Molecular analysis of strains showing mutant phenotypes led to the identification of hupK hoxV , hypC1, hypC2, hypD, hypE, and hynD genes.. In-frame deletion muta-genesis showed that HypC
Trang 1Accessory proteins functioning selectively and pleiotropically in the
Gergely Maro´ti, Barna D Fodor, Ga´bor Ra´khely, A´kos T Kova´cs, Solmaz Arvani and Korne´l L Kova´cs Institute of Biophysics, Biological Research Center, Hungarian Academy of Sciences, and Department of Biotechnology,
University of Szeged, Hungary
There are at least two membrane-bound (HynSL and
HupSL) and one soluble (HoxEFUYH)
[NiFe]hydrogen-ases in Thiocapsa roseopersicina BBS, a purple sulfur
photosynthetic bacterium Genes coding for accessory
pro-teins that participate in the biosynthesis and maturation of
hydrogenases seem to be scattered along the chromosome
Transposon-based mutagenesis was used to locate the
hydrogenase accessory genes Molecular analysis of strains
showing mutant phenotypes led to the identification of hupK
(hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes The
roles of hynD, hupK and the two hypC genes were
investigated in detail The putative HynD was found to be a
hydrogenase-specific endoprotease type protein,
participa-ting in the maturation of the HynSL enzyme HupK plays an
important role in the formation of the functionally active membrane-bound [NiFe]hydrogenases, but not in the bio-synthesis of the soluble enzyme In-frame deletion muta-genesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme The lack of either HypC protein drastically reduced the activity of every hydrogenase Hence both HypCs might participate in the maturation of [NiFe]hydrogenases Homologous comple-mentation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2metabolism of T roseopersicina
Keywords: hydrogenase; accessory genes; pleiotropic; metalloenzymes; [NiFe]center biosynthesis
Hydrogenases (EC class 1.12.1) [1]have the capability to
reduce protons or oxidize molecular hydrogen They are
ancient metalloenzymes present in many archaea and
bacteria, as well as occasionally in eukaryotes Some
microorganisms are known to contain several distinct
hydrogenase enzymes [2]that vary in their cellular location
Two major groups of hydrogenases are distinguished
according to their metal content, the Fe and the [NiFe]
hydrogenases [1–3] The [NiFe] hydrogenases are composed
of at least two subunits The small subunit transfers
electrons via Fe–S clusters, while the large subunit contains
the unique heterobinuclear [NiFe]metallocentre, which is
the catalytic site In the active centre two CN and one CO
ligands are associated with the Fe atom [4] The formation
of an active hydrogenase requires a complex maturation
process, including the incorporation of metal ions (Fe, Ni)
and CO and CN ligands in the active centre, the orientation
of the Fe–S clusters within the small subunit, and the proteolytic cleavage of the C-terminal end of the large subunit by an endoprotease [5,6] Several steps in this maturation process have recently become understood The HypF and HypE proteins were proven to play a key role in providing the CO and CN ligands from carbamoyl phosphate [7–9] A complex of two other pleiotropic accessory gene products, the HypC and HypD proteins has been assumed to carry the iron atom during ligand formation and the assembled Fe-complex is somehow transferred to the C-terminal part of the hydrogenase large subunit as the HypC–HypD proteins dissociate [10–12] There are additional accessory proteins, which are essential
in the synthesis of mature [NiFe]hydrogenases although their particular role in the hydrogenase biosynthesis is less clear at this time Some of these proteins are pleiotropic, as they participate in the biosynthesis of each [NiFe]hydro-genase present in the cell Other accessory proteins are specific enzymes, that play a role only in the formation of a single hydrogenase [6]
In Ralstonia eutropha, the hypA, hypB and hypF genes are duplicated and any of the cognate gene products can mature the hydrogenases in this strain [13] It is an intriguing question, why two copies of the pleiotropic enzymes are needed, if one of them is sufficient to carry out the biological function? Remarkably, the chaperon-like entity, HypC, is a pleiotropic protein, although two variants of this protein have been identified in Escherichia coli HypC is indispens-able for the maturation of the hydrogenase 3 in E coli, although it can replace the function of the similar chaperon-type protein, HybG, in the maturation of hydrogenase 1 but not of hydrogenase 2 [14] There are also two copies of the
Correspondence to K L Kova´cs, Department of Biotechnology,
University of Szeged, H-6726 Szeged, Temesva´ri krt 62, Hungary.
Fax: + 36 62 544 352, Tel.: + 36 62 544 351,
E-mail: kornel@nucleus.szbk.u-szeged.hu
Enzymes: Hydrogenases (EC 1.12.1).
