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Journal of Bacteriology, July 2001, p. 4323-4329, Vol. 183, No. 14
Department of Biological Sciences, Faculty of
Chemistry and Biology, University of Santiago,1
and Institute of Biomedical Sciences, Faculty of Medicine,
University of Chile,3 Santiago, Chile, and
Laboratory of Chemical Biology, CNRS, Marseille,
France2
Received 6 September 2000/Accepted 20 April 2001
A 1.3-kb insertion sequence, termed ISAfe1 (U66426), from
Acidithiobacillus ferrooxidans ATCC 19859 is described.
ISAfe1 exhibits the features of a typical bacterial insertion sequence. It has 26-bp, imperfectly matched, terminal inverted repeats and an
open reading frame (ORF) that potentially encodes a transposase (TPase)
of 404 amino acids (AAB07489) with significant similarity to members of
the ISL3 family of insertion sequences. A potential ribosome-binding
site and potential Acidithiobacillus
ferrooxidans, formerly Thiobacillus ferrooxidans
(24), is a gram-negative bacterium that has been shown to
be active in the solubilization of copper and in the processing of
refractory gold ores in bioleaching operations (reviewed in references
21 and 36). It is also a major contributor to acid mine
drainage in copper and coal mines and in certain natural environments.
It is a chemolithotroph, deriving energy and electrons from the
oxidation of ferrous iron and/or sulfur and various reduced sulfur
compounds at pH 2 to 4, using oxygen as the ultimate electron acceptor
(22). It fixes CO2 by the Calvin-Bassham
scheme. It can also anaerobically oxidize hydrogen at pH 5.5 (15). Recently, the almost complete genome sequence of
A. ferrooxidans was used to detect and inventory the genes
involved in amino acid metabolism (40).
A mutant of A. ferrooxidans ATCC 19859 has been isolated
that is able to switch reversibly, and with high frequency, between a
wild-type state, in which it can oxidize both ferrous iron and sulfur
compounds, and a mutant state, in which it has lost the capacity to
oxidize iron (39). This phenomenon resembles other states
of instability associated with the transposition of insertion sequences
that have been described in other organisms and led us to investigate
whether phenotypic switching might similarly be explained in A. ferrooxidans.
Evidence was recently presented (5) that implicated a
member of the so-called family 1 repetitive elements (50)
in phenotypic switching. This repetitive element was tentatively
identified as an insertion sequence and termed IST1 (renamed ISAfe1
here). Phenotypic switching was shown to be correlated with the high frequency insertion and excision of ISAfe1 into, and out of, the resB gene (5). ResB encodes a
cytochrome c-type maturation protein (reviewed in reference
45), and a model was proposed in which insertion of ISAfe1
into resB eliminated the capacity of ResB to
satisfactorily mature a c-type cytochrome and that this resulted, in turn, in the loss of the ability to oxidize iron but
not sulfur (5).
In order to describe and explain the phenomenon of phenotypic
switching, we carried out a partial molecular characterization of
ISAfe1. We further demonstrate that ISAfe1 can promote plasmid integration and resolution in E. coli, opening up the future
possibility of exploiting E. coli to test experimentally
certain characteristics of this insertion sequence.
Bacterial strains and media.
Strains and plasmids used in
this study are listed in Table 1.
A. ferrooxidans ATCC 19859 was grown on Mackintosh medium or
in modified 9K-ferrous iron medium (50). E. coli was grown in Luria-Bertani (LB) medium (30).
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.14.4323-4329.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
ISAfe1, an ISL3 Family Insertion Sequence from
Acidithiobacillus ferrooxidans ATCC 19859
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
10 and 35 promoter sites for the TPase ORF were
identified, and a +1 transcriptional start site was detected
experimentally. A potential outwardly directed 35 site was identified
in the right inverted repeat of ISAfe1. A second ORF (ORF B), of
unknown function, was found on the complementary strand with
significant similarity to ORF 2 of ISAe1 from Ralstonia eutropha. Southern blot analyses demonstrated that ISAfe1-like elements can be found in multiple copies in a variety of A. ferrooxidans strains and that they exhibit transposition. A codon
adaptation index (CAI) analysis of the TPase of ISAfe1 indicates that
is has a CAI of 0.726 and can be considered well adapted to its host, suggesting that ISAfe1 might be an ancient resident of A. ferrooxidans. Analysis of six of its target sites of insertion in
the genome of A. ferrooxidans ATCC 19859 indicates a
preference for 8-bp pseudopalindromic sequences, one of which resembles
the termini of its inverted repeats. Evidence is presented here that is
consistent with the possibility that ISAfe1 can promote both plasmid
cointegrate formation and resolution in E. coli.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
TABLE 1.
