Removal of phosphoglycolate in hyperthermophilic archaea.

Many organisms that utilize the Calvin-Benson-Bassham (CBB) cycle for autotrophic growth harbor metabolic pathways to remove and/or salvage 2-phosphoglycolate, the product of the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). It has been presumed that the occurrence of 2-phosphoglycolate salvage is linked to the CBB cycle, and in particular, the C2 pathway to the CBB cycle and oxygenic photosynthesis. Here, we examined 2-phosphoglycolate salvage in the hyperthermophilic archaeon Thermococcus kodakarensis, an obligate anaerobe that harbors a Rubisco that functions in the pentose bisphosphate pathway. T. kodakarensis harbors enzymes that have the potential to convert 2-phosphoglycolate to glycine and serine, and their genes were identified by biochemical and/or genetic analyses. 2-phosphoglycolate phosphatase activity increased 1.6-fold when cells were grown under microaerobic conditions compared to anaerobic conditions. Among two candidates, TK1734 encoded a phosphatase specific for 2-phosphoglycolate, and the enzyme was responsible for 80% of the 2-phosphoglycolate phosphatase activity in T. kodakarensis cells. The TK1734 disruption strain displayed growth impairment under microaerobic conditions, which was relieved upon addition of sodium sulfide. In addition, glycolate was detected in the medium when T. kodakarensis was grown under microaerobic conditions. The results suggest that T. kodakarensis removes 2-phosphoglycolate via a phosphatase reaction followed by secretion of glycolate to the medium. As the Rubisco in T. kodakarensis functions in the pentose bisphosphate pathway and not in the CBB cycle, mechanisms to remove 2-phosphoglycolate in this archaeon emerged independent of the CBB cycle.

Sequestration and efflux largely account for cadmium and copper resistance in the deep-sea Nitratiruptor sp. SB155-2 (phylum Campylobacterota).

In deep-sea hydrothermal vent environments, metal-enriched fluids and sediments abound, making these habitats ideal to study metal resistance in prokaryotes. In this investigation, we employed transcriptomics and shotgun proteomics with scanning transmission electron microscopy and energy-dispersive x-ray spectroscopy (STEM-EDX) to better understand mechanisms of tolerance for cadmium (Cd) and copper (Cu) at stress-inducing concentrations in Nitratiruptor sp. SB155-2 (phylum Campylobacterota). Transcriptomic profiles were remarkably different in the presence of these two metals, displaying 385 (19%) and 629 (31%) differentially transcribed genes (DTG) in the presence of Cd(II) and Cu(II), respectively, while only 7% of differentially transcribed (DT) genes were shared, with genes for non-specific metal transporters and genes involved in oxidative stress-response predominating. Transcriptomic and proteomic analyses confirmed that metal-specific DT pathways under Cu(II) stress, including those involving sulfur, cysteine, and methionine, are likely required for high-affinity efflux systems, while flagella formation and chemotaxis were over-represented under Cd(II) stress. Consistent with these differences, STEM-EDX analysis revealed that polyphosphate-like granules (pPLG), the formation of CdS particles, and the periplasmic space are crucial for Cd(II) sequestration. Overall, this study provides new insights regarding metal-specific adaptations of Campylobacterota to deep-sea hydrothermal vent environments.

Fundidesulfovibrio magnetotacticus sp. nov., a sulphate-reducing magnetotactic bacterium, isolated from sediments and freshwater of a pond.