Note: Preliminary results were presented at the Biohydrogen 2002
Conference, Ede-Wageningen, NL, April 21–24, 2002 and reviewed
in Kova´cs, K L., Fodor, B., Kova´cs, A´ T., Csana´di, G., Maro´ti,
G., Balogh, J., Arvani, S & Ra´khely, G (2002) Hydrogenases,
accessory genes and the regulation of [NiFe]hydrogenase biosynthesis
in Thiocapsa roseopersicina Int J Hydrogen Energy 27, 1463–1469.
(Received 29 January 2003, revised 12 March 2003,
accepted 24 March 2003)
Trang 2HypC family members in Ralstonia eutropha, Rhodobacter
capsulatusand Rhizobium leguminosarum [2]
Thiocapsa roseopersicina BBS is a mesophilic purple
sulfur photosynthetic bacterium, containing at least two
membrane-bound (HynSL, and HupSL) [15,16]and a
soluble (HoxEFUYH) (G Ra´khely, Gy Csana´di,
G Maro´ti, B D Fodor & K L Kova´cs, unpublished
observations) [NiFe]hydrogenase No accessory genes
could be identified in the vicinity of the hynSL genes [16]
and the structural genes of the soluble hydrogenase
Downstream from the hupSL structural genes, accessory
genes (hupDHI ) and the hupR gene (corresponding to the
regulator of a two component regulatory system) were
found [15] The lack of accessory genes in the vicinity of the
structural genes is uncommon, as auxiliary genes tend to
form gene clusters in most microorganisms harboring
hydrogenase enzymes [1,2,6,17,18]
Our aim was to find and characterize the accessory genes
needed for the maturation of functionally active
hydro-genases in T roseopersicina and to understand their
physio-logical roles The determination of the specificity of the
accessory proteins is a challenging exercise in this
micro-organism because of the presumed large number of
hydro-genase-related genes A transposon-based mutagenesis
system and a reliable screening method has been established
for T roseopersicina [19] The genetic approach was
devel-oped further for producing in-frame deletion mutants in this
strain Here we show the molecular characterization of the
T roseopersicina mutant strains, where the hydrogenase
biosynthesis is affected specifically and/or pleiotropically
Materials and methods
Bacterial strains and plasmids
Strains and plasmids are listed in Table 1 T roseopersicina
strains were grown photoautotrophically in Pfennig’s
min-eral medium, under anaerobic conditions, in liquid cultures
with continuous illumination at 27–30C for 4–5 days [20]
Plates were solidified with 7 gÆL)1Phytagel (Sigma) [21]and
supplemented with acetate (2 gÆL)1) when selecting for
transconjugants The plates were incubated in anaerobic
jars using the AnaeroCult (Merck) system for two weeks
Escherichia colistrains were maintained on LB-agar plates
Antibiotics were used in the following concentrations
(lgÆmL)1): E coli: ampicillin (100), kanamycin (25),
tetra-cyclin (20); for T roseopersicina: kanamycin (25),
strepto-mycin (5), gentastrepto-mycin (5)
Conjugation
The conjugation was carried out as described in [19]
Transposon mutagenesis
The mini transposon delivery plasmid pUT/mini-Tn5Km
[23]was mobilized from E coli S17-1(kpir) to T
roseo-persicina BBS One hundred colonies were randomly
selected after each mating and screened for a
hydro-genase-deficient phenotype [19] In this work, the M442,
M1250, M4711, M646 and the M1343 mutants were chosen
for detailed molecular analysis
DNA manipulations, PCR, sequencing, Southern blot and sequence analysis
Preparation of genomic DNA, plasmids, cloning and Southern blots were done according to general practice [26], or the manufacturers’ instructions PCR was carried out in a PTC-150 MiniCycler (MJ Research) Sequencing of both strands was done using an automatic Applied Biosys-tems 373 Stretch DNA sequencer The searches in the NBRF, SwissProt, combined EMBL/GenBank and Prosite databases were carried out with the various BLAST programs (http://www3.ncbi.nlm.nih.gov/BLAST/) Mul-tiple alignments were performed with the CLUSTALW
program (DNASIS MAXv1.0, Hitachi Genetic System)
Isolation of the hydrogenase-related genes Partial genomic libraries were prepared from the various mutants in pBluescript SK+ and ampicillin/kanamycin resistant clones were selected A list of the positive clones is given in Table 1 (see also Fig 1) The sequenced genes and regions has been deposited in the GenBank, under the accession numbers AY152822 and AY152823
