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Journal of Bacteriology, May 2001, p. 2817-2822, Vol. 183, No. 9
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.9.2817-2822.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Interplay between the Specific Chaperone-Like
Proteins HybG and HypC in Maturation of Hydrogenases 1, 2, and 3 from Escherichia coli
Melanie
Blokesch,
Axel
Magalon, and
August
Böck*
Lehrstuhl für Mikrobiologie der
Universität München, D-80638 Munich, Germany
Received 4 December 2000/Accepted 9 February 2001
 |
ABSTRACT |
The hybG gene product from Escherichia coli
has been identified as a chaperone-like protein acting in the
maturation of hydrogenases 1 and 2. It was shown that HybG forms a
complex with the precursor of the large subunit of hydrogenase 2. As
with HypC, which is the chaperone-like protein involved in hydrogenase
3 maturation, the N-terminal cysteine residue is crucial for complex
formation. Introduction of a deletion into hybG abolished
the generation of active hydrogenase 2 but only quantitatively reduced
hydrogenase 1 activity since HypC could replace HybG in this function.
In contrast, HybG could not take over the role of HypC in a
hypC genetic background. Overproduction of HybG,
especially of the variants with the replaced N-terminal cysteine
residue, strongly interfered with hydrogenase 3 maturation, apparently
by titrating some other component(s) of the maturation machinery. The
results indicate that the three hydrogenase isoenzymes not only are
interacting at the functional level but are also interconnected during
the maturation process.
| |
INTRODUCTION |
Under anaerobic growth conditions,
Escherichia coli is able to synthesize three [NiFe]
hydrogenases, designated hydrogenase 1, 2, and 3 (2, 3, 28,
29). Hydrogenases 1 and 2 are uptake hydrogenases which couple
H2 oxidation to fumarate reduction, whereas hydrogenase 3 is a gas-evolving isoenzyme and an operational component of the formate
hydrogenlyase complex (25, 28). The structural genes for
hydrogenases 1 and 2 are located in the hya and
hyb transcriptional units (22-24), whereas
those coding for hydrogenase 3 are members of the hyc operon
(5). Genes for a fourth hydrogenase (hyf) have
been identified (1) but they do not appear to be expressed
under the experimental conditions tested (27).
Apart from the structural genes, a set of seven genes has been
identified whose products are involved in the synthesis and insertion
of the [NiFe] metal center and maturation of the enzymes. Six of them
were designated hyp (for hydrogenase pleiotropically acting
genes) since mutations in most of them (hypB,
hypD, hypE, and hypF) affect the
maturation of all three hydrogenases (15, 18). The product
of the seventh maturation gene is an endopeptidase which removes a
C-terminal extension from each of the large subunits. As the cleavage
reaction is a specific process, three different endopeptidases (HyaD,
HybD, and HycI) are involved in the maturation of the large subunits of
hydrogenases 1, 2, and 3, respectively (11, 24, 26).
When the genes coding for HypA and HypC were inactivated it was found
that the mutations only affected the maturation of hydrogenase 3 (15). It was speculated that this apparent nonpleiotropic function could be due to the existence of two genes in the
hyb operon, namely, hybF and hybG,
whose derived amino acid sequences displayed similarity to those of
hypA and hypC, respectively (15, 22). HybF thus may be the homolog of HypA and HybG may be that of HypC, and they could be involved in the maturation of hydrogenases 1 and 2.
The function of the hypC gene product has attracted
considerable attention recently (10, 19). It was found
that HypC is able to form a stable complex with the precursor of the
large subunit (HycE) during the maturation process (10).
The formation of the complex required the function of two residues,
namely, the N-terminal cysteine residue of HypC (the N-terminal
methionine is posttranslationally removed) and the first cysteine
residue of the N-terminal motif (Cys241) involved in [NiFe]
coordination (19). Since HypC forms a complex only with
the precursor of the large subunit of hydrogenase 3 and not with the
mature one, its function was tentatively envisioned as that of a
specific chaperone, keeping the protein in a folding state amenable to metal insertion (10). Because the metal center of [NiFe]
hydrogenases is located at the interface between the small and the
large subunits (12, 30), an additional function of HypC
could also reside in the prevention of the association during the
maturation process (20). A putative catalytic role,
however, is also possible.
