Zichu Yang ¹ Tyler Helmann ² Mael Baudin ³ Karl Schreiber ³ Zhongmeng Bao ² Paul Stodghill ² Adam Deutschbauer ³ Jennifer D. Lewis ³ Bryan Swingle ^2*

• ¹ Plant Pathology and Plant-Microbe Biology Section, School of Integrative Plant Science, Cornell University, Ithaca, New York, United States
• ² Emerging Pests and Pathogens Research Unit, Robert W. Holley Center for Agriculture and Health, Agricultural Research Service (USDA), Ithaca, United States
• ³ Department of Plant & Microbial Biology, College of Natural Resources, University of California, Berkeley, Berkeley, California, United States

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) is a plant pathogenic bacterium that possesses complicated motility regulation pathways including a typical chemotaxis system. A significant portion of our understanding about the genes functioning in Pst DC3000 motility is based on comparison to other bacteria. This leaves uncertainty about whether gene functions are conserved, especially since specific regulatory modules can have opposite functions in sets of Pseudomonas. In this study, we used a competitive selection to enrich for mutants with altered swimming motility and used random barcode transposon-site sequencing (RB-TnSeq) to identify genes with significant roles in swimming motility. Besides many of the known or predicted chemotaxis and motility genes, our method identified PSPTO_0406 (dipA), PSPTO_1042 (chrR) and PSPTO_4229 (hypothetical protein) as novel motility regulators. PSPTO_0406 is a homolog of dipA, a known cyclic di-GMP degrading enzyme in P. aeruginosa. PSPTO_1042 is part of an extracytoplasmic sensing system that controls gene expression in response to reactive oxygen species, suggesting that PSPTO_1042 may function as part of a mechanism that enables Pst DC3000 to alter motility when encountering oxidative stressors. PSPTO_4229 encodes a protein containing an HD-related output domain (HDOD), but with no previously identified functions.We found that deletion and overexpression of PSPTO_4229 both reduce swimming motility, suggesting that its function is sensitive to expression level. We used the overexpression phenotype to screen for nonsense and missense mutants of PSPTO_4229 that no longer reduce swimming motility and found a pair of conserved arginine residues that are necessary for motility suppression. Together these results provide a global perspective on regulatory and structural genes controlling flagellar motility in Pst DC3000.