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Kolody, BC, McCrow JP, Allen ZL, Aylward FO, Fontanez KM, Moustafa A, Moniruzzaman M, Chavez FP, Scholin CA, Allen EE, Worden AZ, Delong EF, Allen AE.  2019.  Diel transcriptional response of a California Current plankton microbiome to light, low iron, and enduring viral infection. The ISME Journal.   10.1038/s41396-019-0472-2   Abstract

Phytoplankton and associated microbial communities provide organic carbon to oceanic food webs and drive ecosystem dynamics. However, capturing those dynamics is challenging. Here, an in situ, semi-Lagrangian, robotic sampler profiled pelagic microbes at 4 h intervals over ~2.6 days in North Pacific high-nutrient, low-chlorophyll waters. We report on the community structure and transcriptional dynamics of microbes in an operationally large size class (>5 μm) predominantly populated by dinoflagellates, ciliates, haptophytes, pelagophytes, diatoms, cyanobacteria (chiefly Synechococcus), prasinophytes (chiefly Ostreococcus), fungi, archaea, and proteobacteria. Apart from fungi and archaea, all groups exhibited 24-h periodicity in some transcripts, but larger portions of the transcriptome oscillated in phototrophs. Periodic photosynthesis-related transcripts exhibited a temporal cascade across the morning hours, conserved across diverse phototrophic lineages. Pronounced silica:nitrate drawdown, a high flavodoxin to ferredoxin transcript ratio, and elevated expression of other Fe-stress markers indicated Fe-limitation. Fe-stress markers peaked during a photoperiodically adaptive time window that could modulate phytoplankton response to seasonal Fe-limitation. Remarkably, we observed viruses that infect the majority of abundant taxa, often with total transcriptional activity synchronized with putative hosts. Taken together, these data reveal a microbial plankton community that is shaped by recycled production and tightly controlled by Fe-limitation and viral activity.

Podell, S, Blanton JM, Neu A, Agarwal V, Biggs JS, Moore BS, Allen EE.  2019.  Pangenomic comparison of globally distributed Poribacteria associated with sponge hosts and marine particles. ISME Journal. 13:468-481.   10.1038/s41396-018-0292-9   Abstract

Candidatus Poribacteria is a little-known bacterial phylum, previously characterized by partial genomes from a single sponge host, but never isolated in culture. We have reconstructed multiple genome sequences from four different sponge genera and compared them to recently reported, uncharacterized Poribacteria genomes from the open ocean, discovering shared and unique functional characteristics. Two distinct, habitat-linked taxonomic lineages were identified, designated Entoporibacteria (sponge-associated) and Pelagiporibacteria (free-living). These lineages differed in flagellar motility and chemotaxis genes unique to Pelagiporibacteria, and highly expanded families of restriction endonucleases, DNA methylases, transposases, CRISPR repeats, and toxin-antitoxin gene pairs in Entoporibacteria. Both lineages shared pathways for facultative anaerobic metabolism, denitrification, fermentation, organosulfur compound utilization, type IV pili, cellulosomes, and bacterial proteosomes. Unexpectedly, many features characteristic of eukaryotic host association were also shared, including genes encoding the synthesis of eukaryotic-like cell adhesion molecules, extracellular matrix digestive enzymes, phosphoinositol-linked membrane glycolipids, and exopolysaccharide capsules. Complete Poribacteria 16S rRNA gene sequences were found to contain multiple mismatches to "universal" 16S rRNA gene primer sets, substantiating concerns about potential amplification failures in previous studies. A newly-designed primer set corrects these mismatches, enabling more accurate assessment of Poribacteria abundance in diverse marine habitats where it may have previously been overlooked.

Schorn, MA, Jordan PA, Podell S, Blanton JM, Agarwal V, Biggs JS, Allen EE, Moore BS.  2019.  Comparative genomics of cyanobacterial symbionts reveals distinct, specialized metabolism in tropical Dysideidae sponges. mBio. 10:e00821-19.   10.1128/mBio.00821-19   Abstract

Marine sponges have long been recognized as valuable sources of secondary metabolites and are renowned as petri-dishes of the sea, providing a home for many symbiotic microorganisms. These ancient, soft-bodied, sessile organisms are often unable to defend themselves against predators and may benefit from chemical defenses provided by their symbionts. Symbiotic bacteria associated with sponges are often difficult to cultivate, and some strains have resisted cultivation for decades, representing a widely untapped source for natural product discovery. Many sponges of the Dysideidea family are well documented to be chemically talented, often containing high levels of polybrominated diphenyl ethers (PBDEs), polychlorinated compounds, a variety of sesquiterpenes, and numerous other classes of small molecules. This group of sponges has also been reported to harbor a high percentage of an uncultured filamentous cyanobacterium, Hormoscilla spongeliae. Advances in sequencing and assembly technologies now facilitate the reconstruction of high quality genomes from metagenomic samples, allowing analysis of the genomes of previously inaccessible microbes. Here, comparative genomic analyses of two phylogenetically distinct Hormoscilla populations reveals shared deficiencies in essential pathways, hinting at possible reasons for their uncultivable status, as well as differing biosynthetic machinery for the production of specialized metabolites. Notably, one symbiont population contains an expanded PBDE pathway, while the other lacks the pathway completely. Genome mining in the non-PBDE producing symbiont uncovered a unique pathway for the biosynthesis of aeruginoside-like compounds, the dysinosins. The hybrid sequencing and assembly approach utilized here allows, for the first time, a comprehensive look into the genomes of these elusive sponge symbionts.

