Leveraging Environmental Complexity to Define Bacterial Gene Function
Abstract
Research Overview and Significance The ability of bacterial cells to execute complex processes in their environment is directly determined by the proteins and RNAs encoded within their genomes. Genomics research programs have cataloged millions of bacterial genes and the three-dimensional structures of thousands of proteins. However, data that define the cellular functions of uncharacterized genes and proteins in these large datasets have been slow to emerge. Indeed, nearly one-quarter of cataloged protein domain families lack any annotated function. To advance understanding of bacterial biology, there is a critical need to fill this gap in our understanding of gene function. An innovative workflow that directly addresses this problem is proposed here. This new approach combines modern mutagenesis and DNA sequencing methods with environmental cultivation approaches that favor ecological complexity and geochemistry over standard laboratory media. The data resulting from the experiments proposed will 1) provide molecular-level understanding of a newly-discovered cell envelope regulator uncovered using this environmental cultivation approach, and will 2) enable future in-depth functional annotation of many uncharacterized bacterial genes. These proposed studies will inform multiple areas of microbiology research including bacterial physiology, synthetic biology, bacterial pathogenesis, and environmental microbiology Ñ including the study of microbial communities. Given the fundamental impact of new gene function information, the work has the potential to inform efforts to improve soldier protection and performance on multiple fronts. Objectives Observable defects in growth, morphology, metabolism, or gene expression of mutant strains provide a powerful experimental handle to characterize gene function. However, it is often a major challenge to uncover cultivation conditions in which a gene disruption results in an obvious defect. This research team has constructed a barcoded pool of transposon mutants in Caulobacter crescentus, a Gram-negative bacterium that is common in freshwater and soil. Through cultivation of this collection of insertional mutants in bona fide lake freshwater, they identified C. crescentus genes that are dispensible under standard laboratory cultivation conditions, but are important for growth in its natural environment. Subsequent studies of a set of signal transduction genes with related growth defects in freshwater led to the discovery of ntrZ, a previously uncharacterized hypothetical gene. NtrZ was recently demonstrated to be a protein that affects C. crescentus cell envelope function by modulating biochemical activity of the NtrY-NtrX two-component regulatory system. Given the success of these initial experiments the following two independent research objectives are proposed: 1) Use classic molecular genetic and biochemical approaches to characterize the molecular mechanism by which NtrZ regulates the NtrY-NtrX signaling system and thereby influences the biology of a Gram negative cell envelope barrier. 2) Conduct time-series cultivation experiments on barcoded pools of C. crescentus mutants in a well-characterized freshwater system to discover and define new gene functions.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jun 30, 2022
- Source ID
- W911NF2210105
Entities
People
- Sean Crosson
Organizations
- Army Contracting Command
- Michigan State University
- United States Army