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Journal of Bacteriology, April 2000, p. 2269-2276, Vol. 182, No. 8
Laboratoire de Chimie Bactérienne,
Institut de Biologie Structurale et de Microbiologie, C.N.R.S.,
13402 Marseille Cedex 20, France
Received 22 November 1999/Accepted 21 January 2000
To construct Thiobacillus ferrooxidans mutants by
marker exchange mutagenesis, a genetic transfer system is required. The transfer of broad-host-range plasmids belonging to the incompatibility groups IncQ (pKT240 and pJRD215), IncP (pJB3Km1), and IncW (pUFR034) from Escherichia coli to two private T. ferrooxidans strains (BRGM1 and Tf-49) and to two collection
strains (ATCC 33020 and ATCC 19859) by conjugation was analyzed. To
knock out the T. ferrooxidans recA gene, a mobilizable
suicide plasmid carrying the ATCC 33020 recA gene disrupted
by a kanamycin resistance gene was transferred from E. coli
to T. ferrooxidans ATCC 33020 by conjugation under the best
conditions determined. The two kanamycin-resistant clones, which have
retained the kanamycin-resistant phenotype after growth for several
generations in nonselective medium, were shown to have the kanamycin
resistance gene inserted within the recA gene, indicating
that the recA:: Thiobacillus ferrooxidans
is an acidophilic chemolithoautotrophic bacterium that obtains its
energy from oxidation of ferrous iron (Fe2+) to ferric iron
(Fe3+) or of reduced sulfur compounds to sulfuric acid. Its
widespread application in mineral leaching and metal remediation has
made it an attractive microorganism to study. Considerable progress has
been made in studying the biochemistry and molecular biology of
T. ferrooxidans in recent years (27, 33).
However, the absence of genetic tools has impaired the understanding of
the physiology of this microorganism. The study of mutants in which a
protein of interest is no longer synthesized can help to establish its
function, but there have been no reports of the construction of
T. ferrooxidans mutants. The construction by marker exchange mutagenesis of null mutants would be possible if a reliable genetic transfer system between Escherichia coli and T. ferrooxidans were available.
Introduction of plasmids into T. ferrooxidans by
electrotransformation (17) and conjugation (23)
has been reported. The plasmids electroporated by Kusano et al.
(17) consisted of the T. ferrooxidans mer operon,
determining resistance to mercury ions, cloned either into the
broad-host-range plasmid pKT240 (IncQ group) or into a cryptic T. ferrooxidans natural plasmid carrying the pUC18 vector. Of the 30 independent T. ferrooxidans private strains tested, only one
(Y4-3) gave transformants. The efficiency of electrotransformation was
low (120 to 200 mercury-resistant colonies per µg of plasmid DNA). On
the other hand, Peng et al. (23) reported the genetic
transfer of broad-host-range IncP plasmids (RP4, R68.45,
RP1::Tn501, and pUB307) by conjugation and the
mobilization of a broad-host-range IncQ plasmid (pJRD215) with the aid
of an RP4 plasmid to seven T. ferrooxidans private strains.
Kanamycin resistance was used as the selection marker. The
physiological states of both the donor and the recipient, and the
mating time, have been shown to be important. The apparent transfer
frequency of the large self-transmissible IncP plasmids was
10 The RecA protein plays an essential role in homologous genetic
recombination, DNA repair, induction of the SOS response, and initiation of stable DNA replication (29). The RecA protein is thought to be ubiquitous in eubacteria and is among the most conserved proteins across bacterial organisms (15). The
recA gene from the T. ferrooxidans ATCC 33020 strain has been cloned (14, 25), sequenced (26),
and expressed in E. coli (14, 25, 26). Both
recombinase activity and SOS response were restored in an E. coli
recA mutant by the T. ferrooxidans recA gene (25, 26), showing that RecA has similar activities in T. ferrooxidans and E. coli.
In this paper, the influence of different factors on the transfer
frequency of IncQ plasmids from E. coli to T. ferrooxidans ATCC 33020 has been analyzed. This study was extended
to three other T. ferrooxidans strains (ATCC 19859, BRGM1,
and Tf-49) and to IncP and IncW plasmids. The feasibility of a marker
exchange mutagenesis program has been tested in the ATCC 33020 strain
with the construction of a recA mutant.
