Diversity, Abundance, and Activity of Archaeal Populations in Oil-Contaminated Groundwater Accumulated at the Bottom of an Underground Crude Oil Storage Cavity

Fluorescence in situ hybridization has shown that cells labeled with an Archaea-specific probe (ARCH915) accounted for approximately 10% of the total cell count in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity. Although chemical analyses have revealed vigorous consumption of nitrate in cavity groundwater, the present study found that the methane production rate was higher than the nitrate consumption rate. To characterize the likely archaeal populations responsible for methane production in this system, fragments of 16S ribosomal DNA (rDNA) were amplified by PCR using eight different combinations of universal and Archaea-specific primers. Sequence analysis of 324 clones produced 23 different archaeal sequence types, all of which were affiliated with the kingdom Euryarchaeota. Among them, five sequence types (KuA1, KuA6, KuA12, KuA16, and KuA22) were obtained in abundance. KuA1 and KuA6 were closely related to the known methanogens Methanosaeta concilii (99% identical) and Methanomethylovorans hollandica (98%), respectively. Although no closely related organism was found for KuA12, it could be affiliated with the family Methanomicrobiaceae. KuA16 and KuA22 showed substantial homology only to some environmental clones. Both of these branched deeply in the Euryarchaeota, and may represent novel orders. Quantitative competitive PCR showed that KuA12 was the most abundant, accounting for ~50% of the total archaeal rDNA copies detected. KuA1 and KuA16 also constituted significant proportions of the total archaeal rDNA copies (7 and 17%, respectively). These results suggest that limited species of novel archaea were enriched in the oil storage cavity. An estimate of specific methane production rates suggests that they were active methanogens.     

Characterization of heterotrophic prokaryote subgroups in the Sfax coastal solar salterns by combining flow cytometry cell sorting and phylogenetic analysis

Here, we combined flow cytometry (FCM) and phylogenetic analyses after cell sorting to characterize the dominant groups of the prokaryotic assemblages inhabiting two ponds of increasing salinity: a crystallizer pond (TS) with a salinity of 390 g/L, and the non-crystallizer pond (M1) with a salinity of 200 g/L retrieved from the solar saltern of Sfax in Tunisia. As expected, FCM analysis enabled the resolution of high nucleic acid content (HNA) and low nucleic acid content (LNA) prokaryotes. Next, we performed a taxonomic analysis of the bacterial and archaeal communities comprising the two most populated clusters by phylogenetic analyses of 16S rRNA gene clone library. We show for the first time that the presence of HNA and LNA content cells could also be extended to the archaeal populations. Archaea were detected in all M1 and TS samples, whereas representatives of Bacteria were detected only in LNA for M1 and HNA for TS. Although most of the archaeal sequences remained undetermined, other clones were most frequently affiliated to Haloquadratum and Halorubrum. In contrast, most bacterial clones belonged to the Alphaproteobacteria class (Phyllobacterium genus) in M1 samples and to the Bacteroidetes phylum (Sphingobacteria and Salinibacter genus) in TS samples.     

Microbial Diversity in Maras Salterns, a Hypersaline Environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 × 10⁶ to 3 × 10⁶ cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4′,6′-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon “Haloquadra walsbyi,” although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the γ-proteobacterium “Pseudomonas halophila” DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the “P. halophila” cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

Diversity and Community Structure of Archaea in Deep Subsurface Sediments from the Tropical Western Pacific

Archaeal 16S rRNA gene clone libraries using PCR amplicons from eight different layers of the MD06-3051 core were obtained from the tropical Western Pacific sediments. A total of 768 clones were randomly selected, and 264 representative clones were sequenced by restriction fragment length polymorphism. Finally, 719 valid clones and 104 operational taxonomic units were identified after chimera-check and ≥97% similarity analysis. The phylogenetic analysis of 16S rDNA sequences obtained from sediment samples were very diverse and showed stratification with depth. Majority of the members were most closely related to uncultivated groups and physiologically uncharacterized assemblages. All phylotypes were affiliated with Crenarchaeota (76%) and Euryarchaeota (24%), respectively. Deep-sea archaeal group (DSAG, 41% of total clones) and miscellaneous crenarchaeotic group (MCG, 29% of total clones) belonging to Crenarchaeota were the most predominant archaeal 16S rDNA phylotypes in clone libraries. Phylotypes in this study shared high similarity with those in subsurface sediments from Peru Margin sites, which indicated that different geographical zones might host similar members of archaeal populations based on similar sedimentary environments. In our study, members of DSAG and MCG seemed to dominate certain layers of the nonhydrate sediments, suggesting a wide ecophysiological adaptation than previously appreciated. The spatial distribution and community structure of these groups might vary with the different geochemical gradients of the environment.     

