AimsThe environmental pathogen Streptococcus uberis causes intramammary infections in dairy cows. Because biofilm growth might contribute to Strep.uberis mastitis, we conducted a biological screen to identify genes potentially involved in the regulation of biofilm growth. Methods and ResultsBy screening a transposon mutant library of Strep.uberis, we determined that the disruption of 13 genes (including hasA, coaC, clpP, miaA, nox and uidA) led to increased biofilm formation. One of the genes (SUB1382) encoded a homologue of the LiaR response regulator (RR) of the Bacillus subtilis two-component signalling system (TCS). Electrophoretic mobility shift assays revealed that DNA binding by LiaR was greatly enhanced by phosphorylation. Two-dimensional differential in-gel electrophoresis analyses of the liaR mutant and the parental Strep.uberis strain revealed five differentially produced proteins with at least a 15-fold change in relative abundance (P<005). ConclusionsThe DNA-binding protein LiaR is a potential regulator of biofilm formation by Strep.uberis. Significance and Impact of the StudySeveral molecular primary and downstream targets involved in biofilm formation by Strep.uberis were identified. This provides a solid foundation for further studies on the regulation of biofilm formation in this important pathogen.