Periplasmic Hydrogenases in the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough 0.000003 -3.77 cytoplasmic Carbon Monoxide hydrogenase, CooU subunit DVU2290 0.000008 -3.40 cytoplasmic Carbon Monoxide hydrogenase, CooL subunit DVU2288 0.00024 -3.09 cytoplasmic Carbon Monoxide hydrogenase, CooX subunit DVU2289 0.000008 -2.86 cytoplasmic Carbon Monoxide hydrogenase, CooK subunit DVU2287 0.000001 2.04 cytoplasmic Ech hydrogenase, EchF subunit DVU0429 0.00044 -2.59 cytoplasmic Carbon Monoxide hydrogenase, CooM subunit DVU2286 0.00462 0.94 periplasmic [NiFeSe] hydrogenase, large subunit (hysA) DVU1918 0.000267 2.24 cytoplasmic Ech hydrogenase, EchA subunit DVU0434 N/A* cytoplasmic Ech hydrogenase, EchB subunit DVU0433 0.000005 2.56 cytoplasmic Ech hydrogenase, EchC subunit DVU0432 0.000002 2.08 cytoplasmic Ech hydrogenase, EchD subunit DVU0431 0.00016 2.06 cytoplasmic Ech hydrogenase, EchE subunit DVU0430 No significant Expression change periplasmic [NiFeSe] hydrogenase, small subunit (hysB) DVU1917 0.0059 -0.73 periplasmic [NiFe] hydrogenase, large subunit, isozyme 2 (hynA) DVU2526 No significant Expression change putative [Fe] hydrogenase DVU1771 0.0081 -0.71 L-lactate dehydrognase (ldh) DVU2451 0.0023 -0.83 periplasmic [NiFe] hydrogenase, small subunit, isozyme 2 (hynB) DVU2525 No significant Expression change periplasmic [NiFe] hydrogenase, large subunit, isozyme 1 (hynA) DVU1922 0.0322 -0.52 periplasmic [NiFe] hydrogenase, small subunit, isozyme 1 (hynB) DVU1921 0.000381 1.54 periplasmic [Fe] hydrogenase, small subunit (hydB) DVU1770 0.000679 1.21 periplasmic [Fe] hydrogenase, large subunit (hydA) DVU1769 P value Log 2 ratio Annotation Gene Sean Caffrey, Hyung-Soo Park and Gerrit Voordouw- Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada 1SP Sulfate-reducing bacteria (SRB), which are ubiquitously present in anaerobic soils and sediments, can derive energy for growth by coupling sulfate reduction with the oxidation of molecular hydrogen (H 2 ). H 2 is cleaved by one of several periplasmic hydrogenases (Hases) to generate a proton-motive force. D. vulgaris contains an iron-only [Fe] Hase, two nickel-iron [NiFe] Hases and a nickel-iron-selenium [NiFeSe] Hase. The roles of these multiple periplasmic Hases remain to be fully elucidated. In view of their different kinetic properties the [Fe] Hase may be effective at high H 2 concentrations, whereas the nickel-containing Hases may be required for growth at low H 2 partial pressures. Growth of a hyn-1 mutant lacking genes for [NiFe]-1 Hase and a hyd mutant missing the [Fe] Hase genes were compared to the wild-type at 30 o C in 1 L bottles containing 600 mL of culture in an anaerobic culturing system with 50%, 5%, or 0.5% (vol/vol) H 2 atmospheres. The H 2 was supplied by bubbling without stirring the liquid phase. When cultured at 50% H 2 the wild-type and hyn-1 strain had similar doubling times around 17 h, while the hyd mutant strain’s doubling time was much longer at 36 h. At 5% H 2 the doubling time of the wild-type and hyn-1 strains were around 38 hours and the hyd strain’s doubling time was 55 h. As the H 2 concentration was dropped further to 0.5% the difference in the doubling times continued to diminish with the hyn-1 and wild-type strains possessing a doubling time of close to 155 h while the hyd strain’s doubling time increased to 184 h. This trend suggests that the [Fe] Hase is relatively more important at higher H 2 partial pressures and that as the H 2 concentration is dropped further the growth rates of the hyn-1 and hyd mutants will become similar. Preliminary microarray data from the wild-type strain suggests that as the H 2 concentration is decreased the relative abundance of the [Fe] and [NiFe]-1 transcripts are slightly decreased, while the amount of the [NiFeSe] Hase mRNA is increased. This data provides additional support for the premise that the [Fe] Hase is the most important periplasmic Hase when D. vulgaris is grown at high H 2 concentrations. Organism Importance o SRB are ubiquitous in anaerobic soils and sediments o SRB have a significant function in global carbon and sulfur cycles o the production of HS - by Sulfate Reducing Bacteria has a considerable economic impact on the petroleum and mining industries o Desulfovibrio vulgaris has become a model SRB its genome has been completely sequenced full genome microarrays are available (courtesy of Dr. Jizhong Zhou, University of Oklahoma) Hydrogenases o catalyses H 2 ↔ 2H + + 2e - o widely prevalent in Archaea and Bacteria otwo common classes: Iron-only [Fe], Nickel-containing [NiFe] and [NiFeSe] o D. vulgaris contains 4 periplasmic Hydrogenases: 1 [Fe], 2 [NiFe] and 1 [NiFeSe] (Figure 1) o each class of hydrogenases has unique kinetic properties (Table 1) [Fe] hydrogenase has high specific activity and low Km [NiFe] has lower specific activity and high Km o consequently: the [Fe] hydrogenase maybe more effective at high H 2 concentrations and the [NiFe] maybe require for efficient growth on lower H 2 partial pressures to determine whether each of the periplasmic hydrogenases are utilized differentially when D. vulgaris is grown at various H 2 partial pressures establish growth rates for D. vulgaris when grown with 5%, 0.5% and 0.05% Hydrogen ascertain whether hydrogenase genes are differentially expressed as H 2 partial pressure is changed Table 1. Specific activity and Km values for Hydrogen Utilization and production and the effect of CO and NO inhibition on Hydrogenase activity for the thee types of hydrogenases found in Desulfovibrio spp. 1 Not Sensitive >>0.02 μM 1 μM 467 n.d. 120 [NiFeSe] Not Sensitive >>0.02 μM 20-30 μM 440 1 μM 1500 [NiFe] Sensitive 0.02-0.04 μM 0.1 μM 4800 100 μM 50000 [Fe] NO 2 - NO Inhibition (amount needed for 50% decrease in activity) CO Inhibition (amount needed for 50% decrease in activity) Specific Activity Evolution μmol H 2 /min/mg protein Km Uptake Specific activity Uptake μmol H 2 /min/mg protein Hydrogenase Type •At hydrogen partial pressures above 1% the [Fe] hydrogenase is important for D. vulgaris growth. The [NiFe]-1 hydrogenase is not of critical importance under any growth conditions tested. •As the hydrogen partial pressure is dropped the difference in growth rates between the two mutant strains and wild-type is decreased. The growth rates become similar below 1% H 2 . •Since neither the [NiFe]-1 nor the [Fe] hydrogenase are needed for growth below 1% H 2 the other nickel-containing hydrogenases are sufficient under these conditions. •Expression data shows that the D. vulgaris hydrogenases are under active transcriptional regulation and respond to changes in the electron donor. •The [NiFe] and Carbon monoxide hydroganases appear to be more important in lactate metabolism than hydrogen metabolism. •Lower hydrogen partial pressures (0.05%) must be tested to see if the [NiFe]-1 hydrogenase is important for efficient growth at very low hydrogen concentrations. •construction of mutant strains missing the [NiFe]-2 and [NiFeSe] hydrogenases •Additional array studies at 0.5% and 0.05% hydrogen are required to determine if changes in the hydrogen concentration change the relative expression levels of the different classes of hydrogenases Table 3: hydrogenase expression data for D. vulgaris cells grown on 50% hydrogen versus D. vulgaris cells grown on lactate gas flow system. Log 2 ratio is 50% H 2 /gDNA versus Lactate/gDNA. Growth Conditions (Figure 2) o