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Applied and Environmental Microbiology, February 2000, p. 671-677, Vol. 66, No. 2
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Cytochrome c3 Mutants of
Desulfovibrio desulfuricans
Barbara J.
Rapp-Giles,1
Laurence
Casalot,1
R. Samuel
English,2
Joseph A.
Ringbauer Jr.,1
Alain
Dolla,3 and
Judy D.
Wall1,*
Biochemistry Department, University of
Missouri
Columbia, Columbia, Missouri 65211,1
College of Health Sciences, Roanoke, Virginia
24016,2 and BIP-IBSM, Centre
National de la Recherche Scientifique, 13402 Marseilles Cedex 20, France3
Received 19 August 1999/Accepted 24 November 1999
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ABSTRACT |
To explore the physiological role of tetraheme cytochrome
c3 in the sulfate-reducing bacterium
Desulfovibrio desulfuricans G20, the gene encoding the
preapoprotein was cloned, sequenced, and mutated by plasmid
insertion. The physical analysis of the DNA from the strain
carrying the integrated plasmid showed that the insertion was
successful. The growth rate of the mutant on lactate with sulfate was
comparable to that of the wild type; however, mutant cultures did not
achieve the same cell densities. Pyruvate, the oxidation product
of lactate, served as a poor electron source for the mutant.
Unexpectedly, the mutant was able to grow on hydrogen-sulfate
medium. These data support a role for tetraheme cytochrome
c3 in the electron transport pathway from
pyruvate to sulfate or sulfite in D. desulfuricans G20.
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INTRODUCTION |
The anaerobic sulfate-reducing
bacteria have several low-potential c-type cytochromes that
are located in the periplasm and presumably function in electron
transfer events. Several members of the genus Desulfovibrio
have been shown to have three cytochromes in this class, monoheme
cytochrome c553, tetraheme cytochrome c3, and a high-molecular-weight cytochrome
c (Hmc) with 16 hemes (20, 21). Of these
cytochromes, the most abundant, and that for which the most
structural information has been gathered, is tetraheme
cytochrome c3 (8, 21).
The physiological role of tetraheme cytochrome
c3 remains enigmatic. This cytochrome has been
reported to interact effectively with an almost unrealistic list of
electron donors and acceptors. In vivo, c3 has
been suggested to be the redox partner of the periplasmic hydrogenases
(5, 22), a role supported by the finding that this
cytochrome is tightly associated with hydrogenases during purification
of the latter (17). A model in which tetraheme cytochrome
c3 shuttles electrons from hydrogenases to the
various polyheme cytochromes (Hmc, nine-heme cytochrome, or octaheme
cytochrome c3) has been proposed (2, 15,
18). In addition to having periplasmically located redox
partners, tetraheme cytochrome c has been shown to form
functional complexes with a number of cytosolic electron carriers,
including ferredoxin (9), rubredoxin (28), and
flavodoxin (16). In vitro studies have shown that
tetraheme cytochrome c3 and an [NiFe]
hydrogenase will mediate reduction of metals, including Fe(III),
Cr(VI), and U(VI), with hydrogen as the electron donor (13).
The reactive nature of tetraheme cytochrome c3
makes biochemical characterization of redox partners and
functional analysis problematic; therefore, a genetic approach
was initiated. As a first step, it was necessary to determine whether
this cytochrome is essential for cell growth. Construction of a strain
with an interrupted gene encoding tetraheme cytochrome
c3 (cycA) allowed confirmation that
this cytochrome is not required for growth with lactate as the primary
source of carbon and reductant and with sulfate as the terminal
electron acceptor. However, the mutant grew poorly, if at all, on
pyruvate-sulfate medium. We infer that the first two electrons from
lactate bypass tetraheme cytochrome c3 while
those from pyruvate pass through that cytochrome for sulfate reduction.
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MATERIALS AND METHODS |
Bacterial strains and plasmids.
The sulfate-reducing
bacterium Desulfovibrio desulfuricans G20 is a spontaneously
nalidixic acid-resistant derivative of the wild-type strain G100A
(34) that is also cured of the endogenous cryptic plasmid
pBG1 (33). The Escherichia coli strains used as
hosts for cloning and for donors in conjugations were DH5
(11) and HB101 (7). Vectors and plasmids used are
listed in Table 1.
Growth of cells.
Routine cultures of D. desulfuricans were grown anaerobically in lactate (30 mM)-sulfate
(50 mM) medium (LS medium) (24). The headspace of cultures
contained the prepurified nitrogen atmosphere of the anaerobic chamber,
which had small amounts of hydrogen (<4 nmol/cm3). When
the lactate in the medium was replaced by pyruvate, the concentration
used was 20 mM. When thiosulfate replaced sulfate, a 50 mM
concentration was used. E. coli cultures were routinely grown with vigorous aeration in LC medium (1% [wt/vol] tryptone, 0.5% [wt/vol] Bacto Yeast Extract [Difco, Detroit, Mich.], and 0.5% [wt/vol] NaCl). Media were solidified by the addition of 1.5%
(wt/vol) agar. Concentrations of antibiotics used for D. desulfuricans were 200 µg of nalidixic acid/ml and 175 µg of
kanamycin/ml; for E. coli, kanamycin was used at 50 µg/ml,
ampicillin was used at 100 µg/ml, and chloramphenicol was used at 30 µg/ml.
Growth of
D. desulfuricans on hydrogen was accomplished in a
defined hydrogen-sulfate medium containing 20 mM
Na
2SO
4, 50 mM
sodium acetate, 20 mM
NH
4Cl, 1 mM CaCl
2, 3 mM MgCl
2, 1.5 mM KH
2PO
4,
and 1.5 ml of a trace element
solution (
25) modified to increase
the FeCl
2
concentration to 42 mM. After sterilization, Na
2S was
added
to a 2.5 mM final concentration to reduce the medium. Finally,
20 ml of
a sterile solution of 1 M sodium phosphate and 0.1 M
NaHCO
3, pH 7.0, was added per liter. Cultures to be tested
for
hydrogen-dependent growth were taken directly from frozen stocks
and inoculated (10% [vol/vol] inoculum) into hydrogen-sulfate
medium
with a headspace slightly pressurized with H
2 for 24 h
of growth, which allowed the consumption of residual substrates.
