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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. Proceedings of the National Academy of Sciences of the United States of America. 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.

Agarwal, V, Blanton JM, Podell S, Taton A, Schorn MA, Busch J, Lin Z, Schmidt EW, Jensen PR, Paul VJ, Biggs JS, Golden JW, Allen EE, Moore BS.  2017.  Metagenomic discovery of polybrominated diphenyl ether biosynthesis by marine sponges. Nat Chem Biol. advance online publication: Nature Publishing Group, a division of Macmillan Publishers Limited. All Rights Reserved.   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.

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.

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. Adv Online Pub(9 Dec 2016)   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.   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. Aug 25; 4(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. Frontiers in Microbiology. 5   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.

Plominsky, AM, Delherbe N, Ugalde JA, Allen EE, Blanchet M, Ikeda P, Santibañez F, Hanselmann K, Ulloa O, De la Iglesia R, von Dassow P, Astorga M, Gálvez MJ, González ML, Henríquez-Castillo C, Vaulot D, Lopes do Santos A, van den Engh G, Gimpel C, Bertoglio F, Delgado Y, Docmac F, Elizondo-Patrone C, Narváez S, Sorroche F, Rojas-Herrera M, Trefault N.  2014.  Metagenome sequencing of the microbial community of a solar saltern crystallizer pond at Cáhuil Lagoon, Chile. Genome Announcements. 2   10.1128/genomeA.01172-14   AbstractWebsite

Cáhuil Lagoon in central Chile harbors distinct microbial communities in various solar salterns that are arranged as interconnected ponds with increasing salt concentrations. Here, we report the metagenome of the 3.0- to 0.2-µm fraction of the microbial community present in a crystallizer pond with 34% salinity.

Agarwal, V, Gamal AEA, Yamanaka K, Poth D, Kersten RD, Schorn M, Allen EE, Moore BS.  2014.  Biosynthesis of polybrominated aromatic organic compounds by marine bacteria. Nature Chemical Biology. 10:640-647.   10.1038/nchembio.1564   Abstract

Polybrominated diphenyl ethers (PBDEs) and polybrominated bipyrroles are natural products that bioaccumulate in the marine food chain. PBDEs have attracted widespread attention because of their persistence in the environment and potential toxicity to humans. However, the natural origins of PBDE biosynthesis are not known. Here we report marine bacteria as producers of PBDEs and establish a genetic and molecular foundation for their production that unifies paradigms for the elaboration of bromophenols and bromopyrroles abundant in marine biota. We provide biochemical evidence of marine brominases revealing decarboxylative-halogenation enzymology previously unknown among halogenating enzymes. Biosynthetic motifs discovered in our study were used to mine sequence databases to discover unrealized marine bacterial producers of organobromine compounds

Coates, CR, Podell S, Korobeynikov A, Lapidus A, Pevzner P, Sherman DH, Allen EE, Gerwick L, Gerwick WH.  2014.  Characterization of cyanobacterial hydrocarbon composition and distribution of biosynthetic pathways. PLoS One. 9(1):e85140.   10.1371/journal.pone.0085140   Abstract

Cyanobacteria possess the unique capacity to naturally produce hydrocarbons from fatty acids. Hydrocarbon compositions of thirty-two strains of cyanobacteria were characterized to reveal novel structural features and insights into hydrocarbon biosynthesis in cyanobacteria. This investigation revealed new double bond (2- and 3-heptadecene) and methyl group positions (3-, 4- and 5-methylheptadecane) for a variety of strains. Additionally, results from this study and literature reports indicate that hydrocarbon production is a universal phenomenon in cyanobacteria. All cyanobacteria possess the capacity to produce hydrocarbons from fatty acids yet not all accomplish this through the same metabolic pathway. One pathway comprises a two-step conversion of fatty acids first to fatty aldehydes and then alkanes that involves a fatty acyl ACP reductase (FAAR) and aldehyde deformylating oxygenase (ADO). The second involves a polyketide synthase (PKS) pathway that first elongates the acyl chain followed by decarboxylation to produce a terminal alkene (olefin synthase, OLS). Sixty-one strains possessing the FAAR/ADO pathway and twelve strains possessing the OLS pathway were newly identified through bioinformatic analyses. Strains possessing the OLS pathway formed a cohesive phylogenetic clade with the exception of three Moorea strains and Leptolyngbya sp. PCC 6406 which may have acquired the OLS pathway via horizontal gene transfer. Hydrocarbon pathways were identified in one-hundred-forty-two strains of cyanobacteria over a broad phylogenetic range and there were no instances where both the FAAR/ADO and the OLS pathways were found together in the same genome, suggesting an unknown selective pressure maintains one or the other pathway, but not both.