Bacterial strains and plasmids.
Strains and plasmids used in
this study are listed in Table 1.
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Construction and Characterization of a
recA Mutant of Thiobacillus ferrooxidans by
Marker Exchange Mutagenesis
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
-Km mutated allele was
transferred from the suicide plasmid to the chromosome by homologous
recombination. These mutants exhibited a slightly reduced growth rate
and an increased sensitivity to UV and 
irradiation compared to the wild-type strain. However, the T. ferrooxidans recA mutants
are less sensitive to these physical DNA-damaging agents than the recA mutants described in other bacterial species,
suggesting that RecA plays a minor role in DNA repair in T. ferrooxidans.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
5 to 107, depending on the plasmid, and
the apparent mobilization frequency of the IncQ pJRD215 plasmid was
about 105.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Bacteria and plasmids used in this study
Media and growth conditions. The E. coli growth medium was Luria-Bertani medium (21) except for conjugation experiments (see below). The composition of the T. ferrooxidans 9K liquid medium has been reported previously (5). The 2:2 and DOP solid media are described in references 24 and 19, respectively.
Conjugation.
Initial conjugation experiments were performed
according to the method of Peng et al. (23). The conjugation
experiments were optimized as presented in Results. The modified mating
procedure was as follows. The E. coli donor strains were
grown at 37°C until late exponential growth phase in 2:2 basal salt
medium (2:2 liquid medium pH 5.2 to 5.4 without an energy source)
supplemented with 0.5% (wt/vol) yeast extract and one antibiotic
selective for the plasmid that they contained. The T. ferrooxidans recipient strains were usually cultured in 9K sulfur
liquid medium (pH 3.5) at 30°C for 5 days to stationary phase. The
cells were collected by centrifugation. T. ferrooxidans
cells were washed three times with 2:2 basal salt medium to remove the
sulfur precipitates. For matings, donor and recipient cells were
combined in a 1:2 ratio. From this cell suspension (approximately
2 × 109 cells per ml), 0.1 ml was spotted on 2:2
solid medium (0.6% agar) supplemented with 0.05% (wt/vol) yeast
extract, 0.5 × 104 M diaminopimelic acid, and
0.05% (wt/vol) Na2S2O3. After 3 days of incubation at 30°C, the cells were harvested and suspended in
1.5 ml of 2:2 basal salt medium. The number of viable recipient bacteria was obtained by plating on 2:2 or DOP solid medium.
Transconjugants were selected at 30°C on DOP solid medium containing
200 µg of kanamycin/ml, with recipient bacteria being counterselected
by kanamycin and donor bacteria by pH and the absence of a carbon source in the selective medium. The plates were incubated at 30°C for
10 to 15 days. The frequencies of plasmid transfer are expressed as the
"apparent transfer frequency," that is, the number of
transconjugants scored on selective medium per recipient colony scored
on nonselective medium after the mating period.
Stability analysis. A single colony of T. ferrooxidans conjugant was grown to late exponential phase in 9K ferrous iron liquid medium without antibiotic, diluted 103-fold in fresh 9K ferrous iron medium, and grown again to late exponential phase. The last two steps were repeated three times. An aliquot was taken at the beginning of each new culture, diluted, and plated on 2:2 solid medium with or without kanamycin. The plasmid stability was calculated as the ratio between the number of colonies observed in the presence and absence of antibiotic.
Construction of a recA mutant of T. ferrooxidans ATCC 33020.
The pNG23 plasmid, carrying the
ATCC 33020 recA and alaS genes cloned into pUC19
(Table 1), was digested with HindIII and religated to
delete the alaS gene. The resulting plasmid,
pUC19recA, replicates in E. coli but not in
T. ferrooxidans. The blunt-ended HindIII
fragment carrying the Kmr gene from the pHP45-Km plasmid
(13) was inserted into the SmaI site of the
pUC19recA plasmid. The resulting plasmid,
pUC19recA::-Km, carries the recA
gene disrupted by a 3.3-kb fragment as shown by restriction analyses,
PCR, Southern hybridizations, and sequence determination. This fragment
consists of the -Km interposon, as expected, but also of an
additional 1.3-kb fragment which corresponds to an internal region of
the -Km HindIII fragment. The 1.6-kb BamHI
fragment from pUC18mob, corresponding to the mobilization region of the RP4 plasmid, was cloned into the ScaI
site of the ampicillin resistance gene of the
pUC19recA::-Km plasmid. This plasmid, called
pUC19recA::-Kmmob, was transformed
into E. coli S17-1 with kanamycin resistance selection. The
mobilization ability of
pUC19recA::-Kmmob was checked by
transferring it by conjugation from E. coli S17-1 to
E. coli CGSC7330; the selection was for tetracycline and
kanamycin resistance.
