Cloning of the Serratia marcescens hasF gene encoding the Has ABC exporter outer membrane component: a TolC analogue.
The Serratia marcescens haemophore HasA is secreted by an ABC exporter comprising three envelope proteins. The ABC protein (ATP-binding cassette) HasD and the MFP protein (membrane fusion protein) HasE but not the outer membrane component have been isolated previously. In Escherichia coli, TolC, the outer membrane component of the haemolysin transporter, can form a hybrid exporter with HasD and HasE. This hybrid secretes HasA and the very similar metalloproteases from S. marcescens and Erwinia chrysanthemi. By analogy, the genuine exporter was predicted to secrete metalloproteases. The hasF gene was thus cloned from S. marcescens into an E. coli tolC mutant carrying hasD and hasE genes, by screening for a proteolytic phenotype on skimmed-milk plates. hasF encodes a protein sharing 74% identity with the E. coli TolC protein. Anti-TolC antibodies cross-reacted with a protein with an apparent molecular weight of 53 kDa in E. coli expressing hasF and in S. marcescens. hasF is unlinked to the has cluster and, unlike the has operon, is not iron regulated. hasF complements some of the tolC phenotypes, including drug- and detergent sensitivities and haemolysin secretion but not colicin E1 uptake. This suggests that the various functions of TolC could correspond to distinct domains on the protein.
Binet
R
R
Unité de Physiologie Cellulaire, Institut Pasteur, URA 1300, CNRS, Paris, France. rbinet@pasteur.fr or cwander@pasteur.fr
Wandersman
C
C
D018528:ATP-Binding Cassette Transporters / Q000235:genetics; D000251:Adenosine Triphosphatases; D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000235:genetics* / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; C093956:HasA protein, Serratia marcescens; C103984:HasF protein, bacteria; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; C073126:tolC protein, E coli; D000251:Adenosine Triphosphatases; C093959:HasD protein, Serratia marcescens