Podell, S, Emerson JB, Jones CM, Ugalde JA, Welch S, Heidelberg KB, Banfield JF, Allen EE.  2014.  Seasonal fluctuations in ionic concentrations drive microbial succession in a hypersaline lake community. ISME Journal. 8(5):979-990.   10.1038/ismej.2013.221   Abstract

Microbial community succession was examined over a two year period using spatially and temporally coordinated water chemistry measurements, metagenomic sequencing, phylogenetic binning, and de novo metagenomic assembly in the extreme hypersaline habitat of Lake Tyrrell, Victoria Australia. Relative abundances of Haloquadratum-related sequences were positively correlated with co-varying concentrations of potassium, magnesium, and sulfate, but not sodium, chloride, or calcium ions, while relative abundances of Halorubrum, Haloarcula, Halonotius, Halobaculum, and Salinibacter-related sequences correlated negatively with Haloquadratum and these same ionic factors. Nanohaloarchaea and Halorhabdus-related sequence abundances were inversely correlated with each other, but not other taxonomic groups. These data, along with predicted gene functions from nearly-complete assembled population metagenomes, suggest different ecological phenotypes for Nanohaloarchaea and Halorhabdus-related strains versus other community members. Nucleotide percent G+C compositions were consistently lower in community metagenomic reads from summer versus winter samples. The same seasonal G+C trends were observed within taxonomically binned read subsets from each of seven different genus-level archaeal groups. Relative seasonal abundances were also linked to percent G+C for assembled population genomes. Together, these data suggest that extreme ionic conditions may exert selective pressure on archaeal populations at the level of genomic nucleotide composition, thus contributing to seasonal successional processes. Despite the unavailability of cultured representatives for most of the organisms identified in this study, effective coordination of physical and biological measurements has enabled discovery and quantification of unexpected taxon-specific, environmentally mediated factors influencing microbial community structure.

Podell, S, Ugalde JA, Narasingarao P, Banfield JF, Heidelberg KB, Allen EE.  2013.  Assembly- Driven Community Genomics of a Hypersaline Microbial Ecosystem. Plos One. 8(4):e61692.   10.1371/journal.pone.0061692   AbstractWebsite

Microbial populations inhabiting a natural hypersaline lake ecosystem in Lake Tyrrell, Victoria, Australia, have been characterized using deep metagenomic sampling, iterative de novo assembly, and multidimensional phylogenetic binning. Composite genomes representing habitat-specific microbial populations were reconstructed for eleven different archaea and one bacterium, comprising between 0.6 and 14.1% of the planktonic community. Eight of the eleven archaeal genomes were from microbial species without previously cultured representatives. These new genomes provide habitat-specific reference sequences enabling detailed, lineage-specific compartmentalization of predicted functional capabilities and cellular properties associated with both dominant and less abundant community members, including organisms previously known only by their 16S rRNA sequences. Together, these data provide a comprehensive, culture-independent genomic blueprint for ecosystem-wide analysis of protein functions, population structure, and lifestyles of co-existing, co-evolving microbial groups within the same natural habitat. The "assembly-driven'' community genomic approach demonstrated in this study advances our ability to push beyond single gene investigations, and promotes genome-scale reconstructions as a tangible goal in the quest to define the metabolic, ecological, and evolutionary dynamics that underpin environmental microbial diversity.

