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Journal of Bacteriology, August 2001, p. 4918-4926, Vol. 183, No. 16
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.16.4918-4926.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Isolation and Characterization of a Shewanella
putrefaciens MR-1 Electron Transport Regulator
etrA Mutant: Reassessment of the Role of
EtrA
Tamara M.
Maier and
Charles R.
Myers*
Department of Pharmacology and Toxicology,
Medical College of Wisconsin, Milwaukee, Wisconsin 53226
Received 19 October 2000/Accepted 23 May 2001
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ABSTRACT |
Shewanella putrefaciens MR-1 has emerged as a good
model to study anaerobic respiration and electron transport-linked
metal reduction. Its remarkable respiratory plasticity suggests the potential for a complex regulatory system to coordinate electron acceptor use in the absence of O2. It had previously been
suggested that EtrA (electron transport regulator A), an analog of Fnr
(fumarate nitrate regulator) from Escherichia coli, may
regulate gene expression for anaerobic electron transport. An
etrA knockout strain (ETRA-153) was isolated from
MR-1 using a gene replacement strategy. Reverse transcription-PCR
analysis of total RNA demonstrated the loss of the etrA
mRNA in ETRA-153. ETRA-153 cells retained the ability to grow on all
electron acceptors tested, including fumarate, trimethylamine
N-oxide (TMAO), thiosulfate, dimethyl sulfoxide, ferric
citrate, nitrate, and O2, as well as the ability to reduce ferric citrate, manganese(IV), nitrate, and nitrite. EtrA is therefore not necessary for growth on, or the reduction of, these electron acceptors. However, ETRA-153 had reduced initial growth rates on
fumarate and nitrate but not on TMAO. The activities for fumarate and
nitrate reductase were lower in ETRA-153, as were the levels of
fumarate reductase protein and transcript. ETRA-153 was also deficient
in one type of ubiquinone. These results are in contrast to those
previously reported for the putative etrA mutant METR-1. Molecular analysis of METR-1 indicated that its etrA
gene is not interrupted; its reported phenotype was likely due to the
use of inappropriate anaerobic growth conditions.
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TEXT |
Shewanella
putrefaciens MR-1 is a nonfermentative, facultatively anaerobic
bacterium originally isolated from anaerobic sediments in Oneida Lake,
N.Y. (32). In the absence of O2,
MR-1 can utilize several electron acceptors for anaerobic respiration,
including iron(III), manganese(IV), thiosulfate, fumarate, nitrate,
trimethylamine N-oxide (TMAO), dimethyl sulfoxide (DMSO),
and others. The ability to utilize many of these anaerobic electron
acceptors is absent in aerobically grown cells (23, 25,
32). Several of MR-1's cytochromes are synthesized only under
anaerobic conditions (24), and menaquinone levels are
increased in cells grown under anaerobic conditions (26).
These results imply that MR-1 has the ability to sense the presence or
absence of O2 and to regulate the expression of
multiple electron transport components necessary for anaerobic respiration. However, little is known about the regulation of these
components in MR-1.
The ability to regulate electron transport components is essential to
enable rapid adaptation to changing environments and to prevent the
waste of cellular resources. For example, Fnr (fumarate nitrate
regulator) from Escherichia coli transcriptionally activates genes necessary for anaerobic growth while repressing others needed for
aerobic growth (5, 6). Fnr is a positive regulator for the
transcription of fumarate and nitrate reductase genes in E. coli (9, 12, 20, 46). Several analogs to Fnr have
been described, including (i) CAP (catabolite gene activator protein), which regulates transcription in response to glucose availability in
E. coli (5, 17, 43); (ii) ANR (anaerobic
regulation of arginine deiminase and nitrate reduction) from
Pseudomonas aeruginosa (4); (iii) and HlyX
(hemolysin regulator) from Actinobacillus pleuropneumoniae
(18). One possible regulator in MR-1 is EtrA (electron
transport regulator A), which has 73.6% identity to E. coli
Fnr on the basis of amino acid alignments (41). A
study describing the isolation of a putative etrA
mutant, METR-1, concluded that the loss of EtrA rendered the
cells unable to grow under anaerobic conditions on several
electron acceptors, including nitrite, thiosulfate, sulfite,
TMAO, DMSO, Fe(III), and fumarate (41). METR-1 was
isolated using a strategy in which a vector derived from pACYC184 was
used to interrupt etrA in the chromosome (41);
this strategy relied on the presumption that pACYC184 would not
replicate freely in MR-1. A subsequent report clearly demonstrated that
pACYC184 does freely replicate in MR-1 (31) and is
therefore not suitable for gene replacement or gene interruption strategies. This finding calls into question the phenotype reported for
METR-1 (41) and prompted the reinvestigation of the role of EtrA in MR-1.
