Gene, 1993 Dec 8, 134(2), 223 - 7 Cloning and expression of a gene encoding gelonin, a ribosome-inactivating protein from Gelonium multiflorum; Nolan PA et al.; A cDNA copy of the gel gene, encoding gelonin (Gel), has been cloned from the seeds of the Asian plant, Gelonium multiflorum . Gel is a type-I ribosome-inactivating protein which has been produced in Escherichia coli as a secreted protein under the transcriptional control of the Salmonella typhimurium araB promoter and linked to the pectate lyase (pelB) leader sequence from Erwinia carotovora . Recombinant, soluble Gel (re-Gel) can be recovered from the E . coli culture supernatant at a yield of greater than 1 mg/ml, and it inhibits protein synthesis in vitro to the same extent as the native protein isolated from plant seeds. Plant Mol Biol, 1993 Dec, 23(5), 1061 - 5 Isolation and characterization of pollen-specific maize genes with sequence homology to ragweed allergens and pectate lyases; Turcich MP et al.; A cDNA clone (Zm58.1) was isolated by differential screening from a cDNA library made to mature Zea mays pollen, and shown to be pollen-specific by RNA blot analysis . When this partial-length clone was used to probe a genomic library, a similar but distinct pollen-specific genomic clone (68% sequence identity) was isolated (Zm58.2) . The putative proteins coded for by these two clones show sequence homology to several flower-expressed gene products from various plant species, including known pollen allergens from short ragweed (Ambrosia artemisiifolia), and to pectate lyases from the plant pathogenic bacteria Erwinia spp . The two genes map to different chromosomes. J Bacteriol, 1993 Dec, 175(24), 7958 - 67 HrpI of Erwinia amylovora functions in secretion of harpin and is a member of a new protein family; Wei ZM et al.; HrpI, a 78-kDa protein, functions in the secretion of harpin, a proteinaceous elicitor of the hypersensitive response from Erwinia amylovora . The predicted amino acid sequence of HrpI is remarkably similar to that of LcrD of Yersinia species, the first member of a recently described protein family . Other proteins of the family are MixA from Shigella flexneri, InvA from Salmonella typhimurium, FlhA from Caulobacter crescentus, HrpI from Pseudomonas syringae pv . syringae, HrpO from Pseudomonas solanacearum, and HrpC2 from Xanthomonas campestris pv . vesicatoria . Cells of E . amylovora containing mutated hrpI genes or cells of Escherichia coli containing the cloned hrp gene cluster with mutated hrpI produce but do not export harpin . When similar cells with functional hrpI genes were grown at 25 degrees C, but not at 37 degrees C, harpin was exported to the culture supernatant . Direct evidence that HrpI is involved in the secretion of a virulence protein has been offered . Two other loci of the hrp gene cluster are involved in the regulation of harpin, and four other loci also are involved in the secretion of harpin . Since harpin and other proteins likely to be secreted by the LcrD family of proteins lack typical signal peptides, their secretion mechanism is distinct from the general protein export pathway. Plant J, 1993 Nov, 4(5), 833 - 40 Functional expression of the Erwinia uredovora carotenoid biosynthesis gene crtl in transgenic plants showing an increase of beta-carotene biosynthesis activity and resistance to the bleaching herbicide norflurazon; Misawa N et al.; Among the enzymes involved in carotenoid biosynthesis, phytoene desaturase is considered to be a rate-limiting enzyme in this pathway and is also the target of many bleaching herbicides . This enzyme shows diversity concerning its function and amino acid homology among various organisms . The phytoene desaturase gene crtl of Erwinia uredovora was expressed, the 5'-region of which was fused to the sequence for the transit peptide of a pea Rubisco small subunit, in tobacco plants under the control of the CaMV 35S promoter . This chimeric gene product was targeted into chloroplasts and processed in the transgenic plants . The production and processing of the corresponding protein could be demonstrated by Western blotting . Immunogold localization showed that the location of the gene product Crtl was preferentially in the thylakoids . A radioactive labeling study using the leaves demonstrated enhanced activity for the synthesis of beta-carotene . In addition, the transgenic tobacco acquired elevated resistance to the bleaching herbicide norflurazon. Mol Gen Genet, 1993 Nov, 241(3-4), 341 - 50 Molecular analysis of the major cellulase (CelV) of Erwinia carotovora: evidence for an evolutionary "mix-and-match" of enzyme domains; Cooper VJ et al.; The structural gene for the major cellulase of Erwinia carotovora subspecies carotovora (Ecc) was isolated and expressed in Escherichia coli . Sequencing of the gene (celV) revealed a typical signal sequence and two functional domains in the enzyme; a catalytic domain linked by a short proline/threonine-rich linker to a cellulose-binding domain (CBD) . The deduced amino acid sequence of the catalytic domain showed homology with cellulases of Family A, including enzymes from Bacillus spp . and Erwinia chrysanthemi CelZ, whereas the CBD showed homology with cellulases from several diverse families, supporting a "mix-and-match" hypothesis for evolution of this domain . Analysis of the substrate specificity of CelV showed it to be an endoglucanase with some exoglucanase activity . The pH optimum is about 7.0 and the temperature optimum about 42 degrees C . CelV is secreted by Ecc and by the taxonomically related Erwinia carotovora subspecies atroseptica (Eca) but not by E . coli . Overproduction of the enzyme from multicopy plasmids in Ecc appears to overload the secretory mechanism. J Bacteriol, 1993 Nov, 175(22), 7321 - 8 Identification of two components of the Serratia marcescens metalloprotease transporter: protease SM secretion in Escherichia coli is TolC dependent; Letoffe S et al.; The Serratia marcescens metalloprotease (protease SM) belongs to a family of proteins secreted from gram-negative bacteria by a signal peptide-independent pathway which requires a specific transporter consisting of three proteins: two in the inner membrane and one in the outer membrane . The prtDSM and prtESM genes encoding the two S . marcescens inner membrane components were cloned and expressed in Escherichia coli . Their nucleotide sequence revealed high overall homology with the two analogous inner membrane components of the Erwinia chrysanthemi protease secretion apparatus and lower, but still significant, homology with the two analogous inner membrane components of the E . coli hemolysin transporter . When expressed in E . coli, these two proteins, PrtDSM and PrtESM, allowed the secretion of protease SM only in the presence of TolC protein, the outer membrane component of the hemolysin transporter. Haematologica, 1993 Nov-Dec, 78(6 Suppl 2), 57 - 60 Erwinia- and E . coli-derived L-asparaginase have similar effects on hemostasis . Pilot study in 10 patients with acute lymphoblastic leukemia; Castaman G et al.; BACKGROUND . L-asparaginase (L-ase) affects the synthesis of several hemostatic factors, including the naturally occurring clotting inhibitors antithrombin III, protein C and S, and thromboembolic events are a well-recognized complication of the hypercoagulable state resulting from this treatment . Recently, in addition to the widely used E . Coli-derived L-ase, a new L-ase derived from Erwinia Carotovora has been introduced into clinical studies . This formulation seems to have lesser side-effects, but data on its effects on the hemostatic system are very limited . We report here the hemostatic changes observed in 10 adult patients with acute lymphoblastic leukemia (ALL) treated with Erwinia- or with E . Coli-derived L-ase . METHODS AND RESULTS . The decreases (percentages of basal level) of antithrombin III, protein C and plasminogen (functional assays) were similar in both groups of patients (about 50% of the basal value 6 or 8 days after starting L-ase) and so were the number of instances in which the level of these proteins fell below 70% of normal . On the contrary, the fibrinogen level (PT-derived method) was less severely decreased by Erwinia L-ase (34% of basal value for E . Coli L-ase versus 58% for Erwinia L-ase; P = 0.048) and there were more instances in which the fibrinogen level was less than 100 mg/dL than with E . Coli L-ase (40% versus 16%; P = 0.051) . Thrombin-AT-III complexes (ELISA) showed similar pattern during both treatments . Two patients treated with Erwinia L-ase had moderate hyperglycemia, requiring insulin treatment but not L-ase discontinuation . No allergic reaction or pancreatitis was observed . A patient in the Erwinia L-ase-treated group developed deep venous thrombosis on day 7 after the start of L-ase and was treated with subcutaneous heparin (12,500 U x 2/day), with prompt improvement . CONCLUSION . This study shows that the two L-ase formulations have similar effects on hemostasis . Patients treated with Erwinia L-ase may develop thrombotic symptoms, as already reported for E . Coli-L-ase. Gene, 1993 Oct 29, 133(1), 115 - 8 Characterization of the prtA and prtB genes of Erwinia chrysanthemi EC16; Boyd C et al.; Two tandem metalloprotease-encoding structural genes, prtA and prtB, were sequenced from Erwinia chrysanthemi EC16 . These were highly homologous to previously reported genes from the same bacteria, as well as to three other metalloprotease-encoding genes from enteric bacteria . The three tandem prt structural genes from strain EC16 were closely linked to a cluster of genes previously found to be essential for extracellular secretion of the metalloproteases. J Bacteriol, 1993 Oct, 175(19), 6082 - 8 Penicillin-binding proteins from Erwinia amylovora: mutants lacking PBP2 are avirulent; Milner JS et al.; Radiolabelled penicillin G was used to examine penicillin-binding proteins (PBPs) from Erwinia amylovora (OT1) . This procedure identified seven PBPs with molecular masses ranging from 22 to 83 kDa . E . amylovora PBPs were compared with those from Escherichia coli (JM101) and from two spherical, avirulent TnphoA mutants derived from OT1 . Radiolabelled penicillin G bound to only six proteins from the spherical mutants which lacked a 69-kDa PBP . The spherical mutants could be complemented by the cloned E . coli pbpA-rodA operon, which restored both cell shape and virulence to apple seedlings . This suggested that the E . amylovora 69-kDa PBP is probably the functional equivalent of the E . coli PBP2 protein . Southern blot analysis using the E . coli rodA and pbpA genes as radiolabelled probes showed that TnphoA had inserted into the E . amylovora equivalent of the E . coli rodA-pbpA operon . Southern blots to chromosomal DNAs of the two spherical mutants, using the cloned hrp and dsp genes from E . amylovora as radiolabelled probes, confirmed that the TnphoA insertions were not located in the region of the E . amylovora chromosome postulated to encode known virulence factors . Both of the spherical TnphoA mutants synthesized amounts of extracellular polysaccharide equivalent to those synthesized by the wild-type strain (OT1), were resistant to lysis in distilled water and to lysozyme, and elicited the hypersensitive response on nonhost plants . These results indicate a possible role for cell shape in the virulence of this plant pathogen. Arch Biochem Biophys, 1993 Oct, 306(1), 152 - 7 Purification and enzymatic characterization of the geranylgeranyl pyrophosphate synthase from Erwinia uredovora after expression in Escherichia coli; Wiedemann M et al.; Geranylgeranyl pyrophosphate (GGPP) synthase from Erwinia uredovora was overexpressed in Escherichia coli and purified to homogeneity from solubilized inclusion bodies . In this protein the first 13 N-terminal amino acids were replaced by 16 other amino acids resulting from the cloning vector pUC18 . Nevertheless, the enzyme showed activity after purification which could be stimulated sixfold by appropriate activation conditions . The homogeneous enzyme was used to study substrate and product specificity as well as to determine Km values for isopentenyl pyrophosphate, dimethylallyl pyrophosphate (DMAPP), geranyl pyrophosphate (GPP), and farnesyl pyrophosphate (FPP) . Reaction rates and Km values indicate that FPP and GPP are the genuine allylic substrates for GGPP synthase, but not DMAPP . Independent of the allylic substrate employed, GGPP was the only reaction product of the enzymatic reaction. Gene, 1993 Sep 6, 131(1), 17 - 25 Characterization and overexpression of the pem gene encoding pectin methylesterase of Erwinia chrysanthemi strain 3937; Laurent F et al.; The pem gene encoding the pectin methylesterase (PME) of Erwinia chrysanthemi strain 3937 was subcloned and its nucleotide sequence determined . The gene contains an open reading frame of 1098 bp and codes for a protein of 366 amino acids (aa) . The mature 37-kDa form of the protein is 342 aa long and has a calculated isoelectric point of 9.64 . A plasmid was constructed to overproduce PME: a DNA fragment carrying pem was amplified by the polymerase chain reaction and cloned downstream from the pL promoter of the lambda phage, in a high-copy-number plasmid . In an Escherichia coli strain transformed with this plasmid, an increase in PME production of more than 60-fold was obtained, compared with the wild-type Er . chrysanthemi strain . PME represents about 5% of the total protein content of the cells . Comparison of this PME sequence with six PMEs from prokaryotic or eukaryotic organisms showed six highly conserved segments whose possible role in enzyme activity are discussed. J Clin Oncol, 1993 Sep, 11(9), 1780 - 6 Comparative pharmacokinetic