genetic and biochemical analysis of protein export from ... · isozymes of 35-5, 36‘5 and 41k (k...

y. Cell Sci. Suppl. II, 59-72 (1989)Printed in Great Britain © The Company of Biologists Limited 1989

59

Genetic and biochemical analysis of protein export from Xanthomonas campestris

J . M . DO W , M . J . D A N I E L S , F . D U M S , P. C . T U R N E R a n d C. G O U G H The Sainsbury Laboratory, John Innes Institute, Colney Lane, Norwich NR4 7UH, UK

SummaryXanthomonas campestris pv. campestris, a Gram-negative phytopathogen, produces a number of extracellular enzymes which can degrade components of the host plant cell. Some non-pathogenic mutants, derived by chemical mutagenesis, were found to be defective in the export but not the synthesis of a number of these enzymes. The pathogenicity and export lesions in one such mutant, strain 8288, could be complemented by a cosmid clone pIJ3000 from the Xanthomonas library. Mutagenesis of pIJ3000 with the transposon Tn5 followed by recombination into the corresponding region of the chromosome has revealed a cluster of 6 to 8 genes whose function is required for enzyme export. Sequence analysis of part of the cluster has revealed two open reading frames that would encode proteins with extensive hydrophobic domains. Export-defective mutants retain the normally exported enzymes in the periplasmic space. These forms have the same molecular weight as the extracellular forms, suggesting that the signal sequence has been properly processed. The results are consistent with a mechanism of sequential translocation across cytoplasmic and outer membrane via the periplasm. The second translocation step may be mediated by the products of the export genes. This may be a common export mechanism amongst Gram-negative bacteria but other mechanisms do exist, sometimes in parallel in the same cell, and these are briefly reviewed.

IntroductionConsiderable attention has been focused on the secretion of proteins which will eventually be localised in the inner or outer membranes or the periplasm of Gram negative bacteria. Comparatively little is known about the export of proteins to the medium from these organisms. One notable exception is the export of haemolysin from Escherichia coli which is discussed in detail in another chapter of this volume (Gray et al. 1989). This paucity of information is perhaps because very few proteins are exported in the most studied species E. coli and Salmonella typhimurium. In contrast Gram-negative phytopathogens, particularly from the genera Pseudomonas y Xanthomonas and Erwinia produce a range of extracellular enzymes which can degrade host tissue components. These include pectinases, cellulases, amylases and proteases (Kotoujansky, 1987; Collmer & Keen, 1986; Daniels et al. 1988). It is perhaps not surprising that the process of export of these enzymes has been shown to be necessary for pathogenesis in all cases studied (Andro et al. 1984; Dow et al. 1987). This process is specific in that it is not accompanied by either ‘leakage’ of periplasmic marker proteins or by cell death. In this paper we present an overview of our work on the biochemistry and genetics of the protein export machinery and exported proteins of Xanthomonas campestris. We discuss this work with reference

Key words: export, Xanthomonas, bacteria.

60 J. M. Dow and others

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Fig. 1. Time course of induction of PG L in the cells (O----- O) and the medium(A ----- A ) of strains of Xanthomonas campestris after addition of polygalacturonateinducer (PGA) to 24 h cultures. Strain 8004 is the wild-type, strain 8288 a chemically- derived mutant and strains 8315 and 8335 transposon insertion mutants. (+ ) , export- competent strain; ( —), export-defective strain. All transposon insertion mutants with Tn5 inserted within the 10 kb pathogenicity /out gene cluster carried on pi J 3000 have responses similar to strain 8315 (but see text). All transposon insertion mutants with Tn5 inserted outside this region have responses similar to strain 8335.

to results from other Gram-negative organisms including Erwinia spp. (which are closely related to E. coli) and animal and human pathogens. This body of information suggests the existence of an export mechanism via the periplasm which is common to many genera, and also the presence of multiple pathways or mechanisms within single bacterial species.