-Kmmob plasmid was
then transferred from E. coli S17-1 to T. ferrooxidans ATCC 33020 under the conditions given above except
that the mating time was extended to 5 days. The selective medium was
the 2:2 medium containing 200 µg of kanamycin/ml. The kanamycin
plates were incubated at 30°C for 4 weeks.
Growth curves of the wild-type and recA ATCC 33020 strains. To determine the growth rate of the wild-type and recA mutant strains, liquid media were inoculated with fresh cultures of ATCC 33020 and recA mutant 5 and shaken at 30°C until the cultures reached the stationary phase. Samples were removed every day, diluted, and plated on solid 2:2 medium. The number of CFU was plotted against the incubation time.
UV and irradiation.
ATCC 33020 and recA
mutant derivatives were grown to mid-exponential phase in 9K liquid
medium with ferrous iron as an energy source. Aliquots (50 µl) of
100 to 106 dilutions were spread on 2:2 or
DOP solid medium. For UV irradiation, the plates were exposed to UV
light (254 nm) at a dose rate of about 1.5 J/m2/s for 5 to
25 s. For irradiation, the plates were irradiated with
60Co at a dose rate of 147 and 35 Gy/min. The plates were
incubated for at least 2 weeks at 30°C. Survival was determined as
the ratio of CFU per milliliter after irradiation to CFU per milliliter before irradiation.
DNA manipulations. General techniques were performed according to standard procedures (3) or the manufacturers' recommendations. Ultrapure plasmid DNA was obtained using the Wizard DNA purification system from Promega. T. ferrooxidans genomic DNA was prepared as previously described (5).
PCR.
The 475-bp fragment from the rusticyanin gene of
T. ferrooxidans was amplified with the oligonucleotides
RUSNM (5'-GGCACGCTGGATTCCACATGGAAAGAGGCG-3') and RCX
(5'-CCACTCGAGCCTTGACAATGATTTTACCAAACATACC-3'). The presence of E. coli cells was detected by the amplification of a 396- and a 570-bp fragment of the regulatory region of the operon encoding the major nitrate reductase of E. coli (6) with
the oligonucleotide pairs S1 (5'-CACGGTTGGTATTGAGAAGC-3')
plus S2 (5'-CGCCGGATTTCATTAAGAGC-3') and S4
(5'-GCCTGCTTAAAGCTTTTCGC-3') plus 64G
(5'-TCCCCATCACTCTTGATCGTTATC-3'). The oligonucleotides KMTN5
(5'-CGATGCGCTGCGAATCGG-3') and AKMTN5 (5'-GCAGCTGTGCTCGACGTTG-3') were used to amplify a 531-bp
fragment of the kanamycin resistance gene from Tn5
(pJRD215), and the oligonucleotides KM1
(5'-AAGATCCTGGTATCGGTCTGC-3') and KM2
(5'-AACATGGCAAAGGTAGCG-3') were used to amplify a 524-bp
fragment of the kanamycin resistance gene from Tn903
(pKT240, pJB3Km1, and pUFR034). The presence of the ampicillin
resistance gene of the pKT240 and pJB3Km1 plasmids was tested for by
amplification of a 633-bp fragment with the oligonucleotides AAMP
(5'-CCGTGTCGCCCTTATTCCC-3') and AMP3
(5'-TGGTCCTGCAACTTTATCCGCC-3'). To amplify the region of the
ATCC 33020 recA gene which overlaps the SmaI site
where the -Km interposon was inserted, the oligonucleotides RECA2
(5'-CGATGACGATGAGGTCC-3') and RECA3
(5'-AAGGATGGTTACCCCTCG-3') were chosen. The insertion
junctions between the recA gene and the -Km cassette were
obtained by amplification of the DNA from Kmr clone 5 between oligonucleotides hybridizing on one side of the recA
SmaI site, in which the -Km cassette had been inserted RECA4 (5'-CGGCTCGCTGGGTCTGG-3') or ARECA5
(5'-CTGACAACTGGCTATGGC-3'), and an oligonucleotide
corresponding to the end of the -Km cassette, CKMTN5
(5'-GGAGTGGGGAGGCACGATGG-3').