In the present study, the function of HybG during formation of the
three hydrogenase isoenzymes from Escherichia coli was analyzed. It is shown that HybG is the specific chaperone-like protein
of hydrogenases 1 and 2 and that there is cross-interaction of the
activities of HybG and HypC and also with other components of the
hydrogenase maturation machinery.
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MATERIALS AND METHODS |
Bacterial strains, plasmids, and mutagenesis.
The bacterial
strains and plasmids employed in this study are listed in Table
1. E. coli DH5
was used as
a host for plasmid construction and maintenance. The primers listed in
Table 2 were used for the generation of
plasmids carrying the hybG gene or derivatives thereof. For
this purpose, the hybG gene was amplified via PCR with the
aid of the primers HybF1 and YghU1 using chromosomal DNA of strain
MC4100 as the template. The resulting PCR products were cloned into the
EcoRV-restricted vectors pACYC184 and pBR322, leading
to plasmids pAHBG and pBHBG, respectively. On these plasmids, the expression of the hybG gene is under the control of the
tet promoter. To replace the cysteine residue of HybG with
alanine and serine, site-directed mutagenesis was performed on plasmid pBHBG as described previously (19) using the primer pairs
HybGC2A-HybG1 and HybGC2S-HybG1, respectively. The fusion between the
hybG gene and the sequence coding for the StreptagII was
generated by PCR amplification of hybG on the plasmid pAHBG
with the oligonucleotides HybF1 and HybG-StrepII. The resulting PCR
product was then cloned into the EcoRV restriction site of
pBR322.
The strains DHB-G (MC4100
hybG), CDH103 (MC4100
hya hyc hybG), and NHC
(MC4100
hybG hypC) were constructed
following
the method of Hamilton et al. (
13) with the aid
of the pMAK
plasmid system. An in-frame deletion of 225 bp was
introduced
into
hybG by inverse PCR on pAHBG using the
oligonucleotides HybG3'
and HybG5' (Table
2), yielding the plasmid
pA
HBG. After restriction
of pA
HBG with
BamHI a 525-bp
fragment, which includes the mutant
hybG gene, was subcloned
into the
BamHI-restricted pMAK700 vector.
The resulting
plasmid was designated pM
HBG and was utilized to
transfer the
in-frame deletion onto the chromosome of the strains
MC4100, HDK103,
and DHP-C, leading to mutants DHB-G, CDH103, and
NHC,
respectively.
Growth conditions.
E. coli cells were grown
anaerobically at 37°C in a buffered rich medium, TGYEP
(4), containing 15 mM sodium formate. As supplements, 1 µM sodium molybdate, 1 µM sodium selenite, and 5 µM nickel
chloride were used. When needed for maintenance of plasmids or strains,
the medium was supplemented with 30 µg of chloramphenicol, 50 µg of
ampicillin, or 50 µg of kanamycin sulfate per ml. At an optical
density at 600 nm of 1.0 the cells were harvested and stored at
20°C. The crude extracts were prepared as described previously
(10).
Polyacrylamide gel electrophoresis and Western blotting.
Proteins were separated on sodium dodecyl sulfate (SDS)-10%
polyacrylamide gels according to the method of Laemmli
(17) or on 10% nondenaturating gels as specified by
Drapal and Böck (10). Thirty micrograms of total
protein was applied per lane.
For Western blot analysis, the proteins were transferred onto
nitrocellulose membranes and the blots were reacted with antibodies
raised against the large subunit of hydrogenase 2 (HybC) (1:1,500)
or
hydrogenase 3 (HycE) (1:1,000). The polyclonal antiserum directed
against StreptagII was purchased from the Institut für
Bioanalytik
(Göttingen, Germany) and used at a dilution of
1:2,000.
To visualize the H
2-dependent benzyl viologen (BV)
reduction activity, the nondenaturating gels were incubated in sodium
phosphate
buffer (100 mM, pH 7.2) containing 0.5 mM BV and 1 mM
triphenyltetrazolium
chloride in a glove box under an atmosphere of
95% N
2 and 5% H
2 for 24 h
(
3). One hundred micrograms of total protein was applied
per
lane.
