Morphological and Phylogenetic Characterizations of Freshwater Thioploca Species from Lake Biwa, Japan, and Lake Constance, Germany

Filamentous, gliding, sulfide-oxidizing bacteria of the genus Thioploca were found on sediments in profundal areas of Lake Biwa, a Japanese freshwater mesotrophic lake, and were characterized morphologically and phylogenetically. The Lake Biwa Thioploca resembled morphologically Thioploca ingrica, a brackish water species from a Danish fjord. The diameters of individual trichomes were 3 to 5.6 μm; the diameters of complete Thioploca filaments ranged from 18 to 75 μm. The cell lengths ranged from 1.2 to 3.8 μm. In transmission electron microscope specimens stained with uranyl acetate, dense intracellular particles were found, which did not show any positive signals for phosphorus and sulfur in an X-ray analysis. The 16S rRNA gene of the Thioploca from Lake Biwa was amplified by using newly designed Thioploca-specific primers (706-Thioploca, Biwa160F, and Biwa829R) in combination with general bacterial primers in order to avoid nonspecific amplification of contaminating bacterial DNA. Denaturing gradient gel electrophoresis (DGGE) analysis of the three overlapping PCR products resulted in single DGGE bands, indicating that a single 16S rRNA gene had been amplified. With the same method, the Thioploca from Lake Constance was examined. The 16S rRNA sequence was verified by performing fluorescence in situ hybridization targeted at specific motifs of the Lake Biwa Thioploca. Positive signals were obtained with the bacterial probe EUB-338, the γ-proteobacterial probe GAM42a, and probe Biwa829 targeting the Lake Biwa Thioploca. Based on the nearly complete 16S rRNA sequence and on morphological similarities, the Thioploca from Lake Biwa and the Thioploca from Lake Constance are closely related to T. ingrica and to each other.     

Community Structure of Bacteria Associated with Sheaths of Freshwater and Brackish Thioploca Species

Bacterial communities associated with sheaths of Thioploca spp. from two freshwater lakes (Lake Biwa, Japan, and Lake Constance, Germany) and one brackish lake (Lake Ogawara, Japan) were analyzed with denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene fragments. The comparison between the DGGE band patterns of bulk sediment and Thioploca filaments of Lake Biwa suggested the presence of specific bacterial communities associated with Thioploca sheaths. As members of sheath-associated communities, bacteria belonging to Bacteroidetes were detected from the samples of both freshwater lakes. A DGGE band from Thioploca of Lake Biwa, belonging to candidate division OP8, was quite closely related to another DGGE band detected from that of Lake Constance. In contrast to the case of freshwater lakes, no bacterium of Bacteroidetes or OP8 was detected from Thioploca of Lake Ogawara. However, two DGGE bands from Lake Ogawara, belonging to Chloroflexi, were quite closely related to a DGGE band from Lake Constance. Two DGGE bands obtained from Lake Biwa were closely related to phylogenetically distant dissimilatory Fe(III)-reducing bacteria. Cloning analyses for a dissimilatory sulfite reductase gene were performed on the same samples used for DGGE analysis. The results of the analyses suggest that sheaths of freshwater/brackish Thioploca have little ecological significance for the majority of sulfate reducers.     

Colorless sulfur bacteria <Emphasis Type="Italic">Thioploca</Emphasis> from different sites in Lake Baikal

The colorless sulfur bacteria Thioploca spp. found in Lake Baikal are probably a marker for the influx of subterranean mineralized fluids. Bacteria act as a biological filter; by consuming sulfide in their metabolism, they detoxicate it and maintain the purity of Lake Baikal’s water. The bacteria were investigated by various techniques. According to analysis of the 16S rRNA gene fragment, Thioploca sp. from Frolikha Bay, Baikal belongs to the clade of freshwater species found in Lake Biwa and Lake Constance; it is most closely related to Thioploca ingrica.     

