A genome-wide analysis of desferrioxamine mediated iron uptake in Erwinia spp. reveals genes exclusive of the Rosaceae infecting strains.

Erwinia amylovora is the etiological agent of fire blight, a devastating disease which is a global threat to commercial apple and pear production. The Erwinia genus includes a wide range of different species belonging to plant pathogens, epiphytes and even opportunistic human pathogens. The aim of the present study is to understand, within the Erwinia genus, the genetic differences between phytopathogenic strains and those strains not reported to be phytopathogenic. The genes related to the hydroxamate siderophores iron uptake have been considered due to their potential druggability. In E. amylovora siderophore-mediated iron acquisition plays a relevant role in the progression of Fire blight. Here we analyzed the taxonomic relations within Erwinia genus and the relevance of the genes related to the siderophore-mediated iron uptake pathway. The results of this study highlight the presence of a well-defined sub-group of Rosaceae infecting species taxonomically and genetically related with a high number of conserved core genes. The analysis of the complete ferrioxamine transport system has led to the identification of two genes exclusively present in the Rosaceae infecting strains.

Erwinia teleogrylli sp. nov., a Bacterial Isolate Associated with a Chinese Cricket.

A bacterial isolate (SCU-B244T) was obtained in China from crickets (Teleogryllus occipitalis) living in cropland deserted for approximately 10 years. The isolated bacteria were Gram-negative, facultatively anaerobic, oxidase-negative rods. A preliminary analysis of the 16S rRNA gene sequence indicated that the strain belongs to either the genus Erwinia or Pantoea. Analysis of multilocus sequence typing based on concatenated partial atpD, gyrB and infB gene sequences and physiological and biochemical characteristics indicated that the strain belonged to the genus Erwinia, as member of a new species as it was distinct from other known Erwinia species. Further analysis of the 16S rRNA gene showed SCU-B244T to have 94.71% identity to the closest species of that genus, Erwinia oleae (DSM 23398T), which is below the threshold of 97% used to discriminate bacterial species. DNA-DNA hybridization results (5.78±2.52%) between SCU-B244T and Erwinia oleae (DSM 23398T) confirmed that SCU-B244T and Erwinia oleae (DSM 23398T) represent different species combined with average nucleotide identity values which range from 72.42% to 74.41. The DNA G+C content of SCU-B244T was 55.32 mol%, which also differs from that of Erwinia oleae (54.7 to 54.9 mol%). The polyphasic taxonomic approach used here confirmed that the strain belongs to the Erwinia group and represents a novel species. The name Erwinia teleogrylli sp. nov. is proposed for this novel taxon, for which the type strain is SCU-B244T (= CGMCC 1.12772T = DSM 28222T = KCTC 42022T).

MORPHOLOGICAL HETEROGENEITY OF TEMPERATE ERWINIOPHAGE 59.

This paper is devoted to the phenomenon of morphological heterogeneity within the population of the temperate erwiniophage 59, which does not have analogies among other bacterial viruses. AIM: To investigate the basic properties of erwiniophage 59 heteroge- neous population obtained from different isogenic strains of amilovora-like bacterium Erwinia "horticola" (Eho). METHODS: Erwiniophage 59 was obtained by propagation on its traditional host Eho 450, as well as on its three isogenic strains and a related bacterium E. "horticola" 60. Physical and chemical properties of the phage particles were studied using centrifugation in CsCl-gradients, electrophoresis in agarose gels, electron micros- copy, restriction analysis ofDNA and SDS-PA GE of the virion polypeptides. RESULTS: It was shown that the pool oftemperatephage 59 is a heterogeneous population consisting of two phage types when propagated on the mentioned hosts. These types are discrete and have buoyant-density difference of 0.02 g/cm3, that allowed to separate subpopulations for a de- tailed investigation. The subpopulation with the higher density was determined as the au- thentic bacteriophage 59 (subpopulation II with capsid diameter of55.36 nm). The capsid diameter of the subpopulation Iparticles equals 51.16 nm. Both types ofparticles do not differ by DNA size and have identical restriction patterns. Based on the SmaI-restriction analysis it may be concluded that the DNA packaging remains unchangeable and is carried out according to the headfull packaging mechanism. However; the subpopulation I differs from the original one by a relative content of some polypeptides. Curiously, the subpopulations I and II have different values of lysogenization and spontaneous induction frequencies. CONCLUSIONS: An unusual type of morphological heterogeneity ofthe phage 59 particles was observedfor thefirst time; this heterogeneity is associated with the presence of two equimolar subpopulations with different physical and chemical parametrs of the virions. Morphological heterogeneity of temperate erwiniophage 59 significantly differs from such of the classical coliphages as T4, P1 and the phage system P2-P4.

