Identification and Characterization of the Sulfazecin Monobactam Biosynthetic Gene Cluster

Abstract

The monobactams, exemplified by the natural product sulfazecin, are the only class of β-lactam antibiotics not inactivated by metallo-β-lactamases, which confer bacteria with extended-spectrum β-lactam resistance. We screened a transposon mutagenesis library from Pseudomonas acidophila ATCC 31363 and isolated a sulfazecin-deficient mutant that revealed a gene cluster encoding two non-ribosomal peptide synthetases (NRPSs), a methyltransferase, a sulfotransferase, and a dioxygenase. Three modules and an aberrant C-terminal thioesterase (TE) domain are distributed across the two NRPSs. Biochemical examination of the adenylation (A) domains provided evidence that L-2,3-diaminopropionate, not L-serine as previously thought, is the direct source of the β-lactam ring of sulfazecin. ATP/PPi exchange assay also revealed an unusual substrate selectivity shift of one A domain when expressed with or without the immediately upstream condensation domain. Gene inactivation analysis defined a cluster of 13 open reading frames sufficient for sulfazecin production, precursor synthesis, self-resistance, and regulation. The identification of a key intermediate supported a proposed NRPS-mediated mechanism of sulfazecin biosynthesis and β-lactam ring formation distinct from the nocardicins, another NRPS-derived subclass of monocyclic β-lactam. These findings will serve as the basis for further biosynthetic research and potential engineering of these important antibiotics.

Keywords: antibiotic; biosynthesis; metallo-β-lactamase; natural product; non-ribosomal peptide synthetase; sulfazecin; sulfotransferase; β-lactam.

Figure 1. β-Lactam Structures

Examples of monobactams and representative structures of the other β-lactam antibiotic families.

Figure 2. Sulfazecin Production and Biosynthetic Gene Cluster

(A) UPLC-HRMS analysis of wild-type P. acidophila (left) and P. acidophila Sul⁻-7 mutant (right). Insets are E. coli ESS growth-inhibition assays. (B) Sulfazecin gene cluster from cosmid clone 1H4. The colored ORFs indicate the putative minimal gene cluster for sulfazecin biosynthesis, precursor synthesis, regulation, and resistance. The domain organization of the NRPSs is also shown, and the amino acid substrate selectively activated by each A domain. The AKN domain at the N terminus of SulI indicates the predicted adenylylsulfate kinase.

Figure 3. ATP/PPi Exchange Assays of A1T1, A2T2, A3T3, and C2A2T2

(A) ATP/PPi exchange assay of A1T1. (B) ATP/PPi exchange assay of A3T3. (C) ATP/PPi exchange assay of A2T2. (D) ATP/PPi exchange assay of C2A2T2. Blank, no enzyme; no amino acids, control.

Figure 4. Construction of Gene Disruptants

Construction of Gm-GFP integrant (P. acidophila KGmM-3)and unmarked plasmid-free(P.acidophila ΔsulL) mutants of sulL, and analysis for sulfazecin production by nitrocefin assays of each strain (Figure S5).

Figure 5. UPLC-HRMS Chromatograms and Antimicrobial Analyses of Products and Intermediates in Different P. acidophila Strains

(A) P. acidophila FGmH-11, sulfazecin-deficient knockout. (B) Sulfazecin produced in P. acidophila FGmH-11 supplemented with L-2,3-Dap. (C) Desmethoxysulfazecin intermediate purified from the wild-type P. acidophila. (D) Multiple sequence alignment of SulTE against TE domains in sulfazecin-like BGCs, and in PKS and NRPS TEs having high-resolution crystal structures. The active-site catalytic residue is labeled in red (*), and the GXSXG or AXCXG motifs are highlighted. SulTE, P. acidophila ATCC 31363; 31532, C. violesium 31532; DM48, B. gladioli ATCC 25417 DM48; E264, B. thailandensis E264; Y049, B. pseudomallei MSHR684 Y049; BP3921g, B. pseudomallei BP_3921g; 3QIT, curacin biosynthesis; 3ILS, aflatoxin PksA; 3QMV, prodiginine biosyn-thesis; 3LCR, tautomycetin biosynthesis; 1MNA, pikromycin biosynthesis.

Figure 6. The Proposed Biogenetic Pathway to Sulfazecin

(A) Biosynthesis of L-2,3-diaminopropionic acid (Dap). (B) Structure of nocardicin G, the first β-lactam containing intermediate in the pathway to nocardicin A. (C) Alternative possible biosynthetic routes to sulfazecin. PAPS, 3′-phosphoadenosine-5′-phosphosulfate; SAM, S-adenosyl methionine; TE, thioesterase; α-KG, α-ketoglutaric acid; L-phosphoSer, L-phosphoserine.