All materials used in this study were from sources previously described
(35). A list of the bacteria and plasmids that were used
in this study is presented in Table 1.
For molecular biology experiments, S. putrefaciens strains
were grown aerobically at room temperature (23 to 25°C) or at 30°C
on Luria-Bertani (LB) medium, pH 7.4 (42). E. coli strains were grown aerobically at 37°C on LB medium. Growth
media were supplemented with appropriate antibiotics when required,
including chloramphenicol at 35 µg ml
1, tetracycline at 20 µg
ml1, and kanamycin at 50 µg
ml1; for some experiments, other concentrations
were used where indicated. For other applications, S. putrefaciens was grown at room temperature either
aerobically or anaerobically as previously described
(24) in M1 defined medium (33)
supplemented with 15 mM lactate, vitamin-free Casamino Acids (0.1 g
liter1), and an appropriate electron acceptor.
Growth on ferric citrate was done using LM medium (27)
supplemented with 15 mM lactate, 2 mM sodium bicarbonate, and 10 mM
ferric citrate.
DNA manipulations performed in this study were done according to
standard techniques (42) as described previously
(35). Electroporation of pACYC184 into MR-1 was done as
described previously (31) with the following exceptions:
the voltage was 1.15 kV and, immediately after electroporation, the
cells were resuspended in 0.5 ml of LB broth, incubated at room
temperature or 30°C on a gyrotary shaker at 180 rpm for 2 h prior to being plated on appropriate media. Primers used in PCR,
inverse PCR, and colony PCR are listed in Table
2. The primers were designed using the genomic sequence for MR-1 (The Institute for Genomic Research [TIGR]). We assigned base numbers to etrA so that +1
corresponds to the first base of the start codon and 753 corresponds to
the last base of its stop codon; negative numbers refer to sequence upstream of etrA. DNA sequencing was performed either by
thermal cycle DNA sequencing with custom primers as previously
described (35) or with an ABI Prism BigDye Terminator
Cycle Sequencing Ready Reaction Kit by an automated single-capillary
method (Applied Biosystems ABI Prism 310). Computer-assisted sequence
analysis and comparisons were done with MacVector software (Accelrys,
San Diego, Calif.).
Construction of an etrA insertion mutation.
Construction of a plasmid for gene replacement of etrA was
completed using the strategy described by Myers and Myers
(35). A 1,532-bp fragment containing the entire
etrA gene plus 5' and 3' flanking sequences was generated by
PCR of MR-1 genomic DNA using primers E1 and E2 (Table 2). This PCR
product was cloned into pCR2.1-TOPO, generating pTOPO/etrA. Inverse PCR
(37) of pTOPO/etrA using primers E3 and E4 (Table 2)
generated TOPO/etrA(
246), a 5.2-kb fragment that is missing 246 bp
of internal etrA sequence. The 2.1-kb kanamycin resistance
(Kmr) gene from pUT/mini-Tn5Km was
generated by PCR with primer K1. Following digestion with
ClaI, the Kmr gene was ligated to the
TOPO/etrA(246) fragment, generating pTOPO/etrA:Km. A 3.4-kb DNA
fragment containing the etrA gene interrupted by the
Kmr gene was generated by PCR using pTOPO/etrA:Km
as the template and primers E1 and E2. This fragment was blunt ended
and ligated into the SmaI site of the suicide vector
pEP185.2, generating pDSEPetrA, which was electroporated into the donor
strain E. coli S17-1
pir. Throughout, appropriate analyses
(restriction digests, PCR, DNA sequencing) were done to verify that the
expected constructs were obtained.
