Mycothiol [2-(N-acetylcysteinyl)amido-2-deoxy-␣-D-glucopyranosyl-(131)-myo-inositol] (MSH. Since this novel thiol is more resistant than glutathione to heavy-metal ion-catalyzed oxidation, it seems likely to be the antioxidant thiol used by aerobic gram-positive bacteria that do not produce glutathione (GSH). In the present study we sought to define the spectrum of organisms that produce MSH. GSH was absent in all actinomycetes and some of the other gram-positive bacteria studied. Surprisingly, the streptococci and enterococci contained GSH, and some strains appeared to synthesize it rather than import it from the growth medium. MSH was found at significant levels in most actinomycetes examined. Among the actinobacteria four Micrococcus species produced MSH, but MSH was not found in representatives of the Arthrobacter, Agromyces, or Actinomyces genera. Of the nocardioforms examined, Nocardia, Rhodococcus, and Mycobacteria spp. all produced MSH. In addition to the established production of MSH by streptomycetes, we found that Micromonospora, Actinomadura, and Nocardiopsis spp. also synthesized MSH. Mycothiol production was not detected in Propionibacterium acnes or in representative species of the Listeria, Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Clostridium genera. Examination of representatives of the cyanobacteria, purple bacteria, and spirochetes also gave negative results, as did tests of rat liver, bonito, Candida albicans, Neurospora crassa, and spinach leaves. The results, which indicate that MSH production is restricted to the actinomycetes, could have significant implications for the detection and treatment of infections with actinomycetes, especially those caused by mycobacteria.

We observed the assembly of a nucleus-like structure in bacteria during viral infection. Using fluorescence microscopy and cryo-electron tomography, we showed that Pseudomonas chlororaphis phage 201ϕ2-1 assembled a compartment that separated viral DNA from the cytoplasm. The phage compartment was centered by a bipolar tubulin-based spindle, and it segregated phage and bacterial proteins according to function. Proteins involved in DNA replication and transcription localized inside the compartment, whereas proteins involved in translation and nucleotide synthesis localized outside. Later during infection, viral capsids assembled on the cytoplasmic membrane and moved to the surface of the compartment for DNA packaging. Ultimately, viral particles were released from the compartment and the cell lysed. These results demonstrate that phages have evolved a specialized structure to compartmentalize viral replication.

Bacillithiol (BSH), the α-anomeric glycoside of L-cysteinyl-D-glucosamine with L-malic acid, is a major low-molecular-weight thiol in Bacillus subtilis and related bacteria. Here, we identify genes required for BSH biosynthesis and provide evidence that the synthetic pathway has similarities to that established for the related thiol (mycothiol) in the Actinobacteria. Consistent with a key role for BSH in detoxification of electrophiles, the BshA glycosyltransferase and BshB1 deacetylase are encoded in an operon with methylglyoxal synthase. BshB1 is partially redundant in function with BshB2, a deacetylase of the LmbE family. Phylogenomic profiling identified a conserved unknown function protein (COG4365) as a candidate cysteine-adding enzyme (BshC) that cooccurs in genomes also encoding BshA, BshB1, and BshB2. Additional evolutionarily linked proteins include a thioredoxin reductase homolog and two thiol:disulfide oxidoreductases of the DUF1094 (CxC motif) family. Mutants lacking BshA, BshC, or both BshB1 and BshB2 are devoid of BSH. BSH is at least partially redundant in function with other low-molecular-weight thiols: redox proteomics indicates that protein thiols are largely reduced even in the absence of BSH. At the transcriptional level, the induction of genes controlled by two thiol-based regulators (OhrR, Spx) occurs normally. However, BSH null cells are significantly altered in acid and salt resistance, sporulation, and resistance to electrophiles and thiol reactive compounds. Moreover, cells lacking BSH are highly sensitive to fosfomycin, an epoxidecontaining antibiotic detoxified by FosB, a prototype for bacillithiol-Stransferase enzymes.Bacillus subtilis | glutathione | glutaredoxin | mycothiol | thioredoxin

