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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.


Mobility shift assay of ArsD.(A) EMSA of ArsD binding to proR and proN. Indicated amount of each DNA fragment binds with (+) or without (−) ArsD. M is a 100 bp DNA ladder with representative sizes indicated. (B) Sequence alignment of ArsDs from Bacillus sp. CDB3 (AAD51848.1), E. coli pR773 (AAA93060) and Acidiphilium multivorum pKW301 (BAA24821). Accession numbers are in parentheses. The stars indicate conserved Cys in all three sequences and shadow indicates those not present in Bacillus sp. CDB3 ArsD.
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fig-5: Mobility shift assay of ArsD.(A) EMSA of ArsD binding to proR and proN. Indicated amount of each DNA fragment binds with (+) or without (−) ArsD. M is a 100 bp DNA ladder with representative sizes indicated. (B) Sequence alignment of ArsDs from Bacillus sp. CDB3 (AAD51848.1), E. coli pR773 (AAA93060) and Acidiphilium multivorum pKW301 (BAA24821). Accession numbers are in parentheses. The stars indicate conserved Cys in all three sequences and shadow indicates those not present in Bacillus sp. CDB3 ArsD.

Mentions: An EMSA was also carried out using recombinant CDB3 ArsD protein to test its ability to bind DNA. Such a binding affinity was not detected so the results did not show a mobility shift of proR DNA after reaction with the ArsD protein (Fig. 5A) indicating that CDB3 ArsD is probably not a repressor. A sequence alignment between CDB3 ArsD and two biochemically characterised repressor ArsDs from E. coli R773 (Wu & Rosen, 1993) and Acidiphilium multivorum AIU 301 pKW301 (Suzuki et al., 1998) showed a distinct dissimilarity (Fig. 5B). Compared with the other two proteins, CDB3 ArsD does not possess the two C-terminal vicinal cysteine pairs, Cys112–Cys113 and Cys119–Cys120.


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

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

Mobility shift assay of ArsD.(A) EMSA of ArsD binding to proR and proN. Indicated amount of each DNA fragment binds with (+) or without (−) ArsD. M is a 100 bp DNA ladder with representative sizes indicated. (B) Sequence alignment of ArsDs from Bacillus sp. CDB3 (AAD51848.1), E. coli pR773 (AAA93060) and Acidiphilium multivorum pKW301 (BAA24821). Accession numbers are in parentheses. The stars indicate conserved Cys in all three sequences and shadow indicates those not present in Bacillus sp. CDB3 ArsD.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig-5: Mobility shift assay of ArsD.(A) EMSA of ArsD binding to proR and proN. Indicated amount of each DNA fragment binds with (+) or without (−) ArsD. M is a 100 bp DNA ladder with representative sizes indicated. (B) Sequence alignment of ArsDs from Bacillus sp. CDB3 (AAD51848.1), E. coli pR773 (AAA93060) and Acidiphilium multivorum pKW301 (BAA24821). Accession numbers are in parentheses. The stars indicate conserved Cys in all three sequences and shadow indicates those not present in Bacillus sp. CDB3 ArsD.
Mentions: An EMSA was also carried out using recombinant CDB3 ArsD protein to test its ability to bind DNA. Such a binding affinity was not detected so the results did not show a mobility shift of proR DNA after reaction with the ArsD protein (Fig. 5A) indicating that CDB3 ArsD is probably not a repressor. A sequence alignment between CDB3 ArsD and two biochemically characterised repressor ArsDs from E. coli R773 (Wu & Rosen, 1993) and Acidiphilium multivorum AIU 301 pKW301 (Suzuki et al., 1998) showed a distinct dissimilarity (Fig. 5B). Compared with the other two proteins, CDB3 ArsD does not possess the two C-terminal vicinal cysteine pairs, Cys112–Cys113 and Cys119–Cys120.

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.