Mutants of Xanthomonas defective in enzyme export (Out- mutants)To investigate the genetic and biochemical basis of pathogenicity and enzyme export in Xanthomonas, we derived mutants defective in these functions by chemical mutagenesis (Daniels et al. 1984«,b). These mutants were originally selected as being non-pathogenic; subsequently some of these non-pathogenic mutants were shown to be export defective. Xanthomonas produces polygalacturonate lyase (P G L, a pectolytic activity), carboxymethylcellulase (CM Case), protease and amylase activities. Although the synthesis of these enzymes was unimpaired, the mutants failed to translocate these normally extracellular proteins to the medium (Dow et al. 1987). Such mutants have been called out mutants (Andro et al. 1984; Thurn & Chatterjee, 1985). The results of an experiment on the synthesis and export of P G L are shown in Fig. 1. P G L was induced within 2h of addition of polygalacturonate inducer to 24 h cultures of wild-type strain 8004; over 75 % of the activity was found in the culture medium, although there was appreciable activity associated with the

Protein export from Xanthomonas 61

I ▼ ▼ ▼▼ y w w __ jv ^ v v | | W |^jr ▼▼▼ VVT ▼ ▼ |__R B R B B B B R

1— I 1 kb

Fig. 2. Physical map of the Xanthomonas DNA insert in pIJ3000. Restriction sites for £coRI(R) and Ba/raHI(B) within the region cloned in pIJ3000 are indicated. Filled triangles represent T n j insertions which do not prevent symptom production or enzyme export when introduced into the wild-type genome; open triangles represent Tn5 insertions which produce a non-pathogenic, export-defective phenotype when in the genome.

cells. Similarly rapid induction was seen with out mutant 8288, but only about 20 % of the activity was found in the medium (Fig. 1). We attempted to complement the pathogenicity lesion in mutant 8288 with members of a genomic library of X. campestris cloned in the broad host range cosmid vector p L A F R l (Daniels et al. 1984a). One recombinant plasmid pIJ3000 containing a defined fragment of 25-8kb of wild-type Xanthomonas D N A was capable of restoring pathogenicity to strain 8288 by complementation of the mutation. This plasmid concomitantly restored enzyme export; almost all of the P G L was translocated to the culture medium within the time course of this experiment (Fig. 1). Similar results were seen with amylase, CMCase and protease distributions. These results suggested that plasmid pIJ3000 carries a gene or genes involved in the export of a number of extracellular enzymes and that these genes are important in pathogenicity.

Several genes for protein export are clustered in plJ3000The number and location of the pathogenicity/out genes carried on the 25-8 kb insert of Xanthomonas D N A in pIJ3000 was determined using gene disruption with the transposable element Tn5 (Turner et al. 1984, 1985). Transposon mutagenesis of pIJ3000, followed by recombination of the mutations into the corresponding sites of the chromosome of the wild-type, generated a series of mutants with Tn5 inserted at different defined sites within the 25-8 kb region of the genome (Fig. 2). Mutants with insertions within a particular 10 kb region were non-pathogenic whereas insertions outside this region had no detectable effects. This region was considered to be a cluster of pathogenicity genes split by a single insertion of Tn5 (which does not result in loss of pathogenicity) into two sub-regions of 4-2 and 5-8kb.

Representative pathogenic and non-pathogenic mutants were tested for their ability to synthesise and export P G L , CMCase and amylase. With one exception the Out- phenotype correlated with the Path- phenotype (Fig. 1). The exception was the single Tn5 insertion between the two regions which showed a Path"1" and Out- phenotype under the experimental conditions described. However, in experiments using turnip cell walls as inducer, this mutant behaved like an Out+ strain (Dow et al. 1987). The reasons for this difference in behaviour are not known. Nevertheless, the fact that single Tn5 insertions in any part of the 10 kb region could eliminate the export, but not the synthesis, of a number of enzymes strongly suggests a

62 jf. M. Dow and others

common mechanism for their export involving the participation of a number of out gene products.

D N A sequence analysis has been carried out on the 3-7kb Bam H I-EcoRI fragment of the left hand sub-region (Dums & Daniels, unpublished). This has revealed two complete open reading frames (O R Fs 2 and 3) and one partial open reading frame (O RF1). These are arranged in the order O RF 1-2-3 from the Bam H I site to the iicoRI site with the S' end of each O R F to the left. The complete O RFs would code for proteins of molecular weight 43 674 and 49 006 which contain large hydrophobic domains. This suggests that they may be intrinsic membrane proteins (Fig. 3). In contrast the partial O RF would code for a largely hydrophilic protein fragment of molecular weight 20 192. Neither of the proteins encoded by the full O RFs contains an apparent signal sequence; they have very little homology with any other proteins in the database (J. Wootton, personal communication). We do not yet know whether these genes are transcribed as an operon; the intragenic regions are short and do not contain sequences homologous to promoter regions from a number of bacteria. However, we know very little of the structure of promoters in Xanthomonas.