Strains and plasmids used in this study
Construction of plasmids. Construction of pTf85. A member of family 1 repeated DNA from A. ferrooxidans ATCC 19859 was cloned into pBR322, and the resulting plasmid was designated pTf11 (50). An internal SphI fragment of pTf1 1 was subcloned into pBR322 and termed pTf1 1-sph. pTf1 1-sph was subsequently used as a probe in a Southern blot against genomic DNA of A. ferrooxidans ATCC 19859 cleaved with BamHI. The genomic DNA was derived both from the wild-type strain and from the phenotypic switching mutant of this strain. Among the 20 to 25 hybridizing bands a 2.8-kb band was identified as hybridizing only in the DNA prepared from the phenotypic switching mutant strain. This band was excised and cloned into pBR322 and termed pTf85. pTf85 contains one copy of ISAfe1 inserted into the resB gene (5).
pACYC184-ISAfe1 was constructed as follows: a BamHI fragment carrying the intact ISAfe1 from pTf85 was cloned into the BamHI site of pACYC184 selecting for chloramphenicol resistance (Cmr) and screening for the loss of ampicillin resistance in Escherichia coli to yield the plasmid pACYC184-ISAfe1.Conjugation experiments. Donor and recipient strains were grown in LB medium, supplemented with the appropriate antibiotics (tetracycline and chloramphenicol, 25 µg/ml; streptomycin, 100 µg/ml) until they reached the middle of the exponential phase. The donor and the recipient strains were mixed in a 1:1 ratio and incubated at 37°C for 2 h without agitation. Suitable dilutions were plated on LB agarose supplemented either with tetracycline and streptomycin (concentrations were as described above) to determine the conjugation frequency or with tetracycline, streptomycin, and chloramphenicol to determine the cointegrate frequency. The presence of ISAfe1 in the transconjugants was detected by PCR amplification using the following inwardly directed primers derived from ISAfe1: A (5'-GGGGGTAGAATGCTGTGG) and B (5'-ATTGGTAATCTGGCTTTCGA). PCR amplification was carried out as follows: 2 min and 30 s at 94°C, followed by 30 cycles at 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s, and then 2 min and 30 s at 72°C.
DNA sequencing.
DNA sequencing and DNA manipulations were
carried out by standard procedures (38). Sequencing
reactions were carried out by using the Sequenase reagents kit (USB
Corp.) with [
-35S]dATP (Amersham). Plasmids
were prepared as described earlier (20).
Southern hybridization. Chromosomal DNA was prepared as previously described (50) and digested with restriction enzymes (New England Biolabs). The resulting DNA fragments were resolved by agarose gel electrophoresis and transferred to nylon membranes by Southern blotting. Prehybridization was carried out for 2 h at 42°C in 6× SSPE (1× SSPE is 0.18 M NaCl, 10 mM NaHPO4, and 1 mM EDTA), 0.4% sodium dodecyl sulfate (SDS) 1× BFP (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% bovine serum albumin), 20% formamide, and 0.1 mg of salmon sperm DNA per ml. Membrane washes were done at 42°C, 60°C, and 75°C in 4× SSPE-0.2% SDS. Hybridization was accomplished by the addition of denatured, nick-translated probe (2 × 109 to 4 × 109 cpm), and incubation was carried out at 42°C for at least 16 h. The membranes were washed using the following procedure: two 5-min incubations at room temperature in 0.3 M NaCl, 0.06 M Tris (pH 7.5), and 0.002 M EDTA; two 30-min incubations at 60°C in 0.3 M NaCl, 0.06 M Tris (pH 7.5), and 0.002 M EDTA; two incubations at room temperature in 0.03 M NaCl, 0.006 M Tris (pH 7.5), and 0.0002 M EDTA. The membranes were air dried, and autoradiography was accomplished using X-Omat AR film (Kodak).
