Arch Biochem Biophys, 1998 Apr 1, 352(1), 37 - 43 The Leu-3 residue of Serratia marcescens metalloprotease inhibitor is important in inhibitory activity and binding with Serratia marcescens metalloprotease; Bae KH et al.; Serratia marcescens metalloprotease inhibitor (SmaPI) is a proteinase inhibitor toward Serratia marcescens metalloprotease (SMP) . In sequential deletion analysis of the N-terminal region of the SmaPI, SmaPIs starting at Ser-2 and Leu-3 residues, respectively, had nearly a full inhibitory activity toward SMP . However, SmaPI starting at Ala-4 residue showed severely decreased inhibitory activity . Furthermore, kinetic analysis demonstrated that SmaPI starting at the Ala-4 residue had an inhibition constant for SMP approximately fourfold higher than that of wild-type SmaPI . The interactions of Leu-3 with SMP contribute 0.73 kcal mol-1 to the overall stability of the SMP-SmaPI complex (8.44 kcal mol-1) . To elucidate the detailed role of the Leu-3 residue in inhibitory activity of SmaPI, several site-directed mutations were introduced . The inhibitory activities of Leu-3 mutants in which the Leu-3 has been converted to Ala, Asp, Gly, Ile, Lys, Phe, or Pro were correlated with the hydrophobicities of substituted amino acids . About 0.3 kcal mol-1 is attributable to the side chain of the Leu-3 residue in the binding with SMP . From these results, it is suggested that (i) in contrast with the Erwinia chrysanthemi inhibitor, Gly-1 and Ser-2 of SmaPI are not critical and (ii) the hydrophobicity of Leu-3 may be important in its inhibitory activity and binding with SMP . Mol Cells, 1998 Feb 28, 8(1), 27 - 35 Cloning and sequencing of the celA gene encoding CMCase of Erwinia carotovora subsp . carotovora LY34; Park YW et al.; The phytopathogenic Erwinia carotovora subsp . carotovora LY34 secretes multiple isozymes of the plant cell wall-disintegrating enzyme, endoglucanases . Genomic DNA from Ecc LY34 was digested with Sau3AI and ligated into the BamHI site of pBluescript II SK+ . One of the E . coli clones containing a Sau3AI fragment of Ecc genomic DNA hydrolyzed carboxymethyl cellulose and was shown to contain the 2.2 kb BamHI restriction fragment, which was subcloned to generate pLYCA100 named as celA . The structural organization of a celA gene encoding 387 amino acids consists of an open reading frame (ORF) of 1161 bp starting with an ATG start codon and followed by a TAA stop codon . CelA protein contained a typical catalytic domain, interdomain, cellulose binding domain, and prokaryotic signal peptide of 32 amino acids . Since the deduced amino acid sequences of CelA protein was very similar to those of CelV of Erwinia carotovora subsp . carotovora SCC3193 enzyme and to those of CelN of Erwinia atroceptica enzyme, it belongs to the cellulase family 5 . The apparent molecular mass of CelA protein was calculated to be 39 kDa by carboxymethylcellulose-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (CMC-SDS-PAGE) . Activity staining of carboxymethyl cellulase (CMCase) in sodium dodecyl sulfate polyacrylamide gel containing 0.1% CMC revealed that the cloned isozyme comigrated with a corresponding isozyme produced by Ecc LY34 . The CelA had a calculated pI of 5.42 . The optimum pH was 7 and the optimum temperature was about 45 degrees C. J Bacteriol, 1998 Apr, 180(8), 2244 - 7 Erwinia amylovora secretes DspE, a pathogenicity factor and functional AvrE homolog, through the Hrp (type III secretion) pathway; Bogdanove AJ et al.; Erwinia amylovora was shown to secrete DspE, a pathogenicity factor of 198 kDa and a functional homolog of AvrE of Pseudomonas syringae pv . tomato . DspE was identified among the supernatant proteins isolated from cultures grown in an hrp gene-inducing minimal medium by immunodetection with a DspE-specific antiserum . Secretion required an intact Hrp pathway. Mol Plant Microbe Interact, 1998 Apr, 11(4), 270 - 6 The exuT gene of Erwinia chrysanthemi EC16: nucleotide sequence, expression, localization, and relevance of the gene product; Haseloff BJ et al.; Galacturonic acid (GalUA) is a major component of pectin and polygalacturonic acid in the plant cell wall . In the phytopathogen Erwinia chrysanthemi, the uptake of molecules derived from degradation of these polymers is an important early step in the events preceding induction of pectinases, ultimately leading to plant tissue maceration . Uptake systems for GalUA and dimers of GalUA have been described and shown to be inducible in E . chrysanthemi . The GalUA uptake gene (exuT) was cloned and sequenced . Nucleotide sequence analysis identified an open reading frame encoding a 345-amino-acid polypeptide with a calculated mass of 37,825 Da . This polypeptide is predicted to be an integral membrane protein based on its high nonpolar amino acid content and hydropathic profile . Localization studies with the labeled polypeptide in the T7-RNA polymerase system also suggest that ExuT is a membrane protein . This evidence is further supported by the observation of hybrid ExuT-PhoA proteins in the bacterial cytoplasmic membrane following immunoblot analysis . Northern (RNA) analysis indicated that the gene is inducible in the presence of the monomer, GalUA . A targeted mutation in the exuT gene affected the utilization of GalUA as a role carbon source for growth . Maceration of potato tuber tissue by this mutant was delayed and reduced, when compared with the parental strain EC16. EMBO J, 1998 Feb 16, 17(4), 936 - 44 The SecB chaperone is involved in the secretion of the Serratia marcescens HasA protein through an ABC transporter; Delepelaire P et al.; The secretion pathways of the heme-binding protein HasA from Serratia marcescens and of the metalloproteases A, B, C and G from Erwinia chrysanthemi have been reconstituted in Escherichia coli . They are secreted in a single step from the cytoplasm across both membranes of the Gram-negative envelope, after recognition of their specific C-terminal secretion signal by their cognate ABC transporter . We report strong evidence that both HasA and the metalloproteases bind the SecB chaperone involved in the export of several envelope proteins via the Sec pathway . We also show that the secretion of the HasA protein is strongly dependent upon SecB in the reconstituted system, whereas that of the proteases is not . HasA secretion in the original host is strongly inhibited by a protein known to interfere with E.coli SecB function . We propose that the proteins secreted by the ABC pathway may have to be unfolded for efficient secretion. J Bacteriol, 1998 Mar, 180(6), 1431 - 7 External loops at the C terminus of Erwinia chrysanthemi pectate lyase C are required for species-specific secretion through the out type II pathway; Lindeberg M et al.; The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins . Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown . The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway . However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins . The functional cluster of E . chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E . chrysanthemi, but not E . carotovora, Pels . We exploited the high sequence similarity between E . chrysanthemi PelC and E . carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pell . The differential secretion of these hybrid proteins by E . coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC . We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning. FEMS Microbiol Lett, 1998 Feb 15, 159(2), 275 - 81 The rpoS gene of Erwinia carotovora: gene organization and functional expression in E . coli; Calcutt MJ et al.; rpoS homologues were identified in several Erwinia species using Escherichia coli rpoS sequences as probes . The rpoS gene from Erwinia carotovora was cloned and the deduced amino acid sequence had 91% identity to E . coli RpoS . The latter sigma factor regulates the stationary phase inducible HPII catalase activity of E . coli . In an E . coli rpoS mutant, the E . carotovora rpoS gene was also able to regulate synthesis of this catalase . The presence of a similar catalase in E . carotovora suggests that the structural gene for this may be part of the rpoS 'regulon' in Erwinia also . This study also showed that there are several differences in the gene organization of the rpoS region of the E . coli and E . carotovora chromosomes. Appl Environ Microbiol, 1998 Mar, 64(3), 914 - 21 Biochemical and genetic characterization of an extracellular protease from Pseudomonas fluorescens CY091; Liao CH et al.; Pseudomonas fluorescens CY091 cultures produce an extracellular protease with an estimated molecular mass of 50 kDa . Production of this enzyme (designated AprX) was observed in media containing CaCl2 or SrCl2 but not in media containing ZnCl2, MgCl2, or MnCl2 . The requirement of Ca2+ (or Sr2+) for enzyme production was concentration dependent, and the optimal concentration for production was determined to be 0.35 mM . Following ammonium sulfate precipitation and ion-exchange chromatography, the AprX in the culture supernatant was purified to near electrophoretic homogeneity . Over 20% of the enzyme activity was retained in the AprX sample which had been heated in boiling water for 10 min, indicating that the enzyme is highly resistant to heat inactivation . The enzyme activity was almost completely inhibited in the presence of 1 mM 1,10-phenanthroline, but only 30% of the activity was inhibited in the presence of 1 mM EGTA . The gene encoding AprX was cloned from the genome of P . fluorescens CY091 by isolating cosmid clones capable of restoring the protease production in a nonproteolytic mutant of strain CY091 . The genomic region of strain CY091 containing the aprX gene was located within a 7.3-kb DNA fragment . Analysis of the complete nucleotide sequence of this 7.3-kb fragment revealed the presence of a cluster of genes required for the production of extracellular AprX in P . fluorescens and Escherichia coli . The AprX protein showed 50 to 60% identity in amino acid sequence to the related proteases produced by Pseudomonas aeruginosa and Erwinia chrysanthemi . Two conserved sequence domains possibly associated with Ca2+ and Zn2+ binding were identified . Immediately adjacent to the aprX structural gene, a gene (inh) encoding a putative protease inhibitor and three genes (aprD, aprE, and aprF), possibly required for the transport of AprX, were also identified . The organization of the gene cluster involved in the synthesis and secretion of AprX in P . fluorescens CY091 appears to be somewhat different from that previously demonstrated in P . aeruginosa and E . chrysanthemi. Proc Natl Acad Sci U S A, 1998 Feb 3, 95(3), 1325 - 30 Homology and functional similarity of an hrp-linked pathogenicity locus, dspEF, of Erwinia amylovora and the avirulence locus avrE of Pseudomonas syringae pathovar tomato; Bogdanove AJ et al.; The "disease-specific" (dsp) region next to the hrp gene cluster of Erwinia amylovora is required for pathogenicity but not for elicitation of the hypersensitive reaction . A 6.6-kb apparent operon, dspEF, was found responsible for this phenotype . The operon contains genes dspE and dspF and is positively regulated by hrpL . A BLAST search revealed similarity in the dspE gene to a partial sequence of the avrE locus of Pseudomonas syringae pathovar tomato . The entire avrE locus was sequenced . Homologs of dspE and dspF were found in juxtaposed operons and were designated avrE and avrF . Introduced on a plasmid, the dspEF locus rendered P . syringae pv . glycinea race 4 avirulent on soybean . An E . amylovora dspE mutant, however, elicited a hypersensitive reaction in soybean . The avrE locus in trans restored pathogenicity to dspE strains of E . amylovora, although restored strains were low in virulence . DspE and AvrE are large (198 kDa and 195 kDa) and hydrophilic . DspF and AvrF are small (16 kDa and 14 kDa) and acidic with predicted amphipathic alpha helices in their C termini; they resemble chaperones for virulence factors secreted by type III secretion systems of animal pathogens. J Biotechnol, 1997 Dec 3, 58(3), 177 - 85 Synthesis of atypical cyclic and acyclic hydroxy carotenoids in Escherichia coli transformants; Albrecht M et al.; A total of eight different hydroxy carotenoids were produced in transformants of the non-carotenogenic bacterium Escherichia coli . They include the acyclic 1-hydroxyneurosporene, 1-hydroxylycopene, 1,1'-dihydroxylycopene and demethylspheroidene as well as the cyclic 3-hydroxy-beta-zeacarotene, 7,8-dihydrozeaxanthin, 3 or 3'-7,8-dihydro-beta-carotene and 1'-hydroxy-gamma-carotene . Most of these uncommon carotenoids are found only in trace amounts in natural sources . For the synthesis of all the carotenoids mentioned above, E . coli was transformed with a combination of up to three compatible plasmids, which contained several carotenogenic genes from Erwinia uredovora and two Rhodobacter species . Their function in the pathway leading to the individual carotenoids was outlined . Finally, growth conditions were optimized for production of the hydroxy carotenoids in amounts which are suitable for their isolation and purification. Microbiology, 1998 Jan, 144 ( Pt 1), 201 - 9 A pheromone-independent CarR protein controls carbapenem antibiotic synthesis in the opportunistic human pathogen Serratia marcescens; Cox AR et al.; Strain ATCC 39006 of Serratia marcescens makes the same carbapenem, (5R)-carbapen-2-em-3-carboxylic acid (Car), as the Erwinia carotovora strain GS101 . Unlike E . carotovora, where the onset of production occurs in the late-exponential phase of growth in response to the accumulation of the small diffusible pheromone N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), in S . marcescens carbapenem is produced throughout the growth phase and does not appear to involve any diffusible pheromone molecule . Two cosmids capable of restoring antibiotic production in E . carotovora group I carbapenem mutants were isolated from an S . marcescens gene library . These cosmids were shown to contain a homologue of the E . carotovora carR gene, encoding a CarR protein with homology to the LuxR family of transcriptional regulators . The S . marcescens carR was subcloned and shown to be capable of complementing in trans, in the absence of OHHL, an E . carotovora carR carI double mutant, releasing the heterologous E . carotovora host from pheromone dependence for carbapenem production . The apparent OHHL-independence of the S . marcescens CarR explains the constitutive nature of carbapenem production in this strain of S . marcescens. Plant Mol Biol, 1997 Oct, 35(3), 331 - 41 Geranylgeranyl pyrophosphate synthase encoded by the newly isolated gene GGPS6 from Arabidopsis thaliana is localized in mitochondria; Zhu XF et al.; We have cloned a new geranylgeranyl pyrophosphate (GGPP) synthase gene, designated GGPS6, from Arabidopsis thaliana genomic DNA . Nucleotide sequence analysis revealed that the GGPS6 gene contains an open reading frame coding for a protein of 343 amino acid residues with a calculated molecular mass of 37,507 Da . Also, the gene is not interrupted by an intron . The predicted amino acid sequence of the GGPS6 gene shows significant homology (34.0-57.7%) with other GGPP synthases from Arabidopsis . The GGPS6 protein contains a N-terminal signal peptide which is thought to function as an organelle targeting sequence . In fact, the GGPS6-GFP fusion protein was found to be localized exclusively in mitochondria when expressed in tobacco BY-2 cells . In vitro analysis of the enzyme activity as well as genetic complementation analysis with Erwinia uredovora crt gene cluster expressed in Escherichia coli showed that the GGPS6 gene most certainly encodes a GGPP synthase catalyzing the conversion of farnesyl pyrophosphate to GGPP. Eur J Biochem, 1997 Nov 15, 250(1), 55 - 62 Elucidation of the structure of the core region and the complete structure of the R-type lipopolysaccharide of Erwinia carotovora FERM P-7576; Fukuoka S et al.; An R-type lipopolysaccharide (LPS) from Erwinia carotovora strain FERM P-7576 was studied after strong alkaline degradation and mild acid hydrolysis . The resulting products were analyzed by fast-atom bombardment mass spectrometry, one- and two-dimensional 1H and 13C NMR spectroscopy, dephosphorylation and methylation analysis . The following structure was proposed for the core region of the LPS: {formula in text} where Hep is L-glycero-D-manno-heptose and Kdo is 3-deoxy-D-manno-octulosonic acid . Some LPS species lack the beta-D-Galp residue or the beta-D-Galp-(1-->7)-alpha-Hepp disaccharide . With the known structures of lipid A {Fukuoka, S., Kamishima, H., Nagawa, Y., Nakanishi, H., Ishikawa, K., Niwa, Y., Tamiya, E . & Karube, I . (1992) Arch . Microbiol . 157, 311-318} and the core moiety, the complete LPS structure was established and confirmed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry of the native and O-deacylated LPS. Plant Physiol, 1998 Jan, 116(1), 69 - 80 The tree-dimensional structure of aspergillus niger pectin lyase B at 1.7-A resolution; Vitali J et al.; The three-dimensional structure of Aspergillus niger pectin lyase B (PLB) has been determined by crystallographic techniques at a resolution of 1.7 A . The model, with all 359 amino acids and 339 water molecules, refines to a final crystallographic R factor of 16.5% . The polypeptide backbone folds into a large right-handed cylinder, termed a parallel beta helix . Loops of various sizes and conformations protrude from the central helix and probably confer function . The largest loop of 53 residues folds into a small domain consisting of three antiparallel beta strands, one turn of an alpha helix, and one turn of a 3(10) helix . By comparison with the structure of Erwinia chrysanthemi pectate lyase C (PelC), the primary sequence alignment between the pectate and pectin lyase subfamilies has been corrected and the active site region for the pectin lyases deduced . The substrate-binding site in PLB is considerably less hydrophilic than the comparable PelC region and consists of an extensive network of highly conserved Trp and His residues . The PLB structure provides an atomic explanation for the lack of a catalytic requirement for Ca2+ in the pectin lyase family, in contrast to that found in the pectate lyase enzymes . Surprisingly, however, the PLB site analogous to the Ca2+ site in PelC is filled with a positive charge provided by a conserved Arg in the pectin lyases . The significance of the finding with regard to the enzymatic mechanism is discussed. J Toxicol Environ Health A, 1998 Jan 9, 53(1), 5 - 18 Effect of inhalation of organic dust-derived microbial agents on the pulmonary phagocytic oxidative metabolism of guinea pigs; Milanowski J; The effect of inhalation exposure of various biological agents associated with organic dusts on the function of guinea pigs pulmonary phagocytes was investigated . Agents included antigens of Erwinia herbicola, Thermoactinomyces vulgaris, and Aspergillus fumigatus; endotoxin of Erwinia herbicola; bacterial protease; and a fungal glucan preparation . Pulmonary parameters monitored in this study were cellular differential counts from bronchoalveolar lavage, and superoxide anion and/or hydrogen peroxide production by phagocytic cells . Most of the agents caused an influx of neutrophils, lymphocytes, and red blood cells to the lung, and an enhancement of secretion of reactive oxygen species by pulmonary phagocytes . However, the relative magnitude of the inflammatory response varied greatly among these biological agents . In general, antigens of Erwinia herbicola and Aspergillus fumigatus were most potent, while bacterial protease was least effective. Biochemistry, 1997 Dec 23, 36(51), 16074 - 86 Solution structure of the cellulose-binding domain of the endoglucanase Z secreted by Erwinia chrysanthemi; Brun E et al.; Two-dimensional proton nuclear magnetic resonance spectroscopy has been used to determine the three-dimensional structure of the 62 amino acid C-terminal cellulose-binding domain (CBD) of the endoglucanase Z (CBDEGZ), secreted by Erwinia chrysanthemi . An experimental data set comprising 958 interproton nOe-derived restraints was used to calculate 23 structures . The calculated structures have an average root-mean-square deviation between Cys4 and Cys61 of 0.91 +/- 0.11 A for backbone atoms and 1.18 +/- 0.12 A for the heavy atoms . The CBDEGZ exhibits a skiboot shape based mainly on a triple antiparallel beta-sheet perpendicular to a less-ordered summital loop . Three aromatic rings (Trp18, Trp43, and Tyr44) are localized on one face of the protein and are exposed to the solvent in a conformation compatible with a cellulose-binding site . Based on its original folding, we have been able to relate the CBD sequence to those of several domains of unknown function occurring in several bacterial chitinases as well as other proteins . This study also provides a structural basis for analyzing the secretion-related information specific to the CBDEGZ. Mol Gen Genet, 1997 Nov, 256(6), 611 - 9 Genetics of sorbitol metabolism in Erwinia amylovora and its influence on bacterial virulence; Aldridge P et al.; A chromosomal DNA fragment from Erwinia amylovora was identified that complemented a deletion mutant in the gut(srl) operon of Escherichia coli . The E . amylovora srl operon on the cloned fragment was localized by transposon mutagenesis . A DNA fragment including the srl genes of E . amylovora was sequenced and found to contain six open reading frames (ORFs) . These ORFs were highly homologous to genes of the gut operon of E . coli . No large gene was found that encoded a protein equivalent to GutA of E . coli; instead two ORFs with extensive similarity to GutA were identified in the E . amylovora srl operon . All transposon insertions were mapped by PCR analysis, and several insertions in a plasmid bearing the srl operon were unable to complement a mutation in the E . coli gutD gene . All E . amylovora srl mutants could be complemented by introducing the sorbitol operon from E . coli . The direction of transcription was confirmed by analysis of lacZ fusions . Expression of the srl operon in E . amylovora was high in the presence of sorbitol in the medium and was repressed by glucose . Mutants with a sorbitol deficiency were still virulent on slices of immature pears, but were unable to cause significant fire blight symptoms on apple shoots . Since sorbitol is used for carbohydrate transport in host plants of E . amylovora, this sugar alcohol may be an important factor in determining host specificity for the fire blight pathogen. Plasmid, 1997, 38(3), 141 - 7 Cloning and characterization of the ori region of pSW1200 of Erwinia stewartii: similarity with plasmid P1; Fu JF et al.; The ori region of an Erwinia stewartii plasmid, pSW1200 (106 kb), has been cloned and sequenced . This region consists of a gene encoding a protein which has 91% similarity and 73% identity with the RepA protein of bacteriophage P1 . The ori region also consists of eight copies of 19-bp iterons which are highly homologous to the iterons of P1 . Similar to plasmid P1, pSW1200 replicon has a copy number of approximately 1 . On the other hand, the copy number increases about ninefold if three of the iterons located downstream from repA gene are deleted . We also demonstrate that pGEM-5Z consisting of a copy of P1 iteron is incompatible with a pSW1200 derivative, pSW1201, suggesting that pSW1200 and P1 DNA are incompatible and both belong to the IncY group. Biochem Biophys Res Commun, 1997 Dec 29, 241(3), 636 - 41 Characterization of Erwinia carotovora subsp . carotovora LY34 endo-1,4-beta-glucanase genes and rapid identification of their gene products; Park YW et al.; Genomic DNA of the phytopathogenic Erwinia carotovora subsp . carotovora LY34 was partially digested with Sau3AI, ligated into the BamHI site of pBlue-script II SK+, and introduced into E . coli . Two clones that were able to hydrolyse carboxymethylcellulose were selected . 1.5 kb and 1.2 kb fragments containing the celA and celB genes, respectively, were subcloned and sequenced . The celA and celB genes had open reading frames of 1,161 bp and 792 bp encoding 487 and 264 amino acid residues with calculated molecular weights of 42,003 Da and 29,890 Da, respectively . Each, CelA and CelB, carried a typical prokaryotic signal peptide of 32 and 36 amino acid residues, respectively . The apparent molecular masses of the proteins when expressed in E . coli cells were approximately 39 kDa (CelA) and 26 kDa (CelB) as assessed by CMC-SDS-PAGE . Activity staining of CMCase in an SDS-PAGE gel containing 0.1% CMC revealed that the cloned endoglucanase isozymes comigrated with the corresponding ones present in Erwinia carotovora subsp . carotovora LY34. Gene, 1997 Nov 20, 202(1-2), 45 - 51 Cloning, sequence and expression of the pel gene from an Amycolata sp; Bruhlmann F et al.; The pel gene from an Amycolata sp . encoding a pectate lyase (EC 4.2.2.2) was isolated by activity screening a genomic DNA library in Streptomyces lividans TK24 . Subsequent subcloning and sequencing of a 2.3 kb BamHI BglII fragment revealed an open reading frame of 930 nt corresponding to a protein of 29,660 Da . The overall G + C content for the coding region was 65%, with a strong G + C preference in the third (wobble) codon position (93%) . A putative ribosome-binding site 5'-GGGAG-3' preceded the translational start codon by 7 base pairs . The Amycolata pectate lyase contains a signal peptide of 26 amino acids, that is cleaved after the sequence Ala-Thr-Ala . The size of the deduced protein as well as its N-terminal amino-acid sequence match the wild-type pectate lyase from the Amycolata sp . Expression of the pel gene in S . lividans TK24 resulted in high pectate lyase activity in the culture supernatant, concomitant with the appearance of a dominant protein band on a sodium dodecyl polyacrylamide gel at 30 kDa . No pectate lyase activity was detected in E . coli BL21 with the pel gene under the strong T7 promotor . The deduced amino-acid sequence showed 40% identity with PelE from Erwinia chrysanthemi and the pectate lyase from Glomerella cingulata . The Amycolata pectate lyase clearly belongs to the pectate lyase superfamily, sharing all functional amino acids and likely has a similar structural topology as Pels from Erwinia chrysanthemi and Bacillus subtilis. Mol Microbiol, 1997 Dec, 26(5), 1071 - 82 Antagonistic effect of CRP and KdgR in the transcription control of the Erwinia chrysanthemi pectinolysis genes; Nasser W et al.; The main virulence factors of the phytopathogenic bacteria Erwinia chrysanthemi are pectinases that cleave pectin, a major constituent of the plant cell wall . The cyclic AMP receptor protein (CRP) was identified as the main activator of the pectinolysis genes . Gel shift and DNase I footprinting experiments showed that the purified E . chrysanthemi CRP protein binds specifically to the promoter regions of seven pectinolysis genes (pelB, pelC, pelD, pelE, ogl, kduI and kdgT) whose expression is positively regulated in vivo by CRP . In contrast, no interaction was observed between CRP and the promoter-operator region of pelA, whose expression is negatively regulated in vivo by CRP . Primer extension experiments demonstrated that each of the pelB, pelC, pelE and kduI genes is expressed from a unique sigma70 promoter, whereas ogl and kdgT possess three and two