Mycothiol is the major thiol present in most actinomycetes and is produced from the pseudodisaccharide 1D-myo-inosityl 2-acetamido-2-deoxy-␣-D-glucopyranoside (GlcNAc-Ins). A transposon mutant of Mycobacterium smegmatis shown to be GlcNAc-Ins and mycothiol deficient was sequenced to identify a putative glycosyltransferase gene designated mshA. The ortholog in Mycobacterium tuberculosis, Rv0486, was used to complement the mutant phenotype.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs, either of which is sufficient for vegetative growth. The class Ia RNR is encoded by the nrdAB genes, and the class II RNR is encoded by nrdJ, which is coexpressed with nrdR. We previously showed that the Streptomyces coelicolor nrdR gene encodes a protein, NrdR, which represses transcription of both sets of RNR genes. NrdR is a member of a highly conserved family of proteins that is confined exclusively to prokaryotes. In this report, we describe a physical and biochemical characterization of the S. coelicolor NrdR protein and show that it is a zinc-ATP/dATP-containing protein that binds to the promoter regions of both Streptomyces RNR operons. The NrdR N terminus contains a zinc ribbon motif that is necessary for binding to the upstream regulatory region of both RNR operons. The latter contains two 16-bp direct repeat sequences, termed NrdR boxes, which are located proximal to, or overlap with, the promoter regions. These experiments support the view that NrdR controls the transcription of RNR genes by binding to the NrdR box sequences. We also show that the central NrdR ATP cone domain binds ATP and dATP and that mutations that abolish ATP/dATP binding significantly reduce DNA binding, suggesting that the ATP cone domain may allosterically regulate NrdR binding. We conclude that NrdR is a widely conserved regulator of RNR genes, binding to specific sequence elements in the promoter region and thereby modulating transcription.Ribonucleotide reductases (RNRs) provide the building blocks for DNA synthesis and repair in all living cells (20). They are essential because they are the only known de novo pathway for the biosynthesis of deoxyribonucleotides, the immediate precursors for DNA synthesis. Three major classes of RNRs are known (8,15,19). Class I RNRs are oxygen-dependent enzymes that occur in eukaryotes, bacteria, and some viruses, and class II RNRs are oxygen-independent enzymes confined to bacteria and archaea, while class III RNRs are found predominantly in anaerobic bacteria. Despite significant differences in structure and in cofactor requirements, all three enzymes share similar catalytic mechanisms creating a protein radical that initiates reduction of ribonucleotides, and all allosterically regulate the balanced formation of the four deoxyribonucleotides (15,19,24).Previously we reported that Streptomyces spp., gram-positive aerobic bacteria that produce a remarkable variety of metabolites and possess a complex life cycle (7, 13), contain class Ia and class II RNRs (5). Either type of RNR is sufficient for vegetative growth (4). In Streptomyces coelicolor, the class Ia and class II RNRs are regulated by coenzyme B12 (adenosylcobalamin) in a reciprocal manner. B12 negatively controls transcription of the class Ia RNR nrdABS genes via a riboswitch mechanism and positively controls the activity of...

SummaryRibonucleotide reductases (RNRs) catalyse the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomyces spp. contain genes coding for two RNRs. We show here that the Streptomyces coelicolor M145 nrdAB genes encoding an oxygendependent class I RNR are co-transcribed with nrdS , which encodes an AraC-like regulatory protein. Likewise, the class II oxygen-independent RNR nrdJ gene forms an operon with a likely regulatory gene, nrdR , which encodes a protein possessing an ATP-cone domain like those present in the allosteric activity site of many class Ia RNRs. Deletions in nrdB and nrdJ had no discernible effect on growth individually, but abolition of both RNR systems, using hydroxyurea to inactivate the class Ia RNR (NrdAB) in the nrdJ deletion mutant, was lethal, establishing that S. coelicolor possesses just two functional RNR systems. The class II RNR (NrdJ) may function to provide a pool of deoxyribonucleotide precursors for DNA repair during oxygen limitation and/or for immediate growth after restoration of oxygen, as the nrdJ mutant was slower in growth recovery than the nrdB mutant or the parent strain. The class Ia and class II RNR genes show complex regulation. The nrdRJ genes were transcribed some five-to sixfold higher than the nrdABS genes in vegetative growth, but when nrdJ was deleted, nrdABS transcription was upregulated by 13-fold. In a reciprocal experiment, deletion of nrdB had little effect on nrdRJ transcription. Deletion of nrdR caused a dramatic increase in transcription of nrdJ and to a less extent nrdABS , whereas disruption of cobN , a gene required for synthesis of coenzyme B12 a cofactor for the class II RNR, caused similar upregulation of transcription of nrdRJ and nrdABS . In contrast, deletion of nrdS had no detectable effect on transcription of either set of RNR genes. These results establish the existence of control mechanisms that sense and regulate overall RNR gene expression.

Biopsy specimens from 23 early stage and 19 tumorstage mycosis fungoides (MF) patients were evaluated for miR-155 expression by real-time qualitative PCR and compared with 15 biopsy specimens from patients with T-cell-rich inflammatory skin diseases. Significant upregulation of miR-155 was found in MF tumors compared with both early-stage MF lesions and controls. There was no difference in miR-155 expression between early-stage and inflammatory dermatoses. Using laser capture microdissection, it was found that miR-155 was significantly higher in the lymphoma cells in tumor stage compared with the intraepidermal lymphocytes in early stage. In contrast, there was no difference in miR-155 expression between the intraepidermal lymphocytes and the dermal lymphocytes in early-stage MF.These findings suggest that although miR-155 expression cannot serve to discriminate early-stage MF from inflammatory dermatoses; however, it is involved in the switch from the indolent early stage into the aggressive tumor stage of the disease.Abbreviations: CTCL, cutaneous T cell lymphoma; MF, mycosis fungoides; SS, sezary syndrome; FFPE, formalin fixed paraffin embedded; LCM, laser capture microdissection; DLE, discoid lupus erythematosus.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotides to deoxyribonucleotides and are essential for de novo DNA synthesis and repair. Streptomycetes contain genes coding for two RNRs. The class Ia RNR is oxygen dependent, and the class II RNR is oxygen independent and requires coenzyme B12. Either RNR is sufficient for vegetative growth. We show here that the Streptomyces coelicolor M145 nrdABS genes encoding the class Ia RNR are regulated by coenzyme B12. The 5-untranslated region of nrdABS contains a 123-nucleotide B12 riboswitch. Similar B12 riboswitches are present in the corresponding regions of eight other S. coelicolor genes. The effect of B12 on growth and nrdABS transcription was examined in a mutant in which the nrdJ gene, encoding the class II RNR, was deleted. B12 concentrations of just 1 g/liter completely inhibited growth of the NrdJ mutant strain. Likewise, B12 significantly reduced nrdABS transcription. To further explore the mechanism of B12 repression, we isolated in the nrdJ deletion strain mutants that are insensitive to B12 inhibition of growth. Two classes of mutations were found to map to the B12 riboswitch. Both conferred resistance to B12 inhibition of nrdABS transcription and are likely to affect B12 binding. These results establish that B12 regulates overall RNR expression in reciprocal ways, by riboswitch regulation of the class Ia RNR nrdABS genes and by serving as a cofactor for the class II RNR.