Article Figures & Data
Figures
- FIG. 1.
Structures and mutagenesis of putative hya and hyb operons. (A) Structure of putative hya operon. Twin-arginine motifs are indicated as vertical black bars. (B) Structure of disrupted hya operon and confirmation of Hya-deficient mutant genotype by Southern blotting. Wild-type and Hya-deficient G. sulfurreducens genomic DNAs were cleaved with PstI and XhoI, blotted, and probed with the 2.4-kb DNA fragment used to introduce the ΔhyaSLB::kan mutation into the genome (see Materials and Methods). Expected radiolabeled bands are 5.28, 3.03, and 2.04 kb for the wild-type strain and 5.28, 1.88, and 1.02 kb for the mutant. (C) Structure of putative hyb operon. Twin-arginine motif sequences are indicated as vertical black bars. (D) Structure of disrupted hyb operon and confirmation of Hyb-deficient mutant genotype by Southern blotting. Wild-type and Hyb-deficient G. sulfurreducens genomic DNAs were cleaved with SmaI, blotted, and probed with the 2.0-kb DNA fragment used to introduce the ΔhybL::cam mutation into the genome (see Materials and Methods). Expected radiolabeled bands are 4.02 and 1.76 kb for the wild-type strain and 4.02 and 1.41 kb for the mutant. (E) In-gel hydrogenase activity of Triton X-114 extracts of membranes prepared from acetate-fumarate cultures of the wild-type and Hya- and Hyb-deficient strains. Nondenaturing polyacrylamide gels were stained for hydrogen-dependent benzyl viologen reductase activity as described in Materials and Methods. Twenty micrograms of solubilized membranes was loaded per lane. Lanes 1 to 3 were incubated under hydrogen, while lanes 4 to 6 were incubated under nitrogen. The horizontal bar indicates the interface of the stacking and resolving gels.
- FIG. 2.
Growth of wild-type and hydrogenase-deficient strains with Fe(III)-citrate as the electron acceptor. (A) Hydrogen-dependent growth of the wild-type and Hya-deficient strains. (B) Hydrogen-dependent growth of the wild-type and Hyb-deficient strains. The experiment was initiated with 2.5% inocula from early-stationary-phase acetate-Fe(III)-citrate-grown cultures. A limiting concentration of acetate (1 mM) was provided as a carbon source. In each panel, wild-type and mutant cultures were inoculated and incubated in parallel. When two transfers are shown, the time of the second transfer (2.5%) is indicated by a vertical arrow. Data are the mean ± standard deviation of triplicate cultures.
- FIG. 3.
Hydrogen-dependent growth of wild-type and hydrogenase-deficient strains in the presence of AQDS and fumarate. Acetate-fumarate-grown cultures were washed in isotonic basal wash medium (22) prior to inoculation to eliminate carryover of acetate and fumarate. Cultures were supplied with a small amount of acetate as a carbon source (0.1 mM for AQDS curves [A and B] and 1 mM for fumarate curves [C and D]). For each panel, wild-type and mutant cultures were inoculated and incubated in parallel. Data are the mean ± standard deviation of triplicate cultures. When two transfers are shown, the time of the second transfer (5%) is indicated by a vertical arrow. Cell densities at time zero were ∼6 × 106/ml for AQDS growth curves (A and B) and ∼2.5 × 106/ml for fumarate growth curves (C and D). For AQDS growth curves, final cell densities at the end of first and second transfers were 4.87 × 107 ± 0.5 × 107 and 3.6 × 107 ± 1.06 × 107/ml, respectively, for the wild-type strain and 3.81 × 107 ± 0.41 × 107 and 2.88 × 107 ± 0.25 × 107/ml, respectively, for the Hya-deficient strain. O.D., optical density.
Tables
- TABLE 1.
Reduction of Fe(III)NTA, AQDS, and fumarate by cell suspensions
Suspensiona and electron donor Mean productionc (μmol/mg/min) ± SD Fe(II)-NTA AHQDSb Succinate Wild type Acetate 0.84 ± 0.17 0.64 ± 0.03 0.182 ± 0.002 Hydrogen 1.48 ± 0.22 1.29 ± 0.19 0.091 ± 0.010 Hya deficient Acetate 1.24 ± 0.05 0.81 ± 0.01 0.189 ± 0.001 Hydrogen 1.83 ± 0.10 1.46 ± 0.05 0.072 ± 0.013 Hyb deficient Acetate 0.95 ± 0.04 0.62 ± 0.05 0.187 ± 0.011 Hydrogen 0.00 ± 0.03 0.02 ± 0.01 0.004 ± 0.004 ↵ a Resting cell suspensions were prepared from late-log-phase acetate-fumarate cultures as described in Materials and Methods.
↵ b AHQDS is the reduced form of AQDS.
↵ c Data are means of triplicate incubations, except for measurements of wild-type reduction of AQDS and Fe(III)-NTA, which are means of six incubations.
