Abstract: A gene encoding hydroxyquinol 1,Zdioxygenase was cloned from 2,4,6-trichlorophenol-degrading Ralstonia (Pseudomonas) pickettii strain DTP0602. Cell-free extracts of Escherichia coli containing a cloned 1.4-kb &I-XhoI DNA fragment of R. pickettii DTP0602 hydroxyquinol 1, Zdioxygenase converted hydroxyquinol into maleylacetate and also degraded 6-chlorohydroxyquinol. The 1.4-kb DNA fragment contained one open reading frame (designated hadO composed of 948 nucleotides. The molecular mass of 34,591 deduced from the gene product (HadC) was in agreement with the size (35 kDa) of the puri6ed HadC protein determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The amino acid sequence of HadC exhibited high homology to that of the hydroxyquinol 1,Zdioxygenase of 2,4,5trichlorophenoxyacetic acid-degrading Burkholderfa cepacia AC1100 (Dauharas, D.L.et al., Appl. Environ. Microbial., 61, 1279-1289,1995). The active enzyme had a molecular mass of 68 kDa, suggesting that it is functional as a homodimer. The enzyme
also catalyzed the oxidation of pyrogallol and 3-methylcatechol, possible intermediates in the degradation of 2,4,6+ichlorophenol, in addition to 6-chlorohydroxyquinol and hydroxyquinol. The dioxygenase catalyzed both ortho- and meta-cleavage of 3-methylcatechol.
Abstract:Ralstonia sp. KNl-1OA is a strain capable of degrading trichloroethylene (TCE) constitutively due to the tac promoter (Ptac)integrated upstream of the phenol hydroxylase genes (phy) in its chromosome. The expression of Ptac was analyzed using luxAB of Vibrio harveyi as a reporter. After determining the nucleotide sequence of phyABCDE required for TCE degradation, a IuxAB-encoding fragment was integrated downstream of phyE by homologous recombination in strain KNl-lOA, obtaining strain KNl-lOA-LX. In the same manner, the luxAB-encodIng fragment was integrated into the chromosome of the wild-type strain, KNl. The resultant strain KNl-LX was used to analyze the gene expression caused by phenol induction. The expression induced by Ptac was compared to that by phenol induction. Although the level of luxAB expression led by Ptac was almost equal to that induced by phenol, the TCE degradation rate by the Ptac-carrying KNl-lOA-LX was markedly slower than that by the phenol-induced KNl-LX. These results suggest that an important gene for TCE degradation was not transcribed by Ptac in KNl-lOA-LX. The nucleotide sequence analysis showed the existence of a small gene, phy2, upstream of phyA, and Ptac was found to be integrated into the middle of phyZ in KNl-lOA-LX. The effect of phyZ on TCE degradation was examined by using recombinant strains expressing phyABCDE with or without phyZ in a plasmid. The coexistence of phyZ markedly accelerated TCE degradation. Through an exhaustive expression analysis, it was demonstrated that the chromosomal integration of Ptac was a very attractive method for high and stable production of phenol hydroxylase for TCE degradation.
Abstract: A newly isolated gene from Ralstonia sp. M1, encoding an esterase, was cloned in Escherichia coli and its nucleotide sequence determined. The 1.6kb insert revealed one complete open reading frame, predicted to encode an esterase (320 aa, 34.1kDa) with a pI of 9.86. EstR contained a putative oxyanion hole H36 G37, a conserved pentapeptide G103 HSLG107 and a conserved catalytic His265
and Asp237. The EstR sequence shared 64–70 and 44–48% identity with the hydrolases/acyltransferases from Burkholderia strains and from Ralstonia strains, respectively, 44 and 38% identity with the lactone-speciWc esterase from Pseudomonas Xuorescens and Mesorhizobium loti, respectively. The esterase EstR was expressed with a high level of 41mg/g wet cells. The Ni–NTA-puriWed esterase EstR showed an optimal activity in the temperature range 60–65°C and pH range 7.5–9.0 towards p-nitrophenyl caproate. The enzyme was found to be highly resistant to many organic solvents especially induced by ethanolamine. Metal ions showed slight effect on esterase activity. The inhibitor phenylmethanesulfonyl Xuoride inhibited strongly the esterase. Triton X-45 induced the activation of EstR, but other detergents slightly to strongly decreased or completely inhibited. Among tested p-NP esters, caproate was the most preferential substrate of this esterase.
