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 ultramicrobacteria classified as actinobacteria from five freshwater habitats in Europe and Asia

We describe the first freshwater members of the class Actinobacteria that have been isolated. Nine ultramicro-size (<0.1 microm(3)) strains were isolated from five freshwater habitats in Europe and Asia. These habitats represent a broad spectrum of ecosystems, ranging from deep oligotrophic lakes to shallow hypertrophic lakes. Even when the isolated strains were grown in very rich media, the cell size was <0.1 microm(3) and was indistinguishable from the cell sizes of bacteria belonging to the smaller size classes of natural lake bacterioplankton. Hybridization of the isolates with oligonucleotide probes and phylogenetic analysis of the 16S rRNA gene sequences of the isolated strains revealed that they are affiliated with the class Actinobacteria and the family Microbacteriaceae. The previously described species with the highest levels of sequence similarity are Clavibacter michiganensis and Rathayibacter tritici, two phytopathogens of terrestrial plants. The 16S rRNA gene sequences of the nine isolates examined are more closely related to cloned sequences from uncultured freshwater bacteria than to the sequences of any previously isolated bacteria. The nine ultramicrobacteria isolated form, together with several uncultured bacteria, a diverse phylogenetic cluster (Luna cluster) consisting exclusively of freshwater bacteria. Isolates obtained from lakes that are ecologically different and geographically separated by great distances possess identical 16S rRNA gene sequences but have clearly different ecophysiological and phenotypic traits. Predator-prey experiments demonstrated that at least one of the ultramicro-size isolates is protected against predation by the bacterivorous nanoflagellate Ochromonas sp. strain DS.     

Complete genome sequence of Polynucleobacter necessarius subsp. asymbioticus type strain (QLW-P1DMWA-1(T))

Polynucleobacter necessarius subsp. asymbioticus strain QLW-P1DMWA-1(T) is a planktonic freshwater bacterium affiliated with the family Burkholderiaceae (class Betaproteobacteria). This strain is of interest because it represents a subspecies with cosmopolitan and ubiquitous distribution in standing freshwater systems. The 16S-23S ITS genotype represented by the sequenced strain comprised on average more than 10% of bacterioplankton in its home habitat. While all strains of the subspecies P. necessarius asymbioticus are free-living freshwater bacteria, strains belonging to the only other subspecies, P. necessarius subsp. necessarius are obligate endosymbionts of the ciliate Euplotes aediculatus. The two subspecies of P. necessarius are the instances of two closely related subspecies that differ in their lifestyle (free-living vs. obligate endosymbiont), and they are the only members of the genus Polynucleobacter with completely sequenced genomes. Here we describe the features of P. necessarius subsp. asymbioticus, together with the complete genome sequence and annotation. The 2,159,490 bp long chromosome with a total of 2,088 protein-coding and 48 RNA genes is the first completed genome sequence of the genus Polynucleobacter to be published and was sequenced as part of the DOE Joint Genome Institute Community Sequencing Program 2006.     

Polynucleobacter bacteria in the brackish-water species Euplotes harpa (Ciliata Hypotrichia)

We have found a Polynucleobacter bacterium in the cytoplasm of Euplotes harpa, a species living in a brackish-water habitat, with a cirral pattern not corresponding to that of the freshwater Euplotes species known to harbor this type of bacteria. The symbiont has been found in three strains of the species, obtained by clonal cultures from ciliates collected in different geographic regions. The 16S rRNA gene sequence of this bacterium identifies it as a member of the beta-proteobacterial genus Polynucleobacter. This sequence shares a high similarity value (98.4-98.5%) with P. necessarius, the type species of the genus, and is associated with 16S rRNA gene sequences of environmental clones and bacterial strains included in the Polynucleobacter cluster (>95%). An oligonucleotide probe was designed to corroborate the assignment of the retrieved sequence to the symbiont and to detect similar bacteria rapidly. Antibiotic experiments showed that the elimination of the bacteria stops the reproductive cycle in E. harpa, as has been shown for the freshwater Euplotes species.     

