Mol Microbiol, 2001 Sep, 41(5), 1125 - 32 Two transporters, TogT and TogMNAB, are responsible for oligogalacturonide uptake in Erwinia chrysanthemi 3937; Hugouvieux-Cotte-Pattat N et al.; Erwinia chrysanthemi causes soft rot of plants by secreting pectinases which cleave pectin, a polysaccharide cementing the plant cell wall constituents . We demonstrated that two transporters mediate the uptake of the extracellularly formed oligomers in E . chrysanthemi . TogMNAB, a multicomponent transporter member of the ATP-binding cassette (ABC) superfamily, is only partially responsible for the uptake of pectic oligomers . Its action is completed by that of the second transporter, TogT, a member of the glycoside-pentoside-hexuronide (GPH) family (TC no . 2.2) which includes transporters involved in the uptake of complex sugars, mostly oligosaccharides and glycosides . Each transport system, TogMNAB and TogT, is able to independently mediate the transport of oligogalacturonides and the simultaneous inactivation of both is necessary to give a total absence of growth with pectin as the carbon source . The togT gene constitutes an independent transcriptional unit . Its expression is induced in the presence of pectic derivatives and it is subject to catabolite repression . In vitro, the repressor KdgR and the activator CRP both interact directly with the togT regulatory region . The decreased pathogenicity of single and double togT, togM mutants indicated that a deficiency in uptake of pectic oligomers leads to reduced bacterial multiplication which, in turn, limits plant maceration. Mol Microbiol, 2001 Sep, 41(5), 1113 - 23 Identification of TogMNAB, an ABC transporter which mediates the uptake of pectic oligomers in Erwinia chrysanthemi 3937; Hugouvieux-Cotte-Pattat N et al.; The bacterium Erwinia chrysanthemi, which causes soft rot disease on various plants, is able to use pectin as a carbon source for growth . Knowledge of the critical step in pectin catabolism which allows the entry of pectic oligomers into the cells is scarce . We report here the first example of a transport system involved in the uptake of pectic oligomers . The TogMNAB transporter of E . chrysanthemi is a member of the ATP-binding cassette (ABC) superfamily . TogM and TogN are homologous to the inner membrane components, TogA exhibits the signature of ABC ATPases and TogB shows similarity with periplasmic ligand-binding proteins . The TogMNAB transporter is a new member of the carbohydrate uptake transporter-1 family (CUT1, TC no . 3.1.1), which is specialized in the transport of complex sugars . The four genes, togM, togN, togA and togB, are apparently co-transcribed in a large operon which also includes the pectate lyase gene pelW . The transcription of the tog operon is induced in the presence of pectic derivatives and is affected by catabolite repression . It is controlled by the KdgR repressor and the CRP activator . The TogMNAB system is able to provide Escherichia coli with the ability to transport oligogalacturonides . In E . chrysanthemi, the TogMNAB system seems to play a major role in switching on the induction of pectin catabolism . TogB also acts as a specific receptor for chemotaxis towards oligogalacturonides . The decreased capacity of maceration of a togM mutant indicates the importance of transport and/or attraction of oligogalacturonides for E . chrysanthemi pathogenicity. Mol Plant Microbe Interact, 2001 Sep, 14(9), 1035 - 42 Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora; Mae A et al.; Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity . We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora . In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner . We suggest that E . carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses . To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL . This was accomplished by ectopic expression in tobacco of the E . carotovora gene expI, which is responsible for OHL biosynthesis . We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants . The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E . carotovora . The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E . carotovora. Genome Biol . 2001;2(8):RESEARCH0030 . Epub 2001 Jul 27. The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa; Calhoun DH et al.; BACKGROUND: Chorismate mutases of the AroQ homology class are widespread in the Bacteria and the Archaea . Many of these exist as domains that are fused with other aromatic-pathway catalytic domains . Among the monofunctional AroQ proteins, that from Erwinia herbicola was previously shown to have a cleavable signal peptide and located in the periplasmic compartment . Whether or not this might be unique to E . herbicola was unknown . RESULTS: The gene coding for the AroQ protein was cloned from Salmonella typhimurium, and the AroQ protein purified from both S . typhimurium and Pseudomonas aeruginosa was shown to have a periplasmic location . The periplasmic chorismate mutases (denoted *AroQ) are shown to be a distinct subclass of AroQ, being about twice the size of cytoplasmic AroQ proteins . The increased size is due to a carboxy-terminal extension of unknown function . In addition, a so-far novel aromatic aminotransferase was shown to be present in the periplasm of P . aeruginosa . CONCLUSIONS: Our analysis has detected a number of additional *aroQ genes . The joint presence of *AroQ, cyclohexadienyl dehydratase and aromatic aminotransferase in the periplasmic compartment of P . aeruginosa comprises a complete chorismate-to-phenylalanine pathway and accounts for the "hidden overflow pathway" to phenylalanine described previously. Appl Environ Microbiol, 2001 Sep, 67(9), 4070 - 6 Rapid identification and differentiation of the soft rot erwinias by 16S-23S intergenic transcribed spacer-PCR and restriction fragment length polymorphism analyses; Toth IK et al.; Current identification methods for the soft rot erwinias are both imprecise and time-consuming . We have used the 16S-23S rRNA intergenic transcribed spacer (ITS) to aid in their identification . Analysis by ITS-PCR and ITS-restriction fragment length polymorphism was found to be a simple, precise, and rapid method compared to current molecular and phenotypic techniques . The ITS was amplified from Erwinia and other genera using universal PCR primers . After PCR, the banding patterns generated allowed the soft rot erwinias to be differentiated from all other Erwinia and non-Erwinia species and placed into one of three groups (I to III) . Group I comprised all Erwinia carotovora subsp . atroseptica and subsp . betavasculorum isolates . Group II comprised all E . carotovora subsp . carotovora, subsp . odorifera, and subsp . wasabiae and E . cacticida isolates, and group III comprised all E . chrysanthemi isolates . To increase the level of discrimination further, the ITS-PCR products were digested with one of two restriction enzymes . Digestion with CfoI identified E . carotovora subsp . atroseptica and subsp . betavasculorum (group I) and E . chrysanthemi (group III) isolates, while digestion with RsaI identified E . carotovora subsp . wasabiae, subsp . carotovora, and subsp . odorifera/carotovora and E . cacticida isolates (group II) . In the latter case, it was necessary to distinguish E . carotovora subsp . odorifera and subsp . carotovora using the alpha-methyl glucoside test . Sixty suspected soft rot erwinia isolates from Australia were identified as E . carotovora subsp . atroseptica, E . chrysanthemi, E . carotovora subsp . carotovora, and non-soft rot species . Ten "atypical" E . carotovora subsp . atroseptica isolates were identified as E . carotovora subsp . atroseptica, subsp . carotovora, and subsp . betavasculorum and non-soft rot species, and two "atypical" E . carotovora subsp . carotovora isolates were identified as E . carotovora subsp . carotovora and subsp . atroseptica. Planta, 2001 Mar, 212(4), 635 - 9 Enhanced disease resistance conferred by expression of an antimicrobial magainin analog in transgenic tobacco; Li Q et al.; Magainins are a group of short peptides originally isolated from frog skin and thought to function as a natural defense mechanism against infection due to their antimicrobial properties . The engineered magainin analog peptide Myp30 was found to inhibit spore germination of the oomycete, Peronospora tabacina (Adam) in vitro, and the growth of a bacterial pathogen Erwinia carotovora subsp . carotovora (Jones) . Transgenic tobacco (Nicotiana tabacum L.) plants expressing Myp30 were evaluated for resistance to these pathogens . The expression of the peptide only to an extracellular location resulted in significant reduction in sporulation and lesion size due to P . tabacina infection . A significant increase in resistance to the bacterial pathogen was also observed regardless of the targeting location of the peptide. J Food Prot, 2001 Aug, 64(8), 1110 - 5 Analysis of native microflora and selection of strains antagonistic to human pathogens on fresh produce; Liao CH et al.; The native microflora of three types of produce (green bell peppers, Romaine lettuce, and prepeeled baby carrots) and two types of sprouting seeds (alfalfa and clover) were investigated . Aerobic plate count (APC) for each produce or seed type as determined on Pseudomonas agar F (PAF) with incubation at 28 degrees C was in the range of 4 to 7 log CFU per g of tissue or seed . There was no significant difference (P > or = 0.05) in APC when the determinations were made with three agar media including PAF, brain heart infusion agar, and plate count agar . However, the APC as determined from plates that were incubated at 28 degrees C was significantly (P < or = 0.05) higher than with incubation at 37 degrees C . Fluorescent pseudomonads accounted for 23 to 73% of APC and 6 to 18% of APC recovered from carrots, pepper, and lettuce were pectolytic . Forty-eight strains of pectolytic bacteria were randomly isolated and identified, respectively, as members of the genera of Pseudomonas, Erwinia, Bacillus, Xanthomonas, or Flavobacterium . Lactic acid bacteria and/or yeast were consistently isolated from baby carrots, lettuce, and sprouting seeds (alfalfa or clover) but not from green bell peppers . Approximately 120 strains of indigenous microflora were tested for their ability to inhibit the growth of Salmonella Chester, Listeria monocytogenes, Escherichia coli, or Erwinia carotovora subsp . carotovora on PAF . Six isolates capable of inhibiting the growth of at least one pathogen were isolated and identified, respectively, as Bacillus spp . (three strains), Pseudomonas aeruginosa (one strain), Pseudomonas fluorescens (strain A3), and yeast (strain D1) . When green pepper disks were inoculated with strains A3 and D1, the growth of Salmonella Chester and L . monocytogenes on the disks was reduced by 1 and 2 logs, respectively, over a period of 3 days . Application of strains A3 and D1 as potential biopreservatives for enhancing the quality and safety of fresh produce is discussed. J Mol Biol, 2001 Jul 27, 310(5), 1055 - 66 Type II protein secretion in gram-negative pathogenic bacteria: the study of the structure/secretion relationships of the cellulase Cel5 (formerly EGZ) from Erwinia chrysanthemi; Chapon V et al.; Erwinia chrysanthemi, a Gram-negative plant pathogen, secretes the cellulase Cel5 (formerly EGZ) via the type II secretion pathway (referred to as Out) . Cel5 is composed of two domains, a large N-terminal catalytic domain (390 amino acid residues) and a small C-terminal cellulose-binding domain (62 amino acid residues) separated by a linker region . A combination of mutagenesis and structural analysis permitted us to investigate the structure/secretion relationships with respect to the catalytic domain of Cel5 . The 3D structure of the catalytic domain was solved by molecular replacement at 2.3 A resolution . Cel5 exhibits the (beta/alpha)8 structural fold and two extra-barrel features . Our previous genetic study based upon tRNA-mediated suppression allowed us to predict positions of importance in the molecule in relation to structure and catalysis . Remarkably, all of the predictions proved to be correct when compared with the present structural information . Mutations of Arg57, which is located at the heart of the catalytic domain, allowed us to test the consequences of structural modifications on the secretion efficiency . The results revealed that secretability imposes remarkably strong constraints upon folding . In particular, an Arg-to-His mutation yielded a species that folded to a stable conformation close to, but distinct from the wild-type, which however was not secretable . We discuss the relationships between folding of a protein in the periplasm, en route to the cell exterior, and presentation of secretion information . We propose that different solutions have been selected for type II secreted exoproteins in order to meet the constraints imposed by their interaction with their respective secretion machineries . We propose that evolutionary pressure has led to the adaptation of different secretion motifs for different type II exoproteins. Mol Plant Microbe Interact, 2001 Aug, 14(8), 962 - 8 Type III secretion contributes to the pathogenesis of the soft-rot pathogen Erwinia carotovora: partial characterization of the hrp gene cluster; Rantakari A et al.; The virulence of soft-rot Erwinia species is dependent mainly upon secreted enzymes such as pectinases, pectin lyases, and proteases that cause maceration of plant tissue . Some soft-rot Erwinia spp . also harbor genes homologous to the hypersensitive reaction and pathogenesis (hrp) gene cluster, encoding components of the type III secretion