Phenylalanine in the pore of the Erwinia ligand-gated ion channel modulates picrotoxinin potency but not receptor function.

The Erwinia ligand-gated ion channel (ELIC) is a bacterial homologue of eukaryotic Cys-loop ligand-gated ion channels. This protein has the potential to be a useful model for Cys-loop receptors but is unusual in that it has an aromatic residue (Phe) facing into the pore, leading to some predictions that this protein is incapable of ion flux. Subsequent studies have shown this is not the case, so here we probe the role of this residue by examining the function of the ELIC in cases in which the Phe has been substituted with a range of alternative amino acids, expressed in Xenopus oocytes and functionally examined. Most of the mutations have little effect on the GABA EC50, but the potency of the weak pore-blocking antagonist picrotoxinin at F16'A-, F16'D-, F16'S-, and F16'T-containing receptors was increased to levels comparable with those of Cys-loop receptors, suggesting that this antagonist can enter the pore only when residue 16' is small. T6'S has no effect on picrotoxinin potency when expressed alone but abolishes the increased potency when combined with F16'S, indicating that the inhibitor binds at position 6', as in Cys-loop receptors, if it can enter the pore. Overall, the data support the proposal that the ELIC pore is a good model for Cys-loop receptor pores if the role of F16' is taken into consideration.

Efficient production of lycopene in Saccharomyces cerevisiae by expression of synthetic crt genes from a plasmid harboring the ADH2 promoter.

Lycopene is an effective antioxidant proposed as a possible treatment for some cancers and other degenerative human conditions. This study aims at generation of a yeast strain (Saccharomyces cerevisiae) of efficient productivity of lycopene by overexpressing synthetic genes derived from crtE, crtB and crtI genes of Erwinia uredovora. These synthetic genes were constructed in accordance with the preferred codon usage in S. cerevisiae but with no changes in amino acid sequences of the gene products. S. cerevisiae cells were transformed with these synthetic crt genes, whose expression was regulated by the ADH2 promoter, which is de-repressed upon glucose depletion. The RT-PCR and Western blotting analyses indicated that the synthetic crt genes were efficiently transcribed and translated in crt-transformed S. cerevisiae cells. The highest level of lycopene in one of the transformed lines was 3.3mglycopene/g dry cell weight, which is higher than the previously reported levels of lycopene in other microorganisms transformed with the three genes. These results suggest the excellence of using the synthetic crt genes and the ADH2 promoter in generation of recombinant S. cerevisiae that produces a high level of lycopene. The level of ergosterol was reversely correlated to that of lycopene in crt-transformed S. cerevisiae cells, suggesting that two pathways for lycopene and ergosterol syntheses compete for the use of farnesyl diphosphate.

Erwinia piriflorinigrans sp. nov., a novel pathogen that causes necrosis of pear blossoms.

Eight Erwinia strains, isolated from necrotic pear blossoms in València, Spain, were compared with reference strains of Erwinia amylovora and Erwinia pyrifoliae, both of which are pathogenic to species of pear tree, and to other species of the family Enterobacteriaceae using a polyphasic approach. Phenotypic analyses clustered the novel isolates into one phenon, distinct from other species of the genus Erwinia, showing that the novel isolates constituted a homogeneous phenotypic group. Rep-PCR profiles, PCR products obtained with different pairs of primers and plasmid contents determined by restriction analysis showed differences between the novel strains and reference strains of E. amylovora and E. pyrifoliae. Phylogenetic analysis of 16S rRNA, gpd and recA gene sequences showed that the eight novel strains could not be assigned to any recognized species. On the basis of DNA-DNA hybridization studies, the novel isolates constituted a single group with relatedness values of 87-100  % to the designated type strain of the group, CFBP 5888(T). Depending on the method used, strain CFBP 5888(T) showed DNA-DNA relatedness values of between 22.7 and 50  % to strains of the closely related species E. amylovora and E. tasmaniensis. The DNA G+C contents of two of the novel strains, CFBP 5888(T) and CFBP 5883, were 51.1 and 50.5 mol%, respectively. On the basis of these and previous results, the novel isolates represent a novel species of the genus Erwinia, for which the name Erwinia piriflorinigrans sp. nov. is proposed. The type strain is CFBP 5888(T) (=CECT 7348(T)).