Diversity of bacterial community in freshwater of Woopo wetland

Diversity of bacterial community in water layer of Woopo wetland was investigated. Cultivable bacterial strains were isolated by the standard dilution plating technique and culture-independent 16S rRNA gene clones were obtained directly from DNA extracts of a water sample. Amplified rDNA restriction analysis (ARDRA) was applied onto both of the isolates and 16S rRNA gene clones. Rarefaction curves, coverage rate and diversity indices of ARDRA patterns were calculated. Representative isolates and clones of all the single isolate/clone phylotype were partially sequenced and analyzed phylogenetically. Sixty-four and 125 phylotypes were obtained from 203 bacterial isolates and 235 culture-independent 16S rRNA gene clones, respectively. Bacterial isolates were composed of 4 phyla, of which Firmicutes (49.8%) and Actinobacteria (32.0%) were predominant. Isolates were affiliated with 58 species. Culture-independent 16S rRNA gene clones were composed of 8 phyla, of which Proteobacteria (62.2%), Actinobacteria (15.5%), and Bacteroidetes (13.7%) were predominant. Diversity of 16S rRNA gene clones originated from cultivation-independent DNA extracts was higher than that of isolated bacteria.     

Bacterioplankton community composition along a salinity gradient of sixteen high-mountain lakes located on the Tibetan Plateau, China

The influence of altitude and salinity on bacterioplankton community composition (BCC) in 16 high-mountain lakes located at altitudes of 2,817 to 5,134 m on the Eastern Qinghai-Xizang (Tibetan) Plateau, China, spanning a salinity gradient from 0.02% (freshwater) to 22.3% (hypersaline), was investigated. Three different methods, fluorescent in situ hybridization, denaturing gradient gel electrophoresis (DGGE) with subsequent band sequencing, and reverse line blot hybridization (RLB) with probes targeting 17 freshwater bacterial groups, were used for analysis of BCC. Furthermore, the salt tolerances of 47 strains affiliated with groups detected in or isolated from the Tibetan habitats were investigated. Altitude was not found to influence BCC significantly within the investigated range. Several groups of typical freshwater bacteria, e.g., the ACK-M1 cluster and the Polynucleobacter group, were detected in habitats located above 4,400 m. Salinity was found to be the dominating environmental factor controlling BCC in the investigated lakes, resulting in only small overlaps in the BCCs of freshwater and hypersaline lakes. The relative abundances of different classes of Proteobacteria showed a sharp succession along the salinity gradient. Both DGGE and RLB demonstrated that a few freshwater bacterial groups, e.g., GKS98 and LD2, appeared over wide salinity ranges. Six freshwater isolates affiliated with the GKS98 cluster grew in ecophysiological experiments at maximum salinities of 0.3% to 0.7% (oligosaline), while this group was detected in habitats with salinities up to 6.7% (hypersaline). This observation indicated ecologically significant differences in ecophysiological adaptations among members of this narrow phylogenetic group and suggested ecological significance of microdiversity.     

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.     

"Bring to lab" of 19 novel species among 60 isolates retrieved from a freshwater pond

We report here on the cultivation of numerous novel bacterial species from a eutrophic freshwater pond. A total of 60 strains, 15 strains per each culture medium, were obtained from the surface of a eutrophic freshwater pond by employing a conventional dilution-plating method with four different kinds of culture media, including R2A, 1/10R2A, PCA, and 1/10PCA. Among the 60 strains isolated, 27 strains showed less than 97% 16S rRNA gene sequence similarities to validly published species, and thus they are considered to comprise 19 novel species. Of the 27 strains assigned to the novel species, the majority of the strains (20 strains) were affiliated with the Alphaproteobacteria and Betaproteobacteria. The remaining 7 strains were affiliated with the Gammaproteobacteria, Firmicutes, Actinobacteria, and Deinococci. Because we have isolated 19 novel species from a usual freshwater pond using a conventional culturing technique, our results suggest that an unexplored ecosystem, even if it looks like a common ecosystem found elsewhere, harbors diverse unidentified microbes, which will be definitely further characterized.     

Identification of novel Archaea in bacterioplankton of a boreal forest lake by phylogenetic analysis and fluorescent in situ hybridization(1)

We report here on novel groups of Archaea in the bacterioplankton of a small boreal forest lake studied by the culture-independent analysis of the 16S rRNA genes amplified directly from lake water in combination with fluorescent in situ hybridization (FISH). Polymerase chain reaction products were cloned and 28 of the 160 Archaea clones with around 900-bp-long 16S rRNA gene inserts, were sequenced. Phylogenetic analysis, including 642 Archaea sequences, confirmed that none of the freshwater clones were closely affiliated with known cultured Archaea. Twelve Archaea sequences from lake Valkea Kotinen (VAL) belonged to Group I of uncultivated Crenarchaeota and affiliated with environmental sequences from freshwater sediments, rice roots and soil as well as with sequences from an anaerobic digestor. Eight of the Crenarchaeota VAL clones formed a tight cluster. Sixteen sequences belonged to Euryarchaeota. Four of these formed a cluster together with environmental sequences from freshwater sediments and peat bogs within the order Methanomicrobiales. Five were affiliated with sequences from marine sediments situated close to marine Group II and three formed a novel cluster VAL III distantly related to the order Thermoplasmales. The remaining four clones formed a distinct clade within a phylogenetic radiation characterized by members of the orders Methanosarcinales and Methanomicrobiales on the same branch as rice cluster I, detected recently on rice roots and in anoxic bulk soil of flooded rice microcosms. FISH with specifically designed rRNA-targeted oligonucleotide probes revealed the presence of Methanomicrobiales in the studied lake. These observations indicate a new ecological niche for many novel 'non-extreme' environmental Archaea in the pelagic water of a boreal forest lake.     

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.     

Global phylogeography of pelagic Polynucleobacter bacteria: Restricted geographic distribution of subgroups,isolation by distance and influence of climate

The free‐living planktonic freshwater bacterium Polynucleobacter necessarius subspecies asymbioticus (> 99% 16S rRNA similarity) represents a taxon with a cosmopolitan distribution and apparently ubiquitous occurrence in lentic freshwater habitats. We tested for intra‐taxon biogeographic patterns by combining cultivation‐independent and cultivation methods. A culture collection of 204 strains isolated from globally distributed freshwater habitats (Arctic to Antarctica) was investigated for phylogeographic patterns based on sequences of two markers, the 16S–23S internal transcribed spacers and the glutamine synthetase gene (glnA). Genetic distance between isolates showed significant geographic distance‐decay patterns for both markers, suggesting that an isolation‐by‐distance mechanism influences the global phylogeography. Furthermore, a couple of subgroups showed restricted geographic distributions. Strains of one subgroup were exclusively obtained from tropical sites on four continents (pantropical subgroup). Cultivation‐independent methods were used to confirm the restricted geographic distributions of two subgroups. The pantropical taxon could be detected in 63% of investigated tropical habitats but not in any of 121 European freshwater samples. Physiological tests indicated that almost all strains of the pantropical subgroup failed to grow at temperatures of 4°C, while strains affiliated with other subgroups showed good growth at this temperature. This suggests that thermal adaptation is involved in phylogeographic structuring of the global Polynucleobacter population.     

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.     

河北九莲城淖尔可培养放线菌多样性及抗菌活性筛选

【目的】勘探干涸的九莲城淖尔土壤放线菌多样性并进行活性筛选,以期发现药用微生物资源,为新抗生素的发现奠定基础。【方法】采用15种分离培养基,以稀释涂布法分离放线菌;根据分离菌株的16S rRNA基因序列同源性分析放线菌多样性;发酵液经乙酸乙酯萃取,菌丝体经丙酮浸提,获得提取浓缩物样品;样品通过纸片扩散法进行抗菌活性初筛;抗菌阳性菌株采用PCR技术进行Ⅰ型聚酮合酶(PKS I)KS域、Ⅱ型聚酮合酶(PKS II)KS域和非核糖体多肽合成酶(NRPS)A结构域抗生素生物合成基因的检测。【结果】从11份盐湖土壤样品中分离纯化到251株放线菌,其分布于放线菌纲的10个目15个科31个属,其中优势菌属为链霉菌属和拟诺卡氏菌属;251株放线菌中包括57株耐(嗜)盐放线菌,其优势菌属为拟诺卡氏菌属(22株)和涅斯捷连科氏菌属(15株)。基于16S r RNA基因序列的系统发育分析显示,菌株J11Y309为糖霉菌科潜在新属,菌株J12GA03为分枝杆菌科潜在新种。96株放线菌活性检测结果显示,56株至少对1株检定菌具有抗菌活性,阳性率为58.3%;56株有活性的放线菌中,47株至少含有1种抗生素生物合成基因,其中17株同时具有3种抗生素生物合成基因。【结论】干涸的九莲城淖尔土壤中含有较为丰富的药用放线菌资源,具有从中发现放线菌新物种和新抗生素的潜力。     

Dilution-to-Extinction Culturing of Psychrotolerant Planktonic Bacteria from Permanently Ice-covered Lakes in the McMurdo Dry Valleys,Antarctica

