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