functional promoters respectively . The position of the CRP binding site relative to the transcription start site suggests that CRP acts as a primary activator at the pelB (via the CRP binding site 1), pelC, pelE, pelD, kdgTP1 and oglP2 promoters . In contrast, transcription at the kduI, oglP1 promoters seems to require another transcriptional activator in synergy with CRP . Investigation of the simultaneous binding of CRP and KdgR, the main repressor of pectinolysis genes, to the regulatory regions of pelB, pelC, pelD, pelE, ogl, kduI and kdgT genes showed that binding of KdgR is preferential and exclusive in the case of ogl and kdgT, whereas the binding of these two regulators is independent in the case of pelB, pelC, pelD, pelE and kduI . Taken together, our data suggest that the antagonistic effects of CRP and KdgR on the expression of the pectinolysis genes occur by different mechanisms, including direct competition between the two regulators or between the repressor and RNA polymerase for the occupation of a common DNA region on the target genes. Mol Microbiol, 1997 Dec, 26(5), 1057 - 69 DspA, an essential pathogenicity factor of Erwinia amylovora showing homology with AvrE of Pseudomonas syringae, is secreted via the Hrp secretion pathway in a DspB-dependent way; Gaudriault S et al.; In Erwinia amylovora, the dsp region, required for pathogenicity on the host plant but not for hypersensitive elicitation on tobacco, is separated from the hrp region by 4 kb . The genetic analysis reported in this paper showed that this 4kb region is not required for pathogenicity on pear seedlings . The environmental conditions allowing expression of a dsp::lacZ fusion were examined: expression was barely detected in rich medium at 30 degrees C, and the highest expression was observed in M9 galactose minimal medium at 25 degrees C . A dsp::uidA fusion appeared to be expressed only in a HrpL-proficient strain, indicating that the dsp region, like the hrp region, is positively controlled via the alternative a factor HrpL . Sequence analysis revealed that the dsp cluster encodes two genes, dspA (5517 bp) and dspB (420 bp), and that the insertions leading to the dsp::lacZ and the dsp::uidA fusions were within dspA . A HrpL-dependent promoter sequence (GGAACC-N15-CAACA) was identified upstream of dspA, and primer extension analysis detected four transcriptional starts 7, 8, 9 and 10 bp downstream of this sequence . A sigma70 promoter sequence (TTGCCC-N16-GATAAT) was observed upstream of dspB . The functionality of this second promoter was confirmed by complementation analysis . This promoter allowed constitutive expression of dspB, as measured by the expression of a dspB::uidA fusion in rich medium . In M9 galactose medium, however, HrpL was shown to activate dspB, as expression of the dspB::uidA fusion was twofold higher in a HrpL+ background than in a HrpL- background . Transposon insertions in either dspA or dspB led to a non-pathogenic phenotype . Thus, both DspA and DspB were required for E . amylovora pathogenicity, as dspB could be expressed independently of dspA . DspA and DspB were visualized as polypeptides with apparent sizes of 190 kDa and 15.5 kDa, respectively, when encoded in the T7 polymerase/promoter system . DspA, which showed homology with the protein predicted from the partial sequence of Pseudomonas syringae pv . tomato avrE transcriptional unit III, was shown to be secreted into the external medium via the Hrp secretion pathway . DspB was predicted to be acidic, like the Syc chaperone of Yersinia . A chaperone role for DspB was suggested further by the fact that DspA secretion required a functional DspB protein. FEMS Microbiol Lett, 1997 Dec 1, 157(1), 59 - 65 Characterization of a periplasmic peptidyl-prolyl cis-trans isomerase in Erwinia chrysanthemi; Pissavin C et al.; The main determinant of the plant pathogen Erwinia chrysanthemi virulence is the production of extracellular enzymes, mainly pectate lyases . Adjacent to a pectate lyase encoding locus, we identified the gene rotA supposed to encode a folding catalyst . Overproduction of the protein and assay of activity using a synthetic substrate, confirmed that rotA encodes a periplasmic peptidyl-prolyl cis-trans isomerase . rotA disruption provokes no change in cell morphology, cell viability, growth rate or stability of the extracellular and periplasmic proteins . In addition, this mutation does not alter the activity of the pectate lyases, their stability in the periplasm during the transitory step of secretion or their recognition by the Out secretory system . rotA expression was followed using a rotA::uidA transcriptional fusion . Some environmental conditions, such as temperature variations and nitrogen starvation, modulate rotA expression . In contrast to the E . coli rotA gene, the E . chrysanthemi rotA possesses only one promoter and is not controlled by the CRP global regulator. Mol Microbiol, 1997 Nov, 26(3), 545 - 56 Analysis of the carbapenem gene cluster of Erwinia carotovora: definition of the antibiotic biosynthetic genes and evidence for a novel beta-lactam resistance mechanism; McGowan SJ et al.; Members of two genera of Gram-negative bacteria, Serratia and Erwinia, produce a beta-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid . We have reported previously the cloning and sequencing of the genes responsible for production of this carbapenem in Erwinia carotovora . These genes are organized as an operon, carA--H, and are controlled by a LuxR-type transcriptional activator, encoded by the linked carR gene . We report in this paper the genetic dissection of this putative operon to determine the function of each of the genes . We demonstrate by mutational analysis that the products of the first five genes of the operon are involved in the synthesis of the carbapenem molecule . Three of these, carABC, are absolutely required . In addition, we provide evidence for the existence of a novel carbapenem resistance mechanism, encoded by the CarF and carG genes . Both products of these overlapping and potentially translationally coupled genes have functional, N-terminal signal peptides . Removal of these genes from the Erwinia chromosome results in a carbapenem-sensitive phenotype . We assume that these novel beta-lactam resistance genes have evolved in concert with the biosynthetic genes to ensure 'self-resistance' in the Erwinia carbapenem producer. J Bacteriol, 1997 Dec, 179(23), 7369 - 78 An exo-poly-alpha-D-galacturonosidase, PehB, is required for wild-type virulence of Ralstonia solanacearum; Huang Q et al.; Ralstonia solanacearum, which causes bacterial wilt disease of many plant species, produces several extracellular plant cell wall-degrading enzymes that are suspected virulence factors . These include a previously described endopolygalacturonase (PG), PehA, and two exo-PGs . A gene encoding one of the exo-PGs, pehB, was cloned from R . solanacearum K60 . The DNA fragment specifying PehB contained a 2,103-bp open reading frame that encodes a protein of 74.2 kDa with a typical N-terminal signal sequence . The cloned pehB gene product cleaves polygalacturonic acid into digalacturonic acid units . The amino acid sequence of pehB resembles that of pehX, an exo-PG gene from Erwinia chrysanthemi, with 47.2% identity at the amino acid level . PehB also has limited similarity to plant exo-PGs from Zea mays and Arabidopsis thaliana . The chromosomal pehB genes in R . solanacearum wild-type strain K60 and in an endo-PG PehA- strain were replaced with an insertionally inactivated copy of pehB . The resulting mutants were deficient in the production of PehB and of both PehA and PehB, respectively . The pehB mutant was significantly less virulent than the wild-type strain in eggplant virulence assays using a soil inoculation method . However, the pehA mutant was even less virulent, and the pehA pehB double mutant was the least virulent of all . These results suggest that PehB is required for a wild-type level of virulence in R . solanacearum although its individual role in wilt disease development may be minor . Together with endo-PG PehA, however, PehB contributes substantially to the virulence of R . solanacearum. J Bacteriol, 1997 Dec, 179(23), 7321 - 30 Pectate lyase PelI of Erwinia chrysanthemi 3937 belongs to a new family; Shevchik VE et al.; Erwinia chrysanthemi 3937 secretes five major isoenzymes of pectate lyases encoded by the pel4, pelB, pelC, pelD, and pelE genes and a set of secondary pectate lyases, two of which, pelL and pelZ, have been already identified . We cloned the pelI gene, encoding a ninth pectate lyase of E . chrysanthemi 3937 . The pelI reading frame is 1,035 bases long, corresponding to a protein of 344 amino acids including a typical amino-terminal signal sequence of 19 amino acids . The purified mature PelI protein has an isoelectric point of about 9 and an apparent molecular mass of 34 kDa . PelI has a preference for partially methyl esterified pectin and presents an endo-cleaving activity with an alkaline pH optimum and an absolute requirement for Ca2+ ions . PelI is an extracellular protein secreted by the Out secretory pathway of E . chrysanthemi . The PelI protein is very active in the maceration of plant tissues . A pelI mutant displayed reduced pathogenicity on chicory leaves, but its virulence did not appear to be affected on potato tubers or Saintpaulia ionantha plants . The pelI gene constitutes an independent transcriptional unit . As shown for the other pel genes, the transcription of pelI is dependent on various environmental conditions . It is induced by pectic catabolic products and affected by growth phase, oxygen limitation, temperature, nitrogen starvation, and catabolite repression . Regulation of pelI expression appeared to be dependent on the three repressors of pectinase synthesis, KdgR, PecS, and PecT, and on the global activator of sugar catabolism, cyclic AMP receptor protein . A functional KdgR binding site was identified close to the putative pelI promoter . Analysis of the amino acid sequence of PelI revealed high homology with a pectate lyase from Erwinia carotovora subsp . carotovora (65% identity) and low homology with pectate lyases of the phytopathogenic fungus Nectria haematococca (Fusarium solani) . This finding indicates that PelI belongs to pectate lyase class III . Using immunoblotting experiments, we detected PelI homologs in various strains of E . chrysanthemi and E . carotovora subsp . carotovora but not in E . carotovora subsp . atroseptica. Eur J Pediatr, 1997 Nov, 156(11), 848 - 50 Changes in coagulation and fibrinolysis in childhood acute lymphoblastic leukaemia re-induction therapy using three different asparaginase preparations; Nowak-Gottl U et al.; Recently we reported the influence of two different Escherichia coli asparaginase (ASP) preparations on fibrinolytic proteins in childhood acute lymphoblastic leukaemia (ALL) demonstrating a significant association between ASP activity and haemostatic alterations . The present study was designed for prospective evaluation of coagulation and fibrinolytic parameters in leukaemic children receiving different ASP preparations during the course of re-induction . Forty leukaemic children receiving ASP (Medac: n = 13; Bayer: n = 10; Erwinia: n = 17) at 3-day intervals during re-induction were enrolled in this study . Blood samples for coagulation studies were obtained before each ASP administration together with serum samples for pharmacokinetic monitoring . Compared with Medac ASP 10,000 IU/m2, patients receiving Bayer ASP or Erwinia ASP showed significantly higher fibrinogen values . Antithrombin and plasminogen showed normal values in children after Erwinia ASP . Alpha2-antiplasmin and D-Dimer were no different in the groups studied . Neither side-effects, nor sustained asparagine depletion was observed in the majority of children treated with Erwinia ASP . Conclusion: Data of this study show a down-regulation of coagulation proteins in children treated with Medac ASP, less pronounced in patients after Bayer or Erwinia ASP . Since children treated with Erwinia ASP showed no adequate asparagine depletion during the course of ASP therapy, a dose adjustment should be discussed to guarantee asparagine depletion, the specific metabolic therapy for ALL. J Bacteriol, 1997 Nov, 179(22), 6994 - 7003 General secretion pathway (eps) genes required for toxin secretion and outer membrane biogenesis in Vibrio cholerae; Sandkvist M et al.; The general secretion pathway (GSP) of Vibrio cholerae is required for secretion of proteins including chitinase, enterotoxin, and protease through the outer membrane . In this study, we report the cloning and sequencing of a DNA fragment from V . cholerae, containing 12 open reading frames, epsC to -N, which are similar to GSP genes of Aeromonas, Erwinia, Klebsiella, Pseudomonas, and Xanthomonas spp . In addition to the two previously described genes, epsE and epsM (M . Sandkvist, V . Morales, and M . Bagdasarian, Gene 123: 81-86, 1993; L . J . Overbye, M . Sandkvist, and M . Bagdasarian, Gene 132:101-106, 1993), it is shown here that epsC, epsF, epsG, and epsL also encode proteins essential for GSP function . Mutations in the eps genes result in aberrant outer membrane protein profiles, which indicates that the GSP, or at least some of its components, is required not only for secretion of soluble proteins but also for proper outer membrane assembly . Several of the Eps proteins have been identified by use of the T7 polymerase-promoter system in Escherichia coli . One of them, a pilin-like protein, EpsG, was analyzed also in V . cholerae and found to migrate as two bands on polyacrylamide gels, suggesting that in this organism it might be processed