Changes in 15N/14N in the soil microbial biomass during nitrogen (N) mineralization have been hypothesized to influence 15N/14N in soil organic matter among ecosystem sites. However, a direct experimental test of this mechanism has not yet been performed. To evaluate the potential control of microbial N mineralization on the natural N isotope composition, we cultured fungi (Aspergillus oryzae) in five types of media of varying C:N ratios of 5, 10, 30, 50, and 100 for 4 d, and tracked changes in δ15N in the microbial biomass, NH4+, and dissolved organic N (DON: glycine) over the course of the experiment. High rates of NH4+ excretion from A. oryzae were accompanied by an increase in δ15N in the microbial biomass in low C:N media (i.e., C/N<30). In contrast, NH4+ was strongly retained in higher C/N treatments with only minor (i.e., <1 ‰) changes being detected in δ15N in the microbial biomass. Differences in δ15N in the microbial biomass were attributed to the loss of low-δ15N NH4+ in low, but not high C/N substrates. We also detected a negative linear correlation between microbial nitrogen use efficiency (NUE) and Δ15N (δ15N-biomass–δ15N-glycine). These results suggest an isotope effect during NH4+ excretion in relatively N-repleted environments in which microbial NUE is low, which may explain the vertical patterns of organic matter δ15N in soil profiles.
Endofungal bacteria are widespread within the phylum Mucoromycota, and these include Burkholderiaceae-related endobacteria (BRE). However, the prevalence of BRE in Mortierellomycotinan fungi and their phylogenetic divergence remain unclear. Therefore, we examined the prevalence of BRE in diverse species of Mortierella. We surveyed 238 isolates of Mortierella spp. mainly obtained in Japan that were phylogenetically classified into 59 species. BRE were found in 53 isolates consisting of 22 species of Mortierella. Among them, 20 species of Mortierella were newly reported as the fungal hosts of BRE. BRE in a Glomeribacter-Mycoavidus clade in the family Burkholderiaceae were separated phylogenetically into three groups. These groups consisted of a group containing Mycoavidus cysteinexigens, which is known to be associated with M. elongata, and two other newly distinguishable groups. Our results demonstrated that BRE were harbored by many species of Mortierella and those that associated with isolates of Mortierella spp. were more phylogenetically divergent than previously reported.
Bacterial interspecies interactions in the oral cavity influence the structural development of cariogenic biofilms and dental caries. Visualization of the biofilm architecture and bacterial localization within biofilms is essential for understanding bacterial interactions. We herein demonstrated that the spatial localization of Streptococcus mutans within dual-species biofilms was altered in a manner that depended on the partner. Furthermore, we found that these biofilms influenced the survival of S. mutans against disinfectants. The present results provide information on how S. mutans interact with other bacteria in multi-species cariogenic biofilms.
Branched nonylphenol (BNP), a degradation product of nonylphenol polyethoxylates, exerts estrogenic effects on various organisms. The genes underlying BNP degradation by Sphingobium amiense DSM 16289T were analyzed by complete genome sequencing and compared with those of the versatile BNP-degrading Sphingobium cloacae JCM 10874T. An opdA homolog (opdADSM16289) encoding BNP degradation activity was identified in DSM 16289T, in contrast with JCM 10874T, possessing both the opdA homolog and nmoA. The degradation profile of different BNP isomers was examined by Escherichia coli transformants harboring opdADSM16289, opdAJCM10874, and nmoAJCM10874 to characterize and compare the expression activities of these genes.
Nitrous oxide (N2O) is a powerful greenhouse gas; however, limited information is currently available on the microbiomes involved in its sink and source in seagrass meadow sediments. Using laboratory incubations, a quantitative PCR (qPCR) analysis of N2O reductase (nosZ) and ammonia monooxygenase subunit A (amoA) genes, and a metagenome analysis based on the nosZ gene, we investigated the abundance of N2O-reducing microorganisms and ammonia-oxidizing prokaryotes as well as the community compositions of N2O-reducing microorganisms in in situ and cultivated sediments in the non-eelgrass and eelgrass zones of Lake Akkeshi, Japan. Laboratory incubations showed that N2O was reduced by eelgrass sediments and emitted by non-eelgrass sediments. qPCR analyses revealed that the abundance of nosZ gene clade II in both sediments before and after the incubation as higher in the eelgrass zone than in the non-eelgrass zone. In contrast, the abundance of ammonia-oxidizing archaeal amoA genes increased after incubations in the non-eelgrass zone only. Metagenome analyses of nosZ genes revealed that the lineages Dechloromonas-Magnetospirillum-Thiocapsa and Bacteroidetes (Flavobacteriia) within nosZ gene clade II were the main populations in the N2O-reducing microbiome in the in situ sediments of eelgrass zones. Sulfur-oxidizing Gammaproteobacteria within nosZ gene clade II dominated in the lineage Dechloromonas-Magnetospirillum-Thiocapsa. Alphaproteobacteria within nosZ gene clade I were predominant in both zones. The proportions of Epsilonproteobacteria within nosZ gene clade II increased after incubations in the eelgrass zone microcosm supplemented with N2O only. Collectively, these results suggest that the N2O-reducing microbiome in eelgrass meadows is largely responsible for coastal N2O mitigation.