Constructions for complementations Homologous complementations were performed using pBBR1MCS-5 based vectors [24] On the pM4710 template the following primers were used to amplify the 949 bp PCR fragment carrying the hynD gene: HYDAZ04: 5¢-ATCGG GATACCGAGACACAT-3¢, HYDAZ05: 5¢-AATGGGT TGAACGAGAGTCG-3¢
First, this fragment was cloned into the HincII-digested pBluescribe plasmid (pHDS), then it was recloned into pBBR1MCS-5, as an SphI–SacI fragment (pBRHynD) pBRHupK was constructed by cloning a 2936 bp ApaI– ClaI fragment, containing the hupK gene with its regulatory region, into the ApaI–ClaI-digested pBBR1MCS-5 vector pBRC1 was obtained by inserting the 1753 bp EcoRI– PstI fragment, containing the hypC1 gene, from pM42-5 (pM42-5: the 6.3 kb NotI–BamHI fragment of the pM42-1 was cloned into the pBluescript SK+ NotI–BamHI sites) into EcoRI–PstI-digested pBBR1MCS-5 pBRC2 was pro-duced by insertion of the 552 bp RsaI fragment, containing the hypC2 gene from pM47-13, into SmaI-digested pBBR1MCS-5 pBRCDE homologous complementation vector was constructed in three steps The 1753 bp EcoRI– PstI fragment from pM42-5 was cloned into the EcoRI–PstI digested pBBR1MCS-5, which yielded the pBRC1 con-struct The 293 bp PstI–BamHI fragment (part of the hypD gene), derived from pM1250, was ligated into the PstI– BamHI-digested pBRC1 (pBRCT2) The 1703 bp BamHI fragment (downstream region of the hypD gene and the entire hypE gene) from pM42-8 was transferred into the BamHI-digested pBRCT2, yielding pBRCDE pBRKCDE homologous complementation vector was also constructed
in three steps The 2936 bp ApaI–ClaI fragment (harboring the hupK gene) from pM42-5 was inserted into ApaI–ClaI-digested pBBR1MCS-5, producing pBRHupK pBRKT2 was obtained by cloning the 1069 bp ClaI–BamHI fragment (containing the hypC1and the 5¢ region of the hypD gene) from pM12-50 into the ClaI–BamHI-digested pBRHupK
Trang 3pBRKT2 was digested with BamHI, and the 1703 bp
BamHI fragment from pM42-8 was built into this vector
(pBRKCDE) The homologous complementation
con-structs were transformed into E coli S17-1(kpir) strain, then
conjugated into the appropriate T roseopersicina strains
In-frame deletion mutagenesis
The in-frame deletion vector constructs derived from the
pK18mobsacB vector [25] For deletion of the hupK gene,
the 932 bp EcoRV–Eco47III fragment of pM42-5
(down-stream region of the hupK) was inserted into the SmaI site of pK18mobsacB (pDHuKA) The polished 878 bp BglI fragment from pM42-5 (the upstream homologous region) was ligated into the HindIII digested/blunted pDHuKA, resulting in pDHuK For removal of hypC1 and hypC2 genes, the pDC1 and pDC2 in-frame deletion constructions were created as follows The blunted 1423 bp SacI fragment (the downstream region of hypC1) was cloned from pM42-5 into the SmaI-digested pK18mobsacB (pDC1A) The upstream region of hypC1was amplified with the TRHC101 (5¢-GTTATCCTGAAGCGCGATCA-3¢) and TRHC102
Table 1 Strains and plasmids used in this study.
Thiocapsa roseopersicina
Escherichia coli
S17-1(kpir) 294 (recA pro res mod) Tp r , Sm r (pRP4-2-Tc::Mu-Km::Tn7), kpir [23]
XL1-Blue MRF¢ D(mcrA)183, D(mcrCB-hsdSMR-mrr)173, endA1, supE44, thi-1, recA1,
gyrA96, relA1 lac [F¢ proAB lacI q
ZDM15 Tn10 (Tetr)]
Stratagene
Plasmids
pUTKm Amp r ; Tn5-based mini transposon delivery plasmid with Km r [23]
pBRCDE Gm r , pBBR1MCS-5 carrying the hypC 1 , hypD and hypE genes gene This work
pBRKT2 Gm r , pBBR1MCS-5 carrying the hupK, hypC 1 genes and the 5¢ region of the hypD gene This work
pBRKCDE Gm r , pBBR1MCS-5 carrying the hupK, hypC 1 , hypD and hypE genes This work
pDC1 Kmr, in-frame up and downstream homologous regions of hypC 1 in pK18mobsacB This work
pDC2 Km r , in-frame up and downstream homologous regions of hypC 2 in pK18mobsacB This work
pDHuK Km r , in-frame up and downstream homologous regions of hupK in pK18mobsacB This work
pM12-50 4.3 kb SalI fragment harboring the transposon from M1250 in pBluescript SK(+) This work
pM42-1 8.1 kb BamHI fragment harboring the transposon from M442 in pBluescript SK(+) This work
pM42-8 3.5 kb PstI fragment harboring the transposon from M442 in pBluescript SK(+) This work
pM47-10 7 kb SphI fragment containing the transposon from M4711 in pBluescribe(+) This work
a G Ra´khely, Gy Csana´di, G Maro´ti, B D Fodor & K L Kovacs.
