Quantification of virus genes provides evidence for seed-bank populations of phycodnaviruses in Lake Ontario,Canada

Using quantitative PCR, the abundances of six phytoplankton viruses DNA polymerase (polB) gene fragments were estimated in water samples collected from Lake Ontario, Canada over 26 months. Four of the polB fragments were most related to marine prasinoviruses, while the other two were most closely related to cultivated chloroviruses. Two Prasinovirus-related genes reached peak abundances of >1000 copies ml⁻¹ and were considered ‘high abundance'', whereas the other two Prasinovirus-related genes peaked at abundances <1000 copies ml⁻¹ and were considered ‘low abundance''. Of the genes related to chloroviruses, one peaked at ca 1600 copies ml⁻¹, whereas the other reached only ca 300 copies ml⁻¹. Despite these differences in peak abundance, the abundances of all genes monitored were lowest during the late fall, winter and early spring; during these months the high abundance genes persisted at 100–1000 copies ml⁻¹ while the low abundance Prasinovirus- and Chlorovirus-related genes persisted at fewer than ca 100 copies ml⁻¹. Clone libraries of psbA genes from Lake Ontario revealed numerous Chlorella-like algae and two prasinophytes demonstrating the presence of candidate hosts for all types of viruses monitored. Our results corroborate recent metagenomic analyses that suggest that aquatic virus communities are composed of only a few abundant populations and many low abundance populations. Thus, we speculate that an ecologically important characteristic of phycodnavirus communities is seed-bank populations with members that can become numerically dominant when their host abundances reach appropriate levels.     

Viral Abundance, Decay, and Diversity in the Meso- and Bathypelagic Waters of the North Atlantic

To elucidate the potential importance of deep-water viruses in controlling the meso- and bathypelagic picoplankton community, the abundance, decay rate, and diversity of the virioplankton community were determined in the meso- and bathypelagic water masses of the eastern part of the subtropical North Atlantic. Viral abundance averaged 1.4 × 10⁶ ml⁻¹ at around 100 m of depth and decreased only by a factor of 2 at 3,000 to 4,000 m of depth. In contrast, picoplankton abundance decreased by 1 order of magnitude to the Lower Deep Water (LDW; 3,500- to 5,000-m depth). The virus-to-picoplankton ratio increased from 9 at about 100 m of depth to 110 in the LDW. Mean viral decay rates were 3.5 × 10⁻³ h⁻¹ between 900 m and 2,750 m and 1.1 × 10⁻³ h⁻¹ at 4,000 m of depth, corresponding to viral turnover times of 11 and 39 days, respectively. Pulsed-field gel electrophoresis fingerprints obtained from the viral community between 2,400 m and 4,000 m of depth revealed a maximum of only four bands from 4,000 m of depth. Based on the high viral abundance and the low picoplankton production determined via leucine incorporation, we conclude that the viral production calculated from the viral decay is insufficient to maintain the high viral abundance in the deep North Atlantic. Rather, we propose that substantial allochthonous viral input or lysogenic or pseudolysogenic production is required to maintain the high viral abundance detected in the meso- and bathypelagic North Atlantic. Consequently, deep-water prokaryotes are apparently far less controlled in their abundance and taxon richness by lytic prokaryotic phages than the high viral abundance and the virus-to-picoplankton ratio would suggest.     

Potential Importance of Fish Predation and Zooplankton Grazing on Natural Populations of Freshwater Bacteria

