Selenium is involved in regulation of periplasmic hydrogenase gene expression in Desulfovibrio vulgaris Hildenborough

Abstract

Desulfovibrio vulgaris Hildenborough is a good model organism to study hydrogen metabolism in sulfate-reducing bacteria. Hydrogen is a key compound for these organisms, since it is one of their major energy sources in natural habitats and also an intermediate in the energy metabolism. The D. vulgaris Hildenborough genome codes for six different hydrogenases, but only three of them, the periplasmic-facing [FeFe], [FeNi]1, and [FeNiSe] hydrogenases, are usually detected. In this work, we studied the synthesis of each of these enzymes in response to different electron donors and acceptors for growth as well as in response to the availability of Ni and Se. The formation of the three hydrogenases was not very strongly affected by the electron donors or acceptors used, but the highest levels were observed after growth with hydrogen as electron donor and lowest with thiosulfate as electron acceptor. The major effect observed was with inclusion of Se in the growth medium, which led to a strong repression of the [FeFe] and [NiFe]1 hydrogenases and a strong increase in the [NiFeSe] hydrogenase that is not detected in the absence of Se. Ni also led to increased formation of the [NiFe]1 hydrogenase, except for growth with H2, where its synthesis is very high even without Ni added to the medium. Growth with H2 results in a strong increase in the soluble forms of the [NiFe]1 and [NiFeSe] hydrogenases. This study is an important contribution to understanding why D. vulgaris Hildenborough has three periplasmic hydrogenases. It supports their similar physiological role in H2 oxidation and reveals that element availability has a strong influence in their relative expression.

FIG. 1.

Activity-stained gels of crude cell extracts (50 μg) from D. vulgaris Hildenborough cells grown in lactate-sulfate (L/S), hydrogen-sulfate (H₂/S), lactate-thiosulfate (L/T), pyruvate-sulfate (P/S), and pyruvate (P). Fe, medium containing only Fe; Ni, medium containing Fe+Ni; Se, medium containing Fe+Ni+Se. Results were reproduced in triplicate experiments. FeFe, [FeFe] Hase; NiFe, [NiFe]₁ Hase; NiFeSe, [NiFeSe] Hase. (a) Gel comparing crude cell extracts of cells grown in Fe and Fe+Ni; (b) gel comparing crude cell extracts of cells grown in Fe+Ni and Fe+Ni+Se. The Fe+Ni extracts are repeated in both gels, because comparison of band intensities should only be performed within the same gel.

FIG. 2.

Activity-stained gels of membrane (M) and soluble (S) extracts (50 μg) from D. vulgaris Hildenborough cells grown in lactate-sulfate (a) and hydrogen-sulfate (b). Se, medium containing Fe+Ni+Se; Ni, medium containing Fe+Ni.

FIG. 3.

Hydrogen production activity (in units per milligram) of soluble (a) and membrane (b) extracts of D. vulgaris Hildenborough cells grown in lactate-sulfate (L/S), hydrogen-sulfate (H₂/S), lactate-thiosulfate (L/T), pyruvate-sulfate (P/S), and pyruvate (P) in the presence of Fe (gray bars), Fe+Ni (black bars), or Fe+Ni+Se (striped bars).

FIG. 4.

Western blots of D. vulgaris Hildenborough crude extracts from cells grown in lactate-sulfate (L/S); hydrogen-sulfate (H₂/S); lactate-thiosulfate (L/T); pyruvate-sulfate (P/S); pyruvate (P); in medium containing only iron (Fe); in medium containing iron and nickel (Ni); or in medium containing iron, nickel, and selenium (Se), using antibodies against the D. vulgaris Hildenborough [FeFe] (A), [NiFe]₁ (B), and [NiFeSe] Hases (C). The amounts used for immunodetection were 20 μg of crude extracts for [FeFe] Hase antibodies and 5 μg for [NiFe]₁ and [NiFeSe] Hase antibodies.

FIG. 5.

Densitometric analysis of immunoblots depicted in Fig. 4 using antibodies against [FeFe] Hase (A), [NiFe]₁ Hase (B), and [NiFeSe] Hase (C). Medium containing only iron (gray bars), iron plus nickel (black bars), or iron plus nickel plus selenium (stripe bars) was used. Values are normalized to the L/S medium in Fe-only conditions in panel A, Fe+Ni conditions in panel B, and Fe+Ni+Se conditions in panel C. Shown are the averages of two experiments.

FIG. 6.

RT-PCR analysis of the transcription of the genes encoding the D. vulgaris Hildenborough [FeFe], [NiFe]₁, and [NiFeSe] Hases on L/S medium. Fe, medium containing Fe; Ni, medium containing Fe+Ni; Se, medium containing Fe+Ni+Se; M, 100-bp DNA ladder. (A) hydA ([FeFe] Hase large subunit) transcription; (B) hynA-1 ([NiFe] Hase large subunit) transcription; (C) hysA ([NiFeSe] Hase large subunit) transcription. RT-PCRs were done with 100 ng of total RNA.