Amycolatopsis aidingensis sp. nov., a Halotolerant Actinobacterium, Produces New Secondary Metabolites

A novel actinobacterium, strain YIM 96748T, was isolated from a saline soil sample collected from the south bank of Aiding Lake in Xinjiang Province, Northwest China. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain YIM 96748T is closely related to Amycolatopsis cihanbeyliensis BNT52T (98.9%) and Amycolatopsis jiangsuensis KLBMP 1262T (97.2%). The DNA–DNA relatedness between strain YIM 96748T and its closest type strain A. cihanbeyliensis BNT52T was 59.6%. The average nucleotide identity between strain YIM 96748T and its neighbor strain was 88.97%. Based on the genotypic and phenotypic characteristics, it is concluded that strain YIM 96748T represents a novel species of the genus Amycolatopsis, whose name was proposed as Amycolatopsis aidingensis sp. nov. The type strain is YIM 96748T. To investigate the biosynthetic potential of producing secondary metabolites, the complete genome of YIM 96748T was sequenced and analyzed. The complete genome sequence of YIM 96748T consists of a 7,657,695-bp circular chromosome, comprising 7,162 predicted genes with a DNA G + C content of 70.21 mol%. Fifty-one putative biosynthetic gene clusters of secondary metabolites were found, including the antibacterial/antitumor agent TLN-05220, the antibacterial agent nocardicin A, the antifungal agent nystatin A1, and the osmolyte ectoine. The investigation of the secondary metabolites of A. aidingensis YIM96748T led to the discovery of two new phenylpropyl acetate enantiomers, amycoletates A (1) and B (2), and five known compounds: 4-hydroxy phenethyl acetate (3), 2-p-acetoxyphenylethanol (4), (S)-ethyl indole-3-lactate (5), (R)-ethyl indole-3-lactate (6), and p-hydroxybenzoic acid (7). One of the gene clusters 14, 36, and 43, which contain a single module of polyketide synthase, might be responsible for the biosynthesis of compounds 1 and 2 from compound 7 as a precursor. Further studies, including the one strain many compounds approach (OSMAC) and genetic modification, are needed to explore novel compounds from this talented halophilic Amycolatopsis strain.

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

ABSTRACT Nocardicin A is a monocyclic β-lactam isolated from the actinomycete Nocardia uniformis, which shows moderate activity against a broad spectrum of gram-negative bacteria. Within the biosynthetic gene cluster of nocardicin A, nocR encodes a 583-amino-acid protein with high similarity to a class of transcriptional regulators known as streptomyces antibiotic regulatory proteins. Insertional inactivation of this gene resulted in a mutant showing morphology and growth characteristics similar to the wild type, but one that did not produce detectable levels of nocardicin A or the early precursor p-hydroxybenzoyl formate. Similar disruptions of nocD, nocE, and nocO yielded mutants that maintained production of nocardicin A at levels similar to the wild-type strain. In trans complementation of the nocR::apr mutant partially restored the wild-type phenotype. Transcriptional analysis of the nocR::apr mutant using reverse transcription-PCR found an absence of mRNA transcripts for the early-stage nocardicin A biosynthetic genes. In addition, transcription of the genes responsible for the biosynthesis of the nonproteinogenic p-hydroxyphenylglycine (pHPG) precursor was attenuated on the nocR disruption mutant. NocR was heterologously expressed and purified from Escherichia coli as an N-terminal maltose binding protein-tagged fusion protein. DNA binding assays demonstrated that NocR is a DNA binding protein, targeting the 126-bp intergenic region between nocF and nocA. Within this intergenic region is the likely binding motif, a direct hexameric repeat, TGATAA, with a 5-bp spacer. These experiments establish NocR as a positive transcriptional regulator of the nocardicin A biosynthetic pathway, coordinating the initial steps of nocardicin A biosynthesis to the production of its pHPG precursor.

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

ABSTRACT The nocardicins are a family of monocyclic β-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 β-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.