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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.

No MeSH data available.


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Multiple sequence alignments of (A) two HtrA-type membrane-bound quality control proteases Bmg4479 and Bmg4817 from B. megaterium and the HtrA (BSORF: BG12608) and HtrB (BSORF: BG14155) proteins from B. subtilis; (B) two peptidoglycan-binding proteins Bmg4247 and Bmg0510 from B. megaterium and the YocH (BSORF: BG13521) protein from B. subtilis.
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Figure 11: Multiple sequence alignments of (A) two HtrA-type membrane-bound quality control proteases Bmg4479 and Bmg4817 from B. megaterium and the HtrA (BSORF: BG12608) and HtrB (BSORF: BG14155) proteins from B. subtilis; (B) two peptidoglycan-binding proteins Bmg4247 and Bmg0510 from B. megaterium and the YocH (BSORF: BG13521) protein from B. subtilis.

Mentions: As shown in Figure 5, a protein spot was identified to be a homologue of both HtrA and HtrB proteins from B. subtilis, showing 50% and 48% sequence identity, respectively. This protein is designated as Bmg4479 in the strain-specific protein database "bmegMEC.v2". We found another homologous protein presenting in "bmegMEC.v2" as Bmg4817 which has 43% and 42% sequence identity to the B. subtilis HtrA and HtrB proteins, respectively. The multiple sequence alignment shown in Figure 11a reveals sequence consensus in the middle region and in the C-termini. The sequence of Bmg4479 is apparently incomplete in its N-termini. Since the coding sequence is at the end of a contig, the missing part is obviously not yet sequenced. Thus, B. megaterium seems also to have two HtrA-type proteins. Bmg4817 was not among the protein spots identified in this study. Probably its expression was suppressed by Bmg4479, since it has been reported that transcriptions of the homologous genes htrA and htrB in B. subtilis are negatively cross-regulated [32,33].


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)

Multiple sequence alignments of (A) two HtrA-type membrane-bound quality control proteases Bmg4479 and Bmg4817 from B. megaterium and the HtrA (BSORF: BG12608) and HtrB (BSORF: BG14155) proteins from B. subtilis; (B) two peptidoglycan-binding proteins Bmg4247 and Bmg0510 from B. megaterium and the YocH (BSORF: BG13521) protein from B. subtilis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 11: Multiple sequence alignments of (A) two HtrA-type membrane-bound quality control proteases Bmg4479 and Bmg4817 from B. megaterium and the HtrA (BSORF: BG12608) and HtrB (BSORF: BG14155) proteins from B. subtilis; (B) two peptidoglycan-binding proteins Bmg4247 and Bmg0510 from B. megaterium and the YocH (BSORF: BG13521) protein from B. subtilis.
Mentions: As shown in Figure 5, a protein spot was identified to be a homologue of both HtrA and HtrB proteins from B. subtilis, showing 50% and 48% sequence identity, respectively. This protein is designated as Bmg4479 in the strain-specific protein database "bmegMEC.v2". We found another homologous protein presenting in "bmegMEC.v2" as Bmg4817 which has 43% and 42% sequence identity to the B. subtilis HtrA and HtrB proteins, respectively. The multiple sequence alignment shown in Figure 11a reveals sequence consensus in the middle region and in the C-termini. The sequence of Bmg4479 is apparently incomplete in its N-termini. Since the coding sequence is at the end of a contig, the missing part is obviously not yet sequenced. Thus, B. megaterium seems also to have two HtrA-type proteins. Bmg4817 was not among the protein spots identified in this study. Probably its expression was suppressed by Bmg4479, since it has been reported that transcriptions of the homologous genes htrA and htrB in B. subtilis are negatively cross-regulated [32,33].

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