Hideomi Itoh

Bacterial population succession and adaptation affected by insecticide application and soil spraying history

Although microbial communities have varying degrees of exposure to environmental stresses such as chemical pollution, little is known on how these communities respond to environmental disturbances and how past disturbance history affects these community-level responses. To comprehensively understand the effect of organophosphorus insecticide application on microbiota in soils with or without insecticide-spraying history, we investigated the microbial succession in response to the addition of fenitrothion [O,O-dimethyl O-(3-methyl-p-nitrophenyl) phosphorothioate, abbreviated as MEP] by culture-dependent experiments and deep sequencing of 16S rRNA genes. Despite similar microbial composition at the initial stage, microbial response to MEP application was remarkably different between soils with and without MEP-spraying history. MEP-degrading microbes more rapidly increased in the soils with MEP-spraying history, suggesting that MEP-degrading bacteria might already exist at a certain level and could quickly respond to MEP re-treatment in the soil. Culture-dependent and -independent evaluations revealed that MEP-degrading Burkholderia bacteria are predominant in soils after MEP application, limited members of which might play a pivotal role in MEP-degradation in soils. Notably, deep sequencing also revealed that some methylotrophs dramatically increased after MEP application, strongly suggesting that these bacteria play a role in the consumption and removal of methanol, a harmful derivative from MEP-degradation, for better growth of MEP-degrading bacteria. This comprehensive study demonstrated the succession and adaptation processes of microbial communities under MEP application, which were critically affected by past experience of insecticide-spraying.

Evidence of Environmental and Vertical Transmission of Burkholderia Symbionts in the Oriental Chinch Bug, Cavelerius saccharivorus (Heteroptera: Blissidae)

ABSTRACT The vertical transmission of symbiotic microorganisms is omnipresent in insects, while the evolutionary process remains totally unclear. The oriental chinch bug, Cavelerius saccharivorus (Heteroptera: Blissidae), is a serious sugarcane pest, in which symbiotic bacteria densely populate the lumen of the numerous tubule-like midgut crypts that the chinch bug develops. Cloning and sequence analyses of the 16S rRNA genes revealed that the crypts were dominated by a specific group of bacteria belonging to the genus Burkholderia of the Betaproteobacteria. The Burkholderia sequences were distributed into three distinct clades: the Burkholderia cepacia complex (BCC), the plant-associated beneficial and environmental (PBE) group, and the stinkbug-associated beneficial and environmental group (SBE). Diagnostic PCR revealed that only one of the three groups of Burkholderia was present in ∼89% of the chinch bug field populations tested, while infections with multiple Burkholderia groups within one insect were observed in only ∼10%. Deep sequencing of the 16S rRNA gene confirmed that the Burkholderia bacteria specifically colonized the crypts and were dominated by one of three Burkholderia groups. The lack of phylogenetic congruence between the symbiont and the host population strongly suggested host-symbiont promiscuity, which is probably caused by environmental acquisition of the symbionts by some hosts. Meanwhile, inspections of eggs and hatchlings by diagnostic PCR and egg surface sterilization demonstrated that almost 30% of the hatchlings vertically acquire symbiotic Burkholderia via symbiont-contaminated egg surfaces. The mixed strategy of symbiont transmission found in the oriental chinch bug might be an intermediate stage in evolution from environmental acquisition to strict vertical transmission in insects.

Isolation of microorganisms involved in reduction of crystalline iron(III) oxides in natural environments

Reduction of crystalline Fe(III) oxides is one of the most important electron sinks for organic compound oxidation in natural environments. Yet the limited number of isolates makes it difficult to understand the physiology and ecological impact of the microorganisms involved. Here, two-stage cultivation was implemented to selectively enrich and isolate crystalline iron(III) oxide reducing microorganisms in soils and sediments. Firstly, iron reducers were enriched and other untargeted eutrophs were depleted by 2-years successive culture on a crystalline ferric iron oxide (i.e., goethite, lepidocrocite, hematite, or magnetite) as electron acceptor. Fifty-eight out of 136 incubation conditions allowed the continued existence of microorganisms as confirmed by PCR amplification. High-throughput Illumina sequencing and clone library analysis based on 16S rRNA genes revealed that the enrichment cultures on each of the ferric iron oxides contained bacteria belonging to the Deltaproteobacteria (mainly Geobacteraceae), followed by Firmicutes and Chloroflexi, which also comprised most of the operational taxonomic units (OTUs) identified. Venn diagrams indicated that the core OTUs enriched with all of the iron oxides were dominant in the Geobacteraceae while each type of iron oxides supplemented selectively enriched specific OTUs in the other phylogenetic groups. Secondly, 38 enrichment cultures including novel microorganisms were transferred to soluble-iron(III) containing media in order to stimulate the proliferation of the enriched iron reducers. Through extinction dilution-culture and single colony isolation, six strains within the Deltaproteobacteria were finally obtained; five strains belonged to the genus Geobacter and one strain to Pelobacter. The 16S rRNA genes of these isolates were 94.8–98.1% identical in sequence to cultured relatives. All the isolates were able to grow on acetate and ferric iron but their physiological characteristics differed considerably in terms of growth rate. Thus, the novel strategy allowed to enrich and isolate novel iron(III) reducers that were able to thrive by reducing crystalline ferric iron oxides.