Neu, AT, Allen EE, Roy K.  2019.  Diversity and composition of intertidal gastropod microbiomes across a major marine biogeographic boundary. Environ Microbiol Rep. 11:434-447.   0.1111/1758-2229.12743   Abstract

Marine biogeographic boundaries act as barriers to dispersal for many animal species, thereby creating distinctive faunas on either side. However, how such boundaries affect the distributions of microbial taxa remains poorly known. To test whether biogeographic boundaries influence the diversity and composition of host-associated microbiota, we analyzed the microbiomes of three species of common intertidal gastropods at two sites separated by the biogeographic boundary at Point Conception (PtC), CA, using 16S rRNA gene sequencing. Our results show that each host species shows microbiome compositional specificity, even across PtC, and that alpha diversity does not change significantly across this boundary for any of the gastropod hosts. However, for two of the host species, beta diversity differs significantly across PtC, indicating that there may be multiple levels of organization of the marine gastropod microbiome. Overall, our results suggest that while biogeographic boundaries do not constrain the distribution of a core set of microbes associated with each host species, they can play a role in structuring the transient portion of the microbiome.

Allemann, MN, Shulse CN, Allen EE.  2019.  Linkage of marine bacterial polyunsaturated fatty acid and long-chain hydrocarbon biosynthesis. Front Microbiol. 10:702.   10.3389/fmicb.2019.00702   Abstract

Various marine gamma-proteobacteria produce omega-3 polyunsaturated fatty acids, such as eicosapentaenoic acid (20:5, EPA) and docosahexaenoic acid (22:6, DHA), which are incorporated into membrane phospholipids. Five genes, designated pfaABCDE, encode the polyketide/fatty acid synthase necessary for production of these long-chain fatty acids. In addition to de novo biosynthesis of EPA and DHA, the “Pfa synthase” is also involved with production of a long-chain polyunsaturated hydrocarbon product (31:9, PUHC) in conjunction with the oleABCD hydrocarbon biosynthesis pathway. In this work, we demonstrate that OleA mediates the linkage between these two pathways in vivo. Co-expression of pfaA-E along with oleA from Shewanella pealeana in Escherichia coli yielded the expected product, a 31:8 ketone along with a dramatic ~10-fold reduction in EPA content. The decrease in EPA content was independent of 31:8 ketone production as co-expression of an OleA active site mutant also led to identical decreases in EPA content. We also demonstrate that a gene linked with either pfa and/or ole operons in diverse bacterial lineages, herein designated pfaT, plays a role in maintaining optimal production of Pfa synthase derived products in Photobacterium and Shewanella species.

Peoples, LM, Grammatopoulou E, Xu X, Osuntokun O, Blanton JM, Allen EE, Nunnally CC, Drazen J, Mayor DJ, Pombrol M, Bartlett DH.  2019.  Microbial community diversity within sediments from two geographically separated hadal trenches. Front Microbiol. 10:347.   10.3389/fmicb.2019.00347   Abstract

Hadal ocean sediments, found at sites deeper than 6,000 m water depth, are thought to 29 contain microbial communities distinct from those at shallower depths due to high hydrostatic 30 pressures and higher abundances of organic matter. These communities may also differ from one 31 other as a result of geographical isolation. Here we compare microbial community composition 32 in surficial sediments of two hadal environments – the Mariana and Kermadec trenches – to 33 evaluate microbial biogeography at hadal depths. Sediment microbial consortia were distinct 34 between trenches, with higher relative sequence abundances of taxa previously correlated with 35 organic matter degradation present in the Kermadec Trench. In contrast the Mariana Trench, and 36 deeper sediments in both trenches, were enriched in taxa predicted to break down recalcitrant 37 material and contained other uncharacterized lineages. At the 97% similarity level sequence38 abundant taxa were not trench-specific and were related to those found in other hadal and abyssal 39 habitats, indicating potential connectivity between geographically isolated sediments. Despite the 40 diversity of microorganisms identified using culture-independent techniques, most isolates 41 obtained under in situ pressures were related to previously identified piezophiles. Members 42 related to these same taxa also became dominant community members when native sediments 43 were incubated under static, long-term, unamended high-pressure conditions. Our results support 44 the hypothesis that there is connectivity between sediment microbial populations inhabiting the 45 Mariana and Kermadec trenches while showing that both whole communities and specific 46 microbial lineages vary between trench of collection and sediment horizon depth. This in situ 47 biodiversity is largely missed when incubating samples within pressure vessels and highlights the 48 need for revised protocols for high-pressure incubations.

