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Microorganisms play a central role in the biology of vinegar flies such as Drosophila suzukii and Drosophila melanogaster: serving as a food source to both adults and larvae, and influencing a range of traits including nutrition, behavior, and development. The niches utilized by the fly species partially overlap, as do the microbiota that sustain them, and interactions among these players may drive the development of crop diseases. To learn more about how the microbiota of one species may affect the other, we isolated and identified microbes from field-caught D. suzukii, and then characterized their effects on D. melanogaster larval development time in the laboratory. We found that the D. suzukii microbiota consistently included both yeasts and bacteria. It was dominated by yeasts of the genus Hanseniaspora, and bacteria from the families Acetobacteraceae and Enterobacteriaceae. Raising D. melanogaster under gnotobiotic conditions with each microbial isolate individually, we found that some bacteria promoted larval development relative to axenic conditions, but most did not have a significant effect. In contrast, nearly all the yeasts tested significantly accelerated larval development. The one exception was Starmerella bacillaris, which had the opposite effect: significantly slowing larval developmental rate. We investigated the basis for this effect by examining whether S. bacillaris cells could sustain larval growth, and measuring the survival of S. bacillaris and other yeasts in the larval gut. Our results suggest S. bacillaris is not digested by D. melanogaster and therefore cannot serve as a source of nutrition. These findings have interesting implications for possible interactions between the two Drosophilia species and their microbiota in nature. Overall, we found that microbes isolated from D. suzukii promote D. melanogaster larval development, which is consistent with the model that infestation of fruit by D. suzukii can open up habitat for D. melanogaster. We propose that the microbiome is an important dimension of the ecological interactions between Drosophila species.

ABSTRACT A priority in gut microbiome research is to develop methods to investigate ecological processes shaping microbial populations in the host from readily accessible data, such as fecal samples. Here, we demonstrate that these processes can be inferred from the proportion of ingested microorganisms that is egested and their egestion time distribution, by using general mathematical models that link within-host processes to statistics from fecal time series. We apply this framework to Drosophila melanogaster and its gut bacterium Acetobacter tropicalis. Specifically, we investigate changes in their interactions following ingestion of a food bolus containing bacteria in a set of treatments varying the following key parameters: the density of exogenous bacteria ingested by the flies (low/high) and the association status of the host (axenic or monoassociated with A. tropicalis). At 5 h post-ingestion, ~35% of the intact bacterial cells have transited through the gut with the food bolus and ~10% are retained in a viable and culturable state, leaving ~55% that have likely been lysed in the gut. Our models imply that lysis and retention occur over a short spatial range within the gut when the bacteria are ingested from a low density, but more broadly in the host gut when ingested from a high density, by both gnotobiotic and axenic hosts. Our study illustrates how time series data complement the analysis of static abundance patterns to infer ecological processes as bacteria traverse the host. Our approach can be extended to investigate how different bacterial species interact within the host to understand the processes shaping microbial community assembly. IMPORTANCE A major challenge to our understanding of the gut microbiome in animals is that it is profoundly difficult to investigate the fate of ingested microbial cells as they travel through the gut. Here, we created mathematical tools to analyze microbial dynamics in the gut from the temporal pattern of their abundance in fecal samples, i.e., without direct observation of the dynamics, and validated them with Drosophila fruit flies. Our analyses revealed that over 5 h after ingestion, most bacteria have likely died in the host or have been egested as intact cells, while some living cells have been retained in the host. Bacterial lysis or retention occurred across a larger area of the gut when flies ingest bacteria from high densities than when flies ingest bacteria from low densities. Our mathematical tools can be applied to other systems, including the dynamics of gut microbial populations and communities in humans.

