Depth distribution of microbial diversity in Mono Lake,a meromictic soda lake in California

We analyzed the variation with depth in the composition of members of the domain Bacteria in samples from alkaline, hypersaline, and currently meromictic Mono Lake in California. DNA samples were collected from the mixolimnion (2 m), the base of the oxycline (17.5 m), the upper chemocline (23 m), and the monimolimnion (35 m). Composition was assessed by sequencing randomly selected cloned fragments of 16S rRNA genes retrieved from the DNA samples. Most of the 212 sequences retrieved from the samples fell into five major lineages of the domain BACTERIA: alpha- and gamma-Proteobacteria (6 and 10%, respectively), Cytophaga-Flexibacter-Bacteroides (19%), high-G+C-content gram-positive organisms (Actinobacteria; 25%), and low-G+C-content gram-positive organisms (Bacillus and Clostridium; 19%). Twelve percent were identified as chloroplasts. The remaining 9% represented beta- and delta-Proteobacteria, Verrucomicrobiales, and candidate divisions. Mixolimnion and oxycline samples had low microbial diversity, with only 9 and 12 distinct phylotypes, respectively, whereas chemocline and monimolimnion samples were more diverse, containing 27 and 25 phylotypes, respectively. The compositions of microbial assemblages from the mixolimnion and oxycline were not significantly different from each other (P = 0.314 and 0.877), but they were significantly different from those of chemocline and monimolimnion assemblages (P < 0.001), and the compositions of chemocline and monimolimnion assemblages were not significantly different from each other (P = 0.006 and 0.124). The populations of sequences retrieved from the mixolimnion and oxycline samples were dominated by sequences related to high-G+C-content gram-positive bacteria (49 and 63%, respectively) distributed in only three distinct phylotypes, while the population of sequences retrieved from the monimolimnion sample was dominated (52%) by sequences related to low-G+C-content gram-positive bacteria distributed in 12 distinct phylotypes. Twelve and 28% of the sequences retrieved from the chemocline sample were also found in the mixolimnion and monimolimnion samples, respectively. None of the sequences retrieved from the monimolimnion sample were found in the mixolimnion or oxycline samples. Elevated diversity in anoxic bottom water samples relative to oxic surface water samples suggests a greater opportunity for niche differentiation in bottom versus surface waters of this lake.     

Spatial and Temporal Patterns in the Microbial Diversity of a Meromictic Soda Lake in Washington State

The microbial community diversity and composition of meromictic Soap Lake were studied using culture-dependent and culture-independent approaches. The water column and sediments were sampled monthly for a year. Denaturing gradient gel electrophoresis of bacterial and archaeal 16S rRNA genes showed an increase in diversity with depth for both groups. Late-summer samples harbored the highest prokaryotic diversity, and the bacteria exhibited less seasonal variability than the archaea. Most-probable-number assays targeting anaerobic microbial guilds were performed to compare summer and fall samples. In both seasons, the anoxic samples appeared to be dominated by lactate-oxidizing sulfate-reducing prokaryotes. High numbers of lactate- and acetate-oxidizing iron-reducing bacteria, as well as fermentative microorganisms, were also found, whereas the numbers of methanogens were low or methanogens were undetectable. The bacterial community composition of summer and fall samples was also assessed by constructing 16S rRNA gene clone libraries. A total of 508 sequences represented an estimated >1,100 unique operational taxonomic units, most of which were from the monimolimnion, and the summer samples were more diverse than the fall samples (Chao1 = 530 and Chao1 = 295, respectively). For both seasons, the mixolimnion sequences were dominated by Gammaproteobacteria, and the chemocline and monimolimnion libraries were dominated by members of the low-G+C-content group, followed by the Cytophaga-Flexibacter-Bacteroides (CFB) group; the mixolimnion sediments contained sequences related to uncultured members of the Chloroflexi and the CFB group. Community overlap and phylogenetic analyses, however, not only demonstrated that there was a high degree of spatial turnover but also suggested that there was a degree of temporal variability due to differences in the members and structures of the communities.     