E. coli S17-1
pir(pDSEPetrA) was mated with MR-1, and MR-1
exconjugants were selected using kanamycin under aerobic conditions
on
defined medium with 15 mM lactate as the electron donor. Colonies
were
screened by colony PCR (
48) using primers E1 and E2; those
lacking the expected wild-type 1.5-kb PCR product were pursued
as
putative insertional mutants. One strain, designated ETRA-153,
met all
criteria expected for an
etrA knockout: (i) it lacked
the
expected 1.5-kb wild-type PCR product for
etrA; (ii) it was
positive for a 3.4-kb PCR product, consistent with
Km
r gene-interrupted
etrA (Fig.
1); (iii) it was negative for a PCR
product using primers C1 and C2 (Table
2), specific to the
cat gene of pEP185.2 (Fig.
1); and (iv) it grew in the
presence of
kanamycin but not in the presence of chloramphenicol. The
lack
of the
cat gene and sensitivity to chloramphenicol
are consistent
with a double-crossover gene replacement. The
etrA gene from ETRA-153
interrupted by the
Km
r gene was cloned into pCR2.1TOPO for
sequencing. The sequence
obtained (using primers E5 and E6 [Table
2])
confirmed that the
etrA gene in ETRA-153 was interrupted by
the Km
r gene at the expected position (data not
shown by request).
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FIG. 1.
Colony PCRs with primers specific for the
etrA gene or the chloramphenicol acetyltransferase gene
(cat) from pEP185.2. Lanes 1 and 2 show reactions with
the etrA primers E1 and E2 (Table 2), and lanes 3 to 5 show reactions with the cat primers C1 and C2 (Table 2).
The templates for PCR were as follows: MR-1 (lanes 1 and 3), ETRA-153
(lanes 2 and 4), and pDSEPetrA (lane 5). The sizes of the DNA markers
(lane M) in kilobases are indicated on the left.
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Characterization and complementation of the etrA
insertion mutant.
Expression of etrA was analyzed by
reverse transcription-PCR (RT-PCR) of total RNA using the Titan One
Tube RT-PCR system (Roche Molecular Biochemicals, Indianapolis, Ind.).
Total RNA was isolated from aerobically grown cells using a hot-phenol
method followed by treatment with RNase-free DNase as previously
described (28). All standard precautions to prevent RNase
contamination were taken (42). Total RNA (2 µg) from
each strain served as the template, using primers E5 and E6. The
expected 662-bp RT-PCR product was present in MR-1 and
MR-1(pVK100) but was absent in ETRA-153(pVK100) (Fig.
2). Complementation of ETRA-153 with
pVKetrA restored the etrA transcript to ETRA-153 (Fig. 2).
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FIG. 2.
Expression of etrA in S.
putrefaciens cells grown aerobically as determined by RT-PCR of
2 µg of total RNA from the following strains: MR-1 (lane 1),
MR-1(pVK100) (lane 2), ETRA-153(pVK100) (lane 3), and
ETRA-153(pVKetrA) (lane 4). The etrA product is
indicated by the arrow at the right. Primers E5 and E6 (Table 2) for
RT-PCR were based on the etrA sequence from the MR-1
genome project (TIGR). The sizes of the DNA markers (lane M) in
kilobases are indicated on the left.
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The ability of ETRA-153 to grow on and reduce various electron
acceptors was investigated in defined medium, sampled once
daily over a
period of 4 days. For most electron acceptors, growth
was assessed
by measuring culture turbidity at 500 nm using a
Beckman DU-64
spectrophotometer. Growth on Fe(III) was assessed
by measuring total
cellular protein (
27). Concentrations of
nitrate,
nitrite, Fe(II), and Mn(II) were determined as previously
described (
36). The growth rates and maximal growth yields
of
ETRA-153(pVK100) were essentially the same as those of MR-1
using
20 mM fumarate, 20 mM TMAO, 5 mM DMSO, 10 mM thiosulfate, 10 mM
ferric citrate, 2 mM nitrate, or O
2 as the
terminal electron acceptor
(data not shown by request).
ETRA-153(pVK100) was able to reduce
10 mM ferric citrate, 2 mM
nitrate, and 5 mM MnO
2 at rates that
were
essentially identical to those of MR-1 (data not shown by
request).