Sequence.
The sequence of the PCR fragments RecA4-CKMTN5 and
ARECA5-CKMTN5, corresponding to the junctions of the -Km cassette
insertion inside the recA gene (see above), were determined
with the Thermo Sequenase II dye terminator cycle-sequencing premix kit
from Amersham. The DNA sequences were compiled and analyzed through the
World Wide Web Netscape facilities.
Southern hybridization.
Genomic DNAs of strain ATCC 33020 and the kanamycin-resistant derivatives were digested with
KpnI and EcoRV restriction endonucleases, electrophoresed on agarose gel, and transferred by capillary blotting to positively charged Hybond-N membranes (Roche Biochemicals). The
kanamycin resistance gene and recA probes were obtained by incorporation of alkali-labile DIG-dUTP (Roche Biochemicals) during PCR
elongation with the oligonucleotides KMTN5 and AKMTN5 (see above) on
one hand and the oligonucleotides RECA3 and RECA2 (see above), which
bracket the recA SmaI site where the -Km interposon was
inserted, on the other hand. The hybridization was carried out under
stringent conditions as recommended by the manufacturer.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Conjugative transfer of the IncP RP4 plasmid and mobilization of
IncQ plasmids from E. coli to T. ferrooxidans.
Although conjugation between E. coli and private T. ferrooxidans strains has been described (23), there is
no report yet of this genetic transfer technique using collection
strains of T. ferrooxidans. We first focused our study on
the ATCC 33020 strain, because we have cloned several genes from this
strain (1, 2, 5, 14), particularly the recA gene
(14). Conjugation experiments between E. coli
HB101 or MOS blue strains carrying the conjugative plasmid RP4 and
T. ferrooxidans ATCC 33020 were carried out according to the
method previously described by Peng et al. (23).
Kanamycin-resistant clones were selected; T. ferrooxidans ATCC 33020 is sensitive to 200 µg of this antibiotic/ml on solid medium. The apparent transfer frequency obtained was lower than 108.
7. The mobilization of plasmid pJRD215
from E. coli S17-1 to the ATCC 19859 strain was also tested.
In that case, the apparent transfer frequency was higher
(104).
Determination of optimal conditions for the mobilization of pJRD215
from E. coli S17-1 to T. ferrooxidans ATCC
33020.
As shown above, the apparent transfer frequency obtained by
the protocol of Peng et al. (23) for T. ferrooxidans ATCC 33020 was lower than 108 in the
case of conjugative plasmids and about 107 in the case of
mobilizable plasmids. We hypothesized that these low frequencies were
due to the completely different growth conditions of the donor and
recipient cells. Indeed, E. coli is a neutrophile, while
T. ferrooxidans is an extreme acidophile; E. coli
is a heterotroph, while T. ferrooxidans is a strict
chemolithoautotroph. Furthermore, E. coli is a fast-growing
microorganism (20-min generation time in Luria-Bertani medium) while
T. ferrooxidans is a slow-growing microorganism (9-h
generation time in 9K medium supplemented with ferrous iron). Transfer
of genetic material by conjugation requires cell-to-cell contacts and
energy for both the donor and the recipient cells. Accordingly, we
sought growth media and mating medium that could minimize differences
in growth conditions, thereby avoiding possible stress during mating.
Rawlings et al. (28) have studied the effect of mixing Luria
agar with inorganic agar medium (pH 4) containing tetrathionate on
E. coli and T. ferrooxidans ATCC 33020 growth and
on the mating efficiency between E. coli cells. They have
noticed that E. coli was able to grow on all media tested except 100% inorganic agar but that the mating efficiency between E. coli cells dropped off as the percentage of inorganic
agar increased. T. ferrooxidans was unable to grow in the
presence of even a low concentration of Luria medium but remained
viable. Because these results suggest that E. coli adapts
more easily to inorganic conditions than T. ferrooxidans to
organic conditions, the E. coli growth medium and the mating
medium used were based on the inorganic medium described by Peng et al.
