Isolation of strains belonging to the cosmopolitan Polynucleobacter necessarius cluster from freshwater habitats located in three climatic zones

More than 40 bacterial strains belonging to the cosmopolitan Polynucleobacter necessarius cluster (Betaproteobacteria) were isolated from a broad spectrum of freshwater habitats located in three climatic zones. Sequences affiliated with the freshwater P. necessarius cluster are among the most frequently detected in studies on bacterial diversity in freshwater ecosystems. Despite this frequent detection with culture-independent techniques and the cosmopolitan occurrence of members affiliated with this cluster, no isolates have been reported thus far. The isolated strains have been obtained from lakes, ponds, and rivers in central Europe, the People's Republic of China, and East Africa by use of the filtration-acclimatization method. The 16S rRNA gene sequences of the isolates are 98.8 to 100% identical to reference sequences obtained by various authors by use of culture-independent methods. The isolates, aerobic heterotrophs, grew on a wide range of standard complex media and formed visible colonies on agar plates. Thus, the previous lack of isolates cannot be explained by a lack of appropriate media. Most of the isolates possess, under a wide range of culture conditions, very small cells (<0.1 micro m(3)), even when grown in medium containing high concentrations of organic substances. Thus, these strains are obligate ultramicrobacteria. The obtained strains have a C-shaped cell morphology which is very similar to that of recently isolated ultramicrobacterial Luna cluster strains (Actinobacteria) and the SAR11 cluster strains (Alphaproteobacteria).     

Specific detection, isolation, and characterization of selected, previously uncultured members of the freshwater bacterioplankton community

High-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. A total of 570 bacterial cultures were obtained by employing the most probable number and MicroDrop techniques. The majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the alpha-Proteobacteria, beta-Proteobacteria, Actinobacteria, Firmicutes, or Flavobacteria-Cytophaga lineage. Correspondingly, the natural bacterioplankton community was analyzed by high-resolution phylogenetic fingerprinting of these five bacterial lineages. 16S rRNA gene fragments were generated for each lineage and subsequently separated by denaturing gradient gel electrophoresis. By the combination of five group-specific PCR protocols, the total number of 16S rRNA gene fingerprints generated from the natural communities was increased sixfold compared to conventional (eubacterial) fingerprinting. Four of the environmental alpha-Proteobacteria 16S rRNA gene sequences obtained from the natural community were found to be identical to those of bacterial isolates. One of these phylotypes was detected in 14 different cultures and hence represented the most frequently cultured bacterium. Three of these 14 strains were characterized in detail. Their complete 16S rRNA gene sequences showed only 93% similarity to that of Sandaracinobacter sibiricus, the closest relative described so far. The novel phylotype of bacterium is a strict aerobe capable of using numerous organic carbon substrates and contains bacteriochlorophyll a bound to two different photosynthetic light-harvesting complexes. Dot blot hybridization revealed that the strains occur in lakes of different trophic status and constitute up to 2% of the microbial community.     

Ecotypes of planktonic actinobacteria with identical 16S rRNA genes adapted to thermal niches in temperate, subtropical, and tropical freshwater habitats

Seven strains with identical 16S rRNA genes affiliated with the Luna2 cluster (Actinobacteria) were isolated from six freshwater habitats located in temperate (Austria and Australia), subtropical (People's Republic of China), and tropical (Uganda) climatic zones. The isolates had sequence differences at zero to five positions in a 2,310-nucleotide fragment of the ribosomal operon, including part of the intergenic spacer upstream of the 16S rRNA gene, the complete 16S rRNA gene, the complete 16S-23S internal transcribed spacer (ITS1), and a short part of the 23S rRNA gene. Most of the few sequence differences found were located in the internal transcribed spacer sequences. Two isolates obtained from habitats in Asia and Europe, as well as two isolates obtained from different habitats in the People's Republic of China, had identical sequences for the entire fragment sequenced. In spite of minimal sequence differences in the part of the ribosomal operon investigated, the strains exhibited significant differences in their temperature response curves (with one exception), as well as pronounced differences in their temperature optima (25.0 to 35.6 degrees C). The observed differences in temperature adaptation were generally in accordance with the thermal conditions in the habitats where the strains were isolated. Strains obtained from temperate zone habitats had the lowest temperature optima, strains from subtropical habitats had intermediate temperature optima, and a strain from a tropical habitat had the highest temperature optimum. Based on the observed temperature responses, we concluded that the strains investigated are well adapted to the thermal conditions in their home habitats. Consequently, these closely related strains represent different ecotypes adapted to different thermal niches.     

Intra- and inter-lake variability of free-living and particle-associated Actinobacteria communities

We have analysed the inter- and intra-lake variability of free-living and particle-associated freshwater Actinobacteria communities in four limnological different lakes of the Mecklenburg Lake District, Northeastern Germany. Denaturing gradient gel electrophoresis (DGGE) specific for Actinobacteria was used to investigate phylogenetic diversity and seasonal dynamics of actinobacterial communities in the epilimnion of all lakes (inter-lake variability) and to assess differences between Actinobacteria communities of the epi-, meta- and hypolimnion of a single lake (intra-lake variability) respectively. DGGE analyses showed significant inter- and intra-lake differences between Actinobacteria communities of all lakes and water layers as well as between free-living and particle-associated Actinobacteria. Phylogenetic inferences of 16S rRNA gene sequences suggest that particular members of particle-associated Actinobacteria were exclusively affiliated to certain actinobacterial lineages. The phylogenetic comparison of 16S rRNA gene sequences of all lakes and water layer, however, indicated the occurrence of almost similar phylogenetic lineages in all studied habitats and suggest high intracluster diversity within already known actinobacterial lineages. Non-metric multidimensional scaling (NMS) ordination analyses and Pearson's product moment correlations revealed several strong correlations between the investigated Actinobacteria communities and various limnological parameters, such as conductivity, total phosphorous, alkalinity or primary production. However, no uniform correlation patterns were found between lakes, water layers and bacterial fractions. These heterogeneous correlation patterns together with the phylogenetic similarities of Actinobacteria communities from different lakes indicate that particular Actinobacteria represent various ecotypes or exhibit a pronounced ecophysiological plasticity.     

Diversity and seasonal dynamics of Actinobacteria populations in four lakes in northeastern Germany

The phylogenetic diversity and seasonal dynamics of freshwater Actinobacteria populations in four limnologically different lakes of the Mecklenburg-Brandenburg Lake District (northeastern Germany) were investigated. Fluorescence in situ hybridization was used to determine the seasonal abundances and dynamics of total Actinobacteria (probe HGC69a) and the three actinobacterial subclusters acI, acI-A, and acI-B (probes AcI-852, AcI-840-1, and AcI-840-2). Seasonal means of total Actinobacteria abundances in the epilimnia of the lakes varied from 13 to 36%, with maximum values of 30 to 58%, of all DAPI (4',6'-diamidino-2-phenylindole)-stained cells. Around 80% of total Actinobacteria belonged to the acI cluster. The two subclusters acI-A and acI-B accounted for 60 to 91% of the acI cluster and showed seasonal means of 49% (acI-B) and 23% (acI-A) in relation to the acI cluster. Total Actinobacteria and members of the clusters acI and acI-B showed distinct seasonal changes in their absolute abundances, with maxima in late spring and fall/winter. In eight clone libraries constructed from the lakes, a total of 76 actinobacterial 16S rRNA gene sequences were identified from a total of 177 clones. The majority of the Actinobacteria sequences belonged to the acI and acIV cluster. Several new clusters and subclusters were found (acSTL, scB1-4, and acIVA-D). The majority of all obtained 16S rRNA gene sequences are distinct from those of already-cultured freshwater Actinobacteria.     

