- [Show abstract] [Hide abstract] ABSTRACT: The Seto Inland Sea is one of the largest semi-enclosed seas of Japan, where inflows of both nutrient-rich freshwater and nutrient-poor open ocean water have a profound influence on oceanographic conditions in the surface layers. To explore the diversity and distribution of unicellular diazotrophic cyanobacteria in this highly complex coastal area, we evaluated the seasonal dynamics of the cyanobacteria in the Seto Inland Sea over a period of 2 years. We obtained sequences belonging to UCYN-A and UCYN-C from the UCYN 16S rRNA gene clone library. The UCYN-C sequences were divided into three major types, referred to as UCYN-C ribotypes 1, 2, and 3; these groups clustered with Cyanothece ATCC51142, Cyanothece SKTU126, and the endosymbiont of Rhopalodia gibba, respectively. Results of qPCR showed that UCYN-A was detected during the early summer season at sampling points with temperatures ranging from 14 to 28 °C and with relatively nitrogen-depleted conditions. The highest abundance of UCYN-C was observed during the winter season, reaching 8.3 × 105nifH gene copies l−1. Our results revealed a widespread presence of unicellular diazotrophic cyanobacteria in the Seto Inland Sea, suggesting their contribution to the biogeochemical cycle.
- [Show abstract] [Hide abstract] ABSTRACT: Ardenticatena maritima strain 110S T is a filamentous bacterium isolated from an iron-rich coastal hydrothermal field, and it is a unique isolate capable of dissimilatory iron or nitrate reduction among the members of the bacterial phylum Chloroflexi . Here, we report the draft genome sequence comprising 3,569,367 bp, containing 3,355 predicted coding sequences (CDSs).
- [Show abstract] [Hide abstract] ABSTRACT: Carboxydotrophic anaerobic thermophiles have been isolated from various hydrothermal environments and are considered to be important carbon monoxide (CO) scavengers or primary producers. However, the ecological factors that influence the distribution, abundance and CO-oxidizing activities of these bacteria are poorly understood. A previous study detected the carboxydotrophic bacteria Carboxydothermus spp. in a hot spring sample and found that they constituted up to 10% of the total bacterial cells. In this study, we investigated environmental features, potential microbial CO-oxidation activities and the abundance of Carboxydothermus spp. in various hot springs to determine environmental factors that affect CO-oxidizers and to see whether Carboxydothermus spp. are common in those environments. We detected potential microbial CO-oxidation activities in samples that showed relatively high values of total organic carbon (TOC), total nitrogen (TN), oxidation-reduction potential (ORP) and soil-water content. The abundance of Carboxydothermus spp. did not correlate with the presence of potential microbial CO-oxidation activities; however, Carboxydothermus spp. were detected in a wide range of environments, suggesting that these bacteria are widely distributed in spite of the relatively low population size. This study implies thermophilic CO-oxidizers occur in a wide range of environments and oxidize CO in somewhat oxidative environments rich in organic matter. © FEMS 2015. All rights reserved. For permissions, please e-mail: firstname.lastname@example.org.
- [Show abstract] [Hide abstract] ABSTRACT: Unlabelled: Microcystis aeruginosa forms toxic cyanobacterial blooms throughout the world where its infectious phages are thought to influence host population dynamics. To assess the cyanophage impact on the host dynamics, we previously monitored Ma-LMM01-type phage abundance using a real-time PCR with a primer set designed based on the sequence of Microcystis phage Ma-LMM01; and we estimated the phage-infected host cell abundance. However, a recent study shows the Ma-LMM01 g91 gene sequence belongs to the smallest group, group III, of the three genotype groups, suggesting Ma-LMM01-type phage abundance was underestimated. Therefore, to re-evaluate the effect of Ma-LMM01-type phages on their hosts, we monitored the abundance of Ma-LMM01-type phages using real-time PCR with a new primer set designed based on the sequences of genotype groups I-III. We found phage abundance between 10(3) and 10(4) ml(-1) using the new primer set in samples where previously these phages were not detected using the old primer set. The frequency of Ma-LMM01-type phage-infected cells to Ma-LMM01-type phage-susceptible host cells may be as high as 30%, suggesting the phages may occasionally affect not only shifts in the genetic composition but also the dynamics of Ma-LMM01-type phage-susceptible host populations. Significance and impact of the study: Phages are one of the factors that may control the ecology of their host blooms. Therefore, it is essential to estimate phage abundance to understand phage impact on host populations. A real-time PCR assay was improved to detect a larger range of Microcystis cyanophages in natural surroundings where no phages were detected using a previous method by re-designing a new primer set based on sequences from three Ma-LMM01-type phage genetic groups. The new method allows us to determine the distribution, dynamics and infection cycle of the phage to help understand the interaction between the phages and the hosts.
