Assembly and function of type III secretory systems.

Type III secretion systems allow Yersinia spp., Salmonella spp., Shigella spp., Bordetella spp., and Pseudomonas aeruginosa and enteropathogenic Escherichia coli adhering at the surface of a eukaryotic cell to inject bacterial proteins across the two bacterial membranes and the eukaryotic cell membrane to destroy or subvert the target cell. These systems consist of a secretion apparatus, made of approximately 25 proteins, and an array of proteins released by this apparatus. Some of these released proteins are "effectors," which are delivered into the cytosol of the target cell, whereas the others are "translocators," which help the effectors to cross the membrane of the eukaryotic cell. Most of the effectors act on the cytoskeleton or on intracellular-signaling cascades. A protein injected by the enteropathogenic E. coli serves as a membrane receptor for the docking of the bacterium itself at the surface of the cell. Type III secretion systems also occur in plant pathogens where they are involved both in causing disease in susceptible hosts and in eliciting the so-called hypersensitive response in resistant or nonhost plants. They consist of 15-20 Hrp proteins building a secretion apparatus and two groups of effectors: harpins and avirulence proteins. Harpins are presumably secreted in the extracellular compartment, whereas avirulence proteins are thought to be targeted into plant cells. Although a coherent picture is clearly emerging, basic questions remain to be answered. In particular, little is known about how the type III apparatus fits together to deliver proteins in animal cells. It is even more mysterious for plant cells where a thick wall has to be crossed. In spite of these haunting questions, type III secretion appears as a fascinating trans-kingdom communication device.

Ralstonia solanacearum produces hrp-dependent pili that are required for PopA secretion but not for attachment of bacteria to plant cells.

As in many other Gram-negative plant pathogenic bacteria, the Ralstonia solanacearum hrp genes are involved in the production of a type III secretion apparatus that allows the translocation of PopA protein to the external medium. Here, we show that hrp genes are also involved in the biogenesis of pili that are mainly composed of the HrpY protein. These pili are produced at one pole of the bacterium and are also released into the external medium where they can form very long straight bundles. An hrpY mutant is defective in pilus production, impaired in interactions with plants and in secretion of the PopA protein but not in attachment to plant cells.

The minimal gene set member msrA, encoding peptide methionine sulfoxide reductase, is a virulence determinant of the plant pathogen Erwinia chrysanthemi.

Peptide methionine sulfoxide reductase (MsrA), which repairs oxidized proteins, is present in most living organisms, and the cognate structural gene belongs to the so-called minimum gene set [Mushegian, A. R. & Koonin, E. V., (1996) Proc. Natl. Acad. Sci. USA 93, 10268-10273]. In this work, we report that MsrA is required for full virulence of the plant pathogen Erwinia chrysanthemi. The following differences were observed between the wild-type and a MsrA- mutant: (i) the MsrA- mutant was more sensitive to oxidative stress; (ii) the MsrA- mutant was less motile on solid surface; (iii) the MsrA- mutant exhibited reduced virulence on chicory leaves; and (iv) no systemic invasion was observed when the MsrA- mutant was inoculated into whole Saintpaulia ionantha plants. These results suggest that plants respond to virulent pathogens by producing active oxygen species, and that enzymes repairing oxidative damage allow virulent pathogens to survive the host environment, thereby supporting the theory that active oxygen species play a key role in plant defense.

Conditions for natural transformation of Ralstonia solanacearum.

The development of competence allowing natural transformation of Ralstonia solanacearum was found to occur during exponential growth and not in response to any excreted factors. Linear DNAs were effectively integrated by recombination requiring a minimum of 50 bp of homologous DNA. Therefore, DNA from other genera and species were ineffective.

The hrp gene locus of Pseudomonas solanacearum, which controls the production of a type III secretion system, encodes eight proteins related to components of the bacterial flagellar biogenesis complex.

