Acquisition of genes through horizontal gene transfer (HGT) allows microbes to rapidly gain new capabilities and adapt to new or changing environments. Identifying widespread HGT regions within multispecies microbiomes can pinpoint the molecular mechanisms that play key roles in microbiome assembly. We sought to identify horizontally transferred genes within a model microbiome, the cheese rind. Comparing 31 newly sequenced and 134 previously sequenced bacterial isolates from cheese rinds, we identified over 200 putative horizontally transferred genomic regions containing 4733 protein coding genes. The largest of these regions are enriched for genes involved in siderophore acquisition, and are widely distributed in cheese rinds in both Europe and the US. These results suggest that HGT is prevalent in cheese rind microbiomes, and that identification of genes that are frequently transferred in a particular environment may provide insight into the selective forces shaping microbial communities.

Microbes commonly use metabolites produced by other organisms to compete effectively with others in their environment. As a result, microbial communities are composed of networks of metabolically interdependent organisms. How these networks evolve and shape population diversity, stability, and community function is a subject of active research. But how did these metabolic interactions develop initially? In particular, how and why are metabolites such as amino acids, cofactors, and nucleobases released for the benefit of others when there apparently is no incentive to do so? Here, we discuss the hypothesis that metabolite provisioning is not itself adaptive but rather can be a natural consequence of other evolved biological functions. We outline two examples of metabolite provisioning as a by-product of other functions by considering cell lysis and regulated metabolite efflux outside their canonical roles and explore their potential to facilitate the emergence of interdependent metabolite sharing.

Microbial communities of fermented food microbiomes typically exhibit predictable patterns of microbial succession. However, the biochemical mechanisms that control the diversity and dynamics of these communities are not well described. Interactions between bacteria and fungi may be one mechanism controlling the development of cheese rind microbiomes. This study characterizes a specific bacterium-fungus interaction previously discovered on cheese rinds between the bacterium Glutamicibacter arilaitensis (formerly Arthrobacter arilaitensis) and fungi of the genus Penicillium and identifies the specialized metabolites produced during cocultures. G. arilaitensis was previously shown to produce an unknown pink pigment in response to the presence of Penicillium. Using a combination of mass spectrometry, nuclear magnetic resonance (NMR), and transcriptome sequencing (RNA-seq), we determined that this pigment production is associated with production of coproporphyrin III. The discovery that coproporphyrin III preferentially bound zinc over other trace metals found in cheese curds highlights the value of using analytical chemistry to confirm identity of predicted chemical species. IMPORTANCE Bacterium-fungus interactions play key roles in the assembly of cheese rind microbial communities, but the molecular mechanisms underlying these interactions are poorly characterized. Moreover, millions of people around the world enjoy eating cheeses and cheese rinds, but our understanding of the diversity of microbial metabolites ingested during cheese consumption is limited. The discovery of zinc coproporphyrin III as the cause of pink pigment production by Glutamicibacter arilaitensis suggests that secretion of this molecule is important for microbial acquisition of trace metals.

Many metagenomic sequencing studies have observed the presence of closely related bacterial species or genotypes in the same microbiome. Previous attempts to explain these patterns of microdiversity have focused on the abiotic environment, but few have considered how biotic interactions could drive patterns of microbiome diversity. We dissected the patterns, processes, and mechanisms shaping the ecological distributions of three closely related Staphylococcus species in cheese rind biofilms. Paradoxically, the most abundant species (S. equorum) is the slowest colonizer and weakest competitor based on growth and competition assays in the laboratory. Through in vitro community reconstructions, we determined that biotic interactions with neighboring fungi help resolve this paradox. Species-specific stimulation of the poor competitor by fungi of the genus Scopulariopsis allows S. equorum to dominate communities in vitro as it does in situ Results of comparative genomic and transcriptomic experiments indicate that iron utilization pathways, including a homolog of the S. aureus staphyloferrin B siderophore operon pathway, are potential molecular mechanisms underlying Staphylococcus-Scopulariopsis interactions. Our integrated approach demonstrates that fungi can structure the ecological distributions of closely related bacterial species, and the data highlight the importance of bacterium-fungus interactions in attempts to design and manipulate microbiomes.

Importance

Decades of culture-based studies and more recent metagenomic studies have demonstrated that bacterial species in agriculture, medicine, industry, and nature are unevenly distributed across time and space. The ecological processes and molecular mechanisms that shape these distributions are not well understood because it is challenging to connect in situ patterns of diversity with mechanistic in vitro studies in the laboratory. Using tractable cheese rind biofilms and a focus on coagulase-negative staphylococcus (CNS) species, we demonstrate that fungi can mediate the ecological distributions of closely related bacterial species. One of the Staphylococcus species studied, S. saprophyticus, is a common cause of urinary tract infections. By identifying processes that control the abundance of undesirable CNS species, cheese producers will have more precise control on the safety and quality of their products. More generally, Staphylococcus species frequently co-occur with fungi in mammalian microbiomes, and similar bacterium-fungus interactions may structure bacterial diversity in these systems.

Microbial interactions are expected to be major determinants of microbiome structure and function. Although fungi are found in diverse microbiomes, their interactions with bacteria remain largely uncharacterized. In this work, we characterize interactions in 16 different bacterial-fungal pairs, examining the impacts of 8 different fungi isolated from cheese rind microbiomes on 2 bacteria (Escherichia coli and a cheese-isolated Pseudomonas psychrophila). Using random barcode transposon-site sequencing with an analysis pipeline that allows statistical comparisons between different conditions, we observed that fungal partners caused widespread changes in the fitness of bacterial mutants compared to growth alone. We found that all fungal species modulated the availability of iron and biotin to bacterial species, which suggests that these may be conserved drivers of bacterial-fungal interactions. Species-specific interactions were also uncovered, a subset of which suggested fungal antibiotic production. Changes in both conserved and species-specific interactions resulted from the deletion of a global regulator of fungal specialized metabolite production. This work highlights the potential for broad impacts of fungi on bacterial species within microbiomes.

Long-term dietary intake influences the structure and activity of the trillions of microorganisms residing in the human gut, but it remains unclear how rapidly and reproducibly the human gut microbiome responds to short-term macronutrient change. Here we show that the short-term consumption of diets composed entirely of animal or plant products alters microbial community structure and overwhelms inter-individual differences in microbial gene expression. The animal-based diet increased the abundance of bile-tolerant microorganisms (Alistipes, Bilophila and Bacteroides) and decreased the levels of Firmicutes that metabolize dietary plant polysaccharides (Roseburia, Eubacterium rectale and Ruminococcus bromii). Microbial activity mirrored differences between herbivorous and carnivorous mammals, reflecting trade-offs between carbohydrate and protein fermentation. Foodborne microbes from both diets transiently colonized the gut, including bacteria, fungi and even viruses. Finally, increases in the abundance and activity of Bilophila wadsworthia on the animal-based diet support a link between dietary fat, bile acids and the outgrowth of microorganisms capable of triggering inflammatory bowel disease. In concert, these results demonstrate that the gut microbiome can rapidly respond to altered diet, potentially facilitating the diversity of human dietary lifestyles.