Expanding the Diversity of Bacterioplankton Isolates and Modeling Isolation Efficacy with Large-Scale Dilution-to-Extinction Cultivation.

Cultivated bacterioplankton representatives from diverse lineages and locations are essential for microbiology, but the large majority of taxa either remain uncultivated or lack isolates from diverse geographic locales. We paired large-scale dilution-to-extinction (DTE) cultivation with microbial community analysis and modeling to expand the phylogenetic and geographic diversity of cultivated bacterioplankton and to evaluate DTE cultivation success. Here, we report results from 17 DTE experiments totaling 7,820 individual incubations over 3 years, yielding 328 repeatably transferable isolates. Comparison of isolates to microbial community data for source waters indicated that we successfully isolated 5% of the observed bacterioplankton community throughout the study; 43% and 26% of our isolates matched operational taxonomic units and amplicon single-nucleotide variants, respectively, within the top 50 most abundant taxa. Isolates included those from previously uncultivated clades such as SAR11 LD12 and Actinobacteria acIV, as well as geographically novel members from other ecologically important groups like SAR11 subclade IIIa, SAR116, and others, providing isolates in eight putatively new genera and seven putatively new species. Using a newly developed DTE cultivation model, we evaluated taxon viability by comparing relative abundance with cultivation success. The model (i) revealed the minimum attempts required for successful isolation of taxa amenable to growth on our media and (ii) identified possible subpopulation viability variation in abundant taxa such as SAR11 that likely impacts cultivation success. By incorporating viability in experimental design, we can now statistically constrain the effort necessary for successful cultivation of specific taxa on a defined medium.IMPORTANCE Even before the coining of the term "great plate count anomaly" in the 1980s, scientists had noted the discrepancy between the number of microorganisms observed under the microscope and the number of colonies that grew on traditional agar media. New cultivation approaches have reduced this disparity, resulting in the isolation of some of the "most wanted" bacterial lineages. Nevertheless, the vast majority of microorganisms remain uncultured, hampering progress toward answering fundamental biological questions about many important microorganisms. Furthermore, few studies have evaluated the underlying factors influencing cultivation success, limiting our ability to improve cultivation efficacy. Our work details the use of dilution-to-extinction (DTE) cultivation to expand the phylogenetic and geographic diversity of available axenic cultures. We also provide a new model of the DTE approach that uses cultivation results and natural abundance information to predict taxon-specific viability and iteratively constrain DTE experimental design to improve cultivation success.

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.

Flexible genomic island conservation across freshwater and marine Methylophilaceae.

The evolutionary trajectory of Methylophilaceae includes habitat transitions from freshwater sediments to freshwater and marine pelagial that resulted in genome reduction (genome-streamlining) of the pelagic taxa. However, the extent of genetic similarities in the genomic structure and microdiversity of the two genome-streamlined pelagic lineages (freshwater "Ca. Methylopumilus" and the marine OM43 lineage) has so far never been compared. Here, we analyzed complete genomes of 91 "Ca. Methylopumilus" strains isolated from 14 lakes in Central Europe and 12 coastal marine OM43 strains. The two lineages showed a remarkable niche differentiation with clear species-specific differences in habitat preference and seasonal distribution. On the other hand, we observed a synteny preservation in their genomes by having similar locations and types of flexible genomic islands (fGIs). Three main fGIs were identified: a replacement fGI acting as phage defense, an additive fGI harboring metabolic and resistance-related functions, and a tycheposon containing nitrogen-, thiamine-, and heme-related functions. The fGIs differed in relative abundances in metagenomic datasets suggesting different levels of variability ranging from strain-specific to population-level adaptations. Moreover, variations in one gene seemed to be responsible for different growth at low substrate concentrations and a potential biogeographic separation within one species. Our study provides a first insight into genomic microdiversity of closely related taxa within the family Methylophilaceae and revealed remarkably similar dynamics involving mobile genetic elements and recombination between freshwater and marine family members.

