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Journal of Bacteriology, February 2000, p. 581-588, Vol. 182, No. 3
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Ralstonia eutropha TF93 Is Blocked in Tat-Mediated
Protein Export
Michael
Bernhard,
Bärbel
Friedrich, and
Roman A.
Siddiqui*
Institut für Biologie,
Humboldt-Universität zu Berlin, 10115 Berlin, Germany
Received 14 September 1999/Accepted 8 November 1999
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ABSTRACT |
Ralstonia eutropha (formerly Alcaligenes
eutrophus) TF93 is pleiotropically affected in the translocation
of redox enzymes synthesized with an N-terminal signal peptide bearing
a twin arginine (S/T-R-R-X-F-L-K) motif. Immunoblot analyses showed
that the catalytic subunits of the membrane-bound [NiFe] hydrogenase
(MBH) and the molybdenum cofactor-binding periplasmic nitrate reductase
(Nap) are mislocalized to the cytoplasm and to the inner membrane,
respectively. Moreover, physiological studies showed that the
copper-containing nitrous oxide reductase (NosZ) was also not
translocated to the periplasm in strain TF93. The cellular localization
of enzymes exported by the general secretion system was unaffected. The
translocation-arrested MBH and Nap proteins were enzymatically active,
suggesting that twin-arginine signal peptide-dependent redox enzymes
may have their cofactors inserted prior to transmembrane export. The
periplasmic destination of MBH, Nap, and NosZ was restored by
heterologous expression of Azotobacter chroococcum tatA
mobilized into TF93. tatA encodes a bacterial Hcf106-like
protein, a component of a novel protein transport system that has
been characterized in thylakoids and shown to translocate folded
proteins across the membrane.
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INTRODUCTION |
Periplasmic enzymes binding redox
cofactors play a central role in alternative energy metabolism of
gram-negative bacteria. In contrast to cytochrome c-type
proteins, various periplasmic enzymes binding constituents, such as the
molybdenum cofactor, a [NiFe] site, copper centers, or iron-sulfur
clusters, contain a conserved, positively charged -S/T-RRXFLK-
(twin-arginine) element within their N-terminal signal peptides,
pointing to a special translocation pathway (2). Very
recently, a system responsible for the membrane targeting and
translocation of [NiFe] hydrogenases, as well as the molybdenum
enzymes dimethylsulfoxide reductase, trimethylamine N-oxide
reductase, and periplasmic nitrate reductase, has been characterized
for Escherichia coli (32, 43). Mutant analyses
suggested that the translocation of the twin-arginine signal
peptide-bearing enzymes proceeds independently of the general secretion
machinery of the cell (Sec), presumably via intermediates which have
their cofactors inserted (31).
Ralstonia eutropha (formerly Alcaligenes
eutrophus [7]) is the host of at least three
periplasmic cofactor-containing enzymes synthesized with an N-terminal
twin-arginine signal peptide: the membrane-bound hydrogenase (MBH)
(involved in energy generation from H2), the periplasmic
nitrate reductase (Nap) (reduces nitrate to nitrite), and the nitrous
oxide reductase (NosZ) (a component of the denitrification pathway).
All three enzymes are encoded in R. eutropha H16 by
megaplasmid-borne genes.
The MBH of R. eutropha is a member of the [NiFe]
hydrogenases (15) composed of a heterodimer (HoxKG) attached
to the periplasmic surface of the inner membrane by a cytochrome
b-type anchor protein (HoxZ) (4, 13). Hydrogen is
activated at the [NiFe] site of HoxG (62 kDa), and the electrons are
transferred via three iron-sulfur clusters within HoxK (35 kDa) to the
physiological electron acceptor HoxZ (4). The small subunit
HoxK contains a twin-arginine signal peptide. A deletion in this region
blocks the membrane targeting of the MBH dimer and leads to the
accumulation of inactive HoxG protein in the cytoplasm (3).
The N-terminal amino acid sequence of the mature HoxG protein is
colinear with the sequence predicted from the nucleotide sequence
(17), indicating the absence of an export-triggering signal
peptide at the N terminus. In contrast, HoxG contains a peptide
extension of 15 amino acids (aa) at the C terminus, which is removed by
a specific protease during [NiFe] cofactor assembly and plays a role
in metal insertion (3). From these results, it was concluded
that the two MBH subunits are cotranslocated in a tandem fashion and
that this process is directed by the twin-arginine signal
peptide-bearing small subunit. This conclusion gained support by a
recent report on hydrogenase-2 of E. coli (27).
The periplasmic nitrate reductase, Nap, belongs to a large family of
respiratory nitrate reductases, which appears to participate in
denitrification, at least in some organisms (1, 28). Nap has
been isolated from R. eutropha as a heterodimeric enzyme
consisting of a 90-kDa subunit (NapA) and a 17-kDa subunit (NapB). NapA
carries the catalytic site and exhibits sequence similarity with
molybdopterin guanine dinucleotide (MGD) binding polypeptides of
bacterial assimilatory nitrate reductases and formate dehydrogenases,
both of which bind an iron-sulfur cluster at the N terminus
(35). In fact, crystal structure analyses of NapA from
Desulfovibrio desulfuricans show two MGD moieties per
polypeptide and a [4Fe-4S] cluster (10). Comparison of the
N-terminal amino acid sequence of the mature R. eutropha
NapA subunit, as determined by Edman degradation, with the predicted
primary structure identified a 29-aa twin-arginine signal peptide in
NapA. NapB, which contains two binding sites for heme c, is
synthesized with an N-terminal signal peptide resembling those required
for translocation by Sec (35).
The periplasmic, copper-dependent nitrous oxide reductase, NosZ, is a
key enzyme of denitrification which converts nitrous oxide to molecular
dinitrogen. The primary structures of NosZ from various bacteria,
including R. eutropha, are highly conserved and are all
characterized by an unusually long N-terminal twin-arginine signal
peptide of 45 to 56 aa (11, 47).
