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ABSTRACT The interconnected and overlapping habitats present in natural ecosystems remain a challenge in determining the forces driving microbial community composition. The cuplike leaf structures of some carnivorous plants, including those of the family Sarraceniaceae, are self-contained ecological habitats that represent systems for exploring such microbial ecology questions. We investigated whether Sarracenia minor and Sarracenia flava cultivate distinct bacterial communities when sampled at the same geographic location and time. This sampling strategy eliminates many abiotic environmental variables present in other studies that compare samples harvested over time, and it could reveal biotic factors driving the selection of microbes. DNA extracted from the decomposing detritus trapped in each Sarracenia leaf pitcher was profiled using 16S rRNA amplicon sequencing. We identified a surprising amount of bacterial diversity within each pitcher, but we also discovered bacteria whose abundance was specifically enriched in one of the two Sarracenia species. These differences in bacterial community representation suggest some biotic influence of the Sarracenia plant on the bacterial composition of their pitchers. Overall, our results suggest that bacterial selection due to factors other than geographic location, weather, or prey availability is occurring within the pitchers of these two closely related plant species. This indicates that specific characteristics of S. minor and S. flava may play a role in fostering distinct bacterial communities. These confined, naturally occurring microbial ecosystems within Sarracenia pitchers may provide model systems to answer important questions about the drivers of microbial community composition, succession, and response to environmental perturbations. IMPORTANCE This study uses amplicon sequencing to compare the bacterial communities of environmental samples from the detritus of the leaf cavities of Sarracenia minor and Sarracenia flava pitcher plants. We sampled the detritus at the same time and in the same geographic location, eliminating many environmental variables present in other comparative studies. This study revealed that different species of Sarracenia contain distinct bacterial members within their pitchers, suggesting that these communities are not randomly established based on environmental factors and the prey pool but are potentially enriched for by the plants’ chemical or physical environment. This study of these naturally occurring, confined microbial ecosystems will help further establish carnivorous pitcher plants as a model system for answering important questions about the development and succession of microbial communities.

ABSTRACT Mass spectrometry imaging (MSI) is becoming an increasingly popular analytical technique to investigate microbial systems. However, differences in the ionization efficiencies of distinct MSI methods lead to biases in terms of what types and classes of molecules can be detected. Here, we sought to increase the molecular coverage of microbial colonies by employing metal-assisted laser desorption/ionization (MetA-LDI) MSI, and we compared our results to more commonly utilized matrix-assisted laser desorption/ionization MALDI MSI. We found substantial (∼67%) overlap in the molecules detected in our analysis of Bacillus subtilis colony biofilms using both methods, but each ionization technique did lead to the identification of a unique subset of molecular species. MetA-LDI MSI tended to identify more small molecules and neutral lipids, whereas MALDI MSI more readily detected other lipids and surfactin species. Putative annotations were made using METASPACE, Metlin, and the BsubCyc database. These annotations were then confirmed from analyses of replicate bacterial colonies using liquid extraction surface analysis tandem mass spectrometry. Additionally, we analyzed B. subtilis biofilms in a polymer-based emulated soil micromodel using MetA-LDI MSI to better understand bacterial processes and metabolism in a native, soil-like environment. We were able to detect different molecular signatures within the micropore regions of the micromodel. We also show that MetA-LDI MSI can be used to analyze microbial biofilms from electrically insulating material. Overall, this study expands the molecular universe of microbial metabolism that can be visualized by MSI. IMPORTANCE Matrix-assisted laser desorption/ionization mass spectrometry imaging is becoming an important technique to investigate molecular processes within microbial colonies and microbiomes under different environmental conditions. However, this method is limited in terms of the types and classes of molecules that can be detected. In this study, we utilized metal-assisted laser desorption/ionization mass spectrometry imaging, which expanded the range of molecules that could be imaged from microbial samples. One advantage of this technique is that the addition of a metal helps facilitate ionization from electrically nonconductive substrates, which allows for the investigation of biofilms grown in polymer-based devices, like soil-emulating micromodels.