Draft genome sequence of the BAL58 Betaproteobacteria representative strain LSUCC0117.

Here, we present the draft genome sequence of strain LSUCC0117, a representative of the abundant aquatic BAL58 Betaproteobacteria group which we isolated from a coastal site in the northern Gulf of Mexico. The genome is estimated at over 99% complete, with a genome size of 2,687,225 bp.

Genome sequences of four agarolytic bacteria from the Bacteroidia and Gammaproteobacteria.

Here we present the genomes of four marine agarolytic bacteria belonging to the Bacteroidota and Proteobacteria. Two genomes are closed and two are in draft form, but all are at least 99% complete and offer new opportunities to study agar-degradation in marine bacteria.

Ecophysiology and genomics of the brackish water adapted SAR11 subclade IIIa.

The Order Pelagibacterales (SAR11) is the most abundant group of heterotrophic bacterioplankton in global oceans and comprises multiple subclades with unique spatiotemporal distributions. Subclade IIIa is the primary SAR11 group in brackish waters and shares a common ancestor with the dominant freshwater IIIb (LD12) subclade. Despite its dominance in brackish environments, subclade IIIa lacks systematic genomic or ecological studies. Here, we combine closed genomes from new IIIa isolates, new IIIa MAGS from San Francisco Bay (SFB), and 460 highly complete publicly available SAR11 genomes for the most comprehensive pangenomic study of subclade IIIa to date. Subclade IIIa represents a taxonomic family containing three genera (denoted as subgroups IIIa.1, IIIa.2, and IIIa.3) that had distinct ecological distributions related to salinity. The expansion of taxon selection within subclade IIIa also established previously noted metabolic differentiation in subclade IIIa compared to other SAR11 subclades such as glycine/serine prototrophy, mosaic glyoxylate shunt presence, and polyhydroxyalkanoate synthesis potential. Our analysis further shows metabolic flexibility among subgroups within IIIa. Additionally, we find that subclade IIIa.3 bridges the marine and freshwater clades based on its potential for compatible solute transport, iron utilization, and bicarbonate management potential. Pure culture experimentation validated differential salinity ranges in IIIa.1 and IIIa.3 and provided detailed IIIa cell size and volume data. This study is an important step forward for understanding the genomic, ecological, and physiological differentiation of subclade IIIa and the overall evolutionary history of SAR11.

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.

Draft Genome Sequence of the Marine Flavobacteriaceae sp. Strain LSUCC0859.

A new marine Flavobacteriaceae sp. strain, LSUCC0859, was isolated off the coast of Louisiana with artificial seawater via high-throughput dilution-to-extinction (DTE) cultivation. The 2,168,862-bp genome sequence provides opportunities to investigate the biology of a poorly understood lineage within the Bacteroidetes.

Ecophysiology of the Cosmopolitan OM252 Bacterioplankton (Gammaproteobacteria).