gassed batch culture at 30ºC with defined media utilizing either Lactate or H 2 as electron donor o gas bubbled at 80 ml/min (H 2 , N 2 , and CO 2 ) is mixed regulated by independent mass flow controllers and is deoxygenated with a copper reduction tube heated to 350ºC Microarray (Figure 3) o utilizing full genome 70mer oligonucluotide arrays o RNA extracted from log phase batch cultures with Trizol/Bacterial Max o indirect Cy3/Cy5 labeling of amino-modified cDNA o reference design utilizing gDNA as reference o each experimental condition consists of 4 arrays (8 spots per gene) o Analysis Normalization •stringent bad channel tolerance and background checking (S/N threshold) •Total Intensity Normalization statistical significance determined by T-test Figure 1: shows environmental hydrogen being oxidized by a periplasmic hydrogenase. The electrons are transported across the membrane and used to reduce sulfate to sulfide. The protons are imported through ATPsynthase to be used in the reduction of sulfate SO 4 2 - APS HSO 3 - HS - ATP Sat Sat Aps AMP 2H + 6H + periplasm cytoplasm 8H + + 8e - 4H 2 Periplasmic Hydrogenases Periplasmic Hydrogenases Synthase ATP Synthase 4H 2 8e - + 8H + 2 lactate 2 acetate + 2 CO 2 2 ATP 2 ADP , Pta, Ack + 2 P i 6e - 2e - Cytoplasmic Hydrogenase ATP ADP + P i Transmembrane Complexes Lactate Oxidation Dsr Figure 2: 5% hydrogen gas is passed through a reduction tub and bubbled into defined media at 30 ºC B 1 B 2 gDNA Organic Acids 50% H 2 5% H 2 0.5% H 2 A 50% Hydrogen versus Lactate log 2 intensity Lactate/gDNA Log2 intensity 50% Hydrogen/gDNA Figure 3: A- gDNA is used as a common reference for the analysis of 50% H 2 , 5% 2 , and 0.5% H 2 B- Using gDNA as the common reference ensures that genes are not lost because they fail to show up in the reference channel. In the gDNA reference channel (B 1) ~98% +/- 4 of the spots are present, but in RNA reference channel (B 2) often less than 70% of the genes are visible. C- Intensity plot of RNA extracted from cells grown with 50% H 2 versus RNA extracted from cells grown with lactate. C 350 C o H 2 184.3 160.0 152.7 0.5 % H 2 54.6 +/- 10.8 39.3 +/- 7.2 38.7 +/- 6.2 5.0 % H 2 36 +/- 6.4 16.9 +/- 3.6 17.0 +/- 6.5 50 % H 2 7.3 6.9 7.0 Lactate [Fe] mutant [NiFe]-1 mutant Wild-type Doubling Time (Hours) Electron Donor 184.3 160.0 152.7 0.5 % H 2 54.6 +/- 10.8 39.3 +/- 7.2 38.7 +/- 6.2 5.0 % H 2 36 +/- 6.4 16.9 +/- 3.6 17.0 +/- 6.5 50 % H 2 7.3 6.9 7.0 Lactate [Fe] mutant [NiFe]-1 mutant Wild-type Doubling Time (Hours) Electron Donor Table 2: Doubling times of wild-type and mutant strains of D. vulgaris grown with either lactate or varying concentrations of hydrogen Growth Studies (Table 2) -under all growth conditions the [Fe] mutant strain’s growth is the most impaired -as the H 2 concentration is decreased the difference in doubling times between the [Fe] and [NiFe]-1 mutant strains decreases (Figure 4) Microarray Analysis 50% H 2 versus Lactate (Table 3) -as shown in Table 3 most of D. vulgaris’ hydrogenases expression changes when the electron donor is changed from lactate to hydrogen *N/A- insufficient spots for statistical analysis 1) Fauque, G., Peck Jr., H.D., Moura, J.J.G., Huynh, B.H., Berlier, Y., DerVertanian, D.V., Teixeira, M., Przybyla, A.E., Lespinat, P.A., Moura, I. and J. Le Gall. 1988. The three classes of hydrogenases from sulfate-reducing bacteria of the Genus Desulfovibrio. FEMS Microbiol. Rev. 54, 299-344. Figure 4: The instantaneous growth rate (h -1 ) of the wild-type and two mutant strains with respect to the log H 2 concentration used as the sole electron donor 0 0.01 0.02 0.03 0.04 0.05 0.06 0.1 1 10 100 log Hydrogen Percentage Instantaneous Growth Rate h-1 DvH [NiFe]-1 [Fe]