Minich, JJ, Petrus S, Michael JD, Michael TP, Knight R, Allen EE.  2019.  Temporal, environmental, and biological drivers of the mucosal microbiome in a wild marine fish, Scomber japonicus. bioRxiv.   10.1101/721555   Abstract

Changing ocean conditions driven by anthropogenic activity may have a negative impact on fisheries by increasing stress and disease with the mucosal microbiome as a potentially important intermediate role. To understand how environment and host biology drives mucosal microbiomes in a marine fish, we surveyed five body sites (gill, skin, digesta, GI, and pyloric caeca) from 229 Pacific chub mackerel, Scomber japonicus, collected across 38 time points spanning one year from the Scripps Institution of Oceanography Pier, making this the largest and longest wild marine fish microbiome survey. Mucosal sites had unique communities significantly different from the surrounding sea water and sediment communities with over 10 times more diversity than sea water alone. Although, external surfaces such as skin and gill were more similar to sea water, digesta was similar to sediment. Both alpha and beta diversity of the skin and gill was explained by environmental and biological factors, especially sea surface temperature, chlorophyll a, and fish age, consistent with an exposure gradient relationship. We verified that seasonal microbial changes were not confounded by migrations of chub mackerel sub-populations by nanopore sequencing a 14 769 bp region of the 16 568 bp mitochondria. A cosmopolitan pathogen, Photobacterium damselae, was prevalent across multiple body sites all year, but highest in the skin, GI, and digesta between June and September. Our study evaluates the extent which the environment and host biology drives mucosal microbial ecology, establishing a baseline for long term monitoring surveys for linking environment stressors to mucosal health of wild marine fish.

Minich, JJ, Humphrey G, Benitez RAS, Sanders J, Swafford A, Allen EE, Knight R.  2018.  High-throughput miniaturized 16S rRNA amplicon library preparation reduces costs while preserving microbiome integrity. mSystems. 3:e00166-18.   10.1128/mSystems.00166-18   Abstract

Next Generation Sequencing technologies have enabled many advances across biology with microbial ecology benefiting primarily through expanded sample sizes. Although the cost of running sequencing instruments has decreased substantially over time, the price of library preparation methods has largely remained unchanged. In this study, we developed a low cost,
miniaturized (5 µL), high-throughput (384-sample), amplicon library preparation method with the Echo 550 acoustic liquid handler. Our method reduces costs of library preparation to $1.42 USD per sample, a 58% reduction compared to existing automated methods and a 21-fold reduction from commercial kits, without compromising sequencing success or distorting the microbial community composition analysis. We further validated the optimized method by sampling five body sites from 46 Pacific chub mackerel fish caught across 16 sampling events over seven months from the Scripps Institution of Oceanography pier in La Jolla, CA. Fish microbiome samples were processed with the miniaturized 5 µL reaction with 0.2 µL of gDNA and the standard 25 µL reaction with 1 µL of gDNA. Between the two methods, alpha diversity was highly correlated (R2>0.95), while distances of technical replicates were much lower than within body site variation (P < 0.0001), further validating the method. The cost savings of implementing the miniaturized library preparation (going from triplicate 25 µL reactions to triplicate 5 µL reactions) are large enough to cover a MiSeq sequencing run for 768 samples, while preserving accurate microbiome measurements.

Guillemette, R, Kaneko R, Blanton JM, Tan J, Witt M, Hamilton S, Allen EE, Medina M, Hamasaki K, Koch BP, Azam F.  2018.  Bacterioplankton drawdown of coral mass-spawned organic matter. ISME Journal. 12:2238-2251.   10.1038/s41396-018-0197-7   Abstract

Coral reef ecosystems are highly sensitive to microbial activities that result from dissolved organic matter (DOM) enrichment of their surrounding seawater. However, the response to particulate organic matter (POM) enrichment is less studied. In a microcosm experiment, we tested the response of bacterioplankton to a pulse of POM from the mass-spawning of Orbicella franksi coral off the Caribbean coast of Panama. Particulate organic carbon (POC), a proxy measurement for POM, increased by 40-fold in seawater samples collected during spawning; 68% of which degraded within 66 h. We hypothesized that the multiple hydrolases that were significantly elevated throughout the microcosms solubilized the spawn-derived POM into DOM. Remarkably, a carbon budget constructed for the 275 µM of degraded POC shows negligible change to the concentration of dissolved organic carbon (DOC), suggesting that the degraded POM was readily utilized. Our sensitivity analysis estimated that bacterial carbon demand (BCD) could have theoretically accounted for a large proportion of the observed POC degradation. Further, using combined bromodeoxyuridine immunocapture and 454 pyrosequencing of the 16S ribosomal RNA gene, we surmise that actively growing bacterial groups were primarily responsible for the degradation of spawn-derived POM. Additionally, Fourier transform ion cyclotron resonance mass spectrometry showed that the DOM pool became enriched with heteroatom containing molecules, a trend that suggests microbial alteration of organic matter. We conclude that coral gametes are highly labile to bacteria and that such large capacity for bacterial degradation and alteration of organic matter has implications for coral reef health and coastal marine biogeochemistry.