Culture-Independent Analysis of Gut Bacteria: the Pig Gastrointestinal Tract Microbiota Revisited

The phylogenetic diversity of the intestinal bacterial community in pigs was studied by comparative 16S ribosomal DNA (rDNA) sequence analysis. Samples were collected from a total of 24 pigs representing a variety of diets, ages, and herd health status. A library comprising 4,270 cloned 16S rDNA sequences obtained directly by PCR from 52 samples of either the ileum, the cecum, or the colon was constructed. In total, 375 phylotypes were identified using a 97% similarity criterion. Three hundred nine of the phylotypes (83%) had a <97% sequence similarity to any sequences in the database and may represent yet-uncharacterized bacterial genera or species. The phylotypes were affiliated with 13 major phylogenetic lineages. Three hundred four phylotypes (81%) belonged to the low-G+C gram-positive division, and 42 phylotypes (11.2%) were affiliated with the Bacteroides and Prevotella group. Four clusters of phylotypes branching off deeply within the low-G+C gram-positive bacteria and one in the Mycoplasma without any cultured representatives were found. The coverage of all the samples was 97.2%. The relative abundance of the clones approximated a lognormal distribution; however, the phylotypes detected and their abundance varied between two libraries from the same sample. The results document that the intestinal microbial community is very complex and that the majority of the bacterial species colonizing the gastrointestinal tract in pigs have not been characterized.     

16S rRNA Sequences and Differences in Bacteria Isolated from the Muztag Ata Glacier at Increasing Depths

Small subunit 16S rRNA sequences, growth temperatures, and phylogenetic relationships have been established for 129 bacterial isolates recovered under aerobic growth conditions from different regions of a 22-m ice core from the Muztag Ata Mountain glacier on the Pamirs Plateau (China). Only 11% were psychrophiles (grew at 2°C or −2°C up to ~20°C), although the majority (82%) were psychrotolerant (grew at 2°C or −2°C up to 37°C). The majority of the isolates had 16S rRNA sequences similar to previously determined sequences, ranging from 85% to 100% identical to database sequences. Based on their 16S rRNA sequences, 42.6% of the isolates were high-G+C (HGC) gram-positive bacteria, 23.3% were γ-Proteobacteria, 14.7% were α-Proteobacteria, 14.7% were Flavobacteria, and 4.7% were low-G+C (LGC) gram-positive bacteria. There were clear differences in the depth distribution, with Proteobacteria, HGC/Cytophaga-Flavobacterium-Bacteroides (CFB), Proteobacteria, LGC/CFB/HGC, Cryobacterium psychrophilum, HGC/CFB, Proteobacteria/HGC/CFB, and HGC/CFB being the predominant isolates from ice that originated from 2.7 to 3.8, 6.2, 7.5, 8.3, 9.0, 9.7, 12.5, and 15.3 m below the surface, respectively. This layered distribution of bacterial isolates presumably reflects both differences in bacteria inhabiting the glacier's surface, differences in bacteria deposited serendipitously on the glacier's surface by wind and snowfall, and nutrient availability within the ice.     

Dissimilatory microbial sulfur and methane metabolism in the water column of a shallow meromictic lake

Lake Harutori is a brackish meromictic lake with a steep physicochemical gradient in shallow water. Anoxic water below the chemocline has been characterized by high concentrations of sulfide (>10 mM) and methane (>1.5 mM). Previously, we reported that uncultured bacteria in the SEEP-SRB1 group were major sulfate reducers in the lake [21], but knowledge of sulfur oxidation and methane metabolism was scarce. In this current study, the Lake Harutori microbial community structure in the mixolimnion (at depths of 1.5 m and 3.0 m), upper chemocline (3.5 m), and monimolimnion (4.5 m) was further investigated by 16S rRNA gene amplicon sequencing and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH). Reads of type I and II methanotrophs were retrieved mainly from 3.5 m and above. Methanotrophic bacteria detected by CARD-FISH accounted for 3.1% of DAPI-stained cells at 3.5 m. Detection frequencies of reads affiliated with the genera Sulfurimonas and Thiomicrorhabdus, which are known to comprise sulfur oxidizers, were relatively high at 3.5 m. Methanogenic archaeal reads were retrieved from the monimolimnion and they affiliated with the genus Methanosaeta. CARD-FISH counts indicated that the cells of Methanosaeta/Methanosarcina/Methanomicrobiales accounted for up to 0.8% of the DAPI-stained cells in the monimolimnion. On the other hand, many of the reads retrieved primarily from the monimolimnion were affiliated with phylogenetically novel uncultured groups.     