or otherwise modified by a prepilin peptidase . We believe that TcpJ prepilin peptidase, which processes the subunit of the toxin-coregulated pilus, TcpA, is not involved in this event . This is supported by the observations that apparent processing of EpsG occurs in a tcpJ mutant of V . cholerae and that, when coexpressed in E . coli, TcpJ cannot process EpsG although the PilD peptidase from Neisseria gonorrhoeae can. Appl Environ Microbiol, 1997 Nov, 63(11), 4604 - 7 A relative of the broad-host-range plasmid RSF1010 detected in Erwinia amylovora; Palmer EL et al.; Streptomycin- and sulfonamide-resistant Erwinia amylovora CA3R from California contained an 8.7-kb plasmid, pEa8.7, with a sulII-strA-strB resistance region; furthermore, PCR, sequencing, hybridization, and restriction analyses showed that pEa8.7 was closely related or identical to broad-host-range plasmid RSF1010 . Although RSF1010 has been found in a variety of bacteria, this is the first report of its presence in plant pathogenic bacteria. Appl Environ Microbiol, 1997 Nov, 63(11), 4421 - 6 Differentiation of Erwinia amylovora strains by pulsed-field gel electrophoresis; Zhang Y et al.; Erwinia amylovora strains, isolated from several host plants in various geographic regions during different years, were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the DNA from lysed, agar-embedded cells with rare-cutting restriction enzymes . The banding patterns obtained with enzyme XbaI digests revealed significant differences among strains from different areas . North American strains E9 and Ea-Rb, a Rubus strain, were highly divergent from other E . amylovora strains . French strains were different from central European and English strains . E . amylovora strains from central Europe and New Zealand had identical PFGE patters, as had strains from Egypt, Greece, and Turkey . PFGE of genomic DNA from American and English strains gave rise to dissimilar patterns . Patterns of some American strains resembled those from strains isolated in other parts of the world . The restriction fragment length polymorphisms observed by PFGE analysis can be used to group strains and may give hints about the course of distribution of the plant disease . From the sizes of the restriction fragments obtained, a molecular mass of approximately 4.5 Mb was calculated for the genome of E . amylovora. J Biol Chem, 1997 Nov 7, 272(45), 28274 - 80 Measurement of Ca2+ fluxes during elicitation of the oxidative burst in aequorin-transformed tobacco cells; Chandra S et al.; We have employed suspension cultured aequorin-transformed tobacco cells to examine the involvement of Ca2+ in signal transduction of the oxidative burst . Use of cultured cells for this purpose was validated by demonstrating that the cells responded to cold shock quantitatively and qualitatively similarly to the intact transgenic plants from which they were derived . Stimulation of the oxidative burst in the cell suspension was achieved by administration of oligogalacturonic acid, Mas-7 (a peptide known to activate G proteins and Ca2+ fluxes), hypo-osmotic stress, or harpin (a protein from the pathogenic bacterium Erwinia amylovora) . The latter failed to promote any detectable increase in cytoplasmic Ca2+ concentration, whereas each of the former three triggered a rapid rise in cytosolic Ca2+ followed by a return within seconds to basal Ca2+ levels . Peak Ca2+ concentrations induced by the former three elicitors were approximately 0.7, 1.4, and 1.3 microM, respectively . Three lines of evidence suggest that the observed Ca2+ pulses are essential to transduction of the oxidative burst signals by their respective elicitors: (i) inhibition of the Ca2+ transients with Ca2+ chelators or Ca2+ channel blockers prevented expression of the oxidative burst, (ii) introduction of exogenous Ca2+ into the same cells initiated the burst even in the absence of other inducers of the response, and (iii) the observed Ca2+ transients often returned to near basal levels well before any H2O2 synthesis could be detected, suggesting that the Ca2+ influx is required to communicate the burst signal but not maintain the defense response . These data suggest that Ca2+ pulses serve frequently, but not invariably, to transduce an oxidative burst signal. J Bacteriol, 1997 Nov, 179(21), 6566 - 72 Cloning and expression of a gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase from Erwinia cypripedii ATCC 29267 in Escherichia coli; Yum DY et al.; We have cloned the gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase (GADH) from Erwinia cypripedii ATCC 29267 in Escherichia coli by performing a direct-expression assay . The positive clone converted D-gluconate to 2-keto-D-gluconate (2KDG) in the culture medium . Nucleotide sequence analysis of the GADH clone revealed that the cloned fragment contained the complete structural genes for a 68-kDa dehydrogenase subunit, a 47-kDa cytochrome c subunit, and a 24-kDa subunit of unknown function and that the genes were clustered with the same transcriptional polarity . Comparison of the deduced amino acid sequences and the NH2-terminal sequences determined for the purified protein indicated that the dehydrogenase, cytochrome c, and 24-kDa subunits contained typical signal peptides of 22, 19, and 42 amino acids, respectively . The molecular masses of the processed subunits deduced from the nucleotide sequences (65, 45, and 20 kDa) coincided well with the molecular masses of subunits estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . In E . cypripedii and recombinant E . coli, the GADH was constitutively formed and the activity of GADH was enhanced more than twofold by addition of D-gluconate to the medium . The holoenzyme glucose dehydrogenase of E . coli was reconstituted by addition of pyrroloquinoline quinone to the culture medium, and the conversion of D-glucose or D-gluconate to 2KDG by recombinant E . coli harboring the cloned GADH gene was attempted in batch culture . The conversion yields for D-glucose were 0.95 mol of 2KDG/mol of D-glucose after 16 h of cultivation, and those for D-gluconate were 0.95 mol of 2KDG/mol of D-gluconate after 12 h of cultivation. J Appl Microbiol, 1997 Oct, 83(4), 485 - 92 Isolation and characterization of a cryptic plasmid from Erwinia citreus ATCC 31623; Bilic M et al.; A multiple-copy plasmid pPZG500 (3.8 kb) was isolated from a phytopathogenic bacterium Erwinia citreus ATCC 31623 . This is the smallest plasmid so far isolated from the genus Erwinia . The plasmid was partially characterized by a set of restriction enzymes and the unique restriction sites were mapped for HindIII, EcoRI, EcoRV and XBaI, while three sites were found for BglII . Nineteen other enzymes did not cut pPZG500 . By deletion analyses minimal regions required for replication (ori) and segregational stability (par) were localized on 1.4 kb EcoRV/BglII and 0.7 kb Bgl/II/EcoRI fragments, respectively . The erythromycin resistance marker (Emr) was cloned into pPZG500 and two plasmid derivatives, pPZG502 and pPZG503, were constructed expressing erythromycin resistance as a good selective marker for recombinant selection in Erw . citreus and Escherichia coli . The segregational stability of both constructed plasmids during 90 generations in E . coli JM109 and Erw . citreus C-4 showed that plasmid pPZG503 lacking the presumptive par region was lost from the population at a higher rate . The results of this study demonstrate that plasmid pPZG500 and derivatives are suitable prerequisites for the construction of useful cloning vector(s) in the genus Erwinia. Biochem Biophys Res Commun, 1997 Oct 9, 239(1), 305 - 9 RpoS dependent overexpression of carotenoids from Erwinia herbicola in OXYR deficient Escherichia coli; Becker-Hapak M et al.; Carotenoid synthesis in Escherichia coli, when transformed with plasmid containing a carotenoid gene cluster from Erwinia herbicola (pPL376), is regulated by RpoS . When the plasmid was transformed into E . coli mutants that were oxyR minus, the intracellular carotenoid concentration dramatically increased from that observed in an oxyR plus allele . The higher carotenoid concentration in these mutants correlated with an increase in rpoS transcription as indicated by beta-galactosidase activity from a rpoS::lacZ promoter fusion . This indication of a higher concentration of carotenoids correlated with an increased resistance to hydrogen peroxide and near-ultraviolet radiation (310-400 nm; near-UV). FEBS Lett, 1997 Sep 22, 415(1), 40 - 4 Avirulence gene D of Pseudomonas syringae pv . tomato may have undergone horizontal gene transfer; Hanekamp T et al.; Avirulence gene D (avrD) is carried on the B-plasmid of the plant pathogen Pseudomonas syringae pv . tomato with plasmid-borne avrD homologs widely distributed among the Pseudomonads . We now report sequences in the soft rot pathogen Erwinia carotovora that cross-hybridize to avrD suggesting a conserved function beyond avirulence . Alternatively, avrD may have been transferred horizontally among species: (i) DNA linked to avrD shows evidence of class II transpositions and contains a novel IS3-related insertion sequence, and (ii) short sequences linked to avrD are similar to pathogenicity genes from a variety of unrelated pathogens . We have also identified the gene cluster that controls B-plasmid stability. Mol Gen Genet, 1997 Sep, 256(1), 72 - 83 Interaction of the regulator proteins RcsA and RcsB with the promoter of the operon for amylovoran biosynthesis in Erwinia amylovora; Kelm O et al.; The RcsA and RcsB proteins of Erwinia amylovora and Escherichia coli were expressed in E . coli and purified . Their DNA-binding activity was examined using a 1-kb DNA region containing the putative promoter of the ams operon of Ew . amylovora, which is responsible for the biosynthesis of the exopolysaccharide amylovoran . Mobility shift assays indicated specific binding of RcsA and RcsB to a region of 78 bp spanning nucleotide positions -578 to -501 relative to the translational start of the first open reading frame of the operon . This region includes stretches of homology to E . coli sigma 70 promoter consensus sequences and to the E . coli cps promoter region . Binding of the Rcs proteins was not found at a JUMPstart consensus, typical for various promoters of polysaccharide gene clusters . DNA-binding activity was not detected for RcsA alone and only high concentrations of RcsB were able to interact with the ams promoter in our assay . The two proteins bind cooperatively at the indicated region of the ams promoter and further evidence is provided showing that the DNA-protein complex formed involves a heterodimer of RcsA and RcsB . The specific activity of RcsA, but not of RcsB, was enhanced when the protein was expressed in E . coli at 28 degrees C, relative to expression at 37 degrees C . In addition, DNA-protein complex formation is affected by temperature . The E . coli RcsA/RcsB proteins bind to the same region of the ams promoter and are able to interact with the Rcs proteins from Ew . amylovora. Int J Syst Bacteriol, 1997 Oct, 47(4), 1061 - 7 Phylogenetic analysis of Erwinia species based on 16S rRNA gene sequences; Kwon SW et al.; The phylogenetic relationships of the type strains of 16 Erwinia species were investigated by performing a comparative analysis of the sequences of the 16S rRNA genes of these organisms . The sequence data were analyzed by the neighbor-joining method, and each branch was supported by moderate bootstrap values . The phylogenetic tree and sequence analyses confirmed that the genus Erwinia is composed of species that exhibit considerable heterogeneity and form four clades that are intermixed with members of other genera, such as Escherichia coli, Klebsiella pneumoniae, and Serratia marcescens . Cluster I includes the type strains of Erwinia herbicola, Erwinia milletiae, Erwinia ananas, Erwinia uredovora, and Erwinia stewartii and corresponds to Dye's herbicola group . Cluster II consists of Erwinia persicinus, Erwinia rhapontici, Erwinia amylovora, and Erwinia cypripedii . Cluster III consists of Erwinia carotovora subspecies and Erwinia chrysanthemi and is characterized by the production of pectate lyases and cellulases . Erwinia salicis, Erwinia rubrifaciens, and Erwinia nigrifluens form the cluster that is most distantly related to other Erwinia species . The data from the sequence analyses are discussed in the context of biochemical and DNA-DNA hybridization data. FEMS Microbiol Lett, 1997 Sep 15, 154(2), 265 - 70 The PecM protein is necessary for the DNA-binding capacity of the PecS repressor, one of the regulators of virulence-factor synthesis in Erwinia chrysanthemi; Praillet T et al.; The pecS regulatory locus is responsible for the down-expression of many virulence genes in Erwinia chrysanthemi . This locus consists of two genes, pecS and pecM, divergently transcribed . Genetic evidence indicates that the PecM protein modulates the regulatory activity of PecS . Purification and characterization of PecS, expressed either from E . coli, from the wild-type E . chrysanthemi strain or from a pecM mutant, showed that the PecS protein produced in these three genetic backgrounds displays the same biochemical properties . Band-shift assay analysis with the three PecS isoforms confirmed the involvement of the PecM protein in modulating the PecS DNA-binding capacity . Moreover, determination of the Kdapp for operator regions of the PecS protein, produced either by the wild-type E . chrysanthemi or by E . coli, reveals similar affinities . Thus, in E . coli, there is likely to be at least one other PecM-like protein able to cross-react with the E . chrysanthemi PecS protein. Biochim Biophys Acta, 1997 Aug 15, 1341(1), 26 - 