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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.


Northern blotting analyses of CDB3 ars1 expression.(A) A diagram of CDB3 ars1 labelled with locations of probes used and possible transcript sizes (kb). (B) Images of northern blot ting using probes 1, 2 and 3. On each blot the left lane is untreated control and right is arsenite-treated at 2 mM/10 min. (C) Images of northern blot ting using probes R and C. On each blot the left lane is untreated control and right is arsenite-treated at 0.5 mM/5 min. (D) Images of northern blot ting using probe 1 of samples treated with 4 mM arsenite for 3 min (left) and 30 min (right).
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fig-1: Northern blotting analyses of CDB3 ars1 expression.(A) A diagram of CDB3 ars1 labelled with locations of probes used and possible transcript sizes (kb). (B) Images of northern blot ting using probes 1, 2 and 3. On each blot the left lane is untreated control and right is arsenite-treated at 2 mM/10 min. (C) Images of northern blot ting using probes R and C. On each blot the left lane is untreated control and right is arsenite-treated at 0.5 mM/5 min. (D) Images of northern blot ting using probe 1 of samples treated with 4 mM arsenite for 3 min (left) and 30 min (right).

Mentions: To examine the transcription pattern of CDB3 ars1, total RNA extracts from arsenite-stressed and control bacterial cells were first analyzed by northern blot analysis using three probes, covering different regions of the cluster (Fig. 1A). While there was no hybridization signal detected in the control RNA sample, arsenite treatment induced the expression of ars1 and a 5.8-kb transcript corresponding to the full cluster length was detected by all three probes (Fig. 1B). This suggests that arsRYCDATorf7orf8 can be transcribed as a single polycistronic mRNA and that there is only one promoter at the 5’-end of the cluster. In addition, probe 1 (covering region RY), but not probes 2 and 3 (covering A and Torf7, respectively), detected two other RNA fragments of approximately 1.9 kb and 1.5 kb. A further northern blot assay (Fig. 1C) demonstrated that probes R and C (covering internal fragments of arsR and arsC, respectively) can both hybridize to the 1.9-kb RNA band, corresponding to the length of arsRYC but the 1.5-kb RNA band was only detected when probe C was applied. It was also observed that the amount of 1.5-kb RNA increased with treatment time, which is evident in the 4 mM arsenite samples: at 3 min the 1.5-kb RNA was not detected but was remarkably abundant after 30 min (Fig. 1D). This suggests that the first three genes, arsRYC, can also be transcribed as a polycistronic unit during ars1 expression and that the inverted repeat between arsC and arsD may function as a read-through transcriptional attenuator. Additionally, the 1.5-kb RNA band was found to lack the arsR gene compared with the 1.9-kb RNA fragment. Thus, it was assumed to be a possible differential mRNA degradation product spanning the YC gene region.


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

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

Northern blotting analyses of CDB3 ars1 expression.(A) A diagram of CDB3 ars1 labelled with locations of probes used and possible transcript sizes (kb). (B) Images of northern blot ting using probes 1, 2 and 3. On each blot the left lane is untreated control and right is arsenite-treated at 2 mM/10 min. (C) Images of northern blot ting using probes R and C. On each blot the left lane is untreated control and right is arsenite-treated at 0.5 mM/5 min. (D) Images of northern blot ting using probe 1 of samples treated with 4 mM arsenite for 3 min (left) and 30 min (right).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-1: Northern blotting analyses of CDB3 ars1 expression.(A) A diagram of CDB3 ars1 labelled with locations of probes used and possible transcript sizes (kb). (B) Images of northern blot ting using probes 1, 2 and 3. On each blot the left lane is untreated control and right is arsenite-treated at 2 mM/10 min. (C) Images of northern blot ting using probes R and C. On each blot the left lane is untreated control and right is arsenite-treated at 0.5 mM/5 min. (D) Images of northern blot ting using probe 1 of samples treated with 4 mM arsenite for 3 min (left) and 30 min (right).
Mentions: To examine the transcription pattern of CDB3 ars1, total RNA extracts from arsenite-stressed and control bacterial cells were first analyzed by northern blot analysis using three probes, covering different regions of the cluster (Fig. 1A). While there was no hybridization signal detected in the control RNA sample, arsenite treatment induced the expression of ars1 and a 5.8-kb transcript corresponding to the full cluster length was detected by all three probes (Fig. 1B). This suggests that arsRYCDATorf7orf8 can be transcribed as a single polycistronic mRNA and that there is only one promoter at the 5’-end of the cluster. In addition, probe 1 (covering region RY), but not probes 2 and 3 (covering A and Torf7, respectively), detected two other RNA fragments of approximately 1.9 kb and 1.5 kb. A further northern blot assay (Fig. 1C) demonstrated that probes R and C (covering internal fragments of arsR and arsC, respectively) can both hybridize to the 1.9-kb RNA band, corresponding to the length of arsRYC but the 1.5-kb RNA band was only detected when probe C was applied. It was also observed that the amount of 1.5-kb RNA increased with treatment time, which is evident in the 4 mM arsenite samples: at 3 min the 1.5-kb RNA was not detected but was remarkably abundant after 30 min (Fig. 1D). This suggests that the first three genes, arsRYC, can also be transcribed as a polycistronic unit during ars1 expression and that the inverted repeat between arsC and arsD may function as a read-through transcriptional attenuator. Additionally, the 1.5-kb RNA band was found to lack the arsR gene compared with the 1.9-kb RNA fragment. Thus, it was assumed to be a possible differential mRNA degradation product spanning the YC gene region.

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.