Northern hybridization. Total RNA was isolated as described previously (49) and stored in H2O in 0.1% diethylpyrocarbonate. The quality of the RNA was checked on a 1.0% agarose gel. A total of 40 mg of total RNA was separated by electrophoresis on a 1.0% agarose-6.8% formaldehyde-morpholinepropanesulfonic acid buffer gel and transferred to a Hybond N (Amersham) nitrocellulose membrane in 20× SSC (1× SSC is 0.15 M NaCl plus 0.015 sodium citrate). RNA was fixed by UV radiation. Hybridization was performed at 65°C in 5× SSC-5× Denhardt's solution-0.5% SDS-0.5 mM EDTA. Blots were washed with 0.5% SSC-0.1% SDS at 65°C. The probe used was an internal SphI-SphI fragment of ISAfe1 derived from the plasmid pTf85 and corresponds to part of the hypothetical coding region of the TPase of ISAfe1.
Primer extension analysis. RNA was isolated as described above for the Northern hybridization experiments. Primer extension was carried out by standard procedures (38) using the following primer: 5'-CCAACCACGGCGGTACCAACCC-3'.
Detection of ISAfe1 insertion sites in the genome. Genomic DNA, prepared from A. ferrooxidans ATCC 19859, was cleaved with the following restriction enzymes, MscI, SmaI, HincII, EcoRV, and StuI, each of which cuts once within ISAfe1. Cleaved DNA was cloned using the Genome Walker Kit (Clontech) according to the manufacturer's instructions and was amplified using the adapter and nested adapter primers of the kit and the following primers and nested primers derived from ISAfe1: 5'-CCTATTCGGGAACGCAACT-3' and 5'-TTCTGGGACCTCAGCTAACTC-3' (both oriented upstream with respect to the transposase [TPase] gene) and 5'-GGCCTACCTGATCCTGG-3' and 5'-TTTATCAACATGGCCTACCTGAT-3' (both oriented downstream). The resulting amplified DNA was sequenced as described above, and the site of insertion of ISAfe1 in the genome was identified by examination of the DNA sequence outside of the known 5' and 3' termini of ISAfe1.
Bioinformatics. The following programs were used: BlastP, BlastN, TBlastN, BlastX, and Psi-Blast (1, 3, 51); Fasta (32); Multalin (14); CLUSTAL W (44). The following additional programs were used: promoter identification (http: //www.fruitfly.org/seq_tools/promoter.html [18]) and Bend DNA (http://www2.icgeb.trieste.it/~dna/bend_it.html).
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A family 1 repetitive element, which had previously been identified as being integrated within the resB gene of A. ferrooxidans ATCC 19859 (5), was cloned into pBR322, generating the plasmid pTf85 (see Materials and Methods). The repetitive element was sequenced (GenBank accession no. U66426) and, as described below, it conforms to the criteria of a bacterial insertion sequence. It was originally termed IST1, but we rename it here ISAfe1, consistent with a recently proposed nomenclature for insertion sequences, in which the insertion sequence descriptor carries the designation "IS" followed by three letters identifying the host plus a unique number (M. Maillon and J. Chandler, personal communication).
ISAfe1 is 1,303 bp in length and exhibits imperfectly matched terminal
inverted repeats of 26 bp with sequence similarity to the terminal
inverted repeats of insertion sequences belonging to the ISL3 family
(Table 2).
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Characterization of the putative TPase. An ORF (ORF A) encoding a hypothetical protein of 404 amino acids and occupying the majority of ISAfe1 was identified (AAB07489). ORF A terminates within the rightward inverted repeat, 14 bp from the 3' terminus of ISAfe1. There is no obvious transcription termination signal. BLASTP and FASTA searches of the nonredundant databases revealed significant similarity of the ISAfe1 putative TPase with the putative TPase ISAE1 (AAC13658.1) of Ralstonia eutrophus (43% identity, 60% positive, expect = 2e86) and other TPases and putative TPases of the ISL3 family of insertion sequences (29). Using the default parameters, BlastP yielded 27 members of the ISL3 family with similarity to the ISAfe1 TPase and a CLUSTAL W alignment, a Blocks alignment, and a comparison of the aligned Blocks with the Blocks in the Lama database has been posted at http://holmeslab.usach.cl/ISL3.html.
