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J Bacteriol. Jul 2001; 183(14): 4323–4329.
PMCID: PMC95323

ISAfe1, an ISL3 Family Insertion Sequence from Acidithiobacillus ferrooxidans ATCC 19859

Abstract

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 −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.

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.

MATERIALS AND METHODS

Bacterial strains and media.

Strains and plasmids used in this study are listed in Table Table1.1. 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).

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 [α-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).

RESULTS

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 (Table2).2).

TABLE 2
Alignment of the 5′ and 3′ termini of ISAfe1 with three members of the ISL3 family

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. (Fig.1).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. (Fig.1).1). The potential −35 site lies within the left terminal inverted repeat.

FIG. 1
5′ Terminus of ISAfe1 showing the putative ATG start (underlined) of the TPase with a potential ribosome-binding site (underlined RBS) and putative −35 and −10 promoter sites (underlined). The +1 start of transcription ...

A potential, outwardly directed −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 (Table3).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.

TABLE 3
Comparison of the potential outward-facing −35 promoter region of ISAfe1 with the outward-facing −35 promoter regions (underlined) in the right terminal IRs of selected insertion sequences (16, 29)

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 (Table4).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.

TABLE 4
Proposed target sites of ISAfe1 in A. ferrooxidans ATCC 19859 and their A+T percent content

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. (Fig.2,2, lanes 2 to 12), together with two strains of the related bacterium Acidithiobacillus thiooxidans (Fig. (Fig.2,2, lanes 13 and 14), reveals the presence of multiple copies of ISAfe1-like sequences in all but one strain (Fig. (Fig.2,2, 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. (Fig.2,2, 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).

FIG. 2
Southern blot analysis of several strains of A. ferrooxidans (lanes 1 to 12) and A. thiooxidans (lanes 13 and 14). Genomic DNA was cut with BamHI and hybridized with 32P-labeled probe derived from an internal SphI fragment of ISAfe1. Lanes: 1, ATTC 23270; ...

A sample of A. ferrooxidans ATCC 13661, received directly from the American Type Culture Collection, was subcloned on solid media, and DNA was prepared from two colonies, termed A and B. The pattern of hybridization of an ISAfe1 specific probe with the DNA of colonies A and B is strikingly different (Fig. (Fig.2,2, compare lanes 2 and 3), suggesting that the strain is not pure at the ATCC source.

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. (Fig.3),3), finding consistent with the idea that ISAfe1 is capable of transposition in A. ferrooxidans ATCC 19859.

FIG. 3
Southern blot analysis of the genomic DNA prepared from eight colonies of A. ferrooxidans and hybridized with α-32P-labeled probe derived from an internal SphI fragment of ISAfe1. Lanes 1 and 8 represent the DNA derived from the original colony, ...

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. (Fig.4).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. Fig.4).4). E. coli NK7379 (designated male strs in Fig. Fig.4)4) carrying the donor plasmid pACYC184-ISAfe1 was conjugated (step B, Fig. Fig.4)4) with the streptomycin-resistant E. coli MC4100 strain (designated female strr in Fig. Fig.4).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.

FIG. 4
Schematic representation of the proposed transposition of ISAfe1 from the plasmid pACYC184-ISAfe1 to the Mini F′Tcr plasmid (step A), followed by cointegrate formation and transfer of the cointegrate by conjugation from the donor male (strs) to ...

In order to determine whether cointegrate resolution could take place, plasmids were isolated from Strr Tcr Cmr conjugants and transformed into E. coli TG1, selecting for Cmr and screening for Tcs (step C, Fig. Fig.4).4). Of 6,064 Cmr transformants analyzed, 6,061 were simultaneously Tcs indicating that the MiniF′Tcr has been lost, while the pACYC184-ISAfe1 replicon has been maintained. These results are in agreement with cointegrate resolution occurring most probably by homologous recombination between the two ISAfe1s present on the cointegrate.

The presence of ISAfe1 in the transconjugants was evaluated by PCR amplification, using ISAfe1 internal primers. The expected product of PCR amplification, if ISAfe1 is present, is 1,130 bp, which was confirmed by agarose gel electrophoresis (Fig. (Fig.5).5).

FIG. 5
Characterization of PCR-amplified products of ISAfe1 in E. coli. Lanes 1 to 3 represent the amplification from three separate colonies of Strs Cmr Tcr transconjugants, lane 4 shows marker DNA, lane 5 shows recipient strain MC4100 prior to conjugation, ...

DISCUSSION

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 (Table2)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 (Table3),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 (Table4),4), which is surprising given the approximately 15 different sites as judged by Southern blot hybridization (Fig. (Fig.2).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 (Table4).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. (Fig.2)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. (Fig.3),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. (Fig.3)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

This work was supported by Fondecyt grant 1980665 and a grant from ECOS/Conicyt C99B05.