studies of three asparaginase preparations; Asselin BL et al.; PURPOSE: As part of pharmacologic studies of asparaginase (ASNase), we determined the half-life of ASNase activity and protein, and the effect of dose, repeated doses, different drug preparations, and hypersensitivity reactions on the half-life (t1/2) of serum ASNase activity . PATIENTS AND METHODS: We measured ASNase activity (spectrophotometric assay) in serum samples obtained from patients with acute lymphoblastic leukemia (ALL) at various times during their therapy with intramuscular ASNase . ASNase protein was measured by enzyme-linked immunoadsorbent assay (ELISA) . RESULTS: Studies following the initial dose of Escherichia coli-derived ASNase demonstrated no difference in apparent t1/2 following 25,000 IU/m2 versus 2,500 IU/m2 (1.24 v 1.35 days, P = .2) . The apparent t1/2s following maintenance doses of E coli ASNase (middle dose t1/2, 1.28 days, or last dose t1/2, 1.14 days) showed no difference when compared with the initial dose of ASNase (P = .3 to .9) . There was no significant difference between the apparent t1/2s of ASNase activity and ASNase protein (n = 8, P = .2 to .6) . The serum t1/2 was 0.65 and 5.73 days for patients receiving Erwinia or polyethylene glycol (PEG)-modified E coli ASNase, respectively, as the induction dose . ASNase activity was undetectable in sera of four patients studied in the week following an anaphylactic reaction to E coli ASNase and the t1/2 was significantly shorter in five patients with a history of allergic reaction to E coli ASNase who were studied following a dose of PEG ASNase, (t1/2, 1.80 days) . CONCLUSION: We conclude that (1) the apparent t1/2 of ASNase is dependent on enzyme preparation used, but is not affected by dose or by repeated use; (2) the apparent t1/2 of E coli ASNase as a protein is the same as the apparent t1/2 of enzymatic activity; and (3) patients who have had a hypersensitivity reaction to E coli ASNase have a decreased apparent t1/2 with both E coli and PEG ASNase. Mol Plant Microbe Interact, 1993 Sep-Oct, 6(5), 553 - 64 Nucleotide sequence and properties of the hrmA locus associated with the Pseudomonas syringae pv . syringae 61 hrp gene cluster; Heu S et al.; The hrmA locus, isolated from Pseudomonas syringae pv . syringae 61, is essential for phenotypic expression of the P . s . pv . syringae 61 hrp cluster in Escherichia coli strains and enables bacteria carrying the hrp/hrm gene cluster to elicit the hypersensitive response (HR) associated with plant disease resistance . The phenotype of P . s . pv syringae 61 hrmA mutants (pathogenicity+, delayed HR) was distinct from that of hrp mutants . The locus was localized to a 3.6-kb BamH1-EcoR1 fragment whose nucleotide sequence was determined . A single open reading frame was identified that encodes for a 41,457-Da protein of unknown biochemical function . Production of the deduced protein product was confirmed by using T7 RNA polymerase-directed expression of the locus and N-terminal sequence analysis of the isolated HrmA . The deduced protein product did not exhibit homology with any of the characterized avr genes or the hrpN product of Erwinia amylovora . Transcription was shown to initiate 37 nucleotides upstream of the translational start from an apparent sigma 70 promoter . Two hrp genes were shown to act as positive transcriptional factors for hrmA expression . Expression of hrmA in P . syringae pv . glycinea race 4 did not exhibit the phenotypic properties of an avr gene or HrpN, but suggested that this locus may serve a regulatory function . A homolog to hrmA was present in strains of only three of the 23 P . syringae pathovars tested. Appl Environ Microbiol, 1993 Sep, 59(9), 3070 - 5 Production of L-dihydroxyphenylalanine in Escherichia coli with the tyrosine phenol-lyase gene cloned from Erwinia herbicola; Foor F et al.; The gene (tutA) encoding tyrosine phenol-lyase from Erwinia herbicola was cloned into Escherichia coli, and fusions to the lac and tac promoters were constructed . The enzyme was expressed at high levels in E . coli in the presence of isopropyl-beta-D-thiogalactopyranoside or lactose as an inducer . L-Dihydroxyphenylalanine was synthesized in high yield from catechol, pyruvate, and ammonia by induced cells. Biochem Biophys Res Commun, 1993 Aug 31, 195(1), 259 - 63 Carotenoid-biosynthesis genes as a genetic marker for the purpose of gene cloning; Liu ST; A cloning vector pSL775 (7.0 kb) was constructed using the carotenoid-biosynthesis genes of Erwinia herbicola Eho13 (ATCC 53489) . This vector contained a ColE1 origin, an ampicillin resistant gene, and a total of 11 single cloning sites: Asp718, AvaI, BamHI, BanII, EcoRV, HindIII, KpnI, MluI, NcoI, NotI, and SmaI . Transforming the vector into an Escherichia coli strain could result in pink clones . On the other hand, insertion of a DNA fragment into one of these cloning sites resulted in nonpigmented clones . The color differential between the two types of colonies could be detected visually on agar medium after culturing the cells at 37 degrees C for 18 hours. FEBS Lett, 1993 Aug 16, 328(3), 275 - 9 A left-handed crossover involved in amidohydrolase catalysis . Crystal structure of Erwinia chrysanthemi L-asparaginase with bound L-aspartate; Miller M et al.; The crystal structure of L-asparaginase from Erwinia chrysanthemi in the presence and absence of L-aspartate was determined at 1.8 A resolution . Conserved residues in a left-handed crossover (a rare occurrence in protein structures) link pairs of dimers into the catalytically active tetrameric form of the enzyme . The structure of ErA containing bound aspartic acid shows that this unusual strand connectivity is an essential part of the active site architecture, responsible for releasing the product of the enzymatic hydrolysis . The orientation of the bound aspartate indicates for the first time a threonine residue as a catalytic nucleophile. Mol Microbiol, 1993 Aug, 9(3), 477 - 85 Erwinia stewartii WtsA, a positive regulator of pathogenicity gene expression, is similar to Pseudomonas syringae pv . phaseolicola HrpS; Frederick RD et al.; Erwinia stewartii contains a large cluster of wts genes that are required by this bacterium for pathogenicity on corn plants . Three complementation groups within the right half of this cluster, wtsA, wtsC, and wtsB, were previously identified . In this study, WtsA was found to be a positive activator of wtsB::lacZ expression . The wtsA locus was sequenced and a single open reading frame is present within the wtsA locus, which has the capacity to encode a 323 amino acid polypeptide . A corresponding 38 kDa protein was observed in Escherichia coli minicells containing the cloned wtsA gene . The predicted WtsA polypeptide has significant similarity to HrpS from Pseudomonas syringae pv . phaseolicola, as well as other members of the NtrC class of prokaryotic regulatory proteins . Similar to other genes activated by NtrC regulators, wtsB::lacZ expression in E . coli was dependent upon rpoN. J Appl Bacteriol, 1993 Aug, 75(2), 149 - 58 A note on the primary structure and expression of an Erwinia carotovora polygalacturonase-encoding gene (peh1) in Escherichia coli and Saccharomyces cerevisiae; Laing E et al.; A 1209-base pair (bp) DNA fragment containing the endopolygalacturonase-encoding gene (peh1) from Erwinia carotovora subsp . carotovora was amplified by the polymerase chain reaction (PCR) technique and expressed in Escherichia coli . The nucleotide sequence of the PCR product was determined and found to be highly homologous to the primary structures of other polygalacturonase-encoding genes . The peh1 DNA fragment encoding the mature polygalacturonase was inserted between two different yeast expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pAMS10 and pAMS11 . These YIp5-derived plasmids were transformed and stably integrated into the genome of a laboratory strain of Saccharomyces cerevisiae . Transcription initiation signals present in these expression-secretion cassettes were derived from the yeast alcohol dehydrogenase (ADC1P) or mating pheromone alpha-factor (MF alpha 1P) gene promoters . The transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T) . Secretion of polygalacturonase was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1S) . Northern blot analysis revealed the presence of peh1 mRNA in the yeast transformants and a polypectate agarose test was used to monitor polygalacturonase production. J Bacteriol, 1993 Aug, 175(15), 4729 - 37 The aroQ-encoded monofunctional chorismate mutase (CM-F) protein is a periplasmic enzyme in Erwinia herbicola; Xia T et al.; Enteric bacteria possess two species of chorismate mutase which exist as catalytic domains on the amino termini of the bifunctional PheA and TyrA proteins . In addition, some of these organisms possess a third chorismate mutase, CM-F, which exists as a small monofunctional protein . The CM-F gene (denoted aroQ) from Erwinia herbicola was cloned and sequenced for the first time . A strategy for selection by functional complementation in a chorismate mutase-free Escherichia coli background was devised by using a recombinant plasmid derivative of pUC18 carrying a Zymomonas mobilis tyrC insert which encodes cyclohexadienyl dehydrogenase . The aroQ gene is 543 bp in length, predicting a 181-residue protein product having a calculated molecular mass of 20,299 Da . The E . herbicola aroQ promoter is recognized by E . coli, and a putative sigma-70 promoter region was identified . N-terminal amino acid sequencing of the purified CM-F protein indicated cleavage of a 20-residue signal peptide . This was consistent with the monomeric molecular mass determined for the enzyme of about 18,000 Da . The native enzyme is a homodimer . The implied translocation of CM-F was confirmed by osmotic shock experiments which demonstrated a periplasmic location . Immunogold electron microscopy indicated a polar localization within the periplasm . Polyclonal antibody raised against E . herbicola CM-F did not cross-react with the CM-F protein from the closely related Serratia rubidaea, as well as from a number of other gram-negative bacteria . Furthermore, when the E . herbicola aroQ gene was used as a probe in Southern blot hybridizations with EcroRI digests of chromosomal DNA from S . rubidaea and other enteric organisms, no hybridization was detected at low stringency . Thus, the aroQ gene appears to be unusually divergent among closely related organisms . The deduced CM-F amino acid sequence did not exhibit compelling evidence for homology with the monofunctional chorismate mutase protein of Bacillus subtilis. Carbohydr Res, 1993 Jul 19, 245(2), 271 - 87 Structure of an extracellular polysaccharide produced by Erwinia chrysanthemi; Gray JS et al.; Erwinia chrysanthemi pv zeae strain SR260, a phytopathogen of corn, produced from lactose an acidic extracellular polysaccharide which was purified and found to consist of L-rhamnose, D-mannose, D-glucose, and D-glucuronic acid in the ratio of 3:1:1:1 . A combination of chemical (carboxyl-group reduction, methylation analysis, periodate oxidation, Smith degradation, and lithium-ethylenediamine degradation) and physical (1 and 2D NMR spectroscopy) methods revealed that the polysaccharide is composed of a hexasaccharide repeating unit 1: {formula: see text} Mol Microbiol, 1993 Jul, 9(2), 383 - 91 Isolation and characterization of hydroxylamine-induced mutations in the Erwinia herbicola ice nucleation gene that selectively reduce warm temperature ice nucleation activity; Gurian-Sherman D et al.; Cells of ice nucleation active bacterial species catalyse ice formation over the temperature range of -2 to -12 degrees C . Current models of ice nucleus structure associate the size of ice nucleation protein aggregates with the temperature at which they catalyse ice formation . To better define the structural features of ice nucleation proteins responsible for the functional heterogeneity of ice nuclei within a genetically homogeneous collection of cells we used in vitro chemical mutagenesis to isolate mutants with reduced ability to nucleate ice at warm assay temperatures but which retain normal or near normal nucleation activity at cold temperatures (WIND, i.e . warm ice nucleus-deficient mutants) . Nearly half of the mutants obtained after hydroxylamine mutagenesis of the iceE gene from Erwinia herbicola had this phenotype . The phenotypes and location of lesions on the genetic map of iceE were determined for a number of mutants . All WIND mutations were restricted to the portion of iceE encoding the repetitive region of the polypeptide . DNA sequencing of two WIND mutants revealed single nucleotide substitutions changing a conserved serine or glycine residue to phenylalanine and serine, respectively . The implications of these findings in structure/function models for the ice nucleation protein are discussed. Mol Microbiol, 1993 Jul, 9(2), 343 - 56 A pleiotropic reduced virulence (Rvi-) mutant of Erwinia carotovora subspecies atroseptica is defective in flagella assembly proteins that are conserved in plant and animal bacterial pathogens; Mulholland V et al.; Erwinia carotovora subsp . atroseptica was mutagenized and assayed for virulence in planta . Those mutants which exhibited reduced virulence (Rvi-) were assayed for growth rate, auxotrophy and extracellular enzyme secretion and seven mutants were found to be wild type for all of these phenotypes . When screened for other phenotypes, two were found to be non-motile . One mutant was complemented for motility by a heterologous gene library . A 2.7kb XmaIII-ClaI complementing fragment was sequenced and the gene products were found to have similarity to flagella biosynthesis gene