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RESULTS |
HybG is required for maturation of hydrogenases 1 and 2.
The
final step of the maturation process of hydrogenases consists of the
proteolytic removal of a C-terminal extension from the precursor of the
large subunit. The assessment of the processing status, therefore,
can be taken as a measure of the function of the components of
the maturation machinery (10, 26).
To study the in vivo maturation role of HybG by this approach, its gene
was inactivated by the introduction of an in-frame
deletion, yielding
strain DHB-G. The
hybG lesion leads to the
blockage of
the maturation of the hydrogenase 2 (pre-HybC) without
affecting
processing of hydrogenase 3 (pre-HycE) (Fig.
1A and
B, lanes 3). In the
hybG hypC double mutant (NHC) (Fig.
1A and
B, lanes 4) neither pre-HybC nor pre-HycE is processed.
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FIG. 1.
Immunoblotting analysis of the precursor and mature
forms of HycE and HybC in the wild-type strain MC4100 and in mutants
with lesions in hybG and hypC. Crude extracts (30 µg protein) were subjected to SDS-polyacrylamide gel electrophoresis
and reacted with antibodies raised against HycE (A) or HybC (B). Lane
1, MC4100; lane 2, HDK200 (hyb); lane 3, DHB-G
(hybG); lane 4, NHC (hybG
hypC) (lane 4). (C) Hydrogenase activity staining of the
same strains is displayed after nondenaturating polyacrylamide gel
electrophoresis.
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|
In order to analyze the influence of
hybG or
hypC
deletions on hydrogenase isoenzymes 1 and 2, cell extracts were
separated
by nondenaturing polyacrylamide gel electrophoresis and the
gels
were analyzed by hydrogenase activity staining (Fig.
1C). Under
these conditions, hydrogenase 3 activity cannot be detected as
previously reported (
28). The results indicate that the
hybG deletion abolishes hydrogenase 2 activity but not that
of hydrogenase
1. Albeit reduced, its activity is on the order of the
level displayed
by a mutant (HDK200) carrying a deletion within the
hyb operon.
The finding that there was still hydrogenase 1 activity present when
the
hyb operon was deleted or when
hybG was
inactivated
suggested the involvement either of HypC or of some other
unknown
homolog in the maturation process. To follow this assumption,
the NHC strain was also analyzed by hydrogenase activity staining
(Fig.
1C, lane 4); the results show that deletion of both
hybG and
hypC completely abolished hydrogenase activity.
Consequently,
the hydrogenase 1 activity displayed by the
hyb or
hybG strains
is most likely due to
the presence of the HypC
protein.
Since both HybG and HypC are able to participate in maturation of
hydrogenase 1, it was important to study whether they also
interact in
the maturation of hydrogenases 2 and 3. For this purpose,
the two
strains CDH103 (
hya hyc hybG)
and DHB-G (
hybG) were
transformed with a plasmid carrying
either
hybG (pAHBG) or
hypC (pJA1021), and the
transformants were analyzed for restoration
of hydrogenase activity
(Fig.
2). It was found that expression
of
hybG from the plasmid led to full restoration of the
activity
of hydrogenase 2 (Fig.
2, lanes 2 and 5) and to an increase of
the activity of isoenzyme 1 (Fig.
2, lane 5). Supply of
hypC
in
trans resulted in an increase of both hydrogenase 1 and 2 activities
(Fig.
2, lanes 3 and 6). Conversely, to analyze whether
pre-HycE
can be matured by HybG in the absence of HypC, the
hypC mutant
(DHP-C) was transformed with plasmid pAHBG,
and the transformants
were analyzed by immunoblotting (Fig.
3A, lane 5). It is evident
that no
processing took place when
hybG was expressed in
trans.
Intriguingly, however, expression of
hypC
in
trans as a control
was unable to fully complement the
hypC mutation (Fig.
3A, lane
4; see below). The results
support the contention that the predominant
role of HybG resides in the
maturation of pre-HybC and that maturation
of isoenzyme 1 can be
supported by both HybG and HypC.
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FIG. 2.
