A Glycolipid Glycosyltransferase with Broad Substrate Specificity from the Marine Bacterium "Candidatus Pelagibacter sp." Strain 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, nonphosphorus 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." strain 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 (DAG) 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 modeling 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 remodeling but also describe an efficient enzymatic tool for the 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 replace phospholipids with nonphosphorus 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." strain HTCC7211. GTcp and its homologs form a group in the GT4 glycosyltransferase family and can synthesize neutral glycolipids (monoglucosyl-1,2-diacyl-sn-glycerol [MGlc-DAG] and monogalactosyl [MGal]-DAG) and monoglucuronic acid diacylglycerol (MGlcA-DAG). We also uncovered 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 remodeling.

Survey of Bacterial Phylogenetic Diversity During the Glacier Melting Season in an Arctic Fjord.

To understand bacterial biogeography in response to the hydrographic impact of climate change derived from the Arctic glacier melting, we surveyed bacterial diversity and community composition using bacterial 16S rRNA gene metabarcoding in the seawaters of Kongsfjorden, Svalbard, during summer 2016. In the present study, bacterial biogeography in the Kongsfjorden seawaters showed distinct habitat patterns according to water mass classification and habitat transition between Atlantic and fjord surface waters. Moreover, we estimated phylogenetic diversity of bacterial communities using the net relatedness, nearest taxon, and beta nearest taxon indices. We found the influence of freshwater input from glacier melting in shaping bacterial assemblage composition through the stochastic model. We further evaluated bacterial contributions to phytoplankton-derived dimethylsulfoniopropionate (DMSP) using a quantitative PCR (qPCR) measurement with demethylation (dmdA) and cleavage (dddP) genes of two fundamentally different processes. Our qPCR results imply that bacterial DMSP degradation follows the Atlantic inflow during summer in Kongsfjorden. These findings suggest that the Atlantic inflow and glacial melting influence bacterial community composition and assembly processes and thus affect the degradation of phytoplankton-derived organic matter in an Arctic fjord.

Structure-Function Analysis Indicates that an Active-Site Water Molecule Participates in Dimethylsulfoniopropionate Cleavage by DddK.

The osmolyte dimethylsulfoniopropionate (DMSP) is produced in petagram quantities in marine environments and has important roles in global sulfur and carbon cycling. Many marine microorganisms catabolize DMSP via DMSP lyases, generating the climate-active gas dimethyl sulfide (DMS). DMS oxidation products participate in forming cloud condensation nuclei and, thus, may influence weather and climate. SAR11 bacteria are the most abundant marine heterotrophic bacteria; many of them contain the DMSP lyase DddK, and their dddK transcripts are relatively abundant in seawater. In a recently described catalytic mechanism for DddK, Tyr64 is predicted to act as the catalytic base initiating the ß-elimination reaction of DMSP. Tyr64 was proposed to be deprotonated by coordination to the metal cofactor or its neighboring His96. To further probe this mechanism, we purified and characterized the DddK protein from Pelagibacter ubique strain HTCC1062 and determined the crystal structures of wild-type DddK and its Y64A and Y122A mutants (bearing a change of Y to A at position 64 or 122, respectively), where the Y122A mutant is complexed with DMSP. The structural and mutational analyses largely support the catalytic role of Tyr64, but not the method of its deprotonation. Our data indicate that an active water molecule in the active site of DddK plays an important role in the deprotonation of Tyr64 and that this is far more likely than coordination to the metal or His96. Sequence alignment and phylogenetic analysis suggest that the proposed catalytic mechanism of DddK has universal significance. Our results provide new mechanistic insights into DddK and enrich our understanding of DMS generation by SAR11 bacteria.IMPORTANCE The climate-active gas dimethyl sulfide (DMS) plays an important role in global sulfur cycling and atmospheric chemistry. DMS is mainly produced through the bacterial cleavage of marine dimethylsulfoniopropionate (DMSP). When released into the atmosphere from the oceans, DMS can be photochemically oxidized into DMSO or sulfate aerosols, which form cloud condensation nuclei that influence the reflectivity of clouds and, thereby, global temperature. SAR11 bacteria are the most abundant marine heterotrophic bacteria, and many of them contain DMSP lyase DddK to cleave DMSP, generating DMS. In this study, based on structural analyses and mutational assays, we revealed the catalytic mechanism of DddK, which has universal significance in SAR11 bacteria. This study provides new insights into the catalytic mechanism of DddK, leading to a better understanding of how SAR11 bacteria generate DMS.