Glutathione is a nearly ubiquitous low-molecular-weight thiol and antioxidant, although it is conspicuously absent from most Gram-positive bacteria. We identify here the structure of bacillithiol, a novel and abundant thiol produced by Bacillus species, Staphylococcus aureus, and Deinococcus radiodurans. Bacillithiol is the α-anomeric glycoside of l-cysteinyl-d-glucosamine with l-malic acid and likely functions as an antioxidant. Bacillithiol, like structurally similar mycothiol, may serve as a substitute for glutathione.

SUMMARY Mycothiol (MSH; AcCys-GlcN-Ins) is the major thiol found in Actinobacteria and has many of the functions of glutathione, which is the dominant thiol in other bacteria and eukaryotes but is absent in Actinobacteria. MSH functions as a protected reserve of cysteine and in the detoxification of alkylating agents, reactive oxygen and nitrogen species, and antibiotics. MSH also acts as a thiol buffer which is important in maintaining the highly reducing environment within the cell and protecting against disulfide stress. The pathway of MSH biosynthesis involves production of GlcNAc-Ins-P by MSH glycosyltransferase (MshA), dephosphorylation by the MSH phosphatase MshA2 (not yet identified), deacetylation by MshB to produce GlcN-Ins, linkage to Cys by the MSH ligase MshC, and acetylation by MSH synthase (MshD), yielding MSH. Studies of MSH mutants have shown that the MSH glycosyltransferase MshA and the MSH ligase MshC are required for MSH production, whereas mutants in the MSH deacetylase MshB and the acetyltransferase (MSH synthase) MshD produce some MSH and/or a closely related thiol. Current evidence indicates that MSH biosynthesis is controlled by transcriptional regulation mediated by σB and σR in Streptomyces coelicolor. Identified enzymes of MSH metabolism include mycothione reductase (disulfide reductase; Mtr), the S-nitrosomycothiol reductase MscR, the MSH S-conjugate amidase Mca, and an MSH-dependent maleylpyruvate isomerase. Mca cleaves MSH S-conjugates to generate mercapturic acids (AcCySR), excreted from the cell, and GlcN-Ins, used for resynthesis of MSH. The phenotypes of MSH-deficient mutants indicate the occurrence of one or more MSH-dependent S-transferases, peroxidases, and mycoredoxins, which are important targets for future studies. Current evidence suggests that several MSH biosynthetic and metabolic enzymes are potential targets for drugs against tuberculosis. The functions of MSH in antibiotic-producing streptomycetes and in bioremediation are areas for future study.

Mycothiol, 1-D-myo-inosityl-2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranoside (MSH), is composed of N-acetylcysteine (AcCys) amide linked to 1-D-myo-inosityl-2-amino-2-deoxy-alpha-D-glucopyranoside (GlcN-Ins) and is the major thiol produced by most actinomycetes. When Mycobacterium smegmatis was treated with the alkylating agent monobromobimane (mBBr), the cellular mycothiol was converted to its bimane derivative (MSmB). The latter was rapidly cleaved to produce GlcN-Ins and the bimane derivative of N-acetylcysteine (AcCySmB), a mercapturic acid that was rapidly exported from the cells into the medium. The other product of cleavage, GlcN-Ins, was retained in the cell and utilized in the resynthesis of mycothiol. The mycothiol S-conjugate amidase (amidase) responsible for cleaving MSmB was purified to homogeneity from M. smegmatis. A value of K(m) = 95 +/- 8 microM and a value of k(cat) = 8 s(-)(1) was determined for the amidase with MSmB as substrate. Activity with 100 microM mycothiol or with the monobromobimane derivative of 1-D-myo-inosityl-2-(L-cysteinyl)amido-2-deoxy-alpha-D-glucopyra nos ide (CySmB-GlcN-Ins) or of 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-(alpha, beta)-D-glucopyranoside (AcCySmB-GlcN) was at least 10(3) lower than with 100 microM MSmB, demonstrating that the amidase is highly specific for S-conjugates of mycothiol. Conjugates of mycothiol with the antibiotic cerulenin, N-ethylmaleimide, 3-(N-maleimidopropionyl)-biocytin, and 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin also exhibited significant activity. The sequence of the amino-terminal 20 residues was determined, and an open reading frame (Rv1082) coding for 288 residues having an identical predicted amino-terminal amino acid sequence was identified in the Mycobacterium tuberculosis genome. The Rv1082 gene (mca) from M. tuberculosis was cloned and expressed in Escherichia coli, and the expressed protein was shown to have substrate specificity similar to the amidase from M. smegmatis. These results indicate that mycothiol and mycothiol S-conjugate amidase play an important role in the detoxification of alkylating agents and antibiotics.

