Prochlorococcus extracellular vesicles: molecular composition and adsorption to diverse microbes.

Extracellular vesicles are small (~50-200 nm diameter) membrane-bound structures released by cells from all domains of life. While vesicles are abundant in the oceans, their functions, both for cells themselves and the emergent ecosystem, remain a mystery. To better characterize these particles - a prerequisite for determining function - we analysed the lipid, protein, and metabolite content of vesicles produced by the marine cyanobacterium Prochlorococcus. We show that Prochlorococcus exports a diverse array of cellular compounds into the surrounding seawater enclosed within discrete vesicles. Vesicles produced by two different strains contain some materials in common, but also display numerous strain-specific differences, reflecting functional complexity within vesicle populations. The vesicles contain active enzymes, indicating that they can mediate extracellular biogeochemical reactions in the ocean. We further demonstrate that vesicles from Prochlorococcus and other bacteria associate with diverse microbes including the most abundant marine bacterium, Pelagibacter. Together, our data point toward hypotheses concerning the functional roles of vesicles in marine ecosystems including, but not limited to, possibly mediating energy and nutrient transfers, catalysing extracellular biochemical reactions, and mitigating toxicity of reactive oxygen species.

Two distinct pools of B12 analogs reveal community interdependencies in the ocean.

Organisms within all domains of life require the cofactor cobalamin (vitamin B12), 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 pseudocobalamin-likely 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.

Quantitative exploration of the contribution of settlement, growth, dispersal and grazing to the accumulation of natural marine biofilms on antifouling and fouling-release coatings.

The accumulation of microbial biofilms on ships' hulls negatively affects ship performance and efficiency while also playing a role in the establishment of even more detrimental hard-fouling communities. However, there is little quantitative information on how the accumulation rate of microbial biofilms is impacted by the balance of the rates of cell settlement, in situ production (ie growth), dispersal to surrounding waters and mortality induced by grazers. These rates were quantified on test panels coated with copper-based antifouling (AF) or polymer-based fouling-release (FR) coatings by using phospholipids as molecular proxies for microbial biomass. The results confirmed the accepted modes of efficacy of these two types of coatings. In a more extensive set of experiments with only the FR coatings, it was found that seasonally averaged cellular production rates were 1.5 ± 0.5 times greater than settlement and the dispersal rates were 2.7 ± 0.8 greater than grazing. The results of this study quantitatively describe the dynamic balance of processes leading to the accumulation of microbial biofilm on coatings designed for ships' hulls.

Quorum Sensing in Marine Microbial Environments.

Quorum sensing (QS) is a form of chemical communication used by certain bacteria that regulates a wide range of biogeochemically important bacterial behaviors. Although QS was first observed in a marine bacterium nearly four decades ago, only in the past decade has there been a rise in interest in the role that QS plays in the ocean. It has become clear that QS, regulated by signals such as acylated homoserine lactones (AHLs) or furanosyl-borate diesters [autoinducer-2 (AI-2) molecules], is involved in important processes within the marine carbon cycle, in the health of coral reef ecosystems, and in trophic interactions between a range of eukaryotes and their bacterial associates. The most well-studied QS systems in the ocean occur in surface-attached (biofilm) communities and rely on AHL signaling. AHL-QS is highly sensitive to the chemical and biological makeup of the environment and may respond to anthropogenic change, including ocean acidification and rising sea surface temperatures.

Precision-engineering the Pseudomonas aeruginosa genome with two-step allelic exchange.

Allelic exchange is an efficient method of bacterial genome engineering. This protocol describes the use of this technique to make gene knockouts and knock-ins, 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 selections 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.

Quorum sensing control of phosphorus acquisition in Trichodesmium consortia.

Colonies of the cyanobacterium Trichodesmium are abundant in the oligotrophic ocean, and through their ability to fix both CO(2) and N(2), have pivotal roles in the cycling of carbon and nitrogen in these highly nutrient-depleted environments. Trichodesmium colonies host complex consortia of epibiotic heterotrophic bacteria, and yet, the regulation of nutrient acquisition by these epibionts is poorly understood. We present evidence that epibiotic bacteria in Trichodesmium consortia use quorum sensing (QS) to regulate the activity of alkaline phosphatases (APases), enzymes used by epibionts in the acquisition of phosphate from dissolved-organic phosphorus molecules. A class of QS molecules, acylated homoserine lactones (AHLs), were produced by cultivated epibionts, and adding these AHLs to wild Trichodesmium colonies collected at sea led to a consistent doubling of APase activity. By contrast, amendments of (S)-4,5-dihydroxy-2,3-pentanedione (DPD)-the precursor to the autoinducer-2 (AI-2) family of universal interspecies signaling molecules-led to the attenuation of APase activity. In addition, colonies collected at sea were found by high performance liquid chromatography/mass spectrometry to contain both AHLs and AI-2. Both types of molecules turned over rapidly, an observation we ascribe to quorum quenching. Our results reveal a complex chemical interplay among epibionts using AHLs and AI-2 to control access to phosphate in dissolved-organic phosphorus.

Possible influence of bacterial quorum sensing on the hydrolysis of sinking particulate organic carbon in marine environments.

A central component of the ocean's biological carbon pump is the export of sinking, photosynthetically derived, particulate organic carbon (POC). Bacteria colonize these particles and produce enzymes that hydrolyse sinking POC thereby acting as one of the major controls on the biological pump. Here we provide evidence that a bacterial cell-cell communication mechanism, quorum sensing (QS), may influence the activity of hydrolytic enzymes on sinking particles. We collected sinking POC from a site off Vancouver Island, Canada and found that it contained acylated homoserine lactones (AHLs), a suite of well-known bacterial communication molecules. Furthermore, we observed that the addition of exogenous AHLs to incubations containing sinking POC affected the activity of key hydrolytic enzymes involved in POC degradation in some cases. Our results suggest that AHL-based QS could play an important role in regulating the degradation of sinking POC and that variability in AHL-triggered POC hydrolysis is a heretofore unrecognized process that impacts the marine biological carbon pump.

Biological formation of ethane and propane in the deep marine subsurface.

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 H(2). Production of C(2) and C(3) 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.

Molecular fossil record of elevated methane levels in late Pleistocene coastal waters.

Accumulating evidence suggests that methane has been released episodically from hydrates trapped in sea floor sediments during many intervals of rapid climate warming. Here we show that sediments from the Santa Barbara Basin deposited during warm intervals in the last glacial period contain molecular fossils that are diagnostic of aerobic and anaerobic methanotrophs. Sediment intervals with high abundances of these compounds indicate episodes of vigorous methanotrophic activity in methane-laden water masses. Signals for anaerobic methanotrophy in 44,100-year-old sediment are evidence for particularly intense methane emissions and suggest that the basin's methane cycle can profoundly affect oxygen budgets in the water column.