Identification of Critical Amino Acid Residues of a Two-Component Sensor Protein for Signal Sensing in Fimbriation via Random Mutant Library Construction.

() utilizes FimA fimbriae to colonize the gingival sulcus and evade the host immune system. The biogenesis of all FimA-related components is positively regulated by the FimS-FimR two-component system, making the FimS sensory protein an attractive target for preventing infection. However, the specific environmental signal received by FimS remains unknown. We constructed random mutant libraries to identify critical amino acid residues for signal sensing by FimS. Optimized error-prone polymerase chain reaction (PCR) was used to introduce a limited number of random mutations in the periplasmic-domain-coding sequence of , and expression vectors carrying various mutants were generated by inverse PCR. More than 500 transformants were obtained from the -knockout strain using the - conjugal transfer system, whereas only ~100 transformants were obtained using electroporation. Four and six transformant strains showed increased and decreased expression, respectively. Six strains had single amino acid substitutions in the periplasmic domain, indicating critical residues for signal sensing by FimS. This newly developed strategy should be generally applicable and contribute to molecular genetics studies of , including the elucidation of structure-function relationships of proteins of interest.