Trang 4(5¢-CTAGACACATGGACAAAAGA-3¢) primers and
the 1441 bp PCR product was cloned into the
HindIII-digested, Klenow filled pDC1A, resulting in pDC1 The
upstream and downstream region of hypC2was amplified
by PCR using Pwo polymerase The following primers were
used: HYDAZ04, HYDAZ05, TRHC201 (5¢-TGAGCA
TGGTCGCAAACACG-3¢), TRHC202 (5¢-GGACGGC
TCGAGGTTTGATC-3¢)
pDC2A was obtained by cloning the HYDAZ04–
HYDAZ05 PCR fragment covering the 949 bp upstream
homologous region of hypC2into the polished SalI site of
the pK18mobsacB vector The 951 bp downstream
homo-logous region was amplified with the TRHC201 and
TRHC202 primers and cloned into the HindIII-digested,
Klenow filled pDC2A (pDC2)
The in-frame deletion constructs were transformed into
E coli S17-1(kpir) strain, then conjugated into T
roseo-persicina BBS, GB11 and GB1121 strains resulting the
in-frame deletion mutants DHKW426 (DhupK BBS),
DHKG517 (DhupK GB11), DC1B (DhypC1BBS), DC1G
(DhypC GB11), DC1H (DhypC GB1121), DC2B (DhypC
BBS), DC2G (DhypC2 GB11), DC2H (DhypC2 GB1121) and DC12B (DhypC1DhypC2BBS) strains Selection for the first recombination event was based on kanamycin resist-ance The selection for the second recombination was based
on the sacB positive selection system In T roseopersicina 3% sucrose was efficient to induce the sacB system [25] The in-frame deletion mutant clones were verified using PCR, Southern analysis and sequencing
RNA isolation, reverse transcription (RT) and PCR RNA was isolated using the TRIzolTM reagent (Gibco BRL), following the manufacturer’s recommendation Prior
to RT-PCR, the RNA was DNase-treated at 37C for
60 min in 40 mMof Tris/HCl (pH¼ 7.5), 20 mMMgCl2,
20 mM CaCl2, 4 U RNase-free DNaseI After phenol/ chloroform extraction and ethanol precipitation, the RNA was dissolved in 20 lL of H2O RT-PCR experiments were carried out as described previously [19] The TRHC102 primer (in hypC1, sequence see above) was used for the reverse transcription and PCR The TRHD04
hypE
A
(8334 bps)
PstI
SalI
NotI
SacI
BamHI
PstI
EcoRV
SacI
ClaI
ClaI
SalI
Eco47III
EcoRI
(5130 bps)
B
BamHI RsaI RsaI BamHIBamHI
Fig 1 Identified hydrogenase accessory genes in the M442, M1250 (A) and M4711 (B) transposon mutant strains PntA is similar to transhydro-genases, orf is a putative conserved protein, tnp seems to encode a transposase Black triangles show the positions where the transposon was inserted The sequences have been deposited with GenBank, accession numbers AY152822 (A) and AY152823 (B).
Trang 5(5¢-TTGCGGTTGTTGAGCCGCTG-3¢) served as the
other primer in PCR Using these primers a 524 bp
fragment could be amplified
Preparation of membrane-associated and soluble
protein fractions ofT roseopersicina
T roseopersicinaculture (300 mL) was harvested in a Sorvall
RC5C centrifuge at 7000 g The cells were suspended in
3 mL of 20 mMK-phosphate buffer (pH 7.0), and sonicated
eight times for 10 s on ice The broken cells were centrifuged
at 10 000 g for 15 min The debris (containing whole cells
and sulfur crystals) was discarded and the supernatant was
centrifuged twice at 100 000 g for 3 h [27] The
ultracentrif-ugation pellet was washed with 20 mMK-phosphate buffer
(pH 7.0) and used as the membrane fraction The
super-natant was considered as the soluble fraction
Hydrogen uptake activity assayin vitro
H2 uptake, coupled to benzylviologen or methylviologen
reduction, was assayed spectrophotometrically at 55C
The harvested cells, membrane or soluble fractions were
suspended in 20 mM K-phosphate buffer (pH 7.0) Two
millilitres of this mixture was placed into a cuvette, 18 lL of
20 mM benzylviologen was added, and the cuvettes were
sealed with SubaSeal stoppers The gas phase was flushed
with N2for 5–10 min and then with H2for 5–10 min
Hydrogen evolution assayin vitro
Sample (0.5 mL) was suspended in 1.2 mL of 20 mM
K-phosphate buffer (pH¼ 7.0) in Hypo-Vials (10 cm3volume,
Pierce) and 1 mL of 1 mM methylviologen was added In
order to measure the activity of the Hyn enzyme selectively,
cells were heat treated at 72C for 30 min prior to the assay
The gas phase was flushed with N2for 10 min, followed by
the anaerobic addition of 0.5 mL of 0.1 gÆmL)1dithionite
Samples were incubated at 40C for 30 min Hydrogen
production was measured by gas chromatograph [19]
Results
Identification and characterization
of the accessory genes
Transposon-based mutagenesis was performed in order to
create a mutant T roseopersicina library and to find the
hydrogenase accessory genes [19] Six of 1600 mutant
colonies showed a hydrogenase-deficient phenotype, five of
which lost all hydrogenase activities and in one case (M646) the hydrogenase activity of the cells was dramatically reduced, but detectable The M442 and M4711 strains were selected for detailed analysis
ThehupK, hypC1, hypD and hypE genes
An approximately 8.1 kb BamHI genomic fragment from the pleiotropic mutant M442 was isolated, subcloned and sequenced The hypC1, hypD and hupK genes were identified
in this clone (Fig 1, Table 2)