Plominsky, AM, Henriquez-Castillo C, Delherbe N, Podell S, Ramirez-Flandes S, Ugalde JA, Santibanez JF, van den Engh G, Hanselmann K, Ulloa O, De la Iglesia R, Allen EE, Trefault N.  2018.  Distinctive archaeal composition of an artisanal crystallizer pond and functional insights into salt-saturated hypersaline environment adaptation. Frontiers in Microbiology. 9:1800.   10.3389/fmicb.2018.01800   AbstractWebsite

Hypersaline environments represent some of the most challenging settings for life on Earth. Extremely halophilic microorganisms have been selected to colonize and thrive in these extreme environments by virtue of a broad spectrum of adaptations to counter high salinity and osmotic stress. Although there is substantial data on microbial taxonomic diversity in these challenging ecosystems and their primary osmoadaptation mechanisms, less is known about how hypersaline environments shape the genomes of microbial inhabitants at the functional level. In this study, we analyzed the microbial communities in five ponds along the discontinuous salinity gradient from brackish to salt-saturated environments and sequenced the metagenome of the salt (halite) precipitation pond in the artisanal Cahuil Solar Saltern system. We combined field measurements with spectrophotometric pigment analysis and flow cytometry to characterize the microbial ecology of the pond ecosystems, including primary producers and applied metagenomic sequencing for analysis of archaeal and bacterial taxonomic diversity of the salt crystallizer harvest pond. Comparative metagenomic analysis of the Cahuil salt crystallizer pond against microbial communities from other salt-saturated aquatic environments revealed a dominance of the archaeal genus Halorubrum and showed an unexpectedly low abundance of Haloquadratum in the Cahuil system. Functional comparison of 26 hypersaline microbial metagenomes revealed a high proportion of sequences associated with nucleotide excision repair, helicases, replication and restriction-methylation systems in all of them. Moreover, we found distinctive functional signatures between the microbial communities from salt-saturated (>30% [w/v] total salinity) compared to sub-saturated hypersaline environments mainly due to a higher representation of sequences related to replication, recombination and DNA repair in the former. The current study expands our understanding of the diversity and distribution of halophilic microbial populations inhabiting salt-saturated habitats and the functional attributes that sustain them.

Minich, JJ, Zhu Q, Xu ZZ, Amir A, Ngochera M, Simwaka M, Allen EE, Zidana H, Knight R.  2018.  Microbial effects of livestock manure fertilization on freshwater aquaculture ponds rearing tilapia (Oreochromis shiranus) and North African catfish (Clarias gariepinus). MicrobiologyOpen. 7:e00716.   10.1002/mbo3.716   Abstract

The majority of seafood is produced from farming, with most finfish coming from freshwater ponds. Across continents, aquaculture is growing fastest in Africa at 11.7 % annual growth, thus improving production for fish farmers while ensuring seafood safety for human consumption is needed. To understand the probiotic or prebiotic effects of fertilizing freshwater ponds with livestock manure, we grew tilapia and catfish together for four weeks under seven manure treatments including layer chicken, broiler chicken, guinea fowl, quail, pig, cow, and standard commercial feed only and evaluated the microbial communities of the manure, water column, tilapia and catfish feces using 16S and 18S rRNA marker genes along with whole genome sequencing. Catfish growth, but not tilapia, was positively associated with microbial activity (P=0.0006, R2=0.4887) and greatest in ponds fertilized with quail manure (ANOVA, P<0.05). Tilapia growth was highest in the broiler manure but not significant while tilapia fecal microbial richness (but not catfish) was positively correlated with microbial activity (P=0.0309, R2=0.2458). Animal manure was unique and influenced the bacterial microbiome in pond water, tilapia gut, and catfish gut and eukaryotic microbiome in pond water and catfish guts (PERMANOVA, P = 0.001). On average, 18.5%, 18.6%, and 45.3% of manure bacteria sOTUs, (sub-operational taxonomic units), were present in the water column, catfish feces, and tilapia feces which comprised 3.7%, 12.8%, and 10.9% of the total microbial richness of the communities, respectively. Antibiotic resistance genes were highest in the manure and water samples followed by tilapia feces and then lowest in catfish feces (P<0.0001). In this study we demonstrate how the bacterial and eukaryotic microbial composition of fish ponds are influenced by livestock specific manure inputs and that the gut microbiome of O. shiranus is more sensitive and responsive than C. gariepinus to these changes. Since only 13 % of the core manure bacteria could be detected in the core pond water and fish gut communities we conclude that animal manure used as fertilizer induces a primarily prebiotic effect on the pond ecosystem rather than a direct probiotic effect on fish. We identify how the tilapia gut microbiome is more influenced by environmental microbes while African catfish growth benefits more from manure fertilization.