Prokaryotic diversity of a Tunisian multipond solar saltern

16S rRNA gene clone libraries were separately constructed from three ponds with different salt concentrations, M2 (15%), TS38 (25%) and S5 (32%), located within a multipond solar saltern of Sfax. The 16S rRNA genes from 216 bacterial clones and 156 archaeal clones were sequenced and phylogenetically analyzed. 44 operational taxonomic units (OTUs) were generated for Bacteria and 67 for Archaea. Phylogenetic groups within the bacterial domain were restricted to Bacteroidetes and Proteobacteria, with the exception that one cyanobacterial OTU was found in the TS38 pond. 85.7, 26.6 and 25.0% of the bacterial OTUs from M2, TS38 and S5 ponds, respectively, are novel. All archaeal 16S rRNA gene sequences were exclusively affiliated with Euryarchaeota. 75.0, 60.0 and 66.7% of the OTUs from, respectively, M2, TS38 and S5 ponds are novel. The result showed that the Tunisian multipond solar saltern harbored novel prokaryotic diversity that has never been reported before for solar salterns. In addition, diversity measurement indicated a decrease of bacterial diversity and an increase of archaeal diversity with rising salinity gradient, which was in agreement with the previous observation for thalassohaline systems. Comparative analysis showed that prokaryotic diversity of Tunisian saltern was higher than that of other salterns previously studied. A. Sghir and E. Ammar have equally contributed to this work.     

Molecular characterization and cytogenetic analysis of highly repeated DNAs of lake trout,Salvelinus namaycush

The chromosomes of lake trout (Salvelinus namaycush) contain a considerable amount of heterochromatin located at the centromeres and/or telomeres of several chromosomes, including a sex-specific block located distally on the X chromosome. In order to investigate further the repetitive DNAs of lake trout, genomic DNA from a female was size fractionated (<600 bp) with the restriction endonuclease AluI and fragments were cloned into the bacteriophage M13. A total of 42 clones were isolated. Relative copy number of individual inserts within the lake trout genome was estimated by Southern analysis. Twelve clones were determined to be highly repetitive and were chosen for further investigation. Inserts of these clones contained sequences similar to the AluI/RsaI, EcoRI/DraI, DraI/BstEII, and MboI/BglII families reported from Arctic char (Salvelinus alpinus). The chromosomal location of several of these fragments was determined in lake trout by fluorescence in situ hybridization (FISH). Two related AluI/RsaI sequences (Type A, 140 bp, and Type B, 120 bp) showed differential hybridization. Type A hybridized to the centromeres of all metacentric as well as several acrocentric chromosomes. Type B hybridized to the centromeres of most acrocentric chromosomes. A sequence with homology to the EcoRI/DraI family hybridized to the centromeres of several acrocentric chromosomes. Sequences with partial similarity to the DraI/BstEII family hybridized to the major rDNA sites (nucleolar organizer regions, NORs) and several minor telomeric sites. The interstitial and telomeric heterochromatin of lake trout, including that of the X chromosome, appears to comprise sequences belonging to the MboI/BglII family.     

Archaeal Diversity in the Haloalkaline Lake Elmenteita in Kenya

A non-culture approach was used to study the archaeal diversity in Lake Elmenteita, Kenya. Five different sampling points were selected randomly within the lake. Wet sediments and water samples were collected from each sampling point. In addition, dry mud cake was collected from three points where the lake had dried. DNA was extracted from these samples and the 16S rRNA genes were amplified using primers described to be Domain-specific for Archaea. Eleven clone libraries were constructed using PCR-amplified 16S rRNA genes. A total of 1,399 clones were picked and analysed via ARDRA. 170 ARDRA patterns were unique and the respective clones were selected for sequencing. 149 clones gave analysable sequences. BLAST analysis showed that 49 belong to the Domain Archaea while the others were either chimera or affiliated to eukaryotic taxa. Comparative sequence analysis of archaeal clones affiliated them to a wide range of genera. The order Halobacteriales was represented by members of the genera Natronococcus, Halovivax, Halobiforma, Halorubrum, and Halalkalicoccus. The highest percentage (46%) of the clones, however, belonged to uncultured members of the Domain Archaea in the order Halobacteriales. The results show that the archaeal diversity in the lake could be higher than previously reported.     

Quantification of ratios of Bacteria and Archaea in methanogenic microbial community by fluorescence in situ hybridization and fluorescence spectrometry

16S rRNA-targeted oligonucleotide probes for Bacteria (Eub338) and Archaea (Arc915) were used for whole-cell, fluorescence in situ hybridization (FISH) to quantify the ratio of these microbial groups in an anaerobic digester. The quantity of specifically bound (hybridized) probe was measured by fluorescence spectrometry and evaluated by analysing the dissociation curve of the hybrids, by the measurement of the binding with a nonsense probe, and by the competitive inhibition of the binding of the labelled probe by the corresponding unlabelled probe. Specific binding of oligonucleotide probes with the biomass of anaerobes was 40–50% of their total binding. The ratio of Arc915 and Eub338 probes hybridized with rRNAs of the cells in anaerobic sludge was 0.50. Measurement of FISH by fluorescence spectrometry appears to be a suitable method for quantification of the microbial community of anaerobes.     

An RFLP marker in tomato linked to the Fusarium oxysporum resistance gene I2

Summary The locus, I2, which in tomato confers resistance against Fusarium oxysporum f. sp. lycopersici race 2, was introgressed into Lycopersicon esculentum from the wild species L. pimpinellifolium (P.I. 126915). We searched for restriction fragment length polymorphisms (RFLPs) between nearly isogenic lines (NILs) in clones that map to the region introgressed from the wild species. Since I2 maps to chromosome 11, we used DNA clones from this chromosome as hybridization probes to Southern blots containing bound DNA of the NILs digested with 23 restriction enzymes. Of the 14 chromosome 11 clones, 9 exhibited polymorphism. These clones were further hybridized to verification filters that contained DNA from resistant and susceptible L. esculentum varieties digested with the enzymes that gave the polymorphism. One clone, TG105, was found to be associated with I2; 19 susceptible lines showed a different RFLP with this probe than 16 resistant lines, including the original L. pimpinellifolium accession used as a source for the resistance gene. These results together with our mapping analysis indicate that TG105 is closely linked to the resistance gene.     

RFLP analysis of mitochondrial DNA from cytoplasmic male-sterile lines of pearl millet

Mitochondrial DNA (mtDNA) from 13 cytoplasmic male-sterile (cms) lines from diverse sources were characterized by Southern blot hybridization to pearl millet and maize mtDNA probes. Hybridization patterns of mtDNA digested with PstI, BamHI, SmaI or XhoI and probed with 13.6-, 10.9-, 9.7- or 4.7-kb pearl millet mtDNA clones revealed similarities among the cms lines 5141 A and ICMA 1 (classified as the S-A1 type of cytoplasm based on fertility restoration patterns), PMC 30A and ICMA 2. The remaining cms lines formed a distinct group, within which three subgroups were evident. Among the maize mitochondiral gene clones used, the coxI probe revealed two distinct groups of cytoplasms similar to the pearl millet mtDNA clones. The atp9 probe differentiated the cms line 81 A4, derived from P. glaucum subsp. monodii, while the coxII gene probe did not detect any polymorphism among the cms lines studied. MtDNA digested with BamHI, PstI or XhoI and hybridized to the atp6 probe revealed distinct differences among the cms lines. The maize atp6 gene clone identified four distinct cytoplasmic groups and four subgroups within a main group. The mtDNA fragments hybridized to the atp6 gene probe with differing intensities, suggesting the presence of more than one copy of the gene in different stoichiometries. Rearrangements involving the coxI and/or rrn18-rrn5 genes (mapped within the pearl millet clones) probably resulted in the S-A1 type of sterility. Rearrangements involving the atp6 gene (probably resulting in chimeric form) may be responsible for male sterility in other cms lines of pearl millet.     

Cloning and structural analyses of partial nuclear rDNA fromBupleurum euphorbioides (Apiaceae)

For the cloning of nuclear ribosomal DNA (rDNA) fromBupleurum euphorbioides (Apiaceae), ten clones were screened by DNA-DNA hybridization method. Among them, two clones were strongly hybridized with a heterologous probe of rice rDNA and with an autologous probe of an internally-transcribed region ofB. euphorbioides amplified by PCR. We sequenced both ends of the two genomic clones aligned with a known sequence of rDNA. ITS2 sequences of the two clones showed 98% and 83% homology with the ITS2 sequence ofB. euphorbioides. Our clones showed 1 bp and 3 bp nucleotide substitutions in the 25S and intergenic spacer regions, respectively, and the ITS1 and 18S regions were both missing. Restriction enzyme sites and the orientation of both clones were analyzed for physical mapping purposes. Apart from the length difference between the two clones, we found restriction site variations in the 25S and intergenic spacer regions.     

