Interactions between primary producers and bacteria impact the physiology of both partners, alter the chemistry of their environment, and shape ecosystem diversity. In marine ecosystems, these interactions are difficult to study partly because the major photosynthetic organisms are microscopic, unicellular phytoplankton. Coastal phytoplankton communities are dominated by diatoms, which generate approximately 40% of marine primary production and form the base of many marine food webs. Diatoms co-occur with specific bacterial taxa, but the mechanisms of potential interactions are mostly unknown. Here we tease apart a bacterial consortium associated with a globally distributed diatom and find that a Sulfitobacter species promotes diatom cell division via secretion of the hormone indole-3-acetic acid, synthesized by the bacterium using both diatom-secreted and endogenous tryptophan. Indole-3-acetic acid and tryptophan serve as signalling molecules that are part of a complex exchange of nutrients, including diatom-excreted organosulfur molecules and bacterial-excreted ammonia. The potential prevalence of this mode of signalling in the oceans is corroborated by metabolite and metatranscriptome analyses that show widespread indole-3-acetic acid production by Sulfitobacter-related bacteria, particularly in coastal environments. Our study expands on the emerging recognition that marine microbial communities are part of tightly connected networks by providing evidence that these interactions are mediated through production and exchange of infochemicals.

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knockins, as well as single nucleotide insertions, deletions and substitutions in Pseudomonas aeruginosa. Unlike other approaches to allelic exchange, this protocol does not require heterologous recombinases to insert or excise selective markers from the target chromosome. Rather, positive and negative selection are enabled solely by suicide vector-encoded functions and host cell proteins. Here, mutant alleles, which are flanked by regions of homology to the recipient chromosome, are synthesized in vitro and then cloned into allelic exchange vectors using standard procedures. These suicide vectors are then introduced into recipient cells by conjugation. Homologous recombination then results in antibiotic resistant single-crossover mutants in which the plasmid has integrated site-specifically into the chromosome. Subsequently, unmarked double-crossover mutants are isolated directly using sucrose-mediated counter-selection. This two-step process yields seamless mutations that are precise to a single base pair of DNA. The entire procedure requires ~2 weeks.

Organisms within all domains of life require the cofactor cobalamin (vitamin B 12 ), which is produced only by a subset of bacteria and archaea. On the basis of genomic analyses, cobalamin biosynthesis in marine systems has been inferred in three main groups: select heterotrophic Proteobacteria, chemoautotrophic Thaumarchaeota, and photoautotrophic Cyanobacteria. Culture work demonstrates that many Cyanobacteria do not synthesize cobalamin but rather produce pseudocobalamin, challenging the connection between the occurrence of cobalamin biosynthesis genes and production of the compound in marine ecosystems. Here we show that cobalamin and pseudocobalamin coexist in the surface ocean, have distinct microbial sources, and support different enzymatic demands. Even in the presence of cobalamin, Cyanobacteria synthesize pseudocobalaminlikely reflecting their retention of an oxygen-independent pathway to produce pseudocobalamin, which is used as a cofactor in their specialized methionine synthase (MetH). This contrasts a model diatom, Thalassiosira pseudonana, which transported pseudocobalamin into the cell but was unable to use pseudocobalamin in its homolog of MetH. Our genomic and culture analyses showed that marine Thaumarchaeota and select heterotrophic bacteria produce cobalamin. This indicates that cobalamin in the surface ocean is a result of de novo synthesis by heterotrophic bacteria or via modification of closely related compounds like cyanobacterially produced pseudocobalamin. Deeper in the water column, our study implicates Thaumarchaeota as major producers of cobalamin based on genomic potential, cobalamin cell quotas, and abundance. Together, these findings establish the distinctive roles played by abundant prokaryotes in cobalamin-based microbial interdependencies that sustain community structure and function in the ocean. vitamin B 12 ) is synthesized by a select subset of bacteria and archaea, yet organisms across all domains of life require it (1-3). In the surface ocean, cobalamin auxotrophs (including most eukaryotic algae) (3) obtain the vitamin through direct interactions with cobalamin producers (3) or breakdown of cobalamin-containing cells (4,5). Interdependencies between marine cobalamin producers and consumers are critical in surface waters where primary productivity can be limited by the availability of cobalamin and the compound is short-lived (1, 6, 7). The exchange of cobalamin in return for organic compounds is hypothesized to underpin mutualistic interactions between heterotrophic bacteria and autotrophic algae (3,6,8,9). The apparent pervasiveness of cobalamin biosynthesis genes in chemoautotrophic Thaumarchaeota and photoautotrophic Cyanobacteria genomes (1, 10, 11) raises the question of whether cobalamin production by these autotrophs may underlie additional, unexplored microbial interactions.Cobalamin is a complex molecule with a central cobalt-containing corrin ring, an α ligand of 5,6-dimethylbenzimidizole (DMB), and a β ligand of either OH-, CN-, Me-, or Ado-(12) (Fig. 1). Pr...

