Alan Collmer

The complete genome sequence of the Arabidopsis and tomato pathogen Pseudomonas syringae pv. tomato DC3000

We report the complete genome sequence of the model bacterial pathogen Pseudomonas syringae pathovar tomato DC3000 (DC3000), which is pathogenic on tomato and Arabidopsis thaliana. The DC3000 genome (6.5 megabases) contains a circular chromosome and two plasmids, which collectively encode 5,763 ORFs. We identified 298 established and putative virulence genes, including several clusters of genes encoding 31 confirmed and 19 predicted type III secretion system effector proteins. Many of the virulence genes were members of paralogous families and also were proximal to mobile elements, which collectively comprise 7% of the DC3000 genome. The bacterium possesses a large repertoire of transporters for the acquisition of nutrients, particularly sugars, as well as genes implicated in attachment to plant surfaces. Over 12% of the genes are dedicated to regulation, which may reflect the need for rapid adaptation to the diverse environments encountered during epiphytic growth and pathogenesis. Comparative analyses confirmed a high degree of similarity with two sequenced pseudomonads, Pseudomonas putida and Pseudomonas aeruginosa, yet revealed 1,159 genes unique to DC3000, of which 811 lack a known function.

Pseudomonas syringae pv. syringae harpinPss: A protein that is secreted via the hrp pathway and elicits the hypersensitive response in plants

The ability of P. syringae to elicit the hypersensitive response in nonhost plants or pathogenesis in hosts is controlled by hrp genes. The P. syringae pv. syringae 61 hrpZ gene encodes harpinPss, a 34.7 kd extracellular protein that elicits hypersensitive necrosis in tobacco and other plants. HarpinPss is heat stable, glycine rich, dissimilar in amino acid sequence to any known protein, produced only in apoplastic fluid-mimicking minimal media, and secreted in a HrpH-dependent manner. The carboxy-terminal 148 amino acid portion of harpinPss contains two directly repeated sequences of GGGLGTP and QTGT and is sufficient and necessary for elicitor activity. The necrosis elicited by harpinPss is an active response of the plant, which can be inhibited by alpha-amanitin, cycloheximide, lanthanum chloride, or sodium vanadate.

Bacterial Pathogens in Plants: Life up against the Wall.

of transposon mutagenesis, broad-host-range cosmid vectors, and marker-exchange mutagenesis to identify and manipulate bacterial genes that have a readily scored phenotype when mutated, conjugated into a related strain, or expressed in Escherichia coli. These approaches have yielded a large inventory of hrp (hypersensitive [esponse and pathogenicity) and avr (eirulence) genes that directly relate to the HR puzzle as we!l as numerous other genes associated with pectic enzyme, toxin, and extracellular polysaccharide (EPS) production. Rather than detail this inventory (which may be fundamentally incomplete; see below), we use representative components to develop a model for bacterial plant pathogenesis that is based on the very recent third development-the discovery that the hrp genes encode a protein secretion system, shared in plant and animal pathogens, that has the potential to transfer virulence proteins into eukaryotic host cells. The necrogenic bacteria have diverse pathogenic personalities with a bewildering array of symptoms and host specificities. The growing evidence that the hrp genes are ubiquitous in these pathogens, controlling early (and generally essential) interactions with plants, provides a unifying entry point for exploring bacterial phytopathogenicity. Hence, after introducing the representative pathogens, we explore the dynamic operation of the Hrp system and then turn briefly to factors such as toxins, EPS, and pectic enzymes that affect the full development of plant disease.

An nptI-sacB-sacR cartridge for constructing directed, unmarked mutations in Gram-negative bacteria by marker exchange-eviction mutagenesis

A technique for marker exchange-eviction mutagenesis that enables the construction of directed, unmarked mutations in Gram-negative bacteria was demonstrated in Erwinia chrysanthemi. The technique employs an nptI-sacB-sacR cartridge that is carried on a 3.8-kb BamHI fragment and confers kanamycin (Km) resistance and sucrose sensitivity (due to the production of levansucrase by sacB) in E. chrysanthemi. The cartridge was inserted into a Sau3A site in a cloned E. chrysanthemi pelC gene (encoding pectate lyase isozyme PLc) and then introduced into the Erwinia genome by gene exchange recombination. The resulting mutant was KmR, sucrose-sensitive, and PLc-deficient. The cartridge was then excised from the plasmid-borne pelC gene by PstI cleavage to leave a 28-bp frame-shifting insertion. The pelC allele containing the 28-bp insertion was exchanged for the chromosomal allele containing the nptI-sacB-sacR cartridge by selection for sucrose tolerance. The resulting E. chrysanthemi mutant was Kms and PLc-deficient. The technique permits the construction of complex strains with many directed mutations without the introduction of a corresponding number of antibiotic resistance markers and should prove useful, for example, in exploring the role of the multiple pel genes in E. chrysanthemi.

