Specific Detection of Dehalococcoides Species by Fluorescence In Situ Hybridization with 16S rRNA-Targeted Oligonucleotide Probes

Dehalococcoides ethenogenes is the only known cultivated organism capable of complete dehalogenation of tetrachloroethene (PCE) to ethene. The prevalence of Dehalococcoides species in the environment and their association with complete dehalogenation of chloroethenes suggest that they play an important role in natural attenuation of chloroethenes and are promising candidates for engineered bioremediation of these contaminants. Both natural attenuation and bioremediation require reliable and sensitive methods to monitor the presence, distribution, and fate of the organisms of interest. Here we report the development of 16S rRNA-targeted oligonucleotide probes for Dehalococcoides species. The two designed probes together encompass 28 sequences of 16S rRNA genes retrieved from the public database. Except D. ethenogenes and CBDB1, all the others are environmental clones obtained from sites contaminated with chlorinated ethenes. They are all closely related and form a unique cluster of Dehalococcoides species. In situ hybridization of probe Dhe1259t with D. ethenogenes strain 195 and two enrichment cultures demonstrated the applicability of the probe to monitoring the abundance of active Dehalococcoides species in these enrichment samples.     

生物素标记寡核苷酸探针探测扩增的病理性突变珠蛋白基因

用末端转移酶催化生物素核苷酸底物(Biotin-ll-dUTP)共价连接在合成的寡核苷酸3’羟基末端,从而合成了两种寡核苷酸探针(β~T_(41-42)及β~A_(41-42))。用它们分别与克隆化扩增的正常和突变的β—珠蛋白基因片段杂变。结果表明该探针都具有与~(32)P探针相似的特异性,其杂交的灵敏度为2—3pg(特异序列)。进而将探测HbS基因的正常和异常两种寡核苷酸19聚体(β~A_6和β~S_6)用~(32)P和生物素分别标记;将HbS杂合子病人的白细胞DNA经聚合酶链反应(PCR)法扩增,并以含正常β—珠蛋白基因的DNA片段作对照,与两种探针分别进行斑点杂交。所得结果完全一致;Hbs杂合子DNA对正常和异常探针都显出杂交信号,而正常DNA只与β~A探针显杂交信号。     

Application of Sequence-Specific Labeled 16S rRNA Gene Oligonucleotide Probes for Genetic Profiling of Cyanobacterial Abundance and Diversity by Array Hybridization

DNA sequence information for the small-subunit rRNA gene (16S rDNA) obtained from cyanobacterial cultures was used to investigate the presence of cyanobacteria and their abundance in natural habitats. Eight planktonic communities developing in lakes characterized by relatively low algal biomass (mesotrophic) and in lakes with correspondingly high biomass (eutrophic) were selected for the study. The organismal compositions of the water samples were analyzed genetically, using multiplex sequence-specific labeling of oligonucleotide probes targeted to 16S rDNA and subsequent hybridization of the labeled probes to their respective complements spotted onto a solid support (DNA array). Ten probes were established to determine the relative abundances of the discernible cyanobacteria encountered in the selected lakes. The probes were generally specific for their targets, as determined through analyses of clone cultures. Reproducible abundance profiles were established for the lakes investigated in the subsequent analyses of natural cyanobacterial communities. The results from the genetic analyses were then compared with information obtained from standard hydrobiological and hydrochemical analyses. Qualitatively, there were relatively good correlations among the groups of organisms (Nostoc, Microcystis, and Planktothrix species) found in the different lakes. The levels of correlation were lower for the quantitative data. This may, however, be due to differences in sample processing technique. The conclusions from these comparisons are that the genetic abundance profiles may provide a foundation for separating and quantifying genetically distinct groups of cyanobacteria in their natural habitats.     

Riboprinting and 16S rRNA Gene Sequencing for Identification of Brewery Pediococcus Isolates

