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Elucidation of the substrate specificity of the ABC transporter Tba uncovers an unsuspected role in the biosynthesis of glycopeptide antibiotics

ABSTRACT: Glycopeptide antibiotics (GPA) such as vancomycin and teicoplanin are essential last-resort antibiotics produced by actinomycetes. Their biosynthesis is encoded within biosynthetic gene clusters, also harboring genes for regulation, and transport. Diverse types of GPAs have been characterized that differ in peptide backbone composition and modification patterns. However, little is known about the ATP-binding cassette (ABC) transporters facilitating GPA export. Employing cutting-edge techniques, we investigated the substrate specificity of GPA ABC-transporters towards the type-I GPA, balhimycin. Phylogenetic analysis and trans-complementation experiments revealed that balhimycin can only be exported by the related type I GPA transporters Tba and Tva (transporter of vancomycin). Molecular dynamics simulations and mutagenesis experiments showed that Tba exhibits specificity towards the peptide backbone rather than the attached modifications. Unexpectedly, deletion or functional inactivation of Tba halted balhimycin biosynthesis. Combined with proximity dependent biotinylation experiments this suggested that the interaction of the active transporter with the biosynthetic machinery is required for compound production.

ORGANISM(S): Amycolatopsis balhimycina

PROVIDER: GSE274067 | GEO | 2025/04/09

REPOSITORIES: GEO

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Project description:The common research strain S. aureus NCTC 8325-4 was subjected to serial passage in increasing concentrations of ramoplanin. The resulting strain, RSPA16, had reduced susceptibility to not only ramoplanin but vancomycin and nisin as well. Electron microscopy revealed that the cell wall of RSPA16 was double the thickness of the susceptible progenitor strain, a phenotype commonly observed in vancomycin intermediate resistant S. aureus (VISA) strains. RSPA16 was also less susceptible to lysis induced by Triton X-100 than its progenitor strain. Transcriptional profiling experiments were performed with NCTC 8325-4 and RSPA16 without antibiotic exposure and exposed to ramoplanin. Increased expression of genes associated with cell wall stress was observed when either strain was treated with ramoplanin. We also observed that treatment with ramoplanin altered of the expression levels of numerous genes encoding proteins involved with amino acid biosynthesis, central metabolic pathways, nucleotide biosynthesis, iron acquisition, ABC transporters and regulation of transcription. Comparison of the transcriptional profiles of RSPA16 and NCTC 8325-4 not exposed to ramoplanin revealed alterations in the expression of levels of several genes involved with biosynthesis of teichoic acids, biosynthesis of peptidoglycan, central metabolism, DNA replication, nucleotide biosynthesis, iron acquisition, ABC transporters, and regulation of transcription. These transcriptional profiles provide insights into the possible sources of the reduced susceptibility of RSPA16 to peptide antibiotics. Mid log phase (OD620 = 0.4) batch TSB cultures of ramoplanin susceptible (NCTC 8325-4) and resistant (RSPA16) strains of S. aureus were exposed to ramoplanin for 30 minutes at 37 C. Untreated controls were performed for each strain as well. The transcriptional response was determined using Affymetrix Gene Chip Arrays. This study also examines the transcriptional alterations which confer reduced susceptibility to ramoplanin as the transcriptional profiles of NCTC 8325-4 and RSPA16 each untreated were compared.

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Elucidation of the substrate specificity of the ABC transporter Tba uncovers an unsuspected role in the biosynthesis of glycopeptide antibiotics

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