This
culture was subsequently used to inoculate (10% [vol/vol]
inoculum)
5 ml of the hydrogen-sulfate medium in a 70-ml serum
bottle. The
headspace atmosphere was made 100% H
2, and the bottles
were incubated horizontally at 37°C. Growth was monitored as the
increase in protein. To test for suppression or reversion of the
cycA mutation during hydrogen growth, 1 ml of the culture
was
used to inoculate LS medium. The resulting cells were used for
periplasmic protein extraction and cytochrome
c3
detection by
Western
analysis.
Analytical procedures.
Hydrogen was determined with a
thermal conductivity detector on a Aerograph gas chromatograph (model
90-P; Varian, Santa Clarita, Calif.) fitted with a column packed with
Molecular Sieve 5A (Supelco, Bellefonte, Pa.). Hydrogen appeared within
1 min of injection. Other gases that were present, including
CO2, H2S, and N2, appeared much
later and, therefore, did not interfere with hydrogen determinations. Utilization of lactate and the appearance of organic acid products were
monitored by high-performance liquid chromatography. Culture filtrates
were analyzed on an Aminex HPX-87H high-performance liquid
chromatograph equipped with a 300- by 7.8-mm column (Bio-Rad), a
Waters Associates (Milford, Mass.) chromatographic pump, and a Waters
model 484 tunable absorbance detector. With 5 mM
H2SO4 as the mobile phase, the organic acids
lactate, pyruvate, and acetate were separated and quantified. For
dry-weight determinations, 100-ml samples were harvested and
washed two times with 10 mM KH2PO4 (pH
7.0). Cell pellets were resuspended in 1-ml volumes of deionized
sterile water and dried until a constant weight was obtained. The
growth yield of cells with lactate as substrate was calculated as the
weight of cells (in grams) produced per mole of lactate consumed.
Isolation of cycA.
Voordouw and coworkers
(32) had reported 21 residues of the N-terminal amino acid
sequence of the mature cytochrome c3 purified from D. desulfuricans. From that protein sequence,
degenerate primers (NT fwd and NT rev [Table
2]) were devised to generate a PCR probe
for the identification of the c3 gene,
cycA. An amplicon of 63 bp was produced that was cloned into
SmaI-digested pBluescript II KS (Table 1). Either the
excised amplicon or the entire plasmid was randomly labeled according
to the protocol supplied with the Prime-a-Gene labeling system
(Promega, Madison, Wis.) and used to identify homologous sequences in
restriction endonuclease-digested chromosomal DNA. Sequencing of a
cloned chromosomal 1.7-kbp PstI fragment that hybridized
with the 63-bp amplicon showed that cycA was interrupted by
the PstI site near its 3' end. The cloned amplicon was also
used to identify a cycA-containing cosmid, pCOS20, from a
library of D. desulfuricans G20 DNA (S. Delgado and J. D. Wall, unpublished data). A 1.5-kbp ApaI fragment was
subcloned from the cosmid into pBC SK, producing pBSCYA. Sequencing of
the fragment revealed the arrangement of open reading frames (ORFs)
(Fig. 1A). For the identification of
cycA, the derived amino acid sequences were compared to
protein sequences in GenBank. Twenty of the amino acid residues
reported for the purified mature cytochrome c3
(32) were found to be part of the deduced polypeptide,
confirming the identification of cycA.
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FIG. 1.
Diagrammatic representation of the D. desulfuricans G20 chromosome region encoding cycA. (A)
Arrangement of genes in wild-type genomic DNA. The vertical arrow
indicates the position of a putative promoter. The black bar indicates
the position of a putative ORF upstream of cycA that is
transcribed left to right, as is cycA in this illustration.
t1 and t2 indicate the locations of possible
transcriptional terminators. (B) Plasmid pBSC2 (Table 1) used for gene
disruption. The SacII-PstI internal
cycA fragment is designated `cycA'. (C)
Plasmid-interrupted chromosomal cycA. The positions of PCR
and sequencing primers, B1 cyc primer and Km primer for the
leftward junction and T3 primer and cyc2 left
primer for the rightward junction, are indicated by arrows. The left
copy of cycA lacks 63 bp of the 3' end of the coding
sequence (which totals 393 bp), while the right copy lacks 71 bp of the
5' end. Note the different scale for panel A versus that for panels B
and C.
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Construction of the integration plasmid.
Plasmid pBGC2 was
prepared from pBluescript II SK, which does not replicate in the
sulfate-reducing bacteria. A 2.7-kbp EcoRV fragment
containing the mob genes from RSF1010 was inserted into pBluescript II SK at the EcoRV site. The resulting plasmid
was doubly digested with SacII and PstI, and an
internal 259-bp fragment from cycA generated with the same
enzymes was inserted. Finally, this composite was opened at its unique
EcoRI site and the kan gene from pUC4KIXX was
introduced, with subsequent selection for kanamycin resistance. The
resulting mutagenic plasmid was designated pBGC2 (Fig. 1B).
Conjugal transfer and selection for plasmid integration.
Conjugation was performed as described previously (1) for
triparental matings with pRK2073 as the helper plasmid. Nalidixic acid
was used to select against E. coli donors, and kanamycin was
used to select for the D. desulfuricans recipients that had integrated the plasmid. After 5 to 7 days, the rare Kmr
colonies that appeared were checked for residual E. coli
contamination and streaked for isolation of single colonies. Potential
mutants were grown in LS medium plus kanamycin, and samples were frozen at 
80°C as stocks for characterization.
Procedures for nucleic acids.
Enzymatic DNA manipulations
were carried out according to the directions of the enzyme suppliers.
Plasmid DNA preparations were made with QIAprep Spin Miniprep kits
(Qiagen, Inc., Santa Clara, Calif.). A Promega Wizard genomic DNA
purification kit was used to obtain chromosomal DNA. In addition, when
needed, CsCl purification of plasmid and chromosomal DNAs was performed as described elsewhere (4). Primer synthesis and standard
dideoxy sequencing were carried out by the Molecular Biology Program
DNA Core of the University of MissouriColumbia. Southern analyses employed Zeta-Probe blotting membranes from Bio-Rad (Hercules, Calif.) and were performed in accordance with the manufacturer's recommended procedure.