The olive fly endosymbiont, "Candidatus Erwinia dacicola," switches from an intracellular existence to an extracellular existence during host insect development.

As polyphagous, holometabolous insects, tephritid fruit flies (Diptera: Tephritidae) provide a unique habitat for endosymbiotic bacteria, especially those microbes associated with the digestive system. Here we examine the endosymbiont of the olive fly [Bactrocera oleae (Rossi) (Diptera: Tephritidae)], a tephritid of great economic importance. "Candidatus Erwinia dacicola" was found in the digestive systems of all life stages of wild olive flies from the southwestern United States. PCR and microscopy demonstrated that "Ca. Erwinia dacicola" resided intracellularly in the gastric ceca of the larval midgut but extracellularly in the lumen of the foregut and ovipositor diverticulum of adult flies. "Ca. Erwinia dacicola" is one of the few nonpathogenic endosymbionts that transitions between intracellular and extracellular lifestyles during specific stages of the host's life cycle. Another unique feature of the olive fly endosymbiont is that unlike obligate endosymbionts of monophagous insects, "Ca. Erwinia dacicola" has a G+C nucleotide composition similar to those of closely related plant-pathogenic and free-living bacteria. These two characteristics of "Ca. Erwinia dacicola," the ability to transition between intracellular and extracellular lifestyles and a G+C nucleotide composition similar to those of free-living relatives, may facilitate survival in a changing environment during the development of a polyphagous, holometabolous host. We propose that insect-bacterial symbioses should be classified based on the environment that the host provides to the endosymbiont (the endosymbiont environment).

A polyphasic approach assigns the pathogenic Erwinia strains from diseased pear trees in Japan to Erwinia pyrifoliae.

AIMS: Bacterial shoot blight of pear in Japan (BSBP) is caused by Erwinia strains which were formerly associated with the species Erwinia amylovora, the causative agent of fire blight. The description of Erwinia pyrifoliae as a pear pathogen in Korea renewed a possible connection of the pear pathogens in both countries. METHODS AND RESULTS: Nucleotide sequence analysis of the 16S rRNA, the house keeping genes gpd and recA, as well as DNA-DNA hybridization kinetics and microbiological assays place the pear pathogens from Japan into the species E. pyrifoliae described as the causative agent of Asian pear blight in Korea. CONCLUSIONS: Erwinia pyrifoliae strains from Korea and the pear pathogenic Erwinia strains from Japan belong taxonomically into the same species, but show slight divergences in nucleotide sequences used for classification. The allocation is not only supported by microbiological properties, but also by a host range restricted to pear observed before by others. SIGNIFICANCE AND IMPACT OF THE STUDY: The data suggest that the BSBP disease observed at the island of Hokkaido was not fire blight and unify BSBP in Japan with the pear pathogenic species E. pyrifoliae from Korea.

Genetic organization of the hrp genes cluster in Erwinia pyrifoliae and characterization of HR active domains in HrpNEp protein by mutational analysis.

