PCC 6803Additional homologues of hypA and hypB are not active in hydrogenase maturation Do¨rte Hoffmann, Kirstin Gutekunst, Monika Klissenbauer, Ru¨diger Schulz-Friedrich and Jens Appel
Trang 1of Synechocystis sp PCC 6803
Additional homologues of hypA and hypB are not active in
hydrogenase maturation
Do¨rte Hoffmann, Kirstin Gutekunst, Monika Klissenbauer, Ru¨diger Schulz-Friedrich and Jens Appel Botanisches Institut, Christian-Albrechts University, Kiel, Germany
The large subunit of heterodimeric NiFe-hydrogenases
contains a metal complex of a nickel and an iron ion
The two are held in close proximity by two disulfide
bridges provided by two cysteine residues of the
pro-tein The iron has two cyanide ions and one carbon
monoxide as ligands, whereas the nickel ion is
coordi-nated by two additional cysteines [1,2] This metal
cen-tre is at the heart of hydrogen oxidation and proton
reduction Its assembly depends upon the presence of
at least six genes collectively called hydrogenase
pleio-tropic (hyp) because of the pleiopleio-tropic effect of their deletion on the synthesis of all hydrogenases in Escheri-chia coli[3] In a last step, a hydrogenase-specific prote-ase cleaves a C-terminal peptide from the protein Many investigations, especially into the processing
of the large subunit of hydrogenase 3 (HycE) from
E coli, have unraveled the role of the proteins enco-ded by the hyp genes [3] Although the origin of the carbon monoxide is still not known, the cells have
to provide carbamoylphosphate for the production of
Keywords
hyp genes; cobalt transport; arginase;
agmatinase; cyanobacteria
Correspondence
J Appel, Botanisches Institut, University of
Kiel, Am Botanischen Garten 1–9,
24118 Kiel, Germany
Fax: +49 431 880 4238
Tel: +49 431 880 4237
E-mail: jappel@bot.uni-kiel.de
(Received 13 May 2006, revised 27 June
2006, accepted 9 August 2006)
doi:10.1111/j.1742-4658.2006.05460.x
Genes homologous to hydrogenase accessory genes are scattered over the whole genome in the cyanobacterium Synechocystis sp PCC 6803 Deletion and insertion mutants of hypA1 (slr1675), hypB1 (sll1432), hypC, hypD, hypE and hypF were constructed and showed no hydrogenase activity Involvement of the respective genes in maturation of the enzyme was con-firmed by complementation Deletion of the additional homologues hypA2 (sll1078) and hypB2 (sll1079) had no effect on hydrogenase activity Thus, hypA1and hypB1 are specific for hydrogenase maturation We suggest that hypA2 and hypB2 are involved in a different metal insertion process The hydrogenase activity of DhypA1 and DhypB1 could be increased by the addition of nickel, suggesting that HypA1 and HypB1 are involved in the insertion of nickel into the active site of the enzyme The urease activity of all the hypA and hypB single- and double-mutants was the same as in wild-type cells Therefore, there seems to be no common function for these two hyp genes in hydrogenase and urease maturation in Synechocystis Similar-ity searches in the whole genome yielded Slr1876 as the best candidate for the hydrogenase-specific protease The respective deletion mutant had no hydrogenase activity Deletion of hupE had no effect on hydrogenase activ-ity but resulted in a mutant unable to grow in a medium containing the metal chelator nitrilotriacetate Growth was resumed upon the addition of cobalt or methionine Because the latter is synthesized by a cobalt-requiring enzyme in Synechocystis, HupE is a good candidate for a cobalt transpor-ter in cyanobactranspor-teria
Abbreviation
hyp, hydrogenase pleiotropic.