To explore the integrity of the left junction of the integrated plasmid
(Fig.
1C), a PCR amplicon was generated with the primer
pair B1
cyc-Km rev (Table
2). The 528-bp product was sequenced
directly or cloned into the pGEM-T vector and sequenced. When
a PCR
product was not obtained with these primers, the DNA from
the putative
mutant was digested with
SacII (Fig.
1C) and recircularized
to generate a Km
r Ap
r plasmid, and the
cycA gene was sequenced from the T3 primer (Promega).
To
examine the right junction of the integrated plasmid (Fig.
1C), the T3
primer located in the plasmid and a primer in the
cycA gene,
cyc2 left (Table
2), were used to produce a 108-bp
PCR
amplicon that was cloned and sequenced. When necessary,
recircularization
of a
KpnI digest of the mutant chromosomal
DNA was performed to
produce a replicon that contained the right
junction, facilitating
sequencing (Fig.
1C). PCRs were carried out in
50 µl of a solution
containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl,
0.1% (wt/vol) Triton
X-100, 2.5 mM MgCl
2, 0.1 mM (each)
deoxynucleoside triphosphate,
50 ng of DNA, 2.5 U of
Taq DNA
polymerase, and 0.4 µM (each) primer.
The thermal cycling profiles
consisted of three cycles of 94°C
at 1 min, 1 min of ramp time, 2.5 min at 37°C, 2.5 min of ramp
time, and 3 min at 72°C, followed by
35 cycles of 1 min at 94°C,
1 min at 50°C, and 1 min at 72°C.
For the preparation of RNA, 10-ml samples of culture were poured onto
3-ml portions of frozen TE buffer (10 mM Tris-1 mM EDTA,
pH 8.0) to
reduce the turnover of mRNA during harvesting. Then
initial isolation
steps were performed as described for the hot
phenol method
(
27). The resulting RNA was then purified with
an RNeasy
mini kit (Qiagen). Northern analysis was performed on
Magna Charge
nylon transfer membranes (Micron Separations, Inc.,
Westborough, Mass.)
by the procedures described by the company.
Transfer of RNA from gels
to membranes was always by capillary
diffusion.
Reverse transcription-PCR (RT-PCR) was performed with a Titan One Tube
RT-PCR kit (Boehringer Mannheim Corp., Indianapolis,
Ind.). The RNA
preparation was treated with RQ1 RNase-free DNase
(Promega) as
described by the manufacturer. The absence of contaminating
DNA was
confirmed by
PCR.
Protein analyses.
Cytochrome c3 was
purified as previously described (32). Polyclonal antibodies
were raised against purified cytochrome c3 in a
female New Zealand White rabbit. Pure protein (50 µg) was emulsified
with 0.5 ml of Freund's complete adjuvant (Sigma) and injected. The
rabbits were boosted once with 50 µg of protein plus incomplete
adjuvant. Western analysis showed that the antiserum interacted almost
exclusively with one polypeptide in the periplasmic extracts (Fig.
2). Total protein concentrations were
determined by Bradford analyses (4) with bovine serum
albumin as a standard.
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FIG. 2.
Immunodetection of cytochrome c3
in periplasmic extracts of D. desulfuricans. Polyclonal
antibodies raised against purified G20 cytochrome
c3 were used to probe periplasmic proteins
separated on a denaturing 10 to 22% (wt/vol) polyacrylamide gel. Lane
1, 1 µg of purified cytochrome c3; lane 2, 15 µg of periplasmic proteins from LS-grown G20; lane 3, 15 µg of
periplasmic proteins from LS-grown mutant I2; lane 4, protein molecular
size standards, with the relative molecular masses (in kilodaltons)
indicated on the right. This figure was digitally manipulated in Adobe
Photoshop 4.0 to omit two lanes containing irrelevant samples.
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The procedure of van der Westen et al. (
30) was used to
prepare periplasmic proteins. Briefly, 50 ml of an
early-stationary-phase
culture of sulfate-reducing bacterial cells was
harvested and
suspended in 1.0 ml of 50 mM Tris-HCl buffer (pH 9)-50
mM EDTA.
The mixture was gently stirred at 0°C for 45 min; the cells
were
removed by centrifugation at 7,000 ×
g for 10 min; and the supernatant,
following a second centrifugation, was used
for the identification
of cytochrome
c3 protein
by Western analysis. After polyacrylamide
gel electrophoresis of the
supernatant on denaturing 10 to 22%
(wt/vol) gradient gels, the
periplasmic proteins were transferred
onto nitrocellulose membranes
(BioTrace NT; Gelman Sciences, Pall
Corporation, Ann Arbor, Mich.) by
the method of Towbin et al.
(
29), with the inclusion of
0.0025% (wt/vol) sodium dodecyl
sulfate in the transfer buffer.
Phosphate-buffered saline (PBS)-Tween
(10 mM
NaH
2PO
4, 150 mM NaCl, 0.3% [vol/vol] Tween
20) with 2%
(wt/vol) nonfat dry milk (PBS-milk) was used to block the
membranes
overnight prior to immunodetection. The membrane was then
incubated
with the primary antibody at a dilution of 1:1,000 in
PBS-milk
for 1.5 h and washed twice (5 min each wash) in each of
three
buffers: PBS-Tween, 1 M NaCl in PBS-Tween, and PBS-milk.
Secondary
antibodies (goat anti-mouse immunoglobulin G and goat
anti-rabbit
immunoglobulin G; Sigma, St. Louis, Mo.) were alkaline
phosphatase
conjugates that were used at a 1:1,000 dilution in PBS-milk
for
a 1- to 2-h incubation. Two 5-min washes with each of five
solutions
(PBS-Tween, 1 M NaCl in PBS-Tween, PBS-Tween, PBS, and 100 mM
Tris [pH 9.5]) were performed sequentially. Color development
was
performed by the method of Blake et al. (
6) with 100 µl
each of nitroblue tetrazolium (33 mg/ml in 70% [vol/vol] dimethyl
sulfoxide; Sigma) and 5-bromo-4-chloro-3-indolylphosphate (17
mg/ml in
100% [vol/vol] dimethyl sulfoxide; Sigma) per 10 ml of
100 mM Tris
buffer (pH 9.5). The stop solution consisted of 10
ml of PBS-Tween
containing 100 µl of 0.5 M
EDTA.