The disease-specific (dsp) region and the hypersensitive response and pathogenicity (hrp) genes, including the hrpW, hrpNEp, and hrpC operons have previously been sequenced in Erwinia pyrifoliae WT3 [Shrestha et al. (2005a)]. In this study, the remaining hrp genes, including the hrpC, hrpA, hrpS, hrpXY, hrpL and hrpJ operons, were determined. The hrp genes cluster (ca. 38 kb) was comprised of eight transcriptional units and contained nine hrc (hrp conserved) genes. The genetic organization of the hrp/hrc genes and their orientation for the transcriptions were also similar to and collinear with those of E. amylovora, showing > or = 80% homologies. However, ORFU1 and ORFU2 of unknown functions, present between the hrpA and hrpS operons of E. amylovora, were absent in E. pyrifoliae. To determine the HR active domains, several proteins were prepared from truncated fragments of the N-terminal and the C-terminal regions of HrpN(Ep) protein of E. pyrifoliae. The proteins prepared from the N-terminal region elicited HR, but not from those of the C-terminal region indicating that HR active domains are located in only N-terminal region of the HrpN(Ep) protein. Two synthetic oligopeptides produced HR on tobacco confirming presence of two HR active domains in the HrpN(Ep). The HR positive N-terminal fragment (HN delta C187) was further narrowed down by deleting C-terminal amino acids and internal amino acids to investigate whether amino acid insertion region have role in faster and stronger HR activity in HrpN(Ep) than HrpN(Ea). The HrpN(Ep) mutant proteins HN delta C187 (D1AIR), HN delta C187 (D2AIR) and HN delta C187 (DM41) retained similar HR activation to that of wild-type HrpN(Ep). However, the HrpN(Ep) mutant protein HN delta C187 (D3AIR) lacking third amino acid insertion region (102 to 113 aa) reduced HR when compared to that of wild-type HrpN(Ep). Reduction in HR elicitation could not be observed when single amino acids at different positions were substituted at third amino acids insertion region. But, substitution of amino acids at L103R, L106K and L110R showed reduction in HR activity on tobacco suggesting their importance in activation of HR faster in the HrpN(Ep) although it requires further detailed analysis.

Transgenic tobacco expressing the hrpN(EP) gene from Erwinia pyrifoliae triggers defense responses against botrytis cinerea.

HrpN(EP), from the gram-negative pathogen, Erwinia pyrifoliae, is a member of the harpin group of proteins, inducing pathogen resistance and hypersensitive cell death in plants. When the hrpN(EP) gene driven by the OsCc1 promoter was introduced into tobacco plants via Agrobacterium-mediated transformation, their resistance to the necrotrophic fungal pathogen, Botrytis cinerea, increased. Resistance to B. cinerea was correlated with enhanced induction of SA-dependent genes such as PR-1a, PR2, PR3 and Chia5, of JA-dependent genes such as PR-1b, and of genes related to ethylene production, such as NT-EFE26, NT-1A1C, DS321, NT-ACS1 and NT-ACS2. However the expression of NPR1, which is thought to be essential for multiple-resistance, did not increase. Since the pattern of expression of defense-related genes in hrpN(EP)-expressing tobacco differed from that in plants expressing hpaG(Xoo) from Xanthomonas oryzae pv. Oryzae, these results suggest that different harpins can affect the expression of different defense-related genes, as well as resistance to different plant pathogens.

Export of the HR eliciting protein, Harpin(Es), of the maize pathogen Erwinia stewartii is species-specific but is independent of the growth temperature.

The extra-cellular export of the HR-eliciting protein, Harpin(Es) of the maize pathogen Erwinia stewartii was studied to find out if the protein needs any species-specific signal for its export and to determine if the export of the protein to the medium is affected in any way by the growth temperature. Based upon the experimental evidence, it was proved that the protein (i.e., Harpin(Es)) does require its own export system (species-specific) to get out of the bacterial cell and can not be exported by the export system of even the very closely related bacterium, Erwinia amylovora. It was also found that the export of Harpin(Es) is, unlike the case of Harpin(Ea) (HR-eliciting protein of Erwinia amylovora), independent of the growth temperature.

Erwinia tasmaniensis sp. nov., a non-phytopathogenic bacterium from apple and pear trees.

Bacteria were isolated from flowers and bark of apple and pear trees at three places in Australia. In Victoria, Tasmania and Queensland, strains with white colonies on nutrient agar were screened for dome-shaped colony morphology on agar with sucrose and were found to be closely related by several criteria. The isolates were not pathogenic on apples or pears. They were characterized by a polyphasic approach including microbiological and API assays as well as fatty acid methyl ester analysis, DNA-DNA hybridization and DNA sequencing. For molecular classification, the 16S rRNA cistron and the conserved genes gpd and recA of these bacteria were investigated. Together with other taxonomic criteria, the results of these studies indicate that the bacteria belong to a novel separate species, which we propose to name Erwinia tasmaniensis sp. nov., with the type strain Et1/99(T) (=DSM 17950(T)=NCPPB 4357(T)). From DNA-DNA hybridization kinetics, microbiological characteristics and nucleotide sequence analyses, this species is related to pathogenic Erwinia species, but also to the epiphytic species Erwinia billingiae.

Mutational analysis of Xanthomonas harpin HpaG identifies a key functional region that elicits the hypersensitive response in nonhost plants.