In this study, we reexamined the H2-oxidizing R. eutropha TF93 (ATCC 17697) strain, which had been reported in
early studies to form a membrane-type of hydrogenase which occurred in
the soluble fraction of the cell (14). We show that due to a
missing or nonfunctional chromosomal factor, the strain is affected not
only in membrane targeting of the twin-arginine signal peptide-bearing [NiFe] hydrogenase but also in the translocation of the periplasmic nitrate reductase and the nitrous oxide reductase. The mislocalized MBH
and Nap proteins were active with artificial electron acceptors and
donors, supporting the interpretation that these metalloenzymes have
their cofactors inserted prior to translocation.
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MATERIALS AND METHODS |
Strains and plasmids.
Bacterial strains and plasmids are
listed in Table 1. Plasmid pGE600 is a
derivative of the mobilizable vector pGE151, carrying the 2.7-kb
BamHI/HindIII fragment of pGY12. DNA
fragments extending from nucleotides 1268 to 1620 and from 1555 to 2006 of the Azotobacter chroococcum tat region (GenBank accession
no. ACU48408) were synthesized by PCR, using plasmid pGY12 as a
template. Amplification of tatA with primer pair
5'-ACCCAAGCTTGCGGAAAAGGCCGCACGGCC-3' and
5'-CGGGATCCAGCAGGAGTTCGCTGAAGCCG-3' produced a 366-bp
fragment containing an additional HindIII and BamHI site. All the PCR amplifications were carried out with
Pfu DNA polymerase (Stratagene). The resulting fragment was
cut with HindIII and BamHI and ligated into
pBluescript SK(+) (pCH800). In the same way, the primers
5'-ACCCAAGCTTGCAAGGTCGAGGAACCGGCCAGG-3' and
5'-CGGGATCCCTGCGCAGCAGGCGCGAGCGC-3' were used for
amplification of a 468-bp fragment containing tatB and
ligated into pBluescript SK(+) (pCH801). The identity of the subcloned
PCR fragments was verified by nucleotide sequence determination. The
HindIII/BamHI fragments containing either
tatA or tatB were excised from the pBluescript
derivatives and cloned into plasmid pGE151, yielding plasmids pGE601
and pGE602, respectively. For nucleotide sequence determination of the
fusion in the tat region (see Fig. 6A), the native 1,638-bp
SphI fragment of pGY12 was cloned into pUC18, yielding
pCH802. E. coli phoA was PCR amplified on a 1.4-kb fragment with the primers 5'-TACATATGAAACAAAGCACTATTGCACTGG-3' and
5'-TATCTAGATTATTTCAGCCCCAGAGCGGC-3', with total DNA as the
template. The PCR product was cut by NdeI and
XbaI and cloned downstream of the strong R. eutropha promoter of the soluble hydrogenase into the
broad-host-range plasmid pEDY309 as previously described
(23), yielding pGE603.
Media and growth conditions.
Lithotrophic cultures of
R. eutropha strains were grown in mineral salts medium under
an atmosphere of hydrogen, carbon dioxide, and oxygen (8:1:1
[vol/vol/vol]) supplemented with 0.8 µM NiCl2 in place
of the standard trace element mixture SL6 (12). Synthetic media for aerobic heterotrophic growth, in order to optimally express
Nap, contained 0.4% (wt/vol) gluconate or 0.4% (wt/vol) fructose and
SL6 as described previously (42). Anaerobic heterotrophic growth, at the expense of 0.2% (vol/vol) nitrate (denitrification), was performed in medium containing fructose as previously described (30). Strains of E. coli were grown in
Luria-Bertani medium (20). Solid media contained 1.5%
(wt/vol) agar. Antibiotics were added as appropriate for R. eutropha (tetracycline, 12.5 µg/ml) and for E. coli
(tetracycline, 12.5 µg/ml; ampicillin, 80 µg/ml).
Conjugative plasmid transfer.
Mobilizable plasmids were
transferred from E. coli S17-1 to R. eutropha by
a spot mating technique (36). Transconjugants were selected
on mineral medium plates containing the appropriate antibiotic under
lithotrophic growth conditions.
DNA techniques.
Standard techniques were used in this study
(29). Plasmid DNA isolation was carried out by the alkaline
lysis procedure and ion-exchange chromatography according to the
manufacturer's instructions (QIAGEN Inc.). DNA and PCR fragments used
in plasmid constructions were isolated from agarose gels by QiaEx
(QIAGEN Inc.). Nucleotide sequence determination of pCH802 was done by the dideoxy chain termination method and by cycle sequencing with sequence-derived fluorescence-labelled primers and the thermostable sequenase kit (Amersham Pharmacia Biotech) in an automatic sequencing device, as recommended by the manufacturer (LICOR).
In vivo expression of tatAB gene products.
Expression of tatA and tatB from plasmids pCH800
and pCH801 was under control of the phage T7 
10 promoter. The
plasmids were transformed into strain NovaBlue(DE3), which carries a
chromosomally encoded T7 polymerase. Synthesis of the
tat-encoded gene products was induced by IPTG
(isopropyl-
-D-thiogalactopyranoside) and labelling with
[35S]methionine followed the procedure previously
described (38).
Isolation of subcellular fractions.
Subcellular fractions
were prepared according to a method described by Bernhard et al.
(4), with modifications. R. eutropha cells (100 ml) were grown in the presence of oxygen either under lithotrophic
conditions for 36 h or heterotrophically on gluconate for 24 to
48 h and then harvested by centrifugation (4,000 × g, 4°C). Cells were washed with 10 ml of 10 mM Tris-HCl, pH 7.5 (4,000 × g, 4°C), and resuspended in 5 ml of 10 mM
Tris-HCl, pH 7.8, containing 0.5 M sucrose. After a 10-min incubation
at 30°C in the presence of 1 mM EDTA, the incubation was continued at
room temperature for 30 min with lysozyme (10 mg/g [wet weight] of cells). The suspension was centrifuged (4,000 × g; 20 min; 4°C), and the supernatant contained the periplasmic fraction.