Trang 2the cyanide ligands [4,5] Corresponding homologues
of the genes of the carbamoylphosphate synthase
genes (carAB) can be found in all cyanobacteria
sequenced to date The carbamoyl group is
trans-ferred to the C-terminal cysteine of HypE This step
is catalysed by HypF via a carbamoyl-AMP
interme-diate In an ATP-dependent dehydration reaction the
C-terminal thiocarboxamide of HypE is then
trans-formed into a thiocyanate, which is suggested to be
the precursor of the cyanide ligands [6,7]
Iron is probably introduced into this process by a
complex of HypC and HypD [8] At this complex, the
coordination of the CN and CO ligands to iron might
be accomplished HypC was also shown to form a
complex with the large subunit of the hydrogenase,
thus serving as a kind of chaperone [9,10] In
Thio-capsa roseopersicina, two homologues of HypC have
been shown to be necessary for the expression of all
three hydrogenases It was discussed that one of them
works as a chaperone and the other could be part of a
complex with HypD [11]
HypA and HypB are responsible for the insertion of
nickel A number of bacterial sequences reveal a
N-terminal domain containing stretches of histidine
residues in HypB These residues have been shown to
bind up to 18 nickel ions per dimer in Bradyrhizobium
japonicum, thus functioning as a storage site [12] The
C-terminal domain has a high similarity to GTPases
HypB of E coli hydrolyses GTP when processing the
large subunit of hydrogenase 3 [13,14] The N-terminal
histidine residue of HypA also takes part in the
pro-cess and seems to play an essential role during nickel
insertion [15] In Helicobacter pylori, HypA and HypB
have been shown to be part of the maturation of
urease also This result is surprising because all the
genes for processing urease are found in the genome
of H pylori [16,17]
In the final step of the maturation process, a
hydrogenase-specific protease cleaves the C-terminal
peptide from the large subunit For each hydrogenase
there is a specific protease, and the uncleaved peptide
seems to stabilize the protein during the maturation
process [3] Bioinformatic analysis suggests that the
two hydrogenases of cyanobacteria also need two
dif-ferent proteases for their proper processing [18]
Synechocystis sp PCC 6803 harbours a single
bidi-rectional NiFe-hydrogenase All the homologues of the
hyp genes are spread over the chromosome as single
genes or in different gene clusters [19] Two
homo-logues of hypA and hypB are present Because of
sequence similarities, we tentatively named the genes
slr1675 hypA1, sll1078 hypA2, sll1432 hypB1 and
sll1079 hypB2 The only hyp genes encoded in the same
gene cluster are hypA2 and hypB2 A similarity search
in the complete genome did not uncover a second copy
of hypC In addition to these genes, slr2135 was anno-tated as hydrogenase accessory (hupE) in the cyano-base (http://www.kazusa.or.jp/cyano/) All the HupE homologues are membrane proteins and are predicted
to contain at least six transmembrane helices The hupEof Rhizobium leguminosarum was hypothesized to encode a nickel transporter [20]
In an attempt to characterize the genes needed for the expression of an active hydrogenase in Synechocys-tis, we deleted all of the hyp genes and hupE, and characterized the corresponding mutants Because it is known that different copies of hypA and hypB can complement each other in Ralstonia eutropha (now renamed Cupriavidus necator) [21], the corresponding double mutants were also constructed
Results and Discussion
Characterization of hyp mutants Deletion and insertion mutants of hypA1 (slr1675), hypA2 (sll1078), hypB1 (sll1432), hypB2 (sll1079), hypC, hypD, hypE, hypF, slr1876, and hupE were cre-ated The vectors constructed and the primers used to amplify the DNA fragments are listed in Tables 1 and
2 After streaking transformants 5–6 times on agar plates, they were tested for proper segregation by Southern blotting or PCR All mutants could be segre-gated completely
Hydrogenase activity measurements revealed that the enzyme was only active in wild-type cells, hypA2::Km, hypB2::Km and DhupE None of the other mutants exhibited hydrogenase activity (Fig 1A) It could thus be concluded that all of the investigated hyp genes, apart from hypA2 and hypB2, are essential for hydrogenase processing in Synechocystis
Similarity searches in the genome of Synechocystis using HoxW of R eutropha and HycI of E coli yielded Slr1876 as the best candidate for a
hydrogenase-speci-fic protease with a similarity of 54 and 43%, respect-ively Because deletion of slr1876 resulted in a mutant without hydrogenase activity, Slr1876 can be tenta-tively assigned to the proteases needed to cleave the C-terminus of the large hydrogenase subunit It seems appropriate to name it HoxW
In order to rule out that any of the mutations affec-ted the transcription of the hydrogenase structural genes, all mutants were tested by RT-PCR A hoxH transcript was detected in all of them (Fig 1B), con-firming that the phenotype of the investigated hyp mutants is not due to an abolished transcription
Trang 3Table 1 Bacterial strains and plasmids used in this study.
