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

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.

Keywords: Arsenic resistance; Operon expression; RNA degradation; Transcription attenuation.

Figure 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).

Figure 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 pAR27HP^Δ. 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.

Figure 3. Mapping of RNA degradation product.

(A) Electropherogram showing the result of primer extension assay. The extension product is indicated by arrowhead and size standards are labelled below. (B) The first 50 nucleotides sequence of CDB3 arsY RNA coding region indicating an inverted repeat (underlined) and 5’-end of 1.5-kb RNA (pointed by diamond).

Figure 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.

Figure 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.