Manipulation of thiocillin variants by prepeptide gene replacement: structure, conformation, and activity of heterocycle substitution mutants

Abstract

Bacillus cereus ATCC 14579 converts the C-terminal 14 residues of a 52-mer prepeptide into a related set of eight variants of the thiocillin subclass of thiazolyl peptide antibiotics by a cascade of post-translational modifications that alter 13 of those 14 residues. We have introduced prepeptide gene variants into a knockout strain to conduct an alanine scan of all 14 progenitor residues, as well as a serine scan of the six cysteine residues that are converted to thiazoles in the mature natural product. No mature scaffolds were detected for the S1A and S10A mutants, consistent with their roles as the source of the pyridine core. In both the alanine and serine scans, only one substitution mutant failed to produce a mature scaffold: cysteine 11. Cysteine to serine mutants gave mixture of dehydrations, aromatizations, and unaltered alcohol side chains depending on position. Overall, substitutions that altered the trithiazolylpyridine core or reduced the conformational rigidity of the 26-membered macrocyclic loop led to loss of antibiotic activity. In total, 21 peptide mutants were cultured, from which production of 107 compounds was observed and 94 compounds, representing 17 structural mutants, were assayed for antibiotic activity. High-resolution NMR solution structures were determined for one mutant and one wild-type compound. These structures demonstrate that the tight conformational rigidity of the natural product is severely disrupted by loss of even a single heterocycle, perhaps accounting for the attendant loss of activity in such mutants.

Figure 1

Examples of thiazolyl peptide antibiotics: Micrococcin P1 (26-member macrocycle), Nosiheptide (26-member macrocycle + 13-member bridge), Thiostrepton (26-member macrocycle + 27-member B-ring), GE2270A (29-member macrocycle), and Berninamycin A (35-member macrocycle).

Figure 2

Gene cluster from B. cereus ATCC 14579 responsible for posttranslational biosynthesis of the thiocillins from a 14 residue prepeptide with a 38 residue leader sequence.

Figure 3

Mechanisms of (A) dehydrobutyrine formation via phosphorylation/elimination as effected by lantibiotic dehydratases; (B) thiazoline formation via cyclodehydration/oxidation and subsequent dehydrogenation to thiazole as with the MccB17 synthase complex; and (C) pyridine formation via putative enzyme-catalyzed [4+2] cycloaddition.

Figure 4

Schematic representation of knockout/knock-in strategy. (A) Excision of four structural gene copies via homologous recombination with a plasmid containing two areas of homology. (B) Knockout strain (exhibiting a 6 base pair scar from the initial knockout) is combined with a second plasmid baring a single area of homology and a single variant copy of the structural gene TclE. (C) Product variant strain is cultured and thiocillins isolated (pictured are comparative HPLC traces of isolates from the wild-type, the knockout, and a wild-type rescue).

Figure 5

Common MS/MS fragmentation pathways observed for Thiocillin variants: (A) C-terminus and (B) between threonines 3 and 4 of the macrocycle subunit. Exempletive mass spectrum of Micrococcin P2 with labeled fragment peaks.

Figure 6

Schematic representation of thiocillin SAR. Substitution of residues backed by red panels result in loss of antibiotic production, while those backed in yellow sustain production but lose activity. Substitutions at positions illustrated in green allow production of active antibiotic analogues.

Figure 7

Anticipated product states of mutant serine side-chains: a) unmodified alcohol, b) dehydroAlanine (dhA), c) oxazoline, and d) oxazole. Sp² centers and non-rotatable bonds are illustrated in red.

Figure 8

HPLC traces (350 nm) of methanolic extracts from 3 day growths of B. cereus C2S mutant. Structures of core regions as determined by LC/MS, MS/MS, and 1D and 2D NMR are depicted adjacent to the individually isolated peaks.

Figure 9

NMR solution phase structures (DMSO-d⁶) of a C2S-alcohol mutant (A and C) and wild-type thiocillin III (B and D). The amide proton of the “bent” amide in thiocillin III is illustrated in white.