As biological sequence data are generated at an ever increasing rate, the role of bioinformatics in biological research also grows. Students must be trained to complete and interpret bioinformatic searches to enable them to effectively utilize the trove of sequence data available. A key bioinformatic tool for sequence comparison and genome database searching is BLAST (Basic Local Alignment Search Tool). BLAST identifies sequences in a database that are similar to the entered query sequence, and ranks them based on the length and quality of the alignment. Our goal was to introduce sophomore and junior level undergraduate students to the basic functions and uses of BLAST with a small group activity lasting a single class period. The activity provides students an opportunity to perform a BLAST search, interpret the data output, and use the data to make inferences about bacterial cell envelope structure. The activity consists of two parts. Part 1 is a handout to be completed prior to class, complete with video tutorial, that reviews cell envelope structure, introduces key terms, and allows students to familiarize themselves with the mechanics of a BLAST search. Part 2 consists of a hands-on, web-based small group activity to be completed during the class period. Evaluation of the activity through student performance assessments suggests that students who complete the activity can better interpret the BLAST output parameters % query coverage and % max identity. While the topic of the activity is bacterial cell wall structure, it could be adapted to address other biological concepts.

Pseudomonas fluorescens Pf0-1 is one of the model organisms for biofilm research. Our previous transposon mutagenesis study suggested a requirement for the de novo purine nucleotide biosynthesis pathway for biofilm formation by this organism. This study was performed to verify that observation and investigate the basis for the defects in biofilm formation shown by purine biosynthesis mutants. Constructing deletion mutations in 8 genes in this pathway, we found that they all showed reductions in biofilm formation that could be partly or completely restored by nucleotide supplementation or genetic complementation. We demonstrated that, despite a reduction in biofilm formation, more viable mutant cells were recovered from the surface-attached population than from the planktonic phase under conditions of purine deprivation. Analyses using scanning electron microscopy revealed that the surface-attached mutant cells were 25 ∼ 30% shorter in length than WT, which partly explains the reduced biomass in the mutant biofilms. The laser diffraction particle analyses confirmed this finding, and further indicated that the WT biofilm cells were smaller than their planktonic counterparts. The defects in biofilm formation and reductions in cell size shown by the mutants were fully recovered upon adenine or hypoxanthine supplementation, indicating that the purine shortages caused reductions in cell size. Our results are consistent with surface attachment serving as a survival strategy during nutrient deprivation, and indicate that changes in the cell size may be a natural response of P. fluorescens to growth on a surface. Finally, cell sizes in WT biofilms became slightly smaller in the presence of exogenous adenine than in its absence. Our findings suggest that purine nucleotides or related metabolites may influence the regulation of cell size in this bacterium.

ABSTRACT Animal-associated bacteria (microbiota) affect host behaviors and physiological traits. To identify bacterial genetic determinants of microbiota-responsive host traits, we employed a metagenome-wide association (MGWA) approach in two steps. First, we measured two microbiota-responsive host traits, development time and triglyceride (TAG) content, in Drosophila melanogaster flies monoassociated with each of 41 bacterial strains. The effects of monoassociation on host traits were not confined to particular taxonomic groups. Second, we clustered protein-coding sequences of the bacteria by sequence similarity de novo and statistically associated the magnitude of the host trait with the bacterial gene contents. The animals had been monoassociated with genome-sequenced bacteria, so the metagenome content was unambiguous. This analysis showed significant effects of pyrroloquinoline quinone biosynthesis genes on development time, confirming the results of a published transposon mutagenesis screen, thereby validating the MGWA; it also identified multiple genes predicted to affect host TAG content, including extracellular glucose oxidation pathway components. To test the validity of the statistical associations, we expressed candidate genes in a strain that lacks them. Monoassociation with bacteria that ectopically expressed a predicted oxidoreductase or gluconate dehydrogenase conferred reduced Drosophila TAG contents relative to the TAG contents in empty vector controls. Consistent with the prediction that glucose oxidation pathway gene expression increased bacterial glucose utilization, the glucose content of the host diet was reduced when flies were exposed to these strains. Our findings indicate that microbiota affect host nutritional status through modulation of nutrient acquisition. Together, these findings demonstrate the utility of MGWA for identifying bacterial determinants of host traits and provide mechanistic insight into how gut microbiota modulate the nutritional status of a model host. IMPORTANCE To understand how certain gut bacteria promote the health of their animal hosts, we need to identify the bacterial genes that drive these beneficial relationships. This task is challenging because the bacterial communities can vary widely among different host individuals. To overcome this difficulty, we quantified how well each of 41 bacterial species protected Drosophila fruit flies from high fat content. The genomes of the chosen bacterial strains were previously sequenced, so we could statistically associate specific bacterial genes with bacterially mediated reduction in host fat content. Bacterial genes that promote glucose utilization were strongly represented in the association, and introducing these genes into the gut bacteria was sufficient to lower the animal’s fat content. Our method is applicable to the study of many other host-microbe interactions as a way to uncover microbial genes important for host health.