However, closer examination of short-term growth, examined
hourly,
demonstrated that ETRA-153(pVK100) has a lower initial
rate of
growth on fumarate and nitrate but that its rate of growth
on TMAO is
identical to that of MR-1 (Fig.
3).
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FIG. 3.
Comparison of levels of short-term growth of various
strains of S. putrefaciens with fumarate (A),
nitrate (B), and TMAO (C). Medium was inoculated with cultures
pregrown anaerobically on the tested substrate. Culture turbidity was
measured over the course of 1 day until ETRA-153(pVK100) reached
growth comparable to that of MR-1. Values are mean ± high and low
values from two parallel but independent experiments for each strain.
O.D., optical density.
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Although ETRA-153 maintained the ability to grow on and reduce
all of the electron acceptors tested, its lower initial growth
rate on
fumarate and nitrate indicated that it may have lower
levels of
fumarate and nitrate reductase. Since
98% of the fumarate
reductase
activity in MR-1 is localized to the soluble fraction
(
23), soluble fractions isolated from cells grown
anaerobically
with 20 mM fumarate were analyzed for fumarate reductase
activity
by monitoring the fumarate-dependent oxidation of reduced
benzyl
viologen under anaerobic conditions as described previously
(
23).
The nitrate reductase activity of washed cells grown
anaerobically
with 2 mM nitrate was determined under anaerobic
conditions. Briefly,
overnight cultures were centrifuged and
resuspended in 0.4 M potassium
phosphate buffer (pH 7.5), with cell
densities equalized to an
optical density of 0.10 at 500 nm. Lactate
and formate were added
to a final concentration of 15 mM each, and the
reaction was started
by adding NaNO
3 (0.5 M) to a
final concentration of 1 mM. Samples
(1 ml) were removed at 0, 15, 30, 45, and 60 min and were immediately
filtered through
0.2-µm-pore-size filters. The filtrates were
immediately
placed on ice until they were analyzed for nitrate.
The fumarate
reductase activity of ETRA-153(pVK100) was 42% less
than that of MR-1 (pVK100) (Fig.
4A). Complementing the
mutant
with pVKetrA restored fumarate reductase activity to the
level
of the wild type (Fig.
4A). Similarly, nitrate reduction by whole
cells of ETRA-153(pVK100) was 35% less than that catalyzed by
MR-1(pVK100) (Fig.
4A); complementing the mutant with pVKetrA
restored the rate of nitrate reduction to that of the wild type
(Fig.
4A). The decreases in nitrate and fumarate reductase activities
correlate with the lower initial rates of growth of ETRA-153 (Fig.
3),
suggesting that EtrA positively, but only partially, regulates
the use
of these electron acceptors in the absence of O
2.
This
is in contrast to results of studies of
E. coli Fnr
mutants that
show nearly complete loss of fumarate and nitrate
reductase activities
under anaerobic conditions (
2,
15).
In one study of four
different Fnr mutants, fumarate reductase
activities were less
than 12% of wild-type levels and nitrate
reductase activities
were less than 4% of wild-type levels
(
15). EtrA appears to
play a more subtle role in
regulation in MR-1 than Fnr in
E. coli.
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FIG. 4.
(A) Analysis of the fumarate reductase activities of
soluble fractions and the nitrate reductase activity of whole cells.
For each panel, the strains were MR-1(pVK100) (first set of bars),
ETRA-153(pVK100) (second set of bars), and ETRA-153(pVKetrA)
(third set of bars). The average activities for MR-1(pVK100) were
14.8 µmol min1 mg of protein1 and 9.4 nmol min1 ml1 for fumarate and nitrate
reductase, respectively. Values are relative means ± standard
deviations (SD) of results of independent experiments
(n = 3 for fumarate reductase;
n = 5 for nitrate reductase). *, statistically
significant to a P of  0.03. (B) Analysis of
fumarate reductase protein of soluble fractions using an antibody
specific for fumarate reductase. Protein levels are relative to those
of MR-1(pVK100) using NIH Image software. Values are
relative means ± SD for soluble fractions isolated from three
different cultures. §, statistically significant from strains 1 and 3 to a P of 0.005;  , statistically significant from
strain 1 to a P of 0.03. (C) RNase protection of 10 µg of RNA isolated from cells grown anaerobically on fumarate with a
probe specific for fumarate reductase. Values are relative to those for
MR-1(pVK100) using NIH Image software and are the means ± SD
of results for three independently grown cultures.  , statistically
significant to a P of 0.02.
