Thermodependence of growth and enzymatic activities implicated in pathogenicity of twoErwinia carotovorasubspecies (Pectobacteriumspp.)

2004 ◽  
Vol 50 (1) ◽  
pp. 19-27 ◽  
Author(s):  
Bruno Smadja ◽  
Xavier Latour ◽  
Sameh Trigui ◽  
Jean François Burini ◽  
Sylvie Chevalier ◽  
...  
Keyword(s):  
Growth Rate ◽  
Extracellular Enzymes ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Optimal Growth ◽  
Ecological Data ◽  
Pectate Lyases ◽  
Lyase Activity ◽  
Active Multiplication

Erwinia carotovora subsp. atroseptica and Erwinia carotovora subsp. carotovora can cause substantial damage to economically important plant crops and stored products. The occurrence of the disease and the scale of the damage are temperature dependent. Disease development consists first of active multiplication of the bacteria in the infection area and then production of numerous extracellular enzymes. We investigated the effects of various temperatures on these two steps. We assayed the specific growth rate and the pectate lyase and protease activities for eight strains belonging to E. carotovora subsp. atroseptica and E. carotovora subsp. carotovora in vitro. The temperature effect on growth rate and on pectate lyase activity is different for the two subspecies, but protease activity appears to be similarly thermoregulated. Our results are in agreement with ecological data implicating E. carotovora subsp. atroseptica in disease when the temperature is below 20 °C. The optimal temperature for pathogenicity appears to be different from the optimal growth temperature but seems to be a compromise between this temperature and temperatures at which lytic activities are maximal.Key words: temperature, Pectobacterium atrosepticum, Pectobacterium carotovorum, growth, pectate lyases, proteases.

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.

Effects of carbon monoxide on cardiac muscle cells in culture

1988 ◽  
Vol 255 (3) ◽  
pp. C291-C296 ◽  
Author(s):  
A. C. Nag ◽  
K. C. Chen ◽  
M. Cheng
Keyword(s):  
Growth Rate ◽  
Cardiac Muscle ◽  
Structural Organization ◽  
Optimal Growth ◽  
Muscle Cells ◽  
Cellular Growth ◽  
Cardiac Muscle Cells ◽  
Cells In Culture ◽  
Significant Difference

Embryonic rat cardiac muscle cells grown in the presence of various tensions of CO (5-95%) without the presence of O2 survived and exhibited reduced cell growth, which was concentration dependent. When cardiac muscle cells were grown in the presence of a mixture of CO (10-20%) and O2 (10-20%), the growth rate of these cells was comparable to that of the control cells. Cardiac myocytes continued to beat when exposed to varying tensions of CO, except in the case of 95% CO. The cells exposed to different concentrations of CO contained fewer myofibrils of different stages of differentiation compared with the control and the culture exposed to a mixture of 20% O2 and 20% CO, with cells that contained abundant, highly differentiated myofibrils. There was no significant difference in the structural organization of mitochondria between the control and the surviving experimental cells. It is evident from the present studies that O2 is required for the optimum in vitro cellular growth of cardiac muscle. Furthermore, CO in combination with O2 at a concentration of 10 or 20% can produce optimal growth of cardiac muscle cells in culture.

Pénétration d'une endo-pectate lyase tritiée dans les cellules corticales de racines de courge

1984 ◽  
Vol 62 (8) ◽  
pp. 1621-1628
Author(s):  
G. F. Vogt ◽  
J. Coulon
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
External Surface ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Cortical Cells ◽  
Pectate Lyases ◽  
Autoradiographic Analysis ◽  
Host Tissues

Erwinia carotovora produces pectate lyases (endo-PGTE) on sterilized beans hypocotyls. Two endo-PGTE fractions were isolated and purified by electrofocusing. The action of these enzymes on the ultrastructure of cortical cells of pumpkin roots was very similar to the action of the whole bacterium. Erwinia carotovora grown on tritiated amino acid supplemented medium produced [3H]endo-PGTE. By incubating the host tissues with 3H-labelled enzymes and by subsequent autoradiographic analysis it was possible to localize the endo-PGTE inside the cells. Thus, it was shown that the enzyme (the complete molecule or only a peptide part thereof) was transported towards the vacuole. It is suggested that the endo-PGTE acts on the filamentous polysaccharide extensions which bind the external surface of the plasmalemma to the cell wall.