(24).
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4 M, a significant increase in the apparent transfer
frequency was obtained (Table 2).
We also tested different donor-to-recipient cell ratios, which is known
to be important for conjugational transfer. As observed in Table 2, the
apparent transfer frequency can vary from 6 × 107
for a 4/1 ratio to 1.2 × 105 for a 1/2 ratio.
By combining all the factors described above, that is, (i) by growing
E. coli in 2:2 liquid medium with 0.5% yeast extract and
growing ATCC 33020 in 9K liquid medium supplemented with sulfur as an
energy source; (ii) by using the 2:2 solid medium with a pH of 4.8, 0.05% thiosulfate, and 0.5 × 104 M DAP as a mating
medium; and (iii) by using a donor-to-recipient cell ratio of 1/2, the
apparent transfer frequency of pJRD215 from E. coli S17-1 to
ATCC 33020 can be increased 102- to 103-fold.
Mobilization of pUFR034 (IncW) and pJB3Km1 (IncP) plasmids from
E. coli S17-1 to T. ferrooxidans ATCC
33020.
Genetic transfer to T. ferrooxidans ATCC 33020 was also tested with the mobilizable plasmids from the IncW (pUFR034)
and IncP (pJB3Km1) incompatibility groups. Under the optimized
conditions described above, the apparent transfer frequency of pJB3Km1
and pUFR034 was about 10 times lower than the frequency obtained with the IncQ plasmids (Table 3).
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Mobilization of IncQ, IncW, and IncP plasmids from E. coli S17-1 to T. ferrooxidans ATCC 19859, BRGM1, and Tf-49. Three other T. ferrooxidans strains were tested as recipients: the two private strains BRGM1 and Tf-49, the latter being one of the strains tested for conjugation by Peng et al. (23), and the ATCC 19859 collection strain, from which several genes have been characterized. The results obtained under the optimized conditions described above are presented in Table 3. The IncP, IncQ, and IncW plasmids tested were all transferred to the four T. ferrooxidans strains tested. The apparent transfer frequency obtained depended on the T. ferrooxidans recipient strain and on the plasmid incompatibility group. More particularly, for all the strains tested, the apparent transfer frequency was highest with the IncQ plasmid.
Transconjugant analyses. For each conjugation experiment performed, eight kanamycin-resistant (Kmr) clones were analyzed to establish that they were true T. ferrooxidans transconjugants.
No contaminating E. coli donor cells were detected by PCR with two oligonucleotide pairs hybridizing to E. coli but not to T. ferrooxidans genomic DNA (Fig. 1). On the other hand, a fragment internal to the rusticyanin-encoding gene, which does not exist in E. coli, was obtained in all cases, indicating that the Kmr clones were indeed T. ferrooxidans cells (Fig. 1).
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Stability analysis.
The stabilities of the different plasmids
tested in the four T. ferrooxidans strains studied were
analyzed as described in Materials and Methods. The IncQ plasmid
pJRD215 was stable, especially in the two collection strains ATCC 19859 and ATCC 33020, with more than 70% retention after 40 generations
without antibiotic selection (Fig. 2A).
On the other hand, the plasmid pUFR034 (IncW) (Fig. 2B) and, more
particularly, the pJB3Km1 (IncP) (Fig. 2C) plasmids, were unstable:
after 40 generations, fewer than 10% of the clones retained pUFR034
(IncW) and all the clones had lost pJFB3Km1 (IncP).
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Construction of a T. ferrooxidans ATCC 33020 recA mutant. To test whether conjugation can be used successfully as a genetic transfer technique to construct null mutants by reverse genetics, we chose the recA gene as a model because (i) construction of recA mutants by marker exchange mutagenesis has already been described in several microorganisms (22); (ii) since the RecA protein from the ATCC 33020 strain has the classical RecA biochemical activities (25, 26), a T. ferrooxidans recA mutant would be expected to have the same properties as the bacterial recA null mutants already characterized; (iii) the recA gene has been shown to be independently transcribed from the downstream essential alaS gene encoding alanyl tRNA synthetase (14), and consequently, recA insertional inactivation will not have a detrimental polar effect on alaS expression; and (iv) a T. ferrooxidans recA mutant is required for further genetic studies to stably maintain recombinant plasmids.