Codon usage. A codon usage table of the ISAfe1 TPase was constructed (data not shown) and compared to the codon usage of the approximately hundred genes of A. ferrooxidans posted at http://www.kuzasa.org/codon. The codon adaptation index (CAI) of the TPase was computed to be 0.762 by the method of Sharp and Li (41).
Characterization of the DNA sequences present in ISAfe1.
A
potential ribosome-binding site was identified by visual inspection 7 bp upstream of the ATG start codon of the putative TPase (Fig.
1). A + 1 transcriptional start site
was identified by primer extension analysis of whole-cell RNA (data not
shown). Additional weak potential +1 transcriptional sites were also
identified that lay outside the insertion sequence (data not shown).
These could represent transcriptional starts from genes that lie
upstream of the ca. 20 copies of ISAfe1 present in the genome. Northern blot analysis of whole-cell RNA failed to reveal a specific band(s) of
RNA that hybridized with an ISAfe1 specific probe (data not shown). It
is possible that the level of ISAfe1-specific mRNA is very low or that
it is unstable. Visual inspection identified potential 10 and 35
sites upstream of, and consistent with, the placement of the +1
transcriptional start site (Fig. 1). The potential 35 site lies
within the left terminal inverted repeat.
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35 promoter site lies within the
right terminal inverted repeat of ISAfe1 12 bp from the 3' terminus and
was matched with other outwardly directed promoters of insertion
sequences (Table 3). No obvious inwardly
directed 10 site lies within the 5' inverted repeat such that a
complete promoter site would be generated on circularization of ISAfe1. Also, no outwardly directed promoter site could be detected within the
5' inverted repeat.
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ORF B. A second ORF (ORF B) was detected on the complementary strand with respect to the ISAfe1 TPase with a potential ATG start site at position 575 with respect to GenBank U66426 and potentially encoding a protein of 156 amino acids. A BlastP search revealed similarity to ORF 2 on the complementary strand of the TPase of ISAe1 (A47041) of R. eutropha. No additional similarities could be detected by Psi-Blast or by Fasta. No obvious promoter or ribosome-binding site could be detected upstream of ORF B by visual inspection nor by analysis using a neural network promoter finding program (18) that has been trained on E. coli promoters.
Target sites.
Six proposed target sites of insertion of ISAfe1
in A. ferrooxidans ATCC 19859 were identified (Table
4). Five of these sites was determined by
random cloning, using the Genome Walker kit (see Materials and
Methods), followed by DNA sequencing using outwardly directed ISAfe1
specific primers. Four of these five sites are palindromic or near
palindromic and are AT-rich (63 to 88% A+T). The sixth site,
5'-GCCATTGGC, was determined by cloning and DNA sequencing
around the insertion of ISAfe1 in resB and is a 9-bp near
palindrome.
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Distribution and number of copies of ISAfe1 in A. ferrooxidans and other organisms.
Southern blot analysis of
DNA prepared from a number of strains of A. ferrooxidans,
isolated from various parts of the world (Fig.
2, lanes 2 to 12), together with two
strains of the related bacterium Acidithiobacillus
thiooxidans (Fig. 2, lanes 13 and 14), reveals the presence of
multiple copies of ISAfe1-like sequences in all but one strain (Fig. 2,
lanes 12). The respective genomic DNAs were cleaved with
BamHI that, in the case of ISAfe1 from A. ferrooxidans ATCC 19859, cuts outside the insertion sequence. If
BamHI also cuts outside of the ISAfe1-like sequences present in the other strains of A. ferrooxidans, then we estimate
that there are about 10 to 30 copies of ISAfe1-like sequences present in the other genomes. A more precise estimate is not possible given
