Microbial Community Changes in a Perturbed Agricultural Soil Investigated by Molecular and Physiological Approaches

Changes in soil microbial activity and diversity after incubation either with nitrogen or with a mixture of methane and air were examined. The perturbation by methane and air were characterized in detail and led to reduced diversity and enrichment of methanotrophs which were identified by denaturing gradient gel electrophoresis and 16S rRNA sequencing.     

Abundance and Diversity of Archaea in Heavy-Metal-Contaminated Soils

The impact of heavy-metal contamination on archaean communities was studied in soils amended with sewage sludge contaminated with heavy metals to varying extents. Fluorescent in situ hybridization showed a decrease in the percentage of Archaea from 1.3% ± 0.3% of 4′,6-diamidino-2-phenylindole-stained cells in untreated soil to below the detection limit in soils amended with heavy metals. A comparison of the archaean communities of the different plots by denaturing gradient gel electrophoresis revealed differences in the structure of the archaean communities in soils with increasing heavy-metal contamination. Analysis of cloned 16S ribosomal DNA showed close similarities to a unique and globally distributed lineage of the kingdom Crenarchaeota that is phylogenetically distinct from currently characterized crenarchaeotal species.     

Phylogenetic relationships within the Alcidae (Charadriiformes: Aves) inferred from total molecular evidence

The Alcidae is a unique assemblage of Northern Hemisphere seabirds that forage by "flying" underwater. Despite obvious affinities among the species, their evolutionary relationships are unclear. We analyzed nucleotide sequences of 1,045 base pairs of the mitochondrial cytochrome b gene and allelic profiles for 37 allozyme loci in all 22 extant species. Trees were constructed on independent and combined data sets using maximum parsimony and distance methods that correct for superimposed changes. Alternative methods of analysis produced only minor differences in relationships that were supported strongly by bootstrapping or standard error tests. Combining sequence and allozyme data into a single analysis provided the greatest number of relationships receiving strong support. Addition of published morphological and ecological data did not improve support for any additional relationship. All analyses grouped species into six distinct lineages: (1) the dovekie (Alle alle) and auks, (2) guillemots, (3) brachyramphine murrelets, (4) synthliboramphine murrelets, (5) true auklets, and (6) the rhinoceros auklet (Cerorhinca monocerata) and puffins. The two murres (genus Uria) were sister taxa, and the black guillemot (Cepphus grylle) was basal to the other guillemots. The Asian subspecies of the marbled murrelet (Brachyramphus marmoratus perdix) was the most divergent brachyramphine murrelet, and two distinct lineages occurred within the synthliboramphine murrelets. Cassin's auklet (Ptychoramphus aleuticus) and the rhinoceros auklet were basal to the other auklets and puffins, respectively, and the Atlantic (Fratercula arctica) and horned (Fratercula corniculata) puffins were sister taxa. Several relationships among tribes, among the dovekie and auks, and among the auklets could not be resolved but resembled "star" phylogenies indicative of adaptive radiations at different depths within the trees.      

Spore-forming thermophilic sulfate-reducing bacteria isolated from north sea oil field waters

Thermophilic sulfate-reducing bacteria were isolated from oil field waters from oil production platforms in the Norwegian sector of the North Sea. Spore-forming rods dominated in the enrichments when lactate, propionate, butyrate, or a mixture of aliphatic fatty acids (C(4) through C(6)) was added as a carbon source and electron donor. Representative strains were isolated and characterized. The isolates grew autotrophically on H(2)-CO(2) and heterotrophically on fatty acids such as formate, propionate, butyrate, caproate, valerate, pyruvate, and lactate and on alcohols such as methanol, ethanol, and propanol. Sulfate, sulfite, and thiosulfate but not nitrate could be used as an electron acceptor. The temperature range for growth was 43 to 78 degrees C; the spores were extremely heat resistant and survived 131 degrees C for 20 min. The optimum pH was 7.0. The isolates grew well in salt concentrations ranging from 0 to 800 mmol of NaCl per liter. Sulfite reductase P582 was present, but cytochrome c and desulfoviridin were not found. Electron micrographs revealed a gram-positive cell organization. The isolates were classified as a Desulfotomaculum sp. on the basis of spore formation, general physiological characteristics, and submicroscopic organization. To detect thermophilic spore-forming sulfate-reducing bacteria in oil field water, polyvalent antisera raised against antigens from two isolates were used. These bacteria were shown to be widespread in oil field water from different platforms. The origin of thermophilic sulfate-reducing bacteria in the pore water of oil reservoirs is discussed.     