We can estimate that there are 6 to 8 genes involved in export within the full gene cluster, although we do not know whether there are other genes required for export in other regions of the chromosome. At least some of these out genes are constitutively transcribed, as shown by work with TnSlac (Turner, unpublished) and a promoter-probe plasmid p IJ3 100 (Osborn et al. 1987; Gough, unpublished), so that they are not co-ordinately regulated with the structural genes for P G L.

Detailed biochemical studies on Out- mutantsIn an attempt to identify the steps in export which are blocked in the Out- mutants, we tried to localise the cell-associated enzymes and make a detailed comparison of the retained forms of the enzymes in Out- mutants with the normally exported forms. These experiments were done using P G L and subsequently CMCase as marker proteins. In addition, the effects of mutations in the out gene cluster on the protein profile of the periplasm and membranes were assessed.

Most of the cell-associated P G L activity of the wild-type was periplasmic as revealed by cell fractionation studies with ED TA . The remaining cellular activity was cytoplasmic with undetectable levels associated with the membranes. In cultures of all Out- mutants tested the major location of P G L was the periplasm; none of the cellular P G L was apparently associated with the membranes (Dow et al. 1987). The P G L activity from the medium of the wild-type could be resolved into three major isozymes of 35-5, 36‘5 and 41K (K = 103M r) by ion-exchange fast protein liquid chromatography (F P L C ). Although these three isoforms had very similar physio- chemical properties, they were genetically and immunologically distinct (Dow et al. unpublished). Individual P G L isozymes from the periplasm of the wild-type were also separated by F P L C . Again there were three isozymes with ion-exchange properties and molecular weights indistinguishable from their extracellular counter-

Protein export from Xanthomonas

A m ino acids

Fig. 3. Hydropathy profiles of proteins encoded by the open reading frames in the .Eco R I-Bam HI fragment of the out gene cluster. The open reading frames are in numerical order from the B am lil to the £coRI restriction sites, although ORF1 is only partial. Positive values are hydrophobic, negative values are hydrophilic. Where the hydropathy of a given amino acid segment is greater than +1-6 there is a high probability that it represents a membrane-spanning region.


M rX 1 0 -3

97 -

66 -

45 -

29 -

Fig. 4. SD S—polyacrylamide gel electrophoresis of PG L isozymes separated by fast protein liquid chromatography, (a) Isozymes from the wild-type. Lanes 1, 3 and 5 and the intracellular isozymes; lanes 2, 4 and 6 are the corresponding extracellular forms. The lower band in lane 3 is not a PG L isozyme as revealed by substrate gel overlay.(b) Isozymes from an export-defective Tn5 mutant. A similar pattern was seen with mutant 8288 (a chemically-derived mutant). Molecular weight standards (in K , where K = 103Mr) are indicated on the left.

parts (Fig. 4). Individual P G L isozymes retained by the Out- mutants had molecular weights and ion-exchange properties indistinguishable from the normally exported forms. If the periplasm is an intermediate compartment in a two step transfer from the cytoplasm to the medium (Pugsley & Schwartz, 1985), we would infer that the second step is blocked in Out- mutants, and that transfer of a protein across the outer membrane does not require cleavage of that protein.

The crude periplasmic fractions from a number of different Out+ and Out- Tn5 mutants grown in peptone-yeast extract-glycerol medium (N YGB medium) were compared by S D S —polyacrylamide gel electrophoresis. There was an accumulation in the Out- strains of a major protein of 53K (Fig. 5). This protein was a CMCase, which was also present to a lesser extent in Out"1" strains. Most other periplasmic proteins were unaffected. When X. campestris is grown in N Y G B, the major


Fig. 5. SDS-polyacrylamide gel electrophoresis of periplasmic fractions from export- competent and export-defective strains. Lane 1: export-defective chemical mutant 8288; lane 2: wild-type strain 8004; lanes 5, 7, 8 and 9: mutants with transposon insertions at different positions within the out gene cluster which eliminate enzyme export; lanes 3 and 6: mutants with transposon insertions outside the out gene cluster where enzyme export is unaffected; lane 4: the single mutant with transposon insertion within the out gene cluster which does not eliminate pathogenicity. The position of the extracellular carboxymethyl- eellulase (C) of S3K (K = 103Mr), is marked on the left.