products from several bacteria . Further similarity was found to a pathogenicity protein from the plant pathogen Xanthomonas campestris pv . glycines and to the Spa pathogenicity proteins of the human pathogen Shigella flexneri, which are involved in the surface presentation of antigens . These studies highlight the emergence of common themes in the molecular strategies employed by both plant and animal bacterial pathogens for the targeting of proteins involved in the elaboration of disease. Mol Plant Microbe Interact, 1993 Jul-Aug, 6(4), 474 - 80 Cloning and transfer of genes for antifungal compounds from Erwinia herbicola to Escherichia coli; Tenning P et al.; Erwinia herbicola CHS1065 produces antifungal compounds highly related to herbicolin A . In 11 Tn5 mutants that have lost the antifungal activity, the transposon insertion is located on a 170-kb plasmid present in CHS1065 . This plasmid was designated pHER1065 . When analyzing these antifungal mutants, it was found that the genes for the biosynthesis of the antifungal compounds were organized in at least two clusters on pHER1065 . Upon insertion of the aphII gene of Tn5 and genes for plasmid mobilization in pHER1065, the plasmid could be stably introduced into Escherichia coli . All the E . coli exconjugants expressed an antifungal activity that was quantitatively and qualitatively comparable to the activity produced by E . herbicola CHS1065 . Amino acid analysis and molecular weight determinations of the antifungal compound produced by CHS1065 were identical to those of herbicolin A. Mol Gen Mikrobiol Virusol, 1993 Jul-Aug, (4), 12 - 5 {Expression of pel genes of Erwinia chrysanthemi ENA49 in Erwinia carotovora var . atroseptica 36A cells}; Babitskaia EV et al.; Erwinia atroseptica 36A cells were transformed by the recombinant plasmid pPL5-1 (a derivative of the vector plasmid pUC19) containing pelb and pelc genes which encode pectate lyases of Erwinia chrysanthemi ENA49 . Synthesis of pectate lyases PLB and PLC determined by the cloned pel genes is constitutive in Erwinia atroseptica 36ApPL5-1 cells and not inducible by sodium polypectate . The major part of these enzymes was accumulated in the periplasmic fraction of Erwinia atroseptica and cells were unable to efficiently secrete the enzymes into the cultural medium . Synthesis and secretion of the native pectate lyases by Erwinia atroseptica harboring the plasmid were as efficient as by the parental cells . The obtained results suggest the high specificity of pectate lyase secretory systems of kindred Erwinias. J Bacteriol, 1993 Jul, 175(13), 4263 - 5 Uptake of galacturonic acid in Erwinia chrysanthemi EC16; San Francisco MJ et al.; Uptake of {14C}galacturonic acid in Erwinia chrysanthemi was found to be stimulated during growth on pectin and its degradation products, saturated digalacturonic acid and galacturonic acid . Cells isolated from macerated potato tissue also showed increased levels of uptake activity for this molecule compared with those showed by glycerol-grown cells . Uptake was found to be an active process, and it displayed saturation kinetics . An Escherichia coli galacturonic acid transport mutant harboring the E . chrysanthemi exuT gene(s) for galacturonic acid uptake was able to transport galacturonic acid but unable to take up the dimer {3H}digalacturonic acid. Science, 1993 Jun 4, 260(5113), 1503 - 7 New domain motif: the structure of pectate lyase C, a secreted plant virulence factor; Yoder MD et al.; Pectate lyases are secreted by pathogens and initiate soft-rot diseases in plants by cleaving polygalacturonate, a major component of the plant cell wall . The three-dimensional structure of pectate lyase C from Erwinia chrysanthemi has been solved and refined to a resolution of 2.2 angstroms . The enzyme folds into a unique motif of parallel beta strands coiled into a large helix . Within the core, the amino acids form linear stacks and include a novel asparagine ladder . The sequence similarities that pectate lyases share with pectin lyases, pollen and style proteins, and tubulins suggest that the parallel beta helix motif may occur in a broad spectrum of proteins. EMBO J, 1993 Jun, 12(6), 2477 - 82 The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa; Jones S et al.; Erwinia carotovora and Pseudomonas aeruginosa secrete exoenzymes that contribute to the pathogenesis of plant and mammalian infections respectively . E.carotovora mutants defective in synthesis of the pectinase, cellulase and protease exoenzymes were isolated and classified into two groups . Group 2 mutants were found to be defective in the production of a small freely diffusible molecule, N-3-(oxohexanoyl)-L-homoserine, lactone (HSL), and were avirulent . Addition of exogenous HSL to these group 2 mutants restores synthesis of the exoenzymes and virulence in planta . Of the exoenzymes of P.aeruginosa the metalloprotease, elastase, is an established virulence determinant . Mutants of P.aeruginosa that are defective in elastase production have been isolated and were again found to fall into two groups . Analogous to the group 2 mutants of E.carotovora, group 2 mutants of P . aeruginosa are defective in the synthesis of HSL and exogenous HSL restores elastase production . HSL has now been linked to the control of bioluminescence in Vibrio fischeri, carbapenem antibiotic production of E.carotovora and the above exoenzyme virulence determinants . This information significantly enhances our understanding of the extent and nature of pheromone mediated gene expression control in prokaryotes. EMBO J, 1993 Jun, 12(6), 2467 - 76 A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora; Pirhonen M et al.; Virulence of the plant pathogen Erwinia carotovora subsp . carotovora is dependent on the production and secretion of a complex arsenal of plant cell wall-degrading enzymes . Production of these exoenzymes is controlled by a global regulatory mechanism . A virulent mutants in one of the regulatory loci, expI, show a pleiotropic defect in the growth phase-dependent transcriptional activation of exoenzyme gene expression . The expI gene encodes a 26 kDa polypeptide that is structurally and functionally related to the luxI gene product of Vibrio fischeri . Functional similarity of expI and luxI has been demonstrated by reciprocal genetic complementation experiments . LuxI controls bioluminescence in V.fischeri in a growth phase-dependent manner by directing the synthesis of the diffusible autoinducer, N-(3-oxohexanoyl) homoserine lactone . E.c . subsp . carotovora expI+ strains or Escherichia coli harboring the cloned expI gene excrete a small diffusible signal molecule that complements the expI mutation of Erwinia as well as a luxI mutation of V.fischeri . This extracellular complementation can also be achieved by E.coli harboring the luxI gene from V.fischeri or by adding the synthetic V.fischeri autoinducer . Both the production of the plant tissue-macerating exoenzymes and the ability of the bacteria to propagate in planta are restored in expI mutants by autoinducer addition . These data suggest that the same signal molecule is employed in control of such diverse processes as virulence in a plant pathogen and bioluminescence in a marine bacterium, and may represent a general mechanism by which bacteria modulate gene expression in response to changing environmental conditions. Indian J Cancer, 1993 Jun, 30(2), 72 - 6 L-asparaginase related hyperglycemia; Iyer RS et al.; L-asparaginase is a valuable chemotherapeutic agent used in the induction of remission and improvement of long term survival in patients with acute lymphoblastic leukemia . Hyperglycemia is a well known side effect of L-asparaginase . Fourteen patients developed hyperglycemia during induction therapy of acute lymphoblastic leukemia with L-asparaginase, prednisolone, vincristine and daunorubicin . Hyperglycemia was observed after a mean of five doses of L-asparaginase (range 2-10) . Seven of fourteen patients had neutropenic related infective episodes . Hyperglycemia resolved in all patients within 12 days (range 4-25) and two patients died of neutropenic septicemia . During reinduction therapy with the same drugs, only one out of ten patients developed hyperglycemia E-coli-L-asparaginase was replaced by Erwinia asparaginase in two patients one of who had recrudescence on further therapy . Close monitoring during L-asparaginase therapy for hyperglycemia will enable prompt recognition and early correction and prevent delay in therapy of acute lymphoblastic leukemia. Mol Gen Genet, 1993 May, 239(1-2), 158 - 68 A gene cluster for amylovoran synthesis in Erwinia amylovora: characterization and relationship to cps genes in Erwinia stewartii; Bernhard F et al.; A large ams gene cluster required for production of the acidic extracellular polysaccharide (EPS) amylovoran by the fire blight pathogen Erwinia amylovora was cloned . Tn5 mutagenesis and gene replacement were used to construct chromosomal ams mutants . Five complementation groups, essential for amylovoran synthesis and virulence in E . amylovora, were identified and designated ams A-E . The ams gene cluster is about 7 kb in size and functionally equivalent to the cps gene cluster involved in EPS synthesis by the related pathogen Erwinia stewartii . Mucoidy and virulence were restored to E . stewartii mutants in four cps complementation groups by the cloned E . amylovora ams genes . Conversely, the E . stewartii cps gene cluster was able to complement mutations in E . amylovora ams genes . Correspondence was found between the amsA-E complementation groups and the cpsB-D region, but the arrangement of the genes appears to be different . EPS production and virulence were also restored to E . amylovora amsE and E . stewartii cpsD mutants by clones containing the Rhizobium meliloti exo A gene. J Photochem Photobiol B, 1993 May, 18(2-3), 245 - 51 Functional identification of al-3 from Neurospora crassa as the gene for geranylgeranyl pyrophosphate synthase by complementation with crt genes, in vitro characterization of the gene product and mutant analysis; Sandmann G et al.; In this work the Neurospora crassa al-3 gene function was determined . Geranylgeranyl pyrophosphate (GGPP) synthase activity was measured in al-2 FGSC 313 and al-3 RP100 FGSC 2082 mutant strains by in vitro synthesis methods . This experiment showed that al-3 RP100 mutant expresses a reduced GGPP synthase activity . The mutated al-3 gene was cloned and sequenced; a single missense mutation was found changing serine into asparagine . Genetic complementation was performed by Escherichia coli transformation, with clusters of crt genes from Erwinia uredovora . Carotenoid accumulation was observed in E . coli transformants when the N . crassa al-3 gene substitutes the GGPP synthase gene (crtE) in the carotenogenic crt cluster . Cell-free studies with E . coli transformants gave direct evidence of the function of the al-3 protein as GGPP synthase and indicated that a short-chain prenylpyrophosphate, such as dimethylallyl pyrophosphate, is the genuine substrate. Mol Microbiol, 1993 May, 8(4), 685 - 95 Characterization of the nucM gene coding for a nuclease of the phytopathogenic bacteria Erwinia chrysanthemi; Moulard M et al.; The gene nucM encoding a nuclease was cloned from a genomic library of Erwinia chrysanthemi . The nucM gene was subcloned, and mutagenized by insertion of a uidA-KanR cartridge . This mutation was introduced by recombination into the Erwinia chrysanthemi chromosome . The nucM mutant lost NucM activity when tested on a DNA plate after 24 hours, but still possessed secondary weak nuclease activity . The nucleotide sequence of nucM was determined . It presents a 798 bp open reading frame, coding for a 266-amino-acid protein, with a predicted molecular mass of 29,910 Da . The deduced NucM protein shows 59% sequence identity with the DNase I precursor from Vibrio cholerae . It contains a typical leader sequence . Experiments of cell fractionation showed that NucM is periplasmic in E . chrysanthemi . The transcription start has been determined by S1 mapping . The -10 and -35 regions do not show homology with consensus sequence of the promoters recognized by sigma 70 . In fact, the promoter seems to be dependent on the sigma 70, but the first transcription nucleotide is unusually far from the -10 region . nucM seems to be expressed constitutively. Mol Plant Microbe Interact, 1993 May-Jun, 6(3), 299 - 308 Characterization of a novel regulatory gene aepA that controls extracellular enzyme production in the phytopathogenic bacterium Erwinia carotovora subsp . carotovora; Liu Y et al.; Erwinia carotovora subsp . carotovora strain Ecc71 produces an array of extracellular enzymes including pectate lyase (Pel), polygalacturonase, cellulase, and protease . In strain Ecc71, these enzymes are coregulated by aepA, which encodes an activator of extracellular protein production (H . Murata, J . L . McEvoy, A . Chatterjee, A . Collmer, and A . K . Chatterjee, Mol . Plant-Microbe Interact, 4:239-246, 1991) . The nucleotide sequence of a 2.7-kb aepA+ DNA segment revealed an open reading frame (ORF) of 1,395 bp which matches with the size of the aepA transcript determined by Northern blot analysis . aepA is predicted to encode a protein of 465 amino acid residues with a molecular mass of approximately 51 kDa and a pI of 6.52 . The occurrence of a putative signal sequence and several hydrophobic domains suggest membrane localization of AepA . An aepA-lacZ operon fusion was constitutively expressed in E . coli (DH5 alpha) but inducible by pectate and celery extract in E . c . subsp . carotovora (AC5006) . These findings suggest that aepA expression may be negatively regulated in E . c . subsp . carotovora . By