Diversity of Freshwater <Emphasis Type="Italic">Thioploca</Emphasis> Species and Their Specific Association with Filamentous Bacteria of the Phylum <Emphasis Type="Italic">Chloroflexi</Emphasis>

Phylogenetic diversity among filamentous sulfur-oxidizing bacteria of the genus Thioploca inhabiting freshwater/brackish environments was analyzed in detail. The 16S rRNA gene sequence of Thioploca found in a freshwater lake in Japan, Lake Okotanpe, was identical to that of Thioploca from Lake Ogawara, a brackish lake. The samples of the two lakes could be differentiated by the sequences of their 23S rRNA genes and 16S–23S rRNA internal transcribed spacer (ITS) regions. The 23S rRNA-based phylogenetic relationships between Thioploca samples from four lakes (Lake Okotanpe, Lake Ogawara, Lake Biwa, and Lake Constance) were similar to those based on the 16S rRNA gene sequences. In addition, multiple types of the ITS sequences were obtained from Thioploca inhabiting Lake Okotanpe and Lake Constance. Variations within respective Thioploca populations were also observed in the analysis of the soxB gene, involved in sulfur oxidation. As major members of the sheath-associated microbial community, bacteria of the phylum Chloroflexi were consistently detected in the samples from different lakes. Fluorescence in situ hybridization revealed that they were filamentous and abundantly distributed within the sheaths of Thioploca.     

The phylogenetic status of Sarcobium lyticum, an obligate intracellular bacterial parasite of small amoebae

A 16S rRNA gene of the obligate intracellular bacterial parasite Sarcobium lyticum was amplified using the polymerase chain reaction in combination with site-specific primers. The amplified DNA was cloned, sequenced and compared with other bacterial 16S rRNA sequences. The analysis revealed that S. lyticum belongs to the gamma subclass of the Proteobacteria and shows the closest relationship to an intracellular Legionella species recovered by amoebal enrichment from the sputum of a patient with pneumonia. S. lyticum could be detected in situ with a fluorescent oligonucleotide probe by whole cell hybridization.     

Digoxigenin-labelled peptide nucleic acid to detect lactobacilli PCR amplicons immobilized on membranes from denaturing gradient gel electrophoresis

AIMS: To develop a digoxigenin (DIG)-labeled peptide nucleic acid (PNA) probe for the detection of Lactobacillus-related genera amongst eubacterial amplicons obtained from vaginal samples using denaturing gradient gel electrophoresis (DGGE) blots. METHODS AND RESULTS: Part of the 16S rRNA gene sequence was used as a target for the PNA probe. After confirming probe specificity using chromosomal DNA from species and isolates that have been detected in the urogenital tract, it was successfully used to detect lactobacilli amplicons generated using eubacterial-specific 16S rRNA gene-targeted primers from vaginal tract samples immobilized on membranes from DGGE. CONCLUSIONS: The Lactobacillus-specific PNA probe could distinguish between DNA fragments from lactobacilli in a DGGE gel from other bacterial species, including those that migrated to a similar position. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of the DIG-labelled PNA probe on blots of eubacterial PCR products from DGGE gels can be used to specifically detect lactobacilli in complex vaginal samples.     

PCR‐DGGE Fingerprinting Analysis of Plankton Communities and Its Relationship to Lake Trophic Status

Plankton communities in eight lakes of different trophic status near Yangtze, China were charac‐terized by using denatured gradient gel electrophoresis (DGGE). Various water quality parameters were also measured at each collection site. Following extraction of DNA from plankton communi‐ties, 16S rRNA and 18S rRNA genes were amplified with specific primers for prokaryotes and eu‐karyotes, respectively; DNA profiles were developed by DGGE. The plankton community of each lake had its own distinct DNA profile. The total number of bands identified at 34 sampling stations ranged from 37 to 111. Both prokaryotes and eukaryotes displayed complex fingerprints composed of a large number of bands: 16 to 59 bands were obtained with the prokaryotic primer set; 21 to 52 bands for the eukaryotic primer set. The DGGE‐patterns were analyzed in relation to water quality parameters by canonical correspondence analysis (CCA). Temperature, pH, alkalinity, and the con‐centration of COD, TP and TN were strongly correlated with the DGGE patterns. The parameters that demonstrated a strong correlation to the DGGE fingerprints of the plankton community differed among lakes, suggesting that differences in the DGGE fingerprints were due mainly to lake trophic status. Results of the present study suggest that PCR‐DGGE fingerprinting is an effective and precise method of identifying changes to plankton community composition, and therefore could be a useful ecological tool for monitoring the response of aquatic ecosystems to environmental perturbations. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Polynucleobacter necessarius, an obligate bacterial endosymbiont of the hypotrichous ciliate Euplotes aediculatus, is a member of the β-subclass of Proteobacteria