The rates of ingestion of natural bacterial assemblages by natural populations of zooplankton (>50 μm in size) were measured during a 19-day period in eutrophic Frederiksborg Slotssø, Denmark, as well as in experimental enclosures (containing 5.3 m³ of lake water). The fish and nutrients of the enclosures were manipulated. In enclosures without fish, large increases in ingestion by zooplankton >140 μm in size were found (up to 3 μg of C liter⁻¹ h⁻¹), compared with values less than 0.3 μg of C liter⁻¹ h⁻¹ in the enclosures with fish and in the open lake. Daphnia cucullata and D. galeata dominated the community of zooplankton of >140 μm. Ingestion rates for zooplankton between 50 and 140 μm decreased after a period of about 8 days, in all enclosures and in the lake, to values below 0.1 μg of C liter⁻¹ h⁻¹. On the last 2 sampling days, somewhat higher values were observed in the enclosures with fish present. The >50-μm zooplankton ingested 48 to 51% of the bacterial net secondary production in enclosures without fish, compared to 4% in the enclosures with added fish. Considering the sum of bacterial secondary production plus biomass change, 35 to 41% of the available bacteria were ingested by zooplankton of >50 μm in the enclosures without fish, compared with 4 to 6% in the enclosures with added fish and 21% in the open lake. Fish predation reduced the occurrence of zookplankton sized >50 μm and thus left a large proportion of the available bacteria to zooplankton sized <50 μm. In fact, there were 4.6 × 10³ to 5.0 × 10³ flagellates (4 to 8 μm in size) ml⁻¹ in the enclosures with fish added as well as in the lake, compared with 0.5 × 10² to 2.3 × 10² ml⁻¹ in the enclosures without fish. This link in the food chain was reduced when fish predation on zooplankton was eliminated and a direct route of dissolved organic matter, via the bacteria to the zooplankton, was established.     

Novel Psychropiezophilic Oceanospirillales Species Profundimonas piezophila gen. nov., sp. nov., Isolated from the Deep-Sea Environment of the Puerto Rico Trench

The diversity of deep-sea high-pressure-adapted (piezophilic) microbes in isolated monoculture remains low. In this study, a novel obligately psychropiezophilic bacterium was isolated from seawater collected from the Puerto Rico Trench at a depth of ∼6,000 m. This isolate, designated YC-1, grew best in a nutrient-rich marine medium, with an optimal growth hydrostatic pressure of 50 MPa (range, 20 to 70 MPa) at 8°C. Under these conditions, the maximum growth rate was extremely slow, 0.017 h⁻¹, and the maximum yield was 3.51 × 10⁷ cells ml⁻¹. Cell size and shape changed with pressure, shifting from 4.0 to 5.0 μm in length and 0.5 to 0.8 μm in width at 60 MPa to 0.8- to 1.0-μm diameter coccoid cells under 20 MPa, the minimal pressure required for growth. YC-1 is a Gram-negative, facultatively anaerobic heterotroph. Its predominant cellular fatty acids are the monounsaturated fatty acids (MUFAs) C_16:1 and C_18:1. Unlike many other psychropiezophiles, YC-1 does not synthesize any polyunsaturated fatty acids (PUFAs). Phylogenetic analysis placed YC-1 within the family of Oceanospirillaceae, closely related to the uncultured symbiont of the deep-sea whale bone-eating worms of the genus Osedax. In common with some other members of the Oceanospirillales, including those enriched during the Deepwater Horizon oil spill, YC-1 is capable of hydrocarbon utilization. On the basis of its characteristics, YC-1 appears to represent both a new genus and a new species, which we name Profundimonas piezophila gen. nov., sp. nov.     

Culturable Heavy Metal-Resistant and Plant Growth Promoting Bacteria in V-Ti Magnetite Mine Tailing Soil from Panzhihua,China

To provide a basis for using indigenous bacteria for bioremediation of heavy metal contaminated soil, the heavy metal resistance and plant growth-promoting activity of 136 isolates from V-Ti magnetite mine tailing soil were systematically analyzed. Among the 13 identified bacterial genera, the most abundant genus was Bacillus (79 isolates) out of which 32 represented B. subtilis and 14 B. pumilus, followed by Rhizobium sp. (29 isolates) and Ochrobactrum intermedium (13 isolates). Altogether 93 isolates tolerated the highest concentration (1000 mg kg⁻¹) of at least one of the six tested heavy metals. Five strains were tolerant against all the tested heavy metals, 71 strains tolerated 1,000 mg kg⁻¹ cadmium whereas only one strain tolerated 1,000 mg kg⁻¹ cobalt. Altogether 67% of the bacteria produced indoleacetic acid (IAA), a plant growth-promoting phytohormone. The concentration of IAA produced by 53 isolates was higher than 20 µg ml⁻¹. In total 21% of the bacteria produced siderophore (5.50–167.67 µg ml⁻¹) with two Bacillus sp. producing more than 100 µg ml⁻¹. Eighteen isolates produced both IAA and siderophore. The results suggested that the indigenous bacteria in the soil have beneficial characteristics for remediating the contaminated mine tailing soil.     