REFERENCES

1. Altschul S, Madden T, Schaffer A, Zhang J, Zhang Z, Miller W, Lipman D. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389–3402. [PMC free article] [PubMed]
2. Arini A, Keller M, Arber W. An antisense RNA in IS30 regulates the translational expression of the transposase. Biol Chem. 1997;378:1421–1431. [PubMed]
3. Bailey T L, Gribskov M. Combining evidence using P-values: application to sequence homology searches. Bioinformatics. 1995;14:48–54. [PubMed]
4. Bender J, Kleckner N. Tn10 insertion specificity is strongly dependent upon sequences immediately adajecent to the target-site consensus sequence. Proc Nat Acad Sci USA. 1992;89:7996–8001. [PMC free article] [PubMed]
5. Cabrejos M E, Zhao H-L, Guacucano M, Bueno S, Levican G, Garcia E, Jedlicki E, Holmes D S. IST1 insertional inactivation of the resB gene: implications for phenotypic switching in Thiobacillus ferrooxidans. FEMS Microbiol Lett. 1999;175:223–229. , [PubMed]
6. Cadiz R, Gaete L, Jedlicki E, Yates J, Holmes D S, Orellana O. Transposition of IST2 in Thiobacillus ferrooxidans. Mol Microbiol. 1994;12:165–170. [PubMed]
7. Calcutt M J, Lavrrar J L, Wise K S. IS1630 of Mycoplasma fermentans, a novel IS30-type insertion element that targets and duplicates inverted repeats of variable length and sequence during insertion. J Bacteriol. 1999;181:7597–7607. [PMC free article] [PubMed]
8. Case C, Roels S, Gonzalez J, Simons E, Simons R. Analysis of the promoters and transcripts involved in IS10 anti-sense RNA control. Gene. 1988;72:219–236. [PubMed]
9. Case C C, Roels S M, Jensen P D, Lee J, Kleckner N, Simons R W. The unusual stability of the IS10 anti-sense RNA is critical for its function and is determined by the structure of its stem-domain. EMBO J. 1989;8:4297–4305. [PMC free article] [PubMed]
10. Chakravarty L, Kittle J, Tuovinen O. Insertion sequence of IS T3091 of Thiobacillus ferrooxidans. Can J Microbiol. 1997;43:503–508. [PubMed]
11. Chang A, Cohen S. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978;134:1141–1156. [PMC free article] [PubMed]
12. Cirillo J D, Barletta R G, Bloom B R, Jacobs W R., Jr A novel transposon trap for mycobacteria: isolation and characterization of IS1096. J Bacteriol. 1991;173:7772–7780. [PMC free article] [PubMed]
13. Clennel A M, Johnston B, Rawlings D E. Structure and function of Tn5467, a Tn21-like transposon located on the Thiobacillus ferrooxidans broad-host-range plasmid pTF-FC2. Appl Environ Microbiol. 1995;61:4223–4229. [PMC free article] [PubMed]
14. Corpet F. Multalin: multiple sequence alignment with hierarchical clustering. Nucleic Acids Res. 1988;16:10881–10890. [PMC free article] [PubMed]
15. Drobner E, Huber H, Stetter K O. Thiobacillus ferrooxidans, a facultative hydrogen oxidizer. Appl Environ Microbiol. 1990;56:2922–2923. [PMC free article] [PubMed]
16. Galas D J, Chandler M. Bacterial insertion sequences. In: Berg D E, Howe M M, editors. Mobile DNA. Washington, D.C.: American Society for Microbiology; 1989. pp. 109–162.
17. Gay N J, Tybulewicz V L, Walker J E. Insertion of transposon Tn7 into the Escherichia coli glmS transcriptional terminator. Biochem J. 1986;234:111–117. [PMC free article] [PubMed]
18. Harley C B, Reynolds R P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987;15:2343–2361. [PMC free article] [PubMed]
19. Hilse R, Hammerschmidt S, Bautch W, Frosch M. Site-specific insertion of IS1301 and distribution in Neisseria meningitidis strains. J Bacteriol. 1996;178:2527–2532. [PMC free article] [PubMed]
20. Holmes D S, Quigley M. A rapid method for the preparation of plasmids. Anal Biochem. 1981;114:193–197. [PubMed]
21. Holmes D S. Biorecovery of metals from mining wastes. In: Martin A M, editor. Bioconversion of waste materials to industrial products. 2nd ed. London, England: Blackie Academic and Professional Press; 1998. pp. 517–545.
22. Ingledew W J. Thiobacillus ferrooxidans, the bioenergetics of an acidiphilic chemoautotroph. Biochim Biophys Acta. 1982;683:149–154. [PubMed]
23. Kallastu A, Horak R, Kivisaar M. Identification and characterization of IS1411, a new insertion sequence which causes transcriptional activation of the phenol degradation genes in Pseudomonas putida. J Bacteriol. 1998;180:5306–5312. [PMC free article] [PubMed]
24. Kelly D P, Wood A P. Reclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., Halothiobacillus gen. nov., and Thermithiobacillus gen. nov. Int J Syst Evol Microbiol. 2000;50:511–516. [PubMed]
