<i>In vivo</i>
 cloning of the pectate lyase and cellulase genes of <i>Erwinia chrysanthemi</i>

Gijsegem, Frédérique Van; Toussaint, Ariane; Schoonejans, Eric

doi:10.1002/j.1460-2075.1985.tb03698.x

Cited by 70 publications

(42 citation statements)

References 24 publications

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“…Although sequence data have not been reported for pel genes from other E. chrysanthemi strains, the EC16 gene coding for the pl 9. pelE gene defined in other strains of E. chrysanthemi (14,28,34). Our pI 9.8 enzyme also conforms to the same pectate lyase isolated from strain EC16 by Thurn et al (Abstract, VI International Conference on Plant Pathogenic Bacteria, Beltsville, Md., 1985) and reported by them to have an isoelectric point of 9.9.…”

Section: Discussionsupporting

confidence: 50%

“…Confirming this, E. coli cells containing the cloned pectate lyase genes macerated plant tissue, albeit less efficiently than E. chrysanthemi (13). Several groups subsequently cloned similar genes from other strains of E. chrysanthemi (5,14,28,34) and the related bacterium Erwinia carotovora (18,29, 40). The genes that we cloned did not cross-hybridize (13), but coded for enzymes with similar physical properties (molecular weights of ca.…”

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confidence: 99%

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Structure of two pectate lyase genes from Erwinia chrysanthemi EC16 and their high-level expression in Escherichia coli

Keen¹,

Tamaki²

1986

J Bacteriol

167

148

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The pelB and pelE genes from Erwinia chrysanthemi EC16, which encode different pectate lyase enzymes, were sequenced and expressed at a high level in Escherichia coli. The genes possessed little similarity to each other in 5' signal regions, signal peptide sequences, coding sequences, or 3' noncoding regions. Both genes contained their own promoters as well as sequences 3' to the coding regions with considerable secondary structure which may function as rho-independent transcriptional termination signals. High-level expression plasmids were constructed with both genes, which led to 20% or more of E. coli cellular protein. The pectate lyases were secreted efficiently to the periplasm and, to ra lesser extent, the culture medium. The mature proteins in E. coli periplasmic fractions were obtained in milligram amounts and high purity with a single-column affinity purification method. E. coli cells which produced high amounts of the pelE protein macerated potato tuber tissue as efficiently as E. chrysanthemi EC16 cells but cells producing high amounts of the pelB protein were less effective. Thus, the pelE gene product is an important pathogenicity factor which solely enables E. coli to cause a soft-rot disease on potato tuber tissue under laboratory conditions. We cloned genes coding for two different pectate lyase (EC 4.2.2.2) enzymes from the phytopathogenic bacterium Erwinia chrysanthemi EC16 (13) and observed their expression in Escherichia coli. Pectate lyases have previously been shown to account largely or entirely for the maceration or soft rotting of plant tissue caused by Erwinia spp. (4). Confirming this, E. coli cells containing the cloned pectate lyase genes macerated plant tissue, albeit less efficiently than E. chrysanthemi (13). Several groups subsequently cloned similar genes from other strains of E. chrysanthemi (5,14,28,34) and the related bacterium Erwinia carotovora (18,29, 40). The genes that we cloned did not cross-hybridize (13), but coded for enzymes with similar physical properties (molecular weights of ca. 40,000 and isoelectric points of 8.8 and 9.8) which both catalyzed the random eliminative cleavage of sodium polypectate. These enzymes were efficiently secreted to the periplasm and, to a lesser extent, the culture medium of E. coli. For reasons discussed below, the cloned DNA fragments appear to contain pelB and pelE, described by others, and the mature proteins which they encode are PLb and PLe, respectively. Plasmids containing our pel genes were named pPL in the previous paper (13), but this designation was found to be already entered in the Plasmid Reference Center (17). Accordingly, our pel gene plasmid constructs have been renamed pPEL, a designation that we have registered in the Plasmid Reference Center (17).The cloned pelB and pelE genes were both regulated by catabolite repression in E. coli (13) but were not induced by sodium polypectate, as occurs in E. chrysanthemi (4). Due to their pathogenic importance in diseases caused by Erwinia spp. and to their regulation propertie...

