BACKGROUND: Protein over-expression in bacteria is still the easiest, cheapest and therefore preferred way to obtain large amounts of proteins for industrial and laboratory scale preparations. Several studies emphasized the importance of understanding cellular and molecular mechanisms triggered by protein over-production in order to obtain higher yield and better quality of the recombinant product. Almost every step leading to a fully functional polypeptide has been investigated, from mRNA stability to the role of molecular chaperones, from aggregation to bottlenecks in the secretory pathway. In this context, we focused on the still poorly addressed relationship between protein production in the cytoplasm and the bacterial envelope, an active and reactive cell compartment that controls interactions with the environment and several major cellular processes. Results available to date show that the accumulation of foreign proteins in the cytoplasm induces changes in the membrane lipids and in the levels of mRNAs for some membrane proteins. However, a direct connection between membrane protein expression levels and soluble/aggregated protein accumulation in the cytoplasm has never been reported. RESULTS: By the use of a combined physiological and proteomic approach, we investigated the effects on the cell membrane of E. coli of the overexpression of two recombinant proteins, the B. cepacia lipase (BCL) and the green fluorescent protein (GFP). Both polypeptides are expressed in the cytoplasm at similar levels but GFP is fully soluble whereas inactive BCL accumulates in inclusion bodies. Growth and viability of the transformed cells were tested in the presence of different drugs. We found that chloramphenycol preferentially inhibited the strain over-producing GFP while SDS was more effective when BCL inclusion bodies accumulated in the cytoplasm. In contrast, both proteins induced a similar response in the membrane proteome, i.e. increased levels of LamB, OmpF, OmpA and TolC. Under all tested conditions, the lipopolysaccharide was not affected, suggesting that a specific rather than a generalized rearrangement of the envelope was induced. CONCLUSIONS: Taking together physiological and biochemical evidence, our work indicates that the E. coli envelope can sense protein over-expression in the cytoplasm and react by modulating the abundance of some membrane proteins, with possible consequences on the membrane traffic of small solutes, i.e. nutrients, drugs and metabolites. Such a response seems to be independent on the nature of the protein being over-expressed. On the other hand both our data reported herein and previous results indicate that membrane lipids may act as a second stress sensor responsive to the aggregation state of the recombinant protein and further contribute to changes in cellular exchanges with the environment.

Villa, R., Lotti, M., & Gatti Lafranconi, P. (2009). Components of the E coli envelope are affected by and can react to protein over-production in the cytoplasm. MICROBIAL CELL FACTORIES, 8(1), 32 [10.1186/1475-2859-8-32].

Components of the E coli envelope are affected by and can react to protein over-production in the cytoplasm

LOTTI, MARINA;
2009

Abstract

BACKGROUND: Protein over-expression in bacteria is still the easiest, cheapest and therefore preferred way to obtain large amounts of proteins for industrial and laboratory scale preparations. Several studies emphasized the importance of understanding cellular and molecular mechanisms triggered by protein over-production in order to obtain higher yield and better quality of the recombinant product. Almost every step leading to a fully functional polypeptide has been investigated, from mRNA stability to the role of molecular chaperones, from aggregation to bottlenecks in the secretory pathway. In this context, we focused on the still poorly addressed relationship between protein production in the cytoplasm and the bacterial envelope, an active and reactive cell compartment that controls interactions with the environment and several major cellular processes. Results available to date show that the accumulation of foreign proteins in the cytoplasm induces changes in the membrane lipids and in the levels of mRNAs for some membrane proteins. However, a direct connection between membrane protein expression levels and soluble/aggregated protein accumulation in the cytoplasm has never been reported. RESULTS: By the use of a combined physiological and proteomic approach, we investigated the effects on the cell membrane of E. coli of the overexpression of two recombinant proteins, the B. cepacia lipase (BCL) and the green fluorescent protein (GFP). Both polypeptides are expressed in the cytoplasm at similar levels but GFP is fully soluble whereas inactive BCL accumulates in inclusion bodies. Growth and viability of the transformed cells were tested in the presence of different drugs. We found that chloramphenycol preferentially inhibited the strain over-producing GFP while SDS was more effective when BCL inclusion bodies accumulated in the cytoplasm. In contrast, both proteins induced a similar response in the membrane proteome, i.e. increased levels of LamB, OmpF, OmpA and TolC. Under all tested conditions, the lipopolysaccharide was not affected, suggesting that a specific rather than a generalized rearrangement of the envelope was induced. CONCLUSIONS: Taking together physiological and biochemical evidence, our work indicates that the E. coli envelope can sense protein over-expression in the cytoplasm and react by modulating the abundance of some membrane proteins, with possible consequences on the membrane traffic of small solutes, i.e. nutrients, drugs and metabolites. Such a response seems to be independent on the nature of the protein being over-expressed. On the other hand both our data reported herein and previous results indicate that membrane lipids may act as a second stress sensor responsive to the aggregation state of the recombinant protein and further contribute to changes in cellular exchanges with the environment.
Articolo in rivista - Articolo scientifico
protein overexpression, stress response, bacterial envelope
English
32
Villa, R., Lotti, M., & Gatti Lafranconi, P. (2009). Components of the E coli envelope are affected by and can react to protein over-production in the cytoplasm. MICROBIAL CELL FACTORIES, 8(1), 32 [10.1186/1475-2859-8-32].
Villa, R; Lotti, M; Gatti Lafranconi, P
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10281/6056
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