New phylotypes of mesophilic filamentous anoxygenic phototrophic bacteria enriched from sulfide-containing environments.

Agar-based solid media with increasing concentrations of organic matter were used to isolate new members of the Chloroflexaceae (phylum Chloroflexi) from mesophilic environments containing sulfide. Inorganic media yielded less than 10% positive enrichments, which were not able to be maintained after repetitive inoculations in fresh medium. The use of casaminoacids and complex organic acid mixtures increased the number of positive enrichments (up to 45%) from both water and sediment samples. Two different green filamentous bacteria, SisoF2 and SalF, could be stably maintained as co-cultures for long periods and their phylogeny inferred from the analysis of complete sequences of the 16S rRNA gene. Ribotype SalF showed a high homology (95-98%) to previously isolated Oscillochloris trichoides strains. The 16S rRNA gene sequence retrieved from culture SisoF2 was largely divergent (< 92% similarity) from any sequence derived from either cultured representatives or environmental samples, suggesting that ribotype SisoF2 may constitute a new genus within the phylum. The presence of the new morphotypes in the environment from where they were enriched was analysed by high-resolution phylogenetic fingerprinting.

Use of the ammonia-oxidizing bacterial-specific phylogenetic probe Nso1225 as a primer for fingerprint analysis of ammonia-oxidizer communities.

Autotrophic ammonia-oxidizing bacteria (AOB) are an essential component of nitrifying wastewater treatment systems. The molecular tools used in group-specific studies are mostly based on the sequence of the 16S rRNA gene, but they have not proved to be fully specific. In this study, the sequence of the FISH probe Nso1225R was used as a reverse primer in order to analyze the AOB composition of several environmental samples by denaturing gradient gel electrophoresis (DGGE). For this purpose, samples from several environments, including aerated reactors, water treatment wetlands, and pilot plants, both aerobic and anaerobic, were analyzed. PCR fragments displayed a DGGE pattern consisting of bands melting between 30 and 40% denaturant, and a series of unresolved bands above 45%, mostly corresponding to AOB and beta-non-AOB, respectively. This second set of bands corresponded to environments subjected to severe oxygen restrictions. AOB sequences showed similarity percentages higher than 92% with those of known beta-AOB. Nso1225R, therefore, proved to be a good molecular phylogenetic marker for AOB samples from well-aerated systems, showing a higher specificity than the group-specific primers used previously.

Assessment of microbial community structure changes by amplified ribosomal DNA restriction analysis (ARDRA).

Amplified ribosomal DNA restriction analysis (ARDRA) is a simple method based on restriction endonuclease digestion of the amplified bacterial 16S rDNA. In this study we have evaluated the suitability of this method to detect differences in activated sludge bacterial communities fed on domestic or industrial wastewater, and subject to different operational conditions. The ability of ARDRA to detect these differences has been tested in modified Ludzack-Ettinger (MLE) configurations. Samples from three activated sludge wastewater treatment plants (WWTPs) with the MLE configuration were collected for both oxic and anoxic reactors, and ARDRA patterns using double enzyme digestions AluI+MspI were obtained. A matrix of Dice similarity coefficients was calculated and used to compare these restriction patterns. Differences in the community structure due to influent characteristics and temperature could be observed, but not between the oxic and anoxic reactors of each of the three MLE configurations. Other possible applications of ARDRA for detecting and monitoring changes in activated sludge systems are also discussed.

Number of triplets in 16S rRNA gene related with pathogenicity of Bacillus spp. and Clostridium spp.

The relation between the number of some trinucleotides in the sequence of 16S rRNA gene and pathogenicity of bacterial species from the genera of Bacillus and Clostridium was revealed. The species of genus Bacillus, which are pathogenic for humans, mammals and insects, have an increased number of AAA and TAT triplets in 16S rRNA gene. Theoretically, these species, B. anthracis and B. cereus for example, may be detected in the specimen by the higher ratio of AAA plus TAT triplets to the number of GGG triplet. Species of genus Clostridium, which are pathogenic for humans and mammals, have a maximum ratio of AAA and TAT triplet numbers. This ratio was higher than 2.6 for pathogenic species and lower than 2.2 for saprophytic ones. These theoretical data may open a new way for detecting pathogenic bacteria through the determination of triplet numbers in the sequences of 16S rRNA or rRNA. However, the mechanism of evolutionary relation between the number of AAA and TAT triplets in the sequence of 16S rRNA gene and the pathogenicity of bacterial species is not known.

Labelled trinucleotides as quantitative probes to identify Bacillus spp. using fluorescent in situ hybridization.

The number of nucleotide triplet repeats in 16 S rRNA sequences can be used for detection and identification of bacteria. Labelled TTT, GGG and ATA triplets were hybridized to the ribonucleic acid of Bacillus subtilis and Bacillus fusiformis whole-cells and the number of such triplets was quantified by synchronous fluorescence spectrometry. Each species was distinctly identified by specific ratios of labelled TTT, GGG and ATA triplets as well as characteristic fluorescence spectra. Notwithstanding the absence of intrinsic specificity, fluorescein-conjugated nucleotide triplet probes appear to be a useful tool for fluorescent spectrometric identification of micro-organisms through the quantitation of trinucleotide repeats.

Taxon-specific content of oligonucleotide triplets in 16S rRNAs of anoxygenic phototrophic and nitrifying bacteria.

