| 1. |
Yoshida A,
Matsuo Y,
Kamio Y,
Izaki K,
( 1992 ) Molecular cloning and sequencing of the extracellular pectate lyase II gene from Erwinia carotovora Er. PMID : 1369060 : DOI : 10.1271/bbb.56.1596 Abstract >>
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.
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2. |
Yoshida A,
Izuta M,
Ito K,
Kamio Y,
Izaki K,
( 1991 ) Cloning and characterization of the pectate lyase III gene of Erwinia carotovora Er. PMID : 1368679 : Abstract >>
A pectate lyase gene III (pel III) of Erwinia carotovora Er was cloned. The gene was expressed independently of a vector promoter in both E. carotovora Er and Escherichia coli. The pel III product was largely excreted in the culture medium of E. carotovora Er, while the product was only exported to the periplasmic space and was not excreted in the medium of E. coli. Nucleotide sequence analysis of pel III disclosed an open reading frame of 1,122 bp encoding a protein of 374 amino acids. The deduced amino acid sequence contained the N-terminal 30 amino acid sequence from the purified pectate lyase III (PL III) indicating the presence of a 22-amino-acid signal peptide. A putative ribosome-binding site was found to be 9 bp upstream of the start codon. The location of pel III was about 5.6 kb downstream of pel I. The PL III showed 80% homology in the amino acid sequence with the PL I of E. carotovora Er.
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3. |
Shirae H,
Yokozeki K,
( 1991 ) Purifications and properties of orotidine-phosphorolyzing enzyme and purine nucleoside phosphorylase from Erwinia carotovora AJ 2992. PMID : 1368721 : Abstract >>
An orotidine-phosphorolyzing enzyme and a purine nucleoside phosphorylase (PNPase) of Erwinia carotovora AJ 2992, which is a potent producer of ribavirin (1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide), an antiviral agent, from orotidine and 1,2,4-triazole-3-carboxamide (TCA), were purified 23-fold and 103-fold, respectively. At each purification step, the orotidine-phosphorolyzing enzyme was always co-purified with an uridine phosphorylase (UPase) and its activity could not be separated from that of the UPase after it showed as a single band on SDS-polyacrylamide gel electrophoresis. These results suggest that this enzyme may be identical with UPase. The purified enzyme had a molecular weight of 68,000 +/- 2,000, and seemed to be a dimer. The optimal temperatures and pH values were 60 degrees C and 6.0 for orotidine phosphorolysis, and 70 degrees C and 7.0 for uridine phosphorolysis. The Michaelis constants for uridine and orotidine were 0.75 mM and 10.87 mM, respectively, at 40 degrees C. The PNPase of E. carotovora AJ 2992 had a molecular weight of 58,000 +/- 2,000 and seemed to be a dimer. The Michaelis constants for inosine and guanosine were 1.92 mM and 1.85 mM, respectively, at 40 degrees C. The PNPase was completely inactivated by p-chloromercuribenzoate.
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4. |
Harada H,
Ishikawa H,
( 1997 ) Phylogenetical relationship based on groE genes among phenotypically related Enterobacter, Pantoea, Klebsiella, Serratia and Erwinia species. PMID : 12501307 : Abstract >>
In an attempt to define the phylogenetical relationship among 17 phenotypically related species of genera Enterobacter, Pantoea, Serratia, Klebsiella and Erwinia, we determined almost all of their groE operon sequences using the polymerase chain reaction direct sequencing method. The number of nucleotide substitutions per site was 0.12+/-0.030. The value was 3.6-fold higher than that of 16S rDNA. As a result, we were successful in constructing molecular phylogenetic trees which had a finer resolution than that based on the 16S rDNA sequences. The phylogenetic trees based on the nucleotide sequences and deduced amino acid sequences of groE operons indicated that the members of genera Enterobacter, Pantoea and Klebsiella were closely related to each other, while Serratia and Erwinia species except Erwinia carotovora, made distinct clades. The close relationship between Enterobacter aerogenes and Klebsiella pneumoniae, that had been suggested by biochemical tests and DNA hybridization, was also supported by our molecular phylogenetic trees.
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5. |
Kim C,
Xuong NH,
Edwards S,
Madhusudan N/A,
Yee MC,
Spraggon G,
Mills SE,
( 2002 ) The crystal structure of anthranilate phosphoribosyltransferase from the enterobacterium Pectobacterium carotovorum. PMID : 12123839 : DOI : 10.1016/s0014-5793(02)02905-8 Abstract >>
The structure of anthranilate phosphoribosyltransferase from the enterobacterium Pectobacterium carotovorum has been solved at 2.4 A in complex with Mn(2+)-pyrophosphate, and at 1.9 A without ligands. The enzyme structure has a novel phosphoribosyltransferase (PRT) fold and displays close homology to the structures of pyrimidine nucleoside phosphorylases. The enzyme is a homodimer with a monomer of 345 residues. Each monomer consists of two subdomains, alpha and alpha/beta, which form a cleft containing the active site. The nature of the active site is inferred from the trapped MnPPi complex and detailed knowledge of the active sites of nucleoside phosphorylases. With the anthranilate (An)PRT structure solved, the structures of all the enzymes required for tryptophan biosynthesis are now known.
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6. |
Dauga C,
( 2002 ) Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies. PMID : 11931166 : DOI : 10.1099/00207713-52-2-531 Abstract >>
Phylogenetic trees showing the evolutionary relatedness of Enterobacteriaceae based upon gyrB and 16S rRNA genes were compared. Congruence among trees of these molecules indicates that the genomes of these species are not completely mosaic and that molecular systematic studies can be carried out. Phylogenetic trees based on gyrB sequences appeared to be more reliable at determining relationships among Serratia species than trees based on 16S rRNA gene sequences. gyrB sequences from Serratia species formed a monophyletic group validated by significant bootstrap values. Serratia fonticola had the most deeply branching gyrB sequence in the Serratia monophyletic group, which was consistent with its atypical phenotypic characteristics. Klebsiella and Enterobacter genera seemed to be polyphyletic, but the branching patterns of gyrB and 16S rRNA gene trees were not congruent. Enterobacter aerogenes was grouped with Klebsiella pneumoniae on the gyrB phylogenetic tree, which supports that this species could be transferred to the Klebsiella genus. Unfortunately, 16S rRNA and gyrB phylogenetic trees gave conflicting evolutionary relationships for Citrobacter freundii because of its unusual gyrB evolutionary process. gyrB lateral gene transfer was suspected for Hafnia alvei. Saturation of gyrB genes was observed by the pairwise comparison of Proteus spp., Providencia alcalifaciens and Morganella morganii sequences. Depending on their level of variability, 16S rRNA gene sequences were useful for describing phylogenetic relationships between distantly related Enterobacteriaceae, whereas gyrB sequence comparison was useful for inferring intra- and some intergeneric relationships.
