On the Origin and Evolution of Microbial Mercury Methylation.

The origin of microbial mercury methylation has long been a mystery. Here, we employed genome-resolved phylogenetic analyses to decipher the evolution of the mercury-methylating gene, hgcAB, constrain the ancestral origin of the hgc operon, and explain the distribution of hgc in Bacteria and Archaea. We infer the extent to which vertical inheritance and horizontal gene transfer have influenced the evolution of mercury methylators and hypothesize that evolution of this trait bestowed the ability to produce an antimicrobial compound (MeHg+) on a potentially resource-limited early Earth. We speculate that, in response, the evolution of MeHg+-detoxifying alkylmercury lyase (encoded by merB) reduced a selective advantage for mercury methylators and resulted in widespread loss of hgc in Bacteria and Archaea.

A consensus protocol for the recovery of mercury methylation genes from metagenomes.

Mercury (Hg) methylation genes (hgcAB) mediate the formation of the toxic methylmercury and have been identified from diverse environments, including freshwater and marine ecosystems, Arctic permafrost, forest and paddy soils, coal-ash amended sediments, chlor-alkali plants discharges and geothermal springs. Here we present the first attempt at a standardized protocol for the detection, identification and quantification of hgc genes from metagenomes. Our Hg-cycling microorganisms in aquatic and terrestrial ecosystems (Hg-MATE) database, a catalogue of hgc genes, provides the most accurate information to date on the taxonomic identity and functional/metabolic attributes of microorganisms responsible for Hg methylation in the environment. Furthermore, we introduce "marky-coco", a ready-to-use bioinformatic pipeline based on de novo single-metagenome assembly, for easy and accurate characterization of hgc genes from environmental samples. We compared the recovery of hgc genes from environmental metagenomes using the marky-coco pipeline with an approach based on coassembly of multiple metagenomes. Our data show similar efficiency in both approaches for most environments except those with high diversity (i.e., paddy soils) for which a coassembly approach was preferred. Finally, we discuss the definition of true hgc genes and methods to normalize hgc gene counts from metagenomes.

DiTing: A Pipeline to Infer and Compare Biogeochemical Pathways From Metagenomic and Metatranscriptomic Data.

Metagenomics and metatranscriptomics are powerful methods to uncover key micro-organisms and processes driving biogeochemical cycling in natural ecosystems. Databases dedicated to depicting biogeochemical pathways (for example, metabolism of dimethylsulfoniopropionate (DMSP), which is an abundant organosulfur compound) from metagenomic/metatranscriptomic data are rarely seen. Additionally, a recognized normalization model to estimate the relative abundance and environmental importance of pathways from metagenomic and metatranscriptomic data has not been organized to date. These limitations impact the ability to accurately relate key microbial-driven biogeochemical processes to differences in environmental conditions. Thus, an easy-to-use, specialized tool that infers and visually compares the potential for biogeochemical processes, including DMSP cycling, is urgently required. To solve these issues, we developed DiTing, a tool wrapper to infer and compare biogeochemical pathways among a set of given metagenomic or metatranscriptomic reads in one step, based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) and a manually created DMSP cycling gene database. Accurate and specific formulae for over 100 pathways were developed to calculate their relative abundance. Output reports detail the relative abundance of biogeochemical pathways in both text and graphical format. DiTing was applied to simulated metagenomic data and resulted in consistent genetic features of simulated benchmark genomic data. Subsequently, when applied to natural metagenomic and metatranscriptomic data from hydrothermal vents and the Tara Ocean project, the functional profiles predicted by DiTing were correlated with environmental condition changes. DiTing can now be confidently applied to wider metagenomic and metatranscriptomic datasets, and it is available at https://github.com/xuechunxu/DiTing.

Development of a free radical scavenging bacterial consortium to mitigate oxidative stress in cnidarians.

