Frida Lise Daae

Soil bacterium DNA was isolated by minor modifications of previously described methods. After purification on hydroxyapatite and precipitation with cetylpyridinium bromide, the DNA was sheared in a French press to give fragments with an average molecular mass of 420,000 daltons. After repeated hydroxyapatite purification and precipitation with cetylpyridinium bromide, high-pressure liquid chromatography analysis showed the presence of 2.1% RNA or less, whereas 5-methylcytosine made up 2.9% of the total deoxycytidine content. No other unusual bases could be detected. The hyperchromicity was 31 to 36%, and the melting curve in 1 X SSC (0.15 M NaCl plus 0.015 M sodium citrate) corresponded to 58.3 mol% G+C. High-pressure liquid chromatography analysis of two DNA samples gave 58.6 and 60.8 mol% G+C. The heterogeneity of the DNA was determined by reassociation of single-stranded DNA, measured spectrophotometrically. Owing to the high complexity of the DNA, the reassociation had to be carried out in 6 X SSC with 30% dimethyl sulfoxide added. Cuvettes with a 1-mm light path were used, and the A275 was read. DNA concentrations as high as 950 micrograms ml-1 could be used, and the reassociation rate of Escherichia coli DNA was increased about 4.3-fold compared with standard conditions. C0t1/2 values were determined relative to that for E. coli DNA, whereas calf thymus DNA was reassociated for comparison. Our results show that the major part of DNA isolated from the bacterial fraction of soil is very heterogeneous, with a C0t1/2 about 4,600, corresponding to about 4,000 completely different genomes of standard soil bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

The community structure of bacterioplankton in meromictic Lake Saelenvannet was examined by PCR amplification of the V3 region of 16S rRNA from microbial communities recovered from various depths in the water column. Two different primer sets were used, one for amplification of DNA from the domain Bacteria and another specific for DNA from the domain Archaea. Amplified DNA fragments were resolved by denaturing gradient gel electrophoresis (DGGE), and the resulting profiles were reproducible and specific for the communities from different depths. Bacterial diversity estimated from the number and intensity of specific fragments in DGGE profiles decreased with depth. The reverse was true for the Archaea, with the diversity increasing with depth. Hybridization of DGGE profiles with oligonucleotide probes specific for phylogenetic groups of microorganisms showed the presence of both sulfate-reducing bacteria and methanogens throughout the water column, but they appeared to be most abundant below the chemocline. Several dominant fragments in the DGGE profiles were excised and sequenced. Among the dominant populations were representatives related to Chlorobium phaeovibrioides, chloroplasts from eukaryotic algae, and unidentified Archaea.

The bacterial and archaeal assemblages at two offshore sites located in polar (Greenland Sea; depth: 50 and 2000 m) and Mediterranean (Ionian Sea; depth 50 and 3000 m) waters were studied by PCR amplification and sequencing of the last 450-500 bp of the 16S rRNA gene. A total of 1621 sequences, together with alignable 16S rRNA gene fragments from the Sargasso Sea metagenome database, were analysed to ascertain variations associated with geographical location and depth. The Ionian 50 m sample appeared to be the most diverse and also had remarkable differences in terms of the prokaryotic groups retrieved; surprisingly, however, many similarities were found at the level of large-scale diversity between the Sargasso database fragments and the Greenland 50 m sample. Most sequences with more than 97% sequence similarity, a value often taken as indicative of species delimitation, were only found at a single location/depth; nevertheless, a few examples of cosmopolitan sequences were found in all samples. Depth was also an important factor and, although both deep-water samples had overall similarities, there were important differences that could be due to the warmer waters at depth of the Mediterranean Sea.

Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously. These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods. Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers). Clone libraries focused on ponds with salinity of 8%, 22% and 32%. Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences. Thus, the use of two sets of primers recovered a higher number of phylotypes. Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences. For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin. Thus, representatives of the alpha-, beta-, gamma- and epsilon-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group (CFB), high-G+C Gram-positive bacteria and cyanobacteria were found. In the 22% salt pond, alpha- and gamma-Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria. From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium. A decrease in the richness of bacterial genera was therefore apparent along the gradient. Archaea behaved quite similarly. In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota. In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae. Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT. Crenarchaeaota were not detected. Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend. This was because diversity in the crystallizers can be considered as 'microdiversity', the co-existence of several closely related clones of Bacteria (the S. ruber cluster) and Archaea (the SPhT cluster). Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant. Both clusters, however, showed a considerable degree of microdiversity. The meaning of such microdiversity remains to be determined.