H-Aquil: a chemically defined cell culture medium for trace metal studies in Vibrios and other marine heterotrophic bacteria.

A variety of trace metals, including prominently iron (Fe) are necessary for marine microorganisms. Chemically defined medium recipes have been used for several decades to study phytoplankton, but similar methods have not been adopted as widely in studies of marine heterotrophic bacteria. Medium recipes for these organisms frequently include tryptone, casamino acids, as well as yeast and animal extracts. These components introduce unknown concentrations of trace elements and organic compounds, complicating metal speciation. Minimal medium recipes utilizing known carbon and nitrogen sources do exist but often have high background trace metal concentrations. Here we present H-Aquil, a version of the phytoplankton medium Aquil adapted for marine heterotrophic bacteria. This medium consists of artificial seawater supplemented with a carbon source, phosphate, amino acids, and vitamins. As in Aquil, trace metals are controlled using the synthetic chelator EDTA. We also address concerns of EDTA toxicity, showing that concentrations up to 100 µM EDTA do not lead to growth defects in the copiotrophic bacterium Vibrio harveyi or the oligotrophic bacterium Candidatus Pelagibacter ubique HTCC1062, a member of the SAR11 clade. H-Aquil is used successfully to culture species of Vibrio, Phaeobacter, and Silicibacter, as well as several environmental isolates. We report a substantial decrease in growth rate between cultures grown with or without added Fe, making the medium suitable for conducting Fe-limitation studies in a variety of marine heterotrophic bacteria.

Manganese Is Essential for PlcP Metallophosphoesterase Activity Involved in Lipid Remodeling in Abundant Marine Heterotrophic Bacteria.

In vast areas of the ocean, microbes must adapt to the availability of scarce nutrients, and a key strategy for reducing the cellular phosphorus (P) quota is to remodel membranes by replacing phospholipids with non-P surrogate lipids. A metallophosphoesterase, PlcP, is essential for lipid remodeling in cosmopolitan marine bacteria of the Roseobacter (e.g., Phaeobacter sp. strain MED193) and SAR11 (e.g., Pelagibacter sp. strain HTCC7211) clades, and transcription of plcP is known to be induced by P limitation. In order to better understand PlcP-mediated lipid remodeling, we sought to characterize PlcP for its metal ion requirement and to determine its selectivity for native bacterial phospholipids. Here, we report the occurrence of a highly conserved binuclear ion center in PlcPs from MED193 and HTCC7211 and show that manganese is the preferred metal for metallophosphoesterase activity. PlcP displayed high activity towards the major bacterial phospholipids, e.g., phosphatidylglycerol but also phosphatidic acid, a key intermediate in phospholipid biosynthesis. In contrast, phosphatidylserine and phosphatidylinositol, both of which are rare lipids in bacteria, are not preferred substrates. These data suggest that PlcP undertakes a generic lipid remodeling role during the cellular response of marine bacteria to P deficiency and that manganese availability may play a key role in regulating the lipid remodeling process.IMPORTANCE Membrane lipids form the structural basis of all cells. In the marine environment, it is well established that phosphorus availability significantly affects lipid composition in cosmopolitan marine bacteria, whereby non-phosphorus-containing lipids are used to replace phospholipids in response to phosphorus stress. Central to this lipid remodeling pathway is a newly identified phospholipase C-type metallophosphoesterase (PlcP). However, little is known about how PlcP activity is regulated. Here, we determined the role of metal ions in regulating PlcP activity and compared PlcP substrate specificities in PlcP enzymes from two model marine bacteria from the marine Roseobacter clade and the SAR11 clade. Our data provide new insights into the regulation of lipid remodeling in these marine bacteria.

