Erwinia

Can J Microbiol, 2004 Nov, 50(11), 951 - 6
Use of 1,4-naphthoquinones for control of Erwinia carotovora; Medina LF et al.; The antimicrobial effect of 5 naphthoquinones was tested against the phytopathogenic bacteria Erwinia carotovora . Disk diffusion tests and determination of minimal inhibitory concentrations (MIC) indicate that the compound naphthazarin (NTZ) has the best antibacterial activity among the naphthoquinones tested . Studies on the mode of action indicate the effect of NTZ was bactericidal at 10 microg/mL . When cultivation was done in the presence of sodium ascorbate, the restoration of E . carotovora growth was observed with 3 microg/mL NTZ, but not when a 10 microg/mL dose was used . The incubation of NTZ with bacterial suspension of E . carotovora resulted in important changes in the absorption spectra of this naphthoquinone, indicating that a redox reaction takes place . These results may suggest that NTZ induces an increase of reactive oxygen species that are toxic to the cell . The compound NTZ was also effective in preventing E . carotovora growth on potato tubers, inhibiting the soft rot development at a concentration of 2 mg/mL.

J Biotechnol, 2005 Feb 9, 115(3), 303 - 6
Effective production of 3,4-dihydroxyphenyl-l-alanine (l-DOPA) with Erwinia herbicola cells carrying a mutant transcriptional regulator TyrR; Koyanagi T et al.; The enzymatic production of 3,4-dihydroxyphenyl-l-alanine (l-DOPA) using Erwinia herbicola cells involves the action of tyrosine phenol-lyase (Tpl, EC 4.1.99.2) . Since Tpl is only synthesized under l-tyrosine-induced conditions, the addition of l-tyrosine to the medium is unavoidable when preparing cells (the enzyme source), but severely impedes the pure preparation of the final product l-DOPA . We circumvented this problem by using recombinant E . herbicola cells carrying a mutant transcriptional regulator TyrR, which is capable of activating the tpl promoter in the absence of l-tyrosine.

J Biol Chem . 2005 Jan 5; {Epub ahead of print}
Altering substrate chain length specificity of an acylhomoserinelactone synthase in bacterial communication; Brader G et al.; Quorum sensing mediated by specific signal compounds (autoinducers) allows bacteria to monitor their cell density and enables a synchronized regulation of target gene sets . The best-studied group of autoinducers are the acyl-homoserine lactones (AHSL) that are central to regulation of virulence in many plant and animal pathogens . Variation of the acyl-side chain of the AHSLs underlies the observed species specificity of this communication system . Here we show that even different strains of the plant pathogen Erwinia carotovora employ different dialects of this language and demonstrate the molecular basis for the acyl-chain length specificity of distinct AHSL synthases . Under physiological concentrations only the cognate AHSL with the "right" acyl-chain is recognized as a signal that will switch on virulence genes . Mutagenesis of the AHSL synthase gene expI(SCC1) identified the changes Met127Thr and Phe69Leu as sufficient to effectively alter ExpI(SCC1) (an N-3-oxohexanoyl-L-homoserine lactone producer) substrate specificity to that of an N-3-oxooctanoyl-L-homoserine lactone producer . Our data identifies critical residues that define the size of the substrate-binding pocket of the AHSL synthase and will help to understand and manipulate this bacterial language.

Ann Agric Environ Med, 2004, 11(2), 329 - 33
A farmer's occupational airborne contact dermatitis masqueraded by coexisting rosacea: delayed diagnosis and legal acknowledgement; Spiewak R et al.; A rare case of coexistence of occupational airborne dermatitis with rosacea is presented in a 41-year-old female farmer . Her first dermatitis symptoms appeared at the age of 10 when she started helping her parents on the farm . Uncovered skin areas of the face, neck, decollete, forearms and the hands gradually became involved . The dermatitis symptoms were provoked by agricultural dusts (especially of flax and dried herbs) . For the subsequent 30 years, the work-related disease remained undiagnosed due to the lack of pre-employment and periodical health check in agriculture . She also suffered from protein contact dermatitis of the hands from cow epithelium . About 20 years after the onset of airborne dermatitis, rosacea developed, possibly secondary to the prolonged treatment . Diagnostic tests carried out at our department confirmed hypersensitivity to occupational allergens: type I allergy to storage mites, moulds, and cow epithelium . A cutaneous late-phase reaction on prick tests and serum precipitins to the bacterium Pantoea agglomerans (Erwinia herbicola) also were found . Among non-occupational hypersensitivities, type I allergy to house dust mites and contact allergy to methylchloroisothiazolinone/methylisothiazolinone (Kathon CG) was found . In connection with these results, the significance of agricultural dusts in farmers' airborne dermatitis is discussed . Also presented are the problems with obtaining acceptance from the State Sanitary Authority for qualification of this case as an occupational disease, which was due to the coexistence of the non-occupational rosacea . Discussed is also the problem of pre-employment exposure to occupational allergens among farmers' children, and the difficulties with delivering occupational health services to self-employed farmers.

J Ethnopharmacol, 2005 Jan 15, 96(3), 461 - 9 Epub 2004 Nov 11.
Anti-fungal and anti-bacterial activity of some herbal remedies from Tanzania; de Boer HJ et al.; Plants are not only important to the millions of people to whom traditional medicine serves as the only opportunity for health care and to those who use plants for various purposes in their daily lives, but also as a source of new pharmaceuticals . During interviews with the Pare people from Northeastern Tanzania, 29 plants that are used for medicinal purposes as well as 41 plants used for non-medicinal purposes were reported . Six medicinally used plants were selected for bioactivity analysis . Extracts of Coccinia adoensis, Cineraria grandiflora, Pavonia urens, Marattia fraxinea, Clutia abyssinica var . usambarica, and Vangueria infausta were made using ethyl acetate, methanol, cold water and boiling water . The antimicrobial activity was tested on Candida albicans, Aspergillus fumigatus, Fusarium culmorum, Staphylococcus aureus, Pseudomonas syringae, and Erwinia amylovora . All plants showed activity against several test organisms.

Planta . 2004 Dec 15; {Epub ahead of print}
The ABI2-dependent abscisic acid signalling controls HrpN-induced drought tolerance in Arabidopsis; Dong HP et al.; HrpN, a protein produced by the plant pathogenic bacterium Erwinia amylovora, has been shown to stimulate plant growth and resistance to pathogens and insects . Here we report that HrpN activates abscisic acid (ABA) signalling to induce drought tolerance (DT) in Arabidopsis thaliana L . plants grown with water stress . Spraying wild-type plants with HrpN-promoted stomatal closure decreased leaf transpiration rate, increased moisture and proline levels in leaves, and alleviated extents of damage to cell membranes and plant drought symptoms caused by water deficiency . In plants treated with HrpN, ABA levels increased; expression of several ABA-signalling regulatory genes and the important effector gene rd29B was induced or enhanced . Induced expression of rd29B, promotion of stomatal closure, and reduction in drought severity were observed in the abi1-1 mutant, which has a defect in the phosphatase ABI1, after HrpN was applied . In contrast, HrpN failed to induce these responses in the abi2-1 mutant, which is impaired in the phosphatase ABI2 . Inhibiting wild-type plants to synthesize ABA eliminated the role of HrpN in promoting stomatal closure and reducing drought severity . Moreover, resistance to Pseudomonas syringae developed in abi2-1 as in wild-type plants following treatment with HrpN . Thus, an ABI2-dependent ABA signalling pathway is responsible for the induction of DT but does not affect pathogen defence under the circumstances of this study.

Plant Cell, 2005 Jan, 17(1), 282 - 94 Epub 2004 Dec 14.
Chlorophyllase 1, a Damage Control Enzyme, Affects the Balance between Defense Pathways in Plants; Kariola T et al.; Accumulation of reactive oxygen species (ROS) is central to plant response to several pathogens . One of the sources of ROS is the chloroplast because of the photoactive nature of the chlorophylls . Chlorophyllase 1 (encoded by AtCLH1) of Arabidopsis thaliana is quickly induced after tissue damage (e.g., caused by the bacterial necrotroph Erwinia carotovora or the necrotrophic fungus Alternaria brassicicola) . RNA interference silencing of AtCLH1 resulted in failure to degrade free chlorophyll after tissue damage and in resistance to E . carotovora . Both inoculation with E . carotovora and exposure to high light caused elevated accumulation of hydrogen peroxide in AtCLH1 silenced plants . This was accompanied by expression of marker genes for systemic acquired resistance and induction of antioxidant defenses . Interestingly, downregulation of AtCLH1 resulted in increased susceptibility to A . brassicicola, resistance to which requires jasmonate signaling . We propose that AtCLH1 is involved in plant damage control and can modulate the balance between different plant defense pathways.

FEMS Microbiol Lett, 2004 Dec 15, 241(2), 179 - 83
The plant pathogen Erwinia amylovora produces acyl-homoserine lactone signal molecules in vitro and in planta; Venturi V et al.; We report for the first time the production of acyl homoserine lactones (AHLs) by Erwina amylovora, an important quarantine bacterial pathogen that causes fire blight in plants . E . amylovora produces one N-acyl homoserine lactone {a N-(3-oxo-hexanoyl)-homoserine lactone or a N-(3-hydroxy-hexanoyl)-homoserine lactone} quorum sensing signal molecule both in vitro and in planta (pear plant) . Given the involvement of AHLs in plant pathogenesis, we speculate that AHL-dependent quorum sensing could play an important role in the regulation of E . amylovora virulence.

Mol Plant Microbe Interact, 2004 Dec, 17(12), 1366 - 75
Identification and characterization of Nip, necrosis-inducing virulence protein of Erwinia carotovora subsp . carotovora; Mattinen L et al.; Erwinia carotovora subsp . carotovora is a gram-negative bacterium that causes soft rot disease of many cultivated crops . When a collection of E . carotovora subsp . carotovora isolates was analyzed on a Southern blot using the harpin-encoding gene hrpN as probe, several harpinless isolates were found . Regulation of virulence determinants in one of these, strain SCC3193, has been characterized extensively . It is fully virulent on potato and in Arabidopsis thaliana . An RpoS (SigmaS) mutant of SCC3193, producing elevated levels of secreted proteins, was found to cause lesions resembling the hypersensitive response when infiltrated into tobacco leaf tissue . This phenotype was evident only when bacterial cells had been cultivated on solid minimal medium at low pH and temperature . The protein causing'the cell death was purified and sequenced, and the corresponding gene was cloned . The deduced sequence of the necrosis-inducing protein (Nip) showed homology to necrosis- and ethylene-inducing elicitors of fungi and oomycetes . A mutant strain of E . carotovora subsp . carotovora lacking the nip gene showed reduced virulence in potato tuber assay but was unaffected in virulence in potato stem or on other tested host plants.

Appl Environ Microbiol, 2004 Dec, 70(12), 7539 - 44
Nucleotide sequences, genetic organization, and distribution of pEU30 and pEL60 from Erwinia amylovora; Foster GC et al.; The nucleotide sequences, genetic organization, and distribution of plasmids pEU30 (30,314 bp) and pEL60 (60,145 bp) from the plant pathogen Erwinia amylovora are described . The newly characterized pEU30 and pEL60 plasmids inhabited strains isolated in the western United States and Lebanon, respectively . The gene content of pEU30 resembled plasmids found in plant-associated bacteria, while that of pEL60 was most similar to IncL/M plasmids inhabiting enteric bacteria.

Mol Biol Rep, 2004 Sep, 31(3), 143 - 9
Cloning, sequencing and partial characterisation of sorbitol transporter (srlT) gene encoding phosphotransferase system, glucitol/sorbitol-specific IIBC components of Erwinia herbicola ATCC 21998; Qazi PH et al.; A DNA fragment of approximately 1500 bp, harbouring the sorbitol transport gene (srlT), was amplified from the chromosomal DNA of Erwinia herbicola ATCC 21998 by PCR and cloned in Escherichia coli JM109 . Degenerate oligonucleotide primers used were designed based on the conserved regions in the gene sequences within the gut operon of E . coli (Gene Bank accession no . J02708) and the srl operon of Erwinia amylovora (Gene Bank accession no . Y14603) . The cloned DNA fragment was sequenced and found to contain an open reading frame of 1473 nucleotides coding for a protein of 491 amino acids, corresponding to a mass of 52410 Da . The nucleotide sequence of this ORF was highly homologous to that of the gutA gene of Escherichia coli gut operon, the srlE gene of Shigella flexrni and the sorbitol transporter gene sequence of Escherichia coli K12 (Gene Bank accession nos . J02708, AE016987 and D90892 respectively) . The protein sequence showed significant homology to that of the phosphotransferase system i.e . the glucitol/sorbitol-specific IIBC components of Escherichia coli and Erwinia amylovora (P56580, O32522) . The cloned DNA fragment was introduced into a pRA90 vector and the recombinant was used for developing srlT mutants of Erwinia herbicola, by homologous recombination . Mutants obtained were unable to grow on minimal medium with sorbitol . The insertion of the pRA90 vector inside the srlT gene sequence of the mutants was confirmed by DNA-DNA hybridisation.

Genetika, 2004 Sep, 40(9), 1194 - 9
{Genetic regulation of pathogenicity and virulence factors in bacteria Erwinia carotovora subsp . atroseptica: phenotypic characteristic of bacteria mutant for the kduD gene}
{Genetic regulation of pathogenicity and virulence factors in bacteria Erwinia carotovora subsp . atroseptica: identification of gene kduD}
{No authors listed}

A mutant that cannot utilize pectin substances of plant cell walls was obtained via insertion of mini-mini-Tn5xylE transposon into the chromosome of phytopathogenic bacteria Erwinia carotovora subsp . atroseptica . The inability of mutant cells to utilize these substrates was caused by a failure to accomplish the catabolism of unsaturated digalacturonic acid (UDA) . Study of enzymatic activities has established that mutant bacteria lost the ability to produce 2,5-diketo-3-deoxygluconate dehydrogenase, an enzyme of intracellular UDA utilization . Molecular cloning of the mutant gene was conducted, and its nucleotide sequence was determined . It was shown that the nucleotide sequence of this gene had an 82% homology with the sequence of Erwinia chrysanthemi EC3937 kduD gene encoding 2,5-diketo-3-deoxygluconate dehydrogenase . The intergene kdul-kduD region in bacteria Erwinia carotovora subsp . atroseptica is shorter in length by 98 nucleotides than the corresponding region of Erwinia chrysanthemi and does not contain promoter sequences . The kduD gene was located at 126.8 min of the Erwinia carotovora subsp . atroseptica genetic map.

Mol Plant Microbe Interact, 2004 Nov, 17(11), 1269 - 78
Involvement of N-acylhomoserine lactones throughout plant infection by Erwinia carotovora subsp . atroseptica (Pectobacterium atrosepticum); Smadja B et al.; Erwinia carotovora subsp . atroseptica is responsible for potato blackleg disease in the field and tuber soft rot during crop storage . The process leading to the disease occurs in two phases: a primary invasion step followed by a maceration step . Bacteria-to-bacteria communication is associated with a quorum-sensing (QS) process based on the production of N-acylhomoserine lactones (HSL) . The role of HSL throughout plant infection was analyzed . To this purpose, HSL produced by a specific E . carotovora subsp . atroseptica wild-type strain, which was particularly virulent on potato, were identified . A derivative of this strain that expressed an HSL lactonase gene and produced low amounts of HSL was generated . The comparison of these strains allowed the evaluation of the role of HSL and QS in disease establishment and development . Bacterial growth and motility; activity of proteins secreted by type I, II, and III systems; and hypersensitive and maceration reactions were evaluated . Results indicated that HSL production and QS regulate only those traits involved in the second stage of the host plant infection (i.e., tissue maceration) and hypersensitive response in nonhost tobacco plants . Therefore, the use of QS quenching strategies for biological control in E . carotovora subsp . atroseptica cannot prevent initial infection and multiplication of this pathogen.

BMC Plant Biol . 2004 Nov 18;4(1):18.
Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohlii Briq; Engprasert S et al.; BACKGROUND: Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway . Geranylgeranyl diphosphate (GGPP) synthase is an important branch point enzyme in terpenoid biosynthesis . Therefore, GGPP synthase is thought to be a key enzyme in biosynthesis of forskolin . Herein we report the first confirmation of the GGPP synthase gene in Coleus forskohlii Briq . RESULTS: The open reading frame for full-length GGPP synthase encodes a protein of 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 kDa . Alignments of C . forskohlii GGPP synthase amino acid sequences revealed high homologies with other plant GGPP synthases . Several highly conserved regions, including two aspartate-rich motifs were identified . Transient expression of the N-terminal region of C . forskohlii GGPP synthase-GFP fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast . Carotenoid production was observed in Escherichia coli harboring pACCAR25DeltacrtE from Erwinia uredovora and plasmid carrying C . forskohlii GGPP synthase . These results suggested that cDNA encoded functional GGPP synthase . Furthermore, C . forskohlii GGPP synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots . CONCLUSION: This investigation proposed that forskolin was synthesised via a non-mevalonate pathway . GGPP synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots.

J Exp Bot, 2005 Jan, 56(409), 81 - 89 Epub 2004 Nov 8.
Metabolic engineering of high carotenoid potato tubers containing enhanced levels of {beta}-carotene and lutein; Ducreux LJ et al.; In order to enhance the carotenoid content of potato tubers, transgenic potato plants have been produced expressing an Erwinia uredovora crtB gene encoding phytoene synthase, specifically in the tuber of Solanum tuberosum L . cultivar Desiree which normally produces tubers containing c . 5.6 mug carotenoid g(-1) DW and also in Solanum phureja L . cv . Mayan Gold which has a tuber carotenoid content of typically 20 mug carotenoid g(-1) DW . In developing tubers of transgenic crtB Desiree lines, carotenoid levels reached 35 mug carotenoid g(-1) DW and the balance of carotenoids changed radically compared with controls: beta-carotene levels in the transgenic tubers reached c . 11 mug g(-1) DW, whereas control tubers contained negligible amounts and lutein accumulated to a level 19-fold higher than empty-vector transformed controls . The crtB gene was also transformed into S . phureja (cv . Mayan Gold), again resulting in an increase in total carotenoid content to 78 mug carotenoid g(-1) DW in the most affected transgenic line . In these tubers, the major carotenoids were violaxanthin, lutein, antheraxanthin, and beta-carotene . No increases in expression levels of the major carotenoid biosynthetic genes could be detected in the transgenic tubers, despite the large increase in carotenoid accumulation . Microarray analysis was used to identify a number of genes that were consistently up- or down-regulated in transgenic crtB tubers compared with empty vector controls . The implications of these data from a nutritional standpoint and for further modifications of tuber carotenoid content are discussed.

Appl Environ Microbiol, 2004 Nov, 70(11), 6800 - 8
Ultrastructural alterations of Erwinia carotovora subsp . atroseptica caused by treatment with aluminum chloride and sodium metabisulfite; Yaganza ES et al.; Aluminum and bisulfite salts inhibit the growth of several fungi and bacteria, and their application effectively controls potato soft rot caused by Erwinia carotovora . In an effort to understand their inhibitory action, ultrastructural changes in Erwinia carotovora subsp . atroseptica after exposure (0 to 20 min) to different concentrations (0.05, 0.1, and 0.2 M) of these salts were examined by using transmission electron microscopy . Plasma membrane integrity was evaluated by using the SYTOX Green fluorochrome that penetrates only cells with altered membranes . Bacteria exposed to all aluminum chloride concentrations, especially 0.2 M, exhibited loosening of the cell walls, cell wall rupture, cytoplasmic aggregation, and an absence of extracellular vesicles . Sodium metabisulfite caused mainly a retraction of plasma membrane and cellular voids which were more pronounced with increasing concentration . Bacterial mortality was closely associated with SYTOX stain absorption when bacteria were exposed to either a high concentration (0.2 M) of aluminum chloride or prolonged exposure (20 min) to 0.05 M aluminum chloride or to a pH of 2.5 . Bacteria exposed to lower concentrations of aluminum chloride (0.05 and 0.1 M) for 10 min or less, or to metabisulfite at all concentrations, did not exhibit significant stain absorption, suggesting that no membrane damage occurred or it was too weak to allow the penetration of the stain into the cell . While mortality caused by aluminum chloride involves membrane damage and subsequent cytoplasmic aggregation, sulfite exerts its effect intracellularly; it is transported across the membrane by free diffusion of molecular SO2 with little damage to the cellular membrane.

Proc R Soc Lond B Biol Sci, 2004 Oct 22, 271(1553), 2171 - 8
Diet-dependent effects of gut bacteria on their insect host: the symbiosis of Erwinia sp . and western flower thrips; de Vries EJ et al.; Studies on bacteria in the gut of insect species are numerous, but their focus is hardly ever on the impact on host performance . We showed earlier that Erwinia bacteria occur in the gut of western flower thrips, most probably acquired during feeding . Here, we investigate whether thrips gain a net benefit or pay a net cost because of these gut bacteria . On a diet of cucumber leaves, the time to maturity is shorter and the oviposition rate is higher in thrips with bacteria than in thrips without (aposymbionts) . When fed on cucumber leaves and pollen, aposymbionts develop faster and lay more eggs . So Erwinia bacteria benefit or parasitize their thrips hosts depending on the diet, which is in accordance with theoretical predictions for fitness of organisms engaged in symbiotic interactions . Possibly, the transmission of gut bacteria has not become strictly vertical because of this diet-dependent fitness variability.

Mikrobiol Z, 2004 May-Jun, 66(3), 22 - 32
{Multiple change of the phenotype, conjugated with the loss of yellow pigmentation of Erwinia herbicola}; Genome-wide identification of plant-upregulated genes of Erwinia chrysanthemi 3937 using a GFP-based IVET leaf array; Department of Plant Pathology, University of California, Riverside 92521, USAA green fluorescent protein-based in vivo expression technology leaf array was used to identify genes in Erwinia chrysanthemi 3937 that were specifically upregulated in plants compared with growth in a laboratory culture medium . Of 10,000 E . chrysanthemi 3937 clones, 61 were confirmed as plant upregulated . On the basis of sequence similarity, these were recognized with probable functions in metabolism (20%), information transfer (15%), regulation (11%), transport (11%), cell processes (11%), and transposases (2%); the function for the remainder (30%) is unknown . Upregulated genes included transcriptional regulators, iron uptake systems, chemotaxis components, transporters, stress response genes, and several already known or new putative virulence factors . Ten independent mutants were constructed by insertions in these plant-upregulated genes and flanking genes . Two different virulence assays, local leaf maceration and systemic invasion in African violet, were used to evaluate these mutants . Among these, mutants of a purM homolog from Escherichia coli (purM::Tn5), and hrpB, hrcJ, and a hrpD homologs from the Erwinia carotovorum hrpA operon (hrpB::Tn5, hrcJ::Tn5, and hrpD::Tn5) exhibited reduced abilities to produce local and systemic maceration of the plant host . Mutants of rhiT from E . chrysanthemi (rhiT::Tn5), and an eutR homolog from Salmonella typhimurium (eutR::TnS) showed decreased ability to cause systemic inva sion on African violet . However, compared with the wild-type E . chrysanthemi 3937, these mutants exhibited no significant differences in local leaf maceration . The pheno type of hrpB::Tn5, hrcC::Tn5, and hrpD::Tn5 mutants further confirmed our previous findings that hrp genes are crucial virulence determinants in E . chrysanthemi 3937.

Mol Plant Microbe Interact, 2004 Sep, 17(9), 943 - 50
Use of a pooled transposon mutation grid to demonstrate roles in disease development for Erwinia carotovora subsp . atroseptica putative type III secreted effector (DspE/A) and helper (HrpN) proteins; Holeva MC et al.; Soft rot Erwinia spp., like other closely related plant pathogens, possess a type III secretion system (TTSS) (encoded by the hrp gene cluster) implicated in disease development . We report the sequence of the entire hrp gene cluster and adjacent dsp genes in Erwinia carotovora subsp . atroseptica SCRI1039 . The cluster is similar in content and structural organization to that in E . amylovora . However, eight putative genes of unknown function located within the E . carotovora subsp . atroseptica cluster do not have homologues in the E . amylovora cluster . An arrayed set of Tn5 insertional mutants (mutation grid) was constructed and pooled to allow rapid isolation of mutants for any given gene by polymerase chain reaction screening . This novel approach was used to obtain mutations in two structural genes (hrcC and hrcV), the effector gene dspE/A, and the helper gene hrpN . An improved pathogenicity assay revealed that these mutations led to significantly reduced virulence, showing that both the putative E . carotovora subsp . atroseptica TTSS-delivered effector and helper proteins are required for potato infection.

Proteomics, 2004 Oct, 4(10), 3177 - 86
The secretome of the plant pathogenic bacterium Erwinia chrysanthemi; Kazemi-Pour N et al.; Erwinia chrysanthemi causes soft-rot diseases of many plants by secreting a battery of enzymes which degrade the plant cell walls . We initiated a proteomic analysis to create a reference map of the E . chrysanthemi secretome . Extracellular proteins were isolated from E . chrysanthemi culture supernatants and resolved by two-dimensional electrophoresis . By analysis of mutants, Western blotting, and matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) 55 spots representing 25 unique proteins were identified . In uninduced conditions, we identified spots corresponding to the cellulase Cel5, the proteases PrtA, PrtB, and PrtC, the flagellin FliC, and some intracellular proteins whose presence probably resulted from spontaneous cell lysis . We identified another secreted protein, AvrL, homologous to an avirulence protein of Xanthomonas campestris . After culture in conditions inducing pectinase production, i.e., in the presence of galacturonate and plant extract, we identified spots corresponding to the endopectate lyases PelA, PelB, PelC, PelD, PelE, PelI, PelL, and PelZ, the pectin acetylesterases PaeX and PaeY, the pectin methylesterase PemA, and the polygalacturonase PehX . In the presence of other inducing compounds, we detected the rhamnogalacturonate lyase RhiE and the esterase FaeD . Analysis of mutants, altered for one type of secretion system, was performed to determine the targets of each system . The type I system Prt was necessary for the secretion of three proteases . No proteins secreted by the type III Hrp system could be detected in E . chrysanthemi supernatants . In addition to the already known substrates (eleven pectinases and one cellulase), this analysis revealed that the type II Out system mediates secretion of the esterase FaeD and of the Avr-like protein AvrL.

Plant Cell Rep . 2004 Sep 16; {Epub ahead of print}
Expression of viral EPS-depolymerase reduces fire blight susceptibility in transgenic pear; Malnoy M et al.; Erwinia amylovora is the causal agent of fire blight of Maloideae . One of the main pathogenicity factors of this bacterium is the exopolysaccharide (EPS) of its capsule . In this paper, we used genetic transformation tools to constitutively express an EPS-depolymerase transgene in the pear ( Pyrus communis L.) cv . Passe Crassane with the aim of decreasing its high susceptibility to fire blight . Expression of the depolymerase gene in 15 independent transgenic clones led, on average, to low depolymerase activity, although relatively high expression was observed at the transcriptional and translational levels . Only two of the transgenic clones (9X and 10M) consistently showed a decrease in fire blight susceptibility in vitro and in the greenhouse . These clones were also among the highest expressers of depolymerase at the RNA and enzyme activity levels . The correlation observed among all transgenic clones between depolymerase expression and fire blight resistance suggested the potential of this strategy.

Mol Cell Probes, 2004 Oct, 18(5), 341 - 8
Genomic DNA detection using cycling probe technology and capillary gel electrophoresis with laser-induced fluorescence; Dickinson Laing T et al.; Cycling probe technology (CPT) is an isothermal DNA analysis method that has been shown to be useful for identifying genetic markers of antibiotic-resistant bacteria in clinical settings . CPT assays have previously employed several assay methods that include polyacrylamide gel electrophoresis and magnetic beads for separations and radioisotopic and colorimetric detection for detection . Capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) is an alternative separation and detection method that offers attributes such as low sample consumption, short analysis times, no radiation hazards and potential for high throughput . We report on the development of a CGE-LIF CPT assay for genomic DNA from Erwinia herbicola and the comparison of this assay with a conventional 32p radioisotopic PAGE CPT assay.Separation and detection of intact and cleaved fluorescein-labeled CPT probe molecules by CGE-LIF was achieved in under 4 min through a gel-filled capillary (7 cm separation length to detector) . Total time, from setup to detection, was about 1 h for the complete assay versus several hours (3-12 h) for the radioisotopic PAGE CPT assay . Similar detection limits of 10(5)-10(6) copies of genomic target DNA were observed with each assay method . This study demonstrated that CGE-LIF CPT is a suitable analysis method for the detection of genomic DNA sequences.

Shi Yan Sheng Wu Xue Bao, 2002 Dec, 35(4), 324 - 8
{Isolation of endophytic bacteria in potato and test of antagonistic action to bacterial ring rot of potato}; Cui L et al.; In this study, two hundred and forty bacterial strains were isolated from inner tissue of potato tubers collected from DaTong, TaiYuan and Inner Mongolia Autonomous regions . On the basis of antagonistic examination in vitro, fifty and five bacteria strains were characterized for antagonistic bacteria to ring rot of potato . It was 22.9 percentage of all bacteria strains . The biggest radius of suppression circle was 13 mm . Nine strains were chosen for their suppression of bacterial ring rot, blackleg and dry rot of potato . These strains were bacteriologically ideatified . Strain 118 was Pseudomonas fluorescens biovar V . Strain 110 was Bacillus pumilus . Strain 085 was Bacillus stearothermophilus . Strain 069 was Erwinia herbicola . Strain 043 was Xanthomomas fragariae . Strain 116 was Curtobacterium . Strains A-10' and T3 were Bacillus . Strain H1-6 was Pseudomonas fluorescens.

J Bacteriol, 2004 Sep, 186(18), 6239 - 47
Mutational analysis of Xanthomonas harpin HpaG identifies a key functional region that elicits the hypersensitive response in nonhost plants; Kim JG et al.; 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.

Planta, 2004 Nov, 220(1), 165 - 71 Epub 2004 Nov.
Plant defense gene promoter enhances the reliability of shiva-1 gene-induced resistance to soft rot disease in potato; Yi JY et al.; PAL5, a tomato (Lycopersicon esculentum Mill.) plant defense gene that encodes phenylalanine ammonia-lyase, is known to respond to a variety of environmental stresses including pathogen infection and wounding . A shiva-1 gene recombinant that encodes a small synthetic antibacterial peptide under the PAL5 gene promoter was transformed into potato (Solanum tuberosum L.) and its ability to induce resistance to Erwinia carotovora was compared with a construct under the control of the constitutive and widely used cauliflower mosaic virus (CaMV) 35S promoter . The shiva-1 peptide, an analog of natural cecropin B, was shown previously to have high bactericidal activity in vitro, but when expressed in vivo under the control of the CaMV 35S promoter, the effects were very inconsistent . As observed previously, in the present studies a few transformants with the CaMV 35S promoter were highly resistant when assayed for susceptibility to soft rot disease . In marked contrast the majority of transformants with the PAL5 gene promoter were highly resistant . More-detailed analyses of the incorporated DNA indicated that most of the transformants with the CaMV 35S promoter contained multiple copies of the transforming DNA while all of the PAL5 recombinants contained single copies . The highly resistant CaMV 35S recombinant also was present as a single copy . The results indicate that, at least in this instance, a constitutive promoter may not be ideal for the effective expression of a foreign gene and suggest that multiple insertions may have negative consequences.

Mol Plant Microbe Interact, 2004 Aug, 17(8), 880 - 7
Potato plants genetically modified to produce N-acylhomoserine lactones increase susceptibility to soft rot erwiniae; Toth IK et al.; Many gram-negative bacteria employ N-acylhomoserine lactones (AHL) to regulate diverse physiological processes in concert with cell population density (quorum sensing {QS}) . In the plant pathogen Erwinia carotovora, the AHL synthesized via the carI/expI genes are responsible for regulating the production of secreted plant cell wall-degrading exoenzymes and the antibiotic carbapen-3-em carboxylic acid . We have previously shown that targeting the product of an AHL synthase gene (yenI) from Yersinia enterocolitica to the chloroplasts of transgenic tobacco plants caused the synthesis in planta of the cognate AHL signaling molecules N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL), which in turn, were able to complement a carI-QS mutant . In the present study, we demonstrate that transgenic potato plants containing the yenI gene are also able to express AHL and that the presence and level of these AHL in the plant increases susceptibility to infection by E . carotovora . Susceptibility is further affected by both the bacterial level and the plant tissue under investigation.

Appl Biochem Biotechnol, 2004 Jul-Sep, 118(1-3), 115 - 32
Characterization of metabolic pathway of linoleic acid 9-hydroperoxide in cytosolic fraction of potato tubers and identification of reaction products; Kimura H et al.; Potato tubers are shown to contain a unique lipoxygenase pathway to form 9-hydroperoxy-10,12-octadecadienoic acid (9-HPODE) from linoleic acid . Here, we report the metabolic pathway of 9-HPODE in the cytosolic fraction and the characterization of enzymes involved in the conversion of metabolites . The analysis of enzymatic reaction products at pH 5.5 revealed the formation of 9-keto-10,12-octadecadienoic acid, 9-hydroxy-10,12-octadecadienoic acid, 9,10-epoxy-11-hydroxy-12-octadecenoic acid, 9,10,13-trihydroxy-11-octadecenoic acid, and 9,12,13-trihydroxy-10-octadecenoic acid . The cytosolic enzymes were separated by anion-exchange chromatography into two fractions E1 and E2, having molecular masses of 66 and 54 kDa, respectively . The enzyme fraction E1 only produced 9-keto-10,12-octadecadienoic acid, whereas E2 formed other products . The enzyme E1 showed higher reactivity with 13- and 9-hydroperoxide of alpha-linolenic acid than 9-HPODE, but no reaction with hydroxy fatty acids . In contrast, the enzyme E2 showed the highest reactivity with 9-HPODE, followed by hydroperoxides of alpha-linolenic acid and arachidonic acid . We also evaluated the antibacterial activity of hydroxy fatty acids against Erwinia carotovora T-29, a bacterium infecting potato tubers . Growth of the bacteria was suppressed more potently with 9- or 13-hydroxy fatty acids than dihydroxy or trihydroxy fatty acids, suggesting a role for the metabolites in the resistance of bacterial infection.

Acta Crystallogr D Biol Crystallogr, 1997 Mar, 53(Pt 2), 197 - 9
Crystallization of cytochrome b(562) from Erwinia chrysanthemi; Wilkinson KW; Cytochrome b(562) from Erwinia chrysanthemi has been crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate as the precipitant . X-ray precession photographs show that the crystals formed belong to either of the enantiomorphic space groups P4(1)2(1)2 or P4(3)2(1)2 with the cell parameters a = b = 98.6 and c = 62.7 A . Estimation of the crystal density and consideration of the possible values for V(m) indicate that there is either a dimer or trimer in the asymmetric unit . Experiments using the synchrotron radiation source at the CCLRC Daresbury Laboratory have shown that the crystals diffract to at least 2.7 A resolution . An analysis of the N-terminal sequence indicates that this cytochrome shows limited homology to the cytochrome b(562) from E . coli . Determination of the structure will therefore allow analysis of the relationship between these two proteins.

Acta Crystallogr D Biol Crystallogr, 1997 May, 53(Pt 3), 256 - 61
Crystallization of Xylanase from Erwinia chrysanthemi: Influence of Heat and Polymeric Substrate; Barba De La Rosa AP; Xylanase from the bacterial plant pathogen Erwinia chrysanthemi (E.C . 3.2.1.8), expressed in E . coli, has been crystallized for X-ray diffraction analysis both in the presence and the absence of its polymeric substrate 4-O-methyl glucuronoxylan . In all cases it was found that the quality, time of appearance, and reproducibility of both the native and complex crystals were significantly enhanced by heating of the protein to 323 K prior to dispensing the crystallization trials . Crystals of the native protein are ideal for X-ray diffraction analysis, producing Bragg reflections beyond 1.5 A resolution with virtually no degradation with time . The native crystals are in space group P2(1), with a = 39.33, b = 49.46, c = 90.85 A and beta = 101.58 degrees . Other polymorphs have also been obtained and their cell parameters determined . Crystallization of the enzyme in the presence of polymeric substrate yields two distinctly different crystals at different concentrations of the xylan . These are thought to be complexes of the protein with stable products of the enzymatic reaction . A similar result had been obtained previously with pancreatic alpha-amylase and its substrate glycogen.

Acta Crystallogr D Biol Crystallogr, 1997 Sep, 53(Pt 5), 612 - 4
Crystallization and preliminary X-ray crystallographic analysis of shikimate kinase from Erwinia chrysanthemi; Krell T; Shikimate kinase from Erwinia chrysanthemi, overexpressed in Escherichia coli has been crystallized by the vapour-diffusion method using sodium chloride as a precipitant . Mass spectrometry was used to confirm the purity of the shikimate kinase and dynamic light scattering was used to assess conditions for the monodispersity of the enzyme . The crystals are tetragonal, space group P4(1)2(1)2 or enantiomorph with cell dimensions a = b = 108.5 and c = 92.8 A (at 100 K) . Native crystals diffract to better than 2.6 A on a synchrotron X-ray source . The asymmetric unit is likely to contain two molecules, corresponding to a packing density of 3.6 A(3) Da(-1).

J Bacteriol, 2004 Aug, 186(16), 5547 - 50
Erwinia chrysanthemi O antigen is required for betaine osmoprotection in high-salt media; Touze T et al.; Cellular components necessary for osmoprotection are poorly known . In this study we show that O antigen is specifically required for the effectiveness of betaines as osmoprotectants for Erwinia chrysanthemi in saline media . The phenotype is correlated with the inability of rfb mutant strains to maintain a high accumulation level of betaines in hypersaline media.

Microbiology, 2004 Aug, 150(Pt 8), 2707 - 14
Expression of bacteriophage phiEa1h lysozyme in Escherichia coli and its activity in growth inhibition of Erwinia amylovora; Kim WS et al.; A 3.3 kb fragment from Erwinia amylovora phage Ea1h in plasmid pJH94 was previously characterized and found to contain an exopolysaccharide depolymerase (dpo) gene and two additional ORFs encoding 178 and 119 amino acids . ORF178 (lyz) and ORF119 (hol) were found to overlap by 19 bp and they resembled genes encoding lysozymes and holins . In nucleotide sequence alignments, lyz had structurally conserved regions with residues important for lysozyme function . The lyz gene was cloned into an expression vector and expressed in Escherichia coli . Active lysozyme was detected only when E . coli cells with the lyz gene and a kanamycin-resistance cassette were grown in the presence of kanamycin . Growth of Erw . amylovora was inhibited after addition of enzyme exceeding a threshold for lysozyme to target cells . When immature pears were soaked in lysates of induced cells, symptoms such as ooze formation and necrosis were retarded or inhibited after inoculation with Erw . amylovora.

Biochem J, 2004 Dec 1, 384(Pt 2), 247 - 53
Kinetic and structural optimization to catalysis at low temperatures in a psychrophilic cellulase from the Antarctic bacterium Pseudoalteromonas haloplanktis; Garsoux G et al.; The cold-adapted cellulase CelG has been purified from the culture supernatant of the Antarctic bacterium Pseudoalteromonas haloplanktis and the gene coding for this enzyme has been cloned, sequenced and expressed in Escherichia coli . This cellulase is composed of three structurally and functionally distinct regions: an N-terminal catalytic domain belonging to glycosidase family 5 and a C-terminal cellulose-binding domain belonging to carbohydrate-binding module family 5 . The linker of 107 residues connecting both domains is one of the longest found in cellulases, and optimizes substrate accessibility to the catalytic domain by drastically increasing the surface of cellulose available to a bound enzyme molecule . The psychrophilic enzyme is closely related to the cellulase Cel5 from Erwinia chrysanthemi . Both kcat and kcat/K(m) values at 4 degrees C for the psychrophilic cellulase are similar to the values for Cel5 at 30-35 degrees C, suggesting temperature adaptation of the kinetic parameters . The thermodynamic parameters of activation of CelG suggest a heat-labile, relatively disordered active site with low substrate affinity, in agreement with the experimental data . The structure of CelG has been constructed by homology modelling with a molecule of cellotetraose docked into the active site . No structural alteration related to cold-activity can be found in the catalytic cleft, whereas several structural factors in the overall structure can explain the weak thermal stability, suggesting that the loss of stability provides the required active-site mobility at low temperatures.