Phaeotheca dimorphospora sp.nov.: description et caractéristiques culturales

1994 ◽  
Vol 72 (6) ◽  
pp. 808-817 ◽  
Author(s):  
Pierre DesRochers ◽  
G. B. Ouellette
Keyword(s):  
Growth Rate ◽  
Growth Temperature ◽  
Type Species ◽  
Optimal Growth ◽  
Outer Wall ◽  
Dutch Elm Disease ◽  
Optimal Growth Temperature ◽  
Ophiostoma Ulmi ◽  
Mother Cells

An unknown fungus isolated from an elm branch and inhibitory against Ophiostoma ulmi in vitro is described as Phaeotheca dimorphospora sp.nov. This dematiaceous deuteromycete propagates by endoconidia released after exfoliation of chlamydospore outer wall, as in mother cells of the type species Phaeotheca fissurella. However, P. dimorphospora differs from the type species by producing hyaline secondary ameroconidia between the endoconidial masses. Other ameroconidia, similar to the secondary ameroconidia, are produced through the chlamydospore outer wall. The optimal growth temperature of P. dimorphospora is 23 °C, whereas it is 15.5 °C for the type species. On media containing a high dextrose concentration (30 g ∙ L−1), colonies of P. dimorphospora are gray and crustose and grow slowly, at least initially. Conversely, on media with a low dextrose concentration (5 or 10 g ∙ L−1) colonies have a faster growth rate and appear whitish or ivory and fluffy. Key words: Phaeotheca dimorphospora, diagnosis, inhibition, Ophiostoma ulmi, Dutch elm disease.

scholarly journals Elevated Temperature Enhances Virulence of Erwinia carotovora subsp. carotovora Strain EC153 to Plants and Stimulates Production of the Quorum Sensing Signal, N-Acyl Homoserine Lactone, and Extracellular Proteins

2005 ◽  
Vol 71 (8) ◽  
pp. 4655-4663 ◽  
Author(s):  
H. Hasegawa ◽  
A. Chatterjee ◽  
Y. Cui ◽  
A. K. Chatterjee
Keyword(s):  
Quorum Sensing ◽  
Extracellular Enzymes ◽  
Rna Binding ◽  
Elevated Temperatures ◽  
Pectate Lyase ◽  
Erwinia Carotovora ◽  
Homoserine Lactone ◽  
Extracellular Proteins ◽  
Acyl Homoserine Lactone ◽  
Tissue Maceration

ABSTRACT Erwinia carotovora subsp. atroseptica, E. carotovora subsp. betavasculorum, and E. carotovora subsp. carotovora produce high levels of extracellular enzymes, such as pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt), and the quorum-sensing signal N-acyl-homoserine lactone (AHL) at 28°C. However, the production of these enzymes and AHL by these bacteria is severely inhibited during growth at elevated temperatures (31.2°C for E. carotovora subsp. atroseptica and 34.5°C for E. carotovora subsp. betavasculorum and most E. carotovora subsp. carotovora strains). At elevated temperatures these bacteria produce high levels of RsmA, an RNA binding protein that promotes RNA decay. E. carotovora subsp. carotovora strain EC153 is an exception in that it produces higher levels of Pel, Peh, Cel, and Prt at 34.5°C than at 28°C. EC153 also causes extensive maceration of celery petioles and Chinese cabbage leaves at 34.5°C, which correlates with a higher growth rate and higher levels of rRNA and AHL. The lack of pectinase production by E. carotovora subsp. carotovora strain Ecc71 at 34.5°C limits the growth of this organism in plant tissues and consequently impairs its ability to cause tissue maceration. Comparative studies with ahlI (the gene encoding a putative AHL synthase), pel-1, and peh-1 transcripts documented that at 34.5°C the RNAs are more stable in EC153 than in Ecc71. Our data reveal that overall metabolic activity, AHL levels, and mRNA stability are responsible for the higher levels of extracellular protein production and the enhanced virulence of EC153 at 34.5°C compared to 28°C.

