Dataset Information

Antimicrobial peptide resistance of Vibrio cholerae results from an LPS modification pathway related to nonribosomal peptide synthetases.

ABSTRACT: The current pandemic El Tor biotype of O1 Vibrio cholerae is resistant to polymyxins, whereas the previous pandemic strain of the classical biotype is polymyxin sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to polymyxins through the glycylation of lipopolysaccharide. Here, we present the mechanistic determination of initial steps in the AlmEFG pathway. We verify that AlmF is an aminoacyl carrier protein and identify AlmE as the enzyme required to activate AlmF as a functional carrier protein. A combination of structural information and activity assays was used to identify a pair of active site residues that are important for mediating AlmE glycine specificity. Overall, the structure of AlmE in complex with its glycyl-adenylate intermediate reveals that AlmE is related to Gram-positive d-alanine/d-alanyl carrier protein ligase, while the trio of proteins in the AlmEFG system forms a chemical pathway that resembles the division of labor in nonribosomal peptide synthetases.

SUBMITTER: Henderson JC

PROVIDER: S-EPMC4520716 | biostudies-literature | 2014 Oct

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Antimicrobial peptide resistance of Vibrio cholerae results from an LPS modification pathway related to nonribosomal peptide synthetases.

Henderson Jeremy C JC Fage Christopher D CD Cannon Joe R JR Brodbelt Jennifer S JS Keatinge-Clay Adrian T AT Trent M Stephen MS

ACS chemical biology 20140818 10

The current pandemic El Tor biotype of O1 Vibrio cholerae is resistant to polymyxins, whereas the previous pandemic strain of the classical biotype is polymyxin sensitive. The almEFG operon found in El Tor V. cholerae confers >100-fold resistance to polymyxins through the glycylation of lipopolysaccharide. Here, we present the mechanistic determination of initial steps in the AlmEFG pathway. We verify that AlmF is an aminoacyl carrier protein and identify AlmE as the enzyme required to activate ...[more]

PMID: 25068415

Similar Datasets

Project description:A 7.5-kbp fragment of chromosomal DNA downstream of the Vibrio cholerae vibriobactin outer membrane receptor, viuA, and the vibriobactin utilization gene, viuB, was recovered from a Sau3A lambda library of O395 chromosomal DNA. By analogy with the genetic organization of the Escherichia coli enterobactin gene cluster, in which the enterobactin biosynthetic and transport genes lie adjacent to the enterobactin outer membrane receptor, fepA, and the utilization gene, fes, the cloned DNA was examined for the ability to restore siderophore synthesis to E. coli ent mutants. Cross-feeding studies demonstrated that an E. coli entF mutant complemented with the cloned DNA regained the ability to synthesize enterobactin and to grow in low-iron medium. Sequence analysis of the cloned chromosomal DNA revealed an open reading frame downstream of viuB which encoded a deduced protein of greater than 2,158 amino acids, homologous to Yersinia sp. HMWP2, Vibrio anguillarum AngR, and E. coli EntF. A mutant with an in-frame deletion of this gene, named vibF, was created with classical V. cholerae strain O395 by in vivo marker exchange. In cross-feeding studies, this mutant was unable to synthesize ferric vibriobactin but was able to utilize exogenous siderophore. Complementation of the mutant with a cloned vibF fragment restored vibriobactin synthesis to normal. The expression of the vibF promoter was found to be negatively regulated by iron at the transcriptional level, under the control of the V. cholerae fur gene. Expression of vibF was not autoregulatory and neither affected nor was affected by the expression of irgA or viuA. The promoter of vibF was located by primer extension and was found to contain a dyad symmetric nucleotide sequence highly homologous to the E. coli Fur binding consensus sequence. A footprint of purified V. cholerae Fur on the vibF promoter, overlapping the Fur binding consensus sequence, was observed using DNase I footprinting. The protein product of vibF is homologous to the multifunctional nonribosomal protein synthetases and is necessary for the biosynthesis of vibriobactin.

Project description:BackgroundNonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (NPS) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including alpha-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi.ResultsPhylogenomic analysis identified nine major subfamilies of fungal NRPSs which fell into two main groups: one corresponds to NPS genes encoding primarily mono/bi-modular enzymes which grouped with bacterial NRPSs and the other includes genes encoding primarily multimodular and exclusively fungal NRPSs. AARs shared a closer phylogenetic relationship to NRPSs than to other acyl-adenylating enzymes. Phylogenetic analyses and taxonomic distribution suggest that several mono/bi-modular subfamilies arose either prior to, or early in, the evolution of fungi, while two multimodular groups appear restricted to and expanded in fungi. The older mono/bi-modular subfamilies show conserved domain architectures suggestive of functional conservation, while multimodular NRPSs, particularly those unique to euascomycetes, show a diversity of architectures and of genetic mechanisms generating this diversity.ConclusionsThis work is the first to characterize subfamilies of fungal NRPSs. Our analyses suggest that mono/bi-modular NRPSs have more ancient origins and more conserved domain architectures than most multimodular NRPSs. It also demonstrates that the alpha-aminoadipate reductases involved in lysine biosynthesis in fungi are closely related to mono/bi-modular NRPSs. Several groups of mono/bi-modular NRPS metabolites are predicted to play more pivotal roles in cellular metabolism than products of multimodular NRPSs. In contrast, multimodular subfamilies of NRPSs are of more recent origin, are restricted to fungi, show less stable domain architectures, and biosynthesize metabolites which perform more niche-specific functions than mono/bi-modular NRPS products. The euascomycete-only NRPS subfamily, in particular, shows evidence for extensive gain and loss of domains suggestive of the contribution of domain duplication and loss in responding to niche-specific pressures.

Dataset Information

Antimicrobial peptide resistance of Vibrio cholerae results from an LPS modification pathway related to nonribosomal peptide synthetases.

Publications

Antimicrobial peptide resistance of Vibrio cholerae results from an LPS modification pathway related to nonribosomal peptide synthetases.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets