Mutations affecting activity and transport of haemolysin in Escherichia coli

Abstract

Temperature-sensitive mutants that exhibit an altered haemolytic phenotype were isolated from Escherichia coli harbouring the plasmid pHly152. Complementation with recombinant plasmids carrying one of the four hly genes (C, A, B or D) allowed localization of the hly(ts) mutations. A ts mutation in hlyC leads to a pro----leu exchange in amino acid position 53 of HlyC. Two ts mutations in HlyA were found in positions 312 (ser----pro) and 315 (thr----ile). Both amino acid exchanges are located in the same hydrophobic domain of HlyA which extends from amino acids 299 to 327. Two different mutations were introduced by site-specific mutagenesis in this hlyA domain: one by an exchange of ala, val to asp, glu (positions 313, 314) altering the hydrophobicity of this region and another which removes most of this hydrophobic portion. Both mutants have entirely lost the haemolytic activity but the mutant haemolysins are still efficiently transported across both membranes when hlyB and hlyD are provided. Functional HlyC is not required for the transport of the mutant haemolysins. Two site-specific mutations at the N-terminal end of hlyA (one at amino acid position 2 leading to a thr----pro exchange and another deleting ile and thr at positions 4 and 5) also do not affect the transport of the altered haemolysins. The thr----pro exchange enhances the haemolytic activity of the corresponding mutant, whereas the ile, thr deletion exhibits little or no effect on the haemolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein secretion and the pathogenesis of bacterial infections

“…HlyC functions as an acyl-transferase to decorate the -amino groups of lysine within HlyA using thioester-linked fatty acids (ACP) as substrate (Hardie et al 1991;Issartel et al 1991). Knockout mutations of hlyC and acp abolish lipid modification and the hemolytic property of HlyA but do not affect secretion of the unmodified polypeptide across the double membrane envelope of E. coli (Ludwig et al 1987). HlyA polypeptide is not cleaved during the secretion process (Felmlee et al 1985a,b).…”

Separable domains define target cell specificities of an RTX hemolysin from Actinobacillus pleuropneumoniae

“…This analysis has identified two domains common to all known RTX toxins. One lies in the N-terminal half of the toxin protein and consists of four hydrophobic regions which may be involved in forming channels in target membranes (28). The second consists of a variable number (9 to 14) of the nonapeptide repeats which are believed to constitute a calcium-binding domain (4,27).…”

E. coli Alpha Hemolysin and Properties

“…This region is predicted to be disordered; and although export of the toxin has been observed to be acylation-independent (Ludwig et al, 1987), as mentioned above, the yield from extracellular transport for proHlyA was lower than that for HlyA. Consequently, covalently bound acyl chains can expose these signal regions and thus facilitate transport.…”

“…Maturation increases the hydrophobicity of the protein, but that property is not required for export (Ludwig et al 1987). E. coli HlyA-related toxins are all secreted across both membranes by the type-I export process employing an uncleaved C-terminal recognition signal (Nicaud et al, 1986), (Stanley et al, 1991), but no N-terminal leader peptide or periplasmic intermediate (Felmlee & Welch, 1988), (Koronakis et al, 1989).…”