To obtain a T. ferrooxidans recA mutant, a mobilizable suicide plasmid carrying the recA gene disrupted with a cassette carrying the kanamycin resistance gene was constructed (see Materials and Methods). This plasmid (pUC19recA::- Kmmob), which
is unable to replicate in T. ferrooxidans, was mobilized
from an E. coli S17-1 strain into strain ATCC 33020 by
conjugation (see Materials and Methods), and kanamycin-resistant
(Kmr) clones were selected. Only five Kmr
clones were obtained from two independent conjugation experiments after
4 weeks of incubation at 30°C. These clones were indeed T. ferrooxidans cells carrying the -Km cassette as shown by PCR analyses (see Materials and Methods). Furthermore, no plasmid had been
detected by plasmid purification and transformation of E. coli, suggesting that either the plasmid had been integrated into
the chromosome by a single-crossover event or that recombination between the suicide plasmid carrying the mutated
recA::-Km allele and the chromosome carrying
the wild-type recA allele had taken place. To increase the
likelihood of this double-crossover event, the five Kmr
clones were subcultured twice in liquid medium for several generations without antibiotic. Under these conditions, three clones (no. 2, 3, and
4) lost their resistance to kanamycin, a result confirmed by PCR
analysis (data not shown) and Southern blot hybridization with a probe
corresponding to an internal fragment of the Kmr gene (Fig.
3A). We conclude that clones 2, 3, and 4 have lost the suicide plasmid. The two other clones (no. 1 and 5) kept
the Kmr phenotype after several generations in the absence
of kanamycin. The presence of the kanamycin resistance gene was
confirmed by PCR analysis (data not shown). On the other hand, the
ampicillin resistance gene could not be detected by PCR in these two
clones. All these results suggest that a recombination event had taken place at the recA locus.
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Characterization of recA mutants.
To determine if
recA is disrupted by the -Km cassette in the putative
recA mutants 1 and 5, genomic DNA was purified from these
clones and compared by Southern blot hybridizations and PCR analyses to
the DNA from the three Kms clones 2, 3, and 4 and to the
DNA from the wild-type ATCC 33020 strain.
-Km cassette had been
inserted within the recA gene in the two Kmr
isolates 1 and 5.
PCR analyses and sequencing of the recA region from
Kmr clones 1 and 5 confirmed the insertion of the -Km
cassette inside the recA gene (data not shown).
Kmr clones 1 and 5 will now be referred as recA
1 and recA 5 mutants.
Properties of the T. ferrooxidans recA mutants. The T. ferrooxidans recA 5 strain showed a slightly reduced growth rate compared to the wild-type strain (data not shown), which is typically observed for recA mutants.
To compare the effectiveness of DNA repair mechanisms in the wild-type strain and recA mutants, we used sensitivity to physical DNA-damaging agents. Sensitivities to irradiation by UV and rays
are presented in Fig. 4. As expected for
recA mutants, recA 1 and recA 5 strains were more sensitive than the parental ATCC 33020 strain to both
UV and radiations.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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This paper reports the first construction by marker exchange mutagenesis of a mutant of the extreme acidophilic T. ferrooxidans. Reverse genetics was made possible by significantly improving the conditions for the transfer of plasmids from E. coli to the ATCC 33020 culture collection strain of T. ferrooxidans by conjugation. The apparent transfer frequency was shown to depend on the donor-to-recipient cell ratio and on the physiological state of the donor and of the recipient cells, two factors which are known to be important for conjugational transfer. The conjugation protocol described in this paper was followed to mobilize plasmids from the three incompatibility groups, IncQ, IncP, and IncW, to four T. ferrooxidans strains, BRGM1, Tf-49, ATCC 19859, and ATCC 33020. Significantly, the IncW pUFR034 and, more particularly, the IncP plasmid pJB3Km1 were unstable in the four strains tested. These plasmids may therefore be used as shuttle vectors for a marker exchange mutagenesis program. On the other hand, the IncQ plasmids, which appeared to be stably maintained, may be useful for construction of expression vectors to introduce heterologous or homologous genes into T. ferrooxidans and for construction of operon fusion vectors to study T. ferrooxidans gene expression.
