CauloKO: an ordered transposon mutant library in Caulobacter crescentus

Genetic screens are powerful approaches to unveiling new biological insights and ordered redundant transposon libraries have emerged as a primary tool for performing screens with known genetic saturation. Newer sequencing approaches based on combinatorial pooling have lowered the cost and time required to generate these libraries. Caulobacter crescentus is a gram-negative bacterium that has served as a model for understanding bacterial physiology with a myriad of genetic tools. To add to this collection of tools, we created CauloKO-the first ordered, transposon library in C. crescentus. CauloKO includes insertion mutants in 86% of all non-essential genes and 77% of all open reading frames of strain CB15. CauloKO insertion mutants were validated using Sanger sequencing. We also present phenotypic analysis of the CauloKO library using a crystal violet screen for biofilm mutants, which both confirmed previous results and identified new mutants for future studies. This combined approach revealed that the CauloKO library shows promise for screening applications, particularly for phenotypes that require monoclonal populations of cells.IMPORTANCEOrdered transposon mutant libraries have dramatically advanced the ability to study other systems, and the potential for combining such a library with the other genome-wide tools available in Caulobacter crescentus makes such a library particularly important for this system. Our work also establishes a novel method for analyzing sequencing data for ordered library construction purposes, and a first-of-its-kind dynamic, online platform for cataloging mutants within the library. Furthermore, our findings further confirmed results of previously published studies, but also identified potential novel regulators of biofilm formation in C. crescentus that will be important starting points for future investigations.