J.-M. Nicaud

Secretion of Haemolysin by Escherichia coli

Escherichia coli secretes very few proteins into the culture medium and it is presumed that the outer membrane constitutes the major barrier to true secretion. The E. coli envelope, whose structure has been extensively reviewed (Nikaido and Nakae 1979; Osborn and Wu 1980; Hall and Silhavy 1981; Lugtenberg and Van Alphen 1983), is shown in Fig. 1. It is composed of an inner and outer membrane which encloses the peptidoglycan or rigid cell wall. The periplasmic space is also located between the membrane layers. This compartment may contain at least 4% of total cell protein (Nossal and Heppel 1966) and may have a quite viscous or gel-like structure (Hobot et al. 1984). The periplasm contains up to 50 distinct polypeptide species (Copeland et al. 1982), the majority of which are concerned with import mechanisms connecting outer membrane pores (porins) with specific inner membrane permeases. It is difficult to estimate the precise volume of the periplasmic space under normal growth conditions since it is not structurally defined.

Genetical and functional organisation of the Escherichia coli haemolysin determinant 2001

SummaryWe have identified gene products corresponding to hlyC, hlyA and hlyD encoded by the Escherichia coli haemolytic determinant 2001 of human origin cloned into the recombinant plasmid pLG570. The product of hlyC is required for the “activation” of the inactive 107K polypeptide encoded by the hlyA gene. the activated 107K protein constitutes the active haemolysin secreted into the medium. hlyB and hlyD are separate regions defined by complementation studies and encode functions essential for the export of haemolysin with hlyD encoding a 53K protein. Complementation studies using subclones and Tn5 insertions into pLG570 have revealed the presence of two major promoters upstream of hlyC and hlyD which transcribe the four hly genes in the same direction. Finally, we were able to reconstitute the complete haemolysin system from three different plasmids encoding hlyC, hlyA and hlyB+hlyD, respectively.

The carboxy-terminal region of haemolysin 2001 is required for secretion of the toxin from Escherichia coli

SummaryAs a first step in the detailed analysis of the mechanism of secretion of haemolysin, we sought to identify sequences or domains within haemolysin A (HlyA) that are essential for its secretion. For this purpose we examined the properties of a deletion and Tn5 insertions into the region of theHlyA gene encoding the C-terminal part of the protein, since both of these are relatively simple to generate. We showed that removal of 27 amino acids from the C-terminus of HlyA is sufficient to inhibit secretion drastically, although the residual polypeptide is still haemolytically active. Cellular fractionation studies showed that haemolytic activity does not accumulate in large amounts within the periplasmic space during normal secretion. More significantly, activity does not appear to accumulate within this compartment when the export functionshlyB andhlyD are removed. These results are consistent with a mechanism in which interaction of the C-terminus of HlyA with the secretion machinery, located in the inner membrane, is followed by direct transfer of haemolysin to the medium.

Characterisation of HlyC and mechanism of activation and secretion of haemolysin from E. coli 2001

In this paper the DNA sequence of the cloned hlyC gene from E. coli 2001 is presented. The gene encodes a protein of 20 kDa which is able to activate the 107 kDa polypeptide encoded by hlyA. This gives rise to a haemolytically active protein which differs from the inactive form in stability and by its migration when analysed by polyacrylamide gel electrophoresis under non‐denaturing conditions. We also show that the inactive form is secreted in the presence of the transport functions hlyB and hlyD. This result rules out any role for the hlyC gene product in the transport of HlyA across the inner membrane

Identification of polypeptides required for the export of haemolysin 2001 from E. coli.

SummaryWe have identified the polypeptides encoded by the haemolysin export genes from a haemolytic determinant 2001 carried by pLG570. This was previously cloned from an E. coli strain, serotype 04 isolated from a human urinary tract infection. Subclones from the recombinant plasmid pLG570 carrying hlyD analysed in vitro and in minicells showed that this gene is transcribed from an independent promoter and encodes a 53 Kd polypeptide. In contrast, detectable levels of the gene products encoded by hlyB were only observed when transcription presumably emanated from a vector promoter. This gene was found to encode at least two polypeptides apparently expressed from alternative translational start sites within a single reading frame. In minicells the major product was a 66 Kd polypeptide whilst after expression in nitro the major product was a 46 Kd polypeptide. Transposon mutagenesis leading to the synthesis of the expected truncated polypeptides was used to confirm the identity of the hlyD and the two hlyB products. Preliminary results suggest that the majority of the 53 Kd polypeptide is located in the inner membrane when cell envelopes from minicells and maxicells were fractionated using sarkosyl, although residual amounts of the 53 Kd polypeptide were also found in the outer membrane.

Regulation of haemolysin synthesis in E. coli determined by HLY genes of human origin

SummaryWe have previously reported the secretion of a 107K polypeptide into the medium from a haemolytic E. coli K12 strain (Mackman and Holland 1984a). In addition, we demonstrated that haemolysin production was correlated with the presence of this polypeptide in the growth medium in a large number of E. coli isolates of human and animal origin (Mackman and Holland 1984b).In this paper we confirm that the 107K polypeptide is indeed haemolysin: both haemolytic activity and the 107K polypeptide show a similar pattern of accumulation during the growth cycle; identical levels are produced in three different growth media; they have the same half-life in minimal medium. The results also show that the expression of haemolysin is not influenced by the growth medium or subject to catabolite repression. However, expression is apparently switched off as cells enter the late exponential phase of growth. Finally, we present data indicating that the previously reported variation in haemolysin production in different media is entirely due to the instability of the haemoolysin itself. Degradation of the 107K polypeptide in the medium was accompanied by the accumulation of a major breakdown product of 60K.

Amplification of synthesis and secretion of haemolysin using a run-away plasmid in Escherichia coli

Abstract Molin and co-workers have described the construction of a ‘run-away’ plasmid, pOU71 which could be useful for the amplification of cloned genes at high temperature when the plasmid replicates to high copy number. In this paper we describe the kinetics of synthesis of a plasmid-coded gene product, β-lactamase, concomitant with pOU71 amplification at 42°C. Maximum amplification was obtained by shifting a culture growing at 30–42°C for 60 min resulting in a 70- to 80-fold amplification for the β-lactamase gene product when the culture was returned to 30°C. The haemolytic determinant LE2001 from an Escherichia coli strain of human origin was cloned into plasmid pOU71 giving rise to plasmid pLG570. Using an identical amplification procedure a 20-fold amplification of the synthesis and secretion of haemolysin was achieved.