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Journal of Bacteriology, February 2009, p. 1066-1077, Vol. 191, No. 3
0021-9193/09/$08.00+0     doi:10.1128/JB.01833-07
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Jeanne M. Davidsen and Craig A. Townsend*

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

Received 20 November 2007/ Accepted 12 November 2008

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.

* Corresponding author. Mailing address: Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218. Phone: (410) 516-7444. Fax: (410) 261-1233. E-mail: ctownsend{at}jhu.edu

{triangledown} Published ahead of print on 21 November 2008.

Journal of Bacteriology, February 2009, p. 1066-1077, Vol. 191, No. 3
0021-9193/09/$08.00+0     doi:10.1128/JB.01833-07
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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