Cytochrome
c3 protein in periplasmic extracts
was also visualized without electrophoretic separation of polypeptides,
by spotting
known quantities of extracted periplasmic protein directly
onto
nitrocellulose membranes. Processing and immunodetection were
as
described for Western
analyses.
Reagents.
Enzymes for DNA manipulation were purchased from
Boehringer Mannheim, Promega, New England Biolabs (Beverly, Mass.), and
Gibco BRL (Gaithersburg, Md.) and were used according to the
manufacturers' instructions. [-32P]dCTP (3,000 Ci/mmol, 10 mCi/ml) was from NEN Life Sciences Products (Boston,
Mass.). Coomassie brilliant blue G-250 for protein staining was from
Eastman Kodak Co. (Rochester, N.Y.). All other chemicals and
biochemicals were purchased from Fisher Scientific (Pittsburgh, Pa.) or
Sigma-Aldrich (St. Louis, Mo.).
Nucleotide sequence accession number.
The sequence of the
cycA gene has been deposited in the GenBank nucleotide
sequence database under accession no. AF205067.
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RESULTS |
Cloning of cycA.
The gene encoding cytochrome
c3 was cloned from D. desulfuricans
G20 on a 1.5-kb ApaI fragment (Fig. 1). When an internal fragment of the cloned gene was used as a probe for chromosomal DNA, a
single hybridizing band was observed, suggesting that one cytochrome
c3 gene was present (data not shown). Sequencing
revealed an ORF of 390 bp from which was deduced a polypeptide of 130 amino acid residues that exhibited the characteristic features of a c3 cytochrome (8) (Fig.
3). Upstream, and apparently transcribed in the same orientation, was part of an ORF whose product has similarity to a methyltransferase. In the 257-bp intergenic region (Fig. 4) were several inverted repeats
that could serve as transcriptional signals. Twenty-four base pairs
after the putative cytochrome stop codon was a region that when
transcribed could form an exact 10-bp stem (9 of the 10 bp are GCs)
with a 4-base loop that might signal transcriptional termination. No
additional ORFs were detected within 160 bp of the 3' end of the
cycA coding sequence. Between the upstream ORF encoding the
putative methyltransferase and cycA were found exact
consensus sequences derived for Desulfovibrio promoters
(21) (TTGACA and TAGGAT [the 35 and 10 sequences, respectively]). These sequences were located 44 bp upstream of the ATG
start codon of cycA and were separated by 17 bp. However, RT-PCR analysis of the mRNA (data not shown) revealed that some transcripts encoding CycA extended as far upstream as 108 bp from the
initiation codon of cycA. The number and location of
promoters for this gene have yet to be determined.
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FIG. 3.
Alignment of D. desulfuricans G20
cytochrome c3 amino acid sequences with those of
other members of the genus. Boxed sequences are the four heme binding
sites. The arrow indicates the apparent cleavage site for the signal
peptide for the D. desulfuricans G20 proapoprotein.
Asterisks mark the conserved histidines that are the sixth axial
ligands to the hemes. DdG20, D. desulfuricans G20 (sequence
deduced from that of the cloned gene); DvHil, D. vulgaris
Hildenborough (sequence deduced from that of the cloned gene [Protein
Identification Resource Database, Johns Hopkins University, PIR
no. A24799]); DvMiy, D. vulgaris Miyazaki (PIR no.
S33874); Ds, D. salexigens (PIR no. A00128); Dg, D. gigas (PIR no. A00126). Levels of identity between the cytochrome
sequence of D. desulfuricans G20 and those of D. vulgaris Hildenborough and Miyazaki, D. salexigens, and D. gigas were 66, 69, 35, and 54%,
respectively.
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FIG. 4.
Sequence of the intergenic region upstream of
cycA. Asterisks indicate the termination codon of the
putative upstream ORF and the start codon of the cycA gene.
Prominent inverted repeats are indicated by arrows. A possible promoter
for cycA, the 35 and 10 regions (separated by 17 bp), is
boxed.
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Cytochrome features recognized in the derived polypeptide include a
23-amino-acid leader sequence for targeting the polypeptide
to the
periplasmic space and four heme binding motifs, two each
of
CX
2CH
and
CX
4CH
sequences. The
N-terminal 20 amino acids
of the mature cytochrome
c3 reported for this strain (
32)
correlate
exactly with residues 24 through 43 of the deduced
polypeptide,
confirming the identity of the
cycA gene. The
21st residue, reported
from the protein sequencing to be a lysine
(
32), was found instead
from the gene sequence to be a
histidine, confirming the more
recent protein sequencing of Aubert et
al. (
3). When compared
with tetraheme cytochromes from
several other
Desulfovibrio strains
(Fig.
3), conservation
of the heme binding sites and the histidines
providing the sixth axial
ligands for the hemes was evident. The
sequence of cytochrome
c3 from
D. desulfuricans G20 was
found
to be 66 and 67% identical to the homologs from
Desulfovibrio vulgaris Hildenborough and Miyazaki,
respectively.
Disruption of cycA by plasmid integration.
To gain
insight into the role of cytochrome c3 in the
metabolism of D. desulfuricans, a mutation of the
cycA gene that eliminated the production of a functional
protein was constructed. A plasmid unable to replicate in D. desulfuricans, pBGC2, containing a 259-bp fragment internal to
cycA, whose integration into the chromosomal cycA
would disrupt the gene and could be detected by the acquisition of
kanamycin resistance encoded on the plasmid, was constructed (Fig. 1B).
The cycA fragment lacked the coding sequences for the signal
sequence at the N terminus and the attachment site for the fourth heme
at the C terminus. Thus, neither of the two copies of cycA
resulting from the plasmid integration encodes a proapopolypeptide that
could be processed into a functional cytochrome.
