Ampicillin amp

The bacterial strains and plasmids used in this study are listed in Table 1. The APEC XM strain (O2:K1) was donated by Dr. Chengping Lu, Nanjing Agricultural University. It was isolated from a duck brain with symptoms of septicemia and meningitis. The clbA deletion mutant and complemented mutant were derived from the APEC XM. All bacteria were grown aerobically on Luria-Bertani (LB) plates or in LB broth at 37°C with agitation (180 rpm), except for the mutants containing the temperature-sensitive plasmid pCP20 or pKD46, which was grown at 30°C. Strains harboring antibiotic resistance genes were cultured in LB containing ampicillin (Amp, 100 μg/mL) (Sangon Biotech, Shanghai, China) or chloramphenicol (Cm, 34 μg/mL) (Sangon Biotech, Shanghai, China) when appropriate. Plasmids pKD3, pKD46 and pCP20 were used for the λ-Red mediated recombination system. pBR322 was used for the construction of the complemented mutant. To determine growth rates, bacteria were incubated at 37°C in LB broth for 24 h with continuous agitation (180 rpm). The number of live bacteria was measured at 1 h intervals by determining the optical density (OD) at 600 nm. The growth curve experiment was performed with three biological replicates.

BamHI restriction endonuclease, T4 DNA ligase, 2×TransStart Fast Fly SuperMix, DMT enzyme and DMT-competent cells were all provided by TransGen Biotech Co., Ltd; Ampicillin (Amp), DNA marker 2000, SanPrep column plasmid DNA Mini-Preps extraction kit and primer synthesis were all provided by Sangon Biotech (Shanghai) Co.,Ltd.; the target gene BCKD and plasmid vector pGEX-4T1 were provided by University of California, Davis; ddH2O and other materials are prepared by the lab of environmental science and engineering, Yangzhou University.

The bacteria and plasmids used in this study are listed in Table 1. The APEC XM strain (O2:K1) was donated by Dr. Chengping Lu, Nanjing Agricultural University. APEC XMΔryhB and APEC XMΔryhB/pryhB constructed in this study were derived from APEC XM. All bacteria were cultured in Luria-Bertani (LB) broth or on LB plates at 37 °C with agitation at 180 rpm. The mutants containing the temperature-sensitive plasmid pCP20 or pKD46 were grown in LB containing ampicillin (Amp, 100 µg/mL) (Sangon Biotech, Shanghai, China) or chloramphenicol (Cm, 34 µg/mL) (Sangon Biotech) when appropriate at 30 °C. For the determination of biofilm formation, APEC XM, APEC XMΔryhB and APEC XMΔryhB/pryhB were statically cultured in biofilm-inducing medium (Oxoid, Altrincham, Cheshire, UK) at 37 °C [58 (link)]. Plasmids pKD3, pKD46 and pCP20 were used for construction of the deletion mutant. Plasmid pBR322 was used for constructing the complemented mutant.