Periplasmic Hydrogenases in the Sulfate-Reducing Bacterium Desulfovibrio vulgaris Hildenborough

0.000003-3.77cytoplasmic Carbon Monoxide hydrogenase, CooU subunitDVU2290

0.000008-3.40cytoplasmic Carbon Monoxide hydrogenase, CooL subunitDVU2288

0.00024-3.09cytoplasmic Carbon Monoxide hydrogenase, CooX subunitDVU2289

0.000008-2.86cytoplasmic Carbon Monoxide hydrogenase, CooK subunitDVU2287

0.0000012.04cytoplasmic Ech hydrogenase, EchF subunitDVU0429

0.00044-2.59cytoplasmic Carbon Monoxide hydrogenase, CooM subunitDVU2286

0.004620.94periplasmic [NiFeSe] hydrogenase, large subunit (hysA)DVU1918

0.0002672.24cytoplasmic Ech hydrogenase, EchA subunitDVU0434

N/A*cytoplasmic Ech hydrogenase, EchB subunitDVU0433

0.0000052.56cytoplasmic Ech hydrogenase, EchC subunitDVU0432

0.0000022.08cytoplasmic Ech hydrogenase, EchD subunitDVU0431

0.000162.06cytoplasmic Ech hydrogenase, EchE subunitDVU0430

No significantExpression changeperiplasmic [NiFeSe] hydrogenase, small subunit (hysB)DVU1917

0.0059-0.73periplasmic [NiFe] hydrogenase, large subunit, isozyme 2 (hynA)DVU2526

No significantExpression changeputative [Fe] hydrogenaseDVU1771

0.0081-0.71L-lactate dehydrognase (ldh)DVU2451

0.0023-0.83periplasmic [NiFe] hydrogenase, small subunit, isozyme 2 (hynB)DVU2525

No significantExpression changeperiplasmic [NiFe] hydrogenase, large subunit, isozyme 1 (hynA)DVU1922

0.0322-0.52periplasmic [NiFe] hydrogenase, small subunit, isozyme 1 (hynB)DVU1921

0.0003811.54periplasmic [Fe] hydrogenase, small subunit (hydB)DVU1770

0.0006791.21periplasmic [Fe] hydrogenase, large subunit (hydA)DVU1769

P valueLog2 ratio AnnotationGene

Sean Caffrey, Hyung-Soo Park and Gerrit Voordouw- Department of Biological Sciences, University of Calgary, Calgary, Alberta, T2N 1N4, Canada1SP

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 (H2). H2 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 H2 concentrations, whereas the nickel-containing Hases may be required for growth at low H2 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 oC in 1 L bottles containing 600 mL of culture in an anaerobic culturing system with 50%, 5%, or 0.5% (vol/vol) H2 atmospheres. The H2 was supplied by bubbling without stirring the liquid phase. When cultured at 50% H2 the wild-type and hyn-1 strain had similar doubling times around 17 h, while the hydmutant strain’s doubling time was much longer at 36 h. At 5% H2 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 H2 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 H2 partial pressures and that as the H2 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 H2concentration 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 H2concentrations.

Organism Importanceo SRB are ubiquitous in anaerobic soils and sedimentso SRB have a significant function in global carbon and sulfur cycleso the production of HS- by Sulfate Reducing Bacteria has a considerable economic impact on the petroleum and mining industrieso 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 H2 ↔ 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 H2concentrations and the [NiFe] maybe require for efficient growth on lower H2partial pressures

to determine whether each of the periplasmic hydrogenases are utilized differentially when D. vulgaris is grown at various H2 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 H2 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 µM1 µM467n.d.120[NiFeSe]

Not Sensitive>>0.02 µM20-30 µM4401 µM1500[NiFe]

Sensitive0.02-0.04 µM0.1 µM4800100 µM50000[Fe]

NO2-NO Inhibition

(amount needed for 50% decrease in activity)

CO Inhibition(amount needed for 50% decrease in activity)

Specific Activity

Evolutionµmol H2/min/mg protein

Km Uptake

Specific activity Uptakeµmol H2/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% H2.

•Since neither the [NiFe]-1 nor the [Fe] hydrogenase are needed for growth below 1% H2the 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. Log2 ratio is 50% H2/gDNA versus Lactate/gDNA.

Growth Conditions (Figure 2)o gassed batch culture at 30ºC with defined media utilizing either Lactate or H2as electron donoro gas bubbled at 80 ml/min (H2, N2, and CO2) 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 arrayso RNA extracted from log phase batch cultures with Trizol/Bacterial Maxo indirect Cy3/Cy5 labeling of amino-modified cDNAo 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

SO42 - APS HSO3

- HS-

ATP

SatSat Aps

AMP

2H+6H+

periplasm

cytoplasm

8H+ + 8e- 4H2

PeriplasmicHydrogenasesPeriplasmic

Hydrogenases

ATP Synthase

ATP Synthase

4H2

8e- + 8H+

2 lactate 2 acetate+ 2 CO2

2 ATP2 ADP

, Pta, Ack

+ 2 Pi

6e-2e-

CytoplasmicHydrogenase

ATP ADP + Pi

TransmembraneComplexes

LactateOxidation

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% H2

5% H2

0.5% H2A

50% Hydrogen versus Lactate

log 2 intensity Lactate/gDNA

Log2

inte

nsity

50%

Hyd

roge

n/gD

NA

Figure 3:

A- gDNA is used as a common reference for the analysis of 50% H2, 5%2, and 0.5% H2

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% H2versus RNA extracted from cells grown with lactate.

C

350 Co

H2

184.3160.0152.70.5 % H2

54.6 +/- 10.839.3 +/- 7.238.7 +/- 6.25.0 % H2

36 +/- 6.416.9 +/- 3.617.0 +/- 6.550 % H2

7.36.97.0Lactate

[Fe] mutant[NiFe]-1 mutantWild-typeDoubling Time (Hours)Electron Donor

184.3160.0152.70.5 % H2

54.6 +/- 10.839.3 +/- 7.238.7 +/- 6.25.0 % H2

36 +/- 6.416.9 +/- 3.617.0 +/- 6.550 % H2

7.36.97.0Lactate

[Fe] mutant[NiFe]-1 mutantWild-typeDoubling 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 H2 concentration is decreased the difference in doubling times between the [Fe] and [NiFe]-1 mutant strains decreases (Figure 4)

Microarray Analysis 50% H2 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 H2concentration 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

Inst

anta

neou

s G

row

th R

ate

h-1

DvH[NiFe]-1[Fe]