Cys-92, Cys-95, and the C-terminal 12 residues of the Vibrio harveyi ferric uptake regulator (Fur) are functionally inessential

Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (Fur_vh) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. Furvh shares 77% overall sequence identity with the Escherichia coli Fur (Fur_Ec) and could complement a mutant of Fur_Ec. Like Fur_Ec, Fur_Vh, possesses two cysteine residues at positions 92 and 95, yet unlike Fur_Ec, in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of Fur_Vh proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of Fur_vh are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of Fur_Vh are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of Fur_Vh is possibly different from that of Fur_Ec; and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of Fur_Vh; and (iii) provided insights into the potential function of the local structure involving C137 and K138.