Functional analysis of the response regulator DegU in Bacillus megaterium DSM319 and comparative secretome analysis of degSU mutants

• Published in: Applied microbiology and biotechnology Vol. 91; no. 3; pp. 699 - 711
• Main Authors: Borgmeier, Claudia, Voigt, Birgit, Hecker, Michael, Meinhardt, Friedhelm
• Format: Journal Article
• Language: English
• Published: Berlin/Heidelberg Springer-Verlag 01.08.2011; Springer; Springer Nature B.V
• Subjects: Amino Acid Sequence; Amylases; Amylases - genetics; Amylases - metabolism; Analysis; Antibiotics; Applied Genetics and Molecular Biotechnology; Bacillus; Bacillus licheniformis; Bacillus megaterium; Bacillus megaterium - genetics; Bacillus megaterium - metabolism; Bacillus subtilis; Bacillus subtilis - genetics; Bacillus subtilis - metabolism; Bacteria; Bacterial Proteins; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological and medical sciences; Biomedical and Life Sciences; biosynthesis; Biotechnology; Cell culture; cell walls; Chemical synthesis; Cloning; culture media; DNA polymerase; E coli; Enzymes; Extracellular Matrix Proteins; Fundamental and applied biological sciences. Psychology; Gene Knockout Techniques; genetics; Hexosyltransferases; Hexosyltransferases - biosynthesis; Hexosyltransferases - genetics; hypersecretion; levansucrase; Life Sciences; Mass Spectrometry; metabolism; Microbial Genetics and Genomics; Microbiology; Mutants; Mutation; Peptide Hydrolases; Peptide Hydrolases - genetics; Peptide Hydrolases - metabolism; phenotype; Phosphorylation; Plasmids; Polymerase Chain Reaction; Polypeptides; Protein Sorting Signals; Protein Sorting Signals - genetics; proteinases; Proteins; proteolysis; Proteome; Proteomics; Sequence Alignment; Sequence Deletion; Sequence Deletion - genetics; Studies; Two-component system; Extracellular proteome; DegSU; Hypersecretion; Degradative enzymes; Enzyme; Proteome; Functional analysis; Regulator; Bacillaceae; Extracellular; Bacillales; Bacteria; Mutation; Bacillus megaterium; Comparative study
• DOI: 10.1007/s00253-011-3302-7
• ISSN: 0175-7598; 1432-0614

We functionally analysed the two-component regulatory system DegSU (historically SacU) in Bacillus megaterium DSM319 by generating a genetic knock out as well as a sacU32 mutation. The latter—known to cause a hypersecretion phenotype in Bacillus subtilis —had no influence on extracellular protease and amylase activity in B. megaterium . Since the B. megaterium DegU complemented a Bacillus licheniformis ∆ degSU mutant, functionality of the protein was proven. Expression of the sacB encoded levansucrase was found to be dependent on DegSU in B. megaterium . Consistently, the fusion of the sacB promoter to gfp revealed a strong increase in GFP-expression in the sacU32 strain. On 2 D-gels of the secretome, a large number of intracellular proteins was seen. The culture medium contained only 42 secreted proteins which can be assigned to polypeptides involved in the metabolism of the cell wall, polypeptides with proteolytic activities and those with unknown functions. Though overall protease activity matches with the wild type, two proteolytic enzymes (Vpr and YwaD) are missing in the secretome of the ∆ degSU strain, while other degradative enzymes are not affected. In line with such findings, no increase of proteolytic or other degradative enzymes was seen in the sacU32 mutant. Thus, compared to B. subtilis and B. licheniformis , the number of extracellular proteins influenced by DegSU is surprisingly low in B. megaterium , a feature, probably advantageous as to the use of the sacU32 mutant for production of secreted proteins.