Upstream from the hupK gene, no hydrogenase-related gene could be identified, but two ORFs showed significant homology to the two-component regulatory system OmpR– EnvZ [28] In T roseopersicina, the hypD gene starts with GUG, and the Tn5 transposon was inserted at bp 792 of the
1146 bp-long ORF As the BamHI fragment from M442 did not contain the whole hypD gene, an overlapping 3.5 kb PstI genomic fragment was cloned and sequenced The hypE-type gene was found downstream from the hypD gene (Fig 1, Table 2) In a separate hydrogenase-deficient mutant group (M1250), the transposon was inserted into the hypE gene No additional accessory genes were found downstream from hypE (data not shown) The 8334 bp-long region was sequenced on both strands
ThehynD and hypC2genes
A 5130 bp-long chromosomal fragment surrounding the transposon in the M4711 nonpleiotropic mutant was sequenced on both strands Two [NiFe]hydrogenase-related ORFs were found The deduced amino acid sequence of the first ORF showed similarity to the HypC proteins (Fig 1, Table 2) and the characteristic motif at the N-terminus of HypCs, namely M-C-(L/I/V)-(G/A)-(L/I/V)-P [10], could also be aligned The second ORF (named hynD) encoded a putative protein, similar to the hydrogenase-specific endo-proteases of other microorganisms [2] Multiple alignment indicated that the putative HynD was similar to the other [NiFe]hydrogenase-processing proteases, after a GTG codon (data not shown) The start codon of the hynD gene could not be identified There was a long stretch (148 aa) upstream from this GTG without ATG in-frame, but the translated sequence was unrelated to any known protein The codon usage of this upstream region is not character-istic of the known codon usage pattern of T roseopersicina (among the 10 codons preceding the GTG, four are preferred at 1–10% frequency in this strain) If hynD starts
at this codon, the putative HynD enzyme consists of 156 amino acids (16.6 kDa) and the transposon is inserted into
Table 2 Identity between the accessory proteins of T roseopersicina and the corresponding proteins from other organisms.
Organism
T roseopersicina HupK (389 aa) HypC 1 (94 aa) HypD (381 aa) HypE (360 aa) HypC 2 (81 aa) HynD (156 aa)
Trang 6the hynD gene at bp 107 of the 471 bp-long gene Thus, the
hypC2and the hynD genes are separated by 120 bp, and
they are in opposite orientation It should be noted that the
C-terminal end of HynD was slightly shorter than those of
its counterparts from other microorganisms
HynD is a processing endopeptidase-like protein
In the wild type T roseopersicina, all hydrogenase activity,
except that related to HynSL, could be eliminated by an
appropriate heat treatment (see Materials and methods)
Only heat labile hydrogenase activity could be detected in a
DhynSL mutant strain (GB11) Likewise, in the mutant, in
which the hynD gene was disrupted by the Tn5 insertion
(M4711), no heat stable hydrogenase activity was
observ-able A series of hydrogenase activity measurements were
performed using the wild type cells, the hynD::Km (M4711),
the DhynSL (GB11) mutants and the complemented M4711
strain Mutants lacking a functional hynD gene (M4711) or
the heat stable [NiFe]hydrogenase, HynSL (GB11), showed
the same behavior in the activity assays (Fig 2)
Comple-mentation of the hynD gene (pBRHynD) restored the heat
stable HynSL hydrogenase activity to the level of the wild
type control As the in silico analysis of the putative HynD
gene product clearly predicted a [NiFe]hydrogenase
processing endopeptidase, it was concluded that HynD is
a protease carrying out the post-translational modification
of the C-terminus of the large subunit [29,30]during the
maturation of the stable HynSL hydrogenase in T
roseo-persicina
Cotranscription ofhupK and hypC1DE
The hupK (hoxV) gene was separated from hypC1 by
194 bp, the start codon of hypD was overlapping with the
stop codon of hypC1, and hypE started 94 bp downstream
from the stop codon of hypD The distances between the
hupK, hypC1Dand hypE genes are compatible with either an
independent transcription of hupK, hypC1Dand/or hypE, or all of these genes could be cotranscribed In order to test this possibility, RT-PCR analysis was performed on total RNA isolated from T roseopersicina An mRNA species contain-ing both the hupK and hypC1 genes was detected, which indicated the common transcriptional regulation of these genes The transcript, however, appeared very weak (Fig 3), and therefore, independent transcription had to
be considered as well The two possibilities were further examined in additional complementation experiments Two constructs were made in order to complement the strain carrying a hypD::Km mutation (M442) The two constructs differed from each other in the hupK gene and its regulatory region One of them contained the hupK-hypC1DE genes (pBRKCDE), and the other one contained only the hypC1DEgenes (pBRCDE) The presence of the pleiotropic hypEgene in the constructs was necessary because of the possible polar effect of the transposon A similar RT-PCR experiment as above showed that the hypD and hypE genes were cotranscribed (data not shown) Both constructions complemented the mutation in hypD::Km, but the comple-mentation was not complete in either case It was signifi-cantly higher when the construct with hupK was used (18% without hupK and 43% with hupK, respectively, Table 3) These results again corroborate the presence of two sets of regulatory elements, one between hupK and hypC1, and one upstream from hupK To some extent, it would explain the low complementation efficacy obtained in the hypC1 complementation experiments, where hupK was omitted from the complementing construct (see above)