Microbial diversity in Maras salterns, a hypersaline environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 x 10(6) to 3 x 10(6) cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4',6'-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon "Haloquadra walsbyi," although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the gamma-proteobacterium "Pseudomonas halophila" DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the "P. halophila" cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

BAC-FISH in wheat identifies chromosome landmarks consisting of different types of transposable elements

Fluorescence in situ hybridization (FISH) has been widely used in the physical mapping of genes and chromosome landmarks in plants and animals. Bacterial artificial chromosomes (BACs) contain large inserts making them amenable for FISH mapping. We used BAC-FISH to study genome organization and evolution in hexaploid wheat and its relatives. We selected 56 restriction fragment length polymorphism (RFLP) locus-specific BAC clones from libraries of Aegilops tauschii (the D-genome donor of hexaploid wheat) and A-genome diploid Triticum monococcum. Different types of repetitive sequences were identified using BAC-FISH. Two BAC clones gave FISH patterns similar to the repetitive DNA family pSc119; one BAC clone gave a FISH pattern similar to the repetitive DNA family pAs1. In addition, we identified several novel classes of repetitive sequences: one BAC clone hybridized to the centromeric regions of wheat and other cereal species, except rice; one BAC clone hybridized to all subtelomeric chromosome regions in wheat, rye, barley and oat; one BAC clone contained a localized tandem repeat and hybridized to five D-genome chromosome pairs in wheat; and four BAC clones hybridized only to a proximal region in the long arm of chromosome 4A of hexaploid wheat. These repeats are valuable markers for defined chromosome regions and can also be used for chromosome identification. Sequencing results revealed that all these repeats are transposable elements (TEs), indicating the important role of TEs, especially retrotransposons, in genome evolution of wheat.Communicated by P.B. Moens     

Candidatus Monilibacter spp., common bulking filaments in activated sludge, are members of Cluster III Defluviicoccus

Two alphaproteobacterial Neisser negative ‘Nostocoida limicola’ morphotypes differing slightly in their trichome diameter and filament regularity were dominant populations in the Bendigo, Victoria, Australia activated sludge community removing phosphorus (P). Neither responded to the FISH probes available for any of the other alphaproteobacterial ‘N. limicola’ morphotypes. Instead both fluoresced with the DF988 FISH probe designed originally to target alphaproteobacterial cluster II Defluviicoccus tetrad forming organisms. A 16S rRNA based clone library from this biomass revealed that the alphaproteobacterial clones grouped closely with Candidatus ‘Monilibacter batavus’ and Defluviicoccus clones in a cluster separate from the existing cluster I and II Defluviicoccus. When a FISH probe was designed against these, it only hybridized to the thinner and less abundant ‘N. limicola’ morphotype. Micromanipulation–RT-PCR was used to selectively recover the main ‘N. limicola’ morphotype and a FISH probe designed against the 16S rRNA clones generated from it showed only this filament fluoresced. From FISH based surveys, both ‘N. limicola’ variants occurred frequently in phosphorus removal activated sludge systems in Australia treating domestic waste. The data suggest that they represent two new strains of Candidatus ‘Monilibacter’, which on this evidence are filamentous members of the genus Defluviicoccus, a potential competitor for the polyphosphate accumulating organisms in these communities.     

A few cosmopolitan phylotypes dominate planktonic archaeal assemblages in widely different oceanic provinces

We compared the phylogenetic compositions of marine planktonic archaeal populations in different marine provinces. Samples from eight different environments were collected at two depths (surface and aphotic zone), and 16 genetic libraries of PCR-amplified archaeal 16S rRNA genes were constructed. The libraries were analyzed by using a three-step hierarchical approach. Membrane hybridization experiments revealed that most of the archaeal clones were affiliated with one of the two groups of marine archaea described previously, crenarchaeotal group I and euryarchaeotal group II. One of the 2,328 ribosomal DNA clones analyzed was related to a different euryarchaeal lineage, which was recently recovered from deep-water marine plankton. In temperate regions (Pacific Ocean, Atlantic Ocean, and Mediterranean Sea) both major groups were found at the two depths investigated; group II predominated at the surface, and group I predominated at depth. In Antarctic and subantarctic waters group II was practically absent. The clonal compositions of archaeal libraries were investigated by performing a restriction fragment length polymorphism (RFLP) analysis with two tetrameric restriction enzymes, which defined discrete operational taxonomic units (OTUs). The OTUs defined in this way were phylogenetically consistent; clones belonging to the same OTU were closely related. The clonal diversity as determined by the RFLP analysis was low, and most libraries were dominated by only one or two OTUs. Some OTUs were found in samples obtained from very distant places, indicating that some phylotypes were ubiquitous. A tree containing one example of each OTU detected was constructed, and this tree revealed that there were several clusters within archaeal group I and group II. The members of some of these clusters had different depth distributions.     