Detection and Isolation of Ultrasmall Microorganisms from a 120,000-Year-Old Greenland Glacier Ice Core

The abundant microbial population in a 3,043-m-deep Greenland glacier ice core was dominated by ultrasmall cells (<0.1 μm³) that may represent intrinsically small organisms or starved, minute forms of normal-sized microbes. In order to examine their diversity and obtain isolates, we enriched for ultrasmall psychrophiles by filtering melted ice through filters with different pore sizes, inoculating anaerobic low-nutrient liquid media, and performing successive rounds of filtrations and recultivations at 5°C. Melted ice filtrates, cultures, and isolates were analyzed by scanning electron microscopy, flow cytometry, cultivation, and molecular methods. The results confirmed that numerous cells passed through 0.4-μm, 0.2-μm, and even 0.1-μm filters. Interestingly, filtration increased cell culturability from the melted ice, yielding many isolates related to high-G+C gram-positive bacteria. Comparisons between parallel filtered and nonfiltered cultures showed that (i) the proportion of 0.2-μm-filterable cells was higher in the filtered cultures after short incubations but this difference diminished after several months, (ii) more isolates were obtained from filtered (1,290 isolates) than from nonfiltered (447 isolates) cultures, and (iii) the filtration and liquid medium cultivation increased isolate diversity (Proteobacteria; Cytophaga-Flavobacteria-Bacteroides; high-G+C gram-positive; and spore-forming, low-G+C gram-positive bacteria). Many isolates maintained their small cell sizes after recultivation and were phylogenetically novel or related to other ultramicrobacteria. Our filtration-cultivation procedure, combined with long incubations, enriched for novel ultrasmall-cell isolates, which is useful for studies of their metabolic properties and mechanisms for long-term survival under extreme conditions.     

Phylogenetic and Physiological Diversity of Microorganisms Isolated from a Deep Greenland Glacier Ice Core

We studied a sample from the GISP 2 (Greenland Ice Sheet Project) ice core to determine the diversity and survival of microorganisms trapped in the ice at least 120,000 years ago. Previously, we examined the phylogenetic relationships among 16S ribosomal DNA (rDNA) sequences in a clone library obtained by PCR amplification from genomic DNA extracted from anaerobic enrichments. Here we report the isolation of nearly 800 aerobic organisms that were grouped by morphology and amplified rDNA restriction analysis patterns to select isolates for further study. The phylogenetic analyses of 56 representative rDNA sequences showed that the isolates belonged to four major phylogenetic groups: the high-G+C gram-positives, low-G+C gram-positives, Proteobacteria, and the Cytophaga-Flavobacterium-Bacteroides group. The most abundant and diverse isolates were within the high-G+C gram-positive cluster that had not been represented in the clone library. The Jukes-Cantor evolutionary distance matrix results suggested that at least 7 isolates represent new species within characterized genera and that 49 are different strains of known species. The isolates were further categorized based on the isolation conditions, temperature range for growth, enzyme activity, antibiotic resistance, presence of plasmids, and strain-specific genomic variations. A significant observation with implications for the development of novel and more effective cultivation methods was that preliminary incubation in anaerobic and aerobic liquid prior to plating on agar media greatly increased the recovery of CFU from the ice core sample.     

Phylogeny of the Defined Murine Microbiota: Altered Schaedler Flora

The “altered Schaedler flora” (ASF) was developed for colonizing germfree rodents with a standardized microbiota. The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis. Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), Lactobacillus salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519) based on phenotypic criteria. 16S rRNA analysis indicated that each of the strains differed from its presumptive identity. The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species. Strain ASF 360 is a novel lactobacillus that clusters with L. acidophilus and Lactobacillus lactis. Strain ASF 519 falls into an unnamed genus containing [Bacteroides] distasonis, [Bacteroides] merdae, [Bacteroides] forsythus, and CDC group DF-3. This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas. The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson. The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group). ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al. Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium plexicaudatum. The 16S rRNA sequence of ASF 492 is identical to that of E. plexicaudatum. Since the type strain and other viable original isolates of E. plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain. Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database. The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.     