Five transcription units of the Pseudomonas solanacearum hrp gene cluster are required for the secretion of the HR-inducing PopA1 protein. The nucleotide sequences of two of these, units 1 and 3, have been reported. Here, we present the nucleotide sequence of the three other transcription units, units 2, 4 and 7, which are together predicted to code for 15 hrp genes. This brings the total number of Hrp proteins encoded by these five transcription units to 20, including HrpB, the positive regulatory protein, and HpaP, which is apparently not required for plant interactions. Among the 18 other proteins, eight belong to protein families regrouping proteins involved in type III secretion pathways in animal and plant bacterial pathogens and in flagellum biogenesis, while two are related solely to proteins involved in secretion systems. For the various proteins found to be related to P. solanacearum Hrp proteins, those in plant-pathogenic bacteria include proteins encoded by hrp genes. For Hrp-related proteins of animal pathogens, those encoded by the spa and mxi genes of Shigella flexneri and of Salmonella typhimurium and by the ysc genes of Yersinia are involved in type III secretion pathways. Proteins involved in flagellum biogenesis, which are related to Hrp proteins of P. solancearum, include proteins encoded by fli and flh genes of S. typhimurium, Bacillus subtilis and Escherichia coli and by mop genes of Erwinia carotovora. P. solanacearum Hrp proteins were also found to be related to proteins of Rhizobium fredii involved in nodulation specificity.

PopA1, a protein which induces a hypersensitivity-like response on specific Petunia genotypes, is secreted via the Hrp pathway of Pseudomonas solanacearum.

This paper describes the identification of a new class of extracellular bacterial proteins, typified by PopA1 and its derivative PopA3, which act as specific hypersensitive response (HR) elicitors. These two heat-stable proteins, with HR-like elicitor activities on tobacco (non-host plant) but without activity on tomato (host plant), have been characterized from the supernatant of the plant pathogenic bacterium Pseudomonas solanacearum strain GMI1000. These two proteins induced the same pattern of response on Petunia, as a function of the genotypes tested. popA, the structural gene for PopA1, maps outside of the hrp gene cluster but belongs to the hrp regulon. The amino acid sequence of PopA1 does not show homology to any characterized proteins. Its secretion is dependent on hrp genes and is followed by stepwise removal of the 93 amino-terminal amino acids, producing the protein PopA3. Petunia lines responsive to PopA3 and its precursors were resistant to infection by strain GMI1000, whereas non-responsive lines were sensitive, suggesting that popA could be an avirulence gene. A popA mutant remained fully pathogenic on sensitive plants, indicating that this gene is not essential for pathogenicity. While lacking PopA1, this mutant, which remained avirulent on tobacco and on resistant Petunia lines, still produced additional extracellular necrogenic compounds. On the basis of both their structural features and the biological properties of the popA mutant, PopA1 and PopA3 clearly differ from hairpins characterized in other plant pathogenic bacteria.

Conservation of secretion pathways for pathogenicity determinants of plant and animal bacteria.

Extracellular proteins of plant and animal bacteria are important in virulence. Many of these are secreted through the type I sec-independent and the type II sec-dependent pathways. Recently, a third distinct pathway, involved in secretion of Yops, has been discovered in Yersinia. This pathway has homology with pathways in plant pathogenic bacteria that are putatively involved in the secretion of proteins active on plant cells, such as harpin and possibly some avr gene products

Genetic analysis of the Erwinia chrysanthemi 3937 chrysobactin iron-transport system: characterization of a gene cluster involved in uptake and biosynthetic pathways.

Twenty of the twenty-two MudII1734 insertions impairing the chrysobactin iron-assimilation system of Erwinia chrysanthemi 3937 were localized to a 50 kbp genomic insert contained in the R-prime plasmid, R'4 (Enard et al., 1988). Using the conjugative plasmid pULB110 (RP4::mini-Mu) and the generalized transducing phage phi EC2, we located this iron-transport region and the two unlinked mutations on the chromosome linkage map. Chrysobactin is a catechol-type siderophore and, as we have previously observed with the entA locus of Escherichia coli, the E. chrysanthemi-derived R'4 was found to complement E. coli entB and entE mutations. A 2.9 kb EcoRi and a 4.8 kb BamHI fragment in the R'4 sharing homology with the E. coli entCEBAP15 operon DNA were subcloned. These fragments were used as DNA/DNA hybridization probes to screen a wild-type gene library, yielding a recombinant cosmid (pEC7) able to complement mutations disrupting the 2,3-dihydroxybenzoic acid biosynthetic pathway in both Erwinia and Escherichia spp. as well as the E. coli entE mutation. Physical mapping of the genomic MudII1734 insertions corresponding to these mutations led to the identification of a cluster of genes confined to a DNA sequence of about 10 kb required for both biosynthetic and receptor functions.

Identification of plant-inducible genes in Erwinia chrysanthemi 3937.

