RESUMEN
The tyrocidines and analogues are cationic cyclodecapeptides [cyclo (D-Phe1-L-Pro2-L-(Phe3/Trp3)-D-(Phe4/Trp4)-L-Asn5-L-Gln6-L-(Tyr7/Phe7/Trp7)-L-Val8-L-(Orn9/Lys9)-L-Leu10], produced together with the neutral linear pentadecapeptide gramicidins, in the antibiotic tyrothricin complex by Brevibacillus parabrevis. Despite discovery 80 years ago, it was still uncertain whether these peptides are secreted or sequestered intracellularly. We resolved this by utilising high resolution electrospray mass spectrometry to confirm the predominantly intracellular sequestration of the peptides in the tyrothricin complex. A "peptidomics" approach allowed us to map the intracellular production of 16 cyclodecapeptides and 6 gramicidins over 16 days of culturing. Gramicidin production remained relatively constant, with Val-gramicidin A the predominant analogue produced throughout the 16 day fermentation period. The tyrothricin cyclodecapeptides have four variable positions and there was a culturing time related shift from the Phe-rich A analogues, containing a L-Phe3-D-Phe4 aromatic dipeptide unit, to the Trp-rich C analogues with L-Trp3-D-Trp4. For the other variable aromatic residue position, Tyr7 was preferentially incorporated above Trp7, with a minor incorporation of Phe7 over the whole culturing period. For the variable basic amino acid residue, there was time-sensitive shift from Orn9 to Lys9 incorporation. Modulation of the cyclodecapeptide profile over time does not correlate with the reported non-ribosomal peptide synthetase affinity, specifically for Trp in the variable aromatic residue positions, indicating additional supply-demand control in the cyclodecapeptides production by B. parabrevis. These novel observations are not only of importance for production and purification of selected peptide analogues from the tyrothricin complex, but also for insight into microbial control of non-ribosomal peptide production that extends beyond the peptide synthetase machinery.
Asunto(s)
Antibacterianos/biosíntesis , Antibacterianos/química , Brevibacillus/crecimiento & desarrollo , Brevibacillus/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Tirotricina/biosíntesis , Tirotricina/química , Sustitución de Aminoácidos , Antibacterianos/metabolismo , Brevibacillus/citología , Fermentación , Factores de TiempoRESUMEN
When using small mole fraction increments to study gramicidins in phospholipid membranes, we found that the phasor dots of intrinsic fluorescence of gramicidin D and gramicidin A in dimyristoyl-sn-glycero-3-phosphocholine (DMPC) unilamellar and multilamellar vesicles exhibit a biphasic change with peptide content at 0.143 gramicidin mole fraction. To understand this phenomenon, we developed a statistical mechanical model of gramicidin/DMPC mixtures. Our model assumes a sludge-like mixture of fluid phase and aggregates of rigid clusters. In the fluid phase, gramicidin monomers are randomly distributed. A rigid cluster is formed by a gramicidin dimer and DMPC molecules that are condensed to the dimer, following particular stoichiometries (critical gramicidin mole fractions, Xcr including 0.143). Rigid clusters form aggregates in which gramicidin dimers are regularly distributed, in some cases, even to superlattices. At Xcr, the size of cluster aggregates and regular distributions reach a local maximum. Before a similar model was developed for cholesterol/DMPC mixtures (Sugar and Chong (2012) J. Am. Chem. Soc. 134, 1164â»1171) and here the similarities and differences are discussed between these two models.