A sulphate-reducing magnetotactic bacterium, designated strain FSS-1T, was isolated from sediments and freshwater of Suwa Pond located in Hidaka, Saitama, Japan. Strain FSS-1T was a motile, Gram-negative and curved rod-shaped bacterium that synthesizes bullet-shaped magnetite (Fe3O4) nanoparticles in each cell. Strain FSS-1T was able to grow in the range of pH 6.5-8.0 (optimum, pH 7.0), 22-34 °C (optimum, 28 °C) and with 0-8.0 g l-1 NaCl (optimum, 0-2.0 g l-1 NaCl). Strain FSS-1T grew well in the presence of 50 µM ferric quinate as an iron source. The major fatty acids were anteiso-C15 : 0, iso-C15 : 0 and anteiso-C17 : 0. The major menaquinone was MK-7 (H2). Strain FSS-1T contained desulfoviridin, cytochrome c 3 and catalase, but did not contain oxidase. Strain FSS-1T used fumarate, lactate, pyruvate, malate, formate/acetate, succinate, tartrate, ethanol, 1-propanol, peptone, soytone and yeast extract as electron donors, while the strain used sulphate, thiosulphate and fumarate as electron acceptors. Fumarate was fermented in the absence of electron acceptors. Analysis of the 16S rRNA gene sequence showed that strain FSS-1T is a member of the genus Fundidesulfovibrio. The gene sequence showed 96.7, 95.0, 92.0, 91.2 and 91.4% similarities to the most closely related members of the genera Fundidesulfovibrio putealis B7-43T, Fundidesulfovibrio butyratiphilus BSYT, Desulfolutivibrio sulfoxidireducens DSM 107105T, Desulfolutivibrio sulfodismutans ThAc01T and Solidesulfovibrio magneticus RS-1T, respectively. The DNA G+C content of strain FSS-1T was 67.5 mol%. The average nucleotide identity value between strain FSS-1T and F. putealis B7-43T was 80.7 %. Therefore, strain FSS-1T represents a novel species within the genus Fundidesulfovibrio, for which the name Fundidesulfovibrio magnetotacticus sp. nov. is proposed (=JCM 32405T=DSM 110007T).

Hadal biosphere: insight into the microbial ecosystem in the deepest ocean on Earth.

Hadal oceans at water depths below 6,000 m are the least-explored aquatic biosphere. The Challenger Deep, located in the western equatorial Pacific, with a water depth of ∼11 km, is the deepest ocean on Earth. Microbial communities associated with waters from the sea surface to the trench bottom (0∼10,257 m) in the Challenger Deep were analyzed, and unprecedented trench microbial communities were identified in the hadal waters (6,000∼10,257 m) that were distinct from the abyssal microbial communities. The potentially chemolithotrophic populations were less abundant in the hadal water than those in the upper abyssal waters. The emerging members of chemolithotrophic nitrifiers in the hadal water that likely adapt to the higher flux of electron donors were also different from those in the abyssal waters that adapt to the lower flux of electron donors. Species-level niche separation in most of the dominant taxa was also found between the hadal and abyssal microbial communities. Considering the geomorphology and the isolated hydrotopographical nature of the Mariana Trench, we hypothesized that the distinct hadal microbial ecosystem was driven by the endogenous recycling of organic matter in the hadal waters associated with the trench geomorphology.

Mariprofundus micogutta sp. nov., a novel iron-oxidizing zetaproteobacterium isolated from a deep-sea hydrothermal field at the Bayonnaise knoll of the Izu-Ogasawara arc, and a description of Mariprofundales ord. nov. and Zetaproteobacteria classis nov.

A novel iron-oxidizing chemolithoautotrophic bacterium, strain ET2T, was isolated from a deep-sea sediment in a hydrothermal field of the Bayonnaise knoll of the Izu-Ogasawara arc. Cells were bean-shaped, curved short rods. Growth was observed at a temperature range of 15-30 °C (optimum 25 °C, doubling time 24 h) and a pH range of 5.8-7.0 (optimum pH 6.4) in the presence of NaCl at a range of 1.0-4.0 % (optimum 2.75 %). The isolate was a microaerophilic, strict chemolithoautotroph capable of growing using ferrous iron and molecular oxygen (O2) as the sole electron donor and acceptor, respectively; carbon dioxide as the sole carbon source; and either ammonium or nitrate as the sole nitrogen source. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that the new isolate was related to the only previously isolated Mariprofundus species, M. ferrooxydans. Although relatively high 16S rRNA gene similarity (95 %) was found between the new isolate and M. ferrooxydans, the isolate was distinct in terms of cellular fatty acid composition, genomic DNA G+C content and cell morphology. Furthermore, genomic comparison between ET2T and M. ferrooxydans PV-1 indicated that the genomic dissimilarity of these strains met the standard for species-level differentiation. On the basis of its physiological and molecular characteristics, strain ET2T (= KCTC 15556T = JCM 30585 T) represents a novel species of Mariprofundus, for which the name Mariprofundus micogutta is proposed. We also propose the subordinate taxa Mariprofundales ord. nov. and Zetaproteobacteria classis nov. in the phylum Proteobacteria.