The spheroplasts were washed and lysed by osmotic shock with 5 ml of 10 mM Tris-HCl, pH 7.8. Cell debris was removed (5,000 × g, 15 min, 4°C), and the membrane and cytoplasmic fractions were
obtained by ultracentrifugation (88,000 × g, 45 min,
4°C). Membranes used for the detection of the MBH were washed three
times and suspended in 50 mM KPO4, pH 7.0. Membranes used
for the detection of NapA were resuspended in 250 mM
NaKPO4, pH 6.5. The periplasmic, cytoplasmic, and membrane fractions were used directly or stored at 
20°C until used for enzyme assays, activity staining, and immunoblotting analysis. The
quality of the subcellular fractions of the lithotrophically grown
cells was monitored by examining the distribution of the cytoplasmic,
NAD-reducing hydrogenase activity of the soluble hydrogenase (SH) of
R. eutropha and immunoblotting with SH-specific antibodies,
as previously described (4). The purity of subcellular fractions of heterotrophically grown cells was controlled by
immunoblotting with antiserum raised against the cytoplasmic marker
flavohemoglobin of R. eutropha (9).
Immunoblot analysis.
Proteins were resolved by
electrophoresis in sodium dodecyl sulfate (SDS)-10% or 12% (wt/vol)
polyacrylamide gels and were transferred to nitrocellulose membranes,
and the immunoblot analysis was done according to a standard protocol
(41). Specific proteins were detected with polyclonal rabbit
antisera and an alkaline phosphatase-labelled goat anti-rabbit
immunoglobulin G (Jackson Immuno Research Laboratories). The proteins
were applied at the following dilutions: anti-flavohemoglobin
(1:5,000), anti-HoxH (1:10,000), anti-HoxG (1:2,000), anti-PhoA
(1:1,000; 5 Prime 
3 Prime, Inc.), anti-nitrite reductase (1:10,000)
(30), and anti-NapA (1:1,000).
Analytical procedures.
SH (hydrogen:NAD+
oxidoreductase [EC 1.12.1.2]) activity was assayed by
spectrophotometric determination of H2-dependent NAD
reduction (14). MBH (ferredoxin:H+
oxidoreductase [EC 1.18.99.1]) activity was determined according to a
previously described method (37), with modifications.
Hydrogenase activities of membranes were determined in
N2-saturated 50 mM KPO4, pH 7.0. Soluble
extracts at pH 5.5 were measured with 0.5 mM methylene blue and 86 µM
H2. One unit of hydrogenase activity was the amount of
enzyme which catalyzed the consumption of 1 µmol of substrate per
min. Nap reductase activity was determined in subcellular fractions
obtained from aerobically grown cells (42) exploiting the
formate-dependent nitrate reduction (35). One unit of Nap
activity was the amount of enzyme which catalyzed the formation of 1 µmol of nitrite per min. Nitrite concentration was determined
colorometrically at 546 nm (18).
In-gel chromogenic detection of hydrogenase activity after native
polyacrylamide gel electrophoresis was done according to
a previously
described method (
4).
c-Type cytochromes were detected after SDS-polyacrylamide
gel electrophoresis of subcellular fractions and specific staining
for
covalently attached heme, as previously described (
39).
Protein determination of cells and subcellular fractions was done by
the method of Lowry et al. (
19).
Determination of nitrous oxide and dinitrogen by gas chromatography was
done as previously described (
9).
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RESULTS AND DISCUSSION |
The membrane-bound hydrogenase of R. eutropha TF93 is
mislocalized to the cytoplasm.
The majority of R. eutropha strains form two enzymes for energy conservation from
molecular hydrogen: a cytoplasmic NAD-dependent SH and an MBH. Both
proteins have been shown to be also present in the natural isolate
R. eutropha TF93; however, the MBH was identified in the
soluble fraction instead of in the membrane. Mislocalization of the
enzyme could not be restored by a megaplasmid exchange using a donor
which synthesized the MBH properly attached to the membrane
(14).
To determine the precise cellular localization of the MBH dimer, we
examined periplasmic, membrane, and cytoplasmic fractions
of
autotrophically grown TF93 cells by immunoblot analysis using
antiserum
raised against the large MBH subunit (HoxG) of
R. eutropha H16. HoxG of TF93 was exclusively found in the cytoplasm (Fig.
1, lane 3), unlike the MBH of strain H16,
which occurred predominantly
in the membrane fraction and only in trace
amounts in the cytoplasm
(Fig.
1, lane 1). Export of the MBH to the
periplasm of TF93 can
be excluded on the basis of the release of the
MBH into the periplasm
by a mutant of H16 impaired in the membrane
anchor HoxZ (Fig.
1, lane 2) (
4). These results
unambiguously show that the MBH
of TF93 is restricted to the cytoplasm,
which was also the case
in a transconjugant, TF140, harboring the
megaplasmid pHG1 of
R. eutropha H16 (Fig.
1, lane 4). This
confirmed that no mutations
in megaplasmid genes are responsible for
the mislocalization of
the MBH in TF93, but pointed to a defective or
missing chromosomally
encoded factor which is required for the proper
targeting of the
MBH to the membrane. Since pHG1 of strain H16 is
better characterized
than the native plasmid pHG2 of TF93, subsequent
experiments were
done with TF140 (Table
1).
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FIG. 1.
Cellular localization of the [NiFe] center-bearing
subunit (HoxG) of MBH in strains of R. eutropha. Equal
amounts of protein (25 µg) were loaded onto the gel except for the
periplasmic fractions (100 µl each), because they contained high
concentrations of lysozyme protein. The subcellular fractions are
indicated: P, periplasm; M, membrane; C, cytoplasm. The strains tested
are as follows: lane 1, H16 (harboring pHG1); lane 2, HF405, carrying
 hoxZ in pHG1; lane 3, TF93 (harboring pHG2); lane 4, TF140 (harboring pHG1).
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R. eutropha TF93 is pleiotropically deficient in
translocation of redox enzymes.
To examine whether the deficiency
of TF93 plays a more general role in the export of cofactor-containing
enzymes, we investigated the localization of Nap and of NosZ in
comparison to proteins which are translocated via the general secretion
system. Subcellular fractions from TF140 cells grown heterotrophically
in synthetic medium in presence of oxygen were analyzed for Nap.
Immunoblots with polyclonal NapA antiserum showed that the catalytic
subunit NapA was trapped in the membrane, whereas both the periplasm
and the cytoplasm were almost free of any cross-reacting material (Fig.
2, lane 1). This clearly pointed to a
pleiotropic nature of the export deficiency in TF140. Identical results
were obtained with the parental TF93 strain (data not shown).
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FIG. 2.
Mislocalization of NapA in TF140 and restoration of NapA
export by tat genes (A) and specificity of the NapA
detection system (B). The subcellular fractions are indicated: P,
periplasm; M, membrane; C, cytoplasm. Nap activity (milliunits per
milligram of protein) is given below the immunoblot. Note, the Nap
activity in the periplasm is given as milliunits per gram (wet weight)
of cells. (A) Lane 1, TF140; lane 2, TF140 harboring tatAB
on pGE600; lane 3, TF140 harboring tatA on pGE601; lane 4, TF140 harboring tatB on pGE602. (B) Lane 1, partially
purified NapA (arrow); lane 2, membrane of TF100 cells; lane 3, membrane of TF100 cells harboring the complete nap genes on
pGE144 (35).
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The second twin-arginine signal peptide-bearing redox enzyme, NosZ, was
investigated on the physiological level. Cells of
TF140 were grown
anaerobically under denitrifying conditions with
nitrate as the
electron acceptor. Nitrous oxide production and
its conversion to
molecular dinitrogen were monitored by gas chromatography.
Figure
3A shows that TF140 accumulated nitrous
oxide as the final
product, indicating that NosZ was incapable of
converting nitrous
oxide. Physiologically active periplasmic NosZ of
R. eutropha,
however, readily converts nitrous oxide, which
only transiently
accumulates in the gas phase (
30). The
result is compatible
with the conclusion that the mislocalization of
NosZ in TF140
impairs its physiological function. Indeed, mutational
analysis
of the NosZ system from
Pseudomonas stutzeri has
shown that the
export to the periplasm is a prerequisite of the protein
to be
physiologically active (
11). Due to the lack of an
appropriate
NosZ antibody, it was not possible to investigate the
cellular
localization of the enzyme. Attempts to use heterologous
antiserum
raised against NosZ by
P. stutzeri (
47)
were not successful.
Nevertheless, the results provide an excellent
explanation for
the failure of
R. eutropha TF93 to produce
dinitrogen gas during
anaerobic nitrate respiration (
44).
Furthermore, the data demonstrate
that the export deficiency in TF93
has an enormous physiological
impact on the cells since it blocks the
function of at least three
independent redox systems.
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FIG. 3.
Blocked nitrous oxide reduction in R. eutropha TF140 (A), which was restored by the expression of
A. chroococcum tatA on pGE601 (B). Growth was performed
anaerobically on fructose containing 10 mM nitrate, and the gaseous
denitrification products were monitored by gas chromatography.  ,
growth of R. eutropha culture at 436 nm;  , nitrous oxide;
 , dinitrogen. OD, optical density.
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To definitely show that the translocation of twin-arginine signal
peptide-dependent redox enzymes is specifically blocked
and that the
translocation of proteins across the cytoplasmic
membrane is not
generally impaired in
R. eutropha TF93, the distribution
of
cytochrome
c-type proteins of this strain was tested.
Periplasmic
and cytoplasmic fractions obtained from cells of TF140
grown aerobically
and anaerobically were subjected to heme
c
staining (Fig.
4).
Heme
c-type
proteins were identified exclusively in the periplasm;
they were absent
in the cytoplasmic fraction (Fig.
4A, lanes 1
and 2). Furthermore,
immunoblot analyses confirmed the periplasmic
localization of the heme
cd1-containing nitrite reductase (data
not
shown) and also of the alkaline phosphatase (PhoA), which
is a
well-known substrate of the general secretory pathway (Sec)
in
E. coli (
25). When heterologously expressed from plasmid
pGE603,
E. coli PhoA is exported to the periplasmic space of
TF140
in the same manner as in an H16 derivative, as expected (Fig.
4B,
lanes 2 and 3, top panel). Purity controls eliminated the
possibility
of a contamination by cytoplasmic proteins (Fig.
4B,
bottom panel).
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FIG. 4.
Periplasmic c-type cytochromes (A) and export
of heterologously expressed E. coli PhoA to the periplasm
(B) in R. eutropha strains. (A) Cytochrome c was
visualized by heme staining of periplasmic (lanes 1, 3, and 5) and
cytoplasmic (lanes 2, 4, and 6; loaded with 100 µg of protein each)
fractions of TF140 cells grown anaerobically on nitrate. The proteins
were separated on SDS-15% (vol/vol) polyacrylamide gels. Lanes 1 and
2, TF140; lanes 3 and 4, TF140 harboring tatAB on pGE600;
lanes 5 and 6, TF140 harboring tatA on pGE601. Prestained
protein markers are on the left and right of the stain. The nature of
the staining at the border of the stacking and the running gel in the
cytoplasmic fractions (lanes 2, 4, and 6) is unclear. (B) Immunoblot
analysis of the periplasmic fractions of autotrophically grown R. eutropha strains with antiserum directed against E. coli PhoA (upper panel) and a purity control with antiserum raised
against the SH protein (anti-HoxH) as a cytoplasmic marker
(4) (lower panel). Lane 1, 1 µg of purified SH; lane 2, periplasm of TF140 expressing a copy of E. coli phoA on
pGE603; lane 3, periplasm of H16 derivative (HF405) expressing a copy
of E. coli phoA on pGE603.
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MBH and Nap are enzymatically active in the translocation-arrested
state.
In the process of cytochrome c maturation, the
polypeptide and the prosthetic group heme c are translocated
separately (reference 40 and references therein).
Thus, neither preapocytochromes nor holocytochrome c
containing the cofactor essential for the catalytic activity is
detectable when export is blocked. Immunochemical studies showed that
cofactor-free apoforms are unstable (21, 22).
The detection of export-blocked MBH and Nap prompted us to examine
whether both are also enzymatically active. MBH activity
was visualized
by in-gel H
2-dependent phenazine methosulfate reduction
and
showed a strong activity staining in the cytoplasmic compartment
of
TF140 (Fig.
5A, lane 3). For H16, only a
faint stain corresponding
to traces of anti-MBH material detected in
the cytoplasm was observed
(Fig.
1 and
5A, lane 1). No staining
appeared in an H16 derivative
which is devoid of the
[NiFe]-containing subunit (HF359;
hoxG)
(Fig.