Genome, 2004 Aug, 47(4), 633 - 8
Assessment of genetic variability of haploids extracted from tetraploid (2n = 4x = 48) Solanum tuberosum; Ercolano MR et al.; The objectives of this study were to assess the genetic variability of haploids (2n = 2x = 24) extracted from tetraploid Solanum tuberosum through 4x x 2x crosses with Solanum phureja . Molecular and phenotypic analyses were performed to fingerprint the genotypes used and to evaluate their potential use in breeding programs . AFLP analysis revealed the presence of specific bands derived from the tetraploid seed parent S . phureja, as well as ex novo originated bands . On average, 210 bands were visualized per genotype, 149 (70%) of which were common to both parental genotypes . The percentage of S . tuberosum specific bands ranged from 25.1% to 18.6%, with an average of 22% . The fraction of genome coming from S . phureja ranged from 1.9% to 6.5%, with an average value of 4% . The percentage of ex novo bands varied from 1.9% to 9.0% . The presence of S . phureja DNA is very interesting because it indicated that S . phureja pollinator is involved in the mechanism of haploid formation . The characterization for resistance to Erwinia carotovora subsp . carotovora and potato virus X (PVX) provided evidence that haploids may express traits that are lacking in the tetraploids they come from, which can be useful for both genetic studies and breeding purposes . It is noteworthy that genotypes combining resistance to both diseases and good pollen stainability were identified . Other possible breeding implications owing to the presence of S . phureja genome in the haploids analyzed are discussed.

J Appl Microbiol, 2004, 97(3), 495 - 503
Cloning and partial characterization of zwittermicin A resistance gene cluster from Bacillus thuringiensis subsp . kurstaki strain HD1; Nair JR et al.; AIM: The study seeks to shed light on the aminopolyol, broad-spectrum antibiotic zwittermicin A gene cluster of Bacillus thuringiensis subsp . kurstaki HD1 and to identify any new uncharacterized genes with an eventual goal to establish a better understanding of the resistance gene cluster . METHODS AND RESULTS: We screened 51 serovars of B . thuringiensis by PCR and identified 12 zmaR-positive strains . The zmaR-positive B . thuringiensis subsp . kurstaki HD1 strain displayed inhibition zones against indicator fungal strain Phytophthora meadii and bacterial strain Erwinia herbicola as well as against Rhizopus sp., Xanthomonas campestris and B . thuringiensis subsp . finitimus . The zmaR gene cluster of strain HD1 was partially cloned using a lambda library and was extensively characterized based on the information available from a study performed on a similar group of genes in Bacillus cereus . CONCLUSIONS: Three of the five genes in the zwittermicin gene cluster, including the zmaR gene, had counterparts in B . cereus, and the other two were new members of the B . thuringiensis zmaR gene cluster . SIGNIFICANCE AND IMPACT OF THE STUDY: The two new genes were extensively analysed and the data is presented . Understanding antifungal activity of B . thuringiensis may help us to design suitable Cry toxin delivery agents with antifungal activity as well as enhanced insecticidal activity .

FEBS Lett, 2004 Jul 30, 571(1-3), 217 - 20
The bacterial type III secretion system-associated pilin HrpA has an unusually long mRNA half-life; Hienonen E et al.; Secondary structures affect mRNA stability and may play a role in protein secretion . We have studied the mRNA of hrpA, which codes for the major structural unit of the type III secretion system-associated pilus of Pseudomonas syringae pv . tomato, Erwinia carotovora and Pseudomonas syringae pv . phaseolicola . We show that hrpA mRNA has an unusually long half-life, approximately 33-47 min . We mapped regions in the transcript that affected hrpA mRNA accumulation . Apparently, sequences at both 5' and 3' ends affect accumulation . Altering the hypothetical, stable GC rich loop structure in the 3' end of the transcript decreased transcript levels.

J Agric Food Chem, 2004 Jul 28, 52(15), 4700 - 4
Fungistatic and bacteriostatic activities of alkamides from Heliopsis longipes roots: affinin and reduced amides; Molina-Torres J et al.; This work demonstrates the fungistatic and bacteriostatic activities of affinin, the main alkamide of Heliopsis longipes (Gray) Blake (Asteraceae) roots and two alkamides obtained by catalytic reduction of affinin: N-isobutyl-2E-decenamide and N-isobutyl-decanamide . The bioactivity was tested against Rhizoctonia solani groups AG3 and AG5, Sclerotium rolfsii, Sclerotium cepivorum, Fusarium sp., Vertcillium sp., phytopathogenic fungi; Phytophthora infestans, a phytopathogenic Chromista; Saccharomyces cerevisiae, a nonphytopathogenic ascomycete; and Escherichia coli, Erwinia carotovora, and Bacillus subtilis, bacteria . Affinin, being the primary component of the lipidic fraction, is expected to be responsible for the fungitoxic activity observed in roots of this plant species . Four of the assayed fungi showed an important sensitivity to the presence of affinin: S . rolfsii, S . cepivorum, P . infestans, and R . solani AG-3 and AG-5, displaying a growth inhibition of 100% . S . cerevisiaeshowed a similar growth inhibition with affinin . None of the alkamides obtained by catalytic reduction of affinin showed a fungitoxic activity . Affinin had a definite negative effect on the growth of E . coli and B . subtilis, but E . carotovora carotovora was not sensitive to the highest dose of affinin assayed . N-Isobutyl-2E-decenamide displayed a higher bacteriostatic activity against E . coli and E . carotovora carotovora . In both cases, this alkamide was more potent than affinin . On the other hand, only N-isobutyl-decanamide displayed a significant activity on the growth of B . subtilis .

Carbohydr Res, 2004 Aug 2, 339(11), 2049 - 53
Structure and hydrodynamic properties of the extracellular polysaccharide from a mutant strain (RA3W) of Erwinia chrysanthemi RA3; Ding Q et al.; The structure of the extracellular polysaccharide (EPS) produced by Erwinia chrysanthemi strain RA3W, a mutant strain of E . chrysanthemi RA3, has been determined using low pressure size-exclusion and anion-exchange chromatographies, high pH anion-exchange chromatography, glycosyl linkage analysis, and 1D (1)H NMR spectroscopy . The polysaccharide is structurally similar, if not identical, to the family of EPS produced by such as E . chrysanthemi strains Ech9, Ech9Sm6, and SR260 . The molecular weight of EPS RA3W by ultracentrifugation (sedimentation equilibrium) and light scattering is compared with those of other E . chrysanthami EPSs, as are the viscometric properties.

Can J Microbiol, 2004 Apr, 50(4), 239 - 49
Endophytic bacterial communities of field-grown potato plants and their plant-growth-promoting and antagonistic abilities; Sessitsch A et al.; To study the effect of plant growth on potato-associated bacteria, the composition and properties of bacteria colonizing the endosphere of field-grown potato were analyzed by a multiphasic approach . The occurrence and diversity of potato-associated bacteria were monitored by a cultivation-independent approach, using terminal restriction fragment length polymorphism analysis of 16S rDNA . The patterns obtained revealed a high heterogeneity of community composition and suggested the existence of plant-specific communities . However, endophytic populations correlated to a certain extent with plant growth performance . Endophytes were also isolated from plants that grew well or grew poorly and were identified by partial sequencing of the 16S rRNA genes . A broad phylogenetic spectrum was found among isolates and differently growing plants hosted different bacterial populations . In an approach to investigate the plant-growth-promoting potential of potato-associated bacteria, a total of 35 bacteria were screened by dual testing for in vitro antagonism towards (i) the fungal pathogens Verticillium dahliae, Rhizoctonia solani, Sclerotinia sclerotiorum, and Phytophthora cactorum and (ii) the bacterial pathogens Erwinia carotovora, Streptomyces scabies, and Xanthomonas campestris . The proportion of isolates with antagonistic activity was highest against Streptomyces sp . (43%) followed by those against Xanthomonas sp . (29%) . As all plants showed more or less severe disease symptoms of scab disease caused by Streptomyces scabies, we assume that the presence of the pathogen induced the colonization of antagonists . The antifungal activity of the isolates was generally low . The biotechnological potential of endophytic isolates assessed by their antagonistic activity and by in vitro production of enzymes, antibiotics, siderophores, and the plant growth hormone indole-1,3-acetic acid was generally high . Overall, seven endophytes were found to antagonize fungal as well as bacterial pathogens and showed a high production of active compounds and were therefore considered promising biological control agents.

Proc Natl Acad Sci U S A, 2004 Jun 29, 101(26), 9927 - 32 Epub 2004 Jun 21.
A family of conserved bacterial effectors inhibits salicylic acid-mediated basal immunity and promotes disease necrosis in plants; DebRoy S et al.; Salicylic acid (SA)-mediated host immunity plays a central role in combating microbial pathogens in plants . Inactivation of SA-mediated immunity, therefore, would be a critical step in the evolution of a successful plant pathogen . It is known that mutations in conserved effector loci (CEL) in the plant pathogens Pseudomonas syringae (the Delta CEL mutation), Erwinia amylovora (the dspA/E mutation), and Pantoea stewartii subsp . stewartii (the wtsE mutation) exert particularly strong negative effects on bacterial virulence in their host plants by unknown mechanisms . We found that the loss of virulence in Delta CEL and dspA/E mutants was linked to their inability to suppress cell wall-based defenses and to cause normal disease necrosis in Arabidopsis and apple host plants . The Delta CEL mutant activated SA-dependent callose deposition in wild-type Arabidopsis but failed to elicit high levels of callose-associated defense in Arabidopsis plants blocked in SA accumulation or synthesis . This mutant also multiplied more aggressively in SA-deficient plants than in wild-type plants . The hopPtoM and avrE genes in the CEL of P . syringae were found to encode suppressors of this SA-dependent basal defense . The widespread conservation of the HopPtoM and AvrE families of effectors in various bacteria suggests that suppression of SA-dependent basal immunity and promotion of host cell death are important virulence strategies for bacterial infection of plants.

Transgenic Res, 2004 Apr, 13(2), 181 - 90
Transgenic potatoes expressing a novel cationic peptide are resistant to late blight and pink rot; Osusky M et al.; Potato is the world's largest non-cereal crop . Potato late blight is a pandemic, foliar wasting potato disease caused by Phytophthora infestans, which has become highly virulent, fungicide resistant, and widely disseminated . Similarly, fungicide resistant isolates of Phytophthora erythroseptica, which causes pink rot, have also become an economic scourge of potato tubers . Thus, an alternate, cost effective strategy for disease control has become an international imperative . Here we describe a strategy for engineering potato plants exhibiting strong protection against these exceptionally virulent pathogens without deleterious effects on plant yield or vigor . The small, naturally occurring antimicrobial cationic peptide, temporin A, was N-terminally modified (MsrA3) and expressed in potato plants . MsrA3 conveyed strong resistance to late blight and pink rot phytopathogens in addition to the bacterial pathogen Erwinia carotovora . Transgenic tubers remained disease-free during storage for more than 2 years . These results provide a timely, sustainable, effective, and environmentally friendly means of control of potato diseases while simultaneously preventing storage losses.

Mol Plant Microbe Interact, 2004 Jun, 17(6), 644 - 53
The Erwinia chrysanthemi EC16 hrp/hrc gene cluster encodes an active Hrp type III secretion system that is flanked by virulence genes functionally unrelated to the Hrp system; Rojas CM et al.; Erwinia chrysanthemi is a host-promiscuous plant pathogen that possesses a type III secretion system (TTSS) similar to that of the host-specific pathogens E . amylovora and Pseudomonas syringae . The regions flanking the TTSS-encoding hrp/hrc gene clusters in the latter pathogens encode various TTSS-secreted proteins . DNA sequencing of the complete E . chrysanthemi hrp/hrc gene cluster and approximately 12 kb of the flanking regions (beyond the previously characterized hecA adhesin gene in the left flank) revealed that the E . chrysanthemi TTSS genes were syntenic and similar (>50% amino-acid identity) with their E . amylovora orthologs . However, the hrp/hrc cluster was interrupted by a cluster of four genes, only one of which, a homolog of lytic transglycosylases, is implicated in TTSS functions . Furthermore, the regions flanking the hrp/hrc cluster lacked genes that were likely to encode TTSS substrates . Instead, some of the genes in these regions predict ABC transporters and methyl-accepting chemotaxis proteins that could have alternative roles in virulence . Mutations affecting all of the genes in the regions flanking or interrupting the hrp/hrc cluster were constructed in E . chrysanthemi CUCPB5047, a mutant whose reduced pectolytic capacity can enhance the phenotype of minor virulence factors . Mutants were screened in witloof chicory leaves and then in potato tubers and Nicotiana clevelandii seedlings . Mu dII1734 insertion in one gene, designated virA, resulted in strongly reduced virulence in all three tests . virA is immediately downstream of hecA, has an unusually low G+C content of 38%, and predicts an unknown protein of 111 amino acids . The E . chrysanthemi TTSS was shown to be active by its ability to translocate AvrPto-Cya (a P . syringae TTSS effector fused to an adenylate cyclase reporter that is active in the presence of eukaryote calmodulin) into N . benthamiana leaf cells . However, VirA(1-61)-Cya was not translocated into plant cells, and virA expression was not affected by mutations in E . chrysanthemi Hrp regulator genes hrpL and hrpS . Thus, the 44-kb region of the E . chrysanthemi EC16 genome that is centered on the hrplhrc cluster encodes a potpourri of virulence factors, but none of these appear to be a TTSS effector.

J Appl Microbiol, 2004, 97(1), 93 - 103
Characterization of Pantoea dispersa UQ68J: producer of a highly efficient sucrose isomerase for isomaltulose biosynthesis; Wu L et al.; AIMS: Isolation, identification and characterization of a highly efficient isomaltulose producer . METHODS AND RESULTS: After an enrichment procedure for bacteria likely to metabolize isomaltulose in sucrose-rich environments, 578 isolates were screened for efficient isomaltulose biosynthesis using an aniline/diphenylamine assay and capillary electrophoresis . An isolate designated UQ68J was exceptionally efficient in sucrose isomerase activity . Conversion of sucrose into isomaltulose by UQ68J (enzyme activity of 90-100 U mg(-1) DW) was much faster than the current industrial strain Protaminobacter rubrum CBS574.77 (41-66 U mg(-1) DW) or a reference strain of Erwinia rhapontici (0.3-0.9 U mg(-1) DW) . Maximum yield of isomaltulose at 78-80% of supplied sucrose was achieved in less than half the reaction time needed by CBS574.77, and the amount of contaminating trehalulose (4%) was the lowest recorded from an isomaltulose-producing microbe . UQ68J is a Gram negative, facultatively anaerobic, motile, noncapsulate, straight rod-shaped bacterium producing acid but no gas from glucose . Based on 16S rDNA analysis UQ68J is closest to Klebsiella oxytoca, but it differs from Klebsiella in defining characteristics and most closely resembles Pantoea dispersa in phenotype . SIGNIFICANCE AND IMPACT OF STUDY: This organism is likely to have substantial advantage over previously characterized sucrose isomerase producers for the industrial production of isomaltulose.

Microb Ecol, 2004 Aug, 48(2), 239 - 45 Epub 2004 May 27.
Recovery of GFP-labeled bacteria for culturing and molecular analysis after cell sorting using a benchtop flow cytometer; Ferrari BC et al.; Exciting opportunities exist for the application of simple fluorescence-activated cell sorting (FACS) to microbiology . The technology is widely available, but critical reports on the efficiency of cell sorting using benchtop instruments are lacking . It is vital that single cell sorting be of the highest purity possible . If purity is compromised detrital material or unwanted cells will be captured along with target cells of interest . Here, the isolation of fluorescent bacteria using a benchtop FACSCalibur-sort flow cytometer is described . The efficiency and purity of isolated cells was determined using fluorescence microscopy, culturing, and molecular analysis . To achieve high purity it was essential that the total event rate did not exceed 300 cells per second . This instrument was capable of recovering >55% sorted Escherichia coli cells, coupled with a purity exceeding 99% . However, the purity of recovered cells was substantially reduced (<25%) when the event rate increased . Cell sorting onto polycarbonate membranes did not reduce the ability of E . coli to form colonies, and sorting of ~1000 E . coli cells was sufficient for 16S rDNA amplification . Additionally, as few as 100 isolated Erwinia sp . carrying the gfp gene were amplified using seminested PCR targeting the single copy gfp gene . With such low numbers of bacteria being required for molecular identification, FACS can be achieved without the requirement for high-speed droplet cell sorters.

J Biol Chem, 2004 Jul 16, 279(29), 30158 - 67 Epub 2004 May 12.
Definition of a consensus DNA-binding site for PecS, a global regulator of virulence gene expression in Erwinia chrysanthemi and identification of new members of the PecS regulon; Rouanet C et al.; In Erwinia chrysanthemi, production of pectic enzymes is modulated by a complex network involving several regulators . One of them, PecS, which belongs to the MarR family, also controls the synthesis of various other virulence factors, such as cellulases and indigoidine . Here, the PecS consensus-binding site is defined by combining a systematic evolution of ligands by an exponential enrichment approach and mutational analyses . The consensus consists of a 23-base pair palindromic-like sequence (C(-11)G(-10)A(-9)N(-8)W(-7)T(-6)C(-5)G(-4)T(-3)A(-2))T(-1)A(0)T(1)(T(2)A(3)C(4)G(5)A(6)N(7)N(8)N(9)C(10)G(11)) . Mutational experiments revealed that (i) the palindromic organization is required for the binding of PecS, (ii) the very conserved part of the consensus (-6 to 6) allows for a specific interaction with PecS, but the presence of the relatively degenerated bases located apart significantly increases PecS affinity, (iii) the four bases G, A, T, and C are required for efficient binding of PecS, and (iv) the presence of several binding sites on the same promoter increases the affinity of PecS . This consensus is detected in the regions involved in PecS binding on the previously characterized target genes . This variable consensus is in agreement with the observation that the members of the MarR family are able to bind various DNA targets as dimers by means of a winged helix DNA-binding motif . Binding of PecS on a promoter region containing the defined consensus results in a repression of gene transcription in vitro . Preliminary scanning of the E . chrysanthemi genome sequence with the consensus revealed the presence of strong PecS-binding sites in the intergenic region between fliE and fliFGHIJKLMNOPQR which encode proteins involved in the biogenesis of flagellum . Accordingly, PecS directly represses fliE expression . Thus, PecS seems to control the synthesis of virulence factors required for the key steps of plant infection.

Int J Food Microbiol, 2004 Jun 1, 93(2), 195 - 208
Partitioning of the variance in the growth parameters of Erwinia carotovora on vegetable products; Shorten PR et al.; The objective of this paper was to estimate and partition the variability in the microbial growth model parameters describing the growth of Erwinia carotovora on pasteurised and non-pasteurised vegetable juice from laboratory experiments performed under different temperature-varying conditions . We partitioned the model parameter variance and covariance components into effects due to temperature profile and replicate using a maximum likelihood technique . Temperature profile and replicate were treated as random effects and the food substrate was treated as a fixed effect . The replicate variance component was small indicating a high level of control in this experiment . Our analysis of the combined E . carotovora growth data sets used the Baranyi primary microbial growth model along with the Ratkowsky secondary growth model . The variability in the microbial growth parameters estimated from these microbial growth experiments is essential for predicting the mean and variance through time of the E . carotovora population size in a product supply chain and is the basis for microbiological risk assessment and food product shelf-life estimation . The variance partitioning made here also assists in the management of optimal product distribution networks by identifying elements of the supply chain contributing most to product variability .

J Microbiol Methods, 2004 Jun, 57(3), 415 - 20
Fast screening method for detection of acyl-HSL-degrading soil isolates; Jafra S et al.; A reliable method was developed for screening of bacteria isolates capable of degrading acyl-HSLs, the signal molecules in quorum-sensing-mediated processes of many Proteobacteria . The microtiter assay was based on the use of a GFP-marked Escherichia coli strain, which fluoresces upon the presence of acyl-HSLs . Measurement of GFP fluorescence with a Molecular Imager FX scanner (fluorometer) detected isolates capable of degrading acyl-HSLs . The potential of this method was demonstrated by isolation of different bacteria from a potato rhizosphere able to inactivate synthetic and natural acyl HSLs produced by Pectobacterium carotovorum subsp . carotovorum (Pcc) (Erwinia carotovora subsp . carotovora (Ecc)).

FEMS Microbiol Lett, 2004 May 1, 234(1), 1 - 8
Stability of short sequence repeats and their application for the characterization of Erwinia amylovora strains; Ruppitsch W et al.; A motif of eight nucleotides (GAATTACA) reiterated 3 to 15 times within the PstI fragment of the pEa29 plasmid was found in Erwinia amylovora strains representing a valuable typing method for this pathogen . The stability of short sequence DNA repeat (SSR) numbers was investigated to determine the suitability of this marker for strain differentiation . The number of SSR units was found to be stable under laboratory and certain stress conditions . This meets the requirements for a suitable genetic marker that should be stable upon cultivation of strains . Therefore, this SSR marker was used for strain differentiation from SSR-3 to SSR-15 and a large number of E . amylovora strains from Austria was screened for their SSR numbers for epidemiological identification purposes . Traceability was possible if strains had very high or very low SSR numbers.

Leukemia, 2004 Jun, 18(6), 1072 - 7
Asparaginase pharmacodynamics differ by formulation among children with newly diagnosed acute lymphoblastic leukemia; Hak LJ et al.; Polyethylene glycol-conjugated (PEG) asparaginase is approved for use in patients who develop allergy to other forms of asparaginase, although its ability to deplete asparagine systemically in patients with hypersensitivity has not been well elucidated . In 53 children with newly diagnosed acute lymphoblastic leukemia, we serially assessed asparagine concentrations in cerebrospinal fluid (CSF) and plasma as well as serum anti-asparaginase antibodies . All patients received native Escherichia coli (Elspar) asparaginase during induction therapy; patients received PEG asparaginase during reinductions when available, and those who developed allergy received Erwinia asparaginase . All eight patients who developed clinical evidence of allergy to asparaginase had anti-asparaginase antibodies . Among patients who had no antibodies, those who received E . coli had lower mean (+/-s.d.) CSF asparagine (0.29+/-0.63, n=9) than those who received PEG (0.77+/-0.82, n=4) (P=0.007) . Results were similar for plasma asparagine . There was no situation where asparagine concentrations were more effectively depleted by PEG than by other preparations . None of the five patients who developed thrombosis had an allergy or antibodies to asparaginase at the time of the thrombosis . We conclude that asparagine concentrations were less effectively depleted by PEG than by E . coli asparaginase at the doses commonly used . The risk of thrombosis may be affected by the intensity of asparaginase exposure.

Can J Microbiol, 2004 Jan, 50(1), 19 - 27
Thermodependence of growth and enzymatic activities implicated in pathogenicity of two Erwinia carotovora subspecies (Pectobacterium spp.); Smadja B et al.; Erwinia carotovora subsp . atroseptica and Erwinia carotovora subsp . carotovora can cause substantial damage to economically important plant crops and stored products . The occurrence of the disease and the scale of the damage are temperature dependent . Disease development consists first of active multiplication of the bacteria in the infection area and then production of numerous extracellular enzymes . We investigated the effects of various temperatures on these two steps . We assayed the specific growth rate and the pectate lyase and protease activities for eight strains belonging to E . carotovora subsp . atroseptica and E . carotovora subsp . carotovora in vitro . The temperature effect on growth rate and on pectate lyase activity is different for the two subspecies, but protease activity appears to be similarly thermoregulated . Our results are in agreement with ecological data implicating E . carotovora subsp . atroseptica in disease when the temperature is below 20 degrees C . The optimal temperature for pathogenicity appears to be different from the optimal growth temperature but seems to be a compromise between this temperature and temperatures at which lytic activities are maximal.

Environ Microbiol, 2004 May, 6(5), 480 - 90
Molecular differentiation of Erwinia amylovora strains from North America and of two Asian pear pathogens by analyses of PFGE patterns and hrpN genes; Jock S et al.; In order to determine a possible genomic divergence of Erwinia amylovora'fruit tree' and raspberry strains from North America, several isolates were differentiated by pulsed-field gel electrophoresis (PFGE) analysis, the size of short DNA sequence repeats (SSRs) and the nucleotide and deduced amino acid sequences of their hrpN genes . By PFGE analysis European strains are highly related, whereas strains from North America were diverse and were further distinguished by the SSR numbers from plasmid pEA29 . The E . amylovora strains from Europe showed identical HrpN sequences in contrast to the American isolates from fruit trees and raspberry . Those were related to each other, but distinguishable by their HrpN patterns . The Asian pear pathogens differed in HrpN among each other and from E . amylovora . Erwinia pyrifoliae isolates and the Erwinia strains from Japan were ordered via their HrpN sequences in agreement with the PFGE patterns . For all three pathogens, dendrograms from PFGE and sequence data indicate an evolutionary diversity within the species in spite of a genetic conservation for parts of the hrpN genes suggesting a long persistence of the Asian pear pathogens in Korea and Japan as well as of fire blight in North America . Some of the divergent American E . amylovora isolates share PFGE patterns with the relatively uniform European strains.

Biotechnol Appl Biochem, 2004 Apr, 39(Pt 2), 215 - 21
One-step purification and kinetic properties of the recombinant L-asparaginase from Erwinia carotovora; Krasotkina J et al.; ECAR-LANS, the recombinant L-asparaginase from Erwinia carotovora, is a prospective therapeutic enzyme for leukaemia treatment . An efficient and economical scheme was developed for the purification, cloning and expression in Eschericha coli of ECAR-LANS . More than 90% purity, complemented with 72% active enzyme recovery, was achieved with a single chromatographic purification step . The activity of purified L-asparaginase was 630 i.u./mg . The ECAR-LANS K (m) value was 98x10(-6) M for the main physiological substrate L-Asn and 3400x10(-6) M for L-Gln . ECAR-LANS was found to have low relative glutaminase activity (1.2%) at physiological concentrations of L-Asn and L-Gln in blood . Kinetic studies of ECAR-LANS showed that the recombinant asparaginase combined the main advantages of Erw . chrysanthemi and E . coli L-asparaginases II, currently used in the treatment of acute lymphoblastic leukaemia.

Annu Rev Phytopathol, 1998, 36, 227 - 48
Management of fire blight: a case study in microbial ecology; Johnson KB et al.; Suppression of the blossom-blight phase of fire blight is a key point in the management of this destructive and increasingly important disease of apple and pear . For blossom infection to occur, the causal bacterium, Erwinia amylovora, needs to increase its population size through an epiphytic phase that occurs on stigmatic surfaces . Knowledge of the ecology of the pathogen on stigmas has been key to the development of predictive models for infection and optimal timing of antibiotic sprays . Other bacterial epiphytes also colonize stigmas where they can interact with and suppress epiphytic growth of the pathogen . A commercially available bacterial antagonist of E . amylovora (BlightBan, Pseudomonas fluorescens A506) can be included in antibiotic spray programs . Integration of bacterial antagonists with chemical methods suppresses populations of the pathogen and concomitantly, fills the ecological niche provided by the stigma with a nonpathogenic, competing microorganism . Further integration of biologically based methods with conventional management of blossom blight may be achievable by increasing the diversity of applied antagonists, by refining predictive models to incorporate antagonist use, and by gaining an improved understanding of the interactions that occur among indigenous and applied bacterial epiphytes, antibiotics, and the physical environment.

Plant Mol Biol, 2003 Nov, 53(4), 493 - 511
Rootstock effects on gene expression patterns in apple tree scions; Jensen PJ et al.; Like many fruit trees, apple trees (Malus pumila) do not reproduce true-to-type from seed . Desirable cultivars are clonally propagated by grafting onto rootstocks that can alter the characteristics of the scion . For example, the M.7 EMLA rootstock is semi-dwarfing and reduces the susceptibility of the scion to Erwinia amylovora, the causal agent of fire blight disease . In contrast, the M.9 T337 rootstock is dwarfing and does not alter fire blight susceptibility of the scion . This study represents a comprehensive comparison of gene expression patterns in scions of the 'Gala' apple cultivar grafted to either M.7 EMLA or M.9 T337 . Expression was determined by cDNA-AFLP coupled with silver staining of the gels . Scions grafted to the M.9 T337 rootstock showed higher expression of a number of photosynthesis-related, transcription/translation-related, and cell division-related genes, while scions grafted to the M.7 EMLA rootstock showed increased stress-related gene expression . The observed differences in gene expression showed a remarkable correlation with physiological differences between the two graft combinations . The roles that the differentially expressed genes might play in tree stature, stress tolerance, photosynthetic activity, fire blight resistance, and other differences conferred by the two rootstocks are discussed.

Res Microbiol, 2004 Mar, 155(2), 71 - 5
Systematic analysis, by the yeast two-hybrid, of protein interaction between components of the type II secretory machinery of Erwinia chrysanthemi; Douet V et al.; Type II systems allow for the secretion of numerous enzymes and toxins in several Gram-negative pathogens . In Erwinia chrysanthemi, 14 Out proteins are necessary for building the type II apparatus . We performed a systematic two-hybrid analysis to test interactions between the periplasmic regions of the Out proteins . Results obtained using this approach suggested that OutJ (a pseudopilin) was able to interact with (i) OutD, the outer membrane secretin, (ii) OutI, mainly located in the periplasm, and (iii) OutL, an inner membrane protein . Taken together, these results suggest that OutJ is involved in multiple partnerships . Implications of these partnerships in the overall architecture of the type II secretion machinery are discussed.

Antimicrob Agents Chemother, 2004 Mar, 48(3), 903 - 8
Tetracycline and streptomycin resistance genes, transposons, and plasmids in Salmonella enterica isolates from animals in Italy; Pezzella C et al.; Fifty-eight multidrug-resistant Salmonella enterica strains of 20 serotypes, isolated from animal sources in Italy, were analyzed for tet(A) and strA-strB, conferring tetracycline and streptomycin resistance, respectively . The strA and strB genes were highly prevalent in Salmonella strains of our collection, being detected in 84% of the streptomycin-resistant strains . In many strains, the strA and strB genes were linked to a particular Tn5393-derivative transposon characterized by the presence of the insertion sequence IS1133, previously identified only in the plant pathogen Erwinia amylovora . Sixty-eight percent of the tetracycline-resistant strains were tet(A) positive, indicating that this gene is widely diffused in Salmonella strains circulating in animals in Italy . Most of the tet(A) genes were localized within a deleted Tn1721 transposon variant . Two prevalent repN and repI1 resistance plasmids were identified in Salmonella isolates of our collection.

Plant Physiol, 2004 Mar, 134(3), 1017 - 26 Epub 2004 Feb 19.
Bacterial volatiles induce systemic resistance in Arabidopsis; Ryu CM et al.; Plant growth-promoting rhizobacteria, in association with plant roots, can trigger induced systemic resistance (ISR) . Considering that low-molecular weight volatile hormone analogues such as methyl jasmonate and methyl salicylate can trigger defense responses in plants, we examined whether volatile organic compounds (VOCs) associated with rhizobacteria can initiate ISR . In Arabidopsis seedlings exposed to bacterial volatile blends from Bacillus subtilis GB03 and Bacillus amyloliquefaciens IN937a, disease severity by the bacterial pathogen Erwinia carotovora subsp . carotovora was significantly reduced compared with seedlings not exposed to bacterial volatiles before pathogen inoculation . Exposure to VOCs from rhizobacteria for as little as 4 d was sufficient to activate ISR in Arabidopsis seedlings . Chemical analysis of the bacterial volatile emissions revealed the release of a series of low-molecular weight hydrocarbons including the growth promoting VOC (2R,3R)-(-)-butanediol . Exogenous application of racemic mixture of (RR) and (SS) isomers of 2,3-butanediol was found to trigger ISR and transgenic lines of B . subtilis that emitted reduced levels of 2,3-butanediol and acetoin conferred reduced Arabidopsis protection to pathogen infection compared with seedlings exposed to VOCs from wild-type bacterial lines . Using transgenic and mutant lines of Arabidopsis, we provide evidence that the signaling pathway activated by volatiles from GB03 is dependent on ethylene, albeit independent of the salicylic acid or jasmonic acid signaling pathways . This study provides new insight into the role of bacteria VOCs as initiators of defense responses in plants.

J Agric Food Chem, 2004 Feb 25, 52(4), 776 - 80
Antifungal activity of beta-asarone from rhizomes of Acorus gramineus; Lee JY et al.; An antifungal substance was isolated from the extract of Acorus gramineus using various chromatographic procedures . The antibiotic was identified as beta-asarone, cis-2,4,5-trimethoxy-1-propenylbenzene, on the basis of the high-resolution EI-mass, NMR, and UV spectral data . Beta-asarone completely inhibited mycelial growth of some plant pathogenic fungi, Cladosporium cucumerinum,Colletotrichum orbiculare, Magnaporthe grisea, and Pythium ultimum, in a range of 0.5-30 microg/mL . The growth of Bacillus subtilis, Erwinia carotovora subsp . carotovora, Ralstonia solanacearum, and Xanthomonas campestris pv . vesicatoria was slightly suppressed by beta-asarone . As the concentration of beta-asarone increased, M . grisea infection was drastically inhibited on rice leaves . Treatment with 500 microg/mL of beta-asarone also greatly suppressed lesion formation of Co . orbiculare on cucumber leaves . This is the first study to demonstrate in vitro and in vivo antifungal activity of beta-asarone against plant fungal pathogens M . grisea and C . orbiculare.

Mol Plant Microbe Interact, 2004 Feb, 17(2), 184 - 94
Analysis of Erwinia chrysanthemi EC16 pelE::uidA, pelL::uidA, and hrpN::uidA mutants reveals strain-specific atypical regulation of the Hrp type III secretion system; Ham JH et al.; The plant pathogen Erwinia chrysanthemi produces a variety of factors that have been implicated in its ability to cause soft-rot diseases in various hosts . These include HrpN, a harpin secreted by the Hrp type III secretion system; PelE, one of several major pectate lyase isozymes secreted by the type II system; and PelL, one of several secondary Pels secreted by the type II system . We investigated these factors in E . chrysanthemi EC16 with respect to the effects of medium composition and growth phase on gene expression (as determined with uidA fusions and Northern analyses) and effects on virulence . pelE was induced by polygalacturonic acid, but pelL was not, and hrpN was expressed unexpectedly in nutrient-rich King's medium B and in minimal salts medium at neutral pH . In contrast, the effect of medium composition on hrp expression in E . chrysanthemi CUCPB1237 and 3937 was like that of many other phytopathogenic bacteria in being repressed in complex media and induced in acidic pH minimal medium . Northern blot analysis of hrpN and hrpL expression by the wild-type and hrpL::omegaCmr and hrpS::omegaCmr mutants revealed that hrpN expression was dependent on the HrpL alternative sigma factor, whose expression, in turn, was dependent on the HrpS putative sigma54 enhancer binding protein . The expression of pelE and hrpN increased strongly in late logarithmic growth phase . To test the possible role of quorum sensing in this expression pattern, the expI/expR locus was cloned in Escherichia coli on the basis of its ability to direct production of acyl-homoserine lactone and then used to construct expI mutations in pelE::uidA, pelL::uidA, and hrpN::uidA Erwinia chrysanthemi strains . Mutation of expI had no apparent effect on the growth-phase-dependent expression of hrpN and pelE, or on the virulence of E . chrysanthemi in witloof chicory leaves . Overexpression of hrpN in E . chrysanthemi resulted in approximately 50% reduction of lesion size on chicory leaves without an effect on infection initiation.

Mol Plant Microbe Interact, 2004 Feb, 17(2), 152 - 61
Importance of opgHXcv of Xanthomonas campestris pv . vesicatoria in host-parasite interactions; Minsavage GV et al.; Tn5 insertion mutants of Xanthomonas campestris pv . vesicatoria were inoculated into tomato and screened for reduced virulence . One mutant exhibited reduced aggressiveness and attenuated growth in planta . Southern blot analyses indicated that the mutant carried a single Tn5 insertion not associated with previously cloned pathogenicity-related genes of X . campestris pv . vesicatoria . The wild-type phenotype of this mutant was restored by one recombinant plasmid (pOPG361) selected from a genomic library of X . campestris pv . vesicatoria 91-118 . Tn3-gus insertion mutagenesis and sequence analyses of a subclone of pOPG361 identified a 1,929-bp open reading frame (ORF) essential for complementation of the mutants . The predicted protein encoded by this ORF was highly homologous to the previously reported pathogenicity-related HrpM protein of Pseudomonas syringae pv . syringae and OpgH of Erwinia chrysanthemi . Based on homology, the new locus was designated opgHXcv . Manipulation of the osmotic potential in the intercellular spaces of tomato leaves by addition of mannitol at low concentrations (25 to 50 mM) compensates for the opgHXcv mutation.

J Appl Microbiol, 2004, 96(3), 535 - 45
Characterization of atypical Erwinia carotovora strains causing blackleg of potato in Brazil; Duarte V et al.; AIMS: To determine the characteristics of bacteria associated with the blackleg disease of potato in Brazil and compare them with species and subspecies of pectolytic Erwinia . METHODS AND RESULTS: Biochemical and physiological characteristics of 16 strains from blackleg-infected potatoes in State of Rio Grande do Sul, Brazil, were determined and differentiated them from all the E . carotovora subspecies and E . chrysanthemi . Pathogenicity and maceration ability of the Brazilian strains were greater than those of E . carotovora subsp . atroseptica, the causal agent of potato blackleg in temperate zones . Analyses of serological reaction and fatty acid composition confirmed that the Brazilian strains differed from E . carotovora subsp . atroseptica, but the sequence of 16S rDNA gene and the 16S-23S intergenic spacer (IGS) region confirmed the Brazilian strains as pectolytic Erwinia . Restriction analysis of the IGS region differentiated the Brazilian strains from the subspecies of E . carotovora and from E . chrysanthemi . A unique SexAI restriction site in the IGS region was used as the basis for a primer to specifically amplify DNA from the Brazilian potato blackleg bacterium in PCR . CONCLUSIONS: The bacterium that causes the blackleg disease of potato in Brazil differs from E . carotovora subsp . atroseptica, the blackleg pathogen in temperate zones . It also differs from other subspecies of E . carotovora and from E . chrysanthemi and warrants status as a new subspecies, which would be appropriately named E . carotovora subsp . brasiliensis . SIGNIFICANCE AND IMPACT OF THE STUDY: The blackleg disease of potato is caused by a different strain of pectolytic Erwinia in Brazil than in temperate potato-growing regions . The Brazilian strain is more virulent than E . carotovora subsp . atroseptica, the usual causal agent of potato blackleg.

Mar Biotechnol (NY), 2000 Nov, 2(6), 522 - 32
Isolation and characterization of novel hydrocarbon-degrading euryhaline consortia from crude oil and mangrove sediments; Piedad Diaz M et al.; Two novel and versatile bacterial consortia were developed for the biodegradation of hydrocarbons . They were isolated from crude oil from the Cormorant Field in the North Sea (MPD-7) and from sediment associated with mangrove roots (MPD-M) . The bacterial consortia were able to degrade both aliphatic and aromatic hydrocarbons in crude oils very effectively in seawater (35 g/L NaCl) and synthetic media containing 0 to 100 g/L NaCl (1.7 M) . Salinities over twice that of normal seawater decreased the biodegradation rates . However, even at the highest salinity biodegradation was significant . Ratios of nC17 to pristane and nC18 to phytane were significantly lowered across the range of salinity . The lowest values were at 0 and 20 g/L (0.34 M) . Phytane was degraded in preference to pristane . The degradation of these compounds was constant over the salinity range, with evidence of a slight increase for consortium MPD-M with increasing salinity . In general, the consortium isolated from mangrove root sediments was more efficient in metabolizing North Sea crude oil than the consortium isolated from Cormorant crude oil . The 5 strains that comprise MPD-M have been tentatively identified as species of the genera Marinobacter, Bacillus, and Erwinia . This is the first report of hydrocarbon-degrading consortia isolated from crude oil and mangrove sediments that are capable of treating oily wastes over such a wide range of salinity.

