Maturation of the lantibiotic subtilin: matrix-assisted laser desorption/ionization time-of-flight mass spectrometry to monitor precursors and their proteolytic processing in crude bacterial cultures.
Bacillus subtilis synthesizes the lanthionine containing 32-amino-acid peptide antibiotic (lanti-biotic) subtilin from a ribosomally generated 56-amino-acid precursor pre-propeptide by extensive posttranslational modifications. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to monitor the production of matured subtilin within crude samples taken from B. subtilis culture media without prior fractionation. The processing reaction of subtilin was blocked with the serine protease inhibitor phenylmethylsulfonyl fluoride and different subtilin precursor peptides in the molecular mass range up to 6220 were observed. Two of these species were isolated by reversed-phase high-performance liquid chromatography (HPLC) and structurally analyzed by post-source decay MALDI-TOFMS. We provide evidence that the precursor species comprise the posttranslational modified C-terminal part of subtilin to which leader peptide moieties with different chain lengths are attached. These antimicrobial-inactive species could be processed to antibiotic-active subtilin by incubation with culture media of different subtilin-nonproducing B. subtilis strains as indicated by a combination of antimicrobial growth assays and MALDI-TOFMS analyses. These achievements are strong evidence for the sensitivity of MALDI-TOFMS methodology that allows straightforward investigations of analytes even in complex mixtures without time-consuming sample preparations.
Stein
Torsten
T
Institut für Mikrobiologie, Johann Wolfgang Goethe-Universität, Marie-Curie-Str. 9, D-60439 Frankfurt/M, Germany. T.Stein@em.uni-frankfurt.de
Entian
Karl-Dieter
KD
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins; D001430:Bacteriocins; D003470:Culture Media; D017077:Culture Media, Conditioned; D010450:Endopeptidases / Q000378:metabolism*; D008969:Molecular Sequence Data; D010455:Peptides; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D001430:Bacteriocins; D003470:Culture Media; D017077:Culture Media, Conditioned; D010455:Peptides; D011480:Protease Inhibitors; D011498:Protein Precursors; D010664:Phenylmethylsulfonyl Fluoride; D010450:Endopeptidases; C002836:subtilin