Abstract An almost full length 16S rRNA gene of the obligate bacterial endosymbiont Polynucleobacter necessarius was amplified using the polymerase chain reaction in combination with site-specific primers. The amplified DNA was directly sequenced and compared with other bacterial 16S rRNA sequences. P. necessarius belongs to the β-subclass of Proteobacteria and shows the closest relationship to Alcaligenes eutrophus, Burkholderia solanacearum , and B. pickettii . In situ hybridization with a specific oligonucleotide probe corroborated the assignment of the retrieved sequence to P. necessarius .     

Crenarchaeota in Lake Michigan sediment.

RNA from Lake Michigan sediment was hybridized with a DNA probe for archaeal 16S rRNA. There was a peak of archaeal rRNA abundance in the oxic zone and another immediately below it. Six contributing species were identified by PCR amplification of extracted DNA with primers specific for archaeal rDNA: two related to Methanosarcina acetivorans and four related to marine crenarchaeotal sequences. rRNA quantification using a DNA probe specific for this crenarchaeotal assemblage showed it is most abundant in the oxic zone, where it accounts for about 10% of total archaeal rRNA.     

Spatio-temporal niche partitioning of closely related picocyanobacteria clades and phycocyanin pigment types in Lake Constance (Germany)

We found that the clade-specific abundance dynamics of Synechococcus type picocyanobacteria in the pelagic and littoral zone macro-habitats of Lake Constance (Germany) challenge the hypothesis of a regular annual succession of picocyanobacteria genotypes in temperate zone lakes. Methods used in this study were quantitative Taq nuclease assays (TNA), denaturing gradient gel electrophoresis (DGGE), a 19-month time series analysis (with two isothermal and two stratified periods) and genotyping of a new littoral phycocyanin (PC)-rich Synechococcus strain collection. The recorded differences between the two macro-habitats and between seasons or years, and the observed effect of water column mixis in winter on the inversion of clade-specific dominance ratios in Lake Constance might explain the known inter-annual differences in abundance and dynamics of the autotrophic picoplankton (APP) in lakes. The APP in Lake Constance shows a high genetic diversity with a low overall abundance, similar to the APP in the Baltic Sea, but different from Lake Biwa in Japan or lakes in the UK. Our results indicate that APP bloom events in both macro-habitats of Lake Constance are driven by phycoerythrin-rich Synechococcus genotypes of the Subalpine Cluster I. DGGE revealed the presence of a diverse periphyton (biofilm) community of the PC-rich Synechococcus pigment type in the littoral zone in early spring, when no such community was detectable in the pelagic habitat. A more sensitive and quantitative approach with TNA, however, revealed an intermittent presence of one PC-rich genotype in the plankton. We discuss the seasonal development of the pelagic and littoral PC-rich community, and while we cannot rule out a strain isolation bias, we found that isolated PC-rich strains from the pelagic habitat have different genotypes when compared to new littoral strains. We also observed littoral substrates colonized by specific PC-rich Synechococcus genotypes.     

Analysis of Bacterial and Fungal Communities in Japanese- and Chinese-Fermented Soybean Pastes Using Nested PCR–DGGE

The microbial diversity of Japanese- and Chinese-fermented soybean pastes was investigated using nested PCR–denaturing gradient gel electrophoresis (DGGE). Five Japanese-fermented soybean paste samples and three Chinese-fermented soybean paste samples were analyzed for bacteria and fungi. Extracted DNA was used as a template for PCR to amplify 16S rRNA and 18S rRNA genes. The nearly complete 16S rRNA and 18S rRNA genes were amplified using universal primers, and the resulting products were subsequently used as a template in a nested PCR to obtain suitable fragments for DGGE. Tetragenococcus halophilus and Staphylococcus gallinarum were found to dominate the bacterial microbiota in Japanese samples, whereas Bacillus sp. was detected as the predominant species in Chinese samples. DGGE analysis of fungi in soybean pastes determined the presence of Aspergillus oryzae and Zygosaccharomyces rouxii in most of the Chinese and Japanese samples. Some differences were observed in the bacterial diversity of Japanese- and Chinese-fermented soybean pastes.     