assaying for the transcript of pel-1, which species a major secreted Pel species in strain Ecc71, and by following the expression of a pel1-lacZ operon fusion we determined that AepA activates pel-1 transcription . The characteristics of aepA including the lack of homology with other prokaryotic regulatory genes indicate that aepA encodes a novel regulatory protein required for extracellular protein production . Whereas homologs of Ecc71 aepA occur in E . c . subsp . carotovora and E . c . subsp . atroseptica strains, activation of exoenzyme production is markedly stimulated by aepA in E . c . subsp . carotovora than in E . c . subsp . atroseptica. Appl Microbiol Biotechnol, 1993 May, 39(2), 181 - 8 Co-expression of an Erwinia chrysanthemi pectate lyase-encoding gene (pelE) and an E . carotovora polygalacturonase-encoding gene (peh1) in Saccharomyces cerevisiae; Laing E et al.; A pectate lyase (PL)-encoding gene (pelE) from Erwinia chrysanthemi and a polygalacturonase (PG)-encoding gene (peh1) from E . carotovora were each inserted between a novel yeast expression-secretion cassette and a yeast gene terminator, and cloned separately into a yeast-centromeric shuttle vector (YCp50), generating recombinant plasmids pAMS12 and pAMS13 . Transcription initiation signals present in the expression-secretion cassette were derived from the yeast alcohol dehydrogenase gene promoter (ADC1P), whereas the transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T) . Secretion of PL and PG was directed by the signal sequence of the yeast mating pheromone alpha-factor (MF alpha 1s) . A pectinase cassette comprising ADC1P-MF alpha 1s-pelE-TRP5T and ADC1P-MF alpha 1s-peh1-TRP5T was subcloned into YCp50, generating plasmid pAMS14 . Subsequently, the dominant selectable Geneticin G418-resistance (GtR) marker, APH1, inserted between the yeast uridine diphosphoglucose 4-epimerase gene promoter (GAL10P) and yeast orotidine-5'-phosphate carboxylase gene terminator (URA3T), was cloned into pAMS14, resulting in plasmid pAMS15 . Plasmids pAMS12, pAMS13 and pAMS14 were transformed into a laboratory strain of Saccharomyces cerevisiae, whereas pAMS15 was stably introduced into two commercial wine yeast strains . DNA-DNA and DNA-RNA hybridization analyses revealed the presence of these plasmids, and the pelE and peh1 transcripts in the yeast transformants, respectively . A polypectate agarose assay indicated the extracellular production of biologically active PL and PG by the S . cerevisiae transformants and confirmed that co-expression of the pelE and peh1 genes synergistically enhanced pectate degradation. Biosci Biotechnol Biochem, 1993 Apr, 57(4), 603 - 6 Large-scale production and purification of an Erwinia ananas ice nucleation protein and evaluation of its ice nucleation activity; Watabe S et al.; The ice nucleation-active protein of Erwinia ananas IN-10 (inaA protein) was over-expressed as inclusion bodies in Escherichia coli in a yield of 15.3 mg of inaA protein from 60 mg of bacterial cells on a dry-matter basis . The inaA protein was purified from the inclusion bodies by solubilization with detergents to obtain a protein preparation free from sugar and lipid . This preparation had a distinct ice nucleation activity, indicating that the inaA protein per se is able to act as a nucleus. Biochem Biophys Res Commun, 1993 Mar 31, 191(3), 1284 - 7 Permanent gases inside healthy and microbially infected cotton fruit during development; Jacks TJ et al.; Permanent gases inside developing cotton fruit (Gossypium hirsutum L.) were, by weight, 46% nitrogen, 29% oxygen, 4% argon and 20% carbon dioxide, whereas plant canopy air assayed at 73% nitrogen, 25% oxygen, 2% argon and 0.3% carbon dioxide . Light exposure, fruit age, and mild infection (Erwinia) had no compositional effect but aggressive infection (Aspergillus) raised carbon dioxide content to 31% by weight and correspondingly lowered oxygen to 17% . Respiration with oxygen replenishment except during aggressive infection accounted for the fruit gas compositions. Gene, 1993 Mar 15, 125(1), 75 - 80 Cloning, sequencing, and expression of a minor protease-encoding gene from Serratia marcescens ATCC21074; Kwon YT et al.; The gene (smp) encoding an extracellular protease (Smp) from Serratia marcescens ATCC21074 has been cloned and expressed in Escherichia coli HB101 . The nucleotide (nt) sequence of the cloned smp gene revealed a single open reading frame of 1056 bp coding for 352 amino acids (aa) (38,479 Da) . The N-terminal aa sequence of Smp excreted from the E . coli host cells revealed that mature Smp consists of 300 aa (32,515 Da) . The deduced aa sequence of Smp showed high overall homology (43%) to the Erwinia carotovora metalloprotease, but low homology (15-20%) to other metalloproteases, including the S . marcescens major metalloprotease . The location for three zinc ligands and the active site for Smp was predicted from other metalloproteases . The biochemical properties of Smp show that this enzyme is a metalloprotease whose activity is optimal at pH 8.0 and 50 degrees C. Mol Biol (Mosk), 1993 Mar-Apr, 27(2), 451 - 60 {Preparation of single-chained antibodies to human ferritin in Escherichia coli}; Bespalov IA et al.; We have cloned cDNA copies of the immunoglobulin heavy and light chain genes from hybridoma cells able to produce antibody against human ferritin . Variable segments of these genes were obtained using the polymerase chain reaction (PCR) . The specific amplifications of ligase reaction products were carried out to combine the variable segments with DNA fragments coding for a peptide-linker and for a signal peptide of the cloned pelB gene of Erwinia carotovora . During the antiferritin single-chain antibody gene expression under the T7 RNA polymerase control in E . coli the processed molecules of recombinant proteins formed aggregates in periplasm . The reversible denaturation in the absence of reducing agents had allowed us to obtain single-chain antibodies with the original binding specificity toward human ferritin. J Antibiot (Tokyo), 1993 Mar, 46(3), 441 - 54 Autoregulation of carbapenem biosynthesis in Erwinia carotovora by analogues of N-(3-oxohexanoyl)-L-homoserine lactone; Chhabra SR et al.; N-(3-Oxohexanoyl)-L-homoserine lactone (HSL) (I) is the autoregulator controlling carbapenem antibiotic biosynthesis in Erwinia carotovora ATCC 39048 . The chemical synthesis and biological evaluation of analogues of HSL are described . These include alterations of chirality, side-chain modifications, ring size and ring hetero atom . A number of compounds are reported which are capable of restoring the phenotype to a HSL negative mutant but at higher concentrations than HSL . A-factor, the autoregulator of streptomycin biosynthesis in Streptomyces griseus, was not active as an inducer of carbapenem biosynthesis in E . carotovora. Mol Plant Microbe Interact, 1993 Mar-Apr, 6(2), 216 - 24 Interaction of Xanthomonas campestris with Arabidopsis thaliana: characterization of a gene from X . c . pv . raphani that confers avirulence to most A . thaliana accessions; Parker JE et al.; Infiltration of leaves of Arabidopsis thaliana accession Columbia with Xanthomonas campestris pathovar campestris leads to bacterial growth and disease symptoms reminiscent of those incited in Brassica plants inoculated under the same conditions . A search among A . thaliana accessions for variation in the reaction phenotype to strains of X . campestris pathovars campestris, aberrans, and raphani showed that there were no clear differential responses between plant accessions to the individual bacterial strains tested . X . c . pv . raphani strain 1067 was avirulent to all A . thaliana accessions tested . A gene was cloned from X . c . pv . raphani 1067 which, when transferred into the virulent X . c . pv . campestris strain 8004, strongly reduced symptom development and bacterial growth in A . thaliana Columbia plants but did not affect virulence to Brassica plants . The gene (denoted avrXca) interacted with all A . thaliana accessions tested except one, Kas-1, which developed disease symptoms and supported growth of the transconjugant to levels similar to those with X . c . pv . campestris 8004 alone . Sequence analysis of avrXca revealed a probable open reading frame encoding a protein of 66,566 Da that has no homology with other known sequences . A sequence motif conserved among hrp genes was identified in the 5' noncoding region of avrXca, and features characteristic of a signal peptide were found in the N-terminal portion of the presumed AvrXca protein . DNA from different phytopathogenic bacteria contained sequences hybridizing with avrXca in related X . campestris pathovars but not in Erwinia or Pseudomonas strains. Mol Microbiol, 1993 Mar, 7(5), 785 - 93 Mutagenesis of cellulase EGZ for studying the general protein secretory pathway in Erwinia chrysanthemi; Py B et al.; Extracellular secretion of endoglucanase Z (EGZ) from Erwinia chrysanthemi is mediated by the so-called Out general secretion pathway and, presumably, involves recognition of EGZ-carried structural information by one or more of the Out proteins . Investigating the relationships between structure and secretability of EGZ was the purpose of the present work . EGZ is made of two independent domains, located at the N- and C-proximal sides, separated by a Ser/Thr-rich region, which are responsible for catalysis and cellulose-binding, respectively . The existence of a secretion region ('targeting signal') was investigated by studying the secretability of modified EGZ derivatives . These resulted from deletion or peptide insertion and were designed by using the domain organization cited above as a guide . Catalytic and/or cellulose-binding tests showed that all proteins exhibited at least a functional EGZ domain while immunoblot analyses confirmed that neither the insertions nor the deletions led to grossly misfolded proteins . In contrast, all of the proteins lost their secretability in E . chrysanthemi . This suggested that at least two secretion motifs existed, one lying within each functional domain . The role of the Ser/Thr-rich linker region was subsequently tested . Accordingly, two proteins containing a linker region whose length was increased by the addition of 8 and 18 additional residues and one protein lacking the linker region were studied . All three exhibited endoglucanase activity and cellulose-binding ability, confirming the independence of the domains within the context of EGZ/polysaccharide interaction . In contrast, none was secreted by E . chrysanthemi.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1993 Feb 15, 268(5), 3033 - 6 High affinity binding of human vitamin K-dependent protein S to a truncated recombinant beta-chain of C4b-binding protein expressed in Escherichia coli; Hardig Y et al.; C4b-binding protein (C4BP) is a plasma glycoprotein that contains independent binding sites for complement component C4b and anticoagulant vitamin K-dependent protein S . C4BP is composed of seven alpha-chains (70 kDa) and one beta-chain (45 kDa) joined by disulfide bonds . In addition to non-repeat regions, the alpha- and beta-chains contain eight and three tandemly arranged modules, respectively, designated short consensus repeats (SCRs) . C4b binds to the alpha-chains while it has been suggested, though not conclusively shown, that the beta-chain binds protein S . A truncated recombinant beta-chain composed of the three SCRs was expressed as a fusion protein with an epitope for a calcium-dependent monoclonal antibody (HPC4) in a procaryotic expression system . A signal peptide from the pelB gene of Erwinia carotovora directed expression to the medium from which the recombinant protein was purified using a HPC4 affinity column . On unreduced polyacrylamide gel electrophoresis in the presence of SDS, the recombinant protein demonstrated two closely spaced bands (M(r) = 19,000 and 20,000), whereas after reduction a broad band with an apparent molecular weight of 25,000 was obtained . On 125I-protein S ligand blotting, only one of the unreduced bands was found to bind protein S . The protein corresponding to this band bound protein S with almost as high affinity as intact C4BP, and it completely inhibited binding of protein S to intact C4BP . After reduction, the protein S binding ability was lost . The multiple carbohydrate side chains present in the native beta-chain of C4BP were apparently not required for protein S binding, as the recombinant beta-chain was not glycosylated . These results demonstrate that the protein S binding site of C4BP is contained within the three SCR modules of its beta-chain. Eur J Biochem, 1993 Feb 15, 212(1), 157 - 65 An allosterically insensitive class of cyclohexadienyl dehydrogenase from Zymomonas mobilis; Zhao G et al.; The key enzyme of tyrosine biosynthesis in many Gram-negative prokaryotes is cyclohexadienyl dehydrogenase . The Zymomonas mobilis gene (tyrC) coding for this enzyme was cloned in Escherichia coli by complementation of a tyrosine auxotroph . The tyrC gene was 882 bp long, encoding a protein with a calculated molecular mass of 32086 Da . The Z . mobilis cyclohexadienyl dehydrogenase expressed in E . coli was purified to electrophoretic homogeneity . The subunit molecular mass of the purified enzyme was 32 kDa as determined by SDS/PAGE . The ratio of the activity of arogenate dehydrogenase to that of prephenate dehydrogenase (approximately 3:1) remained constant throughout purification, and the two activities were therefore inseparable . The genetic and biochemical data obtained demonstrated a single enzyme protein capable of catalyzing either of two reactions . Km values of 0.25 mM and 0.18 mM were obtained from prephenate and L-arogenate, respectively . The Km value obtained for NAD+ (0.09 mM) was the same regardless of whether the enzyme was assayed as arogenate dehydrogenase or as prephenate dehydrogenase . Unlike the corresponding enzyme of Pseudomonas aeruginosa or E . coli, the cyclohexadienyl dehydrogenase of Z . mobilis lacks sensitivity to feedback inhibition by L-tyrosine . A typical NAD(+)-binding domain was found to be located at the N-terminus of the protein . Although the deduced amino-acid sequence of the Z . mobilis cyclohexadienyl dehydrogenase showed relatively low identity (19-32%) with the prephenate dehydrogenases of Bacillus subtilis and Saccharomyces cerevisiae, as well as with the cyclohexadienyl dehydrogenase components of the bifunctional T-proteins of E . coli and Erwinia herbicola, a presumptive motif was identified which may correspond to critical residues of the binding site for cyclohexadienyl substrate molecules . Immediately upstream of tryC a portion of a gene was sequenced and found to exhibit clearcut homology of the deduced amino-acid sequence with the B . subtilis hisH gene product . Thus, the Zymomonas gene organization is reminiscent of the linkage of genes encoding a tryosine-pathway dehydrogenase and a histidine-pathway aminotransferase in B . subtilis. Gene, 1993 Feb 14, 124(1), 145 - 7 Characterization of polygalacturonases; Saarilahti HT; Lei et al . {Gene 117 (1992) 119-124} recently published the nucleotide sequence of the peh gene of Erwinia carotovora subsp . carotovora (Ecc) and a characterization of its product endopolygalacturonase (Peh) . The gene appears highly similar to previously described peh sequences of Ecc {Hinton et al., Mol . Microbiol . 