system . The hrp genes are essential virulence determinants for numerous nonmacerating gram-negative plant pathogens but their role in the virulence of soft-rot Erwinia spp . is not clear . We isolated and characterized 11 hrp genes of Erwinia carotovora subsp . carotovora . Three putative sigmaL-dependent Hrp box promoter sequences were found . The genes were expressed when the bacteria were grown in Hrp-inducing medium . The operon structure of the hrp genes was determined by mRNA hybridization, and the results were in accordance with the location of the Hrp boxes . An E . carotovora strain with mutated hrcC, an essential hrp gene, was constructed . The hrcC- strain was able to multiply and cause disease in Arabidopsis, but the population kinetics were altered so that growth was delayed during the early stages of infection. Mol Plant Microbe Interact, 2001 Aug, 14(8), 931 - 8 Global regulators ExpA (GacA) and KdgR modulate extracellular enzyme gene expression through the RsmA-rsmB system in Erwinia carotovora subsp . carotovora; Hyytiainen H et al.; The production of the main virulence determinants, the extracellular plant cell wall-degrading enzymes, and hence virulence of Erwinia carotovora subsp . carotovora is controlled by a complex regulatory network . One of the global regulators, the response regulator ExpA, a GacA homolog, is required for transcriptional activation of the extracellular enzyme genes of this soft-rot pathogen . To elucidate the mechanism of ExpA control as well as interactions with other regulatory systems, we isolated second-site transposon mutants that would suppress the enzyme-negative phenotype of an expA (gacA) mutant . Inactivation of kdgR resulted in partial restoration of extracellular enzyme production and virulence to the expA mutant, suggesting an interaction between the two regulatory pathways . This interaction was mediated by the RsmA-rsmB system . Northern analysis was used to show that the regulatory rsmB RNA was under positive control of ExpA . Conversely, the expression of rsmA encoding a global repressor was under negative control of ExpA and positive control of KdgR . This study indicates a central role for the RsmA-rsmB regulatory system during pathogenesis, integrating signals from the ExpA (GacA) and KdgR global regulators of extracellular enzyme production in E . carotovora subsp . carotovora. Cancer Chemother Pharmacol, 2001 Jul, 48(1), 77 - 82 Pharmacokinetics of Erwinia asparaginase after intravenous and intramuscular administration; Albertsen BK et al.; PURPOSE: To describe the pharmacokinetics of Erwinia asparaginase (ASNase) after intravenous (i.v.) and intramuscular (i.m.) administration . METHODS: A group of 29 children with newly diagnosed acute lymphoblastic leukemia (ALL) received Erwinia ASNase 30,000 IU/m2 every day for 10 days during multiagent induction therapy . Of these patients . 13 received i.v . therapy and 16 received i.m . therapy . During the reinduction phase the patients received Erwinia ASNase 30,000 IU/m2 twice a week for 2 weeks (Mondays and Thursdays) (8 patients in the i.v.-treated group and 11 patients in the i.m.-treated group) . ASNase activity (spectrophotometric assay) was measured in plasma samples obtained from the patients at various times during therapy . RESULTS: The estimated half-life was 6.4 +/- 0.5 h (n = 13), the absorption rate after i.m . administration was found to limit elimination . The apparent volume of distribution corresponded well with the volume of plasma . The estimated clearance suggested that Erwinia ASNase is a low-clearance drug . Bioavailability after i.m . administration was (mean +/- SEM) 27.0 +/- 4.5% (range 11-61%; n = 12) . CONCLUSIONS: In this study the pharmacokinetic parameters after i.v . and i.m . administration of Erwinia ASNase were determined based on a substantial number of patients . The present findings emphasize the importance of conducting proper pharmacokinetic studies before a new drug or a new preparation of a drug is introduced in a different schedule. Nature, 2001 Jun 14, 411(6839), 813 - 7 Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase; Dong YH et al.; Bacterial cells sense their population density through a sophisticated cell-cell communication system and trigger expression of particular genes when the density reaches a threshold . This type of gene regulation, which controls diverse biological functions including virulence, is known as quorum sensing . Quorum-sensing signals, such as acyl-homoserine lactones (AHLs), are the essential components of the communication system . AHLs regulate virulence gene expression in a range of plant and animal (including human) bacterial pathogens . AHL-producing tobacco restored the pathogenicity of an AHL-negative mutant of Erwinia carotovora . Different bacterial species may produce different AHLs, which vary in the length and substitution of the acyl chain but contain the same homoserine lactone moiety . Here we show that the acyl-homoserine lactonase (AHL-lactonase), a new enzyme from Bacillus sp., inactivates AHL activity by hydrolysing the lactone bond of AHLs . Plants expressing AHL-lactonase quenched pathogen quorum-sensing signalling and showed significantly enhanced resistance to E . carotovora infection . Our results highlight a promising potential to use quorum-sensing signals as molecular targets for disease control, thereby broadening current approaches for prevention of bacterial infections. Carbohydr Res, 2001 Jul 19, 333(4), 295 - 302 Extracellular polysaccharides of modified strains of Erwinia spp; Yang BY et al.; The structure of the extracellular polysaccharide (EPS) produced by Erwinia chrysanthemi strain A2148 has been determined using low pressure size-exclusion and anion-exchange chromatographies, high pH anion-exchange chromatography, glycosyl-linkage analysis, and 1D 1H NMR spectroscopy . The polysaccharide is structurally similar, if not identical, to the EPS produced by E . chrysanthemi strain A350 . A streptomycin-resistant strain of E . chrysanthemi Ech6 (Ech6S(+)) has been generated and has an elevated production of EPS, as does a streptomycin-resistant strain (Ech9Sm6) of E . chrysanthemi Ech9 . These modified E . chrysanthemi spp . have been ribotyped and found to be closely related to their parent strains. Res Microbiol, 2001 Jun, 152(5), 481 - 5 NKBOR, a mini-Tn10-based transposon for random insertion in the chromosome of Gram-negative bacteria and the rapid recovery of sequences flanking the insertion sites in Escherichia coli; Rossignol M et al.; We have constructed an R6K-based suicide vector that permits the random insertion of a mini-transposon named NKBOR into the chromosome of Gram-negative bacteria and the subsequent rapid cloning of sequences flanking the insertion site in Escherichia coli . This mini-transposon contains a conditional R6K plasmid origin of replication, a kanamycin resistance gene and unique restriction sites between the IS10 inverted repeats . NKBOR can be propagated by replication in an E . coli strain containing the R6K replicase pi protein . Alternatively the mini-transposon can be replicated in a pSC 101 derivative that is thermosensitive for its replication so that the mini-transposon acts as a suicide plasmid at nonpermissive temperatures . Efficient NKBOR transposition is ensured by expression of an adjacent transposase gene and has been demonstrated in E . coli, Klebsiella pneumoniae, and Erwinia carotovora . Sequences flanking the insertion sites in these strains can be rapidly recovered and identified in E . coli strains expressing the R6K pi protein. Vet Res, 2001 May-Aug, 32(3-4), 261 - 73 Resistance to trimethoprim and sulfonamides; Skold O; Sulfonamides and trimethoprim have been used for many decades as efficient and inexpensive antibacterial agents for animals and man . Resistance to both has, however, spread extensively and rapidly . This is mainly due to the horizontal spread of resistance genes, expressing drug-insensitive variants of the target enzymes dihydropteroate synthase and dihydrofolate reductase, for sulfonamide and trimethoprim, respectively . Two genes, sul1 and sul2, mediated by transposons and plasmids, and expressing dihydropteroate synthases highly resistant to sulfonamide, have been found . For trimethoprim, almost twenty phylogenetically different resistance genes, expressing druginsensitive dihydrofolate reductases have been characterized . They are efficiently spread as cassettes in integrons, and on transposons and plasmids . One particular gene, dfr9, seems to have originally been selected in the intestine of swine, where it was found in Escherichia coli, on large plasmids in a disabled transposon, Tn5393, originally found in the plant pathogen Erwinia amylovora . There are also many examples of chromosomal resistance to sulfonamides and trimethoprim, with different degrees of complexity, from simple base changes in the target genes to transformational and recombinational exchanges of whole genes or parts of genes, forming mosaic gene patterns . Furthermore, the trade-off, seen in laboratory experiments selecting resistance mutants, showing drug-resistant but also less efficient (increased Kms) target enzymes, seems to be adjusted for by compensatory mutations in clinically isolated drug-resistant pathogens . This means that susceptibility will not return after suspending the use of sulfonamide and trimethoprim. Curr Microbiol, 2001 May, 42(5), 310 - 5 Periplasmic PQQ-dependent glucose oxidation in free-living and symbiotic rhizobia; Bernardelli CE et al.; The expression of the pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenase (GDH) of Rhizobium tropici CIAT899 and Sinorhizobium meliloti RCR2011 was investigated under different nutrient-limiting conditions in continuous cultures, under different conditions of phosphate availability, and in S . meliloti bacteroids . The presence of free PQQ in alfalfa root exudates has also been assayed . It was shown that apo-GDH or holoenzyme was actively synthesized by these rhizobia, with the concomitant production of gluconate from glucose, under certain environmental conditions . GDH activity was also detected in bacteroids from alfalfa root nodules inoculated with either S . meliloti RCR2011 or 102F34 . It was also shown that free PQQ was present in root exudates of alfalfa, but its production is ascribed to the activity of Erwinia sp., a normal contaminant of these seeds. Plant Physiol, 2001 Jun, 126(2), 849 - 60 Jasmonate-dependent induction of indole glucosinolates in Arabidopsis by culture filtrates of the nonspecific pathogen Erwinia carotovora; Brader G et al.; Elicitors from the plant pathogen Erwinia carotovora trigger coordinate induction of the tryptophan (Trp) biosynthesis pathway and Trp oxidizing genes in Arabidopsis . To elucidate the biological role of such pathogen-induced activation we characterized the production of secondary defense metabolites such as camalexin and indole glucosinolates derived from precursors of this pathway . Elicitor induction was followed by a specific increase in 3-indolylmethylglucosinolate (IGS) content, but only a barely detectable accumulation of the indole-derived phytoalexin camalexin . The response is mediated by jasmonic acid as shown by lack of IGS induction in the jasmonate-insensitive mutant coi1-1 . In accordance with this, methyl jasmonate was able to trigger IGS accumulation in Arabidopsis . In contrast, ethylene and salicylic acid seem to play a minor role in the response . They did not trigger alterations in IGS levels, and methyl jasmonate- or elicitor-induced IGS accumulation in NahG and ethylene-insensitive ein2-1 mutant plants was similar as in the wild type . The breakdown products of IGS and other glucosinolates were able to inhibit growth of E . carotovora . The results suggest that IGS is of importance in the defense against bacterial pathogens. Mol Microbiol, 2001 Jun, 40(5), 1129 - 39 Visualization of secreted Hrp and Avr proteins along the Hrp pilus during type III secretion in Erwinia amylovora and Pseudomonas syringae; Jin Q et al.; Pili are required for protein and/or DNA transfer from bacteria to recipient plant or bacterial cells, based on genetic evidence . However, it has never been shown directly that the effector proteins or DNA are localized along or inside the pili in situ . Failure to visualize an association of effector proteins/DNA with pili is the central issue in the debate regarding the exact function of pili in protein and DNA transfer . In this study, a newly developed in situ immunogold labelling procedure enabled visualization of the specific localization of type III effector proteins of Erwinia amylovora and Pseudomonas syringae pv . tomato along the Hrp pilus, but not along the flagellum or randomly in the intercellular space . In contrast, PelE, a pectate lyase secreted via the type II protein secretion system, was not associated with the Hrp pilus . These results provide direct evidence that type III secretion occurs only at the site of Hrp pilus assembly and that the Hrp pilus guides the transfer of effector proteins outside the bacterial cell, favouring the 'conduit/guiding filament' model. Ukr Biokhim Zh, 2000 Nov-Dec, 72(6), 51 - 5 {Characteristics of bacterial lipopolysaccharides depending on extraction method}; Zherebylo OIe et al.; Carbohydrate-containing biopolymers have been isolated from Erwinia carotovora subsp . atroseptica 549 by two methods--the aqueous-phenol and with using physiological solution--with addition and without addition of ethylenediaminetetraacetate (EDTA) . The biopolymers yield from the cells of bacteria are shown to depend on the extraction method . Lipopolysaccharide-protein