scholarly journals Molecular Cloning, Characterization, and Mutagenesis of a pel Gene from Pseudomonas syringae pv. lachrymans Encoding a Member of the Erwinia chrysanthemi PelADE Family of Pectate Lyases

1997 ◽  
Vol 10 (3) ◽  
pp. 369-379 ◽  
Author(s):  
David W. Bauer ◽  
Alan Collmer
Keyword(s):  
Host Range ◽  
Pseudomonas Syringae ◽  
Xanthomonas Campestris ◽  
Pectate Lyase ◽  
Erwinia Chrysanthemi ◽  
Broad Host Range ◽  
Protein Sequence Analysis ◽  
Pectate Lyases ◽  
Dna Libraries ◽  
Cucumber Cotyledons

The pelS gene from Pseudomonas syringae pv. lachrymans 859 was cloned by heterologous expression in nonpectolytic P. syringae pv. syringae BUVS1, using genomic DNA libraries constructed with two novel broad-host-range cosmid vectors, pCPP34 and pCPP47. Screening of P. syringae pv. syringae transconjugants for the ability to pit pectate media at pH 6.0 and 8.5 yielded several overlapping clones of the same DNA region. Ultrathin-layer isoelectric focusing gels, activity-stained with diagnostically buffered substrate overlays, revealed that this region encoded a single pectate lyase (PelS) with a pI of 9.4. pelS was subcloned from cosmid pCPP5020 and sequenced, revealing it to encode a member of the Erwinia chrysanthemi PelADE family, with highest similarity to Pseudomonas viridiflava PelV. A pelS probe hybridized at high stringency in DNA gel blots with total DNA from P. syringae pv. lachrymans strains 859 and Pla5, P. syringae pv. tabaci, P. syringae pv. phaseolicola, P. syringae pv. glycinea, P. fluorescens (marginalis), P. viridiflava, and Xanthomonas campestris pv. campestris, but not with P. syringae pv. pisi, P. syringae pv. syringae, P. syringae pv. tomato, P. syringae pv. papulans, E. chrysanthemi, or Ralstonia (Pseudomonas or Burkholderia) solanacearum. The PelS sequence revealed an N-terminal signal peptide, whose processing in Escherichia coli was confirmed by protein sequence analysis. PelS was similar to E. chrysanthemi PelE in its substrate preference and ability to reduce the viscosity of pectate and to macerate potato tuber tissue. A pelS∷ΩKmr mutation was marker-exchanged into P. syringae pv. lachrymans Pla5. pelS was also subcloned into the broad-host-range expression vector pML122 under control of the vector nptII promoter, and then transformed into P. syringae pv. lachrymans Pla5 to produce a strain overproducing PelS. Necrotic lesions developed in cotyledons following inoculation with all of the P. syringae pv. lachrymans Pla5 derivatives, regardless of their Pel phenotype. However, only cotyledons infected with pelS+ strains showed evidence of maceration and yielded Pel activity upon extraction. In contrast, pelS+ P. syringae pv. syringae BUVS1(pCPP5020) produced no symptoms in cucumber cotyledons. Thus, PelS in P. syringae pv. lachrymans appears to alter the final symptoms in infected cucumber cotyledons without contributing to pathogenicity or altering host range.

scholarly journals Arabidopsis thaliana Expresses Multiple Lines of Defense to Counterattack Erwinia chrysanthemi

2007 ◽  
Vol 20 (7) ◽  
pp. 794-805 ◽  
Author(s):  
Mathilde Fagard ◽  
Alia Dellagi ◽  
Camille Roux ◽  
Claude Périno ◽  
Martine Rigault ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Signaling Pathways ◽  
Oxidative Burst ◽  
Pectate Lyase ◽  
Erwinia Chrysanthemi ◽  
Secreted Proteins ◽  
Type Ii Secretion ◽  
Susceptible Host ◽  
Pectate Lyases ◽  
Lyase Activity

Many taxonomically diverse plant species are attacked by Erwinia chrysanthemi, a member of the causal agents of soft-rotting diseases. Symptom development is due to the collective action of pectin-degrading enzymes secreted by the bacterium through a type II secretion system (T2SS). Using Arabidopsis thaliana as a susceptible host, we show that plants respond to E. chrysanthemi 3937 by expressing cell-wall reactions, production of an oxidative burst, and activation of salicylic acid (SA) and jasmonic acid (JA) or ethylene (ET) signaling pathways. We found that the oxidative burst is mainly generated via the expression of the AtrbohD gene, constitutes a barrier of resistance to bacterial attack, and acts independently of the SA-mediated response. To determine the importance of T2SS-secreted proteins in elicitation of these defenses, we used a T2SS deficient mutant and purified enzymatic preparations of representative members of strain 3937 pectate lyase activity. The T2SS-secreted proteins were responsible only partially for the activation of SA and JA or ET signaling pathways observed after infection with the wild-type bacterium and were not involved in the expression of other identified defense reactions. Our study shows the differential role played by pectate lyases isoenzymes in this process and highlights the complexity of the host immune network, which is finely controlled by the bacterium.