Five Km
r colonies were obtained following conjugal transfer
of the plasmid into strain G20, and Southern analyses were performed
to
confirm the predicted chromosomal structure.
KpnI digests of
the DNA from the presumed mutants and the wild-type parental strain
were probed sequentially with the
cycA internal fragment and
the
Km
r gene,
kan (Fig.
5). The wild-type DNA contained a single
band
of 2.8 kb hybridizing with the
cycA probe, while each
of the possible
mutants had two bands, 4.4 and 6.5 kb, as predicted for
an interrupted
gene. As expected, the Km
r probe did not
hybridize with the wild-type DNA but did hybridize
with a 6.5-kb band
of the mutant DNA. Free plasmid was apparently
not present in the
putative mutants, as evidenced by the absence
of a hybridizing fragment
the size of the
KpnI-digested plasmid,
7.5 kb, in the
Southern analysis and by the inability to recover
plasmid from the
exconjugants. These results confirmed integration
of the plasmid
into the
KpnI DNA fragment containing
cycA,
generating
D. desulfuricans mutants, I1 through
I5, that were structurally
identical.
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FIG. 5.
Confirmation, by Southern analysis, of integration of
the pBSC2 plasmid into the D. desulfuricans G20 chromosome.
Lane 1, G20 total chromosomal DNA digested with KpnI; lanes
2 to 6, total chromosomal DNAs of five kanamycin-resistant strains,
derived after the introduction of pBSC2, digested with KpnI.
Sizes of hybridizing DNA fragments are indicated on the left (in
kilobase pairs). Identical gels were probed with the pCRA amplicon
insert coding for the N-terminal region of the mature
cytochrome c3 (A) or with the kan
cassette from pUC4KIXX (B). KpnI-digested pBSC2
migrated as a 7.5-kbp fragment (not shown). The figure is
representative of six independent Southern analyses.
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Northern analysis of mRNA showed that
cycA transcripts of
about 570 bases were abundant in the wild type but, in I3, were
below
the detection limit of about 10% of the wild-type level.
The presence
of a barely detectable transcript that was about
100 bases longer was
observed in mRNA from I3 (Fig.
6). The
small
amount of longer transcript could be due to readthrough from the
3'-truncated (upstream) copy of the gene that no longer had the
strong
stem-loop structure hypothesized to terminate transcription
of
cycA. These data suggested that cytochrome
c3 is encoded in
a single-gene operon, a
conclusion that is consistent with the
sequence analysis. As a
further test to determine whether
cycA is transcribed
as part of an operon, an RT-PCR to detect transcripts
extending
from the upstream putative methyltransferase-encoding
ORF into
cycA (primers [Table
2], ORF fwd and
cyc2 left)
was
performed. In addition, a second RT-PCR to detected transcripts
extending beyond the strong stem-loop structure at the 3' end
of
cycA (primers, B1
cyc and 1932 rev) was carried
out. Neither
of these analyses gave detectable products, in contrast to
RT-PCR
experiments that allowed detection of the
cycA
(primers, B1
cyc and 1932 rev) or ORF (primers, ORF fwd and
ORF rev) transcripts
alone as controls (data not shown).
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FIG. 6.
Transcription of cycA in D. desulfuricans G20 and constructed strain I3. Total RNA from
mid-exponential-phase cultures of G20 (lane 1) and I3 (lane 2) was
probed with the pCRA amplicon insert. Transcript sizes (shown to the
left) were estimated relative to rRNA migration. The figure is
representative of four independent analyses. This figure was digitally
manipulated in Adobe Photoshop 4.0 to omit one lane containing an
irrelevant sample.
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Mutant instability.
We were surprised to find an abundant
13-kDa heme-staining protein in periplasmic extracts from a subculture
of the cells used for the Northern analysis (data not shown). A
Southern analysis of the DNA from the same cells extracted for
periplasmic proteins confirmed that the plasmid was still integrated
into the gene. To evaluate whether a second tetraheme cytochrome was
now being produced (19), cytochromes were purified from the
periplasm of this strain. Two fractions, representing a monomeric
cytochrome of 13 kDa (isoelectric point, 5.8) and, presumably, a
polymeric version thereof, contained 80% of the periplasmic cytochrome
(A. Dolla, unpublished data). N-terminal sequencing of 21 and 12 residues from the monomeric and polymeric versions, respectively,
established that the proteins were the same as that encoded by the
cycA gene and previously reported as cytochrome
c3 from this bacterium (32). The
remainder of the cytochrome was found in fractions that corresponded to
a putative high-molecular-weight cytochrome and a monoheme cytochrome
c553. Clearly functional cytochrome
c3 was still being produced in the constructed
strain, in spite of the maintenance of the integrated plasmid.
The insertion junctions of the integrated plasmid in I3 (Fig.
1C) were
sequenced to confirm the construction of an interrupted
gene. The 3'
deletion in the upstream gene copy was confirmed;
however, a complete
copy of
cycA was restored downstream. A spontaneous
insertion of 235 bp containing the 5' end of
cycA,
with a concomitant
deletion of 209 bp of pBluescript II SK encoding the
lac promoter
and primer sites for sequencing, had occurred.
The similarity
in the sizes of the deleted and inserted material in
this suppressed
strain, designated B1, accounted for the inability to
observe
the reversion by Southern analysis. In fact, subsequent work
revealed
another suppressor, B5, in which a 106-bp insert restored the
C-terminal coding region of the leftward copy of
cycA (Fig.
1C)
and was accompanied by a deletion of 101 bp to the left of the
gene
conferring kanamycin
resistance.
To determine conditions in which the mutant might be more stable, the
presence of cytochrome
c3 in periplasmic
extracts was
monitored with polyclonal antibodies made against purified
protein.
These antibodies showed no cross-reactivity with other
periplasmic
c-type cytochromes (Fig.
2). With a detection
limit of less than
10 ng of
c3, and with
wild-type G20 producing about 300 ng of
c3/µg
of periplasmic protein in early-stationary-phase cells,
a 30-fold
reduction in this cytochrome could be easily confirmed.
