Evidence for a novel nitrate reductase in the dissimilatory iron-reducing bacterium Geobacter metallireducens

Abstract

Nitrate induced the expression of a membrane-bound nitrate reductase in the strict anaerobe Geobacter metallireducens.A fraction from a DEAE cellulose column which showed nitrate reduetase activity contained polypeptides of M r 18, 36 and 43 K and three c type cytochromes (M r 28, 46 and 68 K). Western and Southern blot analysis revealed no homology between the nitrate reductase from G. metallireducens and the nitrate reductases from respiratory (Escherichia coli) and denitrifying bacteria (Pseudomonas stutzeri, Pseudomonas aeruginosa) which were shown to be related. These data, in addition to this organism's inability to use fumarate or formate, suggest that its nitrate reductase is novel.

Differential cytochrome content and reductase activity in Geospirillum barnesii strain SeS3

“…The methyl viologen spectrophotometric assay of Blumle and Zumft (1991) as modified by Naik et al 1993 was used to test for reductase activity. A reaction mixture containing 2.2 ml bicarbonate buffer (10 mM, pH 7.2), 80 µl methyl viologen (1 mg/ml), and 100 µl of the terminal electron acceptor (from stock solutions prepared as described below) was sparged for 5 min with oxygen-free dinitrogen in a Thunberg cuvette.…”

A heme- C -containing enzyme complex that exhibits nitrate and nitrite reductase activity from the dissimilatory iron-reducing bacterium Geobacter metallireducens

“…Initial biochemical investigations have found that nitrate reductase activity is limited to cells grown on nitrate. This activity is membraneassociated and not solubilized by Triton X 100 or heat, and four c-type cytochromes are overexpressed (Naik et al 1993). Southern blot analysis using narG from E. coli as a probe, and Western blot analyses with antibodies to the holoenzyme from Pseudomonas stutzeri have not revealed a similar type of nitrate reductase in G. metallireducens (Naik et al 1993).…”

“…This activity is membraneassociated and not solubilized by Triton X 100 or heat, and four c-type cytochromes are overexpressed (Naik et al 1993). Southern blot analysis using narG from E. coli as a probe, and Western blot analyses with antibodies to the holoenzyme from Pseudomonas stutzeri have not revealed a similar type of nitrate reductase in G. metallireducens (Naik et al 1993). In addition, cytochrome b has not been detected spectrophotometrically in whole cells, cytoplasmic fractions, membrane fractions, or in preparations of partially purified enzyme (Naik et al 1993).…”

“…Southern blot analysis using narG from E. coli as a probe, and Western blot analyses with antibodies to the holoenzyme from Pseudomonas stutzeri have not revealed a similar type of nitrate reductase in G. metallireducens (Naik et al 1993). In addition, cytochrome b has not been detected spectrophotometrically in whole cells, cytoplasmic fractions, membrane fractions, or in preparations of partially purified enzyme (Naik et al 1993). These data suggest that nitrate reduction in G. metallireducens might occur by an atypical mechanism.…”

Geobacter bremensis sp. nov. and Geobacter pelophilus sp. nov., two dissimilatory ferric-iron-reducing bacteria.

“…The presence of c-type cytochrome(s) was shown by spectral analysis of whole-cell suspensions and membrane fractions of G. metallireducens and G. sulfurreducens (Lovley et al, 1993 ;Naik et al, 1993 ;Caccavo et al, 1994 ;Seeliger et al, 1998). In contrast, cells of P. propionicus lack c-type cytochromes (Schink, 1984).…”