Mutational Analysis of nocK and nocL in the Nocardicin A Producer Nocardia uniformis

Abstract

The nocardicins are a family of monocyclic ␤-lactam antibiotics produced by the actinomycete Nocardia uniformis subsp. tsuyamanensis ATCC 21806. The most potent of this series is nocardicin A, containing a syn-configured oxime moiety, an uncommon feature in natural products. The nocardicin A biosynthetic gene cluster was recently identified and found to encode proteins in keeping with nocardicin A production, including the nocardicin N-oxygenase, NocL, in addition to genes of undetermined function, such as nocK, which bears similarities to a broad family of esterases. The latter was hypothesized to be involved in the formation of the critical ␤-lactam ring. While previously shown to effect oxidation of the 2-amine of nocardicin C to provide nocardicin A, it was uncertain whether NocL was the only N-oxidizing enzyme required for nocardicin A biosynthesis. To further detail the role of NocL in nocardicin production in N. uniformis, and to examine the function of nocK, a method for the transformation of N. uniformis protoplasts to inactivate both nocK and nocL was developed and applied. A reliable protocol is reported to achieve both insertional disruption and in trans complementation in this strain. While the nocK mutant still produced nocardicin A at levels near that seen for wild-type N. uniformis, and therefore has no obvious role in nocardicin biosynthesis, the nocL disruptant failed to generate the oxime-containing metabolite. Nocardicin A production was restored in the nocL mutant upon in trans expression of the gene. Furthermore, the nocL mutant accumulated the biosynthetic intermediate nocardicin C, confirming its role as the sole oxime-forming enzyme required for production of nocardicin A.

Identification and Characterization of NocR as a Positive Transcriptional Regulator of the β-Lactam Nocardicin A inNocardia uniformis

“…Other studies that have shown that different plasmid vectors can produce varying product yields, as noted with studies on daunorubicin-producing Streptomyces peucetius (46) and tylosin-producing Streptomyces fradiae (44). However, when wildtype N. uniformis was transformed with pULVK2T containing no insert, no change in nocardicin A production was observed, consistent with previous in trans complementation experiments (23,24). The current evidence indicates that low expression from the pULVK2T vector may have a limiting effect in N. uniformis.…”

“…Following cloning, the pULVK2 plasmids were transformed into E. coli JM110 cells to provide nonmethylated DNA for protoplast transformation into N. uniformis. Polyethylene glycol-mediated transformation of N. uniformis was performed as described previously (24). N. uniformis pULVK2 vector transformants were selected by an overlay with 200 g/ml kanamycin and 100 g/ml apramycin.…”

“…The protein encoded by nocI is homologous to the Mbt-H family of small proteins that are found in gene clusters of many peptide antibiotics and siderophores, including the CDA and coelichelin biosynthetic clusters of S. coelicolor, and is essential for their biosynthesis (26). Previous experiments indicate that nocK is not essential for nocardicin A biosynthesis (24).…”

“…Three genes, nocF, nocG, and nocN, encode proteins responsible for the biosynthesis of the nonproteinogenic L-p-hydroxyphenylglyine (pHPG) precursor of nocardicin A. Biosynthesis of nocardicin A is initiated by the presumed formation of a tripeptide from pHPG and serine by nonribosomal peptide synthetases (NRPSs) encoded by nocA and nocB. Three genes, nat, nocJ, and nocL, encode tailoring enzymes (23,24,38). The predicted product of nocH is homologous to membrane transport pro-teins and thought to be involved in exporting nocardicin A from the cell.…”

The genus Amycolatopsis: Indigenous plasmids, cloning vectors and gene transfer systems

“…Recent studies have also demonstrated the potential utility of these vectors for mutagenesis studies, which were carried out in Nocardia uniformis subsp. tsuyamanensis ATCC 21806 63,64 .…”