HpaG is a type III-secreted elicitor protein of Xanthomonas axonopodis pv. glycines. We have determined the critical amino acid residues important for hypersensitive response (HR) elicitation by random and site-directed mutagenesis of HpaG and its homolog XopA. A plasmid clone carrying hpaG was mutagenized by site-directed mutagenesis, hydroxylamine mutagenesis, and error-prone PCR. A total of 52 mutants were obtained, including 51 single missense mutants and 1 double missense mutant. The HR elicitation activity was abolished in the two missense mutants [HpaG(L50P) and HpaG(L43P/L50P)]. Seven single missense mutants showed reduced activity, and the HR elicitation activity of the rest of the mutants was similar to that of wild-type HpaG. Mutational and deletion analyses narrowed the region essential for elicitor activity to the 23-amino-acid peptide (H2N-NQGISEKQLDQLLTQLIMALLQQ-COOH). A synthetic peptide of this sequence possessed HR elicitor activity at the same concentration as the HpaG protein. This region has 78 and 74% homology with 23- and 27-amino-acid regions of the HrpW harpin domains, respectively, from Pseudomonas and Erwinia spp. The secondary structure of the peptide is predicted to be an alpha-helix, as is the HrpW region that is homologous to HpaG. The predicted alpha-helix of HpaG is probably critical for the elicitation of the HR in tobacco plants. In addition, mutagenesis of a xopA gene yielded two gain-of-function mutants: XopA(F48L) and XopA(F48L/M52L). These results indicate that the 12 amino acid residues between L39 and L50 of HpaG have critical roles in HR elicitation in tobacco plants.

Surrogate biochemistry: use of Escherichia coli to identify plant cDNAs that impact metabolic engineering of carotenoid accumulation.

Carotenoids synthesized in plants but not animals are essential for human nutrition. Therefore, ongoing efforts to metabolically engineer plants for improved carotenoid content benefit from the identification of genes that affect carotenoid accumulation, possibly highlighting potential challenges when pyramiding traits represented by multiple biosynthetic pathways. We employed a heterologous bacterial system to screen for maize cDNAs encoding products that alter carotenoid accumulation either positively or negatively. Genes encoding carotenoid biosynthetic enzymes from the bacterium Erwinia uredovora were introduced into Escherichia coli cells that were subsequently transfected with a maize endosperm cDNA expression library; and these doubly transformed cells were then screened for altered carotenoid accumulation. DNA sequencing and characterization of one cDNA class conferring increased carotenoid content led to the identification of maize cDNAs encoding isopentenyl diphosphate isomerase. A cDNA that caused a reduced carotenoid content in E. coli was also identified. Based on DNA sequence analysis, DNA hybridization, and further functional testing, this latter cDNA was found to encode the small subunit of ADP-glucose pyrophosphorylase, a rate-controlling enzyme in starch biosynthesis that has been of interest for enhancing plant starch content.

Molecular characterization of natural Erwinia pyrifoliae strains deficient in hypersensitive response.

From necrotic tissue of a Nashi pear tree, 24 Erwinia pyrifoliae strains, found to be identical by pulsed-field gel electrophoresis analysis, were isolated. Thirteen strains were not virulent on immature pears and did not induce a hypersensitive response in tobacco leaves. The defective gene hrpL was complemented with intact genes from E. pyrifoliae and Erwinia amylovora.

[Properties of mutants of bacteria belonging to the genus Erwinia devoid of common components of the phosphoenolpyruvate-dependent phosphotransferase system]. / Svoistva mutantov bakterii roda Erwinia, lishennykh obshchikh komponentov fosfoenolpiruvatzavisimoi fosfotransferaznoi sistemy.

Biochemical consequences of mutational damage to common components of the Erwinia phosphoenolpyruvate-dependent phosphotransferase system (the HPr protein and enzyme I) were studied. The transport of glucose, mannose, fructose, and mannitol in Erwinia was shown to require a preliminary induction of proteins of the phosphotransferase system. A drastic decrease in the rate of the transport of these carbohydrates was observed in ptsI and ptsH mutants. A disturbance in the common components suppresses the synthesis of inducible enzymes (beta-galactosidase, complexes of pectolyases and cellulases) and renders it resistant to catabolite repression by glucose, but mutants were shown to retain intracellular cAMP content. Erwinia mutants devoid of common components of the system lack phytopathogenic features. The appearance of an intact ptsI allele in the cell completely corrected pleiotropic disturbances in these mutants.