Plominsky, AM, Trefault N, Podell S, Blanton JM, De la Iglesia R, Allen EE, von Dassow P, Ulloa O.  2018.  Metabolic potential and in situ transcriptomic profiles of previously uncharacterized key microbial groups involved in coupled carbon, nitrogen, and sulfur cycling in anoxic marine zones. Environmental Microbiology. 20:2727-2742.   10.1111/1462-2920.14109   Abstract

Anoxic marine zones (AMZs) impact biogeochemical cycles at the global scale, particularly the nitrogen cycle. Key microbial players from AMZs have been identified, but the majority remains unrecognized or uncharacterized. Thirty-one single-cell amplified genomes (SAGs) from the eastern tropical North and South Pacific AMZs were sequenced to gain insight into the distribution, metabolic potential and contribution to the community transcriptional profile of these uncharacterized bacterial and archaeal groups. Detailed analyses focused on SAG-bins assigned to three of these groups that presented 79%-100% estimated genome completeness: the putative sulphur-oxidizing Gamaproteobacteria EOSA II clade, a Marinimicrobia member of the recently recognized PN262000N21 clade found to be abundant in AMZ anoxic cores, and a representative of the Marine Benthic Group A Thaumarchaeota. Community-based analyses revealed that these three groups are significantly more abundant and transcriptionally more active in the AMZ microbial communities than previously described phylogenetically related microbial groups. Collectively, these groups have the potential to link biogeochemically relevant processes by coupling the carbon, nitrogen and sulfur cycles. Together, these results increase our understanding of key microbial components inhabiting AMZs and other oxygen-deficient marine environments, enhancing our capacity to predict the impact of the expansion of these ecosystems due to climate change.

Allemann, MN, Allen EE.  2018.  Characterization and application of marine microbial omega-3 polyunsaturated fatty acid synthesis. Methods in Enzymology. 605:3-32.   10.1016/bs.mie.2018.02.018   Abstract

The long-chain omega-3 polyunsaturated fatty acids (n-3 LC-PUFAs) EPA (20:5n-3) and DHA (22:6n-3) are widely recognized as beneficial to human health and development. Select lineages of cosmopolitan marine prokaryotic and eukaryotic microorganisms synthesize these compounds via a unique fatty acid synthase/polyketide synthase mechanism that is distinct from the canonical desaturase/elongase-mediated pathway employed by the majority of eukaryotic single cell microorganisms and metazoans. This “Pfa synthase” mechanism is highly efficient and has been co-opted for the large-scale industrial production of n-3 LC-PUFAs for commercial applications. Both prokaryotic and eukaryotic microbes containing this pathway can be readily isolated from marine environments and maintained in culture under laboratory conditions. Some strains are genetically tractable and have established methods for genetic modification. The discussion and methods presented here should be useful for the exploitation and optimization of n-3 LC-PUFA products from marine microorganisms.

Minich, J, Zhu Q, Janssen S, Hendrickson R, Amir A, Vetter R, Hyde J, Doty M, Stillwell K, Benardini J, Kim J, Allen EE, Venkateswaran K, Knight R.  2018.  KatharoSeq enables high-throughput microbiome analysis from low-biomass samples. mSystems. 3:e00218-17.   10.1128/mSystems.00218-17   Abstract

Microbiome analyses of low-biomass samples are challenging because of
contamination and inefficient amplification, leading many investigators to employ low-
throughput methods with minimal controls. We developed a new automated protocol,
KatharoSeq (from the Greek katharos, “clean”), that outperforms single tube extractions
while processing four times faster. KatharoSeq incorporates positive and negative
controls to reveal the whole bacterial community from inputs of as little as 50 cells, and
correctly identifies 90.6% (S.E. 0.013) of reads from 500 cells. To demonstrate the
broad utility of KatharoSeq, we performed 16S rRNA amplicon (“16S” below) and
shotgun metagenome (“shotgun” below) analyses of the Jet Propulsion Lab Spacecraft
Assembly Facility, SAF, (n=192, 96), 52 rooms from a Neonatal Intensive Care Unit,
NICU, (n=388, 337), and an endangered abalone rearing facility (n=192, 123), obtaining
spatially resolved, unique microbiomes, reproducible across hundreds of samples. The
SAF, our primary focus, contains thirty two sOTUs (sub-OTUs, defined as exact
sequence matches and their inferred variants identified by the deblur algorithm with four
(Acinetobacter lwoffi, Paracoccus marcusii, Mycobacterium sp., and Novosphingobium)
being present in over 75 % of the samples. Using microbial spatial topography, the most
abundant cleanroom contaminant, A. lwoffi, is related to human foot traffic exposure. In
the NICU, we predict a patient disease outcome from the built environment, and in the
abalone facility, we show that microbial communities reflect the marine environment
rather than human input. Consequently, we demonstrate the feasibility and utility of
large-scale, low biomass metagenomics analyses using the KatharoSeq protocol.