complex have been isolated by the sparing method . The purest lipopolysaccharide have been isolated by the aqueousphenol method . The preliminary treatment of cells by EDTA increased the biopolymers output . Carbohydrate containing biopolymers isolated from the cells of bacteria by different methods possess the similar qualitative composition of monosaccharides but they differ in the quantitative content of monosaccharides, spectrum of fatty acids of lipid components as well as in the protein content. Mikrobiol Z, 2001 Jan-Feb, 63(1), 23 - 33 {Adsorbed receptors for Erwinia carotovor subsp . carotovora macromolecular bacteriocins}; Tovkach FI et al.; Study of nature of receptors for macromolecular bacteriocins Erwinia carotovora subsp . carotovora has shown that lipopolysaccharide (LPS) of cell membrane is an attaching structure for them . It has been established that enzymic treatment of LPS preparation with its further deproteinization by phenol is necessary for isolation of biologically active lipopolysaccharide . The process of absorption by LPS has been studied quantitatively and it has been shown that it is a low-efficient receptor as compared with LPS included in the native cell membranes . An approach has been proposed for the first time to the estimation of monosaccharide composition of LPS-receptor based on relations between bacteriocin sensitivity and content of monosaccharides . Study of six strains of E . carotovora subsp . carotovora from different sources has shown that the structure of LPS-receptors includes mannose, fucose, xylose, ramnose and two lipophylic monosaccharides of unknown nature . A conclusion has been made that S-LPS (0-chain) is that part which contains the sites of attachment of macromolecular carotovoricins. Novartis Found Symp, 2001, 236, 219 - 28; discussion 228-32 Biosynthesis of beta-carotene (provitamin A) in rice endosperm achieved by genetic engineering; al-Babili S et al.; To obtain a functioning provitamin A (beta-carotene) biosynthetic pathway in rice endosperm, we introduced in a single, combined transformation effort the cDNAs coding for (1) phytoene synthase (psy) and (2) lycopene beta-cyclase (beta-lcy; both from Narcissus pseudonarcissus and both under control of the endosperm-specific glutelin promoter), with (3) a bacterial phytoene desaturase (crtI, from Erwinia uredovora under constitutive 35S promoter control) . This combination covers the requirements for beta-carotene synthesis, and yellow, beta-carotene-bearing rice endosperm was obtained in the T0 generation . However, further experiments revealed that the presence of beta-lcy was not necessary, since psy and crtI alone were able to drive beta-carotene synthesis as well as the formation of further downstream xanthophylls . This finding could be explained if these downstream enzymes are either constitutively expressed in rice endosperm or are induced by the transformation, e.g . by products derived therefrom . Based on results in N . pseudonarcissus as a model system, a likely hypothesis can be developed that trans lycopene or a trans lycopene derivative acts as an inductor in a kind of feedback mechanism stimulating endogenous carotenogenic genes. Mol Plant Microbe Interact, 2001 Jun, 14(6), 816 - 20 Control of exuT activity for galacturonate transport by the negative regulator ExuR in Erwinia chrysanthemi EC16; Valmeekam V et al.; The negative regulatory protein ExuR in Erwinia chrysanthemi regulates expression of the galacturonate uptake (exuT) and utilization (uxaA, uxaB, uxaC) genes . We cloned and determined the nucleotide sequence of the exuR gene from E . chrysanthemi EC16 . Analysis of the deduced amino acid sequence indicates that this protein possesses a helix-turn-helix motif and belongs to the GntR family of transcriptional repressors . Northern blot analysis and studies with transcriptional fusions of exuT in wild-type and exuR mutant backgrounds indicate that exuT transcription is deregulated in the exuR strain in vivo and in planta . {14C}-galacturonic acid uptake was constitutively high under inducing and noninducing conditions in the exuR mutant . Maximal exuT transcription activity was observed within 8 h of bacterial inoculation into potato tubers, well before any visible symptoms of disease were detected . This suggests that ExuT transport activity in E . chrysanthemi is important in the early stages of disease development. Mol Plant Microbe Interact, 2001 Jun, 14(6), 758 - 67 Essential role of superoxide dismutase on the pathogenicity of Erwinia chrysanthemi strain 3937; Santos R et al.; The sodA gene from Erwinia chrysanthemi strain 3937 was cloned by functional complementation of an Escherichia coli sodA sodB mutant and sequenced . We identified a 639-bp open reading frame, which encodes a protein that is 85% identical to the E . coli manganese-containing superoxide dismutase MnSOD . Promoter elements of this gene were identified by transcriptional mapping experiments . We constructed an E . chrysanthemi deltasodA mutant by reverse genetics . The deltasodA mutation resulted in the absence of a cytoplasmic SOD, which displays the same characteristics as those of MnSOD . The deltasodA mutant was more sensitive to paraquat than the wild-type strain . This mutant could macerate potato tubers, similar to the wild-type strain . In contrast, when inoculated on African violets, the mutant produced, at most, only small necrotic lesions . If the inoculum was supplemented with the superoxide anion-scavenging metalloporphyrin MnTMPyP or purified SOD and catalase, the deltasodA mutant was able to macerate the inoculated zone . Generation of superoxide anion by African violet leaves inoculated with E . chrysanthemi was demonstrated with nitroblue tetrazolium as an indicator . Therefore, at the onset of infection, E . chrysanthemi cells encounter an oxidative environment and require active protective systems against oxidative damages such as MnSOD to overcome these types of conditions. FEBS Lett, 2001 May 25, 497(2-3), 82 - 4 Harpin, a hypersensitive response elicitor from Erwinia amylovora, regulates ion channel activities in Arabidopsis thaliana suspension cells; El-Maarouf H et al.; HrpN, the hypersensitive response elicitor from Erwinia amylovora, stimulated K(+) outward rectifying currents in Arabidopsis thaliana suspension cells . It also decreased anion currents . These data demonstrate the ability of harpin to regulate different plasma membrane ion channels, putative components of signal transduction chains leading to defense responses and programmed cell death. Carbohydr Res, 2001 Jun 4, 332(3), 317 - 23 beta-Elimination of glucosyluronic residues during methylation of an acidic polysaccharide from Erwinia chrysanthemi CU 643; Yun Yang B et al.; The Erwinia chrysanthemi CU643 EPS has a linear hexasaccharide repeating unit in which a 4-linked uronic acid residue is present . The EPS was methylated by either the NaOH-Me2SO-MeI or Li-dimsyl procedure . MALDI-TOF MS analysis of the methylated products indicates that the beta-eliminative degradation occurs during the methylation, as characterized by serial fragments of the hexasaccharide repeating units . The degradation was clearly defined from the methylation of a glucosyluronic-containing pyruvated pentasaccharide. Mol Genet Genomics, 2001 Apr, 265(2), 287 - 92 Quorum sensing controls the synthesis of virulence factors by modulating rsmA gene expression in Erwinia carotovora subsp . carotovora; Koiv V et al.; The plant-pathogenic bacterium Erwinia carotovora subsp . carotovora (Ecc) causes disease mainly by means of a number of extracellular plant cell wall-degrading enzymes (PCWDEs), also referred to as virulence factors . The production of PCWDEs is coordinately activated by the diffusible signal molecule N-acyl-homoserine lactone (HSL) in a population density-dependent manner ("quorum sensing") . ExpI is the enzyme responsible for the synthesis of HSL . The Rsm system negatively regulates the production of PCWDEs . It includes three components: RsmA is an RNA-binding protein which promotes mRNA decay; rsmB is a unique regulator RNA, and RsmC regulates expression of rsmA positively and of rsmB negatively . We report here that in an expI knockout mutant of Ecc strain SCC3193, the levels of rsmA and rsmB RNA are remarkably enhanced in comparison to the wild-type strain, while the level of the rsmC transcript is not affected . The increase in transcription of rsmA in the expI strain represses production of PCWDEs, which in turn leads to the avirulent phenotype of this mutant . In the expI- mutant, addition of exogenous HSL caused repression of rsmA and rsmB transcription to the wild-type level, whereas the expression of rsmC was not affected . Taken together, these data suggest that HSL affects the expression of rsmA, and that this effect is not mediated by RsmC . This specific effect and the previous demonstration that HSL is required for PCWDE production in Ecc support the hypothesis that regulation by quorum sensing in Ecc, in contrast to most other systems already described, requires HSL to repress rsmA transcription, which in turn leads to the activation of PCWDE production . A model is presented that explains how HSL controls the production of PCWDEs by modulating the expression of rsmA. Biochemistry, 2001 May 15, 40(19), 5655 - 64 Structural basis for the activity and substrate specificity of Erwinia chrysanthemi L-asparaginase; Aghaiypour K et al.; Bacterial L-asparaginases, enzymes that catalyze the hydrolysis of L-asparagine to aspartic acid, have been used for over 30 years as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia . Other substrates of asparaginases include L-glutamine, D-asparagine, and succinic acid monoamide . In this report, we present high-resolution crystal structures of the complexes of Erwinia chrysanthemi L-asparaginase (ErA) with the products of such reactions that also can serve as substrates, namely L-glutamic acid (L-Glu), D-aspartic acid (D-Asp), and succinic acid (Suc) . Comparison of the four independent active sites within each complex indicates unique and specific binding of the ligand molecules; the mode of binding is also similar between complexes . The lack of the alpha-NH3(+) group in Suc, compared to L-Asp, does not affect the binding mode . The side chain of L-Glu, larger than that of L-Asp, causes several structural distortions in the ErA active side . The active site flexible loop (residues 15-33) does not exhibit stable conformation, resulting in suboptimal orientation of the nucleophile, Thr15 . Additionally, the delta-COO(-) plane of L-Glu is approximately perpendicular to the plane of gamma-COO(-) in L-Asp bound to the asparaginase active site . Binding of D-Asp to the ErA active site is very distinctive compared to the other ligands, suggesting that the low activity of ErA against D-Asp could be mainly attributed to the low k(cat) value . A comparison of the amino acid sequence and the crystal structure of ErA with those of other bacterial L-asparaginases shows that the presence of two active-site residues, Glu63(ErA) and Ser254(ErA), may correlate with significant glutaminase activity, while their substitution by Gln and Asn, respectively, may lead to minimal L-glutaminase activity. J Mol Biol, 2001 Apr 27, 308(2), 205 - 19 The PDZ domain of OutC and the N-terminal region of OutD determine the secretion specificity of the type II out pathway of Erwinia chrysanthemi; Bouley J et al.; The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete multiple exoproteins by a type II pathway, the Out system . Secretion in Erwinia is species-specific: exoproteins of one species cannot be secreted by the other . We analysed the role of two components of the Out system, the bitopic inner membrane protein OutC and the secretin OutD, in the specific recognition of secreted proteins . We demonstrated that the PDZ domain of OutC determines its secretion specificity towards certain exoproteins . The secretin is the major determinant of specificity of the Out system: OutD of E . carotovora changes the secretion specificity of E . chrysanthemi and enables it to secrete heterologous exoproteins . Construction of chimeric OutD showed that the N-terminal region is the specificity domain of the secretin . Thus, both the PDZ domain of OutC and the N-terminal region of OutD are required for specific recognition of secreted proteins . Systematic analysis of the secretion of several exoproteins demonstrated that different exoproteins secreted by the Out machinery have different requirement for their presumed targeting signals on OutC and OutD . This strongly indicates that diverse exoproteins possess a variable number of targeting signals which are recognised by different regions of OutC and OutD . J Mol Microbiol Biotechnol, 2001 Apr, 3(2), 309 - 18 The PecM protein of the phytopathogenic bacterium Erwinia chrysanthemi, membrane topology and possible involvement in the efflux of the blue pigment indigoidine; Rouanet C et al.; The pecS regulatory locus negatively modulates the expression of many virulence genes in Erwinia chrysanthemi . This locus consists of two genes, pecS and pecM, divergently transcribed . Previous studies have shown that PecS down-regulates the expression of both pecSand pecMgenes and that PecM is required for full PecS activity . Computer-aided hydropathy analysis of PecM predicted the presence of between 8 to 10 potential transmembrane segments . We analyzed the membrane topology of PecM using the beta-lactamase gene fusion system and obtained the following unique characteristics . PecM contains 10 membrane spanning segments, with both the amino and carboxyl termini located in the