Escherichia coli and related Gram-negative bacteria contain an energy-
coupled transport system through the outer membrane which consists of
the proteins TonB, ExbB, ExbD anchored in the cytoplasmic membrane and
receptors in the outer membrane. Differences in the activities of the
Escherichia coli and the Serratia marcescens TonB proteins were used
to identify TonB functional domains. In E. coli TonB segments were rep
laced by equivalent fragments of S. marcescens TonB and the activities
of the resulting chimaeric proteins were determined. In addition, E.
coli TonB was truncated at the C-terminal end, and point mutants were
generated using bisulphite. From the results obtained we draw the foll
owing conclusions: an important site of interaction between TonB and E
xbB is located in the N-terminal region of TonB within or close to the
cytoplasmic membrane since an N-terminal 44-residue fragment of TonB
was stabilized by ExbB and interfered with wild-type TonB activity. In
addition, the activity of a TonB derivative in which histidine residu
e 20 was replaced by arginine was strongly reduced, and a double mutan
t containing arginine-7 to histidine and alanine-22 to threonine subst
itutions displayed an impaired uptake of ferrichrome. Furthermore, the
domain around residue 160 is involved in TonB activity. S. marcescens
TonB segments of this region in E. coli TonB conferred S. marcescens
TonB activities, and E. coli TonB point mutants displayed strongly imp
aired activities for the uptake of colicin B and M and ferric sideroph
ores. Plasmid-encoded tonB mutants of this region showed negative comp
lementation of chromosomal wild-type tonB, and certain tonB mutants su
ppressed colicin B TonB-box mutants. Uptake of colicins required diffe
rent domains in TonB, for colicin B and M around residue 160 and for c
olicin la, a domain closer to the C-terminal end. Tandem duplication o
f the E. coli (EP)X(KP) region by insertion of the S. marcescens (EP)X
(KP) region (38 residues) and replacement of lysine residue 91 by glut
amate did not alter TonB activity so that no evidence was obtained for
this region to be implicated in receptor binding. The aberrant electr
ophoretic mobility of TonB was caused by the proline-rich sequence sin
ce its removal resulted in a normal mobility.