E coli
U169 recA1 endA1 hsdR17(rk, mk+ ) phoA supE44 – thi-1 gyrA96 relA1 Synechocystis
sp PCC 6803
Plasmids
pBlueGM Source of Gm r cassette; amplified with
with Gen-up, Gen-down-primers, digested with SalI and inserted in the SalI site of the pBlueskript SK
Derivative of pBlueskript SK- with the Gmr-cassette from pUC119 [42]
pBluescript SK- pUC19-derivative, with bla- and lacZ gene, Amp r Stratagene, Heidelberg, Germany
3¢-ends; T7- and Sp6-promotors;
lacZ gene; Amp r
Promega, Madison,WI, USA
pKS-CAT pBluescript SK-containing SspI–NaeI fragment of
pBR322 with Cm r cassette inserted into EcoRV site
This study
amplified with NhypA1 and ChypA1-primers
This study
amplified with NhypA2 and ChypA2-primers
This study phypB1 pMOSblue-T vector containing hypB1 (sll1432),
amplified with NhypB1 and ChypB1-primers
This study phypB2 pMOSblue-T vector containing hypB2 (sll1079),
amplified with NhypB2 and ChypB2-primers
This study
amplified with NhypC and ChypC-primers
This study
amplified with NhypD and ChypD-primers
This study
amplified with NhypE and ChypE-primers
This study
amplified with NhypF and ChypF-primers
This study
amplified with NhoxW and ChoxW-primers
This study
hypA1 for homologous recombination and an inserted Cm r cassette
This study
inserted Kmrcassette in the BclI –site
This study
an inserted Sp r cassette in the BalI sites
This study
an inserted Km r cassette in the EcoRI site
This study
homologous recombination and an inserted Gm r cassette
This study
inserted Sp r cassette in the Eco91I site
This study
inserted Kmrcassette in the BclI sites
This study pDF pGEM-T vector with the hypF gene and an inserted Sp r
cassette in the HindIII sites
This study
homologous recombination and an inserted Sp r cassette
This study
Trang 4To perform complementation studies a vector
(pDH1) was constructed that allows the expression of
any ORF under the control of the psbAII promotor
Constructs of all different hyp genes were made
Because pDH1 confers resistance to kanamycin it
could not be used to complement the DhypE mutant
All constructs except those containing hypA2 and
hypB2were able to restore hydrogenase activity to the
wild-type level in the respective mutants This confirms
that the absence of hydrogenase activity was due to
the specific mutation and not to some unpredictable
side effects
Function of hypA and hypB in hydrogenase
maturation
The function of hypA and hypB was investigated in
more detail Because HypA and HypB were shown to
be involved in the insertion of nickel into the active
site of hydrogenases in E coli [13–15], the respective
Synechocystis single- and double-mutants of hypA and
hypBwere grown in medium supplemented with nickel
Because we found nickel to inhibit growth and elicit
cell death at 50 lm, its concentration was not raised
above 15 lm to keep cells growing Hydrogenase
activ-ity could be increased from 0% of the wild-type level
in the DhypA1 and DhypB1 to 19 and 30%, respect-ively (Fig 2) This suggests that hypA1 and hypB1 play
a role in the insertion of Ni2+into the cyanobacterial hydrogenase
As no changes in hydrogenase activity were detected
in hypA2 and hypB2 mutants compared with wild-type cells, and neither was able to complement their respect-ive counterparts in the DhypA1 and DhypB1, the tran-scription of both genes was tested by RT-PCR Both hypA2 and hypB2 were shown to be transcribed, thus ruling out that these additional homologues are silent
in Synechocystis (Fig 3)
In H pylori it was shown that the hydrogenase-processing genes hypA and hypB have a dual func-tion, as they are also necessary for proper processing
of the nickel-containing urease, despite, as in Syn-echocystis, the respective genes ureE and ureG being present in this organism [16,17] The urease activity
of all hypA and hypB mutants (DhypA1,hypA2::Km, DhypA1hypA2::Km, DhypB1,hypB2::Km, DhypB1-hypB2::Km) was investigated An enzyme activity of 0.025 UÆmg)1 protein was measured, showing no dif-ference compared with wild-type cells Interdif-ference of the urease- and hydrogenase-processing pathways in Synechocystis, as described for H pylori, was there-fore excluded
Table 1 (Continued).