cytoplasmic side of the inner membrane . The fourth periplasmic loop, which has a relatively long hydrophilic domain containing 17 amino acid residues, may play an important role in PecM function . The topological model obtained for PecM can be applied to PecM homologues in other bacteria . Measurement of the extrusion of the blue pigment indigoidine by the E . chrysanthemi derivative isogenic mutants pecS, pecM and pecS-pecM revealed that PecM is required for complete efflux of the pigment . Its relation to other efflux systems and its potential physiological role are discussed. Int J Syst Evol Microbiol, 2001 Mar, 51(Pt 2), 723 - 4 Recommended conservation of the names Streptococcus sanguis, Streptococcus rattus, Streptococcus cricetus, and seven other names included in the Approved Lists of Bacterial Names . Request for an opinion; Kilian M; With reference to the first Principle of the International Code of Nomenclature of Bacteria, which emphasizes stability of names, it is proposed that the original names Streptococcus sanguis, Streptococcus rattus, Streptococcus cricetus, Erwinia ananas, Eubacterium tarantellus, Lactobacillus sake, Nitrosococcus oceanus, Pseudomonas betle, Rickettsia canada and Streptomyces rangoon, all included in the Approved Lists of Bacterial Names, be conserved . Request for an Opinion. Biotechnol Prog, 2001 Mar-Apr, 17(2), 311 - 7 Optimization of fermentation conditions for preparation of polygalacturonic acid transeliminase by Erwinia carotovora IFO3830; Ding F et al.; This paper is concerned with optimization of the fermentation conditions for preparation of alkaline pectin lyase (poly-galacturonic acid trans-eliminase, PATE) by Erwinia carotovora IFO3830 (E.C.IFO3830) . The orthogonal matrix method was adopted as the experimental design method for media . The effects of media composition on the growth rate of E.C.IFO3830 are in the order of pectin > KH(2)PO(4)/Na(2)HPO(4) > MgSO(4) > (NH(4))(2)SO(4) > glucose > L-glutamate sodium, and those on PATE activity are in the order of glucose > pectin > L-glutamate sodium > KH(2)PO(4)/Na(2)HPO(4) > MgSO(4) > (NH(4))(2)SO(4) . The strain of E.C.IFO3830 grows quickly and easily near an initial pH of 7.0, and especially at the alkaline range of pH 7.0-8.0 . The growth rate profiles indicate that E.C.IFO3830 grows very quickly; its generation time t(1/2) is about 0.2 h . The appropriate fermentation period is about 10-20 h for a high level productivity of biomass and 25-35 h for high productivity of PATE enzyme. Mol Plant Microbe Interact, 2001 Apr, 14(4), 516 - 26 Effects of the two-component system comprising GacA and GacS of Erwinia carotovora subsp . carotovora on the production of global regulatory rsmB RNA, extracellular enzymes, and harpinEcc; Cui Y et al.; Posttranscriptional regulation mediated by the regulator of secondary metabolites (RSM) RsmA-rsmB pair is the most important factor in the expression of genes for extracellular enzymes and HarpinEcc in Erwinia carotovora subsp . carotovora . RsmA is a small RNA-binding protein, which acts by lowering the half-life of a mRNA species . rsmB specifies an untranslated regulatory RNA and neutralizes the RsmA effect . It has been speculated that GacA-GacS, members of a two-component system, may affect gene expression via RsmA . Because expA, a gacA homolog, and expS (or rpfA), a gacS homolog, have been identified in E . carotovora subsp . carotovora, we examined the effects of these gacA and gacS homologs on the expression of rsmA, rsmB, and an assortment of exoprotein genes . The gacA gene of E . carotovora subsp . carotovora strain 71 stimulated transcription of genes for several extracellular enzymes (pel-1, a pectate lyase gene; peh-1, a polygalacturonase gene; and celV, a cellulase gene), hrpNEcc (an E . carotovora subsp . carotovora gene specifying the elicitor of hypersensitive reaction), and rsmB in GacA+ and GacS+ E . carotovora subsp . carotovora strains . Similarly, the E . carotovora subsp . carotovora gacA gene stimulated csrB (rsmB) transcription in Escherichia coli . A GacS- mutant of E . carotovora subsp . carotovora strain AH2 and a GacA- mutant of E . carotovora subsp . carotovora strain Ecc71 compared with their parent strains produced very low levels of rsmB, pel-1, peh-1, celV, and hrpNEcc transcripts but produced similar levels of rsmA RNA and RsmA protein as well as transcripts of hyperproduction of extracellular enzymes (Hex) hexA, kdgR (repressor of genes for uronate and pectate catabolism), rsmC, and rpoS (gene for Sigma-S, an alternate Sigma factor) . The levels of rsmB, pel-1, peh-1, celV, and hrpNEcc transcripts as well as production of pectate lyase, polygalacturonase, cellulase, protease, and HarpinEcc proteins were stimulated in GacS- and GacA- mutants by GacS+ or GacA+ plasmids, respectively . The GacA effect on exoenzyme genes and hrpNEcc was abrogated in E . carotovora subsp . carotovora mutants deficient in RsmA and RsmC or RsmA, RsmC, and rsmB RNA . The expression of lacZ transcriptional fusions of rsmB of Erwinia amylovora and Erwinia herbicola pv . gypsophilae was markedly reduced in a GacA- and a GacS- mutant of Pseudomonas syringae pv . syringae . Southern blot hybridization revealed the presence of gacA and gacS homologs in all tested strains of soft-rotting Erwinia spp . and several nonsoft-rotting Erwinia species such as E . amylovora, E . rhapontici, E . herbicola, E . stewartii, and E . herbicola pv . gypsophilae . These findings establish that the GacA-GacS system controls transcription of rsmB of E . carotovora subsp . carotovora, E . amylovora, and E . herbicola pv . gypsophilae and support the hypothesis that the effects of this two-component system on extracellular protein production in E . carotovora subsp . carotovora is mediated, at least in part, via the levels of rsmB transcripts. Extremophiles, 2001 Feb, 5(1), 35 - 44 Cloning of two pectate lyase genes from the marine Antarctic bacterium Pseudoalteromonas haloplanktis strain ANT/505 and characterization of the enzymes; Truong LV et al.; A marine Antarctic psychrotolerant bacterium (strain ANT/505), isolated from sea ice-covered surface water from the Southern Ocean, showed pectinolytic activity on citrus pectin agar . The sequencing of the 16S rRNA of isolate ANT/505 indicates a taxonomic affiliation to Pseudoalteromonas haloplanktis . The supernatant of this strain showed three different pectinolytic activities after growth on citrus pectin . By activity screening of a genomic DNA library of isolate ANT/505 in Escherichia coli, two different pectinolytic clones could be isolated . Subcloning and sequencing revealed two open reading frames (ORF) of 1,671 and 1,968 nt, corresponding to proteins of 68 and 75 kDa, respectively . The deduced amino acid sequence of the two ORFs showed homology to pectate lyases from Erwinia chrysanthemi and Aspergillus nidulans . The pectate lyases contain signal peptides of 17 and 26 amino acids that were correctly processed after overexpression in E . coli BL21 . Both enzymes were purified by anionic exchange chromatography . Maximal enzymatic activities for both pectate lyases were observed at 30 degrees C and a pH range of 9 to 10 . The Km values of both lyases for pectate and citrus pectin were 1 g l(-1) and 5 g l(-1), respectively . Calcium was required for activity on pectic substrates, whereas the addition of 1 mM ethylenediaminetetraacetic acid (EDTA) resulted in complete inhibition of the enzymes . These two enzymes represent the first pectate lyases isolated and characterized from a cold-adapted marine bacterium. Plant Physiol, 2001 Apr, 125(4), 2164 - 72 Evidence for the involvement of an oxidative stress in the initiation of infection of pear by Erwinia amylovora; Venisse JS et al.; Involvement of an oxidative burst, usually related to incompatible plant/pathogen interactions leading to hypersensitive reactions, was investigated with Erwinia amylovora, the causal agent of fire blight of Maloideae subfamily of Rosaceae, in interaction with pear (Pyrus communis; compatible situation) and tobacco (Nicotiana tabacum; incompatible situation) . As expected, this necrogenic bacterium induced in tobacco a sustained production of superoxide anion, lipid peroxidation, electrolyte leakage, and concomitant increases of several antioxidative enzymes (ascorbate peroxidases, glutathion reductases, glutathion-S-transferases, and peroxidases), in contrast to the compatible pathogen Pseudomonas syringae pv tabaci, which did not cause such reactions . In pear leaves, however, inoculations with both the disease- and the hypersensitive reaction-inducing bacteria (E . amylovora and P . syringae pv tabaci, respectively) resulted in superoxide accumulation, lipid peroxidation, electrolyte leakage, and enzyme induction at similar rates and according to equivalent time courses . The unexpected ability of E . amylovora to generate an oxidative stress even in compatible situation was linked to its functional hrp (for hypersensitive reaction and pathogenicity) cluster because an Hrp secretion mutant of the bacteria did not induce any plant response . It is suggested that E . amylovora uses the production of reactive oxygen species as a tool to provoke host cell death during pathogenesis to invade plant tissues . The bacterial exopolysaccharide could protect this pathogen against the toxic effects of oxygen species since a non-capsular mutant of E . amylovora induced locally the same responses than the wild type but was unable to further colonize the plant. Plant Physiol, 2001 Apr, 125(4), 1688 - 99 Study of the role of antimicrobial glucosinolate-derived isothiocyanates in resistance of Arabidopsis to microbial pathogens; Tierens KF et al.; Crude aqueous extracts from Arabidopsis leaves were subjected to chromatographic separations, after which the different fractions were monitored for antimicrobial activity using the fungus Neurospora crassa as a test organism . Two major fractions were obtained that appeared to have the same abundance in leaves from untreated plants versus leaves from plants challenge inoculated with the fungus Alternaria brassicicola . One of both major antimicrobial fractions was purified to homogeneity and identified by 1H nuclear magnetic resonance, gas chromatography/electron impact mass spectrometry, and gas chromatography/chemical ionization mass spectrometry as 4-methylsulphinylbutyl isothiocyanate (ITC) . This compound has previously been described as a product of myrosinase-mediated breakdown of glucoraphanin, the predominant glucosinolate in Arabidopsis leaves . 4-Methylsulphinylbutyl ITC was found to be inhibitory to a wide range of fungi and bacteria, producing 50% growth inhibition in vitro at concentrations of 28 microM for the most sensitive organism tested (Pseudomonas syringae) . A previously identified glucosinolate biosynthesis mutant, gsm1-1, was found to be largely deficient in either of the two major antimicrobial compounds, including 4-methylsulphinylbutyl ITC . The resistance of gsm1-1 was compared with that of wild-type plants after challenge with the fungi A . brassicicola, Plectosphaerella cucumerina, Botrytis cinerea, Fusarium oxysporum, or Peronospora parasitica, or the bacteria Erwinia carotovora or P . syringae . Of the tested pathogens, only F . oxysporum was found to be significantly more aggressive on gsm1-1 than on wild-type plants . Taken together, our data suggest that glucosinolate-derived antimicrobial ITCs can play a role in the protection of Arabidopsis against particular pathogens. Int J Pharm, 2001 Apr 17, 217(1-2), 215 - 24 The effect of polysialylation on the immunogenicity and antigenicity of asparaginase: implication in its pharmacokinetics; Fernandes AI et al.; Erwinia carotovora L-asparaginase was conjugated via the epsilon-amino groups of its lysine residues with colominic acid (CA) (polysialic acid) of average molecular mass of 10 kDa by reductive amination in the presence of NaCNBH3 . Polysialylation using 50-, 100- and 250-fold molar excess CA relative to the enzyme led to an increasing proportion of the enzyme's in-amino groups (5.8, 7.6 and 11.3%, respectively) being conjugated to CA . Polysialylated and native (intact) asparaginase were used to immunize mice intravenously . Results (total IgG immune responses) indicate that all preparations elicited antibody production against the enzyme moiety but not against the CA of the conjugates . Moreover, antibody titres appeared highest for the native enzyme and were generally reduced as the degree of polysialylation increased . In other experiments mice pre-immunized with native or polysialylated asparaginase, with anti-asparaginase antibodies in their blood, were injected intravenously with the corresponding enzyme preparations . Results revealed that polysialylation reduces the antigenicity of asparaginase thus leading to circulatory half-lives (t 1/2 beta) that were 3-4-fold greater than that of the native enzyme, and similar to those observed in naive, non-immunized mice . Our data suggest that polysialylation of therapeutic enzymes and other proteins may be useful in maintaining their pharmacokinetics in individuals with antibodies to the therapeutic proteins as a result of chronic treatment. Carbohydr Res, 2001 Mar 9, 331(1), 59 - 67 Pyruvated galactose and oligosaccharides from Erwinia chrysanthemi Ech6 extracellular polysaccharide; Yang BY et al.; The acidic extracellular polysaccharide of Ech6 was depolymerized by fuming HCl . The pyruvated sugars were isolated and characterized by methods that included a combination of low-pressure gel-filtration and high-pH anion-exchange chromatographies, methylation linkage analyses, mass (GC-MS and MALDI-TOF MS) and 