Variance and potential niche separation of microbial communities in subseafloor sediments off Shimokita Peninsula, Japan.

Subseafloor pelagic sediments with high concentrations of organic matter form habitats for diverse microorganisms. Here, we determined depth profiles of genes for SSU rRNA, mcrA, dsrA and amoA from just beneath the seafloor to 363.3 m below the seafloor (mbsf) using core samples obtained from the forearc basin off the Shimokita Peninsula. The molecular profiles were combined with data on lithostratigraphy, depositional age, sedimentation rate and pore-water chemistry. The SSU rRNA gene tag structure and diversity changed at around the sulfate-methane transition zone (SMTZ), whereas the profiles varied further with depth below the SMTZ, probably in connection with the variation in pore-water chemistry. The depth profiles of diversity and abundance of dsrA, a key gene for sulfate reduction, suggested the possible niche separations of sulfate-reducing populations, even below the SMTZ. The diversity and abundance patterns of mcrA, a key gene for methanogenesis/anaerobic methanotrophy, suggested a stratified distribution and separation of anaerobic methanotrophy and hydrogenotrophic or methylotrophic methanogensis below the SMTZ. This study provides novel insights into the relationships between the composition and function of microbial communities and the chemical environment in the nutrient-rich continental margin subseafloor sediments, which may result in niche separation and variability in subseafloor microbial populations.

Distribution of eukaryotic serine racemases in the bacterial domain and characterization of a representative protein in Roseobacter litoralis Och 149.

Two distinct bacterial and eukaryotic serine racemases (SRs) have been identified based on phylogenetic and biochemical characteristics. Although some reports have suggested that marine heterotrophic bacteria have the potential to produce d-serine, the gene encoding bacterial SRs is not found in those bacterial genomes. In this study, using in-depth genomic analysis, we found that eukaryotic SR homologues were distributed widely in various bacterial genomes. Additionally, we selected a eukaryotic SR homologue from a marine heterotrophic bacterium, Roseobacter litoralis Och 149 (RiSR), and constructed an RiSR gene expression system in Escherichia coli for studying the properties of the enzyme. Among the tested amino acids, the recombinant RiSR exhibited both racemization and dehydration activities only towards serine, similar to many eukaryotic SRs. Mg2+ and MgATP enhanced both activities of RiSR, whereas EDTA abolished these enzymatic activities. The enzymatic properties and domain structure of RiSR were similar to those of eukaryotic SRs, particularly mammalian SRs. However, RiSR showed lower catalytic efficiency for L-serine dehydration (kcat/Km=0.094 min(-1) mM(-1)) than those of eukaryotic SRs reported to date (kcat/Km=0.6-21 min(-1) mM(-1)). In contrast, the catalytic efficiency for L-serine racemization of RiSR (kcat/Km=3.14 min(-1) mM(-1)) was 34-fold higher than that of l-serine dehydration. These data suggested that RiSR primarily catalysed serine racemization rather than dehydration.

Cysteine dioxygenase and cysteine sulfinate decarboxylase genes of the deep-sea mussel Bathymodiolus septemdierum: possible involvement in hypotaurine synthesis and adaptation to hydrogen sulfide.