Among the thousands of species that comprise marine bacterioplankton communities, most remain functionally obscure. One key cosmopolitan group in this understudied majority is the OM252 clade of Gammaproteobacteria. Although frequently found in sequence data and even previously cultured, the diversity, metabolic potential, physiology, and distribution of this clade has not been thoroughly investigated. Here, we examined these features of OM252 bacterioplankton using a newly isolated strain and genomes from publicly available databases. We demonstrated that this group constitutes a globally distributed novel genus ("Candidatus Halomarinus"), sister to Litoricola, comprising two subclades and multiple distinct species. OM252 organisms have small genomes (median, 2.21 Mbp) and are predicted obligate aerobes capable of alternating between chemoorganoheterotrophic and chemolithotrophic growth using reduced sulfur compounds as electron donors. Subclade I genomes encode genes for the Calvin-Benson-Bassham cycle for carbon fixation. One representative strain of subclade I, LSUCC0096, had extensive halotolerance and a mesophilic temperature range for growth, with a maximum rate of 0.36 doublings/h at 35°C. Cells were curved rod/spirillum-shaped, ∼1.5 by 0.2 µm. Growth yield on thiosulfate as the sole electron donor under autotrophic conditions was roughly one-third that of heterotrophic growth, even though calculations indicated similar Gibbs energies for both catabolisms. These phenotypic data show that some "Ca. Halomarinus" organisms can switch between serving as carbon sources or sinks and indicate the likely anabolic cost of lithoautotrophic growth. Our results thus provide new hypotheses about the roles of these organisms in global biogeochemical cycling of carbon and sulfur. IMPORTANCE 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). However, further study of these organisms has not occurred. Using another isolated representative (strain LSUCC0096) and publicly available genome sequences from metagenomic and single-cell genomic data sets, we examined the diversity within the OM252 clade and the distribution of these taxa in the world's oceans, reconstructed the predicted metabolism of the group, and quantified growth dynamics in LSUCC0096. Our results generate new knowledge about the previously enigmatic OM252 clade and point toward the importance of facultative chemolithoautotrophy for supporting some clades of ostensibly "heterotrophic" taxa.

Mechanisms driving genome reduction of a novel Roseobacter lineage.

Members of the marine Roseobacter group are key players in the global carbon and sulfur cycles. While over 300 species have been described, only 2% possess reduced genomes (mostly 3-3.5 Mbp) compared to an average roseobacter (>4 Mbp). These taxonomic minorities are phylogenetically diverse but form a Pelagic Roseobacter Cluster (PRC) at the genome content level. Here, we cultivated eight isolates constituting a novel Roseobacter lineage which we named 'CHUG'. Metagenomic and metatranscriptomic read recruitment analyses showed that CHUG members are globally distributed and active in marine pelagic environments. CHUG members possess some of the smallest genomes (~2.6 Mb) among all known roseobacters, but they do not exhibit canonical features of typical bacterioplankton lineages theorized to have undergone genome streamlining processes, like higher coding density, fewer paralogues and rarer pseudogenes. While CHUG members form a genome content cluster with traditional PRC members, they show important differences. Unlike other PRC members, neither the relative abundances of CHUG members nor their relative gene expression levels are correlated with chlorophyll a concentration across the global samples. CHUG members cannot utilize most phytoplankton-derived metabolites or synthesize vitamin B12, a key metabolite mediating the roseobacter-phytoplankton interactions. This combination of features is evidence for the hypothesis that CHUG members may have evolved a free-living lifestyle decoupled from phytoplankton. This ecological transition was accompanied by the loss of signature genes involved in roseobacter-phytoplankton symbiosis, suggesting that relaxation of purifying selection owing to lifestyle shift is likely an important driver of genome reduction in CHUG.

Metagenomics of Antarctic Marine Sediment Reveals Potential for Diverse Chemolithoautotrophy.

The microbial biogeochemical processes occurring in marine sediment in Antarctica remain underexplored due to limited access. Further, these polar habitats are unique, as they are being exposed to significant changes in their climate. To explore how microbes drive biogeochemistry in these sediments, we performed a shotgun metagenomic survey of marine surficial sediment (0 to 3 cm of the seafloor) collected from 13 locations in western Antarctica and assembled 16 high-quality metagenome assembled genomes for focused interrogation of the lifestyles of some abundant lineages. We observe an abundance of genes from pathways for the utilization of reduced carbon, sulfur, and nitrogen sources. Although organotrophy is pervasive, nitrification and sulfide oxidation are the dominant lithotrophic pathways and likely fuel carbon fixation via the reverse tricarboxylic acid and Calvin cycles. Oxygen-dependent terminal oxidases are common, and genes for reduction of oxidized nitrogen are sporadically present in our samples. Our results suggest that the underlying benthic communities are well primed for the utilization of settling organic matter, which is consistent with findings from highly productive surface water. Despite the genetic potential for nitrate reduction, the net catabolic pathway in our samples remains aerobic respiration, likely coupled to the oxidation of sulfur and nitrogen imported from the highly productive Antarctic water column above. IMPORTANCE The impacts of climate change in polar regions, like Antarctica, have the potential to alter numerous ecosystems and biogeochemical cycles. Increasing temperature and freshwater runoff from melting ice can have profound impacts on the cycling of organic and inorganic nutrients between the pelagic and benthic ecosystems. Within the benthos, sediment microbial communities play a critical role in carbon mineralization and the cycles of essential nutrients like nitrogen and sulfur. Metagenomic data collected from sediment samples from the continental shelf of western Antarctica help to examine this unique system and document the metagenomic potential for lithotrophic metabolisms and the cycles of both nitrogen and sulfur, which support not only benthic microbes but also life in the pelagic zone.