A Few Cosmopolitan Phylotypes Dominate Planktonic Archaeal Assemblages in Widely Different Oceanic Provinces

We compared the phylogenetic compositions of marine planktonic archaeal populations in different marine provinces. Samples from eight different environments were collected at two depths (surface and aphotic zone), and 16 genetic libraries of PCR-amplified archaeal 16S rRNA genes were constructed. The libraries were analyzed by using a three-step hierarchical approach. Membrane hybridization experiments revealed that most of the archaeal clones were affiliated with one of the two groups of marine archaea described previously, crenarchaeotal group I and euryarchaeotal group II. One of the 2,328 ribosomal DNA clones analyzed was related to a different euryarchaeal lineage, which was recently recovered from deep-water marine plankton. In temperate regions (Pacific Ocean, Atlantic Ocean, and Mediterranean Sea) both major groups were found at the two depths investigated; group II predominated at the surface, and group I predominated at depth. In Antarctic and subantarctic waters group II was practically absent. The clonal compositions of archaeal libraries were investigated by performing a restriction fragment length polymorphism (RFLP) analysis with two tetrameric restriction enzymes, which defined discrete operational taxonomic units (OTUs). The OTUs defined in this way were phylogenetically consistent; clones belonging to the same OTU were closely related. The clonal diversity as determined by the RFLP analysis was low, and most libraries were dominated by only one or two OTUs. Some OTUs were found in samples obtained from very distant places, indicating that some phylotypes were ubiquitous. A tree containing one example of each OTU detected was constructed, and this tree revealed that there were several clusters within archaeal group I and group II. The members of some of these clusters had different depth distributions.     

Comparison and Characterization of Microbial Communities in Sulfide-rich Wastewater with and without Propidium Monoazide Treatment

A 16S rRNA gene-based culture-independent approach was used to study the bacterial and archaeal communities in a sulfide-rich wastewater. Propidium Monoazide (PMA) treatment was applied to limit the analysis to the fraction of viable cells in environment. A total of 104 and 68 clones respective from bacterial clone library and archaeal library were picked and analyzed by restriction fragment length polymorphism (RFLP). 35 RFLP patterns from bacterial clone library and 10 RFLP patterns from archaeal clone library were unique and the respective clones were selected for sequencing. BLAST analysis and RFLP analysis showed that the bacterial clone library mainly consisted of Gammaproteobacteria (73%), Anaerolineae (6%), Bacilli (5%), Deltaproteobacteria (7%), Clostridia (4%), Bacteroidetes (1%), and Chlorobia (1%); Methanomicrobia (99%) and Thermococci (1%) were the only two lineages of the archaeal domains. This study gave a first insight into the overall microbial structure in a cloth printing and dyeing wastewater treatment plant with high concentration of sulfide and increased knowledge on the applicability of the PMA treatment in combination with PCR-based molecular techniques to analyze only viable cells in microbial ecology.     

Molecular characterization of activated sludge from a seawater‐processing wastewater treatment plant

