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Abstract Studies of microbial interactions often emphasize interactions with large, easily measurable growth differences and short-term ecological outcomes spanning just a few generations. However, more subtle interactions, such as those without obvious phenotypes, may play a significant role in shaping both the short-term ecological dynamics and the long-term evolutionary trajectories of microbial species. We used the cheese rind model microbiome to examine how two fungal species, Penicillium camemberti and Geotrichum candidum, impact global gene expression and genome evolution of the bacterium Pseudomonas carnis LP. Even though fungi had limited impacts on the growth of P. carnis LP, approximately 4–40% of its genome was differentially expressed, depending on the specific fungal partner. When we evolved this Pseudomonas strain alone or in co-culture with each of the fungi, we observed frequent mutations in global regulators of nitrogen regulation, secondary metabolite production, and motility, depending on the fungus. Strikingly, many strains with mutations in the nitrogen regulatory gene ntrB emerged when evolved alone or with G. candidum, but not with P. camemberti. Metabolomic and fitness experiments demonstrate that release of free amino acids by P. camemberti removes the fitness advantages conferred by ntrB mutations. Collectively, these results demonstrate that even in the absence of major short-term growth effects, fungi can have substantial impacts on the transcriptome and genomic evolution of bacterial species. 

SUMMARY Previous comparative and experimental evolution studies have suggested how fungi may rapidly adapt to new environments, but direct observation ofin situselection in fungal populations is rare due to challenges with tracking populations over human time scales. We monitored a population ofPenicillium solitumover eight years in a cheese cave and documented a phenotypic shift from predominantly green to white strains. Diverse mutations in thealb1gene, which encodes the first protein in the DHN-melanin biosynthesis pathway, explained the green to white shift. A similar phenotypic shift was recapitulated with analb1knockout and experimental evolution in laboratory populations. The most common genetic disruption of thealb1genomic region was caused by putative transposable element insertions upstream of the gene. White strains had substantial downregulation in global transcription, with genetically distinct white strains possessing divergent shifts in expression of different biological processes. White strains outcompeted green strains in co-culture, but this competitive advantage was only observed in the absence of light, suggesting that loss of melanin is only adaptive in dark conditions. Our results illustrate how fermented food production by humans provides opportunities for relaxed selection of key fungal traits over short time scales. Unintentional domestication of microbes by cheesemakers may provide opportunities to generate new strains for innovation in traditional cheese production. 

Abstract Evolutionary processes may have substantial impacts on community assembly, but evidence for phylogenetic relatedness as a determinant of interspecific interaction strength remains mixed. In this perspective, we consider a possible role for discordance between gene trees and species trees in the interpretation of phylogenetic signal in studies of community ecology. Modern genomic data show that the evolutionary histories of many taxa are better described by a patchwork of histories that vary along the genome rather than a single species tree. If a subset of genomic loci harbour trait‐related genetic variation, then the phylogeny at these loci may be more informative of interspecific trait differences than the genome background. We develop a simple method to detect loci harbouring phylogenetic signal and demonstrate its application through a proof‐of‐principle analysis ofPenicilliumgenomes and pairwise interaction strength. Our results show that phylogenetic signal that may be masked genome‐wide could be detectable using phylogenomic techniques and may provide a window into the genetic basis for interspecific interactions. 

Abstract Experimental studies of microbial evolution have largely focused on monocultures of model organisms, but most microbes live in communities where interactions with other species may impact rates and modes of evolution. Using the cheese rind model microbial community, we determined how species interactions shape the evolution of the widespread food- and animal-associated bacterium Staphylococcus xylosus. We evolved S. xylosus for 450 generations alone or in co-culture with one of three microbes: the yeast Debaryomyces hansenii, the bacterium Brevibacterium aurantiacum, and the mold Penicillium solitum. We used the frequency of colony morphology mutants (pigment and colony texture phenotypes) and whole-genome sequencing of isolates to quantify phenotypic and genomic evolution. The yeast D. hansenii strongly promoted diversification of S. xylosus. By the end of the experiment, all populations co-cultured with the yeast were dominated by pigment and colony morphology mutant phenotypes. Populations of S. xylosus grown alone, with B. aurantiacum, or with P.solitum did not evolve novel phenotypic diversity. Whole-genome sequencing of individual mutant isolates across all four treatments identified numerous unique mutations in the operons for the SigB, Agr, and WalRK global regulators, but only in the D. hansenii treatment. Phenotyping and RNA-seq experiments highlighted altered pigment and biofilm production, spreading, stress tolerance, and metabolism of S. xylosus mutants. Fitness experiments revealed antagonistic pleiotropy, where beneficial mutations that evolved in the presence of the yeast had strong negative fitness effects in other biotic environments. This work demonstrates that bacterial-fungal interactions can have long-term evolutionary consequences within multispecies microbiomes by facilitating the evolution of strain diversity. 