Phylogenetic Analysis of Bacterial Communities in Mesophilic and Thermophilic Bioreactors Treating Pharmaceutical Wastewater

The phylogenetic diversity of the bacterial communities supported by a seven-stage, full-scale biological wastewater treatment plant was studied. These reactors were operated at both mesophilic (28 to 32°C) and thermophilic (50 to 58°C) temperatures. Community fingerprint analysis by denaturing gradient gel electrophoresis (DGGE) of the PCR-amplified V3 region of the 16S rRNA gene from the domain Bacteria revealed that these seven reactors supported three distinct microbial communities. A band-counting analysis of the PCR-DGGE results suggested that elevated reactor temperatures corresponded with reduced species richness. Cloning of nearly complete 16S rRNA genes also suggested a reduced species richness in the thermophilic reactors by comparing the number of clones with different nucleotide inserts versus the total number of clones screened. While these results imply that elevated temperature can reduce species richness, other factors also could have impacted the number of populations that were detected. Nearly complete 16S rDNA sequence analysis showed that the thermophilic reactors were dominated by members from the β subdivision of the division Proteobacteria (β-proteobacteria) in addition to anaerobic phylotypes from the low-G+C gram-positive and Synergistes divisions. The mesophilic reactors, however, included at least six bacterial divisions, including Cytophaga-Flavobacterium-Bacteroides, Synergistes, Planctomycetes, low-G+C gram-positives, Holophaga-Acidobacterium, and Proteobacteria (α-proteobacteria, β-proteobacteria, γ-proteobacteria and δ-proteobacteria subdivisions). The two PCR-based techniques detected the presence of similar bacterial populations but failed to coincide on the relative distribution of these phylotypes. This suggested that at least one of these methods is insufficiently quantitative to determine total community biodiversity—a function of both the total number of species present (richness) and their relative distribution (evenness).     

Horizontal Gene Transfer of PIB-Type ATPases among Bacteria Isolated from Radionuclide- and Metal-Contaminated Subsurface Soils

Aerobic heterotrophs were isolated from subsurface soil samples obtained from the U.S. Department of Energy's (DOE) Field Research Center (FRC) located at Oak Ridge, Tenn. The FRC represents a unique, extreme environment consisting of highly acidic soils with cooccurring heavy metals, radionuclides, and high nitrate concentrations. Four hundred isolates obtained from contaminated soil were assayed for heavy metal resistance, and a smaller subset was assayed for tolerance to uranium. The vast majority of the isolates were gram-positive bacteria and belonged to the high-G+C- and low-G+C-content genera Arthrobacter and Bacillus, respectively. Genomic DNA from a randomly chosen subset of 50 Pb-resistant (Pb^r) isolates was amplified with PCR primers specific for P_IB-type ATPases (i.e., pbrA/cadA/zntA). A total of 10 pbrA/cadA/zntA loci exhibited evidence of acquisition by horizontal gene transfer. A remarkable dissemination of the horizontally acquired P_IB-type ATPases was supported by unusual DNA base compositions and phylogenetic incongruence. Numerous Pb^r P_IB-type ATPase-positive FRC isolates belonging to the genus Arthrobacter tolerated toxic concentrations of soluble U(VI) (UO₂²⁺) at pH 4. These unrelated, yet synergistic, physiological traits observed in Arthrobacter isolates residing in the contaminated FRC subsurface may contribute to the survival of the organisms in such an extreme environment. This study is, to the best of our knowledge, the first study to report broad horizontal transfer of P_IB-type ATPases in contaminated subsurface soils and is among the first studies to report uranium tolerance of aerobic heterotrophs obtained from the acidic subsurface at the DOE FRC.     

The physical structure and dynamics of a deep,meromictic crater lake (Lac Pavin,France)

The characteristic feature of the physical structure of Lac Pavin is a distinct and permanent chemically induced density increase between about 60 and 70 m depth. This chemocline separates the seasonally mixed mixolimnion from the monimolimnion, which is characterized by elevated temperature and salinity as well as complete anoxia. Previously published box-models of the lake postulated substantial groundwater input at the lake bottom, and consequently a short water residence time in the monimolimnion and high fluxes of dissolved constituents across the chemocline. We present a new view of the physical structure and dynamics of Lac Pavin, which is based on the results of high-resolution CTD profiles, transient and geochemical tracers (tritium, CFCs, helium), and numerical modeling. The CTD profiles indicate the existence of a sublacustrine spring above rather than below the chemocline. A stability analysis of the water column suggests that vertical turbulent mixing in the chemocline is very weak. A numerical one-dimensional lake model is used to reconstruct the evolution of transient tracer distributions over the past 50 years. Model parameters such as vertical diffusivity and size of sublacustrine springs are constrained by comparison with observed tracer data. Whereas the presence of a significant water input to the monimolimnion can clearly be excluded, the input to the mixolimnion – both at the surface and from the indicated sublacustrine spring – cannot be accurately determined. The vertical turbulent diffusivity in the chemocline is well constrained to K 5×10^-8 m² s^-1, about a factor of three below the molecular diffusivity for heat. Assuming thus purely molecular heat transport, the heat flow through the chemocline can be estimated to between 30 and 40 mW m^-2. With respect to dissolved constituents, the very weak turbulent diffusive exchange is equivalent to a stagnant monimolimnion with a residence time of nearly 100 years. Based on these results and vertical concentration profiles of dissolved species, diffusive fluxes between monimolimnion and mixolimnion can be calculated. A large excess of helium with a ³He/⁴He ratio of (9.09 ± 0.01)×10^-6 (6.57 R _a) is present in the monimolimnion, clearly indicating a flux of magmatic gases into the monimolimnion. We calculate a flux of 1.0×10^-12 mol m^-2 s^-1 for mantle helium and infer a flux of 1.2×10^-7 mol m^-2 s^-1 (72 t year^-1) for magmatic CO₂. The monimolimnion appears to be in steady state with respect to these fluxes.     

