Bacterial induction of B cell senescence promotes age-related changes in the gut microbiota.

The elucidation of the mechanisms of ageing and the identification of methods to control it have long been anticipated. Recently, two factors associated with ageing-the accumulation of senescent cells and the change in the composition of gut microbiota-have been shown to play key roles in ageing. However, little is known about how these phenomena occur and are related during ageing. Here we show that the persistent presence of commensal bacteria gradually induces cellular senescence in gut germinal centre B cells. Importantly, this reduces both the production and diversity of immunoglobulin A (IgA) antibodies that target gut bacteria, thereby changing the composition of gut microbiota in aged mice. These results have revealed the existence of IgA-mediated crosstalk between the gut microbiota and cellular senescence and thus extend our understanding of the mechanism of gut microbiota changes with age, opening up possibilities for their control.

Identification of specific and common diagnostic antibody markers for gastrointestinal cancers by SEREX screening using testis cDNA phage library.

The present study was planned to identify novel serum antibody markers for digestive organ cancers. We have used screening by phage expression cloning and identified novel fourteen antigens in this experiment. The presence of auto-antibodies against these antigens in serum specimens was confirmed by western blotting. As for auto-antibodies against fourteen antigens, AlphaLISA (amplified luminescence proximity homogeneous assay) assay was performed in the sera of gastrointestinal cancers patients to confirm the results. Serum antibody levels against these fourteen recombinant proteins as antigens between healthy donors (HD) and esophageal squamous cell carcinoma (ESCC) patients, gastric cancer (GC), or colon cancer (CC) were compared. The serum levels of all fourteen auto-antibodies were significantly higher in ESCC and GC than those of HD. Among those auto-antibodies, except ECSA2 and CCNL2, were also detected significantly higher levels in CC than those of HD. Receiver operating curve (ROC) revealed similar results except CCNL2 in CC. AUC values calculated by ROC were higher than 0.7 in auto-antibodies against TPI1, HOOK2, PUF60, PRDX4, HS3ST1, TUBA1B, TACSTD2, AKR1C3, BAMBI, DCAF15 in ESCC, auto-antibodies against TPI1, HOOK2, PUF60, PRDX4, TACSTD2, AKR1C3, BAMBI, DCAF15 in GC, and auto-antibodies against TPI1, HOOK2, PUF60 in CC. AUC of the combination of HOOK2 and anti-p53 antibodies in ESCC was observed to be as high as 0.8228. Higher serum antibody levels against ten antigens could be potential diagnostic tool for ESCC. Higher serum antibody levels against eight antigens could be potential diagnostic tool for GC, and serum antibody levels against three antigens could be potential diagnostic tool for CC.

Specific clones of Staphylococcus lugdunensis may be associated with colon carcinoma.

Staphylococcus lugdunensis produces a tannase with activity that may be associated with the onset of colon carcinoma. To clarify this feature of colon carcinoma-associated S. lugdunensis, we obtained isolates from healthy subjects and patients with colon adenomas and carcinomas and analyzed their genetic backgrounds. In total, 40 S. lugdunensis isolates from 288 rectal swabs collected between 2002 and 2008 were used. These isolates were classified into four groups according to the diseases of the subjects: healthy (n=13), colon carcinoma (n=13), colon adenoma (n=9), and unknown (n=5). The isolates were also classified by pulsed-field gel electrophoresis (PFGE) and multi-locus sequence typing. In addition, an antimicrobial susceptibility test and detection of resistance genes were performed for all isolates. According to the PFGE analysis, 40 isolates could be classified into five groups. Among the groups, carcinoma and colon adenoma patients were significantly more frequently (40.9%) classified into group D (p<0.05), whereas healthy subjects were more frequently (38.5%) classified into group A. All isolates in group D were typed as ST27, which was clearly different than isolates in the other groups. All isolates were susceptible to the antimicrobial agents tested, including ß-lactams, although seven strains produced ß-lactamase. Our data suggest that a specific clone of S. lugdunensis might be associated with colon carcinoma and colon adenoma. This clone showed high susceptibility to many antimicrobial agents. Therefore, eradication therapy may lead to a decreased risk of colon carcinoma.

Sulfurimonas paralvinellae sp. nov., a novel mesophilic, hydrogen- and sulfur-oxidizing chemolithoautotroph within the Epsilonproteobacteria isolated from a deep-sea hydrothermal vent polychaete nest, reclassification of Thiomicrospira denitrificans as Sulfurimonas denitrificans comb. nov. and emended description of the genus Sulfurimonas.