Ugalde, JA, Gallardo MJ, Belmar C, Munoz P, Ruiz-Tagle N, Ferrada-Fuentes S, Espinoza C, Allen EE, Gallardo VA.  2013.  Microbial life in a fjord: Metagenomic analysis of a microbial mat in Chilean Patagonia. Plos One. 8   10.1371/journal.pone.0071952   AbstractWebsite

The current study describes the taxonomic and functional composition of metagenomic sequences obtained from a filamentous microbial mat isolated from the Comau fjord, located in the northernmost part of the Chilean Patagonia. The taxonomic composition of the microbial community showed a high proportion of members of the Gammaproteobacteria, including a high number of sequences that were recruited to the genomes of Moritella marina MP-1 and Colwellia psycherythraea 34H, suggesting the presence of populations related to these two psychrophilic bacterial species. Functional analysis of the community indicated a high proportion of genes coding for the transport and metabolism of amino acids, as well as in energy production. Among the energy production functions, we found protein-coding genes for sulfate and nitrate reduction, both processes associated with Gammaproteobacteria-related sequences. This report provides the first examination of the taxonomic composition and genetic diversity associated with these conspicuous microbial mat communities and provides a framework for future microbial studies in the Comau fjord.

Emerson, JB, Andrade K, Thomas BC, Norman A, Allen EE, Heidelberg KB, Banfield JF.  2013.  Virus-host and CRISPR dynamics in archaea-dominated hypersaline Lake Tyrrell, Victoria, Australia. Archaea. 2013:370871.   10.1155/2013/370871   AbstractWebsite

The study of natural archaeal assemblages requires community context, namely, a concurrent assessment of the dynamics of archaeal, bacterial, and viral populations. Here, we use filter size-resolved metagenomic analyses to report the dynamics of 101 archaeal and bacterial OTUs and 140 viral populations across 17 samples collected over different timescales from 2007-2010 from Australian hypersaline Lake Tyrrell (LT). All samples were dominated by Archaea (75-95%). Archaeal, bacterial, and viral populations were found to be dynamic on timescales of months to years, and different viral assemblages were present in planktonic, relative to host-associated (active and provirus) size fractions. Analyses of clustered regularly interspaced short palindromic repeat (CRISPR) regions indicate that both rare and abundant viruses were targeted, primarily by lower abundance hosts. Although very few spacers had hits to the NCBI nr database or to the 140 LT viral populations, 21% had hits to unassembled LT viral concentrate reads. This suggests local adaptation to LT-specific viruses and/or undersampling of haloviral assemblages in public databases, along with successful CRISPR-mediated maintenance of viral populations at abundances low enough to preclude genomic assembly. This is the first metagenomic report evaluating widespread archaeal dynamics at the population level on short timescales in a hypersaline system.

Kharbush, JJ, Ugalde JA, Hogle SL, Allen EE, Aluwihare LI.  2013.  Composite bacterial hopanoids and their microbial producers across oxygen gradients in the water column of the California Current. Applied and Environmental Microbiology. Published ahead of print 27 September 2013   doi:10.1128/AEM.02367-13   Abstract

Hopanoids are pentacyclic triterpenoid lipids produced by many prokaryotes as cell membrane components. The structural variations of composite hopanoids, or bacteriohopanepolyols (BHPs), produced by various bacterial genera makes them potentially useful molecular biomarkers of bacterial communities and metabolic processes, in both modern and ancient environments. Building on previous work suggesting that organisms in low-oxygen environments are important contributors to BHP production in the marine water column and that there may be physiological roles for BHPs specific to these environments, this study investigated the relationship between trends in BHP structural diversity and abundance and the genetic
diversity of BHP producers for the first time in a low-oxygen environment of the Eastern Tropical North Pacific. Amplification of the hopanoid biosynthesis gene, squalene hopene cyclase (sqhC), indicated far greater genetic diversity than would be predicted by examining BHP structural diversity alone, and that greater sqhC genetic diversity exists in the marine environment than is represented by cultured representatives and most marine metagenomes. In addition, the genetic relationships in this dataset suggest microaerophilic environments as potential “hot spots” of BHP production. Finally, structural analysis of BHPs showed that an
isomer of the commonly-observed BHP bacteriohopanetetrol (BHT) may be linked to a producer that is more abundant in low-oxygen environments. Results of this study increase the known diversity of BHP producers and provide a detailed phylogeny with implications for the role of hopanoids in modern bacteria as well as the evolutionary history of hopanoid biosynthesis, both of which are important considerations for future interpretations of the marine sedimentary record.