Comparative Genomics of DNA Fragments from Six Antarctic Marine Planktonic Bacteria

Six environmental fosmid clones from Antarctic coastal water bacterioplankton were completely sequenced. The genome fragments harbored small-subunit rRNA genes that were between 85 and 91% similar to those of their nearest cultivated relatives. The six fragments span four phyla, including the Gemmatimonadetes, Proteobacteria (α and γ), Bacteroidetes, and high-G+C gram-positive bacteria. Gene-finding and annotation analyses identified 244 total open reading frames. Amino acid comparisons of 123 and 113 Antarctic bacterial amino acid sequences to mesophilic homologs from G+C-specific and SwissProt/UniProt databases, respectively, revealed widespread adaptation to the cold. The most significant changes in these Antarctic bacterial protein sequences included a reduction in salt-bridge-forming residues such as arginine, glutamic acid, and aspartic acid, reduced proline contents, and a reduction in stabilizing hydrophobic clusters. Stretches of disordered amino acids were significantly longer in the Antarctic sequences than in the mesophilic sequences. These characteristics were not specific to any one phylum, COG role category, or G+C content and imply that underlying genotypic and biochemical adaptations to the cold are inherent to life in the permanently subzero Antarctic waters.     

The Black Water Chemocline of Meromictic Lower Mystic Lake,Massachusetts, U.S.A.

Lower Mystic Lake, Massachusetts, USA, has an anoxic black water layer just below the top of the chemocline (15.5–16.0 m). Bacterial concentrations averaged 10.4 × 10⁶ cells/ml in the black water layer and 4.0 × 10⁶ cells/ml below 17 m. Below the chemocline, microbial concentrations were linearly correlated to the vertical light absorption coefficient, r = 0.82. Phototrophic bacteria were not detected below the top of the chemocline, due to a low PAR that never exceeded 0.0001% surface illumination. Sulfate‐reducing bacteria and methanogens were enriched from the monimolimnion in selective media. Below the chemocline, H₂S concentrations were in excess of 11 mmoles/l and Fe, Mn, CH₄ and CO₂ concentrations were elevated compared to the mixolimnion. Nuisance releases of H₂S occurred from the lake in 1965. Although the monimolimnion remains a highly reduced environment rich in H₂S, the potential of further nuisance releases is small due to the diminished volume of the monimolimnion and the relatively deep chemocline.     

Response of Endophytic Bacterial Communities in Potato Plants to Infection with Erwinia carotovora subsp. atroseptica

The term endophyte refers to interior colonization of plants by microorganisms that do not have pathogenic effects on their hosts, and various endophytes have been found to play important roles in plant vitality. In this study, cultivation-independent terminal restriction fragment length polymorphism analysis of 16S ribosomal DNA directly amplified from plant tissue DNA was used in combination with molecular characterization of isolates to examine the influence of plant stress, achieved by infection with the blackleg pathogen Erwinia carotovora subsp. atroseptica, on the endophytic population in two different potato varieties. Community analysis clearly demonstrated increased bacterial diversity in infected plants compared to that in control plants. The results also indicated that the pathogen stress had a greater impact on the bacteria population than the plant genotype had. Partial sequencing of the 16S rRNA genes of isolated endophytes revealed a broad phylogenetic spectrum of bacteria, including members of the α, β, and γ subgroups of the Proteobacteria, high- and low-G+C-content gram-positive organisms, and microbes belonging to the Flexibacter-Cytophaga-Bacteroides group. Screening of the isolates for antagonistic activity against E. carotovora subsp. atroseptica revealed that 38% of the endophytes protected tissue culture plants from blackleg disease.     

