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Chapter 15 : Methods for Detection of Arsenate-Respiring Bacteria: Advances, Cautions, and Caveats

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Abstract:

This paper presents the results of the efforts to develop and examine the efficacy of culture medium formulation, biochemical probes, and molecular probes for the detection of arsenate-respiring bacteria. The results suggest that, while each approach can be used to assess the presence and activity of these organisms, it is prudent to employ multiple approaches concurrently in order to address the wide range of metabolic capabilities and arsenic tolerances. Different approaches are used for identifying arsenate-respiring bacteria in the environment. The results indicate that media composition can have an impact on the types of organisms that can be enriched for and cultured from arsenic-impacted environments. Ideally, a “matrix” consisting of different media formulations that include an assortment of electron donors and different arsenic concentrations should be employed. This study underscores the need to use a combination of approaches that include geochemical analysis, incubations, and enrichment culture, in concert with biochemical and molecular approaches.

Citation: Stolz J, Berekaa M, Fisher E, Polshyna G, Thangavelu M, Dheer R, Garcia Moyano A, El Assar S, Basu P. 2011. Methods for Detection of Arsenate-Respiring Bacteria: Advances, Cautions, and Caveats, p 283-295. In Stolz J, Oremland R (ed), Microbial Metal and Metalloid Metabolism. ASM Press, Washington, DC. doi: 10.1128/9781555817190.ch15

Key Concept Ranking

Microbial Ecology
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Denaturing Gradient Gel Electrophoresis
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Restriction Fragment Length Polymorphism
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Reverse Transcriptase PCR
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Figures

Image of FIGURE 1
FIGURE 1

Arsenic speciation (as determined by HPLC) in the Ohio River sediment matrix containing 10 mM arsenate with different electron donors: (A) control with no added donor, (B) acetate, (C) hydrogen plus acetate, (D) formate, (E) lactate, and (F) pyruvate. Initial (day 0) and day 7 are shown for uninoculated control (Un.), heat-killed control (H.K.), and experimental (Ex.). Arsenate results are displayed in the gray bars, and arsenite results are displayed in the white bars. 10.1128/9781555817190.ch15.f1

Citation: Stolz J, Berekaa M, Fisher E, Polshyna G, Thangavelu M, Dheer R, Garcia Moyano A, El Assar S, Basu P. 2011. Methods for Detection of Arsenate-Respiring Bacteria: Advances, Cautions, and Caveats, p 283-295. In Stolz J, Oremland R (ed), Microbial Metal and Metalloid Metabolism. ASM Press, Washington, DC. doi: 10.1128/9781555817190.ch15
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Image of FIGURE 2
FIGURE 2

Arsenate speciation (as determined by HPLC) after 7 days in the Ohio River sediment matrix using hydrogen as the electron donor and acetate as the carbon source with different concentrations of As(V): 1 mM (A), 5 mM (B), 10 mM (C), and 20 mM sodium arsenate (D). Un., uninoculated control; H.K., heat-killed control; Ex., experimental. Results are displayed in the gray bars, and arsenite results are displayed in the white bars. An asterisk (*) indicates a concentration below the level of detection. 10.1128/9781555817190.ch15.f2

Citation: Stolz J, Berekaa M, Fisher E, Polshyna G, Thangavelu M, Dheer R, Garcia Moyano A, El Assar S, Basu P. 2011. Methods for Detection of Arsenate-Respiring Bacteria: Advances, Cautions, and Caveats, p 283-295. In Stolz J, Oremland R (ed), Microbial Metal and Metalloid Metabolism. ASM Press, Washington, DC. doi: 10.1128/9781555817190.ch15
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Image of FIGURE 3
FIGURE 3