美洲大蠊成虫肠道可培养细菌多样性研究

【目的】了解美洲大蠊成虫肠道可培养细菌的多样性。【方法】运用纯培养法、数值分类和16S rRNA基因序列的系统发育分析对样品中可培养细菌多样性进行研究。【结果】从NA培养基中分离得到54株细菌,根据形态观察和部分生理生化特性,选取32个代表性菌株进行16S rRNA基因序列的系统发育多样性分析。结果表明,数值分类中的代表菌株在82%相似水平上可分为12个表观群;这些分离菌株代表20个物种,属于4个大的系统发育类群(Proteobacteria,Bacteroidetes,Firmicutes,Actinobacteria)的10个科、15个属。多数菌株属于Proteobacteria门(15株,占46.9%)和Bacteroidetes门(10株,占31.3%)。【结论】美洲大蠊成虫肠道内存在较为丰富的细菌多样性。     

Isolation of Strains Belonging to the Cosmopolitan Polynucleobacter necessarius Cluster from Freshwater Habitats Located in Three Climatic Zones

More than 40 bacterial strains belonging to the cosmopolitan Polynucleobacter necessarius cluster (Betaproteobacteria) were isolated from a broad spectrum of freshwater habitats located in three climatic zones. Sequences affiliated with the freshwater P. necessarius cluster are among the most frequently detected in studies on bacterial diversity in freshwater ecosystems. Despite this frequent detection with culture-independent techniques and the cosmopolitan occurrence of members affiliated with this cluster, no isolates have been reported thus far. The isolated strains have been obtained from lakes, ponds, and rivers in central Europe, the People's Republic of China, and East Africa by use of the filtration-acclimatization method. The 16S rRNA gene sequences of the isolates are 98.8 to 100% identical to reference sequences obtained by various authors by use of culture-independent methods. The isolates, aerobic heterotrophs, grew on a wide range of standard complex media and formed visible colonies on agar plates. Thus, the previous lack of isolates cannot be explained by a lack of appropriate media. Most of the isolates possess, under a wide range of culture conditions, very small cells (<0.1 μm³), even when grown in medium containing high concentrations of organic substances. Thus, these strains are obligate ultramicrobacteria. The obtained strains have a C-shaped cell morphology which is very similar to that of recently isolated ultramicrobacterial Luna cluster strains (Actinobacteria) and the SAR11 cluster strains (Alphaproteobacteria).     

Isolation of novel bacteria, including a candidate division, from geothermal soils in New Zealand

We examined bacterial diversity of three geothermal soils in the Taupo Volcanic Zone of New Zealand. Phylogenetic analysis of 16S rRNA genes recovered directly from soils indicated that the bacterial communities differed in composition and richness, and were dominated by previously uncultured species of the phyla Actinobacteria , Acidobacteria , Chloroflexi , Proteobacteria and candidate division OP10. Aerobic, thermophilic, organotrophic bacteria were isolated using cultivation protocols that involved extended incubation times, low-pH media and gellan as a replacement gelling agent to agar. Isolates represented previously uncultured species, genera, classes, and even a new phylum of bacteria. They included members of the commonly cultivated phyla Proteobacteria , Firmicutes , Thermus/Deinococcus , Actinobacteria and Bacteroidetes , as well as more-difficult-to-cultivate groups. Isolates possessing < 85% 16S rRNA gene sequence identity to any cultivated species were obtained from the phyla Acidobacteria , Chloroflexi and the previously uncultured candidate division OP10. Several isolates were prevalent in 16S rRNA gene clone libraries constructed directly from the soils. A key factor facilitating isolation was the use of gellan-solidified plates, where the gellan itself served as an energy source for certain bacteria. The results indicate that geothermal soils are a rich potential source of novel bacteria, and that relatively simple cultivation techniques are practical for isolating bacteria from these habitats.     