Homologous Recombination in Core Genomes Facilitates Marine Bacterial Adaptation.

Acquisition of ecologically relevant genes is common among ocean bacteria, but whether it has a major impact on genome evolution in marine environments remains unknown. Here, we analyzed the core genomes of 16 phylogenetically diverse and ecologically relevant bacterioplankton lineages, each consisting of up to five genomes varying at the strain level. Statistical approaches identified from each lineage up to ∼50 loci showing anomalously high divergence at synonymous sites, which is best explained by recombination with distantly related organisms. The enriched gene categories in these outlier loci match well with the characteristics previously identified as the key phenotypes of these lineages. Examples are antibiotic synthesis and detoxification in Phaeobacter inhibens, exopolysaccharide production in Alteromonas macleodii, hydrocarbon degradation in Marinobacter hydrocarbonoclasticus, and cold adaptation in Pseudoalteromonas haloplanktis Intriguingly, the outlier loci feature polysaccharide catabolism in Cellulophaga baltica but not in Cellulophaga lytica, consistent with their primary habitat preferences in macroalgae and beach sands, respectively. Likewise, analysis of Prochlorococcus showed that photosynthesis-related genes listed in the outlier loci are found only in the high-light-adapted ecotype and not in the low-light adapted ecotype. These observations strongly suggest that recombination with distant relatives is a key mechanism driving the ecological diversification among marine bacterial lineages.IMPORTANCE Acquisition of new metabolic genes has been known as an important mechanism driving bacterial evolution and adaptation in the ocean, but acquisition of novel alleles of existing genes and its potential ecological role have not been examined. Guided by population genetic theories, our genomic analysis showed that divergent allele acquisition is prevalent in phylogenetically diverse marine bacterial lineages and that the affected loci often encode metabolic functions that underlie the known ecological roles of the lineages under study.

Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium "Candidatus Pelagibacter ubique".

SAR11 bacteria are small, heterotrophic, marine alphaproteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under those conditions are not well understood. To illuminate this issue, we used cryo-electron tomography to study "Candidatus Pelagibacter ubique" strain HTCC1062, a member of the SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization. Frozen-hydrated cells, which were preserved in a life-like state, had an average cell volume (enclosed by the outer membrane) of 0.037 ± 0.011 µm3 Strikingly, the periplasmic space occupied ∼20% to 50% of the total cell volume in log-phase cells and ∼50% to 70% in stationary-phase cells. The nucleoid occupied the convex side of the crescent-shaped cells and the ribosomes predominantly occupied the concave side, at a relatively high concentration of 10,000 to 12,000 ribosomes/µm3 Outer membrane pore complexes, likely composed of PilQ, were frequently observed in both log-phase and stationary-phase cells. Long filaments, most likely type IV pili, were found on dividing cells. The physical dimensions, intracellular organization, and morphological changes throughout the life cycle of "Ca. Pelagibacter ubique" provide structural insights into the functional adaptions of these oligotrophic ultramicrobacteria to their habitat. IMPORTANCE: Bacterioplankton of the SAR11 clade (Pelagibacterales) are of interest because of their global biogeochemical significance and because they appear to have been molded by unusual evolutionary circumstances that favor simplicity and efficiency. They have adapted to an ecosystem in which nutrient concentrations are near the extreme limits at which transport systems can function adequately, and they have evolved streamlined genomes to execute only functions essential for life. However, little is known about the actual size limitations and cellular features of living oligotrophic ultramicrobacteria. In this study, we have used cryo-electron tomography to obtain accurate physical information about the cellular architecture of "Candidatus Pelagibacter ubique," the first cultivated member of the SAR11 clade. These results provide foundational information for answering questions about the cell architecture and functions of these ultrasmall oligotrophic bacteria.

Are ocean currents too slow to counteract SAR11 evolution? A next-generation sequencing, phylogeographic analysis.