Abstract: Directly adjacent to the (tfdT-)tfdCDEF gene cluster for chlorocatechol breakdown on plasmid pJP4 of Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP134, we identified a 0.9-kb DNA element, designated ISJP4, with the typical features of a bacterial insertion sequence. ISJP4 occurs as a single complete copy on plasmid pJP4. About 9 kb away from this copy, in the tfdA–tfdS intergenic region, we found a 71-bp duplication of the ISJP4 right-hand extremity. In addition, we discovered a complete copy of ISJP4 on the chromosome of the R. eutropha JMP134 strain that we use routinely in our laboratory. We suppose that this copy resulted from a recent transposition of the plasmid-borne ISJP4, since it was shown to be lacking from the chromosomes of R. eutropha JMP222 and JMP289, two previously pJP4-cured derivatives of JMP134. By comparing both complete copies and their flanking regions, we could establish that element ISJP4 has a size of 915 bp and is bordered by 18-bp inverted repeats with one mismatch. Based on sequence similarity of its coding regions, ISJP4 could be classified into the IS5 group of the IS4 family of bacterial insertion sequences, where it is mostly related to IS402 of Burkholderia cepacia. A TAA direct repeat, presumably resulting from a duplication of the target site, flanked the chromosomal copy of ISJP4. We could demonstrate that a piece of DNA that is flanked by two complete copies of ISJP4 can be transposed. Even more so, one complete ISJP4 plus its tfdA–tfdS intergenic remnant were sufficient to mediate transposition of intervening DNA. A possible role of ISJP4 in the formation of the tfd pathway genes will be discussed.
Abstract: A G protein a subunit gene (pigpa1) and a G protein b subunit gene (pigpb1) were isolated from the oomycete Phytophthora infestans,/i>, the causal agent of potato late blight. Heterotrimeric G proteins are evolutionary conserved GTP-binding proteins that are composed of a, b, and y subunits and participate in diverse signal transduction pathways. The deduced amino acid sequence of both pigpa1 and pigpb1, showed the typical conserved motifs present in Ga or Gb proteins from other eukaryotes. Southern blot analysis revealed no additional copies of Ga or Gb subunit genes in P. infestans, suggesting that pigpa1 and pigpb1 are single copy genes. By cross-hybridization homologues of gpa1 and gpb1 were detected in other Phythophthora species. Expression analyses revealed that both genes are differentially expressed during asexual development, with the highest mRNA levels in sporangia. In mycelium, no pigpa1 mRNA was detected. Western blot analysis using a polyclonal GPA1 antibody confirmed the differential expression of pigpa1. These expression patterns suggest a role for G-protein-mediated signaling during formation and germination of asexual spores of P. infestans, developmental stages representing the initial steps of the infection process.
Abstract: An 85-kDa β-glucosidase/xylosidase (BGX1) was purified from the axenically grown phytopathogenic oomycete, Phytophthora infestans. The bgx1 gene encodes a predicted 61-kDa protein product which, upon removal of a 21 amino acid leader peptide, accumulates in the apoplastic space. Extensive N-mannosylation accounts for part of the observed molecular mass difference. BGX1 belongs to family 30 of the glycoside hydrolases and is the first such oomycete enzyme deposited in public databases. The bgx1 gene was found in various Phytophthora species, but is apparently absent in species of the related genus, Pythium. Despite significant sequence similarity to human and murine lysosomal glucosylceramidases, BGX1 demonstrated neither glucocerebroside nor galactocerebroside-hydrolyzing activity. The native enzyme exhibited glucohydrolytic activity towards 4-methylumbelliferyl (4-MU) b-Image -glucopyranoside and, to lesser extent, towards 4-MU-Image -xylopyranoside, but not towards 4-MU-b-Image -glucopyranoside. BGX1 did not hydrolyze carboxymethyl cellulose, cellotetraose, chitosan or xylan, suggesting high substrate specificity and/or specific cofactor requirements for enzymatic activity.