A total of 46 brewery and 15 ATCC Pediococcus isolates were ribotyped using a Qualicon RiboPrinter. Of these, 41 isolates were identified as Pediococcus damnosus using EcoRI digestion. Three ATCC reference strains had patterns similar to each other and matched 17 of the brewery isolates. Six other brewing isolates were similar to ATCC 25249. The other 18 P. damnosus brewery isolates had unique patterns. Of the remaining brewing isolates, one was identified as P. parvulus, two were identified as P. acidilactici, and two were identified as unique Pediococcus species. The use of alternate restriction endonucleases indicated that PstI and PvuII could further differentiate some strains having identical EcoRI profiles. An acid-resistant P. damnosus isolate could be distinguished from non-acid-resistant varieties of the same species using PstI instead of EcoRI. 16S rRNA gene sequence analysis was compared to riboprinting for identifying pediococci. The complete 16S rRNA gene was PCR amplified and sequenced from seven brewery isolates and three ATCC references with distinctive riboprint patterns. The 16S rRNA gene sequences from six different brewery P. damnosus isolates were homologous with a high degree of similarity to the GenBank reference strain but were identical to each other and one ATCC strain with the exception of 1 bp in one strain. A slime-producing, beer spoilage isolate had 16S rRNA gene sequence homology to the P. acidilactici reference strain, in agreement with the riboprint data. Although 16S rRNA gene sequencing correctly identified the genus and species of the test Pediococcus isolates, riboprinting proved to be a better method for subspecies differentiation.     

Development and Use of Flow Cytometry for Detection of Airborne Fungi

Traditional methods for the enumeration of airborne fungi are slow, tedious, and rather imprecise. In this study, the possibility of using flow cytometry (FCM) for the assessment of exposure to the fungus aerosol was evaluated. Epifluorescence microscopy direct counting was adopted as the standard for comparison. Setting up of the method was achieved with pure suspensions of Aspergillus fumigatus and Penicillium brevicompactum conidia at different concentrations, and then analyses were extended to field samples collected by an impinger device. Detection and quantification of airborne fungi by FCM was obtained combining light scatter and propidium iodide red fluorescence parameters. Since inorganic debris are unstainable with propidium iodide, the biotic component could be recognized, whereas the preanalysis of pure conidia suspensions of some species allowed us to select the area corresponding to the expected fungal population. A close agreement between FCM and epifluorescence microscopy counts was found. Moreover, data processing showed that FCM can be considered more precise and reliable at any of the tested concentrations.     

Optimization Strategies for DNA Microarray-Based Detection of Bacteria with 16S rRNA-Targeting Oligonucleotide Probes

The usability of the DNA microarray format for the specific detection of bacteria based on their 16S rRNA genes was systematically evaluated with a model system composed of six environmental strains and 20 oligonucleotide probes. Parameters such as secondary structures of the target molecules and steric hindrance were investigated to better understand the mechanisms underlying a microarray hybridization reaction, with focus on their influence on the specificity of hybridization. With adequate hybridization conditions, false-positive signals could be almost completely prevented, resulting in clear data interpretation. Among 199 potential nonspecific hybridization events, only 1 false-positive signal was observed, whereas false-negative results were more common (17 of 41). Subsequent parameter analysis revealed that this was mainly an effect of reduced accessibility of probe binding sites caused by the secondary structures of the target molecules. False-negative results could be prevented and the overall signal intensities could be adjusted by introducing a new optimization strategy called directed application of capture oligonucleotides. The small number of false-positive signals in our data set is discussed, and a general optimization approach is suggested. Our results show that, compared to standard hybridization formats such as fluorescence in situ hybridization, a large number of oligonucleotide probes with different characteristics can be applied in parallel in a highly specific way without extensive experimental effort.     

Oligonucleotide Probes for the Detection of Representatives of the Genus Thermoanaerobacter

Based on the analysis of 16S rRNA nucleotide sequences, oligonucleotide probes were designed for the detection of representatives of the genus Thermoanaerobacter. To increase the specificity of detection, the genus Thermoanaerobacter was divided into three groups. The probe Tab 827 (5"-GCTTCCGCDYCCCACACCTA-3") detected all known representatives of the genus Thermoanaerobacter; the probe Tab_1 844 (5"-TTAACTACGGCACGRAATGCTTC-3") was specific for the first group of species of the genus (T. wiegelii, T. siderophilus, T. sulfurophilus, T. brockii, T. kivui, T. ethanolicus, T. acetoethylicus, and T. thermohydrosulfuricus); the probe Tab_2 424 (5"-CACTAMYGGGGTTTACAACC-3") targeted the second group (T. thermocopriae, T. mathranii, and T. italicus); and the probe Tab_3 184 (5"-TCCTCCATCAGGATGCCCTA-3") was specific for the third group (T. tengcongensis, T. yonseiensis, T. subterraneus, and Carboxydibrachium pacificum, an organism related to the genus Thermoanaerobacter according to its 16S rRNA sequence). The oligonucleotide probes were labeled with Dig-11-dUTP. Hybridization with the probes showed the affiliation with Thermoanaerobacter of several pure cultures that were morphologically similar to representatives of this genus but possessed metabolic features unusual for it (capacity for agarose hydrolysis, anaerobic oxidation of CO, growth at low pH values) or were isolated from habitats previously unknown for Thermoanaerobacter (deep-sea hydrothermal vents).     