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7. |
Nguyen HA,
Tomita T,
Hirota M,
Kaneko J,
Hayashi T,
Kamio Y,
( 2001 ) DNA inversion in the tail fiber gene alters the host range specificity of carotovoricin Er, a phage-tail-like bacteriocin of phytopathogenic Erwinia carotovora subsp. carotovora Er. PMID : 11591670 : DOI : 10.1128/JB.183.21.6274-6281.2001 PMC : PMC100113 Abstract >>
Carotovoricin Er is a phage-tail-like bacteriocin produced by Erwinia carotovora subsp. carotovora strain Er, a causative agent for soft rot disease in plants. Here we studied binding and killing spectra of carotovoricin Er preparations for various strains of the bacterium (strains 645Ar, EC-2, N786, and P7) and found that the preparations contain two types of carotovoricin Er with different host specificities; carotovoricin Era possessing a tail fiber protein of 68 kDa killed strains 645Ar and EC-2, while carotovoricin Erb with a tail fiber protein of 76 kDa killed strains N786 and P7. The tail fiber proteins of 68 and 76 kDa had identical N-terminal amino acid sequences for at least 11 residues. A search of the carotovoricin Er region in the chromosome of strain Er indicated the occurrence of a DNA inversion system for the tail fiber protein consisting of (i) two 26-bp inverted repeats inside and downstream of the tail fiber gene that flank a 790-bp fragment and (ii) a putative DNA invertase gene with a 90-bp recombinational enhancer sequence. In fact, when a 1,400-bp region containing the 790-bp fragment was amplified by a PCR using the chromosomal DNA of strain Er as the template, both the forward and the reverse nucleotide sequences of the 790-bp fragment were detected. DNA inversion of the 790-bp fragment also occurred in Escherichia coli DH5alpha when two compatible plasmids carrying either the 790-bp fragment or the invertase gene were cotransformed into the bacterium. Furthermore, hybrid carotovoricin CGE possessing the tail fiber protein of 68 or 76 kDa exhibited a host range specificity corresponding to that of carotovoricin Era or Erb, respectively. Thus, a DNA inversion altered the C-terminal part of the tail fiber protein of carotovoricin Er, altering the host range specificity of the bacteriocin.
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8. |
Bricker AL,
Diwa A,
( 2000 ) An evolutionarily conserved RNA stem-loop functions as a sensor that directs feedback regulation of RNase E gene expression. PMID : 10817759 : PMC : PMC316614 Abstract >>
RNase E is a key regulatory enzyme that controls the principal pathway for mRNA degradation in Escherichia coli. The cellular concentration of this endonuclease is governed by a feedback mechanism in which RNase E tightly regulates its own synthesis. Autoregulation is mediated in cis by the 361-nucleotide 5' untranslated region (UTR) of rne (RNase E) mRNA. Here we report the determination of the secondary structure of the rne 5' UTR by phylogenetic comparison and chemical alkylation, together with dissection studies to identify the 5' UTR element that mediates autoregulation. Our findings reveal that the structure and function of the rne 5' UTRs are evolutionarily well conserved despite extensive sequence divergence. Within the rne 5' UTRs are multiple RNA secondary structure elements, two of which function in cis to mediate feedback regulation of rne gene expression. The more potent of these two elements is a stem-loop structure containing an internal loop whose sequence is the most highly conserved of any region of the rne 5' UTR. Our data show that this stem-loop functions as a sensor of cellular RNase E activity that directs autoregulation by modulating the degradation rate of rne mRNA in response to changes in RNase E activity.
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9. |
Kõiv V,
Norman-Setterblad C,
Andersson RA,
( 1999 ) Role of RpoS in virulence and stress tolerance of the plant pathogen Erwinia carotovora subsp. carotovora. PMID : 10627052 : DOI : 10.1099/00221287-145-12-3547 Abstract >>
The plant-pathogenic bacterium Erwinia carotovora subsp. carotovora causes plant disease mainly through a number of extracellular plant-cell-wall-degrading enzymes. In this study, the ability of an rpoS mutant of the Er. carotovora subsp. carotovora strain SCC3193 to infect plants and withstand environmental stress was characterized. This mutant was found to be sensitive to osmotic and oxidative stresses in vitro and to be deficient in glycogen accumulation. The production of extracellular enzymes in vitro was similar in the mutant and in the wild-type strains. However, the rpoS mutant caused more severe symptoms than the wild-type strain on tobacco plants and also produced more extracellular enzymes in planta, but did not grow to higher cell density in planta compared to the wild-type strain. When tested on plants with reduced catalase activities, which show higher levels of reactive oxygen species, the rpoS mutant was found to cause lower symptom levels and to have impaired growth. In addition, the mutant was unable to compete with the wild-type strain in planta and in vitro. These results suggest that a functional rpoS gene is needed mainly for survival in a competitive environment and during stress conditions, and not for effective infection of plants.