5A, lane
2). This demonstrated that translocation-arrested
MBH accumulates in
its catalytically active form in the cytoplasm
of TF140. Further
immunoblot analyses showed that the MBH is processed
in TF140, compared
to a mutant in which the protease gene had
been deleted (HF345;
hoxM) (Fig.
5B, compare lanes 1 and 2).
This confirms
that for metal center assembly, the mislocalized
MBH undergoes the same
proteolytic processing at the C terminus
of the [NiFe]-containing
subunit in TF140 as that documented for
the H16 enzyme (
4).
These results are in agreement with the
previous finding of the
partially purified active MBH from the
soluble fraction of
R. eutropha TF93 (
24). Nitrate reductase
assays showed
that the NapA protein trapped in the membrane of
TF140 is also
catalytically active (Fig.
2, lane 1). Only basal
nitrate-reducing
activities were found in the periplasmic and
cytoplasmic fractions
(Fig.
2, lane 1). The nitrate reductase
activity measured is
exclusively due to Nap and not due to the
respiratory membrane-bound
nitrate reductase, which is known to
be expressed anaerobically only
when nitrate serves as the alternative
electron acceptor
(
42). Figure
2B shows that a Nap-negative
variant of TF93
formed no anti-NapA reacting material, and in
consequence, no Nap
activity was trapped in the membrane of the
aerobically grown cells.
However, NapA-specific protein and the
corresponding enzyme activity
were detectable again upon complementation
of the mutant
nap
strain by the
nap genes residing on pGE144 (Fig.
2B, compare
lanes 2 and 3). In summary, the results demonstrate
that MBH and NapA
have their respective cofactors incorporated
in an identical manner
appropriate for the physiological situation
despite their translocation
being blocked.
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FIG. 5.
In-gel detection of MBH-dependent hydrogenase activity
in the cytoplasmic fraction of TF140 (A) and the restoration of MBH
targeting to the membrane by tat genes (B). (A) Lane 1, H16;
lane 2, HF359, defective in the [NiFe]-containing subunit of MBH
(hoxG in pHG1); lane 3, TF140. (B) Analysis of
restoration of MBH translocation was carried out by immunoblot
analysis. S, the soluble extract consisting of the periplasmic and
cytoplasmic fractions; M, the membrane fraction. MBH activity
(milliunits per milligram of protein) is given below the blot. Lane 1, HF345, defective in the MBH-specific protease (hoxM in
pHG1); lane 2, TF140; lane 3, TF140 with tatAB (pGE600);
lane 4, TF140 with tatA (pGE601); lane 5, TF140 with
tatB (pGE602).
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The pretranslocational fashion of cofactor insertion accompanied with
folding of the preproteins precludes translocation by
the Sec system
(
25). In fact, it has been shown recently that
the
twin-arginine signal peptide-dependent translocation of the
trimethylamine
N-oxide reductase proceeds independently of
the
Sec pathway in
E. coli (
31).
A bacterial analog of the thylakoid Hcf106 restores translocation
of mislocalized redox enzymes in R. eutropha.
Interestingly,
a class of plant proteins shares the twin-arginine element (2, 5,
8), which can direct these proteins in the folded state across
the thylakoid membrane in a Sec-independent manner (16). The
import to the lumen of thylakoids is equivalent to export across the
inner membrane of bacterial cells. A component of this
twin-arginine-protein transport system, Hcf106 (30 kDa), was first
identified in maize, and proteins with local similarities have been
deduced also from gene data banks and from sequenced genomes of
bacteria and archaea (33, 34). First attempts to identify
and clone a homologous gene of R. eutropha by virtue of the
local similarities of the primary structures of bacterial Hcf106-like
proteins were unsuccessful.
Hence, we used the
orf4 gene from
A. chroococcum
(
45), whose product had been determined to contain local
similarities to
Hcf106 of maize (
34) for heterologous
complementation.
A. chroococcum contains a membrane-bound
H
2-oxidizing enzyme which is highly
related to the
R. eutropha system (
15). It has been shown that
orf4 is able to complement an
A. chroococcum
mutant with a membrane-bound
hydrogenase activity misassembled to the
soluble fraction, but
the molecular basis remains to be elucidated
(
45). To express
the
A. chroococcum orf4 gene we
cloned the 2.7-kb
BamHI/
HindIII
fragment of
pGY12 into the broad-host-range vector pGE151, yielding
pGE600. In the
course of our studies, we uncovered (by sequence
alignment studies)
that the
orf4 gene product represents a fusion
of two
Hcf106-like proteins (Fig.
6). Nucleotide
sequence determination
of
orf4 on plasmid pGY12 (Fig.
6A)
resulted in the elimination
of a guanine at position 1535 of the
sequence (GenBank accession
no. ACU48404), yielding two new
orf genes,
tatA and
tatB (Fig.
6),
which encode separate proteins as confirmed by Tabor expression
(data
not shown). The primary structure of both products showed
local
similarities to Hcf106 (Fig.
6B). Moreover, the newly annotated
A. chroococcum gene products showed significant similarities
to
the products of the
tat system, recently identified in
the
E. coli genome (
32,
43). Since the product of
the incomplete
orf5 gene, immediately downstream of
tatA and
tatB, also showed
significant primary
sequence identity to a product of the
tat system of
E. coli, the respective
A. chroococcum gene was designated
tatC' (Fig.
6).
tatA and
tatB were
subcloned individually into
pGE151 under the control of the
lac promoter, resulting in plasmids
pGE601 and pGE602,
respectively. Upon mobilization of the cloned
A. chroococcum
genes
tatAB (pGE600),
tatA (pGE601), and
tatB (pGE602)
into strain TF140, the subcellular fractions
of the resulting
transconjugants were tested for the localization of
MBH and of
NapA. Figure
5B shows that the
tatAB- and
tatA-harboring derivatives
targeted the MBH correctly to the
membrane and restored hydrogenase
activity in this fraction. Likewise,
NapA was correctly exported
in the
tatAB- and
tatA-harboring cells, accompanied by the occurrence
of Nap
activity in the periplasm (Fig.
2). Moreover, expression
of
A. chroococcum tatA (on pGE600 and pGE601) restored nitrous
oxide
reduction in TF140; the resulting transconjugants accumulated
dinitrogen in the gas phase, as illustrated representatively for
TF140
harboring a copy of
tatA (Fig.