Environ Toxicol Chem, 2004 Jan, 23(1), 1 - 6
Microbial transformation of pyrethroid insecticides in aqueous and sediment phases; Lee S et al.; Recent studies showed that synthetic pyrethroids (SPs) can move via surface runoff into aquatic systems . Fifty-six of SP-degrading bacteria strains were isolated from contaminated sediments, of which six were evaluated for their ability to transform bifenthrin and permethrin in the aqueous phase and bifenthrin in the sediment phase . In the aqueous phase, bifenthrin was rapidly degraded by strains of Stenotrophomonas acidaminiphila, and the half-life (t1/2) was reduced from >700 h to 30 to 131 h . Permethrin isomers were degraded by Aeromonas sobria, Erwinia carotovora, and Yersinia frederiksenii . Similar to bifenthrin, the t1/2 of cis- and trans-permethrin was reduced by approximately 10-fold after bacteria inoculation . However, bifenthrin degradation by S . acidaminiphila was significantly inhibited in the presence of sediment, and the effect was likely caused by strong adsorption to the solid phase . Bifenthrin t1/2 was 343 to 466 h for a field sediment, and increased to 980 to 1200 h for a creek sediment . Bifenthrin degradation in the inoculated slurry treatments was not greatly enhanced when compared with the noninoculated system . Therefore, although SP-degrading bacteria may be widespread in aquatic systems, adsorption to sediment could render SPs unavailable to the degraders, thus prolonging their persistence.

Appl Environ Microbiol, 2004 Feb, 70(2), 954 - 60
Insecticidal Bacillus thuringiensis silences Erwinia carotovora virulence by a new form of microbial antagonism, signal interference; Dong YH et al.; It is commonly known that bacteria may produce antibiotics to interfere with the normal biological functions of their competitors in order to gain competitive advantages . Here we report that Bacillus thuringiensis suppressed the quorum-sensing-dependent virulence of plant pathogen Erwinia carotovora through a new form of microbial antagonism, signal interference . E . carotovora produces and responds to acyl-homoserine lactone (AHL) quorum-sensing signals to regulate antibiotic production and expression of virulence genes, whereas B . thuringiensis strains possess AHL-lactonase, which is a potent AHL-degrading enzyme . B . thuringiensis did not seem to interfere with the normal growth of E . carotovora; rather, it abolished the accumulation of AHL signal when they were cocultured . In planta, B . thuringiensis significantly decreased the incidence of E . carotovora infection and symptom development of potato soft rot caused by the pathogen . The biocontrol efficiency is correlated with the ability of bacterial strains to produce AHL-lactonase . While all the seven AHL-lactonase-producing B . thuringiensis strains provided significant protection against E . carotovora infection, Bacillus fusiformis and Escherichia coli strains that do not process AHL-degradation enzyme showed little effect in biocontrol . Mutation of aiiA, the gene encoding AHL-lactonase in B . thuringiensis, resulted in a substantial decrease in biocontrol efficacy . These results suggest that signal interference mechanisms existing in natural ecosystems could be explored as a new version of antagonism for prevention of bacterial infections.

Appl Environ Microbiol, 2004 Feb, 70(2), 693 - 703
NorM, an Erwinia amylovora multidrug efflux pump involved in in vitro competition with other epiphytic bacteria; Burse A et al.; Blossoms are important sites of infection for Erwinia amylovora, the causal agent of fire blight of rosaceous plants . Before entering the tissue, the pathogen colonizes the stigmatic surface and has to compete for space and nutrient resources within the epiphytic community . Several epiphytes are capable of synthesizing antibiotics with which they antagonize phytopathogenic bacteria . Here, we report that a multidrug efflux transporter, designated NorM, of E . amylovora confers tolerance to the toxin(s) produced by epiphytic bacteria cocolonizing plant blossoms . According to sequence comparisons, the single-component efflux pump NorM is a member of the multidrug and toxic compound extrusion protein family . The corresponding gene is widely distributed among E . amylovora strains and related plant-associated bacteria . NorM mediated resistance to the hydrophobic cationic compounds norfloxacin, ethidium bromide, and berberine . A norM mutant was constructed and exhibited full virulence on apple rootstock MM 106 . However, it was susceptible to antibiotics produced by epiphytes isolated from apple and quince blossoms . The epiphytes were identified as Pantoea agglomerans by 16S rRNA analysis and were isolated from one-third of all trees examined . The promoter activity of norM was twofold greater at 18 degrees C than at 28 degrees C . The lower temperature seems to be beneficial for host infection because of the availability of moisture necessary for movement of the pathogen to the infection sites . Thus, E . amylovora might employ NorM for successful competition with other epiphytic microbes to reach high population densities, particularly at a lower temperature.

J Appl Microbiol, 2004, 96(2), 302 - 10
Genetic characterization of Erwinia amylovora strains by amplified fragment length polymorphism; Rico A et al.; AIMS: Erwinia amylovora is one of the most important pathogens of pear and apple and is subject to strict quarantine regulations worldwide, although its patterns of dispersal are largely unknown . Previous attempts to fingerprint E . amylovora strains by molecular techniques have detected very little polymorphism because of the high genetic homogeneity of this bacterium . Our aim was to establish and test a typing method to quantify genetic diversity among strains of this plant pathogen . METHODS AND RESULTS: Twenty-two strains from different hosts and geographical locations were examined by PCR fingerprinting with four primers and by amplified fragment length polymorphism (AFLP) with four selected combinations of primers with a single base extension . PCR fingerprinting revealed little polymorphism producing the same amplification patterns for 17 strains, while the combined AFLP patterns yielded 78 polymorphic bands (34% of total bands) and allowed the differentiation of all but two strains . Clustering of strains in the resulting dendrogram was not correlated with host, year or country of isolation, and questions previous genealogies based on PFGE patterns . CONCLUSIONS: The AFLP technique allowed the detection of an unprecedented number of genetic markers in E . amylovora and proved to be the most useful tool so far for discriminating among strains of this pathogen . The results obtained in this study strongly suggest the occurrence of multiple introductions of the pathogen in Spain and other European countries . SIGNIFICANCE AND IMPACT OF THE STUDY: A major limitation in understanding the ecology of fire blight is the lack of typing techniques with a high power of discrimination . This study demonstrates the high resolution and the usefulness of the AFLP technique to differentiate among E . amylovora strains.

Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 190 - 2 Epub 2003 Dec 18.
Crystallization of the pectate lyase PelI from Erwinia chrysanthemi and SAD phasing of a gold derivative; Castang S et al.; The pectate lyase PelI is involved in the degradation of plant tissues by the phytopathogenic bacterium Erwinia chrysanthemi . It has been crystallized from a solution containing PEG 550 in the space group P2(1), with unit-cell parameters a = 61.6, b = 70.7, c = 73.4 A, beta = 112.8 degrees . Crystals diffract to 1.45 A using synchrotron radiation . SAD phases have been computed from a gold-derivative crystal at the wavelength of maximum absorption (L(III) edge).

Appl Microbiol Biotechnol, 2004 May, 64(4), 560 - 7 Epub 2003 Dec 13.
Cloning of the pelA gene from Bacillus licheniformis 14A and biochemical characterization of recombinant, thermostable, high-alkaline pectate lyase; Berensmeier S et al.; The pectate lyase gene pelA from alkaliphilic Bacillus licheniformis strain 14A was cloned and sequenced . The nucleotide sequence corresponded to an open reading frame of 1,026 bp that codes for a 39 amino acid signal peptide and a mature protein with a molecular mass of 33,451 Da . The mature PelA showed significant homology to other pectate lyases belonging to polysaccharide lyase family 1, such as enzymes from different Bacillus spp . and Erwinia chrysanthemi . The pelA gene was expressed in Escherichia coli as a recombinant fusion protein containing a C-terminal His-tag, allowing purification to near homogeneity in a one-step procedure . The values for the kinetic parameters K(m) and Vmax of the fusion protein were 0.56 g/l and 51 micromol/min, respectively . The activity of purified PelAHis was inhibited in the presence of excess substrate . Characterization of product formation revealed unsaturated trigalacturonate as the main product . The yields of unsaturated trigalacturonic acids were further examined for the substrates polygalacturonic acid, citrus pectin and sugar-beet pectin .

J Biol Chem, 2004 Mar 5, 279(10), 9139 - 45 Epub 2003 Dec 11.
The crystal structure of pectate lyase Pel9A from Erwinia chrysanthemi; Jenkins J et al.; The "family 9 polysaccharide lyase" pectate lyase L (Pel9A) from Erwinia chrysanthemi comprises a 10-coil parallel beta-helix domain with distinct structural features including an asparagine ladder and aromatic stack at novel positions within the superhelical structure . Pel9A has a single high affinity calcium-binding site strikingly similar to the "primary" calcium-binding site described previously for the family Pel1A pectate lyases, and there is strong evidence for a common second calcium ion that binds between enzyme and substrate in the "Michaelis" complex . Although the primary calcium ion binds substrate in subsite -1, it is the second calcium ion, whose binding site is formed by the coming together of enzyme and substrate, that facilitates abstraction of the C5 proton from the sacharride in subsite +1 . The role of the second calcium is to withdraw electrons from the C6 carboxylate of the substrate, thereby acidifying the C5 proton facilitating its abstraction and resulting in an E1cb-like anti-beta-elimination mechanism . The active site geometries and mechanism of Pel1A and Pel9A are closely similar, but the catalytic base is a lysine in the Pel9A enzymes as opposed to an arginine in the Pel1A enzymes.

Carbohydr Res, 2003 Nov 14, 338(23), 2763 - 71
Hydrodynamic properties of oxidized extracellular polysaccharides from Erwinia chrysanthemi spp; Yang BY et al.; The molecular weights of the native polysaccharides of Erwinia chrysanthemi strains range from 1.8 to 7.1 x 10(6) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions . The effect on the hydrodynamic properties of the polysaccharides by adding carboxyl groups to increase the charge density is studied, with particular reference to their molecular weight (MW), viscosity and conformation . In general, it is found that periodate oxidation of the extracellular polysaccharides of E . chrysanthemi strains, Ech9Sm6 and Ech6S+, introduces little change in the hydrodynamic properties of the resulting polyaldehydes . However, bromine oxidation at neutral pH of the polyaldehydes results in polycarboxylate biopolymers that show significant reduction in MW and viscosity, but they are still characteristic polyanions.

Mol Plant Microbe Interact, 2003 Dec, 16(12), 1145 - 53
Oxidative burst elicited by Bacillus mycoides isolate Bac J, a biological control agent, occurs independently of hypersensitive cell death in sugar beet; Bargabus RL et al.; Response of sugar beet cultivars C40 and USH11 to syringe infiltration of live and dead Bacillus mycoides isolate Bac J, a biological control agent, and virulent and avirulent isolates of Erwinia carotovora pv . betavasculorum was measured by monitoring systemic acquired resistance control of Cercospora beticola, specific activity of chitinase and beta-glucanase, the oxidative burst, and hypersensitive cell death at the infiltration site . Priming sugar beet with B . mycoides Bac J (1 x 10(8) cells/ml) and avirulent isolates of E . carotovora pv . betavasculorum (1 x 10(6) cells/ml) reduced C . beticola symptoms by nearly 70% on distal, untreated leaves . Systemic resistance responses elicited by live B . mycoides Bac J and avirulent E . carotovora pv . betavasculorum isolates, measured by assays for chitinase and beta-glucanase, were statistically equivalent, and biphasic hydrogen peroxide production was observed . Although similar in timing, the second hydrogen peroxide burst was twofold lower for B . mycoides Bac J than for avirulent E . carotovora pv . betavasculorum . Hypersensitive cell death was elicited by avirulent E . carotovora pv . betavasculorum but not B . mycoides Bac J . An oxidative burst was elicited by spray-applied B . mycoides Bac J under both light and green light conditions, indicating that the signal produced by B . mycoides Bac J was not reliant on the stomata for entry into sugar beet . A working model for signal delivery and systemic resistance induction by B . mycoides Bac J in sugar beet is proposed.

Mol Plant Microbe Interact, 2003 Dec, 16(12), 1106 - 17
GacA, the response regulator of a two-component system, acts as a master regulator in Pseudomonas syringae pv . tomato DC3000 by controlling regulatory RNA, transcriptional activators, and alternate sigma factors; Chatterjee A et al.; Concerted investigations of factors affecting host-pathogen interactions are now possible with the model plant Arabidopsis thaliana and its model pathogen Pseudomonas syringae pv . tomato DC3000, as their whole genome sequences have become available . As a prelude to analysis of the regulatory genes and their targets, we have focused on GacA, the response regulator of a two-component system . The DC3000 gene was cloned by testing for the reversal of phenotypes of an Erwinia GacA- mutant . A GacA- mutant of DC3000 constructed by marker exchange produces much-reduced levels of transcripts of three alternate sigma factors: HrpL, required for the production of effector proteins and their translocation via the type III secretion system; RpoS, required for stress responses and secondary metabolite production; and RpoN, required for an assortment of metabolic processes and expression of hrpL . GacA deficiency also reduces the expression of hrpR and hrpS, which specify enhancer-binding proteins of the NtrC family required for hrpL transcription; ahlI and ahlR, the genes for quorum sensing signal; salA, a regulatory gene known to control virulence; CorS, a sensor kinase; CorR, the cognate response regulator that controls coronatine biosynthetic genes; and rsmB and rsmZ, which specify untranslatable regulatory RNA species . gacA expression itself is regulated by environmental conditions in DC3000, since transcript levels are affected by growth phase and media composition . The observations that high levels of gacA RNA occur in the hrp-inducing medium and GacA deficiency reduces the levels of rpoS expression implicate an important role of GacA in stress responses of DC3000 . Consistent with the effects on hrpL expression, the GacA- mutant produces lower levels of transcripts of avr, hrp, and hop genes controlled by HrpL . In addition, GacA deficiency results in reduced levels of transcripts of several HrpL-independent genes . As would be expected, these effects on gene expression cause drastic changes in bacterial behavior: virulence towards A . thaliana and tomato; multiplication in planta; efficiency of the induction of the hypersensitive reaction (HR); production of pigment and N-acyl-homoserine lactone (AHL), the presumed quorum-sensing signal; and swarming motility . Our findings establish that GacA, located at the top in a regulatory cascade in DC3000, functions as a central regulator by controlling an assortment of transcriptional and posttranscriptional factors.

Proteins, 2003 Dec 1, 53(4), 830 - 9
Tomato pectin methylesterase: modeling, fluorescence, and inhibitor interaction studies-comparison with the bacterial (Erwinia chrysanthemi) enzyme; D'Avino R et al.; The molecular model of Lycopersicon esculentum (tomato) pectin methylesterase (PME) was built by using the X-ray crystal structure of PME from the phytopathogenic bacterium Erwinia chrysanthemi as a template . The overall structure and the position of catalytically important residues (Asp132, Asp 153, and Arg 221, located at the bottom of the active site cleft) are conserved . Instead, loop regions forming the walls of the catalytic site are much shorter and form a less deep cleft, as already revealed by the carrot PME crystal structure . The protein inhibitor of pectin methylesterase (PMEI) isolated from kiwi fruit binds tomato PME with high affinity . Conversely, no complex formation between the inhibitor and PME from E . chrysanthemi is observed, and the activity of this enzyme is unaffected by the presence of the inhibitor . Fluorescence quenching experiments on tomato PME and on PME-PMEI complex suggest that tryptophanyl residues present in the active site region are involved in the interaction and that the inhibitor interacts with plant PME at the level of the active site . We also suggest that the more open active site cleft of tomato PME allows the interaction with the inhibitor . Conversely, the narrow and deep cleft of the active site of E . chrysanthemi PME hinders this interaction . The pH-dependent changes in fluorescence emission intensity observed in tomato PME could arise as the result of protonation of an Asp residue with unusually high pKa, thus supporting the hypothesis that Asp132 acts as acid/base in the catalytic cycle .

Mol Microbiol, 2003 Nov, 50(3), 795 - 807
The PmrA-PmrB two-component system responding to acidic pH and iron controls virulence in the plant pathogen Erwinia carotovora ssp . carotovora; Hyytiainen H et al.; Efficient response to environmental cues is crucial to successful infection by plant-pathogenic bacteria such as Erwinia carotovora ssp . carotovora . The expression of the main virulence genes of this pathogen, encoding extracellular enzymes that degrade the plant-cell wall, is subject to complex regulatory machinery where two-component systems play an important role . In this paper, we describe for the first time the involvement of the PmrA-PmrB two-component system in regulation of virulence in a plant-pathogenic bacterium . Disruption of pmrB resulted in reduced virulence both in potato and in Arabidopsis . This is apparently due to reduced production of the extracellular enzymes . In contrast, a pmrA mutant exhibited increased levels of these enzymes implying negative regulation of the corresponding genes by PmrA . Furthermore, the pmrB but not pmrA mutant exhibited highly increased resistance to the cationic antimicrobial peptide polymyxin B suggesting alterations in cell surface properties of the mutant . A similar increase of polymyxin resistance was detected in the wild type at mildly acidic pH with low Mg2+ . Functional pmrA is essential for bacterial survival on excess iron at acidic pH, regardless of the Mg2+ concentration . We propose that PmrA-PmrB TCS is involved in controlling of bacterial response to external pH and iron and is crucial for bacterial virulence and survival in planta.

Bioresour Technol, 2004 Jan, 91(1), 1 - 29
Development of biobased products; Montgomery R; Research conducted over the past seven years by the biotechnology byproducts consortium (BBC) addresses its mission to investigate the opportunities to add value to agricultural products, byproducts and coproducts and to manage the wastewater arising from agribusinesses in an environmentally favorable way . Since a wide variety of research approaches have been taken, the results are collected in five topic groups: (1) bioremediation that includes anaerobic fermentations of wastes to produce methane and hydrogen, the genetics of methanogenesis and in situ remediation of contaminated aquifer systems, landfill leachates and industrial effluents; (2) land application of fermentation byproducts and their use in animal feeds; (3) biocatalytic studies of transformations of components of corn and soybean oils, peroxidases present in plant products, such as soybean hulls; (4) biochemical reactions for the production of de-icers from industrial water streams, biodiesel production from fats and greases, biodegradable plastics from polymerizable sugar derivatives, single cell foods derived from fungal growth on waste streams, and bacterial polysaccharides from Erwinia species; (5) separation and recovery of components by membrane technologies.

Arch Microbiol, 2003 Dec, 180(6), 490 - 3 Epub 2003 Oct 24.
Purification and characterization of a lactonase from Erwinia cypripedii 314B that hydrolyzes ( S)-5-oxo-2-tetrahydrofurancarboxylic acid; Mochizuki K; A bacterium, strain 314B, able to assimilate ( S)-5-oxo-2-tetrahydrofurancarboxylic acid was isolated from soil and identified as Erwinia cypripedii . A lactonase hydrolyzing ( S)-5-oxo-2-tetrahydrofurancarboxylic acid to l-alpha-hydroxyglutaric acid was purified 63-fold with 2% recovery from crude extracts of this bacterium to homogeneity as judged by SDS-PAGE . The molecular masses estimated by SDS-PAGE and gel filtration were 41 kDa and 79 kDa, respectively . The maximum activity was observed at pH 6.5-7.5 and 35-45 degrees C . The enzyme showed lower activity toward dl-2-oxotetrahydrofuran-4,5-dicarboxylic acid, but did not act on ( R)-5-oxo-2-tetrahydrofurancarboxylic acid and other natural and synthetic lactones tested.

Mol Genet Genomics, 2003 Nov, 270(3), 263 - 72 Epub 2003 Oct 24.
Characterization of the hrp pathogenicity cluster of Erwinia carotovora subsp . carotovora: high basal level expression in a mutant is associated with reduced virulence; Lehtimaki S et al.; Extracellularly targeted proteins are crucial for virulence of gram-negative phytopathogenic bacteria . Erwinia carotovora subsp . carotovora employs the so-called type II (GSP) pathway to secrete a number of pectinases and cellulases, which cause the typical tissue maceration symptoms of soft-rot disease . The type III (hrp) pathway is the major virulence determinant in the genera Pseudomonas, Ralstonia and Xanthomonas, and in non-macerating species of Erwinia . The hrp cluster was recently partially characterized from E . carotovora sp . carotovora, and shown to affect virulence during early stages of infection . Here we have isolated and characterized 15 hrp genes comprising the remaining part of the cluster . The genes hrpL, hrpXY and hrpS were deduced to be transcribed as separate units, whereas the 11 remaining genes from hrpJ to hrcU form a single large operon . The hrpX gene, which codes for the sensory kinase of the two-component regulatory locus hrpXY was insertionally inactivated by placing a transposon (entranceposon) in the gene . The resulting mutant bacterium expresses the hrp genes at high basal level even in a non-inducing medium . This relative overexpression was shown to be due to the hrpX::entranceposon insertion causing enhanced transcription of the downstream hrpY gene . The hrpX(-)-hrpYC mutant bacterium exhibited a slower growth rate and the appearance of disease symptoms in infected Arabidopsis plants was delayed, as compared to the wild-type strain . The need for hrp gene expression for virulence has been documented in both non-macerating plant pathogens and in soft-rotting Erwinia sp . but this is the first demonstration that high basal-level expression of hrp -regulated genes may actually have a negative impact on disease progress in a susceptible host plant.

Curr Microbiol, 2003 Sep, 47(3), 214 - 9
Genus- and isolate-specific real-time PCR quantification of Erwinia on leaf surfaces of English oaks (Quercus robur L.); Heuser T et al.; In order to quantify pathogenic epiphytic bacteria on leaf surfaces of the important European forest tree Quercus robur without time-intensive cultivation and separation of microorganisms, methods were developed to selectively quantify DNA copy numbers of the genus Erwinia in DNA isolated from the leaf surface . By using the combination of the two different real-time PCR techniques SYBR-Green and TaqMan, methods were developed not only to allow quantification of the total DNA copy number of Erwinia on the oak leaf surface, but also to distinguish between two significantly different groups of Erwinia strains . In the present work, these techniques were successfully applied to quantify the copy number of the genus Erwinia and its subgroups compared with the total bacteria number in DNA samples extracted from the upper leaf surface of English oaks collected on the 4th of June 2001 (Julian day 155).

Biotechnol Appl Biochem . 2003 Oct 15; {Epub ahead of print}
One-step purification and kinetic properties of the recombinant L-asparaginase from Erwinia carotovora; Krasotkina J et al.; The recombinant L-asparaginase from Erwinia carotovora (ECAR-LANS) is a perspective therapeutic enzyme for leukemia treatment . Efficient and economical scheme was developed for purification of ECAR-LANS, cloned and expressed in Eschericha coli . More than 90% of purity, complemented with 72% of active enzyme recovery was achieved with the single chromatographic step of purification . The activity of purified L-asparaginase was 630 IU/mg . The ECAR-LANS K m was 98 x 10 -6 M for the main physiological substrate L-Asn and 3400 x 10 -6 M for L-Gln . ECAR-LANS was found to have low relative glutaminase activity (1.2%) at physiological concentrations of L-Asn and L-Gln in the blood . The kinetic studies of ECAR-LANS show that the recombinant asparaginase combines the main advantages of Erwinia chrysanthemi and Eschericha coli L-asparaginases II, currently used in the acute lymphoblastic leukemia treatment.

Biotechnol Adv, 1990, 8(1), 29 - 57
Genetic designs for product formation in recombinant microbes; Murooka Y; Several new bacterial host-vector systems for Klebsiella, Erwinia, Xanthomonas, Nocardia, and Streptomyces have been developed . With these host-vector systems, a strain of Klebsiella, which overproduces the extracellular starch-debranching enzyme, pullulanase, has been developed . The gene for cholesterol oxidase was cloned and used to develop a strain of Streptomyces lividans that extracellularly produces the enzyme, cholesterol oxidase, which is utilized to process cholesterol and diagnostically . The genes for these two enzymes were sequenced, and several interesting facts about their structures and secretory mechanisms were found . For expression of mammalian gene products, the expression vectors . pYM001 to pYM008, containing the lambda P(R)P(L) promoter, which is controlled by a thermolabile repressor, have been developed . The activities of these promoters were compared in various bacterial strains with the galK monitoring system . E . coli promoters, such as lac, trp, tac, lambda P(R), P(L), and P(R)P(L), were found to be expressed in other enteric bacteria and in Bacillus subtilis . With these expression vectors, the vesicular stomatitis virus-nucleocapsid, monkey metallothionein, and human apolipoprotein A1 genes were expressed in E . coli.

Biotechnol Adv, 1988, 6(1), 29 - 37
Approach to maceration mechanism in enzymatic pulping of bast fibers by alkalophilic pectinolytic enzymes produced by Erwinia species; Kobayashi Y et al.; Tissue maceration was generally elucidated by the action of endo-polygalacturonase and endo-pectate or -pectin lyase (endo-PAL or -PNL) . In a process of screening of Erwinia and Pseudomonas strains for enzymatic pulping of pectocellulosic bast fibers, it was found that their PAL productivity was not completely related with defibration activity, i.e., the fact that an E.chrysanthemi strain showed high PAL productivity but possessed rather low defibration activity . Moreover, defibration activity was parallel to the amount of neutral sugars released during pulping . Based on these fact, the maceration or enzymatic pulping of basts was estimated to proceed not only by cleavage of interfiber bonding cause by PAL action but also another factors . Among three possibilities proposed on the maceration mechanism of basts, it was elucidated by a concerted action of PAL and PNL with an aid of xylanase . In addition, a quantitative determinative method of maceration activity toward basts was also presented.

Res Microbiol, 2003 Oct, 154(8), 587 - 92
Identification of Klebsiella oxytoca using a specific PCR assay targeting the polygalacturonase pehX gene; Kovtunovych G et al.; Bacteria of the genus Klebsiella are important opportunistic pathogens responsible for nosocomial infections that are increasingly resistant to antimicrobial agents . Distinctive identification of the species K . oxytoca, K . pneumoniae, K . planticola, K . ornithinolytica and K . terrigena is difficult based on phenotypic tests and misidentifications are frequent in routine clinical microbiology . We developed a specific method to discriminate K . oxytoca from the other species of the genus Klebsiella, based on the PCR amplification of the polygalacturonase (pehX) gene . A PCR amplicon of 344 bp was obtained in all 35 K . oxytoca strains tested, but in none of the 29 K . pneumoniae, 12 K . planticola/K . ornithinolytica and 7 K . terrigena strains tested . The test was also negative for polygalacturonate-degrading species of the genus Erwinia . Analysis of 24 strains designated as K . pneumoniae from international collections (NCTC, PZH) revealed previous misidentification of six K . oxytoca strains . Key biochemical tests fully confirmed the pehX PCR results . The new K . oxytoca identification assay should be useful for both clinical and ecological monitoring of K . oxytoca strains, as well as for controlling the previous identification of collection strains.

Biosci Biotechnol Biochem, 2003 Sep, 67(9), 1950 - 8
Properties of a novel extracellular cell-free ice nuclei from ice-nucleating Pseudomonas antarctica IN-74; Muryoi N et al.; Some ice-nucleating bacterial strains, including Pantoea ananatis (Erwinia uredovora), Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for the ability to shed ice nuclei into the growth medium . A novel ice-nucleating bacterium, Pseudomonas antarctica IN-74, was isolated from Ross Island, Antarctica . Cell-free ice nuclei from P . antarctica IN-74 were different from the conventional cell-free ice nuclei and showed a unique characterization . Cell-free ice nuclei were purified by centrifugation, filtration (0.45 microm), ultrafiltration, and gel filtration . In an ice-nucleating medium in 1 liter of cell culture, maximum growth was obtained with the production of 1.9 mg of cell-free ice nuclei . Ice nucleation activity in these cell-free ice nuclei preparations was extremely sensitive to pH . It was demonstrated that the components of cell-free ice nuclei were protein (33%), saccharide (12%), and lipid (55%), indicating that cell-free ice nuclei were lipoglycoproteins . Also, carbohydrate and lipid stains showed that cell-free ice nuclei contained both carbohydrate and lipid moieties.

Environ Microbiol, 2003 Oct, 5(10), 1016 - 21
The influence of antibiotic production and pre-emptive colonization on the population dynamics of Pantoea agglomerans (Erwinia herbicola) Eh1087 and Erwinia amylovora in planta; Giddens SR et al.; Stigma colonization by Erwinia amylovora is the crucial first step in the development of most fire blight infections in apple and pear trees . Suppression at this point of the disease process by antagonists of E . amylovora, such as Pantoea agglomerans (Erwinia herbicola) strain Eh1087, is a rational approach to control fire blight . We tested the hypothesis that the ability of E . amylovora to compete with Eh1087 for colonization of a stigma is reduced by the potential for Eh1087 to produce the phenazine antibiotic, d-alanylgriseoluteic acid (AGA) . In competition experiments on the stigmas of apple flowers, E . amylovora was significantly less successful against Eh1087 (AGA+) than against EhDeltaAGA (AGA-) . Further experiments to test the importance of pre-emptive colonization of the stigma by either the pathogen or the antagonist suggested that AGA production significantly enhanced the competitiveness of Eh1087 when it was applied at the same time or 24 h before the pathogen . We also found that pre-emptive stigma colonization by either the pathogen or the antagonist resulted in a population that was resilient to subsequent invasion by a second species suggesting that niche exclusion has a dominant influence on the dynamics of bacterial populations on stigmas.

Carbohydr Res, 2003 Sep 10, 338(19), 2025 - 7
Structure of the O-polysaccharide of Erwinia carotovora ssp . carotovora GSPB 436; Senchenkova SN et al.; The O-polysaccharide of a phytopathogenic bacterium, Erwinia carotovora ssp . carotovora GSPB 436, was studied by sugar and methylation analysis, along with 1H and 13C NMR spectroscopy . The following structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established: {carbohydrate structure in text} The O-polysaccharide contains a minor proportion of 4-O-methylrhamnose, which is suggested to terminate the polymer main chain.

Phytochemistry, 2003 Oct, 64(3), 709 - 16
Formation of novel flavonoids in apple (Malusxdomestica) treated with the 2-oxoglutarate-dependent dioxygenase inhibitor prohexadione-Ca; Roemmelt S et al.; Novel flavonoids were formed in young leaves of apple (Malusxdomestica) after treatment with the dioxygenase inhibitor prohexadione-Ca, which is known to reduce the incidence and severity of fire blight caused by Erwinia amylovora and other plant diseases . The compounds were isolated and identified as luteoliflavan, luteoliflavan 5-glucoside, eriodictyol 7-glucoside and 6"-O-trans-p-coumaroyleriodictyol 3'-glucoside . These flavonoids represent a novel biosynthetic pathway in apple leading to the formation of 3-deoxyflavans . Concomitantly, the content of regularly occurring phenylpropanoids is also influenced by prohexadione-Ca with increasing amounts of hydroxycinnamic acids and decreasing flavan-3-ols and flavonols . The altered flavonoid metabolism may be related to the lowered pathogen incidence though the isolated novel flavonoids do not exhibit antibacterial activity.

J Bacteriol, 2003 Oct, 185(19), 5772 - 8
Erwinia chrysanthemi tolC is involved in resistance to antimicrobial plant chemicals and is essential for phytopathogenesis; Barabote RD et al.; TolC is the outer-membrane component of several multidrug resistance (MDR) efflux pumps and plays an important role in the survival and virulence of many gram-negative bacterial animal pathogens . We have identified and characterized the outer-membrane protein-encoding gene tolC in the bacterial plant pathogen Erwinia chrysanthemi EC16 . The gene was found to encode a 51-kDa protein with 70% identity to its Escherichia coli homologue . The E . chrysanthemi gene was able to functionally complement the E . coli tolC gene with respect to its role in MDR efflux pumps . A tolC mutant of E . chrysanthemi was found to be extremely sensitive to antimicrobial agents, including several plant-derived chemicals . This mutant was unable to grow in planta and its ability to cause plant tissue maceration was severely compromised . The tolC mutant was shown to be defective in the efflux of berberine, a model antimicrobial plant chemical . These results suggest that by conferring resistance to the antimicrobial compounds produced by plants, the E . chrysanthemi tolC plays an important role in the survival and colonization of the pathogen in plant tissue.

Lett Appl Microbiol, 2003, 37(4), 340 - 3
Cold storage affects survival and growth of Erwinia amylovora on the calyx of apple; Taylor RK et al.; r.k . taylor and c.n . hale . 2003 . AIMS: To determine the effect of cold storage on the survival of Erwinia amylovora . METHODS AND RESULTS: The survival of E . amylovora was assessed during storage at 2 degrees C . Populations of E . amylovora inoculated into phosphate-buffered saline remained static, whereas in nutrient media populations increased at low temperatures . In contrast, populations of E . amylovora on tissue in the apple calyx decreased during cold storage . CONCLUSIONS: Erwinia amylovora has the ability, in nutrient media, to multiply at low temperatures . However, populations of E . amylovora on tissue in the apple calyx decrease with the time spent in cold storage . SIGNIFICANCE AND IMPACT OF THE STUDY: Cold storage of apples will provide assurance that mature fruit from orchards, free of fire blight, or even with low levels of fire blight, may be exported with a negligible risk of introducing the disease into countries free of fire blight.

Biotechnol Lett, 2003 Jul, 25(14), 1179 - 83
Enhanced conversion of sucrose to isomaltulose by a mutant of Erwinia rhapontici; Ahn SJ et al.; Mutagenesis of Erwinia rhapontici was performed to enhance the production of isomaltulose from sucrose . A mutant strain, BN 68089, was obtained through a screening process involving automated and miniaturized cultivation in Bioscreen C . This high-throughput, miniaturized screening system was optimized to identify the mutant strain, which had a conversion yield (90%) and productivity (194 g l(-1) h(-1)) . The BN 68089 mutant cells were immobilized in sodium alginate and when operated in a packed bed reactor gave a yield of 89% and a productivity of 144 g l(-1) h(-1) of at 30 degrees C, the optimal temperature . Immobilized BN 68089 cells exhibited 8% and 15% higher yield and productivity, respectively, than those of the wild-type strain.

Appl Environ Microbiol, 2003 Sep, 69(9), 5536 - 42
Plasmid transfer from Pseudomonas putida to the indigenous bacteria on alfalfa sprouts: characterization, direct quantification, and in situ location of transconjugant cells; Molbak L et al.; The transfer of the plasmids pJKJ5 and TOL (pWWO) from Pseudomonas putida to the indigenous bacterial community on alfalfa sprouts was studied . Tagging with fluorescent protein markers allowed direct quantification of the introduced donor bacteria and of indigenous bacteria that had received the plasmids . The sprouts were observed for 9 days; during this time alfalfa seeds, inoculated with donor bacteria, developed to edible and subsequently decaying sprouts . The first transconjugants were detected on day 6 after donor inoculation and occurred at frequencies of 3.4 x 10(-4) and 2.0 x 10(-6) transconjugant cells per donor cell for pKJK5::gfp and TOL::gfp, respectively . Confocal laser scanning microscopy revealed that the sprouts were heavily colonized with donors and that most transconjugants were located around the hypocotyl and root areas . Randomly selected members of the indigenous bacterial community from both inoculated and uninoculated sprouts, as well as a representative part of the community that had received the plasmids, were characterized by polymorphisms of PCR-amplified ribosomal DNA (rDNA) spacer regions between the 16S and 23S genes, followed by partial 16S rDNA sequencing . This showed that the initially dominating genera Erwinia and Paenibacillus were gradually replaced by Pseudomonas on the fully developed sprouts . Transconjugants carrying either of the investigated plasmids mainly belonged to the genera Pseudomonas and ERWINIA: The numbers of transconjugant cells did not reach detectable levels until 6 days after the onset of germination, at which point these species constituted the majority of the indigenous bacteria . In conclusion, the alfalfa sprouts provided an environment that allowed noteworthy frequencies of plasmid transfer from P . putida in the absence of selective pressure that could favor the presence of the investigated plasmids.

Luminescence, 2003 Jul-Aug, 18(4), 207 - 13
Expression of firefly luciferase gene in Erwinia amylovora; Gentilomi G et al.; In this study we describe an efficient stable genetic transformation of the phytopathogenic bacterium Erwinia amylovora using a recombinant expression vector encoding the firefly luciferase gene of Photinus pyralis, which is further controlled by IPTG-inducible promoter . Stably transformed E . amylovora cells maintain the same infectivity as the wild-type strain and, after induction with IPTG, produce luciferase . Luminescence produced by the action of luciferase on an exogenous substrate was easily detectable by a simple and rapid bioluminescent assay (BL) . The transformed E . amylovora strain maintains the same high emission level, even after passage in pears, until about 15 days post-infection . Our findings therefore show that the luciferase assay can be conveniently used to follow the bacterial movement in plant tissue and its dissemination in controlled environments.

Mikrobiologiia, 2003 May-Jun, 72(3), 343 - 7
{The bactericidal activity of lectins from nitrogen-fixing bacilli}; Karpunina LV et al.; Lectins I and II isolated from the nitrogen-fixing soil bacterium Paenibacillus polymyxa 1460 were found to be able to suppress the growth of Rhizobium leguminosarum 252 and Bacillus subtilis 36 at nearly all the concentrations tested (from 1 to 10 micrograms/ml) . Lectin I was also inhibitory to Azospirillum brasilense 245 and Erwinia carotovora subsp . citrulis 603, while lectin II exerted bactericidal activity against Xanthomonas campestris B-610 and B-611 and A . brasilense 245 . The bacillar lectins incubated with Rhizobium and Azospirillum cells caused leakage of low-molecular-weight substances from the cells, presumably resulting from impairment of the membrane barrier function . We believe that one of the possible mechanisms of the bacterial growth inhibition by lectins is mediated by the lectin-specific receptors occurring on the bacterial membrane, whose interaction with the lectin molecules induces conformational alterations in the membrane and concurrent malfunction of the metabolism of bacterial cells.

Leukemia, 2003 Aug, 17(8), 1583 - 8
Evaluation of immunologic crossreaction of antiasparaginase antibodies in acute lymphoblastic leukemia (ALL) and lymphoma patients; Wang B et al.; To evaluate how well antibodies to one asparaginase preparation predict or correlate with antibodies to another preparation in acute lymphoblastic leukemia (ALL) and lymphoma patients who did and did not have hypersensitivity reactions during chemotherapy . In all, 24 children with newly diagnosed ALL or lymphoma, who received Escherichia coli asparaginase 10 000 IU/m(2) IM thrice weekly for nine doses as part of multiagent induction and reinduction chemotherapy, and seven monthly doses during the first 7 months of continuation treatment, were studied . Plasma samples were collected at postinduction and at postreinduction . Six of 24 patients had no overt clinical reactions (nonreacting) and received only the E . coli preparation . Of these, 18 patients who had allergic reactions were switched to Erwinia asparaginase . A total of 18 patients had an anaphylactoid reaction to Erwinia asparaginase and were switched to receive polyethylene glycol (PEG) asparaginase . Antibody levels were measured by enzyme-linked immunoadsorbent assay against all the three asparaginase preparations . At postinduction, antibodies against E . coli were higher in reacting patients (0.063+/-0.066) than in nonreacting patients (0.019+/-0.013) (P=0.03) . At postreinduction, anti-Erwinia antibodies were significantly higher in reacting patients (0.431+/-0.727) than in nonreacting patients (0.018+/-0.009) (P=0.007) . Anti-E . coli antibodies correlated with anti-PEG antibodies at postinduction (r=0.714, P<0.001) and at postreinduction (r=0.914, P<0.001), but did not correlate with anti-Erwinia antibodies at postinduction (r=0.119, P=0.580) and at postreinduction (r=0.078, P=0.716) . The results indicate a crossreactivity between patient antibodies raised against natural E . coli and PEG asparaginase but not Erwinia asparaginase.

J Biol Chem, 2003 Oct 3, 278(40), 38352 - 9 Epub 2003 Jul 21.
Biogenesis of Fe-S cluster by the bacterial Suf system: SufS and SufE form a new type of cysteine desulfurase; Loiseau L et al.; Biosynthesis of iron-sulfur clusters (Fe-S) depends on multiprotein systems . Recently, we described the SUF system of Escherichia coli and Erwinia chrysanthemi as being important for Fe-S biogenesis under stressful conditions . The SUF system is made of six proteins: SufC is an atypical cytoplasmic ABC-ATPase, which forms a complex with SufB and SufD; SufA plays the role of a scaffold protein for assembly of iron-sulfur clusters and delivery to target proteins; SufS is a cysteine desulfurase which mobilizes the sulfur atom from cysteine and provides it to the cluster; SufE has no associated function yet . Here we demonstrate that: (i) SufE and SufS are both cystosolic as all members of the SUF system; (ii) SufE is a homodimeric protein; (iii) SufE forms a complex with SufS as shown by the yeast two-hybrid system and by affinity chromatography; (iv) binding of SufE to SufS is responsible for a 50-fold stimulation of the cysteine desulfurase activity of SufS . This is the first example of a two-component cysteine desulfurase enzyme.