This work set out to shed light on the phylogeography of the SAR11 clade of Alphaproteobacteria, which is probably the most abundant group of heterotrophic bacteria on Earth. In particular, we assessed the degree to which empirical evidence (environmental DNA sequences) supports the concept that SAR11 lineages evolve faster than they are dispersed thus generating vicariant distributions, as predicted by recent simulation efforts. We generated 16S rRNA gene sequences from surface seawater collected at the South West Atlantic Ocean and combined these data with previously published sequences from similar environments from elsewhere. Altogether, these data consisted in about 1e6 reads, from which we generated 355,306 high quality sequences of which 95,318 corresponded to SAR11. Quantitative phylogeographic analyses supported the existence of a spatially explicit distribution of SAR11 species and provided evidence in favor of the idea that dispersal limitations significantly contribute to SAR11 radiation throughout the world's oceans. Likewise, pairwise phylogenetic distances between the communities studied here were significantly correlated with the genetic divergences predicted by a previously proposed neutral model. As discussed in the paper, these findings are compatible with the concept that the ocean surface constitutes a homogeneous environment for SAR11, in agreement with previous experimental data. We discuss the implications of this hypothesis in a global change scenario. This is the first study combining high throughput sequencing and phylogenic analysis to study bacterial phylogeography and reporting a distance decay pattern of phylogenetic distances for bacteria.

Selection Maintains Low Genomic GC Content in Marine SAR11 Lineages.

The genomic G+C content of ocean bacteria varies from below 30% to over 60%. This broad range of base composition is likely shaped by distinct mutational processes, recombination, effective population size, and selection driven by environmental factors. A number of studies have hypothesized that depletion of G/C in genomes of marine bacterioplankton cells is an adaptation to the nitrogen-poor pelagic oceans, but they failed to disentangle environmental factors from mutational biases and population history. Here, we reconstructed the evolutionary changes of bases at synonymous sites in genomes of two marine SAR11 populations and a freshwater counterpart with its evolutionary origin rooted in the marine lineage. Although they all have similar genome sizes, DNA repair gene repertoire, and base compositions, there is a stronger bias toward A/T changes, a reduced frequency of nitrogenous amino acids, and an exclusive occurrence of polyamine, opine, and taurine transport systems in the ocean populations, consistent with a greater nitrogen stress in surface oceans compared with freshwater lakes. Furthermore, the ratio of nonsynoymous to synonymous nucleotide diversity is not statistically distinguishable among these populations, suggesting that population history has a limited effect. Taken together, the ecological transition of SAR11 from ocean to freshwater habitats makes nitrogen more available to these organisms, and thus relaxation of purifying selection drove a genome-wide reduction in the frequency of G/C to A/T changes in the freshwater population.

A new method for studying the evolutionary origin of the SAR11 clade marine bacteria.

The free-living SAR11 clade is a globally abundant group of oceanic Alphaproteobacteria, with small genome sizes and rich genomic A+T content. However, the taxonomy of SAR11 has become controversial recently. Some researchers argue that the position of SAR11 is a sister group to Rickettsiales. Other researchers advocate that SAR11 is located within free-living lineages of Alphaproteobacteria. Here, we use the natural vector representation method to identify the evolutionary origin of the SAR11 clade. This alignment-free method does not depend on any model assumptions. With this approach, the correspondence between proteome sequences and their natural vectors is one-to-one. After fixing a set of proteins, each bacterium is represented by a set of vectors. The Hausdorff distance is then used to compute the dissimilarity distance between two bacteria. The phylogenetic tree can be reconstructed based on these distances. Using our method, we systematically analyze four data sets of alphaproteobacterial proteomes in order to reconstruct the phylogeny of Alphaproteobacteria. From this we can see that the phylogenetic position of the SAR11 group is within a group of other free-living lineages of Alphaproteobacteria.

Expansion of Cultured Bacterial Diversity by Large-Scale Dilution-to-Extinction Culturing from a Single Seawater Sample.