After
confirmation by PCR that the junctions between the integrated
plasmid
and the chromosomal DNA of
cycA mutant I2 were as predicted,
I2 was subcultured daily in LS medium plus kanamycin. Detectable
c3 was not found in the periplasm (data not
shown), suggesting
that the majority of the population was mutant. In
the absence
of kanamycin,
c3 protein was
detectable in the third subculture
and the cells were now kanamycin
sensitive, as expected. Loss
of the integrated plasmid in these
revertants was confirmed by
Southern analysis (data not shown). When
the mutant culture was
allowed to enter stationary phase in LS medium
plus kanamycin
and remain nongrowing for 4 to 5 days, the cytochrome
was detected
in the recovered Km
r cells, indicating that
suppression had occurred. Because the
cycA sequences were
not deleted from the mutant chromosomes, recombination
events that
would be able to restore a wild-type
cycA gene could
be
envisioned. Clearly in early work with the mutant, stationary-phase
cultures maintained in kanamycin-containing medium had been subcultured
for experimentation and suppressors had accumulated. Subcultures
to
confirm the transcription studies and all subsequent growth
experiments
with the mutant were started with carefully maintained
exponential-phase cultures and were strictly monitored for suppression
by Western
analyses.
Characterization of cycA mutant I2.
I2 maintained
under growth conditions in LS medium with kanamycin appeared to be
sufficiently stable for a preliminary physiological analysis. The
growth rate of I2 on LS medium was not significantly different from
that of the parental strain (Table 3);
however, the mutant never reached the same cell density (Fig.
7A). These results were not altered when
kanamycin was omitted from the I2 medium. Protein analysis of
early-stationary-phase cultures established that the mutant accumulated
only 70% ± 15% (four determinations) of the protein accumulated by
the wild type. In preliminary analyses, the yield of exponential-phase
cultures of I2 grown on lactate corresponded to about 80% of that of
the wild type (5.4 g · mol
1).
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|
FIG. 7.
Growth curves of D. desulfuricans wild-type
strain G20 ( ) and mutant strain I2 ( ) on LS medium
(representative of 14 trials) (A) and on pyruvate-sulfate
(representative of three trials) (B). The growth temperature was
37°C.
|
|
When I2 was tested with pyruvate as the primary source of carbon and
reductant and sulfate as the terminal electron acceptor,
growth was
barely detectable (Fig.
7B; Table
3). Curiously, preliminary
experiments with pyruvate and thiosulfate as growth substrates
showed
that I2 grew, but two- to threefold slower than the wild
type. The
headspaces of cultures grown to early stationary phase
in different
media were tested for evolved hydrogen. Small amounts
of hydrogen (0.02 µmol/mg of cell protein) were produced in lactate-sulfate
cultures of the wild type, G20, but hydrogen was never detected
in
pyruvate cultures of G20, regardless of the electron acceptor.
In
contrast, 20- to 100-fold-higher hydrogen concentrations were
found in
cultures of I2 growing on LS medium. Even though I2 cultured
with
pyruvate as the electron donor grew poorly, if at all, 4
to 30 µmol
of hydrogen/mg of cell protein had accumulated after
10 h of
incubation. Periplasmic extracts were prepared from mutant
cultures
grown on each of the media to determine that suppressors
or revertants
did not dominate. We infer from the slower growth
of the mutant on
pyruvate that electron transfer from this substrate
involves cytochrome
c3.
To further characterize the utilization of lactate by I2 and the
parental strains, the organic acid content of LS medium was
monitored
with growth. While both strains completely consumed
the lactate,
neither pyruvate nor formate accumulation could be
detected in the two
cultures. There was essentially a stoichiometric
production of acetate
as lactate was consumed (data not
shown).
Growth of the mutant with hydrogen as the electron donor and sulfate as
the terminal acceptor was examined. Growth, as measured
by optical
density or by whole-cell protein, was not abundant
for the wild type;
however, similar growth was detected for I2
(Table
3).
| |
DISCUSSION |
The previous determination of the N-terminal amino acid sequence
of the purified tetraheme cytochrome c3 from
D. desulfuricans G200 (32) allowed the
isolation of the gene cycA from chromosomal DNA. The
complete primary sequence of the proapoprotein was deduced from the
gene sequence, revealing the presence of a signal sequence upstream of
the N terminus of the mature protein that was consistent with the
periplasmic location of the cytochrome. As reported for D. vulgaris Hildenborough (31), a monocistronic transcript
for cycA that was consistent with the position of a
consensus promoter and putative transcriptional terminator was found.
The most commonly held supposition for the function of
Desulfovibrio cytochrome c3 is that
it is an electron transfer partner for periplasmic hydrogenases. It was
reasoned that if hydrogenase null mutants could be created, the
cytochrome that couples with the hydrogenase could also be eliminated
by mutation. Experiments creating knockout mutations in the [NiFe],
NADP-reducing, and/or [Fe] hydrogenase gene of Desulfovibrio
fructosovorans have shown that the loss of these hydrogenases,
singly or in combination, is not lethal and does not impair growth on
hydrogen with sulfate (14, 26; L. Casalot, M. Rousset, P. de Phillip, E. C. Hatchikian, Z. Dermoun, and J. P. Bélaich, Abstr. 5th Int. Conf. Mol. Biol. Hydrogenases, p.
113, 1997). Mutations in an [Fe] hydrogenase and in a [NiFe]
hydrogenase of D. desulfuricans G20 have recently been
constructed and also do not result in dramatic growth phenotypes (J. A. Ringbauer and J. D. Wall, unpublished data). A mutation in the
gene encoding cytochrome c3 was constructed, but
unlike mutations in the hydrogenase operons, the mutation impaired
growth on organic acids but did not prevent growth on hydrogen with sulfate.
The growth phenotypes of the I2 mutant lacking cytochrome
c3, wild-type growth rates on lactate but poor
or no growth on pyruvate, give clues to the metabolic roles of
c3. These substrates provide both carbon and
reductant for the cultures (Fig. 8).
