CONSERVED SEQUENCE MOTIFS IN THE UNORTHODOX BVGS 2-COMPONENT SENSOR PROTEIN OF BORDETELLA-PERTUSSIS

The unorthodox two-component sensor protein BvgS of Bordetella pertuss is contains several interesting sequence motifs of unknown functional relevance, such as a histidine motif in its output domain, which is co nserved among several unorthodox sensor proteins, a putative nucleotid e binding site [Walker box type A] in its linker region, and a region in its periplasmic domain with significant homology to the TonB protei n of Escherichia coli. We investigated potential functions of these se quences by constructing B. pertussis strains that express mutant BvgS derivatives. The His(1172) residue in the output domain was exchanged for Gln, and the Walker motif was mutated either by the replacement of Lys(625) by Arg, or of Gly(624) by Val and Lys(625) by Leu. To analys e the TonB motif, the periplasmic domain of BvgS was replaced with the corresponding domain of EvgS, an E. coli sensor that is highly homolo gous to BvgS but lacks the similarity with TonB. All mutations except the conservative Lys/Arg exchange in the Walker box caused the inactiv ation of BvgS, indicating the functional importance of the conserved m otifs. The activity of the mutant proteins could be restored by comple mentation in trans with various separately expressed, truncated parts of BvgS. Mutations in the BvgS receiver domain could be complemented n ot only by a construct expressing the wild-type receiver and output do mains, but also by the derivative containing the His-Gln exchange. The refore, the histidine motif, although important for BvgS function, is not essential for complementation of BvgS mutants. The mutations in th e Walker box and in the periplasmic domain could be complemented by a truncated BvgS derivative lacking the receiver and output domains. The characterization of a spontaneous revertant of the strain expressing the originally inactive EvgS/BvgS hybrid protein revealed the presence of a mutation in the BvgS linker region, conferring constitutive acti vity on the protein, As TonB energizes transport processes across the outer membrane of E. coli, the strain expressing the constitutive EvgS /BvgS hybrid protein lacking the TonB motif was used in preliminary in vestigations of a possible direct involvement of BvgS in transport pro cesses.