High-throughput cultivation (HTC) based on a dilution-to-extinction method has been applied broadly to the cultivation of marine bacterial groups, which has often led to the repeated isolation of abundant lineages such as SAR11 and oligotrophic marine gammaproteobacteria (OMG). In this study, to expand the phylogenetic diversity of HTC isolates, we performed a large-scale HTC with a single surface seawater sample collected from the East Sea, the Western Pacific Ocean. Phylogenetic analyses of the 16S rRNA genes from 847 putative pure cultures demonstrated that some isolates were affiliated with not-yet-cultured clades, including the OPB35 and Puniceicoccaceae marine group of Verrucomicrobia and PS1 of Alphaproteobacteria. In addition, numerous strains were obtained from abundant clades, such as SAR11, marine Roseobacter clade, OMG (e.g., SAR92 and OM60), OM43, and SAR116, thereby increasing the size of available culture resources for representative marine bacterial groups. Comparison between the composition of HTC isolates and the bacterial community structure of the seawater sample used for HTC showed that diverse marine bacterial groups exhibited various growth capabilities under our HTC conditions. The growth response of many bacterial groups, however, was clearly different from that observed with conventional plating methods, as exemplified by numerous isolates of the SAR11 clade and Verrucomicrobia. This study showed that a large number of novel bacterial strains could be obtained by an extensive HTC from even a small number of samples.

Ribosomal proteins: toward a next generation standard for prokaryotic systematics?

The seminal work of Carl Woese and co-workers has contributed to promote the RNA component of the small subunit of the ribosome (SSU rRNA) as a "gold standard" of modern prokaryotic taxonomy and systematics, and an essential tool to explore microbial diversity. Yet, this marker has a limited resolving power, especially at deep phylogenetic depth and can lead to strongly biased trees. The ever-larger number of available complete genomes now calls for a novel standard dataset of robust protein markers that may complement SSU rRNA. In this respect, concatenation of ribosomal proteins (r-proteins) is being growingly used to reconstruct large-scale prokaryotic phylogenies, but their suitability for systematic and/or taxonomic purposes has not been specifically addressed. Using Proteobacteria as a case study, we show that amino acid and nucleic acid r-protein sequences contain a reliable phylogenetic signal at a wide range of taxonomic depths, which has not been totally blurred by mutational saturation or horizontal gene transfer. The use of accurate evolutionary models and reconstruction methods allows overcoming most tree reconstruction artefacts resulting from compositional biases and/or fast evolutionary rates. The inferred phylogenies allow clarifying the relationships among most proteobacterial orders and families, along with the position of several unclassified lineages, suggesting some possible revisions of the current classification. In addition, we investigate the root of the Proteobacteria by considering the time-variation of nucleic acid composition of r-protein sequences and the information carried by horizontal gene transfers, two approaches that do not require the use of an outgroup and limit tree reconstruction artefacts. Altogether, our analyses indicate that r-proteins may represent a promising standard for prokaryotic taxonomy and systematics.

Ubiquitous filter feeders shape open ocean microbial community structure and function.

The mechanism of mortality plays a large role in how microorganisms in the open ocean contribute to global energy and nutrient cycling. Salps are ubiquitous pelagic tunicates that are a well-known mortality source for large phototrophic microorganisms in coastal and high-latitude systems, but their impact on the immense populations of smaller prokaryotes in the tropical and subtropical open ocean gyres is not well quantified. We used robustly quantitative techniques to measure salp clearance and enrichment of specific microbial functional groups in the North Pacific Subtropical Gyre, one of the largest ecosystems on Earth. We discovered that salps are a previously unknown predator of the globally abundant nitrogen fixer Crocosphaera; thus, salps restrain new nitrogen delivery to the marine ecosystem. We show that the ocean's two numerically dominant cells, Prochlorococcus and SAR11, are not consumed by salps, which offers a new explanation for the dominance of small cells in open ocean systems. We also identified a double bonus for Prochlorococcus, wherein it not only escapes salp predation but the salps also remove one of its major mixotrophic predators, the prymnesiophyte Chrysochromulina. When we modeled the interaction between salp mesh and particles, we found that cell size alone could not account for these prey selection patterns. Instead, the results suggest that alternative mechanisms, such as surface property, shape, nutritional quality, or even prey behavior, determine which microbial cells are consumed by salps. Together, these results identify salps as a major factor in shaping the structure, function, and ecology of open ocean microbial communities.

