(A) Immunodetection of AtaA using an anti-AtaA antiserum against whole cell lysates prepared from Tol 5 WT and the 4140 mutant. (B) Growth curve of Tol 5 WT and the 4140 mutant in LB medium at 28°C, with shaking at 115 rpm. Data are expressed as the mean and SD obtained from 3 independent cultures. (C) Adhesion of Tol 5 WT and the 4140 mutant to a polystyrene surface. The photograph indicates the AZD8931 solubility dmso stained cells adhering to a 48-well plate. Data are expressed as the mean and SEM (n = 3). Statistical significance, *P < 0.01. (D) Autoagglutination this website assay of Tol 5 WT and

the 4140 mutant by the tube-settling assay. The photographs indicate test tubes after a 3-h incubation without agitation. Data are expressed as the mean and SEM (n = 3). check details Statistical significance, *P < 0.001. Although, in this study, we constructed the unmarked ataA mutant by excising a 10-kb segment from the chromosome of Tol 5, our new method can theoretically be used to disrupt a larger gene in other non-competent Gram-negative bacteria because Pósfai

et al. successfully excised 51-kb and 110-kb DNA segments from the chromosome of E. coli K-12 MG1655 by FLP/FRT recombination [29]. Bap (8,620 aa) from Acinetobacter baumannii 307–0294, LapA (8,683 aa) from Pseudomonas fluorescens WCS365, and LapF (6,310 aa) from Pseudomonas putida KT2440 are larger proteins than AtaA (3,630 aa) and play an important role in adhesion to tuclazepam solid surfaces and biofilm formation [11, 14, 15, 31]. Since there was no useful method for introducing an unmarked mutation into large genes encoding them, random transposon mutants have been used to characterize the phenotype

generated by a deficiency of those genes. By using our new unmarked method, such large genes can be easily and efficiently deleted from non-competent Gram-negative bacteria, and mutants that are more appropriate than marked mutants for the analysis of phenotypic changes can be obtained. In addition to functional analyses of large genes, our unmarked method would be effective for the metabolic engineering of bacteria to produce conventional fermentation products, biofuels, medicines, and chemicals by deleting long regions of metabolism-related gene clusters disturbing their production. Conclusion We designed two gene replacement plasmids and developed a new methodology for the construction of an unmarked mutant using the FLP/FRT recombination system. This methodology overcomes the problems associated with introducing an unmarked mutation into a large gene of non-competent Gram-negative bacteria. Using this method, we successfully constructed an unmarked mutant of ataA of Tol 5, which is 10,893 bp long. The plasmids and the methodology should be applicable to a wide range of Gram-negative bacteria except for E. coli and some enterobacteria and are expected to be useful tools to characterize the functions of large genes.