Peoples, LM, Donaldson S, Osuntokun O, Xia Q, Nelson A, Blanton JM, Allen EE, Church MJ, Bartlett DH.  2018.  Vertically distinct microbial communities in the Mariana and Kermadec trenches. PLoS One. 13(4):e0195102.   10.1371/journal.pone.0195102   Abstract

Hadal trenches, oceanic locations deeper than 6500 m, are thought to have distinct microbial communities compared to those at shallower depths due to high hydrostatic pressures, topographical funneling of organic matter, and biogeographical isolation. Here we evaluate the hypothesis that hadal trenches contain unique microbial biodiversity through analyses of the communities present in the bottom waters of the Kermadec and Mariana trenches. Estimates of microbial protein production indicate active populations under in situ hydrostatic pressures and increasing adaptation to pressure with depth. Depth, trench, and size fraction were important drivers of microbial community structure. Many putative hadal bathytypes, such as members of the Marinimicrobia, Rhodobacteraceae, Aquibacter, and Defluviicoccus, are similar to members identified in other trenches. Most of the differences between the two trench microbiomes consists of taxa belonging to the Gammaproteobacteria whose distributions extend throughout the water column. Growth and survival estimates of representative isolates of these taxa under deep-sea conditions suggest they may descend from shallower depths and exist as an abundant but potentially inactive fraction of the hadal zone. We conclude that the distinct pelagic communities residing in these two trenches, and perhaps by extension other trenches, reflect cosmopolitan hadal bathytypes and allochthonous inputs from above.

Leao, T, Castelao G, Korobeynikov A, Monroe EA, Podell S, Glukhov E, Allen EE, Gerwick WH, Gerwick L.  2017.  Comparative genomics uncovers the prolific and distinctive metabolic potential of the cyanobacterial genus Moorea. Proc Natl Acad Sci U.S.A.. 114:3198-3203.   10.1073/pnas.1618556114   AbstractWebsite

Cyanobacteria are major sources of oxygen, nitrogen, and carbon in nature. In addition to the importance of their primary metabolism, some cyanobacteria are prolific producers of unique and bioactive secondary metabolites. Chemical investigations of the cyanobacterial genus Moorea have resulted in the isolation of over 190 compounds in the last two decades. However, preliminary genomic analysis has suggested that genome-guided approaches can enable the discovery of novel compounds from even well-studied Moorea strains, highlighting the importance of obtaining complete genomes. We report a complete genome of a filamentous tropical marine cyanobacterium, Moorea producens PAL, which reveals that about one-fifth of its genome is devoted to production of secondary metabolites, an impressive four times the cyanobacterial average. Moreover, possession of the complete PAL genome has allowed improvement to the assembly of three other Moorea draft genomes. Comparative genomics revealed that they are remarkably similar to one another, despite their differences in geography, morphology, and secondary metabolite profiles. Gene cluster networking highlights that this genus is distinctive among cyanobacteria, not only in the number of secondary metabolite pathways but also in the content of many pathways, which are potentially distinct from all other bacterial gene clusters to date. These findings portend that future genome-guided secondary metabolite discovery and isolation efforts should be highly productive.

Demidenko, A, Akberdin IR, Allemann M, Allen EE, Kalyuzhnaya MG.  2017.  Fatty acid biosynthesis pathways in Methylomicrobium buryatense 5G(B1). Front Microbiol. 7:2167.   10.3389/fmicb.2016.02167   Abstract

Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.

Agarwal, V, Blanton JM, Podell S, Taton A, Schorn MA, Busch J, Lin Z, Schmidt EW, Jensen PR, Biggs JS, Golden JW, Allen EE, Moore BS.  2017.  Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges. Nature Chemical Biology. 13:537-543.   10.1038/nchembio.2330   Abstract

Naturally produced polybrominated diphenyl ethers (PBDEs) pervade the marine environment and structurally resemble toxic man-made brominated flame retardants. PBDEs bioaccumulate in marine animals and are likely transferred to the human food chain. However, the biogenic basis for PBDE production in one of their most prolific sources, marine sponges of the order Dysideidae, remains unidentified. Here, we report the discovery of PBDE biosynthetic gene clusters within sponge-microbiome-associated cyanobacterial endosymbionts through the use of an unbiased metagenome-mining approach. Using expression of PBDE biosynthetic genes in heterologous cyanobacterial hosts, we correlate the structural diversity of naturally produced PBDEs to modifications within PBDE biosynthetic gene clusters in multiple sponge holobionts. Our results establish the genetic and molecular foundation for the production of PBDEs in one of the most abundant natural sources of these molecules, further setting the stage for a metagenomic-based inventory of other PBDE sources in the marine environment.