Actinobacterial 16S rRNA genes from freshwater habitats cluster in four distinct lineages

We analysed the phylogenetic relatedness of 16S rRNA genes from freshwater bacteria affiliated with the class Actinobacteria. A polymerase chain reaction assay was developed to identify reliably rare Actinobacteria-related inserts within 16S rRNA gene clone libraries. In 18 libraries constructed from seven freshwater systems, altogether 63 actinobacterial sequence types were collected from a total of > 1800 clones. Sixty of the newly obtained sequences grouped within four distinct phylogenetic lineages. They constitute approximately 75% of the nearly complete sequences within these clusters that are presently available. A comparison with > 300 sequences from various soil habitats revealed that two of these monophyletic actinobacterial clades (acI and acII) almost exclusively harbour 16S rRNA sequence types from freshwaters and estuaries. This may indicate that such bacteria are not inoculated to freshwaters from terrestrial sources, but are autochthonous components of freshwater microbial assemblages. In contrast, sequence types from freshwaters, marine sediments and soils were clearly mixed in another of the actinobacterial lineages (acIV). Sequence divergence within acIV was the highest of all four lineages (88% minimum similarity), which potentially reflects its radiation across several habitat types. Within the freshwater lineages, groups of essentially identical sequence types were retrieved from geographically distant aquatic systems with strikingly different hydrological and limnological characteristics. This points to the necessity to investigate genotypic variability, in situ abundances and activities of these Actinobacteria in freshwater plankton in greater detail by cultivation-independent techniques.     

环己酮降解细菌的筛选和系统发育分析

通过以环己酮为唯一碳源的选择培养基富集培养和细菌环己酮降解能力的测定,从巴陵石化公司环己酮生产车间排水口的污泥样品中分离到12株降解环己酮性能强的细菌菌株。根据形态观察、部分生理生化试验和16SrRNA基因序列的比对分析,初步确定这些菌株代表8个物种,属于3个大的系统发育类群/门(Actinobacteria,Proteobacteria,Bacteroidetes)的5个科、7个属;大多数菌株与其系统发育关系最密切的典型菌株之间存在一定的遗传差异。结果表明降解环己酮性能强的细菌具有较丰富的系统发育多样性。     

Isolation of uncultivable anaerobic thermophiles of the family Clostridiaceae requiring growth-supporting factors

Novel groups of uncultivable anaerobic thermophiles were isolated from compost by enrichment cultivation in medium with a cell-free extract of Geobacillus toebii. The cell-free extract of G. toebii provided the medium with growth-supporting factors (GSF) needed to cultivate the previously uncultured microorganisms. Twenty-nine GSF-requiring candidates were successfully cultivated, and 16 isolated novel bacterial strains were classified into three different groups of uncultivable bacteria. The similarity among these 16 isolates and a phylogenetic analysis using 16S rRNA gene sequences revealed that these GSF-requiring strains represented novel groups within the family Clostridiaceae.     

Characterization and identification of numerically abundant culturable bacteria from the anoxic bulk soil of rice paddy microcosms.

Most-probable-number (liquid serial dilution culture) counts were obtained for polysaccharolytic and saccharolytic fermenting bacteria in the anoxic bulk soil of flooded microcosms containing rice plants. The highest viable counts (up to 2.5 x 10(8) cells per g [dry weight] of soil) were obtained by using xylan, pectin, or a mixture of seven mono- and disaccharides as the growth substrate. The total cell count for the soil, as determined by using 4', 6-diamidino-2-phenylindole staining, was 4.8 x 10(8) cells per g (dry weight) of soil. The nine strains isolated from the terminal positive tubes in counting experiments which yielded culturable populations that were equivalent to about 5% or more of the total microscopic count population belonged to the division Verrucomicrobia, the Cytophaga-Flavobacterium-Bacteroides division, clostridial cluster XIVa, clostridial cluster IX, Bacillus spp., and the class Actinobacteria. Isolates originating from the terminal positive tubes of liquid dilution series can be expected to be representatives of species whose populations in the soil are large. None of the isolates had 16S rRNA gene sequences identical to 16S rRNA gene sequences of previously described species for which data are available. Eight of the nine strains isolated fermented sugars to acetate and propionate (and some also fermented sugars to succinate). The closest relatives of these strains (except for the two strains of actinobacteria) were as-yet-uncultivated bacteria detected in the same soil sample by cloning PCR-amplified 16S rRNA genes (U. Hengstmann, K.-J. Chin, P. H. Janssen, and W. Liesack, Appl. Environ. Microbiol. 65:5050-5058, 1999). Twelve other isolates, which originated from most-probable-number counting series indicating that the culturable populations were smaller, were less closely related to cloned 16S rRNA genes.     

