Culturable bacteria from Zn- and Cd-accumulating Salix caprea with differential effects on plant growth and heavy metal availability.

AIMS: To characterize bacteria associated with Zn/Cd-accumulating Salix caprea regarding their potential to support heavy metal phytoextraction. METHODS AND RESULTS: Three different media allowed the isolation of 44 rhizosphere strains and 44 endophytes, resistant to Zn/Cd and mostly affiliated with Proteobacteria, Actinobacteria and Bacteroidetes/Chlorobi. 1-Aminocyclopropane-1-carboxylic acid deaminase (ACCD), indole acetic acid and siderophore production were detected in 41, 23 and 50% of the rhizosphere isolates and in 9, 55 and 2% of the endophytes, respectively. Fifteen rhizosphere bacteria and five endophytes were further tested for the production of metal-mobilizing metabolites by extracting contaminated soil with filtrates from liquid cultures. Four Actinobacteria mobilized Zn and/or Cd. The other strains immobilized Cd or both metals. An ACCD- and siderophore-producing, Zn/Cd-immobilizing rhizosphere isolate (Burkholderia sp.) and a Zn/Cd-mobilizing Actinobacterium endophyte were inoculated onto S. caprea. The rhizosphere isolate reduced metal uptake in roots, whereas the endophyte enhanced metal accumulation in leaves. Plant growth was not promoted. CONCLUSIONS: Metal mobilization experiments predicted bacterial effects on S. caprea more reliably than standard tests for plant growth-promoting activities. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteria, particularly Actinobacteria, associated with heavy metal-accumulating Salix have the potential to increase metal uptake, which can be predicted by mobilization experiments and may be applicable in phytoremediation.

Characterization of small GTPases Cdc42 and Rac and the relationship between Cdc42 and actin cytoskeleton in vegetative and ectomycorrhizal hyphae of Suillus bovinus.

This work reports the isolation and molecular characterization of CDC42 and RAC1 cDNAs from the ectomycorrhiza forming filamentous homobasidiomycete Suillus bovinus. Previously, no RAC gene was described from filamentous fungi and no CDC42 gene was described from homobasidiomycetes. Southern hybridization with SbCDC42 and SbRAC1 cDNAs indicated that the S. bovinus genome contains only one CDC42 and one RAC1 gene. The predicted amino acid sequence of SbRaclp is 77% identical with the Rac1B protein of chick, whereas SbCdc42p is most identical with Schizosaccharomyces pombe Cdc42p, showing 88% identity. In the predicted amino acid sequences of SbRaclp and SbCdc42p, the five guanine nucleotide binding regions, switch I and II, and the effector domain are highly identical to those known in other small GTPases. These domain structures suggest that in S. bovinus, SbRac1p and SbCdc42p function as molecular switches regulating the organization of actin cytoskeleton, similar to yeasts and mammals. SbRAC1 and SbCDC42 were expressed in vegetative and ectomycorrhizal hyphae, and SbCdc42p was detected in ectomycorrhiza-forming hyphae if growth and differentiation of the symbiotic hyphae took place. Cdc42p and actin were localized at the tips of S. bovinus vegetative hyphae. Similar to yeast, in filamentous fungi Cdc42p may be necessary to maintain the actin cytoskeleton at hyphal tips, making the polarized growth of the hyphae possible. In developing ectomycorrhiza, Cdc42p and actin were visualized in association with plasma membrane in swollen cells typical to the symbiotic hyphae. The role of Cdc42p and actin in regulation of the growth pattern and morphogenesis of ectomycorrhizal hyphae is discussed.

Molecular characterization of actin genes from homobasidiomycetes: two different actin genes from Schizophyllum commune and Suillus bovinus.

The actin-encoding genes Scact1 and Scact2 of the homobasidiomycete Schizophyllum commune are the first actin genes isolated from higher filamentous fungi. Their isolation shows that homobasidiomycetes have two actin encoding genes instead of one typical to yeasts and filamentous ascomycetes. This result was further confirmed by cloning two actin encoding genes, Sbact1 and Sbact2, from another homobasidiomycete Suillus bovinus. The comparison of the genomic and cDNA sequences of the actin genes showed that Scact1 and Scact2 genes of S. commune contain seven introns, five of which are at the same position in the two genes while S. bovinus actin genes contain nine similarly positioned introns. In the four genes, five intron positions are shared, which indicates a close relationship between the actin encoding genes from S. commune and S. bovinus. Northern hybridization and analysis of two-dimensional immunoblots showed a difference in the expression levels between the two actin genes in each fungus. No actin protein could be detected from S. commune Scact2. The deduced amino acid sequence of the Scact2 gene also differs considerably from any other known actin protein. These data suggest that the Scact2 gene either has a special as yet unidentified function in S. commune life cycle or is a transcribed but no longer translated pseudogene. Scact2 gene has a putative microORF (short open reading frame) and Scact1 gene an intron in the 5'-untranslated region, which could reduce the translational efficiency and increase the transcriptional efficiency of the Scact2 and Scact1 genes, respectively. During mating in S. commune or at formation of ectomycorrhiza in S. bovinus, the expression of actin genes was similar to that in vegetative hyphae. This result suggests that the reorganization of actin cytoskeleton in response to extra- and intracellular signals in higher filamentous fungi could be directly regulated by members of signalling pathways well characterized in yeast and mammalian cells.

Trichoderma reesei prs12 encodes a stress- and unfolded-protein-response-inducible regulatory subunit of the fungal 26S proteasome.

We have cloned a gene, prs12, from the filamentous fungus Trichoderma reesei which encodes a fungal homologue of the mouse and Drosophila regulatory subunit 12 of the 26S proteasome (mov34). Sequencing of both a genomic and a cDNA-clone predicts a 342-aa protein with high overall identity (56-68 %) to the homologous counterparts from human, mammals, Drosophila and Saccharomyces cerevisiae. The predicted protein contains several consensus sequences for phosphorylation, three of which are conserved in all published Prs12p homologues. Its C-terminus is rich in alternating K and E/D, and resembles a potential KEKE-motif. Prs12 exhibits a basal level of transcription during normal growth, but its expression is significantly increased over 60-120 min under conditions of stress evoked by the addition of cadmium ions and hygromycin B. It is also stimulated by the addition of tunicamycin and 2-mercaptoethanol, suggesting its regulation by the presence of unfolded proteins in the endoplasmic reticulum and by hygromycin B. Consistent with this behaviour, motifs in the prs12 5'-upstream sequences show sequence homology with the consensus sequences for general stress response, and for an ER traffic-response element.