4 (1990) 1029-1036; Saarilahti et al., Mol . Microbiol . 4 (1990) 1037-1044} which were not cited in the article . Ecc carries a single peh gene whose product, Peh, is here shown to share similarity with the two Pehs characterized thus far and a Peh-like protein of eukaryotic origin at the amino acid (aa) sequence level . Additionally, a highly conserved region within their C-terminal domains was found to share local similarity with two 13-aa segments of an otherwise distinct exo-poly-alpha-D-galacturonosidase (exo-Peh), suggesting that these segments might be required for enzyme activity in both Peh and exo-Peh. J Protein Chem, 1993 Feb, 12(1), 15 - 22 Pectinase Aspergillus sp . polygalacturonase: multiplicity, divergence, and structural patterns linking fungal, bacterial, and plant polygalacturonases; Stratilova E et al.; Nine forms of Aspergillus sp . polygalacturonase were purified from a commercial preparation of pectinase Rohament P using chromatographies and chromatofocusing . Individual forms differ in isoelectric point, and at least five differ in structure; whereas molecular masses and enzymatic properties are largely identical . Four forms with free alpha-amino groups have identical start positions but internal amino acid replacements . Therefore, the multiplicity is derived from true heterogeneities and not from N-terminal truncations . Peptide analysis of the major polygalacturonase reveals large variations toward the enzyme from other Aspergillus species (72-75% residue differences, depending on species) but additional similarities with the enzyme from bacterial and plant sources (only 66-71% residue differences toward the Erwinia, tomato, and peach enzymes) . Combined with previous data, these facts show polygalacturonase to exhibit extensive multiplicity and much variability, but also unexpected similarities between distantly related forms with conserved functional properties. Mol Microbiol, 1993 Feb, 7(3), 383 - 93 The TonB-dependent ferrichrome receptor FcuA of Yersinia enterocolitica: evidence against a strict co-evolution of receptor structure and substrate specificity; Koebnik R et al.; A Yersinia enterocolitica receptor mutant was isolated which is impaired in ferrichrome uptake . The receptor-encoding gene fcuA was cloned in Escherichia coli K-12 . A fcuA mutant of Y . enterocolitica could be complemented by the cloned DNA fragment . The FcuA-encoding region was sequenced and an open reading frame encoding 758 amino acids including a signal sequence of 36 amino acids was found . FcuA shared 34.6% amino acid sequence homology with FatA, the anguibactin receptor of Vibrio anguillarum, but only 20.6% homology with FhuA, the ferrichrome receptor of E . coli . Since the structure of anguibactin differs strongly from that of ferrichrome there seems to be no co-evolution of receptor structure and substrate specificity . The ferrichrome receptors FcuA from Y . enterocolitica and FhuA from E . coli had slightly different substrate specificities . In contrast to FhuA from E . coli, FcuA from Y . enterocolitica was more stereoselective and failed to transport enantio ferrichrome . Three additional ferrichrome receptors were cloned from Pantoea agglomerans (formerly Erwinia herbicola), Salmonella paratyphi B and Salmonella typhimurium . Their substrate specificity was similar but not identical. J Bacteriol, 1993 Feb, 175(3), 732 - 40 Nucleotide sequence analysis of a transposon (Tn5393) carrying streptomycin resistance genes in Erwinia amylovora and other gram-negative bacteria; Chiou CS et al.; A class II Tn3-type transposable element, designated Tn5393 and located on plasmid pEa34 from streptomycin-resistant strain CA11 of Erwinia amylovora, was identified by its ability to move from pEa34 to different sites in plasmids pGEM3Zf(+) and pUCD800 . Nucleotide sequence analysis reveals that Tn5393 consists of 6,705 bp with 81-bp terminal inverted repeats and generates 5-bp duplications of the target DNA following insertion . Tn5393 contains open reading frames that encode a putative transposase (tnpA) and resolvase (tnpR) of 961 and 181 amino acids, respectively . The two open reading frames are separated by a putative recombination site (res) consisting of 194 bp . Two streptomycin resistance genes, strA and strB, were identified on the basis of their DNA sequence homology to streptomycin resistance genes in plasmid RSF1010 . StrA is separated from tnpR by a 1.2-kb insertion element designated IS1133 . The tnpA-res-tnpR region of Tn5393 was detected in Pseudomonas syringae pv . papulans Psp36 and in many other gram-negative bacteria harboring strA and strB . Except for some strains of Erwinia herbicola, these other gram-negative bacteria lacked insertion sequence IS1133 . The prevalence of strA and strB could be accounted for by transposition of Tn5393 to conjugative plasmids that are then disseminated widely among gram-negative bacteria. Hindustan Antibiot Bull, 1993 Feb-May, 35(1-2), 77 - 86 Fermentative production and isolation of L-asparaginase from Erwinia carotovora, EC-113; Maladkar NK et al.; L-Asparaginase, an enzyme-drug used for the treatment of acute lymphoblastic leukemia was isolated from Erwinia carotovora . The effects of different carbon and nitrogen sources on the fermentative production of the enzyme were studied . Lactose, monosodium glutamate, corn steep liquor, tryptone and yeast extract showed significant stimulation of the production . When L-asparagine (0.2%), a substrate of the enzyme was added to a fermentation medium, a mutant strain EC-113 exhibited 6 times higher production indicating a distinct induction . The enzyme was extracted from the cells and purified about 30 fold to apparent homogeneity employing polyacrylamide gel electrophoresis . The methods used in sequence were DEAE cellulose chromatography, sephadex G-200 gel filtration, hydroxylapatite ion-exchange and affinity chromatography on sepharose CL-6B . The recovery of enzyme was 60% . The purified enzyme showed optimal pH at 8.0 and optimal temperature at 50 degrees C . The Km value of purified enzyme was 1.8 x 10(-5) M . LD50 of purified enzyme in mice by intravenous route was 4,80,000 IU/Kg and repeated treatment at 20,000 IU/Kg by intravenous route did not elicit bone marrow depression or damage to intestinal mucosa . The plasma half life was 14-24 hours and clearance time was 4-5 hours . Purified enzyme shows significant antitumor activity on experimental animal models. J Mol Evol, 1993 Feb, 36(2), 107 - 20 The pheA/tyrA/aroF region from Erwinia herbicola: an emerging comparative basis for analysis of gene organization and regulation in enteric bacteria; Xia T et al.; Extensive knowledge exists in Escherichia coli about the contiguous pheA and aroF-tyrA operons which have opposite transcription orientations and are separated by a bidirectional transcription terminator . The corresponding structural genes and individual components of the terminator and attenuator from Erwinia herbicola have been analyzed from an evolutionary vantage point . A 7.5-kb DNA fragment from E . herbicola carrying the linked pheA, tyrA, and aroF genes was cloned by functional complementation of E . coli auxotrophic requirements . A 3,433-bp segment of DNA consisting of more than half of aroF, all of tyrA, and the entire phenylalanine operon (promoter, leader region encoding the leader peptide and containing the phe attenuator, and pheA) was sequenced . A bidirectional transcription terminator was positioned between the divergently transcribed pheA and tyrA . The adjacent aroF and tyrA genes share a common transcription orientation, consistent with their probable coexistence within an operon . However, tyrA can be expressed efficiently from an internal promoter which appears to lie within the 3' portion of aroF . The gene order is pheA tyrA aroF in E . herbicola, with the same tail-to-tail arrangement of transcription known to exist in E . coli . The pheL coding region of the phe operon was dominated by phenylalanine codons, seven of the 15 amino acid residues of the leader peptide being L-phenylalanine . The E . herbicola pheA and tyrA genes were 1,161 bp and 1,119 bp in length, respectively, and corresponded to deduced gene products having subunit molecular weights of 43,182 and 41,847 . The deduced amino acid sequences of PheA and TyrA were homologous at their N-termini, consistent with a common evolutionary origin of the chorismate mutase domains present at the amino terminus of both PheA and TyrA . A detailed comparison of the E . coli and E . herbicola sequences was made . The pheA, tyrA, and aroF genes of E . herbicola exhibited high overall identity with the counterpart E . coli genes . Within the leader region of the phe operon, the leader peptide coding region was highly conserved . Although the 1:2 and 2':3' stems defining the pause structure and the antiterminator, respectively, were also highly conserved, RNA segment 4 of the attenuator terminator exhibited considerable divergence, as did the distal portion of the attenuator region . Within the span of attenuator region encoding the three stem-loop structures of mRNA secondary configuration, hot spots of base-residue divergence were localized to looped-out regions . No changes occurred which would simultaneously disrupt alternative pairing relationships of secondary configuration . The bidirectional terminator between pheA and tyrA has diverged very substantially.(ABSTRACT TRUNCATED AT 400 WORDS) FEBS Lett, 1993 Jan 11, 315(3), 329 - 34 In vitro expression and activity of lycopene cyclase and beta-carotene hydroxylase from Erwinia herbicola; Hundle BS et al.; The cyclisation of lycopene to beta-carotene and the hydroxylation of beta-carotene to zeaxanthin are common enzymatic steps in the biosynthesis of carotenoids in a wide range of bacteria, fungi, and plants . We have individually expressed in E . coli the two genes coding for these enzymatic steps in Erwinia herbicola . The cyclase and hydroxylase enzymes have apparent molecular weights of 43 kDa and 22 kDa, respectively, as determined by SDS-PAGE . Hydroxylase in vitro activity was obtained only in the cytoplasmic fraction . Cyclase also demonstrated enzyme activity in a crude cell-free lysate, although to a lesser extent. Arch Microbiol, 1993, 159(3), 250 - 6 Purification, partial characterization, and subcellular localization of a 38 kilodalton, calcium-regulated protein of Rhizobium fredii USDA208; Krishman HB et al.; Calcium is essential for the growth of rhizobia and the formation of nitrogen-fixing root-nodules on legumes, but its precise role in these processes remains unknown . We have found that Rhizobium fredii USDA208 accumulates a major 38 kDa protein when grown in media supplemented with 0.3-2 microM Cacl2 . We have purified this protein and raised polyclonal antibodies against it . The protein initially is synthesized as a 40 kDa precursor which subsequently undergoes calcium-dependent processing to give rise to the mature polypeptide . Subcellular and immunocytochemical localization studies indicate that the 38 kDa protein accumulates preferentially in the periplasmic space . Its N-terminal sequence, AETIKIGVAGPMTG, shows significant homology to the N-termini of amino acid binding proteins from the periplasm, including leucine-, isoleucine-, and valine-specific binding proteins of Pseudomonas aeruginosa and Escherichia coli and a leucine-specific binding protein of E . coli . The R . fredii protein does not, however, bind {3H}-leucine . The 38 kDa protein is encoded by the bacterial chromosome . It is absent in several rhizobia other than R . fredii, but antigenically related polypeptides are present in Escherichia coli and Erwinia carotovora subsp . carotovora. J Gen Microbiol, 1993 Jan, 139 ( Pt 1), 1 - 9 Regulation of the expression of a pelA::uidA fusion in Erwinia chrysanthemi and demonstration of the synergistic action of plant extract with polygalacturonate on pectate lyase synthesis; Bourson C et al.; The phytopathogenicity of Erwinia chrysanthemi is chiefly supported by the production