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10. |
Young JM,
Park DC,
( 2007 ) Relationships of plant pathogenic enterobacteria based on partial atpD, carA, and recA as individual and concatenated nucleotide and peptide sequences. PMID : 17451899 : DOI : 10.1016/j.syapm.2007.03.002 Abstract >>
Relationships of the genera in the Enterobacteriaceae containing plant pathogenic species: Brenneria, Dickeya, Enterobacter, Erwinia, Pantoea, Pectobacterium, and Samsonia, were investigated by comparison of their nucleotide and peptide sequences of atpD, carA, recA, and the concatenated sequences. Erwinia spp. and Pantoea spp., with Pectobacterium cypripedii, formed a group distinct from other pathogenic taxa. Pectobacterium, Brenneria, Dickeya, and Samsonia formed a contiguous clade. Samsonia was usually concurrent with Pectobacterium. Most Brenneria were also close to Pectobacterium, suggesting that these three taxa might be better represented as a single genus. Brenneria quercina was not closely associated with other members of this genus and may represent a separate genus. The sequences representing Dickeya were distinct, further supporting the generic status of the taxon. Plant pathogenic Enterobacter spp. display such sequence variability that few definite conclusions as to their specific placement could be made. These data highlight the difficulty of drawing reliable and robust taxonomic conclusions based on comparative analysis of sequence data without some independent criterion to calibrate a scale for diversity.
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11. |
Chatterjee A,
McEvoy JL,
Chambost JP,
Blasco F,
Chatterjee AK,
( 1991 ) Nucleotide sequence and molecular characterization of pnlA, the structural gene for damage-inducible pectin lyase of Erwinia carotovora subsp. carotovora 71. PMID : 1705542 : DOI : 10.1128/jb.173.5.1765-1769.1991 PMC : PMC207328 Abstract >>
In a previous study, pnlA (the DNA damage-inducible structural gene for pectin lyase) of Erwinia carotovora subsp. carotovora 71 was localized to a 1.4-kb DNA segment within a 3.4-kb EcoRI fragment (J. L. McEvoy, H. Murata, and A. K. Chatterjee, J. Bacteriol. 172:3284-3289, 1990). We present here DNA sequence data for a 2.2-kb region revealing an open reading frame of 870 bases, corresponding to a protein (Pnl) of an approximate molecular mass of 32,100 Da and an isoelectric point of 9.92. Although initiation of translation is presumed to occur at the ATG codon, direct protein sequencing revealed alanine as the N-terminal amino acid, probably as a consequence of posttranslational removal of the initiating amino acid. The sequence of the first 20 amino acid residues of Pnl, purified from E. carotovora subsp. carotovora 71, agreed completely with the predicted amino acid sequence of the N-terminal segment. This finding also indicated that Pnl is not subject to processing by a signal peptidase. The transcriptional start site of pnlA was determined to reside 80 bp upstream of the translational start site. Deletion analysis revealed that 218 bp of DNA upstream of the transcriptional start site is sufficient for induction of pnlA by mitomycin C. Within 600 bp upstream of the translational start site, no sequences resembling a LexA binding site (SOS box) or a cyclic AMP receptor protein binding site were found. However, palindromic sequences were detected at -187 and -86 bp relative to the translational start site, and these could be potential sites for the binding of a regulatory protein(s). Comparison of the deduced amino acid sequence for PnlA with that of a Pnl from Aspergillus niger and with those of various pectate lyases of Erwinia species revealed a low degree of homology dispersed throughout the length of the proteins.
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12. |
Lei SP,
Lin HC,
Wang SS,
Higaki P,
Wilcox G,
( 1992 ) Characterization of the Erwinia carotovora peh gene and its product polygalacturonase. PMID : 1644302 : DOI : 10.1016/0378-1119(92)90499-f Abstract >>
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.
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13. |
Schneider KL,
Marrero G,
Alvarez AM,
Presting GG,
( 2011 ) Classification of plant associated bacteria using RIF, a computationally derived DNA marker. PMID : 21533033 : DOI : 10.1371/journal.pone.0018496 PMC : PMC3080875 Abstract >>
A DNA marker that distinguishes plant associated bacteria at the species level and below was derived by comparing six sequenced genomes of Xanthomonas, a genus that contains many important phytopathogens. This DNA marker comprises a portion of the dnaA replication initiation factor (RIF). Unlike the rRNA genes, dnaA is a single copy gene in the vast majority of sequenced bacterial genomes, and amplification of RIF requires genus-specific primers. In silico analysis revealed that RIF has equal or greater ability to differentiate closely related species of Xanthomonas than the widely used ribosomal intergenic spacer region (ITS). Furthermore, in a set of 263 Xanthomonas, Ralstonia and Clavibacter strains, the RIF marker was directly sequenced in both directions with a success rate approximately 16% higher than that for ITS. RIF frameworks for Xanthomonas, Ralstonia and Clavibacter were constructed using 682 reference strains representing different species, subspecies, pathovars, races, hosts and geographic regions, and contain a total of 109 different RIF sequences. RIF sequences showed subspecific groupings but did not place strains of X. campestris or X. axonopodis into currently named pathovars nor R. solanacearum strains into their respective races, confirming previous conclusions that pathovar and race designations do not necessarily reflect genetic relationships. The RIF marker also was sequenced for 24 reference strains from three genera in the Enterobacteriaceae: Pectobacterium, Pantoea and Dickeya. RIF sequences of 70 previously uncharacterized strains of Ralstonia, Clavibacter, Pectobacterium and Dickeya matched, or were similar to, those of known reference strains, illustrating the utility of the frameworks to classify bacteria below the species level and rapidly match unknown isolates to reference strains. The RIF sequence frameworks are available at the online RIF database, RIFdb, and can be queried for diagnostic purposes with RIF sequences obtained from unknown strains in both chromatogram and FASTA format.
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14. |
Nishida T,
Suzuki T,
Ito K,
Kamio Y,
Izaki K,
( 1990 ) Cloning and expression of pectin lyase gene from Erwinia carotovora in Escherichia coli. PMID : 2185758 : DOI : 10.1016/0006-291x(90)92392-d Abstract >>
A pectin lyase (PNL; EC 4.2.2.10) gene of Erwinia carotovora Er was cloned and expressed in Escherichia coli. The analysis of the nucleotide sequence of the 0.6 kb StuI-EcoRI fragment, which was hybridized with the mixed oligonucleotide probe for PNL gene, revealed the presence of an open reading frame (0RF) and correlated exactly with the known N-terminal 18 amino acid sequence of PNL. When a plasmid pTN2159, which has a BamHI-EcoRI fragment containing this ORF, was introduced into E. coli JM109, PNL was not expressed. When a tac-promoter was inserted in front of the ORF, PNL was efficiently expressed in E. coli. Synthesis of PNL by E. coli was also confirmed by immunoblot analysis.