It has been suggested that invertebrates inhabiting deep-sea hydrothermal vent areas use the sulfinic acid hypotaurine, a precursor of taurine, to protect against the toxicity of hydrogen sulfide contained in the seawater from the vent. In this protective system, hypotaurine is accumulated in the gill, the primary site of sulfide exposure. However, the pathway for hypotaurine synthesis in mollusks has not been identified. In this study, we screened for the mRNAs of enzymes involved in hypotaurine synthesis in the deep-sea mussel Bathymodiolus septemdierum and cloned cDNAs encoding cysteine dioxygenase and cysteine sulfinate decarboxylase. As mRNAs encoding cysteamine dioxygenase and cysteine lyase were not detected, the cysteine sulfinate pathway is suggested to be the major pathway of hypotaurine and taurine synthesis. The two genes were found to be expressed in all the tissues examined, but the gill exhibited the highest expression. The mRNA level in the gill was not significantly changed by exposure to sulfides or thiosulfate. These results suggests that the gill of B. septemdierum maintains high levels of expression of the two genes regardless of ambient sulfide level and accumulates hypotaurine continuously to protect against sudden exposure to high level of sulfide.

Palaeoproteomic investigation of an ancient human skeleton with abnormal deposition of dental calculus.

Detailed investigation of extremely severe pathological conditions in ancient human skeletons is important as it could shed light on the breadth of potential interactions between humans and disease etiologies in the past. Here, we applied palaeoproteomics to investigate an ancient human skeletal individual with severe oral pathology, focusing our research on bacterial pathogenic factors and host defense response. This female skeleton, from the Okhotsk period (i.e., fifth to thirteenth century) of Northern Japan, poses relevant amounts of abnormal dental calculus deposition and exhibits oral dysfunction due to severe periodontal disease. A shotgun mass-spectrometry analysis identified 81 human proteins and 15 bacterial proteins from the calculus of the subject. We identified two pathogenic or bioinvasive proteins originating from two of the three "red complex" bacteria, the core species associated with severe periodontal disease in modern humans, as well as two additional bioinvasive proteins of periodontal-associated bacteria. Moreover, we discovered defense response system-associated human proteins, although their proportion was mostly similar to those reported in ancient and modern human individuals with lower calculus deposition. These results suggest that the bacterial etiology was similar and the host defense response was not necessarily more intense in ancient individuals with significant amounts of abnormal dental calculus deposition.

Development of a rapid and highly accurate method for 13C tracer-based metabolomics and its application on a hydrogenotrophic methanogen.

Microfluidic capillary electrophoresis-mass spectrometry (CE-MS) is a rapid and highly accurate method to determine isotopomer patterns in isotopically labeled compounds. Here, we developed a novel method for tracer-based metabolomics using CE-MS for underivatized proteinogenic amino acids. The method consisting of a ZipChip CE system and a high-resolution Orbitrap Fusion Tribrid mass spectrometer allows us to obtain highly accurate data from 1 µl of 100 nmol/l amino acids comparable to a mere 1 [Formula: see text] 104-105 prokaryotic cells. To validate the capability of the CE-MS method, we analyzed 16 protein-derived amino acids from a methanogenic archaeon Methanothermobacter thermautotrophicus as a model organism, and the mass spectra showed sharp peaks with low mass errors and background noise. Tracer-based metabolome analysis was then performed to identify the central carbon metabolism in M. thermautotrophicus using 13C-labeled substrates. The mass isotopomer distributions of serine, aspartate, and glutamate revealed the occurrence of both the Wood-Ljungdahl pathway and an incomplete reductive tricarboxylic acid cycle for carbon fixation. In addition, biosynthesis pathways of 15 amino acids were constructed based on the mass isotopomer distributions of the detected protein-derived amino acids, genomic information, and public databases. Among them, the presence of alternative enzymes of alanine dehydrogenase, ornithine cyclodeaminase, and homoserine kinase was suggested in the biosynthesis pathways of alanine, proline, and threonine, respectively. To our knowledge, the novel 13C tracer-based metabolomics using CE-MS can be considered the most efficient method to identify central carbon metabolism and amino acid biosynthesis pathways and is applicable to any kind of isolated microbe.