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Polyclonal antibodies specific for fumarate reductase
(FumR) were generated against an antigen produced by
recombinant technology.
Briefly, an internal fragment of
fumR encoding a 121-residue fragment
of FumR was
cloned into pBAD/Thio-TOPO (Invitrogen, Carlsbad,
Calif.). Following
arabinose-induced expression in
E. coli TOP10
cells, the
FumR protein fusion was purified using HisBind Quick
resin
(Novagen, Madison, Wis.). The purified protein was used
as an antigen
to generate polyclonal antibodies in New Zealand
White rabbits
(Cocalico Biologicals, Reamstown, Pa.). Purified
immunoglobulin G was
obtained from rabbit sera, nonspecific antibodies
were removed by
absorption with autoclaved
E. coli JM109 cells,
and Western
blotting was done as described previously (
30).
The
specificity of the antibody was confirmed using CMTn-3, a
fumarate reductase-minus mutant (
29) as a negative
control.
The level of FumR protein was 60% lower in
ETRA-153(pVK100) than
in MR-1(pVK100) (Fig.
4B);
complementation with pVKetrA increased
the level of FumR
protein, although it was 35% less than that
seen with
MR-1(pVK100) (Fig.
4B).
In
E. coli, Fnr has a direct role in the transcriptional
regulation of the genes for fumarate and nitrate reductase (
9,
12,
20,
46). Since both the protein and activity levels
for
fumarate reductase were partially reduced in ETRA-153, the
levels of
fumarate reductase (
fumR) mRNA were examined by
RNase
protection assay (RPA III kit; Ambion, Austin, Tex.). Since the
nitrate reductase genes have not yet been identified in MR-1,
the
nitrate reductase transcript could not be examined. A 266-bp
internal fragment of
fumR was cloned into pCR2.1TOPO;
the desired
orientation was verified by PCR. Using M13 primers, a
509-bp fragment
containing the
fumR and flanking 5' and
3' vector DNAs was generated
by PCR; using this fragment as a template,
the antisense
fumR RNA probe was generated using a
MAXIscript kit (Ambion). The probe
was gel purified on a
Tris-borate-EDTA-urea polyacrylamide gel.
RNase protection
assays were done using total RNA (10 µg) and
400 pg of the probe. The
level of the
fumR transcript was 73%
lower in
ETRA-153(pVK100) than in MR-1(pVK100) (Fig.
4C);
complementation
with pVKetrA increased the
fumR
transcript to levels similar to
those of MR-1(pVK100) (Fig.
4C).
To further investigate the phenotype of ETRA-153, subcellular
fractions of fumarate-grown cells were prepared and analyzed.
Cytoplasmic membrane, intermediate membrane, outer membrane (OM),
and
soluble (cytoplasm plus periplasm) fractions were purified
by
an EDTA-lysozyme-Brij protocol as previously described
(
24).
The intermediate membrane is a hybrid of the
cytoplasmic membrane
and the OM (
24). The separation and
purity of these subcellular
fractions were assessed by spectral
cytochrome content analysis
(
24), by membrane
buoyant-density analysis (
24), and with
sodium dodecyl
sulfate (SDS)-polyacrylamide gels (
14,
22)
stained for
protein with Pro-Blue or heme as previously described
(
34). Protein was determined by a modified Lowry method
(
28).
SDS-polyacrylamide gel electrophoresis patterns
confirmed a prominent
separation of the various fractions and
demonstrated that they
were comparable to those of previous experiments
(
23-25,
28,
30,
34). Protein and heme stains of these
fractions were similar
for all strains (data not shown by request).
