yersiniabactin and vibriobactin

The regulatory logic of siderophore biosynthetic genes in bacteria involves the universal repressor Fur, which acts together with iron as a negative regulator. However in other bacteria, in addition to the Fur-mediated mechanism of regulation, there is a concurrent positive regulation of iron transport and siderophore biosynthetic genes that occurs under conditions of iron deprivation. Despite these regulatory differences the mechanisms of siderophore biosynthesis follow the same fundamental enzymatic logic, which involves a series of elongating acyl-S-enzyme intermediates on multimodular protein assembly lines: nonribosomal peptide synthetases (NRPS). A substantial variety of siderophore structures are produced from similar NRPS assembly lines, and variation can come in the choice of the phenolic acid selected as the N-cap, the tailoring of amino acid residues during chain elongation, the mode of chain termination, and the nature of the capturing nucleophile of the siderophore acyl chain being released. Of course the specific parts that get assembled in a given bacterium may reflect a combination of the inventory of biosynthetic and tailoring gene clusters available. This modular assembly logic can account for all known siderophores. The ability to mix and match domains within modules and to swap modules themselves is likely to be an ongoing process in combinatorial biosynthesis. NRPS evolution will try out new combinations of chain initiation, elongation and tailoring, and termination steps, possibly by genetic exchange with other microorganisms and/or within the same bacterium, to create new variants of iron-chelating siderophores that can fit a particular niche for the producer bacterium.

Topics: Amino Acid Sequence; Bacteria; Catechols; Enterobactin; Genes, Bacterial; Molecular Sequence Data; Molecular Structure; Oxazoles; Peptides; Phenols; Sequence Homology, Amino Acid; Siderophores; Thiazoles

The recognition of carrier proteins by multiple catalytic partners occurs in every cycle of chain elongation in the biosynthesis of fatty acids and of the pharmacologically important polyketide and nonribosomal peptide natural products. To dissect the features of carrier proteins that determine specific recognition at distinct points in assembly lines, we have used the two-module Escherichia coli enterobactin synthetase as a model system. Using an entB knockout strain, we developed a selection for growth on iron-limiting medium to evolve aryl carrier protein domains. The aryl carrier proteins from VibB of Vibrio cholerae vibriobactin and HMWP2 of Yersinia pestis yersiniabactin assembly lines were evolved by random mutagenesis to support growth under selection conditions, yielding a convergent set of mutations. Subsequent in vitro biochemical characterizations with partner enzymes EntE, EntF, and Sfp on the evolved VibB aryl carrier protein revealed a approximately 500-fold improvement in reconstituted enterobactin production activity. Mechanistic characterization identified three distinct specific recognition surfaces of VibBArCP for three catalytic partners in enterobactin biosynthesis. Our results suggest that heterologous carrier protein interactions can be engineered with a small number of mutations given a suitable selection scheme and provide insights for reprogramming nonribosomal peptide biosynthesis.

Topics: Carrier Proteins; Catalysis; Catechols; Directed Molecular Evolution; Enterobactin; Escherichia coli Proteins; Humans; Ligases; Multienzyme Complexes; Mutagenesis, Site-Directed; Oxazoles; Phenols; Thiazoles; Vibrio cholerae; Yersinia pestis