Bacteria have been thought to follow only a few well-recognized biochemical pathways when fermenting glucose or other hexoses. These pathways have been chiseled in the stone of textbooks for decades, with most sources rendering them as they appear in the classic 1986 text by Gottschalk. Still, it is unclear how broadly these pathways apply, given that they were established and delineated biochemically with only a few model organisms. Here, we show that well-recognized pathways often cannot explain fermentation products formed by bacteria. In the most extensive analysis of its kind, we reconstructed pathways for glucose fermentation from genomes of 48 species and subspecies of bacteria from one environment (the rumen). In total, 44% of these bacteria had atypical pathways, including several that are completely unprecedented for bacteria or any organism. In detail, 8% of bacteria had an atypical pathway for acetate formation; 21% of bacteria had an atypical pathway for propionate or succinate formation; 6% of bacteria had an atypical pathway for butyrate formation and 33% of bacteria had an atypical or incomplete Embden-Meyerhof-Parnas pathway. This study shows that reconstruction of metabolic pathways - a common goal of omics studies - could be incorrect if well-recognized pathways are used for reference. Furthermore, it calls for renewed efforts to delineate fermentation pathways biochemically.

Fluorescent tracers have been used to measure solute transport, but transport kinetics have not been evaluated by comparison of radiolabeled tracers. Using Streptococcus equinus JB1 and other bacteria, the objective of this study was to determine if a fluorescent analogue of glucose (2-NBDG) would be transported with the same kinetics and transporters as [ 14 C]glucose. We uniquely modified a technique for measuring transport of radiolabeled tracers so that transport of a fluorescent tracer (2-NBDG) could also be measured. Deploying this technique for S. equinus JB1, we could detect 2-NDBG transport quantitatively and within 2 s. We found the V max of 2-NBDG transport was 2.9-fold lower than that for [ 14 C]glucose, and the K m was 9.9-fold lower. Experiments with transport mutants suggested a mannose phosphotransferase system (PTS) was responsible for 2-NBDG transport in S. equinus JB1 as well as Escherichia coli. Upon examination of strains from 12 species of rumen bacteria, only the five that possessed a mannose PTS were shown to transport 2-NBDG. Those five uniformly transported [ 14 C]mannose and [ 14 C]deoxyglucose (other glucose analogues at the C-2 position) at high velocities. Species that did not transport 2-NBDG at detectable velocities did not possess a mannose PTS, though they collectively possessed several other glucose transporters. These results, along with retrospective genomic analyses of previous 2-NBDG studies, suggest that only a few bacterial transporters may display high activity toward 2-NBDG. Fluorescent tracers have the potential to measure solute transport qualitatively, but their bulky fluorescent groups may restrict (i) activity of many transporters and (ii) use for quantitative measurement.

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Few characteristics are more important to a bacterium than the substrates it consumes. It is hard to identify what substrates are consumed by bacteria in natural communities, however, because most bacteria have not been cultured. In this study, we developed a method that uses fluorescent substrate analogs, cell sorting, and DNA sequencing to identify substrates taken up by bacteria. We deployed this method using 2[N-(7-nitrobenz-2-oxa-1,2-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG), a fluorescent glucose analog, and bacteria of the bovine rumen. This method revealed over 40 different bacteria (amplicon sequence variants [ASVs]) from the rumen that take up glucose. Nearly half of these ASVs represent previously uncultured bacteria. We attempted to grow these ASVs on agar media, and we confirmed that nearly two-thirds resisted culture. In coculture experiments, the fluorescent label of 2-NBDG was not transferred to nontarget bacteria by cross-feeding. Because it is not affected by cross-feeding, our method has an advantage over stable isotope probing. Though we focus on glucose, many substrates can be labeled with the fluorophore NBD. Our method represents a new paradigm for identifying substrates used by uncultured bacteria. It will help delineate the niche of bacteria in their environment.IMPORTANCEWe introduce a method for identifying what substrates are consumed by bacteria in natural communities. Our method offers significant improvement over existing methods for studying this characteristic. Our method uses a fluorescently labeled substrate which clearly labels target bacteria (glucose consumers in our case). Previous methods use isotope-labeled substrates, which are notorious for off-target labeling (due to cross-feeding of labeled metabolites). Our method can be deployed with a variety of substrates and microbial communities. It represents a major advance in connecting bacteria to the substrates they take up.