Unlabeled Helper Oligonucleotides Increase the In Situ Accessibility to 16S rRNA of Fluorescently Labeled Oligonucleotide Probes

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083^T. The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.     

Sequence versus Structure for the Direct Detection of 16S rRNA on Planar Oligonucleotide Microarrays

A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays. In this study, we investigated the physical spacing and nucleotide mismatch tolerance between capture and proximal chaperone detector probes that are required to achieve species-specific 16S rRNA detection for the dissimilatory metal and sulfate reducer 16S rRNAs. Microarray specificity was deduced by analyzing signal intensities across replicate microarrays with a statistical analysis-of-variance model that accommodates well-to-well and slide-to-slide variations in microarray signal intensity. Chaperone detector probes located in immediate proximity to the capture probe resulted in detectable, nonspecific binding of nontarget rRNA, presumably due to base-stacking effects. Species-specific rRNA detection was achieved by using a 22-nt capture probe and a 15-nt detector probe separated by 10 to 14 nt along the primary sequence. Chaperone detector probes with up to three mismatched nucleotides still resulted in species-specific capture of 16S rRNAs. There was no obvious relationship between position or number of mismatches and within- or between-genus hybridization specificity. From these results, we conclude that relieving secondary structure is of principal concern for the successful capture and detection of 16S rRNAs on planar surfaces but that the sequence of the capture probe is more important than relieving secondary structure for achieving specific hybridization.     

Sensitive and Specific Detection of Pseudomonas avellanae using Primers based on 16S rRNA Gene Sequences

A rapid polymerase chain reaction (PCR)-based procedure was developed for the detection of Pseudomonas avellanae , the causal agent of hazelnut ( Corylus avellana ) decline in northern Greece and central Italy. The partial sequence of the 16S rRNA gene of P. avellanae strain PD 2390, isolated in central Italy, was compared with the sequence coding for the same gene of P. syringae pv. syringae type-strain LMG 1247_t1. Primers PAV 1 and PAV 22 were chosen, and after the PCR, an amplification product of 762 base pairs was specifically produced only by 40 strains of P. avellanae isolated from northern Greece and central Italy. No other bacterial species among those tested showed an amplification product under optimized PCR conditions. The adding of 4% BLOTTO (10% skim milk powder and 0.2% NaN₃) in the PCR mixture proved essential in order to avoid interference of hazelnut extracts during the amplification. The procedure proved more effective than repetitive PCR with ERIC primer sets in diagnosing apparently healthy hazelnut trees as infected. This technique could be of great help for screening the hazelnut propagative material as well as for monitoring the wild C. avellana trees growing in the woods near the infected hazelnut orchards.     

Detection and Enumeration of Methanotrophs in Acidic Sphagnum Peat by 16S rRNA Fluorescence In Situ Hybridization, Including the Use of Newly Developed Oligonucleotide Probes for Methylocella palustris

Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10⁶ cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10⁴ type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10⁶ type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (±0.2) × 10⁶ cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.     

应用SSP-PCR/SSO方法进行中国辽宁汉族人HLA-DRB1基因的遗传多态性研究

对159名中国辽宁汉族个体的基因组DNA进行分析,共检出42种等位基因,其中以DRB1*09012(12.8%)、*0701(10.7%)、*1501(10.4%) 最为常见,其次为DRB1*1201(7.9%)、*1202(7.5%)、*1101(6.6%)、*0301(5.0%)。并发现辽宁汉族人DRB1等位基因频率与白种人间存在明显差异,揭示不同人种有其自己的主要等位基因。同时对本技术在HLA-DRB1分型应用中的优点进行了讨论。 Abstract　HLA-DRB1 alleles of 159 Chinese donors from Liaoning Han population were determined by using a set of 23 sequence specific oligonucleotide probes directed to various DRB1 alleles. The samples were first amplified and divided into 8 groups by allele/group specific primers. The SSOs enable us to identify 58 different DRB1subtypes. 42 alleles were detected in the study of this population. Among them, the DRB1*09012(12.8%), *0701(10.7%), *1501(10.4%), *1201(7.9%), *1202(7.5%), *1101(6.6%), *0301(5.0%) are the most frequent. The significant difference was found in Chinese northern Han population by comparing the gene frequencies in Caucasoid samples, suggesting that there were principal DRB1 alleles in different races.     