Ecotypes of Planktonic Actinobacteria with Identical 16S rRNA Genes Adapted to Thermal Niches in Temperate, Subtropical, and Tropical Freshwater Habitats

Seven strains with identical 16S rRNA genes affiliated with the Luna2 cluster (Actinobacteria) were isolated from six freshwater habitats located in temperate (Austria and Australia), subtropical (People's Republic of China), and tropical (Uganda) climatic zones. The isolates had sequence differences at zero to five positions in a 2,310-nucleotide fragment of the ribosomal operon, including part of the intergenic spacer upstream of the 16S rRNA gene, the complete 16S rRNA gene, the complete 16S-23S internal transcribed spacer (ITS1), and a short part of the 23S rRNA gene. Most of the few sequence differences found were located in the internal transcribed spacer sequences. Two isolates obtained from habitats in Asia and Europe, as well as two isolates obtained from different habitats in the People's Republic of China, had identical sequences for the entire fragment sequenced. In spite of minimal sequence differences in the part of the ribosomal operon investigated, the strains exhibited significant differences in their temperature response curves (with one exception), as well as pronounced differences in their temperature optima (25.0 to 35.6°C). The observed differences in temperature adaptation were generally in accordance with the thermal conditions in the habitats where the strains were isolated. Strains obtained from temperate zone habitats had the lowest temperature optima, strains from subtropical habitats had intermediate temperature optima, and a strain from a tropical habitat had the highest temperature optimum. Based on the observed temperature responses, we concluded that the strains investigated are well adapted to the thermal conditions in their home habitats. Consequently, these closely related strains represent different ecotypes adapted to different thermal niches.     

Representative freshwater bacterioplankton isolated from Crater Lake, Oregon

High-throughput culturing (HTC) methods that rely on dilution to extinction in very-low-nutrient media were used to obtain bacterial isolates from Crater Lake, Oregon. 16S rRNA sequence determination and phylogenetic reconstruction were used to determine the potential ecological significance of isolated bacteria, both in Crater Lake and globally. Fifty-five Crater Lake isolates yielded 16 different 16S rRNA gene sequences. Thirty of 55 (55%) Crater Lake isolates had 16S rRNA gene sequences with 97% or greater similarity to sequences recovered previously from Crater Lake 16S rRNA gene clone libraries. Furthermore, 36 of 55 (65%) Crater Lake isolates were found to be members of widely distributed freshwater groups. These results confirm that HTC is a significant improvement over traditional isolation techniques that tend to enrich for microorganisms that do not predominate in their environment and rarely correlate with 16S rRNA gene clone library sequences. Although all isolates were obtained under dark, heterotrophic growth conditions, 2 of the 16 different groups showed evidence of photosynthetic capability as assessed by the presence of puf operon sequences, suggesting that photoheterotrophy may be a significant process in this oligotrophic, freshwater habitat.     

Endosymbiosis in statu nascendi: close phylogenetic relationship between obligately endosymbiotic and obligately free-living Polynucleobacter strains (Betaproteobacteria)

Bacterial strains affiliated to the phylogenetically shallow subcluster C (PnecC) of the Polynucleobacter cluster, which is characterized by a minimal 16S rRNA gene sequence similarity of approximately 98.5%, have been reported to occur as obligate endosymbionts of ciliates (Euplotes spp.), as well as to occur as free-living cells in the pelagic zone of freshwater habitats. We investigated if these two groups of closely related bacteria represent strains fundamentally differing in lifestyle, or if they simply represent different stages of a facultative endosymbiotic lifestyle. The phylogenetic analysis of 16S rRNA gene and 16S-23S ITS sequences of five endosymbiont strains from two different Euplotes species and 40 pure culture strains demonstrated host-species-specific clustering of the endosymbiont sequences within the PnecC subcluster. The sequences of the endosymbionts showed characteristics indicating an obligate endosymbiotic lifestyle. Cultivation experiments revealed fundamental differences in physiological adaptations, and determination of the genome sizes indicated a slight size reduction in endosymbiotic strains. We conclude that the two groups of PnecC bacteria represent obligately free-living and obligately endosymbiotic strains, respectively, and do not represent different stages of the same complex life cycle. These closely related strains occupy completely separated ecological niches. To our best knowledge, this is the closest phylogenetic relationship between obligate endosymbionts and obligately free-living bacteria ever revealed.     