Properties of the HupK protein The role of the HupK (HoxV) in the maturation process of the [NiFe]hydrogenases is unknown Conserved regions could be recognized at the N- and C-termini, while the middle portion of the proteins appeared variable The highest homology was found at the C-terminus and,
H 2
hynSL
wt (BBS)
hynD::Km
complemented (M4711+pBRHynD)
hynD::Km
(M4711)
without heat treatment heat treated
(GB11)
0 1 2 3 4 5 6 7 8
Fig 2 Hydrogen evolution activity of the wild
type and the HynD mutant T roseopersicina
strains The samples were or were not
heat-treated before the measurements (Strains
given in Table 1.) It should be noted, that
HynSL is a thermophilic enzyme, i.e its
activity increases with temperature (at least up
to 80 C) [31] Therefore, heat treatment of the
samples probably activates this hydrogenase,
which explains the higher activity of the
heat-treated samples.
Trang 7remarkably, this region showed significant identity to the
HupL (hydrogenase large subunit) proteins as well,
although half of the conserved cysteines were missing [32]
In-frame deletion mutagenesis was used to determine the
specificity of the HupK protein Thirty one amino acid
residues in the truncated HupK originated from the
N-terminus, 37 aa from the C-terminus of the protein and
13 aa came from the multiple cloning site of the
pK18mob-sacBvector The extensively shortened hupK derivative was
cloned into the wild type and DhynSL (GB11) T
roseo-persicinastrains The physiological effects of the mutation
on the hydrogenase enzyme activities were tested in H2
uptake activity assays of each individual [NiFe]hydrogenase
enzyme in T roseopersicina Approximately 90% of both
HynSL and HupSL activity was lost in comparison to the
wild type (Table 4) On the contrary, the soluble fraction
retained almost all of its activity; around 75% of Hox
activity was detectable in the HupK deleted strain, with
respect to the wild type Homologous complementation with the hupK gene (pBRHupK) fully restored the hydrog-enase activity of the cells (Table 3) This has further proven the selectivity of HupK, which is important for the formation of both functionally intact membrane-associated [NiFe]hydrogenases, but it is not involved in the maturation
of the soluble Hox enzyme in this bacterium
The two HypC accessory proteins The role of the putative HypC proteins was studied by in-frame deletion mutagenesis in T roseopersicina Each hypCgene was deleted from the wild type, the GB11 (HynSL minus) and GB1121 (HynSL and HupSL minus) genomes individually In addition, a double hypC mutant strain was also generated from the wild type T roseopersicina BBS (Table 1) Hydrogenase activity assays, in uptake and evolution directions, were carried out both on membrane and soluble fractions of the various mutant strains The absence of HypC1almost completely eliminated the activity
of all [NiFe]hydrogenases: about 3–5% of the activities of both membrane-bound hydrogenases (Hup and Hyn), and 10% of the cytoplasmic (Hox) hydrogenase activity was detectable in the DhypC1 mutant (Table 4) Homologous complementation with the hypC1gene (pBRC1), containing the hypC1upstream region, yielded incomplete restoration
of activity: only 15% of the wild type activity was measurable (Table 3) The low complementation efficacy might be due either to the lack of the putative promoter preceding the hupK gene, or to the absence of the hypD gene
in the complementing construct, i.e an in-frame deletion of hypC1might also have a polar effect on the expression of hypD(M Blokesch, Lehrstuhl fu¨r Mikrobiologie, Universi-ta¨t Mu¨nchen, Germany) The mutation of the hypC2gene also affected all three hydrogenases, the HupSL and the HynSL activities decreased to 9–10% and the soluble Hox hydrogenase retained only 6% of its activity as compared to the wild type Homologous complementation with the hypC2gene (pBRC2) was complete; the wild type Hup, Hyn and Hox activities of these [NiFe]hydrogenases were restored (Table 3) The results indicate that the two related putative proteins cannot replace one another in the matur-ation of the various hydrogenases
Discussion Thiocapsa roseopersicinaharbors at least three hydrogenase enzymes, two of which are attached to the membrane and one that is located in the cytoplasm Thus, it is intriguing and important to explore the functional relationship
250
500
750
1000
bp
Fig 3 RT-PCR analysis of the cotranscription of the hupK and hypC 1
genes M, marker; bp, base pairs; RT+, reverse transcription was
made before PCR reaction; RT–, reverse transcriptase was omitted;
gC, control PCR made on genomic DNA.