Ultra‐high‐sensitivity stable‐isotope probing of rRNA by high‐throughput sequencing of isopycnic centrifugation gradients

Stable isotope probing (SIP) of rRNA directly identifies microorganisms assimilating an isotopically labelled substrate. High-throughput DNA sequencing is available for label screening at high resolution and high sensitivity, yet its effectiveness and validity remain to be clarified. Here, we investigated whether the detection sensitivity of rRNA-SIP could be improved by using Illumina sequencing in place of terminal restriction fragment length polymorphism (T-RFLP) analysis. A dilution series of (13) C-labelled RNA from Escherichia coli (1-0.0001%) and unlabelled RNA from Bacillus subtilis was density separated and fractionated. Illumina sequencing of isopycnic centrifugation gradients was able to detect (13) C-labelled RNA in the heaviest fraction with a buoyant density of 1.798 g ml(-1) even at the mixing ratio of 0.001%, whereas the detection ability of T-RFLP was not lower than 0.5%. Quantitative reverse transcription polymerase chain reaction of the density-separated RNAs showed that (13) C-labelled RNAs at mixing ratios of 0.05-0.001% had definitely accumulated in the heaviest fraction. Consequently, high-throughput sequencing provided up to 500-fold higher sensitivity for screening of (13) C-labelled RNA than T-RFLP. Ultra-high-sensitivity rRNA-SIP represents a clear advance towards a more complete understanding of microbial ecosystem function, including the ecophysiology of rare microorganisms in various natural environments.

Seasonal transition of active bacterial and archaeal communities in relation to water management in paddy soils

Paddy soils have an environment in which waterlogging and drainage occur during the rice growing season. Fingerprinting analysis based on soil RNA indicated that active microbial populations changed in response to water management conditions, although the fundamental microbial community was stable as assessed by DNA-based fingerprinting analysis. Comparative clone library analysis based on bacterial and archaeal 16S rRNAs (5,277 and 5,436 clones, respectively) revealed stable and variable members under waterlogged or drained conditions. Clones related to the class Deltaproteobacteria and phylum Euryarchaeota were most frequently obtained from the samples collected under both waterlogged and drained conditions. Clones related to syntrophic hydrogen-producing bacteria, hydrogenotrophic methanogenic archaea, rice cluster III, V, and IV, and uncultured crenarchaeotal group 1.2 appeared in greater proportion in the samples collected under waterlogged conditions than in those collected under drained conditions, while clones belonging to rice cluster VI related to ammonia-oxidizing archaea (AOA) appeared at higher frequency in the samples collected under drained conditions than in those collected under waterlogged conditions. These results suggested that hydrogenotrophic methanogenesis may become active under waterlogged conditions, whereas ammonia oxidation may progress by rice cluster VI becoming active under drained conditions in the paddy field.

High-Resolution Dynamics of Microbial Communities during Dissimilatory Selenate Reduction in Anoxic Soil

Selenate is one of the most common toxic metal compounds in contaminated soils. Its redox status can be changed by microbial activity, thus affecting its water solubility and soil mobility. However, current knowledge of microbial dynamics has been limited by the low sensitivity of past isolation and identification protocols. Here, high-throughput Illumina sequencing of 16S rRNA genes was applied to monitor the shift of the microorganisms in an anoxic contaminated soil after Se(VI) and acetate amendment. An autoclaved soil with both chemicals and a live soil with acetate alone were used as controls. Preliminary chemical analysis clearly showed the occurrence of biological selenate reduction coupled with acetate oxidation. Principal coordinate analysis and diversity indices of Illumina-derived sequence data showed dynamic succession and diversification of the microbial community in response to selenate reduction. High-resolution phylogenetic analysis revealed that the relative frequency of an operational taxonomic unit (OTU) from the genus Dechloromonas increased remarkably from 0.2% to 36% as a result of Se(VI) addition. Multiple OTUs representing less abundant microorganisms from the Rhodocyclaceae and Comamonadaceae families had significant increases as well. This study demonstrated that these microorganisms are concertedly involved in selenate reduction of the employed contaminated soil under anoxic conditions.

Burkholderia of Plant-Beneficial Group are Symbiotically Associated with Bordered Plant Bugs (Heteroptera: Pyrrhocoroidea: Largidae)

A number of phytophagous stinkbugs (order Heteroptera: infraorder Pentatomomorpha) harbor symbiotic bacteria in a specific midgut region composed of numerous crypts. Among the five superfamilies of the infraorder Pentatomomorpha, most members of the Coreoidea and Lygaeoidea are associated with a specific group of the genus Burkholderia, called the “stinkbug-associated beneficial and environmental (SBE)” group, which is not vertically transmitted, but acquired from the environment every host generation. A recent study reported that, in addition to these two stinkbug groups, the family Largidae of the superfamily Pyrrhocoroidea also possesses a Burkholderia symbiont. Despite this recent finding, the phylogenetic position and biological nature of Burkholderia associated with Largidae remains unclear. Based on the combined results of fluorescence in situ hybridization, cloning analysis, Illumina deep sequencing, and egg inspections by diagnostic PCR, we herein demonstrate that the largid species are consistently associated with the “plant-associated beneficial and environmental (PBE)” group of Burkholderia, which are phylogenetically distinct from the SBE group, and that they maintain symbiosis through the environmental acquisition of the bacteria. Since the superfamilies Coreoidea, Lygaeoidea, and Pyrrhocoroidea are monophyletic in the infraorder Pentatomomorpha, it is plausible that the symbiotic association with Burkholderia evolved at the common ancestor of the three superfamilies. However, the results of this study strongly suggest that a dynamic transition from the PBE to SBE group, or vice versa, occurred in the course of stinkbug evolution.

Dynamic transition of chemolithotrophic sulfur-oxidizing bacteria in response to amendment with nitrate in deposited marine sediments.

Although environmental stimuli are known to affect the structure and function of microbial communities, their impact on the metabolic network of microorganisms has not been well investigated. Here, geochemical analyses, high-throughput sequencing of 16S rRNA genes and transcripts, and isolation of potentially relevant bacteria were carried out to elucidate the anaerobic respiration processes stimulated by nitrate (20 mM) amendment of marine sediments. Marine sediments deposited by the Great East Japan Earthquake in 2011 were incubated anaerobically in the dark at 25∘C for 5 days. Nitrate in slurry water decreased gradually for 2 days, then more rapidly until its complete depletion at day 5; production of N2O followed the same pattern. From day 2 to 5, the sulfate concentration significantly increased and the sulfur content in solid-phase sediments significantly decreased. These results indicated that denitrification and sulfur oxidation occurred simultaneously. Illumina sequencing revealed the proliferation of known sulfur oxidizers, i.e., Sulfurimonas sp. and Chromatiales bacteria, which accounted for approximately 43.5% and 14.8% of the total population at day 5, respectively. These oxidizers also expressed 16S rRNA to a considerable extent, whereas the other microorganisms, e.g., iron(III) reducers and methanogens, became metabolically active at the end of the incubation. Extinction dilution culture in a basal-salts medium supplemented with sulfur compounds and nitrate successfully isolated the predominant sulfur oxidizers: Sulfurimonas sp. strain HDS01 and Thioalkalispira sp. strain HDS22. Their 16S rRNA genes showed 95.2–96.7% sequence similarity to the closest cultured relatives and they grew chemolithotrophically on nitrate and sulfur. Novel sulfur-oxidizing bacteria were thus directly involved in carbon fixation under nitrate-reducing conditions, activating anaerobic respiration processes and the reorganization of microbial communities in the deposited marine sediments.

A Fine-Scale Phylogenetic Analysis of Free-Living Burkholderia Species in Sugarcane Field Soil

The diversity and abundance of Burkholderia species in sugarcane field soils were investigated by a 16S rRNA gene-based approach using genus-specific primers. A total of 365,721 sequences generated by the Illumina MiSeq platform were assigned to the genus Burkholderia. Nearly 58% of these sequences were placed in a previously defined cluster, including stinkbug symbionts. Quantitative PCR analysis revealed a consistent number of 16S rRNA gene copies for Burkholderia species (107 g−1 soil) across the sampled fields. C/N, pH, and nitrate concentrations were important factors shaping the Burkholderia community structure; however, their impacts were not significant considering the overall genus size.

Predominant but Previously-overlooked Prokaryotic Drivers of Reductive Nitrogen Transformation in Paddy Soils, Revealed by Metatranscriptomics

Waterlogged paddy soils possess anoxic zones in which microbes actively induce reductive nitrogen transformation (RNT). In the present study, a shotgun RNA sequencing analysis (metatranscriptomics) of paddy soil samples revealed that most RNT gene transcripts in paddy soils were derived from Deltaproteobacteria, particularly the genera Anaeromyxobacter and Geobacter. Despite the frequent detection of the rRNA of these microbes in paddy soils, their RNT-associated genes have rarely been identified in previous PCR-based studies. This metatranscriptomic analysis provides novel insights into the diversity of RNT microbes present in paddy soils and the ecological function of Deltaproteobacteria predominating in these soils.