recombination and an inserted Gm r cassette
This study
primers N-PsbA2 and C-psbA2
This study
fragment of pSBA2 inserted into KpnI site cut with ScaI religated, partially digested with SalI and religated, Kmr
This study
of the phypA1vector inserted in the SalI ⁄ NdeI site
This study
of the phypA2 vector inserted in the SalI ⁄ NdeI site
This study
of the phypB1 vector inserted in the SalI ⁄ NdeI site
This study
of the phypB2 vector inserted in the SalI ⁄ NdeI site
This study
of the phypC vector inserted in the SalI ⁄ NdeI site
This study
of the phypD vector inserted in the SalI ⁄ NdeI site
This study
of the phypE vector inserted in the SalI ⁄ NdeI site
This study
of the phypF vector inserted in the SalI ⁄ NdeI site
This study
of the phoxW vector inserted in the SalI ⁄ NdeI site
This study
Trang 5Table 2 Primers used for the amplification of deletion constructs and RT-PCR Underlined sequence corresponds to tag.
AGTTGGAACTAGCATCCCTAGAAC
TCCATCAGACTAACTTCGTGCA
AGACTTGGCAGAAATGGGAGTTT
CTGGCCAGGTAGGGCTAGACACA
CCCCTTGGTATTGGGGGAGAGAT
GGACTTGGTGGATTGGCCTGGCA
CCCTTTTTCCACAGG
GGATAATAGGTTGC
AGGCACGAACCCAGT
GTTAGGTGGCGGTACTT
Primer for RT-PCR:
Trang 6Investigations of the immediate vicinity of hypA2
and hypB2 on the chromosome revealed an ORF
annotated as an agmatinase (speB2, sll1077) directly
upstream of hypA2 Studies on arginine catabolism in
Synechocystis could not clearly assign the substrate of
SpeB2 [22] Agmatinases belong to arginase-related
enzymes that catalyse the splitting of guanidinium
groups to urea and amino compounds Arginases are
known to contain a binuclear manganese active site
[23–25] The complete sequences of all available
cyano-bacterial strains show homologues to speB2 in Synechococcus WH 8102, Synechococcus WH 5701, Synechococcus CC 9605, and Synechococcus sp PCC 7002 Strikingly, in all cases, genes highly sim-ilar to hypA and hypB are situated immediately downstream on the chromosome (Fig S1) The trans-ition metal inserted into the active site of SpeB2 might
be manganese, nickel or cobalt Whether HypA2 and HypB2 are metallochaperones involved in the process-ing of this enzyme needs to be further investigated
Table 2 (Continued).
CACTCTCCAAAAACACCATATCCA
Fig 1 (A) Hydrogenase activity of wild-type cells and of deletion mutants of the hydrogenase accessory genes and DhupE (B) RT-PCR with RNA of wild-type and all the deletion mutants of the hyp genes and hoxW For each strain, the reaction including reverse transcription (+) and a negative control without reverse transcription was applied.
