J. Jeffrey Morris

The Black Queen Hypothesis: Evolution of Dependencies through Adaptive Gene Loss

ABSTRACT Reductive genomic evolution, driven by genetic drift, is common in endosymbiotic bacteria. Genome reduction is less common in free-living organisms, but it has occurred in the numerically dominant open-ocean bacterioplankton Prochlorococcus and “Candidatus Pelagibacter,” and in these cases the reduction appears to be driven by natural selection rather than drift. Gene loss in free-living organisms may leave them dependent on cooccurring microbes for lost metabolic functions. We present the Black Queen Hypothesis (BQH), a novel theory of reductive evolution that explains how selection leads to such dependencies; its name refers to the queen of spades in the game Hearts, where the usual strategy is to avoid taking this card. Gene loss can provide a selective advantage by conserving an organism’s limiting resources, provided the gene’s function is dispensable. Many vital genetic functions are leaky, thereby unavoidably producing public goods that are available to the entire community. Such leaky functions are thus dispensable for individuals, provided they are not lost entirely from the community. The BQH predicts that the loss of a costly, leaky function is selectively favored at the individual level and will proceed until the production of public goods is just sufficient to support the equilibrium community; at that point, the benefit of any further loss would be offset by the cost. Evolution in accordance with the BQH thus generates “beneficiaries” of reduced genomic content that are dependent on leaky “helpers,” and it may explain the observed nonuniversality of prototrophy, stress resistance, and other cellular functions in the microbial world.

Facilitation of Robust Growth of Prochlorococcus Colonies and Dilute Liquid Cultures by Helper Heterotrophic Bacteria

ABSTRACT Axenic (pure) cultures of marine unicellular cyanobacteria of the Prochlorococcus genus grow efficiently only if the inoculation concentration is large; colonies form on semisolid medium at low efficiencies. In this work, we describe a novel method for growing Prochlorococcus colonies on semisolid agar that improves the level of recovery to approximately 100%. Prochlorococcus grows robustly at low cell concentrations, in liquid or on solid medium, when cocultured with marine heterotrophic bacteria. Once the Prochlorococcus cell concentration surpasses a critical threshold, the “helper” heterotrophs can be eliminated with antibiotics to produce axenic cultures. Our preliminary evidence suggests that one mechanism by which the heterotrophs help Prochlorococcus is the reduction of oxidative stress.

Dependence of the Cyanobacterium Prochlorococcus on Hydrogen Peroxide Scavenging Microbes for Growth at the Ocean's Surface

The phytoplankton community in the oligotrophic open ocean is numerically dominated by the cyanobacterium Prochlorococcus, accounting for approximately half of all photosynthesis. In the illuminated euphotic zone where Prochlorococcus grows, reactive oxygen species are continuously generated via photochemical reactions with dissolved organic matter. However, Prochlorococcus genomes lack catalase and additional protective mechanisms common in other aerobes, and this genus is highly susceptible to oxidative damage from hydrogen peroxide (HOOH). In this study we showed that the extant microbial community plays a vital, previously unrecognized role in cross-protecting Prochlorococcus from oxidative damage in the surface mixed layer of the oligotrophic ocean. Microbes are the primary HOOH sink in marine systems, and in the absence of the microbial community, surface waters in the Atlantic and Pacific Ocean accumulated HOOH to concentrations that were lethal for Prochlorococcus cultures. In laboratory experiments with the marine heterotroph Alteromonas sp., serving as a proxy for the natural community of HOOH-degrading microbes, bacterial depletion of HOOH from the extracellular milieu prevented oxidative damage to the cell envelope and photosystems of co-cultured Prochlorococcus, and facilitated the growth of Prochlorococcus at ecologically-relevant cell concentrations. Curiously, the more recently evolved lineages of Prochlorococcus that exploit the surface mixed layer niche were also the most sensitive to HOOH. The genomic streamlining of these evolved lineages during adaptation to the high-light exposed upper euphotic zone thus appears to be coincident with an acquired dependency on the extant HOOH-consuming community. These results underscore the importance of (indirect) biotic interactions in establishing niche boundaries, and highlight the impacts that community-level responses to stress may have in the ecological and evolutionary outcomes for co-existing species.

COEXISTENCE OF EVOLVING BACTERIA STABILIZED BY A SHARED BLACK QUEEN FUNCTION

The Black Queen Hypothesis (BQH) was originally proposed to explain the dependence of some marine bacteria on helper organisms for protection from hydrogen peroxide (HOOH). The BQH predicts that selection for the evolutionary loss of leaky functions from individuals can produce commensal or mutualistic interactions. We demonstrated the leakiness of HOOH detoxification by complementing a HOOH‐sensitive Escherichia coli mutant with a plasmid‐encoded HOOH‐detoxifying enzyme, KatG, and then evolving populations founded by this strain in two environments. When HOOH was absent, plasmid‐carrying cells were outcompeted by plasmid‐free segregants, reflecting the high cost of KatG expression. However, plasmid‐carrying and plasmid‐free cells coexisted for at least 1200 generations in three replicate populations evolved in the presence of HOOH, although their relative proportions fluctuated as beneficial mutations arose in one type or the other. Evolved plasmid‐bearing cells reduced the cost of plasmid carriage even as they increased the rate of HOOH removal relative to the ancestor. Meanwhile, plasmid‐free cells remained dependent on HOOH detoxification by the plasmid‐bearing cells. These results demonstrate that partitioning of a Black Queen function can enable the stable coexistence of very similar organisms, even in this most restrictive case where the two types are competing for a single resource.