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15. |
Scott-Craig JS,
Panaccione DG,
Cervone F,
Walton JD,
( 1990 ) Endopolygalacturonase is not required for pathogenicity of Cochliobolus carbonum on maize. PMID : 2152162 : DOI : 10.1105/tpc.2.12.1191 PMC : PMC159966 Abstract >>
A gene (PGN1) encoding extracellular endopolygalacturonase was isolated from the fungal maize pathogen Cochliobolus carbonum race 1. A probe was synthesized by polymerase chain reaction using oligonucleotides based on the endopolygalacturonase amino acid sequence. Genomic and cDNA copies of the gene were isolated and sequenced. The corresponding mRNA was present in C. carbonum grown on pectin but not on sucrose as carbon source. The single copy of PGN1 in C. carbonum was disrupted by homologous integration of a plasmid containing an internal fragment of the gene. Polygalacturonase activity in one transformant chosen for further analysis was 10% or 35% of the wild-type activity based on viscometric or reducing sugar assays, respectively. End product analysis indicated that the residual activity in the mutant was due to an exopolygalacturonase. Pathogenicity on maize of the mutant lacking endopolygalacturonase activity was qualitatively indistinguishable from the wild-type strain, indicating that in this disease interaction endopolygalacturonase is not required. Either pectin degradation is not critical to this interaction or exopolygalacturonase alone is sufficient.
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16. |
Kersey CM,
Agyemang PA,
Dumenyo CK,
( 2012 ) CorA, the magnesium/nickel/cobalt transporter, affects virulence and extracellular enzyme production in the soft rot pathogen Pectobacterium carotovorum. PMID : 21726393 : DOI : 10.1111/j.1364-3703.2011.00726.x Abstract >>
Pectobacterium carotovorum (formerly Erwinia carotovora ssp. carotovora) is a phytopathogenic bacterium that causes soft rot disease, characterized by water-soaked soft decay, resulting from the action of cell wall-degrading exoenzymes secreted by the pathogen. Virulence in soft rot bacteria is regulated by environmental factors, host and bacterial chemical signals, and a network of global and gene-specific bacterial regulators. We isolated a mini-Tn5 mutant of P. carotovorum that is reduced in the production of extracellular pectate lyase, protease, polygalacturonase and cellulase. The mutant is also decreased in virulence as it macerates less host tissues than its parent and is severely impaired in multiplication in planta. The inactivated gene responsible for the reduced virulent phenotype was identified as corA. CorA, a magnesium/nickel/cobalt membrane transporter, is the primary magnesium transporter for many bacteria. Compared with the parent, the CorA(-) mutant is cobalt resistant. The mutant phenotype was confirmed in parental strain P. carotovorum by marker exchange inactivation of corA. A functional corA(+) DNA from P. carotovorum restored exoenzyme production and pathogenicity to the mutants. The P. carotovorum corA(+) clone also restored motility and cobalt sensitivity to a CorA(-) mutant of Salmonella enterica. These data indicate that CorA is required for exoenzyme production and virulence in P. carotovorum.
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17. |
Ohnishi H,
Nishida T,
Yoshida A,
Kamio Y,
Izaki K,
( 1991 ) Nucleotide sequence of pnl gene from Erwinia carotovora Er. PMID : 2018526 : DOI : 10.1016/0006-291x(91)90927-y Abstract >>
The nucleotide sequence of pnl gene encoding pectin lyase (PNL; EC4.2.2.10)from Erwinia carotovora Er was determined. The structural gene of pnl consisted of 942 base pairs. An open reading frame that could encode a 33,700 dalton polypeptide consisting 314 amino acids was assigned. The molecular size of the polypeptide predicted from the amino acid composition was close to the value of PNL determined in E.carotovora Er. The nucleotide sequence of the 5'-flanking region showed the presence of the consensus sequence of ribosome binding site, Pribnow box and the RNA polymerase recognition site in E.carotovora and Escherichia coli. Between the presumed Pribnow box and the ribosome binding site, two pairs of inverted repeats were found. By comparing the predicted amino acid sequences of pnl, several reported bacterial pectate lyases and Aspergillus niger pectin lyase, short regions of homology were found despite the different substrate specificities of these enzymes.
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18. |
Parkinson N,
Stead D,
Bew J,
Heeney J,
Tsror Lahkim L,
Elphinstone J,
( 2009 ) Dickeya species relatedness and clade structure determined by comparison of recA sequences. PMID : 19620370 : DOI : 10.1099/ijs.0.009258-0 Abstract >>
Using sequences from the recA locus, we have produced a phylogeny of 188 Dickeya strains from culture collections and identified species relatedness and subspecies clade structure within the genus. Of the six recognized species, Dickeya paradisiaca, D. chrysanthemi and D. zeae were discriminated with long branch lengths. The clade containing the D. paradisiaca type strain included just one additional strain, isolated from banana in Colombia. Strains isolated from Chrysanthemum and Parthenium species made up most of the clade containing the D. chrysanthemi type strain, and the host range of this species was extended to include potato. The D. zeae clade had the largest number of sequevars and branched into two major sister clades that contained all of the Zea mays isolates, and were identified as phylotypes PI and PII. The host range was increased from six to 13 species, including potato. The recA sequence of an Australian sugar-cane strain was sufficiently distinct to rank as a new species-level branch. In contrast to these species, Dickeya dadantii, D. dianthicola and D. dieffenbachiae were distinguished with shorter branch lengths, indicating relatively closer relatedness. The recA sequence for the type strain of D. dadantii clustered separately from other strains of the species. However, sequence comparison of three additional loci revealed that the D. dadantii type strain grouped together with the six other D. dadantii strains that were sequenced. Analysis of all four loci indicated that the D. dadantii strains were most closely related to D. dieffenbachiae. Three further branches (DUC-1, -2 and -3) were associated with these three species, which all diverged from a common origin and can be considered as a species complex. The large clade containing the D. dianthicola type strain comprised 58 strains and had little sequence diversity. One sequevar accounted for the majority of these strains, which were isolated nearly exclusively from eight hosts from Europe. Isolation of this sequevar on multiple occasions from Dianthus and (more recently) potato demonstrates that this lineage has become established in these species. The D. dadantii clade comprised 11 sequevars, and the known host range of the species was extended from eight to 19 species. New hosts included several ornamental species and potato. The clade DUC-1 was made up exclusively of potato strains originating from Europe, which had identical sequences, whilst DUC-2 strains were isolated mostly from a variety of monocotyledonous species. A single strain from Aglaonema sp. made up DUC-3. A single sequevar constituted the D. dieffenbachiae clade. The phylogenetic method described will provide a simple means for identification to the species and intraspecies level, which will support efforts to control these pathogens based on monitoring and surveillance.