Exclusive localization of carbonic anhydrase in bacteriocytes of the deep-sea clam Calyptogena okutanii with thioautotrophic symbiotic bacteria.

Deep-sea Calyptogena clams harbor thioautotrophic intracellular symbiotic bacteria in their gill epithelial cells. The symbiont fixes CO2 to synthesize organic compounds. Carbonic anhydrase (CA) from the host catalyzes the reaction CO2 + H2O ↔ HCO3(-) + H(+), and is assumed to facilitate inorganic carbon (Ci) uptake and transport to the symbiont. However, the localization of CA in gill tissue remains unknown. We therefore analyzed mRNA sequences, proteins and CA activity in Calyptogena okutanii using expression sequence tag, SDS-PAGE and LC-MS/MS. We found that acetazolamide-sensitive soluble CA was abundantly expressed in the gill tissue of C. okutanii, and the enzyme was purified by affinity chromatography. Mouse monoclonal antibodies against the CA of C. okutanii were used in western blot analysis and immunofluorescence staining of the gill tissues of C. okutanii, which showed that CA was exclusively localized in the symbiont-harboring cells (bacteriocytes) in gill epithelial cells. Western blot analysis and measurement of activity showed that CA was abundantly (26-72% of total soluble protein) detected in the gill tissues of not only Calyptogena clams but also deep-sea Bathymodiolus mussels that harbor thioautotrophic or methanotrophic symbiotic bacteria, but was not detected in a non-symbiotic mussel, Mytilus sp. The present study showed that CA is abundant in the gill tissues of deep-sea symbiotic bivalves and specifically localizes in the cytoplasm of bacteriocytes of C. okutanii. This indicates that the Ci supply process to symbionts in the vacuole (symbiosome) in bacteriocytes is essential for symbiosis.

Brevundimonas abyssalis sp. nov., a dimorphic prosthecate bacterium isolated from deep-subsea floor sediment.

A novel Gram-negative, aerobic, psychrotolerant, alkali-tolerant, heterotrophic and dimorphic prosthecate bacterium, designated strain TAR-001(T), was isolated from deep-sea floor sediment in Japan. Cells of this strain had a dimorphic life cycle and developed an adhesive stalk at a site not coincident with the centre of the cell pole, and the other type of cell, a swarm cell, had a polar flagellum. Colonies were glossy, viscous and yellowish-white in colour. The temperature, pH and salt concentration range for growth were 2-41 °C, pH 6.5-10.0 and 1-4% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences confirmed that strain TAR-001(T) belongs to the family Caulobacteraceae of the class Alphaproteobacteria, and lies between the genus Brevundimonas and the genus Caulobacter. Levels of similarity between the 16S rRNA gene sequence of strain TAR-001(T) and those of the type strains of Brevundimonas species were 93.3-95.7%; highest sequence similarity was with the type strain of Brevundimonas diminuta. Levels of sequence similarity between those of the type strains of Caulobacter species were 94.9-96.0%; highest sequence similarity was with the type strain of Caulobacter mirabilis. The G+C content of strain TAR-001(T) was 67.6 mol%. Q-10 was the major respiratory isoprenoid quinone. The major fatty acids were C18:1ω7c and C16:0, and the presence of 1,2-di-O-acyl-3-O-[D-glucopyranosyl-(1→4)-α-D-glucopyranuronosyl]glycerol suggests strain TAR-001(T) is more closely to the genus Brevundimonas than to the genus Caulobacter. The mean DNA-DNA hybridization levels between strain TAR-001(T) and the type strains of two species of the genus Brevundimonas were higher than that of the genus Caulobacter. On the basis of polyphasic biological features and the 16S rRNA gene sequence comparison presented here, strain TAR-001(T) is considered to represent a novel species of the genus Brevundimonas, for which the name Brevundimonas abyssalis sp. nov. is proposed; the type strain is TAR-001(T) (=JCM 18150(T)=CECT 8073(T)).

