Tuesday, July 31, 2007 - 2:00 PM
S117

Identification of NocR as a Positive Transcriptional Regulator for the Biosynthesis of the ß-lactam Nocardicin A in Nocardia uniformis

Jeanne M. Davidsen and Craig A. Townsend. Chemistry Department, Johns Hopkins University, 3401 N. Charles Street, Baltimore, MD 21218

Nocardicin A is a monocyclic β-lactam, isolated from the actinomycete N. uniformis, which shows moderate activity against a broad spectrum of Gram-negative bacteria.  One of the putative genes identified within the biosynthetic gene cluster of nocardicin A, nocR, encodes 583 amino acid protein, with high similarity to a class of transcriptional regulators known as (Streptomyces Antibiotic Regulatory Proteins) SARPs.  Insertional inactivation of this gene in N. uniformis resulted in a mutant showing morphology and growth characteristics similar to the wild-type, but did not produce detectable levels of nocardicin A or its isolable intermediates.  In trans complementation of the nocR::apra mutant partially restored production of nocardicin A.  Transcriptional analysis, using RT-PCR found an absence of mRNA transcripts for the known nocardicin A biosynthetic genes, nocA, nocB, nat, nocJ, and nocL in the nocR::apra mutant.  In addition, transcription of the biosynthetic genes for the p-hydroxyphenyl glycine (pHPG) precursor nocF, nocG, and nocN were significantly attenuated in the nocR::apra mutant compared to the wild-type.  NocR was heterologously expressed and purified from E.coli as an N-terminal MBP-tagged fusion protein.  Subsequent gel-shift assays demonstrate that NocR, is a DNA binding protein, targeting the 126 bp intergenic region between nocF and nocA.  These experiments establish NocR to be a positive transcriptional regulator of the nocardicin A biosynthetic pathway.

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