A heterologous membrane protein domain fused to the C-terminal ATP-binding domain of HlyB can export Escherichia coli hemolysin.
The hydrophobic-rich NH2-terminal 34 amino acids of a tetracycline resistance determinant (TetC) were fused to the COOH-terminal 240 amino acids of the hemolysin transporter, HlyB, which contains a putative ATP-binding domain. This hybrid protein replaced the NH2-terminal 467-amino-acid portion of HlyB and could still export the Escherichia coli hemolysin (HlyA). Export by the hybrid protein was approximately 10% as efficient as transport by HlyB. Extracellular secretion of HlyA by the TetC-HlyB hybrid required HlyD and TolC. The extracellular and periplasmic levels of beta-galactosidase and beta-lactamase in strains that produced the hybrid were similar to the levels in controls. Thus, HlyA transport was specific and did not appear to be due to leakage of cytoplasmic contents alone. Antibodies raised against the COOH terminus of HlyB reacted with the hybrid protein, as well as HlyB. HlyB was associated with membrane fractions, while the hybrid protein was found mainly in soluble extracts. Cellular fractionation studies were performed to determine whether transport by the hybrid occurred simultaneously across both membranes like wild-type HlyA secretion. However, we found that HlyA was present in the periplasm of strains that expressed the TetC-HlyB hybrid. HlyA remained in the periplasm unless the hlyD and tolC gene products were present in addition to the hybrid.
Thomas
W D
WD
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison 53706.
Wagner
S P
SP
Welch
R A
RA
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism* / Q000502:physiology; D002678:Chimera; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D006461:Hemolysis; D026901:Membrane Transport Proteins; D008954:Models, Biological; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D013347:Subcellular Fractions / Q000737:chemistry; D013753:Tetracycline Resistance / Q000235:genetics
D001426:Bacterial Proteins; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D006460:Hemolysin Proteins; C050837:Hlyb protein, Bacteria; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C077558:TetC-HlyB protein, E coli; D000255:Adenosine Triphosphate