Roughly estimating the diversity of life has for a long time been seen as a challenge. Sequences obtained from the environment by direct amplification are now seen as the sole mean to provide information for at least 99% of the prokaryotes in natural communities. Recent massive parallel sequencing technologies now allow to sequence nearly every sequence resulting from such amplification. Are we ready to deal with such a deluge of data? Using three recent publications (16S rRNA genes amplification in soil and seawater followed by massive parallel sequencing) as case examples, this review analyses the state of the field.
Iodine is an essential trace element for humans and animals because of its important role as a constituent of thyroid hormones. If the anthropogenic iodine-129 (129I, half-life: 1.6×107 years), which is released from nuclear facilities into the environment and has a long half-life, participates in the biogeochemical cycling of iodine, it potentially accumulates in the human thyroid gland and might cause thyroid cancer. Therefore, it is necessary to obtain better information on the behavior of iodine in the environment for accurate safety assessments of 129I. Major pathways of iodine cycling are the volatilization of organic iodine compounds into the atmosphere, accumulation of iodine in living organisms, oxidation and reduction of inorganic iodine species, and sorption of iodine by soils and sediments. Considerable geochemical evidence has indicated that these processes are influenced or controlled by microbial activities, although the precise mechanisms involved are still unclear. This review summarizes current knowledge on interactions between microorganisms and iodine, with special emphasis on newly isolated bacteria possibly contributing to the cycling of iodine on a global scale.
This study investigated seasonal and spatial dynamics of the bacterial community in Gokasho bay with denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA gene fragments. The community structure was related to physico-chemical water conditions in the area examined. The bacterial community clustered into three groups: bacteria collected during January-May; those collected from water at the surface in July and September; and those collected from water at the bottom in July and September and from both depths in November. Canonical correspondence analyses indicated that the seasonal variability in bacterial community was associated with water temperature succession. On the other hand, concentrations of particulate organic matter and nitrite plus nitrate were related to the vertical change in community structure in summer and autumn as well as HNF abundance, suggesting that both top-down and bottom-up control affected the community. The influence of salinity was insignificant though bacterial production was related to salinity. No relationship was observed between the variation in community structure and that in hydrolytic enzyme activity. The results indicate that changes in bacterial activity are not coupled with variation in community structure.
The screening of pollutant degraders by relying solely on cultivation techniques such as liquid enrichment often fails to isolate the actual degraders in the environment. Community analyses by PCR-denaturing gradient gel electrophoresis (DGGE) were performed to isolate bacteria that can degrade 3-chlorobenzoate (3CB) effectively in soil. A forest soil sample was repeatedly dosed with 3CB (500 mg kg-1) to enrich it with indigenous 3CB-degraders, and changes in the bacterial community were monitored by PCR-DGGE of the 16S rRNA gene and benzoate 1,2-dioxygenase alpha subunit gene (benA). Initially, it required about 3 weeks to degrade 3CB in the soil, whereas it took only 3 days after the third dose. With this accelerated degradation, several intensified bands appeared in the DGGE profiles of both 16S rRNA gene and benA. We succeeded in isolating five 3CB-degrading Burkholderia strains corresponding to these bands by direct plating, while most of them were eliminated by liquid enrichment. Inoculation of the strains into the soil demonstrated that the five strains could degrade 3CB effectively in the soil. This study clearly shows significant bias during the liquid enrichment process and the advantage of using PCR-DGGE in screening effective degraders under environmental conditions.
Annual change in the density of sulfur-oxidizing microorganisms on sandstone was enumerated to know the effects on the deterioration of stone materials of the Angkor monuments in Cambodia. Samples were obtained from total 12 stations at the Angkor Wat, Bayon, and Phnom Krom temples between 1998 and 2007. Sulfur-oxidizing microorganisms enumerated in a mineral salts medium supplemented with elemental sulfur as the sole energy source had a density of 101-105 MPN (g sample)-1. The sulfur-oxidizing microorganisms of the samples collected at Angkor Wat have tended to decrease in density since 2002; on the other hand, relatively constant values have been recorded in the samples of Bayon and Phnom Krom. These results suggest that the sulfur-oxidizing microorganisms on the stone play an important role in the decay of the building blocks by excreting sulfuric acid.
We have assayed rates of atmospheric CO and hydrogen uptake, maximum potential CO uptake and the major phylogenetic composition of CO-oxidizing bacterial communities for a variety of volcanic deposits on Miyake-jima, Japan. These deposits represented different ages and stages of plant succession, ranging from unvegetated scoria deposited in 1983 to forest soils on deposits >800 yr old. Atmospheric CO and hydrogen uptake rates varied from -2.0±1.8-6.3±0.1 mg CO m-2 d-1 and 0.0±0.4-2.0±0.2 mg H2 m-2 d-1, respectively, and were similar to or greater than values reported for sites on Kilauea volcano, Hawaii, USA. At one of the forested sites, CO was emitted to the atmosphere, while two vegetated sites did not consume atmospheric hydrogen, an unusual observation. Although maximum potential CO uptake rates were also comparable to values for Kilauea, the relationship between these rates and organic carbon contents of scoria or soil indicated that CO oxidizers were relatively more abundant in Miyake-jima deposits. Phylogenetic analyses based on the large sub-unit gene for carbon monoxide dehydrogenase (coxL) indicated that many novel lineages were present on Miyake-jima, that CO-oxidizing Proteobacteria were prevalent in vegetated sites and that community structure appeared to vary more than composition among sites.