3B). Transfer of
A. chroococcum tatB alone (on pGE602) did not restore any deficiency
observed
in TF140 (Fig.
2,
3, and
5). In contrast, the expression of
the
A. chroococcum tat genes had no effect at all on the
export of
cytochrome
c-type proteins (Fig.
4A). These
results strongly point
to a lesion in a TatA-like protein in
R. eutropha TF93 which appears
to be required for the translocation
of folded twin-arginine signal
peptide-bearing redox enzymes.
View larger version (28K):
[in this window]
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|
FIG. 6.
The tat locus on pGY12 of A. chroococcum according to GenBank accession no. ACU48404, with the
nucleotide sequence correction. (A) The newly annotated genes are in
black. The fragments used for complementation analysis are shown below
with bars, and the corresponding plasmid designations are given at the
left. (B) Alignment of the deduced A. chroococcum TatA and
TatB amino acid sequences, with Hcf106 and Hcf106 analogs from E. coli. Az. c., A. chroococcum; E. c., E. coli; Z. m., Zea mays. Perfect matches are indicated by
asterisks and high and low similarities are indicated by double and
single dots, respectively.
|
|
Although we cannot exclude the possibility that the folding constraints
for MBH and Nap in export-blocked TF93 are different
from the
export-competent cells, the results presented could be
interpreted in
favor of a cytoplasmic assembly rather than a periplasmic
assembly
pathway for twin-arginine signal peptide-bearing redox
enzymes, as
previously proposed (
2). This interpretation also
gains
support by studies indicating that metal insertion has to
take place
before the molybdenum-dependent dimethylsulfoxide reductase
from
Rhodobacter sphaeroides f. sp.
denitrificans
(
46) and hydrogenase-2
from
E. coli
(
26) are translocated. Both enzymes are examples
of
bacterial redox proteins carrying the unusual signal peptide
(
2).
Hcf106-like proteins in targeting and translocation of bacterial
enzymes binding different redox cofactors.
Very recently it was
shown that E. coli contains a Sec-independent transport
apparatus required for the membrane targeting and translocation of
twin-arginine signal peptide-bearing enzymes (31, 32, 43).
The novel system, Tat (twin-arginine translocator, formerly called Mtt
[membrane targeting and translocation]), is encoded by the
tatABCD operon and the unlinked tatE gene
(32). Although controversial, it is now apparent that the
E. coli Tat system comprises at least three gene products
regarded as Hcf106-like proteins, designated TatA (11 kDa), TatB (18 kDa), and TatE (13 kDa) (Fig. 6) (32, 33; A. Chanal,
C. Santini, and L. F. Wu, Letter, Mol. Microbiol.
30:674-676, 1998). Like the corresponding gene products
identified in A. chroococcum, which are 8 (TatA) and 12 (TatB) kDa in size, they are predicted to have a very similar N-terminal membrane-spanning domain, followed by an amphipathic helix
(Fig. 6B). The fourth component identified in E. coli is predicted to constitute a membrane-integral protein, TatC (29 kDa), and
forms the essential core component of the translocation system
(6). Although we have not yet identified any of the corresponding genes in R. eutropha, successful
transcomplementation with a copy of tatA suggests that a
homolog or analog to TatC could be functional in this organism. We
report that A. chroococcum TatA promoted the translocation
of three basically different twin-arginine signal peptide-proteins
carrying the cofactors [NiFe], MGD, and polynuclear copper sites. Our
observation that TatA recognizes a broad range of proteins is
consistent with the very first analysis of bacterial Hcf106-like
proteins in E. coli. Mutational analysis has suggested that
they can compensate for each other in the translocation of
molybdoenzymes and [NiFe] hydrogenases to a certain extent, depending
on the enzyme studied (32, 43). One may speculate that the
Hcf106 analogs function in concert as a membrane-bound receptor complex
and thus respond to variations in the tertiary and oligomeric
structures of the different Tat substrates. The question remains
whether the Hcf106 analogs are sufficient to select, proofread, and
guide the various metalloproteins through a core component. From this
point of view, it is noteworthy that the formation of physiologically
active Tat-dependent enzymes requires individual sets of auxiliary
proteins. We have shown that eight accessory genes are involved in the
energy generation by the MBH in R. eutropha. However, the
functions of six of these gene products are still unknown
(3). Even much less is known about accessory genes of the
nap cluster and the nos locus of R. eutropha. Work is in progress to elucidate whether those accessory gene products assist the coordination of cofactor insertion and Tat-mediated translocation.
| |
ACKNOWLEDGMENTS |
We gratefully thank Geoffrey Yates for plasmid pGY12. The work of
Hubert Schröder to provide NapA-specific antiserum is highly acknowledged. We thank Ursula Stegert and Christine Reinemann for
technical assistance and Edward Schwartz for critically reading the manuscript.
The work was supported by grants from the Deutsche
Forschungsgemeinschaft (to R.A.S. and B.F.) and the Fonds der
Chemischen Industrie (to B.F.).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Institut
für Biologie der Humboldt-Universität zu Berlin,
Chausseestrasse 117, 10115 Berlin, Germany. Phone: 49-30-2093-8109. Fax: 49-30-2093-8102. E-mail:
roman.siddiqui{at}rz.hu-berlin.de.
| |
REFERENCES |
| 1.
|
Bedzyk, L.,
T. Wang, and R. W. Ye.
1999.
The periplasmic nitrate reductase in Pseudomonas sp. strain G-179 catalyzes the first step of denitrification.
J. Bacteriol.
181:2802-2806[Abstract/Free Full Text].
|
| 2.
|
Berks, B. C.
1996.
A common export pathway for proteins binding complex redox cofactors?
Mol. Microbiol.
22:393-404[CrossRef][Medline].
|
| 3.
|
Bernhard, M.,
E. Schwartz,
J. Rietdorf, and B. Friedrich.
1996.
The Alcaligenes eutrophus membrane-bound hydrogenase gene locus encodes functions involved in maturation and electron transport coupling.
J. Bacteriol.
178:4522-4529[Abstract/Free Full Text].
|
| 4.
|
Bernhard, M.,
B. Benelli,
A. Hochkoeppler,
D. Zannoni, and B. Friedrich.
1997.