应用SSP-PCR/SSO方法进行中国辽宁汉族人HLA-DRB1基因的遗传多态性研究

对１５９名中国辽宁汉族个体的基因组ＤＮＡ进行分析,共检出４２种等位基因,其中以ＤＲＢ１０９０１２（１２．８％）、０７０１（１０．７％）、１５０１（１０．４％）最为常见,其次为ＤＲＢ１１２０１（７９％）、１２０２（７５％）、１１０１（６６％）、０３０１（５．０％）。并发现辽宁汉族人ＤＲＢ１等位基因频率与白种人间存在明显差异,揭示不同人种有其自己的主要等位基因。同时对本技术在ＨＬＡ－ＤＲＢ１分型应用中的优点进行了讨论     

鸡球虫18S rRNA基因序列的测定与分析

为了利用18S rRNA基因进行鸡球虫系统进化分析,对巨型艾美耳球虫(Eimeria maxima)、柔嫩艾美耳球虫(E.tenella)、堆形艾美耳球虫(E.acervulina)3种共8个不同来源的虫株,分别提取总DNA进行18S rRNA基因的扩增和测序;将得到的序列登录GenBank进行同源性和趋异性分析,并结合GenBank中其它原虫的18S rRNA基因序列构建进化树.结果显示扩增获得8株鸡球虫18S rRNA基因长度为1746～1756 bp,序列比对显示同种不同株间的同源性大于不同种间的同源性,其中3株E.maxima株间同源性在98.7%～99.3%之间,4株E.tenella株间同源性在99.7%～99.9%之间;不同种间同源性为96.5%～98.1%,其中E.maxima与E.tenclla的遗传距离最大,为0.038;E.maxima与E.acervulina的遗传距离最小,为0.021.顶复器门9个不同属所构建的进化树结果显示,E.imeria和等孢属(Isospora)聚为一支,说明亲缘关系比较近.与GertBank中其它5株不同鸡球虫的18S rRNA基因共同构建的进化树显示,3株E.maxima聚为一支,与E.brunetti、E.mitis、E.mivati、E.praecox和E.acervulina聚为一大分支;4株E.tenella与1株E.necatrix共同形成一个分支,说明E.tenella与E.necattix的亲缘关系最近.本研究证实了在鸡球虫系统进化研究中,18S rRNA基因不仅可以区分不同种,而且有可能成为区分同种不同株的理想靶基因.     

Design and Performance of rRNA Targeted Oligonucleotide Probes for in Situ Detection and Phylogenetic Identification of Microorganisms Inhabiting Acid Mine Drainage Environments

At Iron Mountain, CA, there is an extreme occurrence of acid mine drainage (AMD). This is a result of past mining activity that has exposed a sulfide ore body to weathering and microbial activity. This study presents seven new oligonucleotide probes for the detection of microorganisms at this AMD site by fluorescent in situ hybridization. In the design of these probes we have accounted for a large body of 16S rRNA sequence data recently compiled by us. This was obtained by PCR and cloning directly from environmental DNA and was mostly represented by novel sequences. The probes were developed to include detection of novel and uncultivated organisms. This includes detection for the Thermoplasmales group, a new group of Leptospirillum, the genus Sulfobacillus, the Acidiphilium genus, Acidimicrobium and relatives, and for organisms within the delta Proteobacteria. These probes have been used to examine the abundance and distribution of organisms, including novel and uncultivated taxa, and to clarify their potential contributions to AMD production at the site. We anticipate that these probes will be useful tools for exploration of the microbiology of other natural acidic environments and bioleaching systems.     

Composition of the Landfill Microbial Community as Determined by Application of Domain- and Group-Specific 16S and 18S rRNA-Targeted Oligonucleotide Probes