Temporal Changes and Altitudinal Distribution of Aerobic Anoxygenic Phototrophs in Mountain Lakes

Aerobic anoxygenic phototrophs (AAPs) are bacteriochlorophyll a-containing microorganisms that use organic substrates for growth but can supplement their energy requirements with light. They have been reported from various marine and limnic environments; however, their ecology remains largely unknown. Here infrared epifluorescence microscopy was used to monitor temporal changes in AAPs in the alpine lake Gossenköllesee, located in the Tyrolean Alps, Austria. AAP abundance was low (10³ cells ml⁻¹) until mid-July and reached a maximum of ∼1.3 × 10⁵ cells ml⁻¹ (29% of all prokaryotes) in mid-September. We compared the studied lake with other mountain lakes located across an altitudinal gradient (913 to 2,799 m above sea level). The concentration of dissolved organic carbon and water transparency seem to be the main factors influencing AAP abundance during the seasonal cycle as well as across the altitudinal gradient. While the AAP populations inhabiting the alpine lakes were composed of intensely pigmented large rods (5 to 12 μm), the lakes below the tree line were inhabited by a variety of smaller morphotypes. Analysis of pufM diversity revealed that AAPs in Gossenköllesee were almost exclusively Sphingomonadales species, which indicates that AAP communities inhabiting alpine lakes are relatively homogeneous compared to those in low-altitude lakes.     

Estimating Bacterioplankton Production by Measuring [3H]thymidine Incorporation in a Eutrophic Swedish Lake

Bacterioplankton abundance, [³H]thymidine incorporation, ¹⁴CO₂ uptake in the dark, and fractionated primary production were measured on several occasions between June and August 1982 in eutrophic Lake Norrviken, Sweden. Bacterioplankton abundance and carbon biomass ranged from 0.5 × 10⁹ to 2.4 × 10⁹ cells liter⁻¹ and 7 to 47 μg of C liter⁻¹, respectively. The average bacterial cell volume was 0.185 μm³. [³H]thymidine incorporation into cold-trichloroacetic acid-insoluble material ranged from 12 × 10⁻¹² to 200 × 10⁻¹² mol liter⁻¹ h⁻¹. Bacterial carbon production rates were estimated to be 0.2 to 7.1 μg of C liter⁻¹ h⁻¹. Bacterial production estimates from [³H]thymidine incorporation and ¹⁴CO₂ uptake in the dark agreed when activity was high but diverged when activity was low and when blue-green algae (cyanobacteria) dominated the phytoplankton. Size fractionation indicated negligible uptake of [³H]thymidine in the >3-μm fraction during a chrysophycean bloom in early June. We found that >50% of the ³H activity was in the >3-μm fraction in late August; this phenomenon was most likely due to Microcystis spp., their associated bacteria, or both. Over 60% of the ¹⁴CO₂ uptake in the dark was attributed to algae on each sampling occasion. Algal exudate was an important carbon source for planktonic bacteria. Bacterial production was roughly 50% of primary production.     