Table 3 H 2 uptake activities in homologous complementation experiments The results are given as a percentage compared to the T roseopersicina wild type strain.
Complementing gene Plasmid hupKD, BBS (DHKW426) hypC 1 D, BBS (DC1B) hypC 2 D, BBS (DC2B) hypD::Km (M442)
Trang 8between the biosynthesis and maturation of the various
hydrogenases Mini Tn5 transposon mutagenesis was used
to identify the hydrogenase accessory genes required for the
maturation of the [NiFe]hydrogenase enzymes in this
particular strain Six independent mutant strains were
isolated from a library of 1600 colonies [19] Besides the
previously identified hypF gene [19], detailed molecular
investigation of the mutant strains resulted in the
identifi-cation of one locus containing the hupK-hypC1DEaccessory
genes and another one, where the hypC2 and hynD genes
were found The organization of the accessory genes in this
bacterium is unusual, as the corresponding genes are
frequently organized into large gene clusters in other
organisms [2,6,17] In order to examine the specificity of
the auxiliary proteins, hydrogenase deletion mutant strains
were generated (G Ra´khely, Gy Csana´di, G Maro´ti, B D
Fodor & K L Kova´cs, unpublished observations), and the
effect of the accessory genes was studied through
hydro-genase activity assay measurements In three mutants the
transposon was inserted into the hypD or the hypE gene
abolishing all hydrogenase activities in the cells The
corresponding gene products have obviously fundamental
roles in the formation of any [NiFe]hydrogenase The
physiological functions of the HynD, HupK and HypC1
and HypC2proteins were investigated in detail
The hynD gene of T roseopersicina showed a high level of
homology to the ORFs encoding the specific endoproteases
of the [NiFe]hydrogenases of other bacteria These
proteases have a function in one of the last steps of
hydrogenase maturation, when the C-terminal end of the
precursor large subunit polypeptide is cleaved, as soon as
the [NiFe]heterobinuclear center with its diatomic ligands
[2,6,29,30]has been successfully assembled and inserted into
the active site of the enzyme Downstream from the hupSLC
genes, the hupD gene was identified, which also encodes a
related putative protein, likely to be involved in the
processing of the HupL subunit [15] It is plausible to
assume that HynD is involved in the maturation of the
HynL protein Indeed, in the strain harboring the Tn5
transposon-inactivated hynD gene no HynSL enzyme
activity could be detected HynSL activity was completely restored by hynD complementation
The location of the hupK gene, upstream from hypC1DE,
is somewhat surprising because this gene has been found in the hup operon of other organisms [2] The distance between hupK and hypC1 raised the question of whether hupK-hypC1DE constituted a single operon or whether the transcription of hupK was regulated separately from hypC1DE Homologous complementation experiments clearly indicated that the hypC1DE genes had their own regulatory element, independent from that of the hupK, but they could also be transcribed from the promoter of the hupKgene RT-PCR analysis between the hupK and hypC1 corroborated these conclusions The role of HupK is ambiguous in the strains studied so far In R eutropha, deletion of hoxV (hupK) reduced the activity of the membrane-bound hydrogenase to 30% compared to the wild type [33] On the contrary, inactivation of hupK led to the accumulation of the immature form of the inactive hydrogenase subunits in R leguminosarum [34] In T roseo-persicinathe activities of both membrane-associated [NiFe] hydrogenases (HynSL and HupSL) decreased dramatically
in the absence of the HupK protein, whereas the soluble HoxEFUYH enzyme remained apparently unaffected Remarkably, this protein does not occur in all microbes containing [NiFe]hydrogenase, hence the role of the HupK protein is still uncertain It resembles the large subunit of the [NiFe]hydrogenases, therefore HupK has been suggested to function as a scaffolding protein during metal cofactor assembly [32] Although our study did not uncover the precise function of HupK, this was the first demonstration that it made a selection among the various [NiFe]hydro-genases in the cell, and participated in the biosynthesis of the membrane-bound ones
HypC is a small, chaperon-like protein that participates in two protein complexes, and thus a dual function has been assigned to it HypC interacted with the large subunit of the hydrogenase 3 (HycE) in E coli [10]and it was recently shown to form a complex with the HypD protein [12] In the model based on the observations in E coli, first the HypC– HypD complex is formed, where the Fe gets liganded by CO and two CN with the involvement of HypF and HypE [9] Then HypC, equipped with the Fe-CO-(CN)2complex, is transferred to the HycE subunit with the concomitant dissociation of HypD [12] HypC selectively interacts with hydrogenase 3 and it can take over the functions of the homologous HybG in processing the hydrogenase 1 to some extent in E coli [14] The molecular phenotype of HypC mutations is strikingly different in T roseopersicina In our case, both HypC proteins are important for the maturation
