Rendimiento a la canal, %

In E. coli, exported proteins either remain in the periplasm, or go to the outer membrane. The information required for this sorting is carried in the sequence o f the mature protein (Benson et al 1984). In Gram negative bacteria, such as Erwinia spp., which are capable of secreting certain proteins from the periplasm to the culture medium, the situation is more complicated. The second step of secretion, which involves several accessory proteins, is discussed in later sections (1.7, 1.8).

1.6 ONE-STEP SECRETION.

The one-step pathway allows a protein to be moved from the bacterial cytoplasm to the culture medium in a single step, through 'pores': fusion zones between the inner and outer membranes (Lory et al 1983). Most of the proteins secreted by this pathway are highly homologous. Examples include Pasteurella haemotytica leukotoxin (Strathdee and Lo 1989), Bordetella pert us is adenyl cyclase-haemolysin (Glaser et al 1988), colicin V: secreted by E. coli (Gilson et al 1990), Actinobacillus

pleuropneumoniae (Gygi et al 1990), Morganella morganii (Koronakis et al 1987) and Proteus vulgaris (Koronakis et al 1988). The best characterized example is the secretion of a-haemolysin by E. coli (discussed below). Other proteins, secreted by the same pathway, but not homologous to a-haemolysin are Serratia marcesens metalloprotease (Nakahama et al 1986), Pseudomonas aeruginosa alkaline protease (Guzzo et al 1991) and Erwinia spp. proteases (Letoffe et al 1990, 1991). The rest of this section will concentrate on the secretion of a-haemolysin by E. coli.

a-haemolysin does not cross the membrane via the general export pathway. The protein lacks an N-terminal signal sequence and is not subject to proteolytic cleavage (Hartlein et al 1983). Secretion is independent of the Sec apparatus (Holland et al 1990, Gentschev et al 1990), and does not proceed via a periplasmic intermediate (Gray et al 1986, Koronakis et al 1989). Figure 1.6a shows the components of the one- step secretory apparatus.

Figure 1.6a

Apparatus for the O ne-Step Secretion of Haemolysin

mmt

P e rip la s m

....

A D P + P A D P + P

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The structural gene for a-haemolysin (hlyA), and the genes encoding the secretory proteins (hlyB,C,D) map to a contiguous 7.5 Kb fragment o f DNA and are co­ expressed (Noegel et al 1979, Hartlein el al 1983, Welch et al 1983, Mackman et al

1985).

The HlyA protein is post-translationally modified by the cytoplasmic protein HlyC: a step which is required for a-haemolysin toxicity, but not for secretion (Nicaud et al 1985). Secretion is then directed by a 'signal sequence' at the C-terminus of HlyA (Nicaud et al 1986), which interacts with the HlyB and HlyD proteins. The C-terminal 'signal sequence' is 113 amino acid residues long, and may be analogous to the presequences required for import into mitochondria (Koronakis et al 1989). Secretion does not require the entire signal sequence. It has been proposed that the final 27 residues are essential (Koronakis et al 1989). However, various point mutations in this region had little effect on secretion and it was found that just 4 residues, dispersed throughout the last 46, are vital for optimal secretion (Kenny et al 1992). These 'critical contact' residues are believed to be required for the interaction between HlyA and the membrane translocator. Fusion proteins have been generated, in which the HlyA C-terminal 'signal sequence' directs the secretion of other proteins such as LacZ (Kenny et al 1991). LacZ cannot normally be secreted, as it folds into a secretion- incompetent conformation in the cytoplasm. The ability of the HlyA 'signal sequence' to secrete LacZ therefore suggests that HlyA may be folded prior to secretion.

Secretion is thought to involve direct interaction between the HlyA 'signal sequence', HlyB and possibly HlyD. This may require 'spacers' in HlyA to maintain a protein conformation in which the signal is optimally exposed for interaction with HlyB (Kenny et al 1991).

Gene fusion and cellular fractionation studies have shown HlyB and HlyD to be associated with the inner membrane (Mackman et al 1985, Wang et al 1991), although unlike classical inner membrane proteins, they also fractionate to the outer membrane in small amounts (Wang et al 1991). Topology studies using TnlacZ and TnphoA have suggested that HlyB has eight membrane-spanning domains (Wang et al 1991,

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Gentschev and Goebel 1992), while HlyD has only one (Wang et a l 1991). The two proteins are thought to interact, to form a transmembrane channel.

HlyB, having ATP-binding domains (Higgins et al 1986, Gerlach et al 1986), is thought to provide energy for translocation, although an electrochemical gradient across the membrane is also required during the passage o f the C-terminal 'signal sequence' (Koronakis et al 1991). HlyB belongs to a group o f bacterial transport proteins (all of which bind ATP), which transport a diverse range o f molecules including polypeptides and polysaccharides (Blight and Holland 1990). HlyD homologues have so far only been identified in systems which transport large polypeptides, suggesting that this protein might determine specificity (Blight and Holland 1990).