scholarly journals Isolation of Polyvalent Bacteriophages by Sequential Multiple-Host Approaches

2015 ◽  
Vol 82 (3) ◽  
pp. 808-815 ◽  
Author(s):  
Pingfeng Yu ◽  
Jacques Mathieu ◽  
Mengyan Li ◽  
Zhaoyi Dai ◽  
Pedro J. J. Alvarez
Keyword(s):  
Host Range ◽  
Pseudomonas Syringae ◽  
Burst Size ◽  
Broad Host Range ◽  
Ecological Significance ◽  
Content Type ◽  
Narrow Host Range ◽  
Isolation Methods ◽  
Culture Independent ◽  
K 12

ABSTRACTMany studies on phage biology are based on isolation methods that may inadvertently select for narrow-host-range phages. Consequently, broad-host-range phages, whose ecological significance is largely unexplored, are consistently overlooked. To enhance research on such polyvalent phages, we developed two sequential multihost isolation methods and tested both culture-dependent and culture-independent phage libraries for broad infectivity. Lytic phages isolated from activated sludge were capable of interspecies or even interorder infectivity without a significant reduction in the efficiency of plating (0.45 to 1.15). Two polyvalent phages (PX1 of thePodoviridaefamily and PEf1 of theSiphoviridaefamily) were characterized in terms of adsorption rate (3.54 × 10−10to 8.53 × 10−10ml/min), latent time (40 to 55 min), and burst size (45 to 99 PFU/cell), using different hosts. These phages were enriched with a nonpathogenic host (Pseudomonas putidaF1 orEscherichia coliK-12) and subsequently used to infect model problematic bacteria. By using a multiplicity of infection of 10 in bacterial challenge tests, >60% lethality was observed forPseudomonas aeruginosarelative to uninfected controls. The corresponding lethality forPseudomonas syringaewas ∼50%. Overall, this work suggests that polyvalent phages may be readily isolated from the environment by using different sequential hosts, and this approach should facilitate the study of their ecological significance as well as enable novel applications.

scholarly journals Expanding Small-Molecule Functional Metagenomics through Parallel Screening of Broad-Host-Range Cosmid Environmental DNA Libraries in Diverse Proteobacteria

2010 ◽  
Vol 76 (5) ◽  
pp. 1633-1641 ◽  
Author(s):  
Jeffrey W. Craig ◽  
Fang-Yuan Chang ◽  
Jeffrey H. Kim ◽  
Steven C. Obiajulu ◽  
Sean F. Brady
Keyword(s):  
Small Molecules ◽  
Host Range ◽  
Small Molecule ◽  
Metagenomic Library ◽  
Environmental Dna ◽  
Culture Broth ◽  
Screening Methods ◽  
Broad Host Range ◽  
Functional Metagenomics ◽  
Dna Libraries

ABSTRACT The small-molecule biosynthetic diversity encoded within the genomes of uncultured bacteria is an attractive target for the discovery of natural products using functional metagenomics. Phenotypes commonly associated with the production of small molecules, such as antibiosis, altered pigmentation, or altered colony morphology, are easily identified from screens of arrayed metagenomic library clones. However, functional metagenomic screening methods are limited by their intrinsic dependence on a heterologous expression host. Toward the goal of increasing the small-molecule biosynthetic diversity found in functional metagenomic studies, we report the phenotypic screening of broad-host-range environmental DNA libraries in six different proteobacteria: Agrobacterium tumefaciens, Burkholderia graminis, Caulobacter vibrioides, Escherichia coli, Pseudomonas putida, and Ralstonia metallidurans. Clone-specific small molecules found in culture broth extracts from pigmented and antibacterially active clones, as well as the genetic elements responsible for the biosynthesis of these metabolites, are described. The host strains used in this investigation provided access to unique sets of clones showing minimal overlap, thus demonstrating the potential advantage conferred on functional metagenomics through the use of multiple diverse host species.

scholarly journals Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1

1984 ◽  
Vol 3 (13) ◽  
pp. 3323-3328 ◽  
Author(s):  
M.J. Daniels ◽  
C.E. Barber ◽  
P.C. Turner ◽  
M.K. Sawczyc ◽  
R.J.W. Byrde ◽  
...  
Keyword(s):  
Host Range ◽  
Xanthomonas Campestris ◽  
Broad Host Range ◽  
Xanthomonas Campestris Pv Campestris

scholarly journals The Exopolygalacturonate Lyase PelW and the Oligogalacturonate Lyase Ogl, Two Cytoplasmic Enzymes of Pectin Catabolism in Erwinia chrysanthemi 3937