Microbial ecology of northern Gulf of Mexico estuarine waters.

Estuarine and coastal ecosystems are of high economic and ecological importance, owing to their diverse communities and the disproportionate role they play in carbon cycling, particularly in carbon sequestration. Organisms inhabiting these environments must overcome strong natural fluctuations in salinity, nutrients, and turbidity, as well as numerous climate change-induced disturbances such as land loss, sea level rise, and, in some locations, increasingly severe tropical cyclones that threaten to disrupt future ecosystem health. The northern Gulf of Mexico (nGoM) along the Louisiana coast contains dozens of estuaries, including the Mississippi-Atchafalaya River outflow, which dramatically influence the region due to their vast upstream watershed. Nevertheless, the microbiology of these estuaries and surrounding coastal environments has received little attention. To improve our understanding of microbial ecology in the understudied coastal nGoM, we conducted a 16S rRNA gene amplicon survey at eight sites and multiple time points along the Louisiana coast and one inland swamp spanning freshwater to high brackish salinities, totaling 47 duplicated Sterivex (0.2-2.7 µm) and prefilter (>2.7 µm) samples. We cataloged over 13,000 Amplicon Sequence ariants (ASVs) from common freshwater and marine clades such as SAR11 (Alphaproteobacteria), Synechococcus (Cyanobacteria), and acI and Candidatus Actinomarina (Actinobacteria). We observed correlations with freshwater or marine habitats in many organisms and characterized a group of taxa with specialized distributions across brackish water sites, supporting the hypothesis of an endogenous brackish-water community. Additionally, we observed brackish-water associations for several aquatic clades typically considered marine or freshwater taxa, such as SAR11 subclade II, SAR324, and the acI Actinobacteria. The data presented here expand the geographic coverage of microbial ecology in estuarine communities, help delineate the native and transitory members of these environments, and provide critical aquatic microbiological baseline data for coastal and estuarine sites in the nGoM.IMPORTANCEEstuarine and coastal waters are diverse ecosystems influenced by tidal fluxes, interconnected wetlands, and river outflows, which are of high economic and ecological importance. Microorganisms play a pivotal role in estuaries as "first responders" and ecosystem architects, yet despite their ecological importance, they remain underrepresented in microbial studies compared to open ocean environments. This leads to substantial knowledge gaps that are important for understanding global biogeochemical cycling and making decisions about conservation and management strategies in these environments. Our study makes key contributions to the microbial ecology of estuarine and coastal habitats in the northern Gulf of Mexico. Our microbial community data support the concept of a globally distributed, core brackish microbiome and emphasize previously underrecognized brackish-water taxa. Given the projected worsening of land loss, oil spills, and natural disasters in this region, our results will serve as important baseline data for researchers investigating the microbial communities found across estuaries.

Synergistic effect of biochar with gypsum, lime, and farm manure on the growth and tolerance in rice plants under different salt-affected soils.