25. Kimura M, Yamaguchi I. Convergent transcription units and their promoters at both ends of pot2, an inverted repeat transposon from the rice blast fungus. J Biochem. 1998;124:268–273. [PubMed]
26. Kung S, Chang J, Chow W. Molecular and genetic characterization of an Alcaligenes eutrophus insertion element. J Bacteriol. 1992;174:8023–8029. [PMC free article] [PubMed]
27. Lampe D J, Grant T E, Robertson H M. Factors affecting transposition of the HimarI mariner transposon in vitro. Genetics. 1998;149:179–187. [PMC free article] [PubMed]
28. Lane D, Harrison A, Stahl D, Pace B, Giovannoni S, Olsen G, Pace N. Evolutionary relationships among sulfur and iron oxidizing bacteria. J Bacteriol. 1992;174:269–278. [PMC free article] [PubMed]
29. Mahillon J, Chandler M. Insertion sequences. Microbiol Mol Biol Rev. 1998;62:725–774. [PMC free article] [PubMed]
30. Miller J H. A short course in bacterial genetics: a laboratory manual and handbook for Escherichia coli and related bacteria. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1972.
31. Mollet B, Iida S, Arber W. An active variant of the prokaryotic transposable element IS903 carries anamber stop codon in the middle of an open reading frame. Mol Gen Genet. 1985;199:534–536. [PubMed]
32. Pearson W, Lipman D. Improved tools for biological sequence comparison. Proc Natl Acad Sci USA. 1988;85:2444–2448. [PMC free article] [PubMed]
33. Pepe C M, Maslesa-Galic S, Simons R W. Decay of the IS10 antisense RNA by 3′ exoribonucleases: evidence that RNase II stabilizes RNA-OUT against PNPase attack. Mol Microbiol. 1994;13:1133–1142. [PubMed]
34. Prere M F, Chandler M, Fayet O. Transposition in Shigella dysenteriae: isolation and analysis of IS911, a new member of the IS3 group of insertion sequences. J Bacteriol. 1990;172:4090–4099. [PMC free article] [PubMed]
35. Rak B, von Reutern M. Insertion element IS5 contains a third gene. EMBO J. 1984;3:807–811. [PMC free article] [PubMed]
36. Rawlings D E, editor. Biomining: theory, microbes, and industrial processes. Berlin, Germany: Springer-Verlag; 1997.
37. Salyers A A, Shoemaker N B, Stevens A M, Li L-Y. Conjugative transposons an unusual and diverse set of integrated gene transfer elements. Microbiol Rev. 1995;59:579–590. [PMC free article] [PubMed]
38. Sambrook J, Fritsch E F, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 1989.
39. Schrader J, Holmes D. Phenotypic switching of Thiobacillus ferrooxidans. J Bacteriol. 1988;170:3915–3923. [PMC free article] [PubMed]
40. Selkov E, Overbeek R, Kogan Y, Chu L, Vonstein V, Holmes D, Silver S, Fonstein M. Functional analysis of gapped microbial genomes: principles with examples from Thiobacillus ferrooxidans. Proc Natl Acad Sci USA. 2000;97:3509–3514. [PMC free article] [PubMed]
41. Sharp P M, Li W-H. The codon adaptation index—a measure of directional synonymous codon usage bias, and its potential applications. Nucleic Acids Res. 1987;15:1281–1295. [PMC free article] [PubMed]
42. Sherratt D. Tn3 and related transposable elements: site-specific recombination and transposition. In: Berg D E, Howe M M, editors. Mobile DNA. Washington, D.C.: American Society for Microbiology; 1989. pp. 163–184.
43. Stibitz S. IS481 and IS1002 of Bordetella pertussis. J Bacteriol. 1998;180:4963–4966. [PMC free article] [PubMed]
44. Thompson J, Higgins D, Gibson T. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994;22:4673–4680. [PMC free article] [PubMed]
45. Thony-Meyer L. Biogenesis of respiratory cytochromes in bacteria. Microbiol Mol Biol Rev. 1997;61:337–376. [PMC free article] [PubMed]
46. Timmerman K P, Tu C P. Complete sequence of IS3. Nucleic Acids Res. 1985;13:2127–2139. [PMC free article] [PubMed]
47. Vertes A A, Asai Y, Inui M, Kobayashi M, Kurusu Y, Yukawa H. Transposon mutagenesis of coryneform bacteria. Mol Gen Genet. 1994;245:397–405. [PubMed]
48. Way J C, Davis M A, Morisato D, Roberts D, Kleckner N. New Tn10 derivatives for transposon mutagenesis and for construction of lacZ operon fusions by transposition. Gene. 1984;32:369–379. [PubMed]
49. Yates J R, Holmes D S. The use of genetic probes to detect microorganisms in biomining operations. J Ind Microbiol. 1986;1:129–135.
50. Yates J R, Holmes D S. Two families of repeated DNA in Thiobacillus ferrooxidans. J Bacteriol. 1987;169:1861–1870. [PMC free article] [PubMed]
51. Zhang J, Madden T L. PowerBLAST: a new network application for interactive or automated sequence analysis and annotation. Genome Res. 1997;7:649–656. [PMC free article] [PubMed]

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