Biochemistry, 2003 Jul 22, 42(28), 8411 - 22
First crystallographic structure of a xylanase from glycoside hydrolase family 5: implications for catalysis; Larson SB et al.; The room-temperature structure of xylanase (EC 3.2.1.8) from the bacterial plant pathogen Erwinia chrysanthemi expressed in Escherichia coli, a 45 kDa, 413-amino acid protein belonging to glycoside hydrolase family 5, has been determined by multiple isomorphous replacement and refined to a resolution of 1.42 A . This represents the first structure of a xylanase not belonging to either glycoside hydrolase family 10 or family 11 . The enzyme is composed of two domains similar to most family 10 xylanases and the alpha-amylases . The catalytic domain (residues 46-315) has a (beta/alpha)(8)-barrel motif with a binding cleft along the C-terminal side of the beta-barrel . The catalytic residues, Glu165 and Glu253, determined by correspondence to other family 5 and family 10 glycoside hydrolases, lie inside this cleft on the C-terminal ends of beta-strands 4 and 7, respectively, with an O(epsilon)2...O(epsilon)1 distance of 4.22 A . The smaller domain (residues 31-43 and 323-413) has a beta(9)-barrel motif with five of the strands interfacing with alpha-helices 7 and 8 of the catalytic domain . The first 13 N-terminal residues form one beta-strand of this domain . Residues 44, 45, and 316-322 form the linkers between this domain and the catalytic domain.

Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1339 - 42 Epub 2003 Jun 27.
Effect of mutations in the T1.5 loop of pectate lyase A from Erwinia chrysanthemi EC16; Dehdashti SJ et al.; Pectate lyase A (PelA) is a pectate-degrading enzyme secreted by plant pathogens . PelA from Erwinia chrysanthemi has 61% amino-acid identity and a conserved structural similarity to pectate lyase E (PelE) . Although similar in structure and sequence, the enzymatic characteristics of PelA differ from those for PelE . A structural alignment of PelA and PelE reveals differences in the T1.5 loop . The sequence of the T1.5 loop in PelA was mutated to the homologous sequence in PelE . The crystal structure of the PelA T1.5 mutant has been solved to 1.6 and 2.9 A resolution . The enzymatic and structural properties of the T1.5 mutant are discussed.

Acta Crystallogr D Biol Crystallogr, 2003 Jul, 59(Pt 7), 1256 - 8 Epub 2003 Jun 27.
Expression, purification, crystallization and preliminary X-ray crystallographic studies of a psychrophilic cellulase from Pseudoalteromonas haloplanktis; Violot S et al.; The Antarctic psychrophile Pseudoalteromonas haloplanktis produces a cold-active cellulase . To date, a three-dimensional structure of a psychrophilic cellulase has been lacking . Crystallographic studies of this cold-adapted enzyme have therefore been initiated in order to contribute to the understanding of the molecular basis of the cold adaptation and the high catalytic efficiency of the enzyme at low and moderate temperatures . The catalytic core domain of the psychrophilic cellulase CelG from P . haloplanktis has been expressed, purified and crystallized and a complete diffraction data set to 1.8 A has been collected . The space group was found to be P2(1)2(1)2(1), with unit-cell parameters a = 135.1, b = 78.4, c = 44.1 A . A molecular-replacement solution, using the structure of the mesophilic counterpart Cel5A from Erwinia chrysanthemi as a search model, has been found.

Mol Microbiol, 2003 Jul, 49(2), 347 - 57
The Erwinia chrysanthemi phoP-phoQ operon plays an important role in growth at low pH, virulence and bacterial survival in plant tissue; Llama-Palacios A et al.; We have studied the role of acidic pH as a barrier for the colonization of the plant apoplast by Erwinia chrysanthemi . A minitransposon containing a promoterless reporter gene, gus, was used for random mutagenesis of the bacterial genome . An acid-sensitive mutant, named BT119, was isolated and had the following differential features with respect to the wild-type strain: (i) inability to grow at pH </= 5.5; (ii) decreased survival at acid pH and in plant tissues; (iii) increased susceptibility to antimicrobial peptides; (iv) decreased virulence in chicory leaves and pear fruits; (v) reduced polygalacturonase production; and (vi) reduced ability to alkalinize chicory tissues after infection . The sequence of the interrupted gene was highly similar to the phoQ gene, which is involved in environmental sensing in several bacteria, such as Yersinia pseudotuberculosis, Erwinia carotovora, Salmonella typhimurium and Escherichia coli and thus, this designation was used for the E . chrysanthemi system . This gene was induced at low Mg(2+) concentrations and in planta . These results suggest that E . chrysanthemi PhoP-PhoQ system plays an important role in bacterial survival in plant tissues during the initial infection stages.

Plant Mol Biol, 2003 May, 52(1), 177 - 89
A novel potato defence-related alcohol:NADP+ oxidoreductase induced in response to Erwinia carotovora; Montesano M et al.; Identification of Solanum tuberosum genes responsive to culture filtrates (CF) from Erwinia carotovora subsp . carotovora led to the isolation of a full-length cDNA with high sequence similarity to several alcohol dehydrogenases . Accumulation of transcripts corresponding to this defence-related alcohol dehydrogenase (drd-1) was rapidly induced in CF-treated and wounded plants . The gene was also responsive to molecules involved in defence signalling such as salicylic acid, methyl jasmonate and ethylene . To elucidate the biochemical function of DRD-1, its cDNA was expressed in Escherichia coli . Enzymatic assays revealed that DRD-1 is an alcohol:NADP+ oxidoreductase with preference for various aromatic and aliphatic aldehydes . The enzyme exhibited high activity with several aldehydes including 2-methoxybenzaldehyde, 3-methoxybenzaldehyde, salicylaldehyde, o-vanillin, cinnamaldehyde, hydrocinnamaldehyde, hexanal and octanal . Identification of the reaction product by thin-layer chromatography confirmed the reduction of aldehydes to alcohols . Enzymatic activity measured with 2-methoxybenzaldehyde as a substrate was increased in salicylic acid- or methyl jasmonate-treated plants . These data suggest that DRD-1 may play an important role in potato defence response to Erwinia carotovora.

Biochemistry, 2003 Jul 1, 42(25), 7836 - 47
Inhibition and alternate substrate studies on the mechanism of carbapenam synthetase from Erwinia carotovora; Gerratana B et al.; The Erwinia carotorova carA, carB, and carC gene products are essential for the biosynthesis of (5R)-carbapen-2-em-3-carboxylic acid, the simplest carbapenem beta-lactam antibiotic . CarA (hereafter named carbapenam synthetase) has been proposed to catalyze formation of (3S,5S)-carbapenam-3-carboxylic acid from (2S,5S)-5-carboxymethyl proline based on characterization of the products of fermentation experiments in Escherichia coli cells transformed with pET24a/carB and pET24a/carAB, and on sequence homology to beta-lactam synthetase, an enzyme that catalyzes formation of a monocyclic beta-lactam ring with concomitant ATP hydrolysis . In this study, we have purified recombinant carbapenam synthetase and shown in vitro that it catalyzes the ATP-dependent formation of (3S,5S)-carbapenam-3-carboxylic acid from (2S,5S)-5-carboxymethyl proline . The kinetic mechanism is Bi-Ter where ATP is the first substrate to bind followed by (2S,5S)-5-carboxymethyl proline and PPi is the last product released based on initial velocity, product and dead-end inhibition studies . The reactions catalyzed by carbapenam synthetase with different diastereomers of the natural substrate and with alternate alpha-amino diacid substrates were studied by HPLC, ESI mass spectrometry, and steady-state kinetic analysis . On the basis of these results, we have proposed a role for each moiety of (2S,5S)-5-carboxymethyl proline for binding to the active site of carbapenam synthetase . Coupled enzyme assays of AMP and pyrophosphate release in the reactions catalyzed by carbapenam synthetase with adipic and glutaric acid, which lack the alpha-amino group, in the presence and absence of hydroxylamine support the formation of an acyladenylate intermediate in the catalytic cycle.

Lett Appl Microbiol, 2003, 37(1), 45 - 50
Survivability and long-term preservation of bacteria in water and in phosphate-buffered saline; Liao CH et al.; AIMS: To evaluate the suitability of using sterile water and phosphate-buffered saline (PBS) for preservation of bacteria pathogenic to plants or humans . METHODS AND RESULTS: The stationary-phase bacterial cells collected from rich agar media were transferred to 10 ml of sterile water or PBS (pH 7.2) containing KH2PO4, 15.44 microm; NaCl, 1.55 mm; Na2HPO4, 27.09 microm in a screw-cap tube . The tubes were sealed with parafilm membranes and stored in the dark and at room temperature . Almost all the bacteria tested (148 strains), including Pseudomonas fluorescens, P . viridiflava, Erwinia spp., Xanthomonas campestris, Cytophaga johnsonae, Salmonella spp., Yersinia enterocolitica, Escherichia coli O157:H7, Listeria monocytogenes and Staphylococcus aureus, survived in water for at least several months and up to 16 years . A vast majority of the Gram-negative bacteria tested survived equally well in water and in PBS for at least 30 weeks . However, the populations of two Gram-positive bacteria {G(+)}, L . monocytogenes and Staph . aureus, declined more rapidly in water than in PBS . CONCLUSIONS: Plant- and human-pathogenic bacteria can be preserved in pure water or PBS for several years . G(+) bacteria appear to survive better in PBS than in water . SIGNIFICANCE AND IMPACT OF THE STUDY: The method described here is a simple and economical means for preservation of bacterial cultures, which is especially useful for laboratories not equipped with the lyophilizer or ultra-low freezer . Long-term survival of food-borne pathogens in water underlines the importance of water as a potential vehicle for transmitting the diseases.

Leuk Lymphoma, 2003 May, 44(5), 879 - 82
Successful treatment with Erwinia L-asparaginase for recurrent natural killer/T cell lymphoma; Matsumoto Y et al.; We describe a patient with natural killer (NK)/T cell lymphoma who relapsed after autologous peripheral blood stem cell transplantation (auto-PBSCT) and was successfully treated with Escherichia coli (E . coli) and Erwinia L-asparaginase . A 38-year-old male patient with ulcerated tumor at the left thigh was diagnosed as having nasal type NK/T cell lymphoma on the basis of histopathological and flowcytometric findings of tumor, revealing diffuse infiltration of atypical lymphoid cells into blood vessels and expression of CD7 and CD56 antigens, but not CD3 . He had tumor infiltration in the bone marrow and at the right lower lung field . After five cycles of CHOP (cyclophosphamide, doxorubicin, vincristine and prednisolone) therapy, the patient achieved complete remission and received high-dose chemotherapy with auto-PBSCT, although the tumor recurred in the right leg 10 months later . Despite salvage chemotherapy, followed by local irradiation and surgical amputation, a tumor recurred at the left upper gingiva 10 days after . Using E . coli L-asparaginase (6000 U/m2/day), the tumor regressed, fever was alleviated and the serum lactate dehydrogenase decreased to normal range after several days . The asparagine synthetase expression in tumor cells was immunohistochemically negative on paraffin-embedded tissues . Because of the anaphylactoid reaction developing after E . coli L-asparaginase, alternative Erwinia L-asparaginase (6000 U/m2/day) was administered, resulting in regression of tumor and fever lysis . L-asparaginase is a promising agent for the treatment of NK/T cell lymphoma.

Comp Biochem Physiol B Biochem Mol Biol, 2003 Jun, 135(2), 385 - 95
Distinct sucrose isomerases catalyze trehalulose synthesis in whiteflies, Bemisia argentifolii, and Erwinia rhapontici; Salvucci ME; Isomaltulose {alpha-D-glucopyranosyl-(1,6)-D-fructofuranose} and trehalulose {alpha-D-glucopyranosyl-(1,1)-D-fructofuranose} are commercially valuable sucrose-substitutes that are produced in several microorganisms by the palI gene product, a sucrose isomerase . Trehalulose also occurs in the silverleaf whitefly, Bemisia argentifoli, as the major carbohydrate in the insect's honeydew . To determine if the enzyme that synthesizes trehalulose in whiteflies was similar to the well-characterized sucrose isomerase from microbial sources, the properties of the enzymes from whiteflies and the bacterium, Erwinia rhapontici, were compared . Partial purification of both enzymes showed that the enzyme from whiteflies was a 116 kD membrane-associated polypeptide, in contrast to the enzyme from E . rhapontici, which was soluble and 66 kD . The enzyme from E . rhapontici converted sucrose to isomaltulose and trehalulose in a 5:1 ratio, whereas the enzyme from whiteflies produced only trehalulose . Unlike the E . rhapontici enzyme, the whitefly enzyme did not convert isomaltulose to trehalulose, but both enzymes catalyzed the transfer of fructose to trehalulose using sucrose as the glucosyl donor . The results indicate that trehalulose synthase from whiteflies is structurally and functionally distinct from the sucrose isomerases described in bacteria . The whitefly enzyme is the first reported case of an enzyme that converts sucrose to exclusively trehalulose.

Appl Environ Microbiol, 2003 Jun, 69(6), 3573 - 9
A C35 carotenoid biosynthetic pathway; Umeno D et al.; Upon coexpression with Erwinia geranylgeranyldiphosphate (GGDP) synthase in Escherichia coli, C(30) carotenoid synthase CrtM from Staphylococcus aureus produces novel carotenoids with the asymmetrical C(35) backbone . The products of condensation of farnesyldiphosphate and GDP, C(35) structures comprise 40 to 60% of total carotenoid accumulated . Carotene desaturases and carotene cyclases from C(40) or C(30) pathways accepted and converted the C(35) substrate, thus creating a C(35) carotenoid biosynthetic pathway in E . coli . Directed evolution to modulate desaturase step number, together with combinatorial expression of the desaturase variants with lycopene cyclases, allowed us to produce at least 10 compounds not previously described . This result highlights the plastic and expansible nature of carotenoid pathways and illustrates how combinatorial biosynthesis coupled with directed evolution can rapidly access diverse chemical structures.

Microbiology, 2003 Jun, 149(Pt 6), 1581 - 91
Genetic and biochemical characterization of PrtA, an RTX-like metalloprotease from Photorhabdus; Bowen DJ et al.; Proteases play a key role in the interaction between pathogens and their hosts . The bacterial entomopathogen Photorhabdus lives in symbiosis with nematodes that invade insects . Following entry into the insect, the bacteria are released from the nematode gut into the open blood system of the insect . Here they secrete factors which kill the host and also convert the host tissues into food for the replicating bacteria and nematodes . One of the secreted proteins is PrtA, which is shown here to be a repeats-in-toxin (RTX) alkaline zinc metalloprotease . PrtA has high affinity for artificial substrates such as casein and gelatin and can be inhibited by zinc metalloprotease inhibitors . The metalloprotease also shows a calcium- and temperature-dependent autolysis . The prtA gene carries the characteristic RTX repeated motifs and predicts high similarity to proteases from Erwinia chrysanthemi, Pseudomonas aeruginosa and Serratia marcescens . The prtA gene resides in a locus encoding both the protease ABC transporter (prtBCD) and an intervening ORF encoding a protease inhibitor (inh) . PrtA activity is detectable 24 h after artificial bacterial infection of an insect, suggesting that the protease may play a key role in degrading insect tissues rather than in overcoming the insect immune system . Purified PrtA also shows cytotoxicity to mammalian cell cultures, supporting its proposed role in bioconversion of the insect cadaver into food for bacterial and nematode development.

Biochim Biophys Acta, 2003 May 30, 1648(1-2), 43 - 54
Effects of salts on the function and conformational stability of shikimate kinase; Cerasoli E et al.; The unfolding of shikimate kinase (SK) from Erwinia chrysanthemi by urea and its subsequent refolding on dilution of the denaturing agent has been studied in detail {Eur . J . Biochem . 269 (2002) 2124} . Comparison of the effects of urea on the enzyme with those of guanidinium chloride (GdmCl) and NaCl indicated that chloride ions significantly weakened the binding of shikimate . This finding prompted a more detailed examination of the effects of salts on the structure, function and stability of the enzyme; the effects of NaCl and Na(2)SO(4) were investigated in detail . These salts have very small effects on the secondary structure as judged by far UV CD circular dichroism (CD), although the near UV CD spectra suggest that some limited changes in the environment of aromatic amino acids may occur . Both salts inhibit SK activity and analysis of the kinetic and substrate binding parameters point to a complex mechanism for the inhibition . Inclusion of salts leads to a marked stabilisation against unfolding of the enzyme by urea . When the enzyme is unfolded by incubation in 4 M urea, addition of NaCl or Na(2)SO(4) leads to a relatively slow refolding of the enzyme as judged by the regain of native-like CD and fluorescence . In addition, the refolded enzyme can bind shikimate, though more weakly than the native enzyme . However, the refolded enzyme does not appear to be capable of binding nucleotides, nor does it possess detectable catalytic activity . The refolding process brought about by addition of salt in the presence of 4 M urea is not associated with any change in the fluorescence of the probe 8-anilino-1-naphthalenesulfonic acid (ANS), indicating that an intermediate formed by hydrophobic collapse is unlikely to be significantly populated . The results point to both specific and general effects of salts on SK . These are discussed in the light of the structural information available on the enzyme.

Mikrobiologiia, 2003 Mar-Apr, 72(2), 199 - 205
{The study of Erwinia carotovora bacteriocins in the nalidixic acid resistant Erwinia carotovora}; Tovkach FI et al.; A novel approach is proposed for the study of the macromolecular bacteriocins of Erwinia carotovora (MCTVs) . The approach lies in that the bacteriocinogeny of pectolytic erwinia is studied using a lawn of a bacterial mutant resistant to nalidixic acid, an inducer of MCTVs . The high efficiency of this approach was demonstrated by studying carotovoricins in 104 different E . carotovora strains, 88% of which bear MCTVs, distinguished by the morphology of zones of induced lysis on a lawn of susceptible cells, the lysis pattern, and some other characteristics . Preliminary studies by this approach showed that there is no correlation between the occurrence of MCTVs in particular E . carotovora strains and the habitat of the host plants from which these strains were isolated . There are grounds to believe that the approach proposed can also be used for investigating bacterial lysogeny.

Plasmid, 2003 May, 49(3), 233 - 52
Nucleotide sequence based characterizations of two cryptic plasmids from the marine bacterium Ruegeria isolate PR1b; Zhong Z et al.; Two plasmids, 76 and 148 kb in size, isolated from Ruegeria strain PR1b were entirely sequenced . These are the first plasmids to be characterized from this genus of marine bacteria . Sequence analysis revealed a biased distribution of function among the putative proteins encoded on the two plasmids . The smaller plasmid, designated pSD20, encodes a large number of putative proteins involved in polysaccharide biosynthesis and export . The larger plasmid, designated pSD25, primarily encodes putative proteins involved in the transport of small molecules and in DNA mobilization . Sequence analysis revealed uncommon potential replication systems on both plasmids . pSD25, the first repABC-type replicon isolated from the marine environment, actually contains two repABC-type replicons . pSD20 contains a complex replication region, including a replication origin and initiation protein similar to iteron-containing plasmids (such as pSW500 from the plant pathogen Erwinia stewartii) linked to putative RepA and RepB stabilization proteins of a repABC-type replicon and is highly homologous to a plasmid from the phototrophic bacterium Rhodobacter sphaeroides . Given the nature of the putative proteins encoded by both plasmids it is possible that these plasmids enhance the metabolic and physiological flexibility of the host bacterium, and thus its adaptation to the marine sediment environment.

Mol Plant Microbe Interact, 2003 May, 16(5), 389 - 97
Peh production, flagellum synthesis, and virulence reduced in Erwinia carotovora subsp . carotovora by mutation in a homologue of cytR; Matsumoto H et al.; Erwinia carotovora subsp . carotovora is a causal agent of soft-rot diseases in a wide variety of plants . Here, we have isolated a new regulatory factor involved in the virulence of E . carotovora subsp . carotovora by in vivo insertional mutagenesis using a transposon Tn5 . The gene was homologous to cytR encoding a transcriptional repressor of nucleoside uptake and catabolism genes in Escherichia coli, Salmonella typhimurium, and Vibrio cholerae . Phenotypic characterization of a nonpolar deletion mutant of the cytR homologue (delta cytR) revealed that the delta cytR mutant produced a reduced level of polygalacturonase (Peh) and lost its motility compared to that in the parental strain . With electron microscopy, the delta cytR mutant was shown to be aflagellate . Furthermore, the expression of fliA and fliC (encoding sigma28 and flagellin, respectively) was also reduced in delta cytR mutant . The virulence of delta cytR mutant was reduced in Chinese cabbage and potato compared to that of the parental strain . These results suggest that the CytR homologue of E . carotovora subsp . carotovora positively controls Peh production and flagellum synthesis and plays an important role in its pathogenicity.

J Bacteriol, 2003 May, 185(10), 3091 - 100
PaeX, a second pectin acetylesterase of Erwinia chrysanthemi 3937; Shevchik VE et al.; Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls . Pectin is a complex polysaccharide . The main chain is constituted of galacturonate residues, and some of them are modified by methyl and/or acetyl esterification . Esterases are necessary to remove these modifications and, thus, to facilitate the further degradation of the polysaccharidic chain . In addition to PaeY, the first pectin acetylesterase identified in the E . chrysanthemi strain 3937, we showed that this bacterium produces a second pectin acetylesterase encoded by the gene paeX . The paeX open reading frame encodes a 322-residue precursor protein of 34,940 Da, including a 21-amino-acid signal peptide . Analysis of paeX transcription, by using gene fusions, revealed that it is induced by pectic catabolic products and affected by catabolite repression . The expression of paeX is regulated by the repressor KdgR, which controls all the steps of pectin catabolism; by the repressor PecS, which controls most of the pectinase genes; and by catabolite regulatory protein, the global activator of sugar catabolism . The paeX gene is situated in a cluster of genes involved in the catabolism and transport of pectic oligomers . In induced conditions, the two contiguous genes kdgM, encoding an oligogalacturonate-specific porin, and paeX are both transcribed as an operon from a promoter proximal to kdgM, but transcription of paeX can also be uncoupled from that of kdgM in noninduced conditions . PaeX is homologous to the C-terminal domain of the Butyrivibrio fibriosolvens xylanase XynB and to a few bacterial esterases . PaeX contains the typical box (GxSxG) corresponding to the active site of the large family of serine hydrolases . Purified PaeX releases acetate from various synthetic substrates and from sugar beet pectin . The PaeX activity increased after previous depolymerization and demethylation of pectin, indicating that its preferred substrates are nonmethylated oligogalacturonides . PaeX is mostly found in the periplasmic space of E . chrysanthemi . These data suggest that PaeX is mainly involved in the deacetylation of esterified oligogalacturonides that enter the periplasm by the KdgM porin.

Planta, 2003 May, 217(1), 75 - 83 Epub 2003 Feb 11.
Enhanced resistance to Phytophthora infestans and Alternaria solani in leaves and tubers, respectively, of potato plants with decreased activity of the plastidic ATP/ADP transporter; Conrath U et al.; Recently, it has been reported that tubers of transgenic potato ( Solanum tuberosum L.) plants with decreased activity of the plastidic ATP/ADP transporter (AATP1) contain less starch, despite having an increased glucose level {P . Geigenberger et al . (2001) Plant Physiol 125:1667-1678} . The metabolic alterations correlated with enhanced resistance to the bacterium Erwinia carotovora . Here it is shown that transgenic potato tubers, possessing less starch yet increased glucose levels due to the expression of a cytoplasm-localized yeast invertase, exhibit drastic susceptibility to E . carotovora . In addition, it is demonstrated that AATP1 anti-sense tubers show an increased capacity to ward off the pathogenic fungus Alternaria solani . In contrast to AATP1 anti-sense tubers, the corresponding leaf tissue does not show changes in carbohydrate accumulation . However, upon elicitor treatment, AATP1 anti-sense leaves possess an increased capacity to release H(2)O(2) and activate various defence-related genes, reactions that are associated with substantially delayed appearance of disease symptoms caused by Phytophthora infestans . Grafting experiments between AATP1 anti-sense plants and wild-type plants indicate the presence of a signal that is generated in AATP1 rootstocks and primes wild-type scions for potentiated activation of cellular defence responses in leaves . Together, the results suggest that (i) the enhanced pathogen tolerance of AATP1 anti-sense tubers is not due to "high sugar resistance", (ii) the increased disease resistance of AATP1 anti-sense tubers is effective against different types of pathogen and (iii) a systemic signal induced by antisensing the plastidic ATP/ADP transporter in potato tubers confers increased resistance to pathogens.

Meded Rijksuniv Gent Fak Landbouwkd Toegep Biol Wet, 2002, 67(2), 361 - 8
Bacteriocin Serratine-P as a biological tool in the control of fire blight Erwinia amylovora; Schoofs H et al.; Fire blight, caused by the bacterium Erwinia amylovora (Burill Winslow et al.), is the most important bacterial disease in European pear growing . It can cause a lot of damage in some countries on apple and on pear trees in orchards and also in the fruit tree nurseries . In Belgium, the disease is present since 1972 . Control of fire blight in Belgian fruit orchards is made on a broad basis of measurements in and around the fruit trees . The use of an antibiotic is allowed for application only during the primary blossom period under strict controlled regulations . The use of antobiotics in agriculture is strongly discussed on the European level today and will probably disappear in the near future . Therefore, the research on fire blight control concentrates on the possibilities of biological control with antagonistic bacteria such as Pantoea agglomerans (Erwinia herbicola), Bacillus subtilis or Pseudomonas syringae strain A 506 . The use of Serratine-P, a phage tail-like bacteriocin, produced by Serratia plymiticum, shows an interesting antibacterial activity against Erwinia amylovora . Its mode of action consists in the perforation of the cytoplasmic membrane of the target cell, inducing perturbations in cellular exchanges and a final lysis of the bacterial cell . In this paper some trials are discussed on the use of Serratine-P at different doses and on different infection types on pear trees . The results indicate interesting protection possibilities on blossom- and fruit infections.

Appl Environ Microbiol, 2003 Apr, 69(4), 2133 - 8
Bacteriophages of Erwinia amylovora; Gill JJ et al.; Fifty bacteriophage isolates of Erwinia amylovora, the causal agent of fire blight, were collected from sites in and around the Niagara region of southern Ontario and the Royal Botanical Gardens, Hamilton, Ontario . Forty-two phages survived the isolation, purification, and storage processes . The majority of the phages in the collection were isolated from the soil surrounding trees exhibiting fire blight symptoms . Only five phages were isolated from infected aerial tissue in pear and apple orchards . To avoid any single-host selection bias, six bacterial host strains were used in the initial isolation and enrichment processes . Molecular characterization of the phages with a combination of PCR and restriction endonuclease digestions showed that six distinct phage types, described as groups 1 to 6, were recovered . Ten phage isolates were related to the previously characterized E . amylovora PEa1, with some divergence of molecular markers between phages isolated from different sites . A study of the host ranges of the phages revealed that certain types were unable to efficiently lyse some E . amylovora strains and that some isolates were able to lyse the epiphytic bacterium Pantoea agglomerans . Representatives from the six molecular groups were studied by electron microscopy to determine their morphology . The phages exhibited distinct morphologies when examined by an electron microscope . Group 1 and 2 phages were tailed and contractile, and phages belonging to groups 3 to 6 had short tails or openings with thin appendages . Based on morphotypes, the bacteriophages of E . amylovora were placed in the order Caudovirales, in the families Myoviridae and PODOVIRIDAE:

Appl Microbiol Biotechnol, 2003 Feb, 60(6), 713 - 9 Epub 2002 Dec 21.
Surrogate biochemistry: use of Escherichia coli to identify plant cDNAs that impact metabolic engineering of carotenoid accumulation; Gallagher CE et al.; 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.

J Org Chem, 2003 Apr 4, 68(7), 2889 - 94
Enantiopure synthesis of all four stereoisomers of carbapenam-3-carboxylic acid methyl ester; Avenoza A et al.; The retro-Dieckmann reaction has been used as a stereodivergent synthetic tool on N-Boc-7-azabicyclo{2.2.1}heptan-2-one-1-carboxylic acid methyl ester to obtain enantiopure trans- and cis-5-(carboxymethyl)pyrrolidine-2-carboxylic acid methyl esters . These disubstituted pyrrolidines have been used as starting materials to develop concise and straightforward syntheses of all four stereoisomers of carbapenam-3-carboxylic acid methyl esters . In this way, we have confirmed unequivocally the stereochemistry of two carbapenams isolated from strains of Serratia and Erwinia species.

Mol Genet Genomics, 2003 Mar, 268(6), 739 - 49 Epub 2003 Feb 22.
Instability of short-sequence DNA repeats of pear pathogenic Erwinia strains from Japan and Erwinia amylovora fruit tree and raspberry strains; Jock S et al.; An array of short-sequence DNA repeats (SSRs) occurs in the plasmid pEA29 of the fire blight pathogen Erwinia amylovora . A large number of "fruit tree" strains, mainly from Central and Western Europe, were screened for their SSR numbers, and the analyses were extended to five raspberry strains from North America and six pear pathogenic Erwinia strains from Japan . The repeat ATTACAGA present in all E . amylovorastrains was found to be reiterated 3 to 15 times . The Japanese strains contained the major repeat sequence GGATTCTG, which was reiterated 16 to 24 times . ATTACAGG, which resembles the SSR of E . amylovora, was reiterated two or three times . In a novel approach, sequencing gels were used to visualize the rare occurrence of shorter arrays (down to three repeats) in E . amylovoraand the Japanese Erwinia strains . Changes in the repeat numbers in E . amylovora were observed repeatedly when the bacteria had been exposed to stress conditions . The repeat structures of homo- and heteroduplices of PCR-amplified repeats were also analyzed by cleavage of annealed molecules with the single-strand-specific endonuclease from bacteriophage T4 . Not only heteroduplexes, but also homoduplexes showed non-matching regions in the SSRs, which could arise from transient formation of loops due to strand slippage during the assays.

Mol Plant Microbe Interact, 2003 Mar, 16(3), 249 - 60
The regulatory cascade that activates the Hrp regulon in Erwinia herbicola pv . gypsophilae; Nizan-Koren R et al.; The pathogenicity of Erwinia herbicola pv . gypsophilae (Ehg) is dependent on a plasmid (pPATH(Ehg)) that harbors the hrp gene cluster and additional virulence genes . The hrp regulatory cascade of Ehg comprises an hrpXY operon encoding a two-component system; hrpS encoding a transcriptional factor of the NtrC family and hrpL encoding an alternative sigma factor . Results obtained suggest the following signal transduction model for activating the Hrp regulon: phosphorylated HrpY activates hrpS, HrpS activates hrpL, and HrpL activates genes containing "hrp box" promoter . This model was supported by studies on the effects of mutations in the regulatory genes on pathogenicity and complementation analysis . Nonpolar mutations in hrpX did not affect virulence or transcription of downstream genes . Site-directed mutagenesis of the conserved aspartate 57 in HrpY suggested that its phosphorylation is crucial for activating the hrp regulatory cascade . Studies on the effects of mutations in the hrp regulatory genes on transcriptional activity of downstream genes or of their isolated promoters in planta showed dependency of hrpS expression on active HrpY, of hrpL expression on active HrpS, and of hrpN or hrpJ expression on active HrpL . These results were also partially supported by overexpression of regulatory genes under in vitro conditions . The hrpXY is constitutively expressed with high basal levels under repressive conditions, in contrast to hrpS and hrpL, which exhibit low basal expression levels and are environmentally regulated.

Mol Plant Microbe Interact, 2003 Mar, 16(3), 226 - 37
Comparative study of regulatory mechanisms for pectinase production by Erwinia carotovora subsp . carotovora and Erwinia chrysanthemi; Matsumoto H et al.; The production of pectinase, the major virulence determinant of soft-rot Erwinia species, is controlled by many regulatory factors . We focused on the major regulatory proteins, KdgR, CRP, Pir, and PecS, characterized mainly in E . chrysanthemi, and tested for their presence and function in the control of pectate lyase (Pel) and polygalacturonase (Peh) production in E . carotovora subsp . carotovora . Homologues of kdgR and crp but not of pir and pecS were detected by Southern blot analyses in E . carotovora subsp . carotovora . In fact, KdgR and CRP homologues of E . carotovora subsp . carotovora had high amino acid identities to those of E . chrysanthemi, including a complete match of the hypothetical helix-turn-helix DNA-binding motif . However, in Western blot analyses using anti-Pir (E . chrysanthemi) antibodies, a cross-reacting protein was present in both Erwinia species, although Pel production in E . carotovora subsp . carotovora was not further stimulated by adding plant extract into the medium containing PGA (polygalacturonic acid) in which hyperinduction by Pir has been reported in E . chrysanthemi EC16 . When plasmids that contained each of these regulatory genes from E . chrysanthemi were introduced into E . carotovora subsp . carotovora, Pel production was controlled as predicted from their roles in E . chrysanthemi, except for PecS . PecS exerted a positive control in E . carotovora subsp . carotovora, in contrast to a negative control in E . chrysanthemi . DNA-binding assays demonstrated that KdgR, CRP, Pir, and PecS of E . chrysanthemi and KdgR and CRP homologues of E . carotovora subsp . carotovora could bind to the promoter regions of pel-1, pel-3, and peh of E . carotovora subsp . carotovora . Taken together, KdgR and CRP homologues of E . carotovora subsp . carotovora may regulate Pel and Peh production as in E . chrysanthemi . However, the presence of Pir and PecS homologues in E . carotovora subsp . carotovora was not identified in this study, though these proteins of E . chrysanthemi were functional on the promoter regions of the pectinase genes of E . carotovora subsp . carotovora.

Mol Plant Microbe Interact, 2003 Mar, 16(3), 179 - 87
Erwinia carotovora subsp . carotovora and Erwinia-derived elicitors HrpN and PehA trigger distinct but interacting defense responses and cell death in Arabidopsis; Kariola T et al.; We have used an hrp-positive strain of the soft rot pathogen Erwinia carotovora subsp . carotovora to elucidate plant responses to this bacterial necrotroph . Purified virulence determinants, harpin (HrpN) and polygalacturonase (PehA), were used as tools to facilitate this analysis . We show that HrpN elicits lesion formation in Arabidopsis and tobacco and triggers systemic resistance in Arabidopsis . Establishment of resistance is accompanied by the expression of salicylic acid (SA)-dependent, but also jasmonate/ethylene (JA/ET)-dependent, marker genes PR1 and PDF1.2, respectively, suggesting that both SA-dependent and JA/ET-dependent defense pathways are activated . Use of pathway-specific mutants and transgenic NahG plants show that both pathways are required for the induction of resistance . Arabidopsis plants treated simultaneously with both elictors PehA, known to trigger only JA/ET-dependent defense signaling, and HrpN react with accelerated and enhanced induction of the marker genes PR1 and PDF1.2 both locally and systemically . This mutual amplification of defense gene expression involves both SA-dependent and JA/ET-dependent defense signaling . The two elicitors produced by E . carotovora subsp . carotovora also cooperate in triggering increased production of superoxide and lesion formation.

Biochemistry, 2003 Mar 25, 42(11), 3359 - 65
Bacterial phytoene synthase: molecular cloning, expression, and characterization of Erwinia herbicola phytoene synthase; Iwata-Reuyl D et al.; Phytoene synthase (PSase) catalyzes the condensation of two molecules of geranylgeranyl diphosphate (GGPP) to give prephytoene diphosphate (PPPP) and the subsequent rearrangement of the cyclopropylcarbinyl intermediate to phytoene . These reactions constitute the first pathway specific step in carotenoid biosynthesis . The crtB gene encoding phytoene synthase was isolated from a plasmid containing the carotenoid gene cluster in Erwinia herbicola and cloned into an Escherichia coli expression system . Upon induction, recombinant phytoene synthase constituted 5-10% of total soluble protein . To facilitate purification of the recombinant enzyme, the structural gene for PSase was modified by site-directed mutagenesis to incorporate a C-terminal Glu-Glu-Phe (EEF) tripepetide to allow purification by immunoaffinity chromatography on an immobilized monoclonal anti-alpha-tubulin antibody YL1/2 column . Purified recombinant PSase-EEF gave a band at 34.5 kDa upon SDS-PAGE . Recombinant PSase-EEF was then purified to >90% homogeneity in two steps by ion-exchange and immunoaffinity chromatography . The enzyme required Mn(2+) for activity, had a pH optimum of 8.2, and was strongly stimulated by detergent . The concentration of GGPP needed for half-maximal activity was approximately 35 microM, and a significant inhibition of activity was seen at GGPP concentrations above 100 microM . The sole product of the reaction was 15,15'-Z-phytoene.

J Biol Chem, 2003 May 16, 278(20), 17993 - 8001 Epub 2003 Mar 12.
SufA from Erwinia chrysanthemi . Characterization of a scaffold protein required for iron-sulfur cluster assembly; Ollagnier-de Choudens S et al.; SufA is a component of the recently discovered suf operon, which has been shown to play an important function in bacteria during iron-sulfur cluster biosynthesis and resistance to oxidative stress . The SufA protein from Erwinia chrysanthemi, a Gram-negative plant pathogen, has been purified to homogeneity and characterized . It is a homodimer with the ability to assemble rather labile {2Fe-2S} and {4Fe-4S} clusters as shown by Mossbauer spectroscopy . These clusters can be transferred to apoproteins such as ferredoxin or biotin synthase during a reaction that is not inhibited by bathophenanthroline, an iron chelator . Cluster assembly in these proteins is much more efficient when iron and sulfur are provided by holoSufA than by free iron sulfate and sodium sulfide . We propose the function of SufA is that of a scaffold protein for {Fe-S} cluster assembly and compare it to IscA, a member of the isc operon also involved in cluster biosynthesis in both prokaryotes and eukaryotes . Mechanistic and physiological implications of these results are also discussed.

Mol Cell Probes, 2003 Feb, 17(1), 25 - 32
Detection of type III secretion genes as a general indicator of bacterial virulence; Stuber K et al.; Type III secretion systems of Gram-negative bacteria are specific export machineries for virulence factors which allow their translocation to eukaryotic cells . Since they correlate with bacterial pathogenicity, their presence is used as a general indicator of bacterial virulence . By comparing the genetic relationship of the major type III secretion systems we found the family of genes encoding the inner-membrane channel proteins represented by the Yersinia enterocolitica lcrD (synonym yscV) and its homologous genes from other species an ideal component for establishing a general detection approach for type III secretion systems . Based on the genes of the lcrD family we developed gene probes for Gram-negative human, animal and plant pathogens . The probes comprise lcrD from Y . enterocolitica, sepA from enteropathogenic Escherichia coli, invA from Salmonella typhimurium, mxiA from Shigella sonnei, as well as hrcV from Erwinia amylovora . In addition we included as a control probe the flhA gene from E . coli K-12 to validate our approach . FlhA is part of the flagellar export apparatus which shows a high degree of similarity with type III secretions systems, but is not involved in pathogenicity . The probes were evaluated by screening a series of pathogenic as well as non-pathogenic bacteria . The probes detected type III secretion in pathogens where such systems were either known or were expected to be present, whereas no positive hybridization signals could be found in non-pathogenic Gram-negative bacteria . Gram-positive bacteria were devoid of known type III secretion systems . No interference due to the genetic similarity between the type III secretion system and the flagellar export apparatus was observed . However, potential type III secretion systems could be detected in bacteria where no such systems have been described yet . The presented approach provides therefore a useful tool for the assessment of the virulence potential of bacterial isolates of human, animal and plant origin . Moreover, it is a powerful means for a first safety assessment of poorly characterized strains intended to be used in biotechnological applications .

FEBS Lett, 2003 Feb 27, 537(1-3), 198 - 202
Involvement of three pathogenicity factors of Erwinia amylovora in the oxidative stress associated with compatible interaction in pear; Venisse JS et al.; Erwinia amylovora, the causal agent of fire blight of Maloideae, induces in its susceptible host plants an oxidative burst as does an incompatible pathogen . In this paper we present evidence that the elicitation of this phenomenon is the result of the combined action of two Hrp effectors of the bacteria, HrpN and DspA . We also confirmed that desferrioxamine, the siderophore of E . amylovora, is necessary for the bacteria to tolerate high levels of hydrogen peroxide . Two other pathogenicity factors of the bacteria, the HrpW effector and the capsule, do not seem to play any role in the elicitation of the oxidative burst nor in the protection of the bacteria.