1999 ◽  
Vol 181 (13) ◽  
pp. 3912-3919 ◽  
Author(s):  
Vladimir E. Shevchik ◽  
Guy Condemine ◽  
Janine Robert-Baudouy ◽  
Nicole Hugouvieux-Cotte-Pattat
Keyword(s):  
Yersinia Pseudotuberculosis ◽  
Divalent Cations ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Erwinia Chrysanthemi ◽  
Pectate Lyases ◽  
Acid Protein ◽  
Lyase Activity ◽  
Bacterial Cytoplasm ◽  
High Degree

ABSTRACT Erwinia chrysanthemi 3937 secretes into the external medium several pectinolytic enzymes, among which are eight isoenzymes of the endo-cleaving pectate lyases: PelA, PelB, PelC, PelD, and PelE (family 1); PelI (family 4); PelL (family 3); and PelZ (family 5). In addition, one exo-cleaving pectate lyase, PelX (family 3), has been found in the periplasm of E. chrysanthemi. The E. chrysanthemi 3937 gene kdgC has been shown to exhibit a high degree of similarity to the genes pelY ofYersinia pseudotuberculosis and pelB ofErwinia carotovora, which encode family 2 pectate lyases. However, no pectinolytic activity has been assigned to the KdgC protein. After verification of the corresponding nucleotide sequence, we cloned a longer DNA fragment and showed that this gene encodes a 553-amino-acid protein exhibiting an exo-cleaving pectate lyase activity. Thus, the kdgC gene was renamed pelW. PelW catalyzes the formation of unsaturated digalacturonates from polygalacturonate or short oligogalacturonates. PelW is located in the bacterial cytoplasm. In this compartment, PelW action could complete the degradation of pectic oligomers that was initiated by the extracellular or periplasmic pectinases and precede the action of the cytoplasmic oligogalacturonate lyase, Ogl. Both cytoplasmic pectinases, PelW and Ogl, seem to act in sequence during oligogalacturonate depolymerization, since oligomers longer than dimers are very poor substrates for Ogl but are good substrates for PelW. The estimated number of binding subsites for PelW is three, extending from subsite −2 to +1, while it is probably two for Ogl, extending from subsite −1 to +1. The activities of the two cytoplasmic lyases, PelW and Ogl, are dependent on the presence of divalent cations, since both enzymes are inhibited by EDTA. In contrast to the extracellular pectate lyases, Ca2+ is unable to restore the activity of PelW or Ogl, while several other cations, including Co2+, Mn2+, and Ni2+, can activate both cytoplasmic lyases.

scholarly journals Commensal Effect of Pectate Lyases Secreted fromDickeya dadantiion Proliferation ofEscherichia coliO157:H7 EDL933 on Lettuce Leaves

2010 ◽  
Vol 77 (1) ◽  
pp. 156-162 ◽  
Author(s):  
Akihiro Yamazaki ◽  
Jin Li ◽  
William C. Hutchins ◽  
Lixia Wang ◽  
Jincai Ma ◽  
...  
Keyword(s):  
Pectate Lyase ◽  
Type Ii Secretion ◽  
Broad Host Range ◽  
Deficient Mutant ◽  
In Planta ◽  
Enzyme Activity Assay ◽  
Dickeya Dadantii ◽  
Pectate Lyases ◽  
Σ Factor ◽  
Tissue Maceration

ABSTRACTThe outbreaks caused by enterohemorrhagicEscherichia coliO157:H7 on leafy greens have raised serious and immediate food safety concerns. It has been suggested that several phytopathogens aid in the persistence and proliferation of the human enteropathogens in the phyllosphere. In this work, we examined the influence of virulence mechanisms ofDickeya dadantii3937, a broad-host-range phytopathogen, on the proliferation of the human pathogenE. coliO157:H7 EDL933 (EDL933) on postharvest lettuce by coinoculation of EDL933 withD. dadantii3937 derivatives that have mutations in virulence-related genes. A type II secretion system (T2SS)-deficient mutant ofD. dadantii3937, A1919 (ΔoutC), lost the capability to promote the multiplication of EDL933, whereas Ech159 (ΔrpoS), a stress-responsive σ factor RpoS-deficient mutant, increased EDL933 proliferation on lettuce leaves. A spectrophotometric enzyme activity assay revealed that A1919 (ΔoutC) was completely deficient in the secretion of pectate lyases (Pels), which play a major role in plant tissue maceration. In contrast to A1919 (ΔoutC), Ech159 (ΔrpoS) showed more than 2-fold-greater Pel activity than the wild-typeD. dadantii3937. Increased expression ofpelD(encodes an endo-pectate lyase) was observed in Ech159 (ΔrpoS)in planta. These results suggest that the pectinolytic activity ofD. dadantii3937 is the dominant determinant of enhanced EDL933 proliferation on the lettuce leaves. In addition, RpoS, the general stress response σ factor involved in cell survival in suboptimal conditions, plays a role in EDL933 proliferation by controlling the production of pectate lyases inD. dadantii3937.

