Rapid enumeration of sulphate-reducing bacteria from aquatic environments using real-time PCR

Kondo, Ryou; Shigematsu, Kotaro; Butani, Junki

doi:10.3800/pbr.3.180

Cited by 37 publications

(37 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…In the only other study specifically investigating dsrA genes from BBD lesions, Barneah et al (2007) similarly observed sequences closely related to Desulfovibrio species and retrieved no representatives from other genera of SRB, implying the dominance of Desulfovibrio in BBD lesions. Studies investigating the microbial diversity of BBD lesions from different geographical regions using 16S rRNA gene profiling techniques have also identified Desulfovibrio affiliated sequences as constituents of the BBD mat (Cooney et al, 2002;Frias-Lopez et al, 2002, 2004 A quantitative qRT-PCR assay targeting the dsrA gene with primers DSR1-F þ and DSR-R, was adapted from Kondo et al (2008) and LeLoup et al (2007). The qPCR assay was modified through the use of the TaqMan chemistry (Applied Biosystems, Foster City, CA, USA).…”

Section: Resultsmentioning

confidence: 99%

“…The metabolic capability of dissimilatory sulfate reduction is widely distributed within both bacterial and archeal phyla and the enzyme responsible (dissimilatory sulfite reductase; DSR) is highly conserved and ubiquitous in all known sulfate-reducing prokaryotes and, therefore, ideal for assessing the diversity of these populations within environmental samples (Wagner et al, 1998(Wagner et al, , 2005Leloup et al, 2007;Kondo et al, 2008). This study modified an established quantitative real-time PCR (RT-PCR) approach, targeting the gene coding for the a-subunit of the DSR gene (dsrA) (Leloup et al, 2007;Kondo et al, 2008) to assess both the diversity and relative abundance of SRB within microbial samples collected from CP, intermediate stages (IM) and BBD coral disease lesions. Owing to the variability in bacterial population diversity and sampled biomass within individual coral lesions, the SRB population was determined as a percentage of the total bacterial population by including a quantitative RT-PCR assay targeting the bacterial 16S rRNA gene (Nadkarni et al, 2002).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Changes in sulfate-reducing bacterial populations during the onset of black band disease

2010

View full text Add to dashboard Cite

Factors that facilitate the onset of black band disease (BBD) of corals remain elusive, though anoxic conditions under the complex microbial mat and production of sulfide are implicated in necrosis of underlying coral tissues. This study investigated the diversity and quantitative shifts of sulfatereducing bacterial (SRB) populations during the onset of BBD using real-time PCR (RT-PCR) and cloning approaches targeting the dissimilatory (bi)sulfite reductase (dsrA) gene. A quantitative-PCR (qPCR) assay targeting the 16S rRNA gene also provided an estimate of total bacteria, and allowed the relative percentage of SRB within the lesions to be determined. Three Montipora sp. coral colonies identified with lesions previously termed cyanobacterial patches (CPs) (comprising microbial communities unlike those of BBD lesions), were tagged and followed through time as CP developed into BBD. The dsrA-targeted qPCR detected few copies of the gene in the CP samples (o65 per ng DNA), though copy numbers increased in BBD lesions (42500 per ng DNA). SRB in CP samples were less than 1% of the bacterial population, though represented up to 7.5% of the BBD population. Clone libraries also demonstrated a shift in the dominant dsrA sequences as lesions shifted from CP into BBD. Results from this study confirm that SRB increase during the onset of BBD, likely increasing sulfide concentrations at the base of the microbial mat and facilitating the pathogenesis of BBD.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Changes in sulfate-reducing bacterial populations during the onset of black band disease

2010

View full text Add to dashboard Cite

show abstract

“…Each reaction contained 1 µL of brackish or freshwater wetland DNA template and was conducted using the PerfeCTa SYBR Green FastMix (Quanta BioSciences). For the mcrA qPCR, primer details and thermocycling conditions in West et al (2012) were replicated except that we employed a fluorescent detection step at 78 • C for 20 s. For the dsrA qPCR primer, details and thermocycling conditions in Kondo et al (2008) were replicated. Melting curves for both mcrA and dsrA were run to ensure the absence of non-specific amplification.…”

Section: Microbial Analysesmentioning

confidence: 99%

“…We amplified mcrA using primers detailed in Luton et al (2002) and thermocycling conditions in West et al (2012), and dsrA by replicating primer details and thermocycling conditions in Kondo et al (2008). After amplification, we used gel electrophoresis and an Invitrogen PureLink Quick Gel Extraction Kit (Invitrogen, Carlsbad, CA, USA) to isolate the mcrA and dsrA amplicons.…”