Continuous hydrogen peroxide production by organic buffers in phytoplankton culture media

We investigated the production of hydrogen peroxide (HOOH) in illuminated seawater media containing a variety of zwitterionic buffers. Production rates varied extensively among buffers, with 4‐(2‐hydroxyethyl)1‐piperazineethanesulfonic acid (HEPES) highest and N‐Tris(hydroxymethyl)methyl‐3‐aminopropanesulfonic acid (TAPS) among the lowest. The rate of HOOH accumulation was remarkably consistent over many days, and increased linearly with buffer concentration, natural seawater concentration, and light level. Concentrations of HEPES commonly used in culture media (1–10 mM) generated enough HOOH to kill the axenic Prochlorococcus strain VOL1 during growth in enriched seawater media at lower, environmentally realistic cell concentrations and/or under high light exposure. We also demonstrated that HEPES can be used experimentally to study the biological effects of chronic exposure to sublethal levels of HOOH such as may be experienced by light‐exposed microorganisms.

Degradation of hydrogen peroxide at the ocean’s surface: the influence of the microbial community on the realized thermal niche of Prochlorococcus

Prochlorococcus, the smallest and most abundant phytoplankter in the ocean, is highly sensitive to hydrogen peroxide (HOOH), and co-occurring heterotrophs such as Alteromonas facilitate the growth of Prochlorococcus by scavenging HOOH. Temperature is also a major influence on Prochlorococcus abundance and distribution in the ocean, and studies in other photosynthetic organisms have shown that HOOH and temperature extremes can act together as synergistic stressors. To address potential synergistic effects of temperature and HOOH on Prochlorococcus growth, high- and low-temperature-adapted representative strains were cultured at ecologically relevant concentrations under a range of HOOH concentrations and temperatures. Higher concentrations of HOOH severely diminished the permissive temperature range for growth of both Prochlorococcus strains. At the permissive temperatures, the growth rates of both Prochlorococcus strains decreased as a function of HOOH, and cold temperature increased susceptibility of photosystem II to HOOH-mediated damage. Serving as a proxy for the natural community, co-cultured heterotrophic bacteria increased the Prochlorococcus growth rate under these temperatures, and expanded the permissive range of temperature for growth. These studies indicate that in the ocean, the cross-protective function of the microbial community may confer a fitness increase for Prochlorococcus at its temperature extremes, especially near the ocean surface where oxidative stress is highest. This interaction may play a substantial role in defining the realized thermal niche and habitat range of Prochlorococcus with respect to latitude.

The impact of elevated CO 2 on Prochlorococcus and microbial interactions with ‘helper’ bacterium Alteromonas

Prochlorococcus is a globally important marine cyanobacterium that lacks the gene catalase and relies on ‘helper’ bacteria such as Alteromonas to remove reactive oxygen species. Increasing atmospheric CO2 decreases the need for carbon concentrating mechanisms and photorespiration in phytoplankton, potentially altering their metabolism and microbial interactions even when carbon is not limiting growth. Here, Prochlorococcus (VOL4, MIT9312) was co-cultured with Alteromonas (strain EZ55) under ambient (400 p.p.m.) and elevated CO2 (800 p.p.m.). Under elevated CO2, Prochlorococcus had a significantly longer lag phase and greater apparent die-offs after transfers suggesting an increase in oxidative stress. Whole-transcriptome analysis of Prochlorococcus revealed decreased expression of the carbon fixation operon, including carboxysome subunits, corresponding with significantly fewer carboxysome structures observed by electron microscopy. Prochlorococcus co-culture responsive gene 1 had significantly increased expression in elevated CO2, potentially indicating a shift in the microbial interaction. Transcriptome analysis of Alteromonas in co-culture with Prochlorococcus revealed decreased expression of the catalase gene, known to be critical in relieving oxidative stress in Prochlorococcus by removing hydrogen peroxide. The decrease in catalase gene expression was corroborated by a significant ~6-fold decrease in removal rates of hydrogen peroxide from co-cultures. These data suggest Prochlorococcus may be more vulnerable to oxidative stress under elevated CO2 in part from a decrease in ecosystem services provided by heterotrophs like Alteromonas. This work highlights the importance of considering microbial interactions in the context of a changing ocean.