Soil salinization and sodication harm soil fertility and crop production, especially in dry regions. To combat this, using biochar combined with gypsum, lime, and farm manure is a promising solution for improving salt-affected soils. In a pot experiment, cotton stick biochar (BC) was applied at a rate of 20 t/ha in combination with gypsum (G), lime (L), and farm manure (F) at rates of 5 and 10 t/ha. These were denoted as BCG-5, BCL-5, BCF-5, BCG-10, BCL-10, and BCF-10. Three different types of soils with electrical conductivity (EC) to sodium adsorption ratio (SAR) ratios of 2.45:13.7, 9.45:22, and 11.56:40 were used for experimentation. The application of BCG-10 led to significant improvements in rice biomass, chlorophyll content, and overall growth. It was observed that applying BCG-10 to soils increased the membrane stability index by 75% in EC:SAR (2.45:13.7), 97% in EC:SAR (9.45:22), and 40% in EC:SAR (11.56:40) compared to respective control treatments. After BCG-10 was applied, the hydrogen peroxide in leaves dropped by 29%, 23%, and 21% in EC:SAR (2.45:13.7), EC:SAR (9.45:22), and EC:SAR (11.56:40) soils, relative to their controls, respectively. The application of BCG-10 resulted in glycine betaine increases of 60, 119, and 165% in EC: SAR (2.45:13.7), EC: SAR (9.45:22), and EC: SAR (11.56:40) soils. EC: SAR (2.45:13.7), EC: SAR (9.45:22), and EC: SAR (11.56:40) soils all had 70, 109, and 130% more ascorbic acid in BCG-10 applied treatment, respectively. The results of this experiment show that BCG-10 increased the growth and physiological traits of rice plants were exposed to different levels of salt stress. This was achieved by lowering hydrogen peroxide levels, making plant cells more stable, and increasing non-enzymatic activity.

The AEGEAN-169 clade of bacterioplankton is synonymous with SAR11 subclade V (HIMB59) and metabolically distinct.

Bacterioplankton of the SAR11 clade are the most abundant marine microorganisms and consist of numerous subclades spanning order-level divergence (Pelagibacterales). The assignment of the earliest diverging subclade V (a.k.a. HIMB59) to the Pelagibacterales is highly controversial, with multiple recent phylogenetic studies placing them completely separate from SAR11. Other than through phylogenomics, subclade V has not received detailed examination due to limited genomes from this group. Here, we assessed the ecogenomic characteristics of subclade V to better understand the role of this group in comparison to the Pelagibacterales. We used a new isolate genome, recently released single-amplified genomes and metagenome-assembled genomes, and previously established SAR11 genomes to perform a comprehensive comparative genomics analysis. We paired this analysis with the recruitment of metagenomes spanning the open ocean, coastal, and brackish systems. Phylogenomics, average amino acid identity, and 16S rRNA gene phylogeny indicate that SAR11 subclade V is synonymous with the ubiquitous AEGEAN-169 clade and support the contention that this group represents a taxonomic family. AEGEAN-169 shared many bulk genome qualities with SAR11, such as streamlining and low GC content, but genomes were generally larger. AEGEAN-169 had overlapping distributions with SAR11 but was metabolically distinct from SAR11 in its potential to transport and utilize a broader range of sugars as well as in the transport of trace metals and thiamin. Thus, regardless of the ultimate phylogenetic placement of AEGEAN-169, these organisms have distinct metabolic capacities that likely allow them to differentiate their niche from canonical SAR11 taxa. IMPORTANCE One goal of marine microbiologists is to uncover the roles various microorganisms are playing in biogeochemical cycles. Success in this endeavor relies on differentiating groups of microbes and circumscribing their relationships. An early-diverging group (subclade V) of the most abundant bacterioplankton, SAR11, has recently been proposed as a separate lineage that does not share a most recent common ancestor. But beyond phylogenetics, little has been done to evaluate how these organisms compare with SAR11. Our work leverages dozens of new genomes to demonstrate the similarities and differences between subclade V and SAR11. In our analysis, we also establish that subclade V is synonymous with a group of bacteria established from 16S rRNA gene sequences, AEGEAN-169. Subclade V/AEGEAN-169 has clear metabolic distinctions from SAR11 and their shared traits point to remarkable convergent evolution if they do not share a most recent common ancestor.

Phosphate-related genomic islands as drivers of environmental adaptation in the streamlined marine alphaproteobacterial HIMB59.