1H NMR (1D and 2D) spectroscopies . The following pyruvated sugars were obtained: 4,6-O-(1-carboxyethylidene)-D-Galp; 4,6-O-(1-carboxyethylidene)- alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3)-D-Galp; 4,6-O-(1-carboxyethylidene)-alpha-D-Galp-(1-->4)-alpha-D-GlcAp- (1-->3)-alpha-D-Galp-(1-->3)-L-Fucp; 4,6-O-(1-carboxyethylidene)-alpha-D-Galp-(1-->4)-beta-D-GlcAp-(1-->3) -alpha-D-Galp-(1-->3)-L-{beta-D-Glcp-(1-->4)}-Fucp . These oligosaccharides present potential haptenes for the development of specific antibodies and confirm the partial structure proposed previously for the extracellular polysaccharide from Erwinia chrysanthemi Ech6 {Yang, B . Y.; Gray, J . S . S.; Montgomery, R . Int . J . Biol . Macromol., 1994, 16, 306-312}. Int J Hyg Environ Health, 2001 Mar, 203(3), 201 - 4 Hospital infestation by the cluster fly, Pollenia rudis sensu stricto Fabricius 1794 (Diptera: Calliphoridae), and its possible role in transmission of bacterial pathogens in Germany; Faulde M et al.; The potential of the cluster fly, Pollenia rudis sensu stricto, to transmit bacterial pathogens was investigated during a mass infestation that took place in a German hospital . Cluster flies were individually examined for mesophilic bacteria carried on the exoskeleton . Bacterial growth could only be detected by using the enrichment culture technique to increase sensitivity, but not by direct intoculation of fly samples to agar plates . All 50 cluster fly samples that were tested carried opportunistic aerobic mesophilic Bacillus spp., whereas 41 fly samples were positive for Erwinia spp., 16 samples for Erwinia amylovara, 24 samples for Stenotrophomonas maltophilia, and 4 samples for Flavobacterium odoratum . Staphylococcus lugdunensis and Pseudomonas aeruginosa were found in 5 samples . No bacteriologically sterile cluster fly samples were obtained . The whole bacterial pattern found on P . rudis s . s . is known for its potential to cause opportunistic and/or nosocomial infections in humans . The results obtained led to the assumption that mass infestations of cluster flies occurring in sensitive areas, especially in hospitals, may cause a low, but not neglectable health threat due to mechanical transmission of bacterial pathogens. Mol Plant Microbe Interact, 2001 Mar, 14(3), 431 - 6 Genetic organization of the hrp gene cluster and dspAE/BF operon in Erwinia herbicola pv . gypsophilae; Mor H et al.; Erwinia herbicola pv . gypsophilae induces gall formation in gypsophila that is dependent on the existence of a pathogenicity plasmid (pPATHEhg) . We previously demonstrated the presence of several hrp genes on this plasmid . By employing transposon mutagenesis and sequencing, a functional hrp gene cluster on the pPATHEhg has now been characterized completely . The hrp genes of E . herbicola pv . gypsophilae are remarkably similar to and colinear with those of Erwinia amylovora and Pantoea stewartii and generally showed 60 to 90% nucleotide or deduced amino acid identity . E . herbicola pv . gypsophilae, however, lacks hrpW, which is present in E . amylovora . Additionally, E . herbicola pv . gypsophilae mutants deficient in harpin production retained pathogenicity and were slightly reduced in their ability to elicit a hypersensitive response (HR) in tobacco . The "disease specific" region, dspA/EB/F, exhibited 60 to 74% identity with the dspA/EB/F loci of E . amylovora and P . stewartii, respectively . Mutations in dspA/E abolished pathogenicity of E . herbicola pv . gypsophilae but not HR elicitation on tobacco . Inactivation of HrpL reduced plant-induced transcription of dspA/E by three orders, indicating Hrp-dependent regulation. Mol Plant Microbe Interact, 2001 Mar, 14(3), 386 - 93 Relative effects on virulence of mutations in the sap, pel, and hrp loci of Erwinia chrysanthemi; Lopez-Solanilla E et al.; We constructed strains of Erwinia chrysanthemi EC16 with multiple mutations involving three virulence systems in this bacterium, namely pel (coding for the major pectate lyases pelABCE), hrp (hypersensitive response and pathogenicity), and sap (sensitivity to antimicrobial peptides) . The relative effects on virulence of those mutations have been analyzed on potato tubers and chicory leaves . In potato tubers, the sap mutation (BT105) had a greater effect in the reduction of the virulence than the pel (CUCPB5006) and hrp (CUCPB5039) mutations . This reduction was similar to that observed in the pel-hrp double mutant (CUCPB5037) . The analysis of the strains affected in Pel-Sap (BT106), Hrp-Sap (BT107), and Pel-Hrp-Sap (BT108) suggested that the effects of these mutations are additive . In chicory leaves, the mutation in the sap locus appeared to have a greater effect than in potato tubers . The competitive indices of strains BT105, UM1005 (Pel-), CUCPB5039, and CUCPB5037 have been estimated in vivo and in vitro . These results indicate that the mutation in the hrp locus can be complemented in vivo by coinfection, whereas the mutations in pel and sap cannot. J Bacteriol, 2001 Apr, 183(8), 2425 - 30 Cloning and characterization of the gene cluster for palatinose metabolism from the phytopathogenic bacterium Erwinia rhapontici; Bornke F et al.; Erwinia rhapontici is able to convert sucrose into isomaltulose (palatinose, 6-O-alpha-D-glucopyranosyl-D-fructose) and trehalulose (1-O-alpha-D-glucopyranosyl-D-fructose) by the activity of a sucrose isomerase . These sucrose isomers cannot be metabolized by plant cells and most other organisms and therefore are possibly advantageous for the pathogen . This view is supported by the observation that in vitro yeast invertase activity can be inhibited by palatinose, thus preventing sucrose consumption . Due to the lack of genetic information, the role of sucrose isomers in pathogenicity has not been evaluated . Here we describe for the first time the cloning and characterization of the palatinose (pal) genes from Erwinia rhapontici . To this end, a 15-kb chromosomal DNA fragment containing nine complete open reading frames (ORFs) was cloned . The pal gene products of Erwinia rhapontici were shown to be homologous to proteins involved in uptake and metabolism of various sugars from other microorganisms . The palE, palF, palG, palH, palK, palQ, and palZ genes were oriented divergently with respect to the palR and palI genes, and sequence analysis suggested that the first set of genes constitutes an operon . Northern blot analysis of RNA extracted from bacteria grown under various conditions implies that the expression of the palI gene and the palEFGHKQZ genes is oppositely regulated at the transcriptional level . Genes involved in palatinose uptake and metabolism are down regulated by sucrose and activated by palatinose . Palatinose activation is inhibited by sucrose . Functional expression of palI and palQ in Escherichia coli revealed sucrose isomerase and palatinase activity, respectively. J Invertebr Pathol, 2001 Feb, 77(2), 129 - 37 Growth and transmission of gut bacteria in the Western flower thrips, Frankliniella occidentalis; de Vries EJ et al.; The Western flower thrips (Frankliniella occidentalis), a polyphagous insect with global distribution, has a permanent association with a near Erwinia species TAC bacterium in its hindgut . Since this bacterium is able to grow outside the thrips, it is a facultative symbiont that is not completely dependent on the host . In this study we address the question of how the association is maintained and how bacteria are transmitted to newly hatched thrips larvae . Bacteria are passed on to new thrips via the food source . No evidence was found for vertical transmission from mother to offspring via the egg . Gut bacteria show unlimited growth during the larval (feeding) stages, and in the second instar stage 100% of the larvae become infected with high numbers of bacteria . In the prepupal and pupal stage, the number of bacteria declines, but increases again during the adult phase . A method to rear aposymbiotic (bacteria-free) thrips is described which enables studies on the impact of bacteria on the fitness of thrips . EMBO Rep, 2001 Mar, 2(3), 244 - 8 An inner membrane platform in the type II secretion machinery of Gram-negative bacteria; Py B et al.; The type II secretion machinery allows most Gram-negative bacteria to deliver virulence factors into their surroundings . We report that in Erwinia chrysanthemi, GspE (the putative NTPase), GspF, GspL and GspM constitute a complex in the inner membrane that is presumably used as a platform for assembling other parts of the secretion machinery . The GspE-GspF-GspL-GspM complex was demonstrated by two methods: (i) co-immunoprecipitation of GspE-GspF-GspL with antibodies raised against either GspE or GspF; (ii) interactions in the yeast two-hybrid system between GspF and GspE, GspF and GspL, GspL and GspM . GspL was found to have an essential role in complex formation . We propose a model in which the GspE-GspF-GspL-GspM proteins constitute a building block within the secretion machinery on top of which another building block, referred to as a pseudopilus, assembles . By analogy, we predict that a similar platform is required for the biogenesis of the type IV pilus. Mol Microbiol, 2001 Feb, 39(4), 960 - 72 SoxR-dependent response to oxidative stress and virulence of Erwinia chrysanthemi: the key role of SufC, an orphan ABC ATPase; Nachin L et al.; Erwinia chrysanthemi causes soft-rot disease in a great variety of plants . In addition to the depolymerizing activity of plant cell wall-degrading enzymes, iron acquisition and resistance to oxidative stress contribute greatly to the virulence of this pathogen . Here, we studied the pin10 locus originally thought to encode new virulence factors . The sequence analysis revealed six open reading frames that were homologous to the Escherichia coli sufA, sufB, sufC, sufD, sufS and sufE genes . Sequence similarity searching predicted that (i) SufA, SufB, SufD, SufS and SufE proteins are involved in iron metabolism and possibly in Fe-S cluster assembly; and (ii) SufC is an ATPase of an ABC transporter . The reverse transcription-polymerase chain reaction procedure showed that the sufABCDSE genes constitute an operon . Expression of a sufB:uidA fusion was found to be induced in iron-deficient growth conditions and to be repressed by the iron-sensing Fur repressor . Each of the six suf genes was inactivated by the insertion of a cassette generating a non-polar mutation . The intracellular iron level in the sufA, sufB, sufC, sufS and sufE mutants was higher than in the wild type, as assessed by increased sensitivity to the iron-activated antibiotic streptonigrin . In addition, inactivation of sufC and sufD led to increased sensitivity to paraquat . Virulence tests showed that sufA and sufC mutants exhibited reduced ability to cause maceration of chicory leaves, whereas a functional sufC gene was necessary for the bacteria to cause systemic invasion of Saintpaulia ionantha . The E . coli sufC homologue was inactivated by reverse genetic . This mutation was found to modify the soxR-dependent induction of soxS gene expression . We discuss the possibility that SufC is a versatile ATPase that can associate either with the other Suf proteins to form a Fe-S cluster-assembling machinery or with membrane proteins encoded elsewhere in the chromosome to form an Fe-S ABC exporter . Overall, these results stress the importance of the connection between iron metabolism and oxidative stress during the early steps of infection by E . chrysanthemi. Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3454 - 9 Heterogeneous transcription of an indoleacetic acid biosynthetic gene in Erwinia herbicola on plant surfaces; Brandl MT et al.; We investigated the spatial pattern of expression of ipdC, a plant inducible gene involved in indoleacetic acid biosynthesis in Erwinia herbicola, among individual cells on plants to gain a better understanding of the role of this phenotype in the epiphytic ecology of bacteria and the factors involved in the regulation of ipdC . Nonpathogenic E . herbicola strain 299R harboring a transcriptional fusion of ipdC to gfp was inoculated onto bean plants, recovered from individual leaves 48 h after inoculation, and subjected to fluorescence in situ hybridization using a 16S rRNA oligonucleotide probe specific to strain 299R . Epifluorescence images captured through a rhodamine filter were used to distinguish the 5carboxytetramethylrhodamine-labeled cells of strain 299R from other leaf microflora . Quantification of the green fluorescence intensity of individual cells by analysis of digital images revealed that about 65% of the 299R cells recovered from bean leaves had higher ipdC expression than in culture . Additionally, 10% of the cells exhibited much higher levels of green fluorescence than the median fluorescence intensity, indicating that they are more heterogeneous with respect to ipdC expression on plants than in culture . Examination of 299R cells in situ on leaf surfaces by confocal laser scanning microscopy after fluorescence in situ hybridization of cells on leaf samples showed that even cells that were in close proximity exhibited dramatically different green fluorescence intensities, and thus, were in a physical or chemical microenvironment that induced differential expression of ipdC. Proc Natl Acad Sci U S A, 2001 Mar 13, 98(6), 3446 - 53 Appetite of an epiphyte: quantitative monitoring of bacterial sugar consumption in the phyllosphere; Leveau JH et al.; We report here the construction, characterization, and application of a bacterial bioreporter for fructose and sucrose that was designed to monitor the availability of these sugars to microbial colonizers