Concentrations and isotopic compositions of ethane and propane in cold, deeply buried sediments from the southeastern Pacific are best explained by microbial production of these gases in situ. Reduction of acetate to ethane provides one feasible mechanism. Propane is enriched in 13 C relative to ethane. The amount is consistent with derivation of the third C from inorganic carbon dissolved in sedimentary pore waters. At typical sedimentary conditions, the reactions yield free energy sufficient for growth. Relationships with competing processes are governed mainly by the abundance of H2. Production of C2 and C3 hydrocarbons in this way provides a sink for acetate and hydrogen but upsets the general belief that hydrocarbons larger than methane derive only from thermal degradation of fossil organic material.ethanogenesis ͉ hydrocarbon gases ͉ marine sediments ͉ propanogenesis ͉ stable carbon isotopes L eg 201 of the Ocean Drilling Program was dedicated to the study of microbial life in deeply buried marine sediments (1, 2). Cores were obtained from open-ocean sites in the Equatorial Pacific, where sediments deposited 40 million years ago are underlain by seafloor basalts through which oxygenated seawater is flowing, and from the Peruvian Margin, where drilling penetrated sediments up to 15 million years old (Fig. 1). Temperatures in sediments ranged from 2°C to 25°C. All sites are isolated from reservoirs of fossil hydrocarbons. At both openocean and ocean-margin sites, treatment of sediments with strong base released ethane and propane (Fig. 2). When the treatment was repeated with fresh sediment and isotopically labeled water (␦D ϭ ϩ4000‰), no excess deuterium appeared in the ethane or propane. Therefore, we conclude that the hydrocarbons were strongly sorbed, indigenous constituents of the sediment and did not derive from a chemical reaction between the strong base and an organic substrate.Earlier reports describe sediments offshore Peru (3) and Spitsbergen (4), from which similar mixtures of hydrocarbons could be released by treatment with hot solutions of phosphoric acid. In each case, the carbon-isotopic compositions and abundance ratios (C 1 ͞C 2ϩ ) led to reluctant suggestions that the gases must be of thermogenic origin and thus have migrated into the unconsolidated seafloor sediments: ''the [postulated] migration of C 2ϩ hydrocarbons. . . is somehow related to these fluids [brines that might have flowed from one basin to another]'' (3); and ''. . . elevated seepages [of thermogenic hydrocarbons] occurred irregularly but are not currently active. . . it remains speculative whether the detected hydrocarbon anomalies are related to reservoirs and͞or active source rocks'' (4). No mechanism for sorbing the putatively migrated hydrocarbons more strongly than indigenous microbial products has been offered.Ethane and propane with similar isotopic characteristics and abundance ratios have recently been reported in Cretaceous marine shales in the Western Canadian sedimentary basin (5). Previous work has also pointe...