Modified linker-PCR primers facilitate complete sequencing of DGGE DNA fragments

Modified linker-PCR primers were developed to enable complete sequencing of a DGGE band in one reaction. Commonly used bacterial and archaeal 16S rRNA gene PCR-DGGE primers were modified to contain linkers and sequencing primers. This protocol does not involve additional stages, and improves retrieval of sequence from DGGE bands by approximately 23%.     

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.     

Identification of and spatio-temporal differences between microbial assemblages from two neighboring sulfurous lakes: comparison by microscopy and denaturing gradient gel electrophoresis

The microbial assemblages of Lake Cisó and Lake Vilar (Banyoles, northeast Spain) were analyzed in space and time by microscopy and by performing PCR-denaturing gradient gel electrophoresis (DGGE) and sequence analysis of 16S rRNA gene fragments. Samples obtained from different water depths and at two different times of the year (in the winter during holomixis and in the early spring during a phytoplankton bloom) were analyzed. Although the lakes have the same climatic conditions and the same water source, the limnological parameters were different, as were most of the morphologically distinguishable photosynthetic bacteria enumerated by microscopy. The phylogenetic affiliations of the predominant DGGE bands were inferred by performing a comparative 16S rRNA sequence analysis. Sequences obtained from Lake Cisó samples were related to gram-positive bacteria and to members of the division Proteobacteria. Sequences obtained from Lake Vilar samples were related to members of the Cytophaga-Flavobacterium-Bacteroides phylum and to cyanobacteria. Thus, we found that like the previously reported differences between morphologically distinct inhabitants of the two lakes, there were also differences among the community members whose morphologies did not differ conspicuously. The changes in the species composition from winter to spring were also marked. The two lakes both contained sequences belonging to phototrophic green sulfur bacteria, which is consistent with microscopic observations, but these sequences were different from the sequences of cultured strains previously isolated from the lakes. Euryarchaeal sequences (i.e., methanogen- and thermoplasma-related sequences) also were present in both lakes. These euryarchaeal group sequences dominated the archaeal sequences in Lake Cisó but not in Lake Vilar. In Lake Vilar, a new planktonic population related to the crenarchaeota produced the dominant archaeal band. The phylogenetic analysis indicated that new bacterial and archaeal lineages were present and that the microbial diversity of these assemblages was greater than previously known. We evaluated the correspondence between the abundances of several morphotypes and DGGE bands by comparing microscopy and sequencing results. Our data provide evidence that the sequences obtained from the DGGE fingerprints correspond to the microorganisms that are actually present at higher concentrations in the natural system.     

Case study of the distribution of mucosa-associated Bifidobacterium species, Lactobacillus species, and other lactic acid bacteria in the human colon

The distribution of mucosa-associated bacteria, bifidobacteria and lactobacilli and closely related lactic acid bacteria, in biopsy samples from the ascending, transverse, and descending parts of the colon from four individuals was investigated by denaturing gradient gel electrophoresis (DGGE). Bifidobacterial genus-specific, Lactobacillus group-specific, and universal bacterial primers were used in a nested PCR approach to amplify a fragment of the 16S rRNA gene. DGGE profiles of the bifidobacterial community were relatively simple, with one or two amplicons detected at most sampling sites in the colon. DGGE profiles obtained with Lactobacillus group-specific primers were complex and varied with host and sampling site in the colon. The overall bacterial community varied with host but not sampling site.     

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.     

Modified linker-PCR primers facilitate complete sequencing of DGGE DNA fragments

Modified linker-PCR primers were developed to enable complete sequencing of a DGGE band in one reaction. Commonly used bacterial and archaeal 16S rRNA gene PCR-DGGE primers were modified to contain linkers and sequencing primers. This protocol does not involve additional stages, and improves retrieval of sequence from DGGE bands by ≈ 23%.     