of the phyllosphere . Plasmid pP(fruB)-gfp{AAV} carries the Escherichia coli fruB promoter upstream from the gfp{AAV} allele that codes for an unstable variant of green fluorescent protein (GFP) . In Erwinia herbicola, this plasmid brings about the accumulation of GFP fluorescence in response to both fructose and sucrose . Cells of E . herbicola (pP(fruB)-gfp{AAV}) were sprayed onto bean plants, recovered from leaves at various time intervals after inoculation, and analyzed individually for GFP content by quantitative analysis of digital microscope images . We observed a positive correlation between single-cell GFP accumulation and ribosomal content as determined by fluorescence in situ hybridization, indicating that foliar growth of E . herbicola occurred at the expense of fructose and/or sucrose . One hour after inoculation, nearly all bioreporter cells appeared to be actively engaged in fructose consumption . This fraction dropped to approximately 11% after 7 h and to approximately 1% a day after inoculation . This pattern suggests a highly heterogeneous availability of fructose to individual E . herbicola cells as they colonize the phyllosphere . We estimated that individual cells were exposed to local initial fructose abundances ranging from less than 0.15 pg fructose to more than 4.6 pg. J Bacteriol, 2001 Apr, 183(7), 2249 - 58 SirA orthologs affect both motility and virulence; Goodier RI et al.; The sirA gene of Salmonella enterica serovar Typhimurium encodes a two-component response regulator of the FixJ family that has a positive regulatory influence on the expression of type III secretion genes involved with epithelial cell invasion and the elicitation of bovine gastroenteritis . SirA orthologs in Pseudomonas, Vibrio, and Erwinia control the expression of distinct virulence genes in these genera, but an evolutionarily conserved target of SirA regulation has never been identified . In this study we tested the hypothesis that sirA may be an ancient member of the flagellar regulon . We examined the effect of a sirA mutation on transcriptional fusions to flagellar promoters (flhD, fliE, fliF, flgA, flgB, fliC, fliD, motA, and fliA) while using fusions to the virulence gene sopB as a positive control . SirA had only small regulatory effects on all fusions in liquid medium (less than fivefold) . However, in various types of motility agar plates, sirA was able to activate a sopB fusion by up to 63-fold while repressing flagellar fusions by values exceeding 100-fold . Mutations in the sirA orthologs of Escherichia coli, Vibrio cholerae, Pseudomonas fluorescens, and Pseudomonas aeruginosa result in defects in either motility or motility gene regulation, suggesting that control of flagellar regulons may be an evolutionarily conserved function of sirA orthologs . The implications for our understanding of virulence gene regulation in the gamma Proteobacteria are discussed. J Clin Oncol, 2001 Mar 1, 19(5), 1297 - 303 Effect of protracted high-dose L-asparaginase given as a second exposure in a Berlin-Frankfurt-Münster-based treatment: results of the randomized 9102 intermediate-risk childhood acute lymphoblastic leukemia study--a report from the Associazione Italiana Ematologia Oncologia Pediatrica; Rizzari C et al.; PURPOSE: To assess in a randomized study the therapeutic effect of the addition of high-dose L-asparaginase (HD ASP) in the context of a Berlin-Frankfurt-Munster (BFM)-based chemotherapy regimen for intermediate risk (IR) childhood acute lymphoblastic leukemia (ALL) . PATIENTS AND METHODS: From March 1991 to April 1995, a total of 705 patients, with 59% of the cohort of patients fewer than 15 years old, with newly diagnosed non-B ALL, enrolled onto the Associazione Italiana Ematologia Oncologia Pediatrica (AIEOP) ALL-91 study, were assigned to the IR group . Patients in remission at the beginning of the reinduction phase were randomized either to the standard treatment (SD ASP arm) or the experimental treatment (HD ASP arm; weekly intramuscular administration of HD ASP 25,000 IU/m(2) repeated for a total of 20 weeks) . Most of the patients (90%) were treated with Erwinia chrysanthemi L-asparaginase product . RESULTS: Among the 610 patients randomized to the SD ASP arm (n = 322) or to the HD ASP arm (n = 288), relapse occurred at a median time of 24 months after randomization in 76 (24%) and in 64 children (22%), respectively . Most of the relapses occurred in the marrow (100 isolated, 21 combined) . There was no significant difference between the disease-free survival in the two treatment arms (P =.64), with estimated values at 7 years from randomization of 72.4% (SE 3.1) v 75.7% (SE 2.6) in the SD ASP and HD ASP arms, respectively . CONCLUSION: No advantage was observed for IR ALL children treated with BFM-based intensive chemotherapy who received protracted E chrysanthemi HD ASP during reinduction and the early continuation phase. Appl Environ Microbiol, 2001 Mar, 67(3), 1308 - 17 Biological sensor for sucrose availability: relative sensitivities of various reporter genes; Miller WG et al.; A set of three sucrose-regulated transcriptional fusions was constructed . Fusions p61RYTIR, p61RYlac, and p61RYice contain the scrR sucrose repressor gene and the promoterless gfp, lacZ, and inaZ reporter genes, respectively, fused to the scrY promoter from Salmonella enterica serovar Typhimurium . Cells of Erwinia herbicola containing these fusions are induced only in media amended with sucrose, fructose, or sorbose . While a large variation in sucrose-dependent reporter gene activity was observed in cells harboring all gene fusions, fusions to the inaZ reporter gene yielded a much wider range of activity and were responsive to lower levels of sucrose than either lacZ or gfp . The lacZ reporter gene was found to be more efficient than gfp, requiring approximately 300-fold fewer cells for a detectable response over all concentrations of sucrose . Similarly, inaZ was found to be more efficient than lacZ, requiring 30-fold fewer cells at 1.45 microM sucrose and 6,100-fold fewer cells at 29 mM sucrose for a quantifiable response . The fluorescence of individual cells containing p61RYTIR was quantified following epifluorescence microscopy in order to relate the fluorescence exhibited by populations of cells in batch cultures with that of individual cells in such cultures . While the mean fluorescence intensity of a population of individual cells increased with increasing concentrations of sucrose, a wide range of fluorescence intensity was seen among individual cells . For most cultures the distribution of fluorescence intensity among individual cells was log-normally distributed, but cells grown in intermediate concentrations of sucrose exhibited two distinct populations of cells, one having relatively low fluorescence and another with much higher fluorescence . When cells were inoculated onto bean leaves, whole-cell ice nucleation and gfp-based biological sensors for sucrose each indicated that the average concentration of sucrose on moist leaf surfaces was about 20 microM . Importantly, the variation in green fluorescent protein fluorescence of biosensor cells on leaves suggested that large spatial variations in sugar availability occur on leaves. Appl Environ Microbiol, 2001 Mar, 67(3), 1198 - 209 Acyl-homoserine lactone production is more common among plant-associated Pseudomonas spp . than among soilborne Pseudomonas spp; Elasri M et al.; A total of 137 soilborne and plant-associated bacterial strains belonging to different Pseudomonas species were tested for their ability to synthesize N-acyl-homoserine lactones (NAHL) . Fifty-four strains synthesized NAHL . Interestingly, NAHL production appears to be more common among plant-associated than among soilborne Pseudomonas spp . Indeed, 40% of the analyzed Pseudomonas syringae strains produced NAHL which were identified most often as the short-chain NAHL, N-hexanoyl-L-homoserine lactone, N-(3-oxo-hexanoyl)-homoserine lactone, and N-(3-oxo-octanoyl)-L-homoserine lactone (no absolute correlation between genomospecies of P . syringae and their ability to produce NAHL could be found) . Six strains of fluorescent pseudomonads, belonging to the species P . chlororaphis, P . fluorescens, and P . putida, isolated from the plant rhizosphere produced different types of NAHL . In contrast, none of the strains isolated from soil samples were shown to produce NAHL . The gene encoding the NAHL synthase in P . syringae pv . maculicola was isolated by complementation of an NAHL-deficient Chromobacterium mutant . Sequence analysis revealed the existence of a luxI homologue that we named psmI . This gene is sufficient to confer NAHL synthesis upon its bacterial host and has strong homology to psyI and ahlI, two genes involved in NAHL production in P . syringae pv . tabaci and P . syringae pv . syringae, respectively . We identified another open reading frame that we termed psmR, transcribed convergently in relation to psmI and partly overlapping psmI; this gene encodes a putative LuxR regulatory protein . This gene organization, with luxI and luxR homologues facing each other and overlapping, has been found so far only in the enteric bacteria Erwinia and Pantoea and in the related species P . syringae pv . tabaci. Environ Microbiol, 2000 Apr, 2(2), 203 - 15 hexA of Erwinia carotovora ssp . carotovora strain Ecc71 negatively regulates production of RpoS and rsmB RNA, a global regulator of extracellular proteins, plant virulence and the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone; Mukherjee A et al.; The soft-rotting bacterium, Erwinia carotovora ssp . carotovora (E . c . carotovora), produces an array of extracellular enzymes (= exoenzymes), including pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel) and protease (Prt), as well as HarpinEcc, the elicitor of hypersensitive reaction (HR) . The production of these exoenzymes and HarpinEcc responds to plant products and the quorum-sensing signal {N-(3-oxohexanoyl)-L-homoserine lactone; OHL} and is subject to both transcriptional and post-transcriptional regulation . hexA of E . c . carotovora strain Ecc71 (hereafter hexA71), like that of another E . c . carotovora strain, negatively controls the production of exoenzymes, OHL and virulence in E . c . carotovora strain Ecc71 . In addition to exoenzymes, HexA71 negatively regulates the expression of hrpNEcc, the structural gene for HarpinEcc . Exoenzyme overproduction is abolished by OHL deficiency in a HexA- and Ohll- double mutant, indicating that HexA and OHL are components of a common regulatory pathway controlling exoenzyme production . HexA71 negatively affects RpoS, as the levels of this alternative sigma factor are higher in the HexA- mutant than in the HexA+ strain . However, a HexA- and RpoS double mutant produces higher levels of exoenzymes and transcripts of pel-1, peh-1 and celVgenes than the HexA- and RpoS+ parent . Thus, the elevated levels of RpoS protein in the HexA- mutant do not account for exoenzyme overproduction . The following evidence associates for the first time the phenotypic changes in the HexA mutant to overproduction of rsmB RNA, a global regulator of exoenzymes, HarpinEcc, OHL and secondary metabolites . Analyses of rsmB transcripts and expression of an rsmB-lacZoperon fusion in E . c . carotovora strain Ecc71 revealed that HexA71 negatively regulates transcription of rsmB . Multiple copies of hexA71+ DNA suppress various phenotypes, including exoenzyme production in E . c . carotovora strain Ecc71, and concomitantly inhibit the production of rsmB, pel-1, peh-1, celV and hrpNEcc transcripts . Multiple copies of rsmB+ DNA, on the other hand, stimulate exoenzyme production by relieving the negative effects of a chromosomal copy of hexA+ . The occurrence of hexA homologues and the negative effect of the dosage of hexA71 DNA on rsmB transcripts were also detected in other E . c . carotovora strains as well as Erwinia carotovora atroseptica and Erwinia carotovora betavasculorum . Extrapolating from the findings with LrhA, the Escherichia coli homologue of HexA, and the presence of sprE homologues in E . carotovora subspecies, we propose that HexA71 controls several regulatory pathways in E . carotovora including rsmB transcription and the production of SprEEcc which, in turn, affects RpoS levels . A model is presented that integrates the findings presented here and our current knowledge of the major regulatory network that controls exoprotein production in soft-rotting Erwinia carotovora subspecies. Acta Crystallogr D Biol Crystallogr, 2001 Mar, 57(Pt 3), 369 - 77 Structures of two highly homologous bacterial L-asparaginases: a case of enantiomorphic space groups; Jaskolski M et al.; Quasi-enantiomorphic crystals of the Y25F mutant of Escherichia coli L-asparaginase and of the native Erwinia chrysanthemi L-asparaginase were obtained in the hexagonal space groups P6(5)22 and P6(1)22, respectively . The structures of these highly homologous enzymes were solved by molecular replacement and were refined with data extending to 2.2-2.5 A . These structures were compared with each other, as well as with other L-asparaginase structures previously observed with different crystal packing . It is concluded that the observed phenomenon, which is rare, was most likely to have arisen by chance. J Bacteriol, 2001 Mar, 183(6), 2093 - 100 Functional characterization of a novel xylanase from a corn strain of Erwinia chrysanthemi; Hurlbert JC et al.; A beta-1,4-xylan hydrolase (xylanase A) produced by Erwinia chrysanthemi D1 isolated from corn was analyzed with respect to its secondary structure and enzymatic function . The pH and temperature optima for the enzyme were