Various DNA extraction methods are often used interchangeably for the characterization of microbial communities despite indications that different techniques produce disparate results. The microbiomes of two ascidian species were herein characterized using two common DNA extraction kits, the DNeasy Blood and Tissue Kit (Qiagen) and the PowerSoil DNA Isolation Kit (Mo Bio Laboratories), followed by next-generation (Illumina) sequencing of partial 16S rRNA genes. Significant differences were detected in microbial community diversity and structure between ascidian species, but not between kits, suggesting similar recovery of biological variation and low technical variation between the two extraction methods for ascidian microbiome characterization.
The phylum Aquificae comprises chemolithoautotrophic thermophilic to hyperthermophilic bacteria, in which the nitrogenase reductase gene (nifH) has been reported. However, nitrogen-fixing activity has not yet been demonstrated in members of this deeply branching bacterial phylum. We isolated two thermophilic diazotrophic strains from chemosynthetic microbial communities in slightly alkaline hot springs (≥70°C) in Nakabusa, Nagano Prefecture, Japan. A phylogenetic analysis based on 16S rRNA genes identified these strains as members of the genus Hydrogenobacter within Aquificae. Their NifH sequences showed 96.5 and 97.4% amino acid sequence identities to that from Hydrogenobacter thermophilus TK-6. Nitrogenase activity, measured by acetylene reduction, was confirmed in both strains at 70°C. These novel strains grew under semi-aerobic conditions by using CO2 as the sole carbon source and N2 as the sole nitrogen source in media containing hydrogen and/or thiosulfate. To the best of our knowledge, this is the first demonstration of active nitrogen fixation in thermophilic bacteria at 70°C and in the phylum Aquificae.
Plant growth-promoting bacteria (PGPB) inhabit the rhizosphere of plants and are capable of enhancing plant growth through a number of mechanisms. A strain of Pseudomonas fluorescens DUS1-27 was identified as a potential PGPB candidate based on its ability to increase the growth of Brassica napus L. (canola) over that of uninoculated control plants in a soil-based system. The same P. fluorescens isolate was found to reduce plant growth in a hydroponic growth system, with plants showing the symptoms of a microbe-associated molecular pattern (MAMP) response to the bacteria. The amperometric quantification of H2O2, fluorescence-based total peroxidase assays, and quantification of catalase gene expression levels using qRT-PCR revealed that oxidative stress reduced plant growth in the hydroponic system. The addition of the cyanobacterium Nostoc punctiforme (known to have high catalase activity levels) in the hydroponic system as a co-inoculant reduced oxidative stress (49.7% decrease in H2O2 concentrations) triggered by the addition of P. fluorescens DUS1-27, thereby enabling plants to grow larger than uninoculated control plants. These results show the advantage of inoculating with multiple bacteria to promote plant growth and, for the first time, demonstrate that N. punctiforme beneficially assists plants under oxidative stress through its catalase activity in planta.
We herein report on the dynamics of a sulfate-reducing bacteria (SRB) community structure in the surface sediment of a seasonally hypoxic enclosed bay for two consecutive years (2012 and 2013). The uppermost (0–5 mm) and subsurface (5–10 mm) layers of sediment were examined with a terminal-restriction fragment length polymorphism (T-RFLP) analysis based on the dissimilatory sulfite reductase (dsrA) gene. The SRB community significantly differed between the two sediment layers over the sampling period. This difference was mainly attributed to operational taxonomic units (OTUs) that were unique to either of the sediment layers. However, nearly 70% of total OTUs were shared between the two layers, with a few predominating. Therefore, no significant shift was observed in the SRB community structure under varying dissolved oxygen (DO) conditions in bottom water overlying the sediment surface. An additional analysis of 16S rRNA gene amplicon sequences, conducted for three uppermost sediment samples (July, August, and September in 2012), revealed that Desulfococcus, a member of SRB with high tolerance to oxygen, was the predominant Deltaproteobacteria across the uppermost sediment samples. Based on the predominance of shared OTUs across the SRB community in the sediment (0–10 mm) regardless of bottom-water DO, some SRB that are physiologically tolerant of a wide range of DO conditions may have dominated and masked changes in responsive SRB to DO concentrations. These results suggest that the SRB community structure in the enclosed bay became stable under repeated cycles of seasonal hypoxia, but may be compromised if the severity of hypoxia increases in the future.