Trang 7Effect of deletion of hupE on metal uptake
Homologues of hupE⁄ ureJ are widespread among
bacteria and are frequently found in hydrogenase or
urease gene clusters Because urease and hydrogenase
are nickel-dependent enzymes, the encoded proteins
were suggested to belong to a family of nickel⁄ cobalt
transporters with six to seven transmembrane helices,
and were subsequently shown to transport nickel in
the proteobacterium Rhodopseudomonas palustris
[26,27] Moreover, because hupE is annotated as a
hydrogenase accessory gene in the cyanobase, we
investigated the effect of its deletion on hydrogenase
and urease activity and metal transport
Surprisingly, DhupE did not show a difference com-pared with wild-type cells concerning its hydrogenase activity (Fig 1A) or its urease activity (data not shown), although the nickel content of the BG-11 med-ium used was beyond the detection limit (1.7 nm) Moreover, its growth did not differ from that of wild-type cells under normal conditions (Fig 4) We there-fore used the metal chelator nitrilotriacetate added to the medium to compare the growth of mutant and wild-type cells Whereas the growth rate of wild-type cells was reduced to about half that of the control, the mutant was no longer able to grow at all, supporting the hypothesis that HupE is a metal transporter (Fig 4)
As shown in Fig 4A, the growth inhibitory effect
of nitrilotriacetate in case of the hupE mutant was partially antagonized by the addition of methionine, whereas the effect on wild-type cells was only margi-nal In Synechocystis, methionine is synthesized by the methionine synthase MetH, which catalyses a cobal-amin-dependent methyl transfer from methyl tetra-hydrofolate to homocysteine In the total genome no other enzymes catalysing the last step of methionine synthesis, for example MetE, could be found There-fore, this experiment suggests that the mutant is suffering from cobalt limitation It also shows that nickel-dependent enzymes like hydrogenase and urease are not essential for growth in Synechocystis, because under these conditions trace amounts of nickel should
be masked quantitatively by nitrilotriacetate This is in accordance with previous results showing that lack of NiFe-hydrogenase has no influence on growth in Syn-echocystis [28] and that deletion of the urease yields viable mutants in Synechococcus sp PCC 7002 [29] Growth of the mutant was also resumed by the addition of cobalt at a concentration of 100 lm This
is in clear contrast to wild-type cells, in which no
Fig 2 Hydrogenase activity of cultures of wild-type cells and the
hypA- and hypB-deletion mutants grown with the addition of nickel
up to a concentration of 15 l M
Fig 3 Agarose gel electrophoresis of RT-PCRs of transcripts of hypB1, hypB2, hypA2B2, ureG and ureC RT-PCR of wild-type RNA was performed as described in the Experimental procedures For each RT-PCR a reaction including reverse transcription (+), and a negative con-trol without reverse transcription (–) was applied On the left marker bands are indicated.
Trang 8difference could be detected in the presence of
nitrilo-triacetate with or without the addition of cobalt
(Fig 4B) By adding 200 lm cobalt, the growth of the
mutant could be increased further, but above this
con-centration toxic effects began to show, and no increase
in growth rate could be attained at 300 lm cobalt A
growth-inhibitory effect was detectable at 200 lm in
wild-type cells (data not shown) Therefore, DhupE is
dependent on additional supplementation, whereas
wild-type cells do not suffer from cobalt limitation
The addition of nickel to the medium supplemented
with nitrilotriacetate increased growth of both the
hupEdeletion mutant and wild-type cells Addition of
100 lm Ni2+allowed wild-type cells to grow almost as
fast as controls, whereas the growth-inhibitory effect
of nitrilotriacetate could not be abolished in DhupE
However, by adding 200 and 300 lm, growth could be
continuously increased Above 300 lm Ni2+no further
increase could be elicited, probably because of toxic
effects (data not shown) Because nickel is bound by
nitrilotriacetate with a six times higher affinity than
cobalt [30], the addition of nickel shifts the binding
equilibrium of the cobalt in the medium to higher
con-centrations of the free ion The added nickel also
allows the expression of urease and hydrogenase in the
cells and leads to an increased growth rate in wild-type cells Taking these results into consideration, we assume that HupE is a cobalt transporter Whether HupE is also able to transport nickel remains to be shown Nevertheless, it should be concluded from our results that there is another uptake pathway apart from HupE for nickel in Synechocystis that allows the mutant to take up nickel at concentrations < 1.7 nm Recent bioinformatic analysis of a large set of bacterial genomes suggests that the five genes sll0381 to sll0385 encode an ATP-dependent nickel transporter in Syn-echocystis [31]
Transport of cobalt by HupE is supported by a suggested vitamin B12-dependent riboswitch which was detected upstream of its gene [32] This type of riboswitch is thought to be involved in the vitamin B12-dependent transcriptional regulation of downstream genes Our growth analysis supports the suggestion that HupE is needed for the expression of a functional methionine synthase that is dependent on vitamin B12 These results are also very interesting regarding investigations of the demand for cobalt in marine cyanobacteria [33,34] It is plausible that the investi-gated strains Prochlorococcus and Synechococcus need
a specific cobalt transporter Similarity searches in the
Fig 4 Growth curves of wild-type cells and DhupE in the absence or presence of nitrilotriacetate (NTA) (A) Growth of wild-type cells and DhupE in the presence of nitrilotriacetate supplemented with 0.25 l M methionine (B) Wild-type cells and DhupE grown in the presence of nitrilotriacetate and different cobalt concentrations (C) Wild-type cells and DhupE grown in the presence of nitrilotriacetate and different nickel concentrations The control curves in normal medium without additions are shown for comparison.
