ACTIVITY DOMAINS OF THE TONB PROTEIN

Citation
I. Traub et al., ACTIVITY DOMAINS OF THE TONB PROTEIN, Molecular microbiology, 8(2), 1993, pp. 409-423
Citations number
34
Categorie Soggetti
Biology,Microbiology
Journal title
ISSN journal
0950382X
Volume
8
Issue
2
Year of publication
1993
Pages
409 - 423
Database
ISI
SICI code
0950-382X(1993)8:2<409:ADOTTP>2.0.ZU;2-U
Abstract
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.