Whole-Genome Sequence Analysis of Pseudomonas syringae pv. phaseolicola 1448A Reveals Divergence among Pathovars in Genes Involved in Virulence and Transposition

Pseudomonas syringae pv. phaseolicola, a gram-negative bacterial plant pathogen, is the causal agent of halo blight of bean. In this study, we report on the genome sequence of P. syringae pv. phaseolicola isolate 1448A, which encodes 5,353 open reading frames (ORFs) on one circular chromosome (5,928,787 bp) and two plasmids (131,950 bp and 51,711 bp). Comparative analyses with a phylogenetically divergent pathovar, P. syringae pv. tomato DC3000, revealed a strong degree of conservation at the gene and genome levels. In total, 4,133 ORFs were identified as putative orthologs in these two pathovars using a reciprocal best-hit method, with 3,941 ORFs present in conserved, syntenic blocks. Although these two pathovars are highly similar at the physiological level, they have distinct host ranges; 1448A causes disease in beans, and DC3000 is pathogenic on tomato and Arabidopsis. Examination of the complement of ORFs encoding virulence, fitness, and survival factors revealed a substantial, but not complete, overlap between these two pathovars. Another distinguishing feature between the two pathovars is their distinctive sets of transposable elements. With access to a fifth complete pseudomonad genome sequence, we were able to identify 3,567 ORFs that likely comprise the core Pseudomonas genome and 365 ORFs that are P. syringae specific.

Genomewide identification of Pseudomonas syringae pv. tomato DC3000 promoters controlled by the HrpL alternative sigma factor.

The ability of Pseudomonas syringae pv. tomato DC3000 to parasitize tomato and Arabidopsis thaliana depends on genes activated by the HrpL alternative sigma factor. To support various functional genomic analyses of DC3000, and specifically, to identify genes involved in pathogenesis, we developed a draft sequence of DC3000 and used an iterative process involving computational and gene expression techniques to identify virulence-implicated genes downstream of HrpL-responsive promoters. Hypersensitive response and pathogenicity (Hrp) promoters are known to control genes encoding the Hrp (type III protein secretion) machinery and a few type III effector proteins in DC3000. This process involved (i) identification of 9 new virulence-implicated genes in the Hrp regulon by miniTn5gus mutagenesis, (ii) development of a hidden Markov model (HMM) trained with known and transposon-identified Hrp promoter sequences, (iii) HMM identification of promoters upstream of 12 additional virulence-implicated genes, and (iv) microarray and RNA blot analyses of the HrpL-dependent expression of a representative subset of these DC3000 genes. We found that the Hrp regulon encodes candidates for 4 additional type III secretion machinery accessory factors, homologs of the effector proteins HopPsyA, AvrPpiB1 (2 copies), AvrPpiC2, AvrPphD (2 copies), AvrPphE, AvrPphF, and AvrXv3, and genes associated with the production or metabolism of virulence factors unrelated to the Hrp type III secretion system, including syringomycin synthetase (SyrE), Nɛ-(indole-3-acetyl)-l-lysine synthetase (IaaL), and a subsidiary regulon controlling coronatine production. Additional candidate effector genes, hopPtoA2, hopPtoB2, and an avrRps4 homolog, were preceded by Hrp promoter-like sequences, but these had HMM expectation values of relatively low significance and were not detectably activated by HrpL.

Genomewide identification of proteins secreted by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato DC3000.