extracellular protein (representing about 90 % of the total) is a CMCase of 53K. This protein is synthesised with an N-terminal sequence of 25 amino acids which is structurally similar to signal sequences from bacteria (Watson, 1984). This was determined by a comparison of the N-terminal amino acid sequence of the purified protein with that predicted from the nucleotide sequence of the structural gene, cloned by expression in the non-cellulolytic X. campestris pv. translucens (Gough et al. unpublished). Although we have not conclusively shown that the periplasmic and extracellular CM Cases are identical, these preliminary data suggest that the signal sequence of the exported cellulase is properly processed in both the wild-type and Out- mutants in passage from the cytoplasm to the periplasm. The P G L genes in Xanthomonas also probably encode proteins with signal sequences, as has been


shown for several pectate lyase genes in Erwinia spp. (Keen & Tamaki, 1986; Lei eta l. 1987).

No major differences were seen in the membrane proteins between the various Out- and Out+ strains used in these experiments, so that lesions in the cluster of out genes do not have gross effects on membrane assembly or composition. Overall, we can also infer that the out gene products are not among the more populous proteins of the inner or outer membranes or the periplasm.

A common export mechanism via the periplasm in Gram-negative bacteriaOut- mutants with similar properties to those described here for Xanthomonas have been derived in Erwinia, Pseudomonas and Aeromonas spp. by mutagenesis with chemicals, transposable elements and phage Mu derivatives (Andro et al. 1984; Thurn & Chatterjee, 1985; Howard & Buckley, 1983; J i et al. 1987; Wretlind & Pavlovskis, 1984; Lindgren & Wretlind, 1987). The export of a number of enzymes is co-ordinately affected, although protease is unaffected in Erwinia spp., and may proceed by a parallel mechanism (see below). These mutants similarly retain the properly processed proteins in the periplasmic space. Some periplasmic or outer membrane proteins are missing in some Out- mutants (Ji et al. 1987; Thurn & Chatterjee, 1985; Howard & Buckley, 1983) and it is tempting to speculate that these proteins are the products of some of the out genes. Out genes have been cloned from Erwinia chrysanthemi and E. carotovora subsp. carotovora (Boccara et al. 1986; Gibson et al. 1988; Murata et al. 1988) by complementation of Out- mutants and the use of transposon tagging. There are at least four out genes in E. chrysanthemi, located in several different regions of the chromosome (Boccara et al. 1986; Murata et al. i988). Out genes from E. chrysanthemi but notX . campestris can complement Out- mutants of E. carotovora.

The phenotypic similarities between these Out- mutants and the participation of a number of out gene products which may encode periplasmic or outer membrane proteins suggest the existence of a mechanism of export that is common to many Gram-negative bacteria. The simplest model consistent with these results is that of a two step translocation mechanism (Pugsley & Schwartz, 1985; Fig. 6). The first step would be the transfer of the protein via the signal sequence-dependent pathway from the cytoplasm to the periplasm, accompanied by signal sequence cleavage. The second step would involve transfer across the outer bacterial membrane mediated by the out gene products which may form a multi-protein complex of transmembrane and periplasmic proteins. This step would not require further cleavage of the protein. Mutation of the out genes would then lead to periplasmic accumulation of properly processed proteins because of the absence of a functional complex. Alternative models involving direct translocation from the cytoplasm to the medium through junctions between the inner and outer membranes have been proposed. Here the out gene products would form a protein complex spanning both membranes, perhaps through the zones of adhesion (‘Bayer’ junctions) between the


out gene products form a ‘pore’ across outer membrane

TWO-STEP MECHANISM DIRECT TRANSLOCATION

Fig. 6. Possible mechanisms of protein export to the medium either via a two-step mechanism involving a periplasmic intermediate or by direct transfer to the medium. Mutations in out genes would lead to accumulation of a properly processed periplasmic protein either through the absence of a functional ‘pore’ in the outer membrane or by the re-routing of proteins because of the absence of junctions between inner and outer membranes.

inner and outer membranes (Bayer, 1979). Disruption of the complex following mutation of the out genes may only allow routing to the cytoplasm either via the remaining gene products (Fig. 6) or possibly by re-routing via the sec pathway. Although consistent with the data on haemolysin and exotoxin A export (see below), this model is difficult to reconcile with the considerable periplasmic activities of exported proteins seen in wild-type Xanthomonas and Erwinia and direct evidence of periplasmic intermediates in other Gram-negative bacteria (Hirst & Holmgren, 19876; Fecycz & Campbell, 1985).