We have demonstrated that conjugation under the conditions described in
this study can be used successfully as a genetic transfer technique to
construct null mutants by reverse genetics. The knockout of the ATCC
33020 recA gene was confirmed by both molecular and physiological approaches. Evidence of recA gene disruption
by the -Km cassette includes the results of Southern hybridizations, PCR analyses, and sequencing on both sides of the -Km cassette. Moreover, as expected for recA mutants (22), the
knockout mutants exhibit slightly reduced growth rates and are more
sensitive to UV and irradiation compared to the parental strain. It
is worth mentioning that the T. ferrooxidans recA mutants
were not as sensitive to UV and irradiation as recA
mutants described in other bacterial species. One could then speculate
whether another recA gene is present in the T. ferrooxidans ATCC 33020 strain. This is unlikely because only one
recA gene has been found by Southern hybridization (this
paper and reference 25) and only one recA
gene has been cloned by complementation of an E. coli recA
mutant (25). Another possibility is that RecA-dependent DNA
repair is a minor pathway in T. ferrooxidans compared to
other repair mechanisms. In E. coli, the UV and irradiation-induced DNA lesions are primarily removed by nucleotide and
base excision repair processes, respectively (see reference
30 and references therein). However, if the
replication fork encounters a lesion before repair has taken place,
replication stalls or collapses. Replication restarts only from
recombination intermediates generated by RecA and accessory proteins,
such as RecFOR (gap repair) and RecBCD (double-strand break repair)
(18; see references 9, 20, and
30 and references therein). Therefore, RecA is
absolutely required for DNA repair at the level of the replication fork
in E. coli. Because this bacterium is a fast-growing microorganism, RecA plays a key role in the survival of exponentially growing cells exposed to UV or irradiation. Accordingly, the basal
level of RecA protein is high, and recA gene transcription is increased further by induction of the SOS response (see reference 32 and references therein). Because the T. ferrooxidans generation time is long (9 h at 30°C) but its
replication rate is likely similar to that of E. coli (about
1,000 nucleotides per s [16]), it is reasonable to
assume that in T. ferrooxidans, prereplicative repair
processes (nucleotide and base excision repair) have sufficient time to
repair most DNA damage before the arrival of the replication fork.
Postreplication RecA-dependent recombinational repair processes would
therefore play a minor role. In agreement with this hypothesis, the
basal level of RecA protein in the T. ferrooxidans ATCC
33020 strain is low (25) and the recA gene is not
induced by DNA damaging agents (26).
The construction of a T. ferrooxidans recA mutant should facilitate future genetic studies of this chemolithoautotrophic acidophilic microorganism by allowing the stable maintenance of plasmids in which homologous or heterologous genes have been cloned. Furthermore, the allelic replacement procedure used to produce the recA mutant should be applicable to the construction of null mutants of the genes encoding proteins whose physiological function have yet to be determined.
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ACKNOWLEDGMENTS |
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We owe special thanks to P. Moreau for fruitful advice and
suggestions and also to A. Bengrine for helpful discussions. We thank
J. DeMoss for critical reading of the manuscript. We acknowledge J. M. Blatny for the gift of the pJB3Km1 plasmid and the
Escherichia coli Genetic Stock Center for strain CGSC7330.
We are indebted to M. Kasmaier from C.E.A. (Cadarache, France) who
welcomed us for the radiation experiments. We are grateful to the
Centre de Sequençage de l'ADN (I.B.S.M., L.C.B., Marseille).
Z.L. acknowledges A. Klier (Institut Pasteur, Paris, France) and the support of a CIES grant in the context of an agreement between the University of Shandong (China) and Université Paris 7 (France). This work was partly supported by A.D.E.M.E., B.R.G.M., CO.GE.MA., and by an ACC-SV from the C.N.R.S.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratoire de Chimie Bactérienne, Institut de Biologie Structurale et de Microbiologie, C.N.R.S., 31 chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Phone: (33) 491 164 146. Fax: (33) 491 718 914. E-mail: bonnefoy{at}ibsm.cnrs-mrs.fr.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, April 2000, p. 2269-2276, Vol. 182, No. 8
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