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.
Davidsen
Jeanne M
JM
Department of Chemistry, Johns Hopkins University, Baltimore, MD 21218, USA.
Townsend
Craig A
CA
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D015964:Gene Expression Regulation, Bacterial; D005998:Glycine / Q000031:analogs & derivatives / Q000378:metabolism; D007769:Lactams / Q000378:metabolism*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009615:Nocardia / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D007769:Lactams; D014157:Transcription Factors; C027318:nocardicin; C023010:4-hydroxyphenylglycine; D005998:Glycine