Hydrogenase activity analysis of crude extracts of the
hybG strains CDH103 and DHB-G transformed with plasmids
carrying hybG and hypC genes, respectively. Crude
extracts (100 µg of protein) were subjected to nondenaturating
polyacrylamide gel electrophoresis and stained for
H2-dependent BV reduction activity as indicated in
Materials and Methods. Lane 1, CDH103; lane 2, CDH103/pAHBG
(hybG); lane 3, CDH103/pJA1021 (hypC); lane 4, DHB-G; lane 5, DHB-G/pAHBG (hybG); lane 6, DHB-G/pJA1021
(hypC).
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FIG. 3.
Immunoblotting analysis of the precursor and mature
forms of HycE. Crude extracts (30 µg protein) were subjected to
SDS-polyacrylamide gel electrophoresis and reacted with antibodies
raised against HycE. (A) Transformants of the hypC strain
(DHP-C) expressing either hypC or hybG genes from
a plasmid were analyzed. Lane 1, MC4100; lane 2, HD705; lane 3, DHP-C;
lane 4, DHP-C/pJA1021 (hypC); lane 5, DHP-C/pAHBG
(hybG). (B) Transformants of MC4100 expressing
hypC and different hybG variants from a plasmid
were tested. Lane 1, MC4100; lane 2, HD705; lane 3, MC4100/pBHBG
(hybG); lane 4, MC4100/pBC2A (hybG[C2A]); lane
5, MC4100/pBC2S (hybG[C2S]); lane 6, MC4100/pBHBG/pJA16
(hybG hypBCDE); lane 7, MC4100/pJA16 (hypBCDE);
lane 8, MC4100/pJA1021 (hypC).
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|
Overproduction of HybG interferes with the activity of HypC in
maturation of hydrogenase 3.
Overproduction of HypC in the
hypC genetic background did not fully substitute for the
function of a hypC copy located at its indigenous
chromosomal site (Fig. 3A, lane 4). This unexpected but intriguing
finding could be the consequence of a polarity effect of the mutation
on the expression of some downstream gene(s) or the effect of titration
of some other component(s) of the maturation machinery. To
differentiate between these possibilities, strain MC4100 was
transformed with plasmids carrying either hypC, hybG, or one
of the hybG variants constructed (see below), and processing of pre-HycE was studied by immunoblotting (Fig. 3B). It is evident that
expression of hybG in trans impeded pre-HycE
processing (Fig. 3B, lane 3) and the inhibition was augmented when the
N-terminal cysteine of HybG was replaced by alanine or serine (Fig. 3B,
lanes 4 and 5). Introduction of a second plasmid carrying the
hypBCDE genes counteracted the inhibition exerted by the
overproduction of HybG (Fig. 3B, lane 6). In contrast, expression of
hypC from a plasmid did not interfere with pre-HycE
maturation when the chromosomal hypC gene copy was present
(Fig. 3B, lane 8).
HybG forms a complex with pre-HybC.
To analyze whether HybG
indeed has a chaperone-like function in the maturation of the large
subunit of hydrogenase 2, its ability to form a stable complex with
pre-HybC was studied. Since antibodies directed against HybG were not
available, a fusion between hybG at its 3' end and the
StreptagII sequence was constructed, and a commercially available
anti-StreptagII serum was used to localize the gene fusion product in
nondenaturating polyacrylamide gels (Fig.
4). A HybG-pre-HybC complex could
be detected both by anti-StreptagII antibodies (Fig. 4A, lane 3)
and by anti-HybC antibodies (Fig. 4B, lanes 2 and 3). HybG was unable
to enter the complex when its N-terminal cysteine was replaced by an
alanine or serine residue (Fig. 4A and B, lanes 4 and 5). These HybG
variants were also unable to generate hydrogenase 1 and 2 activities (Fig. 4C, lanes 4 and 5).
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FIG. 4.
Immunoblotting analysis of HybG-pre-HybC complex
formation in transformants expressing different HybG variants from a
plasmid. (A and B) Crude extracts (30 µg protein) were analyzed by
Western blotting after nondenaturating polyacrylamide gel
electrophoresis with antisera directed against StreptagII (A) and HybC
(B). (C) Corresponding hydrogenase activity staining. Lanes 1, DHB-G;
lanes 2, DHB-G/pBHBG; lanes 3, DHB-G/pBHBG-Strep; lanes 4, DHB-G/pBC2A-Strep; lanes 5, DHB-G/pBC2S-Strep.