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Section: Discussionsupporting

confidence: 50%

mentioning

confidence: 99%

Structure of two pectate lyase genes from Erwinia chrysanthemi EC16 and their high-level expression in Escherichia coli

Keen¹,

Tamaki²

1986

J Bacteriol

167

148

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“…pULB110, a kanamycin-sensitive derivative of RP4 : : mini-Mu, was used to generate R-prime derivatives containing bacterial DNA (van Gijsegem et al, 1985). Mating between the recipient E. coli strain JC11305 and Er.…”

Section: Methodsmentioning

confidence: 99%

Tol-Pal proteins are critical cell envelope components of Erwinia chrysanthemi affecting cell morphology and virulence

Dubuisson

Vianney

Hugouvieux‐Cotte‐Pattat

et al. 2005

Microbiology

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The tol-pal genes are necessary for maintaining the outer-membrane integrity of Gram-negative bacteria. These genes were first described in Escherichia coli, and more recently in several other species. They are involved in the pathogenesis of E. coli, Haemophilus ducreyi, Vibrio cholerae and Salmonella enterica. The role of the tol-pal genes in bacterial pathogenesis was investigated in the phytopathogenic enterobacterium Erwinia chrysanthemi, assuming that this organism might be a good model for such a study. The whole Er. chrysanthemi tol-pal region was characterized. Tol-Pal proteins, except TolA, showed high identity scores with their E. coli homologues. Er. chrysanthemi mutants were constructed by introducing a uidA-kan cassette in the ybgC, tolQ, tolA, tolB, pal and ybgF genes. All the mutants were hypersensitive to bile salts. Mutations in tolQ, tolA, tolB and pal were deleterious for the bacteria, which required high concentrations of sugars or osmoprotectants for their viability. Consistent with this observation, they were greatly impaired in their cell morphology and division, which was evidenced by observations of cell filaments, spherical forms, membrane blebbing and mislocalized bacterial septa. Moreover, tol-pal mutants showed a reduced virulence in a potato tuber model and on chicory leaves. This could be explained by a combination of impaired phenotypes in the tol-pal mutants, such as reduced growth and motility and a decreased production of pectate lyases, the major virulence factor of Er. chrysanthemi. INTRODUCTIONThe Tol-Pal system of Gram-negative bacteria is required for outer-membrane stability (Lazzaroni et al., 1999). It comprises five envelope proteins, TolQ, TolR, TolA, TolB and Pal, which form two complexes. The TolQ, TolR and TolA inner-membrane proteins interact via their transmembrane domains (Derouiche et al., 1995;Lazzaroni et al., 1995). The b-propeller domain of the periplasmic protein TolB is responsible for its interaction with Pal (Ray et al., 2000). TolB also interacts with the outer-membrane peptidoglycan-associated proteins Lpp and OmpA (Cascales et al., 2002;Clavel et al., 1998). TolA undergoes a conformational change in response to changes in the protonmotive force (Germon et al., 2001), and interacts with Pal in an energy-dependent manner (Cascales et al., 2001). The Cterminal periplasmic domain of TolA also interacts with the N-terminal domain of TolB (Dubuisson et al., 2002;Walburger et al., 2002).The transcriptional organization of the E. coli tol-pal genes has been characterized. The genes ybgC (orf1), tolQ, tolR, tolA and tolB, and pal and ybgF (orf2) form two operons (Muller & Webster, 1997); a large ybgC-ybgF transcript has also been postulated (Vianney et al., 1996). ybgC and ybgF encode proteins of unknown function located in the cytoplasm and the periplasm, respectively (Clavel et al., 1996;Sun & Webster, 1987). Inactivation of these two ORFs induces no obvious phenotype in E. coli. In contrast, mutations in the tol-pal genes cause the disruption of outermembr...