A simple extraction method and real-time PCR with SYBR-Green I were combined to monitor Microcystis 16S rRNA gene (16S rDNA) concentrations over a wide range. A Fast DNA SPIN Kit (MP Biomedicals) was used to extract rDNA quantitatively. Real-time PCR amplified Microcystis rDNA for quantification with the forward primer Micro229f, which was newly designed by us and was highly specific for Microcystis, and the reverse universal primer 342r. The method developed here can detect Microcystis at concentrations as low as 3 cells mL-1. The rDNA concentration and cell count were highly correlated in the range from 1.2×104 to 1.1×106 copies mL-1 and from 9.5×102 to 1.0×105 cells mL-1, respectively, in a canal where Microcystis algal blooms occur annually. The Microcystis rDNA concentration in Lake Kasumigaura was measured by the application of our method to water samples collected monthly from April 2004 to March 2006. Microcystis was not detected by microscopy from January to June 2005, except in May, but our method detected 1.0×103 to 1.0×104 copies mL-1 of Microcystis rDNA during this period. This result clearly showed that our method is useful for clarifying the annual fluctuation in Microcystis concentration, especially when concentrations are low.
Effects of organic fertilizers with and without the application of chemical fertilizers for seven years as part of a wheat-pearl millet cropping sequence on soil chemical properties and the establishment of nitrogen fixing bacteria in the rhizosphere were examined. The application of farmyard manure, poultry manure, and sugarcane filter cake alone or in combination with chemical fertilizers improved the soil organic C, total N, P, and K status. Larger populations of Azotobacter chroococcum and Rhizobium leguminosarum biovar trifolii in the rhizosphere of wheat and Egyptian clover respectively, were maintained in soils receiving organic fertilizers either alone or in combination with chemical fertilizers than in soils given chemical fertilizers alone.
The prokaryotic communities in deep subseafloor sediment collected during Ocean Drilling Program (ODP) Leg 204 from the South Hydrate Ridge (SHR) on the Cascadia Margin were analyzed by 16S rRNA gene clone sequencing and a fluorescent quantitative PCR technique. The microbial communities came from sites with contrasting geological characteristics on the SHR: sites 1244 and 1245 (located on the flank of the ridge, hydrate-rich sediment) and site 1251 (located on the slope basin of SHR, hydrate-poor sediment). The overall copy numbers of the 16S rRNA gene, and the proportion of archaeal 16S rRNA gene in all 16S rRNA gene community in sediment were larger on the slope basin than on the flank of the SHR. Archaeal community structure around the sulfate-methane transition zone at site 1251 (4.5 mbsf) was intensively investigated using two different PCR primer sets. A relatively abundant distribution of the 16S rRNA gene sequences related to mesophilic methanogen of the genus Methanoculleus was identified at a depth of 43.2 mbsf, and suggested that the methanogens occur in relatively shallow zones of sediment. This study demonstrated that the subseafloor microbial communities shown by 16S rRNA gene clone analyses were not directly associated with subseafloor methane hydrate deposits.
Hawaiian volcanic deposits offer ideal opportunities to assess methanotrophic bacterial colonization of new substrates, and to determine the relative significance of methanotrophy during ecosystem succession. Activity and molecular ecological surveys indicated that significant methanotrophic activity was restricted to vegetated ecosystems characterized by closed-canopy forests and significant soil accumulation. In these systems, atmospheric methane oxidation rates (0.7-1.8 mg CH4 m-2 d-1) were comparable to the lower end of values reported for continental soils. No trends in activity related to deposit age or type were evident at ambient or elevated methane levels. Analyses of clone libraries based on particulate methane monooxygenase and ammonia monooxygenase (pmoA/amoA) genes revealed largely novel sequences, with distinct assemblages for each of two sites. Remarkably, sequences from a 300-yr old forest soil were most closely related to sequences from Arctic soils. Collectively, the evidence indicates that methanotrophs colonize volcanic substrates slowly and likely depend on interactions with plant and other microbial communities.