Cytochrome spectra
of the various subcellular fractions indicated that
the cytochrome
content and distribution of ETRA-153(pVK100) were
similar to those
of MR-1(pVK100) (data not shown). Using an
antibody specific for
the OM cytochrome OmcA
(
30), Western blotting of OM fractions
of
MR-1(pVK100), ETRA-153(pVK100), and ETRA-153(pVKetrA)
showed
no significant differences among these strains in the levels of
OmcA (data not shown). CymA, a tetraheme cytochrome
c, is
required
for growth on and reduction of nitrate, fumarate, Fe(III), and
Mn(IV) (
28,
35); since ETRA-153 grew well on these
electron
acceptors, EtrA is not required for the expression of
cymA. A
more subtle role for EtrA in the expression of
cymA cannot be
excluded at this
time.
Since the levels of various quinones are affected by growth under
aerobic versus anaerobic conditions (
26), the quinone
contents of the various strains were examined by thin-layer
chromatography
(TLC). Quinones were extracted from cells by a method
adapted
from the work of Kröger and Dadák (
13)
as previously described
(
35). Quinones were resolved by
TLC as described previously
(
7) on Merck Kieselgel 60 F
254 plates (20 by 20 cm, 0.25 mm
thick)
developed with petroleum ether-diethyl ether (9/1 [vol/vol]).
The
plates were examined under reflective UV light (wavelength,
254 nm).
All strains were similar in their contents of menaquinones
and most
ubiquinone species. However, ETRA-153(pVK100) was deficient
in one
of the ubiquinone species that was present in all other
strains tested
(Fig.
5). The deficiency in one type of
ubiquinone
seen in ETRA-153 (Fig.
5) indicates that EtrA may play a
role
in the regulation of ubiquinone synthesis. However, this role
appears to be minor since the levels of menaquinones and the other
ubiquinones were similar to those of the wild type. The presence
of
multiple ubiquinone bands on TLC is due to variation in the
lengths of
the membrane-anchoring isoprenyl side chains. The length
of a side
chain is determined by the enzyme polyprenyl diphosphate
synthase, and
p-hydroxybenzoate polyprenyltransferase catalyzes
the
attachment of various isoprenyl chain substrates to
p-hydroxybenzoate
(
40). However, it has been
shown that the exact length of the
isoprenyl side chain is not crucial
for function (
38,
39).
This implies that a deficiency in
one species of ubiquinone will
not likely affect growth since other
forms can effectively substitute.
This may explain why ETRA-153 can
grow on all tested electron
acceptors despite being deficient in one
particular ubiquinone.
In
E. coli, the ratios of aerobic to
anaerobic quinone were not
altered in an Fnr deletion mutant,
indicating that Fnr is not
involved in the regulation of quinone
synthesis (
44).
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FIG. 5.
Thin-layer chromatogram of quinones isolated from
S. putrefaciens cells grown anaerobically with
fumarate as the electron acceptor. Lanes 1 and 2 were loaded with
ubiquinone-10 and menaquinone-4 standards, respectively. The
other lanes were loaded with quinone extracts (equivalent to 0.10 g [wet weight] of cells) isolated from cells of the following
strains: MR-1 (lane 3), MR-1(pVK100) (lane 4), ETRA-153(pVK100)
(lane 5), ETRA-153(pVKetrA) (lane 6), MR-1(pACYC184) (lane 7),
and METR-1 (lane 8). The samples were loaded at the bottom, and
migration was upward. The positions of various quinones are indicated
at the right. I, methylmenaquinone; II, menaquinone; III-VI, various
ubiquinones. The identities of these quinones were previously verified
by high-pressure liquid chromatography with diode array
detection (35).
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Reexamination of the putative etrA mutant
METR-1.
In 1993, Saffarini and Nealson (41) described
the putative etrA mutant METR-1 and concluded that
etrA was necessary for anaerobic growth on nitrite,
thiosulfate, sulfite, TMAO, DMSO, Fe(III), and fumarate
(41). This conclusion is in contrast to the results
reported here for ETRA-153, which grew readily on all of these electron
acceptors. METR-1 was putatively constructed by site-specific
insertion through a single crossover between etrA in the
genome and plasmid pSETR-3 (a small fragment of etrA in the
vector pACYC184) (41). This approach presumed that
pACYC184 could not replicate in MR-1 and could be maintained only by
insertion into the genome. However, it has since been shown that
pACYC184 can readily replicate in MR-1 (31); this finding
raised the question as to how the strategy of Saffarini and Nealson
could have been successfully used for site-specific insertional mutagenesis.