Paerl, RW, Bouget F-Y, Lozano J-C, Verge V, Schatt P, Allen EE, Palenik BP, Azam F.  2016.  Use of plankton-derived vitamin B1 precursors, especially thiazole-related precursor, by key marine picoeukaryotic phytoplankton. ISME Journal. 11:753-765.   10.1038/ismej.2016.145   Abstract

Several cosmopolitan marine picoeukaryotic phytoplankton are B1 auxotrophs requiring exogenous vitamin B1 or precursor to survive. From genomic evidence, representatives of picoeukaryotic phytoplankton (Ostreococcus and Micromonas spp.) were predicted to use known thiazole and pyrimidine B1 precursors to meet their B1 demands, however, recent culture-based experiments could not confirm this assumption. We hypothesized these phytoplankton strains could grow on precursors alone, but required a thiazole-related precursor other the well-known and extensively tested 4-methyl-5-thiazoleethanol. This hypothesis was tested using bioassays and co-cultures of picoeukaryotic phytoplankton and bacteria. We found that specific B1-synthesizing proteobacteria and phytoplankton are sources of a yet-to-be chemically identified thiazole-related precursor(s) that, along with pyrimidine B1 precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine, can support growth of Ostreococcus spp. (also Micromonas spp.) without B1. We additionally found that the B1-synthesizing plankton do not require contact with picoeukaryotic phytoplankton cells to produce thiazole-related precursor(s). Experiments with wild-type and genetically engineered Ostreococcus lines revealed that the thiazole kinase, ThiM, is required for growth on precursors, and that thiazole-related precursor(s) accumulate to appreciable levels in the euphotic ocean. Overall, our results point to thiazole-related B1 precursors as important micronutrients promoting the survival of abundant phytoplankton influencing surface ocean production and biogeochemical cycling.

Traller, JC, Cokus SJ, Lopez DA, Gaidarenko O, Smith SR, McCrow JP, Gallaher SD, Podell S, Thompson M, Cook O, Morselli M, Jaroszewicz A, Allen EE, Allen AE, Merchant SS, Pellegrini M, Hildebrand M.  2016.  Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype. Biotechnology for Biofuels. 9:258.   10.1186/s13068-016-0670-3   Abstract

Improvement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom Cyclotella cryptica, which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids.
We sequenced the nuclear genome and methylome of this oleaginous diatom to identify the genetic traits that enable substantial accumulation of triacylglycerol. The genome is comprised of highly methylated repetitive sequence, which does not significantly change under silicon starved lipid induction, and data further suggests the primary role of DNA methylation is to suppress DNA transposition. Annotation of pivotal glycolytic, lipid metabolism, and carbohydrate degradation processes reveal an expanded enzyme repertoire in C. cryptica that would allow for an increased metabolic capacity toward triacylglycerol production. Identification of previously unidentified genes, including those involved in carbon transport and chitin metabolism, provide potential targets for genetic manipulation of carbon flux to further increase its lipid phenotype. New genetic tools were developed, bringing this organism on a par with other microalgae in terms of genetic manipulation and characterization approaches.
Functional annotation and detailed cross-species comparison of key carbon rich processes in C. cryptica highlights the importance of enzymatic subcellular compartmentation for regulation of carbon flux, which is often overlooked in photosynthetic microeukaryotes. The availability of the genome sequence, as well as advanced genetic manipulation tools enable further development of this organism for deployment in large-scale production systems.

Hogle, SL, Bundy RM, Blanton JM, Allen EE, Barbeau KA.  2016.  Copiotrophic marine bacteria are associated with strong iron-binding ligand production during phytoplankton blooms. Limnology and Oceanography Letters. 1:36-43.   10.1002/lol2.10026   Abstract

Although marine bacteria were identified nearly two decades ago as potential sources for strong iron-binding organic ligands detected in seawater, specific linkages between ligands detected in natural water and the microbial community remain unclear. We compared the production of different classes of iron-binding ligands, dissolved iron and macronutrient concentrations, and phytoplankton and bacterioplankton assemblages in a series of iron amended 6-d incubations. Incubations with high iron additions had near complete macronutrient consumption and higher phytoplankton biomass compared with incubations with low iron additions, but both iron treatments were dominated by diatoms. However, we only detected the strongest ligands in high-iron treatments, and strong iron-binding ligands were generally correlated with an increased abundance of copiotrophic bacteria, particularly Alteromonas strains. Ultimately, these robust correlations suggest a potential linkage between copiotrophic bacteria and strong iron-binding ligand production after iron fertilization events in the marine environment.