34 Polysialylated asparaginase: preparation, activity and pharmacokinetics; Fernandes AI et al.; Erwinia carotovora L-asparaginase was coupled covalently to colominic acid, a low molecular mass polysialic acid, by reductive amination . Depending on the molar ratios of colominic acid-asparaginase (50:1, 100:1 and 250:1), polysialylated constructs contained 4.2-8.1 molecules of colominic acid per molecule of enzyme . Such constructs retained most (82-86%) of the initial asparaginase activity and also maintained the Km values of the native enzyme towards the substrate asparagine . On exposure to (mouse) blood plasma at 37 degrees C, polysialylated asparaginase constructs exhibited resistance to proteolysis with 65-83% of the initial enzyme activity still present after 6 h . In contrast, most of the native enzyme was inactivated under the same conditions . In vivo experiments with intravenously injected mice revealed a significant increase in the half-life of the polysialylated asparaginase over that observed with the native enzyme . Such an increase was greatest (250%, about 38 h) for the construct with the highest degree of polysialylation . Results suggest that polysialylation of asparaginase and other proteins may provide an alternative means to improve their effective use in therapeutics. Biochemistry, 1997 Aug 19, 36(33), 10067 - 72 In vitro and in vivo redox states of the Escherichia coli periplasmic oxidoreductases DsbA and DsbC; Joly JC et al.; DsbC is a periplasmic protein of Escherichia coli that was previously identified by a genetic selection that rescued sensitivity to dithiothreitol in Tn10 mutagenized cells . The Erwinia chrysanthemi dsbC gene was identified in a previous genetic screen to restore motility in a dsbA null strain . In order to analyze the biochemical role of E . coli DsbC, the protein was overexpressed, purified, and compared with DsbA in terms of disulfide isomerization, thiol oxidation, and in vivo redox state . In vitro, DsbC and DsbA have an equivalent kcat for disulfide isomerization with the model substrate, misfolded insulin-like growth factor-1 . However, DsbA is a more effective oxidant than DsbC of protein dithiols . In vivo, DsbA is found exclusively in the oxidized state in wild-type strains grown in rich media . On the other hand, in vivo DsbC has one pair of cysteines oxidized and one pair reduced . DsbD is required to maintain this reduced pair of cysteines, confirming previous genetic results . A dsbC deletion strain showed decreases in the production of some, but not all, heterologous proteins containing multiple disulfide bonds . Notably, those proteins affected by the dsbC deletion do not have the cysteines paired consecutively. J Bacteriol, 1997 Aug, 179(15), 4909 - 18 Characterization of the pecT control region from Erwinia chrysanthemi 3937; Castillo A et al.; Erwinia chrysanthemi synthesizes and secretes pectate lyases that attack components of the plant cell wall and, therefore, play a major role in the pathogenesis of soft rot disease . We isolated a new mutant (designated pec-1), by Tn5 mutagenesis, that displays weak pectate lyase production and decreased motility and mucoidicity . Maceration and pathogenicity tests done on different plant organs showed that the pec-1 strain displays a reduced virulence compared to that of the parental strain . The Tn5 insertion was localized between the pelL and the out loci and defines a new regulatory region . Sequencing of the pec-1::Tn5 insertion revealed that pec-1 is tightly linked to the pecT regulatory gene that also controls pectate lyase synthesis . Moreover, the pecT mutation is dominant over the pec-1 mutation, suggesting that these two loci are involved in the same regulatory network . We demonstrated, by Northern blot analysis, that the pec-1::Tn5 insertion provokes derepression of pecT transcription and defines a cis-acting element . Introduction of the pecT gene in trans of a pecT::uidA fusion induced a decrease of pecT::uidA transcription, indicating a negative autoregulation . Band shift experiments confirmed that the PecT repressor specifically interacts with the pecT regulatory region . We also demonstrated that the PecT protein interacts with the regulatory region of the pelD gene encoding a pectate lyase . Therefore, the abolition of the pecT autoregulation in the pec-1 mutant provokes an overproduction of the PecT repressor that is responsible for the decrease of pectate lyase synthesis . Mutagenesis of the pecT regulatory region revealed the presence of two sites in which insertions reproduced the pec-1 phenotype . This result suggests that pecT autoregulation requires the presence of two functional operator sites . From this study, we propose that the PecT repressor binds to these two sites, generating a loop that blocks pecT transcription. J Bacteriol, 1997 Aug, 179(15), 4754 - 60 Lipase secretion by bacterial hybrid ATP-binding cassette exporters: molecular recognition of the LipBCD, PrtDEF, and HasDEF exporters; Akatsuka H et al.; Serratia marcescens secretes several proteins, such as the lipase LipA, the metalloprotease PrtA, and the heme-binding protein HasA, which is required for heme acquisition, through two N-terminal signal peptide-independent systems that are classified as bacterial ATP-binding cassette (ABC) exporters . One is the ABC exporter for HasA, consisting of the ABC protein HasD, the membrane fusion protein (MFP) HasE, and the outer membrane protein (OMP) HasF . The second, composed of LipB (an ABC protein), LipC (an MFP), and LipD (an OMP), promotes secretion of LipA and PrtA in Escherichia coli recombinant clones . PrtA, which shows homology to the Erwinia chrysanthemi metalloproteases, is efficiently secreted by E . coli cells carrying the E . chrysanthemi ABC exporter PrtD (ABC protein)-PrtE (MFP)-PrtF (OMP) . The existence of distinct systems in this bacterium and of various substrates for these systems allowed the study of protein secretion by heterologous Has, Lip, and Prt systems and by Has-Lip and Lip-Prt hybrid exporters in the genuine host as well as in E . coli . For that purpose, lipB-, lipC-, and lipD-deficient mutants were isolated from S . marcescens 8000 and their secretion of LipA and PrtA was analyzed . This demonstrated that a unique exporter, the Lip apparatus, in S . marcescens secretes both LipA and PrtA . Hybrid exporters were tested for secretion of HasA and LipA . The LipB-HasE-HasF exporter allowed secretion of LipA but not HasA, showing that the ABC protein LipB is responsible for the substrate specificity . LipA, HasA, and E . chrysanthemi PrtC were secreted via heterologous exporters and via some hybrid exporters . Analysis of secretion via hybrid exporters showed that specific interactions occur between MFPs and OMPs in these systems . These genetic experiments demonstrated that specific interactions between the ABC protein and the MFP are required for the formation of active exporters. J Biol Chem, 1997 Jul 11, 272(28), 17502 - 10 The RNA molecule CsrB binds to the global regulatory protein CsrA and antagonizes its activity in Escherichia coli; Liu MY et al.; The RNA-binding protein CsrA (carbon storage regulator) is a new kind of global regulator, which facilitates specific mRNA decay . A recombinant CsrA protein containing a metal-binding affinity tag (CsrA-H6) was purified to homogeneity and authenticated by N-terminal sequencing, matrix-assisted laser desorption/ionization time of flight mass spectrometry, and other studies . This protein was entirely contained within a globular complex of approximately 18 CsrA-H6 subunits and a single approximately 350-nucleotide RNA, CsrB . cDNA cloning and nucleotide sequencing revealed that the csrB gene is located downstream from syd in the 64-min region of the Escherichia coli K-12 genome and contains no open reading frames . The purified CsrA-CsrB ribonucleoprotein complex was active in regulating glg (glycogen biosynthesis) gene expression in vitro, as was the RNA-free form of the CsrA protein . Overexpression of csrB enhanced glycogen accumulation in E . coli, a stationary phase process that is repressed by CsrA . Thus, CsrB RNA is a second component of the Csr system, which binds to CsrA and antagonizes its effects on gene expression . A model for regulatory interactions in Csr is presented, which also explains previous observations on the homologous system in Erwinia carotovora . A highly repeated nucleotide sequence located within predicted stem-loops and other single-stranded regions of CsrB, CAGGA(U/A/C)G, is a plausible CsrA-binding element. Int J Biol Macromol, 1997 Jul, 20(4), 251 - 8 The solution molecular weight and shape of the bacterial exopolysaccharides amylovoran and stewartan; Jumel K et al.; Amylovoran, the acidic exopolysaccharide (EPS) of Erwinia amylovora, and stewartan, the capsular EPS of E . stewartii, were characterized by analytical ultracentrifugation and by size exclusion chromatography connected to dual detection of light scattering and mass . The average molecular weights of amylovoran and stewartan were determined as 1.0 x 10(6) and 1.7 x 10(6) Da, with polydispersity values (Mw/M(n)) of 1.5 and 1.4, respectively . Based on the sugar composition and their molecular weight, both exopolysaccharides consist of approximately 1000 repeating units per molecule, this suggests a similar mechanism for chain length determination during biosynthesis of EPS in both organisms. J Appl Microbiol, 1997 Jul, 83(1), 17 - 24 Analysis of conductance responses during depolymerization of pectate by soft rot Erwinia spp . and other pectolytic bacteria isolated from potato tubers; Fraaije BA et al.; Different bacteria isolated from potato tubers were screened for their pectolytic properties by examining pitting in polypectate agar, recording conductance responses in polypectate medium and performing potato tuber soft rot tests . For bacteria found positive in conductimetry, the role of polygalacturonase (PG) and pectate lyase (PL) in the generation of conductance changes in a polygalacturonic acid (PGA) medium was further analysed using enzyme activity staining after gel electrophoresis and high-performance anion exchange chromatography . The extent of the conductance changes during depolymerization of PGA was dependent on the amounts of galacturonate monomers and oligomers accumulated in the medium . In comparison with an unidentified saprophyte and a Klebsiella strain, both mainly having PL activity, soft rot Erwinia spp . rapidly produced larger conductance responses, due to a combined action of multiple forms of PG and PL . The responses of Erwinia spp . were initially associated with the accumulation of large amounts of monomers and saturated dimers to heptamers, due to PG activity . Subsequently, as well as monomers and saturated dimers, large amounts of unsaturated dimers were also detected, due to PL activity . The role of PG as an important conductimetric factor was also demonstrated for a pectinase preparation derived from Aspergillus niger . Besides detection, automated conductimetric assays in pectate media may also be useful for monitoring of pectolytic activity in pectinase preparations and for screening of pectolytic activity of microorganisms under different media and growth conditions. Plant Physiol, 1997 Jul, 114(3), 1071 - 6 A cDNA clone highly expressed in ripe banana fruit shows homology to pectate lyases; Dominguez-Puigjaner E et al.; A cDNA clone (Ban17), encoding a protein homologous to pectate lyase, has been isolated from a cDNA library from climacteric banana fruit by means of differential screening . Northern analysis showed that Ban17 mRNA is first detected in early climacteric fruit, reaches a steady-state maximum at the climacteric peak, and declines thereafter in overripe fruit . Accumulation of the Ban17 transcript can be induced in green banana fruit by exogenous application of ethylene . The demonstrates that expression of this gene is under hormonal control, its induction being regulated by the rapid increase in ethylene production at the onset of ripening . The deduced amino acid sequence derived from the Ban17 cDNA shares significant identity with pectate lyases from pollen and plant pathogenic bacteria of the genus Erwinia . Similarity to bacterial pectate lyases that were proven to break down the pectic substances of the plant cell wall suggest that Ban17 might play a role in the loss of mesocarp firmness during fruit ripening. Mol Plant Microbe Interact, 1997 Jul, 10(5), 677 - 82 The presence of hrp genes on the pathogenicity-associated plasmid of the tumorigenic bacterium Erwinia herbicola pv . gypsophilae; Nizan R et al.; The pathogenicity-associated plasmid (pPATH) of Erwinia herbicola pv . gypsophilae (Ehg), which is present only in pathogenic strains, contains a gene cluster encoding indole-3-acetic acid and cytokinin biosynthesis . The transposon-reporter Tn3-Spice was used to generate nonpathogenic mutants on two overlapping cosmids, pLA150 and pLA352, of the pPATH . A cluster of such mutations, which spanned 16 kb, mapped approximately 15 kb from the gene cluster involved in phytohormone biosynthesis . Non-pathogenic mutants also failed to elicit the hypersensitive reaction (HR) on tobacco . Pathogenicity and HR were restored concomitantly to these mutants by in trans complementation with wild-type Ehg DNA . A 3.8-kb HindIII DNA fragment that complemented the hrp mutants was sequenced and six complete and two partial open reading frames (ORFs) were identified . Comparison of the deduced amino acid sequences of the eight ORFs showed striking homology and co-linearity with hrp genes of E . amylovora as well as with other plant and mammalian pathogenic bacterial genes encoding proteins of the