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“…Plasmid pULB110, a kanamycin-sensitive RP4::mini-Mu derivative (41), was used to generate R-prime derivatives containing bacterial DNA. Matings between recipient and donor strains carrying plasmids were performed by spreading 0.2 ml of overnight cultures of the strains on 63 minimal medium plates and incubating for 5 h at 30°C.…”

Section: Methodsmentioning

confidence: 99%

Osmoregulated Periplasmic Glucan Synthesis Is Required for Erwinia chrysanthemi Pathogenicity

Page

Altabe

Hugouvieux‐Cotte‐Pattat

et al. 2001

J Bacteriol

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Erwinia chrysanthemi is a phytopathogenic enterobacterium causing soft rot disease in a wide range of plants. Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the gram-negative bacterial envelope. We cloned the opgGH operon of E. chrysanthemi, encoding proteins involved in the glucose backbone synthesis of OPGs, by complementation of the homologous locus mdoGH of Escherichia coli. OpgG and OpgH show a high level of similarity with MdoG and MdoH, respectively, and mutations in the opgG or opgH gene abolish OPG synthesis. The opg mutants exhibit a pleiotropic phenotype, including overproduction of exopolysaccharides, reduced motility, bile salt hypersensitivity, reduced protease, cellulase, and pectate lyase production, and complete loss of virulence. Coinoculation experiments support the conclusion that OPGs present in the periplasmic space of the bacteria are necessary for growth in the plant host.Osmoregulated periplasmic glucans (OPGs) are a family of oligosaccharides found in the periplasmic space of gram-negative bacteria. Their two common features are the presence of glucose as the sole constituent sugar and their increased level in media of low osmolarity (5).Members of the family Enterobacteriaceae and related bacteria synthesize a family of linear and branched OPGs that are variously substituted. The linear backbone is constituted by glucose units joined by ␤,1-2 linkages, and the branches are made of one glucose unit linked to the main chain by a ␤,1-6 linkage. In Escherichia coli, the backbone, containing 7 to 13 glucose units, is substituted with phosphoglycerol, phosphoethanolamine, and succinyl residues (19). In Erwinia chrysanthemi, the backbone contains 5 to 12 glucose units substituted with succinyl and acetyl residues (9), and in Pseudomonas syringae, the backbone, consisting of 6 to 13 glucose units, is not substituted (38). In E. coli, the OPG backbone is synthesized by the products of the mdoGH operon located in the vicinity of pyrC, a gene involved in the biosynthesis of uracil (6). In this bacterium, the defect in OPG synthesis does not confer an easily selectable phenotype in laboratory conditions. Thus, the mdoGH locus was cloned using the linked selectable genetic marker pyrC (23).Many factors are involved in the virulence of pathogenic bacteria, and OPGs appear to be among them. In P. syringae pv. syringae, the causal agent of brown spot disease of the common bean (Phaseolus vulgaris), the hrpM mutant, obtained after transposon mutagenesis, was isolated because it failed to incite disease on the host plant and to cause the hypersensitive reaction on a non-host plant such as tobacco (29). The hrpM mutant does not synthesize OPGs, and the hrpM locus complements the OPG synthesis defect of the mdoH200::Tn10 mutant of E. coli. The amino acid sequences of HrpM and MdoH are 75.5% identical and 87.5% similar (25). More recently, a transposon insertion in a gene similar to hrpM/mdoH was isolated because it severely reduces the virulence in Pseudomonas aeruginosa PA14, an opp...

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In vivo cloning of the pectate lyase and cellulase genes of Erwinia chrysanthemi

Cited by 70 publications

References 24 publications

Structure of two pectate lyase genes from Erwinia chrysanthemi EC16 and their high-level expression in Escherichia coli

Structure of two pectate lyase genes from Erwinia chrysanthemi EC16 and their high-level expression in Escherichia coli

Tol-Pal proteins are critical cell envelope components of Erwinia chrysanthemi affecting cell morphology and virulence

Osmoregulated Periplasmic Glucan Synthesis Is Required for Erwinia chrysanthemi Pathogenicity

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