Ugalde, JA, Narasingarao P, Kuo S, Podell S, Allen EE.  2013.  Draft genome sequence of "Candidatus Halobonum tyrrellensis" strain G22 isolated from the hypersaline waters of Lake Tyrrell, Australia. Genome Announc. 1(6):e01001-13.   10.1128/genomeA.01001-13   Abstract

We report the draft 3.675-Mbp genome sequence of “Candidatus Halobonum tyrrellensis” strain G22, a novel halophilic archaeon isolated from the surface hypersaline waters of Lake Tyrrell, Australia. The availability of the first genome from the “Candidatus Halobonum” genus provides a new genomic resource for the comparative genomic analysis of halophilic Archaea.

Summers, MM, Katz S, Allen EE, Rouse GW.  2013.  Association of rhizobia with a marine polychaete. Environmental Microbiology Reports. 5:492-498.   10.1111/1758-2229.12043   AbstractWebsite

We report the presence of Mesorhizobium, a genus best known for its nitrogen-fixing symbiosis with terrestrial legumes, associated with the marine polychaete Meganerilla bactericola (Annelida: Nerillidae). Abundant epibionts were previously described as coating the exterior of M. bactericola, which is found within the anoxic sulfide-oxidizing microbial mats of the Santa Barbara Basin, California, USA. 16S rRNA investigation of the bacterial community associated with this polychaete discovered the presence of bacteria belonging to Mesorhizobium. We identified these bacteria using phylogenetic analyses of 16S rRNA and three additional functional genes, nifH, atpD and recA, and group-specific fluorescence in situ hybridization (FISH).

Emerson, JB, Thomas BC, Andrade K, Allen EE, Heidelberg KB, Banfield JF.  2012.  Dynamic Viral Populations in Hypersaline Systems as Revealed by Metagenomic Assembly. Applied and Environmental Microbiology. 78:6309-6320.   10.1128/aem.01212-12   AbstractWebsite

Viruses of the Bacteria and Archaea play important roles in microbial evolution and ecology, and yet viral dynamics in natural systems remain poorly understood. Here, we created de novo assemblies from 6.4 Gbp of metagenomic sequence from eight community viral concentrate samples, collected from 12 h to 3 years apart from hypersaline Lake Tyrrell (LT), Victoria, Australia. Through extensive manual assembly curation, we reconstructed 7 complete and 28 partial novel genomes of viruses and virus-like entities (VLEs, which could be viruses or plasmids). We tracked these 35 populations across the eight samples and found that they are generally stable on the timescale of days and transient on the timescale of years, with some exceptions. Cross-detection of the 35 LT populations in three previously described haloviral metagenomes was limited to a few genes, and most previously sequenced haloviruses were not detected in our samples, though 3 were detected upon reducing our detection threshold from 90% to 75% nucleotide identity. Similar results were obtained when we applied our methods to haloviral metagenomic data previously reported from San Diego, CA: 10 contigs that we assembled from that system exhibited a variety of detection patterns on a timescale of weeks to 1 month but were generally not detected in LT. Our results suggest that most haloviral populations have a limited or, possibly, a temporally variable global distribution. This study provides high-resolution insight into viral biogeography and dynamics and it places "snapshot" viral metagenomes, collected at a single time and location, in context.

Ziemert, N, Podell S, Penn K, Badger JH, Allen EE, Jensen PR.  2012.  The Natural Product Domain Seeker NaPDoS: A Phylogeny Based Bioinformatic Tool to Classify Secondary Metabolite Gene Diversity. Plos One. 7   10.1371/journal.pone.0034064   AbstractWebsite

New bioinformatic tools are needed to analyze the growing volume of DNA sequence data. This is especially true in the case of secondary metabolite biosynthesis, where the highly repetitive nature of the associated genes creates major challenges for accurate sequence assembly and analysis. Here we introduce the web tool Natural Product Domain Seeker (NaPDoS), which provides an automated method to assess the secondary metabolite biosynthetic gene diversity and novelty of strains or environments. NaPDoS analyses are based on the phylogenetic relationships of sequence tags derived from polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) genes, respectively. The sequence tags correspond to PKS-derived ketosynthase domains and NRPS-derived condensation domains and are compared to an internal database of experimentally characterized biosynthetic genes. NaPDoS provides a rapid mechanism to extract and classify ketosynthase and condensation domains from PCR products, genomes, and metagenomic datasets. Close database matches provide a mechanism to infer the generalized structures of secondary metabolites while new phylogenetic lineages provide targets for the discovery of new enzyme architectures or mechanisms of secondary metabolite assembly. Here we outline the main features of NaPDoS and test it on four draft genome sequences and two metagenomic datasets. The results provide a rapid method to assess secondary metabolite biosynthetic gene diversity and richness in organisms or environments and a mechanism to identify genes that may be associated with uncharacterized biochemistry.

