Bacterial degradation of poly(trans-1,4-isoprene) (gutta percha).

Gutta percha, the trans-isomer of polyisoprene, is being used for several technical applications due to its resistance to biological degradation. In the past, several attempts to isolate micro-organisms capable of degrading chemically pure poly(trans-1,4-isoprene) have failed. This is the first report on axenic cultures of bacteria capable of degrading gutta percha. From about 100 different habitats and enrichment cultures, six bacterial strains were isolated which utilize synthetic poly(trans-1,4-isoprene) as sole carbon and energy source for growth. All isolates were assigned to the genus Nocardia based on 16S rRNA gene sequences. Four isolates were identified as strains of Nocardia nova (L1b, SH22a, SEI2b and SEII5a), one isolate was identified as a strain of Nocardia jiangxiensis (SM1) and the other as a strain of Nocardia takedensis (WE30). In addition, the type strain of N. takedensis obtained from a culture collection (DSM 44801(T)) was shown to degrade poly(trans-1,4-isoprene). Degradation of poly(trans-1,4-isoprene) by these seven strains was verified in mineralization experiments by determining the release of CO(2). All seven strains were also capable of mineralizing poly(cis-1,4-isoprene) and to use this polyisoprenoid as a carbon and energy source for growth. Mineralization of poly(trans-1,4-isoprene) after 80 days varied from 3 % (strain SM1) to 54 % (strain SEI2b) and from 34 % (strain L1b) to 43 % (strain SH22a) for the cis-isomer after 78 days. In contrast, Gordonia polyisoprenivorans strain VH2, which was previously isolated as a potent poly(cis-1,4-isoprene)-degrading bacterium, was unable to degrade poly(trans-1,4-isoprene). Scanning electron microscopy revealed cavities in solid materials prepared from poly(trans-1,4-isoprene) and also from poly(cis-1,4-isoprene) after incubation with N. takedensis strain WE30 or with N. nova strain L1b, whereas solid poly(trans-1,4-isoprene) material remained unaffected if incubated with G. polyisoprenivorans strain VH2 or under sterile conditions.

Identification and characterization of genes from Streptomyces sp. strain K30 responsible for clear zone formation on natural rubber latex and poly(cis-1,4-isoprene) rubber degradation.

Streptomyces sp. strain K30 was isolated from soil next to a city high way in Münster (Germany) according to its ability to degrade natural and synthetic poly(cis-1,4-isoprene) rubber and to form clear zones on natural rubber latex agar plates. The clear zone forming phenotype was used to clone the responsible gene by phenotypic complementation of a clear zone negative mutant. An open reading frame (lcp) of 1,191 bp was identified, which was preceded by a putative signal sequence and restored the capability to form clear zones on natural rubber latex in the mutant. The putative translation product exhibited strong homologies (50% aa identity) to a putative secreted protein from Streptomyces coelicolor strain A3(2), another clear zone forming strain. Heterologous expression of lcp of Streptomyces sp. strain K30 in Streptomyces lividans strain TK23 enabled the latter to form clear zones on latex-overlay agar plates and to accumulate a degradation product of about 12 kDa containing aldehyde groups. Two ORFs putatively encoding a heterodimeric molybdenum hydroxylase (oxiAB) were identified downstream of lcp in Streptomyces sp. strain K30 strain which exerted a positive effect on clear zone formation and enabled the strain to oxidize the resulting aldehydes. Heterologous expression of a fragment harboring lcp plus oxiAB in S. lividans TK23 resulted in accumulation of aldehydes only in the presence of 10 mM tungstate. Determination of protein content during cultivation on poly(cis-1,4-isoprene) revealed an increase of the cellular protein, and gel permeation chromatography analysis indicated a shift of the molecular weight distribution of the rubber to lower values in the transgenic S. lividans strains and in the wild type, thus confirming utilization and degradation of rubber. Therefore, for the first time, genes responsible for clear zone formation on natural rubber latex and synthetic cis-1,4-polyisoprene degradation in Gram-positive bacteria were identified and characterized.

CPTF1, a CREB-like transcription factor, is involved in the oxidative stress response in the phytopathogen Claviceps purpurea and modulates ROS level in its host Secale cereale.

CPTF1, a transcription factor with significant homology to ATF/CREB bZIP factors, was identified during an expressed sequence tag (EST) analysis of in planta-expressed genes of the phytopathogen Claviceps purpurea. Using a gene-replacement approach, deletion mutants of cptf1 were created. Expression studies in axenic culture showed that the H2O2-inducible gene cpcat1 (encoding a secreted catalase) had a reduced basal expression level and no longer responded to oxidative stress in the delta cptf1 mutant. Biochemical analyses indicated that CPTF1 is a general regulator of catalase activity. Delta cptf1 mutants showed significantly reduced virulence on rye. Electron microscopical in situ localization revealed significant amounts of H2O2 in delta cptf1-infected rye epidermal tissues, indicating that the plant tissue displayed an oxidative burst-like reaction, an event not detected in wild-type infections. These data indicate that CPTF1 is involved not only in oxidative stress response in the fungus but also in modulation of the plant's defense reactions.