that some of the more intensely hybridizing bands may harbor more than
one copy of the insertion sequence and some of the less intensely
hybridizing bands may contain truncated ISAef1-like elements. In the
case of A. thiooxidans, about 30 to 40 hybridizing bands can
be detected, but the majority of these hybridize less intensely than
those of A. ferrooxidans (Fig. 2, lanes 13 and 14). This
could be due to a possibly weaker similarity of their insertion
sequences to ISAfe1 and/or BamHI might cleave one or more
times within their insertion sequences reducing the target size for
hybridization. No hybridization could be detected to another
iron-oxidizing bacterium, Leptospirullum ferrooxidans, nor
to the acidophilic heterotrophs, Acidophilum cryptum and
A. organovorans (data not shown).
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Possible transposition of ISAfe1 in A. ferrooxidans.
A single colony of A. ferrooxidans
ATCC 19859 was isolated from solid iron-containing media and was
disrupted in a vortex blender. Dispersed cells were plated on solid
iron-containing media, and DNA was prepared from 25 isolated colonies.
The DNA was cut with BamHI, and a Southern blot was prepared
and hybridized with 32P-labeled probe specific for ISAfe1.
Of the 25 colonies, 7 exhibited band differences compared to the
starting pattern of hybridization (Fig.
3), finding consistent with the idea that
ISAfe1 is capable of transposition in A. ferrooxidans ATCC
19859.
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Transposition of ISAfe1 in E. coli.
The ability of
ISAfe1 to transpose in E. coli was evaluated by monitoring
its ability to generate replicon fusion (Fig.
4). The nonmobilizable
pACYC184-ISAfe1 plasmid was used as the donor replicon (see
Materials and Methods) and the conjugative MiniF'Tcr devoid
of transposable elements as the recipient replicon (48). As a control, a pACYC184 derivative (Tcs) was used in place
of the pACYC184-ISAfe1. The donor plasmids were introduced by
transformation into the streptomycin-sensitive (Strs)
E. coli NK7379, which harbored the conjugative episome
MiniF'Tcr, selecting for chloramphenicol and tetracycline
resistance. To determine whether transposition of ISAfe1 from
pACYC184-ISAfe1 to MiniF'Tcr could take place, the
formation of cointegrates was evaluated (step A, Fig. 4). E. coli NK7379 (designated male strs in Fig. 4) carrying
the donor plasmid pACYC184-ISAfe1 was conjugated (step B, Fig. 4) with
the streptomycin-resistant E. coli MC4100 strain (designated
female strr in Fig. 4). On the one hand, Strr
and Tcr conjugants were selected to determine the
conjugation frequency, and on the other hand Strr,
Tcr, and Cmr exconjugants were selected to
check for cointegrate formation. Cointegrates (Strr,
Tcr, and Cmr) were obtained with the
pACYC184-ISAfe1 but not with the pACYC184 Tcs derivative.
The transposition frequency, corresponding to the number of
Strr Tcr Cmr conjugants relative to
the number of Strr Tcr exconjugants, was
estimated to be about 10
7. Because pACYC184 is a
nonmobilizable vector, chloramphenicol-resistant exconjugants can only
be obtained by replicon fusion between the conjugative
MiniF'Tcr and the pACYC184-ISAfe1 plasmid. Replicon fusions
are formed by homologous recombination between the two replicons.
Because there is no homology between the donor and the recipient
replicons and because the only transposable element present on the two
replicons is the ISAfe1, the most likely way that recombination can
take place is after a transposition event in which ISAfe1 present on the pACYC184-ISAfe1 plasmid has transposed to the
MiniF'Tcr.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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A BlastP analysis of the nonredundant database suggests that
ISAfe1 is a member of the ISL3 family of insertion sequences. The
similarity of the inverted terminal repeats of ISAfe1 with those of the
ISL3 family (Table 2) is consistent with this designation. A +1
transcription initiation site of the ISAfe1 TPase was mapped in ISAfe1
by primer extension. Putative 35 and 10 promoter regions were
identified by visual inspection and by a neural network program trained
on E. coli sigma 70 promoter sites (18). The
spacing between the proposed 35 and 10 regions is 18 bp, which is
consistent with the average spacing in E. coli sigma 70 promoter sites (18). There is no compendium of known sigma
70-type promoter sites for A. ferrooxidans from which
consensus 35 and 10 sites could be derived for comparison with the
suggested sites for the ISAfe1 TPase promoter. However, the recently
published, almost-complete genome sequence of A. ferrooxidans should expedite the identification of promoter sites
(40). Failure to detect ISAfe1 TPase mRNA by Northern
blotting suggests that not many copies of the TPase mRNA exist per
cell, which could be explained by a weak promoter, although other
reasons, such as RNase sensitivity of the TPase mRNA, could also
explain this result.
