Molecular analysis of the grd operon coding for genes of the glycine reductase and of the thioredoxin system from Clostridium sticklandii

Graentzdoerffer, Andrea; Pich, Andreas; Andreesen, Jan R.

doi:10.1007/s002030000232

Cited by 24 publications

(20 citation statements)

References 43 publications

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“…The locations of the SeCys residues in the C. difficile 630 GR amino acid sequences are conserved with respect to these residues in GRs from C. sticklandii and E. acidaminophilum. The gene order from grdX to grdC is also identical to that from C. sticklandii (17). It should be noted that a gene predicted to encode proline aminopeptidase (proP) is located at the end of the grd genes (Fig.…”

Section: Resultsmentioning

confidence: 77%

“…Genes encoding subunits of glycine reductase (denoted grd) are clustered together in an apparent operon, based on close linkage of the open reading frames in both C. difficile and C. sticklandii. Two of the genes, grdA and grdB, contain an in-frame UGA codon, suggesting that these two genes encode the two previously identified selenocysteine-containing subunits of GR (17). The locations of the SeCys residues in the C. difficile 630 GR amino acid sequences are conserved with respect to these residues in GRs from C. sticklandii and E. acidaminophilum.…”

Section: Resultsmentioning

confidence: 90%

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Analysis of Proline Reduction in the Nosocomial Pathogen Clostridium difficile

et al. 2006

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Clostridium difficile, a proteolytic strict anaerobe, has emerged as a clinically significant nosocomial pathogen in recent years. Pathogenesis is due to the production of lethal toxins, A and B, members of the large clostridial cytotoxin family. Although it has been established that alterations in the amino acid content of the growth medium affect toxin production, the molecular mechanism for this observed effect is not yet known. Since there is a paucity of information on the amino acid fermentation pathways used by this pathogen, we investigated whether Stickland reactions might be at the heart of its bioenergetic pathways. Growth of C. difficile on Stickland pairs yielded large increases in cell density in a limiting basal medium, demonstrating that these reactions are tied to ATP production. Selenium supplementation was required for this increase in cell yield. Analysis of genome sequence data reveals genes encoding the protein components of two key selenoenzyme reductases, glycine reductase and D-proline reductase (PR). These selenoenzymes were expressed upon the addition of the corresponding Stickland acceptor (glycine, proline, or hydroxyproline). Purification of the selenoenzyme D-proline reductase revealed a mixed complex of PrdA and PrdB (SeCys-containing) proteins. PR utilized only D-proline but not L-hydroxyproline, even in the presence of an expressed and purified proline racemase. PR was found to be independent of divalent cations, and zinc was a potent inhibitor of PR. These results show that Stickland reactions are key to the growth of C. difficile and that the mechanism of PR may differ significantly from that of previously studied PR from nonpathogenic species.

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Section: Resultsmentioning

confidence: 77%

Section: Resultsmentioning

confidence: 90%

Analysis of Proline Reduction in the Nosocomial Pathogen Clostridium difficile

et al. 2006

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“…10 -25-ml cultures were used, and the cells were lysed in 20 mg of lysozyme ml Ϫ1 for 10 min. Northern hybridizations were performed as described (26).…”

Section: Uptakementioning

confidence: 99%

Tungstate Uptake by a Highly Specific ABC Transporter inEubacterium acidaminophilum

Makdessi

Andreesen

Pich

2001

Journal of Biological Chemistry

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The Gram-positive anaerobe Eubacterium acidaminophilum contains at least two tungsten-dependent enzymes: viologen-dependent formate dehydrogenase and aldehyde dehydrogenase.185 W-Labeled tungstate was taken up by this organism with a maximum rate of 0.53 pmol min ؊1 mg ؊1 of protein at 36°C. The uptake was not affected by equimolar amounts of molybdate. The genes tupABC coding for an ABC transporter specific for tungstate were cloned in the downstream region of genes encoding a tungsten-containing formate dehydrogenase. The substrate-binding protein, TupA, of this putative transporter was overexpressed in Escherichia coli, and its binding properties toward oxyanions were determined by a native polyacrylamide gel retardation assay. Only tungstate induced a shift of TupA mobility, suggesting that only this anion was specifically bound by TupA. If molybdate and sulfate were added in high molar excess (>1000-fold), they were also slightly bound by TupA. The K d value for tungstate was determined to be 0.5 M. The genes encoding the tungstate-specific ABC transporter exhibited highest similarities to putative transporters from Methanobacterium thermoautotrophicum, Haloferax volcanii, Vibrio cholerae, and Campylobacter jejuni. These five transporters represent a separate phylogenetic group of oxyanion ABC transporters as evident from analysis of the deduced amino acid sequences of the binding proteins. Downstream of the tupABC genes, the genes moeA, moeA-1, moaA, and a truncated moaC have been identified by sequence comparison of the deduced amino acid sequences. They should participate in the biosynthesis of the pterin cofactor that is present in molybdenum-and tungstencontaining enzymes except nitrogenase.

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“…Treponema denticola, an established resident of the oral cavity, performs Stickland reactions via the selenoprotein glycine reductase (32). Glycine reductase is composed of a multiprotein complex that contains two separate selenoproteins, termed selenoprotein A and selenoprotein B (1,7,8,15,16). This complex of proteins converts glycine to acetyl phosphate by using inorganic phosphate and the reducing potential from thioredoxin.…”

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confidence: 99%