Biochem J, 2003 Jun 1, 372(Pt 2), 329 - 34
Topology of the Erwinia chrysanthemi oligogalacturonate porin KdgM; Pellinen T et al.; The Erwinia chrysanthemi oligogalacturonate-specific monomeric porin, KdgM, does not present homology with any porins of known structure . A model of this protein, based on sequence similarity and the amphipathy profile, was constructed . The model depicts a beta-barrel composed of 14 antiparallel beta-strands . The accuracy of this model was tested by the chemical labelling of cysteine residues introduced by site-directed mutagenesis . The protein has seven surface-exposed loops . They are rather small with the exception of one, loop L6 . Deletion of this loop allowed the entry of maltopentaose into the bacteria, a molecule too large to enter through the wild-type KdgM . Loop L6 could fold back into the lumen of the pore and play the role of the constriction loop L3 of general porins . With 14 transmembrane segments, the KdgM porin family could represent the smallest porin characterized to date.

J Bacteriol, 2003 Mar, 185(5), 1642 - 9
Rhamnogalacturonate lyase RhiE is secreted by the out system in Erwinia chrysanthemi; Laatu M et al.; Supernatants of rhamnose-induced Erwinia chrysanthemi strain 3937 cultures contain a principal secreted protein named RhiE . A rhiE mutant has been found among a set of rhamnose-induced MudI1681 lacZ fusions . RhiE is a 62-kDa protein that has rhamnogalacturonate lyase activity on rhamnogalacturonan I (RG-I) . It does not require a divalent cation for its activity and has an optimal pH of 6.0 . rhiE expression is strongly induced in the presence of rhamnose but is also regulated by PecT and Crp, two regulators of the transcription of pectinolytic enzyme genes . RhiE is secreted through the type II Out secretion pathway . RhiE has no disulfide bond . The absence of RhiE secretion in a dsb mutant indicated that disulfide bond formation is required for the biogenesis of the secretion apparatus . RhiE was searched for in several E . chrysanthemi strains by using antibodies, and it was found to be present in one-third of the strains tested . However, the reduced virulence of the rhiE mutant indicates that degradation of the RG-I region of pectin is important for full virulence of E . chrysanthemi.

J Forensic Sci, 2003 Jan, 48(1), 122 - 6
Stable-isotope fingerprints of biological agents as forensic tools; Horita J et al.; Naturally occurring stable isotopes of light elements in chemical and biological agents may possess unique "stable-isotope fingerprints" depending on their sources and manufacturing processes . To test this hypothesis, two strains of bacteria (Bacillus globigii and Erwinia agglomerans) were grown under controlled laboratory conditions . We observed that cultured bacteria cells faithfully inherited the isotopic composition (hydrogen, carbon, and nitrogen) of media waters and substrates in predictable manners in terms of bacterial metabolism and that even bacterial cells of the same strain, which grew in media water and substrates of different isotopic compositions, have readily distinguishable isotopic signatures . These "stable-isotopic fingerprints" of chemical and biological agents can be used as forensic tools in the event of biochemical terrorist attacks.

Wei Sheng Wu Xue Bao, 2002 Aug, 42(4), 465 - 70
{Type III secretion system in Se9R can recognize and secret harpin in Erwinia amylovora}; Wei G et al.; Bacterial pathogens of both animals and plants use type III secretion machines to secret virulence proteins . The signal that leads to tye type III secretion is encoded as stem loop structure in the messenger RNA . Harpin, produced by Erwinia anylovora is secreted via type III secretion . Erwinia carotovora Se9R and Erwinia amylovora belong to the same genus and they use type III secretion machines to secret virulence proteins . We used PCR to amplify hrpN from pCPP430 which harbors hrpN gene cluster, and cloned it to pGEM-T vector to get pWGF1, which was then chemically transformed into DH5 alpha (pCPP430hrpN-) and electrotransformed into Se9R . DH5 alpha (pCPP430hrpN-/pWGF1) induced hypersensitive response on tomato leaf and Se9R(pWGF1) showed significantly reduced virulence than Se9R on Chinese cabbage leaf . Western-blotting analysis of the CFEP of Se9R(pWGF1) showed production of harpin protein . These results suggest the type III secretion machine in Se9R can recognize secretion singal in the gene of harpin in Erwinia amylovora and secret it as a biologically native form.

EMBO J, 2003 Feb 3, 22(3), 427 - 37
SufC: an unorthodox cytoplasmic ABC/ATPase required for {Fe-S} biogenesis under oxidative stress; Nachin L et al.; Proteins containing {Fe-S} clusters perform essential functions in all domains of life . Previously, we identified the sufABCDSE operon as being necessary for virulence of the plant pathogen Erwinia chrysanthemi . In addition, we collected preliminary evidence that the sufABCDSE operon might be involved in the assembly of {Fe-S} clusters . Of particular interest are the sufB, sufC and sufD genes, which are conserved among Eubacteria, Archaea, plants and parasites . The present study establishes SufC as an unorthodox ATPase of the ABC superfamily that is located in the cytosol, wherein it interacts with both SufB and SufD . Moreover, under oxidative stress conditions, SufC was found to be necessary for the activity of enzymes containing oxygen-labile {Fe-S} clusters, but dispensable for glutamate synthase, which contains an oxidatively stable {Fe-S} cluster . Lastly, we have shown SufBCD to be essential for iron acquisition via chrysobactin, a siderophore of major importance in virulence . We discuss a model wherein the SufBCD proteins contribute to bacterial pathogenicity via their role in the assembly of {Fe-S} clusters under oxidative stress and iron limitation.

Wei Sheng Wu Xue Bao, 2000 Oct, 40(5), 475 - 81
{Studies on gene knocking out of 2-keto aldose reductases from Erwinia sp . SCB125}; Chen F et al.; Based on the reported gene sequences, the segments containing 2-keto aldose reductase (2-KRA and B) genes were amplified by PCR from the plasmids and Erwinia sp . SCB125 each for gene expression and gene knocking out . Then cloning them into expression vector pBL and successfully expressing them with high enzyme activity in E . coli DH5 alpha . After their enzyme activities were proved, the work on gene knock out followed . Introducing the knock-out vector which distribute unstably during the cell division to the host strains Erwinia sp . SCB125 . Screening firstly by the positive marker, one resistance which resulted from the expression of the resistance gene inserting inside the reductase genes and the negative marker, another resistance which outside the reductase genes in the vector . The strains selected out will be tested by further study . This work was the bases of blocking the pathway metabolism and constructing a recombinant strain that can produce 2-KLG directly from D-glucose by one-step fermentation.

J Biol Chem, 2003 Apr 4, 278(14), 12271 - 7 Epub 2003 Jan 22.
Characterization and implications of Ca2+ binding to pectate lyase C; Herron SR et al.; Ca(2+) is essential for in vitro activity of Erwinia chrysanthemi pectate lyase C (PelC) . Crystallographic analyses of 11 PelC-Ca(2+) complexes, formed at pH 4.5, 9.5, and 11.2 under varying Ca(2+) concentrations, have been solved and refined at a resolution of 2.2 A . The Ca(2+) site represents a new motif for Ca(2+), consisting primarily of beta-turns and beta-strands . The principal differences between PelC and the PelC-Ca(2+) structures at all pH values are the side-chain conformations of Asp-129 and Glu-166 as well as the occupancies of four water molecules . According to calculations of pK(a) values, the presence of Ca(2+) and associated structural changes lower the pK(a) of Arg-218, the amino acid responsible for proton abstraction during catalysis . The Ca(2+) affinity for PelC is weak, as the K(d) was estimated to be 0.132 (+/-0.004) mm at pH 9.5, 1.09 (+/-0.29) mm at pH 11.2, and 5.84 (+/-0.41) mm at pH 4.5 from x-ray diffraction studies and 0.133 (+/-0.045) mm at pH 9.5 from intrinsic tryptophan fluorescence measurements . Given the pH dependence of Ca(2+) affinity, PelC activity at pH 4.5 has been reexamined . At saturating Ca(2+) concentrations, PelC activity increases 10-fold at pH 4.5 but is less than 1% of maximal activity at pH 9.5 . Taken together, the studies suggest that the primary Ca(2+) ion in PelC has multiple functions.

Appl Environ Microbiol, 2003 Jan, 69(1), 679 - 82
Molecular characterization of natural Erwinia pyrifoliae strains deficient in hypersensitive response; Jock S et al.; 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.

Appl Environ Microbiol, 2003 Jan, 69(1), 673 - 8
Degradation and transformability of DNA from transgenic leaves; Ceccherini M et al.; The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum . Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene . These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues . Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.

Folia Microbiol (Praha), 2002, 47(5), 473 - 6
Production and properties of asparaginase from a new Erwinia sp; Borkotaky B et al.; Asparaginase production by a mesophilic strain of Erwinia sp . was examined; the maximum of activity was found at 40 degrees C and pH 8.5 . Among the various carbon sources, mannitol was shown to be the best for production of activity . Inorganic nitrogen sources were better than the organic ones . The enzyme activity was not inhibited by 10 mmol/L metal ions (Na+, K+, Mg2+, Ca2+, Ba2+, Co2+, Ni2+, Zn2+); the activity was strongly inhibited by addition of EDTA . L-Arginine, DL-alanine, L-asparagine and L-glutamine stimulated the L-asparaginase production by 3.9, 1.7, 4.3 and 4.0 fold, respectively . The combination of L-arginine, L-asparagine and L-glutamine synergistically stimulated the asparaginase up to 5.8 fold.

J Gen Appl Microbiol, 1998 Dec, 44(6), 405 - 413
Enhanced production of extracellular ice nucleators from Erwinia herbicola; Li J et al.; The effects of growth conditions and chemical or physical treatments on the production of extracellular ice nucleators (ECINs) by Erwinia herbicola cells were investigated . The spontaneous release of ECINs, active at temperatures higher than -4 degrees C, into the environment depended on culture conditions, with optimal production when cells were grown in yeast extract to an early stationary phase at temperatures below 22 degrees C . ECINs were vesicular, released from cell surfaces with sizes ranging from 0.1 to 0.3 &mgr;m as determined by ultrafiltration and transmission electron microscopy . Protein profiles of ECIN fractions during bacterial growth were examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), and Ina proteins were detected by Western blotting . ECIN production was enhanced 5-fold when cells were treated with EDTA and 20- to 30-fold when subjected to sonication . These conditions provide a means for large-scale preparationage> ECINs by E . herbicola.

Acta Crystallogr D Biol Crystallogr, 2003 Jan, 59(Pt 1), 84 - 92 Epub 2002 Dec 19.
Atomic resolution structure of Erwinia chrysanthemi L-asparaginase; Lubkowski J et al.; An X-ray structure of L-asparaginase from Erwinia chrysanthemi (ErA) has been refined at 1 A resolution to an R factor of below 0.1, using data collected on a synchrotron source . With four molecules of the enzyme consisting of 327 amino acids each, this crystal contains one of the largest asymmetric units of a protein refined to date at atomic resolution . Previously, structures of ErA and of related enzymes from other bacterial sources have been refined at resolutions not exceeding 1.7 A; thus, the present structure represents a very significant improvement in the quality of the available models of these proteins and should provide a good basis for future studies of the conformational variability of proteins, identification of subtle conformational features and corroboration of the stereochemical libraries, amongst other things . L-Asparaginases, which are enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid, have been used for over 30 y as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia, although the details of the enzymatic reaction and substrate specificity have not yet been completely elucidated . This atomic resolution structure is a step in that direction.

Carbohydr Res, 2002 Nov 29, 337(24), 2469 - 80
Extracellular polysaccharides of Erwinia futululu, a bacterium associated with a fungal canker disease of Eucalyptus spp; Yang BY et al.; Extracellular polysaccharides (EPSs) produced by an Erwinia spp . associated with a fungal canker disease of Eucalyptus were fractionated into two polysaccharides, one that was identified with that produced by Erwinia stewartii . The other has a similar structure, but with one terminal Glc residue replaced by pyruvic acid to give 4,6-O-{(R)-1-carboxyethylidene)-Galp . Their structures were determined using a combination of chemical and physical techniques including methylation analysis, periodate oxidation, low-pressure gel filtration and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy . The new polysaccharides, identified as EPS Futululu FF-1 and FF-2, have the following structures:The molecular weights of the polysaccharides range from 1.3-2.1x10(6) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.

Mol Plant Microbe Interact, 2002 Dec, 15(12), 1204 - 12
Modulation of defense responses of Malus spp . during compatible and incompatible interactions with Erwinia amylovora; Venisse JS et al.; Erwinia amylovora is the causal agent of fire blight, a disease affecting members of subfamily Maloideae . In order to analyze mechanisms leading to compatible or incompatible interactions, early plant molecular events were investigated in two genotypes of Malus with contrasting susceptibility to fire blight, after confrontation with either E . amylovora or the incompatible tobacco pathogen Pseudomonas syringae pv . tabaci . Many defense mechanisms, including generation of an oxidative burst and accumulation of pathogenesis-related proteins, were elicited in both resistant and susceptible genotypes by the two pathogens at similar rates and according to an equivalent time course . This elicitation was linked with the functional hypersensitive reaction and pathogenicity (hrp) cluster of E . amylovora, because an hrp secretion mutant did not induce such responses . However, a delayed induction of several genes of various branch pathways of the phenylpropanoid metabolism was recorded in tissues of the susceptible genotype challenged with the wild-type strain of E . amylovora, whereas these genes were quickly induced in every other plant-bacteria interaction, including interactions with the hrp secretion mutant . This suggests the existence of hrp-independent elicitors of defense in the fire blight pathogen as well as hrp-dependant mechanisms of suppression of these nonspecific inductions.

Microbiology, 2002 Dec, 148(Pt 12), 4015 - 24
Genetics of biosynthesis and structure of the capsular exopolysaccharide from the Asian pear pathogen Erwinia pyrifoliae; Kim WS et al.; Erwinia pyrifoliae is a novel bacterial pathogen, which causes Asian pear blight and is related to Erwinia amylovora, the causative agent of fire blight . E . pyrifoliae produces exopolysaccharide (EPS) related to amylovoran in its sugar composition and sugar linkages . This was shown by degradation of the EPS with a viral depolymerase, and by methylation analysis and ESI/MS . The structure of the repeating units was confirmed by (1)H-NMR spectra . The EPS of E . pyrifoliae carried side chains, which were mainly terminated by acetyl and pyruvyl residues as found previously for amylovoran . On the other hand, a second side chain with glucose found for up to 65% of the repeating units of amylovoran was completely absent . The nucleotide sequences of five genes of the cps cluster of E . pyrifoliae encoding proteins for EPS synthesis were characterized and displayed a high homology with the corresponding ams genes . Similar functions of the gene products are assumed . As for ams mutants of E . amylovora, a cpsB mutant of E . pyrifoliae did not synthesize EPS and did not produce ooze on slices of immature pears or symptoms on pear seedlings . The cps mutant was complemented for EPS synthesis and virulence on pear slices with a gene cluster of E . amylovora that included amsB.

Phytochemistry, 2003 Jan, 62(1), 101 - 7
Benzoquinone, the substance essential for antibacterial activity in aqueous extracts from succulent young shoots of the pear Pyrus spp; Jin S et al.; Aqueous extracts of the tissue of succulent young shoots of the pear Pyrus spp . exhibited strong antibacterial activity against the bacterium Erwinia amylovora bv . 4 . This activity was investigated quantitatively by a newly developed bioassay method . It was found that the activity changed with the age of the tissue . Extracts of the youngest leaves and stems from the shoot tops showed the strongest activity, and the activity decreased with age of the leaves and stems . The activity also changed with increase in time after preparation of the extract, increasing rapidly in the first hour after preparation, reaching a maximum at about 4 h, and then decreasing slowly . The substance essential for the antibacterial activity was isolated from the extract by steam distillation in vacuo and through charcoal powder column chromatography . It was identified as benzoquinone (2,5-cyclohexadiene-1,4-dione) by NMR-spectra, mass spectra and HPLC analysis . The phenolic metabolism from arbutin to hydroquinone and then to benzoquinone in the aqueous extracts was analyzed quantitatively by HPLC . The changes in the contents of benzoquinone in the extracts of leaves and stems with tissue aging and with increase in time after preparation of the extracts paralleled the changes in antibacterial activity as determined by the quantitative bioassay.

Plant J, 2002 Dec, 32(5), 749 - 62
Spatio-temporal expression of patatin-like lipid acyl hydrolases and accumulation of jasmonates in elicitor-treated tobacco leaves are not affected by endogenous levels of salicylic acid; Dhondt S et al.; We have previously isolated three tobacco genes (NtPat) encoding patatin-like proteins, getting rapidly induced during the hypersensitive response (HR) to tobacco mosaic virus, in advance to jasmonate accumulation . NtPAT enzymes are lipid acyl hydrolases that display high phospholipase A2 (PLA2) activity and may mobilize fatty acid precursors of oxylipins . Here, we performed a detailed study of NtPat gene regulation under various biotic and abiotic stresses . PLA2 activity was poorly induced in response to drought, wounding, reactive oxygen intermediates, salicylic acid (SA) or methyl-jasmonate (MJ) whereas the ethylene (ET) precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), provoked a moderate induction . In contrast, PLA2 activity was strongly induced when ACC was combined with MJ, and in response to the bacterium Erwinia carotovora or to the fungus Botrytis cinerea, as well as to treatment with beta-megaspermin, a cell death-inducing protein elicitor . A simplified system based on the infiltration of beta-megaspermin into leaves was used to dissect the spatio-temporal activation of PLA2 activity with regards to the accumulation of jasmonates and to the influence of endogenous SA . NtPat-encoded PLA2 activity was rapidly induced in the infiltrated zone before the appearance of cell death and with some delay in the surrounding living cells . A massive accumulation of 12-oxo-phytodienoic and jasmonic acids occurred in the elicitor-infiltrated zone, but only low levels were detectable outside this area . A similar picture was found in SA-deficient plants, showing that in tobacco, accumulation of jasmonates is not affected by the concomitant HR-induced build-up of endogenous SA . Finally, ET-insensitive plants showed a weakened induction of PLA2 activity outside the elicitor-infiltrated tissue.

Appl Environ Microbiol, 2002 Dec, 68(12), 6182 - 92
Relatedness of chromosomal and plasmid DNAs of Erwinia pyrifoliae and Erwinia amylovora; McGhee GC et al.; The plant pathogen Erwinia pyrifoliae has been classified as a separate species from Erwinia amylovora based in part on differences in molecular properties . In this study, these and other molecular properties were examined for E . pyrifoliae and for additional strains of E . amylovora, including strains from brambles (Rubus spp.) . The nucleotide composition of the internal transcribed spacer (ITS) region was determined for six of the seven 16S-23S rRNA operons detected in these species with a 16S rRNA gene probe . Each species contained four operons with a tRNA(Glu) gene and two with tRNA(Ile) and tRNA(Ala) genes, and analysis of the operons from five strains of E . amylovora indicated a high degree of ITS variability among them . One tRNA(Glu)-containing operon from E . pyrifoliae Ep1/96 was identical to one in E . amylovora Ea110, but three tRNA(Glu) operons and two tRNA(Ile) and tRNA(Ala) operons from E . pyrifoliae contained unique nucleotide changes . When groEL sequences were used for species-specific identification, E . pyrifoliae and E . amylovora were the closest phylogenetic relatives among a set of 12 bacterial species . The placement of E . pyrifoliae distinct from E . amylovora corroborated molecular hybridization data indicating low DNA-DNA similarity between them . Determination of the nucleotide sequence of plasmid pEP36 from E . pyrifoliae Ep1/96 revealed a number of presumptive genes that matched genes previously found in pEA29 from E . amylovora and similar organization for the genes and origins of replication . Also, pEP36 and pEA29 were incompatible with clones containing the reciprocal origin regions . Finally, the ColE1-like plasmid pEP2.6 from strain Ep1/96 contained sequences found in small plasmids in E . amylovora strains IL-5 and IH3-1.

Antonie Van Leeuwenhoek, 2002 Aug, 81(1-4), 223 - 31
The regulation of virulence in phytopathogenic Erwinia species: quorum sensing, antibiotics and ecological considerations; Whitehead NA et al.; Erwinia carotovora is a Gram-negative bacterial phytopathogen that causes soft-rot disease and potato blackleg . The organism is environmentally widespread and exhibits an opportunistic plant pathogenesis . The ability to secrete multiple plant cell wall-degrading enzymes is a key virulence trait and exoenzyme production is responsive to multiple environmental and physiological cues . One important cue is the cell population density of the pathogen . Cell density is monitored via an acylated homoserine lactone (acyl HSL) signalling molecule, which is thought to diffuse between Erwinia cells in a process now commonly known as 'quorum sensing' . This molecule also acts as the chemical communication signal controlling production of a broad-spectrum beta-lactam antibiotic (1-carbapen-2-em-3-carboxylic acid; carbapenem) synthesised in concert with exoenzyme elaboration, possibly for niche defence . In antibiotic production control, quorum sensing acts at the level of transcriptional activation of the antibiotic biosynthetic cluster . This is achieved via a dedicated LuxR-type protein, CarR that is bound to the signalling molecule . The molecular relay connecting acyl HSL production and exoenzyme induction is not clear, despite the identification of a multitude of global regulatory genes, including those of the RsmA/rsmB system, impinging on enzyme synthesis . Quorum sensing control mediated by acyl HSLs is widespread in Gram-negative bacteria and is responsible for the regulation of diverse phenotypes . Although there is still a paucity of meaningful information on acyl HSL availability and in-situ biological function, there is growing evidence that such molecules play significant roles in microbial ecology.

Mol Plant Microbe Interact, 2002 Oct, 15(10), 1058 - 68
PopP1, a new member of the YopJ/AvrRxv family of type III effector proteins, acts as a host-specificity factor and modulates aggressiveness of Ralstonia solanacearum; Lavie M et al.; A functional analysis of an 11-kb-long region of the genome of the plant-pathogenic bacterium Ralstonia solanacearum, previously identified as an alternative codon usage region (ACUR), reveals that it was probably acquired through horizontal gene transfer . This ACUR encodes an insertion sequence and eight potential proteins, one of which is partially homologous with a host-specificity factor from a plant-pathogenic Erwinia sp., and another, PopP1, which is homologous to members of the YopJ/AvrRxv family of type III-secreted bacterial effectors controlling interaction between bacteria and their hosts . The analysis of mutants affecting all except one of the genes identified in the ACUR showed that only the popP1-deficient strain had an altered phenotype in plant infection tests . This mutant strain became pathogenic to a Petunia line that is resistant to the wild-type strain . Therefore, popP1 behaves as a typical avirulence gene . We demonstrate that PopP1 protein is secreted and that secretion of this protein requires a functional type III-secretion pathway . In contrast to the structural genes for other type III-secreted proteins identified in R . solanacearum, transcription of the popP1 gene is not coregulated with transcription of hrp genes but is constitutive.

J Am Chem Soc, 2002 Nov 20, 124(46), 13709 - 15
Direct structure determination using residual dipolar couplings: reaction-site conformation of methionine sulfoxide reductase in solution; Beraud S et al.; Residual dipolar couplings (RDC) from partially aligned molecules provide long-range structural data and are thus particularly well adapted to rapid structure validation or protein fold recognition . Extensive measurements in two alignment media can also provide precise de novo structure from RDC alone . We have applied a novel combination of these approaches to the study of methionine sulfoxide reductase (MsrA) from Erwinia chrysanthemi, a 27 kDa enzyme essential for repairing oxidative stress damage . The tertiary fold was initially validated by comparing backbone RDC to expected values from the crystal structure of the homologous MsrA from Escherichia coli . Good agreement was found throughout the chain, verifying the overall topology of the molecule, with the exception of the catalytically important peptide P196-L202, where strong and systematic RDC violation was observed . No evidence for local differential mobility in this region was detected, implying that the structure of the strand differs in the two molecules . We have therefore applied the de novo approach meccano to determine the conformation of this peptide using only RDC . A single conformation is found that is in agreement with all measured data . The aligned peptide can be docked onto the expected covalence of the rest of the template molecule while respecting its strictly defined relative orientation . In contrast to the structure of MsrA from E . coli, the reactive side chain of Cys200 is oriented toward the interior of the molecule and therefore closer to the catalytic Cys53, obviating the need for previously proposed conformational reorganization prior to formation of this disulfide intermediate . This analysis requires only backbone assignment and uses unambiguously assigned and readily measurable structural data, thereby greatly economizing investigation time compared to established nuclear Overhauser effect- (nOe-) based structure calculation methods.

J Bacteriol, 2002 Dec, 184(23), 6690 - 9
Evolution of the C30 carotenoid synthase CrtM for function in a C40 pathway; Umeno D et al.; The C30 carotene synthase CrtM from Staphylococcus aureus and the C40 carotene synthase CrtB from Erwinia uredovora were swapped into their respective foreign C40 and C30 biosynthetic pathways (heterologously expressed in Escherichia coli) and evaluated for function . Each displayed negligible ability to synthesize the natural carotenoid product of the other . After one round of mutagenesis and screening, we isolated 116 variants of CrtM able to synthesize C40 carotenoids . In contrast, we failed to find a single variant of CrtB with detectable C30 activity . Subsequent analysis revealed that the best CrtM mutants performed comparably to CrtB in an in vivo C40 pathway . These mutants showed significant variation in performance in their original C30 pathway, indicating the emergence of enzymes with broadened substrate specificity as well as those with shifted specificity . We discovered that Phe 26 alone determines the specificity of CrtM . The plasticity of CrtM with respect to its substrate and product range highlights the potential for creating further new carotenoid backbone structures.

Appl Environ Microbiol, 2002 Nov, 68(11), 5704 - 10
Characterization of serracin P, a phage-tail-like bacteriocin, and its activity against Erwinia amylovora, the fire blight pathogen; Jabrane A et al.; Serratia plymithicum J7 culture supernatant displayed activity against many pathogenic strains of Erwinia amylovora, the causal agent of the most serious bacterial disease of apple and pear trees, fire blight, and against Klebsiella pneumoniae, Serratia liquefaciens, Serratia marcescens, and Pseudomonas fluorescens . This activity increased significantly upon induction with mitomycin C . A phage-tail-like bacteriocin, named serracin P, was purified from an induced culture supernatant of S . plymithicum J7 . It was found to be the only compound involved in the antibacterial activity against sensitive strains . The N-terminal amino acid sequence analysis of the two major subunits (23 and 43 kDa) of serracin P revealed high homology with the Fels-2 prophage of Salmonella enterica, the coliphages P2 and 168, the phiCTX prophage of Pseudomonas aeruginosa, and a prophage of Yersinia pestis . This strongly suggests a common ancestry for serracin P and these bacteriophages.

Anal Biochem, 2002 Oct 1, 309(1), 117 - 26
Analytical validation of a microplate reader-based method for the therapeutic drug monitoring of L-asparaginase in human serum; Lanvers C et al.; The enzyme L-asparaginase (ASNASE), which hydrolyzes L-asparagine (L-Asn) to ammonia and L-aspartic acid (L-Asp), is commonly used for remission induction in acute lymphoblastic leukemia . To correlate ASNASE activity with L-Asn reduction in human serum, sensitive methods for the determination of ASNASE activity are required . Using L-aspartic beta-hydroxamate (AHA) as substrate we developed a sensitive plate reader-based method for the quantification of ASNASE derived from Escherichia coli and Erwinia chrysanthemi and of pegylated E . coli ASNASE in human serum . ASNASE hydrolyzed AHA to L-Asp and hydroxylamine, which was determined at 710 nm after condensation with 8-hydroxyquinoline and oxidation to indooxine . Measuring the indooxine formation allowed the detection of 2 x 10(-5)U ASNASE in 20 microl serum . Linearity was observed within 2.5-75 and 75-1,250 U/L with coefficients of correlation of r(2)>0.99 . The coefficients of variation for intra- and interday variability for the three different ASNASE enzymes were 1.98 to 8.77 and 1.73 to 11.0% . The overall recovery was 101+/-9.92% . The coefficient of correlation for dilution linearity was determined as r(2)=0.986 for dilutions up to 1:20 . This method combined with sensitive methods for the quantification of L-Asn will allow bioequivalence studies and individualized therapeutic drug monitoring of different ASNASE preparations.

J Agric Food Chem, 2002 Oct 23, 50(22), 6371 - 7
In vitro efficacy of plant volatiles for inhibiting the growth of fruit and vegetable decay microorganisms; Utama IM et al.; The effects of acetaldehyde, benzaldehyde, cinnamaldehyde, ethanol, benzyl alcohol, nerolidol, 2-nonanone, beta-ionone, and ethyl formate vapors on the growth of Rhizopus stolonifer, Penicillium digitatum, Colletotrichum musae, Erwinia carotovora, and Pseudomonas aeruginosa on agar medium were evaluated . The aldehydes were found to be the strongest growth inhibitors and the most lethal to the fungal spores and mycelia and bacterial cells . The average minimum inhibitory concentrations (MICs) of aldehydes that were germicidal to decay microorganisms were 0.28, 0.49, and 0.88 mmol per Petri dish, for cinnamaldehyde, benzaldehyde, and acetaldehyde, respectively . Ethanol also inhibited growth completely, but the MIC, which was 14.6 mmol per Petri dish, was significantly higher than those of the aldehydes . Ethanol can be considered germistatic because the alcohol does not inhibit germination of spores completely; it completely controlled only mycelial growth . The ketones tended to be effective only on P . digitatum and C . musae, whereas ethyl formate was not effective except on P . digitatum . The concentration of a volatile compound in the headspace of the Petri dish and its diffusion into the medium largely determined its efficacy against decay microorganisms.

J Enzyme Inhib Med Chem, 2002 Feb, 17(1), 37 - 43
Inhibition of hydrolytic enzyme activities and plant pathogen growth by invertase inhibitors; Isla MI et al.; The invertase inhibitory protein isolated from Cyphomandra betacea Sendt and Solanum tuberosum inhibited the invertase activity from different species, genera and even plant family . Furthermore, proteinaceous inhibitors are not invertase specific; fungal, bacterial and higher plant enzymes including polygalacturonase, pectinase, pectin lyase, alpha-L-arabinofuranosidase and beta-glucosidase are also shown to be inhibited . Both inhibitors exhibited an in vitro antibacterial action against phytopathogenics strains of Xanthomonas campestris pvar vesicatoria CECT 792, Pseudomonas solanacearum CECT 125, Pseudomonas corrugata CECT 124, Pseudomonas syringae and Erwinia carotovora var carotovora.

FEBS Lett, 2002 Sep 25, 528(1-3), 130 - 2
Beta-aspartylpeptides as substrates of L-asparaginases from Escherichia coli and Erwinia chrysanthemi; Kelo E et al.; L-Asparaginase is known to catalyze the hydrolysis of L-asparagine to L-aspartic and ammonia, but little is known about its action on peptides . When we incubated L-asparaginases purified either from Escherichia coli or Erwinia chrysanthemi - commonly used as chemotherapeutic agents because of their antitumour activity - with eight small beta-aspartylpeptides such as beta-aspartylserineamide, beta-aspartylalanineamide, beta-aspartylglycineamide and beta-aspartylglycine, we found that both L-asparaginases could catalyze the hydrolysis of five of them yielding L-aspartic acid and amino acids or peptides . Our data show that L-asparaginases can hydrolyze beta-aspartylpeptides and suggest that L-asparaginase therapy may affect the metabolism of beta-aspartylpeptides present in human body.

Proc Natl Acad Sci U S A, 2002 Oct 1, 99(20), 13142 - 7 Epub 2002 Sep 23.
HecA, a member of a class of adhesins produced by diverse pathogenic bacteria, contributes to the attachment, aggregation, epidermal cell killing, and virulence phenotypes of Erwinia chrysanthemi EC16 on Nicotiana clevelandii seedlings; Rojas CM et al.; Erwinia chrysanthemi is representative of a broad class of bacterial pathogens that are capable of inducing necrosis in plants . The E . chrysanthemi EC16 hecA gene predicts a 3,850-aa member of the Bordetella pertussis filamentous hemagglutinin family of adhesins . A hecATn7 mutant was reduced in virulence on Nicotiana clevelandii seedlings after inoculation without wounding . Epifluorescence and confocal laser-scanning microscopy observations of hecA and wild-type cells expressing the green fluorescent protein revealed that the mutant is reduced in its ability to attach and then form aggregates on leaves and to cause an aggregate-associated killing of epidermal cells . Cell killing also depended on production of the major pectate lyase isozymes and the type II, but not the type III, secretion pathway in E . chrysanthemi . HecA homologs were found in bacterial pathogens of plants and animals and appear to be unique to pathogens and universal in necrogenic plant pathogens . Phylogenetic comparison of the conserved two-partner secretion domains in the proteins and the 16S rRNA sequences in respective bacteria revealed the two datasets to be fundamentally incongruent, suggesting horizontal acquisition of these genes . Furthermore, hecA and its two homologs in Yersinia pestis had a G+C content that was 10% higher than that of their genomes and similar to that of plant pathogenic Ralstonia, Xylella, and Pseudomonas spp . Our data suggest that filamentous hemagglutinin-like adhesins are broadly important virulence factors in both plant and animal pathogens.

Mikrobiologiia, 2002 Jul-Aug, 71(4), 467 - 74
{In vitro self assembly of supermolecular structures after spontaneous disassembly of carotovoricins}; Tonkach FI; The self-assembly of supramolecular structures (empty sheaths and polysheaths of the macromolecular Erwinia carotovora bacteriocins) was studied by electron microscopy in the course of 1- to 2-year incubation of phage particles at 4 degrees C . This study showed that the empty sheaths and polysheaths of the bacteriocins of eight E . carotovora strains spontaneously assemble at the self-assembly centers (or crystallization centers), which have a diameter of 26-65 nm and contain a dense proteinaceous material . The self-assembly center consists of two components, a primer and the structural protein of contracted sheaths . Empty sheaths assembled in the crystallization centers are polar structures synthesized through the stepwise head-to-tail polymerization of monomeric units . The supramolecular structures of two E . carotovora 62A bacteriocins are assembled in a different way . At the early stages of their self-assembly, a reticular structure is formed, which then transforms into very long polysheaths composed of monomers . Along with polysheaths, rounded or lamplike structures 33-117 nm in size composed of the subunits of contracted sheath are produced . Carotovoricins may serve as suitable objects for the study of the self-assembly of elementary biological structures.

Plant Cell, 1997 Apr, 9(4), 547 - 557
Salicylic Acid Interferes with Tobacco Mosaic Virus Replication via a Novel Salicylhydroxamic Acid-Sensitive Mechanism; Chivasa S et al.; Salicylic acid (SA) induces resistance to all plant pathogens, including bacteria, fungi, and viruses, but the mechanism by which SA engenders resistance to viruses is not known . Pretreatment of tobacco mosaic virus (TMV)-susceptible (nn genotype) tobacco tissue with SA reduced the levels of viral RNAs and viral coat protein accumulating after inoculation with TMV . Viral RNAs were not affected equally, suggesting that SA treatment interferes with TMV replication . Salicylhydroxamic acid (SHAM), an inhibitor of the mitochondrial alternative oxidase, antagonized both SA-induced resistance to TMV in nn genotype plants and SA-induced acquired resistance in resistant (NN genotype) tobacco . SHAM did not inhibit induction of the PR-1 pathogenesis-related protein or induction of resistance to Erwinia carotovora or Botrytis cinerea by SA . This indicates that SA induces resistance to TMV via a novel SHAM-sensitive signal transduction pathway (potentially involving alternative oxidase), which is distinct from that leading to resistance to bacteria and fungi.

Mol Plant Microbe Interact, 2002 Sep, 15(9), 971 - 80
Regulation of Erwinia carotovora hrpL(Ecc) (sigma-L(Ecc)), which encodes an extracytoplasmic function subfamily of sigma factor required for expression of the HRP regulon; Chatterjee A et al.; In Erwinia carotovora subsp . carotovora (Ecc) strain 71 (Ecc71), HrpL(Ecc), an alternate sigma factor of the extracytoplasmic function subfamily, plays a central role in the expression of the hrp (hypersensitive reaction and pathogenicity) regulon . We document here that sigma-54 (RpoN) is required for full expression of hrpL(Ecc) and that HrpS, in conjunction with sigma-54, activates hrpL(Ecc) transcription . We also made the novel observation that integration host factor is required for the activation of the hrpL(Ecc) promoter . Our findings reveal that the RsmA/rsmB RNA-mediated post-transcriptional system, known to control extracellular enzyme and harpin production, affects hrpL(Ecc) expression as well . For example, hrpL(Ecc) RNA levels are barely detected in an RsmB- strain . Conversely, hrpL(Ecc) mRNA levels are much higher in RsmA- bacteria than in the RsmA+ parent . This effect is due to RsmA-promoted decay of hrpL(Ecc) RNA . Moreover, the following regulators known to control the production of either RsmA, rsmB RNA, or both also affect hrpL(Ecc) expression: GacA (response regulator of a two-component system), KdgR (an IcII type repressor), HexA (a LysR type repressor), RsmC (a putative transcriptional adapter) . Based upon the data now available for Ecc and extrapolating from the evidence in other systems, we propose a tentative model that depicts the Hrp regulatory system of Ecc and explains the basis for coregulation of extracellular enzyme production and expression of the Hrp regulon.

Plant Physiol, 1994 Nov, 106(3), 849 - 862
The Three-Dimensional Structure of Pectate Lyase E, a Plant Virulence Factor from Erwinia chrysanthemi; Lietzke SE et al.; The three-dimensional structure of pectate lyase E (PelE) has been determined by crystallographic techniques at a resolution of 2.2 A . The model includes all 355 amino acids but no solvent, and refines to a crystallographic refinement factor of 20.6% . The polypeptide backbone folds into a large right-handed cylinder, termed a parallel {beta} helix . Loops of various sizes and conformations protrude from the central helix and probably confer function . A putative Ca2+-binding site as well as two cationic sites have been deduced from the location of heavy atom derivatives . Comparison of the PelE and recently determined pectate lyase C (PelC) structures has led to identification of a putative polygalacturonate-binding region in PelE . Structural differences relevant to differences in the enzymatic mechanism and maceration properties of PelE and PelC have been identified . The comparative analysis also reveals a large degree of structural conservation of surface loops in one region as well as an apparent aromatic specificity pocket in the amino-terminal branch . Also discussed is the sequence and possible functional relationship of the pectate lyases with pollen and style plant proteins.

Plant Physiol, 1994 Jan, 104(1), 119 - 125
Expression of Erwinia uredovora Phytoene Desaturase in Synechococcus PCC7942 Leading to Resistance against a Bleaching Herbicide; Windhovel U et al.; The gene coding for phytoene desaturase of the bacterium Erwinia uredovora (crtI) was inserted into the chromosome of the cyanobacterium Synechococcus PCC7942 strain R2-PIM8 . For expression of crtI in the heterologous host, two constructs with different promoters were introduced into Synechococcus . In the first, crtI was fused to the 5{prime} region of the psbA gene of the xanthophycean microalga Bumilleriopsis filiformis . The second construct carried crtI inserted downstream of the neomycin phosphotransferase II gene (nptII) from the transposon Tn5 . Expression of crtI under the control of the respective promoter was shown by immunodetection of the gene product . The functionality of the heterologously expressed phytoene desaturase CRTI in the transformants was demonstrated by enzymic assays . The transformants acquired very strong resistance toward the bleaching herbicide norflurazon.

Plant Physiol, 1993 Aug, 102(4), 1341 - 1344
Harpin, An Elicitor of the Hypersensitive Response in Tobacco Caused by Erwinia amylovora, Elicits Active Oxygen Production in Suspension Cells; Baker CJ et al.; Active oxygen (AO) production and a K+/H+ exchange response (XR) are two concurrent early events associated with incompatible plant-bacteria interactions that result in a hypersensitive response (HR) . Recently, a protein, termed harpin, produced by Erwinia amylovora has been reported to be the elicitor responsible for the HR caused by this pathogen . Although both the bacterium and harpin are reported to induce XR in tobacco (Nicotiana tabacum) cell suspensions, there have been no reports regarding the concurrent production of AO in this system . Here we report that E . amylovora stimulates the AO response, whereas an E . amylovora mutant that does not produce harpin does not elicit the AO response . In addition, a cell-free preparation of harpin induces AO production . This study indicates that harpin may be the bacterial elicitor of the XR and AO responses during the development of E . amylovora-induced HR.