Stress protein of cyanobacteria CP36: interaction with photoactive complexes and formation of supramolecular structures

Cyanobacterium Anacystis nidulans R2, Synechocystis sp. PCC 6803 (wild-type strain and mutants Delta2 and Delta3 lacking PSII and PSI, respectively), and Synechocystis sp. BO 9201 synthesize the pigment--protein complex CP36 (CPIV-4, CP43') under iron deficiency in the medium. Accumulation of CP36 is accompanied by structural reorganizations in the photosynthetic membranes. Integrating mean times of excitation relaxation (quenching) are 2.2 nsec (CP36), 1 nsec (PSI), and 420 psec (PSII in Fm state). The energy migration between CP36 and the photosystems can be described by a model of a one-layer ring of CP36 around core-complexes. The excitation from CP36 to PSI is transferred within <10 psec. The energy transfer from CP36 to PSII occurs during 170 psec. Cells with low content of CP36 probably contain only a latent fraction of unbound to phycobilisomes PSII which is the analog of PSIIbeta of higher plants. In PSI there are four binding sites for CP36 monomers per RC. PSII can bind up to 32 molecules of CP36 per RC. Cells with a large amount of CP36 contain monomer form of PSII core-complex which can bind eight tetramers of CP36 (8 binding sites). In conditions of iron deficiency only one monomer of a dimer PSII core-complex is destroyed and released chlorophyll is accumulated in CP36. Accumulation of CP36 in A. nidulans cells can be accompanied by membrane stacking which is similar to the stacking in chlorophyll b-containing organisms. The stacking can occur in the region of localization of PSII latent fraction bound to CP36. The membrane stacking shields PSII stromal surfaces from the aqueous phase for activation of electron transfer on the acceptor side of PSII.     

Methanotrophic diversity in an agricultural soil as evaluated by denaturing gradient gel electrophoresis profiles of pmoA, mxaF and 16S rDNA sequences

Molecular methods were used to characterize the diversity of a methanotrophic population in an agricultural soil. For this purpose we have used DGGE analysis of functional and phylogenetic markers. Functional markers utilised comprised the pmoA-gene coding for the -subunit of the particulate methane monooxygenase (pMMO) present in all known methanotrophs and the mxaF-gene coding for the -subunit of methanol dehydrogenase (MDH) present in all Gram-negative methylotrophs. In addition, we have used 16S rDNA as a phylogenetic marker. DGGE patterns of an enrichment culture, and sequencing of major DGGE bands obtained with the bacterial specific primers showed that the community structure was dominated by methanotrophic populations related to Methylobacter sp. and Methylomicrobium sp. The PCR products amplified with the functional primer sets were related to both type I and type II methanotrophs. We also designed a new pmoA-targeting primer set which could be used in a nested protocol to amplify PCR-products from DNA extracted directly from the soil.     

Phenotypic diversity and antibiotic resistance in soil bacterial communities

The community structure in two different agricultural soils has been investigated. Phenotypic diversity was assessed by applying BIOLOG-profiles on a total of 208 bacterial isolates. Diversity indices were calculated from cluster analysis of the BIOLOG data. The bacterial isolates were also evaluated for resistance towards six different antibiotics, mercury resistance and the presence of plasmids. The presence of tetracycline-resistant determinants class A to E among Gram-negative bacteria was analysed with DNA probes. The distribution of tetracycline resistance markers among colonies growing on non-selective and tetracycline-selective plates were compared. The phenotypic approach demonstrated some difference in the diversity within the two soils. The frequency of antibiotic resistance isolates was high in both soils, whereas the frequency of mercury resistance differed significantly. We found no correlation between plasmid profiles and antibiotic resistance patterns. We found all the tetracycline resistance determinants except class B, indicating that the diversity of the tetracycline resistance determinants was complex in populations of resident soil bacteria under no apparent selective pressure for the genes in question.     

Mutations in the VNTR of the carboxyl-ester lipase gene (CEL) are a rare cause of monogenic diabetes

We have previously shown that heterozygous single-base deletions in the carboxyl-ester lipase (CEL) gene cause exocrine and endocrine pancreatic dysfunction in two multigenerational families. These deletions were found in the first and fourth repeats of a variable number of tandem repeats (VNTR), which has proven challenging to sequence due to high GC-content and considerable length variation. We have therefore developed a screening method consisting of a multiplex PCR followed by fragment analysis. The method detected putative disease-causing insertions and deletions in the proximal repeats of the VNTR, and determined the VNTR-length of each allele. When blindly testing 56 members of the two families with known single-base deletions in the CEL VNTR, the method correctly assessed the mutation carriers. Screening of 241 probands from suspected maturity-onset diabetes of the young (MODY) families negative for mutations in known MODY genes (95 individuals from Denmark and 146 individuals from UK) revealed no deletions in the proximal repeats of the CEL VNTR. However, we found one Danish patient with a short, novel CEL allele containing only three VNTR repeats (normal range 7–23 in healthy controls). This allele co-segregated with diabetes or impaired glucose tolerance in the patient’s family as six of seven mutation carriers were affected. We also identified individuals who had three copies of a complete CEL VNTR. In conclusion, the CEL gene is highly polymorphic, but mutations in CEL are likely to be a rare cause of monogenic diabetes.