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PubMed2002

Characterization of PG2, an early endoPG produced by Sclerotinia sclerotiorum, expressed in yeast.

Sclerotinia sclerotiorum, a plant pathogenic ascomycete, contains a neutral endopolygalacturonase (endoPG) subfamily of genes that was previously isolated. We report here that pg2, a member of this subfamily, is early and strongly expressed during the first steps of pathogenesis of sunflower cotyledons. The corresponding protein, PG2, was produced in the heterologous Kluyveromyces lactis system and purified. Characterization of the recombinant enzyme revealed a narrow pH activity curve with an optimal pH of 4.5. Hydrolysis of polygalacturonic acid by PG2 resulted in the accumulation of oligomers ranging from 2- to 9-mer. This degradation profile indicates a random attack on the polymer and demonstrates an endo-mode of action. These results provide evidence that pg2 contributes to the infection process during the early phase of host colonization.

Ascomycota↗
PubMed1978

Pectolytic enzymes in Rhizobium.

A sensitive pectin agar plate assay was used to demonstrate low levels of pectolytic enzymes in infective and noninfective strains of Rhizobium. The possible relation of this characteristic to legume infection is discussed.

Agar↗
PubMed1979

Pectinolytic enzymes of large rumen treponemes.

Large spiral organisms isolated from the rumen of cattle produced and released into the external environment a complex of pectinolytic enzymes, consisting mainly of poly(1,4-alpha-D-galacturonide) lyase (EC 4.2.2.2, formerly EC 4.2.99.3), most active at pH 8.0 to 9.0, and another enzyme acting at pH below 7.0, probably a poly(1,4-alpha-D-galacturonide) glycanohydrolase (EC 3.2.1.15). The mixture of enzymes degraded polygalacturonate to saturated and unsaturated monogalacturonates as the end products. A pectin pectylhydrolase (pectinesterase) (EC 3.1.1.11) was also present in the clarified cultures.

Animals↗
PubMed1980

Small pectinolytic spirochetes from the rumen.

Two strains of small spirochetes utilizing pectin as sole source of energy for growth were isolated from the rumen of cattle and partially characterized. The pectinolytic enzyme produced by one of the organisms was classified as a poly-1,4-alpha-D-galacturonide lyase (EC 4.2.2.2). A pectinesterase (EC 3.1.1.11) was also produced.

Animals↗
PubMed1985

Cloning and expression in Escherichia coli of pectinase genes of Erwinia carotovora subsp. carotovora.

Genes coding for an endo-pectate lyase, an exo-pectate lyase, and an endopolygalacturonase of Erwinia carotovora subsp. carotovora Ecc71 were cloned in Escherichia coli HB101, using the cosmid pHC79. The products of the cloned pectinase genes paralleled their counterparts in strain Ecc71 in isoelectric mobility, mode of substrate degradation, and ability to macerate potato tuber tissue.

Cloning, Molecular↗
PubMed1995

Biochemical analysis of starch degradation by Ruminobacter amylophilus 70.

Ruminobacter amylophilus is an obligate anaerobe that uses only alpha-linked glucose molecules (i.e., maltose, maltodextrins, and starch) as a source of energy, making it an excellent model for the study of bacterial starch degradation. Constitutive amylase, amylopectinase, and pullulanase activities were found in intracellular and extracellular fractions of R. amylophilus. However, extracellular activities apparently resulted from cell lysis. Both soluble and membrane-bound polysaccharidase activities were detected. Most of the soluble polysaccharidase activity partitioned with the periplasmic cell fraction. No alpha-glucosidase or maltase activity was detected in either the cellular or extracellular fraction. In addition, intact cells of R. amylophilus bound U-14C-starch. This binding could be saturated and was constitutive and sensitive to proteinase K, indicating protein or protein complex mediation. Competition experiments showed that these starch-binding sites had equally high affinities for starch and maltodextrins larger than maltotriose. The sites had a reduced affinity for maltose and virtually no affinities for glucose and nonstarch polysaccharides. These findings suggest that R. amylophilus binds starch molecules to the cell surface as an initial step in transporting the molecule through the outer membrane and into the periplasmic space. Extracellular polysaccharides do not appear to be involved in starch degradation.

Amylases↗
PubMed1999

Cloning and partial characterization of endopolygalacturonase genes from Botrytis cinerea.

