary structure and long-transcript levels.(A) Predicted mRNA secondary structure of the C–D intergenic region in CDB3 ars1 and altered nucleotides to abolish the hairpin structure. The four regions of the two stem-loops are indicated by symbols I, II, III and IV. (B) Expression levels of the long transcript in untreated (control) and arsenite-treated E. coli AW3110 strains harbouring pAR27 or pAR27HPΔ. The arsenite treatment was at 0.5 mM for 5 min and qPCR was carried out using RT-DF/RT-DR primer pairs. Each data point corresponds to average copies of ArsD cDNA (copies/µL) and the error bars indicate standard deviation of three independent measurements.
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fig-2: mRNA secondary structure and long-transcript levels.(A) Predicted mRNA secondary structure of the C–D intergenic region in CDB3 ars1 and altered nucleotides to abolish the hairpin structure. The four regions of the two stem-loops are indicated by symbols I, II, III and IV. (B) Expression levels of the long transcript in untreated (control) and arsenite-treated E. coli AW3110 strains harbouring pAR27 or pAR27HPΔ. The arsenite treatment was at 0.5 mM for 5 min and qPCR was carried out using RT-DF/RT-DR primer pairs. Each data point corresponds to average copies of ArsD cDNA (copies/µL) and the error bars indicate standard deviation of three independent measurements.

Mentions: To examine the role of inverted repeats between arsC and arsD, which can form a hairpin structure, the intergenic DNA sequence was altered by mutagenesis to eliminate the repeats (Fig. 2A). A transcription study was carried out in E. coli AW3110, which was shown to produce the two RNA transcripts when transformed by CDB3 ars1 (data not shown). RNA samples isolated from AW3110 strains harbouring pAR27 (ars1) or pAR27HPΔ (hairpin knockout mutant) with or without arsenite treatment were reverse transcribed and analysed using qPCR assay. The results showed that in the mutant strain, expression levels of the long eight-gene transcript increased more than one fold over that in the control strain (Fig. 2B), suggesting that the hairpin structure does function as a transcriptional terminator which can be read through under some conditions.


Transcriptional and posttranscriptional regulation of Bacillus sp. CDB3 arsenic-resistance operon ars1.

Yu X, Zheng W, Bhat S, Aquilina JA, Zhang R - PeerJ (2015)

mRNA secondary structure and long-transcript levels.(A) Predicted mRNA secondary structure of the C–D intergenic region in CDB3 ars1 and altered nucleotides to abolish the hairpin structure. The four regions of the two stem-loops are indicated by symbols I, II, III and IV. (B) Expression levels of the long transcript in untreated (control) and arsenite-treated E. coli AW3110 strains harbouring pAR27 or pAR27HPΔ. The arsenite treatment was at 0.5 mM for 5 min and qPCR was carried out using RT-DF/RT-DR primer pairs. Each data point corresponds to average copies of ArsD cDNA (copies/µL) and the error bars indicate standard deviation of three independent measurements.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4562236&req=5

fig-2: mRNA secondary structure and long-transcript levels.(A) Predicted mRNA secondary structure of the C–D intergenic region in CDB3 ars1 and altered nucleotides to abolish the hairpin structure. The four regions of the two stem-loops are indicated by symbols I, II, III and IV. (B) Expression levels of the long transcript in untreated (control) and arsenite-treated E. coli AW3110 strains harbouring pAR27 or pAR27HPΔ. The arsenite treatment was at 0.5 mM for 5 min and qPCR was carried out using RT-DF/RT-DR primer pairs. Each data point corresponds to average copies of ArsD cDNA (copies/µL) and the error bars indicate standard deviation of three independent measurements.
Mentions: To examine the role of inverted repeats between arsC and arsD, which can form a hairpin structure, the intergenic DNA sequence was altered by mutagenesis to eliminate the repeats (Fig. 2A). A transcription study was carried out in E. coli AW3110, which was shown to produce the two RNA transcripts when transformed by CDB3 ars1 (data not shown). RNA samples isolated from AW3110 strains harbouring pAR27 (ars1) or pAR27HPΔ (hairpin knockout mutant) with or without arsenite treatment were reverse transcribed and analysed using qPCR assay. The results showed that in the mutant strain, expression levels of the long eight-gene transcript increased more than one fold over that in the control strain (Fig. 2B), suggesting that the hairpin structure does function as a transcriptional terminator which can be read through under some conditions.

Bottom Line: Compared to other ars gene clusters, regulation of the Bacillus sp.CDB3 ars1 operon is more complex.It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Biological Sciences, University of Wollongong , Wollongong, NSW , Australia.

ABSTRACT
Bacillus sp. CDB3 possesses a novel eight-gene ars cluster (ars1, arsRYCDATorf7orf8) with some unusual features in regard to expression regulation. This study demonstrated that the cluster is a single operon but can also produce a short three-gene arsRYC transcript. A hairpin structure formed by internal inverted repeats between arsC and arsD was shown to diminish the expression of the full operon, thereby probably acting as a transcription attenuator. A degradation product of the arsRYC transcript was also identified. Electrophoretic mobility shift analysis demonstrated that ArsR interacts with the ars1 promoter forming a protein-DNA complex that could be impaired by arsenite. However, no interaction was detected between ArsD and the ars1 promoter, suggesting that the CDB3 ArsD protein may not play a regulatory role. Compared to other ars gene clusters, regulation of the Bacillus sp. CDB3 ars1 operon is more complex. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons.

No MeSH data available.