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| |
DISCUSSION |
The complex between HypC and the precursor of the large subunit of
hydrogenase 3 has been demonstrated to be the key intermediate in the
maturation process (10). Its formation is an early step since mutants defective in each other gene involved in maturation accumulate the complex (10) and since its dissolution only
precedes the final step, namely, endoproteolytic removal of the
C-terminal extension of pre-HycE (20). In view of the high
sequence similarity between HypC and HybG (~77%), a similar function
was assumed for HybG in the maturation of hydrogenase 2 and possibly
also of isoenzyme 1 (15, 22).
The in silico predictions now have been proven biochemically: HybG
forms a complex with the precursor of the large subunit of hydrogenase
2 and as with HypC the N-terminal cysteine residue appears to be
directly involved in complex formation. It is still open whether this
residue solely has a structural role in the interaction of the two
proteins or whether its role is catalytic, e.g., in some redox reaction
taking place during insertion of the metal(s) or the addition of the CO
or CN ligands identified in the active site of hydrogenases (9,
14, 31).
Intriguingly, HybG has a dual function in that it is also required for
maturation of hydrogenase 1. However, whereas the function of HybG is
indispensable for the maturation of hydrogenase 2, its involvement in
hydrogenase 1 maturation can be partially taken over by HypC. The
evidence is that a hybG mutant contains significant levels of hydrogenase 1 activity which can be augmented by
overproducing HypC in trans. Also, a double mutant lacking
both HybG and HypC is unable to form the three hydrogenase isoenzymes
in a processed and active form.
The specificity and the interactions of the various maturation
components are summarized in Fig. 5.
HypB, HypD, HypE, and HypF are required for the synthesis of all three
hydrogenase isoenzymes, since they are possibly involved in general
reactions like nickel acquisition and donation (21) or
CO-CN ligand biosynthesis. Others, such as HybG and HypC, are shared
between two of the systems or they are specific like the endopeptidases
(HyaD, HybD, and HycI), processing the precursors of the large subunits
after the metals have been inserted. It will be interesting to study
the consequences of these interconnections under different
physiological conditions.
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FIG. 5.
Network of involvement of the auxiliary proteins in the
formation of hydrogenases 1, 2, and 3. Dotted lines leading away from
HybF indicate postulated functions.
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|
The results of this study are also relevant for the discussion of the
physiological role of the three hydrogenase isoenzymes. Whereas the
function of hydrogenase 2 as an uptake enzyme and that of hydrogenase 3 as a fermentative gas-evolving one are undisputed, that of hydrogenase
1 is not fully understood (3, 16). If it is indeed
H2 recycling the results would add another argument for
such a role since the two enzymes would not only be interacting functionally but also be connected via a common component during the
maturation process.
When overexpressed in trans, hybG interfered with
hydrogenase 3 maturation in an otherwise wild-type genetic
background: the inhibition was particularly pronounced in the case of
the HybG variants in which the N-terminal cysteine was altered. A
plausible explanation is that the overproduced HybG sequesters some
other component(s) of the processing machinery, thereby forming
unproductive processing intermediates in the case of the N-terminally
altered variants. The fact that coexpression of the hypBCDE
genes alleviated the inhibition by the HybG variants provides support
for this contention.
| |
ACKNOWLEDGMENTS |
We thank A. Paschos and E. Zehelein for the kind donation of
antiserum directed against HybC.
This work was supported by a research fellowship from the Alexander von
Humboldt Foundation to A.M. and by grants from the Deutsche
Forschungsgemeinschaft and the Fonds der Chemischen Industrie to A.B.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institute of
Genetics and Microbiology, University of Munich, Maria-Ward-Strasse 1a, D-80638 Munich, Germany. Phone: 49-89-21806120. Fax: 49-89-21806122. E-mail: august.boeck{at}lrz.uni-muenchen.de.
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Journal of Bacteriology, May 2001, p. 2817-2822, Vol. 183, No. 9
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.9.2817-2822.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
