Thiamine metabolism genes in diatoms are not regulated by thiamine despite the presence of predicted riboswitches

Thiamine pyrophosphate (TPP), an essential co-factor for all species, is biosynthesised through a metabolically expensive pathway regulated by TPP riboswitches in bacteria, fungi, plants and green algae. Diatoms are microalgae responsible for approximately 20% of global primary production. They have been predicted to contain TPP aptamers in the 3'UTR of some thiamine metabolism-related genes, but little is known about their function and regulation. We used bioinformatics, antimetabolite growth assays, RT-qPCR, targeted mutagenesis and reporter constructs to test whether the predicted TPP riboswitches respond to thiamine supplementation in diatoms. Gene editing was used to investigate the functions of the genes with associated TPP riboswitches in Phaeodactylum tricornutum. We found that thiamine-related genes with putative TPP aptamers are not responsive to thiamine or its precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), and the targeted mutation of the TPP aptamer in the HMP-P synthase (THIC) does not deregulate thiamine biosynthesis in P. tricornutum. Through genome editing we established that PtSSSP is necessary for thiamine uptake and that PtTHIC is essential for thiamine biosynthesis. Our results highlight the importance of experimentally testing bioinformatic aptamer predictions and provide new insights into the thiamine metabolism shaping the structure of marine microbial communities with global biogeochemical importance.

Bacterial Nanotubes as Intercellular Linkages in Marine Assemblages

Several types of bacterial appendages, e.g., pili and fimbriae, are known for their role in promoting interactions and aggregation with particles and bacteria in the ocean. First discovered in Bacillus subtilis and Escherichia coli, but novel to marine bacteria, bacterial nanotubes are hollow tubular structures connecting cell pairs that allow for the internal transport of cytoplasmic metabolites across the connecting structure. While the significance of nanotubes in exchange of cytoplasmic content has been established in non-marine bacteria, their occurrence and potential ecological significance in marine bacteria has not been reported. Using multiple high-resolution microscopy methods (atomic force microscopy, scanning, and transmission electron microscopy), we have determined that marine bacteria isolates and natural assemblages from nearshore upper ocean waters can express bacterial nanotubes. In marine isolates Pseudoalteromonas sp. TW7 and Alteromonas sp. ALTSIO, individual bacterial nanotubes measured 50–160 nm in width and extended 100–600 nm between connected cells. The spatial coupling of different cells via nanotubes can last for at least 90 min, extending the duration of interaction events between marine bacteria within natural assemblages. The nanomechanical properties of bacterial nanotubes vary in adhesion and dissipation properties, which has implication for structural and functional variability of these structures in their ability to stick to surfaces and respond to mechanical forces. Nanotube frequency is low among cells in enriched natural assemblages, where nanotubes form short, intimate connections, <200 nm, between certain neighboring cells. Bacterial nanotubes can form the structural basis for a bacterial ensemble and function as a conduit for cytoplasmic exchange (not explicitly studied here) between members for multicellular coordination and expression. The structural measurements and nanomechanical analyses in this study also extends knowledge about the physical properties of bacterial nanotubes and their consequences for marine microenvironments. The discovery of nanotube expression in marine bacteria has significant potential implications regarding intimate bacterial interactions in spatially correlated marine microbial communities.

An integrated metagenomic and metabolite profiling study of hydrocarbon biodegradation and corrosion in navy ships

Naval vessels regularly mix fuel and seawater as ballast, a practice that might exacerbate fuel biodegradation and metal biocorrosion. To investigate, a metagenomic characterization and metabolite profiling of ballast from U.S. Navy vessels with residence times of 1-, ~20-, and 31 weeks was conducted and compared with the seawater used to fill the tanks. Aerobic Gammaproteobacteria differentially proliferated in the youngest ballast tank and aerobic-specific hydrocarbon degradation genes were quantitatively more important compared to seawater or the other ballast tanks. In contrast, the anaerobic Deltaproteobacteria dominated in the eldest ballast fluid with anaerobic-specific hydrocarbon activation genes being far more prominent. Gene activity was corroborated by detection of diagnostic metabolites and corrosion was evident by elevated levels of Fe, Mn, Ni and Cu in all ballast samples relative to seawater. The findings argue that marine microbial communities rapidly shift from aerobic to anaerobic hydrocarbonoclastic-dominated assemblages that accelerate fuel and infrastructure deterioration.

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.

Diversity, prevalence, and expression of cyanase genes (cynS) in planktonic marine microorganisms

Cyanate is utilized by many microbes as an organic nitrogen source. The key enzyme for cyanate metabolism is cyanase, converting cyanate to ammonium and carbon dioxide. Although the cyanase gene cynS has been identified in many species, the diversity, prevalence, and expression of cynS in marine microbial communities remains poorly understood. Here, based on the full-length cDNA sequence of a dinoflagellate cynS and 260 homologs across the tree of life, we extend the conserved nature of cyanases by the identification of additional ultra-conserved residues as part of the modeled holoenzyme structure. Our phylogenetic analysis showed that horizontal gene transfer of cynS appears to be more prominent than previously reported for bacteria, archaea, chlorophytes, and metazoans. Quantitative analyses of marine planktonic metagenomes revealed that cynS is as prevalent as ureC (urease subunit alpha), suggesting that cyanate plays an important role in nitrogen metabolism of marine microbes. Highly abundant cynS transcripts from phytoplankton and nitrite-oxidizing bacteria identified in global ocean metatranscriptomes indicate that cyanases potentially occupy a key position in the marine nitrogen cycle by facilitating photosynthetic assimilation of organic N and its remineralisation to NO3 by the activity of nitrifying bacteria.

Extracellular ribosomal RNA provides a window into taxon-specific microbial lysis

Microbes are by far the dominant biomass in the world's oceans and drive biogeochemical cycles that are critical to life on Earth. The composition of marine microbial communities is highly dynamic spatially and temporally, with consequent effects on their functional roles. In part, these changes in composition result from viral lysis, which is taxon-specific and estimated to account for about half of marine microbial mortality. Here we determined taxon-specific cell lysis of prokaryotes in coastal seawater by sequencing extracellular and cellular ribosomal RNA (rRNA). We detected lysis in about 15% of the 16946 prokaryotic amplicon sequence variants (ASVs) identified, and lysis of up to 34% of the ASVs within a water sample. High lysis was most commonly associated with rare but typically highly productive bacteria, while relatively low lysis was more common in taxa that are often abundant, consistent with the proposed model of "kill the winner", and the idea that less abundant taxa generally experience higher relative lysis than dominant taxa. These results provide an explanation to the long-standing conundrum of why highly productive bacteria that are readily isolated from seawater are often in very low abundance.

Ecophysiology of the Cosmopolitan OM252 Bacterioplankton ( Gammaproteobacteria )

Marine microbial communities are teeming with understudied taxa due to the sheer numbers of species in any given sample of seawater. One group, the OM252 clade of Gammaproteobacteria , has been identified in gene surveys from myriad locations, and one isolated organism has even been genome sequenced (HIMB30).

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.

Diverse DNA modification in marine prokaryotic and viral communities

Chemical modifications of DNA, including methylation, play an important role in prokaryotes and viruses. However, our knowledge of the modification systems in environmental microbial communities, typically dominated by members not yet cultured, is limited. Here, we conducted 'metaepigenomic' analyses by single-molecule real-time sequencing of marine microbial communities. In total, 233 and 163 metagenomic assembly genomes (MAGs) were constructed from diverse prokaryotes and viruses, respectively, and 220 modified motifs and 276 DNA methyltransferases (MTases) were identified. Most of the MTases were not associated with the defense mechanism. The MTase-motif correspondence found in the MAGs revealed 10 novel pairs, and experimentally confirmed the catalytic specificities of the MTases. We revealed novel alternative motifs in the methylation system that are highly conserved in Alphaproteobacteria, illuminating the co-evolutionary history of the methylation system and host genome. Our findings highlight diverse unexplored DNA modifications that potentially affect the ecology and evolution of prokaryotes and viruses.

Marine Community Metabolomes Carry Fingerprints of Phytoplankton Community Composition

ABSTRACT Phytoplankton transform inorganic carbon into thousands of biomolecules that represent an important pool of fixed carbon, nitrogen, and sulfur in the surface ocean. Metabolite production differs between phytoplankton, and the flux of these molecules through the microbial food web depends on compound-specific bioavailability to members of a wider microbial community. Yet relatively little is known about the diversity or concentration of metabolites within marine plankton. Here, we compare 313 polar metabolites in 21 cultured phytoplankton species and in natural planktonic communities across environmental gradients to show that bulk community metabolomes reflect the chemical composition of the phytoplankton community. We also show that groups of compounds have similar patterns across space and taxonomy, suggesting that the concentrations of these compounds in the environment are controlled by similar sources and sinks. We quantify several compounds in the surface ocean that represent substantial understudied pools of labile carbon. For example, the N-containing metabolite homarine was up to 3% of particulate carbon and is produced in high concentrations by cultured Synechococcus, and S-containing gonyol accumulated up to 2.5 nM in surface particles and likely originates from dinoflagellates or haptophytes. Our results show that phytoplankton composition directly shapes the carbon composition of the surface ocean. Our findings suggest that in order to access these pools of bioavailable carbon, the wider microbial community must be adapted to phytoplankton community composition. IMPORTANCE Microscopic phytoplankton transform 100 million tons of inorganic carbon into thousands of different organic compounds each day. The structure of each chemical is critical to its biological and ecosystem function, yet the diversity of biomolecules produced by marine microbial communities remained mainly unexplored, especially small polar molecules which are often considered the currency of the microbial loop. Here, we explore the abundance and diversity of small biomolecules in planktonic communities across ecological gradients in the North Pacific and within 21 cultured phytoplankton species. Our work demonstrates that phytoplankton diversity is an important determinant of the chemical composition of the highly bioavailable pool of organic carbon in the ocean, and we highlight understudied yet abundant compounds in both the environment and cultured organisms. These findings add to understanding of how the chemical makeup of phytoplankton shapes marine microbial communities where the ability to sense and use biomolecules depends on the chemical structure.