The surface crust that caps highly weathered banded iron formations (BIFs) supports a unique ecosystem that is a post-mining restoration priority in iron ore areas. Geochemical evidence indicates that biological processes drive the dissolution of iron oxide minerals and contribute to the ongoing evolution of this duricrust. However, limited information is available on present-day biogeochemical processes in these systems, particularly those that contribute to the precipitation of iron oxides and, thus, the cementation and stabilization of duricrusts. Freshly formed iron precipitates in water bodies perched on cangas in Karijini National Park, Western Australia, were sampled for microscopic and molecular analyses to understand currently active microbial contributions to iron precipitation in these areas. Microscopy revealed sheaths and stalks associated with iron-oxidizing bacteria. The iron-oxidizing lineages Sphaerotilus, Sideroxydans, and Pedomicrobium were identified in various samples and Leptothrix was common in four out of five samples. The iron-reducing bacteria Anaeromyxobacter dehalogens and Geobacter lovleyi were identified in the same four samples, with various heterotrophs and diverse cyanobacteria. Given this arid, deeply weathered environment, the driver of contemporary iron cycling in Karijini National Park appears to be iron-reducing bacteria, which may exist in anaerobic niches through associations with aerobic heterotrophs. Overall oxidizing conditions and Leptothrix iron-oxidizers contribute to net iron oxide precipitation in our sampes, rather than a closed biogeochemical cycle, which would result in net iron oxide dissolution as has been suggested for canga caves in Brazil. Enhancements in microbial iron oxide dissolution and subsequent reprecipitation have potential as a surface-crust-ecosystem remediation strategy at mine sites.
Chemosynthetic microbial communities develop and form dense cell aggregates in slightly alkaline sulfidic hot springs in the temperature range of 70–86°C at Nakabusa, Japan. Nitrogenase activity has recently been detected in the microbial communities collected. To identify possible members capable of nitrogen fixation, we examined the diversities of 16S rRNA and nitrogenase reductase (NifH) gene sequences in four types of chemosynthetic communities with visually different colors and thicknesses. The results of a 16S rRNA gene analysis indicated that all four microbial communities had similar bacterial constituents; the phylum Aquificae was the dominant member, followed in abundance by Thermodesulfobacteria, Firmicutes, and Thermotogae. Most of the NifH sequences were related to sequences reported in hydrothermal vents and terrestrial hot springs. The results of a phylogenetic analysis of NifH sequences revealed diversity in this gene among the communities collected, distributed within 7 phylogenetic groups. NifH sequences affiliated with Aquificae (Hydrogenobacter/Thermocrinis) and Firmicutes (Caldicellulosiruptor) were abundant. At least two different energy metabolic pathways appeared to be related to nitrogen fixation in the communities analyzed; aerobic sulfur/hydrogen-oxidizing bacteria in Aquificae and fermentative bacteria in Firmicutes. The metabolic characteristics of these two dominant phyla differed from those previously inferred from nitrogenase activity assays on chemosynthetic communities, which were associated with hydrogen-dependent autotrophic sulfate reduction. These assays may correspond to the observed NifH sequences that are distantly related to the known species of Thermodesulfovibrio sp. (Nitrospirae) detected in the present study. The activities of nitrogen-fixing organisms in communities may depend on redox states as well as the availability of electron donors, acceptors, and carbon sources.
Gluconacetobacter diazotrophicus is a plant growth-promoting bacterium that is used as a bioinoculant. Phosphate (Pi) modulates intracellular polyphosphate (polyP) levels in Escherichia coli, affecting cellular fitness and biofilm formation capacity. It currently remains unclear whether environmental Pi modulates polyP levels in G. diazotrophicus to enhance fitness in view of its technological applications. In high Pi media, cells accumulated polyP and degraded it, thereby improving survival, tolerance to environmental stressors, biofilm formation capacity on abiotic and biotic surfaces, and competence as a growth promoter of strawberry plants. The present results support the importance of Pi and intracellular polyP as signals involved in the survival of G. diazotrophicus.
