Metagenomic analysis provides functional insights into seasonal change of a non-cyanobacterial prokaryotic community in temperate coastal waters.

The taxonomic compositions of marine prokaryotic communities are known to follow seasonal cycles, but functional metagenomic insights into this seasonality is still limited. We analyzed a total of 22 metagenomes collected at 11 time points over a 14-month period from two sites in Sendai Bay, Japan to obtain seasonal snapshots of predicted functional profiles of the non-cyanobacterial prokaryotic community. Along with taxonomic composition, functional gene composition varied seasonally and was related to chlorophyll a concentration, water temperature, and salinity. Spring phytoplankton bloom stimulated increased abundances of putative genes that encode enzymes in amino acid metabolism pathways. Several groups of functional genes, including those related to signal transduction and cellular communication, increased in abundance during the mid- to post-bloom period, which seemed to be associated with a particle-attached lifestyle. Alternatively, genes in carbon metabolism pathways were generally more abundant in the low chlorophyll a period than the bloom period. These results indicate that changes in trophic condition associated with seasonal phytoplankton succession altered the community function of prokaryotes. Our findings on seasonal changes of predicted function provide fundamental information for future research on the mechanisms that shape marine microbial communities.

Identification of a dual orange/far-red and blue light photoreceptor from an oceanic green picoplankton.

Photoreceptors are conserved in green algae to land plants and regulate various developmental stages. In the ocean, blue light penetrates deeper than red light, and blue-light sensing is key to adapting to marine environments. Here, a search for blue-light photoreceptors in the marine metagenome uncover a chimeric gene composed of a phytochrome and a cryptochrome (Dualchrome1, DUC1) in a prasinophyte, Pycnococcus provasolii. DUC1 detects light within the orange/far-red and blue spectra, and acts as a dual photoreceptor. Analyses of its genome reveal the possible mechanisms of light adaptation. Genes for the light-harvesting complex (LHC) are duplicated and transcriptionally regulated under monochromatic orange/blue light, suggesting P. provasolii has acquired environmental adaptability to a wide range of light spectra and intensities.

Development of a time-series shotgun metagenomics database for monitoring microbial communities at the Pacific coast of Japan.

Although numerous metagenome, amplicon sequencing-based studies have been conducted to date to characterize marine microbial communities, relatively few have employed full metagenome shotgun sequencing to obtain a broader picture of the functional features of these marine microbial communities. Moreover, most of these studies only performed sporadic sampling, which is insufficient to understand an ecosystem comprehensively. In this study, we regularly conducted seawater sampling along the northeastern Pacific coast of Japan between March 2012 and May 2016. We collected 213 seawater samples and prepared size-based fractions to generate 454 subsets of samples for shotgun metagenome sequencing and analysis. We also determined the sequences of 16S rRNA (n = 111) and 18S rRNA (n = 47) gene amplicons from smaller sample subsets. We thereafter developed the Ocean Monitoring Database for time-series metagenomic data ( http://marine-meta.healthscience.sci.waseda.ac.jp/omd/ ), which provides a three-dimensional bird's-eye view of the data. This database includes results of digital DNA chip analysis, a novel method for estimating ocean characteristics such as water temperature from metagenomic data. Furthermore, we developed a novel classification method that includes more information about viruses than that acquired using BLAST. We further report the discovery of a large number of previously overlooked (TAG)n repeat sequences in the genomes of marine microbes. We predict that the availability of this time-series database will lead to major discoveries in marine microbiome research.

Dynamics of seagrass bed microbial communities in artificial Chattonella blooms: A laboratory microcosm study.

The influence of algicidal and growth-inhibiting bacteria in a seagrass (Zostera marina) bed, and their capability of controlling blooms of the fish-killing raphidophyte flagellate, Chattonella antiqua, were examined in laboratory microcosm experiments. Bacterial communities in seawater collected from the seagrass bed and Z. marina biofilm suppressed artificial Chattonella blooms in the presence of their natural competitors and predators. Phylogenetic analysis suggest that considerable numbers of bacteria that suppress Chattonella, including algicidal or growth-inhibiting bacteria isolated from seagrass biofilm and seawater from the seagrass bed, are members of Proteobacteria that can decompose lignocellulosic compounds. A direct comparison of partial 16S rRNA gene sequences (500 bp) revealed that the growth-limiting bacterium (strain ZM101) isolated from Z. marina biofilm belonged to the genus Phaeobacter (Alphaproteobacteria) showed 100% similarity with strains of growth-limiting bacteria isolated from seawater of both the seagrass bed and nearshore region, suggesting that the origin of these growth-limiting bacteria are the seagrass biofilm or seawater surrounding the seagrass bed. This study demonstrates that Chattonella growth-limiting bacteria living on seagrass biofilm and in the adjacent seawater can suppress Chattonella blooms, suggesting the possibility of Chattonella bloom prevention through restoration, protection, or introduction of seagrass in coastal areas.

A Preliminary Metagenome Analysis Based on a Combination of Protein Domains.

Metagenomic data have mainly been addressed by showing the composition of organisms based on a small part of a well-examined genomic sequence, such as ribosomal RNA genes and mitochondrial DNAs. On the contrary, whole metagenomic data obtained by the shotgun sequence method have not often been fully analyzed through a homology search because the genomic data in databases for living organisms on earth are insufficient. In order to complement the results obtained through homology-search-based methods with shotgun metagenomes data, we focused on the composition of protein domains deduced from the sequences of genomes and metagenomes, and we utilized them in characterizing genomes and metagenomes, respectively. First, we compared the relationships based on similarities in the protein domain composition with the relationships based on sequence similarities. We searched for protein domains of 325 bacterial species produced using the Pfam database. Next, the correlation coefficients of protein domain compositions between every pair of bacteria were examined. Every pairwise genetic distance was also calculated from 16S rRNA or DNA gyrase subunit B. We compared the results of these methods and found a moderate correlation between them. Essentially, the same results were obtained when we used partial random 100 bp DNA sequences of the bacterial genomes, which simulated raw sequence data obtained from short-read next-generation sequences. Then, we applied the method for analyzing the actual environmental data obtained by shotgun sequencing. We found that the transition of the microbial phase occurred because the seasonal change in water temperature was shown by the method. These results showed the usability of the method in characterizing metagenomic data based on protein domain compositions.

Spatial variation of bacterial community composition at the expiry of spring phytoplankton bloom in Sendai Bay, Japan.

In order to characterize how bacterial communities are propagated over spatial scales in a coastal area, the bacterial community composition was examined along with a transect line set in a bay at an expiry of spring phytoplankton bloom. Four distinctive bacterial communities were found within the bay by a fingerprinting method of 16S rRNA gene amplicons. The most widely distributed one was distributed in the surface and middle layers at whole area of the bay. The water was characterized by low inorganic nutrients concentration and high bacterial abundance, suggesting that the bacterial community had been developed in the bloom. Pyrosequencing analyses of the gene amplicons indicated that Rhodobacteriaceae and Flavobacteriaceae were abundant in the bacterial community, though the most abundant bacterial taxon was SAR11. The second group was distributed in the bottom water at the coastal side of the bay where considerably high Chl. a concentration was observed, probably because of the sedimentation of phytoplankton bloom. The community diversity was high and Alteromonadaceae, Saprospiraceae, and some families of Actinobacter existed more in this community than the others. The third group was distributed in the deep water near the border with the outside of the bay. The ratio of SAR11 was the highest in this community; besides, Burkholderianceae and Rhodospilliraceae existed in relatively high abundances. Another bacterial community having intermediate characters was observed in the middle to bottom layers around a central part of the bay where vertical water mixing was observed. These findings suggest that spatially different bacterial communities were formed under the influences of phytoplankton bloom and/or hydrographic events such as oceanic seawater intrusion of the bay.

Phylogenetic diversity of ammonia-oxidizing archaea and bacteria in biofilters of recirculating aquaculture systems.

We constructed ammonia monooxygenase alpha subunit (amoA) gene clone libraries of ammonia-oxidizing archaea (AOA) and bacteria (AOB) from three biofiltration tanks used for closed marine fish culture systems. The number of operational taxonomic units (OTUs) found in any one place was 76%-80% of the total OTUs in each tank for AOA and 100% for AOB when OUTs were defined on the basis of a 5% nucleotide difference. In a phylogenetic tree, all of the AOA amoA sequences fell into a cluster, which contained Candidatus Nitrosopumilus maritimus. All of the AOB amoA sequences were related to the Nitrosospira lineage. These results indicated that different ammonia oxidizers were present in different tanks, but that the dominant phylogenetic types were stable. In a biofiltration tank to which a high concentration of ammonium chloride was added periodically to condition the biofilter materials, most of the AOA amoA sequences were different from the dominant one observed in the fish culture tanks. The AOB amoA sequences were also different, and were similar to those of Nitrosomonas aestuarii. These findings suggest that high concentration ammonia loads have a considerable affect on ammonia-oxidizer community composition.

Comparison of sulphate-reducing bacterial communities in Japanese fish farm sediments with different levels of organic enrichment.

Fish farm sediments receive a large amount of organic matter from uneaten food and fecal material. This nutrient enrichment, or organic pollution, causes the accumulation of sulphide in the sediment from the action of sulphate-reducing bacteria (SRB). We investigated the effect of organic enrichment around coastal fish farms comparing the SRB community structure in these sediments. Sediment samples with different levels of organic pollution classified based upon the contents of acid-volatile sulphide and chemical oxygen demand were collected at three stations on the coast of western Japan. The SRB community composition was assessed using PCR amplification, cloning, sequencing and phylogenetic analysis of the dissimilatory sulphite reductase b-subunit gene (dsrB) fragments using directly extracted sediment DNA. Sequencing of the cloned PCR products of dsrB showed the existence of different SRB groups in the sediments. The majority of dsrB sequences were associated with the families Desulfobacteraceae and Desulfobulbaceae. Clones related to the phylum Firmicutes were also detected from all sediment samples. Statistical comparison of sequences revealed that community compositions of SRB from polluted sediments significantly differed from those of moderately polluted sediments and unpolluted sediments (LIBSHUFF, p<0.05), showing a different distribution of SRB in the fish farm sediments. There is evidence showing that the organic enrichment of sediments influences the composition of SRB communities in sediments at marine fish farms.

Distribution of ammonia-oxidizing archaea and bacteria in the surface sediments of Matsushima Bay in relation to environmental variables.

Ammonia oxidization is the first and a rate-limiting step of nitrification, which is often a critical process in nitrogen removal from estuarine and coastal environments. To clarify the correlation of environmental conditions with the distribution of ammonia oxidizers in organic matter-rich coastal sediments, ammonia-oxidizing archaea (AOA) and bacteria (AOB) ammonia monooxygenase alpha subunit gene (amoA) abundance was determined in sediments of Matsushima Bay located in northeast Japan. The AOA and AOB amoA copy numbers ranged from 1.1×10(6) to 1.7×10(7) and from 7.1×10(5) to 7.6×10(6) copies g(-1) sediment, respectively. AOA and AOB amoA abundance was negatively correlated with dissolved oxygen levels in the bottom water. AOA amoA abundance was also correlated with total phosphorus levels in the sediments. On the other hand, no significant relationship was observed between the amoA abundance and ammonium, organic matter (ignition loss), or acid volatile sulfide-sulfur levels in the sediments. These results show the heterogeneous distribution of ammonia oxidizers by the difference in environmental conditions within the bay. Moreover, AOA amoA diversity was relatively low in the area of high AOA amoA abundance, suggesting the variability of AOA community composition.

Seasonal and spatial variation of bacterial community structure in river-mouth areas of Gokasho bay, Japan.

This study investigated seasonal and spatial dynamics of the bacterial community in Gokasho bay with denaturing gradient gel electrophoresis (DGGE) profiles of PCR-amplified 16S rRNA gene fragments. The community structure was related to physico-chemical water conditions in the area examined. The bacterial community clustered into three groups: bacteria collected during January-May; those collected from water at the surface in July and September; and those collected from water at the bottom in July and September and from both depths in November. Canonical correspondence analyses indicated that the seasonal variability in bacterial community was associated with water temperature succession. On the other hand, concentrations of particulate organic matter and nitrite plus nitrate were related to the vertical change in community structure in summer and autumn as well as HNF abundance, suggesting that both top-down and bottom-up control affected the community. The influence of salinity was insignificant though bacterial production was related to salinity. No relationship was observed between the variation in community structure and that in hydrolytic enzyme activity. The results indicate that changes in bacterial activity are not coupled with variation in community structure.