of pectate lyase isoenzymes, encoded by the pel genes . One of these enzymes, PelA, encoded by the pelA gene, seems to represent only a small part of the total pectate lyase activity, but is required for full bacterial pathogenicity . To study the regulation of pelA gene expression, a pelA::uidA gene fusion was constructed . Expression of this fusion was analysed under various growth conditions and in various genetic backgrounds . Whatever the culture conditions, the pelA gene was weakly expressed . Moreover, pelA expression seems not to be regulated by the pecS gene product, but essentially by the kdgR gene product . In many plant-associated bacteria, genes involved in pathogenicity are induced by certain plant compounds . In this work, we demonstrate that E . chrysanthemi pel genes are induced in the presence of plant extracts, but only in synergy with known pectate lyase inducers (KDG: 2-keto-3-deoxygluconate; DKII: 2,5-diketo-3-deoxygluconate) . However, different pel genes did not exhibit the same sensitivity to plant signal molecules . Partial purification of inducing plant compounds suggested that plant extracts contain at least one molecule involved in pectate lyase induction . This compound is thermoresistant, and has a low molecular mass and a very hydrophilic behaviour. Mol Microbiol, 1993 Jan, 7(1), 141 - 50 Involvement of lipopolysaccharide in the secretion of Escherichia coli alpha-haemolysin and Erwinia chrysanthemi proteases; Wandersman C et al.; The presence of the alpha-haemolysin secretion genes sensitizes Escherichia coli to vancomycin, a glycopeptide antibiotic that is normally excluded from the Gram-negative envelope (owing to its large size) (M(r) 1400) . The selection of vancomycin mutants in strains carrying such genes was found to be a very powerful method for selecting non-haemolytic mutants . In this way, mutations in the known secretion genes, hlyB, hlyD and tolC, were obtained . However additional mutations mapped in genes rfaH and galU which are required for lipopolysaccharide (LPS) biosynthesis . Mutations in rfaH and galU strongly reduced alpha-haemolysin secretion as well as the secretion of Erwinia chrysanthemi proteases in E . coli without affecting their synthesis . These mutations markedly lowered the content of TolC protein, required for haemolysin secretion and also of the PrtF protein necessary for protease secretion . These results raise the possibility that LPS is involved in the correct incorporation of the TolC and PrtF proteins into the cell envelope. Biomater Artif Cells Immobilization Biotechnol, 1993, 21(3), 323 - 33 Free and microencapsulated Erwinia herbicola for the production of tyrosine; Lloyd-George I et al.; Erwinia herbicola (ATCC 21434) was grown in a medium which caused the cells to induce tyrosine phenol-lyase (TPL) activity . Whole cells of Erwinia herbicola were then microencapsulated within alginate-poly-L-lysine-alginate membraned microcapsules (diameter 800 microns) . In a rotary shaker-incubator with a 1.9 cm horizontal throw, an agitation rate of at least 240 revolutions per minute (rpm) was required before the TPL activity of the microencapsulated cells was equal to that of the free cells . The TPL activity of the cells, whether free or microencapsulated, could be used for the conversion of ammonia, pyruvate and phenol into tyrosine at 37 degrees C . The results indicate that free cells and microencapsulated cells effect the conversion of these reactants to tyrosine equally well if the agitation rate is 240 rpm . In liver failure the concentrations of both ammonia, phenol and pyruvate are elevated . Hence the TPL activity of microencapsulated Erwinia herbicola could possibly find application in a novel approach for the removal of toxic phenol and ammonia during liver failure. Adv Biochem Eng Biotechnol, 1993, 50, 1 - 19 Genetically engineered microorganisms to rescue plants from frost injury; Dar GH et al.; Ice nucleation active bacteria belonging to genera Pseudomonas, Xanthomonas and Erwinia contribute to frost damage to plants by initiating the formation of ice in plants that would otherwise supercool and avoid the damaging ice formation . The biological control of frost injury can be achieved by the application of non-ice nucleation active bacteria to the plant surfaces before they become colonized by Ice+ species . ice genes have been cloned from Pseudomonas and isogenic Ice- derivatives constructed via genetic manipulations . These genetically engineered microorganisms (GEMs) have been released into the environment to control the frost damage . The incidence of frost injury to the plants has, thereby, been reduced by 50-85% during natural frosts . These GEMs do not survive in soil and show no aerial dispersal in the environment. Pneumonol Alergol Pol, 1993, 61(11-12), 598 - 605 {Pathogenic action of Erwinia herbicola . II . In vivo investigations}; Milanowski J et al.; The pulmonary response of guinea pigs to inhalation of antigen and endotoxin of Erwinia herbicola was investigated . Pulmonary parameters monitored in this study were breathing rate, cellular differential counts from bronchoalveolar lavage fluid, superoxide anion and interleukin-1 (IL-1) production by alveolar macrophages . Both agents caused an increase in breathing rate; an influx of neutrophils, lymphocytes, and red blood cells to the lung; and an enhancement of secretion of superoxide anions and IL-1 by pulmonary macrophages . The results indicate significant role of extrinsic bacilli Erwinia herbicola in respiratory pathology and show new mechanisms of their action. Pneumonol Alergol Pol, 1993, 61(11-12), 592 - 7 {Pathogenic action of Erwinia herbicola . I . In vitro investigations}; Milanowski J et al.; The effects of antigen and endotoxin of Erwinia herbicola on physiological status of alveolar macrophages were studied in vitro . The studies comprised determination of the cytotoxicity of antigens + and their effects on superoxide anion and interleukin-1 (IL-1) generation by alveolar macrophages . It was found that tested agents were cytotoxic for alveolar macrophages in large concentrations (from 1000 micrograms/ml . In lower concentrations (1-10 micrograms/ml) they stimulated the activity of alveolar macrophages, causing significant increase in superoxide anion(O2) and IL-1 generation . Superoxide anion and IL-1 production may be of importance in pathogenesis of organic dust-induced lung diseases and our results indicate that agents associated with these diseases induced their production. J Chem Technol Biotechnol, 1993, 58(1), 71 - 6 Some implications of structural collapse during freeze-drying using Erwinia caratovora L-asparaginase as a model; Adams GD et al.; When the enzyme Erwinia caratovora L-asparaginase was freeze-dried in mixtures of lactose and sodium chloride, biological activity and protein structure were preserved during drying . However, by altering the ratios of the excipients in the formulation it was possible to obtain products which were pharmaceutically acceptable or unacceptable as assessed by the criteria of dried cake appearance, moisture content or ease of reconstitution. FEMS Microbiol Lett, 1992 Dec 15, 79(1-3), 161 - 7 Small molecule-mediated density-dependent control of gene expression in prokaryotes: bioluminescence and the biosynthesis of carbapenem antibiotics; Williams P et al.; Sophisticated signal transduction systems enable prokaryotes to sense their growth environment and mount an appropriate adaptive response . Signal transduction and gene regulation through the phosphorylation of two regulatory components is now recognised as one of the major global regulatory networks in bacteria . However, not all types of sensor-regulator circuits relay information via phosphoryl transfer . The Vibrio fischeri LuxR protein which has previously been characterised as a member of the response-regulator superfamily responds to a small diffusible signal molecule N-(3-oxohexanoyl)homoserine lactone (HSL) . Biosynthesis of HSL in V . fischeri is dependent on the expression of the luxI gene . Until recently, the role of HSL as an 'autoinducer' was thought to be restricted to V . fischeri and a few related marine bacteria in which it controls the onset of bioluminescence . However, we have discovered that a diverse group of terrestrial bacteria: (1) produce HSL; (2) possess genes analogous to luxI; and (3) exhibit cell density-dependent induction of bioluminesence when transformed with a recombinant plasmid carrying V . fischeri lux genes but lacking luxI . In one of these, Erwinia carotovora, HSL is shown to mediate the cell density-dependent biosynthesis of a carbapenem antibiotic. Biochem J, 1992 Dec 15, 288 ( Pt 3), 997 - 1004 N-(3-oxohexanoyl)-L-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora; Bainton NJ et al.; Erwinia carotovora A.T.C.C . 39048 produces the antibiotic 1-carbapen-2-em-3-carboxylic acid . A number of mutants with a carbapenem-non-producing phenotype were selected as part of an investigation into the molecular and genetic basis of carbapenem biosynthesis . Cross-feeding studies revealed that the mutants fell into two discrete groups . Group 1 mutants were found to secrete a diffusible low-molecular-mass compound which restored carbapenem production in group 2 mutants . This compound was isolated from the spent culture supernatant of a group 1 mutant using solvent extraction, hydrophobic-interaction chromatography and silica-gel chromatography, and finally purified by reverse-phase semipreparative h.p.l.c . M.s . and n.m.r . spectroscopy revealed that the compound was N-(3-oxohexanoyl)homoserine lactone . Both D- and L-isomers were synthesized, and subsequent analysis by c.d . established that the natural product has the L-configuration . Although carbapenem production was restored by both isomers, dose-response curves indicated that the L-isomer has greater activity, with an induction threshold of about 0.5 micrograms/ml . N-(3-Oxohexanoyl)-L-homoserine lactone is, therefore, an autoregulator of carbapenem biosynthesis rather than a biosynthetic intermediate . This compound is already known for its role in autoinduction of bioluminescence in the marine bacterium Vibrio fischeri . It is also structurally-related to the A- and I-factors which are known to regulate production of antibiotics in some Streptomyces species . Its association in this work with the regulation of carbapenem biosynthesis implies a broader role for autoregulator-controlled gene expression in prokaryotes. Mol Gen Genet, 1992 Dec, 236(1), 125 - 34 Nucleotide sequence analysis and comparison of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida; Garriga X et al.; The complete nucleotide sequences of the lexA genes from Salmonella typhimurium, Erwinia carotovora, Pseudomonas aeruginosa and Pseudomonas putida were determined; the DNA sequences of the lexA genes from these bacteria were 86%, 76%, 61% and 59% similar, respectively, to the Escherichia coli K12 gene . The predicted amino acid sequences of the S . typhimurium, E . carotovora and P . putida LexA proteins are 202 residues long whereas that of P . aeruginosa is 204 . Two putative LexA repressor binding sites were localized upstream of each of the heterologous genes, the distance between them being 5 bp in S . typhimurium and E . carotovora, as in the lexA gene of E . coli, and 3 bp in P . putida and P . aeruginosa . The first lexA site present in the lexA operator of all five bacteria is very well conserved . However, the second lexA box is considerably more variable . The Ala-84--Gly-85 bond, at which the LexA repressor of E . coli is cleaved during the induction of the SOS response, is also found in the LexA proteins of S . typhimurium and E . carotovora . Likewise, the amino acids Ser-119 and Lys-156 are present in all of these three LexA repressors . These residues also exist in the LexA proteins of P . putida and P . aeruginosa, but they are displaced by 4 and 6 residues, respectively . Furthermore, the structure and sequence of the DNA-binding domain of the LexA repressor of E . coli are highly conserved in the S . typhimurium, E . carotovora, P . aeruginosa and P . putida LexA proteins. Gene, 1992 Dec 1, 122(1), 27 - 34 Construction of a Tn7-lux system for gene expression studies in gram-negative bacteria; Shen H et al.; A Tn7-lux system was developed for gene expression studies in Gram- bacteria . The plasmids constructed, pHSK728 and pHSK729, have the following features: (1) a promoterless Vibrio fischeri lux operon as a reporter system; (2) multiple cloning sites (MCS) ahead of the lux operon, in opposite orientation for the cloning of promoter fragments; (3) a transcriptional terminator ahead of the MCS and translational stop codons in all reading frames before the translational start of the luxC gene; (4) a streptomycin/spectinomycin-resistance encoding gene as a selection marker; and (5) Tn7 border sequences flanking the above elements, permitting the transposition of lux fusion constructs into bacterial genomes . The system was tested using the Escherichia coli lac promoter as well as the differentially regulated promoters of the avrD gene from Pseudomonas syringae pv . tomato and the pelE gene of Erwinia chrysanthemi EC16 . Southern blot analysis showed that all fusion constructs had integrated into the host genomes in a single-copy, site-specific manner . The promoters of the avrD and pelE genes resulted in little or no light production when bacteria were grown in rich culture media, but high levels of induction were observed when the bacteria were grown in plant tissues . These results demonstrated that the Tn7-lux system provided a simple, sensitive assay of promoter activity in Gram- bacteria. Protein Expr Purif, 1992 Dec, 3(6), 453 - 60 Expression and purification of a soluble tissue factor fusion protein with an epitope for an unusual calcium-dependent antibody; Rezaie AR et al.; The use of bacterial signal peptides to target recombinant mammalian proteins to the periplasmic space of Escherichia coli (to promote proper disulfide bond formation) has met with variable success . We report the design and use of a bacterial expression vector to direct recombinant fusion proteins to the periplasmic space of E . coli: it contains the signal peptide from the pelB gene of Erwinia carotovora linked to a small peptide epitope for an unusual calcium-dependent antibody (HPC4) . HPC4 binds to the epitope in a Ca(2+)-dependent manner, but the epitope itself does not bind Ca2+ . We have used this system to express a biologically active, soluble form of tissue factor, the protein responsible for triggering the blood clotting cascade . Soluble tissue factor was secreted into the culture medium at 1-2 mg/liter, from which it could be readily purified using immobilized HPC4 antibody . The HPC4 epitope could be removed by digestion with thrombin or factor Xa, although a free amino terminus was not required for function since soluble tissue factor was equally active with the epitope still in place . This vector/epitope system permits large-scale expression and purification of recombinant soluble tissue factor and should be generally applicable to the isolation of other recombinant proteins . Furthermore, the epitope confers Ca(2+)-dependent binding of the fusion protein to HPC4 antibody while avoiding the creation of a new metal binding site on the fusion protein itself . Tb3+ can bind in this Ca2+ site near Trp, allowing this site to serve as a means of attaching a fluorescent probe to tissue factor. Gene, 1992 Nov 2, 121(1), 47 - 54 Sequence of a cluster of genes controlling synthesis and secretion of alkaline protease in Pseudomonas aeruginosa: relationships to other secretory pathways; Duong F et al.; A genetic locus implicated in the synthesis and secretion of alkaline protease (APR) in Pseudomonas aeruginosa has been previously described {Guzzo et al., J . Bacteriol . 172 (1990) 942-948} . The nucleotide sequence of the DNA fragment encoding these functions was determined and revealed the existence of five open reading frames: aprA, the structural gene encoding APR; aprI, which encodes a protease inhibitor; and aprD, aprE, aprF whose products are involved in protease secretion . The AprD, AprE and AprF proteins share significant homology with proteins implicated in secretion of Erwinia chrysanthemi proteases and Escherichia coli alpha-haemolysin . These results provide further evidence for the existence of a specialized secretory system widespread among Gram- bacteria. Gene, 1992 Nov 2, 121(1), 35 - 45 Synthesis and secretion of an Erwinia chrysanthemi pectate lyase in Saccharomyces cerevisiae regulated by different combinations of bacterial and yeast promoter and signal sequences; Laing E et al.; Nine different expression-secretion cassettes, comprising novel combinations of yeast and bacterial gene promoters and secretion signal sequences, were constructed and evaluated . A pectate lyase-encoding gene (pelE) from Erwinia chrysanthemi was inserted between each one of these expression-secretion cassettes and a yeast gene terminator, generating recombinant yeast-integrating shuttle plasmids pAMS1 through pAMS9 . These YIp5-derived plasmids were transformed and stably integrated into the genome of a laboratory strain of Saccharomyces cerevisiae, and the pectate lyase production was monitored . Transcription initiation signals for pelE expression were derived from the yeast alcohol dehydrogenase (ADC1P), the yeast mating pheromone alpha-factor (MF alpha 1P) and the Bacillus amyloliquefaciens alpha-amylase (AMYP) gene promoters . The transcription termination signals were derived from the yeast tryptophan synthase gene terminator (TRP5T) . Secretion of pectate lyase (PLe) was directed by the signal sequences of the yeast mating pheromone alpha-factor (MF alpha 1S), B . amyloliquefaciens alpha-amylase (AMYS) and Er . chrysanthemi pectate lyase (pelES) . The ADC1P-MF alpha 1S expression-secretion system proved to be the most efficient control cassette for the expression of pelE and the secretion of PLe in S . cerevisiae. Appl Environ Microbiol, 1992 Nov, 58(11), 3522 - 6 Sensitive and species-specific detection of Erwinia amylovora by polymerase chain reaction analysis; Bereswill S et al.; Detection and identification of the fire blight pathogen, Erwinia amylovora, can be accurately done by polymerase chain reaction (PCR) analysis in less than 6 h . Two oligomers derived from a 29-kb plasmid which is common to all strains of E . amylovora were used to amplify a 0.9-kb fragment of the plasmid . By separation of the PCR products on agarose gel, this fragment wa specifically detected when E . amylovora DNA was present in the amplification assay . It was not found when DNA from other plant-pathogenic bacteria was used for the assay . A visible band specific to the 0.9-kb fragment was produced with DNA from fewer than 100 E . amylovora cells . A signal of similar strength was also obtained from E . amylovora cell lysates in the presence of the mild detergent Tween 20 . Signals were weaker when bacteria were added to the PCR mixture without the detergent . As with results obtained from hybridization experiments using pEA29 DNA< the PCR signal was obtained with E . amylovora isolates from various geographic regions . This technique could also be used for detection of the fire blight pathogen in extracts of tissue obtained from infected plant material. Mol Microbiol, 1992 Nov, 6(21), 3199 - 211 Some of the out genes involved in the secretion of pectate lyases in Erwinia chrysanthemi are regulated by kdgR; Condemine G et al.; The out genes of Erwinia chrysanthemi are required for the translocation across the outer membrane of pectate lyases and cellulases . We present the characterization and the nucleotide sequence of five genes of the out cluster . The products of outS, B, C, D and E have significant homology with the PulS, B, C, D and E proteins necessary to the secretion of pullulanase in Klebsiella pneumoniae . An open reading frame, outT, located between outB and outC has no homology with the pul cluster but is involved in secretion . outC, outD and outE form an operon while outS, outB and outT constitute independent transcription units . outT and the outCDE operon are regulated by kdgR, the negative regulatory gene controlling pectinase production . outB and outS seem to be expressed constitutively. J Bacteriol, 1992 Nov, 174(22), 7385 - 97 Analysis of eight out genes in a cluster required for pectic enzyme secretion by Erwinia chrysanthemi: sequence comparison with secretion genes from other gram-negative bacteria; Lindeberg M et al.; Many extracellular proteins produced by Erwinia chrysanthemi require the out gene products for transport across the outer membrane . In a previous report (S . Y . He, M . Lindeberg, A . K . Chatterjee, and A . Collmer, Proc . Natl . Acad . Sci . USA 88:1079-1083, 1991) cosmid pCPP2006, sufficient for secretion of Erwinia chrysanthemi extracellular proteins by Escherichia coli, was partially sequenced, revealing four out genes sharing high homology with pulH through pulK from Klebsiella oxytoca . The nucleotide sequence of eight additional out genes reveals homology with pulC through pulG, pulL, pulM, pulO, and other genes involved in secretion by various gram-negative bacteria . Although signal sequences and hydrophobic regions are generally conserved between Pul and Out proteins, four out genes contain unique inserts, a pulN homolog is not present, and outO appears to be transcribed separately from outC through outM . The sequenced region was subcloned, and an additional 7.6-kb region upstream was identified as being required for secretion in E . coli . out gene homologs were found on Erwinia carotovora cosmid clone pAKC651 but were not detected in E . coli . The outC-through-outM operon is weakly induced by polygalacturonic acid and strongly expressed in the early stationary phase . The out and pul genes are highly similar in sequence, hydropathic properties, and overall arrangement but differ in both transcriptional organization and the nature of their induction. Res Microbiol, 1992 Nov-Dec, 143(9), 857 - 67 A fourth metalloprotease gene in Erwinia chrysanthemi; Ghigo JM et al.; Erwinia chrysanthemi, a Gram-negative phytopathogenic bacterium, was previously shown to secrete 3 related extracellular metalloproteases, A, B and C via a specific signal-peptide-independent pathway . A new gene (prtG) encoding a fourth, 52-kDa metalloprotease was identified on the same recombinant cosmid (pEW1) that carries the genes for the previously described proteases (prtA, prtB and prtC), for the specific secretion factors (prtD, prtE and prtF) and for a protease inhibitor (inh) cloned from E . chrysanthemi B374 . The predicted sequence of PrtG was similar to those of PrtA, PrtB and PrtC, its secretion required PrtD, PrtE and PrtF; its secretion signal was located at the C terminus but its proteolytic activity was distinct from that of the 3 other proteases . Results presented here suggest that prtG could be the first gene of an operon that includes inh, prtD, prtE and prtF. J Biol Chem, 1992 Oct 5, 267(28), 19891 - 5 Expression in Escherichia coli, purification, and reactivation of the recombinant Erwinia uredovora phytoene desaturase; Fraser PD et al.; A plasmid has been constructed by cloning the complete crtI gene encoding phytoene desaturase from Erwinia uredovora behind the lac Z promoter of pUC18 resulting in a reading frame for the full polypeptide with additional 9 amino acids at the N terminus . This plasmid mediated the overexpression of phytoene desaturase in transformed Escherichia coli . The overexpressed enzyme was sequestrated into inclusion bodies requiring urea treatment for solubilization . Purification to homogeneity was subsequently performed on a DEAE-cellulose column and by SDS-polyacrylamide gel electrophoresis . The purification scheme allowed the isolation of 5.3 mg of homogeneous desaturase protein from 100 ml of E . coli cell suspension . On SDS-polyacrylamide gel electrophoresis an apparent molecular mass of 56.2 kDa was determined . An antiserum raised against phytoene desaturase cross-reacted with the expressed protein and was employed to monitor the isolation steps . Upon removal of urea, desaturase activity was restored . The isolated desaturase catalyzed the conversion of 15-cis-phytoene to trans-lycopene as well as to bisdehydrolycopene . FAD was involved in desaturation, whereas NAD and NADP were inhibitory . This is the first time that a membrane-integrated carotenogenic enzyme has been purified and finally obtained in an active state. Proc Natl Acad Sci U S A, 1992 Oct 1, 89(19), 9321 - 5 Functional expression of zeaxanthin glucosyltransferase from Erwinia herbicola and a proposed uridine diphosphate binding site; Hundle BS et al.; Erwinia herbicola, a nonphotosynthetic bacterium, is yellow colored due to the accumulation of unusually polar carotenoids, primarily mono- and diglucosides of zeaxanthin . We have cloned and expressed the gene for the enzyme that catalyzes the glucosylation of zeaxanthin . The enzyme has an apparent molecular mass of 45 kDa on an SDS/polyacrylamide gel, which is consistent with its calculated molecular mass . In vitro enzymatic activity was demonstrated using UDP-{14C}glucose and zeaxanthin as substrates . The product zeaxanthin diglucoside and its intermediate monoglucoside were identified by thin layer chromatography . The optimum pH and temperature ranges of the enzyme are 7.0-7.5 and 32-37 degrees C, respectively . A hydropathy plot indicates no apparent membrane-spanning regions, and biochemical experiments suggest that the enzyme is weakly membrane-associated . The amino acid sequence derived from the zeaxanthin glucosyltransferase gene shows a small region of high similarity with other glucuronosyl- and glucosyltransferases that use either UDP-activated glucuronic acid or a sugar as one of their substrates . Based on these similarities, we propose that this conserved sequence is part of the UDP binding site. Biosci Biotechnol Biochem, 1992 Oct, 56(10), 1596 - 600 Molecular cloning and sequencing of the extracellular pectate lyase II gene from Erwinia carotovora Er; Yoshida A et al.; Erwinia carotovora Er produces three extra-cellular pectate lyases (PL I, II, and III) . The gene for pectate lyase II (pelII) of E . carotovora Er was cloned and expressed both in Escherichia coli and E . carotovora Er . Localization experiments in E . coli showed that PL II was exclusively in the cytoplasmic space, while PL II was excreted into the culture medium . The complete nucleotides of the pelII gene were sequenced and found to include one open reading frame of 1122 bp coding for a protein of 374 amino acid residues . From comparison of the N-terminal amino acid sequence between the purified PL II and the deduced protein from the nucleotide sequence we reached the conclusion that the mature protein is composed of 352 amino acids with a calculated molecular weight of 38,169 and is preceded by a typical signal sequence of 22 amino acid residues . PL II had 90.1% and 82.9% homologies with PL I and PL III in amino acid sequence, respectively. Mol Plant Microbe Interact, 1992 Sep-Oct, 5(5), 384 - 9 hrp genes of Pseudomonas solanacearum are homologous to pathogenicity determinants of animal pathogenic bacteria and are conserved among plant pathogenic bacteria; Gough CL et al.; The majority of bacterial plant diseases are caused by members of three