Glutathione is the predominant low-molecular-weight peptide thiol present in living organisms and plays a key role in protecting cells against oxygen toxicity. Until now, glutathione synthesis was thought to occur solely through the consecutive action of two physically separate enzymes, ␥-glutamylcysteine ligase and glutathione synthetase. In this report we demonstrate that Listeria monocytogenes contains a novel multidomain protein (termed GshF) that carries out complete synthesis of glutathione. Evidence for this comes from experiments which showed that in vitro recombinant GshF directs the formation of glutathione from its constituent amino acids and the in vivo effect of a mutation in GshF that abolishes glutathione synthesis, results in accumulation of the intermediate ␥-glutamylcysteine, and causes hypersensitivity to oxidative agents. We identified GshF orthologs, consisting of a ␥-glutamylcysteine ligase (GshA) domain fused to an ATP-grasp domain, in 20 gram-positive and gram-negative bacteria. Remarkably, 95% of these bacteria are mammalian pathogens. A plausible origin for GshF-dependent glutathione biosynthesis in these bacteria was the recruitment by a GshA ancestor gene of an ATP-grasp gene and the subsequent spread of the fusion gene between mammalian hosts, most likely by horizontal gene transfer.Glutathione (␥-glutamyl-cysteinyl-glycine) (GSH) is the predominant low-molecular-weight peptide thiol present in living organisms. In bacteria it plays a pivotal role in many metabolic processes, chief among which are thiol redox homeostasis, protection against reactive oxygen species, protein folding, and provision of electrons via NADPH to reductive enzymes, such as ribonucleotide reductase. Low-molecular-weight nonribosomal peptides are assembled by the action of versatile multimodular enzymes termed nonribosomal peptide synthetases (NRPS) (22, 37) or through the consecutive actions of individual enzymes. GSH synthesis is a prime example of the latter, and GSH is made in a highly conserved two-step ATP-dependent process by two unrelated peptide bond-forming enzymes (21). The ␥-carboxyl group of L-glutamate and the amino group of L-cysteine are ligated by the enzyme ␥-glutamylcysteine ligase (encoded by gshA) to give ␥-glutamylcysteine, which is then condensed with glycine in a reaction catalyzed by glutathione synthetase (encoded by gshB) to form GSH. Most grampositive bacteria do not contain GSH (9). However, a broad survey of the distribution of thiols in microorganisms revealed that several species of gram-positive bacteria, including Listeria, streptococci, and enterococci, produce significant amounts of GSH (23). The source of GSH in these bacteria has remained a puzzle, since their genomes do not contain a canonical gshB gene. The recent paper of Copley and Dhillon provides a clue to the origin of this GSH (7). These authors identified in the genomes of Listeria monocytogenes, Listeria innocua, Clostridium perfringens, and Pasteurella multocida an open reading frame (ORF) that is predicted to co...

Glycome analysis of Alzheimer’s patients reveals interplay between glycosylation pathways and suggests novel biomarkers.

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.

It is increasingly clear that transcription factors play versatile roles in turning genes “on” or “off” depending on cellular context via the various transcription complexes they form. This poses a major challenge in unraveling combinatorial transcription complex codes. Here we use the powerful genetics of Drosophila combined with microarray and bioinformatics analyses to tackle this challenge. The nuclear adaptor CHIP/LDB is a major developmental regulator capable of forming tissue-specific transcription complexes with various types of transcription factors and cofactors, making it a valuable model to study the intricacies of gene regulation. To date only few CHIP/LDB complexes target genes have been identified, and possible tissue-dependent crosstalk between these complexes has not been rigorously explored. SSDP proteins protect CHIP/LDB complexes from proteasome dependent degradation and are rate-limiting cofactors for these complexes. By using mutations in SSDP, we identified 189 down-stream targets of CHIP/LDB and show that these genes are enriched for the binding sites of APTEROUS (AP) and PANNIER (PNR), two well studied transcription factors associated with CHIP/LDB complexes. We performed extensive genetic screens and identified target genes that genetically interact with components of CHIP/LDB complexes in directing the development of the wings (28 genes) and thoracic bristles (23 genes). Moreover, by in vivo RNAi silencing we uncovered novel roles for two of the target genes, xbp1 and Gs-alpha, in early development of these structures. Taken together, our results suggest that loss of SSDP disrupts the normal balance between the CHIP-AP and the CHIP-PNR transcription complexes, resulting in down-regulation of CHIP-AP target genes and the concomitant up-regulation of CHIP-PNR target genes. Understanding the combinatorial nature of transcription complexes as presented here is crucial to the study of transcription regulation of gene batteries required for development.

Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.Ribonucleotide reductases (RNRs) are essential enzymes in all living cells, providing the only known de novo pathway for the biosynthesis of deoxyribonucleotides, the immediate precursors of DNA synthesis and repair. RNRs catalyze the controlled reduction of all four ribonucleotides to maintain a balanced pool of deoxyribonucleotides during the cell cycle (29). Three main classes of RNRs are known. Class I RNRs are oxygen-dependent enzymes, class II RNRs are oxygen-independent enzymes, and class III RNRs are oxygen-sensitive enzymes. Class I RNRs are divided into two subclasses, subclasses Ia and Ib.