Representative Freshwater Bacterioplankton Isolated from Crater Lake, Oregon

High-throughput culturing (HTC) methods that rely on dilution to extinction in very-low-nutrient media were used to obtain bacterial isolates from Crater Lake, Oregon. 16S rRNA sequence determination and phylogenetic reconstruction were used to determine the potential ecological significance of isolated bacteria, both in Crater Lake and globally. Fifty-five Crater Lake isolates yielded 16 different 16S rRNA gene sequences. Thirty of 55 (55%) Crater Lake isolates had 16S rRNA gene sequences with 97% or greater similarity to sequences recovered previously from Crater Lake 16S rRNA gene clone libraries. Furthermore, 36 of 55 (65%) Crater Lake isolates were found to be members of widely distributed freshwater groups. These results confirm that HTC is a significant improvement over traditional isolation techniques that tend to enrich for microorganisms that do not predominate in their environment and rarely correlate with 16S rRNA gene clone library sequences. Although all isolates were obtained under dark, heterotrophic growth conditions, 2 of the 16 different groups showed evidence of photosynthetic capability as assessed by the presence of puf operon sequences, suggesting that photoheterotrophy may be a significant process in this oligotrophic, freshwater habitat.     

Specific Detection, Isolation, and Characterization of Selected, Previously Uncultured Members of the Freshwater Bacterioplankton Community

High-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. A total of 570 bacterial cultures were obtained by employing the most probable number and MicroDrop techniques. The majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the α-Proteobacteria, β-Proteobacteria, Actinobacteria, Firmicutes, or Flavobacteria-Cytophaga lineage. Correspondingly, the natural bacterioplankton community was analyzed by high-resolution phylogenetic fingerprinting of these five bacterial lineages. 16S rRNA gene fragments were generated for each lineage and subsequently separated by denaturing gradient gel electrophoresis. By the combination of five group-specific PCR protocols, the total number of 16S rRNA gene fingerprints generated from the natural communities was increased sixfold compared to conventional (eubacterial) fingerprinting. Four of the environmental α-Proteobacteria 16S rRNA gene sequences obtained from the natural community were found to be identical to those of bacterial isolates. One of these phylotypes was detected in 14 different cultures and hence represented the most frequently cultured bacterium. Three of these 14 strains were characterized in detail. Their complete 16S rRNA gene sequences showed only 93% similarity to that of Sandaracinobacter sibiricus, the closest relative described so far. The novel phylotype of bacterium is a strict aerobe capable of using numerous organic carbon substrates and contains bacteriochlorophyll a bound to two different photosynthetic light-harvesting complexes. Dot blot hybridization revealed that the strains occur in lakes of different trophic status and constitute up to 2% of the microbial community.     

Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an abundant group of actinobacteria