Abstract: The primary structure of syringicin (syr), a new acidic α-elicitin, isolated from culture filtrates of Phytophthora syringae, causal agent of citrus fruit rot, has been determined using a combined approch based on Edman degradation and MALDI-MS (TTCTT TQQTA AYVAL VSILS DSSFN QCATD SGYSM LTATA LPTTA QYKLM CASTA CKTMI TKIVS LNAPD CELTV PTSGL VLNVY SYANG FSSTC ASL). Syr has 98 amino acids with a Mr of 10194.6±0.2, which was determined by electrospray ionisation-mass spectrometry (ES-MS) and in agreement with three disulphide bridges, located between Cys3-Cys71, Cys27-Cys56 and Cys51-Cys95. Syr induces a hypersensitive response and electrolyte leakage in tobacco. These are characteristic elicitor properties of the group and in agreement with the molecular mechanism recently proposed for this kind of protein. Finally, its possible applications in biological agriculture and biomedicine are briefly discussed.
Abstract: Differential expression of pal1 and hmgr2 was investigated using northern blot analysis in two potato cultivars (Russet Burbank (RB), susceptible and Kennebec (KB), moderately tolerant) after inoculation with two Phytophthora infestans isolates from the formerly (US-1) and currently predominant genotypes (US-8). The accumulation of pal1 transcripts was weaker in response to US-8 as compared to US-1 and occurred earlier in KB than in RB. The stronger expression of pal1 in response to US-1, as compared to US-8, is suggested to be due to defense gene suppression by the latter. No apparent strong accumulation of hmgr2 transcripts was recorded in RB as compared to KB inoculated with either US-1 or US-8. The induction of pal1 and hmgr2 was first observed in un-inoculated (proximal) close to the inoculated leaflets, then in un-inoculated (distal) leaflets of leaves adjacent to the inoculated leaf, and finally in local inoculated leaflets. The stronger expression of the two genes in proximal and distal leaflets, as compared to the local site of inoculation suggests the translocation of signal(s) from this site to healthy parts of the plant.
Abstract: The time-course and the spatial accumulation of PR-proteins pr-1 and pr-5 gene transcripts were investigated in two potato cultivars differing in their levels of susceptibility to late blight, caused by Phytophthora infestans (Mont.) de Bary. Cultivars Russet Burbank (RB, susceptible) and Kennebec (KB, moderately tolerant) were inoculated with either P. infestans genotype US-1 (old lineage) or US-8 (new lineage). A strong induction of both genes was detected in both cultivars inoculated with either P. infestans genotype, as compared to the healthy-controls. The accumulation of transcripts from both genes occurred earlier in KB than in RB leaflets. By comparing the two P. infestans isolates tested, a stronger and earlier induction of both PR genes was recorded in response to US-1 as compared to US-8. The spatio-temporal profiling of pr-1 and pr-5 genes expression showed a strong and early accumulation of transcripts at the local infection site, a late and intermediate level of induction at the proximal site, and no or very weak induction at a distal site remote from the infection site. These results show that pr-1 and pr-5 genes both are related to the defense mechanisms of potato to late blight, and that the higher infection success of P. infestans US-8 as compared to US-1 might be due to the late and/or the weak induction of these defense genes.
Abstract: The differential expression of four Phytophthora cinnamomi elicitin genes was analysed by Real Time RT-PCR. In in vitro cultures, the b-cinnamomin gene showed the highest level of expression, the b-cinnamomin gene (b-cin ) was the most inducible, and the HAE transcripts were in low abundance. Transcription of all the elicitins was active during the active growth of the pathogen when infecting cork oak (Quercus suber) roots, and as host colonization progressed, the level of b-cin expression fell, while that of b-cin rose. In an antisense transgenic strain, the silencing of b-cin also negatively affected the expression of other elicitin genes in the cluster. The reduced in planta growth of the b-cin knock-out is related to the altered pattern of elicitin gene expression, supporting the idea that one of the functions of elicitins is related, directly or indirectly, with pathogenesis.
Abstract