Trang 9marine Synechococcus WH 8102 and the
Prochlorococ-cusstrains available at cyanobase revealed the presence
of orthologues to hupE in all their genomes
Experimental procedures
Cultivation and growth experiments
Synechocystis sp PCC 6803 was grown in BG-11 medium
as described previously [28] Transformants were selected
on BG-11 agar plates containing 50 lgÆmL)1 kanamycin,
25 lgÆmL)1 chloramphenicol, 20 lgÆmL)1 spectinomycin or
5 lgÆmL)1 gentamicin, respectively Total segregation was
checked by PCR and Southern hybridization For growth
experiments cultures were bubbled with air
Cloning procedures
DNA cloning and PCR amplification were performed using
standard procedures [35] In order to construct the deletion
mutants (Table 1) the primers listed in Table 2 were used
to amplify DNA fragments Using two different strategies,
an antibiotic resistance cassette was transferred into the
respective gene for the following homologous
recombina-tion into the genome The hypB1 and hypB2 genes were
cloned into the pMOSblue-T vector (Amersham, Freiburg,
Germany) and hypA2, hypD, hypE and hypF were cloned
into the pGEM-T vector (Promega, Madison, WI, USA)
The respective antibiotic resistance cassette was then
inser-ted after restriction digests The mutants of hypA1, hypC
and hoxW were produced through a PCR fusion, adapted
from Chenchick et al [36], of 300 bp PCR products made
from the regions up- and downstream of the gene with a
resistance cassette The PCR fusion (step 1, 95C for
1 min; step 2, 95C for 30 s; step 3, 60 C for 5 min; step
4, 68C for 25 min; 30 cycles from step 2 to step 4) was
accomplished by complementary overhangs of the primers
P2 and P3 (Table 2) with the antibiotic resistance cassette
To express the hyp genes in a different locus a vector was
constructed using the constructed pIGA vector of Kunert
et al [37] For this purpose the psbAII promotor was
ampli-fied with the primers FpsbA2 and RpsbA2 (Table 1) The
resulting fragment was cut with KpnI and ligated in the KpnI
site of pIGA This vector was digested with ScaI and
religat-ed The resulting vector was partially digesting with SalI and
after blunt ending with Klenow religated to yield the vector
pMK1 In the NdeI and SalI of the pDH1 the amplified
ORFs of the different hyp genes were inserted All constructs
were sequenced before transformation in Synechocystis
Southern blot hybridization
For Southern analyses, genomic DNA was isolated from
Synechocystis cells grown on agar plates Cells were
resuspended in 100 lL TE buffer (10 mm Tris, 1 mm Na2 -EDTA, pH 8.0) and the suspension was supplemented with
an equal volume of glass beads (0.5 mm diameter), 2 lL of
a 10% (w⁄ v) SDS solution and 100 lL phenol ⁄ chloro-form⁄ isoamylalcohol (25 : 24 : 1 v ⁄ v ⁄ v) The mixture was vortexed three times for 10 s and then centrifuged at
10 000 g for 10 min The supernatant was extracted once with phenol⁄ chloroform ⁄ isoamylalcohol and twice with chloroform⁄ isoamylalcohol (24 : 1 v ⁄ v) and centrifuged at
10 000 g for 2 min, respectively The DNA was precipitated
by the addition of 1⁄ 10 volume sodium acetate (3 m,
pH 6.5) and 2.5· volume of 100% ethanol for 2 h at )20 C After centrifugation (15 700 g for 15 min at )8 C), the pellet was washed with 70% ethanol Then the pellet was dried in a vacuum centrifuge and resuspended in 20 lL
TE buffer overnight at 4C Southern hybridization was carried out with the Dig-system (Roche Diagnostics GmbH, Mannheim, Germany) as described by the manufacturer
