Article Figures & Data
Figures
- FIG. 1.
The ars gene cluster of Halobacterium sp. strain NRC-1. A 14-kb region of pNRC100 (bp 132000 to144000) (8) from wild-type Halobacterium sp. strain NRC-1 is shown containing the genes putatively responsible for arsenic resistance in this organism. Genes and open reading frames are shown as arrows to scale, with rightward or leftward transcriptional direction indicated. The positions of two IS elements flanking the gene cluster are indicated by boxes.
- FIG. 2.
Gene knockout strategy and strain construction. (A) Map of plasmid pSK400 (10), a Litmus 18 derivative containing the Halobacterium sp. strain NRC-1 ura3 gene used as the knockout vector. The position of target gene fragments containing 5′ and 3′ flanking regions cloned into the PvuII site in the 3′ region of the lacZα gene fragment (Z) is shown above pSK400. (B) PCR assay of transformants grown on dropout SKURA medium (plasmid integrants) using appropriate ars primers (arsA reverse and arsC reverse for SK402 and 2599 reverse and 2603 forward for SK421) and bla forward and reverse primers. Positive isolates containing both PCR fragments are visible (two of five for SK402 and two of three for SK421). (C) PCR assays of isolates grown on 5-FOA plates that result from plasmid excision and deletion of ars genes (ΔarsADRC for strain SK402, lanes 1 to 3, and ΔarsB for strain SK421, lanes 4 to 6). Lanes 1 and 4 contain PCR products with a positive control (tbpC primers), lanes 2 and 5 contain products with ura3 gene primers, and lanes 3 and 6 contain products with ars primers (as in panel B).
- FIG. 3.
Arsenite, arsenate, and antimonite resistance of Halobacterium sp. strains NRC-1 (solid symbols) and SK402 (open symbols). Growth with different concentrations of sodium arsenite (A), sodium arsenate (B) and antimony potassium tartrate (C) is plotted. Original cultures of each single colony from NRC-1 and SK402 were induced with 1 μM arsenite and diluted 1:500 into fresh broth with the indicated concentrations of As(III), As(V), or Sb(III). Cell growth was monitored at OD600 for 14 days with incubation at 42°C and shaking at 200 rpm. ▪ and □, no addition; ▴ and ▵, 0.1 mM arsenite (A), 10 mM arsenate (B), or 0.05 mM antinomite (C); • and ○, 0.15 mM arsenite (A), 20 mM arsenate (B), or 0.1 mM antinomite (C).
- FIG. 4.
Arsenic resistance of Halobacterium strains NRC-1, SK402, and SK431. Original cultures of each single colony from NRC-1, SK402, and SK431 were induced with 1 μM arsenite and diluted 1:500 into fresh broth with 0.1 mM arsenite. Cell growth was monitored at OD600 for 14 days with incubation at 42°C and shaking at 200 rpm. For Halobacterium sp. strain NRC-1, ▴ represents no addition and ▵ represents 0.1 mM As(III). For Halobacterium sp. strain SK402, ▪ represents no addition and □ represents 0.1 mM As(III). For Halobacterium sp. strain SK431, • represents no addition and ○ represents 0.1 mM As(III).
- FIG. 5.
Northern blot analysis of ars genes in Halobacterium sp. strain NRC-1. Panels contain hybridization results for arsB, arsR, arsD, arsA, arsR2, and arsM are labeled. RNA was isolated from Halobacterium sp. strain NRC-1 grown in either standard CM medium (lane 1), medium supplemented with 25 μM As(III) (lane 2), or medium supplemented with 10 μM Sb(III) (lane 3). The arsC transcript was not detected by Northern blot (data not shown). The approximate molecular sizes of the transcripts were as follows (nucleotides): 1,800 for arsB, 400 for arsR, 300 for arsD, 1,950 for arsA, 400 for arsR2, and 800 for arsM. The panel labeled rRNA shows a stained gel as a control for the amount of RNA loaded.
- FIG. 6.
RT-PCR analysis of arsC gene expression in Halobacterium sp. strain NRC-1. A 2% agarose gel is shown after electrophoresis and staining with ethidium bromide. Lanes 1 to 7 contain PCR products using arsC primers. Lanes 1, 2, and 3 contain RT-PCR products from RNAs (after DNase I treatment) purified from Halobacterium sp. strain NRC-1 cultured in CM medium, 25 μM As(III), and 10 μM Sb(III), respectively. Arrows indicate the positions of the major RT-PCR products in lanes 2 and 3. Lanes 4, 5, and 6 contain PCR products using the same RNAs as in lanes 1, 2, and 3, respectively, after DNase I treatment but without RT. Lane 7 contains a positive control PCR fragment using genomic DNA from Halobacterium sp. strain NRC-1 as a template. Lanes 8 to 10 contain RT-PCR products using the same RNAs as in lanes 1, 2, and 3, respectively, but with universal 16S rRNA gene primers. Lanes 11 to 13 contain RT-PCR products using the same RNAs as in lanes 1, 2, and 3, respectively, with archaeal 16S rRNA gene primers. Lane 14 contains a 100-bp ladder as a size marker.
Tables
- TABLE 1.
Oligonucleotides used for gene knockouts
Gene and orientation Sequence (5′ → 3′) arsA reverse CGGTCACCTCAGTGATCGTC arsA inverse CTGCGAATCCCCTGTCGAGACGA arsC inverse GCGCGTTGTAGACCTGTTCG arsC reverse AACAGACACCACCGGAACGC 2599 reverse CTACGCGAAGAACTGTGCCA arsB 5′ inverse CGCTGACATGGTGCCACGGT arsB 3′ inverse TGGGGCGTCTAAGGCGGATA 2603 forward ATGGAACAAGTGTGGGCAGA 5175 reverse CTATTTGAAATCCTGAAAGA arsM 3′ inverse GACTGACCGGCCGAATCG arsM 5′ inverse CATGGTTGCCTCGCAGGT 5176 forward ATGGTTCAGGACGCAACCGC ura3 forward ATGAGCTTCGTCGAGGAACT ura3 reverse CTACCGGTGGCGGTTCAGGC bla forward ATGAGTATTCAACATTTCCG bla reverse TTACCAATGCTTAATCAGTG tbpC forward ATGACGGTCGAGATTGCGAA tbpC reverse TTAAACTAATTCTTGGACTT - TABLE 2.
Oligonucleotides used for making probes
Gene and orientation Sequence (5′ → 3′) arsA forward ATGACTGCTACACAAACGCC arsA reverse TCACGACACCGTCACCTCCT arsD forward ATGACTCAACTCACCCTGTA arsD reverse TTACGCTTCCTGTGGGTCTG arsR forward ATGTCATCGACCGAGCGGTT arsR reverse TCATCTGCGGGATCCGTTCA arsC forward ATGTCTGCTGATTCGACGAC arsC reverse TTAGTCGTCGGGATTGAACT arsB forward ATGTCAGCGGTGTCACCACC arsB reverse TTAGACGCCCCAGAAGGCCG arsM forward ATGGTTCAGGACGCAACC arsM reverse CTATCCGATTCTATTTGAAA arsR2 forward ATGGTTCAGGACGCAACCGC arsR2 reverse TTACTCATGGTTGCCTCGCA Universal 16S 338F CTCCTACGGGAGGCAGCAG Universal 16S 784R GGACTACCAGGGTATCTAATCC Archaeal 16S 27F TCCGGTTGATCCTGCCGGAG Archaeal 16S 685R TTACGGGATTTCACTCCTAC