- [Show abstract] [Hide abstract] ABSTRACT: Nitric oxide reductase (NOR) catalyzes the reduction of nitric oxide to generate nitrous oxide. We recently reported on the crystal structure of a quinol-dependent NOR (qNOR) from Geobacillus stearothermophilus [Y. Matsumoto, T. Tosha, A.V. Pisliakov, T. Hino, H. Sugimoto, S. Nagano, Y. Sugita and Y. Shiro, Nat. Struct. Mol. Biol. 19 (2012) 238–246], and suggested that a water channel from the cytoplasm, which is not observed in cytochrome c-dependent NOR (cNOR), functions as a pathway transferring catalytic protons. Here, we further investigated the functional and structural properties of qNOR, and compared the findings with those for cNOR. The pH optimum for the enzymatic reaction of qNOR was in the alkaline range, whereas Pseudomonas aeruginosa cNOR showed a higher activity at an acidic pH. The considerably slower reduction rate, and a correlation of the pH dependence for enzymatic activity and the reduction rate suggest that the reduction process is the rate-determining step for the NO reduction by qNOR, while the reduction rate for cNOR was very fast and therefore is unlikely to be the rate-determining step. A close examination of the heme/non-heme iron binuclear center by resonance Raman spectroscopy indicated that qNOR has a more polar environment at the binuclear center compared with cNOR. It is plausible that a water channel enhances the accessibility of the active site to solvent water, creating more polar environment in qNOR. This structural feature could control certain properties of the active site, such as redox potential, which could explain the different catalytic properties of the two NORs. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.
- [Show abstract] [Hide abstract] ABSTRACT: Nitric oxide reductase (NOR) catalyzes the generation of nitrous oxide (N2 O) via the reductive coupling of two nitric oxide (NO) molecules at a heme/non-heme Fe center. We report herein on the structures of the reduced and ligand-bound forms of cytochrome c-dependent NOR (cNOR) from Pseudomonas aeruginosa at a resolution of 2.3-2.7 Å, to elucidate structure-function relationships in NOR, and compare them to those of cytochrome c oxidase (CCO) that is evolutionarily related to NOR. Comprehensive crystallographic refinement of the CO-bound form of cNOR suggested that a total of four atoms can be accommodated at the binuclear center. Consistent with this, binding of bulky acetaldoxime (CH3 -CH=N-OH) to the binuclear center of cNOR was confirmed by the structural analysis. Active site reduction and ligand binding in cNOR induced only ~0.5 Å increase in the heme/non-heme Fe distance, but no significant structural change in the protein. The highly localized structural change is consistent with the lack of proton-pumping activity in cNOR, because redox-coupled conformational changes are thought to be crucial for proton pumping in CCO. It also permits the rapid decomposition of cytotoxic NO in denitrification. In addition, the shorter heme/non-heme Fe distance even in the bulky ligand-bound form of cNOR (~4.5 Å) than the heme/Cu distance in CCO (~5 Å) suggests the ability of NOR to maintain two NO molecules within a short distance in the confined space of the active site, thereby facilitating N-N coupling to produce a hyponitrite intermediate for the generation of N2 O. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
- [Show abstract] [Hide abstract] ABSTRACT: Carbon monoxide dehydrogenase-I (CODH-I) from the CO-utilizing bacterium Carboxydothermus hydrogenoformans are expected to be utilized as a part of reproducible carbon dioxide photoreduction system. However, the over-expression system for CODH-I remains to be constructed. CODH-I constitutes a hydrogenase/CODH gene cluster including a gene encoding a Ni-insertion accessory protein, CooC (cooC3). Through co-expression of CooC3, we found an over-expression system with higher activity. The Rec-CODH-I with the co-expression exhibits 8060 U/mg which was approximately threefold than that without co-expression (2270 U/mg). In addition, co-expression resulted in Ni(2+) content increase; the amount of Ni atoms of Rec-CODH-I was approximately thrice than that without co-expression.
- [Show abstract] [Hide abstract] ABSTRACT: The cyanobacterium, Microcystis aeruginosa, contains a large number of defense genes ( Makarova et al., 2011); thus, it is a good model to study the co-evolution of phage and bacteria. Here, we isolated and characterized two phage-resistant M. aeruginosa mutants that came from a phage intermediate-sensitive culture. To determine the mutation conferring resistance, a protein expression pattern analysis was performed comparing phage-sensitive and -resistant sub-strains using SDS-PAGE. There were no apparent differences in expression patterns in the soluble fraction; however, a ∼90 kDa protein in the hydrophobic fraction from the phage-sensitive sub-strain was observed. Using a successive thermal asymmetric interlaced-PCR, the entire sequence encoding the protein, assigned ISP90, as well as its neighboring regions (ca. 7.8 kb) was determined. ISP90 contained no conserved domains and was predicted to be a membrane-associated protein. No mutations were detected in the nucleotide sequences coding ISP90 and diversification of ISP90 regions within this species were observed. Diversification of ISP90 regions within this species suggests a possible genomic island that may be subjected to selective pressures from phages. The ISP90 sequence involving phage resistance/sensitivity contributes to the understanding of co-evolution between M. aeruginosa and phages.
- [Show abstract] [Hide abstract] ABSTRACT: Bacteriophages rapidly diversify their genes through co-evolution with their hosts. We hypothesize that gene diversification of phages leads to locality in phages genome. To test this hypothesis, we investigated the genetic diversity and composition of Microcystis cyanophages using 104 sequences of Ma-LMM01-type cyanophages from two geographically distant sampling sites. The intergenetic region between the ribonucleotide reductase genes nrdA and nrdB was used as the genetic marker. This region contains the host-derived auxiliary metabolic genes nblA, an unknown function gene g04, and RNA ligase gene g03. The sequences obtained were conserved in the Ma-LMM01 gene order and contents. Although the genetic diversity of the sequences was high, it varied by gene. The genetic diversity of nblA was the lowest, suggesting that nblA is a highly significant gene that does not allow mutation. In contrast, g03 sequences had many point mutations. RNA ligase is involved in the counter-host's phage defense mechanism, suggesting that phage defense also plays an important role for rapid gene diversification. The maximum parsimony network and phylogenic analysis showed the sequences from the two sampling sites were distinct. These findings suggest Ma-LMM01-type phages rapidly diversify their genomes through co-evolution with hosts in each location and eventually provided locality of their genomes.
- [Show abstract] [Hide abstract] ABSTRACT: Clustered regularly interspaced short palindromic repeat (CRISPR) confers adaptive immunity against phages via sequence fragments (spacers) derived from mobile genetic elements (MGEs), thus serving as a memory of the past host-phage co-evolution. To understand co-evolutionary dynamics in natural settings, we examined CRISPR diversity in 94 isolates of Microcystis aeruginosa from a small eutrophic pond. Fifty-two isolates possessed the CRISPR and were classified into 22 different CRISPR-related genotypes, suggesting stable coexistence of multiple genotypes with different phage susceptibility. Seven CRISPR-related genotypes showed variation of spacers at the leader-end of the CRISPR, indicating active spacer addition from MGEs. An abundant phylotype (based on internal transcribed spacer (ITS) of rRNA gene) contained different CRISPR spacer genotypes with the same CRISPR-associated cas2 gene. These data suggest that selective phage infection and possibly plasmid transfer may contribute to maintenance of multiple genotypes of M. aeruginosa and rapid co-evolution within a host-phage combination may be driven by increased contact frequency. Forty-two isolates lacked detectable CRISPR loci. Relative abundance of the CRISPR-lacking genotypes in the population suggests that the CRISPR loss may be selected for enhanced genetic exchange.
Dataset: Supplementary Material
- [Show abstract] [Hide abstract] ABSTRACT: Physicochemical characteristics and archaeal and bacterial community structures in an iron-rich coastal hydrothermal field, where the temperature of the most active hot spot reaches above 100 ｰ C, were investigated to obtain fundamental information on microbes inhabiting a coastal hydrothermal field. The environmental settings of the coastal hydrothermal field were similar in some degree to those of deep-sea hydrothermal environments because of its emission of H2, CO2, and sulfide from the bottom of the hot spot. The results of clone analyses based on the 16S rRNA gene led us to speculate the presence of a chemo-synthetic microbial ecosystem, where chemolithoautotrophic thermophiles, primarily the bacterial order Aquificales, function as primary producers using H2 or sulfur compounds as their energy source and CO2 as their carbon source, and the organic compounds synthesized by them support the growth of chemoheterotrophic thermophiles, such as members of the order Thermales and the family Desulfurococcaceae. In addition, the dominance of members of the bacterial genus Herbaspirillum in the high temperature bottom layer led us to speculate the temporal formation of mesophilic zones where they can also function as primary producing or nitrogen-fixing bacteria.
- [Show abstract] [Hide abstract] ABSTRACT: A unique [Ni-Fe-S] cluster (C-cluster) constitutes the active center of Ni-containing carbon monoxide dehydrogenases (CODHs). His(261), which coordinates one of the Fe atoms with Cys(295), is suggested to be the only residue required for Ni coordination in the C-cluster. To evaluate the role of Cys(295), we constructed CODH-II variants. Ala substitution for the Cys(295) substitution resulted in the decrease of Ni content and didn't result in major change of Fe content. In addition, the substitution had no effect on the ability to assemble a full complement of [Fe-S] clusters. This strongly suggests Cys(295) indirectly and His(261) together affect Ni-coordination in the C-cluster.
- [Show abstract] [Hide abstract] ABSTRACT: Aeropyrum spp are aerobic, heterotrophic, and hyperthermophilic marine archaea. There are two closely related Aeropyrum species, Aeropyrum camini and Aeropyrum pernix, which are isolated from geographically distinct locations. Recently, we compared their genome sequences to determine their genomic variation. They possess highly conserved small genomes, reflecting their close relationship. The entire genome similarity may result from their survival strategies in adapting to extreme environmental conditions. Meanwhile, synteny disruptions were observed in some regions including clustered regularly interspaced short palindromic repeats elements. Further, the largest portion of their non-orthologous genes were genes in the two proviral regions of A. pernix (Aeropyrum pernix spindle-shaped virus 1 and Aeropyrum pernix ovoid virus 1) or ORFans considered to be derived from viruses. Our data shows that genomic diversification of Aeropyrum spp may be substantially induced by viruses. This suggests that Aeropyrum spp may have a large pan-genome that can be extended by viruses, while each of the species shares a highly conserved small genome specializing for extreme environments.
- [Show abstract] [Hide abstract] ABSTRACT: The increasing number of genome sequences of archaea and bacteria show their adaptation to different environmental conditions at the genomic level. Aeropyrum spp. are aerobic and hyperthermophilic archaea. Aeropyrum camini was isolated from a deep-sea hydrothermal vent, and Aeropyrum pernix was isolated from a coastal solfataric vent. To investigate the adaptation strategy in each habitat, we compared the genomes of the two species. Shared genome features were a small genome size, a high GC content, and a large portion of orthologous genes (86 to 88%). The genomes also showed high synteny. These shared features may have been derived from the small number of mobile genetic elements and the lack of a RecBCD system, a recombinational enzyme complex. In addition, the specialized physiology (aerobic and hyperthermophilic) of Aeropyrum spp. may also contribute to the entire-genome similarity. Despite having stable genomes, interference of synteny occurred with two proviruses, A. pernix spindle-shaped virus 1 (APSV1) and A. pernix ovoid virus 1 (APOV1), and clustered regularly interspaced short palindromic repeat (CRISPR) elements. Spacer sequences derived from the A. camini CRISPR showed significant matches with protospacers of the two proviruses infecting A. pernix, indicating that A. camini interacted with viruses closely related to APSV1 and APOV1. Furthermore, a significant fraction of the nonorthologous genes (41 to 45%) were proviral genes or ORFans probably originating from viruses. Although the genomes of A. camini and A. pernix were conserved, we observed nonsynteny that was attributed primarily to virus-related elements. Our findings indicated that the genomic diversification of Aeropyrum spp. is substantially caused by viruses.
- [Show abstract] [Hide abstract] ABSTRACT: Elongation factor-1alpha (EF-1alpha) and elongation factor-like (EFL) proteins are functionally homologous to one another, and are core components of the eukaryotic translation machinery. The patchy distribution of the two elongation factor types across global eukaryotic phylogeny is suggestive of a 'differential loss' hypothesis that assumes that EF-1alpha and EFL were present in the most recent common ancestor of eukaryotes followed by independent differential losses of one of the two factors in the descendant lineages. To date, however, just one diatom and one fungus have been found to have both EF-1alpha and EFL (dual-EF-containing species). In this study, we characterized 35 new EF-1alpha/EFL sequences from phylogenetically diverse eukaryotes. In so doing we identified 11 previously unreported dual-EF-containing species from diverse eukaryote groups including the Stramenopiles, Apusomonadida, Goniomonadida, and Fungi. Phylogenetic analyses suggested vertical inheritance of both genes in each of the dual-EF lineages. In the dual-EF-containing species we identified, the EF-1alpha genes appeared to be highly divergent in sequence and suppressed at the transcriptional level compared to the co-occurring EFL genes. According to the known EF-1alpha/EFL distribution, the differential loss process should have occurred independently in diverse eukaryotic lineages, and more dual-EF-containing species remain unidentified. We predict that dual-EF-containing species retain the divergent EF-1alpha homologues only for a sub-set of the original functions. As the dual-EF-containing species are distantly related to each other, we propose that independent re-modelling of EF-1alpha function took place in multiple branches in the tree of eukaryotes.
- [Show abstract] [Hide abstract] ABSTRACT: In bacteria, 70S ribosomes (consisting of 30S and 50S subunits) dimerize to form 100S ribosomes, which were first discovered in Escherichia coli. Ribosome modulation factor (RMF) and hibernation promoting factor (HPF) mediate this dimerization in stationary phase. The 100S ribosome is translationally inactive, but it dissociates into two translationally active 70S ribosomes after transfer from starvation to fresh medium. Therefore, the 100S ribosome is called the 'hibernating ribosome'. The gene encoding RMF is found widely throughout the Gammaproteobacteria class, but is not present in any other bacteria. In this study, 100S ribosome formation in six species of Gammaproteobacteria and eight species belonging to other bacterial classes was compared. There were several marked differences between the two groups: (i) Formation of 100S ribosomes was mediated by RMF and short HPF in Gammaproteobacteria species, similar to E. coli, whereas it was mediated only by long HPF in the other bacterial species; (ii) RMF/short HPF-mediated 100S ribosome formation occurred specifically in stationary phase, whereas long HPF-mediated 100S ribosome formation occurred in all growth phases; and (iii) 100S ribosomes formed by long HPF were much more stable than those formed by RMF and short HPF.
- [Show abstract] [Hide abstract] ABSTRACT: A hydrogenogenic, carboxydotrophic marine bacterium strain KKC1(T) was isolated from a sediment core sample taken from a submerged marine caldera. Cells were non-motile 1.0-3.0 μm straight rods and Gram negative often observed with round endospores. Growth range was 55-68 °C, pH 5.2-9.3, and 0.8-14% (w/v) salinity. Optimum growth occurred at 65 °C, pH 7.0-7.5, and 2.4% salinity with a doubling time of 3.7 h. The isolate grew chemolithotrophically producing H2 from carbon monoxide (CO) oxidation with reduction of various electron acceptors, e.g., sulfite, thiosulfate, fumarate, ferric iron, and AQDS. KKC1(T) grew heterotrophically on pyruvate, lactate, fumarate, glucose, fructose, and mannose with thiosulfate as an electron acceptor. When grown mixotrophically on CO and pyruvate, C16:0 comprised almost half of the total cellular fatty acids. The G + C content was 50.6 mol%. The 16S rRNA gene sequence of KKC1(T) was most closely related to members of the genus Moorella with similarity ranging 91-89%. Based on physiological and phylogenetic novelty of the isolate, we propose the isolate as a new genus and species with the name Calderihabitans maritimus gen. nov., sp. nov.; the type strain is KKC1(T) (=DSM 26464(T) =NBRC 109353(T)).
- [Show abstract] [Hide abstract] ABSTRACT: Viruses influence the abundance of host populations through virus-mediated host cell lysis. Viruses contribute to the generation and maintenance of host diversity, which also results in viral diversity throughout their co-evolution. Here, to determine the phage gene diversification throughout co-evolution of host and phage in a natural environment, we investigated the genetic diversity and temporal changes in Microcystis cyanophage populations using a total of 810 sequences of the Ma-LMM01-type cyanophage tail sheath gene (g91) from 2006 to 2011 in a natural pond. The sequences obtained were highly diverse and assigned to 419 different genotypes (GT1-GT419) clustered at 100% nucleotide sequence similarity. A maximum parsimony network showed the genotypes were largely divided into three sequence groups, which were dominated by major genotypes (more than 24 sequences: GT2, GT53, and GT163 in group I; GT25 in group II; and GT1 in group III). These major genotypes co-existed and oscillated throughout the sampling periods, suggesting the Microcystis-cyanophage co-evolution was partly driven by a negative frequency-dependent selection. Meanwhile, the high viral genetic diversity observed was derived from a large number of the variants of each major and moderately-frequent genotype (including 7 to 18 sequences: GT7, GT26, GT56, GT149, and GT182 in group I; GT152 in group II) (1-2 nucleotide substitutions). The variants almost always co-occurred with their origins. This manner of variant emergence suggests increased contact frequency with a host-phage population promotes rapid co-evolution in an arms race.
- [Show abstract] [Hide abstract] ABSTRACT: CO-oxidizing bacteria, termed carboxydotrophs, have been assumed to play an important role in microbial environment as “CO-scavengers”. However, little is known about their ecology, especially in hydrothermal environments. In this study we focused on the members of the genus Carboxydothermus, the most studied thermophilic carboxydotrophs found in hot springs, which harbor unique sets of CO metabolisms. Carbon monoxide dehydrogenases (CODHs) are key enzymes of CO oxidation among these bacteria. We designed a real-time PCR primer set to amplify a conserved region of the gene encoding the CODH-II (cooS-II), and quantified Carboxydothermus in environmental samples. Real-time PCR primers developed in this study specifically amplified cooS-II partial sequences of 165-bp in length. The highest copy number of cooS-II obtained was 9.45 × 105 copies/ g sediment from a hot spring, equivalent to 10 % of Bacterial 16 S rRNA gene copies, while some samples were under detection level (< 1.0 × 102 copies). Apparently, the distribution of cooS-II was related to environmental temperature and microbial populations.
Nihon UniversityEdo, Tōkyō, Japan
Kioto, Kyōto, Japan
- Division of Applied Biosciences
Japan Agency for Marine-Earth Science Technology
Йокосука, Kanagawa, Japan
- Institute of Biogeosciences