Correction: Cultivation and genomics of the first freshwater SAR11 (LD12) isolate.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Complete Genome Sequence of Marinobacterium sp. Strain LSUCC0821, Isolated from the Coastal Gulf of Mexico.

Here, we present the complete genome sequence of Marinobacterium sp. strain LSUCC0821, isolated from the coastal Gulf of Mexico with artificial seawater using high-throughput dilution-to-extinction (DTE) cultivation. The 2.36-Mbp circularized genome sequence has 2,231 predicted genes, a 91.5% coding density, and a GC content of 47.8%.

Draft Genome Sequence of Novel Coastal Bacterium LSUCC0115 from the MWH-UniPo Clade, Order Burkholderiales, Class Betaproteobacteria.

Here, we present the draft genome of LSUCC0115, a novel coastal Gulf of Mexico bacterioplankton isolate within the order Burkholderiales LSUCC0115 has the metabolic potential for aerobic heterotrophy, phototrophy, and lithoautotrophy, as well as genes for flagellar assembly and quorum sensing.

Draft Genome Sequence of Strain LSUCC0112, a Gulf of Mexico Representative of the Oligotrophic Marine Gammaproteobacteria.

Here, we present the draft genome sequence of strain LSUCC0112, a cultured representative from the Gulf of Mexico that is phylogenetically close to the OM182 clade within oligotrophic marine Gammaproteobacteria LSUCC0112 shows the potential for aerobic heterotrophy, glycogen synthesis, flagellar motility, and assimilatory sulfate reduction.

Draft Genome Sequence of Strain LSUCC0057, a Member of the SAR92 Clade of Gammaproteobacteria.

We present the draft genome sequence of strain LSUCC0057, a member of the SAR92 clade of Gammaproteobacteria, isolated from coastal waters near Buras, LA. The genome contains proteorhodopsin and indicates the potential for aerobic heterotrophy, assimilatory sulfate reduction, and carotenoid biosynthesis.

Comparative genomics of 16 Microbacterium spp. that tolerate multiple heavy metals and antibiotics.

A total of 16 different strains of Microbacterium spp. were isolated from contaminated soil and enriched on the carcinogen, hexavalent chromium [Cr(VI)]. The majority of the isolates (11 of the 16) were able to tolerate concentrations (0.1 mM) of cobalt, cadmium, and nickel, in addition to Cr(VI) (0.5-20 mM). Interestingly, these bacteria were also able to tolerate three different antibiotics (ranges: ampicillin 0-16 µg ml-1, chloramphenicol 0-24 µg ml-1, and vancomycin 0-24 µg ml-1). To gain genetic insight into these tolerance pathways, the genomes of these isolates were assembled and annotated. The genomes of these isolates not only have some shared genes (core genome) but also have a large amount of variability. The genomes also contained an annotated Cr(VI) reductase (chrR) that could be related to Cr(VI) reduction. Further, various heavy metal tolerance (e.g., Co/Zn/Cd efflux system) and antibiotic resistance genes were identified, which provide insight into the isolates' ability to tolerate metals and antibiotics. Overall, these isolates showed a wide range of tolerances to heavy metals and antibiotics and genetic diversity, which was likely required of this population to thrive in a contaminated environment.