found to be pH 6.0 and 35 degrees C, with a secondary structure under those conditions that consists of approximately 10 to 15% alpha-helices . The enzyme was still active at temperatures higher than 40 degrees C and at pHs of up to 9.0 . The loss of enzymatic activity at temperatures above 45 degrees C was accompanied by significant loss of secondary structure . The enzyme was most active on xylan substrates with low ratios of xylose to 4-O-methyl-D-glucuronic acid and appears to require two 4-O-methyl-D-glucuronic acid residues for substrate recognition and/or cleavage of a beta-1,4-xylosidic bond . The enzyme hydrolyzed sweetgum xylan, generating products with a 4-O-methyl-glucuronic acid-substituted xylose residue one position from the nonreducing terminus of the oligoxyloside product . No internal cleavages of the xylan backbone between substituted xylose residues were observed, giving the enzyme a unique mode of action in the hydrolysis compared to all other xylanases that have been described . Given the size of the oligoxyloside products generated by the enzyme during depolymerization of xylan substrates, the function of the enzyme may be to render substrate available for other depolymerizing enzymes instead of producing oligoxylosides for cellular metabolism and may serve to produce elicitors during the initiation of the infectious process. Leukemia, 2000 Dec, 14(12), 2257 - 66 Long-term results of three randomized trials (58831, 58832, 58881) in childhood acute lymphoblastic leukemia: a CLCG-EORTC report . Children Leukemia Cooperative Group; Vilmer E et al.; We present here the long-term results of three randomized clinical trials conducted on children with newly diagnosed acute lymphoblastic leukemia (ALL) between 1983 and 1998 by the Children Leukemia Cooperative Group (CLCG) from EORTC . In study 58831/32, the overall event-free survival (EFS) rates (+/- s.e.) at 6 and 10 years were 66% +/- 1.8% and 65% +/- 1.8%, respectively, and the risk of isolated central nervous system (CNS) relapse was 6% +/- 1% and 7% +/- 1%, respectively . In patients with a standard risk of relapse the omission of cyclophosphamide had no adverse effect on disease-free survival rates at 10 years (trial 58831) . In medium- and high-risk patients the omission of radiotherapy did not increase the risk of CNS or systemic relapse (trial 58832) . In study 58881 (1989-1998) the overall EFS rate at 8 years was 68.4% +/- 1.2% and the risk of isolated CNS relapse was 4.2%+/-0.5% . In this trial which adressed three randomized questions, the following results were obtained: the combination of cytarabine at high doses with methotrexate at high doses during interval therapy did not improve prognosis . The addition of 6-mercaptopurine iv during maintenance increased the risk of late relapse . E . coli asparaginase was more toxic and has a higher efficacy than Erwinia asparaginase . Leukocyte counts >100 x 10(9)/l, specific genetic abnormalities, a poor initial response to steroids or a high level of minimal residual disease at early time points were consistently associated with an adverse prognosis in the 58881 trial. Environ Microbiol, 1999 Dec, 1(6), 535 - 47 The hexY genes of Erwinia carotovora ssp . carotovora and ssp . atroseptica encode novel proteins that regulate virulence and motility co-ordinately; Shih YL et al.; Mutations located in a new gene, hexY, in Erwinia carotovora ssp . carotovora (Ecc) and ssp . atroseptica (Eca) cause strong upregulation of production of exoenzyme virulence factors and motility . The hexY gene encodes a novel 14.4 kDa protein with no known homologues . The hexY mRNA transcript has an unusually long (525bp) 5' untranslated region, which may be important for post-transcriptional regulation . An elevated level of transcription of two exoenzyme genes, pelCand celV, was observed in the HexY mutant background . The levels of cellulase and protease in a HexY mutant were independent of the presence of PGA, suggesting a role for HexY in the induction of these enzymes seen upon PGA addition . Electron microscopy revealed that HexY cells were hyperflagellated, perhaps contributing to the hypermotility phenotype of this mutant . The HexY mutant M5 exhibited enhanced maceration capacity on potato tubers . Therefore, the hexY gene and its gene product may define another level of regulation of virulence determinants in Ecc and Eca. Cell Microbiol, 1999 Sep, 1(2), 131 - 41 Visualization of harpin secretion in planta during infection of apple seedlings by Erwinia amylovora; Perino C et al.; Erwinia amylovora is a Gram-negative pathogenic bacterium that infects pear and apple trees as well as other plants from the Rosaceae family . E . amylovora pathogenicity is dependent on a functional Hrp type III secretion system . Harpin, a protein playing a major role in virulence, has been shown to be exported in vitro via the type III secretion apparatus . The data presented here focus on harpin detection in planta after infection of apple seedlings with the wild-type strain CFPB 1430 . Using a specific harpin antiserum, harpin was not detected inside the host plant cells, but was found associated with the bacteria and secreted . The extracellular localization of harpin is in agreement with the physiological effects induced by purified harpin when applied as an exogenous elicitor . Harpin was not found associated with the host plant cell wall, a result that weakens its postulated role in cell wall loosening . A differential labelling was observed at the bacterial level: for some bacteria, harpin was exclusively cytoplasmic, whereas in others, it appeared as small patches over the bacterial outer membrane or associated with extracellular linear structures . All the bacteria present within the same area were similarly labelled, suggesting co-ordination in the secretion process . All observations suggest that harpin is synthesized in the bacterial cytoplasm and that secretion occurs from this cytoplasmic pool upon sensing of a plant or bacterial signal. J Microbiol Methods, 2001 Feb 1, 44(1), 69 - 77 The use of 16S and 16S-23S rDNA to easily detect and differentiate common Gram-negative orchard epiphytes; Jeng RS et al.; The identification of Gram-negative pathogenic and non-pathogenic bacteria commonly isolated from an orchard phylloplane may result in a time consuming and tedious process for the plant pathologist . The paper provides a simple "one-step" protocol that uses the polymerase chain reaction (PCR) to amplify intergenic spacer regions between 16S and 23S genes and a portion of 16S gene in the prokaryotic rRNA genetic loci . Amplified 16S rDNA, and restriction fragment length polymorphisms (RFLP) following EcoRI digestion produced band patterns that readily distinguished between the plant pathogen Erwinia amylovora (causal agent of fire blight in pear and apple) and the orchard epiphyte Pantoea agglomerans (formerly E . herbicola) . The amplified DNA patterns of 16S-23S spacer regions may be used to differentiate E . amylovora at the intraspecies level . Isolates of E . amylovora obtained from raspberries exhibited two major fragments while those obtained from apples showed three distinct amplified DNA bands . In addition, the size of the 16S-23S spacer region differs between Pseudomonas syringae and Pseudomonas fluorescens . The RFLP pattern generated by HaeIII digestion may be used to provide a rapid and accurate identification of these two common orchard epiphytes. J Mol Biol, 2001 Jan 26, 305(4), 951 - 60 Three-dimensional structure of Erwinia chrysanthemi pectin methylesterase reveals a novel esterase active site; Jenkins J et al.; Most structures of neutral lipases and esterases have been found to adopt the common alpha/beta hydrolase fold and contain a catalytic Ser-His-Asp triad . Some variation occurs in both the overall protein fold and in the location of the catalytic triad, and in some enzymes the role of the aspartate residue is replaced by a main-chain carbonyl oxygen atom . Here, we report the crystal structure of pectin methylesterase that has neither the common alpha/beta hydrolase fold nor the common catalytic triad . The structure of the Erwinia chrysanthemi enzyme was solved by multiple isomorphous replacement and refined at 2.4 A to a conventional crystallographic R-factor of 17.9 % (R(free) 21.1 %) . This is the first structure of a pectin methylesterase and reveals the enzyme to comprise a right-handed parallel beta-helix as seen in the pectinolytic enzymes pectate lyase, pectin lyase, polygalacturonase and rhamnogalacturonase, and unlike the alpha/beta hydrolase fold of rhamnogalacturonan acetylesterase with which it shares esterase activity . Pectin methylesterase has no significant sequence similarity with any protein of known structure . Sequence conservation among the pectin methylesterases has been mapped onto the structure and reveals that the active site comprises two aspartate residues and an arginine residue . These proposed catalytic residues, located on the solvent-accessible surface of the parallel beta-helix and in a cleft formed by external loops, are at a location similar to that of the active site and substrate-binding cleft of pectate lyase . The structure of pectin methylesterase is an example of a new family of esterases . Cryobiology, 2000 Nov, 41(3), 195 - 203 Characterization and properties of intracellular proteins after cold acclimation of the ice-nucleating bacterium Pantoea agglomerans (Erwinia herbicola) IFO12686; Koda N et al.; The ice-nucleating bacterium Pantoea agglomerans (Erwinia herbicola) IFO12686 (INA(+)) responds to a decrease in temperature by the induction of proteins . The pattern of protein bands from strain IFO12686 following a shift in temperature from 30 to 12 degrees C could be divided into four major groups: (1) increasing protein bands, (2) decreasing protein bands, (3) increasing--decreasing protein bands, and (4) almost constant protein bands . We identified a cryoprotective function in the increasing protein band found in strain IFO12686 . The increasing protein bands that followed a reduction in temperature were considered to have an important role in cold acclimation or adaptation . We showed that these proteins possessed cryoprotective activity when tested against the freeze-labile enzyme lactate dehydrogenase . The strain IFO12686 had greater cryotolerance than Pa . agglomerans IAM1595 (INA(-)), and the degree of cryotolerance was increased by cold acclimation . Plant Physiol, 2001 Feb, 125(2), 1126 - 38 Characterization of maize cytochrome P450 monooxygenases induced in response to safeners and bacterial pathogens; Persans MW et al.; Plants use a diverse array of cytochrome P450 monooxygenases in their biosynthetic and detoxification pathways . To determine the extent to which various maize P450s are induced in response to chemical inducers, such as naphthalic anhydride (NA), triasulfuron (T), phenobarbital, and bacterial pathogens (Erwinia stuartii, Acidovorax avenae), we have analyzed the response patterns of seven P450 transcripts after treatment of seedlings with these inducers . Each of these P450 transcripts has distinct developmental, tissue-specific, and chemical cues regulating their expression even when they encode P450s within the same biosynthetic pathway . Most notably, the CYP71C1 and CYP71C3 transcripts, encoding P450s in the DIMBOA biosynthetic pathway, are induced to the same level in response to wounding and NA treatment of younger seedlings and differentially in response to NA/T treatment of younger seedlings and NA and NA/T treatment of older seedlings . NA and T induce expression of both CYP92A1 and CYP72A5 transcripts in older seedling shoots, whereas phenobarbital induces CYP92A1 expression in older seedling shoots and highly induces CYP72A5 expression in young and older seedling roots . Expressed sequence tag (EST) 6c06b11 transcripts, encoding an undefined P450 activity, are highly induced in seedling shoots infected with bacterial pathogens. J Bacteriol, 2001 Feb, 183(4), 1346 - 58 Subset of hybrid eukaryotic proteins is exported by the type I secretion system of Erwinia chrysanthemi; Palacios JL et al.; Erwinia chrysanthemi exports degradative enzymes by using a type I protein secretion system . The proteases secreted by this system lack an N-terminal signal peptide but contain a C-terminal secretion signal . To explore the substrate specificity of this system, we have expressed the E . chrysanthemi transporter system (prtDEF genes) in Escherichia coli and tested the ability of this ABC transporter to export hybrid proteins carrying C-terminal fragments of E . chrysanthemi protease B . The C terminus contains six glycine-rich repeated motifs, followed by two repeats of the sequences DFLV and DIIV . Two types of hybrid proteins were assayed for transport, proteins with the 93-residue-protease-B C terminus containing one glycine-rich repeat and both hydrophobic terminal repeats and proteins with the 181-residue C terminus containing all repeat motifs . Although the shorter C terminus is unable to export the hybrids, the longer C terminus can promote the secretion of hybrid proteins with N termini as large as 424 amino acids, showing that the glycine-rich motifs are required for the efficient secretion of these hybrids . However, the secretion of hybrids occurs only if these proteins do not carry disulfide bonds in their mature structures . These latter results suggest that disulfide bond formation can occur prior to or during the secretion . Disulfide bonds may prevent type I secretion of hybrids . One simple hypothesis to explain these results is that the type I channel is too narrow to permit the export of proteins with secondary structures stabilized by disulfide bonds. Appl Environ Microbiol, 2001 Feb, 67(2), 598 - 607 Analysis of the type IV fimbrial-subunit gene fimA of Xanthomonas hyacinthi: application in PCR-mediated detection of yellow disease in Hyacinths; van Doorn J et al.; A sensitive and specific detection method was developed for Xanthomonas hyacinthi; this method was based on amplification of a subsequence of the type IV fimbrial-subunit gene fimA from strain S148 . The fimA gene was amplified by PCR with degenerate DNA primers designed by using the N-terminal and C-terminal amino acid sequences of trypsin fragments of FimA . The nucleotide sequence of fimA was determined and compared with the nucleotide sequences coding for the fimbrial subunits in other type IV fimbria-producing bacteria, such as Xanthomonas campestris pv . vesicatoria, Neisseria gonorrhoeae, and Moraxella bovis . In a PCR internal primers JAAN and JARA, designed by using the nucleotide sequences of the variable central and C-terminal region of fimA, amplified a 226-bp DNA fragment in all X . hyacinthi isolates . This PCR was shown to be pathovar specific, as assessed by testing 71 Xanthomonas pathovars and bacterial isolates belonging to other genera, such as Erwinia and Pseudomonas . Southern hybridization experiments performed with the labelled 226-bp DNA amplicon as a probe suggested that there is only one structural type IV fimbrial-gene cluster in X . hyacinthi . Only two Xanthomonas translucens pathovars cross-reacted weakly in PCR . Primers amplifying a subsequence of the fimA gene of X . campestris pv . vesicatoria (T . Ojanen-Reuhs, N . Kalkkinen, B . Westerlund-Wikstrom, J . van Doorn, K . Haahtela, E.