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Identification of the biosynthetic gene cluster for the pacidamycin group of peptidyl nucleoside antibiotics.

ABSTRACT: Pacidamycins are a family of uridyl tetra/pentapeptide antibiotics that act on the translocase MraY to block bacterial cell wall assembly. To elucidate the biosynthetic logic of pacidamcyins, a putative gene cluster was identified by 454 shotgun genome sequencing of the producer Streptomyces coeruleorubidus NRRL 18370. The 31-kb gene cluster encodes 22 proteins (PacA-V), including highly dissociated nonribosomal peptide synthetase (NRPS) modules and a variety of tailoring enzymes. Gene deletions confirmed that two NRPSs, PacP and PacO, are required for the biosynthesis of pacidamycins. Heterologous expression and in vitro assays of PacL, PacO, and PacP established reversible formation of m-Tyr-AMP, l-Ala-AMP, and diaminopropionyl-AMP, respectively, consistent with the amino acids found in pacidamycin scaffolds. The unusual Ala(4)-Phe(5) dipeptidyl ureido linkage was formed during in vitro assays containing purified PacL, PacJ, PacN, and PacO. Both the genetic and enzymatic studies validate identification of the biosynthetic genes for this subclass of uridyl peptide antibiotics and provide the basis for future mechanistic study of their biosynthesis.

SUBMITTER: Zhang W

PROVIDER: S-EPMC2947877 | biostudies-literature | 2010 Sep

REPOSITORIES: biostudies-literature

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Identification of the biosynthetic gene cluster for the pacidamycin group of peptidyl nucleoside antibiotics.

Zhang Wenjun W Ostash Bohdan B Walsh Christopher T CT

Proceedings of the National Academy of Sciences of the United States of America 20100908 39

Pacidamycins are a family of uridyl tetra/pentapeptide antibiotics that act on the translocase MraY to block bacterial cell wall assembly. To elucidate the biosynthetic logic of pacidamcyins, a putative gene cluster was identified by 454 shotgun genome sequencing of the producer Streptomyces coeruleorubidus NRRL 18370. The 31-kb gene cluster encodes 22 proteins (PacA-V), including highly dissociated nonribosomal peptide synthetase (NRPS) modules and a variety of tailoring enzymes. Gene deletions ...[more]

PMID: 20826445

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Project description:UnlabelledAnalysis of the genome sequence of Pseudomonas aeruginosa PA14 revealed the presence of an operon encoding an ABC-type transporter (NppA1A2BCD) showing homology to the Yej transporter of Escherichia coli. The Yej transporter is involved in the uptake of the peptide-nucleotide antibiotic microcin C, a translation inhibitor that targets the enzyme aspartyl-tRNA synthetase. Furthermore, it was recently shown that the Opp transporter from P. aeruginosa PAO1, which is identical to Npp, is required for uptake of the uridyl peptide antibiotic pacidamycin, which targets the enzyme translocase I (MraY), which is involved in peptidoglycan synthesis. We used several approaches to further explore the substrate specificity of the Npp transporter. Assays of growth in defined minimal medium containing peptides of various lengths and amino acid compositions as sole nitrogen sources, as well as Biolog Phenotype MicroArrays, showed that the Npp transporter is not required for di-, tri-, and oligopeptide uptake. Overexpression of the npp operon increased susceptibility not just to pacidamycin but also to nickel chloride and the peptidyl nucleoside antibiotic blasticidin S. Furthermore, heterologous expression of the npp operon in a yej-deficient mutant of E. coli resulted in increased susceptibility to albomycin, a naturally occurring sideromycin with a peptidyl nucleoside antibiotic. Additionally, heterologous expression showed that microcin C is recognized by the P. aeruginosa Npp system. Overall, these results suggest that the NppA1A2BCD transporter is involved in the uptake of peptidyl nucleoside antibiotics by P. aeruginosa PA14.ImportanceOne of the world's most serious health problems is the rise of antibiotic-resistant bacteria. There is a desperate need to find novel antibiotic therapeutics that either act on new biological targets or are able to bypass known resistance mechanisms. Bacterial ABC transporters play an important role in nutrient uptake from the environment. These uptake systems could also be exploited by a Trojan horse strategy to facilitate the transport of antibiotics into bacterial cells. Several natural antibiotics mimic substrates of peptide uptake routes. In this study, we analyzed an ABC transporter involved in the uptake of nucleoside peptidyl antibiotics. Our data might help to design drug conjugates that may hijack this uptake system to gain access to cells.

Dataset Information

Identification of the biosynthetic gene cluster for the pacidamycin group of peptidyl nucleoside antibiotics.

Publications

Identification of the biosynthetic gene cluster for the pacidamycin group of peptidyl nucleoside antibiotics.

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