To investigate these discrepancies, further experiments were done with
METR-1. Colony PCR with primers E7 and E8 (Table
2)
designed to amplify
etrA indicated that METR-1 does not have an
interrupted
etrA gene; it yielded the same 844-bp product as MR-1
(data
not shown by request). Plasmid DNA was isolated from METR-1,
and
restriction digests with
SalI and
EcoRV indicated
that the
strain contains a 7.8-kb plasmid. An
NcoI digest
yielded two fragments
of approximately 3.6 and 4.1 kb (data not shown).
No plasmid should
be present if METR-1 is a true single-crossover
mutant; MR-1 does
not contain this 7.8-kb plasmid. The plasmid
in METR-1 is almost
twice as large as expected for pACYC184 (4.2 kb) or
pSETR3 (4
kb). Its two
NcoI fragments were subcloned into
pCALc (Stratagene,
La Jolla, Calif.), and the DNA sequences of the ends
of these
fragments were determined using primers S1 and S2 (Table
2).
The larger
NcoI fragment contained sequence that matched
pACYC184,
whereas the smaller
NcoI fragment contained
sequence that matched
etrA of MR-1 (TIGR MR-1 genome
project) (data not shown). Since
only the ends of the fragments were
sequenced, the identity of
the "extra" DNA is unknown. These
results are consistent with
the demonstrated ability of pACYC184 to
replicate freely in MR-1
(
31).
While this molecular analysis indicates that the
etrA gene
was not interrupted in METR-1, it does not provide a rationale
for the
reported phenotype of METR-1 (
41). Four factors may
have
contributed to the reported phenotype: (i) no control strains
were
compared with METR-1; (ii) qualitative growth measurements,
such as
visual turbidity and end product odor formation, were
used for some
electron acceptors, which limits the detection of
growth changes that
fall between the extremes; (iii) low concentrations
of electron
acceptors (2 mM) were used, which may not have been
sufficient to
support robust growth; and (iv) tetracycline was
used to select for the
tetracycline resistance (Tc
r) marker of pACYC184
(tetracycline may markedly compromise anaerobic
growth because
resistance is encoded by an energy-intensive class
C tetracycline
efflux pump) (
8,
16,
19). Levels of growth
of METR-1 and
MR-1(pACYC184) were similar when fumarate (20 mM)
was nonlimiting
(Fig.
6). The growth of
MR-1(pACYC184) was more
rapid and reached a higher maximal density
when the medium contained
chloramphenicol (35 µg
ml
1) than when it contained tetracycline (16 µg ml
1); this demonstrated that tetracycline
can partially impede growth
under anaerobic conditions. In contrast,
all strains grew very
poorly under the conditions used by Saffarini and
Nealson (
41);
i.e., when fumarate was limiting (2 mM), no
significant increase
in turbidity was observed over 8 days (Fig.
6).
These conditions
are clearly inadequate to support growth, even of
MR-1, but explain
why METR-1 was reported as negative for growth on
fumarate (
41).
Growth on TMAO, DMSO, and
O
2 was also investigated; similar to
fumarate-grown cells, both METR-1 and MR-1(pACYC184) grew well
and
to similar extents on TMAO (20 mM) and DMSO (5 mM) but grew
poorly when
placed under limiting electron acceptor conditions
(2 mM) (data not
shown by request). Growth on O
2 was evident under
all tested conditions, but maximal turbidity was less when the
strains
were grown with tetracycline than when they were grown
with
chloramphenicol (data not shown by request). In summary,
tetracycline-grown cells showed reduced growth, which is consistent
with energy expenditure for the tetracycline efflux pump, leaving
less
energy for cell growth. Also, there was little or no growth
over 8 days
when electron acceptors were limiting (2 mM); therefore,
an electron
acceptor concentration of 2 mM is insufficient to
adequately assess
quantitative growth differences between various
strains in liquid
culture.
View larger version (40K):
[in this window]
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|
FIG. 6.
