Oligonucleotide Microarray for 16S rRNA Gene-Based Detection of All Recognized Lineages of Sulfate-Reducing Prokaryotes in the Environment

Abstract

For cultivation-independent detection of sulfate-reducing prokaryotes (SRPs) an oligonucleotide microarray consisting of 132 16S rRNA gene-targeted oligonucleotide probes (18-mers) having hierarchical and parallel (identical) specificity for the detection of all known lineages of sulfate-reducing prokaryotes (SRP-PhyloChip) was designed and subsequently evaluated with 41 suitable pure cultures of SRPs. The applicability of SRPPhyloChip for diversity screening of SRPs in environmental and clinical samples was tested by using samples from periodontal tooth pockets and from the chemocline of a hypersaline cyanobacterial mat from Solar Lake (Sinai, Egypt). Consistent with previous studies, SRP-PhyloChip indicated the occurrence of Desulfomicrobium spp. in the tooth pockets and the presence of Desulfonema-and Desulfomonile-like SRPs (together with other SRPs) in the chemocline of the mat. The SRP-PhyloChip results were confirmed by several DNA microarrayindependent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).

Application of COMPOCHIP Microarray to Investigate the Bacterial Communities of Different Composts

“…Of the 30,258 individual probetarget hybridization reactions performed (hybridization of 82 reference microorganisms), there were only 22 falsenegative signals (0.07%) and 188 false-positive signals seen (0.62%). These hybridization results are statistically similar to those obtained by other authors [8,33,39]. Certain probes were found to bind non-specifically with several targets, these probes being the Alphaproteobacteria KO 240 (9), Low G+C KO 319 (17), Nitrospira KO 295 (7), Thermomonospora chromogena KO 330 (7), and Actinomyces KO 342 (6).…”

“…PCR amplifications were performed in a ThermoHybaid PCR Express thermalcycler in 50-μl volumes, with each standard reaction mix containing a final concentration of 1× reaction buffer [ enhancer (Peqlab, Germany), 1.25 U BioTherm™ DNA polymerase (GeneCraft, Münster, Germany), and sterile water. In addition, 10 mM tetramethylammonium chloride was included in reactions to enhance the specificity [33]. Five microliters of extracted compost DNA (diluted 1/60) was applied directly to the PCR reaction mix.…”

“…Single-stranded Cy5-labeled PCR product (500 ng) was vacuum-dried and resuspended in 20 μl of a hybridization buffer consisting of 5× SSC, 1% blocking reagent (Roche, Mannheim, Germany), 0.02% sodium dodecyl sulfate (SDS), 0.1% n-laurylsarcosine, and 5% formamide [33]. One microliter of a 50-nM Cy5-labeled control oligonucleotide (Electronic supplementary material, Table 1) was added to each tube, and the mixture was denatured for 10 min at 95°C, before being placed on ice.…”

“…The median foreground and background signals for all spots were determined. The signal-to-noise ratio (SNR) for all spots was calculated according to the following calculation: Signals were assumed to be positive if a SNR value of ≥2 was obtained [33]. Statistical analyses of data were performed using the program Canoco 4.5 [46].…”

“…For example, the Burkholderia probe KO 234 was found to hybridize with Saccharomonospora DNA, despite six basepair differences in the probe target sequence, and Clostridium bifermentans DNA hybridized with the Clostridium butyricum KO 377 probe, despite ten mismatches in the probe target sequence. Because microarray experiments use the one set of conditions for hybridization and washing, false positives are not an uncommon event [33]. Both cultured and uncultured/unknown microorganisms can cause the generation of false-positive signals.…”