Functional and structural role of the cytochrome b subunit of the membrane-bound hydrogenase complex of Alcaligenes eutrophus H16.
Eur. J. Biochem.
248:179-186[Medline].
|
| 5.
|
Bogsch, E.,
S. Brink, and C. Robinson.
1997.
Pathway specificity for a pH-dependent precursor thylakoid lumen protein is governed by a `Sec-avoidance' motif in the transfer peptide and a `Sec-incompatible' mature protein.
EMBO J.
16:3851-3859[CrossRef][Medline].
|
| 6.
|
Bogsch, E. G.,
F. Sargent,
N. R. Stanley,
B. C. Berks,
C. Robinson, and T. Palmer.
1998.
An essential component of a novel bacterial protein export system with homologues in plastids and mitochondria.
J. Biol. Chem.
273:18003-18006[Abstract/Free Full Text].
|
| 7.
|
Brim, H.,
M. Heyndrickx,
P. de Vos,
A. Wilmotte,
D. Springael,
H. G. Schlegel, and M. Mergeay.
1999.
Amplified rDNA restriction analysis and further genotypic characterisation of metal-resistant soil bacteria and related facultative hydrogenotrophs.
Syst. Appl. Microbiol.
22:258-268[Medline].
|
| 8.
|
Chaddock, A. M.,
A. Mant,
I. Karnauchov,
S. Brink,
R. G. Herrmann,
R. B. Klösgen, and C. Robinson.
1995.
A new type of signal peptide: central role of a twin-arginine motif in transfer signals for the pH-dependent thylakoidal protein translocase.
EMBO J.
14:2715-2722[Medline].
|
| 9.
|
Cramm, R.,
R. A. Siddiqui, and B. Friedrich.
1994.
Primary sequence and evidence for a physiological function of the flavohemoprotein of Alcaligenes eutrophus.
J. Biol. Chem.
269:7349-7354[Abstract/Free Full Text].
|
| 10.
|
Dias, J. M.,
M. E. Than,
A. Humm,
R. Huber,
G. P. Bourenkov,
H. D. Bartunik,
S. Bursakov,
J. Calvete,
J. Caldeira,
C. Carneiro,
J. J. Moura,
I. Moura, and M. J. Romao.
1999.
Crystal structure of the first dissimilatory nitrate reductase at 1.9 Å solved by MAD methods.
Structure
7:65-79[Medline].
|
| 11.
|
Dreusch, A.,
D. M. Burgisser,
C. W. Heizmann, and W. G. Zumft.
1997.
Lack of copper insertion into unprocessed cytoplasmic nitrous oxide reductase generated by an R20D substitution in the arginine consensus motif of the signal peptide.
Biochim. Biophys. Acta
1319:311-318[Medline].
|
| 12.
|
Eberz, G., and B. Friedrich.
1991.
Three trans-acting regulatory functions control hydrogenase synthesis in Alcaligenes eutrophus.
J. Bacteriol.
173:1845-1854[Abstract/Free Full Text].
|
| 13.
|
Eismann, K.,
K. Mlejnek,
D. Zipprich,
M. Hoppert,
H. Gerberding, and F. Mayer.
1995.
Antigenic determinants of the membrane-bound hydrogenase in Alcaligenes eutrophus are exposed toward the periplasm.
J. Bacteriol.
177:6309-6312[Abstract/Free Full Text].
|
| 14.
|
Friedrich, B.,
C. Hogrefe, and H. G. Schlegel.
1981.
Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus.
J. Bacteriol.
147:198-205[Abstract/Free Full Text].
|
| 15.
|
Friedrich, B., and E. Schwartz.
1993.
Molecular biology of hydrogen utilization in aerobic chemolithotrophs.
Annu. Rev. Microbiol.
47:351-383[CrossRef][Medline].
|
| 16.
|
Hynds, P. J.,
D. Robinson, and C. Robinson.
1998.
The Sec-independent twin-arginine translocation system can transport both tightly folded and malfolded proteins across the thylakoid membrane.
J. Biol. Chem.
273:34868-34874[Abstract/Free Full Text].
|
| 17.
|
Kortlüke, C.,
K. Horstmann,
E. Schwartz,
M. Rohde,
R. Binsack, and B. Friedrich.
1992.
A gene complex coding for the membrane-bound hydrogenase of Alcaligenes eutrophus H16.
J. Bacteriol.
174:6277-6289[Abstract/Free Full Text].
|
| 18.
|
Lowe, R. H., and H. J. Evans.
1964.
Preparation and some properties of soluble nitrate reductase from Rhizobium japonicum.
Biochim. Biophys. Acta
85:377-389.
|
| 19.
|
Lowry, O. H.,
N. J. Rosebrough,
A. L. Farr, and R. J. Randall.
1951.
Protein measurement with the Folin phenol reagent.
J. Biol. Chem.
193:265-275[Free Full Text].
|
| 20.
|
Miller, J. H.
1972.
Experiments in molecular genetics.
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 21.
|
Page, M. D., and S. J. Ferguson.
1989.
A bacterial c-type cytochrome can be translocated to the periplasm as an apo form; the biosynthesis of cytochrome cd1 (nitrite reductase) from Paracoccus denitrificans.
Mol. Microbiol.
3:653-661[CrossRef][Medline].
|
| 22.
|
Page, M. D., and S. J. Ferguson.
1990.
Apo forms of cytochrome c550 and cytochrome cd1 are translocated to the periplasm of Paracoccus denitrificans in the absence of haem incorporation caused either mutation or inhibition of haem synthesis.
Mol. Microbiol.
4:1181-1192[CrossRef][Medline].
|
| 23.
|
Pierik, A.,
M. Schmelz,
O. Lenz,
B. Friedrich, and S. P. J. Albracht.
1998.
Characterization of the active site of a hydrogen sensor from Alcaligenes eutrophus.
FEBS Lett.
438:231-235[CrossRef][Medline].
|
| 24.
|
Podzuweit, H. G.,
K. Schneider, and H. Knüttel.
1987.
Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain.
Biochim. Biophys. Acta
905:435-446[Medline].
|
| 25.
|
Pugsley, A. P.