Cultivation and genomics of the first freshwater SAR11 (LD12) isolate.

Evolutionary transitions between fresh and salt water happen infrequently among bacterioplankton. Within the ubiquitous and highly abundant heterotrophic Alphaproteobacteria order Pelagibacterales (SAR11), most members live in marine habitats, but the LD12 subclade has evolved as a unique freshwater lineage. LD12 cells occur as some of the most dominant freshwater bacterioplankton, yet this group has remained elusive to cultivation, hampering a more thorough understanding of its biology. Here, we report the first successful isolation of an LD12 representative, strain LSUCC0530, using high-throughput dilution-to-extinction cultivation methods, and its complete genome sequence. Growth experiments corroborate ecological data suggesting active populations of LD12 in brackish water up to salinities of ~5. LSUCC0530 has the smallest closed genome thus far reported for a SAR11 strain (1.16 Mbp). The genome affirms many previous metabolic predictions from cultivation-independent analyses, like a complete Embden-Meyerhof-Parnas glycolysis pathway, but also provides novel insights, such as the first isocitrate dehydrogenase in LD12, a likely homologous recombination of malate synthase from outside of the SAR11 clade, and analogous substitutions of ion transporters with others that occur throughout the rest of the SAR11 clade. Growth data support metagenomic recruitment results suggesting temperature-based ecotype diversification within LD12. Key gene losses for osmolyte uptake provide a succinct hypothesis for the evolutionary transition of LD12 from salt to freshwater. For strain LSUCC0530, we propose the provisional nomenclature Candidatus fonsibacter ubiquis.

Draft Genome Sequence of Strain LSUCC0135, an Early Diverging Member of the Order Methylophilales in the Phylum Betaproteobacteria.

We present the draft genome of Methylophilales sp. strain LSUCC0135, isolated using high-throughput dilution-to-extinction culturing methods from the coast of Freshwater City, Louisiana, USA. The genome indicates metabolic flexibility for differing oxygen concentrations and electron donors.

Biogeochemical and Microbial Variation across 5500 km of Antarctic Surface Sediment Implicates Organic Matter as a Driver of Benthic Community Structure.

Western Antarctica, one of the fastest warming locations on Earth, is a unique environment that is underexplored with regards to biodiversity. Although pelagic microbial communities in the Southern Ocean and coastal Antarctic waters have been well-studied, there are fewer investigations of benthic communities and most have a focused geographic range. We sampled surface sediment from 24 sites across a 5500 km region of Western Antarctica (covering the Ross Sea to the Weddell Sea) to examine relationships between microbial communities and sediment geochemistry. Sequencing of the 16S and 18S rRNA genes showed microbial communities in sediments from the Antarctic Peninsula (AP) and Western Antarctica (WA), including the Ross, Amundsen, and Bellingshausen Seas, could be distinguished by correlations with organic matter concentrations and stable isotope fractionation (total organic carbon; TOC, total nitrogen; TN, and δ(13)C). Overall, samples from the AP were higher in nutrient content (TOC, TN, and NH4 (+)) and communities in these samples had higher relative abundances of operational taxonomic units (OTUs) classified as the diatom, Chaetoceros, a marine cercozoan, and four OTUs classified as Flammeovirgaceae or Flavobacteria. As these OTUs were strongly correlated with TOC, the data suggests the diatoms could be a source of organic matter and the Bacteroidetes and cercozoan are grazers that consume the organic matter. Additionally, samples from WA have lower nutrients and were dominated by Thaumarchaeota, which could be related to their known ability to thrive as lithotrophs. This study documents the largest analysis of benthic microbial communities to date in the Southern Ocean, representing almost half the continental shoreline of Antarctica, and documents trophic interactions and coupling of pelagic and benthic communities. Our results indicate potential modifications in carbon sequestration processes related to change in community composition, identifying a prospective mechanism that links climate change to carbon availability.