Genome sequence of a novel deep-sea vent epsilonproteobacterial phage provides new insight into the co-evolution of Epsilonproteobacteria and their phages.

Epsilonproteobacteria are among the predominant primary producers in deep-sea hydrothermal vent ecosystems. However, phages infecting deep-sea vent Epsilonproteobacteria have never been isolated and characterized. Here, we successfully isolated a novel temperate phage, NrS-1, that infected a deep-sea vent chemolithoautotrophic isolate of Epsilonproteobacteria, Nitratiruptor sp. SB155-2, and its entire genome sequence was obtained and analyzed. The NrS-1 genome is linear, circularly permuted, and terminally redundant. The NrS-1 genome is 37,159 bp in length and contains 51 coding sequences. Five major structural proteins including major capsid protein and tape measure protein were identified by SDS-PAGE and mass spectrometry analysis. NrS-1 belongs to the family Siphoviridae, but its sequence and genomic organization are distinct from those of any other previously known Siphoviridae phages. Homologues of genes encoded in the NrS-1 genome were widely distributed among the genomes of diverse Epsilonproteobacteria. The distribution patterns had little relation to the evolutionary traits and ecological and physiological differentiation of the host epsilonproteobacterial species. The widespread occurrence of phage genes in diverse Epsilonproteobacteria supports early co-evolution between temperate phages and Epsilonproteobacteria prior to the divergence of their habitats and physiological adaptation.

Geofilum rubicundum gen. nov., sp. nov., isolated from deep subseafloor sediment.

A novel, facultatively anaerobic bacterium (strain JAM-BA0501(T)) was isolated from a deep subseafloor sediment sample at a depth of 247 m below seafloor off the Shimokita Peninsula of Japan in the north-western Pacific Ocean (Site C9001, 1180 m water depth). Cells of strain JAM-BA0501(T) were gram-negative, filamentous, non-spore-forming and motile on solid medium by gliding. Phylogenetic analysis based on the 16S rRNA gene sequence of strain JAM-BA0501(T) indicated a distant relationship to strains representing genera within the order Bacteroidales, such as Alkaliflexus imshenetskii Z-7010(T) (91.1 % similarity), Marinilabilia salmonicolor ATCC 19041(T) (86.2 %) and Anaerophaga thermohalophila Fru22(T) (89.3 %). The new isolate produced isoprenoid quinones with menaquinone MK-7 as the major component, and the predominant fatty acids were iso-C(15 : 0) and anteiso-C(15 : 0). The DNA G+C content of the isolate was 42.9 mol%. Based on its taxonomic distinctiveness, strain JAM-BA0501(T) is considered to represent a novel species of a new genus within the family Marinilabiliaceae, for which the name Geofilum rubicundum gen. nov., sp. nov. is proposed. The type strain of Geofilum rubicundum is JAM-BA0501(T) ( = JCM 15548(T)  = NCIMB 14482(T)).

Proteomic Time-Course Analysis of the Filamentous Anoxygenic Phototrophic Bacterium, Chloroflexus aurantiacus, during the Transition from Respiration to Phototrophy.

Chloroflexus aurantiacus is a filamentous anoxygenic phototrophic bacterium that grows chemotrophically under oxic conditions and phototrophically under anoxic conditions. Because photosynthesis-related genes are scattered without any gene clusters in the genome, it is still unclear how this bacterium regulates protein expression in response to environmental changes. In this study, we performed a proteomic time-course analysis of how C. aurantiacus expresses proteins to acclimate to environmental changes, namely the transition from chemoheterotrophic respiratory to photoheterotrophic growth mode. Proteomic analysis detected a total of 2520 proteins out of 3934 coding sequences in the C. aurantiacus genome from samples collected at 13 time points. Almost all proteins for reaction centers, light-harvesting chlorosomes, and carbon fixation pathways were successfully detected during the growing phases in which optical densities and relative bacteriochlorophyll c contents increased simultaneously. Combination of proteomics and pigment analysis suggests that the self-aggregation of bacteriochlorophyllide c could precede the esterification of the hydrophobic farnesyl tail in cells. Cytoplasmic subunits of alternative complex III were interchanged between oxic and anoxic conditions, although membrane-bound subunits were used for both conditions. These data highlight the protein expression dynamics of phototrophy-related genes during the transition from respiration to phototrophy.

