Identifying Gene Functions in E. Coli, G. Hansenii, and Z. Mobilis via a Model-Enabled Gene Search

Undergraduate #106
Discipline: Biological Sciences
Subcategory: Microbiology/Immunology/Virology

Audrey Haines - University of Minnesota, Minneapolis, MN
Co-Author(s): Jennifer L. Reed and Shu Pan, University of Wisconsin, Madison, WI

Understanding bacterial metabolism is important for many biotechnological and medical applications; however, the functions of many metabolic genes in most useful organisms are unknown. This project aims to link genes to essential functions in Escherichia coli, which has a wide range of biotech applications, Gluconacetobacter hansenii, which is important in biocellulose synthesis, and Zymomonas mobilis, which is an industrial ethanol producer. Model-Enabled Gene Search (MEGS) (1) is a novel method for finding missing genes corresponding to essential functions in bacteria. MEGS uses an E. coli model to design E. coli strains and media conditions such that missing metabolic reactions in G. hansenii and Z. mobilis can rescue E. coli growth. The missing reactions in G. hansenii and Z. mobilis metabolism are essential; thus, we hypothesized that G. hansenii and Z. mobilis carry functionally analogous genes with different gene sequences. G. hansenii lacked the panD gene, which is a key component of the β-alanine synthesis in E. coli. Z. mobilis was missing the ubiC gene, part of the ubiquinone synthesis pathway. Both organisms were missing the pdxB gene, which is responsible for pyridoxal 5′-phosphate synthesis in E. coli. Additionally, it is known that E. coli carries a second, unknown, valine exporter gene (2), analogous to the previously discovered ygaZH gene. This project aims to identify this gene as well. MEGS utilizes the Datsenko-Wanner (3) method to make E. coli knockouts for the E. coli genes listed above. G. hansenii, Z. mobilis, and E. coli genomic libraries were then transformed into the lethal knockouts to ‘rescue’ E. coli mutants that were missing the genes listed above. The cells that were able to grow in minimal media were then sequenced to identify which genes could functionally replace the E. coli genes. Genes analogous to the missing E. coli sequences were discovered for panD and pdxB in G. hansenii and for pdxB and ubiC in Z. mobilis. They will be added to the existing metabolic models for those bacteria and will contribute to improving out understanding of how metabolic pathways within the organisms can be engineered for beneficial purposes. Future experimentation will include fine-tuning the approach to find the ygaZH analog, and making growth curves of the rescued E. coli cells using 96-well plates to confirm these results.

References: 1. Pan et al. (Submitted) 2. Park et al. PNAS. 2007: 104, 19 3. Datsenko & Wanner. Proc Natl Acad Sci. 2000;97(12):6640-5

Funder Acknowledgement(s): Thank you to the Reed group and to the University of Wisconsin REU in the Chemistry of Materials for Renewable Energy. Funding for this research was supported by the National Science Foundation through the EFRI-REM program under award number EFRI-1240268.

Faculty Advisor: Jennifer L. Reed, reed@engr.wisc.edu

Role: I took essential functions that lacked known genes in G. hansenii and Z. mobilis and did all of the necessary experimentation to discover new sequences responsible for the aforementioned metabolic functions.