Periplasmic intermediates in protein exportEvidence for a periplasmic intermediate in phosphatase export from Pseudomonas aeruginosa has come from a kinetic study of appearance of phosphatase activity in cytoplasmic, periplasmic and extracellular fractions folowing de-repression (Poole & Hancock, 1983). However, no detailed comparison of the periplasmic and extracellular activities was made, so that the data are inconclusive. Cholera toxin and heat- labile enterotoxin transiently enter the periplasm of Vibrio cholerae during their secretion to the medium: this has been shown by the use of polymyxin B to release periplasmic proteins (Hirst & Holmgren, 1987a). In this case, the existence of specific export functions to translocate the toxins across the outer membrane, although suspected, has not yet been demonstrated. A cell-associated precursor of


extracellular protease 1 of P. aeruginosa is located primarily in the periplasmic space (Fecycz & Campbell, 1985). The precursor has the same molecular weight as the mature enzyme, but is inactive probably due to the presence of a non-covalently bound inhibitor. An interesting speculation is that interaction with the inhibitor, which maintains the precursor in a more hydrophobic form than the active enzyme, presents the precursor in an export-competent fashion to the outer membrane (Fecycz & Campbell, 1985). Thus, in addition to being a device to prevent premature activation of the protease, the periplasmic inhibitor could be seen as an out gene product. The existence of ‘binding’ proteins, which would maintain an extended or unfolded state of a periplasmic intermediate after translocation across the cytoplasmic membrane, could answer the puzzling problem of how the proteins are unfolded, as might be expected, to cross the outer membrane. The binding protein and enzyme precursor would have to be dissociated by interaction with the outer membrane, and also presumably by experimental conditions used to release periplasmic proteins, to explain the common observation of the presence of active enzymes in that compartment. Howard & Buckley (1985) have shown that a processed intermediate of aerolysin, trapped within the cells of Aeromonas by treatment with the uncoupler CCCP, could not be released by osmotic shock as would be expected of a soluble intermediate, but could be degraded by protease applied to the shocked cells. In addition, the precursor was completely degraded by the added protease, unlike the mature toxin, or toxin accumulated in Out- mutants. This again suggests that the conformation of the periplasmic intermediate is not that of the mature protein and that interactions with the membranes or other periplasmic proteins may hold it in an export-competent conformation. In marked contrast, there is evidence that sub-units of the heat-labile enterotoxin of Vibrio cholerae assemble in the periplasm in a stable quaternary structure before secretion (Hirst & Holmgren, 19876). This has led to speculation that proteins cross the outer membrane in a folded state, a mechanism wholly different from protein translocation across any other biological membrane (Hirst & Holmgren, 19876). Such controversies may be expected in the light of a multiplicity of export mechanisms already revealed (see below). Only further research will reveal if this mechanism is widespread among Gram-negative bacteria.

The source of energy for this second translocation step may come from the Donnan potential created by the unique ionic environment of the periplasm, or by coupling of the energy states of the cytoplasmic and outer membranes by a gene product equivalent to Ton B in E. coli (Hammond et al. 1984). There is also a considerable difference in concentration of exported proteins between the periplasmic and external sides of the outer membrane (Hirst & Holmgren, 19876) which presumably contributes to the thermodynamic driving force for export.

Multiple pathways and mechanisms of export existMultiple mechanisms or pathways of protein export for different proteins may exist


within the same cell. In Erwinia, protease export is not impaired in mutants defective in the export of cellulase and pectinase (Andro et al. 1984; Thurn & Chatterjee, 1985). In addition, a 16 kb D N A fragment carrying the structural gene for protease from Erwinia chyrsanthemi, when expressed in E. coli, also allows export of the protease from the cell (Barras et al. 1986). This is a specific effect since the export of P G L , whose synthesis was directed by another piece of cloned D N A in the same cell, was not affected. Thus, there may be a separate gene or genes encoding protease export functions which must be closely linked to the protease structural gene. Similar conclusions were reached by Wandersman et al. (1987) using another strain of E. chrysanthemi; structural genes for two proteases, a protease inhibitor and functions involved in protease synthesis or export or both, were present on an 8-5 kb D N A fragment.