bacterial genera, Pseudomonas, Xanthomonas, and Erwinia . The identification and characterization of mutants that have lost the abilities to provoke disease symptoms on a compatible host and to induce a defensive hypersensitive reaction (HR) on an incompatible host have led to the discovery of clusters of hrp genes (hypersensitive reaction and pathogenicity) in phytopathogenic bacteria from each of these genera . Here, we report that predicted protein sequences of three hrp genes from Pseudomonas solanacearum show remarkable sequence similarity to key virulence determinants of animal pathogenic bacteria of the genus Yersinia . We also demonstrate DNA homologies between P . solanacearum hrp genes and hrp gene clusters of P . syringae pv . phaseolicola, Xanthomonas campestris pv . campestris, and Erwinia amylovora . By comparing the role of the Yersinia determinants in the control of the extracellular production of proteins required for pathogenicity, we propose that hrp genes code for an export system that might be conserved among many diverse bacterial pathogens of plants and animals but that is distinct from the general export pathway. Appl Environ Microbiol, 1992 Sep, 58(9), 2792 - 8 Loss of allosteric control but retention of the bifunctional catalytic competence of a fusion protein formed by excision of 260 base pairs from the 3' terminus of pheA from Erwinia herbicola; Xia T et al.; A bifunctional protein denoted as the P protein and encoded by pheA is widely present in purple gram-negative bacteria . This P protein carries catalytic domains that specify chorismate mutase (CM-P) and prephenate dehydratase . The instability of a recombinant plasmid carrying a pheA insert cloned from Erwinia herbicola resulted in a loss of 260 bp plus the TAA stop codon from the 3' terminus of pheA . The plasmid carrying the truncated pheA gene (denoted pheA*) was able to complement an Escherichia coli pheA auxotroph . pheA* was shown to be a chimera composed of the residual 5' part of pheA (901 bp) and a 5-bp fragment from the pUC18 vector . The new fusion protein (PheA*) retained both chorismate mutase and prephenate dehydratase activities . PheA* had a calculated subunit molecular weight of 33,574, in comparison to the 43,182-molecular-weight subunit size of PheA . The deletion did not affect the ability of PheA* to assume the native dimeric configuration of PheA . Both the CM-P and prephenate dehydratase components of PheA* were insensitive to L-phenylalanine inhibition, in contrast to the corresponding components of PheA . L-Phenylalanine protected both catalytic activities of PheA from thermal inactivation, and this protective effect of L-phenylalanine upon the PheA* activities was lost . PheA* was more stable than PheA to thermal inactivation; this was more pronounced for prephenate dehydratase than for CM-P . In the presence of dithiothreitol, the differential resistance of PheA* prephenate dehydratase to thermal inactivation was particularly striking.(ABSTRACT TRUNCATED AT 250 WORDS) J Bacteriol, 1992 Aug, 174(16), 5471 - 4 Genetic evidence for an activator required for induction of pectin lyase in Erwinia carotovora subsp . carotovora by DNA-damaging agents; McEvoy JL et al.; In Erwinia carotovora subsp . carotovora 71, the induction of pectin lyase (Pnl), the bacteriocin carotovoricin (Ctv), and cellular lysis (Lss) requires a RecA function . We obtained mutants wherein a pleiotropic defect, i.e., the lack of induction with mitomycin C, is not restored by the recA+ DNA . From a genomic library of strain 71, a cosmid (pAKC280) that restored induction of Pnl, Ctv, and Lss by mitomycin C was isolated . The activator function, designated Rdg for regulator of damage-inducible genes, was localized by subcloning and insertional mutagenesis to a 2.6-kb region within a 6.7-kb EcoRI fragment . An rdg-lacZ operon fusion was inducible by mitomycin C in RecA+ but not RecA- derivatives of E . carotovora subsp . carotovora 71 and Escherichia coli . A RecA+ E . coli strain carrying only a PnlA+ plasmid was not inducible for Pnl production; however, when both a PnlA+ plasmid and a Rdg+ plasmid were present, the transcription of pnlA and the production of the enzyme were activated by mitomycin C . The size of the pnlA transcript produced in E . coli was identical to that of the transcript produced by E . carotovora subsp . carotovora 71, suggesting that the same promoter and termination sequences were being utilized in these bacteria. Gene, 1992 Aug 1, 117(1), 119 - 24 Characterization of the Erwinia carotovora peh gene and its product polygalacturonase; Lei SP et al.; The peh gene, encoding polygalacturonase (Peh), was identified in Erwinia carotovora strain EC and cloned in Escherichia coli . Recombinant Peh (re-Peh) was purified from E . coli strain 706 containing peh on a recombinant plasmid . The activity of the re-Peh protein is optimal at pH 5.5 . The N-terminal and internal amino acid (aa) sequences of re-Peh were determined and compared to the aa sequence deduced from the nucleotide (nt) sequence of the cloned peh . The re-Peh has no similarity, based on either the nt sequences or the deduced aa sequences, to pectate lyases from the same Er . carotovora strain or other organisms. J Bacteriol, 1992 Aug, 174(15), 4920 - 7 Secretion of CyaA-PrtB and HlyA-PrtB fusion proteins in Escherichia coli: involvement of the glycine-rich repeat domain of Erwinia chrysanthemi protease B; Letoffe S et al.; Protease B from Erwinia chrysanthemi was shown previously to have a C-terminal secretion signal located downstream of a domain that contains six glycine-rich repeats . This domain is conserved in all known bacterial proteins secreted by the signal peptide-independent pathway . The role of these repeats in the secretion process is controversial . We compared the secretion processes of various heterologous polypeptides fused either directly to the signal or separated from it by the glycine-rich domain . Although the repeats are not involved in the secretion of small truncated protease B carboxy-terminal peptides, they are required for the secretion of higher-molecular-weight fusion proteins . Secretion efficiency was also dependent on the size of the passenger polypeptide. Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 6761 - 4 The crtE gene in Erwinia herbicola encodes geranylgeranyl diphosphate synthase; Math SK et al.; A cluster of genes essential for the biosynthesis of carotenoids in Erwinia herbicola has been isolated and characterized {Armstrong, G.A., Alberti, M . & Hearst, J . E . (1990) Proc . Natl . Acad . Sci . USA 87, 9975-9979} . Related gene clusters are found in other carotenoid-producing bacteria . Two of these genes, crtB and crtE, have been assigned to enzymes responsible for conversion of geranylgeranyl diphosphate (GGPP) to prephytoene diphosphate and prephytoene diphosphate to phytoene, respectively . We isolated crtE from the Er . herbicola cluster by PCR amplification and cloned the coding region into the Escherichia coli expression vector pARC306N . Es . coli JM101 was transformed with the expression plasmid, and transformants were assayed for GGPP synthase and phytoene synthase activity . Extracts from JM101/pSM145 accumulated {14C}GGPP when incubated with {14C}isopentenyl diphosphate and farnesyl diphosphate, whereas similar incubations with {3H}GGPP did not yield prephytoene diphosphate or phytoene . Thus, crtE encodes GGPP synthase. Mol Microbiol, 1992 Aug, 6(16), 2363 - 76 Analysis of the regulation of the pelBC genes in Erwinia chrysanthemi 3937; Hugouvieux-Cotte-Pattat N et al.; Erwinia chrysanthemi secretes five major isoenzymes of pectate lyases encoded by the pelABCDE genes . The nucleotide sequence of the region surrounding the pelB gene of E . chrysanthemi 3937 was determined, including the regulatory regions involved in pelB and pelC expression . Analysis of the transcripts showed that transcription of pelB or pelC gave, in both cases, only one transcript . The transcription initiation sites of both pelB and pelC were precisely determined as well as the position of the transcription termination of pelB . The pelB and pelC promoters are very similar, showing a good homology with the -35 consensus region but low homology with the -10 consensus . In both cases a KdgR-box overlaps the -35 region . The pelC gene may have two KdgR operators . Moreover, the pelB and pelC genes are preceded by other sequences presenting the typical symmetry of operator sites that could be involved in more specific regulations . Comparison of E . chyrsanthemi pel regulatory regions revealed three classes of homology: pelA, pelB-pelC and pelD-pelE . The sole regulatory sequence conserved among the three classes corresponds to the KdgR-binding site . Moreover, all the pel regulatory regions are AT-rich in contrast to the coding regions which are GC-rich . Gel retardation experiments with fragments overlapping the pelB or pelC regulatory regions demonstrated that the KdgR protein specifically binds to these regions . Other proteins probably also interact with these DNA fragments . Transcription of pelB terminates in a region corresponding to a GC-rich inverted repeat followed by a run of T residues, typical of rho-independent transcription termination sites . Moreover, preliminary results imply that a region adjacent to pelC provoke, directly or indirectly, the repression of pelB and pelC expression. J Appl Bacteriol, 1992 Aug, 73(2), 114 - 9 Haemagglutinins and fimbriae of soft rot Erwinias; Wallace A et al.; Strains of phytopathogenic soft rot Erwinia spp . were examined for haemagglutinin (HA) production . Mannose-sensitive HA was found only in five of 15 strains of E . carotovora subsp . carotovora . Mannose-resistant HA (MRHA) was found in 12 of 15 strains of E.c . carotovora, ten of 13 strains of E.c . subsp . atroseptica and the single strain of E.c . subsp . betavasculorum, as well as all seven strains of E . chrysanthemi . MRHA, detectable only in a microtitre tray HA assay was of either broad- or narrow-spectrum activity when examined against blood of seven different animal species and could be inhibited by the beta-galactoside asialofetuin . Fimbriae of ca 10 nm diameter were found on MRHA(+) bacteria E.c . carotovora and E.c . atroseptica. Science, 1992 Jul 3, 257(5066), 85 - 8 Harpin, elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora; Wei ZM et al.; A proteinaceous elicitor of the plant defense reaction known as the hypersensitive response was isolated from Erwinia amylovora, the bacterium that causes fire blight of pear, apple, and other rosaceous plants . The elicitor, named harpin, is an acidic, heat-stable, cell-envelope-associated protein with an apparent molecular weight of 44 kilodaltons . Harpin caused tobacco leaf lamina to collapse and caused an increase in the pH of bathing solutions of suspension-cultured tobacco cells . The gene encoding harpin (hrpN) was located in the 40-kilobase hrp gene cluster of E . amylovora, sequenced, and mutated with Tn5tac1 . The hrpN mutants were not pathogenic to pear, did not elicit the hypersensitive response, and did not produce harpin. Gene, 1992 Jul 1, 116(1), 87 - 91 A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia; Bainton NJ et al.; Micro-organisms have evolved complex and diverse mechanisms to sense environmental changes . Activation of a sensory mechanism typically leads to alterations in gene expression facilitating an adaptive response . This may take several forms, but many are mediated by response-regulator proteins . The luxR-encoded protein (LuxR) has previously been characterised as a member of the response-regulator superfamily and is known to respond to the small diffusible autoinducer signal molecule N-(beta-ketocaproyl) homoserine lactone (KHL) . Observed previously in only a few marine bacteria, we now report that KHL is in fact produced by a diverse group of terrestrial bacteria . In one of these (Erwinia carotovora), we show that it acts as a molecular control signal for the expression of genes controlling carbapenem antibiotic biosynthesis . This represents the first substantive evidence to support the previous postulate that the lux autoinducer, KHL, is widely involved in bacterial signalling. Gene, 1992 Jul 1, 116(1), 27 - 33 Expression of the Erwinia carotovora polygalacturonase-encoding gene in Bacillus subtilis: role of signal peptide fusions on production of a heterologous protein; Hemila H et al.; The pehA gene encoding an endopolygalacturonase (pectinase) of Erwinia carotovora subsp . carotovora has been cloned previously {Saarilahti et al., Mol . Microbiol . 4 (1990) 1037-1044} . We expressed pehA in Bacillus subtilis using a secretion vector based on the promoter and signal sequence of the alpha-amylase (Amy)-encoding gene, amyE, from Bacillus amyloliquefaciens . To test whether the location of the junction between the secretion vector and pehA affects the protein yield, we made four different junctions . Two constructs contained an intact Amy signal sequence, whereas the other two were fusions between the Amy signal sequence and the polygalacturonase (PG) signal sequence . There was approximately fourfold variation in the production efficiency of B . subtilis strains carrying the different constructs . The most efficient construct contained the N-terminal and hydrophobic regions of the Amy signal peptide joined to the C terminus of PG signal peptide . This construct produced, in a shake flask culture, 0.8 g of polygalacturonase per liter of growth medium . In a pulse-chase experiment, the signal peptide of the most efficient construct was rapidly cleaved while cleavage was slow in the other constructs . Our results suggest that fusions containing intact signal peptides, which are