Characterization of HasB, a Serratia marcescens TonB-like protein specifically involved in the haemophore-dependent haem acquisition system

In Gram-negative bacteria, the TonB-ExbB-ExbD inner membrane multiprotein c omplex is required for active transport of diverse molecules through the ou ter membrane. We present evidence that Serratia marcescens, like several ot her Gram-negative bacteria, has two TonB proteins: the previously character ized TonB(SM), and also HasB, a newly identified component of the has opero n that encodes a haemophore-dependent haem acquisition system. This system involves a soluble extracellular protein (the HasA haemophore) that acquire s free or haemoprotein-bound haem and presents it to a specific outer membr ane haemophore receptor (HasR). TonB(SM) and HasB are significantly similar and can replace each other for haem acquisition. However, TonB(SM), but no t HasB, mediates iron acquisition from iron sources other than haem and hae moproteins, showing that HasB and TonB(SM) only display partial redundancy. The reconstitution in Escherichia coli of the S. marcescens Has system dem onstrated that haem uptake is dependent on the E. coli ExbB, ExbD and TonB proteins and that HasB is non-functional in E. coli. Nevertheless, a mutati on in the HasB transmembrane anchor domain allows it to replace TonB(EC) fo r haem acquisition. As the change affects a domain involved in specific Ton B(EC)-ExbB(EC) interactions, HasB may be unable to interact with ExbB(EC), and the HasB mutation may allow this interaction. In E. coli, the HasB muta nt protein was functional for haem uptake but could not complement the othe r TonB(EC)-dependent functions, such as iron siderophore acquisition, and p hage DNA and colicin uptake. Our findings support the emerging hypothesis t hat TonB homologues are widespread in bacteria, where they may have specifi c functions in receptor-ligand uptake systems.