Nitrate-dependent regulation of acetate biosynthesis and nitrate respiration by Clostridium thermoaceticum - PubMed (original) (raw)

Nitrate-dependent regulation of acetate biosynthesis and nitrate respiration by Clostridium thermoaceticum

A F Arendsen et al. J Bacteriol. 1999 Mar.

Abstract

Nitrate has been shown to shunt the electron flow in Clostridium thermoaceticum from CO2 to nitrate, but it did not influence the levels of enzymes involved in the Wood-Ljungdahl pathway (J. M. Fröstl, C. Seifritz, and H. L. Drake, J. Bacteriol. 178:4597-4603, 1996). Here we show that under some growth conditions, nitrate does in fact repress proteins involved in the Wood-Ljungdahl pathway. The CO oxidation activity in crude extracts of nitrate (30 mM)-supplemented cultures was fivefold less than that of nitrate-free cultures, while the H2 oxidation activity was six- to sevenfold lower. The decrease in CO oxidation activity paralleled a decrease in CO dehydrogenase (CODH) protein level, as confirmed by Western blot analysis. Protein levels of CODH in nitrate-supplemented cultures were 50% lower than those in nitrate-free cultures. Western blots analyses showed that nitrate also decreased the levels of the corrinoid iron-sulfur protein (60%) and methyltransferase (70%). Surprisingly, the decrease in activity and protein levels upon nitrate supplementation was observed only when cultures were continuously sparged. Northern blot analysis indicates that the regulation of the proteins involved in the Wood-Ljungdahl pathway by nitrate is at the transcriptional level. At least a 10-fold decrease in levels of cytochrome b was observed with nitrate supplementation whether the cultures were sparged or stoppered. We also detected nitrate-inducible nitrate reductase activity (2 to 39 nmol min-1 mg-1) in crude extracts of C. thermoaceticum. Our results indicate that nitrate coordinately represses genes encoding enzymes and electron transport proteins in the Wood-Ljungdahl pathway and activates transcription of nitrate respiratory proteins. CO2 also appears to induce expression of the Wood-Ljungdahl pathway genes and repress nitrate reductase activity.

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Figures

FIG. 1

CODH activity in crude extracts of C. thermoaceticum grown in the presence of different concentrations of nitrate. Solid circles represent C. thermoaceticum cultures grown in stoppered vials under 100% CO2; open circles represent cultures grown in CO2-sparged vials.

FIG. 2

Western blot analysis of total cell protein of C. thermoaceticum, grown in the absence (−) or presence (+) of nitrate (30 mM). Cells were grown under CO2 in stoppered vials or in CO2-sparged vials. Equal amounts (16 μg) of total cell protein were loaded on an SDS–12.5% gel and subsequently blotted onto a nitrocellulose filter. The blots were hybridized with polyclonal antibodies raised against CODH, MeTr, or CFeSP.

FIG. 3

Western blot analysis of total cell protein of C. thermoaceticum, grown in the absence (−) or presence (+) of nitrate (30 mM). Cells were grown in vials which were sparged with either 1% CO, 1% H2, 5% H2, or 0.1% NO (gases balanced with CO2). Equal amounts (16 μg) of total cell protein were loaded on an SDS–12.5% gel and subsequently blotted onto a nitrocellulose filter. The blots were hybridized with polyclonal antibodies raised against CODH, MeTr, or CFeSP.

FIG. 4

Northern blot analysis of isolated total mRNA (1 μg in each lane) of C. thermoaceticum, grown in the absence (−) or presence (+) of nitrate (30 mM). Cells were grown under CO2 in stoppered (stopp.) vials or in CO2-sparged vials. The blots were hybridized with a 200-bp digoxigenin-labeled DNA probe against the gene for the α or β subunit of CODH, α or β subunit of CFeSP, or MeTr. Equal loading was confirmed by comparing intensities of rRNA on a formaldehyde gel. The nitrate-dependent decrease in mRNA levels in CO2-sparged cultures was observed in three independent experiments.

FIG. 5

Reduced-minus-oxidized spectra of C. thermoaceticum membranes (1 mg/ml). Cells were grown in the absence or presence of nitrate in vials that were stoppered under a CO2 atmosphere (a) or sparged with CO2 (b), CO (c), H2 (d), or NO (e). OD, optical density.

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Nitrate-dependent regulation of acetate biosynthesis and nitrate respiration by Clostridium thermoaceticum - PubMed (original) (raw)