Plant Physiol, 1997 Oct, 115(2), 853 - 861
Rapid and Transient Activation of a Myelin Basic Protein Kinase in Tobacco Leaves Treated with Harpin from Erwinia amylovora; Adam AL et al.; Harpins are bacterial protein elicitors that induce hypersensitive response-like necrosis when infiltrated into nonhost plants such as tobacco (Nicotiana tabacum L.) (Z.-M . Wei, R.J . Laby, C.H . Zumoff, D.W . Bauer, S.Y . He, A . Collmer, S.V . Beer {1992} Science 257: 85-88) . Activity of a 49-kD Mg2+-dependent and Ca2+-independent kinase in tobacco leaves increased 50-fold 15 min after infiltration of harpin from Erwinia amylovora (harpinEa) . Much less pronounced and more transient activation was detected in water-infiltrated leaves . Biochemical characteristics of the harpinEa-activated protein kinase (HAPK) activity are consistent with those of the mitogen-activated protein kinase family . HAPK is cytosolic and phosphorylates myelin basic protein on serine/threonine residues . Treatment with a protein tyrosine phosphatase completely eliminated HAPK activity, suggesting that tyrosine phosphorylation is required for posttranslational activation . Sustained HAPK activation after cycloheximide treatment implies that HAPK may be negatively regulated by a translation-dependent mechanism . The extracellular Ca2+ chelator EGTA or the protein kinase inhibitor K252a, infiltrated in planta together with harpinEa, partially blocked HAPK activation . The Ca2+-channel blocker La3+ had no effect on HAPK activation, suggesting that phosphorylation events precede and/or do not depend on the entry of extracellular Ca2+ into the cell . These results suggest that early signal transduction events during harpinEa- induced hypersensitive response elicitation depend in part on the activation of HAPK.

J Econ Entomol, 2002 Aug, 95(4), 797 - 802
Pear transformed with a lytic peptide gene for disease control affects nontarget organism, pear psylla (Homoptera: Psyllidae); Puterka GJ et al.; The biology and behavior of pear psylla, Cacopsylla pyricola Foerster, on a transgenic clone of 'Bartlett' pear, Pyrus communis L., containing a synthetic antimicrobial gene, D5C1, was compared with that of a nontransgenic parental clone to determine whether there were any nontarget effects . The gene construct also contained the marker gene nptII (aminoglycoside 3'-phosphotransferase II) that encodes for antibiotic resistance to identify transformed plants . The purpose of the original transformation was to enhance pear resistance to the bacterial disease fireblight caused by Erwinia amylovora (Burr.) Winslow et al . The biology and behavior of pear psylla on a transgenic clone were compared with a nontransgenic parental pear clone in short- (< or = 7-d) and long-term (32-d) studies . Short-term studies indicated pear psylla adults preferred to settle and oviposit, and nymphs fed more and developed slightly faster, on transgenic pear compared with nontransgenic pear . In contrast, a long-term study on psylla colony development showed considerably fewer eggs, nymphs, and adults were produced on transgenic pear . Although adults reared on transgenic pear did not have weight affected, females produced fewer eggs and nymphal hatch was significantly reduced on the transgenic pear clone . Our results suggest that pear psylla biology and behavior are initially enhanced on this transgenic pear clone . However, chronic exposure of psylla populations to transformed pear plants that express the nptII marker and lytic peptide genes had detrimental effects on pear psylla reproductive biology . Field studies would be required to determine the specific effects of each gene on pear psylla biology and behavior and whether these effects would be expressed under natural conditions . The four-fold reduction in psylla population levels that resulted on this disease resistant transgenic pear line would be an added benefit to a pear integrated pest management (IPM) program . Overall, this study demonstrates that genetically altering plants to control one particular organism can have unintentional yet beneficial effects against other nontarget pest organisms in agricultural crops.

Arch Pharm (Weinheim), 2002 Jun, 335(6), 251 - 61
A new synthesis of oxadiazole, thiazolidinone, N-phthalimidoamino carbonyl and arylidene derivatives with potential antimicrobial activity; Hassaneen HM et al.; Condensation of carbohydrazide derivatives Ia, b with dimethyl acetylenedicarboxylate and acetylenedicarboxylic acid yielded benzofuran derivatives II a-d . Reaction of Ib with aromatic aldehydes formed products III a-d . Treatment of compounds III a-d with mercaptoacetic acid yielded the cyclocondensation products (IVa-d) . Phthalic anhydride reacted with compounds (Ia, b)to form products (Va, b) . It has been found that both khellin and visnagin (VIa, b)react with aromatic aldehydes to give arylidene derivatives (VIIa-e) . Condensation of diphenyl nitrilamine with 2-arylidene furochromones VII derivatives afforded cyclo-adducts (VIII a-i) . The antibacterial activities of the selected compounds were tested against Staphylococcus aureus, B . subtilis, E . coli, Pseudomonas, Salmonella and Erwinia with good results.

Mikrobiol Z, 2002 May-Jun, 64(3), 20 - 6
{Method for studying horizontal transfer of plasmids in Erwinia carotovora}; Gorb TE et al.; A simple method to determine frequency characteristics of the horizontal transfer of antibiotic resistance by transconjugative plasmids of various incompatibility groups into Erwinia carotovora strains has been developed . The proposed method for selection of E . carotovora transconjugates provides for the use of selective medium with pectin and corresponding antibiotic, which permits carrying out reliable counterselection of the donor (Escherichia, Salmonella) . The proposed method helped to study horizontal transfer of plasmids RP4 (Inc P), R 391 (Inc J) and pKM101 (Inc N) to some E . carotovora strains and to demonstrate their inheritance as extrachromosomal DNA in Erwinia cells.

Mikrobiol Z, 2002 Mar-Apr, 64(2), 65 - 81
{Comparative study of properties of temperate erwiniophages 49 and 59}; Tovkach FI et al.; Molecular-biological properties of two relative temperate erwiniophages 49 and 59 have been comparatively studied . The both phages are highly specific with respect to sensitive bacteria and lyse only inconsiderable quantity of amylovora-like strains of Erwinia horticola . It has been established that erwiniophages are distinguished by the basic parameters of a single reproduction cycle in the cells of common host E . horticola 450 . Considerable differences between phages have been also found in the areas of genomes responsible for the establishment and maintenance of lysogenic state in the cells of the bacterium-host . Study of structure polypeptides has confirmed the identity of capsids and tails of phages 49 and 59 . It has been shown that phage 49 has another, as compared to phage 59, basal plate, which availability destabilises the phage tail and leads to virion destruction under various physical effects . Virion DNA of phages 49 and 59 are of the same size--47.9 kbp, but differ as to GC-content . Using the restriction analysis it has been shown that genome of phage 49, as well as the genome of phage 59, is permuted, but its permutation is of discrete character . The fact of recombination interaction between erwiniophages 49 and 59 has been established . It is supposed that phage 49 is the recombination (hybrid) derivative of phage 59 and unknown phage, or prophage, genetic module . The given recombination, probably, took place under the persistence of different phages in the general polylysogenic system of E . horticola.

Mol Plant Microbe Interact, 2002 Aug, 15(8), 764 - 73
Constitutive expression of hrap gene in transgenic tobacco plant enhances resistance against virulent bacterial pathogens by induction of a hypersensitive response; Ger MJ et al.; Hypersensitive response-assisting protein (HRAP) has been previously reported as an amphipathic plant protein isolated from sweet pepper that intensifies the harpin(Pss)-mediated hypersensitive response (HR) . The hrap gene has no appreciable similarity to any other known sequences, and its activity can be rapidly induced by incompatible pathogen infection . To assess the function of the hrap gene in plant disease resistance, the CaMV 35S promoter was used to express sweet pepper hrap in transgenic tobacco . Compared with wild-type tobacco, transgenic tobacco plants exhibit more sensitivity to harpin(Pss) and show resistance to virulent pathogens (Pseudomonas syringae pv . tabaci and Erwinia carotovora subsp . carotovora) . This disease resistance of transgenic tobacco does not originate from a constitutive HR, because endogenous level of salicylic acid and hsr203J mRNA showed similarities in transgenic and wildtype tobacco under noninfected conditions . However, following a virulent pathogen infection in hrap transgenic tobacco, hsr203J was rapidly induced and a micro-HR necrosis was visualized by trypan blue staining in the infiltration area . Consequently, we suggest that the disease resistance of transgenic plants may result from the induction of a HR by a virulent pathogen infection.

Plant Physiol, 2002 Aug, 129(4), 1607 - 15
Inhibition of the plastidic ATP/ADP transporter protein primes potato tubers for augmented elicitation of defense responses and enhances their resistance against Erwinia carotovora; Linke C et al.; Tubers of transgenic potato (Solanum tuberosum) plants with decreased activity of the plastidic ATP/ADP transporter AATP1 display reduced levels of starch, modified tuber morphology, and altered concentrations of primary metabolites . Here, we demonstrate that the spontaneous production of hydrogen peroxide, the endogenous content of salicylic acid, and the levels of mRNAs of various defense-related genes are similar in tuber discs of wild-type and AATP1(St) antisense plants . However, upon challenging the tissue with fungal elicitors or culture supernatants of the soft rot-causing pathogen Erwinia carotovora subsp . atroseptica, the AATP1(St) antisense tubers exhibit highly potentiated activation of defense responses when compared with wild-type tissue . The augmented defense responses comprise enhanced accumulation of transcripts of five defense-related genes (beta-1,3-GLUCANASE B2 and A1, CHITINASE B3 and A2, and Phe AMMONIA-LYASE) and enhanced elicitation (up to 21-fold) of the early hydrogen peroxide burst . The potentiated activation of cellular defense responses in AATP1(St) antisense tubers is not accompanied by a precedent increase in endogenous salicylic acid levels, but is associated with a strongly enhanced resistance of the tissue to E . carotovora . From these results, we conclude that inhibition of primary metabolic reactions induces a primed state that sensitizes the potato tubers for improved elicitation of various cellular defense responses, which likely contribute to enhanced E . carotovora resistance.

Genetika, 2002 Jul, 38(7), 904 - 10
{Properties of mutants of bacteria belonging to the genus Erwinia devoid of common components of the phosphoenolpyruvate-dependent phosphotransferase system}; Datsenko KA et al.; 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.

Cancer Chemother Pharmacol, 2002 Aug, 50(2), 117 - 20 Epub 2002 Jun 15.
Anti-Erwinia asparaginase antibodies during treatment of childhood acute lymphoblastic leukemia and their relationship to outcome: a case-control study; Klug Albertsen B et al.; PURPOSE: A case-control study was performed to determine whether patients who had been treated with Erwinia asparaginase as part of their treatment for childhood acute lymphoblastic leukemia (ALL) and who showed relapsed of their disease more often developed anti-asparaginase antibodies than patients who remained in remission . METHODS: A group of 13 patients who showed relapsed of their disease (median follow-up 35 months) were randomly matched with control patients of the same risk group (two control patients to each case), who had received therapy of the same intensity during the same period (median follow-up 70 months) . Anti- Erwinia asparaginase antibodies were measured (ELISA method) during maintenance therapy after asparaginase treatment (30,000 IU/m(2) daily for 10 days in all patients plus twice weekly for 2 weeks in intermediate-risk and high-risk ALL patients) . RESULTS: The overall incidence of anti- Erwinia asparaginase antibodies was 8% (3 of 39 patients) . There was no statistically significant difference in the incidence of antibody formation between patients who had suffered relapse (1 of 13) and those who had not (2 of 26) . In two of the three patients who developed antibodies, the antibodies disappeared after some time, whereas one patient had measurable antibody levels for more than a year after asparaginase therapy . CONCLUSIONS: In this study, the development of anti-Erwinia asparaginase antibodies was rare and was unrelated to the risk of relapse.

Annu Rev Phytopathol, 2002, 40, 443 - 65 Epub 2002 Feb 20.
Antibiotic use in plant agriculture; McManus PS et al.; Antibiotics have been used since the 1950s to control certain bacterial diseases of high-value fruit, vegetable, and ornamental plants . Today, the antibiotics most commonly used on plants are oxytetracycline and streptomycin . In the USA, antibiotics applied to plants account for less than 0.5% of total antibiotic use . Resistance of plant pathogens to oxytetracycline is rare, but the emergence of streptomycin-resistant strains of Erwinia amylovora, Pseudomonas spp., and Xanthomonas campestris has impeded the control of several important diseases . A fraction of streptomycin-resistance genes in plant-associated bacteria are similar to those found in bacteria isolated from humans, animals, and soil, and are associated with transfer-proficient elements . However, the most common vehicles of streptomycin-resistance genes in human and plant pathogens are genetically distinct . Nonetheless, the role of antibiotic use on plants in the antibiotic-resistance crisis in human medicine is the subject of debate.

Appl Environ Microbiol, 2002 Aug, 68(8), 3919 - 24
Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis; Lee SJ et al.; Gram-negative bacteria can communicate with each other by N-acyl homoserine lactones (AHLs), which are quorum-sensing autoinducers . Recently, the aiiA gene (encoding an enzyme catalyzing the degradation of AHL) has been cloned from Bacillus sp . strain 240B1 . During investigations in the course of the ongoing Bacillus thuringiensis subsp . morrisoni genome project, an aiiA homologue gene in the genome sequence was found . These results led to consideration of the possibility of the widespread existence of the gene in B . thuringiensis . aiiA homologue genes were found in 16 subspecies of B . thuringiensis, and their sequences were determined . Comparison of the Bacillus sp . strain 240B1 aiiA gene with the B . thuringiensis aiiA homologue genes showed high homologies of 89 to 95% and 90 to 96% in the nucleotide sequence and deduced amino acid sequence, respectively . Among the subspecies of B . thuringiensis having an aiiA gene, the subspecies aizawai, galleriae, kurstaki, kyushuensis, ostriniae, and subtoxicus were shown to degrade AHL . It was observed that recombinant Escherichia coli producing AiiA proteins also had AHL-degrading activity and could also attenuate the plant pathogenicity of Erwinia carotovora . These results indicate that insecticidal B . thuringiensis strains might have potential to compete with gram-negative bacteria in natural ecosystems by autoinducer-degrading activity.

J Biotechnol, 2002 Jun 13, 96(1), 119 - 24
Potato tubers as bioreactors for palatinose production; Bornke F et al.; Palatinose (isomaltulose, 6-O-alpha-D-glucopyranosyl-D-fructose) is a structural isomer of sucrose with very similar physico-chemical properties . Due to its non-cariogenicity and low calorific value it is an ideal sugar substitute for use in food production . Palatinose is produced on an industrial scale from sucrose by an enzymatic rearrangement using immobilized bacterial cells . To explore the potential of transgenic plants as alternative production facilities for palatinose, a chimeric sucrose isomerase gene from Erwinia rhapontici under control of a tuber-specific promoter was introduced into potato plants . The enzyme catalyses the conversion of sucrose into palatinose . Expression of the palI gene within the apoplast of transgenic tubers led to a nearly quantitative conversion of sucrose into palatinose . Despite the soluble carbohydrates having been altered within the tubers, growth of palI expressing transgenic potato plants was indistinguishable from wild type plants . Therefore, expression of a bacterial sucrose isomerase provides a valid tool for high level palatinose production in storage tissues of transgenic crop plants.

Mol Microbiol, 2002 Aug, 45(3), 769 - 83
Characterization of a novel phenazine antibiotic gene cluster in Erwinia herbicola Eh1087; Giddens SR et al.; Erwinia herbicola strain Eh1087 produces the broad-spectrum phenazine antibiotic D-alanylgriseoluteic acid (AGA) . In this report, a cluster of 16 ehp (Erwinia herbicola phenazine) plasmid genes required for the production of AGA by Eh1087 is described . The extent of the gene cluster was revealed by the isolation of 82 different Eh1087 AGA- mutants, all found to possess single mini-Tn5lacZ2 insertions within a 14 kbp DNA region . Additional transposon insertions that did not affect antibiotic production by Eh1087 were created to define the boundaries of the gene cluster . The size and location of genes between these boundaries were derived from a combination of DNA sequence analyses, minicell protein analyses and the correlation between mutation position and the production of coloured AGA intermediates by many ehp mutants . Precursor-feeding and complementation experiments resulted in 15 ehp genes being assigned to one of four functional groups according to their role in the synthesis of AGA . Group 1 is required for the synthesis of the phenazine nucleus in the form of antibiotic precursor one (AP1, phenazine-1,6-dicarboxylic acid) . Group 2 is responsible for conversion of AP1 to AP2, which is subsequently modified to AP3 (griseoluteic acid) and exported by the group 3 gene products . Group 4 catalyses the addition of D-alanine to AP3 to create AGA, independently of groups 1, 2 and 3 . A gene that is divergently transcribed from the 15 AGA synthesis ehp genes confers resistance to AGA.

Plant Mol Biol, 2002 Sep, 50(1), 129 - 42
Expression of a bacterial carotene hydroxylase gene (crtZ) enhances UV tolerance in tobacco; Gotz T et al.; Carotenoids are essential components of the photosynthetic apparatus involved in plant photoprotection . To investigate the protective role of zeaxanthin under high light and UV stress we have increased the capacity for its biosynthesis in tobacco plants (Nicotiana tabacum L . cv . Samsun) by transformation with a heterologous carotenoid gene encoding beta-carotene hydroxylase (crtZ) from Erwinia uredovora under constitutive promoter control . This enzyme is responsible for the conversion of beta-carotene into zeaxanthin . Although the total pigment content of the transgenics was similar to control plants, the transformants synthesized zeaxanthin more rapidly and in larger quantities than controls upon transfer to high-intensity white light . Low-light-adapted tobacco plants were shown to be susceptible to UV exposure and therefore chosen for comparative analysis of wild-type and transgenics . Overall effects of UV irradiation were studied by measuring bioproductivity and pigment content . The UV exposed transformed plants maintained a higher biomass and a greater amount of photosynthetic pigments than controls . For revelation of direct effects, photosynthesis, pigment composition and chlorophyll fluorescence were examined immediately after UV treatment . Low-light-adapted plants of the crtZ transgenics showed less reduction in photosynthetic oxygen evolution and had higher chlorophyll fluorescence levels in comparison to control plants . After 1 h of high-light pre-illumination and subsequent UV exposure a greater amount of xanthophyll cycle pigments was retained in the transformants . In addition, the transgenic plants suffered less lipid peroxidation than the wild-type after treatment with the singlet-oxygen generator rose bengal . Our results indicate that an enhancement of zeaxanthin formation in the presence of a functional xanthophyll cycle contributes to UV stress protection and prevention of UV damage.

Mikrobiologiia, 2002 May-Jun, 71(3), 359 - 67
{Defective lysogeny in Erwinia carotovora}; Tovkach FI; The electron microscopic study of several Erwinia carotovora strains showed that the SOS-induced cells of this pectolytic phytopathogenic bacterium produce particular phage parts (tails, heads, and baseplates) but do not assemble them into fully functional phage particles . E . carotovora cells produced several times greater amounts of phage tails in response to induction by mitomycin C than in response to induction by nalidixic acid . The tails were 128-192 nm in length and 13-21 nm in diameter . Phage heads were characterized by four discrete ranges of diameters: 18, 55-59, 66-75, and 92-98 nm . The diameters of phage baseplates varied from 39 to 53 nm, depending on the particular strain . It was shown that cells of the same species may contain several different types of phage tails and heads . The structural organization of phage tails and baseplates in the nalidixic acid-induced lysate of E . carotovora J2 was studied in more detail . The data obtained suggest that pectolytic phytopathogenic erwinia are characterized by defective polylysogeny.

Mol Plant Microbe Interact, 2002 Jul, 15(7), 709 - 16
The presence of diverse IS elements and an avrPphD homologue that acts as a virulence factor on the pathogenicity plasmid of Erwinia herbicola pv . gypsophilae; Guo M et al.; The pathogenicity of Erwinia herbicola pv . gypsophilae (Ehg) and Erwinia herbicola pv . betae (Ehb) is dependent on a native plasmid (pPATH(Ehg) or pPATH(Ehb)) that harbors the hrp gene cluster, genes encoding type III effectors, phytohormones, biosynthetic genes, and several copies of IS1327 . Sequence analysis of the hrp-flanking region in pPATH(Ehg) (cosmid pLA150) revealed a cluster of four additional IS elements designated as ISEhel, ISEhe2, ISEhe3, and ISEhe4 . Two copies of another IS element (ISEhe5) were identified on the upstream region of the indole-3-acetic acid operon located on the same cosmid . Based on homology of amino acids and genetic organization, ISEhe1 belongs to the IS630 family, ISEhe2 to the IS5 family, ISEhe3 and ISEhe4 to different groups of the IS3 family, and ISEhe5 to the IS1 family . With the exception of ISEhe4, one to three copies of all the other IS elements were identified only in pathogenic strains of Erwinia herbicola pv . gypsophilae and Erwinia herbicola pv . betae whereas ISEhe4 was present in both pathogenic and nonpathogenic strains . An open reading frame that exhibited high identity (89% in amino acids) to AvrPphD of Pseudomonas syringae pv . phaseolicola was present within the cluster of IS elements . An insertional mutation in the AvrPphDEh, reduced gall size in gypsophila by approximately 85% . In addition, remnants of known genes from four different bacteria were detected on the same cosmid.

Mol Plant Microbe Interact, 2002 Jul, 15(7), 619 - 29
Microarray profiling of Erwinia chrysanthemi 3937 genes that are regulated during plant infection; Okinaka Y et al.; Microarray technology was used to identify genes in Erwinia chrysanthemi 3937 that are specifically up- or down-regulated in a plant host compared with growth in laboratory culture medium . Several genes were plant down-regulated, and almost all of them were homologues of well-known housekeeping genes, such as those encoding metabolic functions, oxidative phosphorylation components, and transcription or translation processes . On the other hand, almost all of the plant up-regulated genes were involved with specialized functions, including already known or new putative virulence factors, anaerobiosis, iron uptake, transporters or permeases, xenobiotic resistance, chemotaxis, and stress responses to reactive oxygen species and heat . A substantial number of the plant up-regulated genes do not appear to be directly involved in damaging the host, but are probably important in adapting the pathogen to the host environment . We constructed insertion mutations in several of the plant up-regulated E . chrysanthemi 3937 genes . Among these, mutations of Bacillus subtilis pps1, Escherichia coli purU, and Pseudomonas aeruginosa pheC homologues reduced virulence on African violet leaves . Thus, new insights were obtained into genes important in bacterial virulence.

Can J Microbiol, 2002 May, 48(5), 387 - 98
Molecular characterization of DNA encoding 16S-23S rRNA intergenic spacer regions and 16S rRNA of pectolytic Erwinia species; Fessehaie A et al.; Sequences of 16S rDNAs and the intergenic spacer (IGS) regions between the 16S and 23S rDNA of bacterial strains from genus Erwinia were determined . Comparison of 16S rDNA sequences from different species and subspecies clearly revealed intraspecies-subspecies homology and interspecies heterogeneity . Phylogenetic analyses of 16S rDNA sequence data revealed that Erwinia spp . formed a discrete monophyletic clade with moderate to high bootstrap values . PCR amplification of the 16S-23S rDNA regions using primers complementary to the 3' end of 16S and 5' end of 23S rRNA genes generated two DNA fragments . The small 16S-23S rDNA IGS regions of Erwinia spp . examined in this study varied considerably in size and nucleotide sequence . Multiple sequence alignment and phylogenetic analysis of small IGS sequence data showed a consistent relationship among the test strains that was roughly in agreement with the 16S rDNA data that reflected the accepted species and subspecies structure of the taxon . Sequence data derived from the large IGS resolved the strains into coherent groups; however, the sequence information would not allow any phylogenetic conclusion, because it failed to reflect the accepted species structure of the test strains.

J Bacteriol, 2002 Aug, 184(15), 4089 - 95
RsmA and the quorum-sensing signal, N-{3-oxohexanoyl}-L-homoserine lactone, control the levels of rsmB RNA in Erwinia carotovora subsp . carotovora by affecting its stability; Chatterjee A et al.; RsmA (for regulator of secondary metabolism), RsmC, and rsmB RNA, the components of a posttranscriptional regulatory system, control extracellular protein production and pathogenicity in Erwinia carotovora subsp . carotovora . RsmA, an RNA binding protein, acts as a negative regulator by promoting message decay . rsmB RNA, on the other hand, acts as a positive regulator by neutralizing the effect of RsmA . RsmC modulates the levels of RsmA and rsmB RNA by positively regulating rsmA and negatively controlling rsmB . The level of rsmB RNA is substantially higher in RsmA(+) bacteria than in RsmA(-) mutants . We show that rsmB RNA is more stable in the presence of RsmA than in its absence . RsmA does not stimulate the expression of an rsmB-lacZ transcriptional fusion; in fact, the beta-galactosidase level is somewhat higher in RsmA(-) bacteria than in RsmA(+) bacteria . We also investigated the basis for increased levels of rsmA and rsmB RNAs in the absence of the quorum-sensing signal, N-{3-oxohexanoyl}-L-homoserine lactone (OHL) . The absence of OHL activates transcription of rsmA but not of rsmB . Instead, increased stability of rsmB RNA in the presence of RsmA accounts for the elevated levels of the rsmB RNA in OHL(-) bacteria . Mutant studies disclosed that while RsmA, OHL, and RsmC control the levels of rsmB RNA, high levels of rsmB RNA occur in the absence of RsmC or OHL only in RsmA(+) bacteria, indicating a critical role for RsmA in modulating the levels of rsmB RNA . The findings reported here firmly establish that the quorum-sensing signal is channeled in E . carotovora subsp . carotovora via the rsmA-rsmB posttranscriptional regulatory system.

Ann Bot (Lond), 2002 Mar, 89(3), 245 - 53
Altering plant-microbe interaction through artificially manipulating bacterial quorum sensing; Fray RG; Many bacteria regulate diverse physiological processes in concert with their population size . Bacterial cell-to-cell communication utilizes small diffusible signal molecules, which the bacteria both produce and perceive . The bacteria couple gene expression to cell density by eliciting a response only when the signalling molecules reach a critical threshold (a point at which the population is said to be 'quorate') . The population as a whole is thus able to modify its behaviour as a single unit . Amongst Gram-negative bacteria, the quorum sensing signals most commonly used are N-acylhomoserine lactones (AHLs) . It is now apparent that AHLs are used for regulating diverse behaviours in epiphytic, rhizosphere-inhabiting and plant pathogenic bacteria and that plants may produce their own metabolites that interfere with this signalling . Transgenic plants that produce high levels of AHLs or which can degrade bacterial-produced AHLs have been made . These plants have dramatically altered susceptibilities to infection by pathogenic Erwinia species . In addition, such plants will prove useful tools in determining the roles of AHL-regulated density-dependent behaviour in growth promoting, biological control and pathogenic plant-associated bacterial species.

FEMS Microbiol Lett, 2002 Jun 4, 211(2), 247 - 52
Characterization of Bacillus strains from apple and pear trees in South Africa antagonistic to Erwinia amylovora; Jock S et al.; In order to find reasons for the absence of fire blight in most countries of the Southern hemisphere, bark samples from apple and pear trees in orchards of the Western Cape region in South Africa were extracted for bacteria which could be antagonistic to Erwinia amylovora . Screening was done in the late growth season and mainly Gram-positive bacteria were isolated . Approximately half of them produced growth inhibition zones on a lawn of E . amylovora . Most isolates were classified as Bacillus megaterium by microbiological assays and in API 50 test systems . They were visualized in the light microscope as non-motile large rods . These strains may not be responsible for the absence of fire blight in orchards, but they may indicate unfavourable climatic conditions for Gram-negative bacteria including E . amylovora . They may reduce the ability of E . amylovora to establish fire blight and could also be useful for application in biological disease control.

Genetika, 2002 May, 38(5), 622 - 8
{Isolation and genetic study of Erwinia mutants devoid of common components of the phosphoenolpyruvate-dependent phosphotransferase system}; Datsenko KA et al.; Mutants of bacteria belonging the genus Erwinia (Erwinia chrysanthemi and Erwinia carotovora) with pleiotropic disturbances in the utilization of many substrates were obtained through chemical and transposon mutagenesis . Genetic studies revealed that these mutants had defective ptsI or ptsH genes responsible for the synthesis of common components of the phosphoenolpyruvate-dependent phosphotransferase system, enzyme I and the HPr protein, respectively . The ptsI+ allele in both Erwinia species was cloned in vivo . Mapping of obtained mutations indicated that the ptsI and ptsH genes of E . chrysanthemi do not constitute a linkage group . The ptsI gene is located at 100 min of the chromosomal map, whereas the ptsH gene is located at 175 min . Sequencing of a portion of the E . chrysanthemi ptsI gene showed that a product of the cloned DNA region had up to 68% homology with the N terminus of Escherichia coli enzyme I.

J Appl Microbiol, 2002, 93(1), 144 - 8
Interaction of ozone and negative air ions to control micro-organisms; Fan L et al.; AIMS: The aims of this study were to investigate the effect of ozone and/or negative air ions (NAI) on the viability of bacteria . METHODS AND RESULTS: Dilute cell suspensions of Pseudomonas fluorescens, Erwinia carotovora pv . carotovora and Escherichia coli were inoculated onto agar and subsequently exposed to ozone and/or NAI . Ozone concentration was maintained at 100 +/- 5 nl l-1 and NAI at 106 ml-1 . When exposed to a combination of ozone and NAI, viability among all three bacterial species decreased more rapidly when they were inoculated onto potato dextrose agar (PDA) than onto nutrient agar (NA) . A subsequent test examined the effect of ozone and NAI alone or in combination on the bacteria inoculated onto PDA only . Treatment with NAI alone had no killing effect on any of the bacterial species . However, a strong interaction between ozone and NAI was observed . Pseudomonas fluorescens was most susceptible to the combined treatment . Cell viability was reduced to 0.7% after 6 h, while 76% of the cells remained viable when exposed to ozone alone . Viability of Erwinia carotovora pv . carotovora was reduced to 4% after 6 h in the combined treatment compared with 69% when exposed to ozone alone . Escherichia coli was relatively more resistant to the combined treatment; viability was reduced to 40% after 11 h compared with 70% in the ozone alone treatment . CONCLUSIONS: A strong synergism between ozone and NAI on bacterial cell death was found, but the degree of this effect varied depending on bacterial species . SIGNIFICANCE AND IMPACT OF THE STUDY: The synergism of ozone with NAI may provide an effective method of reducing food-borne disease and decay of fresh produce.

Mol Microbiol, 2002 Jun, 44(6), 1651 - 65
Identification of XcpP domains that confer functionality and specificity to the Pseudomonas aeruginosa type II secretion apparatus; Gerard-Vincent M et al.; Gram-negative bacteria have evolved several types of secretion mechanisms to release proteins into the extracellular medium . One such mechanism, the type II secretory system, is a widely conserved two-step process . The first step is the translocation of signal peptide-bearing exoproteins across the inner membrane . The second step, the translocation across the outer membrane, involves the type II secretory apparatus or secreton . The secretons are made up of 12-15 proteins (Gsp) depending on the organism . Even though the systems are conserved, heterologous secretion is mostly species restricted . Moreover, components of the secreton are not systematically exchangeable, especially with distantly related microorganisms . In closely related species, two components, the GspC and GspD (secretin) family members, confer specificity for substrate recognition and/or secreton assembly . We used Pseudomonas aeruginosa as a model organism to determine which domains of XcpP (GspC member) are involved in specificity . By constructing hybrids between XcpP and OutC, the Erwinia chrysanthemi homologue, we identified a region of 35 residues that was not exchangeable . We showed that this region might influence the stability of the XcpYZ secreton subcomplex . Remarkably, XcpP and OutC have domains, coiled-coil and PDZ, respectively, which exhibit the same function but that are structurally different . Those two domains are exchangeable and we provided evidence that they are involved in the formation of homomultimeric complexes of XcpP.

J Mol Biol, 2002 Jun 7, 319(3), 779 - 89
Crystal structure of shikimate kinase from Mycobacterium tuberculosis reveals the dynamic role of the LID domain in catalysis; Gu Y et al.; Shikimate kinase (SK) and other enzymes in the shikimate pathway are potential targets for developing non-toxic antimicrobial agents, herbicides, and anti-parasite drugs, because the pathway is essential in the above species but is absent from mammals . The crystal structure of Mycobacterium tuberculosis SK (MtSK) in complex with MgADP has been determined at 1.8 A resolution, revealing critical information for the structure-based design of novel anti-M . tuberculosis agents . MtSK, with a five-stranded parallel beta-sheet flanked by eight alpha-helices, has three domains: the CORE domain, the shikimate-binding domain (SB), and the LID domain . The ADP molecule is bound with its adenine moiety sandwiched between the side-chains of Arg110 and Pro155, its beta-phosphate group in the P-loop, and the alpha and beta-phosphate groups hydrogen bonded to the guanidinium group of Arg117 . Arg117 is located in the LID domain, is strictly conserved in SK sequences, is observed for the first time to interact with any bound nucleotide, and appears to be important in both substrate binding and catalysis . The crystal structure of MtSK (this work) and that of Erwinia chrysanthemi SK suggest a concerted conformational change of the LID and SB domains upon nucleotide binding . (c) 2002 Elsevier Science Ltd.

Acta Crystallogr D Biol Crystallogr, 2002 Jun, 58(Pt 6 Pt 2), 1008 - 15 Epub 2002 May 29.
Structure of pectate lyase A: comparison to other isoforms; Thomas LM et al.; Pectate lyase A is a virulence factor secreted by the plant-pathogenic bacteria Erwinia chrysanthemi . The enzyme cleaves the glycosidic bond of pectate polymers by a calcium-dependent beta-elimination mechanism . The crystal structure of pectate lyase A from E . chrysanthemi EC16 has been determined in two crystal forms, monoclinic C2 to 1.8 A and rhombohedral R3 to 2.1 A . The protein structure is compared with two other pectate lyase isoforms from E . chrysanthemi EC16, pectate lyase C and pectate lyase E . Pectate lyase A is unique as it is the only acidic pectate lyase and has end products that are significantly more varied in length in comparison to those of the other four major pectate lyase isozymes . Differences and similarities in polypeptide trace, size and volume of the active-site groove and surface electrostatics are discussed.

Mol Plant Microbe Interact, 2002 May, 15(5), 472 - 80
hrp genes of Erwinia chrysanthemi 3937 are important virulence factors; Yang CH et al.; We developed improved virulence assays for Erwinia chrysanthemi 3937 on African violet varieties and devised a new method for the construction of precise bacterial gene knockouts . These methods were tested by constructing mutations in genes suspected to be involved with plant interactions . The virulence of the hrpG and hrcC mutant strains (both gene products presumed to be involved in protein secretion) was greatly reduced on leaves of semitolerant African violet varieties . An hrpN mutant strain produced delayed symptoms on African violet leaves and an hrpN delta pel (delta pel = five major pectate lyase genes deleted) double mutant was nonpathogenic . The hrcC and hrpG mutants did not produce a rapid hypersensitive response (HR) in tobacco, unlike the wild-type bacterium, and the hrpN mutant gave a reduced HR . The results, therefore, establish the importance of hrp genes in the virulence of E . chrysanthemi and their ability to elicit HR on nonhosts . The data also suggest that other effector proteins secreted by the Hrp system are required for full virulence and HR elicitation.

Planta, 2002 Jun, 215(2), 205 - 9 Epub 2002 Mar 09.
Natural variability in the Arabidopsis response to infection with Erwinia carotovora subsp . carotovora; Aguilar I et al.; The natural variation in the response of Arabidopsis thaliana (L.) Heynh . to Erwinia carotovora subsp . carotovora has been studied in seven ecotypes and two mutants . The susceptibility of all the plant types was investigated by (i) macroscopic symptoms, (ii) fluorescence microscopy using green fluorescent protein (GFP) and (iii) bacterial growth in planta . Although all the plants were susceptible to the bacterium, there was no correlation in the degree of infection as ascertained by the three methods . The induction, upon infection, of several genes known to be involved in defense was analyzed by RNA blot hybridization . The patterns of expression of these genes differed according to the genotype . These results suggest that both salicylic and jasmonic acid play a role in the response of Arabidopsis to this bacterium.

Appl Opt, 2002 May 1, 41(13), 2541 - 5
Detection of bacterial infection of agave plants by laser-induced fluorescence; Cervantes-Martinez J et al.; Greenhouse-grown plants of Agave tequilana Weber var . azul were inoculated with Erwinia carotovora, the causal agent of stem soft rot . We investigated the laser-induced fluorescence (LIF) of agave plants to determine whether LIF can be used as a noninvasive sensing tool for pathological studies . The LIF technique was also investigated as a means of detecting the effect of the polyamine biosynthesis inhibitor beta-hydroxyethylhydrazine as a bactericide against the pathogenic bacterium Erwinia carotovora . A He-Ne laser at 632.8 nm was used as the excitation source, and in vivo fluorescence emission spectra were recorded in the 660-790-range . Fluorescence maxima were at 690 and 740 nm . The infected plants that were untreated with the bactericide showed a definite increase in fluorescence intensity at both maxima within the first three days after infection . Beginning on the fifth day, a steady decrease in fluorescence intensity was observed, with a greater effect at 740 than at 690 nm . After 30 days there was no fluorescence . The infected plants that had been treated with the bactericide showed no significant change in fluorescence compared with that of the uninfected plants . The ratio of fluorescence intensities was determined to be F 690 nm/F 740 nm for all treatments . These studies indicate that LIF measurements of agave plants may be used for the early detection of certain types of disease and for determining the effect of a bactericide on bacteria . The results also showed that fluorescence intensity ratios can be used as a reliable indicator of the progress of disease.

J Bacteriol, 2002 Jun, 184(11), 3135 - 41
Functional analysis of the Erwinia herbicola tutB gene and its product; Katayama T et al.; The tutB gene, which lies just downstream of tpl, has been cloned from Erwinia herbicola, and its product was analyzed . Despite its high sequence similarity to tryptophan transporters, TutB was found to be a tyrosine-specific transporter . Tryptophan acted as a competitive inhibitor of tyrosine transport . Unlike the tryptophanase operon, the tpl and tutB genes do not constitute an operon.

Biosci Biotechnol Biochem, 2002 Feb, 66(2), 444 - 7
Temperature-dependent production of carotovoricin Er and pectin lyase in phytopathogenic Erwinia carotovora subsp . carotovora Er; Nguyen HA et al.; The production of pectin lyase (Pnl) and carotovoricin (Ctv), as well as cell lysis in the plant-pathogenic bacterium Erwinia carotovora subsp . carotovora Er are induced by mitomycin C . Here, Pnl and Ctv production and cell lysis were found to be temperature-dependent . The optimal temperature for Pnl production was 30 degrees C . However, the optimal temperature for both Ctv production and cell lysis was 23 degrees C, at which Pnl production was reduced to 47% of the maximum . These data suggest the possible existence of novel regulation system(s) for the production of Pnl and Ctv, and cell lysis, in addition to the well-documented regulation system of recA, rdgA, and rdgB genes.

Microbiology, 2002 May, 148(Pt 5), 1367 - 78
Sample sequencing of a selected region of the genome of Erwinia carotovora subsp . atroseptica reveals candidate phytopathogenicity genes and allows comparison with Escherichia coli; Bell KS et al.; Genome sequencing is making a profound impact on microbiology . Currently, however, only one plant pathogen genome sequence is publicly available and no genome-sequencing project has been initiated for any species of the genus Erwinia, which includes several important plant pathogens . This paper describes a targeted sample sequencing approach to study the genome of Erwinia carotovora subsp . atroseptica (Eca), a major soft-rot pathogen of potato . A large insert DNA (approx . 115 kb) library of Eca was constructed using a bacterial artificial chromosome (BAC) vector . Hybridization and end-sequence data revealed two overlapping BAC clones that span an entire hrp gene cluster . Random subcloning and one-fold sequence coverage (>200 kb) across these BACs identified 25 (89%) of 28 hrp genes predicted from the orthologous hrp cluster of Erwinia amylovora . Regions flanking the hrp cluster contained orthologues of known or putative pathogenicity operons from other Erwinia species, including dspEF (E . amylovora), hecAB and pecSM (E . chrysanthemi), sequences similar to genes from the plant pathogen Xylella fastidiosa, including haemagglutinin-like genes, and sequences similar to genes involved in rhizobacterium-plant interactions . Approximately 10% of the sequences showed strongest nucleotide similarities to genes in the closely related model bacterium and animal pathogen Escherichia coli . However, the positions of some of these genes were different in the two genomes . Approximately 30% of sequences showed no significant similarity to any database entries . A physical map was made across the genomic region spanning the hrp cluster by hybridization to the BAC library and to digested BAC clones, and by PCR between sequence contigs . A multiple genome coverage BAC library and one-fold sample sequencing are an effective combination for extracting useful information from important regions of the Eca genome, providing a wealth of candidate novel pathogenicity genes for functional analyses . Other genomic regions could be similarly targeted.