Botrytis cinerea is a plant-pathogenic fungus infecting over 200 different plant species. We use a molecular genetic approach to study the process of pectin degradation by the fungus. Recently, we described the cloning and characterization of an endopolygalacturonase (endoPG) gene from B. cinerea (Bcpg1) which is required for full virulence. Here we describe the cloning and characterization of five additional endoPG-encoding genes from B. cinerea SAS56. The identity at the amino acid level between the six endoPGs of B. cinerea varied from 34 to 73%. Phylogenetic analysis, by using a group of 35 related fungal endoPGs and as an outgroup one plant PG, resulted in the identification of five monophyletic groups of closely related proteins. The endoPG proteins from B. cinerea SAS56 could be assigned to three different monophyletic groups. DNA blot analysis revealed the presence of the complete endoPG gene family in other strains of B. cinerea, as well as in other Botrytis species. Differential gene expression of the gene family members was found in mycelium grown in liquid culture with either glucose or polygalacturonic acid as the carbon source.

Blotting, Southern↗
PubMed1999

Use of green fluorescent protein to detect expression of an endopolygalacturonase gene of Colletotrichum lindemuthianum during bean infection.

The 5' noncoding region of clpg2, an endopolygalacturonase gene of the bean pathogen Colletotrichum lindemuthianum, was fused to the coding sequence of a gene encoding a green fluorescent protein (GFP), and the construct was introduced into the fungal genome. Detection of GFP accumulation by fluorescence microscopy examination revealed that clpg2 was expressed at the early stages of germination of the conidia and during appressorium formation both in vitro and on the host plant.

Colletotrichum↗
PubMed1999

Comparison of bacterial community structures in the rhizoplane of tomato plants grown in soils suppressive and conducive towards bacterial wilt.

It has been reported that the growth of Ralstonia solanacearum is suppressed at the rhizoplane of tomato plants and that tomato bacterial wilt is suppressed in plants grown in a soil (Mutsumi) in Japan. To evaluate the biological factors contributing to the suppressiveness of the soil in three treated Mutsumi soils (chloroform fumigated soil; autoclaved soil mixed with intact Mutsumi soil; and autoclaved soil mixed with intact, wilt-conducive Yamadai soil) infested with R. solanacearum, we bioassayed soil samples for tomato bacterial wilt. Chloroform fumigation increased the extent of wilt disease. More of the tomato plant samples wilted when mixed with Yamadai soil than when mixed with Mutsumi soil. Consequently, the results indicate that the naturally existing population of microorganisms in Mutsumi soil was significantly able to reduce the severity of bacterial wilt of tomato plants. To characterize the types of bacteria present at the rhizoplane, we isolated rhizoplane bacteria and classified them into 22 groups by comparing their 16S restriction fragment length polymorphism patterns. In Yamadai soil a single group of bacteria was extremely predominant (73.1%), whereas in Mutsumi soil the distribution of the bacterial groups was much more even. The 16S rDNA sequence analysis of strains of dominant groups suggested that gram-negative bacteria close to the beta-proteobacteria were most common at the rhizoplane of the tomato plants. During in vitro assays, rhizoplane bacteria in Mutsumi soil grew more vigorously on pectin, one of the main root exudates of tomato, compared with those in Yamadai soil. Our results imply that it is difficult for the pathogen to dominate in a diversified rhizobacterial community that thrives on pectin.

Bacteria↗
PubMed2000

Lack of host specialization in Aspergillus flavus.

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22 degrees C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton balls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.

Animals↗
PubMed2001

pH signaling in Sclerotinia sclerotiorum: identification of a pacC/RIM1 homolog.

Sclerotinia sclerotiorum acidifies its ambient environment by producing oxalic acid. This production of oxalic acid during plant infection has been implicated as a primary determinant of pathogenicity in this and other phytopathogenic fungi. We found that ambient pH conditions affect multiple processes in S. sclerotiorum. Exposure to increasing alkaline ambient pH increased the oxalic acid accumulation independent of carbon source, sclerotial development was favored by acidic ambient pH conditions but inhibited by neutral ambient pH, and transcripts encoding the endopolygalacturonase gene pg1 accumulated maximally under acidic culture conditions. We cloned a putative transcription factor-encoding gene, pac1, that may participate in a molecular signaling pathway for regulating gene expression in response to ambient pH. The three zinc finger domains of the predicted Pac1 protein are similar in sequence and organization to the zinc finger domains of the A. nidulans pH-responsive transcription factor PacC. The promoter of pac1 contains eight PacC consensus binding sites, suggesting that this gene, like its homologs, is autoregulated. Consistent with this suggestion, the accumulation of pac1 transcripts paralleled increases in ambient pH. Pac1 was determined to be a functional homolog of PacC by complementation of an A. nidulans pacC-null strain with pac1. Our results suggest that ambient pH is a regulatory cue for processes linked to pathogenicity, development, and virulence and that these processes may be under the molecular regulation of a conserved pH-dependent signaling pathway analogous to that in the nonpathogenic fungus A. nidulans.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of an endopolygalacturonase from Fusarium oxysporum expressed during early stages of infection.