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19. |
Mavrodi DV,
Peever TL,
Mavrodi OV,
Parejko JA,
Raaijmakers JM,
Lemanceau P,
Mazurier S,
Heide L,
Blankenfeldt W,
Weller DM,
Thomashow LS,
( 2010 ) Diversity and evolution of the phenazine biosynthesis pathway. PMID : 20008172 : DOI : 10.1128/AEM.02009-09 PMC : PMC2813009 Abstract >>
Phenazines are versatile secondary metabolites of bacterial origin that function in biological control of plant pathogens and contribute to the ecological fitness and pathogenicity of the producing strains. In this study, we employed a collection of 94 strains having various geographic, environmental, and clinical origins to study the distribution and evolution of phenazine genes in members of the genera Pseudomonas, Burkholderia, Pectobacterium, Brevibacterium, and Streptomyces. Our results confirmed the diversity of phenazine producers and revealed that most of them appear to be soil-dwelling and/or plant-associated species. Genome analyses and comparisons of phylogenies inferred from sequences of the key phenazine biosynthesis (phzF) and housekeeping (rrs, recA, rpoB, atpD, and gyrB) genes revealed that the evolution and dispersal of phenazine genes are driven by mechanisms ranging from conservation in Pseudomonas spp. to horizontal gene transfer in Burkholderia spp. and Pectobacterium spp. DNA extracted from cereal crop rhizospheres and screened for the presence of phzF contained sequences consistent with the presence of a diverse population of phenazine producers in commercial farm fields located in central Washington state, which provided the first evidence of United States soils enriched in indigenous phenazine-producing bacteria.
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20. |
Kim HS,
Ma B,
Perna NT,
Charkowski AO,
( 2009 ) Phylogeny and virulence of naturally occurring type III secretion system-deficient Pectobacterium strains. PMID : 19411432 : DOI : 10.1128/AEM.01336-08 PMC : PMC2704834 Abstract >>
Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers.
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21. |
Olarte-Lozano M,
Mendoza-Nuñez MA,
Pastor N,
Segovia L,
Folch-Mallol J,
Martínez-Anaya C,
( 2014 ) PcExl1 a novel acid expansin-like protein from the plant pathogen Pectobacterium carotovorum, binds cell walls differently to BsEXLX1. PMID : 24755657 : DOI : 10.1371/journal.pone.0095638 PMC : PMC3995998 Abstract >>
Microbial expansins act on plant cell walls similarly to plant expansins, albeit their loosening activity levels are tenfold lesser compared to plant expansins. We report the characterization of an expansin-like gene from the plant pathogen Pectobacterium carotovorum, named exl1. PcExl1 is an acidic protein that binds cellulose (Avicel), and weakens filter paper. The acidic nature of PcExl1 confers different binding properties when compared to Bacillus subtilis BsEXLX1, which is a basic protein. PcExl1 binding to wheat cell wall increased when acidic components were depleted, reaching a similar level to the binding to Avicel, indicating that cellulose is the target of PcExl1.
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22. |
Welte CU,
de Graaf RM,
van den Bosch TJ,
Op den Camp HJ,
van Dam NM,
Jetten MS,
( 2016 ) Plasmids from the gut microbiome of cabbage root fly larvae encode SaxA that catalyses the conversion of the plant toxin 2-phenylethyl isothiocyanate. PMID : 26234684 : DOI : 10.1111/1462-2920.12997 Abstract >>
Cabbage root fly larvae (Delia radicum) cause severe crop losses (? 50%) of rapeseed/ canola and cabbages used in the food and biofuel industries. These losses occur despite the fact that cabbages produce insecticidal toxins such as isothiocyanates. Here we describe the cabbage root fly larval gut microbiome as a source of isothiocyanate degrading enzymes. We sequenced the microbial gut community of the larvae and analysed phylogenetic markers and functional genes. We combined this with the isolation of several microbial strains representing the phylogenetic distribution of the metagenome. Eleven of those isolates were highly resistant towards 2-phenylethyl isothiocyanate, a subset also metabolized 2-phenylethyl isothiocyanate. Several plasmids appeared to be shared between those isolates that metabolized the toxin. One of the plasmids harboured a saxA gene that upon transformation gave resistance and enabled the degradation of 2-phenylethyl isothiocyanate in Escherichia coli. Taken together, the results showed that the cabbage root fly larval gut microbiome is capable of isothiocyanate degradation, a characteristic that has not been observed before, and may help us understand and design new pest control strategies.
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23. |
Trollinger D,
Berry S,
Belser W,
Keen NT,
( N/A ) Cloning and characterization of a pectate lyase gene from Erwinia carotovora EC153. PMID : 2520159 : Abstract >>
A pel gene cloned from strain EC153 of Erwinia carotovora encoded a pectate lyase that macerated plant tissue with moderate efficiency. This gene, called pel153, was sequenced and found to possess considerable homology with a pectate lyase gene from Yersinia pseudotuberculosis. The Yersinia protein, however, was truncated at the carboxyl terminal end relative to the Erwinia gene product and had a lower isoelectric point. The Erwinia pel153 gene was overexpressed in cells of Escherichia coli, and a 56-kDa protein was observed on sodium dodecyl sulfate-polyacrylamide gels. This compares with a molecular weight of 61 kDa for the mature, secreted protein as determined from sequencing data. Southern blot analysis disclosed the presence of the pel153 gene in three different strains of E. carotovora, but mutation of the gene in strain EC153 did not affect its ability to soft-rot potato tubers.