A possible outwardly directed 35 region was identified in ISAfe1
(Table 3), lying within the right inverted repeat 12 bp from the 3'
end, and is placed in such a way that it could form a hybrid promoter
with an appropriately located 10 region outside the insertion
sequence. The ability of numerous insertion sequences to form hybrid
promoters that can control the expression of downstream genes or, via
circle formation, form self-hybrid promoters, is well established
(reviewed in references 16 and 29).
Inspection of ISAfe1 does not reveal an obvious hybrid promoter if the
insertion sequence were to circularize.
Target sites. Extensive analysis of a number of cloned ISAfe1 insertions yielded only six different potential target sites (Table 4), which is surprising given the approximately 15 different sites as judged by Southern blot hybridization (Fig. 2). It is possible that some of the hybridizing bands on the Southern blot represent mutated or truncated copies of ISAfe1 that do not yield products with the PCR primers used to detect and sequence the putative insertion sites.
Five of the six proposed target sites of ISAfe1 exhibit 8-bp palindromic or pseudopalindromic symmetry (Table 4). Several other insertion sequences, such as IS1301 (19), IS481 (43), Tn7 (17), and IS1630 (7) have also been shown to prefer palindromic or near-palindromic sequences as sites of integration and target site duplication, and 8-bp target sites have been reported for the ISL3 family (29). Four of the six sites of ISAfe1 show a strong preference for AT-rich DNA (average, 78.5% A+T) and an AT preference has been noted for other members of the ISL3 family (29). In contrast to the other five evaluated cases, the site 5'-GCCATTGGC is a 9-bp palindrome. This is the site in the resB gene in which insertion of ISAfe1 has been postulated to cause a mutation that is associated with a the loss of iron-oxidizing ability (5). Reversion of this mutant to wild type is associated with the spontaneous loss of ISAfe1 from resB and is accompanied by the removal of the target site duplication, restoring the wild-type DNA sequence (5). The high frequency transposition of ISAfe1 into resB and the mechanism for precise excision require explanations. Our analysis reveals that the target site of insertion into resB is the pseudopalindromic sequence 5'-GCCATTGGC, of which the 3'-terminal GGC resembles the first three bases of ISAfe1. Similarity of the target site with its terminal inverted repeats has also been reported for IS911 (34) and for IS481 (43), and it was proposed that this might promote a preference for entry into such sites. The frequency and preference for insertion of some insertion sequences are also known to be affected by negative supercoiling, as in the case of Tn10 (5) and the presence of bendable DNA in the case of the Himar1 transposon (27). However, the program Bendit failed to detect bendable DNA in the region of insertion of ISAfe1 in resB.ORF B. ORF B on the complementary strand to the ISAfe1 TPase has significant similarity to ORF 2 on the complementary strand of the related family ISL3 insertion sequence ISAe1 of R. eutropha. The function of ORF 2 remains unknown (26). Several other insertion sequences, such as IS30 (2), IS10 (8, 9, 33), and pot2 (25), have been shown to encode antisense RNA on the opposite strand to the TPase. The antisense RNA of IS30 contains an ORF which has been shown not to be translated at detectable levels (2). It has been speculated that insertion sequence antisense RNA may function in the control of expression of TPase. Other insertion sequences, such as IS5 (35), IS903 (31), and IS3 (46), have complementary strand ORFs, but it has not been determined if they are transcribed into antisense RNA, and their function remains unknown. The absence of an obvious ribosome-binding site and promoter for the ORF B suggests that it might not be transcribed and translated, although it may just reflect our inability to identify these regulatory sequences in the poorly defined context of A. ferrooxidans genes.
Distribution of ISAfe1 in other strains of A. ferrooxidans and other bacteria: mobility of ISAfe1 within A. ferrooxidans and codon usage. The distribution of ISAfe1-like sequences in diverse strains of A. ferrooxidans and in A. thiooxidans (Fig. 2) suggests that an ancestral ISAfe1 invaded the acidithiobacilli before the separation of A. ferrooxidans and T. thiooxidans, but after the separation of these from the leptospirilli. According to ribosomal DNA analysis, A. ferrooxidans and T. thiooxidans are closely related, but L. ferrooxidans is only distantly related (28). The favorably high CAI of 0.762 calculated for the TPase of ISAfe1 is indicative of a gene well adapted to its host and is consistent with the view of an ancient relationship between ISAfe1 and A. ferrooxidans.
A comparison of the positions of ISAfe1 in the genome of several clonal derivatives of A. ferrooxidans ATCC 19859, as judged by Southern blot hybridization (Fig. 3), is consistent with the idea that at least several ISAfe1 sequences are capable of transposition, although recombination between insertion sequences leading to rearrangements and deletions could also explain the results.Transposition of ISAfe1 in E. coli. The observation that ISAfe1 can promote cointegrate formation and resolution in E. coli can be explained if ISAfe1 first transposed, by a nonreplicative cut-and-paste mechanism (reviewed in reference 29), from the donor plasmid pACYC184-ISAfe1 to the conjugative MiniF'Tcr plasmid, followed by cointegrate formation between the target plasmid and one of the original donor plasmids. Resolution of the cointegrate could then take place by recombination between the two copies of ISAfe1, restoring the initial donor molecule with one copy of the element and the target molecule with the second copy. Alternatively, cointegrate formation could take place with duplication of ISAfe1 by replicative transposition as has been shown to occur in the Tn3 family transposons and Mu phage (reviewed in reference 92).
Transposition has been investigated for several members of the ISL3 family. IS31831 exhibits cointegrate formation and resolution (47) and IS1096 was shown to contain a putative resolvase (12). It was suggested that IS1411 might transpose via circle formation (23), as has been shown to occur during the transfer of conjugative plasmids (37). Although Southern blotting experiments (Fig. 3) suggest that ISAfe1 is capable of transposition in its native host, A. ferrooxidans, nothing is known about the mechanism of transposition. The demonstration that ISAfe1 is capable of frequent and reversible insertion into the resB gene (5, 39) is also consistent with the idea that ISAfe1 is capable of transposition in its natural host. There is only one previous report describing the transposition of an insertion sequence within A. ferrooxidans. Transposition of IST2, a member of the IS256 family, was shown to transpose via a mechanism that does not involve recombination (6). Also, there is only one previous report of the transposition of an A. ferrooxidans insertion sequence in the heterologous host E. coli, in which IS3091, a member of the IS30 family, was shown to be capable of cointegrate formation (10). Tn5467, an A. ferrooxidans composite transposon, was shown to contain a partial transposase gene and a resolvase gene, but transposition was not detected in E. coli (13). It is hoped that, with two reported cases of possible transposition of A. ferrooxidans insertion sequences in E. coli at hand (reference 10 and this study), future work will lead to the elucidation of the mechanism(s) of insertion sequence transposition, at least in the heterologous host E. coli.| |
ACKNOWLEDGMENTS |
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This work was supported by Fondecyt grant 1980665 and a grant from ECOS/Conicyt C99B05.
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FOOTNOTES |
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* Corresponding author. Mailing address: Laboratory of Bioinformatics and Molecular Microbiology, Faculty of Chemistry and Biology, University of Santiago (USACH), 3363, Avenida Bernardo O'Higgins, Santiago, Chile. Phone: 56-2-681-2575, x797. Fax: 56-2-681-2108. E-mail: dsholmes{at}hotmail.com.
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Journal of Bacteriology, July 2001, p. 4323-4329, Vol. 183, No. 14
0021-9193/01/$04.00+0 DOI: 10.1128/JB.183.14.4323-4329.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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