Anaerobic growth of METR-1 and MR-1(pACYC184) using
fumarate as the electron acceptor. Two variations of media were
compared: M1 medium (pH 7.4) with 15 mM lactate, 20 mM fumarate, and
either tetracycline (16 µg ml1) or chloramphenicol (35 µg ml1) (M) and M1 medium (pH 7.9) with 30 mM lactate,
2 mM fumarate, and either tetracycline (8 µg ml1) or
chloramphenicol (30 µg ml1) (SN). SN refers to
conditions reported by Saffarini and Nealson (41). METR-1
was not examined with chloramphenicol because it is Tcr
only.
|
|
Concluding remarks.
Although EtrA plays a subtle role in MR-1
anaerobic gene regulation, other regulatory components are likely
involved as well. This is not surprising considering that themes of
cooperative regulation and redundancy are common in living organisms.
Given the complex respiratory diversity displayed by MR-1, it is
possible that there are multiple regulatory elements involved in the
expression of various electron transport components. For example,
multiple cytochromes are localized to the outer membrane of MR-1 under anaerobic conditions (24), but little is known about the
regulatory mechanisms involved in the expression or localization of
these various cytochromes. However, cytochrome spectra and
SDS-polyacrylamide gel electrophoresis analysis of subcellular
fractions of ETRA-153 indicated that, relative to MR-1, there were no
major changes in cytochrome content or distribution. This implies that
EtrA plays no significant role in the global regulation of cytochrome production and that other as yet uncharacterized regulators are likely
involved in the control of cytochromes expressed under anaerobic conditions.
Since it has been suggested that MR-1 may have more than one Fnr-like
element (
41), the MR-1 genomic fragments compiled
by TIGR
were searched using MacVector for other possible analogs
to Fnr or
EtrA. Both nucleotide and amino acid searches were completed.
No other
open reading frames with significant homology to
etrA were
identified in the MR-1 genome. Queries based on ANR (
Pseudomonas aeruginosa, GenBank accession number
M98276;
Pseudomonas fluorescens,
GenBank accession number
AF053611), HlyX (
Actinobacillus pleuropneumoniae,
GenBank
accession number
M80712), and Fnr (
Vibrio cholerae,
GenBank accession number
AF244992) in the MR-1 genome showed
significant homology with EtrA, but no other convincing alignments
were
obtained. Although MR-1 inevitably has other regulatory elements,
it
appears that they are not obviously homologous with Fnr or
EtrA.
Also, the sequence upstream of the fumarate reductase gene in MR-1 was
searched (using MacVector software) for the presence
of a possible Fnr
consensus sequence (TTGATnnnnATCAA) (where n
is any
base) (
4,
21), allowing for differences in the
spacing
of the half-sites; this consensus sequence was not found in the
8 kb immediately upstream of the fumarate reductase gene. At 3.9
kb
upstream of the fumarate reductase gene, the sequence
T
CGATcttc
CTCAA
is present, which has two
variations from the Fnr-binding consensus
sequence (bold letters). At
7.2 kb upstream of the fumarate reductase
gene, the sequence
TTGA
CaataAT
TAA is present, which
also has two
variations from the Fnr consensus sequence (bold
letters). Although
these two sites may potentially be recognition
sites for EtrA, this is
only speculative. The consensus sequence
for EtrA is not known and may
be different than that for
Fnr.
| |
ACKNOWLEDGMENTS |
This work was supported by National Institutes of Health grant
R01GM50786 to C.R.M. The graduate fellowship for T. M. Maier was
provided in part by the Robert G. Zach Family Foundation.
We are grateful to V. L. Miller for graciously providing pEP185.2,
to D. Frank for providing pUT/mini-Tn5Km, and to J. M. Myers for providing expertise and advice in the laboratory and for
initial studies with anti-FumR. We thank the Protein & Nucleic Acid
Shared Facility of the Medical College of Wisconsin for performing automated nucleic acid sequencing for this study.
The preliminary genomic sequence data was obtained from TIGR through
their website at http://www.tigr.org. Sequencing of S. putrefaciens MR-1 genomic DNA by TIGR was accomplished with
support from the Department of Energy.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Dept. of
Pharmacology and Toxicology, Medical College of Wisconsin, 8701 Watertown Plank Rd., Milwaukee, WI 53226. Phone: (414) 456-8593. Fax:
(414) 456-6545. E-mail: cmyers{at}mcw.edu.
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