The microbial community composition of colonized cotton and leachate samples from a landfill was quantified using small subunit (SSU) rRNA probes (quantitative rRNA hybridization). Relative quantification of bacteria, eukaryotes, and archaea revealed variations in the landfill microbial community between samples from different areas of the landfill site and indicated the presence of potentially novel archaea. Anaerobic fungi were quantified in rumen fluid samples but were not sufficiently abundant for direct detection in the landfill samples.Molecular ecological studies should now be focused on assigning functions to the recognized microbial assemblages in any given environment. However, with respect to landfill microbiology, even the identity and abundance of the indigenous microbiota are barely understood. This is in part due to the unfeasibility of obtaining representative samples of the highly heterogeneous solid waste fraction, together with the inability to retrieve waste samples other than leachate from capped landfill sites (2). PCR-based molecular ecological studies of landfill leachate have nevertheless enabled the direct detection of species known to be involved in primary degradation of cellulose, the main carbon source in landfill (29), and more recently, taxa that had previously been thought to occur only in the herbivore gastrointestinal tract, i.e., anaerobic fungi (16) and fibrobacters (20). Landfill microcosms or leachate bioreactors have also been used to provide source material for DNA and culture-based analyses (3, 4), but the composition of these microbial communities has not been directly compared to those of the landfill sites themselves. In the field of molecular microbial ecology, the overreliance on analysis of sequences generated by PCR amplification of environmental DNA extracts has recently been questioned (13), primarily on the basis of the recognized variation in amplification efficiency with different DNA templates, a shortcoming which also applies to quantitative PCR techniques. Furthermore, there is evidence that probe-based methodologies for the detection of rRNA genes in environmental microarrays reveal a greater diversity of microbial taxa than does the traditional sequencing of clones from PCR amplification products (9).In this study, domain- and group-specific oligonucleotide probes were applied in slot blot hybridization experiments to quantify different obligately anaerobic microbial groups associated with cellulose degradation in rumen fluid, landfill leachate, and landfill microcosm RNA samples. The identity and molecular ecology of microorganisms capable of cellulose hydrolysis in the rumen are relatively well understood, and these samples therefore provided a reference point for evaluation of this approach as well as for providing fresh information on the abundance of anaerobic fungi in rumen fluid. Two landfill leachate microcosms were constructed with material from different waste cells within the same landfill site. Dewaxed cotton string (19) was incubated in each microcosm, and the RNA extracted from the cotton biofilm and the planktonic leachate-associated community was subjected to quantitative hybridization with domain-targeted probes and probes specific for particular subgroups of cellulolytic microorganisms.The almost entire small subunit (SSU) (16S/18S) rRNA gene in DNA extracted from each bacterial, archaeal, and eukaryotic control strain was amplified using the primer sets pA and pH′, 1Af and 1404R, NS1-Euk and Univ 1390, respectively (10, 21, 30, 33), and Phusion high-fidelity (HF) DNA polymerase (Finnzymes). PCRs were performed in 50-μl volumes containing the following: 0.2 mM each primer, 0.2 mM each deoxynucleoside triphosphate (dNTP), 1× Phusion HF buffer (Finnzymes), 3% dimethyl sulfoxide (DMSO), 1× bovine serum albumin (BSA), 1 unit Phusion HF DNA polymerase (Finnzymes) and double-distilled water (ddH₂O). PCR cycling conditions were as follows: 98°C for 45 s, 30 cycles of 98°C for 10 s, 30 s at the specific annealing temperature for each primer set, 72°C for 20 s, and a final extension of 72°C for 8 min. PCR amplification products were cloned into Escherichia coli JM109 cells (Promega) by using the pGEM-T Easy vector system I (Promega) according to the manufacturer''s protocol. Plasmid DNA was extracted and sequenced in both orientations by Macrogen, Inc. (South Korea). Cloned DNA sequences were assembled into contigs by using PreGap 4 and Gap 4 software (25), and base calling was visually checked using the sequencing traces. Reference rRNAs were synthesized from linearized plasmid DNA (3 μg) by using either T7 or SP6 RNA polymerase (Promega), and plasmid DNA was digested using a Turbo DNA-free kit (Ambion).To determine the specificity of an oligonucleotide probe for its target group, each probe was hybridized against a panel of 32 reference rRNAs from control strains, which included representatives of phylogenetic groups indigenous to landfill and rumen environments, and control rRNAs possessing 1- to 4-bp mismatches within the probe target site. Stock reference rRNAs (100 ng μl⁻¹) were denatured by adding 3 volumes of 2% (vol/vol) glutaraldehyde in 50 mM NaH₂PO₄ (pH 7.0), followed by incubation for 10 min at room temperature. The rRNA was then diluted to 2 ng μl⁻¹ by using a hybridization sample buffer (RNase-free water containing 1 μg ml⁻¹ poly[A] [Sigma] and 0.004% bromophenol blue). A 100-μl volume of each reference rRNA (200 ng) was blotted onto positively charged nylon membranes (Hybond) by using a slot blot device (Bio-Rad) under slight vacuum. Membranes were air dried, and rRNA was fixed to membranes by using a Stratalinker 2400 UV cross-linker (Stratagene). DNA oligonucleotide probes were 5′-end labeled with ³²P by using a T4 polynucleotide kinase (New England Biolabs) and [γ-³²P]ATP (Perkin Elmer) (8). Membranes were placed in glass hybridization tubes (Hybaid) and 100 μl hybridization buffer (0.9 M NaCl, 50 mM NaH₂PO₄ [pH 7.0], 5 mM EDTA, 10× Denhardt''s solution, 0.5% SDS, and 0.1 mg poly[A] ml⁻¹) added per cm² of membrane. Membranes were prehybridized for 2 h at 40°C. A labeled probe (400 μl) was subsequently added and hybridized at 40°C for 16 to 18 h in a Techne hybridizer HB-1 (Techne) and then washed for 15 min at 40°C in wash buffer (1× SSC [0.15 M NaCl plus 0.0015 M sodium citrate], 1% SDS) and washed two subsequent times for 30 min each at the optimum wash temperature (T_w) for each probe (Table ​(Table1).1). Phosphor screens (Molecular Dynamics) were used to visualize membranes, and hybridization signals were determined using a Storm 860 scanner (Molecular Dynamics) and quantified using TotalLab TL100 software (Nonlinear Dynamics).

