Thiols in Nitric Oxide Synthase-Containing Nocardia sp. Strain NRRL 5646

Nocardia sp. strain NRRL 5646 contains the first nitric oxide (NO) synthase (NOS) enzyme system found in the bacterial world (3). This enzyme catalyzes a complex process by which arginine is oxidized to NO and citrulline. The roles of this novel Nocardia NOS system have been investigated (3, 16, 17, 21, 31). One facet of this work that became interesting was an evaluation of possible roles of thiols in modulating NO toxicity, serving as stable storage forms for NO, or in trafficking of NO (11, 18, 28, 36). Evidence is gathered in this study that thiols are produced by Nocardia sp. strain NRRL 5646, and mycothiol (MSH) in particular may play an important role in the detoxification, if not the pathogenicity and regulation, of NO in NOS-containing bacteria (15, 26, 27). In this regard, Nocardia sp. strain NRRL 5646 represents a prototype counterpart model of glutathione (GSH) and S-nitrosoglutathione metabolism in eukaryotes (4, 6, 9-11, 13, 18, 19, 32).

For analysis, Nocardia sp stain. NRRL 5646 was grown in soybean flour-glucose medium in a two-stage process (3, 16, 17, 31). Cells harvested after 48 h from fully grown second-stage cultures were pelleted by centrifugation (8,000 × g for 20 min) and subjected to French pressure homogenization (12,000 lb/in²) and subsequent centrifugation (100,000 × g for 60 min). Extracts filtered through 0.22-μm Millex-GV membranes were used for determinations of protein (1), thiols such as cysteine, GSH, and nitrosothiols (RSNO) formed when thiols react with NO produced by NOS in this organism.

HgCl₂ converted RSNO in cell extracts to thiols plus nitrogen oxides that reacted with 2,3-diaminonaphthalene to form 2,3-naphthotriazole, a product determined by fluorescence spectrometry (12, 20). Analyses showed 2.50 ± 0.17 pmol NO₂⁻/g (dry weight) of cells, reflective of RSNO concentrations present in cell extracts. Analysis of the same extracts by Ellman's method (7) using 5,5′-dithio-bis(2-nitrobenzoic acid) indicated that 3.04 ± 0.02 μmol thiol/g (dry weight) of cells were present in Nocardia cell extracts. Although individual RSNO could not be identified, Nocardia contained RSNO corresponding to about 1/1,000 of the total measured thiols.

Specific Nocardia thiols were determined by N-(1-pyrenyl) maleimide (NPM) (29, 33) and monobromobimane (mBBr) (25) derivatization and subsequent high-performance liquid chromatography (HPLC) analysis. A modified NPM derivatization/HPLC method (33) showed that γ-glutamyl-l-cysteine was the only cell extract component that corresponded to standards, and its concentration was estimated to be 0.14 ± 0.03 μmol/g (dry weight) of cells. Nocardia thiols were also measured using a modified mBBr derivatization/HPLC method (25) to show bimane-derivatized totals of 2.2 ± 0.2 μmol of MSH (see Fig. 2), 0.14 ± 0.02 μmol of γ-glutamyl-l-cysteine, and 0.067 ± 0.008 μmol l-cysteine per gram (dry weight) of cells. γ-Glutamyl-l-cysteine and MSH-bimane (MSmB) derivatives nearly coeluted with a reagent-derived peak.

MSmB (Fig. 1) was isolated from 106 g of Nocardia cells that were extracted with aqueous acetonitrile and reacted with mBBr (23, 33). Half the extract was subjected to preparative HPLC, where peaks eluting at 25.3 min were pooled and dried to give 3.9 mg of >98% pure (HPLC) MSmB for spectral analysis. High-resolution mass spectrometry gave m/z 699.2171 for C₂₇H₄₀N₄O₁₄SNa, [MNa⁺] (calculated 699.2159), and an optical rotation of [α]²⁵_D + 26° (c 0.1, H₂O).

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FIG. 1.

Structures of MSH and MSmB.

Nuclear magnetic resonance (NMR) and other spectral properties were comparable with those for MSmB isolated from Streptomyces (23) and Mycobacterium (23) and to MSH recently synthesized in our laboratory (21). NMR spectra contained signals for all structural moieties including d-myo-inositol, d-glucosamine, N-acetyl-l-cysteine, and bimane, and analyses confirmed how these moieties were linked together. Key proton NMR signals corresponding to the 5″-methyl group of N-acetyl-l-cysteine and methyl groups at positions 7b, 9b, and 10b of bimane were clearly evident between 1.8 and 2.5 ppm. One anomeric proton signal for d-glucosamine (5.12 ppm; J_H-1 = 3.7 Hz) was consistent with an α-glycosidic linkage. By ¹³C-NMR, all four methyl groups were observed between 8 and 25 ppm, and the spectrum also showed one anomeric carbon signal at 101.9 ppm for d-glucosamine.