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24. |
Zhao XJ,
( 1990 ) DNA sequence analysis of the recA genes from Proteus vulgaris, Erwinia carotovora, Shigella flexneri and Escherichia coli B/r. PMID : 2274037 : DOI : 10.1007/bf00633842 Abstract >>
The complete nucleotide sequences of the recA genes from Escherichia coli B/r, Shigella flexneri, Erwinia carotovora and Proteus vulgaris were determined. The DNA sequence of the coding region of the E. coli B/r gene contained a single nucleotide change compared with the E. coli K12 gene sequence whereas the S. flexneri gene differed at 7 residues. In both cases, the predicted proteins were identical in primary structure to the E. coli K12 RecA protein. The DNA sequences of the recA genes from E. carotovora and P. vulgaris were 80% and 74% homologous, respectively, to the E. coli K12 gene. The predicted amino acid sequences of the E. carotovora and P. vulgaris RecA proteins were 91% and 85% identical respectively, to that of E. coli K12. The RecA proteins from both P. vulgaris and E. carotovora diverged significantly in sequence in the last 50 residues whereas they showed striking conservation throughout the first 300 amino acids which include an ATP-binding region and a subunit interaction domain. A putative LexA repressor binding site was localized upstream of each of the heterologous genes.
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25. |
( 1996 ) Genetic organization of a DNA-processing region required for mobilization of a non-self-transmissible plasmid, pEC3, isolated from Erwinia carotovora subsp. carotovora. PMID : 8621089 : DOI : 10.1016/0378-1119(95)00806-3 Abstract >>
A non-self-transmissible multiple-copy plasmid, pEC3, isolated from the phytopathogenic bacterium, Erwinia carotovora subsp. carotovora, can be mobilized by an IncP-type plasmid. The hybrid plasmid vector, pETC3, constructed from pEC3 by fusion to markers conferring TcR and CmR, was transferred by conjugation from Escherichia coli (Ec) to various genera of Enterobacteriaceae and to other genera of Gram(-) bacteria which included Xanthomonas, Agrobacterium and Rhizobium. Deletion analysis and successive subcloning of pEC3 revealed that a cis-acting locus, oriT and a trans-acting locus, mob, were involved in mobilization of pEC3. Five open reading frames (ORFs) were found in the mob region, of which four were identified as mobA, B, C and D. The mobA gene overlapped with mobC, B, D and ORF1 that were transcribed polycistronically from upstream from mobC. The nature of the four products of mob genes, MobA, B, C and D, was verified by use of the T7 promoter system in Ec.
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26. |
( 1996 ) Transkingdom transfer of the phosphoglucose isomerase gene. PMID : 8875859 : Abstract >>
Previous analysis of the gene encoding phosphoglucose isomerase (Pgi) suggests that this gene may have been transferred between a eukaryote and a bacterium. However, excluding the alternative hypothesis of ancient gene duplication has proven difficult because of both insufficient sampling of taxa and an earlier misidentification of a bacterial Pgi sequence. This paper presents a phylogenetic analysis of published complete Pgi sequences together with analysis of new partial Pgi sequences from six species of bacteria. The data identify a group of bacterial Pgi sequences, including sequences from Escherichia coli and Haemophilus influenzae, which are more closely related to eukaryotic Pgi sequences than to other bacterial sequences. The topology of gene trees constructed using several different methods are all consistent with the hypothesis of lateral gene transfer and not ancient gene duplication. Furthermore, an estimate of a molecular clock for Pgi dates the divergence of the E. coli and H. influenzae sequences from the animal sequences to between 470 and 650 million years ago, well after other estimates of the divergence between eukaryotes and bacteria. This study provides the most convincing evidence to date of the transkingdom transfer of a nuclear gene.
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27. |
Heikinheimo R,
Flego D,
Pirhonen M,
Karlsson MB,
Eriksson A,
Mäe A,
Kõiv V,
Palva ET,
( N/A ) Characterization of a novel pectate lyase from Erwinia carotovora subsp. carotovora. PMID : 7756691 : Abstract >>
The pectate lyase (Pel, EC 4.2.2.2) isoenzyme profile of Erwinia carotovora subsp. carotovora was characterized by isoelectric focusing, and the corresponding genes coding for four different exported Pels were cloned. The nucleotide sequence of the pelB gene encoding one of these isoenzymes was determined and was shown to contain 1,040-bp open reading frame coding for a 37,482-Da protein with a putative cleavable amino terminal signal peptide. Overexpression and selective labeling experiments with the pelB clone demonstrated the synthesis of a 35-kDa polypeptide, which is in accordance with the deduced size of the processed PelB. The predicted amino acid sequence of PelB was very similar to that of Pel-3 of another E.c. subsp. carotovora strain 71, but showed no similarity to other previously characterized pectinolytic enzymes. The pelB gene is located next to the previously characterized pehA gene encoding an endopolygalacturonase. The two genes are divergently transcribed from a common control region and are subject to similar global regulation by the central virulence regulator expI. Inactivation of pelB did not appear to reduce the virulence of the mutant strain, suggesting that pelB does not have a major role in pathogenicity. Unlike other Pels, PelB required partially methyl esterified pectin as substrate suggesting that PelB represents a novel isoform of pectate lyase.
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28. |
( 1994 ) Nucleotide sequence and expression of a novel pectate lyase gene (pel-3) and a closely linked endopolygalacturonase gene (peh-1) of Erwinia carotovora subsp. carotovora 71. PMID : 8074530 : PMC : PMC201682 Abstract >>
Our previous genetic analysis (J. W. Willis, J. K. Engwall, and A. K. Chatterjee, Phytopathology 77:1199-1205, 1987) had revealed a tight linkage between pel-3 (pel, pectate lyase gene) and peh-1 (peh, polygalacturonase gene) within the chromosome of Erwinia carotovora subsp. carotovora 71. Nucleotide sequencing, transcript assays, and expression of enzymatic activities in Escherichia coli have now confirmed that a 3,500-bp segment contains the open reading frames (ORFs) for Pel-3 and Peh-1. The 1,041-bp pel-3 ORF and the 1,206-bp peh-1 ORF are separated by a 579-bp sequence. The genes are transcribed divergently from their own promoters. In E. coli and E. carotovora subsp. carotovora 71, peh-1 is better expressed than pel-3. However, plant signals activate the expression of both the genes in E. carotovora subsp. carotovora. A consensus integration host factor (IHF)-binding sequence upstream of pel-3 appears physiologically significant, since pel-3 promoter activity is higher in an E. coli IHF+ strain than in an IHF- strain. While peh-1 has extensive homology with plant and bacterial peh genes, pel-3 appears not to have significant homology with the pel genes belonging to the pelBC, pelADE, or periplasmic pel families. Pel-3 also is unusual in that it is predicted to contain an ATP- and GTP-binding site motif A (P-loop) not found in the other Pels.