The export of haemolysin from E. coli requires the action of two genes encoding export functions located immediately 3' of the structural gene for haemolysin (Gray et al. 1989). Haemolysin is unlike most of the proteins exported from bacteria in that there is no N-terminal signal sequence. A C-terminal part of the protein is required for transport, which may occur directly from the cytoplasm to the medium. Transfer directly from the cytoplasm to the medium has also been suggested for exotoxin A export from Pseudomonas aeruginosa (Lory et al. 1983). In this case a signal sequence is present which can direct transfer of the cloned gene product to the periplasm of E. coli via the secA dependent pathway (Douglas et al. 1987). It is probable that specific export genes are also required in this case.

Pullulanase (a starch debranching enzyme) produced by Klebsiella pneumoniae has a N-terminal signal sequence which is cleaved prior to fatty acylation of the N-terminal cysteine of the mature protein (D ’Enfert et al. 1987). These steps are sufficient to translocate the enzyme from the cytoplasm to the outer membrane but not to the outer surface when the gene is expressed in E. coli. Specific secretory functions are required for transfer to the outer surface and hence to the medium. The genes encoding these functions have been cloned and are located on both sides of the structural gene (D ’Enfert et al. 1987). The fatty acid on the N terminus may allow a strong association with the membranes such that no ‘soluble’ periplasmic intermediate can be found.

In contrast to the mechanisms already described, there are at least two examples where distinct export functions are not required, and all the information for export resides within the precursor protein itself. The extracellular IgA protease of Neisseria gonorrhoeae and serine protease of Serratia marcescens are both exported by E. coli carrying the cloned protease structural genes (Pohlner et al. 1987; Yanagida et al. 1986). In these cases no specific out gene function is required, although export does depend on the signal sequence-dependent export pathway. Both proteins have a N-terminal signal sequence and a C-terminal domain required for export. Both sequences are cleaved during the formation of the mature extracellular protease. The C-terminal ‘helper’ domain may serve as a pore for the export of the remainder of the protein through the outer membrane from the periplasm.


Concluding remarksThe specific C-terminal domain of haemolysin required for its export can also direct or allow the export of a normally non-exportable protein (a hybrid of beta- galactosidase and Omp F lacking a signal sequence) when fused to its C terminus (Mackman et al. 1987). As yet no such topogenic sequences have been identified in the majority of exported proteins. Fusion of the N-terminal regions of these exported proteins to ‘reporter’ proteins usually allows translocation of the hybrid protein to the periplasm but not to the medium (see for example Collmer et al. 1986). This may be because the fusion protein lacks further topogenic sequences or that it folds in the periplasm in an export-incompetent fashion so that three-dimensional signals are not properly generated or the protein cannot subsequently be unfolded for passage across the outer membrane. Genetic manipulation of the cloned structural genes for these extracellular enzymes to produce truncated forms or proteins with specific amino acid replacements, perhaps coupled with immunological assay of the distribution of the engineered proteins (if they do not retain enzymatic activity), should help to identify possible sorting signals for the second translocation step.

From a biotechnological aspect, the success of using the C-terminal domain of haemolysin to direct the export of cloned gene products from E. coli is very exciting. As the general applicability of this export system to all proteins is not yet known, work on identifying targeting signals in other exported proteins and reconstruction of the export systems themselves is certainly warranted. It is not yet known whether all of the genes whose function is required for protein export in Erwinia or Xanthomo- nas have been described. If the inventory of Erwinia out genes is complete, it should be possible to reconstruct a functional Erwinia export system in Escherichia coli.

Location of the gene products of the out genes should now be possible by immunological methods. This may help to confirm the existence of a periplasmic/ outer membrane complex of proteins involved in export as envisaged in the two-step model. Spatial inter-relationships between different out gene products could also be investigated by the use of chemical cross-linking agents. Since the process of export of enzymes is apparently necessary for the pathogenesis of a number of animal and plant pathogens, the further potential exists for using this export apparatus as a target for specific antibiotic drug action.

The work described in this paper was supported by the Agricultural and Food Research Council (via a grant-in-aid to the John Innes Institute and a studentship to C .G .), the Commission of the European Community (F .D .) and the Gatsby Foundation (P .C .T .).

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genetic and biochemical analysis of protein export from ... · isozymes of 35-5, 36‘5 and 41k (k...

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