(John Cronan, Director; Jonathan Sweedler, co-PI)
Headed by microbiologist Prof. John Cronan and analytical chemist Prof Jonathan Sweedler, both at the University of Illinois, Urbana-Champaign, the EFI Microbiology Core uses a variety of techniques to correlate functions discovered in vitro to physiological roles in vivo. The Microbiology Core investigates functional predictions made by the Computation Core that were corroborated experimentally in the Bridging Projects and Structure Core. Additional in vivo evidence informs the initial functional assignment, and more often than not uncovers interesting and unknown biology in the process. The Microbiology Core coordinates its efforts with the other EFI groups, including the Superfamily/Genome, Computation, Structure, and Protein Cores as well as the Bridging Projects, to focus their efforts on enzymes that are most likely to be conducive to in vivo characterization and yield the richest information.
The Microbiology Core’s strategies generally fall into the following categories: bacterial genetics, transcriptional analyses, and metabolite analyses. Depending on the nature of the organism and proposed pathway, multiple approaches are often employed or used in concert. Targets investigated by the Microbiology Core are limited to those from genetically tractable organisms. This stipulation decreases the number of targets that can be investigated because no genetic techniques exist for the majority of sequenced bacteria and even when available, methods are often labor intensive and slow. Despite these drawbacks, the ability to culture and manipulate organisms allows for employment of immensely powerful tools such as construction of null (knockout) and/or overexpression strains. Growth of such strains, especially in comparison to wild-type, enables evaluation of phenotypes associated with a given target (e.g. rate and extent of growth, morphology, pigmentation, etc.). To facilitate the display of phenotypes, the Microbiology Core has assembled large phenotypic screening libraries with varying carbon, nitrogen, phosphorus and sulfur sources.
Beyond these staple techniques, the Microbiology Core also uses reverse transcription PCR (RT-PCR) and RNAseq to identify increased or decreased expression levels of mRNAs for genes of interest. Results of such studies are especially useful in illuminating other genes likely to be functional in the same pathway, because they frequently display similar expression profiles as the initial target of interest. Metabolite analysis is also a powerful tool for examining potential cellular pathways. The Microbiology Core often employs targeted or untargeted mass spectrometry to examine how metabolite pools are perturbed, for example, by feeding a putative precursor metabolite.
The strengths and necessity of the Microbiology Core were borne out early in the EFI with the undertaking of a model project that investigated a divergent RuBisCO-like protein (RLP) from Rhodospirillum rubrum. Although an in vitro reaction had been determined for the R. rubrum RLP, the physiological context and necessity for the reaction, an unusual isomerization of a methylthio-containing 5-carbon sugar, was completely unknown. Curiously, the rubrum RLP is similar to Bacilli RLPs that function in a methionine salvage pathway, but R. rubrum lacks many of the enzymes in this pathway. Microbiology Core experiments with R. rubrum cells fed an upstream metabolite not only revealed the presence of the metabolites predicted from the observed in vitro reaction, but also showed flux of the carbon backbone through the non-mevalonate pathway for isoprenoid biosynthesis. This untargeted metabolomics analysis was able to confirm the hypothesized novel methionine salvage pathway and, more importantly, provide new biological insight into the downstream metabolites and pathways. This work has was published in Nature Chemical Biology (Erb et al. 2012). This project developed methodologies that ultimately provided evidence placing the proposed chemical reaction in a biological context, thereby completing the functional assignment and leading to the discovery of unexpected and exciting new biology.