In a search for cosmopolitan phylogenetic clusters of freshwater bacteria, we recovered a total of 190 full and partial 16S ribosomal DNA (rDNA) sequences from three different lakes (Lake Gossenk?llesee, Austria; Lake Fuchskuhle, Germany; and Lake Baikal, Russia). The phylogenetic comparison with the currently available rDNA data set showed that our sequences fall into 16 clusters, which otherwise include bacterial rDNA sequences of primarily freshwater and soil, but not marine, origin. Six of the clusters were affiliated with the alpha, four were affiliated with the beta, and one was affiliated with the gamma subclass of the Proteobacteria; four were affiliated with the Cytophaga-Flavobacterium-Bacteroides group; and one was affiliated with the class Actinobacteria (formerly known as the high-G+C gram-positive bacteria). The latter cluster (hgcI) is monophyletic and so far includes only sequences directly retrieved from aquatic environments. Fluorescence in situ hybridization (FISH) with probes specific for the hgcI cluster showed abundances of up to 1.7 x 10(5) cells ml(-1) in Lake Gossenk?llesee, with strong seasonal fluctuations, and high abundances in the two other lakes investigated. Cell size measurements revealed that Actinobacteria in Lake Gossenk?llesee can account for up to 63% of the bacterioplankton biomass. A combination of phylogenetic analysis and FISH was used to reveal 16 globally distributed sequence clusters and to confirm the broad distribution, abundance, and high biomass of members of the class Actinobacteria in freshwater ecosystems.     

Species diversity of magnetotactic bacteria from the Ol’khovka River, Russia

A fraction of magnetotactic bacteria was isolated by magnetic separation from the water and silt samples collected from the Ol’khovka River (Kislovodsk, Russia). A 16S rRNA clone library was obtained from the total DNA of the fraction by PCR amplification and molecular cloning. Phylogenetic analysis of 67 16S rRNA gene sequences of randomly selected clones demonstrated that two phylotypes of magnetotactic bacteria were present in the library: the first phylotype consisted of 42 sequences and the second one included only one sequence. The remaining 24 sequences belonged to non-magnetotactic bacteria. According to the results of phylogenetic analysis, both phylotypes were magnetotactic cocci; the predominant sequences were almost identical to the 16S rRNA sequence of the freshwater coccus TB24 (X81185.1) identified earlier among the magnetotactic bacteria isolated from Lake Chiemsee (Bavaria). The phylotype represented by a single sequence formed a separate branch in the dendrogram, with 97% similarity between its sequence and that of TB24. The discovered phylotypes formed with the sequences of uncultured freshwater magnetotactic cocci a separate branch within the class Alphaproteobacteria and presumably belonged to a separate family within the recently described order Magnetococcales. Despite the fact that phylogenetic analysis of the 16S rRNA clone library did not reveal any phylotypes of magnetotactic spirilla, after the secondary enrichment of the fraction of magnetotactic bacteria using the “race track” technique, a new strain of magnetotactic spirilla, Magnetospirillum SO-1, was isolated. The closest relative of strain SO-1 was the previously described magnetotactic spirillum Magnetospirillum magneticum AMB-1.     

The filtration-acclimatization method for isolation of an important fraction of the not readily cultivable bacteria

We developed a novel method, the filtration-acclimatization method (FAM), which enables the isolation and cultivation of an important fraction of the bacterial diversity, which is not cultivable by standard methods. The method consists of a filtration step, which removes most of the readily cultivable bacteria able to overgrow slowly growing bacteria, and an acclimatization procedure that provides a slow transition from the low environmental substrate concentrations to the high concentration of standard microbial media. So far, we isolated in total 65 strains from surface freshwater habitats by utilizing FAM. The isolates are affiliated with Actinobacteria, Alpha-, Betaproteobacteria, Bacteroidetes, and Spirochaeta. All isolates are pure cultures and form visible colonies on agar plates with high substrate concentrations. For further analysis, strains sharing more than a 97% 16S rRNA gene sequence similarity were grouped into one taxon. Based on sequence similarities, 88% of the obtained taxa can be considered to be undescribed species (<97% similarity to closest species). The highest similarity value of the taxa to the respective closest related species ranged from 87.7% to 99.8%, and was on average 94.5%. For comparison we isolated, by direct plating of water samples on a rich agar medium, a similar number of taxa. Amongst these taxa the percentage of taxa, which can be considered to be undescribed species, was only half of the percentage found for the taxa isolated by FAM. More importantly, it was amongst the taxa obtained by the standard method no taxon that was closer related to an uncultured bacterium than to an isolate, while 56% of the taxa isolated by FAM were closely related to uncultured bacteria.