The toxicity of three organophosphorous nematicides, imicyafos, fosthiazate and cadusafos, to non-target organisms in soil was evaluated. Imicyafos and fosthiazate had no significant inhibitory effect on the growth of fungal (Fusarium oxysporum f. sp. lactucae, Rhizoctonia solani and Trichoderma viride) and bacterial (Ralstonia solanacearum and Pseudomonas fluorescens) strains in media at 12.5 to 200 mg L-1. Cadusafos, however, significantly inhibited the growth of all these strains except R. solanacearum. A pot test was conducted using a soil naturally infested with Pratylenchus penetrans, and treated with imicyafos or fosthiazate, which are less toxic to non-target organisms. The density of P. penetrans decreased to less than 10% of the control level after exposure to imicyafos and fosthiazate at 3 kg active ingredient ha-1, the conventional dose. No significant effect was observed on the density of free-living nematodes, cellulose decomposition activity, microbial biomass evaluated with the ATP method and number of ammonia oxidizers between the soil treated with imicyafos or fosthiazate and the untreated control soil. Our results revealed that imicyafos and fosthiazate effectively suppressed a plant-parasitic nematode, P. penetrans, but had little impact on free-living nematodes and the soil microbial community.
Denitrification activity and bacterial community constituents were investigated in both well-drained and poorly drained soils of a temperate forest in central Japan by 15N tracer experiments and a cloning-sequencing approach. Denitrification activity was much higher in wet soil than in dry soil, based on 15N15N (30N2) and 15N15NO (46N2O) production. Labeled nitrate (15NO3-) was immediately reduced to 30N2 in wet soil, whereas it was only reduced to 46N2O in dry soil. Thus, the wet soil at the lower end of the catchment is a functional site for the scavenging for NO3- and N2O. Nitrite reductase gene (nirK and nirS) fragments from these soils were PCR amplified, cloned, and sequenced. Both nirK and nirS fragments were detected in the wet soil, whereas only nirK fragments were detected in the dry soil. All the nirK and nirS clones showed less than 90% similarity to known clones. Numerous operational taxonomic units for nirK and nirS were found in the wet soil. Considerable diversification within the largest clade on the nirK phylogenetic tree, which contained no known sequence, was observed in wet soil. Thus, a wet soil environment can provide both the habitat and conditions for the expression of denitrification activity.
Anabaena cells develop regular one-dimensional filaments through cell division in planes parallel to each other. A gcvP mutant displayed morphological defects such as filaments with sharp bends and/or branching and irregular cell clumps. The defects probably result from depletion of S-adenosylmethionine (AdoMet), because they were rescued by the application of methionine, an AdoMet precursor, and because sinefungin, a strong inhibitor of AdoMet-dependent methyltransferases, caused morphological abnormalities in wild-type Anabaena similar to those of the mutant. AdoMet-dependent methylation is involved in the spatial regulation of cell polarity in Anabaena.
The relative isolation and unique physical properties of the Savusavu Hot Springs in Fiji may yield unique thermophiles. This study was conducted to determine the presence of aerobic thermophilic bacteria in these hot springs. A total of 104 thermophilic bacterial isolates were characterized and using Thermus and Bacillus strains as controls, 58% of the isolates were identified as Anoxybacillus flavithermus, 19% as Geobacillus stearothermophilus/Bacillus licheniformis, 10% as Thermus sp. TG153 and 10% as Thermus sp. TG206. Four isolates were unique in their molecular patterns suggesting there may be novel bacteria in the Savusavu hot springs.
The adsorption of DNA by an andosol was much greater than that by a fluvisol or acrisol. The decrease in soil organic matter on treatment with hydrogen peroxide (H2O2) little affected DNA adsorption per weight of the particles, implying that the organic matter is not involved in the adsorption. The acid oxalate treatment of the H2O2-treated soils decreased DNA adsorption. Among soil constituents, acid oxalate-extractable materials such as allophane and noncrystalline Al and Fe oxides are likely to make a major contribution to the adsorption of DNA.
The molecular analysis of ciliates for identification and phylogeny is usually conducted through PCR amplification and DNA sequencing, with DNA extracted from a large number of cells. Therefore, the analysis of ciliates is limited to only those species that can be cultured. We propose a single-cell PCR procedure to overcome the difficulty in the analysis of unculturable species. The procedure has been tested on 6 Stichotrichia strains and uncultured Levicoleps biwae cells, targeting 18S rRNA gene sequences, after carrying out microscopic observations and obtaining photographic records. This procedure enables the systematic analysis of unculturable ciliate strains in natural environments by linking the morphology and genotype of a single cell.
Twenty seven soil bacterial isolates able to grow on cinnamic acid or ferulic acid as a sole carbon and energy source were examined for the ability to utilize other phenylpropanoids and phylogenetic interrelationships. A 16S rRNA gene sequence analysis revealed that 26 of the isolates were closely related to Pseudomonas putida biovar A and classified into 6 OTUs: cinnamic acid utilization-positive (OTUs, CN-1 to CN-3) and -negative groups (FR-1 to FR-3). P. putida NBRC 14164T and JCM 13061 belonged to FR-3. A probable reclassification of P. putida was discussed in relation to the utilization of phenylpropanoids.