The ability of Pseudomonas syringae pv. tomato DC3000 to be pathogenic on plants depends on the Hrp (hypersensitive response and pathogenicity) type III protein secretion system and the effector proteins it translocates into plant cells. Through iterative application of experimental and computational techniques, the DC3000 effector inventory has been substantially enlarged. Five homologs of known avirulence (Avr) proteins and five effector candidates, encoded by genes with putative Hrp promoters and signatures of horizontal acquisition, were demonstrated to be secreted in culture and/or translocated into Arabidopsis in a Hrp-dependent manner. These 10 Hrp-dependent outer proteins (Hops) were designated HopPtoC (AvrPpiC2 homolog), HopPtoD1 and HopPtoD2 (AvrPphD homologs), HopPtoK (AvrRps4 homolog), HopPtoJ (AvrXv3 homolog), HopPtoE, HopPtoG, HopPtoH, HopPtoI, and HopPtoS1 (an ADP-ribosyltransferase homolog). Analysis of the enlarged collection of proteins traveling the Hrp pathway in P. syringae revealed an export-associated pattern of equivalent solvent-exposed amino acids in the N-terminal five positions, a lack of Asp or Glu residues in the first 12 positions, and amphipathicity in the first 50 positions. These characteristics were used to search the unfinished DC3000 genome, yielding 32 additional candidate effector genes that predicted proteins with Hrp export signals and that also possessed signatures of horizontal acquisition. Among these were genes encoding additional ADP-ribosyltransferases, a homolog of SrfC (a candidate effector in Salmonella enterica), a catalase, and a glucokinase. One ADP-ribosyltransferase and the SrfC homolog were tested and shown to be secreted in a Hrp-dependent manner. These proteins, designated HopPtoS2 and HopPtoL, respectively, bring the DC3000 Hrp-secreted protein inventory to 22.

Assay methods for pectic enzymes

Publisher Summary The characterization and purification of a pectic enzyme is often complicated by the presence of other pectic enzymes produced by the source microorganism. This chapter describes three assay procedures that address this problem. Pectic enzymes from Erwinia spp. is used as examples in the chapter. The assays are readily adapted to the analysis of pectic enzyme complexes of other organisms and can be used with crude preparations. Results from plant tissue extracts should be interpreted cautiously, however, because of the prevalence of pectic enzyme inhibitors. The activity stains in the first procedure enable rapid approximation of the number and type of pectic enzymes in the sample and can guide purification strategies, particularly when used in conjunction with titration curves. The second and third procedures permit quantitative assay of hydrolytic and β-eliminative pectic enzymes, respectively. The action patterns of purified pectic enzymes are determined by viscometric and reaction product analyses; oligogalacturonides are resolved by paper chromatography or thin-layer chromatography and detected with bromphenol blue or thiobarbituric acid spray reagent.