Lakes in the McMurdo Dry Valleys of Antarctica are characterized by a permanent ice cover and little or no anthropogenic influence. Although bacterial cultures have been obtained from these habitats, recent culture-independent studies indicate that the most abundant microbes in these systems are not yet cultivated. By using dilution-to-extinction cultivation methods with sterilized and nutrient-amended lake water as media, we isolated 148 chemotrophic psychrotolerant bacterial cultures from fresh surface water of Lake Fryxell and the east lobe of Lake Bonney and the hypersaline, suboxic bottom water from the west lobes of Lake Bonney. Screening of the 16S ribosomal ribonucleic acid (rRNA) genes of the cultures by restriction fragment length polymorphism (RFLP) yielded 57 putatively pure psychrotolerant, slow growing cultures grouped into 18 clusters. The sequencing of 16S rRNA genes of randomly selected representatives of each RFLP cluster revealed that the corresponding isolates belong to the Alphaproteobacteria (six RFLP patterns), Betaproteobacteria (six RFLP patterns), Bacteroidetes (four RFLP patterns), and Actinobacteria (two RFLP patterns). Phylogenetic analysis of the sequences showed that the vast majority of the isolates were not closely related to previously described species. Thirteen of 18 RFLP patterns shared a 16S ribosomal deoxyribonucleic acid sequence similarity of 97% or less with the closest described species, and four isolates had a sequence similarity of 93% or less with the nearest described species. Phylogenetic analysis showed that these sequences were representatives of deeply branching organisms in the respective phylum. A comparison of the isolates with 16S rRNA clone libraries prepared from the same environments showed substantial overlap, indicating that dilution-to-extinction culturing in natural lake water media can help isolate some of the most abundant organisms in these perennially ice-covered lakes.     

The phylogenetic and ecological context of cultured and whole genome-sequenced planktonic bacteria from the coastal NW Mediterranean Sea

Microbial isolates are useful models for physiological and ecological studies and can also be used to reassemble genomes from metagenomic analyses. However, the phylogenetic diversity that can be found among cultured marine bacteria may vary significantly depending on the isolation. Therefore, this study describes a set of 136 bacterial isolates obtained by traditional isolation techniques from the Blanes Bay Microbial Observatory, of which seven strains have had the whole genome sequenced. The complete set was compared to a series of environmental sequences obtained by culture-independent techniques (60 DGGE sequences and 303 clone library sequences) previously obtained by molecular methods. In this way, each isolate was placed in both its “ecological” (time of year, nutrient limitation, chlorophyll and temperature values) context or setting, and its “phylogenetic” landscape (i.e. similar organisms that were found by culture-independent techniques, when they were relevant, and when they appeared). Nearly all isolates belonged to the Gammaproteobacteria, Alphaproteobacteria, or the Bacteroidetes (70, 40 and 20 isolates, respectively). Rarefaction analyses showed similar diversity patterns for sequences from isolates and molecular approaches, except for Alphaproteobacteria where cultivation retrieved a higher diversity per unit effort. Approximately 30% of the environmental clones and isolates formed microdiversity clusters constrained at 99% 16S rRNA gene sequence identity, but the pattern was different in Bacteroidetes (less microdiversity) than in the other main groups. Seventeen cases (12.5%) of nearly complete (98–100%) rRNA sequence identity between isolates and environmental sequences were found: nine in the Alphaproteobacteria, five in the Gammaproteobacteria, and three in the Bacteroidetes, indicating that cultivation could be used to obtain at least some organisms representative of the various taxa detected by molecular methods. Collectively, these results illustrated the largely unexplored potential of culturing on standard media for complementing the study of microbial diversity by culture-independent techniques and for obtaining phylogenetically distinct model organisms from natural seawater.     

Bacterial isolates from the bryozoan Membranipora membranacea: influence of culture media on isolation and antimicrobial activity

From specimens of the bryozoan Membranipora membranacea collected in the Baltic Sea, bacteria were isolated on four different media, which significantly increased the diversity of the isolated groups. All isolates were classified according to 16S rRNA gene sequence analysis and tested for antimicrobial properties using a panel of five indicator strains and six different media. Each medium featured a unique set of isolated phylotypes, and a phylogenetically diverse collection of isolates was obtained. A total of 96 isolates were assigned to 49 phylotypes and 29 genera. Only one-third of the members of these genera had been isolated previously from comparable sources. The isolates were affiliated with Alpha- and Gammaproteobacteria, Bacilli, and Actinobacteria. A comparable large portion of up to 22 isolates, i.e., 15 phylotypes, probably represent new species. Likewise, 47 isolates (approximately 50%) displayed antibiotic activities, mostly against grampositive indicator strains. Of the active strains, 63.8 % had antibiotic traits only on one or two of the growth media, whereas only 12.7 % inhibited growth on five or all six media. The application of six different media for antimicrobial testing resulted in twice the number of positive hits as obtained with only a single medium. The use of different media for the isolation of bacteria as well as the variation of media considered suitable for the production of antibiotic substances significantly enhanced both the number of isolates obtained and the proportion of antibiotic active cultures. Thus the approach described herein offers an improved strategy in the search for new antibiotic compounds.     