The prokaryotic community composition of activated sludge from a seawater‐processing wastewater treatment plant (Almeria, Spain) was investigated by using the rRNA approach, combining different molecular techniques such as denaturing gradient gel electrophoresis (DGGE), clone libraries and in situ hybridization (FISH and CARD‐FISH). Most of the sequences retrieved in the DGGE and the clone libraries were similar to uncultured members of different phyla. The most abundant sequence recovered from Bacteria in the clone library corresponded to a bacterium from the Deinococcus–Thermus cluster (almost 77% of the clones), and the library included members from other groups such as the Alpha, Gamma and Delta subclasses of Proteobacteria, the Bacteroidetes and Firmicutes. Concerning the archaeal clone library, we basically found sequences related to different orders of methanogenic Archaea, in correspondence with the recovered DGGE bands. Enumeration of DAPI (4′,6‐diamidino‐2‐phenylindole) stained cells from two different activated sludge samples after a mechanical flocculation disruption revealed a mean cell count of 1.6 × 10⁹ ml^?1. Around 94% of DAPI counts (mean value from both samples) hybridized with a Bacteria specific probe. Alphaproteobacteria were the dominant bacterial group (36% of DAPI counts), while Beta‐, Delta‐ and Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes contributed to lower proportions (between 0.5–5.7% of DAPI counts). Archaea accounted only for 6% of DAPI counts. In addition, specific primers for amplification of the amoA (ammonia monooxygenase) gene were used to detect the presence of Beta, Gamma and archaeal nitrifiers, yielding positive amplifications only for Betaproteobacteria. This, together with negative in situ hybridizations with probes for well‐known nitrifiying bacteria, suggests that nitrification is performed by still undetected microorganisms. In summary, the combination of the three approaches provided different and complementary pictures of the real assemblage composition and allowed to get closer to the main microorganisms involved in key processes of seawater‐processing activated sludge.     

Tomato extensin and extensin-like cDNAs: structure and expression in response to wounding

Two tomato cDNA libraries were synthesized from poly(A)⁺ RNAs isolated from unwounded and wounded tomato stems. These cDNA libraries were packaged in gt10 and screened by in situ plaque hybridization with a tomato extensin gene clone (pTom 5.10). Several cDNA clones were identified and isolated from both libraries in this manner and subjected to restriction enzyme digestion, Southern gel blot hybridization, RNA gel blot hybridization, and DNA sequence analyses. From these analyses, the various cDNA clones were found to fall into one of five distinct classes (classes I–V). Class I clones hybridized to a 4.0 kb mRNA which accumulated markedly after wounding and encoded an extensin characterized largely by Ser-(Pro)₄-Ser-Pro-Ser-(Pro)₄-(Tyr)₃-Lys repeats. Class II clones hybridized to a 2.6 kb mRNA which showed no accumulation following wounding and encoded an extensin containing Ser-(Pro)₄-Ser-Pro-Ser-(Pro)₄-Thr-(Tyr)_1–3-Ser repeats. Class III clones hybridized to a 0.6 kb mRNA which greatly accumulated in response to wounding and encoded a glycine-rich protein (GRP) with (Gly)_2–6-Tyr-Pro and(Gly)_2–6-Arg repeats. Class IV clones contained both class I and class III DNA sequences and consequently hybridized to both the 4.0 kb and the 0.6 kb wound-accumulating mRNAs; these clones encoded a portion of a GRP sequence on one DNA strand and encoded a portion of an extensin sequence on the other DNA strand. Class V clones hybridized to a 2.3 kb mRNA which decreased following wounding and encoded a GRP sequence characterized by (Gly)_2–5-Arg repeats.     

Diversity,abundance, and activity of archaeal populations in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity

Fluorescence in situ hybridization has shown that cells labeled with an Archaea-specific probe (ARCH915) accounted for approximately 10% of the total cell count in oil-contaminated groundwater accumulated at the bottom of an underground crude oil storage cavity. Although chemical analyses have revealed vigorous consumption of nitrate in cavity groundwater, the present study found that the methane production rate was higher than the nitrate consumption rate. To characterize the likely archaeal populations responsible for methane production in this system, fragments of 16S ribosomal DNA (rDNA) were amplified by PCR using eight different combinations of universal and Archaea-specific primers. Sequence analysis of 324 clones produced 23 different archaeal sequence types, all of which were affiliated with the kingdom EURYARCHAEOTA: Among them, five sequence types (KuA1, KuA6, KuA12, KuA16, and KuA22) were obtained in abundance. KuA1 and KuA6 were closely related to the known methanogens Methanosaeta concilii (99% identical) and Methanomethylovorans hollandica (98%), respectively. Although no closely related organism was found for KuA12, it could be affiliated with the family METHANOMICROBIACEAE: KuA16 and KuA22 showed substantial homology only to some environmental clones. Both of these branched deeply in the Euryarchaeota, and may represent novel orders. Quantitative competitive PCR showed that KuA12 was the most abundant, accounting for approximately 50% of the total archaeal rDNA copies detected. KuA1 and KuA16 also constituted significant proportions of the total archaeal rDNA copies (7 and 17%, respectively). These results suggest that limited species of novel archaea were enriched in the oil storage cavity. An estimate of specific methane production rates suggests that they were active methanogens.