of all three hydrogenases, i.e both of them have a task in every stage, even if they can partially substitute each other Consequently, both HypCs are truly pleiotropic accessory proteins in T roseopersicina The findings in the two bacteria can be assembled into a generalized [NiFe]hydrogenase maturation scheme if we assume that two HypC proteins are needed in the HypC cycle [12] In our working hypothesis one HypC interacts with HypD, while the other one holds the unprocessed large subunit protein in an open confor-mation Iron binding and ligation occurs on the HypC– HypD complex then this metal complex (possibly without the HypC protein) is transferred to the HypC–unprocessed
Table 4 Hydrogenase activities of the wild type and in-frame deletion
mutant T roseopersicina strains H 2 uptake activities were measured on
the membrane and soluble fractions, respectively The results are given
in percentage activity compared to the wild type strain (100%).
Experimental error was within 10% For the description of the strains,
see Table 1.
Inactivated genes Strain
Activity
Trang 9large subunit complex formed independently The HypCs
involved in the two separate steps can be the same proteins
or homologous counterparts, which may have dissimilar
affinities to the HypD and to the unprocessed large subunit
of the [NiFe]hydrogenases The difference in the affinity may
determine the specificity of the various HypC chaperons
There are at least two considerations, which are
compat-ible with a HypC cycle involving two (iso)enzymes On the
one hand, all known HypC type proteins share the
N-terminal highly conserved region
M-C-(L/I/V)-(G/A)-(L/I/V)-P [10], which is the sequence element essential for the
interaction with both target proteins [12] In our model, this
interaction is made possible without competition for the
same binding site between the HypD and the unprocessed
large subunit as only the iron complex is transferred from the
HypC–HypD complex to the HypC–unprocessed large
subunit assembly On the other hand, it should be noted
that there are two copies of the small chaperon-like protein in
every [NiFe]hydrogenase-containing microorganism
stud-ied in detail, e.g in E coli HypC and HybG [14], in
R eutrophaHypC and HoxL [33,35], in R leguminosarum
[36], R capsulatus and Bradyrhizobium japonicum [2]HypC
and HupF, and in T roseopersicina HypC1and HypC2 Our
model offers a function for both chaperons Experimental
evidence that supports the cooperativity-based model are as
follows First, in T roseopersicina both HypC proteins are
required for the biosynthesis of each hydrogenase A similar
situation was observed in R eutropha [33,35]where a
mutation in either the hypC or in the homologous hoxL
resulted in the dramatic reduction but not the complete loss
of membrane-bound hydrogenase activity Second, it was
shown in E coli that the HypC–preHycE complex exists on
HypD–background [12] This demonstrated the independent
formation of the HypC–HypD and the HypC–preHycE
complexes in E coli also Third, the distinct affinity of the
two chaperon-like proteins, HypC and HybG, to the target
protein was demonstrated in E coli, when both HybG and
HypC proteins were expressed in HybG–background and
only the HybG–HypD complex was detectable, although
this experiment was not evaluated quantitatively [12] It
should be noted that this is only a working hypothesis, which
can interpret the data obtained in various microbes, but
further validation of the universal nature of the model is
necessary Experiments to test this model and to identify the
intermediates in the various T roseopersicina mutants are in
progress
In summary, HupK is selectively involved in the
biosyn-thesis of the various [NiFe]hydrogenases In contrast, both
HypCs are truly pleiotropic proteins, which are very
important for the maturation of all [NiFe]hydrogenases
We propose that the two HypCs might have distinct
functions in the maturation process, and they can replace
each other to some extent
Acknowledgements
This research is supported by EU 5th Framework Programme projects
(QLK5-1999-01267, QLK3-2000-01528, QLK3-2001-01676,
ICA1-CT-2000-70026) and by domestic sources (OTKA, FKFP, OMFB, OM
KFHA´T, NKFP) International collaboration through the EU
network COST Action 841 is greatly appreciated.
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