A novel mesophilic bacterium, strain GO25(T), was isolated from a nest of hydrothermal vent polychaetes, Paralvinella sp., at the Iheya North field in the Mid-Okinawa Trough. Cells were motile short rods with a single polar flagellum. Growth was observed between 4 and 35 degrees C (optimum 30 degrees C; 13-16 h doubling time) and between pH 5.4 and 8.6 (optimum pH 6.1). The isolate was a facultatively anaerobic chemolithoautotroph capable of growth using molecular hydrogen, elemental sulfur or thiosulfate as the sole energy source, carbon dioxide as the sole carbon source, ammonium or nitrate as the sole nitrogen source and elemental sulfur, thiosulfate or yeast extract as the sole sulfur source. Strain GO25(T) represents the first deep-sea epsilonproteobacterium capable of growth by both hydrogen and sulfur oxidation. Nitrate or molecular oxygen (up to 10 % partial pressure) could serve as the sole electron acceptor to support growth. Metabolic products of nitrate reduction shifted in response to the electron donor provided. The G+C content of genomic DNA was 37.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the novel isolate belonged to the genus Sulfurimonas and was most closely related to Sulfurimonas autotrophica OK10(T) (96.3 % sequence similarity). DNA-DNA hybridization demonstrated that the novel isolate could be differentiated genotypically from Sulfurimonas autotrophica OK10(T). On the basis of the physiological and molecular properties of the novel isolate, the name Sulfurimonas paralvinellae sp. nov. is proposed, with strain GO25(T) (=JCM 13212(T)=DSM 17229(T)) as the type strain. Thiomicrospira denitrificans DSM 1251(T) (=ATCC 33889(T)) is phylogenetically associated with Sulfurimonas autotrophica OK10(T) and Sulfurimonas paralvinellae GO25(T). Based on the phylogenetic relationship between Thiomicrospira denitrificans DSM 1251(T), Sulfurimonas autotrophica OK10(T) and Sulfurimonas paralvinellae GO25(T), we propose the reclassification of Thiomicrospira denitrificans as Sulfurimonas denitrificans comb. nov. (type strain DSM 1251(T)=ATCC 33889(T)). In addition, an emended description of the genus Sulfurimonas is proposed.

Host-symbiont relationships in hydrothermal vent gastropods of the genus Alviniconcha from the Southwest Pacific.

Hydrothermal vent gastropods of the genus Alviniconcha are unique among metazoans in their ability to derive their nutrition from chemoautotrophic gamma- and epsilon-proteobacterial endosymbionts. Although host-symbiont relationships in Alviniconcha gastropods from the Central Indian Ridge in the Indian Ocean and the Mariana Trough in the Western Pacific have been studied extensively, host-symbiont relationships in Alviniconcha gastropods from the Southwest Pacific remain largely unknown. Phylogenetic analysis using mitochondrial cytochrome c oxidase subunit I gene sequences of host gastropods from the Manus, North Fiji, and Lau Back-Arc Basins in the Southwest Pacific has revealed a new host lineage in a Alviniconcha gastropod from the Lau Basin and the occurrence of the host lineage Alviniconcha sp. type 2 in the Manus Basin. Based on 16S rRNA gene sequences of bacterial endosymbionts, two gamma-proteobacterial lineages and one epsilon-proteobacterial lineage were identified in the present study. The carbon isotopic compositions of the biomass and fatty acids of the gastropod tissues suggest that the gamma- and epsilon-proteobacterial endosymbionts mediate the Calvin-Benson cycle and the reductive tricarboxylic acid cycle, respectively, for their chemoautotrophic growth. Coupling of the host and symbiont lineages from the three Southwest Pacific basins revealed that each of the Alviniconcha lineages harbors different bacterial endosymbionts belonging to either the gamma- or epsilon-Proteobacteria. The host specificity exhibited in symbiont selection provides support for the recognition of each of the host lineages as a distinct species. The results from the present study also suggest the possibility that Alviniconcha sp. types 1 and 2 separately inhabit hydrothermal vent sites approximately 120 m apart in the North Fiji Basin and 500 m apart in the Manus Basin.

Biogeographical distribution and diversity of microbes in methane hydrate-bearing deep marine sediments on the Pacific Ocean Margin.