Because the oxidation of lactate gives rise to pyruvate, clearly
pyruvate is a ready carbon source for I2. Therefore, the lack of growth on pyruvate-sulfate must reflect a block in electron transport from
pyruvate to sulfate. The accumulation of hydrogen in the headspace of
the I2 cultures suggests one of three alternatives: (i) cytochrome
c3 is a component of the electron transfer
pathway from pyruvate to sulfate, and impaired
c3 results in release of excess reductant as
hydrogen (Fig. 8); (ii) electrons from pyruvate participate in
intracellular hydrogen cycling (17), and cytochrome c3 functions with a periplasmic hydrogenase
to allow reoxidation of the cytoplasmically produced hydrogen; or (iii)
cytochrome c3 functions in both the preceding
processes. We consider alternative explanation (i) to be the most
likely, since growth rates on hydrogen-sulfate are not sufficient to
account for the rapid growth of the wild type via recycling hydrogen
from pyruvate-sulfate. Explanation (ii) is made less likely by the
observation of growth of the mutant on hydrogen-sulfate. Growth of
mutant I2 on pyruvate-thiosulfate, while slower than that of the wild
type, did occur. Thus, cytochrome c3 may be
involved in that electron pathway, but it is not essential. Although
molar growth yields might be informative in distinguishing these
possibilities, the quantification would be confounded by any
reutilization of hydrogen, by the occurrence of suppressors, and if
compensation by other c-type cytochromes occurs.
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|
FIG. 8.
Diagrammatic representation of organic acid oxidation
and electron flow to sulfate as the terminal electron acceptor for
D. desulfuricans G20. The asterisk indicates the region of
the metabolic pathway likely to be affected by the absence of
cytochrome c3 in mutant I2, causing poor growth
on pyruvate with a buildup of reductant that is released as hydrogen.
Arrows do not imply single-electron-transfer components.
|
|
The inability of I2 to grow on pyruvate contributes to the lower cell
yield observed when I2 is grown on lactate. Because of this altered
metabolism, it is logical that suppressed derivatives would accumulate
in stationary-phase LS cultures of I2, as we have observed. We conclude
that cytochrome c3 is not essential for growth
on lactate but is necessary for efficient use of reductant from
pyruvate. Therefore, the electron transfer pathways to sulfate from
these two substrates have one or more unshared components. A
cycA deletion is being constructed to further characterize
this pathway.
| |
ACKNOWLEDGMENTS |
We thank Nancy David, Peter Tipton, and Robert Kunz for technical
advice and assistance.
This work was supported in part by the Basic Energy Research Program
and the Natural and Accelerated Bioremediation Research Program of the
U.S. Department of Energy through grants DE-FG02-87ER13713 and DE
FG02-97ER62495, respectively; the Missouri Agricultural Experiment
Station; and the Centre National de la Recherche Scientifique.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Biochemistry
Department, 117 Schweitzer Hall, University of MissouriColumbia,
Columbia, MO 65211. Phone: (573) 882-8726. Fax: (573) 882-5635. E-mail: wallj{at}missouri.edu.
| |
REFERENCES |
| 1.
|
Argyle, J. L.,
B. J. Rapp-Giles, and J. D. Wall.
1992.
Plasmid transfer by conjugation in Desulfovibrio desulfuricans.
FEMS Microbiol. Lett.
94:255-262[CrossRef].
|
| 2.
| Aubert, C., M. Brugna, A. Dolla, M. Bruschi, and
M. T. Giudici-Orticoni. A sequential electron transfer from
hydrogenases to cytochromes in sulfate-reducing bacteria. Biochim.
Biophys. Acta, in press.
|
| 3.
|
Aubert, C.,
G. Leroy,
P. Bianco,
E. Forest,
M. Bruschi, and A. Dolla.
1998.
Characterization of the cytochromes C from Desulfovibrio desulfuricans.
Biochem. Biophys. Res. Commun.
242:213-218[CrossRef][Medline].
|
| 4.
|
Ausubel, F. M.,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith, and K. Struhl (ed.).
1987.
Current protocols in molecular biology.
Green Publishing Associates, New York, N.Y.
|
| 5.
|
Bianco, P.,
J. Haladjian,
M. Bruschi, and F. Guerlesquin.
1992.
Reactivity of [Fe] and [Ni-Fe-Se] hydrogenases with their oxido-reduction partner, the tetraheme cytochrome c3.
Biochem. Biophys. Res. Commun.
189:633-639[CrossRef][Medline].
|
| 6.
|
Blake, M. S.,
K. H. Johnston,
G. J. Russell-Jones, and E. C. Gotschlich.
1984.
A rapid, sensitive method for detection of alkaline phosphatase-conjugated anti-antibody on Western blots.
Anal. Biochem.
136:175-179[CrossRef][Medline].
|
| 7.
|
Boyer, H. W., and D. Roulland-Dussoix.
1969.
A complementation analysis of the restriction and modification of DNA in Escherichia coli.
J. Mol. Biol.
41:459-472[CrossRef][Medline].
|
| 8.
|
Coutinho, I. B., and A. V. Xavier.
1994.
Tetraheme cytochromes.
Methods Enzymol.
243:119-140[Medline].
|
| 9.
|
Dolla, A.,
G. Leroy,
F. Guerlesquin, and M. Bruschi.
1991.
Identification of the site of interaction between cytochrome c3 and ferredoxin using peptide mapping of the cross-linked complex.
Biochim. Biophys. Acta
1058:171-177[Medline].
|
| 10.
|
Figurski, D. H., and D. R. Helinski.
1979.
Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.
Proc. Natl. Acad. Sci. USA
76:1648-1652[Abstract/Free Full Text].
|
| 11.
|
Hanahan, D.
1983.
Studies on transformation of Escherichia coli with plasmids.
J. Mol. Biol.
166:557-580[Medline].
|
| 12.
|
Kim, C. H.,
D. R. Helinski, and G. Ditta.
1986.
Overlapping transcription of the nifA regulatory gene in Rhizobium meliloti.
Gene
50:141-148[CrossRef][Medline].
|
| 13.
|
Lovley, D. R.
1993.
Metal-reducing microorganisms.
Annu. Rev. Microbiol.
47:263-290[CrossRef][Medline].
|
| 14.
|
Malki, S.,
G. de Luca,
M. L. Fardeau,
M. Rousset,
J. P. Bélaich, and Z. Dermoun.
1997.
Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans.
Arch. Microbiol.
167:38-45[CrossRef][Medline].
|
| 15.
|
Matias, P. M.,
L. M. Saraiva,
C. M. Soares,
A. V. Coelho,
J. Legall, and M. A. Carrondo.
1999.
Nine-haem cytochrome c from Desulfovibrio desulfuricans ATCC 27774: primary sequence determination, crystallographic refinement at 1.8 and modeling studies of its interaction with the tetrahaem cytochrome c3.
J. Biol. Inorg. Chem.
4:478-494[CrossRef][Medline].
|
| 16.
|
Palma, P. N.,
I. Moura,
J. LeGall,
J. van Beeumen,
J. E. Wampler, and J. J. G. Moura.
1994.
Evidence for a ternary complex formed between flavodoxin and cytochrome c3. 1H-NMR and molecular modeling studies.
Biochemistry
33:6394-6407[CrossRef][Medline].
|
| 17.
|
Peck, H. D., Jr.
1993.
Bioenergetic strategies of the sulfate-reducing bacteria, p. 41-76.
In
J. M. Odom, and R. Singleton, Jr. (ed.), The sulfate-reducing bacteria: contemporary perspectives. Springer-Verlag, New York, N.Y.
|
| 18.
|
Pereira, I. A. C.,
C. V. Romao,
A. V. Xavier,
J. Legall, and M. Teixiera.
1998.
Electron transfer between hydrogenases and mono- and multiheme cytochromes in Desulfovibrio ssp.
J. Biol. Inorg. Chem.
3:494-498[CrossRef].
|
| 19.
|
Pieulle, L.,
J. Haladjian,
J. Bonicel, and E. C. Hatchikian.
1996.
Biochemical studies of the c-type cytochromes of the sulfate reducer Desulfovibrio africanus. Characterization of two tetraheme cytochromes c3 with different specificity.
Biochim. Biophys. Acta
1273:51-61[Medline].
|
| 20.
|
Pollock, W. B. R.,
M. Loutfi,
M. Bruschi,
B. J. Rapp-Giles,
J. D. Wall, and G. Voordouw.
1991.
Cloning, sequencing, and expression of the gene encoding the high-molecular-weight cytochrome c from Desulfovibrio vulgaris Hildenborough.
J. Bacteriol.
173:220-228[Abstract/Free Full Text].
|
| 21.
|
Pollock, W. B. R., and G. Voordouw.
1994.
Molecular biology of c-type cytochromes from Desulfovibrio vulgaris Hildenborough.
Biochimie
76:554-560[Medline].
|
| 22.
|
Postgate, J. R.
1984.
The sulphate-reducing bacteria, 2nd ed.
Cambridge University Press, Cambridge, United Kingdom.
|
| 23.
|
Priefer, U. B.,
R. Simon, and A. Pühler.
1985.
Extension of the host range of Escherichia coli vectors by incorporation of RSF1010 replication and mobilization functions.
J. Bacteriol.
163:324-330[Abstract/Free Full Text].
|
| 24.
|
Rapp, B. J., and J. D. Wall.
1987.
Genetic transfer in Desulfovibrio desulfuricans.
Proc. Natl. Acad. Sci. USA
84:9128-9130[Abstract/Free Full Text].
|
| 25.
|
Rousset, M.,
L. Casalot,
B. J. Rapp-Giles,
Z. Dermoun,
P. de Philip,
J. P. Bélaich, and J. D. Wall.
1998.
New shuttle vectors for the introduction of cloned DNA in Desulfovibrio.
Plasmid
39:114-122[CrossRef][Medline].
|
| 26.
|
Rousset, M.,
Z. Dermoun,
M. Chippaux, and J. P. Bélaich.
1991.
Marker exchange mutagenesis of the hydN genes in Desulfovibrio fructosovorans.
Mol. Microbiol.
5:1735-1740[CrossRef][Medline].
|
| 27.
|
Scherrer, K.
1969.
Isolation and sucrose gradient analysis of RNA, p. 413-432.
In
K. Habel, and E. Salsman (ed.), Fundamental techniques in virology. Academic Press, New York, N.Y.
|
| 28.
|
Stewart, D. E., and J. E. Wampler.
1991.
Molecular dynamics simulations of the cytochrome c3-rubredoxin complex from Desulfovibrio vulgaris.
Proteins Struct. Funct. Genet.
11:142-152[CrossRef][Medline].
|
| 29.
|
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].
|
| 30.
|
van der Westen, H.,
S. G. Mayhew, and C. Veeger.
1978.
Separation of hydrogenase from intact cells of Desulfovibrio vulgaris.
FEBS Lett.
86:122-126[CrossRef][Medline].
|
| 31.
|
Voordouw, G., and S. Brenner.
1986.
Cloning and sequencing of the gene encoding cytochrome c3 from Desulfovibrio vulgaris Hildenborough.
Eur. J. Biochem.
159:347-352[Medline].
|
| 32.
|
Voordouw, G.,
W. B. R. Pollock,
M. Bruschi,
F. Guerlesquin,
B. J. Rapp-Giles, and J. D. Wall.
1990.
Functional expression of Desulfovibrio vulgaris Hildenborough cytochrome c3 in Desulfovibrio desulfuricans G200 after conjugational gene transfer from Escherichia coli.
J. Bacteriol.
172:6122-6126[Abstract/Free Full Text].
|
| 33.
|
Wall, J. D.,
B. J. Rapp-Giles, and M. Rousset.
1993.
Characterization of a small plasmid from Desulfovibrio desulfuricans and its use for shuttle vector construction.
J. Bacteriol.
175:4121-4128[Abstract/Free Full Text].
|
| 34.
|
Weimer, P. J.,
M. J. Van Kavelaar,
C. B. Michel, and T. K. Ng.
1988.
Effect of phosphate on the corrosion of carbon steel and on the composition of corrosion products in two-stage continuous cultures of Desulfovibrio desulfuricans.
Appl. Environ. Microbiol.
54:386-396[Abstract/Free Full Text].
|
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Abstract
Introduction
