Kristina L. Hillesland

Metabolic modeling of a mutualistic microbial community

The rate of production of methane in many environments depends upon mutualistic interactions between sulfate‐reducing bacteria and methanogens. To enhance our understanding of these relationships, we took advantage of the fully sequenced genomes of Desulfovibrio vulgaris and Methanococcus maripaludis to produce and analyze the first multispecies stoichiometric metabolic model. Model results were compared to data on growth of the co‐culture on lactate in the absence of sulfate. The model accurately predicted several ecologically relevant characteristics, including the flux of metabolites and the ratio of D. vulgaris to M. maripaludis cells during growth. In addition, the model and our data suggested that it was possible to eliminate formate as an interspecies electron shuttle, but hydrogen transfer was essential for syntrophic growth. Our work demonstrated that reconstructed metabolic networks and stoichiometric models can serve not only to predict metabolic fluxes and growth phenotypes of single organisms, but also to capture growth parameters and community composition of simple bacterial communities.

Rapid evolution of stability and productivity at the origin of a microbial mutualism

Mutualistic interactions are taxonomically and functionally diverse. Despite their ubiquity, however, the basic ecological and evolutionary processes underlying their origin and maintenance are poorly understood. A major reason for this is the lack of an experimentally tractable model system. We examine the evolution of an experimentally imposed obligate mutualism between sulfate-reducing and methanogenic microorganisms that have no known history of previous interaction. Twenty-four independent pairings (cocultures) of the bacterium Desulfovibrio vulgaris and the archaeon Methanococcus maripaludis were established and followed for 300 community doublings in two environments, one allowing for the development of a heterogeneous distribution of resources and the other not. Evolved cocultures grew up to 80% faster and were up to 30% more productive (biomass yield per mole of substrate) than the ancestors. The evolutionary process was marked by periods of significant instability leading to extinction of two of the cocultures, but it resulted in more stable, efficient, and productive mutualisms for most replicated pairings. Comparisons of evolved cocultures with those assembled from one evolved mutualist and one ancestral mutualist showed that evolution of both species contributed to improved productivity. Surprisingly, however, overall improvements in growth rate and yield were less than the sum of the individual contributions, suggesting antagonistic interactions between mutations from the coevolved populations. Physical constraints on the transfer of metabolites in the evolution environment affected the evolution of M. maripaludis, but not of D. vulgaris. Together, these results demonstrate that challenges can imperil nascent obligate mutualisms and demonstrate the evolutionary responses that enable their persistence and future evolution.

How sulphate-reducing microorganisms cope with stress: lessons from systems biology

Sulphate-reducing microorganisms (SRMs) are a phylogenetically diverse group of anaerobes encompassing distinct physiologies with a broad ecological distribution. As SRMs have important roles in the biogeochemical cycling of carbon, nitrogen, sulphur and various metals, an understanding of how these organisms respond to environmental stresses is of fundamental and practical importance. In this Review, we highlight recent applications of systems biology tools in studying the stress responses of SRMs, particularly Desulfovibrio spp., at the cell, population, community and ecosystem levels. The syntrophic lifestyle of SRMs is also discussed, with a focus on system-level analyses of adaptive mechanisms. Such information is important for understanding the microbiology of the global sulphur cycle and for developing biotechnological applications of SRMs for environmental remediation, energy production, biocorrosion control, wastewater treatment and mineral recovery.

Comparative analysis of Myxococcus predation on soil bacteria.

ABSTRACT Predator-prey relationships among prokaryotes have received little attention but are likely to be important determinants of the composition, structure, and dynamics of microbial communities. Many species of the soil-dwelling myxobacteria are predators of other microbes, but their predation range is poorly characterized. To better understand the predatory capabilities of myxobacteria in nature, we analyzed the predation performance of numerous Myxococcus isolates across 12 diverse species of bacteria. All predator isolates could utilize most potential prey species to effectively fuel colony expansion, although one species hindered predator swarming relative to a control treatment with no growth substrate. Predator strains varied significantly in their relative performance across prey types, but most variation in predatory performance was determined by prey type, with Gram-negative prey species supporting more Myxococcus growth than Gram-positive species. There was evidence for specialized predator performance in some predator-prey combinations. Such specialization may reduce resource competition among sympatric strains in natural habitats. The broad prey range of the Myxococcus genus coupled with its ubiquity in the soil suggests that myxobacteria are likely to have very important ecological and evolutionary effects on many species of soil prokaryotes.