Functional analysis of H(2)O(2)-generating systems in Botrytis cinerea: the major Cu-Zn-superoxide dismutase (BCSOD1) contributes to virulence on French bean, whereas a glucose oxidase (BCGOD1) is dispensable.

SUMMARY The oxidative burst, a transient and rapid accumulation of reactive oxygen species (ROS), is a widespread defence mechanism of higher plants against pathogen attack. There is increasing evidence that the necrotrophic fungal pathogen Botrytis cinerea itself generates ROS, and that this capability could contribute to the virulence of the fungus. Two potential H(2)O(2)-generating systems were studied with respect to their impact on the interaction of B. cinerea and its host plant Phaseolus vulgaris. A Cu-Zn-superoxide dismutase gene (bcsod1) and a putative glucose oxidase gene (bcgod1) were cloned and characterized, and deletion mutants were created using a gene-replacement methodology. Whereas the Deltabcgod1-mutants displayed normal virulence on bean leaves, the Deltabcsod1 mutants showed a significantly retarded development of lesions, indicating that the Cu-Zn SOD-activity is an important single virulence factor in this interaction system. Whether dismutation of (fungal or host) superoxide, or generation of H(2)O(2) (or both), are important for pathogenesis in this system remains to be elucidated.

CPMK2, an SLT2-homologous mitogen-activated protein (MAP) kinase, is essential for pathogenesis of Claviceps purpurea on rye: evidence for a second conserved pathogenesis-related MAP kinase cascade in phytopathogenic fungi.

Cpmk2, encoding a mitogen-activated protein (MAP) kinase from the ascomycete Claviceps purpurea, is an orthologue of SLT2 from Saccharomyces cerevisiae, the first isolated from a biotrophic, non-appressorium-forming pathogen. Deletion mutants obtained by a gene replacement approach show impaired vegetative properties (no conidiation) and a significantly reduced virulence, although they retain a limited ability to colonize the host tissue. Increased sensitivity to protoplasting enzymes indicates that the cell wall structure of the mutants may be altered. As the phenotypes of these mutants are similar to those observed in strains of the rice pathogen, Magnaporthe grisea, that have been deprived of their MAP kinase gene mps1, the ability of cpmk2 to complement the defects of delta mps1 was investigated. Interestingly, the C. purpurea gene, under the control of its own promoter, was able to complement the M. grisea mutant phenotype: transformants were able to sporulate and form infection hyphae on onion epidermis and were fully pathogenic on barley leaves. This indicates that, despite the differences in infection strategies, which include host and organ specificity, mode of penetration and colonization of host tissue, CPMK2/MPS1 defines a second MAP kinase cascade (after the Fus3p/PMK1 cascade) essential for fungal pathogenicity.

Polygalacturonase is a pathogenicity factor in the Claviceps purpurea/rye interaction.

Claviceps purpurea is a biotrophic, organ-specific pathogen of grasses and cereals, attacking exclusively young ovaries. We have previously shown that its mainly intercellular growth is accompanied by degradation of pectin, and that two endopolygalacturonase genes (cppg1/cppg2) are expressed throughout all stages of infection. We report here on a functional analysis of these genes using a gene-replacement approach. Mutants lacking both polygalacturonase genes are not affected in their vegetative properties, but they are nearly nonpathogenic on rye. Complementation of the mutants with wild-type copies of cppg1 and cppg2 fully restored pathogenicity, proving that the endopolygalacturonases encoded by cppg1 and cppg2 represent pathogenicity factors in the interaction system C. purpurea/Secale cereale, the first unequivocally identified so far in this system.

Functional analysis of an extracellular catalase of Botrytis cinerea.

Summary There is evidence that the necrotrophic fungal pathogen Botrytis cinerea is exposed to oxidative processes within plant tissues. The pathogen itself also generates active oxygen species and H(2)O(2) as pathogenicity factors. Our aim was to study how the pathogen may defend itself against cellular damage caused by the accumulation of H(2)O(2) and the role of an extracellular catalase in its detoxification during the infection of tomato and bean plants by B. cinerea. Chloronaphthol staining followed by light microscopy showed that H(2)O(2) accumulates in the infection zone in tomato and bean leaves. An extracellular catalase gene (denominated Bccat2) was cloned from B. cinerea. Exposure of mycelium to H(2)O(2) in liquid culture resulted in increased Bccat2 mRNA levels in a concentration-dependent manner. Bccat2 mRNA was detected at early stages of tomato leaf infection, suggesting that B. cinerea experiences oxidative stress. Bccat2-deficient mutants were generated by transformation-mediated gene disruption. Mutants were more sensitive then the wild-type strain to H(2)O(2)in vitro, but they partly compensated for the absence of BcCAT2 by activating other protective mechanisms in the presence of H(2)O(2). Bccat2-deficient mutants did not display a consistent reduction of virulence on bean and tomato leaves. Cerium chloride staining of infected leaf tissue for ultrastructural studies showed that Bccat2-deficient mutants were exposed to H(2)O(2) comparably to the wild-type. The results suggest that B. cinerea is a robust pathogen adapted to growing in hostile oxidizing environments in host tissues.