Global abundance of planktonic heterotrophic protists in the deep ocean

The dark ocean is one of the largest biomes on Earth, with critical roles in organic matter remineralization and global carbon sequestration. Despite its recognized importance, little is known about some key microbial players, such as the community of heterotrophic protists (HP), which are likely the main consumers of prokaryotic biomass. To investigate this microbial component at a global scale, we determined their abundance and biomass in deepwater column samples from the Malaspina 2010 circumnavigation using a combination of epifluorescence microscopy and flow cytometry. HP were ubiquitously found at all depths investigated down to 4000 m. HP abundances decreased with depth, from an average of 72±19 cells ml⁻¹ in mesopelagic waters down to 11±1 cells ml⁻¹ in bathypelagic waters, whereas their total biomass decreased from 280±46 to 50±14 pg C ml⁻¹. The parameters that better explained the variance of HP abundance were depth and prokaryote abundance, and to lesser extent oxygen concentration. The generally good correlation with prokaryotic abundance suggested active grazing of HP on prokaryotes. On a finer scale, the prokaryote:HP abundance ratio varied at a regional scale, and sites with the highest ratios exhibited a larger contribution of fungi molecular signal. Our study is a step forward towards determining the relationship between HP and their environment, unveiling their importance as players in the dark ocean''s microbial food web.     

Comparison of the Seasonal Variations of Synechococcus Assemblage Structures in Estuarine Waters and Coastal Waters of Hong Kong

Seasonal variation in the phylogenetic composition of Synechococcus assemblages in estuarine and coastal waters of Hong Kong was examined through pyrosequencing of the rpoC1 gene. Sixteen samples were collected in 2009 from two stations representing estuarine and ocean-influenced coastal waters, respectively. Synechococcus abundance in coastal waters gradually increased from 3.6 × 10³ cells ml⁻¹ in March, reaching a peak value of 5.7 × 10⁵ cells ml⁻¹ in July, and then gradually decreased to 9.3 × 10³ cells ml⁻¹ in December. The changes in Synechococcus abundance in estuarine waters followed a pattern similar to that in coastal waters, whereas its composition shifted from being dominated by phycoerythrin-rich (PE-type) strains in winter to phycocyanin-only (PC-type) strains in summer owing to the increase in freshwater discharge from the Pearl River and higher water temperature. The high abundance of PC-type Synechococcus was composed of subcluster 5.2 marine Synechococcus, freshwater Synechococcus (F-PC), and Cyanobium. The Synechococcus assemblage in the coastal waters, on the other hand, was dominated by marine PE-type Synechococcus, with subcluster 5.1 clades II and VI as the major lineages from April to September, when the summer monsoon prevailed. Besides these two clades, clade III cooccurred with clade V at relatively high abundance in summer. During winter, the Synechococcus assemblage compositions at the two sites were similar and were dominated by subcluster 5.1 clades II and IX and an undescribed clade (represented by Synechococcus sp. strain miyav). Clade IX Synechococcus was a relatively ubiquitous PE-type Synechococcus found at both sites, and our study demonstrates that some strains of the clade have the ability to deal with large variation of salinity in subtropical estuarine environments. Our study suggests that changes in seawater temperature and salinity caused by the seasonal variation of monsoonal forcing are two major determinants of the community composition and abundance of Synechococcus assemblages in Hong Kong waters.     

An Inexpensive,Accurate, and Precise Wet-Mount Method for Enumerating Aquatic Viruses

Viruses affect biogeochemical cycling, microbial mortality, gene flow, and metabolic functions in diverse environments through infection and lysis of microorganisms. Fundamental to quantitatively investigating these roles is the determination of viral abundance in both field and laboratory samples. One current, widely used method to accomplish this with aquatic samples is the “filter mount” method, in which samples are filtered onto costly 0.02-μm-pore-size ceramic filters for enumeration of viruses by epifluorescence microscopy. Here we describe a cost-effective (ca. 500-fold-lower materials cost) alternative virus enumeration method in which fluorescently stained samples are wet mounted directly onto slides, after optional chemical flocculation of viruses in samples with viral concentrations of <5 × 10⁷ viruses ml⁻¹. The concentration of viruses in the sample is then determined from the ratio of viruses to a known concentration of added microsphere beads via epifluorescence microscopy. Virus concentrations obtained by using this wet-mount method, with and without chemical flocculation, were significantly correlated with, and had precision equivalent to, those obtained by the filter mount method across concentrations ranging from 2.17 × 10⁶ to 1.37 × 10⁸ viruses ml⁻¹ when tested by using cultivated viral isolates and natural samples from marine and freshwater environments. In summary, the wet-mount method is significantly less expensive than the filter mount method and is appropriate for rapid, precise, and accurate enumeration of aquatic viruses over a wide range of viral concentrations (≥1 × 10⁶ viruses ml⁻¹) encountered in field and laboratory samples.     

