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Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase.

Wang W, Hollmann R, Deckwer WD - Proteome Sci (2006)

Bottom Line: High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected.The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320.At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemical Engineering, Technical University Braunschweig, GBF/TU-BCE, Mascheroder Weg 1, D-38124 Braunschweig, Germany. wwa@gbf.de

ABSTRACT
High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320), both carrying a heterologous dextransucrase (dsrS) gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction) the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available. High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses. The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the mutated genes in WH320 in addition to the known lacZ. In view of the results of this proteomic study it seems that at high cell density conditions and hence low growth rates MS941, in contrast to WH320, does not maintain a vegetative growth which is essential for the expression of the foreign dsrS gene by using the xylA promoter. It is conceivable that applications of a promoter which is highly active under nutrient-limited cultivation conditions is necessary, at least for MS941, for the overexpression of recombinant genes in such B. megaterium fed-batch cultivation process. However to obtain a heterologous protein in secreted and properly folded form stills remains a big challenge.

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Expression profiles of the preprotein translocae SecA subunit, the membrane-anchored ATP-dependent protease FtsH and Bmg4479, a HtrA-type membrane-bound quality control protease.
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Figure 10: Expression profiles of the preprotein translocae SecA subunit, the membrane-anchored ATP-dependent protease FtsH and Bmg4479, a HtrA-type membrane-bound quality control protease.

Mentions: As depicted in Figure 10, for WH320 SecA level was relatively constant within the experimental error range for the first three samples. It reduced then to a certain extent during the time of high-level DsrS expression (see Table 1). According to its signal peptide sequence DsrS is destined to be translocated via the SecA-dependent pathway. The result indicated that DsrS overexpression did not bring about a concomitant increase but rather, if any, a decrease in SecA expression. Similar to the general heterologous protein secretion problems associated with Bacillus species, significant accumulation of DsrS in the cytoplasm was identified for WH320 in high cell density cultivation [[6], and this work]. This could lead to secretion stresses at the cell membrane and might, in return, pose an obstacle to SecA expression. It has been reported that high-level expression of a heterologous α-amylase in B. subtilis resulted in a massive accumulation of the α-amylase precursor in the cytoplasm and did not lead to an increase in the amount of SecA [25].


Comparative proteomic analysis of high cell density cultivations with two recombinant Bacillus megaterium strains for the production of a heterologous dextransucrase.

Wang W, Hollmann R, Deckwer WD - Proteome Sci (2006)

Expression profiles of the preprotein translocae SecA subunit, the membrane-anchored ATP-dependent protease FtsH and Bmg4479, a HtrA-type membrane-bound quality control protease.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1622742&req=5

Figure 10: Expression profiles of the preprotein translocae SecA subunit, the membrane-anchored ATP-dependent protease FtsH and Bmg4479, a HtrA-type membrane-bound quality control protease.
Mentions: As depicted in Figure 10, for WH320 SecA level was relatively constant within the experimental error range for the first three samples. It reduced then to a certain extent during the time of high-level DsrS expression (see Table 1). According to its signal peptide sequence DsrS is destined to be translocated via the SecA-dependent pathway. The result indicated that DsrS overexpression did not bring about a concomitant increase but rather, if any, a decrease in SecA expression. Similar to the general heterologous protein secretion problems associated with Bacillus species, significant accumulation of DsrS in the cytoplasm was identified for WH320 in high cell density cultivation [[6], and this work]. This could lead to secretion stresses at the cell membrane and might, in return, pose an obstacle to SecA expression. It has been reported that high-level expression of a heterologous α-amylase in B. subtilis resulted in a massive accumulation of the α-amylase precursor in the cytoplasm and did not lead to an increase in the amount of SecA [25].

Bottom Line: High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected.The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320.At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biochemical Engineering, Technical University Braunschweig, GBF/TU-BCE, Mascheroder Weg 1, D-38124 Braunschweig, Germany. wwa@gbf.de

ABSTRACT
High cell density cultivations were performed under identical conditions for two Bacillus megaterium strains (MS941 and WH320), both carrying a heterologous dextransucrase (dsrS) gene under the control of the xylA promoter. At characteristic points of the cultivations (end of batch, initial feeding, before and after induction) the proteome was analyzed based on two dimensional gel electrophoresis and mass spectrometric protein identification using the protein database "bmegMEC.v2" recently made available. High expression but no secretion of DsrS was found for the chemical mutant WH320 whereas for MS 941, a defined protease deficient mutant of the same parent strain (DSM319), not even expression of DsrS could be detected. The proteomic analysis resulted in the identification of proteins involved in different cellular pathways such as in central carbon and overflow metabolism, in protein synthesis, protein secretion and degradation, in cell wall metabolism, in cell division and sporulation, in membrane transport and in stress responses. The two strains exhibited considerable variations in expression levels of specific proteins during the different phases of the cultivation process, whereas induction of DsrS production had, in general, little effect. The largely differing behaviour of the two strains with regard to DsrS expression can be attributed, at least in part, to changes observed in the proteome which predominantly concern biosynthetic enzymes and proteins belonging to the membrane translocation system, which were strongly down-regulated at high cell densities in MS941 compared with WH320. At the same time a cell envelope-associated quality control protease and two peptidoglycan-binding proteins related to cell wall turnover were strongly expressed in MS941 but not found in WH320. However, to further explain the very different physiological responses of the two strains to the same cultivation conditions, it is necessary to identify the mutated genes in WH320 in addition to the known lacZ. In view of the results of this proteomic study it seems that at high cell density conditions and hence low growth rates MS941, in contrast to WH320, does not maintain a vegetative growth which is essential for the expression of the foreign dsrS gene by using the xylA promoter. It is conceivable that applications of a promoter which is highly active under nutrient-limited cultivation conditions is necessary, at least for MS941, for the overexpression of recombinant genes in such B. megaterium fed-batch cultivation process. However to obtain a heterologous protein in secreted and properly folded form stills remains a big challenge.

No MeSH data available.


Related in: MedlinePlus