Using artificial systems to explore the ecology and evolution of symbioses

The web of life is weaved from diverse symbiotic interactions between species. Symbioses vary from antagonistic interactions such as competition and predation to beneficial interactions such as mutualism. What are the bases for the origin and persistence of symbiosis? What affects the ecology and evolution of symbioses? How do symbiotic interactions generate ecological patterns? How do symbiotic partners evolve and coevolve? Many of these questions are difficult to address in natural systems. Artificial systems, from abstract to living, have been constructed to capture essential features of natural symbioses and to address these key questions. With reduced complexity and increased controllability, artificial systems can serve as useful models for natural systems. We review how artificial systems have contributed to our understanding of symbioses.

Erosion of functional independence early in the evolution of a microbial mutualism

Significance Nature is full of species that cooperate in mutually beneficial interactions to survive. Some are completely dependent on such relationships. How and why does this specialization evolve? We show that as the bacterium Desulfovibrio vulgaris evolved for 1,000 generations in conditions forcing cooperation with the archaeon Methanococcus maripaludis, it lost a key metabolic trait that would be required for it to grow alone in most environments. Large subpopulations lacking the capacity to respire sulfate evolved in 13 of 21 replicates. Such striking parallel evolution suggests a trade-off between performance in the mutualistic environment and maintaining the flexibility to survive alone. This result may explain why sulfate reducers share a common ancestor with many species specialized for cooperation with methanogens. Many species have evolved to function as specialized mutualists, often to the detriment of their ability to survive independently. However, there are few, if any, well-controlled observations of the evolutionary processes underlying the genesis of new mutualisms. Here, we show that within the first 1,000 generations of initiating independent syntrophic interactions between a sulfate reducer (Desulfovibrio vulgaris) and a hydrogenotrophic methanogen (Methanococcus maripaludis), D. vulgaris frequently lost the capacity to grow by sulfate respiration, thus losing the primary physiological attribute of the genus. The loss of sulfate respiration was a consequence of mutations in one or more of three key genes in the pathway for sulfate respiration, required for sulfate activation (sat) and sulfate reduction to sulfite (apsA or apsB). Because loss-of-function mutations arose rapidly and independently in replicated experiments, and because these mutations were correlated with enhanced growth rate and productivity, gene loss could be attributed to natural selection, even though these mutations should significantly restrict the independence of the evolved D. vulgaris. Together, these data present an empirical demonstration that specialization for a mutualistic interaction can evolve by natural selection shortly after its origin. They also demonstrate that a sulfate-reducing bacterium can readily evolve to become a specialized syntroph, a situation that may have often occurred in nature.

Experimental evolution of a microbial predator's ability to find prey

Foraging theory seeks to explain how the distribution and abundance of prey influence the evolution of predatory behaviour, including the allocation of effort to searching for prey and handling them after they are found. While experiments have shown that many predators alter their behaviour phenotypically within individual lifetimes, few have examined the actual evolution of predatory behaviour in light of this theory. Here, we test the effects of prey density on the evolution of a predators searching and handling behaviours using a bacterial predator, Myxococcus xanthus. Sixteen predator populations evolved for almost a year on agar surfaces containing patches of Escherichia coli prey at low or high density. Improvements in searching rate were significantly greater in those predators that evolved at low prey density. Handling performance also improved in some predator populations, but prey density did not significantly affect the magnitude of these gains. As the predators evolved greater foraging proficiency, their capacity diminished to produce fruiting bodies that enable them to survive prolonged periods of starvation. More generally, these results demonstrate that predators evolve behaviours that reflect at least some of the opportunities and limitations imposed by the distribution and abundance of their prey.

