Vergleichende Analyse der Sec- und Tat-abhängigen sekretorischen Proteingewinnung mit Gram-positiven Bakterien als Wirtsorganismen

Meißner, Daniel

Dissertation / PhD Thesis/Book

Vergleichende Analyse der Sec- und Tat-abhängigen sekretorischen Proteingewinnung mit Gram-positiven Bakterien als Wirtsorganismen

Meißner, D. (Corresponding author)FZJ*

2006
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag Jülich
ISBN: 3-89336-427-7

Jülich : Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag, Schriften des Forschungszentrums Jülich. Reihe Lebenswissenschaften / Life Sciences 26, 140 S. (2006) = Universtät Düsseldorf, Diss., 2005

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Please use a persistent id in citations: http://hdl.handle.net/2128/2465

Abstract: For secretory protein recovery in Gram-positive bacteria, the Sec pathway has so far been used exclusively, where the secretion of heterologous substrates is inefficient in many cases, since they often only fold very slowly or wrongly after membrane passage and thus become victims of extracellular proteases. In the Tat pathway, the substrates are already folded in the cytoplasm, so that folding problems of heterologous substrates after translocation are avoided. The Tat pathway, for which investigations concerning its biotechnological potential have only just begun, may thus be an alternative to the Sec pathway in the secretory recovery of heterologous proteins. This has been examined in more detail in the present study using the Gram-positive bacteria $\textit{Staphylococcus carnosus, Corynebacterium glutamicum}$ and $\textit{Bacillus subtilis}$, where at the beginning only $\textit{B. subtilis}$ was known to have a functional Tat system. For $\textit{S. carnosus}$ and $\textit{C. glutamicum}$ this was shown here for the first time. In characterizing the Tat systems it was found that under the conditions tested here the Tat pathway in $\textit{C. glutamicum}$, in clear contrast to the situation in $\textit{S. carnosus}$, plays an important role for both growth and metabolism. For a comparative assessment of the secretion properties of these bacteria, the Tat-dependent secretion of different fusion proteins was examined. In $\textit{S. carnosus}$ it was found that the CGTase and GFP model proteins get stuck in the cell wall and are not secreted. In the case of the CGTase, the high molecular weight of 70 kDa apparently limits cell wall passage, and in the case of GFP (25 kDa) secretion is not prevented by the value, but obviously by electrostatic interactions between GFP and the cell wall. In $\textit{C. glutamicum}$ and $\textit{B. subtilis}$, in contrast, it was shown that GFP and also another substrate (MalE) are secreted with high yield in the Tat-dependent manner. A localization of the proteins has revealed that only a small amount is stuck in the cell wall here and that, consequently, the interactions between GFP or MalE and the cell wall of $\textit{B. subtilis}$ and $\textit{C. glutamicum}$ are obviously not as strong as in the case of GFP in $\textit{S. carnosus}$. Moreover, a comparison of the Tat- and Sec-dependent secretion of MalE in $\textit{C glutamicum}$ has shown that a clearly higher yield is achieved via the Tat pathway. This is apparently a consequence of the higher stability of the MalE protein exported via the Tat pathway. Furthermore, it has been demonstrated by the observation that GFP is secreted in $\textit{C. glutamicum}$ in the active form that the Tat pathway might be used above all as a secretion pathway for those proteins which like GFP require cytosolic folding factors and thus cannot be secreted in the active form via the Sec pathway. A Tat-dependent secretion of GFP in $\textit{B. subtilis}$ and $\textit{C. glutamicum}$ was mediated both with heterologous and with homologous Tat signal peptides. Depending on the fusion protein used, however, different yields were achieved, for which apparently the stability of the respective mRNA and/or of the fusion protein is decisive. Moreover, it has also been shown in $\textit{C. glutamicum}$ that the medium used also has a great influence on yield, and it is also assumed here that the stability of the mRNA and/or the fusion protein, which varies depending on the medium, is responsible for this. The findings obtained make it clear that there is no general system for heterologous protein secretion, but that for each protein to be secreted it must first be verified according to the tool-box principle in what fusion, in what host and under what conditions the highest yields are achieved. Tat signal peptides in contrast to Sec signal peptides cannot be generally exchanged. It was thus also observed in this study that the Tat signal peptides of PhoD from $\textit{B. subtilis}$ and $\textit{C. glutamicum}$ obviously do not mediate an export of GFP in $\textit{E. coli}$ and $\textit{S. carnosus}$, which suggests species-specific differences in signal peptide recognition by the respective Tat translocases. This is not the case for the TorA signal peptide, since it can mediate a Tat-dependent export in all the organisms examined here. Apparently, the PhoD signal peptides exhibit properties which prevent a recognition by the Tat translocases in $\textit{E. coli}$ and $\textit{S. carnosus}$.

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