scholarly journals RNA chaperones Hfq and ProQ play a key role in the virulence of the plant pathogenic bacterium Dickeya dadantii

2021 ◽  
Author(s):  
Simon Leonard ◽  
Camille Villard ◽  
William Nasser ◽  
Sylvie Reverchon ◽  
Florence Hommais
Keyword(s):  
Extracellular Enzymes ◽  
Pectate Lyase ◽  
Soft Rot ◽  
Pathogenic Bacterium ◽  
Growth Defect ◽  
Dickeya Dadantii ◽  
Genes Encoding ◽  
Lyase Activity ◽  
Rna Chaperones ◽  
Post Transcriptional Regulation

Dickeya dadantii is an important pathogenic bacterium that infects a number of crops including potato and chicory. While extensive works have been carried out on the control of the transcription of its genes encoding the main virulence functions, little information is available on the post-transcriptional regulation of these functions. We investigated the involvement of the RNA chaperones Hfq and ProQ in the production of the main D. dadantii virulence functions. Phenotypic assays on the hfq and proQ mutants showed that inactivation of hfq resulted in a growth defect, a modified capacity for biofilm formation and strongly reduced motility, and in the production of degradative extracellular enzymes (proteases, cellulase and pectate lyases). Accordingly, the hfq mutant failed to cause soft rot on chicory leaves. The proQ mutant had reduced resistance to osmotic stress, reduced extracellular pectate lyase activity compared to the wild-type strain, and reduced virulence on chicory leaves. Most of the phenotypes of the hfq and proQ mutants were related to the low amounts of mRNA of the corresponding virulence factors. Complementation of the double mutant hfq-proQ by each individual protein and cross-complementation of each chaperone suggested that they might exert their effects via partially overlapping but different sets of targets. Overall, it clearly appeared that the two Hfq and ProQ RNA chaperones are important regulators of pathogenicity in D. dadantii. This underscores that virulence genes are regulated post transcriptionally by non-coding RNAs.

scholarly journals HrpW of Erwinia amylovora, a New Harpin That Contains a Domain Homologous to Pectate Lyases of a Distinct Class

1998 ◽  
Vol 180 (19) ◽  
pp. 5203-5210 ◽  
Author(s):  
Jihyun F. Kim ◽  
Steven V. Beer
Keyword(s):  
Plant Cell Wall ◽  
Erwinia Amylovora ◽  
Pectate Lyase ◽  
Hypersensitive Reaction ◽  
Host Tissue ◽  
Wild Type ◽  
Pectate Lyases ◽  
Heat Stable ◽  
Lyase Activity ◽  
Type Strains

ABSTRACT Harpins, such as HrpN of Erwinia amylovora, are extracellular glycine-rich proteins that elicit the hypersensitive reaction (HR). We identified hrpW of E. amylovora, which encodes a protein similar to known harpins in that it is acidic, rich in glycine and serine, and lacks cysteine. A putative HrpL-dependent promoter was identified upstream ofhrpW, and Western blot analysis of hrpL mutants indicated that the production of HrpW is regulated by hrpL. HrpW is secreted via the Hrp (type III) pathway based on analysis of wild-type strains and hrp secretion mutants. When infiltrated into plants, HrpW induced rapid tissue collapse, which required active plant metabolism. The HR-eliciting activity was heat stable and protease sensitive. Thus, we concluded that HrpW is a new harpin. HrpW of E. amylovora consists of two domains connected by a Pro and Ser-rich sequence. A fragment containing the N-terminal domain was sufficient to elicit the HR. Although no pectate lyase activity was detected, the C-terminal region of HrpW is homologous to pectate lyases of a unique class, suggesting that HrpW may be targeted to the plant cell wall. Southern analysis indicated that hrpW is conserved among several Erwiniaspecies, and hrpW, provided in trans, enhanced the HR-inducing ability of a hrpN mutant. However, HrpW did not increase the virulence of a hrpN mutant in host tissue, and hrpW mutants retained the wild-type ability to elicit the HR in nonhosts and to cause disease in hosts.

