Transport of hemolysin across the outer membrane of Escherichia coli requires two functions

Abstract

MATERIALS AND METHODS Bacterial strains. E. coli K-12(pHlyl52) and the Tn3 mutants of pHlyl52 have been described previously (20, 21). E. coli 605 carrying TnS in the chromosome was given to us by A. Piihler. Plasmids. The recombinant plasmid pANN202-312, carrying the whole hly determinant of pHlyl52, and its derivatives have been recently described (8). The vector plasmid pUR222 was a gift from B. Muller Hill. Cleavage with restriction enzymes and in vitro recombination. Restriction enzyme cleavage, in vitro recombination, and transformation were carried out as described previously (8, 20). Transposon mutagenesis of pHlyl52 and pANN202-312 by Tn5. Plasmid pHlyl52 was transferred by conjugation from E. coli K-12(pHlyl52) into E. coli 605(TnS). In a second mating E. coli 605 (pHlyl52) was crossed with E. coli HB101 rpsL as described previously (9), and transconjugant colonies were selected on nutrient broth agar containing 25 1Lg of streptomycin and 30 ,ug of kanamycin per ml. Those colonies which did not exhibit the alpha-hemolytic phenotype and produced no external hemolysin in liquid medium but still showed production of internal hemolysin were taken for further analysis and designated Hly,j-/ Hlyin' mutants. Tn5 insertions into the hly determinant of pANN202-312 (8) were obtained by transforming pANN202-312 into E. coli 605 and growing this strain for more than 50 generations at 30°C.

The synthesis and function of the Escherichia coli hemolysin and related RTX exotoxins

“…1 we present a complete summation of the biology of the E. coli hemolysin. The four hly genes and their respective proteins were first identified through the efforts of the Goebel laboratory in Wurzburg and our laboratory [13][14][15][16]. Recently, Wandersman's laboratory has shown that TolC is necessary for export of the hemolysin [17].…”

Structure of TolC, the outer membrane component of the bacterial type I efflux system, derived from two‐dimensional crystals

“…Distinct from this is the so-called type I export process, whereby translocation of protein toxins, proteases and nodulation factors across both membranes is achieved directly, in an 'all-or-nothing' fashion without a periplasmic intermediate Guzzo et al, 1990;Glaser et al, 1988;Letoffe et al, 1990;Rivilla et al, 1995;Koronakis et al, 1989;Koronakis and Hughes, 1993); the substrate either crosses both membranes or remains in the cytoplasm. This process requires an inner-membrane translocator, comprising an ABC exporter and an accessory protein speculated to act in a bridging role (Wagner et al, 1983;Dinh et al, 1994), and the outer membrane protein TolC , which has been reported to be able to form ion-permeable channels (Benz et al, 1993). Homologues of these components are also responsible for the efflux of small toxic compounds, including antibiotics (Nagel de Zwaig and Luria, 1967;Davies and Reeves, 1975;Nikaido, 1994;Li et al, 1995;Ma et al, 1994;Poole et al, 1993;Fralick, 1996), and are required for invasive virulence (Stone and Miller, 1995), emphasizing the medical importance of this export mechanism.…”

Construction of chromosomally encoded secreted hemolysin fusion proteins by use of mini-TnhlyAs transposon

“…At present three independent secretion systems have been iden-ti¢ed and characterized in these bacteria. The Escherichia coli hemolysin (HlyA) secretion apparatus is a prototype of the type I secretion system consisting of two cytoplasmic membrane proteins HlyB and HlyD and the outer membrane protein TolC [2,3]. The Hly secretion apparatus recognizes a signal sequence (HlyA s ), located at the C-terminus of hemolysin (HlyA), that comprises the last 46 [4] to 62 amino acids of HlyA [5].…”