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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.


Binding motifs and mobility shift assays of ArsR.(A) Putative promoter region in ars1. The inverted repeat is marked by inverted arrows lines . The putative ribosomal binding site (RBS), −35 and −10 boxes and start codon of arsR are indicated. (B) Examination of ArsR binding. Left: ArsR binding with proR. The amounts of DNA fragment and protein used were indicated above the panel. Right: ArsR binding with proN. ArsR was incubated with proN at equal and much higher concentrations than that used for proR. (C) Effect of arsenite (Left) and arsenate (Right) on ArsR-DNA complex. Each reaction contains 0 .1 mM proR and 0.75 mM of ArsR (+) or 0 mM of ArsR (−).The arsenic concentrations in each reaction are shown on top of panel. Lane M is a 100 bp DNA ladder with representative sizes indicated. (D) Multiple ArsR sequences alignment . Representative homologues (accession numbers in parentheses) are from E. coli R773 (P15905), Bacillus sp. CDB3 (AF178758), Acidithiobacillus ferrooxidans (AAF69241) and Corynebacterium glutamicum (YP_225794.1). The identified or predicted metalloid binding cysteines are highlighted in shadow.
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fig-4: Binding motifs and mobility shift assays of ArsR.(A) Putative promoter region in ars1. The inverted repeat is marked by inverted arrows lines . The putative ribosomal binding site (RBS), −35 and −10 boxes and start codon of arsR are indicated. (B) Examination of ArsR binding. Left: ArsR binding with proR. The amounts of DNA fragment and protein used were indicated above the panel. Right: ArsR binding with proN. ArsR was incubated with proN at equal and much higher concentrations than that used for proR. (C) Effect of arsenite (Left) and arsenate (Right) on ArsR-DNA complex. Each reaction contains 0 .1 mM proR and 0.75 mM of ArsR (+) or 0 mM of ArsR (−).The arsenic concentrations in each reaction are shown on top of panel. Lane M is a 100 bp DNA ladder with representative sizes indicated. (D) Multiple ArsR sequences alignment . Representative homologues (accession numbers in parentheses) are from E. coli R773 (P15905), Bacillus sp. CDB3 (AF178758), Acidithiobacillus ferrooxidans (AAF69241) and Corynebacterium glutamicum (YP_225794.1). The identified or predicted metalloid binding cysteines are highlighted in shadow.

Mentions: The CDB3 ArsR protein is homologous to the ars operon regulator of Bacillus subtilis Skin element with 89% similarity (Bhat et al., 2011), suggesting a potential repressor function in Bacillus sp. CDB3. Sequence analysis of the ars1 promoter region identified a 22-bp inverted repeat (Fig. 4A), probably acting as repressor binding site. With the recombinant CDB3 ArsR protein produced and purified from E. coli, an electrophoretic mobility shift assay (EMSA) demonstrated that the ArsR protein retarded the mobility of a 167-bp DNA fragment (proR) containing the promoter region of ars1 by forming a DNA-protein complex (Fig. 4B left). At a fixed concentration (0.1 mM) of DNA, the binding was enhanced along with increasing concentrations (0.37–1.5 mM) of ArsR. However, there was no interaction observed when the protein was incubated with a non-specific DNA fragment (proN) even at 25.5 mM (255-fold concentration as that used for proR) (Fig. 4B right), which confirmed the binding specificity between ArsR and the ars1 promoter. Since the transcription of ars1 can be induced by arsenic, interruption of the binding by arsenic compounds was assessed. As shown in Fig. 4C (left), with the concentration of arsenite increased the protein-DNA complex was gradually disrupted and a complete separation occurred at 50 mM. However, arsenate did not affect the ArsR-DNA complex even at a concentration of 80 mM (Fig. 4C right). Therefore, arsenite, but not arsenate, is capable of binding CDB3 ArsR, leading to dissociation from the ars1 promoter.