type III secretion system . Limited DNA sequencing at various sites on the remaining 11-kb region of pLA352 also showed high identity to Hrp proteins of E . amylovora, E . stewartii, and Pseudomonas syringae . These results suggest that hrp genes are mandatory for gall formation by E . herbicola pv . gypsophilae. Mol Plant Microbe Interact, 1997 Jul, 10(5), 635 - 45 Characterization of acquired resistance in lesion-mimic transgenic potato expressing bacterio-opsin; Abad MS et al.; The lesion-mimic mutants of certain plants display necrotic lesions resembling those of the hypersensitive response and activate local and systemic defense responses in the absence of pathogens . We have engineered a lesion-mimic phenotype in transgenic Russet Burbank potato plants through constitutive expression of a bacterio-opsin (bO) proton pump derived from Halobacterium halobium . Transgenic potato plants exhibiting a lesion-mimic phenotype had increased levels of salicylic acid and overexpressed several pathogenesis-related messenger RNAs, all hallmarks of systemic acquired resistance (SAR) . The lesion-mimic plants also displayed enhanced resistance to the US1 isolate (A1 mating type) of a fungal pathogen, Phytophthora infestans, a causal agent of late blight disease . In contrast, little resistance was observed against the US8 isolate (A2 mating type) of this pathogen . Furthermore, a majority of the transgenic plants displaying the lesion-mimic phenotype had increased susceptibility to potato virus X . The tubers of these plants were not resistant to the bacterial pathogen Erwinia carotovora . These results indicate that expression of bO can result in the activation of defense responses in transgenic potato plants and show for the first time that bO expression can confer resistance to a pathogenic fungus . However, our results also demonstrate that like SAR, this "engineered" resistance is likely to be limited to certain pathogens and particular cultivars. Gene, 1997 Jun 11, 192(1), 39 - 43 Longus pilus of enterotoxigenic Escherichia coli and its relatedness to other type-4 pili--a minireview; Giron JA et al.; Longus is a long pilus produced by human enterotoxigenic Escherichia coli (ETEC) which shares significant structural and biochemical features with class-B type-4 pili . These pili include the toxin-coregulated pilus (TCP) of Vibrio cholerae, the bundle-forming pilus (BFP) of enteropathogenic E . coli and both longus and the colonization factor antigen III (CFA/III) of ETEC . These pili are produced under defined growth conditions indicating that they are under the control of different regulatory elements . While TCP is chromosomally encoded, the remaining pili are encoded on large virulence plasmids . Longus and CFA/III are closely related pili although certain DNA and protein differences also exist between them . This may account for the differences in the regulation, surface presentation, antigenicity, and prevalence of these two pilins among ETEC . Neighboring lngA, a second open reading frame termed lngB was found which encodes a protein with significant homology to proteins which are part of a type-II secretory system such as XcpV, OutC, and PulO of Pseudomonas aeruginosa, Erwinia chrysanthemi, and Klebsiella pneumoniae, respectively . This suggests that lngB may be an accessory gene involved in biogenesis of longus. Gene, 1997 Jun 11, 192(1), 7 - 11 Protein secretion by Gram-negative bacterial ABC exporters--a review; Binet R et al.; One of the strategies used by Gram-negative bacteria to secrete proteins across the two membranes which delimit the cells is sec-independent and dedicated to proteins lacking an N-terminal signal peptide . Most of these proteins display a C-terminal secretion signal located in the last 60 amino acids (aa) . Using one Erwinia chrysanthemi protease, PrtG, secreted by such a pathway it was shown that the smallest C-terminal sequence allowing efficient secretion contains the last 29 aa of PrtG and that low but significant secretion can be promoted by the last 15 aa of PrtG . Moreover, the extreme C-terminal motif, consisting of a negatively charged aa followed by several hydrophobic aa must be exposed and is conserved amongst many proteins following this pathway . This secretion system depends on ABC protein-mediated exporters, which consist of three cell envelope proteins: two inner membrane proteins, an ATPase (the ABC protein), a membrane fusion protein (MFP) and an outer membrane polypeptide . These Gram-negative bacterial protein exporters are dedicated to the secretion of one or several closely related proteins belonging to the toxin, protease and lipase families . The genes encoding the three secretion proteins and the exoproteins are usually all linked, consistent with the specificity of the systems . Er . chrysanthemi metalloproteases B and C and Serratia marcescens hemoprotein HasA are secreted by such homologous pathways and interact with the ABC protein . Interaction between the ABC protein and its substrate has also been evidenced by studies on protease and HasA hybrid transporters obtained by combining components from each system . Association between hemoprotein HasA and the three exporter secretion proteins was demonstrated by affinity chromatography on hemin agarose on which the substrate remained bound with the three secretion proteins . The three components' association was ordered and substrate binding was required for the formation of this multiprotein complex. EMBO J, 1997 Jun 2, 16(11), 3007 - 16 Specific interaction between OutD, an Erwinia chrysanthemi outer membrane protein of the general secretory pathway, and secreted proteins; Shevchik VE et al.; OutD is an outer membrane component of the main terminal branch of the general secretory pathway (GSP) in Erwinia chrysanthemi . We analyzed the interactions of OutD with other components of the GSP (Out proteins) and with secreted proteins (PelB, EGZ and PemA) . OutD is stabilized by its interaction with another GSP component, OutS . The 62 C-terminal amino acids of OutD are necessary for this interaction . In vivo formation of OutD multimers, up to tetramers, was proved after the dissociation in mild conditions of the OutD aggregates formed in the outer membrane . Thus, OutD could form a channel-like structure in the outer membrane . We showed that OutD is stabilized in vivo when co-expressed with Out-secreted proteins . This stabilization results from the formation of complexes that were detected in experiments of co-immunoprecipitation and co-sedimentation in sucrose density gradients . The presence of the N-terminal part of OutD is required for this interaction . The interaction between OutD and the secreted protein PelB was confirmed in vitro, suggesting that no other component of the GSP is required for this recognition . No interaction was observed between the E . carotovora PelC and the E . chrysanthemi OutD . Thus, the interaction between GspD and the secreted proteins present in the periplasm could be the key to the specificity of the secretion machinery and a trigger for that process. Mol Microbiol, 1997 Jun, 24(6), 1285 - 301 Identification of a bacterial pectin acetyl esterase in Erwinia chrysanthemi 3937; Shevchik VE et al.; Erwinia chrysanthemi causes soft-rot diseases of various plants by enzymatic degradation of the pectin in plant cell walls . The structural complexity of pectin requires the combined action of several pectinases for its efficient breakdown . Three types of pectinases have so far been identified in E . chrysanthemi: two pectin methyl esterases (PemA, PemB), a polygalacturonase (PehX), and eight pectate lyases (PelA, PelB, PelC, PelD, PelE, PelL, PelZ, PelX) . We report in this paper the analysis of a novel enzyme, the pectin acetyl esterase encoded by the paeY gene . No bacterial form of pectin acetyl esterases has been described previously, while plant tissues and some pectinolytic fungi were found to produce similar enzymes . The paeY gene is present in a cluster of five pectinase-encoding genes, pelA-pelE-pelD-paeY-pemA . The paeY open reading frame is 1650 bases long and encodes a 551-residue precursor protein of 60704Da, including a 25-amino-acid signal peptide . PaeY shares one region of homology with a rhamnogalacturonan acetyl esterase of Aspergillus aculeatus . To characterize the enzyme, the paeY gene was overexpressed and its protein product was purified . PaeY releases acetate from sugar-beet pectin and from various synthetic substrates . Moreover, the enzyme was shown to act in synergy with other pectinases . The de-esterification rate by PaeY increased after previous demethylation of the pectins by PemA and after depolymerization of the pectin by pectate lyases . In addition, the degradation of sugar-beet pectin by pectate lyases is favoured after the removal of methyl and acetyl groups by PemA and PaeY, respectively . The paeY gene was first identified on the basis of its regulation, which shares several characteristics with that of other pectinases . Analysis of the paeY transcription, using gene fusions, revealed that it is induced by pectic catabolic products and is affected by growth phase, oxygen limitation and catabolite repression . Regulation of paeY expression appears to be dependent on the KdgR repressor, which controls all the steps of pectin catabolism, and on the catabolite regulatory protein (CRP), the global activator of sugar catabolism . The contiguous pelD, paeY and pemA genes are transcribed as an operon from a promoter proximal to pelD which allows the regulation by KdgR and CRP . However, transcription can be interrupted at the intra-operon Rho-independent terminator situated between pelD and paeY . The paeY mutant inoculated into Saintpaulia plants was less invasive than the wild-type E . chrysanthemi strain 3937, demonstrating the important role of PaeY in the soft-rot disease. Biochem J, 1997 Jun 1, 324 ( Pt 2), 421 - 6 Expression of an exogenous isopentenyl diphosphate isomerase gene enhances isoprenoid biosynthesis in Escherichia coli; Kajiwara S et al.; Escherichia coli expressing the Erwinia carotenoid biosynthesis genes, crtE, crtB, crtI and crtY, form yellow-coloured colonies due to the presence of beta-carotene . This host was used as a visible marker for evaluating regulatory systems operating in isoprenoid biosynthesis of E . coli . cDNAs enhancing carotenoid levels were isolated from the yeast Phaffia rhodozyma and the green alga Haematococcus pluvialis . Nucleotide sequence analysis indicated that they coded for proteins similar to isopentenyl diphosphate (IPP) isomerase of the yeast Saccharomyces cerevisiae . Determination of enzymic activity confirmed the identity of the gene products as IPP isomerases . The corresponding gene was isolated from the genomic library of S . cerevisiae based on its nucleotide sequence, and was confirmed to have the same effect as the above two IPP isomerase genes when introduced into the E . coli transformant accumulating beta-carotene . In the three E . coli strains carrying the individual exogenous IPP isomerase genes, the increases in carotenoid levels are comparable to the increases in IPP isomerase enzyme activity with reference to control strains possessing the endogenous gene alone . These results imply that IPP isomerase forms an influential step in isoprenoid biosynthesis of the prokaryote E . coli, with potential for the efficient production of industrially useful isoprenoids by metabolic engineering. Appl Environ Microbiol, 1997 Jun, 63(6), 2258 - 65 Detection of RTX toxin genes in gram-negative bacteria with a set of specific probes; Kuhnert P et al.; The family of RTX (RTX representing repeats in the structural toxin) toxins is composed of several protein toxins with a characteristic nonapeptide glycine-rich repeat motif . Most of its members were shown to have cytolytic activity . By comparing the genetic relationships of the RTX toxin genes we established a set of 10 gene probes to be used for screening as-yet-unknown RTX toxin genes in bacterial species . The probes include parts of apxIA, apxIIA, and apxIIIA from Actinobacillus pleuropneumoniae, cyaA from Bordetella pertusis, frpA from Neisseria meningitidis, prtC from Erwinia chrysanthemi, hlyA and elyA from Escherichia coli, aaltA from Actinobacillus actinomycetemcomitans and lktA from Pasteurella haemolytica . A panel of pathogenic and nonpathogenic gram-negative bacteria were investigated for the presence of RTX toxin genes . The probes detected all known genes for RTX toxins . Moreover, we found potential RTX toxin genes in several pathogenic bacterial species for which no such toxins are known yet . This indicates that RTX or RTX-like toxins are widely distributed among pathogenic gram-negative bacteria . The probes generated by PCR and the hybridization method were optimized to allow broad-range screening for RTX toxin genes in one step . This included the binding of unlabelled probes to a nylon filter and subsequent hybridization of the filter with labelled genomic DNA of the strain to be tested . The method constitutes a powerful tool for the assessment of the potential pathogenicity of poorly characterized strains intended to be used in biotechnological applications . Moreover, it is useful for the detection of already-known or new RTX toxin genes in bacteria of medical importance. J Bacteriol, 1997 Jun, 179(11), 3500 - 8 The cyclic AMP receptor protein is the main activator of pectinolysis genes in Erwinia chrysanthemi; Reverchon S et al.; The main virulence factors of the phytopathogenic bacterium Erwinia chrysanthemi are pectinases that cleave pectin, a major constituent of the plant cell wall . Although physiological studies suggested that pectinase production in