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GENETICS AND MOLECULAR BIOLOGY

Arsenic Resistance in Halobacterium sp. Strain NRC-1 Examined by Using an Improved Gene Knockout System

Gejiao Wang, Sean P. Kennedy, Sabeena Fasiludeen, Christopher Rensing, Shiladitya DasSarma
Gejiao Wang
1Department of Soil, Water, and Environmental Science, University of Arizona, Tucson, Arizona 85721
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Sean P. Kennedy
2Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202
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Sabeena Fasiludeen
2Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202
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Christopher Rensing
1Department of Soil, Water, and Environmental Science, University of Arizona, Tucson, Arizona 85721
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  • For correspondence: dassarma@umbi.umd.edu rensingc@ag.arizona.edu
Shiladitya DasSarma
2Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, Maryland 21202
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  • For correspondence: dassarma@umbi.umd.edu rensingc@ag.arizona.edu
DOI: 10.1128/JB.186.10.3187-3194.2004
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  • FIG. 1.
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    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.
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    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.
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    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.
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    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.
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    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.
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    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

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  • TABLE 1.

    Oligonucleotides used for gene knockouts

    Gene and orientationSequence (5′ → 3′)
    arsA reverseCGGTCACCTCAGTGATCGTC
    arsA inverseCTGCGAATCCCCTGTCGAGACGA
    arsC inverseGCGCGTTGTAGACCTGTTCG
    arsC reverseAACAGACACCACCGGAACGC
    2599 reverseCTACGCGAAGAACTGTGCCA
    arsB 5′ inverseCGCTGACATGGTGCCACGGT
    arsB 3′ inverseTGGGGCGTCTAAGGCGGATA
    2603 forwardATGGAACAAGTGTGGGCAGA
    5175 reverseCTATTTGAAATCCTGAAAGA
    arsM 3′ inverseGACTGACCGGCCGAATCG
    arsM 5′ inverseCATGGTTGCCTCGCAGGT
    5176 forwardATGGTTCAGGACGCAACCGC
    ura3 forwardATGAGCTTCGTCGAGGAACT
    ura3 reverseCTACCGGTGGCGGTTCAGGC
    bla forwardATGAGTATTCAACATTTCCG
    bla reverseTTACCAATGCTTAATCAGTG
    tbpC forwardATGACGGTCGAGATTGCGAA
    tbpC reverseTTAAACTAATTCTTGGACTT
  • TABLE 2.

    Oligonucleotides used for making probes

    Gene and orientationSequence (5′ → 3′)
    arsA forwardATGACTGCTACACAAACGCC
    arsA reverseTCACGACACCGTCACCTCCT
    arsD forwardATGACTCAACTCACCCTGTA
    arsD reverseTTACGCTTCCTGTGGGTCTG
    arsR forwardATGTCATCGACCGAGCGGTT
    arsR reverseTCATCTGCGGGATCCGTTCA
    arsC forwardATGTCTGCTGATTCGACGAC
    arsC reverseTTAGTCGTCGGGATTGAACT
    arsB forwardATGTCAGCGGTGTCACCACC
    arsB reverseTTAGACGCCCCAGAAGGCCG
    arsM forwardATGGTTCAGGACGCAACC
    arsM reverseCTATCCGATTCTATTTGAAA
    arsR2 forwardATGGTTCAGGACGCAACCGC
    arsR2 reverseTTACTCATGGTTGCCTCGCA
    Universal 16S 338FCTCCTACGGGAGGCAGCAG
    Universal 16S 784RGGACTACCAGGGTATCTAATCC
    Archaeal 16S 27FTCCGGTTGATCCTGCCGGAG
    Archaeal 16S 685RTTACGGGATTTCACTCCTAC
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Arsenic Resistance in Halobacterium sp. Strain NRC-1 Examined by Using an Improved Gene Knockout System
Gejiao Wang, Sean P. Kennedy, Sabeena Fasiludeen, Christopher Rensing, Shiladitya DasSarma
Journal of Bacteriology Apr 2004, 186 (10) 3187-3194; DOI: 10.1128/JB.186.10.3187-3194.2004

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Arsenic Resistance in Halobacterium sp. Strain NRC-1 Examined by Using an Improved Gene Knockout System
Gejiao Wang, Sean P. Kennedy, Sabeena Fasiludeen, Christopher Rensing, Shiladitya DasSarma
Journal of Bacteriology Apr 2004, 186 (10) 3187-3194; DOI: 10.1128/JB.186.10.3187-3194.2004
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KEYWORDS

Arsenic
halobacterium

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