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

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

Binding motifs and mobility shift assays of ArsR.(A) Putative promoter region in ars1. The inverted repeat is marked by inverted arrows lines . The putative ribosomal binding site (RBS), −35 and −10 boxes and start codon of arsR are indicated. (B) Examination of ArsR binding. Left: ArsR binding with proR. The amounts of DNA fragment and protein used were indicated above the panel. Right: ArsR binding with proN. ArsR was incubated with proN at equal and much higher concentrations than that used for proR. (C) Effect of arsenite (Left) and arsenate (Right) on ArsR-DNA complex. Each reaction contains 0 .1 mM proR and 0.75 mM of ArsR (+) or 0 mM of ArsR (−).The arsenic concentrations in each reaction are shown on top of panel. Lane M is a 100 bp DNA ladder with representative sizes indicated. (D) Multiple ArsR sequences alignment . Representative homologues (accession numbers in parentheses) are from E. coli R773 (P15905), Bacillus sp. CDB3 (AF178758), Acidithiobacillus ferrooxidans (AAF69241) and Corynebacterium glutamicum (YP_225794.1). The identified or predicted metalloid binding cysteines are highlighted in shadow.
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Related In: Results  -  Collection

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fig-4: Binding motifs and mobility shift assays of ArsR.(A) Putative promoter region in ars1. The inverted repeat is marked by inverted arrows lines . The putative ribosomal binding site (RBS), −35 and −10 boxes and start codon of arsR are indicated. (B) Examination of ArsR binding. Left: ArsR binding with proR. The amounts of DNA fragment and protein used were indicated above the panel. Right: ArsR binding with proN. ArsR was incubated with proN at equal and much higher concentrations than that used for proR. (C) Effect of arsenite (Left) and arsenate (Right) on ArsR-DNA complex. Each reaction contains 0 .1 mM proR and 0.75 mM of ArsR (+) or 0 mM of ArsR (−).The arsenic concentrations in each reaction are shown on top of panel. Lane M is a 100 bp DNA ladder with representative sizes indicated. (D) Multiple ArsR sequences alignment . Representative homologues (accession numbers in parentheses) are from E. coli R773 (P15905), Bacillus sp. CDB3 (AF178758), Acidithiobacillus ferrooxidans (AAF69241) and Corynebacterium glutamicum (YP_225794.1). The identified or predicted metalloid binding cysteines are highlighted in shadow.
Mentions: The CDB3 ArsR protein is homologous to the ars operon regulator of Bacillus subtilis Skin element with 89% similarity (Bhat et al., 2011), suggesting a potential repressor function in Bacillus sp. CDB3. Sequence analysis of the ars1 promoter region identified a 22-bp inverted repeat (Fig. 4A), probably acting as repressor binding site. With the recombinant CDB3 ArsR protein produced and purified from E. coli, an electrophoretic mobility shift assay (EMSA) demonstrated that the ArsR protein retarded the mobility of a 167-bp DNA fragment (proR) containing the promoter region of ars1 by forming a DNA-protein complex (Fig. 4B left). At a fixed concentration (0.1 mM) of DNA, the binding was enhanced along with increasing concentrations (0.37–1.5 mM) of ArsR. However, there was no interaction observed when the protein was incubated with a non-specific DNA fragment (proN) even at 25.5 mM (255-fold concentration as that used for proR) (Fig. 4B right), which confirmed the binding specificity between ArsR and the ars1 promoter. Since the transcription of ars1 can be induced by arsenic, interruption of the binding by arsenic compounds was assessed. As shown in Fig. 4C (left), with the concentration of arsenite increased the protein-DNA complex was gradually disrupted and a complete separation occurred at 50 mM. However, arsenate did not affect the ArsR-DNA complex even at a concentration of 80 mM (Fig. 4C right). Therefore, arsenite, but not arsenate, is capable of binding CDB3 ArsR, leading to dissociation from the ars1 promoter.

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.