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29. |
Chatterjee A,
Liu Y,
Chatterjee AK,
( N/A ) Nucleotide sequence of a pectate lyase structural gene, pel1 of Erwinia carotovora subsp. carotovora strain 71 and structural relationship of pel1 with other pel genes of Erwinia species. PMID : 7772808 : Abstract >>
Of the various exoproteins secreted by Erwinia carotovora subsp. carotovora strain 71, Pel1 is the major pectate lyase species with tissue macerating activity. Nucleotide sequencing of a 2.2-kb pel1+ DNA segment revealed a 1,122 base pair open reading frame which could encode pre-Pel1 of 374 amino acid residues. A signal peptide of 22 amino acid residues is present within the NH2-terminal region of pre-Pel1. Transcription of pel1 was initiated at the guanine residue 111 base pairs upstream of the start codon. Consensus sequences for the binding of KdgR, a negative regulatory factor known to control some of the E. chrysanthemi pectinases, flank the promoter of pel1. Although pel1 belongs to the pelBC family, it is more closely related to the pel genes of E. carotovora than to the pelBC genes of E. chrysanthemi.
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30. |
Ohnishi H,
Nikaidou N,
Kamio Y,
Izaki K,
( 1994 ) Analysis of promoter region of the pectin lyase gene from Erwinia carotovora Er. PMID : 7764549 : Abstract >>
A pectin lyase defective mutant was constructed from Erwinia carotovora Er by transposon Tn5 insertion mutagenesis to analyze the promoter region of the pnl gene, which had been cloned. The promoter of pnl is between -140 and -74 upstream of the structural gene of pnl and appears not to be regulated by Lex A.
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31. |
Largen M,
Mills SE,
Rowe J,
Yanofsky C,
( 1978 ) Purification and properties of a third form of anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase from the Enterobacteriaceae. PMID : 338606 : Abstract >>
Anthranilate-5-phosphoribosylpyrophosphate phosphoribosyltransferase was purified from the bacterium Erwinia carotovora, a member of the Enterobacteriaceae. The enzyme was homogeneous according to the criteria of gel electrophoresis and NH2-terminal amino acid sequence analysis. The molecular weight of the enzyme as determined on a calibrated Sephadex G-200 column was 67,000 +/- 2,000. Sodium dodecyl sulfate-polyacrylamide gels gave a subunit molecular weight of 40,000 +/- 1,000, suggesting that the enzyme was a dimer. A comparison of the NH2-terminal sequence of the enzyme with the (previously determined) homologue from Serratia marcescens, a monomer with a molecular weight of 45,000, showed that the larger Serratia subunit came into register with amino acid 14 of the Erwinia subunit. The register for the length of the known overlap, 26 amino acids, was highly conserved.
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32. |
Rosanas A,
Barbé J,
Gibert I,
( 1995 ) Cloning and sequencing of the gyrA gene from the plant pathogen Erwinia carotovora. PMID : 7642123 : DOI : 10.1016/0378-1119(95)00267-a Abstract >>
The gyrA gene of Erwinia carotovora subsp. carotovora has been cloned and sequenced. The deduced protein possessed 86% identity with the Escherichia coli GyrA protein. E. carotovora gyrA was also shown to complement an E. coli gyrA43ts mutation.
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33. |
van den Bosch TJM,
Tan K,
Joachimiak A,
Welte CU,
( 2018 ) Functional Profiling and Crystal Structures of Isothiocyanate Hydrolases Found in Gut-Associated and Plant-Pathogenic Bacteria. PMID : 29752272 : DOI : 10.1128/AEM.00478-18 PMC : PMC6029094 Abstract >>
Isothiocyanates (ITCs) are produced by cruciferous plants to protect them against herbivores and infection by microbes. These compounds are of particular interest due to their antimicrobial and anticarcinogenic properties. The breakdown of ITCs in nature is catalyzed by isothiocyanate hydrolases (ITCases), a novel family within the metallo-�]-lactamase (MBL)-fold superfamily of proteins. saxA genes that code for ITCases are particularly widespread in insect- and plant-associated bacteria. Enzymatic characterization of seven phylogenetically related but distinct ITCases revealed similar activities on six selected ITCs, suggesting that phylogenetic diversity does not determine the substrate specificity of ITCases. X-ray crystallography studies of two ITCases sharing 42% amino acid sequence identity revealed a highly conserved tertiary structure. Notable features of ITCases include a hydrophobic active site with two Zn2+ ions coordinating water/hydroxide and a flexible cap that is implicated in substrate recognition and covers the active site. This report reveals the function and structure of the previously uncharacterized family of isothiocyanate hydrolases within the otherwise relatively well-studied superfamily of metallo-�]-lactamases.IMPORTANCE This study explores a newly discovered protein in the �]-lactamase superfamily, namely, SaxA, or isothiocyanate hydrolase. Isothiocyanates are defensive compounds found in many cabbage-related crop plants and are currently being investigated for their antimicrobial and anticarcinogenic properties. We show that isothiocyanate hydrolases are responsible for the breakdown of several of these plant defensive chemicals in vitro and suggest their potential for mitigating the beneficial effects of isothiocyanates in crop protection and cancer prevention.