Vertical Distribution of Archaea and Bacteria in a Meromictic Lake as Determined by Fluorescent In Situ Hybridization

The prokaryotic cells distribution in the water column of the coastal saline meromictic Lake Faro (Messina, Italy) was investigated by microscopic counting techniques. Water samples were collected at a central station from the surface to the bottom, when waters were characterized by a marked stratification. A “red-water” layer, caused by a dense growth of photosynthetic sulfur bacteria, was present at a depth of 15 m, defining a transition area between oxic (mixolimnion) and anoxic (monimolimnion) layers. Fluorescently labeled 16S rRNA oligonucleotide, group-specific probes were used to determine the abundance of Bacteria and Archaea, and their subgroups, Green Sulfur Bacteria (GSB), Sulfate Reducing Bacteria (SRB), Cyanobacteria and Chromatium okenii, and Crenarchaeota and Euryarchaeota, as key elements of the microbial community. Bacteria decreased from surface to bottom, while Archaea increased with depth and reached the maximum value at 30 m, where they outnumbered the Bacteria. Bacteria and picophytoplankton prevailed in the mixolimnion. At the chemocline high numbers of prokaryotic cells were present, mainly represented by Cyanobacteria, Chromatium okenii and Euryarchaeota. GSB, SRB, and Crenarchaeota prevailed below the chemocline. Although Archaea constitute a minor fraction of microbial community, they could represent active contributors to the meromictic Lake Faro ecosystem.     

Succession of Bacterial Community Structure along the Changjiang River Determined by Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

Bacterial community structure along the Changjiang River (which is more than 2,500 km long) was studied by using denaturing gradient gel electrophoresis (DGGE) and clone library analysis of PCR-amplified 16S ribosomal DNA (rDNA) with universal bacterial primer sets. DGGE profiles and principal-component analysis (PCA) demonstrated that the bacterial community gradually changed from upstream to downstream in both 1998 and 1999. Bacterial diversity, as determined by the Shannon index (H′), gradually decreased from upstream to downstream. The PCA plots revealed that the differences in the bacterial communities among riverine stations were not appreciable compared with the differences in two adjacent lakes, Lake Dongting and Lake Poyang. The relative stability of the bacterial communities at the riverine stations was probably due to the buffering action of the large amount of water flowing down the river. Clone library analysis of 16S rDNA revealed that the dominant bacterial groups changed from β-proteobacteria and the Cytophaga-Flexibacter-Bacteroides group upstream to high-G+C-content gram-positive bacteria downstream and also that the bacterial community structure differed among the stations in the river and the lakes. The results obtained in this study should provide a reference for future changes caused by construction of the Three Gorges Dam.     

Vertical distributions of stable isotopic compositions and bacteriochlorophyll homologues in suspended particulate matter in saline meromictic Lake Abashiri

We determined chloropigment composition as well as stable carbon and nitrogen isotopic compositions of dissolved and particulate species in saline meromictic Lake Abashiri. We observed a sharp peak of bacteriochlorophyll e in a narrow redox boundary zone and the upper monimolimnion, indicating a dense population of brown-colored strains of green sulfur bacteria around the chemocline. Nitrogen isotopic records of particulate nitrogen and dissolved ammonium suggested that the green sulfur bacteria in the redox boundary zone assimilated either ammonium or dinitrogen through the nitrogen fixation pathway. In the anoxic monimolimnion, several lines of evidence suggest that a major portion of particulate organic matter originated from the overlying mixolimnion and redox boundary zone.     