Unified nomenclature for broadly conserved hrp genes of phytopathogenic bacteria

Genes of plant-pathogenic bacteria controlling hypersensitive response (HR) elicitation and pathogenesis were designated ‘hrp’ by Lindgren et al. in 1986 (J Bacteriol 168: 512–522). hrp genes have been characterized in several species of the four major genera of Gramnegative plant pathogens, Erwinia, Pseudomonas, Ralstonia (a new proposed genus including Pseudomonas solanacearum) and Xanthomonas. To date, hrp genes have been found mainly in large clusters, and they have been shown to be conserved physically and, in many cases, functionally among different bacteria. Hybridization studies and genetic analyses have revealed the presence of functional hrp genes even in species that are not typically observed to elicit an HR, such as Erwinia chrysanthemi and Erwinia stewartii, suggesting that hrp genes may be common to all Gram-negative plant pathogens, possibly excluding Agrobacterium spp. Current knowledge of hrp genes has been reviewed by Bonas (1994, Curr Top Microbiol Immunol 192: 79–98) and by Van Gijsegem et al. (1995, In Pathogenesis and Host–Parasite Specificity in Plant Diseases: Histopathological, Biochemical, Genetic and Molecular Basis. Volume 1. (Kohmoto et al., eds); Oxford: Pergamon Press, pp. 273–292). The nucleotide sequences of four hrp gene clusters, those of Ralstonia solanacearum (previously P. solanacearum) (Genin et al., 1992, Mol Microbiol 6: 3065–3076; Gough et al., 1992, Mol Plant–Microbe Interact 5: 384–389; Gough et al., 1993, Mol Gen Genet 239: 378–392; Van Gijsegem et al., 1995, Mol Microbiol 15: 1095–1114), Erwinia amylovora (Bogdanove et al., 1996, J Bacteriol 178: 1720– 1730; Wei and Beer, 1993, J Bacteriol 175: 7958–7967; Wei and Beer, 1995, J Bacteriol 177: 6201–6210; Wei et al., 1992, Science 257: 85–88; S. V. Beer, unpublished), Pseudomonas syringae pv. syringae (Huang et al., 1992, J Bacteriol 174: 6878–6885; Huang et al., 1993, Mol Plant–Microbe Interact 6: 515–520; Huang et al., 1995, Mol Plant–Microbe Interact 8: 733–746; Lidell and Hutcheson, 1994, Mol Plant–Microbe Interact 7: 488–497; Preston et al., 1995, Mol Plant–Microbe Interact 8: 717–732; Xiao et al., 1994, J Bacteriol 176: 1025–1036), and Xanthomonas campestris pv. vesicatoria (Fenselau et al., 1992, Mol Plant–Microbe Interact 5: 390–396; Fenselau and Bonas, 1995, Mol Plant–Microbe Interact 8: 845–854; U. Bonas, unpublished), have been largely determined. These clusters each contain more than twenty genes, many of which encode components of a novel proteinsecretion pathway designated ‘type III’. It has been shown directly that various extracellular proteins involved in pathogenesis and defence elicitation by plantpathogenic bacteria utilize this pathway (Arlat et al., 1994, EMBO J 13: 543–553; He et al., 1993, Cell 73: 1255–1266; Wei and Beer, 1993, ibid.), and the pathway is known to function in the export of virulence factors from the animal pathogens Salmonella typhimurium, Shigella flexneri, and Yersinia entercolitica, Yersinia pestis, and Yersinia pseudotuberculosis (for reviews, see Salmond and Reeves, 1993, Trends Biochem Sci 18: 7–12; and Van Gijsegem et al., 1993, Trends Microbiol 1: 175– 180). Nine type III secretion genes are conserved among all four of the plant pathogens listed above and among the animal pathogens. Based on sequence analysis and some experimental evidence, they are believed to encode one outer-membrane protein, one outer-membrane-associated lipoprotein, five inner-membrane proteins, and two cytoplasmic proteins, one of which is a putative ATPase. All of the predicted gene products, except the outer-membrane protein, show significant similarity to components of the flagellar biogenesis complex (for reviews see Blair, 1995, Annu Rev Microbiol 49: 489–522; and Bischoff and Ordal, 1992, Mol Microbiol 6: 23–28). We herein refer to the hrp-encoded type III pathway as the ‘Hrp pathway’. Because hrp genes have been characterized independently in diverse plant-pathogenic bacteria, hrp gene nomenclature differs in different species, and it is not always consistent even within the same organism. Different designations are used for homologous genes, and, even worse, the same designation is used for different genes in different organisms. For example, hrpI of E. amylovora is homologous with hrpC2 of X. campestris pv. vesicatoria and hrpO of R. solanacearum, and the homologue in P. syringae pv. syringae appears in the literature both as hrpI and as hrpJ2. Also, ‘hrpN ’ in R. solanacearum designates a secretion-pathway gene, whereas in E. amylovora, ‘hrpN ’ designates the gene encoding the elicitor harpin. Furthermore, in many bacteria the number of known hrp genes approaches 26. In anticipation of exhausting the alphabet, some authors chose to designate hrp genes with a letter and a number, creating the potential for confusion of distinct genes with alleles of the same gene. For hrp gene researchers, the current nomenclature is at best inconvenient; for other scientists, it is bewildering. Another problem exists: accumulation of knowledge about the structure of hrp loci has outpaced the accumulation of Molecular Microbiology (1996) 20(3), 681–683

Genomic mining type III secretion system effectors in Pseudomonas syringae yields new picks for all TTSS prospectors

Many bacterial pathogens of plants and animals use a type III secretion system (TTSS) to deliver virulence effector proteins into host cells. Because effectors are heterogeneous in sequence and function, there has not been a systematic way to identify the genes encoding them in pathogen genomes, and our current inventories are probably incomplete. A pre-closure draft sequence of Pseudomonas syringae pv. tomato DC3000, a pathogen of tomato and Arabidopsis, has recently supported five complementary studies which, collectively, identify 36 TTSS-secreted proteins and many more candidate effectors in this strain. These studies demonstrate the advantages of combining experimental and computational approaches, and they yield new insights into TTSS effectors and virulence regulation in P. syringae, potential effector targeting signals in all TTSS-dependent pathogens, and strategies for finding TTSS effectors in other bacteria that have sequenced genomes.