ABSTRACT
In a previous work, we demonstrated that the Mycobacterium tuberculosis Rv2358-furB operon is induced by zinc. In this study, the orthologous genes from Mycobacterium smegmatis mc(2)155 were inactivated and mutants analyzed. Rv2358 protein was purified and found to bind upstream of the Rv2358 gene. Binding was inhibited by Zn(2+) ions.
Subject(s)
Bacterial Proteins/genetics , Gene Deletion , Mycobacterium smegmatis/genetics , Mycobacterium tuberculosis/genetics , Transcription Factors/genetics , Zinc/metabolism , Bacterial Proteins/metabolism , Base Sequence , Molecular Sequence Data , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/metabolism , Operon/physiology , Promoter Regions, Genetic/physiology , Protein Binding/genetics , Transcription Factors/metabolismABSTRACT
The Mycobacterium tuberculosis genome encodes two ferric uptake regulator homologues, furA and furB, the function of which is under investigation. Using Mycobacterium smegmatis as a model system, we investigated the transcriptional pattern of Rv(Ms)2358-furB genes. Transcripts covering the two genes could be identified by northern blotting and by reverse transcriptase PCR. The transcriptional start point was mapped at one base upstream of the Ms2358 start codon by the RACE technique. By cloning M. smegmatis and M. tuberculosis DNA regions upstream of a reporter gene, we demonstrated the presence of one promoter, located immediately upstream of the Rv(Ms)2358 gene. Promoter induction was tested on several cultures grown under different conditions of pH and temperature, and in the presence of different concentrations of metallic ions. The promoter was found to be specifically induced by zinc. The recombinant M. tuberculosis FurB protein typically contained two zinc ions per protein monomer. Complete removal of zinc could not be obtained, even with strong denaturation treatment. Our data are in favour of the hypothesis that Rv2358 and FurB are transcriptional regulators involved in zinc homeostasis.
Subject(s)
Bacterial Proteins/genetics , Mycobacterium tuberculosis/genetics , Operon/drug effects , Repressor Proteins/genetics , Zinc/pharmacology , Amino Acid Sequence , Bacterial Proteins/chemistry , Base Sequence , Cloning, Molecular , DNA, Bacterial/chemistry , DNA, Bacterial/genetics , Molecular Sequence Data , Mycobacterium smegmatis/drug effects , Mycobacterium smegmatis/genetics , Mycobacterium smegmatis/metabolism , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/metabolism , Operon/genetics , Promoter Regions, Genetic/genetics , Promoter Regions, Genetic/physiology , RNA, Bacterial/chemistry , RNA, Bacterial/genetics , Random Amplified Polymorphic DNA Technique , Repressor Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, DNA , Transcription, Genetic/drug effects , Transcription, Genetic/genetics , Transcription, Genetic/physiology , Zinc/metabolismABSTRACT
The enzyme NAD(+) synthetase (NadE) catalyzes the last step of NAD biosynthesis. Given NAD vital role in cell metabolism, the enzyme represents a valid target for the development of new antimycobacterial agents. In the present study we expressed and purified two putative forms of Mycobacterium tuberculosis NAD(+) synthetase, differing in the polypeptide chain length (NadE-738 and NadE-679). Furthermore, we evaluated several systems for the heterologous expression and large scale purification of the enzyme. In particular, we compared the efficiency of production, the yield of purification, and the catalytic activity of recombinant enzyme in different hosts, ranging from Escherichia coli strains to cultured High Five (Trichoplusia ni BTI-TN-5B1-4) insect cells. Among the systems assayed, we found that the expression of a thioredoxin-NadE fusion protein in E. coli Origami(DE3) is the best system in obtaining highly pure, active NAD(+) synthetase. The recombinant enzyme maintained its activity even after proteolytic cleavage of thioredoxin moiety. Biochemical evidence suggests that the shorter form (NadE-679) may be the real M. tuberculosis NAD(+) synthetase. These results enable us to obtain a purified product for structure-function analysis and high throughput assays for rapid screening of compounds which inhibit enzymatic activity.