The deep subseafloor biosphere is among the least-understood habitats on Earth, even though the huge microbial biomass therein plays an important role for potential long-term controls on global biogeochemical cycles. We report here the vertical and geographical distribution of microbes and their phylogenetic diversities in deeply buried marine sediments of the Pacific Ocean Margins. During the Ocean Drilling Program Legs 201 and 204, we obtained sediment cores from the Peru and Cascadia Margins that varied with respect to the presence of dissolved methane and methane hydrate. To examine differences in prokaryotic distribution patterns in sediments with or without methane hydrates, we studied >2,800 clones possessing partial sequences (400-500 bp) of the 16S rRNA gene and 348 representative clone sequences (approximately 1 kbp) from the two geographically separated subseafloor environments. Archaea of the uncultivated Deep-Sea Archaeal Group were consistently the dominant phylotype in sediments associated with methane hydrate. Sediment cores lacking methane hydrates displayed few or no Deep-Sea Archaeal Group phylotypes. Bacterial communities in the methane hydrate-bearing sediments were dominated by members of the JS1 group, Planctomycetes, and Chloroflexi. Results from cluster and principal component analyses, which include previously reported data from the West and East Pacific Margins, suggest that, for these locations in the Pacific Ocean, prokaryotic communities from methane hydrate-bearing sediment cores are distinct from those in hydrate-free cores. The recognition of which microbial groups prevail under distinctive subseafloor environments is a significant step toward determining the role these communities play in Earth's essential biogeochemical processes.

Enzymatic and genetic characterization of carbon and energy metabolisms by deep-sea hydrothermal chemolithoautotrophic isolates of Epsilonproteobacteria.

The carbon and energy metabolisms of a variety of cultured chemolithoautotrophic Epsilonproteobacteria from deep-sea hydrothermal environments were characterized by both enzymatic and genetic analyses. All the Epsilonproteobacteria tested had all three key reductive tricarboxylic acid (rTCA) cycle enzymatic activities--ATP-dependent citrate lyase, pyruvate:ferredoxin oxidoreductase, and 2-oxoglutarate:ferredoxin oxidoreductase--while they had no ribulose 1,5-bisphosphate carboxylase (RubisCO) activity, the key enzyme in the Calvin-Benson cycle. These results paralleled the successful amplification of the key rTCA cycle genes aclB, porAB, and oorAB and the lack of success at amplifying the form I and II RubisCO genes, cbbL and cbbM. The combination of enzymatic and genetic analyses demonstrates that the Epsilonproteobacteria tested use the rTCA cycle for carbon assimilation. The energy metabolisms of deep-sea Epsilonproteobacteria were also well specified by the enzymatic and genetic characterization: hydrogen-oxidizing strains had evident soluble acceptor:methyl viologen hydrogenase activity and hydrogen uptake hydrogenase genes (hyn operon), while sulfur-oxidizing strains lacked both the enzyme activity and the genes. Although the energy metabolism of reduced sulfur compounds was not genetically analyzed and was not fully clarified, sulfur-oxidizing Epsilonproteobacteria showed enzyme activity of a potential sulfite:acceptor oxidoreductase for a direct oxidation pathway to sulfate but no activity of AMP-dependent adenosine 5'-phosphate sulfate reductase for a indirect oxidation pathway. No activity of thiosulfate-oxidizing enzymes was detected. The enzymatic and genetic characteristics described here were consistent with cellular carbon and energy metabolisms and suggest that molecular tools may have great potential for in situ elucidation of the ecophysiological roles of deep-sea Epsilonproteobacteria.

Novel chemoautotrophic endosymbiosis between a member of the Epsilonproteobacteria and the hydrothermal-vent gastropod Alviniconcha aff. hessleri (Gastropoda: Provannidae) from the Indian Ocean.

The hydrothermal-vent gastropod Alviniconcha aff. hessleri from the Kairei hydrothermal field on the Central Indian Ridge houses bacterium-like cells internally in its greatly enlarged gill. A single 16S rRNA gene sequence was obtained from the DNA extract of the gill, and phylogenetic analysis placed the source organism within a lineage of the epsilon subdivision of the Proteobacteria. Fluorescence in situ hybridization analysis with an oligonucleotide probe targeting the specific epsilonproteobacterial subgroup showed the bacterium densely colonizing the gill filaments. Carbon isotopic homogeneity among the gastropod tissue parts, regardless of the abundance of the endosymbiont cells, suggests that the carbon isotopic composition of the endosymbiont biomass is approximately the same as that of the gastropod. Compound-specific carbon isotopic analysis revealed that fatty acids from the gastropod tissues are all (13)C enriched relative to the gastropod biomass and that the monounsaturated C(16) fatty acid that originates from the endosymbiont is as (13)C enriched relative to the gastropod biomass as that of the epsilonproteobacterial cultures grown under chemoautotrophic conditions. This fractionation pattern is most likely due to chemoautotrophy based on the reductive tricarboxylic-acid (rTCA) cycle and subsequent fatty acid biosynthesis from (13)C-enriched acetyl coenzyme A. Enzymatic characterization revealed evident activity of several key enzymes of the rTCA cycle, as well as the absence of ribulose-1,5-bisphosphate carboxylase/oxygenase activity in the gill tissue. The results from anatomic, molecular phylogenetic, bulk and compound-specific carbon isotopic, and enzymatic analyses all support the inference that a novel nutritional strategy relying on chemoautotrophy in the epsilonproteobacterial endosymbiont is utilized by the hydrothermal-vent gastropod from the Indian Ocean. The discrepancies between the data of the present study and those of previous ones for Alviniconcha gastropods from the Pacific Ocean imply that at least two lineages of chemoautotrophic bacteria, phylogenetically distinct at the subdivision level, occur as the primary endosymbiont in one host animal type.

Molecular phylogenetic and isotopic evidence of two lineages of chemoautotrophic endosymbionts distinct at the subdivision level harbored in one host-animal type: the genus Alviniconcha (Gastropoda: Provannidae).

The hydrothermal-vent gastropod Alviniconcha hessleri from the Alice Springs deep-sea hydrothermal field in the Mariana Back-Arc Basin in the Western Pacific houses an intracellular bacterial endosymbiont in its gill. Although enzymatic analysis has revealed that the endosymbiont is a sulfur-oxidizing chemoautotroph using the Calvin-Benson cycle for the fixation of carbon dioxide, the phylogenetic affiliation of, and the trophic relationship of A. hessleri with, the chemoautotrophic endosymbiont remains undetermined. A single 16S rRNA gene sequence was obtained from the DNA extract of the gill, and phylogenetic analysis placed the source organism within the lineage of the gamma subdivision of the Proteobacteria that consists of many chemoautotrophic endosymbionts of marine invertebrates. Fluorescence in situ hybridization analysis showed the bacterium densely colonizing the gill filaments. The fatty acid profile of the symbiont-free mantle contains the high level of the 16:1 fatty acid originating from the endosymbiont, which indicates that the endosymbiont cells are digested by, and incorporated into, the host. Compound-specific carbon isotopic analysis revealed that fatty acids from the gastropod tissues are all (13)C-depleted relative to the gastropod biomass. This fractionation pattern is consistent with chemoautotrophy based on the Calvin-Benson cycle and subsequent fatty-acid biosynthesis from (13)C-depleted acetyl coenzyme A. The results from the present study are clearly different from those from our previous study for A. aff. hessleri from the Indian Ocean that harbors a chemoautotrophic endosymbiont belonging to the epsilon subdivision of the Proteobacteria, which mediates the reductive tricarboxylic acid cycle for carbon fixation. Thus, it is concluded here that two lineages of chemoautotrophic bacteria, phylogenetically distinct at the subdivision level, occur as the primary endosymbiont in one host-animal type, which is unknown for the other metazoans.

Bacterial community shift along a subsurface geothermal water stream in a Japanese gold mine.

Change of bacterial community occurring along a hot water stream in the Hishikari gold mine, Japan, was investigated by applying a combination of various culture-independent techniques. The stream, which is derived from a subsurface anaerobic aquifer containing plentiful CO2, CH4, H2, and NH4+, emerges in a mine tunnel 320 m below the surface providing nutrients for a lush microbial community that extends to a distance of approximately 7 m in the absence of sunlight-irradiation. Over this distance, the temperature decreases from 69 degrees C to 55 degrees C, and the oxidation-reduction potential increases from -130 mV to +59 mV. In the hot upper reaches of the stream, the dominant phylotypes were: 1) a deeply branching lineage of thermophilic methane-oxidizing gamma-Proteobacteria, and 2) a thermophilic hydrogen- and sulfur-oxidizing Sulfurihydrogenibium sp. In contrast, the prevailing phylotypes in the middle and lower parts of the stream were closely related to ammonia-oxidizing Nitrosomonas and nitrite-oxidizing Nitrospira spp.. Changes in the microbial metabolic potential estimated by competitive PCR analysis of genes encoding the enzymes, particulate methane monooxygenase (pmoA), ammonia monooxygenase (amoA), and putative nitrite oxidoreductase (norB), also substantiated the community shift indicated by 16S rRNA gene analysis. The diversity of putative norB lineages was assessed for the first time in the hot water environment. Estimation of dominant phylotypes by whole-cell fluorescent in situ hybridization and changes in inorganic nitrogen compounds such as decreasing ammonium and increasing nitrite and nitrate in the mat-interstitial water along the stream were consistent with the observed transition of the bacterial community structure in the stream.