Eur J Biochem, 2002 Apr, 269(8), 2124 - 32
The refolding of type II shikimate kinase from Erwinia chrysanthemi after denaturation in urea; Cerasoli E et al.; Shikimate kinase was chosen as a convenient representative example of the subclass of alpha/beta proteins with which to examine the mechanism of protein folding . In this paper we report on the refolding of the enzyme after denaturation in urea . As shown by the changes in secondary and tertiary structure monitored by far UV circular dichroism (CD) and fluorescence, respectively, the enzyme was fully unfolded in 4 m urea . From an analysis of the unfolding curve in terms of the two-state model, the stability of the folded state could be estimated as 17 kJ.mol-1 . Approximately 95% of the enzyme activity could be recovered on dilution of the urea from 4 to 0.36 m . The results of spectroscopic studies indicated that refolding occurred in at least four kinetic phases, the slowest of which (k = 0.009 s-1) corresponded with the regain of shikimate binding and of enzyme activity . The two most rapid phases were associated with a substantial increase in the binding of 8-anilino-1-naphthalenesulfonic acid with only modest changes in the far UV CD, indicating that a collapsed intermediate with only partial native secondary structure was formed rapidly . The relevance of the results to the folding of other alpha/beta domain proteins is discussed.

Med Pediatr Oncol, 2002 May, 38(5), 310 - 6
Antibody formation during intravenous and intramuscular therapy with Erwinia asparaginase; Albertsen BK et al.; BACKGROUND: Determination of the frequency of antibody formation during first and second exposure to Erwinia asparaginase after i.v . and i.m . administration . PROCEDURE: Thirty-nine children with newly diagnosed acute lymphoblastic leukemia (ALL) were included in this prospective study . Antibodies were determined (ELISA method) in plasma from these patients on specific days during and after therapy with 30,000 IU/m(2) i.v . or i.m . every day for ten days during the induction phase (first exposure) . For 19 children, antibodies were measured in plasma during and after the re-induction phase (second exposure) following treatment with 30,000 IU/m(2) i.v . or i.m . twice a week for two weeks (Mondays and Thursdays) . On the same days of therapy, enzyme activity (spectrophotometric method) and the concentration of asparagine (HPLC) was determined . RESULTS: During the first exposure, none of the patients developed anti-Erwinia asparaginase antibodies . During the second exposure, one patient (1 of 8 patients) treated intravenously developed antibodies, which were associated with disappearance of enzyme activity and reappearance of asparagine . Three of eleven patients developed antibodies of pharmacokinetic importance after i.m . therapy . None of the children had any clinical symptoms of hypersensitivity . CONCLUSIONS: The formation of antibodies and subsequently altered pharmacokinetics of Erwinia asparaginase seemed to be of importance only during a second period of asparaginase therapy .

J Bacteriol, 2002 May, 184(10), 2664 - 73
PehN, a polygalacturonase homologue with a low hydrolase activity, is coregulated with the other Erwinia chrysanthemi polygalacturonases; Hugouvieux-Cotte-Pattat N et al.; Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants . E . chrysanthemi also produces some hydrolases that cleave pectin . Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-alpha-D-galacturonosidases, have been characterized . These enzymes liberate digalacturonides from the nonreducing end of pectin . We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases . PehN has a low hydrolase activity on polygalacturonate and on various pectins . PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX . However, removal of the pehN gene does not significantly alter the virulence of E . chrysanthemi . Regulation of pehN transcription was analyzed by using gene fusions . Like other pectinase genes, pehN transcription is dependent on several environmental conditions . It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions . The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism . The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN . The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region . The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined . Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription . In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase . This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.

Appl Environ Microbiol, 2002 May, 68(5), 2261 - 8
Response of endophytic bacterial communities in potato plants to infection with Erwinia carotovora subsp . atroseptica; Reiter B et al.; The term endophyte refers to interior colonization of plants by microorganisms that do not have pathogenic effects on their hosts, and various endophytes have been found to play important roles in plant vitality . In this study, cultivation-independent terminal restriction fragment length polymorphism analysis of 16S ribosomal DNA directly amplified from plant tissue DNA was used in combination with molecular characterization of isolates to examine the influence of plant stress, achieved by infection with the blackleg pathogen Erwinia carotovora subsp . atroseptica, on the endophytic population in two different potato varieties . Community analysis clearly demonstrated increased bacterial diversity in infected plants compared to that in control plants . The results also indicated that the pathogen stress had a greater impact on the bacteria population than the plant genotype had . Partial sequencing of the 16S rRNA genes of isolated endophytes revealed a broad phylogenetic spectrum of bacteria, including members of the alpha, beta, and gamma subgroups of the Proteobacteria, high- and low-G+C-content gram-positive organisms, and microbes belonging to the Flexibacter-Cytophaga-Bacteroides group . Screening of the isolates for antagonistic activity against E . carotovora subsp . atroseptica revealed that 38% of the endophytes protected tissue culture plants from blackleg disease.

Appl Environ Microbiol, 2002 May, 68(5), 2120 - 32
Simultaneous transport of two bacterial strains in intact cores from Oyster, Virginia: biological effects and numerical modeling; Dong H et al.; The transport characteristics of two adhesion-deficient, indigenous groundwater strains, Comamonas sp . strain DA001 and Erwinia herbicola OYS2-A, were studied by using intact sediment cores (7 by 50 cm) from Oyster, Va . Both strains are gram-negative rods (1.10 by 0.56 and 1.56 by 0.46 microm, respectively) with strongly hydrophilic membranes and a slightly negative surface charge . The two strains exhibited markedly different behaviors when they were transported through granular porous sediment . To eliminate any effects of physical and chemical heterogeneity on bacterial transport and thus isolate the biological effect, the two strains were simultaneously injected into the same core . DA001 cells were metabolically labeled with (35)S and tagged with a vital fluorescent stain, while OYS2-A cells were metabolically labeled with (14)C . The fast decay of (35)S allowed deconvolution of the two isotopes (and therefore the two strains) . Dramatic differences in the transport behaviors were observed . The breakthrough of DA001 and the breakthrough of OYS2-A both occurred before the breakthrough of a conservative tracer (termed differential advection), with effluent recoveries of 55 and 30%, respectively . The retained bacterial concentration of OYS2-A in the sediment was twofold higher than that of DA001 . Among the cell properties analyzed, the statistically significant differences between the two strains were cell length and diameter . The shorter, larger-diameter DA001 cells displayed a higher effluent recovery than the longer, smaller-diameter OYS2-A cells . CXTFIT modeling results indicated that compared to the DA001 cells, the OYS2-A cells experienced lower pore velocity, higher porosity, a higher attachment rate, and a lower detachment rate . All these factors may contribute to the observed differences in transport.

Environ Microbiol, 2002 Feb, 4(2), 106 - 14
Following spread of fire blight in Western, Central and Southern Europe by molecular differentiation of Erwinia amylovora strains with PFGE analysis; Jock S et al.; Fire blight has been detected recently in several areas of northern Spain and north-eastern Italy . To follow spread of the disease within Europe, more than 120 Erwinia amylovora strains isolated from 1957-1900 in England, France, Germany, The Netherlands, Belgium, Poland, Italy and Spain were assayed using pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA after XbaI digestion . Pattern types Pt1 and Pt4 were found for strains from England . Pt1 was also found in central Europe and eastern France, Pt4 in western France . Pt2 appeared first in Egypt, from where strains with this pattern disseminated northwards as far as into the Balkans . Pt3 was typical for northern France and Belgium . Strains from Spain displayed the pattern types Pt3 and Pt4 . In Italy, Pt2 was found in the south-eastern areas, Pt3 in the north-eastern areas, and Pt1 was found very recently in orchards adjacent to the Austrian border, together with Pt3 . Despite barely controlled trade with fire blight host plants and associated plant products within Europe, the PFGE patterns of the E . amylovora isolates were ordered indicating sequential spread . On the other hand, the appearance of Pt3 in northern Italy and central Spain can be explained by the import of contaminated plants by nurseries.

Mol Microbiol, 2002 Apr, 44(1), 233 - 44
Functional assembly of the foreign carotenoid lycopene into the photosynthetic apparatus of Rhodobacter sphaeroides, achieved by replacement of the native 3-step phytoene desaturase with its 4-step counterpart from Erwinia herbicola; Garcia-Asua G et al.; Photosynthetic organisms synthesize a diverse range of carotenoids . These pigments are important for the assembly, function and stability of photosynthetic pigment-protein complexes, and they are used to quench harmful radicals . The photosynthetic bacterium Rhodobacter sphaeroides was used as a model system to explore the origin of carotenoid diversity . Replacing the native 3-step phytoene desaturase (CrtI) with the 4-step enzyme from Erwinia herbicola results in significant flux down the spirilloxanthin pathway for the first time in Rb . sphaeroides . In Rb . sphaeroides, the completion of four desaturations to lycopene by the Erwinia CrtI appears to require the absence of CrtC and, in a crtC background, even the native 3-step enzyme can synthesize a significant amount (13%) of lycopene, in addition to the expected neurosporene . We suggest that the CrtC hydroxylase can intervene in the sequence of reactions catalyzed by phytoene desaturase . We investigated the properties of the lycopene-synthesizing strain of Rb . sphaeroides . In the LH2 light-harvesting complex, lycopene transfers absorbed light energy to the bacteriochlorophylls with an efficiency of 54%, which compares favourably with other LH2 complexes that contain carotenoids with 11 conjugated double bonds . Thus, lycopene can join the assembly pathway for photosynthetic complexes in Rb . sphaeroides, and can perform its role as an energy donor to bacteriochlorophylls.

Carbohydr Res, 2002 Apr 17, 337(8), 731 - 42
Extracellular polysaccharides of a bacterium associated with a fungal canker disease of Eucalyptus sp; Yang BY et al.; Extracellular polysaccharides (EPSs) produced by an Erwinia sp associated with a fungal canker disease of Eucalyptus were fractionated into one polysaccharide that was identified with that produced by Erwinia chrysanthemi strains SR260, Ech1, and Ech9, and the other distinctively different from any other EPS produced by E . chrysanthemi strains so far studied . Their structures were determined using a combination of chemical and physical techniques including methylation analysis, low pressure gel-filtration, and anion-exchange chromatographies, high-pH anion-exchange chromatography, mass spectrometry and 1D and 2D 1H NMR spectroscopy . The new polysaccharide, identified as EPS Teranera, has the following structure: {structure: see text} The molecular weights of the polysaccharides range from 3.2-6.2 x 10(5) and their hydrodynamic properties are those of polydisperse, polyanionic biopolymers with pseudoplastic, non-thixotropic flow characteristics in aqueous solutions.

Mikrobiol Z, 2002 Jan-Feb, 64(1), 11 - 9
{Study of population dissociation of collection strains of Erwinia carotovora}; Tovkach FI et al.; Unusual dissociants of Erwinia carotovora subsp . atroseptica (EAT) population that emerge with high frequency (18-94%) during long-term storage of cultures were found . The dissociants (Poe-phenotype) differ from parental forms by slow growth and long development of the colour of colonies on EMB agar though they are not Lac-mutants . More than 60% of Poe-mutants of strain EAT 39A are characterized by 8- and 150-fold decrease of stability to erythromycin and oleandomicin, correspondingly . They are also sensitive to colicin-like carotovoricin, which is induced by mytomicin C, to carotovoricin of initial strain EAT 39A and to autocin, which they produce themselves during induction . A multiple phenotype of these dissociants (dissociants of the second type) is labeled: PoeOmSErSBnSAu . Its appearance as well as the appearance of pure Poe-variants is not associated with usual mutations and a loss of residential plasmids by cells . Population dissociants of the both types--Poe and PoeApSOmS--were also obtained from E . carotovora subsp . carotovora (J2) during growth at supraoptimal temperature . Poe-variants are more often observed in representatives of subspecies atroseptica than carotovora and can be useful when studying pathogeneity of these practically important erwinias.

Blood, 2002 Apr 15, 99(8), 2734 - 9
Comparison of Escherichia coli-asparaginase with Erwinia-asparaginase in the treatment of childhood lymphoid malignancies: results of a randomized European Organisation for Research and Treatment of Cancer-Children's Leukemia Group phase 3 trial; Duval M et al.; Asparaginase is an enzyme used in the treatment of acute lymphoblastic leukemia and lymphoblastic lymphoma in children . It has minimal bone marrow toxicity . Its major side effects are anaphylaxis, pancreatitis, diabetes, coagulation abnormalities, and thrombosis, especially intracranial . It is derived from 2 different sources: Escherichia coli and Erwinia chrysanthemi . Nonrandomized clinical studies have suggested a similar efficacy of these 2 types of asparaginases and a lower toxicity for Erwinia-asparaginase . The European Organisation for Research and Treatment of Cancer-Children's Leukemia Group (EORTC-CLG) 58881 trial randomized 700 children with acute lymphoblastic leukemia or lymphoblastic lymphoma to either E coli- or Erwinia-asparaginase at the same dosage of 10 000 IU/m(2) twice weekly to compare toxicity and efficacy . Coagulation abnormalities were more frequent in the E coli-asparaginase than in the Erwinia-asparaginase arm of the study (30.2% versus 11.9%, P <.0001) . The incidence of other toxicity was not significantly different . In the Erwinia-asparaginase arm, more patients failed to achieve complete remission (4.9% versus 2.0%; P =.038) and the relapse rate was higher, leading to shorter event-free survival (hazard ratio,1.59; 95% CI, 1.23-2.06; P =.0004) . The estimate of event-free survival rate (SE) at 6 years was 59.8% (2.6%) versus 73.4% (2.4%) . Overall survival rate at 6 years was also lower in the Erwinia-asparaginase arm at 75.1% (2.3%) versus 83.9% (2.0%), P =.002 . With the dose scheduling used in this protocol, E coli-asparaginase induced more coagulation abnormalities but was superior to Erwinia-asparaginase for the treatment of childhood lymphoid malignancies.

Mol Microbiol, 2002 Feb, 43(3), 733 - 48
H-NS-dependent activation of pectate lyases synthesis in the phytopathogenic bacterium Erwinia chrysanthemi is mediated by the PecT repressor; Nasser W et al.; Production of the main virulence determinant pectate lyases (Pels) of the phytopathogenic bacterium Erwinia chrysanthemi is modulated by a complex regulatory network involving the repressor proteins KdgR, PecS and PecT and the activator systems Pir, ExpI-ExpR and CRP . Of these regulators, CRP and PecT are particularly important since the absence of CRP or a slight overproduction of PecT leads to a drastic reduction in synthesis of Pel species . Recently, it has been shown that production of Pel species is strongly reduced in an E . chrysanthemi hns mutant, suggesting an activator function of the nucleoid-associated protein H-NS in the expression of the pel genes . Here, we report that the reduced synthesis of Pel species in the hns mutant results from a negative control, exerted by H-NS, on the transcription of the regulatory gene pecT . This H-NS/PecT cascade regulation is one of the first elucidations of a positive effect of H-NS on target gene expression . Moreover, we found that H-NS also represses the expression of expI, expR and pel genes . H-NS control is the result of H-NS binding to extended regions within the pecT, expI, expR and pel genes . Investigation of the simultaneous binding of CRP, RNA polymerase (RNAP) and H-NS on the pelD gene revealed that these three proteins form a nucleoprotein com-plex . Together, these data indicate that, by exerting a negative control at multiple levels, H-NS plays a crucial role in the E . chrysanthemi pel regulatory network.

Mol Microbiol, 2002 Feb, 43(3), 585 - 96
A putative three-dimensional targeting motif of polygalacturonase (PehA), a protein secreted through the type II (GSP) pathway in Erwinia carotovora; Palomaki T et al.; Intramolecular information specifying protein secretion through the type II (GSP) pathway of Gram-negative bacteria was investigated . Two regions of the polygalacturonase (PehA) of Erwinia carotovora containing residues proposed to be included in a targeting motif were located, one close to the C-terminus between residues 342 and 369 and another between residues 84 and 135 in the large central loops . The regions were required together to promote secretion . Further residues in the middle of the protein were required for proper positioning of the regions, suggesting that they were both involved in interaction with the GSP . To our knowledge, this is the first time that a possible three-dimensional targeting motif has been defined . At least one of the motifs comprises a cluster on the surface of the protein . The two motifs are structurally dissimilar, suggesting that there are two distinct recognition regions in the GSP apparatus . Finally, we propose that the targeting motifs are of a complex conformational nature with some variability accommodated, as illustrated by the observation that many mutations exhibited no clear phenotype individually but, in combination, severely compromised secretion.

Appl Environ Microbiol, 2002 Apr, 68(4), 1624 - 30
The ybiT gene of Erwinia chrysanthemi codes for a putative ABC transporter and is involved in competitiveness against endophytic bacteria during infection; Llama-Palacios A et al.; We investigated the role in bacterial infection of a putative ABC transporter, designated ybiT, of Erwinia chrysanthemi AC4150 . The deduced sequence of this gene showed amino acid sequence similarity with other putative ABC transporters of gram-negative bacteria, such as Escherichia coli and Pseudomonas aeruginosa, as well as structural similarity with proteins of Streptomyces spp . involved in resistance to macrolide antibiotics . The gene contiguous to ybiT, designated as pab (putative antibiotic biosynthesis) showed sequence similarity with Pseudomonas and Streptomyces genes involved in the biosynthesis of antibiotics . A ybiT mutant (BT117) was constructed by marker exchange . It retained full virulence in potato tubers and chicory leaves, but it showed reduced ability to compete in planta against the wild-type strain or against selected saprophytic bacteria . These results indicate that the ybiT gene plays a role in the in planta fitness of the bacteria.

Appl Environ Microbiol, 2002 Apr, 68(4), 1499 - 508
Application of amplified fragment length polymorphism fingerprinting for taxonomy and identification of the soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi; Avrova AO et al.; The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops . However, the taxonomy of these pathogens, particularly at subspecies level, is unclear . An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness . Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (clusters 1 to 4) resulted . Cluster 1 contained Erwinia carotovora subsp . carotovora (subclusters 1a and 1b) and Erwinia carotovora subsp . odorifera (subcluster 1c) strains, while cluster 2 contained Erwinia carotovora subsp . atroseptica (subcluster 2a) and Erwinia carotovora subsp . betavasculorum (subcluster 2b) strains . Clusters 3 and 4 contained Erwinia carotovora subsp . wasabiae and E . chrysanthemi strains, respectively . While E . carotovora subsp . carotovora and E . chrysanthemi showed a high level of molecular diversity (23 to 38% mean similarity), E . carotovora subsp . odorifera, E . carotovora subsp . betavasculorum, E . carotovora subsp . atroseptica, and E . carotovora subsp . wasabiae showed considerably less (56 to 76% mean similarity), which may reflect their limited geographical distributions and/or host ranges . The species- and subspecies-specific banding profiles generated from the AFLPs allowed rapid identification of unknown isolates and the potential for future development of diagnostics . AFLP fingerprinting was also found to be more differentiating than other techniques for typing the soft rot erwinias and was applicable to all strain types, including different serogroups.

Mikrobiologiia, 2002 Jan-Feb, 71(1), 82 - 8
{Study of Erwinia carotovora phage resistance with the use of temperate bacteriophage ZF40}; Tovkach FI; The causes of the unique phage resistance of the pectinolytic phytopathogenic strains of Erwinia carotovora were studied with the use of temperate bacteriophage ZF40 . It was shown that, in these bacteria, the bacteriophage-cell interaction can be substantially blocked at the adsorption level . An adequate indicator for studying the temperate bacteriophages of erwinias was developed on the basis of mutants resistant to macromolecular bacteriocins . Various restriction-modification systems, which influence cell resistance to bacteriophages, were revealed for the first time in E . carotovora . The phage resistance was shown to be determined by the wide occurrence of homoimmune temperate viruses in pectinolytic erwinias.

Mol Microbiol, 1997 Jul, 25(1), 135 - 46
The Rhizobium leguminosarum prsDE genes are required for secretion of several proteins, some of which influence nodulation, symbiotic nitrogen fixation and exopolysaccharide modification; Finnie C et al.; NodO is a secreted protein from Rhizobium leguminosarum bv . viciae with a role in signalling during legume nodulation . A Tn5-induced mutant was identified that was defective in NodO secretion . As predicted, the secretion defect decreased pea and vetch nodulation but only when the nodE gene was also mutated . This confirms earlier observations that NodO plays a particularly important role in nodulation when Nod factors carrying C18:1 (but not C18:4) acyl groups are the primary signalling molecules . In addition to NodO secretion and nodulation, the secretion mutant had a number of other characteristics . Several additional proteins including at least three Ca2+-binding proteins were not secreted by the mutant and this is thought to have caused the pleiotropic phenotype . The nodules formed by the secretion mutant were unable to fix nitrogen efficiently; this was not due to a defect in invasion because the nodule structures appeared normal and nodule cells contained many bacteroids . The mutant formed sticky colonies and viscous liquid cultures; analysis of the acidic exopolysaccharide revealed a decrease in the ratio of reducing sugars to total sugar content, indicating a longer chain length . The use of a plate assay showed that the mutant was defective in an extracellular glycanase activity . DNA sequencing identified the prsDE genes, which are homologous to genes encoding protease export systems in Erwinia chrysanthemi and Pseudomonas aeruginosa . An endoglycanase (Egl) from Azorhizobium caulinodans may be secreted from R . leguminosarum bv . viciae in a prsD-dependent manner . We conclude that the prsDE genes encode a Type I secretion complex that is required for the secretion of NodO, a glycanase and probably a number of other proteins, at least one of which is necessary for symbiotic nitrogen fixation.

Plant Physiol, 2002 Mar, 128(3), 951 - 61
Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection; Berrocal-Lobo M et al.; The peptide snakin-2 (StSN2) has been isolated from potato (Solanum tuberosum cv Jaerla) tubers and found to be active (EC(50) = 1-20 microM) against fungal and bacterial plant pathogens . It causes a rapid aggregation of both Gram-positive and Gram-negative bacteria . The corresponding StSN2 cDNA encodes a signal sequence followed by a 15-residue acidic sequence that precedes the mature StSN2 peptide, which is basic (isoelectric point = 9.16) and 66 amino acid residues long (molecular weight of 7,025) . The StSN2 gene is developmentally expressed in tubers, stems, flowers, shoot apex, and leaves, but not in roots, or stolons, and is locally up-regulated by wounding and by abscisic acid treatment . Expression of this gene is also up-regulated after infection of potato tubers with the compatible fungus Botritys cinerea and down-regulated by the virulent bacteria Ralstonia solanacearum and Erwinia chrysanthemi . These observations are congruent with the hypothesis that the StSN2 is a component of both constitutive and inducible defense barriers.

Planta, 2002 Mar, 214(5), 708 - 16 Epub 2001 Nov 29.
Altered lignin structure and resistance to pathogens in spi 2-expressing tobacco plants; Elfstrand M et al.; The physiological role of the Norway spruce { Picea abies (L.) Karst.} spi 2 gene, encoding a defense-related cationic peroxidase was examined in transgenic tobacco (Nicotiana tabacum L.) . Expression of spi 2, under control of the 35S promoter, in tobacco plants resulted in higher total peroxidase activities . The phenotype of the spi 2-transformed lines was normal . The spi 2-transformed lines displayed lignin levels similar to levels in the control line, but with some alteration in lignin histochemistry and structure . These changes were associated with reduced flexibility of the tobacco stems . The defense against pathogenic microorganisms was altered in the transgenic tobacco plants compared with control plants . High peroxidase activities increased the susceptibility to the pathogenic oomycete Phytophthora parasitica var . nicotianae, but increased the ability of the tobacco plants to suppress growth of the pathogenic bacterium Erwinia carotovora.

Microbiology, 2002 Mar, 148(Pt 3), 835 - 42
Regulation of the expression of prtW::gusA fusions in Erwinia carotovora subsp . carotovora; Marits R et al.; Erwinia carotovora subsp . carotovora, a Gram-negative phytopathogenic bacterium, secretes an extracellular metalloprotease, PrtW . Previous results demonstrated that protease activity is necessary for the normal progression of disease symptoms caused by this bacterium . The present study revealed that the prtW gene constitutes an independent transcriptional unit . It is demonstrated that introduction of the prtW(+) plasmid in trans into the prtW(-) mutant restores the protease activity in this strain . Gene fusions to the gusA (beta-glucuronidase) reporter were employed to analyse the transcription of prtW . The transcription of prtW is dependent on many environmental signals . When the bacteria were grown in the presence of potato extract, the expression of the protease gene was markedly higher at the beginning of the exponential phase of growth than that observed when cells were grown in the presence of polygalacturonate (PGA) . Analysis of the promoter revealed that an essential regulatory region resided between 371 and 245 bp 5' of the translational start site . As this sequence showed no homology to the KdgR box it may be involved in the binding of an unknown negative regulator protein in E . carotovora subsp . carotovora . The differential responses of prtW expression to potato extract and to PGA appeared to be dependent on the KdgR repressor and the response regulator ExpA . According to the results presented here, it is conceivable that the multiple regulatory network allows flexibility in the expression of the prtW gene during different stages of infection.

J Nutr, 2002 Mar, 132(3), 506S - 510S
Golden Rice: introducing the beta-carotene biosynthesis pathway into rice endosperm by genetic engineering to defeat vitamin A deficiency; Beyer P et al.; To obtain a functioning provitamin A (beta-carotene) biosynthetic pathway in rice endosperm, we introduced in a single, combined transformation effort the cDNA coding for phytoene synthase (psy) and lycopene beta-cyclase (beta-lcy) both from Narcissus pseudonarcissus and both under the control of the endosperm-specific glutelin promoter together with a bacterial phytoene desaturase (crtI, from Erwinia uredovora under constitutive 35S promoter control) . This combination covers the requirements for beta-carotene synthesis and, as hoped, yellow beta-carotene-bearing rice endosperm was obtained in the T(0)-generation . Additional experiments revealed that the presence of beta-lcy was not necessary, because psy and crtI alone were able to drive beta-carotene synthesis as well as the formation of further downstream xanthophylls . Plausible explanations for this finding are that these downstream enzymes are constitutively expressed in rice endosperm or are induced by the transformation, e.g., by enzymatically formed products . Results using N . pseudonarcissus as a model system led to the development of a hypothesis, our present working model, that trans-lycopene or a trans-lycopene derivative acts as an inductor in a kind of feedback mechanism stimulating endogenous carotenogenic genes . Various institutional arrangements for disseminating Golden Rice to research institutes in developing countries also are discussed.

Appl Environ Microbiol, 2002 Mar, 68(3), 1297 - 304
Purification and characterization of an extracellular protease from Xenorhabdus nematophila involved in insect immunosuppression; Caldas C et al.; Xenorhabdus nematophila, a bacterium pathogenic for insects associated with the nematode Steinernema carpocapsae, releases high quantities of proteases, which may participate in the virulence against insects . Zymogram assays and cross-reactions of antibodies suggested that two distinct proteases were present . The major one, protease II, was purified and shown to have a molecular mass of 60 kDa and an estimated isoelectric point of 8.5 . Protease II digested the chromogenic substrate N-tosyl-Gly-Pro-Arg-paranitroanilide (pNA) with V(max) and K(m) values of 0.0551 microM/min and 234 microM, respectively, and the substrate DL-Val-Leu-Arg-pNA with V(max) and K(m) values of 0.3830 microM/min and 429 microM, respectively . Protease II activity was inhibited 93% by Pefabloc SC and 45% by chymostatin . The optimum pH for protease II was 7, and the optimum temperature was 23C . Proteolytic activity was reduced by 90% at 60 degrees C for 10 min . Sequence analysis was performed on four internal peptides that resulted from the digestion of protease II . Fragments 29 and 45 are 75 and 68% identical to alkaline metalloproteinase produced by Pseudomonas aeruginosa . Fragment 29 is 79% identical to a metalloprotease of Erwinia amylovora and 75% identical to the protease C precursor of Erwinia chrysanthemi . Protease II showed no toxicity to hemocytes but destroyed antibacterial activity on the hemolymph of inoculated insects' larvae and reduced 97% of the cecropin A bacteriolytic activity.

Eur J Cancer, 2002 Mar, 38 Suppl 4, S44 - 9
The Children Leukemia Group: 30 years of research and achievements; Otten J et al.; The EORTC Children Leukemia Group (CLG) is part of the offspring of the EORTC Hemopathies Working Party which in 1978 split into a paediatric and to an 'adult' branch . At that time, the Berlin-Frankfurt-Munster (BFM) designed by H . Riehm for acute lymphoblastic leukaemia (ALL) appeared much more efficacious than all others and the CLG decided to adapt that treatment strategy for its own clinical trials . The main results of these may be summarised as follows:for standard risk patients, the deletion of cyclophosphamide from consolidation and reconsolidation courses does not jeopardise the patient's outcomefor medium- and high-risk patients receiving high-dose methotrexate (MTX), cranial radiotherapy is superfluouswith the dose scheduling of the BFM regimen, E-Coli L-Asparaginase is more efficacious than Erwinia L-Asparaginasethe addition of monthly intravenous (i.v.) 6-mercaptopurine to conventional maintenance chemotherapy is detrimentalthe assessment by quantitative polymerase chain reaction (PCR) of minimal residual disease at completion of induction is feasible in a cooperative setting and can be used as a powerful and independent prognostic factor.The CLG also conducted clinical studies of acute myeloblastic leukaemia . Since 1989, lymphoblastic non-Hodgkin's lymphomas have been treated within the ALL trials . The CLG collaborates with other Groups within the I-BFM Study Group and participants in the meta-analytic studies conducted by the Oxford team by the Oxford Children ALL Collaborative Group.

Planta, 2002 Jan, 214(3), 356 - 64
High-level production of the non-cariogenic sucrose isomer palatinose in transgenic tobacco plants strongly impairs development; Bornke F et al.; Palatinose (isomaltulose, 6-O-alpha-D-glucopyranosyl-D-fructose) is a structural isomer of sucrose which is produced from sucrose by some bacterial strains as a reserve material during periods of low carbon availability . The ability to synthesise palatinose is not only advantageous for the bacteria but is also of industrial interest since palatinose is used as a sucrose substitute in food production . To explore the possibility of palatinose production in plants a recently isolated sucrose isomerase gene (palI; EC 5.4.99.11) from Erwinia rhapontici {F . Bornke et al . (2001) J Bacteriol 183: 2425-2430} was cloned into a plant expression vector between the constitutive 35S CaMV promoter and the octopine synthase polyadenylation signal . To allow secretion of the protein into the apoplast the signal peptide of the potato proteinase inhibitor II was N-terminally fused to the pall coding region . Expression of the protein was verified by northern and western blot analyses . Efficient secretion of the protein was demonstrated by palI detection in intercellular fluids . Transgenic plants expressing palI accumulated high levels of palatinose . As a consequence, transgenic plants showed severe phenotypic alterations . Young leaves were curled and developed bleached areas during maturation . Flowers were misshapen and sterile . Based on nonaqueous fractionation experiments palatinose was found in several subcellular compartments, indicating limited membrane transport of the sugar . In contrast to results obtained with short-term feeding experiments, no evidence for palatinose-mediated regulation of photosynthetic or defence genes could be obtained in the transgenic palI-expressing tobacco plants . Based on our results we conclude that plants can efficiently be used as bioreactors for the production of palatinose . Furthermore, tissue-specific expression of palI should allow carbon allocation to specific tissues and/or cell-types to be modulated.

Plant J, 2001 Dec, 28(6), 663 - 70
Infra-red thermography revealed a role for mitochondria in pre-symptomatic cooling during harpin-induced hypersensitive response; Boccara M et al.; The establishment of Erwinia amylovora harpin-induced hypersensitive response (HR) in Nicotiana sylvestris was followed by infra-red thermography (IRT) . Three to four hours after elicitation, the temperature decreased in the harpin-infiltrated zone associated to stomatal opening . The marked drop in temperature which reached 2 degrees C and preceded necrosis symptoms for several hours, is thus likely caused by higher transpiration . Neither of these effects was observed in a respiratory mutant, affected in complex I structure and function and over-expressing alternative oxidase, indicating that they are directly or indirectly mediated by mitochondrial function . However, as the HR establishment was similar in both wild type and mutant, cell death was either uncorrelated with the observed epidermal changes or occurred by a different signalling pathway in the two genotypes . IRT revealed a novel aspect of plant-pathogen interactions and could be applied to screen for mutants affected in elicitor signalling and/or for respiratory mutants.

J Bacteriol, 2002 Mar, 184(5), 1340 - 8
Two novel type III-secreted proteins of Xanthomonas campestris pv . vesicatoria are encoded within the hrp pathogenicity island; Noel L et al.; The Hrp type III protein secretion system (TTSS) is essential for pathogenicity of gram-negative plant pathogen Xanthomonas campestris pv . vesicatoria . cDNA-amplified fragment length polymorphism and reverse transcription-PCR analyses identified new genes, regulated by key hrp regulator HrpG, in the regions flanking the hrp gene cluster . Sequence analysis revealed genes encoding HpaG, a predicted leucine-rich repeat-containing protein, the lysozyme-like HpaH protein, and XopA and XopD, which are similar in sequence to Hpa1 from Xanthomonas oryzae pv . oryzae and PsvA from Pseudomonas syringae, respectively . XopA and XopD (Xanthomonas outer proteins) are secreted by the Xanthomonas Hrp TTSS and thus represent putative effector proteins . Mutations in xopA, but not in xopD, resulted in reduced bacterial growth in planta and delayed plant reactions in susceptible and resistant host plants . Since the xopD promoter contains a putative hrp box, which is characteristic of hrpL-regulated genes in P . syringae and Erwinia spp., the gene was probably acquired by horizontal gene transfer . Interestingly, the regions flanking the hrp gene cluster also contain insertion sequences and genes for a putative transposase and a tRNA(Arg) . These features suggest that the hrp gene cluster of X . campestris pv . vesicatoria is part of a pathogenicity island.

Br J Haematol, 2001 Dec, 115(4), 983 - 90
Comparison of intramuscular therapy with Erwinia asparaginase and asparaginase Medac: pharmacokinetics, pharmacodynamics, formation of antibodies and influence on the coagulation system; Albertsen BK et al.; Asparaginase comes from different biological sources and the various preparations have different pharmacokinetic properties, and their tendency to induce side-effects is different . Erwinia asparaginase (ASNase) has a shorter half-life than the Escherichia coli preparations, and it has been reported to be less immunogenic than the E . coli preparations and to induce fewer coagulation disorders . Children with newly diagnosed acute lymphoblastic leukaemia (ALL) were included in this study . Twenty-seven patients were treated with Erwinia ASNase (induction therapy 30.000 IU/m2/d i.m . for 10 d, and re-induction therapy 30.000 IU/m2 twice a week for 2 weeks) and 15 were treated with ASNase Medac (induction therapy 1.000 IU/m2/d i.m . for 10 d, and re-induction therapy 5.000 IU/m2 i.m . twice a week for 2 weeks) . Blood samples were drawn to determine enzyme activity, l-asparagine, anti-asparaginase antibodies, and coagulation parameters . After i.m . administration, Erwinia ASNase displayed a protracted absorption phase compared to ASNase Medac . The mean bioavailability after i.m . administration was 27% for Erwinia ASNase and 45% for ASNase Medac respectively . Mean trough enzyme activities during induction therapy were Erwinia ASNase 1748 IU/l and ASNase Medac 272 IU/l, and during re-induction therapy Erwinia ASNase 83 IU/l and ASNase Medac 147 IU/l . We conclude that in this setting, therapy with ASNase Medac resulted in sufficient treatment during both phases of therapy, whereas treatment with Erwinia ASNase resulted in unnecessarily intense therapy during the induction phase and insufficient treatment during the re-induction phase . There was no significant difference in the incidence of antibody formation, and therapy with Erwinia ASNase resulted in a more pronounced influence on the coagulation parameters than therapy with ASNase Medac.

Microbiology, 2002 Feb, 148(Pt 2), 583 - 95
Genotyping of bacteria belonging to the former Erwinia genus by PCR-RFLP analysis of a recA gene fragment; Waleron M et al.; Genotypic characterization, based on the analysis of restriction fragment length polymorphism of the recA gene fragment PCR product (recA PCR-RFLP), was performed on members of the former Erwinia genus . PCR primers deduced from published recA gene sequences of Erwinia carotovora allowed the amplification of an approximately 730 bp DNA fragment from each of the 19 Erwinia species tested . Amplified recA fragments were compared using RFLP analysis with four endonucleases (AluI, HinfI, TasI and Tru1I), allowing the detection of characteristic patterns of RFLP products for most of the Erwinia species . Between one and three specific RFLP groups were identified among most of the species tested (Erwinia amylovora, Erwinia ananas, Erwinia cacticida, Erwinia cypripedii, Erwinia herbicola, Erwinia mallotivora, Erwinia milletiae, Erwinia nigrifluens, Erwinia persicina, Erwinia psidii, Erwinia quercina, Erwinia rhapontici, Erwinia rubrifaciens, Erwinia salicis, Erwinia stewartii, Erwinia tracheiphila, Erwinia uredovora, Erwinia carotovora subsp . atroseptica, Erwinia carotovora subsp . betavasculorum, Erwinia carotovora subsp . odorifera and Erwinia carotovora subsp . wasabiae) . However, in two cases, Erwinia chrysanthemi and Erwinia carotovora subsp . carotovora, 15 and 18 specific RFLP groups were detected, respectively . The variability of genetic patterns within these bacteria could be explained in terms of their geographic origin and/or wide host-range . The results indicated that PCR-RFLP analysis of the recA gene fragment is a useful tool for identification of species and subspecies belonging to the former Erwinia genus, as well as for differentiation of strains within E . carotovora subsp . carotovora and E . chrysanthemi.

Appl Environ Microbiol, 2002 Feb, 68(2), 705 - 12
Efficient insertion mutagenesis strategy for bacterial genomes involving electroporation of in vitro-assembled DNA transposition complexes of bacteriophage mu; Lamberg A et al.; An efficient insertion mutagenesis strategy for bacterial genomes based on the phage Mu DNA transposition reaction was developed . Incubation of MuA transposase protein with artificial mini-Mu transposon DNA in the absence of divalent cations in vitro resulted in stable but inactive Mu DNA transposition complexes, or transpososomes . Following delivery into bacterial cells by electroporation, the complexes were activated for DNA transposition chemistry after encountering divalent metal ions within the cells . Mini-Mu transposons were integrated into bacterial chromosomes with efficiencies ranging from 10(4) to 10(6) CFU/microg of input transposon DNA in the four species tested, i.e., Escherichia coli, Salmonella enterica serovar Typhimurium, Erwinia carotovora, and Yersinia enterocolitica . Efficiency of integration was influenced mostly by the competence status of a given strain or batch of bacteria . An accurate 5-bp target site duplication flanking the transposon, a hallmark of Mu transposition, was generated upon mini-Mu integration into the genome, indicating that a genuine DNA transposition reaction was reproduced within the cells of the bacteria studied . This insertion mutagenesis strategy for microbial genomes may be applicable to a variety of organisms provided that a means to introduce DNA into their cells is available.

