Coordinate transcription and physical linkage of domains in surfactin synthetase are not essential for proper assembly and activity of the multienzyme complex.
Bacterial peptide synthetases have two common features that appear to be strictly conserved. 1) The enzyme subunits are co-regulated at both transcriptional and translational level. 2) The organization of the different enzymatic domains constituting the enzyme fulfills the "colinearity rule" according to which the order of the domains along the chromosome parallels their functional hierarchy. Considering the high degree of conservation of these features, one would expect that mutations such as transcription uncoupling and domain dissociations, deletions, duplications, and reshuffling would result in profound effects on the quality and quantity of synthesized peptides. To start testing this hypothesis, we designed two mutants. In one mutant, the operon structure of surfactin synthetase was destroyed, thus altering the concerted expression of the enzyme subunits. In the other mutant, the thioesterase domain naturally fused to the last amino acid binding domain of surfactin was physically dissociated and independently expressed. When the lipopeptides secreted by the mutant Bacillus subtilis strains were purified and characterized, they appeared to be expressed approximately at the same level of the wild type surfactin and to be identical to it, indicating that specific domain-domain interactions rather than coordinated transcription and translation play the major role in determining the correct assembly and activity of peptide synthetases.
Guenzi
E
E
Department of Molecular Biology, Chiron S.p.A., Via Fiorentina, 1 53100, Siena, Italy.
Galli
G
G
Grgurina
I
I
Pace
E
E
Ferranti
P
P
Grandi
G
G
D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008074:Lipoproteins / Q000032:analysis; D013058:Mass Spectrometry; D009154:Mutation / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D009876:Operon / Q000235:genetics; D010453:Peptide Synthases / Q000737:chemistry / Q000235:genetics*; D014176:Protein Biosynthesis / Q000235:genetics; D014158:Transcription, Genetic / Q000235:genetics
D001426:Bacterial Proteins; D008074:Lipoproteins; D010453:Peptide Synthases; C069542:surfactin synthetase