Edwardsiella tarda is a gram-negative broad-host-range pathogen that causes hemorrhagic septicemia in many commercially important fish species.Its ability to adapt to and thrive in diverse environments outside and inside of its hosts prompts us to investigate the roles of the previously identified 33 putative two-component signal transduction systems (TCSs) in E.tarda.In this work, we successfully constructed deletion mutations in each of the response regulator genes, suggesting that none of the TCSs are essential for cell viability in E.tarda.The mutants were further examined for roles in biofilm formation, antibiotic resistance, stress response, expression and secretion of proteins involved in either the type Ⅲ secretion system (T3SS) or type Ⅵ secretion system (T6SS), as well as virulence.Through these assays,we identified four regulators of biofilm development, two regulators of antibiotic resistance, and four regulators involved in stress responses.We found that two regulators, EsrB and PhoP, have codependent and independent contributions to E.tarda virulence.Mutation of EsrB resulted in the complete loss of both the T3SS and T6SS proteins, while PhoP partially regulated the expression of T3SS and T6SS genes through EsrB, and was essential for resistance to antimicrobial peptides.