Can J Microbiol, 2001 Dec, 47(12), 1126 - 31
The operon for cytokinin biosynthesis of Erwinia herbicola pv . gypsophilae contains two promoters and is plant induced; Guo M et al.; The operon for cytokinin biosynthesis in the gall-forming bacterium Erwinia herbicola pv . gypsophilae (Ehg) has been previously shown to reside on an indigenous plasmid (pPATH(Ehg)) that is mandatory for pathogenicity . This operon consists of two genes: the first open reading frame (pre-etz) is of unknown function, whereas the second one (etz) encodes for isopentenyl transferase . Northern hybridization performed with the wild-type strain Ehg824-1 grown in Luria-Bertani broth demonstrated two transcripts of which an etz-specific transcript (1.0 kb) was predominant . Fusion of upstream DNA fragments of both pre-etz and etz to the ice nucleation reporter gene inaZ in pVSP61 showed high ice nucleation activity in both cultures, confirming the presence of two independent promoters . An increase of 1-1.5 orders in transcriptional activity of these promoters was observed following inoculation of gypsophila cuttings . Mutants of Ehg824-1 were generated by insertion of inaZ into pre-etz and etz using the transposon reporter Tn3-Spice . An increase of about two orders in transcriptional activity was recorded with both mutants following inoculation of gypsophila or bean cuttings . A similar induction was also observed when the bacteria were applied to the leaf surface of these plants . Unlike other virulence genes present on the pPATH(Ehg), neither pre-etz nor etz was regulated by the adjacent hrp gene cluster.

J Bacteriol, 2002 Feb, 184(4), 1163 - 71
Nonenzymatic turnover of an Erwinia carotovora quorum-sensing signaling molecule; Byers JT et al.; The production of virulence factors and carbapenem antibiotic in the phytopathogen Erwinia carotovora is under the control of quorum sensing . The quorum-sensing signaling molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), accumulates in log-phase culture supernatants of E . carotovora but diminishes in concentration during the stationary phase . In this study, we show that the diminution in OHHL was not due to sequestration of the ligand by the cells, although some partitioning did occur . Rather, it was caused by degradation of the molecule . The rate of stationary-phase degradation of OHHL was as rapid as the rate of log-phase accumulation of the ligand, but it was nonenzymatic and led to a decrease in the expression of selected genes known to be under the control of quorum sensing . The degradation of OHHL was dependent on the pH of the supernatant, which increased as the growth curve progressed in cultures grown in Luria-Bertani medium from pH 7 to approximately 8.5 . OHHL became unstable over a narrow pH range (pH 7 to 8) . Instability was increased at high temperatures even at neutral pH but could be prevented at the growth temperature (30 degrees C) by buffering the samples at pH 6.8 . These results may provide a rationale for the observation that an early response of plants which are under attack by Erwinia is to activate a proton pump which alkalizes the site of infection to a pH of >8.2.

Proc Natl Acad Sci U S A, 2002 Jan 22, 99(2), 1092 - 7
Evaluation of transgenic tomato plants expressing an additional phytoene synthase in a fruit-specific manner; Fraser PD et al.; Phytoene synthase from the bacterium Erwinia uredovora (crtB) has been overexpressed in tomato (Lycopersicon esculentum Mill . cv . Ailsa Craig) . Fruit-specific expression was achieved by using the tomato polygalacturonase promoter, and the CRTB protein was targeted to the chromoplast by the tomato phytoene synthase-1 transit sequence . Total fruit carotenoids of primary transformants (T(0)) were 2-4-fold higher than the controls, whereas phytoene, lycopene, beta-carotene, and lutein levels were increased 2.4-, 1.8-, and 2.2-fold, respectively . The biosynthetically related isoprenoids, tocopherols plastoquinone and ubiquinone, were unaffected by changes in carotenoid levels . The progeny (T(1) and T(2) generations) inherited both the transgene and phenotype . Determination of enzyme activity and Western blot analysis revealed that the CRTB protein was plastid-located and catalytically active, with 5-10-fold elevations in total phytoene synthase activity . Metabolic control analysis suggests that the presence of an additional phytoene synthase reduces the regulatory effect of this step over the carotenoid pathway . The activities of other enzymes in the pathway (isopentenyl diphosphate isomerase, geranylgeranyl diphosphate synthase, and incorporation of isopentenyl diphosphate into phytoene) were not significantly altered by the presence of the bacterial phytoene synthase.

J Bacteriol, 2002 Feb, 184(3), 654 - 65
Characterization of indigoidine biosynthetic genes in Erwinia chrysanthemi and role of this blue pigment in pathogenicity; Reverchon S et al.; In the plant-pathogenic bacterium Erwinia chrysanthemi production of pectate lyases, the main virulence determinant, is modulated by a complex network involving several regulatory proteins . One of these regulators, PecS, also controls the synthesis of a blue pigment identified as indigoidine . Since production of this pigment is cryptic in the wild-type strain, E . chrysanthemi ind mutants deficient in indigoidine synthesis were isolated by screening a library of Tn5-B21 insertions in a pecS mutant . These ind mutations were localized close to the regulatory pecS-pecM locus, immediately downstream of pecM . Sequence analysis of this DNA region revealed three open reading frames, indA, indB, and indC, involved in indigoidine biosynthesis . No specific function could be assigned to IndA . In contrast, IndB displays similarity to various phosphatases involved in antibiotic synthesis and IndC reveals significant homology with many nonribosomal peptide synthetases (NRPS) . The IndC product contains an adenylation domain showing the signature sequence DAWCFGLI for glutamine recognition and an oxidation domain similar to that found in various thiazole-forming NRPS . These data suggest that glutamine is the precursor of indigoidine . We assume that indigoidine results from the condensation of two glutamine molecules that have been previously cyclized by intramolecular amide bond formation and then dehydrogenated . Expression of ind genes is strongly derepressed in the pecS background, indicating that PecS is the main regulator of this secondary metabolite synthesis . DNA band shift assays support a model whereby the PecS protein represses indA and indC expression by binding to indA and indC promoter regions . The regulatory link, via pecS, between indigoidine and virulence factor production led us to explore a potential role of indigoidine in E . chrysanthemi pathogenicity . Mutants impaired in indigoidine production were unable to cause systemic invasion of potted Saintpaulia ionantha . Moreover, indigoidine production conferred an increased resistance to oxidative stress, indicating that indigoidine may protect the bacteria against the reactive oxygen species generated during the plant defense response.

Int J Food Microbiol, 2001 Dec 30, 71(2-3), 197 - 210
Effect of high oxygen modified atmosphere packaging on microbial growth and sensorial qualities of fresh-cut produce; Jacxsens L et al.; The application of High Oxygen Atmospheres (HOA) (i.e . > 70% O2) for packaging ready-to-eat vegetables was evaluated as an alternative technique for low O2 Equilibrium Modified Atmosphere (EMA) packaging (3% O2-5% CO2-balance N2) for respiring products . Comparative experiments between both techniques were performed in-vitro and in-vivo . Typical spoilage causing microorganisms (Pseudomonas fluorescens, Candida lambica), the moulds Botrytis cinerea, Aspergillus flavus and the opportunistic psychrotrophic human pathogenic microorganism associated with refrigerated minimally processed vegetables . Aeromonas caviae (HG4), showed a retarded growth during the conducted in-vitro studies at 4 degrees C in 70%, 80% and 95% O2 as examples of HOA compared to the in-vitro experiments in 5% O2 (as example of EMA packaging) and the effect was more pronounced in 95% O2 . The effect of the high O2-concentrations on the human pathogen Listeria monocytogenes resulted in an extended lag phase (95% O2) . The plant pathogen Erwinia carotovora was increasingly stimulated by increasing high O2-concentrations . During a storage experiment of three types of ready-to-eat vegetables (mushroom slices, grated celeriac and shredded chicory endive), which are sensitive to enzymatic browning and microbial spoilage, the effect of EMA and HOA (95% O2-5% N2) on their quality and shelf life was compared . High O2 atmospheres were found to be particularly effective in inhibiting enzymatic browning of the tested vegetables . Also, the microbial quality was better as a reduction in yeast growth was observed . The HOA can be applied as an alternative for low O2 modified atmospheres for some specific types of ready-to-eat vegetables, sensitive to enzymatic browning and spoilage by yeasts.

Bioseparation, 2001, 10(1-3), 73 - 85
Direct process integration of cell disruption and fluidised bed adsorption in the recovery of labile microbial enzymes; Bierau H et al.; The practical feasibility and generic applicability of the direct integration of cell disruption by bead milling with the capture of intracellular products by fluidised bed adsorption has been demonstrated . Pilot-scale purification of the enzyme L-asparaginase from unclarified Erwinia chrysanthemi disruptates exploiting this novel approach yielded an interim product which rivalled or bettered that produced by the current commercial process employing discrete operations of alkaline lysis, centrifugal clarification and batch adsorption . In addition to improved yield and quality of product, the process time during primary stages of purification was greatly diminished . Two cation exchange adsorbents, CM HyperD LS (Biosepra/Life Technologies) and SP UpFront (custom made SP form of a prototype stainless steel/agarose matrix, UpFront Chromatography) were physically and biochemically evaluated for such direct product sequestration . Differences in performance with regard to product capacity and adsorption/desorption kinetics were demonstrated and are discussed with respect to the design of adsorbents for specific applications . In any purification of L-asparaginase (pI = 8.6), product-debris interactions commonly diminish the recovery of available product . It was demonstrated herein, that immediate disruptate exposure to a fluidised bed adsorbent promoted concomitant reduction of product in the liquid phase, which clearly counter-acted the product-debris interactions to the benefit of product yield.

Mikrobiol Z, 2001 May-Jun, 63(3), 43 - 50
{Epiphytic phase of Erwinia amylovora and Pseudomonas syringae pv . syringae on orchard weeds}; Gvozdiak RI et al.; Epiphyte phase of phytopathogenic bacteria Erwinia amylovora and Pseudomonas syringae pv . syringae on the fruit garden weeds has been studied . It has been shown that healthy weeds of the fruit-tree stands can be an ecologic niche for Erwinia amylovora and Pseudomonas syringae pv . syringae which gives them an opportunity to survive as epiphytes . Strains of Pseudomonas syringae pv . syringae were isolated from seven studied weeds (47-49%) during the whole vegetation period (March-October) . Strains of Erwinia amylovora distributed on the leaves of Arctium lappa L., Amarantus reflexus L . and Tripleurospermum inodorum (L) Sch . Vir . in the period of the disease intensive development on the pear-tree (June-August) . Cells of Erwinia amylovora were isolated from 12-14% of selected weeds.

Mikrobiologiia, 2001 Nov-Dec, 70(6), 804 - 10
{Isolation and preliminary characterization of cryptic plasmids from Erwinia carotovora}; Tovkach FI; Of the fifty-two Erwinia carotovora strains studied, sixteen were found to contain extrachromosomal DNA (plasmids) from 2.5 to 129 kbp in size . Some E . carotovora strains bore two to five different plasmids . Experiments showed that the cryptic plasmids of erwinia are not responsible for their resistance to antibiotics and are not involved in the synthesis of macromolecular colicin-like carotovoricins . At the same time, one of the E . carotovora strains, 13A, augmented the production of carotovoricin after curing from one of its plasmids, 47.7-kbp pCA 6-2 . Three E . carotovora subsp . carotovora strains and one E . carotovora subsp . atroseptica strain contained large 129-kbp plasmids, which may play a role in the ecology of phytopathogenic pectinolytic erwinia.

J Biol Chem, 2002 Mar 8, 277(10), 7936 - 44 Epub 2001 Dec 28.
The oligogalacturonate-specific porin KdgM of Erwinia chrysanthemi belongs to a new porin family; Blot N et al.; The phytopathogenic Gram-negative bacteria Erwinia chrysanthemi secretes pectinases, which are able to degrade the pectic polymers of plant cell walls, and uses the degradation products as a carbon source for growth . We characterized a major outer membrane protein, KdgM, whose synthesis is strongly induced in the presence of pectic derivatives . The corresponding gene was characterized . Analysis of transcriptional fusions showed that the kdgM expression is controlled by the general repressor of pectinolytic genes, KdgR, by the repressor of hexuronate catabolism genes, ExuR, by the pectinase gene repressor, PecS, and by catabolite repression via the cyclic AMP receptor protein (CRP) transcriptional activator . A kdgM mutant is unable to grow on oligogalacturonides longer than trimers, and its virulence is affected . Electrophysiological experiments with planar lipid bilayers showed that KdgM behaves like a voltage-dependent porin that is slightly selective for anions and that exhibits fast block in the presence of trigalacturonate . In contrast to most porins, KdgM seems to be monomeric . KdgM has no homology with currently known porins, but proteins similar to KdgM are present in several bacteria . Therefore, these proteins might constitute a new family of porin channels.

Biochim Biophys Acta, 2001 Dec 17, 1550(2), 117 - 28
Do bacterial L-asparaginases utilize a catalytic triad Thr-Tyr-Glu?
Aghaiypour K, Wlodawer A, Lubkowski J.
The structures of Erwinia chrysanthemi L-asparaginase (ErA) complexed with the L- and D-stereoisomers of the suicide inhibitor, 6-diazo-5-oxy-norleucine, have been solved using X-ray crystallography and refined with data extending to 1.7 A . The distances between the Calpha atoms of the inhibitor molecules and the hydroxyl oxygen atoms of Thr-15 and Tyr-29 (1.20 and 1.60 A, respectively) clearly indicate the presence of covalent bonds between these moieties, confirming the nucleophilic role of Thr-15 during the first stage of enzymatic reactions and also indicating direct involvement of Tyr-29 . The factors responsible for activating Tyr-29 remain unclear, although some structural changes around Ser-254', Asp-96, and Glu-63, common to both complexes, suggest that those residues play a function . The role of Glu-289' as the activator of Tyr-29, previously postulated for the closely related Pseudomonas 7A L-glutaminase-asparaginase, is not confirmed in this study, due to the lack of interactions between these residues in these complexes and in holoenzymes . The results reported here are consistent with previous reports that mutants of Escherichia coli L-asparaginase lacking Glu-289 remain catalytically active and prove the catalytic roles of both Thr-15 and Tyr-29, while still leaving open the question of the exact mechanism resulting in the unusual chemical properties of these residues.

Biochim Biophys Acta, 2001 Dec 5, 1568(2), 162 - 70
Molecular cloning and sequencing of the gene encoding an exopolygalacturonase of a Bacillus isolate and properties of its recombinant enzyme; Sawada K et al.; An exopolygalacturonase (exo-PGase; EC 3.2.1.82) was found in the culture broth of a Bacillus isolate . The gene encoding the exo-PGase, pehK, was cloned by polymerase chain reaction using mixed primers designed from N-terminal and internal amino acid (aa) sequences of the enzyme (PehK) . The determined nucleotide (nt) sequence of pehK revealed a 2940 bp open reading frame (980 aa) that encoded a putative signal sequence (27 aa) and a mature protein (953 aa; 103810 Da) . The recombinant enzyme was purified to homogeneity from a culture broth of Bacillus subtilis harboring a pehK-containing plasmid . It had a molecular mass of 105 kDa and a pI value of 5.0 . The maximum activity was observed at pH 8 and 55 degrees C in Tris-HCl buffer . The degradation products from polygalacturonic or oligogalacturonic acids were digalacturonic acid, like the exo-PGases, PehX of Erwinia chrysanthemi and PehB of Ralstonia solanacearum . The deduced aa sequence of PehK exhibited moderate homology to those of PehX and PehB with approx . 30% identity for both . High homology was observed in a suitably aligned internal region of the three enzymes (65% identity), and some of the conserved aa residues appeared to form the catalytic core of the enzymes.

J Immunol, 2001 Dec 15, 167(12), 6920 - 3
Enteric bacteria counteract lipopolysaccharide induction of antimicrobial peptide genes; Lindmark H et al.; The humoral immunity of Drosophila involves the production of antimicrobial peptides, which are induced by evolutionary conserved microbial molecules, like LPS . By using Drosophila mbn-2 cells, we found that live bacteria, including E . coli, Salmonella typhimurium, Erwinia carotovora, and Pseudomonas aeruginosa, prevented LPS from inducing antimicrobial peptide genes, while Micrococcus luteus and Streptococcus equi did not . The inhibitory effect was seen at bacterial levels from 20 per mbn-2 cell, while antimicrobial peptides were induced at lower bacterial concentrations (< or =2 bacteria per cell) also in the absence of added LPS . Gel shift experiment suggests that the inhibitory effect is upstream or at the level of the activation of the transcription factor Relish, a member of the NF-kappaB/Rel family . The bacteria have to be in physical contact with the cells, but not phagocytosed, to prevent LPS induction . Interestingly, the inhibiting mechanism is, at least for E . coli, independent of the type III secretion system, indicating that the inhibitory mechanism is unrelated to the one earlier described for YopJ from Yersinia.

Biotechnol Prog, 2001 Nov-Dec, 17(6), 1014 - 9
Optimization of fermentation reaction conditions for preparation of PATE enzyme by Erwinia carotovora IFO3830 by TFCCRD method; Ding F et al.; The fermentation conditions for preparation of polygalacturonic acid transeliminase (PATE) enzyme by Erwinia carotovora IFO3830 were optimized for seed ratio, vibration rate, and temperature by the TFCCRD method . The results indicated that the optimum fermentation conditions for E . carotovora IFO 3830 were that seed ratio, vibration rate, and temperature were 5% v/v, 113 min(-1), and 29 degrees C, respectively.

FEMS Microbiol Lett, 2001 Nov 27, 205(1), 131 - 8
Halogenated furanones from the red alga, Delisea pulchra, inhibit carbapenem antibiotic synthesis and exoenzyme virulence factor production in the phytopathogen Erwinia carotovora; Manefield M et al.; The plant pathogen Erwinia carotovora regulates expression of virulence factors and antibiotic production via an N-3-oxohexanoyl-L-homoserine lactone (3-oxo-C6-HSL) dependent quorum sensing mechanism . The marine alga Delisea pulchra produces halogenated furanones known to antagonise 3-oxo-C6-HSL activity . We have tested the effects of a halogenated furanone on the production of carbapenem, cellulase and protease in E . carotovora . Despite differences in the regulatory mechanisms controlling carbapenem and exoenzyme production each was inhibited by the algal metabolite . We present evidence to suggest that the furanone dependent inhibition of carbapenem production is a result of the disruption of the 3-oxo-C6-HSL dependent expression of the carABCDEFGH operon.

Int J Med Microbiol, 2001 Nov, 291(5), 353 - 60
Overexpresssion of a Legionella pneumophila homologue of the E . coli regulator csrA affects cell size, flagellation, and pigmentation; Fettes PS et al.; Legionella pneumophila is an inhabitant of the aquatic environment and the causative agent of a bacterial pneumonia . We identified the presence of an L . pneumophila homologue of csrA of E . coli and rsmA of Erwinia carotovora, genes which regulate gene expression by destabilising mRNA and which have been shown to relate to environmental fitness and pathogenicity . The Legionella csrA was able to complement a csrA-negative mutant of E . coli . Overproduction of csrA in L . pneumophila lead to a reduction of flagellation and pigmentation and an increase in bacterial cell size . csrA overproduction was associated with a reduction of fliA and flaA transcripts . This suggests that similar to E . coli and Erwinia, L . pneumophila csrA is a regulator of gene expression and may contribute to the capability of the pathogen to rapidly adapt to changing environments.

J Mol Biol, 2001 Nov 23, 314(2), 187 - 93
Protease C of Erwinia chrysanthemi: the crystal structure and role of amino acids Y228 and E189; Hege T et al.; PrtC, a metallo-protease secreted by Erwinia chrysanthemi, is a member of the serralysin family and hence belongs to the metzincin superfamily . While the crystal structures of representatives of all metzincin subfamilies have been elucidated in the past, there is still some controversy about the reaction mechanism and the role of certain characteristic amino acids in the active centre . In this study, we probed the role of Tyr228 and Glu189 by site-directed mutagenesis and X-ray crystallography . There is evidence that these residues participate in catalysis, although conflicting hypotheses have been proposed . The crystal structures of wild-type and mutants have been refined to an R(free) of about 0.20 at resolutions of 2.0 A or better . Exchange of Glu189 versus alanine reduces the catalytic efficiency to less than 0.5 % using resorufin casein as substrate and to about 3 % using an assay utilising the thiol ester Ac-Pro-Leu-Gly-{(S)Leu}-Leu-Gly-OEt . The drop in activity is caused by a reduction in k(cat) while the K(M) values are virtually the same . In the resorufin casein assay, the mutant Y228F shows about 3 % of the wild-type activity and in the thiol ester assay this increases to about 56 % . In the latter case, the K(M) value of the mutant is increased from 5.3 mM to 9.0 mM with only little reduction in k(cat) . The different behaviour of this mutant with respect to the two substrates can be explained by a switch in the rate-determining step during catalysis . The study presented here provides clear evidence that Glu189 of the HEXXHXXGXXH motif is the catalytic base, while Tyr228 is more likely involved in substrate binding and the stabilisation of the tetrahedral transition state .

J Mol Biol, 2001 Nov 23, 314(2), 181 - 6
The conserved methionine residue of the metzincins: a site-directed mutagenesis study; Hege T et al.; The metalloprotease clan of the metzincins derive their name from the presence of a conserved methionine residue that is located on the C-terminal side of the zinc-binding consensus sequence HEXXHXXGXXH . This methionine residue is located in a rather divergent part of the primary sequence but is structurally very well conserved . It is located under the pyramidal base of the three histidine residues that coordinate the catalytic zinc ion and is not involved in any direct contact with the metal nor the substrate . In order to clarify its role, this methionine residue (M226) of the protease C from Erwinia chrysanthemi has been mutated to various other amino acids . The mutants M226L, M226A, M226I were sufficiently stable to be isolated, while the mutants M226H, M226S and M226N could not be purified . The kinetic properties of these mutants were analysed . All mutants showed decreased activity, whereby increases in K(M) as well as decreases in k(cat) were observed . The M226L mutant and M226C-E189 K double mutant, which has the catalytic glutamic acid substituted as well, could be crystallised . The structure of the M226L mutant was determined to a resolution of 2.0 A and refined to R(free) of 0.20 . The structure is isomorphous to the wild-type and does not show large differences, with the exception of a very small movement of the zinc-liganding histidine residues . The M226C-E189 K double mutant crystal structure has been refined to an R(free) of 0.20 at 2.1 A resolution . A small rearrangement of the zinc-liganding histidine residues can be detected, which leads to a slightly different zinc coordination and could explain the decrease in activity .

Acta Crystallogr D Biol Crystallogr, 2001 Dec, 57(Pt 12), 1870 - 1 Epub 2001 Nov 21.
Crystallization and preliminary X-ray diffraction analysis of shikimate kinase from Mycobacterium tuberculosis in complex with MgADP; Gu Y et al.; Shikimate kinase (SK) from Mycobacterium tuberculosis (Mt) was overexpressed in Escherichia coli, purified and cocrystallized with MgADP in hanging drops using the vapor-diffusion procedure with PEG 4000 and 2-propanol as precipitants at pH 7.5 . The crystal of MtSK-MgADP, which diffracted to 2.2 A resolution, belonged to space group P3(2)21 or P3(1)21, with unit-cell parameters a = b = 64.01, c = 92.41 A . There was one MtSK molecule in the asymmetric unit . Molecular-replacement trials with the crystal structure of SK from Erwinia chrysanthemi (PDB code 1shk) and adenylate kinase (PDB code 1ake) as search models were not successful . Heavy-atom derivative screening is in progress.

Annu Rev Phytopathol, 2001, 39, 79 - 102
Organization of genes controlling disease resistance in the potato genome; Gebhardt C et al.; Nineteen single dominant genes (R genes) for resistance to viruses, nematodes, and fungi have been positioned on the molecular map of potato using DNA markers . Fourteen of those genes are located in five "hotspots" for resistance in the potato genome . Quantitative trait loci (QTL) for resistance to late blight caused by the oomycete Phytophthora infestans, to tuber rot caused by the bacterium Erwinia carotovora ssp . atroseptica, and to root cyst nematodes have been identified on all 12 potato chromosomes . Some QTL for resistance to different pathogens are linked to each other and/or to resistance hotspots . Based on the genetic clustering with R genes, we propose that some QTL for resistance have a molecular basis similar to single R genes . Mapping potato genes with sequence similarity to cloned R genes of other plants and other defense-related genes reveals linkage between candidate genes, R genes, and resistance QTL . To explain the molecular basis of polygenic resistance in potato we propose (a) genes having structural similarity with cloned R genes and (b) genes involved in the defense response . The "candidate gene approach" enables the identification of markers highly useful for marker-assisted selection in potato breeding.

Annu Rev Phytopathol, 1999, 37, 307 - 334
WITHHOLDING AND EXCHANGING IRON: Interactions Between Erwinia spp . and Their Plant Hosts; Expert D; The critical role of iron in plant host-parasite relationships has been elucidated in diseases as different as the soft rot and fire blight incited by Erwinia chrysanthemi and E . amylovora, respectively . As in animal infections, the role of iron and its ligands in the virulence of plant pathogens seems to be more subtle than might be expected, and is intimately related to the life cycle of the pathogen within its host . This review discusses how iron, because of its unique position in biological systems, controls the activities of these plant pathogens . Molecular studies illustrating the key question of iron acquisition and homeostasis during pathogenesis are described . The production of siderophores by pathogens not only represents a powerful strategy to acquire iron from host tissues but may also act as a protective agent against iron toxicity . The need of the host to bind and possibly sequester the metal during pathogenesis is another central issue . Possible modes of iron competition between plant host and pathogen are considered.

J Biol Chem, 2002 Feb 22, 277(8), 6726 - 32 Epub 2001 Nov 06.
The N terminus of the HasA protein and the SecB chaperone cooperate in the efficient targeting and secretion of HasA via the ATP-binding cassette transporter; Sapriel G et al.; Secretion of the HasA hemophore is mediated by a C-terminal secretion signal as part of an ATP-binding cassette (ABC) pathway in the Gram-negative bacterium Serratia marcescens . We reconstituted the HasA secretion pathway in Escherichia coli . In E . coli, this pathway required three specific secretion functions and SecB, the general chaperone of the Sec pathway that recognizes HasA . The secretion of the isolated C-terminal secretion signal was not SecB-dependent . We have previously shown that intracellular folded HasA can no longer be secreted, and we proposed a step in the secretion process before the recognition of the secretion signal . Here we show that the secretion of a fully functional HasA variant, lacking the first 10 N-terminal amino acids, was less efficient than that of HasA and was SecB-independent . The N terminus of HasA was required, along with SecB, for the efficient secretion of the whole protein . We have also previously shown that HasA inhibits the secretion of metalloproteases from Erwinia chrysanthemi by their specific ABC transporter . Here we show that this abortive interaction between HasA and the E . chrysanthemi metalloprotease ABC transporter required both SecB and the N terminus of HasA . N-terminal fragments of HasA displayed this abortive interaction in vivo and also interacted specifically in vitro with the ABC protein of the Prt system . SecB also interacted specifically in vitro with the ABC protein of the Prt system . Finally, the HasA variant, lacking the first 10 N-terminal amino acids did not display this abortive interaction with the Prt system . We suggest that the N-terminal domain of HasA specifically recognizes the ABC protein in a SecB-dependent fashion, facilitating functional interaction with the C-terminal secretion signal leading to efficient secretion.

J Bacteriol, 2001 Dec, 183(23), 6752 - 62
Predictive and interpretive simulation of green fluorescent protein expression in reporter bacteria; Leveau JH et al.; We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion . The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria . From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures . To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P(A1/O4/O3) in response to increasing concentrations of the inducer IPTG (isopropyl-beta-D-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P(A1/O4/O3) . Aided by the model, we also quantified the proteolytic degradation of GFP{AAV}, GFP{ASV}, and GFP{LVA}, which are popular variants of GFP with reduced stability in bacteria . Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one . The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli . This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants . The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data . In practice, our model will be helpful for prior testing of different combinations of promoter-gfp fusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

J Biol Chem, 2002 Jan 25, 277(4), 2385 - 95 Epub 2001 Nov 01.
Chrysobactin-dependent iron acquisition in Erwinia chrysanthemi . Functional study of a homolog of the Escherichia coli ferric enterobactin esterase; Rauscher L et al.; Under iron limitation, the plant pathogen Erwinia chrysanthemi produces the catechol-type siderophore chrysobactin, which acts as a virulence factor . It can also use enterobactin as a xenosiderophore . We began this work by sequencing the 5'-upstream region of the fct-cbsCEBA operon, which encodes the ferric chrysobactin receptor and proteins involved in synthesis of the catechol moiety . We identified a new iron-regulated gene (cbsH) transcribed divergently relative to the fct gene, the translated sequence of which is 45.6% identical to that of Escherichia coli ferric enterobactin esterase . Insertions within this gene interrupt the chrysobactin biosynthetic pathway by exerting a polar effect on a downstream gene with some sequence identity to the E . coli enterobactin synthase gene . These mutations had no effect on the ability of the bacterium to obtain iron from enterobactin, showing that a functional cbsH gene is not required for iron removal from ferric enterobactin in E . chrysanthemi . The cbsH-negative mutants were less able to utilize ferric chrysobactin, and this effect was not caused by a defect in transport per se . In a nonpolar cbsH-negative mutant, chrysobactin accumulated intracellularly . These defects were rescued by the cbsH gene supplied on a plasmid . The amino acid sequence of the CbsH protein revealed characteristics of the S9 prolyl oligopeptidase family . Ferric chrysobactin hydrolysis was detected in cell extracts from a cbsH-positive strain that was inhibited by diisopropyl fluorophosphate . These data are consistent with the fact that chrysobactin is a d-lysyl-l-serine derivative . Mossbauer spectroscopy of whole cells at various states of (57)Fe-labeled chrysobactin uptake showed that this enzyme is not required for iron removal from chrysobactin in vivo . The CbsH protein may therefore be regarded as a peptidase that prevents the bacterial cells from being intracellularly iron-depleted by chrysobactin.

J Ind Microbiol Biotechnol, 2001 Oct, 27(4), 215 - 9
Comparative evaluation of pectolytic and proteolytic enzyme production by free and immobilized cells of some strains of the phytopathogenic Erwinia chrysanthemi; Muyima NY et al.; Whole cells of the phytopathogenic Erwinia chrysanthemi strains were immobilized in k-carrageenan and grown in high-calcium Xanthomonas campestris medium containing sodium polypectate as carbon source . All the strains used survived immobilization into k-carrageenan beads . Immobilized E . chrysanthemi strains displayed higher pectolytic and proteolytic enzyme activities than free cells in liquid suspension . Carrageenan immobilization techniques could provide a system to mimic the conditions of E . chrysanthemi cells in the infected plant tissue . This could prompt a thorough study of the factors governing the biosynthesis of virulence factors by this bacterium.

Mol Microbiol, 2001 Oct, 42(1), 87 - 99
Glycine betaine loses its osmoprotective activity in a bspA strain of Erwinia chrysanthemi; Touze T et al.; Erwinia chrysanthemi insertion mutants were isolated that grew poorly specifically in the presence of glycine betaine (GB) or its analogues in high-salt media . Transposon insertions were found to affect the bspA gene, which forms an operon including the psd locus coding for phosphatidylserine decarboxylase . Initial GB uptake is not affected by the bspA mutation . However, in high-salt medium, its initial accumulation is followed by a reduced glucose uptake and a release of GB but not a loss of viability . BspA is homologous to the widespread MscS channel, YggB, but does not seem to constitute a mechanosensitive channel . We suggest that BspA is a protein sensing both intracellular GB and the extracellular salt content of the medium, the hypothesis being built on the observation that BspA is necessary to maintain the GB pool during osmoadaptation in high-salt media containing this osmoprotectant.

Br J Clin Pharmacol, 2001 Oct, 52(4), 433 - 7
Monitoring of Erwinia asparaginase therapy in childhood ALL in the Nordic countries; Albertsen BK et al.; AIMS: Evaluation of L-asparaginase therapy in the NOPHO-92 ALL-protocol (treatment protocol of acute lymphoblastic leukaemia of the Nordic Society of Paediatric Haematology and Oncology, initiated in 1992) after intravenous and intramuscular administration of Erwinia asparaginase during induction and re-induction therapy . METHODS: Forty children with newly diagnosed acute lymphoblastic leukaemia received Erwinia asparaginase (30 000 IU/m2 i.v . or i.m.) during induction therapy (every day for 10 days), and 19 children received Erwinia asparaginase (30 000 IU/m2 i.v . or i.m.) during re-induction therapy (twice a week for 2 weeks) . Within the treatment periods asparaginase trough activity (using a spectrophotometric assay) was determined on specific days . The goal of therapy is complete L-asparagine depletion, which asparaginase activities above 100 IU l(-1) have been shown to ensure . Therefore determination of L-asparagine (using a h.p.l.c . method) was performed only in plasma samples with asparaginase activities below 100 IU l(-1) . RESULTS: During induction therapy 92.2% of the trough enzyme activities were above 500 IU l(-1) for the i.v.-treated patients, and 92.4% of the trough enzyme activities were above 500 IU l(-1) for the i.m.-treated patients . During re-induction therapy 64.7% of the trough enzyme activities were below 100 IU l(-1) in the i.v.-treated group, and 73.3% of the trough enzyme activities were below 100 IU l(-1) in the i.m.-treated group . For trough enzyme activities below 100 IU l(-1) L-asparagine depletion was complete in two thirds of the samples . CONCLUSIONS: In the NOPHO-92 ALL-protocol L-asparaginase treatment during induction therapy was unnecessarily intense, but during the re-induction phase it appeared inadequate.

Drug Saf, 2001, 24(10), 767 - 79
Prevention and management of antineoplastic-induced hypersensitivity reactions; Zanotti KM et al.; Acute hypersensitivity reactions (HSRs) are an unpredictable and potentially catastrophic complication of treatment with chemotherapeutic agents . Reactions may affect any organ system in the body and range widely in severity from mild pruritus to systemic anaphylaxis . Certain classes of chemotherapeutic agents, such as the taxanes, platinum compounds, asparaginases, and epipodophyllotoxins are commonly associated with HSRs . The clinical characteristics of these high risk agents with respect to HSRs are discussed in this review . Protocols to prevent or reduce the severity of these reactions have been developed, but despite these attempts, HSRs will still happen . Should a reaction occur, it is imperative that it be recognised quickly in order to minimise exposure to the inciting agent and implement appropriate therapeutic and supportive measures . When a patient becomes sensitised to a chemotherapeutic agent, avoidance of re-exposure is the mainstay of future prevention . For sensitised patients who have derived clinically meaningful benefit from a particular agent, however, continuation of treatment with the agent is desirable . Options may include attempting a trial of desensitisation or treatment with a related compound . Virtually all patients demonstrating HSRs to paclitaxel and docetaxel are able to successfully tolerate re-treatment following discontinuation and administration of diphenhydramine and hydrocortisone . Re-treatment has generally been less successful with platinum compounds . with recurrent HSRs occurring in up to 50% of patients following desensitisation protocols . Patients sensitised to asparaginase are often able to tolerate the alternative preparations, Erwinia carotovora asparaginase or polyethylene glycol-modified Escherichia coli asparaginase . There is very little experience with re-treatment following sensitisation to the epipodophyllotoxins . As re-treatment may have serious consequences, careful consideration of the risks and benefits of these strategies is imperative when deciding among these options.

Mol Plant Microbe Interact, 2001 Oct, 14(10), 1223 - 34
Biological activity of harpin produced by Pantoea stewartii subsp . stewartii; Ahmad M et al.; 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.

Mol Plant Microbe Interact, 2001 Oct, 14(10), 1213 - 22
Genetic organization of the Pantoea stewartii subsp . stewartii hrp gene cluster and sequence analysis of the hrpA, hrpC, hrpN, and wtsE operons; Frederick RD et al.; 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.

J Agric Food Chem, 2001 Oct, 49(10), 4662 - 6
Expression of a bacterial ice nucleation gene in a yeast Saccharomyces cerevisiae and its possible application in food freezing processes; Hwang WZ et al.; A 3.6 kb ice nucleation gene (ina) isolated from Erwinia herbicola was placed under control of the galactose-inducible promoter (GAL1) and introduced into Saccharomyces cerevisiae . Yeast transformants showed increased ice nucleation activity over untransformed controls . The freezing temperature of a small volume of water droplets containing yeast cells was increased from approximately -13 degrees C in the untransformed controls to -6 degrees C in ina-expressing (Ina(+)) transformants . Lower temperature growth of Ina(+) yeast at temperatures below 25 degrees C was required for the expression of ice nucleation activity . Shift of temperature to 5-20 degrees C could induce the ice nucleation activity of Ina(+) yeast when grown at 25 degrees C, and maximum ice nucleation activity was achieved after induction at 5 degrees C for approximately 12 h . The effects of Ina(+) yeast on freezing and texturization of several food materials was also demonstrated.

Biochim Biophys Acta, 2001 Nov 1, 1515(1), 12 - 22
Isolation and characterisation of the major outer membrane protein of Erwinia carotovora; El Hamel C et al.; The purified major outer membrane protein (37275 Da) from the psychrotrophic phytopathogen Erwinia carotovora MFCL0 was structurally characterised by MALDI-TOF mass spectrometry, N-terminal microsequencing and DNA sequence determinations, and secondary structure prediction analyses . The deduced amino acid sequence showed 76% and 72% of similarities with the Serratia marcescens and Escherichia coli OmpA proteins respectively . Dendrogram analysis allowed to point out that E . carotovora is close to the genus Serratia . After reconstitution into planar lipid bilayers, this major protein induced ion channels with a major conductance level of 630 pS in 1 M NaCl and a weak cationic selectivity . These functional and structural features allowed to identify this major outer membrane component of E . carotovora as an OmpA-like protein, i.e., a channel-forming protein which could be involved in the infection process of this phytopathogen agent.

J Am Chem Soc, 2001 Oct 17, 123(41), 9935 - 46
Synthesis and biological evaluation of analogues of the antibiotic pantocin B; Sutton AE et al.; Strains of the bacteria Erwinia herbicola produce antibiotics that effectively control E . amylovora, the bacterial pathogen responsible for the plant disease fire blight . Pantocin B was the first of these antibiotics to be characterized, and a flexible synthesis of various analogues is reported . Embedded in the "pseudo-tripeptide" backbone of pantocin B are a methylenediamine and a methyl sulfone, both unusual structural features in natural products . The peptidic nature of pantocin B facilitated a series of structure-activity relationship studies that probed the roles of these functional groups in determining the biological activity of pantocin B . A clear demarcation of the roles between the N- and C-terminal portions of the antibiotic was determined as a result of the structure-activity relationship studies . The N-terminal L-alanyl group is needed for cellular import but not for interaction with the intracellular target, the arginine biosynthetic enzyme N-acetylornithine aminotransferase . The methylenediamine and methyl sulfone portions were found to be essential for antibiotic activity, presumably due to extensive interactions with N-acetylornithine aminotransferase.

J Bacteriol, 2001 Nov, 183(21), 6274 - 81
DNA inversion in the tail fiber gene alters the host range specificity of carotovoricin Er, a phage-tail-like bacteriocin of phytopathogenic Erwinia carotovora subsp . carotovora Er; Nguyen HA et al.; Carotovoricin Er is a phage-tail-like bacteriocin produced by Erwinia carotovora subsp . carotovora strain Er, a causative agent for soft rot disease in plants . Here we studied binding and killing spectra of carotovoricin Er preparations for various strains of the bacterium (strains 645Ar, EC-2, N786, and P7) and found that the preparations contain two types of carotovoricin Er with different host specificities; carotovoricin Era possessing a tail fiber protein of 68 kDa killed strains 645Ar and EC-2, while carotovoricin Erb with a tail fiber protein of 76 kDa killed strains N786 and P7 . The tail fiber proteins of 68 and 76 kDa had identical N-terminal amino acid sequences for at least 11 residues . A search of the carotovoricin Er region in the chromosome of strain Er indicated the occurrence of a DNA inversion system for the tail fiber protein consisting of (i) two 26-bp inverted repeats inside and downstream of the tail fiber gene that flank a 790-bp fragment and (ii) a putative DNA invertase gene with a 90-bp recombinational enhancer sequence . In fact, when a 1,400-bp region containing the 790-bp fragment was amplified by a PCR using the chromosomal DNA of strain Er as the template, both the forward and the reverse nucleotide sequences of the 790-bp fragment were detected . DNA inversion of the 790-bp fragment also occurred in Escherichia coli DH5alpha when two compatible plasmids carrying either the 790-bp fragment or the invertase gene were cotransformed into the bacterium . Furthermore, hybrid carotovoricin CGE possessing the tail fiber protein of 68 or 76 kDa exhibited a host range specificity corresponding to that of carotovoricin Era or Erb, respectively . Thus, a DNA inversion altered the C-terminal part of the tail fiber protein of carotovoricin Er, altering the host range specificity of the bacteriocin.

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