Section: Microbial Analysesmentioning

confidence: 99%

Regulators of coastal wetland methane production and responses to simulated global change

et al. 2017

View full text Add to dashboard Cite

Abstract. Wetlands are the largest natural source of methane (CH 4 ) emissions to the atmosphere, which vary along salinity and productivity gradients. Global change has the potential to reshape these gradients and therefore alter future contributions of wetlands to the global CH 4 budget. Our study examined CH 4 production along a natural salinity gradient in fully inundated coastal Alaska wetlands. In the laboratory, we incubated natural sediments to compare CH 4 production rates between non-tidal freshwater and tidal brackish wetlands, and quantified the abundances of methanogens and sulfate-reducing bacteria in these ecosystems. We also simulated seawater intrusion and enhanced organic matter availability, which we predicted would have contrasting effects on coastal wetland CH 4 production. Tidal brackish wetlands produced less CH 4 than non-tidal freshwater wetlands probably due to high sulfate availability and generally higher abundances of sulfate-reducing bacteria, whereas non-tidal freshwater wetlands had significantly greater methanogen abundances. Seawater addition experiments with freshwater sediments, however, did not reduce CH 4 production, perhaps because the 14-day incubation period was too short to elicit a shift in microbial communities. In contrast, increased organic matter enhanced CH 4 production in 75 % of the incubations, but this response depended on the macrophyte species added, with half of the species treatments having no significant effect. Our study suggests that CH 4 production in coastal wetlands, and therefore their overall contribution to the global CH 4 cycle, will be sensitive to increased organic matter availability and potentially seawater intrusion. To better predict future wetland contributions to the global CH 4 budget, future studies and modeling efforts should investigate how multiple global change mechanisms will interact to impact CH 4 dynamics.

show abstract

“…After transportation to the laboratory, within a few hours of sampling, a 50-mL aliquot was filtered through a sterile polycarbonate membrane filter (0.2 µm; Toyo Roshi Kaisha, Tokyo, Japan) to collect the microbial biomass for subsequent nucleic acid purification. Nucleic acids were extracted from the filtered samples using a FastDNA Spin Kit as described previously (16).…”

Section: Sample Collection and Dna Extractionmentioning

confidence: 99%

Comprehensive Detection of Phototrophic Sulfur Bacteria Using PCR Primers That Target Reverse Dissimilatory Sulfite Reductase Gene

et al. 2010

Self Cite

View full text Add to dashboard Cite

A new set of primers for the detection of phototrophic sulfur bacteria in natural environments is described. The primers target the α-subunit of the reverse dissimilatory sulfite reductase gene (dsrA). PCR-amplification resulted in products of the expected size from all the phototrophic strains tested, including purple sulfur and green sulfur bacteria. Seventy-nine clones obtained from environmental DNA using the primers were sequenced and all found to be closely related to the dsrA of purple sulfur bacteria and green sulfur bacteria. This newly developed PCR assay targeting dsrA is rapid and simple for the detection of phototrophic sulfur bacteria in situ and superior to the use of culture-dependent techniques.Key words: meromictic lake, phototrophic sulfur bacteria, reverse dissimilatory sulfite reductase Phototrophic sulfur bacteria constitute a paraphyletic group of metabolically diverse anaerobes capable of utilizing light as an energy source, but that do not evolve molecular oxygen. Widely distributed in nature where oxygen is either absent or present at low concentrations and opposing gradients of sulfide and light exist simultaneously, phototrophic sulfur bacteria use sulfide or other reduced sulfur compounds as electron donors in photosynthesis, and play an important role in sulfur cycling in aquatic systems such as lacustrine environments, where anoxic layers containing reduced sulfur compounds are exposed to light (19). These bacteria supply fixed carbon to chemoorganotrophic organisms, and thus, also have a significant role in the carbon cycle in holomictic and meromictic lakes (26,35).Phototrophic sulfur bacteria are traditionary classified as purple sulfur bacteria within the Gammaproteobacteria and as green sulfur bacteria within the phylum Chlorobi (26, 35). Their polyphyletic nature restricts the concomitant detection of all recognized members using a single 16S rRNA genetargeting probe or primer set in environmental analyses (1,3,8,28,34). Alternative approaches have been developed to detect phototrophic sulfur bacteria using PCR primers that target functional genes coding for key enzymes of sulfur oxidation pathways; ATP sulfurylase, the iron-sulfur flavoprotein adenosine-5'-phosphosulfate (APS) reductase and siroheme sulfite reductase (18,20,21).Dissimilatory sulfite reductase (DsrAB) is a key enzyme in dissimilatory sulfate reduction in sulfate-reducing prokaryotes. The presence of DsrAB with a proposed function in sulfide oxidation has been demonstrated for colorless and purple sulfur bacteria (29,30). Analyses of genome data revealed the presence of the dissimilatory sulfite reductase genes (dsrAB) in phototrophic sulfur bacteria (e.g. reference 22). Moreover, dsrAB sequences of phototrophic sulfur bacteria were conserved and phylogenetically distinct from sulfate-reducing prokaryotes (4). Thus, dsrAB coding for the reverse-operating DsrAB is a potential marker gene for phototrophic sulfur bacteria unlike primers and probes targeted to the 16S rRNA gene (7). Recently, PCR assays have been ...

show abstract

Rapid enumeration of sulphate-reducing bacteria from aquatic environments using real-time PCR

Cited by 37 publications

References 21 publications

Changes in sulfate-reducing bacterial populations during the onset of black band disease

Changes in sulfate-reducing bacterial populations during the onset of black band disease

Regulators of coastal wetland methane production and responses to simulated global change

Comprehensive Detection of Phototrophic Sulfur Bacteria Using PCR Primers That Target Reverse Dissimilatory Sulfite Reductase Gene

Contact Info

Product

Resources

About