T H E HYPERSENSITIVE RESPONSE.

The hypersensitive response (HR), which occurs when a pathogenic bacterium invades a non-host plant, is characterized by the release of a range of toxins, enzymes and antimicrobial phytoalexins, from the plant cells. The responding plant cells die within 8-24 h r, and the multiplication and spread o f the pathogen is arrested (Klement 1982). The genes (designated hrp) required for the initiation of the HR response in

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non-host plants, by various phytopathogenic bacteria, have been reviewed recently (Willis et al 1991). It is thought that in addition to their involvement in HR initiation, hrp genes are required for pathogenicity in host plants, although other genes are also necessary for this (Huang et al 1988).

Pseudomonas syringae pv. syringae, which causes brown spot disease of beans, has a large cluster of hrp genes which have been cloned and characterized (Huang et al 1988, Mukhopadhyay et al 1988, Huang et al 1991, Xiao et al 1992). There are thought to be thirteen hrp genes: probably organized into eight transcriptional units (Xiao et al 1992), and the majority o f these genes are thought to be induced in the presence of plant extracts. Sequence analysis has suggested that some genes of the hrp locus encode membrane-associated proteins (Huang et al 1991), and one Hrp protein is homologous to PulD (Collmer pers. comm.).

Recent studies have revealed similarities between the hrp cluster of Ps. syringae pv. syringae and those of other species, including Ps. solanacearum (Boucher pers. comm.), Ps. syringae pv. phaseolicola (Rahme et al 1991, Panopoulos pers. comm.), Xanthomonas campestris pv. vesicatoria (Bonas pers. comm.) and Yersinia spp. In each case, genes have been identified which are thought to encode membrane-associated or exported proteins, and these are believed to function in the secretion of molecules involved in the interaction with the plant.

1.9.10 FILAMENTOUS PHAGE ASSEMBLY.

A general review of filamentous phage assembly has been published recently (Russel 1991). The following discussion is limited to those areas which are relevant to the study of traffic wardens.

Unlike most bacterial viruses which are assembled in the cell cytoplasm and released by lysis, filamentous phages are continuously assembled and extruded from the cell.

Phage fl, which infects E. coli via F pili, is composed of five phage- encoded proteins. The DNA is enclosed in a hollow tube: formed from approximately 2700

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monomers of major coat protein (pVIII) in a helical array. The minor coat proteins, arranged at the ends of these tubes, are pill and pVI (involved in adsorption and penetration respectively), and pVII and pIX w hich have a role in phage assembly (Russel 1991).

Three other proteins are essential for encapsidation and extrusion o f phage. These are pi and pIV (phage encoded) and the E. coli protein, thioredoxin. Mutations in the genes for pi and pIV do not affect the synthesis o f phage DNA or proteins, but prevent the production of progeny, suggesting that p i and pIV have a role in morphogenesis.

The pIV protein is synthesized with an N-terminal signal sequence. After processing, the mature protein is transiently soluble in the periplasm before integrating into the membrane (Brissette and Russel 1990), although it does not appear to have an anchor sequence. Although fractionation experiments suggest that pIV is localized in both the inner and outer membranes, the protein is thought to be predominantly in the outer membrane (Brissette and Russel 1990). There are some similarities between pIV, which is thought to affect the cytoplasmic membrane, and outer membrane porins (Russel 1991). Little is known about the function of pIV, although it causes E. coli host cells to synthesize Psp (phage shock protein), and is lethal when overexpressed (Brissette et a l 1990). It is possible that the N-terminal domain of the transient periplasmic form of pIV disrupts the cytoplasmic membrane: allowing access for a new phage particle, although this would not account for the estimate that fifty pIV molecules are required for every phage particle assembled (Brissette and Russel 1990).

1.9.11 THE TRAFFIC WARDENS: COMMON THEMES.

Having considered each member of the traffic warden family separately, it is clear that there are some common features. These are important in understanding the evolutionary development and functioning o f th e different systems, and are summarized below

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The genes are probably normally organized as polycistronic operons. Many o f the encoded proteins are predicted to be membrane associated. Many o f the loci encode a cytoplasmic, ATP-binding protein.

Several genes have been identified which encode small, pilin-like proteins with MePhe cleavage sites.

Genes encoding MePhe peptidases have also been identified.

The systems described in the preceding sections are responsible for the transfer of a wide range o f macromolecules across biological membranes, and yet have many features in common. It is clear that a specialized, yet widespread bacterial transport system has been identified. This raises an interesting question as to the evolution of the systems. One possibility is that a mechanism for the transfer o f a phage was captured by host cells, then evolved, acquiring other functions (Dubnau 1991).

1.9.12 TRAFFIC WARDEN FUNCTION IN HETEROLOGOUS SYSTEMS.

As stated previously (1.7.2), the Out systems of Erwinia spp. are species-specific for the enzymes they secrete (He et al 1991a, Py et al 1991b). The abilities of traffic wardens to function in other heterologous systems are outlined below.

Pseudomonas aeruginosa is unable to secrete plasmid-encoded Klebsiella oxytoca pullulanase (de Groot et al 1991).

PulL and PulM are unable to complement mutations in xc p Y and xcpZ (de Groot et al 1991). This might be because the proteins are required to form a complex structure, but cannot interact with proteins from a different species.

When the phage fl pIV gene is expressed in E. coli, it induces the synthesis of phage shock protein (Brissette et al 1990). The same response was observed with cloned pulD, suggesting that pIV and PulD have some function in common (Russel 1991).

PulO has a broad specificity. It complements mutations in xcpA (Bally et al 1992) and processes gonococcal type IV pilin (Dupuy et al 1992). XcpA also processes a

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range of proteins, provided they have the consensus MePhe cleavage site (Strom and Lory 1991).

In summary, although homologous, the different members of the traffic warden family show specificity for the molecules they transport, even though some transport a range of apparently unrelated proteins. No specific recognition signals have yet been identified. However, since there is evidence to suggest that proteins may be folded before being secreted across the outer membrane (Pugsley el al 1990b), recognition could involve a particular 3-dimensional structure. This would obviously be more difficult to identify than a simple signal sequence.

Another possibility which must be considered is that specificity is due to differential regulation of the gene clusters encoding the various systems.