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J. Bacteriol. doi:10.1128/JB.01833-07
Copyright (c) 2008, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

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

Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, 21218, USA

^* To whom correspondence should be addressed. Email: ctownsend{at}jhu.edu.

	Abstract

Nocardicin A is a monocyclic -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, SARPs. 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 disruption of nocD, nocE, and nocO yielded mutants that maintain production of nocardicin A at a level 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 RT-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 non-proteinogenic p-hydroxyphenylglycine (pHPG) precursor was attenuated on the nocR disruption mutant. NocR was heterologously expressed and purified from E. coli as an N-terminal MBP-tagged fusion protein. DNA-binding assays demonstrate 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.