In Pseudomonas fluorescens Pf0-1 the availability of inorganic phosphate (Pi) is an environmental signal that controls biofilm formation through a cyclic dimeric GMP (c-di-GMP) signaling pathway. In low Pi conditions, a c-di-GMP phosphodiesterase (PDE) RapA is expressed, depleting cellular c-di-GMP and causing the loss of a critical outer-membrane adhesin LapA from the cell surface. This response involves an inner membrane protein LapD, which binds c-di-GMP in the cytoplasm and exerts a periplasmic output promoting LapA maintenance on the cell surface. Here we report how LapD differentially controls maintenance and release of LapA: c-di-GMP binding to LapD promotes interaction with and inhibition of the periplasmic protease LapG, which targets the N-terminus of LapA. We identify conserved amino acids in LapA required for cleavage by LapG. Mutating these residues in chromosomal lapA inhibits LapG activity in vivo, leading to retention of the adhesin on the cell surface. Mutations with defined effects on LapD's ability to control LapA localization in vivo show concomitant effects on c-di-GMP-dependent LapG inhibition in vitro. To establish the physiological importance of the LapD-LapG effector system, we track cell attachment and LapA protein localization during Pi starvation. Under this condition, the LapA adhesin is released from the surface of cells and biofilms detach from the substratum. This response requires c-di-GMP depletion by RapA, signaling through LapD, and proteolytic cleavage of LapA by LapG. These data, in combination with the companion study by Navarro et al. presenting a structural analysis of LapD's signaling mechanism, give a detailed description of a complete c-di-GMP control circuit--from environmental signal to molecular output. They describe a novel paradigm in bacterial signal transduction: regulation of a periplasmic enzyme by an inner membrane signaling protein that binds a cytoplasmic second messenger.

The bacterial second messenger bis-(3'-5') cyclic dimeric guanosine monophosphate (c-di-GMP) has emerged as a central regulator for biofilm formation. Increased cellular c-di-GMP levels lead to stable cell attachment, which in Pseudomonas fluorescens requires the transmembrane receptor LapD. LapD exhibits a conserved and widely used modular architecture containing a HAMP domain and degenerate diguanylate cyclase and phosphodiesterase domains. c-di-GMP binding to the LapD degenerate phosphodiesterase domain is communicated via the HAMP relay to the periplasmic domain, triggering sequestration of the protease LapG, thus preventing cleavage of the surface adhesin LapA. Here, we elucidate the molecular mechanism of autoinhibition and activation of LapD based on structure-function analyses and crystal structures of the entire periplasmic domain and the intracellular signaling unit in two different states. In the absence of c-di-GMP, the intracellular module assumes an inactive conformation. Binding of c-di-GMP to the phosphodiesterase domain disrupts the inactive state, permitting the formation of a trans-subunit dimer interface between adjacent phosphodiesterase domains via interactions conserved in c-di-GMP-degrading enzymes. Efficient mechanical coupling of the conformational changes across the membrane is realized through an extensively domain-swapped, unique periplasmic fold. Our structural and functional analyses identified a conserved system for the regulation of periplasmic proteases in a wide variety of bacteria, including many free-living and pathogenic species.