Adaptive evolution resulted in three subtypes of the Riemerella anatipestifer crpR1 gene

The global transcription regulator CRP protein controls diverse cellular processes in many bacteria. Our group previously reported that Tn4531 insertion in FIP52_09345 (designated as crpR1), which encodes a special CRP protein without the HTH motif, attenuated the virulence of Riemerella anatipestifer. Here, sequence analyses indicate that R. anatipestifer crpR1 can be classified into three subtypes (crpR1a, crpR1b, and crpR1c) based on the ORF length. Of 52 strains, 42 carried crpR1a. CrpR1b and CrpR1c contained both the CRP domain and HTH motif, while CrpR1a lacked the HTH motif. The crpR1a sequence had two alleles, and crpR1b of some strains originated from allele 1 of crpR1a, while crpR1c of strain CH3 originated from allele 2 of crpR1a. Point mutations and insertion sequences may play an important role in the evolution of crpR1. Moreover, deletion of crpR1a from strain Yb2 resulted in reduced virulence in ducklings by approximately 1,000-fold, while deletion of the 3'-end of crpR1a (encoding the disordered region) or the downstream AS87_RS10485 (encoding the HTH motif) resulted in reduced virulence of strain Yb2 in ducklings. In addition, mutation of 450C451A in the crpR1a ORF of strain Yb2 to 450G generated the non-pathogenic Yb2 crpR1-450G mutant, with the crpR1 subtype changed to crpR1b. Mutants with the 3' end of the crpR1 ORF in Yb2 crpR1-450G, consistent with that of strain HXb2 or CH3 crpR1, were pathogenic to ducklings. These findings confirm that adaptive evolution resulted in three subtypes of R. anatipestifer crpR1.IMPORTANCEBacterial CRP regulators regulate metabolism, stress resistance, biofilm formation, and pathogenesis. Here, we report that a special CRP in Riemerella anatipestifer, CrpR1a, which lacks the HTH motif, has biological functions. The crpR1a, carried by most R. anatipestifer strains, is the ancestor of crpR1b and crpR1c. Deletion of crpR1a in strain Yb2 or mutation of 450C451A to 450G in this crpR1a ORF leads to significant reduction or loss of virulence, but a single base mutation of 559C > A, 586C > T, or 591T > C in the non-pathogenic mutant Yb2 crpR1-450G crpR1 ORF increases its virulence in ducklings. Moreover, deletion of the C-terminal disordered region of CrpR1a or its downstream HTH motif resulted in reduced virulence of strain Yb2.