Horizontal and Vertical Distribution of Marine Virioplankton: A Basin Scale Investigation Based on a Global Cruise

Despite the fact that marine viruses have been increasingly studied in the last decade, there is little information on viral abundance and distribution on a global scale. In this study, we report on a global-scale survey covering the Pacific, Atlantic, and Indian Oceans on viral distribution using flow cytometry. Viruses were stained with the SYBR Green I, which targets only dsDNA viruses. The average viral abundance was 1.10±0.73×10⁷ ml⁻¹ in global surface oceans and decreased from the areas with high chlorophyll concentration (on average, 1.47±0.78×10⁷ ml⁻¹) to the oligotrophic subtropical gyres (on average, 6.34±2.18×10⁶ ml⁻¹). On a large-spatial-scale, viruses displayed significant relationships with both heterotrophic and autotrophic picoplankton abundance, suggesting that viral distribution is dependent on their host cell abundance. Our study provided a basin scale pattern of marine viral distributions and their relationship with major host cells, indicating that viruses play a significant role in the global marine ecosystem.     

Quantitative Analysis of Cereulide, the Emetic Toxin of Bacillus cereus, Produced under Various Conditions

This paper describes a quantitative and sensitive chemical assay for cereulide, the heat-stable emetic toxin produced by Bacillus cereus. The methods previously available for measuring cereulide are bioassays that give a toxicity titer, but not an accurate concentration. The dose of cereulide causing illness in humans is therefore not known, and thus safety limits for cereulide cannot be indicated. We developed a quantitative and sensitive chemical assay for cereulide based on high-performance liquid chromatography (HPLC) connected to ion trap mass spectrometry. This chemical assay and a bioassay based on boar sperm motility inhibition were calibrated with purified cereulide and with valinomycin, a structurally similar cyclic depsipeptide. The boar spermatozoan motility assay and chemical assay gave uniform results over a wide range of cereulide concentrations, ranging from 0.02 to 230 μg ml⁻¹. The detection limit for cereulide and valinomycin by HPLC-mass spectrometry was 10 pg per injection. The combined chemical and biological assays were used to define conditions and concentrations of cereulide formation by B. cereus strains F4810/72, NC7401, and F5881. Cereulide production commenced at the end of logarithmic growth, but was independent of sporulation. Production of cereulide was enhanced by incubation with shaking compared to static conditions. The three emetic B. cereus strains accumulated 80 to 166 μg of cereulide g⁻¹ (wet weight) when grown on solid medium. Strain NC7401 accumulated up to 25 μg of cereulide ml⁻¹ in liquid medium at room temperature (21 ± 1°C) in 1 to 3 days, during the stationary growth phase when cell density was 2 × 10⁸ to 6 × 10⁸ CFU ml⁻¹. Cereulide production at temperatures at and below 8°C or at 40°C was minimal.     

Abundance and distribution of Ostreococcus sp. in the San Pedro Channel, California, as revealed by quantitative PCR

Ostreococcus is a genus of widely distributed marine phytoplankton which are picoplanktonic in size (<2 mum) and capable of rapid growth. Although Ostreococcus has been detected around the world, little quantitative information exists on its contribution to planktonic communities. We designed and implemented a genus-specific TaqMan-based quantitative PCR (qPCR) assay to investigate the dynamics and ecology of Ostreococcus at the USC Microbial Observatory (eastern North Pacific). Samples were collected from 5 m and the deep chlorophyll maximum (DCM) between September 2000 and August 2002. Ostreococcus abundance at 5 m was generally <5.0 x 10(3) cells ml(-1), with a maximum of 8.2 x 10(4) cells ml(-1). Ostreococcus abundance was typically higher at the DCM, with a maximum of 3.2 x 10(5) cells ml(-1). The vertical distribution of Ostreococcus was examined in March 2005 and compared to the distribution of phototrophic picoeukaryotes (PPE) measured by flow cytometry. The largest contribution to PPE abundance by Ostreococcus was approximately 70% and occurred at 30 m, near the DCM. Despite its relatively low abundance, the depth-integrated standing stock of Ostreococcus in March 2005 was approximately 30 mg C m(-2). Our work provides a new technique for quantifying the abundance of Ostreococcus and demonstrates the seasonal dynamics of this genus and its contribution to picoeukaryote biomass at our coastal sampling station.     

