Kaiserslautern - Fachbereich Biologie
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- Avirulence (1)
- Biotrophy (1)
- Haustoria (1)
- Rust effector (1)
- Streptomyces (1)
- Transferred proteins (1)
- biochemical characterisation (1)
- esterases (1)
- lipases (1)
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Haustoria of the rust fungus pathogen Uromyces fabae deliver RTP1 (Rust Transferred Protein1) into host plant cells. In this work, different heterologous expression systems were used to study RTP1 biological function as well as RTP1 transfer mechanism. The first part of this thesis focused on the identification of the subcellular target compartment of RTP1 in plant cells. In this respect we could identify a functional bipartite nuclear localization signal within RTP1. However, stable and transient expression studies of RTP1 in different plant species, including the host plant Vicia faba, interfered with plant cell vitality but did not result in detection of RTP1 protein. These findings led us to propose that RTP1 interferes with plant gene expression. However, the molecular basis of this interference remains unclear. By deletion studies, we could localize the active region of RTP1 within a 45 amino acid central domain. In the second part of this study, two different lines of approaches were taken to study RTP1 transfer mechanism. First, transient expression of secreted RTP1 (sRTP1) also interfered with plant cell vitality. Addition of an endoplasmic reticulum retention signal abolished sRTP1 interference with plant cell vitality, suggesting that RTP1 can reenter the plant cell from the apoplast after secretion in the absence of the pathogen. We have identified a PEST-like region within RTP1, however, contribution of this region to the stability of RTP1 is not clear. Site directed mutagenesis analysis showed that the PEST-like region is likely to play a role during the transfer of RTP1 through plant plasma membrane. In the second line of approach, we established a recombinant delivery model, using Ustilago maydis/Zea mays pathosystem, to pursue RTP1 translocation into the plant cell. Our results indicate that U. maydis is capable of secreting high amounts of recombinant RTP1, showing similar glycosylation pattern as RTP1 secreted from rust haustoria. Our data propose the use of this model system to study RTP1 domains mediating its entry into the plant cell. Haustoria of the rust fungus pathogen Uromyces fabae deliver RTP1 (Rust Transferred Protein1) into host plant cells. In this work, different heterologous expression systems were used to study RTP1 biological function as well as RTP1 transfer mechanism. The first part of this thesis focused on the identification of the subcellular target compartment of RTP1 in plant cells. In this respect we could identify a functional bipartite nuclear localization signal within RTP1. However, stable and transient expression studies of RTP1 in different plant species, including the host plant Vicia faba, interfered with plant cell vitality but did not result in detection of RTP1 protein. These findings led us to propose that RTP1 interferes with plant gene expression. However, the molecular basis of this interference remains unclear. By deletion studies, we could localize the active region of RTP1 within a 45 amino acid central domain. In the second part of this study, two different lines of approaches were taken to study RTP1 transfer mechanism. First, transient expression of secreted RTP1 (sRTP1) also interfered with plant cell vitality. Addition of an endoplasmic reticulum retention signal abolished sRTP1 interference with plant cell vitality, suggesting that RTP1 can reenter the plant cell from the apoplast after secretion in the absence of the pathogen. We have identified a PEST-like region within RTP1, however, contribution of this region to the stability of RTP1 is not clear. Site directed mutagenesis analysis showed that the PEST-like region is likely to play a role during the transfer of RTP1 through plant plasma membrane. In the second line of approach, we established a recombinant delivery model, using Ustilago maydis/Zea mays pathosystem, to pursue RTP1 translocation into the plant cell. Our results indicate that U. maydis is capable of secreting high amounts of recombinant RTP1, showing similar glycosylation pattern as RTP1 secreted from rust haustoria. Our data propose the use of this model system to study RTP1 domains mediating its entry into the plant cell.
Esterases and lipases are widely used as industrial enzymes and for the synthesis of chiral drugs. Because of their rich secondary metabolism, Streptomyces species offer a relatively untapped source of interesting esterases and lipases. S. coelicolor and S. avermitilis contain 51 genes annotated as esterases and/or lipases. In this study I have cloned 14 different genes encoding for lipolytic enzymes from S. coelicolor (11 genes) and S. avermitilis (four genes). Some of these genes were over-expressed in E. coli. Three of the produced enzymes, which were produced by the genes SCO 7131, SCO6966 and SCO3644, were characterized biochemically and one of them was subjected for directed evolution. The gene estA (locus SCO 7131) was annotated as a putative lipase/esterase in the genome sequence of S. coelicolor A3(2), but does not have a homologue in the genome sequence of S. avermitilis or in other known Streptomyces sequences. estA was cloned and expressed in E. coli as a His-tagged protein. The protein was purified and could be recovered in its non-tagged form after digestion with factor Xa. The relative molecular weight was estimated to be 35.5kDa. The enzyme was only active towards acetate esters and not on larger substrates. It had a stereospecificity towards α-naphathylacetate. It was thermostable, with a half-life at 50C of 4.5 hours. Est A showed stability over pH range 5.5-10, and had optimum pH of 7.5. Its activity was drastically decreased when it was pre-incubated in 10mM PMSF, Cu+2 and Hg+2. It was not very stable in most organic solvents and had only slight enantioselectivity. Est A belongs to the HSL family whose founder member is the human hormone-sensitive lipase. I have developed a protein profile for the HSL family modifying the conserved motifs found by Arpigny and Jaeger (1999). Due to the presence of several HSL members with known 3D structure and good homology to Est A, I was able to make a homology model of Est A. Five different mutants of Est A were produced through site directed mutagenesis: W87F, V158A, W87F/V158A, M162L and S163A. The mutants M162L and S163A did not produce a significant change either in substrate specificity or enzyme kinetics. The mutants V158A and W87F/V158A could act on the larger substrates p-nitrophenylbutyrate and caproate and tributyrin. The mutant V158A had improved thermostability and its t1/2 at 50ºC increased to 24h. The affinity of V158A towards p-nitrophenyacetate increased 6-fold when compared with the wild type, whereas the affinity of W87F decreased 4-fold. Directed evolution of Est A was done through random mutagenesis and ER-PCR. A library of 6336 mutants was constructed and screened for mutants with a broader spectrum of substrate specificity. The mutant XXVF7 did show alteration in the substrate specificity of Est A. The mutant XXVF7 had 5 amino acids changes L76R, L146P, S196G, W213R and L267R. The gene locus SCO 6966 (estB gene) was cloned and expressed in E. coli as a His-tagged protein. It was not possible to remove the His-tag using factor Xa. The tagged protein had a molecular weight 31.9kDa. Est B was active against short chain fatty acid esters (C2-C6). Its optimum temperature was 30ºC and was stable for 1h at temperatures up to 37ºC. The enzyme had maximum activity at pH 8-8.5 and was stable over pH range 7.5-11 for 24h. It was highly sensitive for PMSF, Cu+2 and Hg+2. The enzymatic activity deceased in presence of organic solvents, however it was fairly stable for 1h in 20% organic solvents solutions. A third esterase was produced from the gene locus SCO 3644. This esterase was a thermosensitive one with optimum temperature of 35ºC. The three characterized enzymes included a thermophilic, mesophilic and psychrophilic ones. This indicates the high variation in the characters of Streptomyces lipolytic enzymes and highlighting Streptomyces as a source for esterases and lipases of interesting catalytic activity. This study was an initial trial to provide a strategy for a comprehensive use of genome data.