Reverse and Multiple Stable Isotope Probing to Study Bacterial Metabolism and Interactions at the Single Cell Level

The interactions between microorganisms driven by substrate metabolism and energy flow are important to shape diversity, abundance, and structure of a microbial community. Single cell technologies are useful tools for dissecting the functions of individual members and their interactions in microbial communities. Here, we developed a novel Raman stable isotope probing (Raman-SIP), which uses Raman microspectrosonpy coupled with reverse and, D2O colabeling to study metabolic interactions in a two species community consisting of Acinetobacter baylyi ADP1 and Escherichia coli DH5 alpha-GFP. This Raman-SIP approach is able to detect carbon assimilation and general metabolic activity simultaneously. Taking advantage of Raman shift of single cell Raman spectra (SCRS) mediated by incorporation of stable-isotopic substrates, Raman-SIP with reverse labeling has been applied to detect initially C-13-labeled bands of ADP1 SCRS reverting back to C-12 positions in the presence of C-12 citrate. Raman-SIP with D2O labeling has been employed to probe metabolic activity of single,cells without the need of cell replication. Our results show that E. coli alone in minimal Medium with citrate as the sole carbon source had no metabolic activity, but became metabolically active in the presence of ADP1. Mass spectrometry based metabolite footprint analysis suggests that,putrescine and phenylalanine excreted by ADP1 cells may support the metabolic activity of E. coli. This study demonstrates that Raman-SIP with reverse labeling would be a Useful tool to probe metabolism of any carbon substrate, overcoming limitations when stable isotopic substrates are not readily available. It is also found that Raman-SIP with D2O labeling is a sensitive and reliable approach to distinguish metabolically active cells but not quiescent cells. This novel: approach extends the application of Raman-SIP and demonstrates its potential application as a valuable strategic approach for probing cellular metabolism, metabolic activity, and interactions in microbial communities at the single cell level.