Theoretical evaluation of the content of oligonucleotide triplets AAA, CCC, and UAU in 16S rRNAs of anoxygenic phototrophic bacteria (genera Chlorobium; Chloroflexus; Chromatium: Rhodopseudomonas) and nitrifying bacteria (genera Nitrosococcus, Nitrosomonas, Nitrosolobus, Nitrosovibrio, Nitrospira, Nitrospina, Nitrobacter) showed that the number of the AAA, CCC or UAU triplets in 16S rRNAs specifically corresponds to the genus and species of bacteria. The ratio of AAA and CCC triplet numbers in the sequences of 16S rRNA (AAA/CCC) of anoxygenic phototrophic bacteria was within the range of 0.61 to 2.03, and the ratio of AAA and UAU (AAA/UAU) triplet numbers in the sequence of 16S rRNA was within the range of 2.88 to 12.00. The regions of any genus within the AAA/CCC and AAA/UAU axes did not overlap. The combination of the numbers of nucleotide triplets in 16S rRNA is genus-specific character. The similar data were obtained in the study of a physiological group of nitrifying bacteria. The range of AAA/UAU ratio was from 1.8 to 9.0, and range of AAA/CCC was from 0.9 to 2.6 for this taxon. The number of triplets in 16S rRNAs of the studied taxa was genus- and species-specific character. The biological significance of these data is the evidence that not only the sequence but the number of nucleotide triplets in 16S rRNAs reflects the phylogeny of corresponding taxa.

Effects of gilvin on the composition and dynamics of metalimnetic communities of phototrophic bacteria in freshwater North-American lakes.

The spectral distribution of light reaching the populations of phototrophic bacteria in the metalimnion of stratified lakes is a selective factor determining the community composition. At deep metalimnia, light spectra are enriched in photons of the central part of the spectrum (500-600 nm) and benefit Chromatiaceae, brown-coloured Chlorobiaceae and phyco-erythrine-containing cyanobacteria. Their carotenoids (okenone, spiriloxanthine, isorenieratene) and phycoerythrines allow these phototrophic bacteria to use light from the narrow central spectral wavebands. Otherwise, shallow metalimnetic communities receive light from a wide range (400-800 nm) and their composition is more diverse and usually enriched in green-coloured Chlorobiaceae, which are unable to take advantage of the central part of the spectrum. Gilvin compounds (humic substances dissolved in water), have strong effects on light absorption, especially at shorter wavelengths. Therefore, light spectra in lakes with high gilvin contents are enriched in photons of long wavelengths (> 600 nm). Several Wisconsin lakes with different gilvin contents were studied during the period of summer stratification in 1994. Spectral distribution of light reaching their metalimnia changed with increasing gilvin contents (measured as g(440) ). In the latter, phototrophic metalimnetic bacterial communities were absolutely dominated by green-coloured Chlorobiaceae. Intermediate lakes could experiment changes on their community composition depending on variations in gilvin content, as happened in Little Long lake. The dynamics of this lake was studied during summer 1995. The ratio of green-coloured species in respect to brown-coloured species increased after a sudden increase of gilvin due to strong rainfall. These results agree with the photosynthetic advantage of green-coloured Chlorobiaceae under red-light illumination, inferred from laboratory experiments, and suggest a bacteriochlorophyll-dependent, light-harvesting strategy of these phototrophic sulphur bacteria.

Phylogeny of the genus Chlorobium based on 16S rDNA sequence.

A comparative analysis of 16S ribosomal DNA sequences from 18 strains belonging to different species of the genus Chlorobium has been made in order to investigate their phylogenetic relationships. All studied strains with brown pigmentation, belonging to species C. phaeobacteroides and C. phaeovibrioides, were clustered together and separated from green species. Most of the C. limicola strains formed a consistent group and were clustered next to C. tepidum. But, despite their identical morphology, C. vibrioforme strain 6030 (= DSM 260T) and C. vibrioforme strain 8327 (= DSM 263) were clearly differentiated on the basis of their 16S rDNA sequence. On the other hand, morphologically distinctive strains of C. chlorovibrioides, C. vibrioforme and C. limicola presented sequences with a high degree of similarity. Since morphological characters do not seem to be sufficient to adequately classify this group, these findings help to set up the basis for a revision of the taxonomy of the genus Chlorobium.

Effects of light quality on the physiology and the ecology of planktonic green sulfur bacteria in lakes.

The effect of light quality on the selection of natural populations of Green Sulfur Bacteria (Chlorobiaceae) is considered to be a classic factor in the determination of their ecological niches. From the comparison among phototrophic bacterial populations of lakes, it is shown that brown and green pigmented groups of Chlorobiaceae have a differential distribution depending on depth. Statistical analyses prove that green species, which dominate at shallow oxic/anoxic boundaries, are correlated to light spectra enriched in long wavelengths, while brown ones are found when light spectra are enriched in the central region of the spectrum, as in deeper lake layers. Physiological experiments have been made withChlorobium limicola andC. phaeobacteroides cultures placed under different light quality conditions, in order to verify these hypotheses made on a field data basis. Results show that red and white light has more positive effects on the green bacterium than on the brown. Blue and green light illuminations have opposite consequences. Therefore, the effect of shallow depths and Chromatiaceae shading-which also increases the proportion of long wavelengths in light spectra-benefits the bacteriochlorophyll-based strategies of green species. On the other hand, the carotenoid-based strategies of brown ones are favored by the light climates usually dominant at greater depths. Thus, brown species are considered to be singular adaptations of Chlorobiaceae to depth, where bacteriochlorophyll light-harvesting is strongly limited by light quality.