-L . Nurmiaho-Lassila, K . Wengelink, U . Bonas, and T . K . Korhonen, J . Bacteriol . 179: 1280-1290, 1997) were shown to be pathovar specific, indicating that the fimbrial-subunit sequences are more generally applicable in xanthomonads for detection purposes . Under laboratory conditions, approximately 1,000 CFU of X . hyacinthi per ml could be detected . In inoculated leaves of hyacinths the threshold was 5,000 CFU/ml . The results indicated that infected hyacinths with early symptoms could be successfully screened for X . hyacinthi with PCR. Int J Syst Evol Microbiol, 2000 Nov, 50 Pt 6, 2057 - 68 Phylogenetic relationships of necrogenic Erwinia and Brenneria species as revealed by glyceraldehyde-3-phosphate dehydrogenase gene sequences; Brown EW et al.; Recent examination of the relationships of the dry necrosis-inducing (necrogenic) erwinias using 16S rDNA sequences demonstrated that these bacteria comprise a polyphyletic group and, therefore, have been subdivided into three distinct genera, Erwinia, Brenneria and Pectobacterium, with the classical 'amylovora' group species now being distributed nearly evenly among the first two . To further assess the molecular evolutionary relationships between current necrogenic Erwinia and Brenneria species, as well as between these genera and the exclusively soft-rotting genus Pectobacterium, the glyceraldehyde-3-phosphate dehydrogenase (gapDH) genes from 57 Erwinia and Brenneria isolates along with Pectobacterium type strains were PCR-amplified, sequenced and subjected to phylogenetic analysis . Pairwise alignments of cloned gapDH genes revealed remarkably high interspecies genetic diversity among necrogenic isolates . Four evolutionary clades of necrogenic species were described that assorted more closely to known soft-rotting species than to each other . Interclade comparisons of gapDH nucleotide sequences revealed as much genetic divergence between these four necrogenic clades as existed between necrogenic and soft-rotting clades . An examination of the phylogenetic utility of the gapDH gene in light of current 16S rDNA clustering of these species revealed varying levels of taxonomic congruence between these genes for the structure of Erwinia, Brenneria and Pectobacterium . These analyses suggest that, while gapDH possesses sufficient genetic variation to fully differentiate Erwinia and Brenneria species, the gene may not accurately reflect interspecies taxonomic relatedness among all three phytopathogenic genera. J Bacteriol, 2001 Jan, 183(2), 597 - 603 Disulfide bond in Pseudomonas aeruginosa lipase stabilizes the structure but is not required for interaction with its foldase; Liebeton K et al.; Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif . The lipase contains two cysteine residues which form an intramolecular disulfide bond . Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane . Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed in Escherichia coli, denatured by treatment with urea, and subsequently refolded in vitro . Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase . However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P . aeruginosa . These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase . This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively. Appl Environ Microbiol, 2001 Jan, 67(1), 284 - 92 Pantoea agglomerans strain EH318 produces two antibiotics that inhibit Erwinia amylovora in vitro; Wright SA et al.; Pantoea agglomerans (synonym: Erwinia herbicola) strain Eh318 produces through antibiosis a complex zone of inhibited growth in an overlay seeded with Erwinia amylovora, the causal agent of fire blight . This zone is caused by two antibiotics, named pantocin A and B . Using a genomic library of Eh318, two cosmids, pCPP702 and pCPP704, were identified that conferred on Escherichia coli the ability to inhibit growth of E . amylovora . The two cosmids conferred different antibiotic activities on E . coli DH5alpha and had distinct restriction enzyme profiles . A smaller, antibiotic-conferring DNA segment from each cosmid was cloned . Each subclone was characterized and mutagenized with transposons to generate clones that were deficient in conferring pantocin A and B production, respectively . Mutated subclones were introduced into Eh318 to create three antibiotic-defective marker exchange mutants: strain Eh421 (pantocin A deficient); strain Eh439 (pantocin B deficient), and Eh440 (deficient in both pantocins) . Cross-hybridization results, restriction maps, and spectrum-of-activity data using the subclones and marker exchange mutants, supported the presence of two distinct antibiotics, pantocin A and pantocin B, whose biosynthetic genes were present in pCPP702 and pCPP704, respectively . The structure of pantocin A is unknown, whereas that of pantocin B has been determined as (R)-N-{((S)-2-amino-propanoylamino)-methyl}-2-methanesulfonyl-s uccina mic acid . The two pantocins mainly affect other enteric bacteria, based on limited testing. Appl Environ Microbiol, 2001 Jan, 67(1), 59 - 64 Isolation and characterization of five Erwinia amylovora bacteriophages and assessment of phage resistance in strains of Erwinia amylovora; Schnabel EL et al.; Phages able to infect the fire blight pathogen Erwinia amylovora were isolated from apple, pear, and raspberry tissues and from soil samples collected at sites displaying fire blight symptoms . Among a collection of 50 phage isolates, 5 distinct phages, including relatives of the previously described phages phiEa1 and phiEa7 and 3 novel phages named phiEa100, phiEa125, and phiEa116C, were identified based on differences in genome size and restriction fragment pattern . phiEa1, the phage distributed most widely, had an approximately 46-kb genome which exhibited some restriction site variability between isolates . Phages phiEa100, phiEa7, and phiEa125 each had genomes of approximately 35 kb and could be distinguished by their EcoRI restriction fragment patterns . phiEa116C contained an approximately 75-kb genome . phiEa1, phiEa7, phiEa100, phiEa125, and phiEa116C were able to infect 39, 36, 16, 20, and 40, respectively, of 40 E . amylovora strains isolated from apple orchards in Michigan and 8, 12, 10, 10, and 12, respectively, of 12 E . amylovora strains isolated from raspberry fields (Rubus spp.) in Michigan . Only 22 of 52 strains were sensitive to all five phages, and 23 strains exhibited resistance to more than one phage . phiEa116C was more effective than the other phages at lysing E . amylovora strain Ea110 in liquid culture, reducing the final titer of Ea110 by >95% when added at a ratio of 1 PFU per 10 CFU and by 58 to 90% at 1 PFU per 10(5) CFU. Appl Environ Microbiol, 2001 Jan, 67(1), 6 - 14 Gene integration and expression and extracellular secretion of Erwinia chrysanthemi endoglucanase CelY (celY) and CelZ (celZ) in ethanologenic Klebsiella oxytoca P2; Zhou S et al.; The development of methods to reduce costs associated with the solubilization of cellulose is essential for the utilization of lignocellulose as a renewable feedstock for fuels and chemicals . One promising approach is the genetic engineering of ethanol-producing microorganisms that also produce cellulase enzymes during fermentation . By starting with an ethanologenic derivative (strain P2) of Klebsiella oxytoca M5A1 with the native ability to metabolize cellobiose, the need for supplemental beta-glucosidase was previously eliminated . In the current study, this approach has been extended by adding genes encoding endoglucanase activities . Genes celY and celZ from Erwinia chrysanthemi have been functionally integrated into the chromosome of P2 using surrogate promoters from Zymomonas mobilis for expression . Both were secreted into the extracellular milieu, producing more than 20,000 endoglucanase units (carboxymethyl cellulase activity) per liter of fermentation broth . During the fermentation of crystalline cellulose with low levels of commercial cellulases of fungal origin, these new strains produced up to 22% more ethanol than unmodified P2 . Most of the beneficial contribution was attributed to CelY rather than to CelZ . These results suggest that fungal enzymes with substrate profiles resembling CelY (preference for long-chain polymers and lack of activity on soluble cello-oligosaccharides of two to five glucosyl residues) may be limiting in commercial cellulase preparations. Mol Microbiol, 2000 Nov, 38(4), 673 - 83 Transcriptional regulation of transport and utilization systems for hexuronides, hexuronates and hexonates in gamma purple bacteria; Rodionov DA et al.; The comparative approach is a powerful tool for the analysis of gene regulation in bacterial genomes . It can be applied to the analysis of regulons that have been studied experimentally as well as that of regulons for which no known regulatory sites are available . It is assumed that the set of co-regulated genes and the regulatory signal itself are conserved in related genomes . Here, we use genomic comparisons to study the regulation of transport and utilization systems for sugar acids in gamma purple bacteria Escherichia coli, Salmonella typhi, Klebsiella pneumoniae, Yersinia pestis, Erwinia chrysanthemi, Haemophilus influenzae and Vibrio cholerae . The variability of the operon structure and the location of the operator sites for the main transcription factors are demonstrated . The common metabolic map is combined with known and predicted regulatory interactions . It includes all known and predicted members of the GntR, UxuR/ExuR, KdgR, UidR and IdnR regulons . Moreover, most members of these regulons seem to be under catabolite repression mediated by CRP . The candidate UxuR/ExuR signal is proposed, the KdgR consensus is extended, and new operators for all transcription factors are identified in all studied genomes . Two new members of the KdgR regulon, a hypothetical ATP-dependent transport system OgtABCD and YjgK protein with unknown function, are detected . The former is likely to be the transport system for the products of pectin degradation, oligogalacturonides. Mol Gen Genet, 2000 Oct, 264(3), 233 - 40 Genes of Erwinia amylovora involved in yellow color formation and release of a low-molecular-weight compound during growth in the presence of copper ions; Zhang Y et al.; Most Erwinia amylovora strains form yellow mucoid colonies on solid minimal medium containing asparagine and copper sulfate (MM2Cu) . One exception is the strain Ea25/82, which produces white colonies on MM2Cu agar . This strain was transformed with a genomic library of E . amylovora and yellow colonies were recovered . A 1.5-kb fragment was found to complement strain Ea25/82 for color formation, and subsequent sequencing revealed two ORFs . The smaller ORF132(ycfB) overlapped with the end of the larger ORF253(ycfA) . The putative protein YcfA shows low homology with K+/Na+ channel transporter ATPases . Resistance genes were inserted in both ORFs, and the E . amylovora strains Ea1/79-YA and Ea1/79-YB were created by site-directed mutagenesis . The mutation in ycfB did not affect color formation, whereas the ycfA mutant formed white colonies on MM2Cu . Sequence analysis of the ycf region in strain Ea25/82 revealed a 1-bp alteration in ycfA and no change in ycfB . Stable complementation of Ea25/82 and Ea1/79-YA, however, required both genes . Carotenoids were not detected in E . amylovora grown in the presence of copper ions . On the other hand, copper-independent secretion of a low-molecular-weight compound with an absorption maximum at 340 nm (CP340) was found for strain Ea1/79, but not for Ea25/82 or the mutant Ea1/79-YA . CP340 formed a complex with copper ions, and complementation with plasmids carrying both ycfA and ycfB restored its release from mutant strains . The compound may be connected with the yellow pigment or function in sensing bacterial population densities. Plant Physiol, 2000 Nov, 124(3), 1027 - 38 Linear beta-1,3 glucans are elicitors of defense responses in tobacco; Kla