Physiological and comparative proteomic characterization of Desulfolithobacter dissulfuricans gen. nov., sp. nov., a novel mesophilic, sulfur-disproportionating chemolithoautotroph from a deep-sea hydrothermal vent.

In deep-sea hydrothermal environments, inorganic sulfur compounds are important energy substrates for sulfur-oxidizing, -reducing, and -disproportionating microorganisms. Among these, sulfur-disproportionating bacteria have been poorly understood in terms of ecophysiology and phylogenetic diversity. Here, we isolated and characterized a novel mesophilic, strictly chemolithoautotrophic, diazotrophic sulfur-disproportionating bacterium, designated strain GF1T, from a deep-sea hydrothermal vent chimney at the Suiyo Seamount in the Izu-Bonin Arc, Japan. Strain GF1T disproportionated elemental sulfur, thiosulfate, and tetrathionate in the presence of ferrihydrite. The isolate also grew by respiratory hydrogen oxidation coupled to sulfate reduction. Phylogenetic and physiological analyses support that strain GF1T represents the type strain of a new genus and species in the family Desulfobulbaceae, for which the name Desulfolithobacter dissulfuricans gen. nov. sp. nov. is proposed. Proteomic analysis revealed that proteins related to tetrathionate reductase were specifically and abundantly produced when grown via thiosulfate disproportionation. In addition, several proteins possibly involved in thiosulfate disproportionation, including those encoded by the YTD gene cluster, were also found. The overall findings pointed to a possible diversity of sulfur-disproportionating bacteria in hydrothermal systems and provided a refined picture of microbial sulfur disproportionation.

Loss of genes for DNA recombination and repair in the reductive genome evolution of thioautotrophic symbionts of Calyptogena clams.

BACKGROUND: Two Calyptogena clam intracellular obligate symbionts, Ca. Vesicomyosocius okutanii (Vok; C. okutanii symbiont) and Ca. Ruthia magnifica (Rma; C. magnifica symbiont), have small genomes (1.02 and 1.16 Mb, respectively) with low G+C contents (31.6% and 34.0%, respectively) and are thought to be in an ongoing stage of reductive genome evolution (RGE). They lack recA and some genes for DNA repair, including mutY. The loss of recA and mutY is thought to contribute to the stabilization of their genome architectures and GC bias, respectively. To understand how these genes were lost from the symbiont genomes, we surveyed these genes in the genomes from 10 other Calyptogena clam symbionts using the polymerase chain reaction (PCR). RESULTS: Phylogenetic trees reconstructed using concatenated 16S and 23S rRNA gene sequences showed that the symbionts formed two clades, clade I (symbionts of C. kawamurai, C. laubieri, C. kilmeri, C. okutanii and C. soyoae) and clade II (those of C. pacifica, C. fausta, C. nautilei, C. stearnsii, C. magnifica, C. fossajaponica and C. phaseoliformis). recA was detected by PCR with consensus primers for recA in the symbiont of C. phaseoliformis. A detailed homology search revealed a remnant recA in the Rma genome. Using PCR with a newly designed primer set, intact recA or its remnant was detected in clade II symbionts. In clade I symbionts, the recA coding region was found to be mostly deleted.In the Rma genome, a pseudogene of mutY was found. Using PCR with newly designed primer sets, mutY was not found in clade I symbionts but was found in clade II symbionts. The G+C content of 16S and 23S rRNA genes in symbionts lacking mutY was significantly lower than in those with mutY. CONCLUSIONS: The extant Calyptogena clam symbionts in clade II were shown to have recA and mutY or their remnants, while those in clade I did not. The present results indicate that the extant symbionts are losing these genes in RGE, and that the loss of mutY contributed to the GC bias of the genomes during their evolution.