TABLE 1.

SSU (16S/18S) rRNA oligonucleotide hybridization probes used in this study and their wash temperatures (T_w)

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

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 SRP-PhyloChip 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 microarray-independent techniques, including specific PCR amplification, cloning, and sequencing of SRP 16S rRNA genes and the genes encoding the dissimilatory (bi)sulfite reductase (dsrAB).     

Development of a cob-18S rRNA Gene Real-Time PCR Assay for Quantifying Pfiesteria shumwayae in the Natural Environment

Despite the fact that the heterotrophic dinoflagellate Pfiesteria shumwayae is an organism of high interest due to alleged toxicity, its abundance in natural environments is poorly understood. To address this inadequacy, a real-time quantitative PCR assay based on mitochondrial cytochrome b (cob) and18S rRNA gene was developed and P. shumwayae abundance was investigated in several geographic locations. First, cob and its 5′-end region were isolated from a P. shumwayae culture, revealing three different copies, each consisting of an identical cob coding region and an unidentified region (X) of variable length and sequence. The unique sequences in cob and the X region were then used to develop a P. shumwayae-specific primer set. This primer set was used with reported P. shumwayae-specific 18S primers in parallel real-time PCRs to investigate P.shumwayae abundance from Maine to North Carolina along the U.S. east coast and along coasts in Chile, Hawaii, and China. Both genes generally gave similar results, indicating that this species was present, but at low abundance (mostly <10 cells · ml⁻¹), in all the American coast locations investigated (with the exception of Long Island Sound, where which both genes gave negative results). Genetic variation was detected by use of both genes in most of the locations, and while cob consistently detected P. shumwayae or close genetic variants, some of the 18S PCR products were unrelated to P. shumwayae. We conclude that (i) the real-time PCR assay developed is useful for specific quantification of P. shumwayae, and (ii) P. shumwayae is distributed widely at the American coasts, but normally only as a minor component of plankton even in high-risk estuaries (Neuse River and the Chesapeake Bay).     

ITS-2 and 18S rRNA Gene Phylogeny of Aplysinidae (Verongida, Demospongiae)

18S ribosomal DNA and internal transcribed spacer 2 (ITS-2) full-length sequences, each of which was sequenced three times, were used to construct phylogenetic trees with alignments based on secondary structures, in order to elucidate genealogical relationships within the Aplysinidae (Verongida). The first poriferan ITS-2 secondary structures are reported. Altogether 11 Aplysina sponges and 3 additional sponges (Verongula gigantea, Aiolochroia crassa, Smenospongia aurea) from tropical and subtropical oceans were analyzed. Based on these molecular studies, S. aurea, which is currently affiliated with the Dictyoceratida, should be reclassified to the Verongida. Aplysina appears as monophyletic. A soft form of Aplysina lacunosa was separated from other Aplysina and stands at a basal position in both 18S and ITS-2 trees. Based on ITS-2 sequence information, the Aplysina sponges could be distinguished into a single Caribbean–Eastern Pacific cluster and a Mediterranean cluster. The species concept for Aplysina sponges as well as a phylogenetic history with a possibly Tethyan origin is discussed.Reviewing Editor: Dr. Martin Kreitman