We herein described a new microbial isolation method using the interaction between the floating aquatic plant, duckweed, and microbes. We harvested microbial cells from Japanese loosestrife roots and co-cultivated these cells with aseptic duckweed using artificial inorganic medium for the plant for four weeks. During the co-cultivation, some duckweeds were collected every week, and the roots were used for microbial isolation using a low-nutrient plate medium. As a result, diverse microbial isolates, the compositions of which differed from those of the original source (Japanese loosestrife root), were obtained when the roots of duckweed were collected after 2 weeks of cultivation. We also successfully isolated a wide variety of novel microbes, including two strains within the rarely cultivated phylum, Armatimonadetes. The present study shows that a duckweed-microbe co-cultivation approach together with a conventional technique (direct isolation from a microbial source) effectively obtains more diverse microbes from a sole environmental sample.
Rock outcrops of aged deep-sea seamounts are generally covered with Fe and Mn oxides, known as ferromanganese (Fe–Mn) crusts. Although the presence of microorganisms in Fe–Mn crusts has been reported, limited information is currently available on intra- and inter-variations in crust microbial communities. Therefore, we collected several Fe–Mn crusts in bathyal and abyssal zones (water depths of 1,150–5,520 m) in the Takuyo-Daigo Seamount in the northwestern Pacific, and examined microbial communities on the crusts using culture-independent molecular and microscopic analyses. Quantitative PCR showed that microbial cells were abundant (106–108 cells g–1) on Fe–Mn crust surfaces through the water depths. A comparative 16S rRNA gene analysis revealed community differences among Fe–Mn crusts through the water depths, which may have been caused by changes in dissolved oxygen concentrations. Moreover, community differences were observed among positions within each Fe–Mn crust, and potentially depended on the availability of sinking particulate organic matter. Microscopic and elemental analyses of thin Fe–Mn crust sections revealed the accumulation of microbial cells accompanied by the depletion of Mn in valleys of bumpy crust surfaces. Our results suggest that heterogeneous and abundant microbial communities play a role in the biogeochemical cycling of Mn, in addition to C and N, on crusts and contribute to the extremely slow growth of Fe–Mn crusts.
The hydrothermal vent squat lobster Shinkaia crosnieri Baba & Williams harbors an epibiotic bacterial community, which is numerically and functionally dominated by methanotrophs affiliated with Methylococcaceae and thioautotrophs affiliated with Sulfurovum and Thiotrichaceae. In the present study, shifts in the phylogenetic composition and metabolic function of the epibiont community were investigated using S. crosnieri individuals, which were reared for one year in a tank fed with methane as the energy and carbon source. The results obtained indicated that indigenous predominant thioautotrophic populations, such as Sulfurovum and Thiotrichaceae members, became absent, possibly due to the lack of an energy source, and epibiotic communities were dominated by indigenous Methylococcaceae and betaproteobacterial methylotrophic members that adapted to the conditions present during rearing for 12 months with a supply of methane. Furthermore, the overall phylogenetic composition of the epibiotic community markedly changed from a composition dominated by chemolithotrophs to one enriched with cross-feeding heterotrophs in addition to methanotrophs and methylotrophs. Thus, the composition and function of the S. crosnieri epibiotic bacterial community were strongly affected by the balance between the energy and carbon sources supplied for chemosynthetic production as well as that between the production and consumption of organic compounds.
The deep sea, the largest biosphere on Earth, nurtures a large variety of animals. However, no virus that infects deep-sea animals has been found. We herein report the first full-length RNA viral genome sequence identified from the deep-sea animal, Osedax japonicus, called Osedax japonicus RNA virus 1 (OjRV1). This sequence showed the highest amino acid sequence similarity to a virus of the family Togaviridae. However, the phylogenetic position and genome structure of OjRV1 differed from those of viruses in Togaviridae. These results suggest that OjRV1 belongs to a new virus family and that deep-sea animals may associate with new viruses.
Aerobic ammonia-oxidizing archaea (AOA) play a crucial role in the global nitrogen cycle by oxidizing ammonia to nitrite, and nitric oxide (NO) is a key intermediate in AOA for sustaining aerobic ammonia oxidation activity. We herein heterologously expressed the NO-forming, copper-containing, dissimilatory nitrite reductase (NirK) from Nitrososphaera viennensis and investigated its enzymatic properties. The recombinant protein catalyzed the reduction of 15NO2– to 15NO, the oxidation of hydroxylamine (15NH2OH) to 15NO, and the production of 14-15N2O from 15NH2OH and 14NO2–. To the best of our knowledge, the present study is the first to document the enzymatic properties of AOA NirK.