Allen, LZ, Allen EE, Badger JH, McCrow JP, Paulsen IT, Elbourne LDH, Thiagarajan M, Rusch DB, Nealson KH, Williamson SJ, Venter JC, Allen AE.  2012.  Influence of nutrients and currents on the genomic composition of microbes across an upwelling mosaic. ISME Journal. 6:1403-1414.   10.1038/ismej.2011.201   AbstractWebsite

Metagenomic data sets were generated from samples collected along a coastal to open ocean transect between Southern California Bight and California Current waters during a seasonal upwelling event, providing an opportunity to examine the impact of episodic pulses of cold nutrient-rich water into surface ocean microbial communities. The data set consists of similar to 5.8 million predicted proteins across seven sites, from three different size classes: 0.1-0.8, 0.8-3.0 and 3.0-200.0 mu m. Taxonomic and metabolic analyses suggest that sequences from the 0.1-0.8 mu m size class correlated with their position along the upwelling mosaic. However, taxonomic profiles of bacteria from the larger size classes (0.8-200 mu m) were less constrained by habitat and characterized by an increase in Cyanobacteria, Bacteroidetes, Flavobacteria and double-stranded DNA viral sequences. Functional annotation of transmembrane proteins indicate that sites comprised of organisms with small genomes have an enrichment of transporters with substrate specificities for amino acids, iron and cadmium, whereas organisms with larger genomes have a higher percentage of transporters for ammonium and potassium. Eukaryotic-type glutamine synthetase (GS) II proteins were identified and taxonomically classified as viral, most closely related to the GSII in Mimivirus, suggesting that marine Mimivirus-like particles may have played a role in the transfer of GSII gene functions. Additionally, a Planctomycete bloom was sampled from one upwelling site providing a rare opportunity to assess the genomic composition of a marine Planctomycete population. The significant correlations observed between genomic properties, community structure and nutrient availability provide insights into habitat-driven dynamics among oligotrophic versus upwelled marine waters adjoining each other spatially. The ISME Journal (2012) 6, 1403-1414; doi:10.1038/ismej.2011.201; published online 26 April 2012

Narasingarao, P, Podell S, Ugalde JA, Brochier-Armanet C, Emerson JB, Brocks JJ, Heidelberg KB, Banfield JF, Allen EE.  2012.  De novo metagenomic assembly reveals abundant novel major lineage of Archaea in hypersaline microbial communities. ISME Journal. 6:81-93.   10.1038/ismej.2011.78   AbstractWebsite

This study describes reconstruction of two highly unusual archaeal genomes by de novo metagenomic assembly of multiple, deeply sequenced libraries from surface waters of Lake Tyrrell (LT), a hypersaline lake in NW Victoria, Australia. Lineage-specific probes were designed using the assembled genomes to visualize these novel archaea, which were highly abundant in the 0.1–0.8 μm size fraction of lake water samples. Gene content and inferred metabolic capabilities were highly dissimilar to all previously identified hypersaline microbial species. Distinctive characteristics included unique amino acid composition, absence of Gvp gas vesicle proteins, atypical archaeal metabolic pathways and unusually small cell size (approximately 0.6 μm diameter). Multi-locus phylogenetic analyses demonstrated that these organisms belong to a new major euryarchaeal lineage, distantly related to halophilic archaea of class Halobacteria. Consistent with these findings, we propose creation of a new archaeal class, provisionally named ‘Nanohaloarchaea’. In addition to their high abundance in LT surface waters, we report the prevalence of Nanohaloarchaea in other hypersaline environments worldwide. The simultaneous discovery and genome sequencing of a novel yet ubiquitous lineage of uncultivated microorganisms demonstrates that even historically well-characterized environments can reveal unexpected diversity when analyzed by metagenomics, and advances our understanding of the ecology of hypersaline environments and the evolutionary history of the archaea.