1993.
The complete general secretory pathway in gram-negative bacteria.
Microbiol. Rev.
57:50-108[Abstract/Free Full Text].
|
| 26.
|
Rodrigue, A.,
D. H. Boxer,
M. A. Mandrand-Berthelot, and L.-F. Wu.
1996.
Requirement for nickel of the transmembrane translocation of NiFe-hydrogenase 2 in Escherichia coli.
FEBS Lett.
392:81-86[CrossRef][Medline].
|
| 27.
|
Rodrigue, A.,
A. Chanal,
K. Beck,
M. Müller, and L.-F. Wu.
1999.
Co-translocation of a periplasmic enzyme complex by a hitchhiker mechanism through the bacterial tat pathway.
J. Biol. Chem.
274:13223-13228[Abstract/Free Full Text].
|
| 28.
|
Sabaty, M.,
J. Gagnon, and A. Vermeglio.
1994.
Induction by nitrate of cytoplasmic and periplasmic proteins in the photodenitrifier Rhodobacter sphaeroides forma sp. denitrificans under anaerobic or aerobic condition.
Arch. Microbiol.
162:335-343[CrossRef][Medline].
|
| 29.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 30.
|
Sann, R.,
S. Kostka, and B. Friedrich.
1994.
A cytochrome cd1-type nitrite reductase mediates the first step of denitrification in Alcaligenes eutrophus.
Arch. Microbiol.
161:453-459[Medline].
|
| 31.
|
Santini, C. L.,
B. Ize,
A. Chanal,
M. Müller,
G. Giordano, and L.-F. Wu.
1998.
A novel Sec-independent periplasmic protein translocation pathway in Escherichia coli.
EMBO J.
17:101-112[CrossRef][Medline].
|
| 32.
|
Sargent, F.,
E. G. Bogsch,
N. R. Stanley,
M. Wexler,
C. Robinson,
B. C. Berks, and T. Palmer.
1998.
Overlapping functions of components of a bacterial Sec-independent protein export pathway.
EMBO J.
17:3640-3650[CrossRef][Medline].
|
| 33.
|
Settles, A. M., and R. Martienssen.
1998.
Old and new pathways of protein export in chloroplasts and bacteria.
Trends Cell Biol.
8:494-501[CrossRef][Medline].
|
| 34.
|
Settles, A. M.,
A. Yonetani,
A. Baron,
D. R. Bush,
K. Cline, and R. Martienssen.
1997.
Sec-independent protein translocation by the maize Hcf106 protein.
Science
278:1467-1470[Abstract/Free Full Text].
|
| 35.
|
Siddiqui, R. A.,
U. Warnecke-Eberz,
A. Hengsberger,
B. Schneider,
S. Kostka, and B. Friedrich.
1993.
Structure and function of a periplasmic nitrate reductase in Alcaligenes eutrophus H16.
J. Bacteriol.
175:5867-5876[Abstract/Free Full Text].
|
| 36.
|
Simon, R.,
U. Priefer, and A. Pühler.
1983.
A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram-negative bacteria.
Bio/Technology
1:748-791.
|
| 37.
|
Sweet, W. J.,
J. P. Houchins,
P. R. Rosen, and D. J. Arp.
1980.
Polarographic measurement of H2 in aqueous solutions.
Anal. Biochem.
107:337-340[CrossRef][Medline].
|
| 38.
|
Tabor, S., and C. C. Richardson.
1985.
A bacteriophage T7 polymerase/promoter system for controlled exclusive expression of specific genes.
Proc. Natl. Acad. Sci. USA
82:1074-1078[Abstract/Free Full Text].
|
| 39.
|
Thomas, P. E.,
D. Ryan, and W. Levin.
1976.
An improved staining procedure for the detection of the peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels.
Anal. Biochem.
75:168-176[CrossRef][Medline].
|
| 40.
|
Thöny-Meyer, L.
1997.
Biogenesis of respiratory cytochromes in bacteria.
Microbiol. Mol. Biol. Rev.
61:337-376[Abstract].
|
| 41.
|
Towbin, H.,
T. Staehelin, and J. Gordon.
1979.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:4350-4354[Abstract/Free Full Text].
|
| 42.
|
Warnecke-Eberz, U., and B. Friedrich.
1993.
Three nitrate reductase activities in Alcaligenes eutrophus.
Arch. Microbiol.
159:405-409[CrossRef].
|
| 43.
|
Weiner, J. H.,
P. T. Bilous,
G. M. Shaw,
S. P. Lubitz,
L. Frost,
G. H. Thomas,
J. A. Cole, and R. J. Turner.
1998.
A novel and ubiquitous system for membrane targeting and secretion of cofactor-containing proteins.
Cell
93:93-101[CrossRef][Medline].
|
| 44.
|
Yabuuchi, E.,
Y. Kosako,
I. Yano,
H. Hotta, and Y. Nishiuchi.
1995.
Transfer of two Burkholderia and an Alcaligenes species to Ralstonia gen. nov.: proposal of Ralstonia pickettii (Ralston, Palleroni and Doudoroff 1973) comb. nov., Ralstonia solanacearum (Smith 1896) comb. nov. and Ralstonia eutropha (Davis 1969) comb. nov.
Microbiol. Immunol.
39:897-904[Medline].
|
| 45.
|
Yates, M. G.,
E. M. De Souza, and J. H. Kahindi.
1997.
Oxygen, hydrogen and nitrogen fixation in Azotobacter.
Soil Biol. Biochem.
29:863-869[CrossRef].
|
| 46.
|
Yoshida, Y.,
M. Takai,
T. Satoh, and S. Takami.
1991.
Molybdenum requirement for translocation to the periplasmic space in a photodenitrifier, Rhodobacter sphaeroides f. sp. denitrificans.
J. Bacteriol.
173:3277-3281[Abstract/Free Full Text].
|
| 47.
|
Zumft, W. G.,
A. Dreusch,
S. Löchelt,
H. Cuypers,
B. Friedrich, and B. Schneider.
1992.
Derived amino acid sequences of the nosZ gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues. Implications for the CuA site of N2O reductase and cytochrome-c oxidase.
Eur. J. Biochem.
208:31-40[Medline].
|
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Abstract
Introduction