Fuentes-Valdes, JJ, Plominsky AM, Allen EE, Tamames J, Vasquez M.  2016.  Complete genome sequence of a cylindrospermopsin-producing cyanobacterium, Cylindrospermopsis raciborskii CS505, containing a circular chromosome and a single extrachromosomal element. Genome Announcements. 4:e00823-16.   10.1128/genomeA.00823-16   Abstract

Cylindrospermopsis raciborskii is a freshwater cyanobacterium producing bloom events and toxicity in drinking water source reservoirs. We present the first genome sequence for C. raciborskii CS505 (Australia), containing one 4.1-Mbp chromosome and one 110-Kbp plasmid having G+C contents of 40.3% (3933 genes) and 39.3% (111 genes), respectively.

Andrade, K, Logemann J, Heidelberg KB, Emerson JB, Comolli LR, Hug LA, Probst AJ, Keillar A, Thomas BC, Miller CS, Allen EE, Moreau JW, Brocks JJ, Banfield JF.  2015.  Metagenomic and lipid analyses reveal a diel cycle in a hypersaline microbial ecosystem. ISME Journal. 9:2697-2711.   10.1038/ismej.2015.66   AbstractWebsite

Marine microbial communities experience daily fluctuations in light and temperature that can have important ramifications for carbon and nutrient cycling. Elucidation of such short time scale community-wide dynamics is hindered by system complexity. Hypersaline aquatic environments have lower species richness than marine environments and can be well-defined spatially, hence they provide a model system for diel cycle analysis. We conducted a 3-day time series experiment in a well-defined pool in hypersaline Lake Tyrrell, Australia. Microbial communities were tracked by combining cultivation-independent lipidomic, metagenomic and microscopy methods. The ratio of total bacterial to archaeal core lipids in the planktonic community increased by up to 58% during daylight hours and decreased by up to 32% overnight. However, total organism abundances remained relatively consistent over 3 days. Metagenomic analysis of the planktonic community composition, resolved at the genome level, showed dominance by Haloquadratum species and six uncultured members of the Halobacteriaceae. The post 0.8 mu m filtrate contained six different nanohaloarchaeal types, three of which have not been identified previously, and cryo-transmission electron microscopy imaging confirmed the presence of small cells. Notably, these nano-sized archaea showed a strong diel cycle, with a pronounced increase in relative abundance over the night periods. We detected no eukaryotic algae or other photosynthetic primary producers, suggesting that carbon resources may derive from patchily distributed microbial mats at the sediment-water interface or from surrounding land. Results show the operation of a strong community-level diel cycle, probably driven by interconnected temperature, light abundance, dissolved oxygen concentration and nutrient flux effects.

Tully, BJ, Emerson JB, Andrade K, Brocks JJ, Allen EE, Banfield JF, Heidelberg KB.  2015.  De novo sequences of Haloquadratum walsbyi from Lake Tyrrell, Australia, reveal a variable genomic landscape. Archaea. 2015:875784.   10.1155/2015/875784   AbstractWebsite

Hypersaline systems near salt saturation levels represent an extreme environment, in which organisms grow and survive near the limits of life. One of the abundant members of the microbial communities in hypersaline systems is the square archaeon, Haloquadratum walsbyi. Utilizing a short-read metagenome from Lake Tyrrell, a hypersaline ecosystem in Victoria, Australia, we performed a comparative genomic analysis of H. walsbyi to better understand the extent of variation between strains/subspecies. Results revealed that previously isolated strains/subspecies do not fully describe the complete repertoire of the genomic landscape present in H. walsbyi. Rearrangements, insertions, and deletions were observed for the Lake Tyrrell derived Haloquadratum genomes and were supported by environmental de novo sequences, including shifts in the dominant genomic landscape of the two most abundant strains. Analysis pertaining to halomucins indicated that homologs for this large protein are not a feature common for all species of Haloquadratum. Further, we analyzed ATP-binding cassette transporters (ABC-type transporters) for evidence of niche partitioning between different strains/subspecies. We were able to identify unique and variable transporter subunits from all five genomes analyzed and the de novo environmental sequences, suggesting that differences in nutrient and carbon source acquisition may play a role in maintaining distinct strains/subspecies.

Marietou, A, Nguyen ATT, Allen EE, Bartlett D.  2015.  Adaptive laboratory evolution of Escherichia coli K-12 MG1655 for growth at high hydrostatic pressure. Front Microbiol. 5:749.   10.3389/fmicb.2014.00749   AbstractWebsite

Much of microbial life on Earth grows and reproduces under the elevated hydrostatic pressure conditions that exist in deep-ocean and deep-subsurface environments. In this study adaptive laboratory evolution (ALE) experiments were conducted to investigate the possible modification of the piezosensitive Escherichia coli for improved growth at high pressure. After approximately 500 generations of selection, a strain was isolated that acquired the ability to grow at pressure non-permissive for the parental strain. Remarkably, this strain displayed growth properties and changes in the proportion and regulation of unsaturated fatty acids that indicated the acquisition of multiple piezotolerant properties. These changes developed concomitantly with a change in the gene encoding the acyl carrier protein, which is required for fatty acid synthesis.