Arsenate reductase activity and cross-reactivity with anti-ArrA-1 affinity-purified antibodies. (A) Zymogram (nondenaturing PAGE) showing arsenate reductase activity in (lane 2), Mono Lake strain SLAS-1 (lane 6), and (lane 7), but not (lane 3), Mono Lake strain MLMS1 (lane 4), strain JMM-4 (lane 5), strain SES-3 (lane 8), (lane 9), and strain MIT-13 (lane 10). Lane 1 displays molecular mass standards (206, 115, 98, 54, 37, 29, 20, 7 kDa). (B) Western blot analysis (SDS-PAGE gel) showing cross-reactivity with the anti-ArrA-1 antisera with , and , but not Mono Lake strain SLAS-1, Mono Lake strain MLMS1, strain JMM4, , or The lanes are the same as in panel A. 10.1128/9781555817190.ch15.f3

Citation: Stolz J, Berekaa M, Fisher E, Polshyna G, Thangavelu M, Dheer R, Garcia Moyano A, El Assar S, Basu P. 2011. Methods for Detection of Arsenate-Respiring Bacteria: Advances, Cautions, and Caveats, p 283-295. In Stolz J, Oremland R (ed), Microbial Metal and Metalloid Metabolism. ASM Press, Washington, DC. doi: 10.1128/9781555817190.ch15
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Image of FIGURE 4
FIGURE 4

Detection of ArrA in cells of grown with different terminal electron acceptors. The results show that, although arsenate reductase activity can be detected in nitrate-and DMSO-grown cells, only arsenate-grown cells express ArrA. (A) Zymogram (nondenaturing PAGE) showing arsenate reductase activity in cells grown in arsenate (lane 2), nitrate (lane 3), and DMSO (lane 6) but not fumarate (lane 4) or selenite (lane 5). Lane 1 displays molecular mass standards (206, 115, 98, 54, 37, 29, 20, 7 kDa). (B) Western blot analysis (SDS-PAGE gel) showing presence of ArrA only in cells grown on arsenate. The lanes are the same as in panel A. (C) Duplicate gel of panel B stained with Coomassie blue (indicating that comparable amount of protein was loaded to each lane). Lanes are the same as in panels A and B. 10.1128/9781555817190.ch15.f4

Citation: Stolz J, Berekaa M, Fisher E, Polshyna G, Thangavelu M, Dheer R, Garcia Moyano A, El Assar S, Basu P. 2011. Methods for Detection of Arsenate-Respiring Bacteria: Advances, Cautions, and Caveats, p 283-295. In Stolz J, Oremland R (ed), Microbial Metal and Metalloid Metabolism. ASM Press, Washington, DC. doi: 10.1128/9781555817190.ch15
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Image of FIGURE 5
FIGURE 5

Primers and DGGE of (A) Gene map of showing the relative locations of the primers. (B) DGGE of PCR amplicons using UF1GC/UR3 on a 30 to 70% gradient. Lane 1, ; lane 2, ; lane 3, (C) DGGE of PCR amplicons using UF1GC/UR2 (lanes 1 to 3), UF5GC/UR6 (lanes 4 to 6), and UF5GC/UR8 (lanes 7 to 9) on a 30 to 70% gradient. Lanes 1, 4, and 7 represent ; lanes 2, 5, and 8, ; lanes 3, 6, and 9,

Citation: Stolz J, Berekaa M, Fisher E, Polshyna G, Thangavelu M, Dheer R, Garcia Moyano A, El Assar S, Basu P. 2011. Methods for Detection of Arsenate-Respiring Bacteria: Advances, Cautions, and Caveats, p 283-295. In Stolz J, Oremland R (ed), Microbial Metal and Metalloid Metabolism. ASM Press, Washington, DC. doi: 10.1128/9781555817190.ch15
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Tables

Generic image for table
TABLE 1

PCR primers for amplication and DGGE

Citation: Stolz J, Berekaa M, Fisher E, Polshyna G, Thangavelu M, Dheer R, Garcia Moyano A, El Assar S, Basu P. 2011. Methods for Detection of Arsenate-Respiring Bacteria: Advances, Cautions, and Caveats, p 283-295. In Stolz J, Oremland R (ed), Microbial Metal and Metalloid Metabolism. ASM Press, Washington, DC. doi: 10.1128/9781555817190.ch15

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