For thousands of years, humans have enjoyed the novel flavors, increased shelf-life, and nutritional benefits that microbes provide in fermented foods and beverages. Recent sequencing surveys of ferments have mapped patterns of microbial diversity across space, time, and production practices. But a mechanistic understanding of how fermented food microbiomes assemble has only recently begun to emerge. Using three foods as case studies (surface-ripened cheese, sourdough starters, and fermented vegetables), we use an ecological and evolutionary framework to identify how microbial communities assemble in ferments. By combining in situ sequencing surveys with in vitro models, we are beginning to understand how dispersal, selection, diversification, and drift generate the diversity of fermented food communities. Most food producers are unaware of the ecological processes occurring in their production environments, but the theory and models of ecology and evolution can provide new approaches for managing fermented food microbiomes, from farm to ferment. 

ABSTRACT Potent antimicrobial metabolites are produced by filamentous fungi in pure culture, but their ecological functions in nature are often unknown. Using an antibacterial Penicillium isolate and a cheese rind microbial community, we demonstrate that a fungal specialized metabolite can regulate the diversity of bacterial communities. Inactivation of the global regulator, LaeA, resulted in the loss of antibacterial activity in the Penicillium isolate. Cheese rind bacterial communities assembled with the laeA deletion strain had significantly higher bacterial abundances than the wild-type strain. RNA-sequencing and metabolite profiling demonstrated a striking reduction in the expression and production of the natural product pseurotin in the laeA deletion strain. Inactivation of a core gene in the pseurotin biosynthetic cluster restored bacterial community composition, confirming the role of pseurotins in mediating bacterial community assembly. Our discovery demonstrates how global regulators of fungal transcription can control the assembly of bacterial communities and highlights an ecological role for a widespread class of fungal specialized metabolites. IMPORTANCE Cheese rinds are economically important microbial communities where fungi can impact food quality and aesthetics. The specific mechanisms by which fungi can regulate bacterial community assembly in cheeses, other fermented foods, and microbiomes in general are largely unknown. Our study highlights how specialized metabolites secreted by a Penicillium species can mediate cheese rind development via differential inhibition of bacterial community members. Because LaeA regulates specialized metabolites and other ecologically relevant traits in a wide range of filamentous fungi, this global regulator may have similar impacts in other fungus-dominated microbiomes. 

Bacterial–fungal interactions (BFIs) can shape the structure of microbial communities, but the small molecules mediating these BFIs are often understudied. We explored various optimization steps for our microbial culture and chemical extraction protocols for bacterial–fungal co-cultures, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed that metabolomic profiles are mainly comprised of fungi derived features, indicating that fungi are the key contributors to small molecules in BFIs. LC-inductively coupled plasma MS (LC-ICP-MS) and MS/MS based dereplication using database searching revealed the presence of several known fungal specialized metabolites and structurally related analogues in these extracts, including siderophores such as desferrichrome, desferricoprogen, and palmitoylcoprogen. Among these analogues, a novel putative coprogen analogue possessing a terminal carboxylic acid motif was identified from Scopulariopsis sp. JB370, a common cheese rind fungus, and its structure was elucidated via MS/MS fragmentation. Based on these findings, filamentous fungal species appear to be capable of producing multiple siderophores with potentially different biological roles ( i.e. various affinities for different forms of iron). These findings highlight that fungal species are important contributors to microbiomes via their production of abundant specialized metabolites and that elucidating their role in complex communities should continue to be a priority. 

Fungi and bacteria are commonly found co-occurring both in natural and synthetic microbiomes, but our understanding of fungal–bacterial interactions is limited to a handful of species. Conserved mechanisms of interactions and evolutionary consequences of fungal–bacterial interactions are largely unknown. Our RNA sequencing and experimental evolution data with Penicillium species and the bacterium S. equorum demonstrate that divergent fungal species can elicit conserved transcriptional and genomic responses in co-occurring bacteria. Penicillium molds are integral to the discovery of novel antibiotics and production of certain foods. By understanding how Penicillium species affect bacteria, our work can further efforts to design and manage Penicillium -dominated microbial communities in industry and food production.