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34. |
Niemi O,
Laine P,
Koskinen P,
Pasanen M,
Pennanen V,
Harjunpää H,
Nykyri J,
Holm L,
Paulin L,
Auvinen P,
Palva ET,
Pirhonen M,
( 2017 ) Genome sequence of the model plant pathogen Pectobacterium carotovorum SCC1. PMID : 29276572 : DOI : 10.1186/s40793-017-0301-z PMC : PMC5738896 Abstract >>
Bacteria of the genus Pectobacterium are economically important plant pathogens that cause soft rot disease on a wide variety of plant species. Here, we report the genome sequence of Pectobacterium carotovorum strain SCC1, a Finnish soft rot model strain isolated from a diseased potato tuber in the early 1980's. The genome of strain SCC1 consists of one circular chromosome of 4,974,798 bp and one circular plasmid of 5524 bp. In total 4451 genes were predicted, of which 4349 are protein coding and 102 are RNA genes.
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35. |
( 1998 ) Identification and characterization of the fis operon in enteric bacteria. PMID : 9811652 : PMC : PMC107668 Abstract >>
The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage lambda genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens, E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence for H. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) preceding fis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from -53 to +27 and the region around -116 containing an ihf binding site. Both beta-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.
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36. |
( 1998 ) Functional and structural conservation in the mechanosensitive channel MscL implicates elements crucial for mechanosensation. PMID : 9632260 : DOI : 10.1046/j.1365-2958.1998.00821.x Abstract >>
mscL encodes a channel in Escherichia coli that is opened by membrane stretch force, probably serving as an osmotic gauge. Sequences more or less similar to mscL are found in other bacteria, but the degree of conserved function has been unclear. We subcloned and expressed these putative homologues in E. coli and examined their products under patch clamp. Here, we show that each indeed encodes a conserved mechanosensitive channel activity, consistent with the interpretation that this is an important and primary function of the protein in a wide range of bacteria. Although similar, channels of different bacteria differ in kinetics and their degree of mechanosensitivity. Comparison of the primary sequence of these proteins reveals two highly conserved regions, corresponding to domains previously shown to be important for the function of the wild-type E. coli channel, and a C-terminal region that is not conserved in all species. This structural conservation is providing insight into regions of this molecule that are vital to its role as a mechanosensitive channel and may have broader implications for the understanding of other mechanosensitive systems.
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37. |
( 1998 ) Bacterial production of carbapenems and clavams: evolution of beta-lactam antibiotic pathways. PMID : 9614345 : Abstract >>
Research into two of the four classes of naturally produced beta-lactams--the clavams and carbapenems--has started to throw light upon their biochemical pathways and underlying genetics. Interesting similarities between these two classes, from their joint discovery to an apparently common beta-lactam ring-forming enzyme, are now being revealed.
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38. |
( 1998 ) The rpoS gene of Erwinia carotovora: gene organization and functional expression in E. coli. PMID : 9503622 : DOI : 10.1111/j.1574-6968.1998.tb12872.x Abstract >>
rpoS homologues were identified in several Erwinia species using Escherichia coli rpoS sequences as probes. The rpoS gene from Erwinia carotovora was cloned and the deduced amino acid sequence had 91% identity to E. coli RpoS. The latter sigma factor regulates the stationary phase inducible HPII catalase activity of E. coli. In an E. coli rpoS mutant, the E. carotovora rpoS gene was also able to regulate synthesis of this catalase. The presence of a similar catalase in E. carotovora suggests that the structural gene for this may be part of the rpoS 'regulon' in Erwinia also. This study also showed that there are several differences in the gene organization of the rpoS region of the E. coli and E. carotovora chromosomes.
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39. |
( 1998 ) Two-component regulators involved in the global control of virulence in Erwinia carotovora subsp. carotovora. PMID : 9675890 : DOI : 10.1094/MPMI.1998.11.8.743 Abstract >>
Production of extracellular, plant cell wall degrading enzymes, the main virulence determinants of the plant pathogen Erwinia carotovora subsp. carotovora, is coordinately controlled by a complex regulatory network. Insertion mutants in the exp (extracellular enzyme production) loci exhibit pleiotropic defects in virulence and the growth-phase-dependent transcriptional activation of genes encoding extracellular enzymes. Two new exp mutations, designated expA and expS, were characterized. Introduction of the corresponding wild-type alleles to the mutants complemented both the lack of virulence and the impaired production of plant cell wall degrading enzymes. The expA gene was shown to encode a 24-kDa polypeptide that is structurally and functionally related to the uvrY gene product of Escherichia coli and the GacA response regulator of Pseudomonas fluorescens. Functional similarity of expA and uvrY was demonstrated by genetic complementation. The expA gene is organized in an operon together with a uvrC-like gene, identical to the organization of uvrY and uvrC in E. coli. The unlinked expS gene encodes a putative sensor kinase that shows 92% identity to the recently described rpfA gene product from another E. carotovora subsp. carotovora strain. Our data suggest that ExpS and ExpA are members of two-component sensor kinase and response regulator families, respectively. These two proteins might interact in controlling virulence gene expression in E. carotovora subsp. carotovora.
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40. |
( 1997 ) Analysis of the carbapenem gene cluster of Erwinia carotovora: definition of the antibiotic biosynthetic genes and evidence for a novel beta-lactam resistance mechanism. PMID : 9402024 : DOI : 10.1046/j.1365-2958.1997.6001974.x Abstract >>
Members of two genera of Gram-negative bacteria, Serratia and Erwinia, produce a beta-lactam antibiotic, 1-carbapen-2-em-3-carboxylic acid. We have reported previously the cloning and sequencing of the genes responsible for production of this carbapenem in Erwinia carotovora. These genes are organized as an operon, carA--H, and are controlled by a LuxR-type transcriptional activator, encoded by the linked carR gene. We report in this paper the genetic dissection of this putative operon to determine the function of each of the genes. We demonstrate by mutational analysis that the products of the first five genes of the operon are involved in the synthesis of the carbapenem molecule. Three of these, carABC, are absolutely required. In addition, we provide evidence for the existence of a novel carbapenem resistance mechanism, encoded by the CarF and carG genes. Both products of these overlapping and potentially translationally coupled genes have functional, N-terminal signal peptides. Removal of these genes from the Erwinia chromosome results in a carbapenem-sensitive phenotype. We assume that these novel beta-lactam resistance genes have evolved in concert with the biosynthetic genes to ensure 'self-resistance' in the Erwinia carbapenem producer.
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