Genome-wide association identifies a common variant in the reelin gene that increases the risk of schizophrenia only in women

Sex differences in schizophrenia are well known, but their genetic basis has not been identified. We performed a genome-wide association scan for schizophrenia in an Ashkenazi Jewish population using DNA pooling. We found a female-specific association with rs7341475, a SNP in the fourth intron of the reelin (RELN) gene (p = 2.9 × 10⁻⁵ in women), with a significant gene-sex effect (p = 1.8 × 10⁻⁴). We studied rs7341475 in four additional populations, totaling 2,274 cases and 4,401 controls. A significant effect was observed only in women, replicating the initial result (p = 2.1 × 10⁻³ in women; p = 4.2 × 10⁻³ for gene-sex interaction). Based on all populations the estimated relative risk of women carrying the common genotype is 1.58 (p = 8.8 × 10⁻⁷; p = 1.6 × 10⁻⁵ for gene-sex interaction). The female-specific association between RELN and schizophrenia is one of the few examples of a replicated sex-specific genetic association in any disease.     

Identification of Multiple Genetic Susceptibility Loci in Takayasu Arteritis

Takayasu arteritis is a rare inflammatory disease of large arteries. The etiology of Takayasu arteritis remains poorly understood, but genetic contribution to the disease pathogenesis is supported by the genetic association with HLA-B^∗52. We genotyped ∼200,000 genetic variants in two ethnically divergent Takayasu arteritis cohorts from Turkey and North America by using a custom-designed genotyping platform (Immunochip). Additional genetic variants and the classical HLA alleles were imputed and analyzed. We identified and confirmed two independent susceptibility loci within the HLA region (r² < 0.2): HLA-B/MICA (rs12524487, OR = 3.29, p = 5.57 × 10⁻¹⁶) and HLA-DQB1/HLA-DRB1 (rs113452171, OR = 2.34, p = 3.74 × 10⁻⁹; and rs189754752, OR = 2.47, p = 4.22 × 10⁻⁹). In addition, we identified and confirmed a genetic association between Takayasu arteritis and the FCGR2A/FCGR3A locus on chromosome 1 (rs10919543, OR = 1.81, p = 5.89 × 10⁻¹²). The risk allele in this locus results in increased mRNA expression of FCGR2A. We also established the genetic association between IL12B and Takayasu arteritis (rs56167332, OR = 1.54, p = 2.18 × 10⁻⁸).     

Rectal Carriage of Enterohemorrhagic Escherichia coli O157 in Slaughtered Cattle

Escherichia coli O157:H7 is an important cause of diarrhea, hemorrhagic colitis, and potentially fatal human illness. Cattle are considered a primary reservoir of infection, and recent experimental evidence has indicated that the terminal rectum is the principal site of bacterial carriage. To test this finding in naturally colonized animals, intact rectum samples from 267 cattle in 24 separate lots were obtained immediately after slaughter, and fecal material and mucosal surfaces were cultured for E. coli O157 by direct and enrichment methods. Two locations, 1 and 15 cm proximal to the recto-anal junction, were tested. In total, 35 animals were positive for E. coli O157 at at least one of the sites and 232 animals were negative as determined by all tests. The frequency of isolation and the numbers of E. coli O157 cells were higher at the site closer to the recto-anal junction, confirming our previous experimental findings. We defined low- and high-level carriers as animals with E. coli O157 levels of <1 × 10³ CFU g⁻¹ or <1 × 10³ CFU ml⁻¹ and animals with E. coli O157 levels of ≥1 × 10³ CFU g⁻¹ or ≥1 × 10³ CFU ml⁻¹ in feces or tissues, respectively. High-level carriage was detected in 3.7% of the animals (95% confidence interval, 1.8 to 6.8%), and carriage on the mucosal surface of the terminal rectum was associated with high-level fecal excretion. In summary, our results support previous work demonstrating that the mucosal epithelium in the bovine terminal rectum is an important site for E. coli O157 carriage in cattle. The data also support the hypothesis that high-level fecal shedding (≥1 × 10³ CFU g of feces⁻¹) of enterohemorrhagic E. coli O157 results from colonization of this site.     