scholarly journals The Pseudomonas syringae pv. tomato HrpW Protein Has Domains Similar to Harpins and Pectate Lyases and Can Elicit the Plant Hypersensitive Response and Bind to Pectate

1998 ◽  
Vol 180 (19) ◽  
pp. 5211-5217 ◽  
Author(s):  
Amy O. Charkowski ◽  
James R. Alfano ◽  
Gail Preston ◽  
Jing Yuan ◽  
Sheng Yang He ◽  
...  
Keyword(s):  
Amino Acids ◽  
Cell Wall ◽  
Pseudomonas Syringae ◽  
Plant Cell ◽  
Hypersensitive Response ◽  
Xanthomonas Campestris ◽  
Plant Cell Wall ◽  
Erwinia Carotovora ◽  
Major Constituent ◽  
Pectate Lyases

ABSTRACT The host-specific plant pathogen Pseudomonas syringaeelicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42.9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity inA 230 assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringaepathovars, Pseudomonas viridiflava, Ralstonia(Pseudomonas) solanacearum, andXanthomonas campestris. ΔhrpZ::nptII orhrpW::ΩSpr P. syringaepv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in ahrpZ hrpW double mutant. These features of hrpWand its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.

scholarly journals PehN, a Polygalacturonase Homologue with a Low Hydrolase Activity, Is Coregulated with the Other Erwinia chrysanthemi Polygalacturonases

2002 ◽  
Vol 184 (10) ◽  
pp. 2664-2673 ◽  
Author(s):  
Nicole Hugouvieux-Cotte-Pattat ◽  
Vladimir E. Shevchik ◽  
William Nasser
Keyword(s):  
Rna Polymerase ◽  
Direct Interaction ◽  
Pectate Lyase ◽  
Soft Rot ◽  
Hydrolase Activity ◽  
Erwinia Chrysanthemi ◽  
Receptor Protein ◽  
Glycosyl Hydrolases ◽  
Pectate Lyases ◽  
Almost All

ABSTRACT Erwinia chrysanthemi 3937 secretes an arsenal of pectinolytic enzymes, including at least eight endo-pectate lyases encoded by pel genes, which play a major role in the soft-rot disease caused by this bacterium on various plants. E. chrysanthemi also produces some hydrolases that cleave pectin. Three adjacent hydrolase genes, pehV, pehW, and pehX, encoding exo-poly-α-d-galacturonosidases, have been characterized. These enzymes liberate digalacturonides from the nonreducing end of pectin. We report the identification of a novel gene, named pehN, encoding a protein homologous to the glycosyl hydrolases of family 28, which includes mainly polygalacturonases. PehN has a low hydrolase activity on polygalacturonate and on various pectins. PehN action favors the activity of the secreted endo-pectate lyases, mainly PelB and PelC, and that of the periplasmic exo-pectate lyase PelX. However, removal of the pehN gene does not significantly alter the virulence of E. chrysanthemi. Regulation of pehN transcription was analyzed by using gene fusions. Like other pectinase genes, pehN transcription is dependent on several environmental conditions. It is induced by pectic catabolic products and is affected by growth phase, catabolite repression, osmolarity, anaerobiosis, nitrogen starvation, and the presence of calcium ions. The transcription of pehN is modulated by the repressor KdgR, which controls almost all the steps of pectin catabolism, and by cyclic AMP receptor protein (CRP), the global activator of sugar catabolism. The regulator PecS, which represses the transcription of the pel genes but activates that of pehV, pehW, and pehX, also activates transcription of pehN. The three regulators KdgR, PecS, and CRP act by direct interaction with the pehN promoter region. The sequences involved in the binding of these three regulators and of RNA polymerase have been precisely defined. Analysis of the simultaneous binding of these proteins indicates that CRP and RNA polymerase bind cooperatively and that the binding of KdgR could prevent pehN transcription. In contrast, the activator effect of PecS is not linked to competition with KdgR or to cooperation with CRP or RNA polymerase. This effect probably results from competition between PecS and an unidentified repressor involved in peh regulation.

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