Seasonal and spatial community dynamics in the meromictic Lake Cadagno

The seasonal and spatial variations in the community structure of bacterioplankton in the meromictic alpine Lake Cadagno were examined by temporal temperature gradient gel electrophoresis (TTGE) of PCR-amplified 16S rDNA fragments. Two different amplifications were performed, one specific for the domain Bacteria (Escherichia coli positions 8-536) and another specific for the family Chromatiaceae (E. coli positions 8-1005). The latter was followed by semi-nested reamplification with the bacterial primer set, allowing comparison of the two PCR approaches by TTGE. The TTGE patterns of samples from the chemocline and the anoxic monimolimnion were essentially identical, whereas the oxic mixolimnion displayed distinctively different banding patterns. For samples from the chemocline and the monimolimnion, dominant bands in the Bacteria-specific TTGE profiles comigrated with bands obtained by the semi-nested PCR approach specific for Chromatiaceae. This observation suggested that Chromatiaceae are in high abundance in the anoxic water layer. All dominant bands were excised and sequenced. Changes in the community structure, as indicated by changes in the TTGE profiles, were observed in samples taken at different times of the year. In the chemocline, Chomatium okenii was dominant in the summer months, whereas Amoebobacter purpureus populations dominated in autumn and winter. This change was confirmed by fluorescent in situ hybridization.     

Impact of Protists on the Activity and Structure of the Bacterial Community in a Rice Field Soil

Flooded rice fields have become a model system for the study of soil microbial ecology. In Italian rice fields, in particular, aspects from biogeochemistry to molecular ecology have been studied, but the impact of protistan grazing on the structure and function of the prokaryotic community has not been examined yet. We compared an untreated control soil with a γ-radiation-sterilized soil that had been reinoculated with a natural bacterial assemblage. In order to verify that the observed effects were due to protistan grazing and did not result from sterilization, we set up a third set of microcosms containing sterilized soil that had been reinoculated with natural assemblage bacteria plus protists. The spatial and temporal changes in the protistan and prokaryotic communities were examined by denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) analysis, respectively, both based on the small-subunit gene. Sequences retrieved from DGGE bands were preferentially affiliated with Cercozoa and other bacteriovorous flagellates. Without protists, the level of total DNA increased with incubation time, indicating that the level of the microbial biomass was elevated. Betaproteobacteria were preferentially preyed upon, while low-G+C-content gram-positive bacteria became more dominant under grazing pressure. The bacterial diversity detectable by T-RFLP analysis was greater in the presence of protists. The level of extractable NH₄⁺ was lower and the level of extractable SO₄²⁻ was higher without protists, indicating that nitrogen mineralization and SO₄²⁻ reduction were stimulated by protists. Most of these effects were more obvious in the partially oxic surface layer (0 to 3 mm), but they could also be detected in the anoxic subsurface layer (10 to 13 mm). Our observations fit well into the overall framework developed for protistan grazing, but with some modifications pertinent to the wetland situation: O₂ was a major control, and O₂ availability may have limited directly and indirectly the development of protists. Although detectable in the lower anoxic layer, grazing effects were much more obvious in the partially oxic surface layer.     

Microbial Communities and Fecal Indicator Bacteria Associated with Cladophora Mats on Beach Sites along Lake Michigan Shores

A high biomasses of Cladophora, a filamentous green alga, is found mainly during the summer along the shores of Lake Michigan. In this study, the abundance and persistence of the fecal indicator bacterium Escherichia coli and sulfate-reducing bacteria (SRB) on Cladophora mats collected at Lake Michigan beaches were evaluated using both culture-based and molecular analyses. Additionally, 16S rRNA gene cloning and sequencing were used to examine the bacterial community composition. Overall, E. coli was detected in all 63 samples obtained from 11 sites, and the average levels at most beaches ranged from 2,700 CFU/100 g (wet weight) of Cladophora to 7,500 CFU/100 g of Cladophora. However, three beaches were found to have site average E. coli densities of 12,800, 21,130, and 27,950 CFU/100 g of Cladophora. The E. coli levels in the lake water collected at the same time from these three sites were less than the recommended U.S. Environmental Protection Agency limit, 235 CFU/100 ml. E. coli also persisted on Cladophora mats in microcosms at room temperature for more than 7 days, and in some experiments it persisted for as long as 28 days. The SRB densities on Cladophora mats were relatively high, ranging from 4.4 × 10⁶ cells/g (6.64 log CFU/g) to 5.73 × 10⁶ cells/g (6.76 log CFU/g) and accounting for between 20% and 27% of the total bacterial counts. Partial sequences of the 16S rRNA gene clones revealed a phylogenetically diverse community, in which the Cytophaga-Flavobacterium-Bacteroides cluster and the low-G+C-content gram-positive bacteria were the dominant organisms, accounting for 40% and 12.8%, respectively, of the total clone library. These results further reveal the potential public health and ecological significance of Cladophora mats that are commonly found along the shoreline of Lake Michigan, especially with regard to the potential to harbor microorganisms associated with fecal pollution and odor-causing bacteria.