RNA isolation and RT-PCR
Total RNA from Synechocystis was isolated by phenol– chloroform extraction [38] After precipitating the nucleic acids, the pellet was dried and resuspended in TE buffer
An equal volume of 5 m LiCl was added and the mixture was incubated for 1.5 h at )20 C The DNA-containing supernatant was removed after centrifuging at 18 000 g for
30 min at )8 C The LiCl precipitation of RNA was repeated with the pellet for a second time The RNA pellet was resuspended in a small volume of water and treated with DNase I (Roche, Mannheim, Germany), extracted first with phenol⁄ chloroform ⁄ isoamylalcohol (25 : 24 : 1 v ⁄ v ⁄ v) and then twice with chloroform⁄ isoamylalcohol (24 : 1
v⁄ v) Sodium acetate (3 m, pH 6.5) in an amount equalling
1⁄ 10 of the extract volume and ethanol ()20 C) corres-ponding to 2.5 times of the extract volume was added The mixture was incubated for 30 min at)80 C After centrifu-gation (20 min at 15 000 g at 4C) the RNA pellet was washed with 70% ethanol, dried and resuspended in a small volume of water
cDNA was synthesized with a tagged primer (Table 2) that annealed with 15–19 nucleotides specific to the cor-responding gene and carried a tag of 13–14 nucleotides at its 5¢-end as a target for subsequent PCR according to the method of Cobley et al [39] Five hundred nanograms
of RNA were incubated with 5 pmol primer and 20 nm dNTPs for 5 min at 65C The reaction was chilled
on ice Subsequently, 5· buffer (Invitrogen, Karlsruhe, Germany) and 40 U RNase Inhibitor (MBI Fermentas,
St Leon-Rot, Germany) were added After incubating the mixture at 42C for 2 min, 200 U Superscript II (Invitro-gen) was added In a control reaction the reverse tran-scriptase was replaced by water Reverse transcription was performed at 42C for 50 min The reaction was
Trang 10termin-ated by incubating at 70C for 15 min Then the reaction
was immediately chilled on ice and treated with 3 U
RNaseH (Roche, Mannheim, Germany) An aliquot of
2 lL of the RT reaction was used to amplify the cDNA
with a gene specific and an adapter specific primer
(Table 2)
Hydrogen measurements
Hydrogenase activity was determined by a H2-evolution
assay using dithionite and methylviologen as electron donor
as described by Appel et al [28] but with a Clark-type
elec-trode from Hansatech (DW 1 Liquid Clark elecelec-trode,
Hansa-tech Institute, Norfolk, UK) The electrode was connected
to a lab-made control box with a voltage of)600 mV
Urease measurements
Urease activity was determined according to the method of
Kaltwasser & Schlegel [40] with a 0.04 m Tris buffer
(pH 8.0), 0.8 mm alpha-ketoglutarate, 0.03 m urea and 9 U
glutamate dehydrogenase in the assay mixture of 1 mL
The activity was measured at 366 nm with a
spectropho-tometer (Shimadzu UV-2501PC, Kyoto, Japan) at 30C
Detection of nickel and cobalt
Nickel and cobalt concentrations were determined using
AAnalyst300 (Perkin-Elmer, Boston, MA, USA) with
standards of nickel and cobalt from Johnson & Matthey
(Zurich, Switzerland)
Acknowledgements
Financial support from Linde AG,
Innovations-Stiftung Schleswig-Holstein and Studienstiftung des
deutschen Volkes are gratefully acknowledged Special
thanks to T Eitinger (Humboldt-University, Berlin)
for helpful discussions We thank Sabine Karg and
Monika Schneeweiss for excellent technical assistance
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