NATURAL PRODUCTS BIOTECHNOLOGY

Author: MARTINEZ ARTEAGA BASELGA ROCIO.
Year: 2005.
University: AUTÓNOMA DE MADRID.
Place of defense: CENTRO NACIONAL DE BIOTECNOLOGIA CSIC.
Place of preparation: CENTRONACIONAL DE BIOTECNOLOGIA CSIC (UAM).

Summary: We have demonstrated how the bus in the transcript of one of the group members dcw (ftsZ) causes physiological effects: in addition to not split, the cells lose their viability. FtsZ is a protein essential for the division in Escherichia coli. The absence of this protein leads to the filamentation, and the cells grow but are unable to divide. In the absence of FtsZ, the filaments lose their viability in a time period of two hours. Viability is recovered when the levels of FtsZ are restored to the initial values present in the parental strain. The use of gene arrays has enabled us to analyze the overall pattern of gene expression in three different conditions; expressing ftsZ, in the absence of his speech and restore. Levels of tránscrito group ribosomal genes show a steep decline after the stop in gene expression ftsZ and during the period of restoration are not recovered. During the restoration of gene expression ftsZ, add layers of tránscrito of genes in response to damage to DNA and genes involved in metabolism biosintético (anabolismo nucleotide, and amino acid metabolism Central intermediary). To validate the results of gene arrays were quantified levels tránscrito some relevant genes (eg ftsZ, clpP, uvrC) by RT-PCR. Quantifying levels tránscrito of such genes largely corroborated the results obtained using gene arrays. The transcriptional response observed suggests possible mechanisms that the cell takes to adapt to live in the absence of division and how they can influence the actions of viable include dilution of crops. Once satisfied that the stop in the division provokes a response in the transcript overall, we investigated the opposite effect, ie if the changes in physiological conditions produce an effect on the levels of tránscrito group dcw, the largest group of genes of division and synthesis of peptidoglicano. We analyzed the levels of tránscrito of genes group dcw by RT-PCR to verify whether physiological conditions affecting the expression of these genes. The expression of genes in the group dcw is affected by physiological conditions, so that as they grow exponentially crops, is higher amid rich amid poor. These results show that the speed of growth produces an effect on the levels of tránscrito group dcw.

Author: PALOMINO DEL CASTILLO CARMEN.
Year: 2005.
University: AUTÓNOMA DE MADRID.
Place of defense: CENTRO NACIONAL DE BIOTECNOLOGÍA - CSIC.
Place of preparation: CENTRO NACIONAL DE BIOTECNOLOGÍA.

Summary: Streptomyces lividans is a Gram positive bacterium that secretes large amount of extracellular enzymes (Gilbert et al., 1995). This bacterium has been used as a host system for the production of recombinant proteins (Gilbert et al., 1995), however, both the achievement and improving the production of these proteins, some industrial application, it requires a detailed study and identifying bottlenecks of the mechanisms involved in the secretion of extracellular proteins in Strptomyces. The mechanism of general transport proteins (sec) begins with the recognition and interaction with intracellular machinery sequences aminoterminales of protein secretion. Two main routes have been described in various bacteria on the one hand, the mechanism mediated by SecA / B protein secretion leads to the membrane form postraducional (Beck et al., 2000) and on the other hand, the mechanism mediated by machinery SRP, formed by Ffh and scRNA and receiver FtsY who run proteins to the membrane at the beginning of its synthesis for translocation through the membrane channel or translocón, many of these being integral membrane proteins as in the case of E.coli (Dalvey and Chan, 2005). We have not found any gene homologues SecB in Streptomyces lividans, but they are present, the genes of machinery SRP, ffh, scRNA and ftsY (Palacín et al., 2003). Ffh and scRNA constitute the SRP of S.lividans (Palación et al., 2003). With this work, we encountered an entirely formed by Ffh, FtsY and protein secretion model, alpha-amilasa and agarasa, in the cytoplasm of the cell mutant for pepetidasa signal majority (Y62) (Palomino and Mellado, 2005), where the pattern the secretion is decreased (Palación et al., 2002), indicating that the SRP is involved in the mechanism for secretion of this bacterium. In addition, it was found that the mutant Y62 has an effect on levels of Ffh and FtsY in membrane, as both components are increasing, finding themselves in the form of ocmplejos consisting Ffh, FtsY and preproteína pre-alpha amylase. Thanks to the miscroscopía immunofluorescence we have seen the location of the SRP components distributed to individual sites of the hyphae of the mutant Y62 front of the wild strain diffusely. This location reminded the location microscopic specific translocón coupled with GFP. It has been described a phenomenon accumulation intermediaries formed by Ffh, your receiver and preproteínas, in the membrane of the endoplasmic reticulum of eukaryotic cells when there are no transolocón available (song and cabbage., 2000). A similar situation may be occurring in S.lividans in the absence of peptidasa signal majority, where processing occurs leader peptide can lead to an inefficient temporary block in the translocón. Lastly, Ffh and FtsY coupled with pre-alpha amylase forming a complex membrane are also associated with ATPase membrane SecA, indicating that the proteins are transported to the membrane by SRP required for the action of SecA for translocation.