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 Disulfide Bond in Pseudomonas aeruginosaLipase Stabilizes the Structure but Is Not Required for Interaction with Its Foldase

2001 ◽  
Vol 183 (2) ◽  
pp. 597-603 ◽  
Author(s):  
Klaus Liebeton ◽  
Annette Zacharias ◽  
Karl-Erich Jaeger
Keyword(s):  
Disulfide Bond ◽  
Elevated Temperatures ◽  
Signal Sequence ◽  
Pectate Lyase ◽  
Salt Bridge ◽  
Erwinia Carotovora ◽  
Cysteine Residues ◽  
Intramolecular Disulfide Bond ◽  
Bond Function

ABSTRACT Pseudomonas aeruginosa secretes a 29-kDa lipase which is dependent for folding on the presence of the lipase-specific foldase Lif. The lipase contains two cysteine residues which form an intramolecular disulfide bond. Variant lipases with either one or both cysteines replaced by serines showed severely reduced levels of extracellular lipase activity, indicating the importance of the disulfide bond for secretion of lipase through the outer membrane. Wild-type and variant lipase genes fused to the signal sequence of pectate lyase from Erwinia carotovora were expressed inEscherichia coli, denatured by treatment with urea, and subsequently refolded in vitro. Enzymatically active lipase was obtained irrespective of the presence or absence of the disulfide bond, suggesting that the disulfide bond is required neither for correct folding nor for the interaction with the lipase-specific foldase. However, cysteine-to-serine variants were more readily denatured by treatment at elevated temperatures and more susceptible to proteolytic degradation by cell lysates of P. aeruginosa. These results indicate a stabilizing function of the disulfide bond for the active conformation of lipase. This conclusion was supported by the finding that the disulfide bond function could partly be substituted by a salt bridge constructed by changing the two cysteine residues to arginine and aspartate, respectively.

Growth of Trypanosoma cruzi in vitro: development and application of a continuous-flow culture system

Parasitology ◽  
1982 ◽  
Vol 84 (3) ◽  
pp. 511-526 ◽  
Author(s):  
G. T. Williams ◽  
L. Hudson
Keyword(s):  
Growth Rate ◽  
Trypanosoma Cruzi ◽  
Continuous Flow ◽  
Culture System ◽  
Extreme Values ◽  
Environmental Parameters ◽  
Optimal Growth ◽  
Parasite Growth ◽  
Vitro Development

SummaryThe design and operation of a modular, bacteriological continuous-flow culture system have been adapted for the growth of Trypanosoma cruzi epimastigotes in simple monophasic media. The system was designed to achieve a minimum of 200 days of continuous culture and provision was made for the continuous supply of medium and collection of parasites under sterile conditions. The system provides large quantities of epimastigotes with homogeneous morphology and uniform viability. The system also lends itself tothe analysis of the factors which affect parasite growth. We have examined the effects of changes in environmental parameters on epimastigote growth rate. Optimal growth was observed at 27 °C. The rate ofstirring of the culture had a small but definable effect on the growth rate, which was greatest at 80 r.p.m. Growth was only slightly affected by the level of dissolved oxygen between 10 and 50% saturation, but was inhibited at higher concentrations. Growth was slower at extreme values of pH but showed a broad optimum around pH 7·4.

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