Function of periplasmic hydrogenases in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

Abstract

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen>50% hydrogen>lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.

FIG. 1.

Growth curves for wild-type D. vulgaris in WP medium with lactate (A), 50% (vol/vol) hydrogen (B), or 5% (vol/vol) hydrogen (C) as the sole electron donor for sulfate reduction. The linear scale on the left corresponds to the following, each as a function of time: ▿, sulfate concentration (A to C); ▪ concentration of lactate (A); and ▴, concentration of acetate (A). The logarithmic scale on the right indicates the cell density at OD₆₀₀ (○ and •). The filled circles represent data used for calculation of the specific growth rate constant, k.

FIG. 2.

Specific growth rate constants (k) and stationary-phase cell densities (OD₆₀₀) of wild-type and mutant strains grown with lactate (A and B), 50% (vol/vol) hydrogen (C and D), or 5% (vol/vol) hydrogen (E and F) as the sole electron donor for the reduction of sulfate. The data are averages from at least four independent experiments. Error bars represent 95% confidence intervals.

FIG. 3.

Changes in expression levels of hydA and hysA genes. (A) RT-PCR data for each condition (lactate, 50% H₂, or 5% H₂) were normalized for expression of 16S rRNA and are presented relative to expression with lactate. (B) Microarray hybridization intensity ratios, R_i = <cDNA_i>/<gDNA_i>, were calculated from measured data as described in the text. The log₂R_i values for the hysA and hydA genes are plotted for growth with lactate, 50% (vol/vol) hydrogen, or 5% (vol/vol) hydrogen.