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Early evolution of mutualism is characterized by big and predictable adaptive changes, including the specialization of interacting partners, such as through deleterious mutations in genes not required for metabolic cross-feeding. We sought to investigate whether these early mutations improve cooperativity by manifesting in synergistic epistasis between genomes of the mutually interacting species. Specifically, we have characterized evolutionary trajectories of syntrophic interactions of Desulfovibrio vulgaris (Dv) with Methanococcus maripaludis (Mm) by longitudinally monitoring mutations accumulated over 1000 generations of nine independently evolved communities with analysis of the genotypic structure of one community down to the single-cell level. We discovered extensive parallelism across communities despite considerable variance in their evolutionary trajectories and the perseverance within many evolution lines of a rare lineage of Dv that retained sulfate-respiration (SR+) capability, which is not required for metabolic cross-feeding. An in-depth investigation revealed that synergistic epistasis across pairings of Dv and Mm genotypes had enhanced cooperativity within SR− and SR+ assemblages, enabling their coexistence within the same community. Thus, our findings demonstrate that cooperativity of a mutualism can improve through synergistic epistasis between genomes of the interacting species, enabling the coexistence of mutualistic assemblages of generalists and their specialized variants.

SUMMARYFrequent fluctuations in sulfate availability rendered syntrophic interactions between the sulfate reducing bacteriumDesulfovibrio vulgaris(Dv) and the methanogenic archaeonMethanococcus maripaludis(Mm) unsustainable. By contrast, prolonged laboratory evolution in obligate syntrophy conditions improved the productivity of this community but at the expense of erosion of sulfate respiration (SR). Hence, we sought to understand the evolutionary trajectories that could both increase the productivity of syntrophic interactions and sustain SR. We combined a temporal and combinatorial survey of mutations accumulated over 1000 generations of 9 independently-evolved communities with analysis of the genotypic structure for one community down to the single-cell level. We discovered a high level of parallelism across communities despite considerable variance in their evolutionary trajectories and the perseverance of a rare SR+Dvlineage within many evolution lines. An in-depth investigation revealed that synergistic epistasis acrossDvandMmgenotypes had enhanced cooperativity within SR- and SR+ assemblages, allowing their co-existence asr- andK-strategists, respectively.

Microbial populations can withstand, overcome and persist in the face of environmental fluctuation. Previously, we demonstrated how conditional gene regulation in a fluctuating environment drives dilution of condition-specific transcripts, causing a population of Desulfovibrio vulgaris Hildenborough (DvH) to collapse after repeatedly transitioning from sulfate respiration to syntrophic conditions with the methanogen Methanococcus maripaludis. Failure of the DvH to successfully transition contributed to the collapse of this model community. We investigated the mechanistic basis for loss of robustness by examining whether conditional gene regulation altered heterogeneity in gene expression across individual DvH cells. We discovered that robustness of a microbial population across environmental transitions was attributable to the retention of cells in two states that exhibited different condition-specific gene expression patterns. In our experiments, a population with disrupted conditional regulation successfully alternated between cell states. Meanwhile, a population with intact conditional regulation successfully switched between cell states initially, but collapsed after repeated transitions, possibly due to the high energy requirements of regulation. These results demonstrate that the survival of this entire model microbial community is dependent on the regulatory system's influence on the distribution of distinct cell states among individual cells within a clonal population.

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.

Cellular responses to environmental stimuli are mediated by the co-ordinated activity of multiple control mechanisms, which result in the dynamics of cell function. Communication between different levels of regulation is central for this adaptability. The present study focuses on the interplay between transcriptional regulators and chromatin modifiers to co-operatively regulate transcription in response to a fatty acid stimulus. The genes involved in the β-oxidation of fatty acids are highly induced in response to fatty acid exposure by four gene-specific transcriptional regulators, Oaf (oleate-activated transcription factor) 1p, Pip2p (peroxisome induction pathway 2), Oaf3p and Adr1p (alcohol dehydrogenase regulator 1). In the present study, we examine the interplay of these factors with Htz1p (histone variant H2A.Z) in regulating POT1 (peroxisomal oxoacyl thiolase 1) encoding peroxisomal thiolase and PIP2 encoding the autoregulatory oleate-specific transcriptional activator. Temporal resolution of ChIP (chromatin immunoprecipitation) data indicates that Htz1p is required for the timely removal of the transcriptional repressor Oaf3p during oleate induction. Adr1p plays an important role in the assembly of Htz1p-containing nucleosomes on the POT1 and PIP2 promoters. We also investigated the function of the uncharacterized transcriptional inhibitor Oaf3p. Deletion of OAF3 led to faster POT1 mRNA accumulation than in the wild-type. Most impressively, a highly protected nucleosome structure on the POT1 promoter during activation was observed in the OAF3 mutant cells in response to oleate induction.

Chz1p is a histone chaperone that interacts physically and functionally with the histone variant Htz1p, which has been implicated in establishing and maintaining boundaries between transcriptionally inactive heterochromatin and active euchromatin. To investigate the role of Chz1p in chromatin organization, we performed genome-wide expression arrays and chromatin immunoprecipitations of SIR complex components and modified histones in a CHZ1 deletion strain. Deletion of CHZ1 led to reduced ubiquitination of subtelomere-associated H2B, reduced subtelomeric H3K79 di-methylation, and increased binding of Sir3p, and Sir4p at telomere-distal euchromatin regions, correlating with decreased gene expression in subtelomeric regions. This anti-silencing defect appears to be mediated by enhanced association of de-ubiquitinase Ubp10p with subtelomeric DNA, as detected by chromatin immunoprecipitation analysis. In support of this, we show that deletion of UBP10 can antagonize the subtelomeric silencing phenotype of Deltachz1. Taken together, the results demonstrate a novel role for Chz1p in epigenetic regulation, through H2B de-ubiquitination by Ubp10p.

A high speed flow cytometric cell sorter was modified to maintain a controlled anaerobic environment. This technology enabled coupling of the precise high-throughput analytical and cell separation capabilities of flow cytometry to the assessment of cell viability of evolved lineages of obligate anaerobic organisms from cocultures.