Erwinia species is subjected to catabolite repression, the direct implication of the cyclic AMP receptor protein (CRP) in this regulation has never been demonstrated . To investigate the role of CRP in pectin catabolism, we cloned the E . chrysanthemi crp gene by complementation of an Escherichia coli crp mutation and then constructed E . chrysanthemi crp mutants by reverse genetics . The carbohydrate fermentation phenotype of the E . chrysanthemi crp mutants is similar to that of an E . coli crp mutant . Furthermore, these mutants are unable to grow on pectin or polygalacturonate as the sole carbon source . Analysis of the nucleotide sequence of the E . chrysanthemi crp gene revealed the presence of a 630-bp open reading frame (ORF) that codes for a protein highly similar to the CRP of E . coli . Using a crp::uidA transcriptional fusion, we demonstrated that the E . chrysanthemi CRP represses its own expression, probably via a mechanism similar to that described for the E . coli crp gene . Moreover, in the E . chrysanthemi crp mutants, expression of pectinase genes (pemA, pelB, pelC, pelD, and pelE) and of genes of the intracellular part of the pectin degradation pathway (ogl, kduI, and kdgT), which are important for inducer formation and transport, is dramatically reduced in induced conditions . In contrast, expression of pelA, which encodes a pectate lyase important for E . chrysanthemi pathogenicity, seems to be negatively regulated by CRP . The E . chrysanthemi crp mutants have greatly decreased maceration capacity in potato tubers, chicory leaves, and celery petioles as well as highly diminished virulence on saintpaulia plants . These findings demonstrate that CRP plays a crucial role in expression of the pectinolysis genes and in the pathogenicity of E . chrysanthemi. Mol Microbiol, 1997 May, 24(4), 803 - 14 Mutual control of the PecS/PecM couple, two proteins regulating virulence-factor synthesis in Erwinia chrysanthemi; Praillet T et al.; The Erwinia chrysanthemi pecS mutant displays constitutive production of virulence factors, such as pectinases or cellulases . Complementation of the pecS mutation can be obtained in the presence of the pecS wild-type gene on a low-copy-number plasmid . Moreover, the resulting plasmid decreases the expression of a pecS::uidA chromosomal fusion, indicating the existence of an autoregulation mechanism . This negative autoregulation was confirmed and quantified by analysis of the pecS transcripts using primer-extension experiments . Band-shift assays and DNase I footprinting experiments demonstrated that the PecS protein could bind to the intergenic regulatory region, located between the pecS and pecM genes, with a relatively high affinity (apparent dissociation constant (K'{d}) close to 4nM) . These PecS-binding sites overlap the pecS and pecM promoters . The comparison of these new PecS-binding sites with those previously characterized on the target genes confirms the absence of a consensus . This observation was in accordance with the results of the missing-contact experiments performed on the pecS-pecM intergenic regulatory region and the celZ operator . Concurrently, we demonstrated that the PecS protein negatively controls the expression of the divergently transcribed pecM gene located 400bp upstream from the pecS gene . By following the efficiency of pecS autoregulation in a double E . chrysanthemi pecM-pecS mutant, we established that the PecM protein potentiates PecS activity in vivo. J Pharm Pharmacol, 1997 May, 49(5), 472 - 7 Investigation of physicochemical changes to L-asparaginase during freeze-thaw cycling; Jameel F et al.; L-Asparaginase derived from Erwinia chrysanthemi which is being investigated as an alternative to E . coli for the treatment of lymphoblastic leukaemia has been found in our laboratory to lose activity upon exposure to consecutive freeze-thaw cycles . An investigation was undertaken using several techniques to characterize fully the physicochemical changes L-Asparaginase is undergoing during freeze-thaw cycling leading to the loss of its activity . A total protein assay suggested that the loss of some enzyme activity was a result of protein precipitation . Circular dichroism (CD) studies showed a decrease of alpha-helical structure with a concomitant increase in beta sheet and random coil content, suggesting alterations in the secondary structure leading to unfolding, the first step of denaturation processes . The elution profiles obtained from size-exclusion chromatography (SEC) studies indicated the formation of several species during the process of freezing and thawing . Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) studies showed bands corresponding to 1-3 kDa and 32 kDa, suggesting that some of the species are fragments and shortened monomers resulting from cleavage of monomers . The molecular weight distribution obtained using SEC-linked light scattering indicated a substantial fraction of polydispersed fragments ranging from 900 Da to 3 kDa and a small fraction of aggregates corresponding to 300 kDa . A scheme was proposed to explain the cascade of events leading to the loss of soluble protein and accompanying loss of enzyme activity . Tetramers of the enzyme dissociate into monomers some of which are cleaved into small fragments . The shortened monomers then aggregate and precipitate. Ann Pharmacother, 1997 May, 31(5), 616 - 24 Pegaspargase: an alternative? Holle LM. OBJECTIVE: To review the chemistry, pharmacology, pharmacokinetics, clinical activity, adverse effects, dosage, and administration guidelines for pegaspargase . DATA SOURCES: A MEDLINE search (1980-1996), a CANCERLIT search (1983-1996), and a CURRENT CONTENTS search (1980-1996) using the terms pegaspargase, PEG-asparaginase, PEG-L-asparaginase, polyethylene glycol L-asparaginase, polyethylene glycol conjugated L-asparaginase, and Oncaspar were conducted . STUDY SELECTION AND DATA EXTRACTION: All articles were considered for possible inclusion in this review . Abstracts were included only when they were judged to add critical information not otherwise available in the medical literature . DATA SYNTHESIS: L-Asparaginase has been a main component of treatment regimens for acute lymphocytic leukemia . A key limiting factor of L-asparaginase use has been the development of hypersensitivity to the drug . Recently, a polyethylene glycol (PEG) conjugated form of L-asparaginase, pegaspargase, has been made available . PEG modification of L-asparaginase has been shown to alter the tendency of the enzyme to induce an immune response and to extend the half-life of the drug . The majority of patients with hypersensitivity to the native enzyme preparations tolerate pegaspargase without further clinical hypersensitivity . The adverse effect profile of pegaspargase is similar to that of the native forms of L-asparaginase . The recommended dosage of pegaspargase is 2500 IU/m2 administered by intramuscular or intravenous injection every 2 weeks in combination with other chemotherapeutic agents . CONCLUSIONS: Pegaspargase is a safe, effective alternative to L-asparaginase in patients who have had clinical hypersensitivity reactions to both Escherichia coli- and Erwinia carotovora-derived L-asparaginase . However, pegaspargase should not be routinely substituted for L-asparaginase. Mol Plant Microbe Interact, 1997 May, 10(4), 462 - 71 Molecular characterization and expression of the Erwinia carotovora hrpNEcc gene, which encodes an elicitor of the hypersensitive reaction; Mukherjee A et al.; The nucleotide sequence of hrpNEcc DNA, cloned from Erwinia carotovora subsp . carotovora strain Ecc71, reveals a coding region of 1,068 bp which matches the size of hrpNEcc transcripts . hrpNEcc is predicted to encode a glycine-rich protein of approximately 36 kDa . Like the elicitors of the hypersensitive reaction (HR) produced by E . chrysanthemi (HarpinEch) and E . amylovora (HarpinEa), the deduced 36-kDa protein does not possess a typical signal sequence, but it contains a putative membrane-spanning domain . In Escherichia coli strains overexpressing hrpNEcc, the 36-kDa protein has been identified as the hrpNEcc product by Western blot analysis using anti-HarpinEch antibodies . The 36-kDa protein fractionated from E . coli elicits the HR in tobacco leaves . Moreover, a HrpN- and RsmA- double mutant (RsmA = regulator of secondary metabolites) does not produce this 36-kDa protein or elicit the HR, although this strain, like the RsmA- and HrpN+ bacteria, overproduces extracellular enzymes and macerates celery petioles . These observations demonstrate that hrpNEcc encodes the elicitor of the HR, designated HarpinEcc . The levels of hrpNEcc transcripts are affected in both RsmA+ and RsmA- strains by media composition and carbon sources, although the mRNA levels are substantially higher in the RsmA- strains . The expression of hrpNEcc in Ecc71 is cell density dependent and is activated by the quorum-sensing signal, N-(3-oxohexanoyl)-L-homoserine lactone (OHL) . By contrast, hrpNEcc expression in an RsmA- strain is independent of cell density, and substantial expression occurs in the absence of OHL . The effects of cultural conditions and the occurrence of putative cis-acting sequences, such as consensus sigma 54 promoters and an hrp promoter upstream of the transcriptional start site, indicate that the production of HarpinEcc in wild-type RsmA+ E . carotovora subsp . carotovora is tightly regulated . These observations, taken along with the finding that the HR is caused by RsmA- mutants but not by RsmA+ strains (Cui et al., 1996, Mol . Plant-Microbe Interact . 9:565-573), strongly support the idea that the inability of the wild-type pectolytic E . carotovora subsp . carotovora to elicit the HR is due to the lack of a significant level of HarpinEcc production. Gene, 1997 Apr 21, 189(2), 169 - 74 Cloning, sequencing and expressing the carotenoid biosynthesis genes, lycopene cyclase and phytoene desaturase, from the aerobic photosynthetic bacterium Erythrobacter longus sp . strain Och101 in Escherichia coli; Matsumura H et al.; Two genes which encode the enzymes lycopene cyclase and phytoene desaturase in the aerobic photosynthetic bacterium Erythrobacter longus sp . strain Och101 have been cloned and sequenced . The gene for lycopene cyclase, designated crtY, was expressed in a strain of Escherichia coli which contained the crtE, B, I and Z genes encoding geranylgeranyl pyrophosphate synthase, phytoene synthase, phytoene desaturase, and beta-carotene hydroxylase, respectively . As a result, zeaxanthin production was observed in E . coli transformants . In addition, expression of the E . longus gene crtI for phytoene desaturase in E . coli containing crtE and B resulted in the accumulation of lycopene in transformants . Zeaxanthin and lycopene were also determined by mass spectrum . Nucleotide sequence similarities between E . longus crtY gene and other microbial lycopene cyclase genes are 40.2% (Erwinia herbicola), 37.4% (Erwinia uredovora) and 22.9% (Synechococcus sp.), and those between phytoene desaturase genes are 50.3% (E . herbicola), 54.7% (E . uredovora) and 39.6% (Rhodobacter capsulatus). Microbiology, 1997 Apr, 143 ( Pt 4), 1389 - 94 Identification of genes in Rhizobium leguminosarum bv . trifolii whose products are homologues to a family of ATP-binding proteins; Krol J et al.; The specific interaction between rhizobia and their hosts requires many genes that influence both early and late steps in symbiosis . Three new genes, designated prsD, prsE (protein secretion) and orf3, were identified adjacent to the exo133 mutation in a cosmid carrying the genomic DNA of Rhizobium leguminosarum bv . trifolii TA1 . The prsDE genes share significant homology to the genes encoding ABC transporter proteins PrtDE from Erwinia chrysanthemi and AprDE from Pseudomonas aeruginosa which export the proteases in these bacteria . PrsD shows at least five potential transmembrane hydrophobic regions and a large hydrophilic domain containing an ATP/GTP binding cassette . PrsE has only one potential transmembrane hydrophobic domain in the N-terminal part and is proposed to function as an accessory factor in the transport system . ORF3, like PrtF and AprF, has a typical N-terminal signal sequence but has no homology to these proteins . The insertion of a kanamycin resistance cassette into the prsD gene of the R . leguminosarum bv . trifolii TA1 wild-type strain created a mutant which produced a normal amount of exopolysaccharide but was not effective in the nodulation of clover plants. J Appl Microbiol, 1997 Apr, 82(4), 448 - 54 Mechanisms of biocontrol by Pantoea dispersa of sugar cane leaf scald disease caused by Xanthomonas albilineans; Zhang L et al.; Albicidins, a family of phytotoxins and antibiotics produced by Xanthomonas albilineans, are important in sugar cane leaf scald disease development . The albicidin detoxifying bacterium Pantoea dispersa (syn . Erwinia herbicola) SB1403 provides very effective biocontrol against leaf scald disease in highly susceptible sugar cane cultivars . The P . dispersa gene (albD) for enzymatic detoxification of albicidin has recently been cloned and sequenced . To test the role of albicidin detoxification in biocontrol of leaf scald disease, albD was inactivated in P . dispersa by site-directed mutagenesis . The mutants, which were unable to detoxify albicidin, were less resistant to the toxin and less effective in biocontrol of leaf scald disease than their parent strain . This indicates that albicidin detoxification contributes to the biocontrol capacity of P . dispersa against X . albilineans . Rapid growth and ability to acidify media are other characteristics likely to contribute to the competitiveness of P . dispersa against X . albilineans at wound sites used to invade sugar cane. Mol Plant Microbe Interact, 1997 Apr, 10(3), 407 - 15 Identification of a pathogenicity locus, rpfA, in Erwinia carotovora subsp . carotovora subsp . carotovora that encodes a two-component sensor-regulator protein; Frederick RD et al.; A mutant of Erwinia carotovora subsp . carotovora, AH2552, created by a Mud1 insertion was found to be reduced in plant pathogenicity and deficient in extracellular protease and cellulase activity, although it produced normal levels of pectate lyase and polygalacturonase . A cosmid clone, pEC462, was isolated from a wild-type E . carotovora subsp . carotovora DNA library that concomitantly restored pathogenicity and protease and cellulase activities of AH2552 to wild-type levels when present in trans . The genetic locus that was disrupted in AH2552 by insertion of Mud1 has been designated rpfA, for regulator of pathogenicity factors . Sequencing of the rpfA region identified an open reading frame of 2,787 bp, and the predicted 929-amino acid polypeptide shared high identity with several two-component sensor-regulator proteins: BarA from Escherichia coli, ApdA from Pseudomonas fluorescens, PheN from P . tolaasii, RepA from P . viridiflava, LemA from P . syringae pv . syringae, and RpfC from Xanthomonas campestris pv . campestris . The RpfA locus described in this study encodes a putative sensor kinase protein that is involved in both extracellular protease and cellulase production and the pathogenicity of E . carotovora subsp . carotovora on potato tubers. J Bacteriol, 1997 Apr, 179(8), 2503 - 11 Comparative analysis of the five major Erwinia chrysanthemi pectate lyases: enzyme characteristics and potential inhibitors; Tardy F et al.; In Erwinia chrysanthemi 3937, pectate lyase activity mainly results from the cumulative action of five major isoenzymes, PelA to PelE . Comparison of their amino acid sequences revealed two families, PelB-C and PelA-D-E . Molecular cloning permitted expression of the different pel genes in Escherichia coli and the isolation of each Pel independently from the other isoenzymes . We used similar experimental conditions to overproduce and purify the five Pels in a one-step chromatography method . We analyzed some of the basic enzymatic properties of these five isoenzymes . PelA has a low specific activity compared to the other four enzymes . PelB and PelC have a high affinity for their substrate: about 10-fold higher than the enzymes of the PelA-D-E group . The optimum pH is more alkaline for PelB and PelC (about 9.2) than for PelA, PelD, and PelE (from 8 to 8.8) . Below pH 7, activity was negligible for PelB and PelC, while PelA, PelD, and PelE retained 25 to 30% of their activities . The temperature optima were determined to be 50 degrees C for PelD and PelE, 55 degrees C for PelA, and 60 degrees C for PelB and PelC . Enzymes of the PelB-C group are more stable than those of the PelA-D-E group . Use of substrates presenting various degrees of methylation revealed that PelA, PelD, and PelE are active only for very low levels of methylation, while PelB and PelC are more active on partially methylated pectins (up to 22% for PelC and up to 45% for PelB) . Pectate lyases have an absolute requirement for Ca2+ ions . For the five isoenzymes, maximal activity was obtained at a Ca2+ concentration of 0.1 mM . None of the tested cations (Ba2+, Co2+, Cu2+, Mg2+, Mn2+, Sr2+, Zn2+) can substitute for Ca2+ . At a high concentration (1 mM), most of the divalent cations inhibited pectate lyase activity . In addition, we demonstrated that two compounds present in plant tissues, epicatechin and salicylic acid, inhibit the pectate lyases at a concentration of 0.2 mM. Plant Cell Physiol, 1997 Mar, 38(3), 357 - 61 Cloning and functional expression of a novel geranylgeranyl pyrophosphate synthase gene from Arabidopsis thaliana in Escherichia coli; Zhu XF et al.; A gene encoding a novel geranylgeranyl pyrophosphate (GGPP) synthase from Arabidopsis thaliana has been identified and termed GGPS5 . The gene has been sequenced and expressed in Escherichia coli . The deduced amino acid sequence showed 64.5% and 57.5% identify with a putative GGPP synthase from Arabidopsis and Capsicum annuum, respectively . GGPP enzymatic activity was detected in E . coli cells expressing the GGPS5 gene in two different ways . One was the direct measurement of GGPP synthase activity in cell extracts and the other was the yellow color production of cells when the GGPS5 gene was co-expressed with crtB, crtI, crtY and crtZ genes derived from Erwinia uredovora. Int J Clin Pharmacol Ther, 1997 Mar, 35(3), 96 - 8 Pharmacokinetics and drug monitoring of L-asparaginase treatment; Boos J; The enzyme L-asparaginase is an important component of the treatment protocols for Acute Lymphoblastic Leukemia (ALL) . This enzyme is derived from different biological sources (E . coli and Erwinia chrysanthemi) . An increasing number of hemorrhagic and thrombotic events prompted us to initiate a monitoring program for asparaginase treatment . Different asparaginase preparations were monitored in children on the ALL-BFM induction and reinduction treatment (10,000 U/m2 every 3-4 days) . The different preparations resulted in significantly different trough levels of parameters of asparaginase activity, asparagine depletion, and coagulation . Not even the two preparations from E . coli were interchangeable: In a subsequent study, a mere 2500 U/m2 of the E . coli preparation Asparaginase medac resulted in trough levels comparable to 10,000 U/m2 of Crasnitin . The Erwinia preparation Erwinase, however, did not maintain measurable trough levels at the protocol schedule . We conclude that different L-asparaginase preparations are not readily interchangeable and that changes in the preparation, dosage or schedule require careful observation and possibly pharmacokinetic monitoring. Microbiology, 1997 Mar, 143 ( Pt 3), 713 - 20 The general secretion pathway of Erwinia carotovora subsp . carotovora: analysis of the membrane topology of OutC and OutF; Thomas JD et al.; The out gene cluster of Erwinia carotovora subsp . carotovora (Ecc) encodes the proteins of the type II or general secretory pathway (GSP) apparatus which is required for secretion of pectinase and cellulase . In this study, fusions between Ecc out genes and the topology probe blaM were constructed . The ability of Out protein domains to export BlaM across the cytoplasmic membrane in both Escherichia coli and the cognate host was utilized to confirm the computer-predicted cytoplasmic membrane topology of OutC and OutF, When outC was fused to blaM, the resulting phenotype suggested that the majority of OutC is targeted to the periplasm, typical of a type II bitopic conformation in the cytoplasmic membrane . In contrast, for the outF gene product, three transmembrane regions were identified which connect a large N-terminal cytoplasmic domain, a smaller periplasmic domain, and a large cytoplasmic loop . Fusions between blaM and outD and outE were used to further substantiate the locations of these gene products in the outer membrane and the cytoplasm respectively . The data derived suggest that a number of the Out apparatus components possess domains in the cytoplasm and/or the periplasm with potential for protein-protein interactions which facilitate the secretion of periplasmic enzyme intermediates across the outer membrane to the external milieu. Microbiology, 1997 Mar, 143 ( Pt 3), 705 - 12 Activation of the Erwinia carotovora subsp . carotovora pectin lyase structural gene pnlA: a role for RdgB; Liu Y et al.; The activation of pectin lyase (Pnl) production in Erwinia carotovora subsp . carotovora strain 71 occurs upon DNA damage via a unique regulatory circuit involving recA, rdgA and rdgB . In a similar Pnl-inducible system reconstituted in Escherichia coli, the rdgB product was found to activate the expression of pnlA, the structural gene for pectin lyase . The kinetic data presented here also show that transcription of pnlA followed that of rdgB in Er . carotovora subsp . carotovora, indicating a temporal order of gene expression . By deletion analysis we have localized the promoter/regulatory region within a 66 bp DNA segment upstream of the pnlA transcriptional start site . This region contains the -10 consensus sequence but not the sequences corresponding to the E . coli -35 region . For DNA-binding studies, rdgB was overexpressed in E . coli and a 14 kDa polypeptide was identified as the gene product . RdgB from crude extracts or a purified preparation caused an identical gel mobility shift of a 164 bp DNA segment containing the pnlA promoter/regulatory region . Utilizing DNase I protection assay the RdgB-binding site was localized between nucleotides -29 and -56, i.e . overlapping the position of the putative -35 box . The findings reported here, taken along with our previous observation that the rdgE product is required for pnlA expression, establishes that rdgB encodes a transcriptional factor which specifically interacts with the pnlA promoter/regulatory region. J Bacteriol, 1997 Mar, 179(6), 1880 - 6 Extracellular melibiose and fructose are intermediates in raffinose catabolism during fermentation to ethanol by engineered enteric bacteria; Moniruzzaman M et al.; Contrary to general concepts of bacterial saccharide metabolism, melibiose (25 to 32 g/liter) and fructose (5 to 14 g/liter) accumulated as extracellular intermediates during the catabolism of raffinose (O-alpha-D-galactopyranosyl-1, 6-alpha-D-glucopyranosyl-beta-D-fructofuranoside) (90 g/liter) by ethanologenic recombinants of Escherichia coli B, Klebsiella oxytoca M5A1, and Erwinia chrysanthemi EC16 . Both hydrolysis products (melibiose and fructose) were subsequently transported and further metabolized by all three organisms . Raffinose catabolism was initiated by beta-fructosidase; melibiose was subsequently hydrolyzed to galactose and glucose by alpha-galactosidase . Glucose and fructose were completely metabolized by all three organisms, but galactose accumulated in the fermentation broth with EC16(pLOI555) and P2 . MM2 (a raffinose-positive E . coli mutant) was the most effective biocatalyst for ethanol production (38 g/liter) from raffinose . All organisms rapidly fermented sucrose (90 g/liter) to ethanol (48 g/liter) at more than 90% of the theoretical yield . During sucrose catabolism, both hydrolysis products (glucose and fructose) were metabolized concurrently by EC16(pLOI555) and P2 without sugar leakage . However, fructose accumulated extracellularly (27 to 28 g/liter) at early stages of fermentation with KO11 and MM2 . Sequential utilization of glucose and fructose correlated with a diauxie in base utilization (pH maintenance) . The mechanism of sugar escape remains unknown but may involve downhill leakage via permease which transports precursor saccharides or novel sugar export proteins . If sugar escape occurs in nature with wild organisms, it could facilitate the development of complex bacterial communities which are based on the sequence of saccharide catabolism and the hierarchy of sugar utilization. J Bacteriol, 1997 Mar, 179(5), 1690 - 7 The hrpA and hrpC operons of Erwinia amylovora encode components of a type III pathway that secretes harpin; Kim JF et al.; A 6.2-kb region of DNA corresponding to complementation groups II and III of the Erwinia amylovora hrp gene cluster was analyzed . Transposon mutagenesis indicated that the two complementation groups are required for secretion of harpin, an elicitor of the hypersensitive reaction . The sequence of the region revealed 10 open reading frames in two putative transcription units: hrpA, hrpB, hrcJ, hrpD, and hrpE in the hrpA operon (group III) and hrpF, hrpG, hrcC, hrpT, and hrpV in the hrpC operon (group II) . From promoter regions of the hrpA, hrpC, and hrpN operons, sequences similar to those of the HrpL-dependent promoters of Pseudomonas syringae pathovars were identified with a consensus sequence of 5'-GGAAC-N17-18-CACTNAA-3' . The protein products of seven genes, hrpA, hrcJ, hrpE, hrpF, hrpG, hrcC, and hrpV, were visualized with a T7 polymerase/promoter expression system . HrcC, HrcJ, and HrpT sequences contained potential signal peptides, and HrcC appeared to be envelope associated based on a TnphoA translational fusion . Comparison of deduced amino acid sequences indicated that many of the proteins are homologous to proteins that function in the type III protein secretion pathway . HrcC is a member of the YscC-containing subgroup in the PulD/pIV superfamily of outer membrane proteins . HrcJ is a member of a lipoprotein family that includes YscJ of Yersinia spp., MxiJ of Shigella flexneri, and NolT of Rhizobim fredii . Additional similarities were detected between HrpB and YscI and between HrpE and YscL . HrcJ and HrpE were similar to flagellar biogenesis proteins FliF and FliH, respectively . In addition, HrpA, HrpB, HrcJ, HrpD, HrpE, HrpF, and HrcC showed various degrees of similarity to corresponding proteins of P . syringae . Comparison of hrp clusters with respect to gene organization and similarity of individual proteins confirms that the hrp systems of E . amylovora and P . syringae are closely related to each other and distinct from those of Ralstonia (Pseudomonas) solanacearum and Xanthomonas campestris . Possible implications of extensive similarities between the E . amylovora and P . syringae hrp systems in pathogenesis mechanisms are discussed. J Bacteriol, 1997 Mar, 179(5), 1555 - 62 The lac operon of Lactobacillus casei contains lacT, a gene coding for a protein of the Bg1G family of transcriptional antiterminators; Alpert CA et al.; The 5' region of the lac operon of Lac