Resource level affects relative performance of the two motility systems of Myxococcus xanthus.

The adventurous (A) and social (S) motility systems of the microbial predator Myxococcus xanthus show differential swarming performance on distinct surface types. Under standard laboratory conditions, A-motility performs well on hard agar but poorly on soft agar, whereas the inverse pattern is shown by S-motility. These properties may allow M. xanthus to swarm effectively across a greater diversity of natural surfaces than would be possible with one motility system alone. Nonetheless, the range of ecological conditions under which dual motility enhances effective swarming across distinct surfaces and how ecological parameters affect the complementarity of A-motility and S-motility remain unclear. Here we have examined the role of nutrient concentration in determining swarming patterns driven by dual motility on distinct agar surfaces, as well as the relative contributions of A-motility and S-motility to these patterns. Swarm expansion rates of dually motile (A+S+), solely A-motile (A+S−), and solely S-motile (A−S+) strains were compared on hard and soft agar across a wide range of casitone concentrations. At low casitone concentrations (0–0.1%), swarming on soft agar driven by S-motility is very poor, and is significantly slower than swarming on hard agar driven by A-motility. This reverses at high casitone concentration (1–3.2%) such that swarming on soft agar is much faster than swarming on hard agar. This pattern greatly constrained the ability of M. xanthus to encounter patches of prey bacteria on a soft agar surface when nutrient levels between the patches were low. The swarming patterns of a strain that is unable to produce extracellular fibrils indicate that these appendages are responsible for the elevated swarming of S-motility at high resource levels. Together, these data suggest that large contributions by S-motility to predatory swarming in natural soils may be limited to soft, wet, high-nutrient conditions that may be uncommon. Several likely benefits of S-motility to the M. xanthus life cycle are discussed, including synergistic interactions with A-motility across a wide variety of conditions.

Deletion of the Desulfovibrio vulgaris Carbon Monoxide Sensor Invokes Global Changes in Transcription

The carbon monoxide-sensing transcriptional factor CooA has been studied only in hydrogenogenic organisms that can grow using CO as the sole source of energy. Homologs for the canonical CO oxidation system, including CooA, CO dehydrogenase (CODH), and a CO-dependent Coo hydrogenase, are present in the sulfate-reducing bacterium Desulfovibrio vulgaris, although it grows only poorly on CO. We show that D. vulgaris Hildenborough has an active CO dehydrogenase capable of consuming exogenous CO and that the expression of the CO dehydrogenase, but not that of a gene annotated as encoding a Coo hydrogenase, is dependent on both CO and CooA. Carbon monoxide did not act as a general metabolic inhibitor, since growth of a strain deleted for cooA was inhibited by CO on lactate-sulfate but not pyruvate-sulfate. While the deletion strain did not accumulate CO in excess, as would have been expected if CooA were important in the cycling of CO as a metabolic intermediate, global transcriptional analyses suggested that CooA and CODH are used during normal metabolism.

Mechanism for microbial population collapse in a fluctuating resource environment

Managing trade‐offs through gene regulation is believed to confer resilience to a microbial community in a fluctuating resource environment. To investigate this hypothesis, we imposed a fluctuating environment that required the sulfate‐reducer Desulfovibrio vulgaris to undergo repeated ecologically relevant shifts between retaining metabolic independence (active capacity for sulfate respiration) and becoming metabolically specialized to a mutualistic association with the hydrogen‐consuming Methanococcus maripaludis. Strikingly, the microbial community became progressively less proficient at restoring the environmentally relevant physiological state after each perturbation and most cultures collapsed within 3–7 shifts. Counterintuitively, the collapse phenomenon was prevented by a single regulatory mutation. We have characterized the mechanism for collapse by conducting RNA‐seq analysis, proteomics, microcalorimetry, and single‐cell transcriptome analysis. We demonstrate that the collapse was caused by conditional gene regulation, which drove precipitous decline in intracellular abundance of essential transcripts and proteins, imposing greater energetic burden of regulation to restore function in a fluctuating environment.