We present a method for identifying plant-inducible genes of Erwinia chrysanthemi 3937. Mutagenesis was done with the Mu dIIPR3 transposon, which carries a promoterless neomycin phosphotransferase gene (nptI), so upon insertion, the truncated gene can fuse to E. chrysanthemi promoters. Mutants containing insertions in plant-inducible genes were selected for their sensitivity to kanamycin on minimal plates and for their acquired resistance to this antibiotic when an S. ionantha plant extract was added to kanamycin minimal plates. The selection allowed the identification of E. chrysanthemi promoters inducible by host factors present in the S. ionantha plant extract. Using this method, we isolated 30 mutants and characterized 10 of them. Two mutants were defective in cation uptake, one was defective in the galacturonate degradation pathway, and another was altered in the production of the acidic pectate lyase. The functions of the other mutated genes are still unknown, but we show that most of them are involved in pathogenicity.

Relationship between the pel genes of the pelADE cluster in Erwinia chrysanthemi strain B374.

In this paper, we have used filter hybridization and nucleotide sequencing to analyse the relationship between the three genes of the pelADE cluster in the Erwinia chrysanthemi (Ech) strain B374. This cluster encodes for three of the five pectate lyase proteins that are involved in the maceration and soft-rotting of plant tissue, an important trait in Ech pathogenicity. Southern hybridization revealed homology between each of the three pel genes. A 3560 bp DNA fragment containing the pelE and pelD genes was sequenced. These two genes show extensive homology in the coding regions but only low homology in the 5' and 3' non-coding regions. However both genes exhibit sequences homologous to the Escherichia coli CAP-binding site consensus sequence upstream of the start codon and an inverted repeat sequence which may act as a rho-independent transcriptional terminator after the translational stop. The pel genes of Ech B374 were also compared with the already sequenced pel genes of EC16, another Ech strain.

Chromosomal mapping of the pel and cel genes in Erwinia chrysanthemi strain B374.

Using the RP4::mini-Mu in vivo cloning technique, van Gijsegem et al. (1985) isolated several pel and cel genes of Erwinia chrysanthemi (Ech) B374 strain. We have localized these genes on the Ech chromosome by co-transfer mapping of MudI1734 insertion mutants and refined the map by co-transposition analysis. This analysis has enabled us to identify another cel gene.

Pseudomonas solanacearum genes controlling both pathogenicity on tomato and hypersensitivity on tobacco are clustered.

A pLAFR3 cosmid clone designated pVir2 containing a 25-kilobase (kb) DNA insert was isolated from a wild-type Pseudomonas solanacearum GMI1000 genomic library. This cosmid was shown to complement all but one of the nine Tn5-induced mutants which have been isolated after random mutagenesis and which have lost both pathogenicity toward tomato and ability to induce hypersensitive reaction (HR) on tobacco (hrp mutants). The insert is colinear with the genome and provides restoration of the HR-inducing ability when transferred into several Tn5-induced hrp mutants, but failed to complement deletion mutants extending on both sides of the pVir2 region. Localized mutagenesis demonstrated that the hrp genes are clustered within a 17.5-kb region of pVir2 and that this cluster probably extends on the genomic region adjacent to the pVir2 insert. A 3-kb region adjacent to the hrp cluster modulates aggressiveness toward tomato but does not control HR-inducing ability. Sequences within the hrp cluster of pVir2 have homology with the genomic DNA of Xanthomonas campestris strains representing eight different pathovars, suggesting that a set of common pathogenicity functions could be shared by P. solanacearum and X. campestris.

Organization of a pectate lyase gene family in Erwinia chrysanthemi.

The pelA, pelD and pelE genes encode three of the five major pectate lyase (PL) isoenzymes (PLa, PLd and PLe) in Erwinia chrysanthemi strains B374 and 3937. These genes were previously isolated from genomic libraries or by in vivo cloning as R' factors promoted by the pULB113 plasmid. They are clustered near purE on the chromosomal map of E. chrysanthemi B374 [Van Gijsegem et al., EMBO J. 4 (1985) 787-792]. Genes pelA, pelD and pelE were subcloned separately into pBR322 derivatives, to test their individuality. It then became possible to select specific mutations in each separated gene. Such mutations were obtained using the transposable bacteriophage MudI1734 that allows the construction of lacZ gene fusions. Subcloning experiments and analysis of MudI1734 insertions permitted us to determine the length of each gene, the transcriptional orientation and the location of the promoter. We concluded that the three genes constitute three independent transcriptional units. They are clustered on a 5-kb DNA fragment, in the order: pelD-pelE-pelA. Genes pelD and pelE are transcribed in the same direction, while the transcription of pelA seems to be divergent. Organization of the pel region was very similar in the two strains B374 and 3937. Moreover, lacZ gene fusions were introduced by marker exchange into the chromosome of E. chrysanthemi B374, giving rise to three strains lacking PLa, PLd or PLe. These fusions allowed us to study the regulation of the mutagenized genes.