Culturable Diversity and Community Fatty Acid Profiling of Sulfate-Reducing Fluidized-Bed Reactors Treating Acidic,Metal-Containing Wastewater

Cultivable strains were identified from sulfate-reducing fluidized-bed reactors (FBR) treating acidic metal-containing wastewater. The FBR-communities were further characterized using culture-independent phenotypic markers, phospholipid fatty acid (PLFA) profiling. After morphological screening of 128 bacterial strains and partial sequencing of 55 strains, 17 distinct phylogenetic types were identified and characterized further. A total of 14 and 6 different bacterial strains were isolated from ethanol- and lactate-fed FBRs, respectively. Sequencing of the 16S rRNA gene showed that these strains were affiliated with members of the δ-Proteobacteria, Firmicutes and Bacteroidetes The strains were affiliated with members of the genera Desulfovibrio, Desulfotomaculum, Desulfobulbus, Desulfitobacterium, Clostridium, Caloramator, Oxobacter, and Bacteroides. Many of the strains were only distantly related to previously described species and, thus, may represent novel species or genera. A number of the strains were not detected in previously employed molecular analyses of the FBR communities, and the major component of each FBR as identified in the molecular analyses were not retrieved as cultures in this study. Most of the SRB, and two of the non-SRB utilized ethanol and lactate as a source of carbon and energy, but none of the isolates grew on acetate, an intermediate in the oxidation of ethanol and lactate. PLFA analysis revealed that the FBR community members contained large amounts of saturated fatty acids. Although the PLFA analysis showed some signatures consistent with sulfate-reducing communities, it did not show any substantial difference in the microbial communities between the reactors, an outcome that was quite contrary to the culture-independent phylogenetic analyses.     

Viability, diversity and composition of the bacterial community in a high Arctic permafrost soil from Spitsbergen, Northern Norway

The viable and non-viable fractions of the bacterial community in a 2347-year-old permafrost soil from Spitsbergen were subjected to a comprehensive investigation using culture-independent and culture-dependent methods. LIVE/DEAD BacLight staining revealed that 26% of the total number of bacterial cells were viable. Quantitatively, aerobic microcolonies, aerobic colony-forming units and culturable anaerobic bacteria comprised a minor fraction of the total number of viable bacteria, which underlines the necessity for alternative cultivation approaches in bacterial cryobiology. Sulfate reduction was detected at temperatures between -2 degrees C and 29 degrees C while methanogenesis was not detected. Bacterial diversity was high with 162 operational taxonomic units observed from 800 16S rDNA clone sequences. The 158 pure cultures isolated from the permafrost soil affiliated with 29 different bacterial genera, the majority of which have not previously been isolated from permafrost habitats. Most of the strains isolated were affiliated to the genera Cellulomonas and Arthrobacter and several of the pure cultures were closely related to bacteria reported from other cryohabitats. Characterization of viable bacterial communities in permafrost soils is important as it will enable identification of functionally important groups together with the as yet undescribed adaptations that bacteria have evolved for surviving subzero temperatures for millennia.     

Identification of phototrophic sulfur bacteria through the analysis of lmwRNA band patterns

Several phototrophic sulfur bacteria were identified preliminarily through the analysis of the low-molecular-weight RNA fraction (lmwRNA) of bacterial cells. This fraction includes the ribosomal 5S RNA and several transfer RNAs. These molecules were separated by high-resolution electrophoresis in polyacrylamide gels, and the resulting band patterns were used as fingerprints for the identification of the organisms. We examined a large number of well-characterized reference strains together with a broad range of purple sulfur bacterial isolates from freshwater and marine environments. A cluster analysis was run using the similarity matrix calculated from the band patterns. Despite the shortcomings of the method, close relatives were clustered together yielding a number of groups consistent with the phylogenetic arrangement established through the analyses of a few available 16S rRNA gene sequences. Thus, the classification obtained gives further support to rearrangement of the group as the analyses of 16S rRNA gene sequences had previously suggested. We conclude that the analysis of lmwRNA band patterns is a rapid and simple tool for grouping and preliminarily identifying new isolates of phototrophic sulfur bacteria. Received: 5 February 1998 / Accepted: 15 June 1998     

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.