The tomato vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici produces an array of pectinolytic enzymes that may contribute to penetration and colonization of the host plant. Here we report the isolation of pg5, encoding a novel extracellular endopolygalacturonase (endoPG) that is highly conserved among different formae speciales of F. oxysporum. The putative mature pg5 product has a calculated molecular mass of 35 kDa and a pI of 8.3 and is more closely related to endoPGs from other fungal plant pathogens than to PG1, the major endoPG of F. oxysporum. Overexpression of pg5 in a bacterial heterologous system produced a 35-kDa protein with endoPG activity. Accumulation of pg5 transcript is induced by citrus pectin and D-galacturonic acid and repressed by glucose. As shown by reverse transcription-PCR, pg5 is expressed by F. oxysporum in tomato roots during the initial stages of infection. Targeted inactivation of pg5 has no detectable effect on virulence toward tomato plants.

Amino Acid Sequence↗
PubMed2003

Degradation and transformability of DNA from transgenic leaves.

The fate of transplastomic (chloroplast genome contains the transgene) tobacco plant DNA in planta was studied when the plant leaves were subjected to decay conditions simulating those encountered naturally, including grinding, incubation with cellulase or enzymes produced by Erwinia chrysanthemi, and attack by the plant pathogen Ralstonia solanacearum. Direct visualization of DNA on agarose gels, gene extraction yield (the number of amplifiable aadA sequences in extracted plant DNA), and the frequency that recipient bacteria can be transformed by plant DNA were used to evaluate the quality and quantity of plant DNA and the transgene. These measurements were used to monitor the physical and biological degradation of DNA inside decaying plant tissues. Our results indicate that while most of the DNA will be degraded inside plant cells, sufficient DNA persists to be released into the soil.

Acinetobacter calcoaceticus↗
PubMed2003

Heterologous expression of the Saccharomyces cerevisiae PGU1 gene in Schizosaccharomyces pombe yields an enzyme with more desirable properties for the food industry.

The Saccharomyces cerevisiae PGU1 gene was successfully expressed in Schizosaccharomyces pombe. The optimum pH and temperature for the recombinant enzyme were 5 and 40 degrees C, respectively, these being around 0.5 U higher and 5 degrees C lower than those shown by the native enzyme. The K(m) value was about fourfold higher than that of the S. cerevisiae enzyme. The recombinant endopolygalacturonase was more efficient in reducing the viscosity of polygalacturonic acid and was also more stable at different pHs and temperatures than the native enzyme.

Culture Media↗
PubMed1982

An exo-poly-alpha-D-galacturonosidase implicated in the regulation of extracellular pectate lyase production in Erwinia chrysanthemi.

Pectic enzymes in the supernatants of Erwinia chrysanthemi cultures in late-logarithmic-phase growth on D-galacturonan were resolved into three components: two pectate lyase isozymes and an exo-poly-alpha-D-galacturonosidase previously unreported in this organism. The hydrolytic enzyme was purified to homogeneity by ammonium sulfate fractionation, preparative electrofocusing in Ultrodex gel, and gel filtration through Ultrogel AcA54. The enzyme had a specific activity of 591 mumol/min per mg of protein, a pI of 8.3, a molecular weight of 67,000, a pH optimum of 6.0, and a Km of 0.05 mM for D-galacturonan. Analyses of reaction mixtures by paper chromatography revealed that the enzyme released only digalacturonic acid from D-galacturonan. The action of the hydrolytic enzyme on D-galacturonan labeled at the nonreducing end by partial digestion with pectate lyase revealed that it rapidly released 4,5-unsaturated digalacturonic acid from 4,5-unsaturated pectic polymers. The production of extracellular exo-poly-alpha-D-galacturonosidase was coordinately regulated with pectate lyase production. The action patterns of the two enzymes appeared complementary in the degradation of pectic polymers to disaccharides that stimulated pectic enzyme production and supported bacterial growth.

Erwinia↗
PubMed1984

Mutants of Erwinia chrysanthemi defective in secretion of pectinase and cellulase.

Erwinia chrysanthemi produced several pectate lyases (EC 4.2.2.2) and endocellulases (EC 3.2.1.4) which were largely secreted into the culture medium. Mutants deficient in the secretion mechanism for these enzymes were obtained by chemical and insertion mutagenesis. Further study of one such mutant revealed that both enzyme activities were retained simultaneously within the periplasmic space.

Cellulase↗