Identification and Characterization of NocR As a Positive Transcriptional Regulator of the Beta-lactam Nocardicin A in Nocardia Uniformis

Nocardicin A is a monocyclic beta-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.