In addition to the Hly proteins, TolC, a minor outer membrane protein is specifically required for a-haemolysin secretion (Wandersman and Delepelaire 1990). TolC may allow the HlyB/HlyD complex to interact with the outer membrane (Wandersman and Delepelaire 1990).

It is proposed that the proteins described above, interact to create a 'revolving door', in which conformational changes in the translocator result in the movement of Hly A across the membrane (Blight and Holland 1990).

There are some differences between the machinery for HlyA secretion and other related systems. The secretion of metalloproteases (PrtB and PrtC) by Ech requires three proteins: PrtD, PrtE and PrtF, homologous to HlyB, HlyD and TolC respectively (Letoffe et al 1990). These are encoded by genes which map upstream of the structural prt genes, and have their own promoters, meaning that regulation must differ from that of the E. coli hly genes (Letoffe et al 1990).

Only limited cross complementation occurs between the different one-step secretory apparati. The Ech Prt export machinery is capable of secreting metalloprotease from Serratio sp., unlike the E. coli a-haemolysin machinery (Letoffe et al 1990). The specificity of the systems is thought to act at the level of recognition of the C-terminal 'signal sequence' by the translocator (Wang et al 1991).

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1.7 EXTRACELLULAR ENZYME SECRETION BY ERWINIA SPP.

1.7.1 ONE-STEP OR TWO-STEP SECRETION?

The first evidence of a specific secretory system in Erwinia spp. was obtained by fractionating Ech cultures and assaying for enzym e activity (Andro et al 1984). Pel and Cel were found to be extracellular, while the marker enzymes Bla and LacZ were localized in the periplasm and cytoplasm respectively, showing that lysis had not occurred. Similar results were later obtained to show that Peh and Prt are also specifically secreted (Wandersman et al 1986).

The study of secretion by Erwinia spp. is based on the analysis of various mutants. Mutagenesis (chemical and insertional) has produced a range o f mutants, defective for extracellular enzymes (Andro et al 1984, T hum and Chatteijee 1985, Salmond et al 1986, Hinton and Salmond 1987). An interesting class of mutants (termed Out') synthesize, but do not secrete Pel and Cel (A ndro et al 1984). The Pel'Cel' phenotype of Out* mutants shows that both enzymes are secreted by the same pathway. Since the secretion o f Prt is not affected in Out* mutants (Andro et al 1984), this must occur via a different pathway.

Prt secretion is independent of the general export (Sec-dependent) pathway. The Erwinia spp. Prt enzymes are synthesized w ithout N-terminal signal sequences, and are thought, like o-haemolysin, to have C-terminal sequences required for secretion (Delepelaire and Wandersman 1990). Sequence analysis revealed homologies between proteins involved in the secretion of Ech P rt, and of E. coli HlyA (Letoffe et al 1990). All the evidence suggests that Erwinia spp. secrete Prt by the one-step mechanism.

The activity of Pel, Cel and Peh accumulates in the periplasm o f Out* mutants (Andro et al 1984, Thum and Chatteijee 1985). This suggests that secretion normally occurs via the periplasm, although it must be considered that mutations might cause a re-routing to the periplasm. However, there is other evidence for a secretory route via the periplasm. A transient build-up of Pel and Cel has been detected in the periplasm of wild-type Ech cells early in the growth curve: before secretion occurs (Andro et al

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1984, Ji et al 1987). The existence of periplasmic intermediates of Pel, Cel and Peh suggests that secretion is via the two-step pathway.

Pulse-chase labelling showed that in Ech, Pel is processed and localized in the periplasm, prior to being translocated across the outer membrane. Almost all o f the Pel synthesized, was secreted into the supernatant within 2 min (H e et al 1991b). Overexpression of Pel led to an accumulation of the enzyme in the periplasm (He et al 1991b). This not only supported the two-step hypothesis, but also showed that the apparatus for secretion across the outer membrane of Ech is saturable.

Genes for the production and secretion (out) of Pel from Ech (see 1.7.2) were introduced into a strain of E. coli which was a temperature sensitive (Ts) Sec mutant (He et al 1991b). At the non-permissive (but not the permissive) temperature, pre-Pei accumulated inside the cell, independent o f the presence o f out genes. This proved that Pel is exported across the inner membrane via the Sec-dependent pathway, and that Pel-processing is dependent on Sec, rather than Out proteins. Entry to the Out pathway is therefore thought to occur only after Sec-dependent export.

The structural genes for Pel from Ech and Ecc have been cloned into E. coli. The enzymes were synthesized, exported and processed as in Erwinia spp., but then remained in the periplasm (Keen et al 1984, Collmer et al 1985, Thum and Chatteijee 1985, Zink and Chatteijee 1985, Ji et al 1987). These results support the hypothesis that the first step in Pel secretion uses the general export pathway present in E. coli, while showing that E. coli is not capable of directing Pel to the supernatant. The existence o f a secretory mechanism in Erwinia spp. which is not present in E. coli makes these bacteria important for the study of Gram negative secretion.