Corals are colonized by symbiotic microorganisms that profoundly influence the animal's health. One noted symbiont is a single-celled alga (in the dinoflagellate family Symbiodiniaceae), which provides the coral with most of its fixed carbon. Thermal stress increases the production of reactive oxygen species (ROS) by Symbiodiniaceae during photosynthesis. ROS can both damage the algal symbiont's photosynthetic machinery and inhibit its repair, causing a positive feedback loop for the toxic accumulation of ROS. If not scavenged by the antioxidant network, excess ROS may trigger a signaling cascade ending with the coral host and algal symbiont disassociating in a process known as bleaching. We use Exaiptasia diaphana as a model for corals and constructed a consortium comprised of E. diaphana-associated bacteria capable of neutralizing ROS. We identified six strains with high free radical scavenging (FRS) ability belonging to the families Alteromonadaceae, Rhodobacteraceae, Flavobacteriaceae and Micrococcaceae. In parallel, we established a consortium of low FRS isolates consisting of genetically related strains. Bacterial whole genome sequences were used to identify key pathways that are known to influence ROS.

Mercury methylation by metabolically versatile and cosmopolitan marine bacteria.

Microbes transform aqueous mercury (Hg) into methylmercury (MeHg), a potent neurotoxin that accumulates in terrestrial and marine food webs, with potential impacts on human health. This process requires the gene pair hgcAB, which encodes for proteins that actuate Hg methylation, and has been well described for anoxic environments. However, recent studies report potential MeHg formation in suboxic seawater, although the microorganisms involved remain poorly understood. In this study, we conducted large-scale multi-omic analyses to search for putative microbial Hg methylators along defined redox gradients in Saanich Inlet, British Columbia, a model natural ecosystem with previously measured Hg and MeHg concentration profiles. Analysis of gene expression profiles along the redoxcline identified several putative Hg methylating microbial groups, including Calditrichaeota, SAR324 and Marinimicrobia, with the last the most active based on hgc transcription levels. Marinimicrobia hgc genes were identified from multiple publicly available marine metagenomes, consistent with a potential key role in marine Hg methylation. Computational homology modelling predicts that Marinimicrobia HgcAB proteins contain the highly conserved amino acid sites and folding structures required for functional Hg methylation. Furthermore, a number of terminal oxidases from aerobic respiratory chains were associated with several putative novel Hg methylators. Our findings thus reveal potential novel marine Hg-methylating microorganisms with a greater oxygen tolerance and broader habitat range than previously recognized.

Insights into the Vertical Stratification of Microbial Ecological Roles across the Deepest Seawater Column on Earth.

The Earth's oceans are a huge body of water with physicochemical properties and microbial community profiles that change with depth, which in turn influences their biogeochemical cycling potential. The differences between microbial communities and their functional potential in surface to hadopelagic water samples are only beginning to be explored. Here, we used metagenomics to investigate the microbial communities and their potential to drive biogeochemical cycling in seven different water layers down the vertical profile of the Challenger Deep (0-10,500 m) in the Mariana Trench, the deepest natural point in the Earth's oceans. We recovered 726 metagenome-assembled genomes (MAGs) affiliated to 27 phyla. Overall, biodiversity increased in line with increased depth. In addition, the genome size of MAGs at ≥4000 m layers was slightly larger compared to those at 0-2000 m. As expected, surface waters were the main source of primary production, predominantly from Cyanobacteria. Intriguingly, microbes conducting an unusual form of nitrogen metabolism were identified in the deepest waters (>10,000 m), as demonstrated by an enrichment of genes encoding proteins involved in dissimilatory nitrate to ammonia conversion (DNRA), nitrogen fixation and urea transport. These likely facilitate the survival of ammonia-oxidizing archaea α lineage, which are typically present in environments with a high ammonia concentration. In addition, the microbial potential for oxidative phosphorylation and the glyoxylate shunt was enhanced in >10,000 m waters. This study provides novel insights into how microbial communities and their genetic potential for biogeochemical cycling differs through the Challenger deep water column, and into the unique adaptive lifestyle of microbes in the Earth's deepest seawater.

Novel insights into the Thaumarchaeota in the deepest oceans: their metabolism and potential adaptation mechanisms.

BACKGROUND: Marine Group I (MGI) Thaumarchaeota, which play key roles in the global biogeochemical cycling of nitrogen and carbon (ammonia oxidizers), thrive in the aphotic deep sea with massive populations. Recent studies have revealed that MGI Thaumarchaeota were present in the deepest part of oceans-the hadal zone (depth > 6000 m, consisting almost entirely of trenches), with the predominant phylotype being distinct from that in the "shallower" deep sea. However, little is known about the metabolism and distribution of these ammonia oxidizers in the hadal water. RESULTS: In this study, metagenomic data were obtained from 0-10,500 m deep seawater samples from the Mariana Trench. The distribution patterns of Thaumarchaeota derived from metagenomics and 16S rRNA gene sequencing were in line with that reported in previous studies: abundance of Thaumarchaeota peaked in bathypelagic zone (depth 1000-4000 m) and the predominant clade shifted in the hadal zone. Several metagenome-assembled thaumarchaeotal genomes were recovered, including a near-complete one representing the dominant hadal phylotype of MGI. Using comparative genomics, we predict that unexpected genes involved in bioenergetics, including two distinct ATP synthase genes (predicted to be coupled with H+ and Na+ respectively), and genes horizontally transferred from other extremophiles, such as those encoding putative di-myo-inositol-phosphate (DIP) synthases, might significantly contribute to the success of this hadal clade under the extreme condition. We also found that hadal MGI have the genetic potential to import a far higher range of organic compounds than their shallower water counterparts. Despite this trait, hadal MDI ammonia oxidation and carbon fixation genes are highly transcribed providing evidence they are likely autotrophic, contributing to the primary production in the aphotic deep sea. CONCLUSIONS: Our study reveals potentially novel adaptation mechanisms of deep-sea thaumarchaeotal clades and suggests key functions of deep-sea Thaumarchaeota in carbon and nitrogen cycling. Video Abstract.

Ancestral niche separation and evolutionary rate differentiation between sister marine flavobacteria lineages.

Marine flavobacteria are specialists for polysaccharide degradation. They dominate in habitats enriched with polysaccharides, but are also prevalent in pelagic environments where polysaccharides are less available. These niches are likely occupied by distinct lineages, but evolutionary processes underlying their niche differentiation remain elusive. Here, genomic analyses and physiological assays indicate that the sister flavobacteria lineages Leeuwenhoekiella and Nonlabens likely explore polysaccharide-rich macroalgae and polysaccharide-poor pelagic niches respectively. Phylogenomic analyses inferred that the niche separation likely occurred anciently and coincided with increased sequence evolutionary rate in Nonlabens compared with Leeuwenhoekiella. Further analyses ruled out the known mechanisms likely driving evolutionary rate acceleration, including reduced selection efficiency, decreased generation time and increased mutation rate. In particular, the mutation rates were determined using an unbiased experimental method, which measures the present-day populations and may not reflect ancestral populations. These data collectively lead to a new hypothesis that an ancestral and transient mutation rate increase resulted in evolutionary rate increase in Nonlabens. This hypothesis was supported by inferring that gains and losses of genes involved in SOS response, a mechanism known to drive transiently increased mutation rate, coincided with evolutionary rate acceleration. Our analyses highlight the evolutionary mechanisms underlying niche differentiation of flavobacteria lineages.

Comparative genomic and metabolic analysis of manganese-oxidizing mechanisms in Celeribacter manganoxidans DY25T: Its adaptation to the environment of polymetallic nodules.

Manganese (Mn) nodule is one of the ubiquitous polymetallic concretions and mainly consists of Mn - Fe oxi-hydroxide precipitations. A primary oxidation of Mn(II) to MnO2, in which microorganisms may play important roles, is followed by agglomeration of MnO2 into nodules. Celeribater manganoxidans DY25T, belonging to family Rhodobacteraceae, has ability to catalyze the formation of MnO2 [1]. The concentration of MnO2 formed by harvested cells reached 7.08 µM after suspended in 10 mM HEPES (pH 7.5). Genomic and physiological characteristics of strain DY25T provided a better understanding of its Mn-oxidizing mechanism. Fifteen genes (including four multicopper oxidases) may be involved in Mn(II)-oxidation, whereas only three of them can promote this process. Sulfur-oxidizing activity was detected, which may be associated with manganese oxidation. Genes involved in import and export of primary elemental ingredients (C, N, P and S) and metallic elements (e.g. Mn) were discovered, demonstrating its potential roles in the biogeochemical cycle.

Proliferation of hydrocarbon-degrading microbes at the bottom of the Mariana Trench.

BACKGROUND: The Mariana Trench is the deepest known site in the Earth's oceans, reaching a depth of ~ 11,000 m at the Challenger Deep. Recent studies reveal that hadal waters harbor distinctive microbial planktonic communities. However, the genetic potential of microbial communities within the hadal zone is poorly understood. RESULTS: Here, implementing both culture-dependent and culture-independent methods, we perform extensive analysis of microbial populations and their genetic potential at different depths in the Mariana Trench. Unexpectedly, we observed an abrupt increase in the abundance of hydrocarbon-degrading bacteria at depths > 10,400 m in the Challenger Deep. Indeed, the proportion of hydrocarbon-degrading bacteria at > 10,400 m is the highest observed in any natural environment on Earth. These bacteria were mainly Oleibacter, Thalassolituus, and Alcanivorax genera, all of which include species known to consume aliphatic hydrocarbons. This community shift towards hydrocarbon degraders was accompanied by increased abundance and transcription of genes involved in alkane degradation. Correspondingly, three Alcanivorax species that were isolated from 10,400 m water supplemented with hexadecane were able to efficiently degrade n-alkanes under conditions simulating the deep sea, as did a reference Oleibacter strain cultured at atmospheric pressure. Abundant n-alkanes were observed in sinking particles at 2000, 4000, and 6000 m (averaged 23.5 µg/gdw) and hadal surface sediments at depths of 10,908, 10,909, and 10,911 m (averaged 2.3 µg/gdw). The δ2H values of n-C16/18 alkanes that dominated surface sediments at near 11,000-m depths ranged from - 79 to - 93‰, suggesting that these sedimentary alkanes may have been derived from an unknown heterotrophic source. CONCLUSIONS: These results reveal that hydrocarbon-degrading microorganisms are present in great abundance in the deepest seawater on Earth and shed a new light on potential biological processes in this extreme environment.

Spatiotemporal Dynamics of Free-Living and Particle-Associated Vibrio Communities in the Northern Chinese Marginal Seas.

Vibrio species are associated with human health and play important roles in the carbon cycle. The interest in the Vibrio ecology in marine pelagic environments has increased in recent years, and the correlations between the Vibrio community structure and various environmental factors have been demonstrated. However, the identification of planktonic Vibrio species and their relationship with particulate matter are unclear. Here, we elucidated the spatiotemporal dynamics of Vibrio diversity and in relation to environmental factors in the northern Chinese marginal seas, which feature complex and ever-changing environmental conditions. Vibrio abundance derived from quantitative PCR analysis was higher in summer (∼1.4 × 106 copies liter-1) than in winter (∼1.9 × 105 copies liter-1). Interestingly, the average amount of free-living (on a 0.22-µm-pore-size filter membrane) Vibrio was higher (∼3.89 times) than that of particle-associated Vibrio (on a 3-µm-pore-size filter membrane), making it likely that the preferential lifestyle of the planktonic Vibrio community was free living. Shifts in Vibrio community composition identified by high-throughput amplicon sequencing of the Vibrio-specific 16S rRNA gene were observed at both spatial and temporal scales, which were mainly driven by temperature, dissolved oxygen, ammonium, salinity, nitrite, and phosphate. The most prominent operational taxonomic units in summer were closely related to Vibrio campbellii and Vibrio caribbeanicus and shifted to those affiliated with Vibrio atlanticus in winter. Our study demonstrated abundant and diverse Vibrio populations in the northern Chinese marginal seas, contributing to a better understanding of their potential ecological roles in these ecosystems.IMPORTANCE The dynamics of Vibrio communities have been shown in many marine habitats that are close to land, including estuary or harbor areas. Here, we investigated the spatiotemporal dynamics of Vibrio populations in the northern Chinese marginal seas, spanning a wide spatial scale. We showed that the abundances of the Vibrio population in the present study were higher than those in most previously studied areas and that Vibrio species are more likely to adopt a free-living lifestyle. Moreover, our results expanded upon previous results by showing a clear shift in the dominant Vibrio species from summer to winter, which was mainly attributable to the reduction in the abundance of dominant species in summer. Overall, this work contributes to the understanding of the ecology of the Vibrio communities in the marginal seas.

Role of RpoN from Labrenzia aggregata LZB033 (Rhodobacteraceae) in Formation of Flagella and Biofilms, Motility, and Environmental Adaptation.

Labrenzia aggregata LZB033 (Rhodobacteraceae), which produces dimethylsulfoniopropionate (DMSP) and reduces nitrate to nitrogen, was isolated from seawater of the East China Sea. Its genome encodes a large number of transcriptional regulators which may be important for its adaptation to diverse marine environments. The alternative σ54 factor (RpoN) is a central regulator of many bacteria, regulating the transcription of multiple genes and controlling important cellular functions. However, the exact role of RpoN in Labrenzia spp. is unknown. In this study, an in-frame rpoN deletion mutant was constructed in LZB033, and the function of RpoN was determined. To systematically identify RpoN-controlled genes, we performed a detailed analysis of gene expression differences between the wild-type strain and the ΔrpoN mutant using RNA sequencing. The expression of 175 genes was shown to be controlled by RpoN. Subsequent phenotypic assays showed that the ΔrpoN mutant was attenuated in flagellar biosynthesis and swimming motility, utilized up to 13 carbon substrates differently, lacked the ability to assimilate malic acid, and displayed markedly decreased biofilm formation. In addition, stress response assays showed that the ΔrpoN mutant was impaired in the ability to survive under different challenge conditions, including osmotic stress, oxidative stress, temperature changes, and acid stress. Moreover, both the DMSP synthesis and catabolism rates of LZB033 decreased after rpoN was knocked out. Our work provides essential insight into the regulatory function of RpoN, revealing that RpoN is a key determinant for LZB033 flagellar formation, motility, biofilm formation, and environmental fitness, as well as DMSP production and degradation.IMPORTANCE This study established an in-frame gene deletion method in the alphaproteobacterium Labrenzia aggregata LZB033 and generated an rpoN gene mutant. A comparison of the transcriptomes and phenotypic characteristics between the mutant and wild-type strains confirmed the role of RpoN in L. aggregata LZB033 flagellar formation, motility, biofilm formation, and carbon usage. Most importantly, RpoN is a key factor for survival under different environmental challenge conditions. Furthermore, the ability to synthesize and metabolize dimethylsulfoniopropionate (DMSP) was related to RpoN. These features revealed RpoN to be an important regulator of stress resistance and survival for L. aggregata LZB033 in marine environments.

Comparative genomic analysis reveals the evolution and environmental adaptation strategies of vibrios.

BACKGROUND: Vibrios are among the most diverse and ecologically important marine bacteria, which have evolved many characteristics and lifestyles to occupy various niches. The relationship between genome features and environmental adaptation strategies is an essential part for understanding the ecological functions of vibrios in the marine system. The advent of complete genome sequencing technology has provided an important method of examining the genetic characteristics of vibrios on the genomic level. RESULTS: Two Vibrio genomes were sequenced and found to occupy many unique orthologues families which absent from the previously genes pool of the complete genomes of vibrios. Comparative genomics analysis found vibrios encompass a steady core-genome and tremendous pan-genome with substantial gene gain and horizontal gene transfer events in the evolutionary history. Evolutionary analysis based on the core-genome tree suggested that V. fischeri emerged ~ 385 million years ago, along with the occurrence of cephalopods and the flourish of fish. The relatively large genomes, the high number of 16S rRNA gene copies, and the presence of R-M systems and CRISPR system help vibrios live in various marine environments. Chitin-degrading related genes are carried in nearly all the Vibrio genomes. The number of chitinase genes in vibrios has been extremely expanded compared to which in the most recent ancestor of the genus. The chitinase A genes were estimated to have evolved along with the genus, and have undergone significant purifying selective force to conserve the ancestral state. CONCLUSIONS: Vibrios have experienced extremely genome expansion events during their evolutionary history, allowing them to develop various functions to spread globally. Despite their close phylogenetic relationships, vibrios were found to have a tremendous pan-genome with a steady core-genome, which indicates the highly plastic genome of the genus. Additionally, the existence of various chitin-degrading related genes and the expansion of chitinase A in the genus demonstrate the importance of the chitin utilization for vibrios. Defensive systems in the Vibrio genomes may protect them from the invasion of external DNA. These genomic features investigated here provide a better knowledge of how the evolutionary process has forged Vibrio genomes to occupy various niches.

Novel Insights Into Bacterial Dimethylsulfoniopropionate Catabolism in the East China Sea.

The compatible solute dimethylsulfoniopropionate (DMSP), made by many marine organisms, is one of Earth's most abundant organosulfur molecules. Many marine bacteria import DMSP and can degrade it as a source of carbon and/or sulfur via DMSP cleavage or DMSP demethylation pathways, which can generate the climate active gases dimethyl sulfide (DMS) or methanthiol (MeSH), respectively. Here we used culture-dependent and -independent methods to study bacteria catabolizing DMSP in the East China Sea (ECS). Of bacterial isolates, 42.11% showed DMSP-dependent DMS (Ddd+) activity, and 12.28% produced detectable levels of MeSH. Interestingly, although most Ddd+ isolates were Alphaproteobacteria (mainly Roseobacters), many gram-positive Actinobacteria were also shown to cleave DMSP producing DMS. The mechanism by which these Actinobacteria cleave DMSP is unknown, since no known functional ddd genes have been identified in genome sequences of Ddd+ Microbacterium and Agrococcus isolates or in any other sequenced Actinobacteria genomes. Gene probes to the DMSP demethylation gene dmdA and the DMSP lyase gene dddP demonstrated that these DMSP-degrading genes are abundant and widely distributed in ECS seawaters. dmdA was present in relatively high proportions in both surface (19.53% ± 6.70%) and bottom seawater bacteria (16.00% ± 8.73%). In contrast, dddP abundance positively correlated with chlorophyll a, and gradually decreased with the distance from land, which implies that the bacterial DMSP lyase gene dddP might be from bacterial groups that closely associate with phytoplankton. Bacterial community analysis showed positive correlations between Rhodobacteraceae abundance and concentrations of DMS and DMSP, further confirming the link between this abundant bacterial class and the environmental DMSP cycling.

Genomic insight into Aquimarina longa SW024 T: its ultra-oligotrophic adapting mechanisms and biogeochemical functions.

BACKGROUND: South Pacific Gyre (SPG) is the largest and clearest gyre in the world, where the concentration of surface chlorophyll a and primary production are extremely low. Aquimarina longa SW024(T) was isolated from surface water of the SPG center. To understand how this bacterium could survive in this ultra-oligotrophic oceanic environment and its function in biogeochemical cycle, we sequenced the genome of A. longa SW024(T) and performed extensive genomic analyses. METHODS: Genomic DNA was extracted and sequenced using Illumina Hiseq 2000 and Miseq platform. Genome annotation, genomic comparison and phylogenetic analyses were performed with the use of multiple bioinformatics tools like: BLAST+ 2.2.24, Glimmer3.0, RAST server, Geneious 4.8.5, ClustalW2 and MEGA5. Physiological and morphological features were tested by bacterial culture, electron microscopy, fluorescence microscopy and exopolysaccharides extraction. RESULTS: Analysis of seven Aquimarina genomes and 30 other genomes of Flavobacteriaceae isolated from seawater showed that most of the strains had low DNA G + C contents, and Aquimarina had larger genomes than other strains. Genome comparison showed varying genomic properties among seven Aquimarina genomes, including genome sizes and gene contents, which may warrant their specific adaptive strategies. Genome of A. longa SW024(T) was further compared with the genomes of two other Aquimarina species which were also isolated from the SPG and A. longa SW024(T) appeared to have much more genes related to replication, recombination and repair. As a copiotroph, A. longa SW024(T) is long in length, and possesses large genome size and diverse transporters. However, it has also evolved many properties to survive in the oligotrophic marine environment. This bacterium grew better on solid medium than in liquid medium, suggesting it may be liable to attach to particle surfaces in order to survive in the nutrient-limiting environment. Gliding motility and the capacity to degrade various polymers possibly allow the bacterium to grow on detritus particles and use polymeric substances as carbon and energy sources. Moreover, genes related to carbon, nitrogen, and sulfur metabolisms were identified, which showed that A. longa SW024(T) might be involved in various elemental cycles. CONCLUSIONS: Genomic comparison of Aquimarina genus exhibits comprehensive capabilities of the strains to adapt to diverse marine environments. The genomic characteristics of A. longa SW024(T) reveal that it evolves various strategies to cope with both copiotrophic and ultra-oligotrophic marine environment, which provides a better understanding of the survival abilities of bacteria in prevalent and even extreme oceanic environments. Furthermore, carbon, nitrogen and sulfur utilization of A. longa SW024(T) may represent its potential functions in the global biogeochemical cycle.