中国典型冻土区土壤可培养细菌多样性

[目的]对比分析中国典型高纬度冻土区和高海拔冻土区土壤可培养细菌的多样性.[方法]采用NM、TSA 、R2A 3种培养基分离培养不同冻土区土壤可培养细菌,用通用引物扩增分离的细菌16S rRNA基因,根据系统发育分析进行鉴定.[结果]从6个样品中得到冻土土壤可培养细菌的菌落数量为4.70×103 -2.57×105 cfu/g(土壤干重),根据不同的菌落形态分离出144株可培养细菌.纯培养物的16S rRNA基因部分序列分析表明:我国高纬度冻土区土壤样品中的细菌分别属于Firmicutes分支(59.52％)、Gammaproteobacteria 分支(38.10％)、Betaproteobacteria分支(2.38％),其中假单胞菌属(Pseudomonas)、芽胞杆菌属(Bacillus)、类芽胞杆菌属(Paenibacillus)的菌株为该区域的三大优势菌群.我国高海拔冻土区土壤样品中分离细菌属于Gammaproteobacteria分支(89.22％)、Firmicutes分支(8.82％)和Bacteroidetes分支(1.96％)o优势菌群为假单胞菌属( Pseudomonas).[结论]我国高纬度冻土区和高海拔冻土区土壤具有较高的可培养细菌多样性;不同类型冻土区土壤可培养细菌群落组成不同.本文研究结果将为我国冻土区土壤细菌资源研究与利用提供理论依据.     

Bacterial and archaeal assemblages in sediments of a large shallow freshwater lake, Lake Taihu, as revealed by denaturing gradient gel electrophoresis

Aims:  To explore the association of microbial community structure with the development of eutrophication in a large shallow freshwater lake, Lake Taihu.
Methods and Results:  The bacterial and archaeal assemblages in sediments of different lake areas were analysed using denaturing gradient gel electrophoresis (DGGE) of amplified 16S rDNA fragments. The bacterial DGGE profiles showed that eutrophied sites, grass-bottom areas and relatively clean sites with a eutrophic (albeit dredged) site are three respective clusters. Fifty-one dominant bacterial DGGE bands were detected and 92 corresponding clones were sequenced, most of which were affiliated with bacterial phylotypes commonly found in freshwater ecosystems. Actinobacteria were detected in the centre of the lake and not at eutrophied sites whereas the opposite was found with respect to Verrucomicrobiales . Twenty-five dominant archaeal DGGE bands were detected and 31 corresponding clones were sequenced, most of which were affiliated with freshwater archaeal phylotypes.
Conclusions:  The bacterial community structures in the sediments of different areas with similar water quality and situation tend to be similar in Taihu Lake.
Significance and Impact of the Study:  This study may expand our knowledge on the relationship between the overall microbial assemblages and the development of eutrophication in the shallow freshwater lake.     

Relationship between bacterioplankton and phytoplankton community dynamics during late spring and early summer in Lake Taihu, China

The relationship between bacterioplankton and phytoplankton community compositions was investigated from April to June, 2005 with samples taken at five different stations in Lake Taihu, China. Bacterioplankton and phytoplankton community compositions as determined by denaturing gradient gel electrophoresis of polymerase chain reaction amplified bacterial 16S rRNA gene fragments (PCR-DGGE) and microscopic examination, respectively, revealed a high degree of diversity. In total, 43 unique bands were identified amongst the DGGE profiles, and 29 dominant phytoplankton populations were observed. The DGGE profiles were clustered into two distinct groups. These two clusters were closely associated with two dominant phytoplanktons, Ulothrix sp. and Microcystis spp., as observed on April 19, May 27 and June 27, 2005, respectively. The relationship between bacterioplankton and phytoplankton community compositions was investigated using Canonical Correspondence Analysis (CCA) with the Shannon-Wiener diversity index of phytoplankton community (Hp), the total cell abundance of phytoplankton (N) and the relative abundance of Microcystis spp. (M) as the variables. The CCA results indicated a 30.9 % correlation coefficient, suggesting a high degree of correlation between bacterioplankton and phytoplankton community compositions.