Molecular Phylogenetic and Biogeochemical Studies of Sulfate-Reducing Bacteria in the Rhizosphere of Spartina alterniflora

The population composition and biogeochemistry of sulfate-reducing bacteria (SRB) in the rhizosphere of the marsh grass Spartina alterniflora was investigated over two growing seasons by molecular probing, enumerations of culturable SRB, and measurements of SO₄²⁻ reduction rates and geochemical parameters. SO₄²⁻ reduction was rapid in marsh sediments with rates up to 3.5 μmol ml⁻¹ day⁻¹. Rates increased greatly when plant growth began in April and decreased again when plants flowered in late July. Results with nucleic acid probes revealed that SRB rRNA accounted for up to 43% of the rRNA from members of the domain Bacteria in marsh sediments, with the highest percentages occurring in bacteria physically associated with root surfaces. The relative abundance (RA) of SRB rRNA in whole-sediment samples compared to that of Bacteria rRNA did not vary greatly throughout the year, despite large temporal changes in SO₄²⁻ reduction activity. However, the RA of root-associated SRB did increase from <10 to >30% when plants were actively growing. rRNA from members of the family Desulfobacteriaceae comprised the majority of the SRB rRNA at 3 to 34% of Bacteria rRNA, with Desulfobulbus spp. accounting for 1 to 16%. The RA of Desulfovibrio rRNA generally comprised from <1 to 3% of the Bacteria rRNA. The highest Desulfobacteriaceae RA in whole sediments was 26% and was found in the deepest sediment samples (6 to 8 cm). Culturable SRB abundance, determined by most-probable-number analyses, was high at >10⁷ ml⁻¹. Ethanol utilizers were most abundant, followed by acetate utilizers. The high numbers of culturable SRB and the high RA of SRB rRNA compared to that of Bacteria rRNA may be due to the release of SRB substrates in plant root exudates, creating a microbial food web that circumvents fermentation.     

Studies on the Inoculation and Competitiveness of a Rhizobium leguminosarum Strain in Soils Containing Indigenous Rhizobia

The competitiveness of a Rhizobium leguminosarum strain was investigated at two separate locations in field inoculation studies on commercially grown peas. The soil at each location (sites I and II) contained an indigenous R. leguminosarum population of ca. 3 × 10⁴ rhizobia per g of soil. At site I it was necessary to use an inoculum concentration as large as 4 × 10⁷ CFU ml⁻¹ (2 × 10⁶ bacteria seed⁻¹) to establish the inoculum strain in the majority of nodules (73%). However, at site II the inoculum strain formed only 33% of nodules when applied at this (10⁷ CFU ml⁻¹) level. Establishment could not be further improved by increasing the inoculum concentration even as high as 10⁹ CFU ml⁻¹ (9.6 × 10⁷ bacteria seed⁻¹). The inoculum strain could be detected at both sites 19 months after inoculation. Analysis by intrinsic antibiotic resistance patterns and plasmid DNA profiles indicated that a dominant strain(s) and plasmid pool existed among the indigenous population at site II. Competition experiments were carried out under laboratory conditions between a dominant indigenous isolate and the inoculum strain. Both strains were shown to be equally competitive.     

Development and Application of a Real-Time PCR Approach for Quantification of Uncultured Bacteria in the Central Baltic Sea

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the “Epsilonproteobacteria” related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 × 10³ to 4.4 × 10⁹ copies ml⁻¹ or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 × 10¹ to 2.2 ×10⁶ copies ml⁻¹ or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml⁻¹. The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment.