Structural and Mechanistic Insights Into Dimethylsulfoxide Formation Through Dimethylsulfide Oxidation

Dimethylsulfide (DMS) and dimethylsulfoxide (DMSO) are widespread in marine environment, and are important participants in the global sulfur cycle. Microbiol oxidation of DMS to DMSO represents a major sink of DMS in marine surface waters. The SAR11 clade and the marine Roseobacter clade (MRC) are the most abundant heterotrophic bacteria in the ocean surface seawater. It has been reported that trimethylamine monooxygenase (Tmm, EC 1.14.13.148) from both MRC and SAR11 bacteria likely oxidizes DMS to generate DMSO. However, the structural basis of DMS oxidation has not been explained. Here, we characterized a Tmm homolog from the SAR11 bacterium Pelagibacter sp. HTCC7211 (Tmm7211). Tmm7211 exhibits DMS oxidation activity in vitro. We further solved the crystal structures of Tmm7211 and Tmm7211 soaked with DMS, and proposed the catalytic mechanism of Tmm7211, which comprises a reductive half-reaction and an oxidative half-reaction. FAD and NADPH molecules are essential for the catalysis of Tmm7211. In the reductive half-reaction, FAD is reduced by NADPH. In the oxidative half-reaction, the reduced FAD reacts with O2 to form the C4a-(hydro)peroxyflavin. The binding of DMS may repel the nicotinamide ring of NADP+, and make NADP+ generate a conformational change, shutting off the substrate entrance and exposing the active C4a-(hydro)peroxyflavin to DMS to complete the oxidation of DMS. The proposed catalytic mechanism of Tmm7211 may be widely adopted by MRC and SAR11 bacteria. This study provides important insight into the conversion of DMS into DMSO in marine bacteria, leading to a better understanding of the global sulfur cycle.

Genomic evidence for inter-class host transition between abundant streamlined heterotrophs by a novel and ubiquitous marine Methylophage

The methylotrophic OM43 clade are Gammaproteobacteria that comprise some of the smallest free-living cells known with highly streamlined genomes. OM43 represents an important microbial link between marine primary production and return of carbon back to the atmosphere. Bacteriophages shape microbial communities and are major drivers of mortality and global marine biogeochemistry. Recent cultivation efforts have brought the first viruses infecting members of the OM43 clade into culture. Here we characterize a novel myophage infecting OM43, called Melnitz. Melnitz was isolated independently on three separate occasions (with isolates sharing >99.95% average nucleotide identity) from water samples from a subtropical ocean gyre (Sargasso Sea) and temperate coastal (Western English Channel) systems. Metagenomic recruitment from global ocean viromes confirmed that Melnitz is globally ubiquitous, congruent with patterns of host abundance. Bacteria with streamlined genomes such as OM43 and the globally dominant SAR11 clade use riboswitches as an efficient method to regulate metabolism. Melnitz encodes a two-piece tmRNA (ssrA), controlled by a glutamine riboswitch, providing evidence that riboswitch use also occurs for regulation during phage infection of streamlined heterotrophs. Virally encoded tRNAs and ssrA found in Melnitz were phylogenetically more closely related to those found within the alphaproteobacterial SAR11 clade and their associated myophages than those within their gammaproteobacterial hosts. This suggests the possibility of an ancestral inter-class host transition event between SAR11 and OM43. Melnitz and a related myophage that infects SAR11 were unable to infect hosts of the SAR11 and OM43, respectively, suggesting host transition rather than a broadening of host range.

Influence of Estuarine Water on the Microbial Community Structure of Patagonian Fjords

Fjords are sensitive areas affected by climate change and can act as a natural laboratory to study microbial ecological processes. The Chilean Patagonian fjords (41–56°S), belonging to the Subantarctic ecosystem (46–60°S), make up one of the world’s largest fjord systems. In this region, Estuarine Water (EW) strongly influences oceanographic conditions, generating sharp gradients of oxygen, salinity and nutrients, the effects of which on the microbial community structure are poorly understood. During the spring of 2017 we studied the ecological patterns (dispersal and oceanographic factors) underlying the microbial community distribution in a linear span of 450 km along the estuarine-influenced Chilean Patagonian fjords. Our results show that widespread microbial dispersion existed along the fjords where bacterioplankton exhibited dependence on the eukaryotic phytoplankton community composition. This dependence was particularly observed under the low chlorophyll-a conditions of the Baker Channel area, in which a significant relationship was revealed between SAR11 Clade III and the eukaryotic families Pyrenomonadaceae (Cryptophyte) and Coccomyxaceae (Chlorophyta). Furthermore, dissolved oxygen and salinity were revealed as the main drivers influencing the surface marine microbial communities in these fjords. A strong salinity gradient resulted in the segregation of the Baker Channel prokaryotic communities from the rest of the Patagonian fjords. Likewise, Microbacteriaceae, Burkholderiaceae and SAR11 Clade III, commonly found in freshwater, were strongly associated with EW conditions in these fjords. The direct effect of EW on the microbial community structure and diversity of the fjords exemplifies the significance that climate change and, in particular, deglaciation have on this marine region and its productivity.

Characterization of a glycolipid glycosyltransferase with broad substrate specificity from the marine bacterium Candidatus Pelagibacter sp. HTCC7211

In the marine environment, phosphorus availability significantly affects the lipid composition in many cosmopolitan marine heterotrophic bacteria, including members of the SAR11 clade and the Roseobacter clade. Under phosphorus stress conditions, non-phosphorus sugar-containing glycoglycerolipids are substitutes for phospholipids in these bacteria. Although these glycoglycerolipids play an important role as surrogates for phospholipids under phosphate deprivation, glycoglycerolipid synthases in marine microbes are poorly studied. In the present study, we biochemically characterized a glycolipid glycosyltransferase (GTcp) from the marine bacterium Candidatus Pelagibacter sp. HTCC7211, a member of the SAR11 clade. Our results showed that GTcp is able to act as a multifunctional enzyme by synthesizing different glycoglycerolipids with UDP-glucose, UDP-galactose, or UDP-glucuronic acid as sugar donors and diacylglycerol as the acceptor. Analyses of enzyme kinetic parameters demonstrated that Mg2+ notably changes the enzyme’s affinity for UDP-glucose, which improves its catalytic efficiency. Homology modelling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GTcp with its GT-B fold. A phylogenetic analysis showed that GTcp and its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodelling, but also describe an efficient enzymatic tool for future synthesis of bioactive molecules. Importance The bilayer formed by membrane lipids serves as the containment unit for living microbial cells. In the marine environment, it has been firmly established that phytoplankton and heterotrophic bacteria can substitute phospholipids with non-phosphorus sugar-containing glycoglycerolipids in response to phosphorus limitation. However, little is known about how these glycoglycerolipids are synthesized. Here, we determined the biochemical characteristics of a glycolipid glycosyltransferase (GTcp) from the marine bacterium Candidatus Pelagibacter sp. HTCC7211. GTcp and its homologs form a group in the GT4 glycosyltransferase family, and can synthesize neutral glycolipids (MGlc-DAG and MGal-DAG) and an acidic glycolipid (MGlcA-DAG). We also uncover the key residues for DAG-binding through molecular docking, site-direct mutagenesis and subsequent enzyme activity assays. Our data provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodelling.

Characterization of a glycolipid glycosyltransferase with broad substrate specificity from the marine bacterium Candidatus Pelagibacter sp. HTCC7211

In the marine environment, phosphorus availability significantly affects the lipid composition in many cosmopolitan marine heterotrophic bacteria, including members of the SAR11 clade and the Roseobacter clade. Under phosphorus stress conditions, non-phosphorus sugar-containing glycoglycerolipids are substitutes for phospholipids in these bacteria. Although these glycoglycerolipids play an important role as surrogates for phospholipids under phosphate deprivation, glycoglycerolipid synthases in marine microbes are poorly studied. In the present study, we biochemically characterized a glycolipid glycosyltransferase (GTcp) from the marine bacterium Candidatus Pelagibacter sp. HTCC7211, a member of the SAR11 clade. Our results showed that GTcp is able to act as a multifunctional enzyme by synthesizing different glycoglycerolipids with UDP-glucose, UDP-galactose, or UDP-glucuronic acid as sugar donors and diacylglycerol as the acceptor. Analyses of enzyme kinetic parameters demonstrated that Mg2+ notably changes the enzyme's affinity for UDP-glucose, which improves its catalytic efficiency. Homology modelling and mutational analyses revealed binding sites for the sugar donor and the diacylglycerol lipid acceptor, which provided insights into the retaining mechanism of GTcp with its GT-B fold. A phylogenetic analysis showed that GTcp and its homologs form a group in the GT4 glycosyltransferase family. These results not only provide new insights into the glycoglycerolipid synthesis mechanism in lipid remodelling, but also describe an efficient enzymatic tool for future synthesis of bioactive molecules.

Diversity of Bacterioplankton and Bacteriobenthos from the Veracruz Reef System, Southwestern Gulf of Mexico

Bacterial diversity was explored among field samples and cultured isolates from coral reefs within the Veracruz Reef System. Bacterioplankton and bacteriobenthos were characterized by pyrosequencing 16S rRNA genes. Identified sequences belonged to the kingdom Bacteria and classified into 33 phyla. Proteobacteria (likely SAR11 clade) dominated in collective field samples, whereas Firmicutes were the most abundant taxa among cultured isolates. Bioinformatic sorting of sequences to family level revealed 223 bacterial families. Pseudomonadaceae, Exiguobacteraceae and Bacillaceae were dominant among cultured isolates. Vibrionaceae, Alteromonadaceae, and Flavobacteriaceae dominated in reef-associated sediments, whereas Rickettsiaceae and Synechoccaceae were more highly represented in the water column. Bacterial communities from sediments were more diverse than from the water column. This study reveals cryptic bacterial diversity among microenvironmental components of marine microbial reef communities subject to differential influence of anthropogenic stressors. Such investigations are critical for constructing scenarios of environmentally induced shifts in bacterial biodiversity and species composition.

Metagenome Mining Reveals Hidden Genomic Diversity of Pelagimyophages in Aquatic Environments

ABSTRACT The SAR11 clade is one of the most abundant bacterioplankton groups in surface waters of most of the oceans and lakes. However, only 15 SAR11 phages have been isolated thus far, and only one of them belongs to the Myoviridae family (pelagimyophages). Here, we have analyzed 26 sequences of myophages that putatively infect the SAR11 clade. They have been retrieved by mining ca. 45 Gbp aquatic assembled cellular metagenomes and viromes. Most of the myophages were obtained from the cellular fraction (0.2 μm), indicating a bias against this type of virus in viromes. We have found the first myophages that putatively infect Candidatus Fonsibacter (freshwater SAR11) and another group putatively infecting bathypelagic SAR11 phylogroup Ic. The genomes have similar sizes and maintain overall synteny in spite of low average nucleotide identity values, revealing high similarity to marine cyanomyophages. Pelagimyophages recruited metagenomic reads widely from several locations but always much more from cellular metagenomes than from viromes, opposite to what happens with pelagipodophages. Comparing the genomes resulted in the identification of a hypervariable island that is related to host recognition. Interestingly, some genes in these islands could be related to host cell wall synthesis and coinfection avoidance. A cluster of curli-related proteins was widespread among the genomes, although its function is unclear. IMPORTANCE SAR11 clade members are among the most abundant bacteria on Earth. Their study is complicated by their great diversity and difficulties in being grown and manipulated in the laboratory. On the other hand, and due to their extraordinary abundance, metagenomic data sets provide enormous richness of information about these microbes. Given the major role played by phages in the lifestyle and evolution of prokaryotic cells, the contribution of several new bacteriophage genomes preying on this clade opens windows into the infection strategies and life cycle of its viruses. Such strategies could provide models of attack of large-genome phages preying on streamlined aquatic microbes.

High contribution ofPelagibacteralesto bacterial community composition and activity in spring blooms off Kerguelen Island (Southern Ocean)

The ecology of Pelagibacterales (SAR11 clade), the most abundant bacterial group in the ocean, has been intensively studied in temperate and tropical ocean regions, but the distribution patterns of this clade remains largely unexplored in the Southern Ocean. Through amplicon sequencing of the 16S rRNA genes, we assessed the contribution of Pelagibacterales to bacterial community composition in the naturally iron fertilized region off Kerguelen Island (Southern Ocean). We investigated the upper 300 m water column at seven sites located in early spring phytoplankton blooms and at one site in HNLC waters. Despite pronounced vertical patterns of the bacterioplankton assemblages, the SAR11 clade had high relative abundances at all depths and sites, averaging 40% (±15%) of the total community relative abundance. Micro-autoradiography combined with CARD-FISH further revealed that the SAR11 clade contributed substantially (45-60% in surface waters) to bacterial biomass production (as determined by3H leucine incorporation). A clear niche partitioning of the further resolved SAR11 subclades was observed with depth layers, but differences among sites were detectable for only a few subclades. Our study provides novel observations of the distribution and contribution to the marine carbon cycle of the SAR11 clade in the cold waters of the Southern Ocean.

Fine stratification of microbial communities through a metagenomic profile of the photic zone

ABSTRACTMost marine metagenomic studies of the marine photic zone analyze only samples taken at one or two depths. However, when the water column is stratified, physicochemical parameters change dramatically over relatively short depth intervals. We sampled the photic water column every 15m depth at a single point of an off-shore Mediterranean site during a period of strong stratification (early autumn) to evaluate the effects of small depth increases on the microbiome. Using genomic assembly and metagenomic read recruitment, we found major shifts in the community structure over small variations of depth, with most microbes showing a distribution limited to layers approximately 30 meters thick (stenobathic). Only some representatives of the SAR11 clade and the Sphingomonadaceae appeared to be eurybathic, spanning a greater range of depths. These results were confirmed by studying a single gene (rhodopsin) for which we also found narrow depth distributions. Our results highlight the importance of considering vertical distribution as a major element when analyzing the presence of marine clades and species or comparing the microbiome present at different locations.