Biological activity of harpin produced by Pantoea stewartii subsp. stewartii.

Pantoea stewartii subsp. stewartii causes Stewart's wilt of sweet corn. A hypersensitive response and pathogenicity (Hrp) secretion system is needed to produce water-soaking and wilting symptoms in corn and to cause a hypersensitive response (HR) in tobacco. Sequencing of the hrp cluster revealed a putative harpin gene, hrpN. The product of this gene was overexpressed in Escherichia coli and shown to elicit the HR in tobacco and systemic resistance in radishes. The protein was designated HrpN(Pnss). Like other harpins, it was heat stable and protease sensitive, although it was three- to fourfold less active biologically than Erwinia amylovora harpin. We used antibodies to purified HrpN(Pnss) to verify that hrpN mutants could not produce harpin. This protein was secreted into the culture supernatant and was produced by strains of P. stewartii subsp. indologenes. In order to determine the importance of HrpN(Pnss) in pathogenesis on sweet corn, three hrpN::Tn5 mutants were compared with the wild-type strain with 50% effective dose, disease severity, response time, and growth rate in planta as parameters. In all tests, HrpN(Pnss) was not required for infection, growth, or virulence in corn or endophytic growth in related grasses.

Genetic organization of the Pantoea stewartii subsp. stewartii hrp gene cluster and sequence analysis of the hrpA, hrpC, hrpN, and wtsE operons.

The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions.

Genetic organization of the hrp gene cluster and dspAE/BF operon in Erwinia herbicola pv. gypsophilae.

Erwinia herbicola pv. gypsophilae induces gall formation in gypsophila that is dependent on the existence of a pathogenicity plasmid (pPATHEhg). We previously demonstrated the presence of several hrp genes on this plasmid. By employing transposon mutagenesis and sequencing, a functional hrp gene cluster on the pPATHEhg has now been characterized completely. The hrp genes of E. herbicola pv. gypsophilae are remarkably similar to and colinear with those of Erwinia amylovora and Pantoea stewartii and generally showed 60 to 90% nucleotide or deduced amino acid identity. E. herbicola pv. gypsophilae, however, lacks hrpW, which is present in E. amylovora. Additionally, E. herbicola pv. gypsophilae mutants deficient in harpin production retained pathogenicity and were slightly reduced in their ability to elicit a hypersensitive response (HR) in tobacco. The "disease specific" region, dspA/EB/F, exhibited 60 to 74% identity with the dspA/EB/F loci of E. amylovora and P. stewartii, respectively. Mutations in dspA/E abolished pathogenicity of E. herbicola pv. gypsophilae but not HR elicitation on tobacco. Inactivation of HrpL reduced plant-induced transcription of dspA/E by three orders, indicating Hrp-dependent regulation.

The association of Western flower thrips, Frankliniella occidentalis, with a near Erwinia species gut bacteria: transient or permanent?

Associations between insects and gut bacteria are ubiquitous. It is possible to make a distinction between permanent associations (called symbiosis), in which the same type of bacteria is present in more than one generation of the insect, and transient associations. Transient bacteria are ingested together with food but do not settle in the insect gut in such a way that they will be passed on to the next generation. In this study, we describe the permanent association between Western flower thrips (Frankliniella occidentalis), a polyphagous insect species that is a major pest worldwide, and one type of gut bacteria. On the basis of direct microscopic observations and counts of bacteria, it was found that thrips from the populations studied contained large numbers of bacteria in their hindgut. Bacteria were isolated from their host and grown on 10 different agar media. The number of bacteria isolated on agar media equaled the number of direct counts. All isolates had the same colony morphology. On the basis of their 16S rDNA sequence these bacteria were identified as Enterobacteriaceae, closely related to Escherichia coli. Isolates tested with API 20E biochemical tests were Erwinia species. This was the only type of bacteria found in all thrips individuals on any of the 10 different agar media. Universal primers, which would potentially pick up DNA from any bacterium present in the insect, were applied on crude DNA extracts from thrips with bacteria. We only found 16S rDNA sequences similar to those of the isolated thrips gut bacteria. The same type of bacteria was present in all life stages of the thrips and was found to persist in the thrips populations for at least 2 years (more than 50 generations).