Identification of nocobactin NA biosynthetic gene clusters in Nocardia farcinica

Abstract

We identified the biosynthetic gene clusters of the siderophore nocobactin NA. The nbt clusters, which were discovered as genes highly homologous to the mycobactin biosynthesis genes by the genomic sequencing of Nocardia farcinica IFM 10152, consist of 10 genes separately located at two genomic regions. The gene organization of the nbt clusters and the predicted functions of the nbt genes, particularly the cyclization and epimerization domains, were in good agreement with the chemical structure of nocobactin NA. Disruptions of the nbtA and nbtE genes, respectively, reduced and abolished the productivity of nocobactin NA. The heterologous expression of the nbtS gene revealed that this gene encoded a salicylate synthase. These results indicate that the nbt clusters are responsible for the biosynthesis of nocobactin NA. We also found putative IdeR-binding sequences upstream of the nbtA, -G, -H, -S, and -T genes, whose expression was more than 10-fold higher in the low-iron condition than in the high-iron condition. These results suggest that nbt genes are regulated coordinately by IdeR protein in an iron-dependent manner. The ΔnbtE mutant was found to be impaired in cytotoxicity against J774A.1 cells, suggesting that nocobactin NA production is required for virulence of N. farcinica.

FIG. 1.

Gene and domain organization of the nbt clusters (A) and structure of nocobactin NA from N. farcinica IFM 10152 (B). Abbreviations: A, adenylation; C, condensation; PCP, peptidyl carrier protein; ArCP, aryl carrier protein; E, epimerization; Cy, cyclization; KS, ketoacyl synthase; AT, acyltransferase; KR, ketoreductase; ACP, acyl carrier protein.

FIG. 2.

Production of salicylate by the heterologous expression of the nbtS gene in S. avermitilis. HPLC conditions and sample preparations are described in Materials and Methods. (a) Authentic standard of salicylate; (b) S. avermitilis; (c) S. avermitilis/pKU251; (d) S. avermitilis/pKUnbtS. The elution peak of salicylate is indicated by an arrow with its retention time.

FIG. 3.

(A) Analyses of nocobactin NA production in the wild-type and mutant strains. a, wild type; b, ΔnbtA mutant; c, ΔnbtA/pNVnbtA mutant; d, ΔnbtE mutant; e, ΔnbtE/pNV mutant with nbtE mutant; f, ΔnbtS mutant; g, ΔnbtS/pNVnbtS mutant; h, ΔnbtS100 μg/ml salicylate _nbtAp; s1, nocobactin NA (n = 7); s2, nocobactin NA (n = 9). Nocobactin NA was detected at 267 nm. The elution peaks of the standards are indicated by arrows with their retention times. White arrowheads indicate the elution peaks of the newly identified metabolites (8.2 and 13.2 min) in the ΔnbtE mutant. UV spectra (B) and proposed structures (C) of the newly identified metabolites are also indicated.

FIG. 4.

Cytotoxic activity of Nocardia strains. J774A.1 cells were infected with the wild-type (b), ΔnbtE (c), and ΔnbtE/pNV_nbtApnbtE (d) strains, and uninfected J774A.1 cells (a) served as the control. Cells were photographed by phase-contrast microscopy 24 h after infection. Magnification, ×200.

FIG. 5.

Proposed biosynthetic pathway of nocobactin NA.