Characterization of C1-metabolizing prokaryotic communities in methane seep habitats at the Kuroshima Knoll, southern Ryukyu Arc, by analyzing pmoA, mmoX, mxaF, mcrA, and 16S rRNA genes.

Samples from three submerged sites (MC, a core obtained in the methane seep area; MR, a reference core obtained at a distance from the methane seep; and HC, a gas-bubbling carbonate sample) at the Kuroshima Knoll in the southern Ryuku arc were analyzed to gain insight into the organisms present and the processes involved in this oxic-anoxic methane seep environment. 16S rRNA gene analyses by quantitative real-time PCR and clone library sequencing revealed that the MC core sediments contained abundant archaea (approximately 34% of the total prokaryotes), including both mesophilic methanogens related to the genus Methanolobus and ANME-2 members of the Methanosarcinales, as well as members of the delta-Proteobacteria, suggesting that both anaerobic methane oxidation and methanogenesis occurred at this site. In addition, several functional genes connected with methane metabolism were analyzed by quantitative competitive-PCR, including the genes encoding particulate methane monooxygenase (pmoA), soluble methane monooxygenase (mmoX), methanol dehydrogenese (mxaF), and methyl coenzyme M reductase (mcrA). In the MC core sediments, the most abundant gene was mcrA (2.5 x 10(6) copies/g [wet weight]), while the pmoA gene of the type I methanotrophs (5.9 x 10(6) copies/g [wet weight]) was most abundant at the surface of the MC core. These results indicate that there is a very complex environment in which methane production, anaerobic methane oxidation, and aerobic methane oxidation all occur in close proximity. The HC carbonate site was rich in gamma-Proteobacteria and had a high copy number of mxaF (7.1 x 10(6) copies/g [wet weight]) and a much lower copy number of the pmoA gene (3.2 x 10(2) copies/g [wet weight]). The mmoX gene was never detected. In contrast, the reference core contained familiar sequences of marine sedimentary archaeal and bacterial groups but not groups specific to C1 metabolism. Geochemical characterization of the amounts and isotopic composition of pore water methane and sulfate strongly supported the notion that in this zone both aerobic methane oxidation and anaerobic methane oxidation, as well as methanogenesis, occur.

Microbial communities associated with geological horizons in coastal subseafloor sediments from the sea of okhotsk.

Microbial communities from a subseafloor sediment core from the southwestern Sea of Okhotsk were evaluated by performing both cultivation-dependent and cultivation-independent (molecular) analyses. The core, which extended 58.1 m below the seafloor, was composed of pelagic clays with several volcanic ash layers containing fine pumice grains. Direct cell counting and quantitative PCR analysis of archaeal and bacterial 16S rRNA gene fragments indicated that the bacterial populations in the ash layers were approximately 2 to 10 times larger than those in the clays. Partial sequences of 1,210 rRNA gene clones revealed that there were qualitative differences in the microbial communities from the two different types of layers. Two phylogenetically distinct archaeal assemblages in the Crenarchaeota, the miscellaneous crenarchaeotic group and the deep-sea archaeal group, were the most predominant archaeal 16S rRNA gene components in the ash layers and the pelagic clays, respectively. Clones of 16S rRNA gene sequences from members of the gamma subclass of the class Proteobacteria dominated the ash layers, whereas sequences from members of the candidate division OP9 and the green nonsulfur bacteria dominated the pelagic clay environments. Molecular (16S rRNA gene sequence) analysis of 181 isolated colonies revealed that there was regional proliferation of viable heterotrophic mesophiles in the volcanic ash layers, along with some gram-positive bacteria and actinobacteria. The porous ash layers, which ranged in age from tens of thousands of years to hundreds of thousands of years, thus appear to be discrete microbial habitats within the coastal subseafloor clay sediment, which are capable of harboring microbial communities that are very distinct from the communities in the more abundant pelagic clays.