Alcaligenes eutrophus CH34 is a facultative chemolithotroph with plasmid-bound resistance to heavy metals.

Alcaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncP1 plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, less than 0.07; Zin+, 12.0; Zin-, 0.6; Cad+, 2.5; and Cad-, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophically and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).

Isolation and characterization of Erwinia chrysanthemi mutants defective in degradation of hexuronates.

Spontaneous and Tn9-induced mutants of Erwinia chrysanthemi were isolated which affect the degradative pathway of galacturonate and ketodeoxygluconate. The mutations were characterized both biochemically and functionally by complementation analysis and localized in the E. chrysanthemi chromosome. The kdgK gene mapped very close to ile, the kdgA gene was between trp and his, and the exuT-uxaC-uxaB-uxaA cluster was linked to thy. The different types of mutants obtained were consistent with an organization of the exu-uxa cluster into two transcription units, one containing the exuT gene, and the other containing the three uxa genes, with the transcription going from uxaC to uxaA.

Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase.

Erwinia chrysanthemi produced several pectate lyases (EC 4.2.2.2) and endocellulases (EC 3.2.1.4) which were largely secreted into the culture medium. Mutants deficient in the secretion mechanism for these enzymes were obtained by chemical and insertion mutagenesis. Further study of one such mutant revealed that both enzyme activities were retained simultaneously within the periplasmic space.

Chromosome transfer and R-prime plasmid formation mediated by plasmid pULB113 (RP4::mini-Mu) in Alcaligenes eutrophus CH34 and Pseudomonas fluorescens 6.2.

Plasmid pULB113 (RP4::mini-Mu), which contains the mini-Mu transposon, promoted both homologous and heterologous gene transfer from Pseudomonas fluorescens 6.2 and Alcaligenes eutrophus CH34. Homologous gene transfer in P. fluorescens 6.2 and A. eutrophus CH34 occurred at a frequency of 10(-4) to 10(-5), and recombinants inherited unselected recessive markers, suggesting a process of chromosome mobilization. Loci involved in autotrophic growth were among those transferred in A. eutrophus. In heterospecific matings, markers were transferred from P. fluorescens to A. eutrophus, Salmonella typhimurium LT2, and Escherichia coli, from A. eutrophus to P. fluorescens, and from Erwinia carotovora subsp. chrysanthemi to A. eutrophus. Heterospecific matings resulted in the formation of R-prime plasmids at frequencies of 10(-7) to 10(-4) per transferred plasmid. When S. typhimurium was the recipient, we observed R-prime plasmids with both restriction-proficient and restriction-deficient strains, although restriction markedly affected the frequency of transfer of pULB113. R-prime plasmids were quite stable, but lost the transposed marker more easily in a rec+ background than in a recA background, suggesting excision of transposed material by reciprocal recombination between flanking copies of mini-Mu. R-prime plasmids could be transferred easily into different recipients and were used in complementation studies. PstI restriction digests of four R-prime plasmids carrying P. fluorescens 6.2 DNA showed a number of additional bands, suggesting that several genes were transposed together with the selected marker on the plasmid.

In vivo cloning of Erwinia carotovora genes involved in the catabolism of hexuronates.

Using the RP4::mini-Mu pULB113 plasmid, an RP4 derivative carrying a deleted Mu prophage which allows the plasmid to pick up any chromosomal DNA segment to form R' plasmids, we cloned all of the genes of Erwinia carotovora involved in the catabolism of the hexuronates and in the transport of these substrates. With the R' plasmids we isolated, we performed complementation analysis and found that, in the Erwinia carotovora strain we used, the genes involved in the catabolism of the hexuronates are clustered in four regions of the chromosome. This genetic organization is compared with that of Escherichia coli K-12.

Formation of F'trp plasmids in Escherichia coli K12.

From strains carrying two different F-prime factors, we recovered F' derivatives that acquired the trp chromosomal region. These F'trp plasmids can be isolated at a frequency of 10(-5) to 10(-6). They were characterized genetically by looking at the size of the trp segment they acquired and at the location of that segment in the parental F' plasmid. Results are discussed in relationship to possible transposition mechanisms.