Figure 2 showed that Nocardia sp. strain NRRL 5646 contained MSH as the major low-molecular-weight thiol plus smaller amounts of γ-glutamyl-l-cysteine and l-cysteine. This is the first report of the characterization of MSmB from a NOS-containing species of Nocardia. Nocardia sp. strain NRRL 5646 contained 2.2 μmol/g (dry weight) of cells of MSH. This value is within the MSH range of 2.7 to 19 μmol/g (dry weight) of cells for other nocardioform bacteria (24). The highest levels of MSH occur in mycobacteria, whereas Nocardia asteroides and Nocardia brasiliensis contained 2.8 and 3.5 μmol/g (dry weight) MSH, respectively (24).

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FIG. 2.

HPLC chromatogram of mBBr-derivatized thiols in Nocardia sp. strain NRRL 5646 cell extracts. C*, endogenous fluorescent compound from cell extracts; R*, reagent-derived peak.

High concentrations of NO and S-nitrosoglutathione (GSNO) are toxic to bacteria (14, 28). Low-molecular-weight RSNO can disrupt cellular function by the transnitrosation of thiol groups in critical proteins (28, 35). However, bacterial cells have evolved apparent defense mechanisms against NO and RSNO as toxic agents. In Escherichia coli, flavorubredoxin with its associated oxidoreductase and flavohemoglobin convert NO to less noxious nitrous oxide and nitrate, respectively (8, 28). E. coli also contains GSH-dependent formaldehyde dehydrogenase (GS-FAD), a GSNO-metabolizing enzyme (22). GS-FAD is a widely distributed and highly conserved class III alcohol dehydrogenase. The enzyme is also known as GSNO reductase in E. coli, in Saccharomyces cerevisiae, and in mouse macrophages (22). GSNO reductase activity appears to be an inherent property of GS-FAD and a major means of regulating RSNO levels in cells.

MSH-dependent formaldehyde dehydrogenase activity in strain NRRL 5646 was measured using a slight modification of a method described previously by Vanophem et al. (34). Crude MSH solution was prepared by boiling cell extracts for 5 min and removing denatured protein and cell debris by centrifugation (40,000 × g for 30 min). The resulting supernatant was lyophilized and redissolved in 20 mM potassium phosphate buffer (pH 7.2). MSH concentrations in extracts were determined by mBBr derivatization and HPLC analysis. MSH-free cell extracts in 20 mM potassium phosphate buffer (pH 7.2) were prepared by passage over a Pharmacia PD-10 desalting column. For MSH-dependent formaldehyde dehydrogenase determinations, reactions were initiated by adding formaldehyde to MSH-free cell extracts containing 1 mM dithiothreitol, 2.5 mM NAD⁺, and 55 μM MSH. One unit of enzyme activity produced 1 μmol NADH,H⁺/min at 25°C. MSH-dependent formaldehyde dehydrogenase activity in Nocardia cell extracts was detected at 0.015 units/mg protein. Enzyme activity was absent without MSH. MSH-dependent formaldehyde dehydrogenase activity is known for Mycobacterium tuberculosis, where MSH-dependent formaldehyde dehydrogenase activity was identified as an S-nitrosomycothiol reductase (35).

Since its first isolation, Nocardia sp. strain NRRL 5646 has not been subjected to molecular identification by 16S rRNA gene analysis. Near-full-length 16S rRNA gene sequences are considered to be a reliable criterion for species identification in Nocardia (2, 30, 37). Moreover, a variable region that exists within the first 500 bp of the gene has been deemed reliable for species differentiation among members of this genus (5). We amplified a 1.4-kb 16S rRNA gene fragment of Nocardia sp. strain NRRL 5646 using eubacterial primers 27f and 1492R and a conventional PCR technique. Automated fluorescence DNA sequencing established a 1,427-base sequence that was found to have the highest identity (98%) to the corresponding regions of N. brasiliensis strain DSM 43758^T (GenBank accession number AF430038.1) and several other N. brasiliensis strains (accessions numbers Z36935, X80608, AY245543, DQ659902, and X80591) by BLAST analysis. No comparative similarities to Rhodococcus species were uncovered. This confirmed the Nocardia designation of strain NRRL 5646 but not at a known species level, since at least 99.2% identity is required for this classification (5). It appears not to be N. brasiliensis, since the signature sequence in the variable region showed a base transversion (149-TTTCAGT-155 in N. brasiliensis versus 146-TTCCAGT-152 in NRRL 5646), a signature sequence that also distinguishes it from Nocardia asteroides, N. farcinica, N. nova, N. otitidiscaviarum, N. pseudobrasiliensis, and N. transvalensis (5). Hence, the 16S sequence of NRRL 5646 most likely represents a new Nocardia species that would require further study, such as a polyphasic approach.

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