IMPORTANCE: These results shed light on the evolutionary strategies of microbes with streamlined genomes to adapt and survive in the oligotrophic conditions that dominate the surface waters of the global ocean. At the individual level, these microbes have been subjected to evolutionary constraints that have led to a more efficient use of nutrients, removing non-essential genes named as "streamlining theory." However, at the population level, they conserve a highly diverse gene pool in flexible genomic islands resulting in polyclonal populations on the same genomic background as an evolutionary response to environmental pressures. Localization of these islands at equivalent positions in the genome facilitates horizontal transfer between clonal lineages. This high level of environmental genomic heterogeneity could explain their cosmopolitan distribution. In the case of the order HIMB59 within the class Alphaproteobacteria, two factors exert evolutionary pressure and determine this intraspecific diversity: phages and the concentration of P in the environment.

The mystery of the ice cold rose-Microbiome of an Arctic winter frost flower.

Under very cold conditions, delicate ice-crystal structures called frost flowers emerge on the surface of newly formed sea ice. These understudied, ephemeral structures include saline brine, organic material, inorganic nutrients, and bacterial and archaeal communities in their brine channels. Hitherto, only a few frost flowers have been studied during spring and these have been reported to be dominated by Rhizobia or members of the SAR11 clade. Here we report on the microbiome of frost flowers sampled during the winter and polar night in the Barents Sea. There was a distinct difference in community profile between the extracted DNA and RNA, but both were dominated by members of the SAR11 clade (78% relative abundance and 41.5% relative activity). The data further suggested the abundance and activity of Cand. Nitrosopumilus, Nitrospinia, and Nitrosomonas. Combined with the inference of marker genes based on the 16S rRNA gene data, this indicates that sulfur and nitrogen cycling are likely the major metabolism in these ephemeral structures.

Spatiotemporal Variation of Microbial Communities in the Ultra-Oligotrophic Eastern Mediterranean Sea.

Marine microbial communities vary seasonally and spatially, but these two factors are rarely addressed together. In this study, the temporal and spatial patterns of the bacterial and archaeal community were studied along a coast-to-offshore transect in the Eastern Mediterranean Sea (EMS) over six cruises, in three seasons of 2 consecutive years. Amplicon sequencing of 16S rRNA genes and transcripts was performed to determine presence and activity, respectively. The ultra-oligotrophic status of the Southeastern Mediterranean Sea was reflected in the microbial community composition dominated by oligotrophic bacterial groups such as SAR11, even at the most coastal station sampled, throughout the year. Seasons significantly affected the microbial communities, explaining more than half of the observed variability. However, the same few taxa dominated the community over the 2-year sampling period, varying only in their degree of dominance. While there was no overall effect of station location on the microbial community, the most coastal site (16 km offshore) differed significantly in community structure and activity from the three further offshore stations in early winter and summer. Our data on the microbial community compositions and their seasonality support previous notions that the EMS behaves like an oceanic gyre.

Exaggerated trans-membrane charge of ammonium transporters in nutrient-poor marine environments.

Transporter proteins are a vital interface between cells and their environment. In nutrient-limited environments, microbes with transporters that are effective at bringing substrates into their cells will gain a competitive advantage over variants with reduced transport function. Microbial ammonium transporters (Amt) bring ammonium into the cytoplasm from the surrounding periplasm space, but diagnosing Amt adaptations to low nutrient environments solely from sequence data has been elusive. Here, we report altered Amt sequence amino acid distribution from deep marine samples compared to variants sampled from shallow water in two important microbial lineages of the marine water column community-Marine Group I Archaea (Thermoproteota) and the uncultivated gammaproteobacterial lineage SAR86. This pattern indicates an evolutionary pressure towards an increasing dipole in Amt for these clades in deep ocean environments and is predicted to generate stronger electric fields facilitating ammonium acquisition. This pattern of increasing dipole charge with depth was not observed in lineages capable of accessing alternative nitrogen sources, including the abundant alphaproteobacterial clade SAR11. We speculate that competition for ammonium in the deep ocean drives transporter sequence evolution. The low concentration of ammonium in the deep ocean is therefore likely due to rapid uptake by Amts concurrent with decreasing nutrient flux.