Proteome and transcriptome based analysis of Bacillus subtilis cells overproducing an insoluble heterologous protein.
Bacillus subtilis and related Bacillus species are frequently used as hosts for the industrial production of recombinant proteins. In this study the cellular response of B. subtilis to the overproduction of an insoluble heterologous protein was investigated. For this purpose PorA, an outer membrane protein from Neisseria meningitidis, which accumulates after overexpression in the cytoplasm of B. subtilis mainly in the form of inclusion bodies, was used. The molecular response to overexpression of porA has been analysed at the transcriptional level using the DNA macro array technique and at the translational level by two-dimensional polyacrylamide gel electrophoresis. It was found that the expression of the heat shock genes of class I (dnaK, groEL and grpE) and class III (clpP and clpC) are increased under overproducing conditions. Furthermore, the protein levels of the two ribosomal proteins RpsB and RplJ are increased in the PorA overproducing cells. The transcriptome analysis indicated that mRNA levels of genes encoding pyrimidine and purine synthesis enzymes but also from ribosomal protein genes have elevated levels under overproducing conditions. Finally, the association of the protease ClpP and its ATPase subunits ClpC and ClpX with the PorA inclusion bodies was demonstrated by means of the immunogold labelling technique.
Jürgen
B
B
Institute of Microbiology, Ernst-Moritz-Arndt-University, Greifswald, Germany.
Hanschke
R
R
Sarvas
M
M
Hecker
M
M
Schweder
T
T
D000251:Adenosine Triphosphatases / Q000096:biosynthesis; D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000096:biosynthesis*; D015153:Blotting, Western; D049071:Endopeptidase Clp; D006360:Heat-Shock Proteins / Q000096:biosynthesis* / Q000235:genetics; D002479:Inclusion Bodies / Q000378:metabolism; D018832:Molecular Chaperones; D009344:Neisseria gonorrhoeae / Q000737:chemistry / Q000235:genetics; D018272:Porins / Q000096:biosynthesis / Q000235:genetics; D020543:Proteome / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis; D012269:Ribosomal Proteins / Q000096:biosynthesis; D012697:Serine Endopeptidases / Q000096:biosynthesis
D001426:Bacterial Proteins; D006360:Heat-Shock Proteins; D018832:Molecular Chaperones; D018272:Porins; D020543:Proteome; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C057699:porin protein, Neisseria; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C115251:DnaK protein, Brevibacillus choshinensis