Isolation and cultivation of a novel sulfate-reducing magnetotactic bacterium belonging to the genus Desulfovibrio.

Magnetotactic bacteria (MTB) synthesize magnetosomes composed of membrane-enveloped magnetite (Fe3O4) and/or greigite (Fe3S4) nanoparticles in the cells. It is known that the magnetotactic Deltaproteobacteria are ubiquitous and inhabit worldwide in the sediments of freshwater and marine environments. Mostly known MTB belonging to the Deltaproteobacteria are dissimilatory sulfate-reducing bacteria that biomineralize bullet-shaped magnetite nanoparticles, but only a few axenic cultures have been obtained so far. Here, we report the isolation, cultivation and characterization of a dissimilatory sulfate-reducing magnetotactic bacterium, which we designate "strain FSS-1". We found that the strain FSS-1 is a strict anaerobe and uses casamino acids as electron donors and sulfate as an electron acceptor to reduce sulfate to hydrogen sulfide. The strain FSS-1 produced bullet-shaped magnetite nanoparticles in the cells and responded to external magnetic fields. On the basis of 16S rRNA gene sequence analysis, the strain FSS-1 is a member of the genus Desulfovibrio, showing a 96.7% sequence similarity to Desulfovibrio putealis strain B7-43T. Futhermore, the magnetosome gene cluster of strain FSS-1 was different from that of Desulfovibrio magneticus strain RS-1. Thus, the strain FSS-1 is considered to be a novel sulfate-reducing magnetotactic bacterium belonging to the genus Desulfovibrio.

Chloroplast acquisition without the gene transfer in kleptoplastic sea slugs, Plakobranchus ocellatus.

Some sea slugs sequester chloroplasts from algal food in their intestinal cells and photosynthesize for months. This phenomenon, kleptoplasty, poses a question of how the chloroplast retains its activity without the algal nucleus. There have been debates on the horizontal transfer of algal genes to the animal nucleus. To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus. Nevertheless, it was confirmed that light illumination prolongs the life of mollusk under starvation. These data presented a paradigm that a complex adaptive trait, as typified by photosynthesis, can be transferred between eukaryotic kingdoms by a unique organelle transmission without nuclear gene transfer. Our phylogenomic analysis showed that genes for proteolysis and immunity undergo gene expansion and are up-regulated in chloroplast-enriched tissue, suggesting that these molluskan genes are involved in the phenotype acquisition without horizontal gene transfer.

Reduced genome of the thioautotrophic intracellular symbiont in a deep-sea clam, Calyptogena okutanii.

Although dense animal communities at hydrothermal vents and cold seeps rely on symbioses with chemoautotrophic bacteria [1, 2], knowledge of the mechanisms underlying these chemosynthetic symbioses is still fragmentary because of the difficulty in culturing the symbionts and the hosts in the laboratory. Deep-sea Calyptogena clams harbor thioautotrophic bacterial symbionts in their gill epithelial cells [1, 2]. They have vestigial digestive tracts and nutritionally depend on their symbionts [3], which are vertically transmitted via eggs [4]. To clarify the symbionts' metabolic roles in the symbiosis and adaptations to intracellular conditions, we present the complete genome sequence of the symbiont of Calyptogena okutanii. The genome is a circular chromosome of 1,022,154 bp with 31.6% guanine + cytosine (G + C) content, and is the smallest reported genome in autotrophic bacteria. It encodes 939 protein-coding genes, including those for thioautotrophy and for the syntheses of almost all amino acids and various cofactors. However, transporters for these substances to the host cell are apparently absent. Genes that are unnecessary for an intracellular lifestyle, as well as some essential genes (e.g., ftsZ for cytokinesis), appear to have been lost from the symbiont genome. Reductive evolution of the genome might be ongoing in the vertically transmitted Calyptogena symbionts.