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Mycothiol (MSH; 1D-myo-inosityl 2-[N-acetyl-L-cysteinyl]amido-2-deoxy-␣-D-glucopyranoside) is the major low-molecular-weight thiol produced by mycobacteria. Mutants of Mycobacterium smegmatis mc2 155 deficient in MSH production were produced by chemical mutagenesis as well as by transposon mutagenesis. One chemical mutant (mutant I64) and two transposon mutants (mutants Tn1 and Tn2) stably deficient in MSH production were isolated by screening for reduced levels of MSH content. The MSH contents of transposon mutants Tn1 and Tn2 were found to be less than 0.1% that of the parent strain, and the MSH content of I64 was found to be 1 to 5% that of the parent strain. All three strains accumulated 1D-myo-inosityl 2-deoxy-␣-D-glucopyranoside to levels 20-to 25-fold the level found in the parent strain. The cysteine:1D-myo-inosityl 2-amino-2-deoxy-␣-D-glucopyranoside ligase (MshC) activities of the three mutant strains were <2% that of the parent strain. Phenotypic analysis revealed that these MSH-deficient mutants possess increased susceptibilities to free radicals and alkylating agents and to a wide range of antibiotics including erythromycin, azithromycin, vancomycin, penicillin G, rifamycin, and rifampin. Conversely, the mutants possess at least 200-fold higher levels of resistance to isoniazid than the wild type. We mapped the mutation in the chemical mutant by sequencing the mshC gene and showed that a single amino acid substitution (L205P) is responsible for reduced MSH production and its associated phenotype. Our results demonstrate that there is a direct correlation between MSH depletion and enhanced sensitivity to toxins and antibiotics. Fig. 1A) is a low-molecular-weight thiol that serves functions in mycobacteria analogous to those of glutathione in gram-negative bacteria and eukaryotes (7). These functions include protection against oxidative damage and inactivation of electrophilic toxins (15,17). The distribution of MSH is limited to gram-positive actinomycetes, and of all the species tested, mycobacteria appear to generate the highest levels of MSH (14). The absence of MSH in mammalian cells suggests that the enzymes involved in the metabolism of MSH may be attractive drug targets for either rational drug design or screening of libraries of inhibitory compounds (25). Mycothiol (MSH;Although studies of MSH biosynthesis are still at an early stage, as shown in Fig. 1B, elements of the overall pathway have been described (1, 4, 16). Moreover, direct measurements of intermediates in bacterial extracts that support the proposed biosynthetic pathway have been reported (1). The genes for the MSH biosynthesis pathway were designated mshA, mshB, mshC, and mshD (16), with the corresponding enzymes labeled as shown in Fig. 1. It has been shown that 1D-myo-inosityl 2-acetamino-2-deoxy-␣-D-glucopyranoside (GlcNAc-Ins) is an intermediate (16), and we presume that its formation is the first dedicated step in MSH biosynthesis. Glucosaminylinositol is formed by deacetylation of GlcNAc-Ins. We have previously identified m...