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Previous Article | Next Article 
Applied and Environmental Microbiology, November 2005, p. 7515-7522, Vol. 71, No. 11
0099-2240/05/$08.00+0 doi:10.1128/AEM.71.11.7515-7522.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Isolation, Sequence Analysis, and Comparison of Two Plasmids (28 and 29 Kilobases) from the Biomining Bacterium Leptospirillum ferrooxidans ATCC 49879
Nicolette J. Coram,
Leonardo J. van Zyl, and
Douglas E. Rawlings*
Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa
Received 9 March 2005/
Accepted 28 June 2005
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ABSTRACT
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Two plasmids, of 28,878 bp and 28,012 bp, were isolated from Leptospirillum ferrooxidans ATCC 49879. Altogether, a total of 67 open reading frames (ORFs) were identified on both plasmids, of which 32 had predicted products with high homology to proteins of known function, while 11 ORFs had predicted products with homology to previously identified proteins of unknown function. Twenty-four ORFs had products with no homologues in the GenBank/NCBI database. An analysis of the ORFs and other features of the two plasmids, the first to be isolated from a bacterium of the genus Leptospirillum, is presented.
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INTRODUCTION
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Leptospirillum ferrooxidans is an iron-oxidizing bacterium that is one of the most metabolically restricted organisms known. It is obligately autotrophic, highly acidophilic (optimum pH, 1.5 to 2.0), and mesophilic (optimum temperature, 35 to 40°C), and as far as is known, uses only one type of electron donor, ferrous iron. L. ferrooxidans and related species of leptospirilli, such as Leptospirillum ferriphilum (2), are frequently found in acid mine drainage waters (23) and other iron-rich acid environments such as the Tinto river (8). Leptospirilli are also able to grow by oxidation of the mineral pyrite and are important players in certain industrial processes used for the recovery of metals from ores (14, 19, 22). Based on 16S rRNA gene sequence information, these bacteria cluster within the little-studied group of bacteria known as Nitrospira (10).
Plasmids are extrachromosomal pieces of self-replicating DNA that constitute a particularly fluid part of the bacterial genome. They gain DNA efficiently by transposition or recombination, and many plasmids are capable of moving themselves and the DNA that they carry horizontally between bacteria by means of conjugation. Plasmids therefore comprise an important part of the horizontal gene pool (27). Given their severe metabolic constraints, the leptospirilli are difficult to work with, and no plasmids from these bacteria have been identified, cloned, or sequenced. Since these bacteria occupy a unique ecological niche, we isolated and sequenced plasmids from leptospirilli with the objective of determining whether they possessed plasmid-associated functions such as a replicon, conjugation or mobilization, and stability systems that were unique or similar to those that had been reported before. We also wished to identify what types of accessory genes were carried by these plasmids. Following a survey of plasmids in 10 strains of L. ferrooxidans and 6 strains of L. ferriphilum, only two plasmids were identified, both of them in Leptospirillum ferrooxidans ATCC 49879 (all strains listed in Table 1 of reference 2 were tested for plasmids [not shown]).
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Two plasmids are present in L. ferrooxidans ATCC 49879.
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L. ferrooxidans ATCC 49879 (previously referred to as R3) was isolated from the Ilba mine in Romania (9). Plasmids were isolated by growing L. ferrooxidans cells in 10 liters of 9K medium (25) at 30°C, harvested at 9,000 rpm for 35 min, washed in 10 ml 9K medium lacking iron, and resuspended in SET buffer (50 mM Tris, 2 mM EDTA, 25% sucrose, pH 8.0). After incubation for 1 h at –20°C, 200 µl of proteinase K (20 mg/ml) was added, and cells were incubated at 4°C for 15 min and lysed with 10% sodium dodecyl sulfate. Plasmids were prepared by the CsCl gradient method (21) and digested with restriction endonucleases that have recognition sites that occur rarely, and the fragments were separated in an agarose gel (Fig. 1). Digestion with XbaI or PacI gave bands of approximately 29 kb, while digestion with PmeI gave a band of approximately 28 kb, and in each case undigested plasmid DNA remained in the well (Fig. 1). XbaI-PmeI or PacI-PmeI double digestion gave 28- to 29-kb double bands, with no DNA remaining in the well. Based on these and other digests, the results were interpreted as indicating that two plasmids were present in the preparation. The 29-kb plasmid had single XbaI, PacI, and PmeI sites, while the 28-kb plasmid had a single PmeI site but no XbaI or PacI sites (also see Fig. 2).
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FIG. 1. Restriction endonuclease analysis of plasmids from L. ferrooxidans ATCC 49879. Lanes 1 and 7, molecular markers of phage lambda DNA cut with SmaI and KpnI, respectively; lane 2, digestion with XbaI; lane 3, digestion with PmeI; lane 4, digestion with PacI; lane 5, digestion with XbaI-PmeI; lane 6, digestion with PacI-PmeI.
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FIG. 2. Maps of p49879.1 (AY941098) and p49879.2 (AY941099) showing ORFs, their potential direction of translation, and a brief designation where available. ORFs with matches shown as hypothetical or uncharacterized proteins are designated as such in the GenBank database. The circular plasmids are shown as linear maps beginning at the unique PacI site of p49879.1 and a nonunique EcoRI site of p49879.2 to avoid splitting any of the open reading frames. Colored arrows indicate ORFs with amino acid sequences that are related and are present on both plasmids (see text for details). Large open triangles indicate where the EZ::TN <R6K ori/KAN-2> transposon was inserted. a, region with 20-bp iterons; b, region with direct and indirect repeats (see Fig. 4A); c, region with direct and indirect repeats downstream of par-like genes (see Fig. 4B).
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Plasmid cloning and DNA sequencing.
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To separate, clone, and propagate the plasmids in Escherichia coli, the EZ::TN <R6Kori/KAN-2> transposon (Epicenter, Madison, Wisconsin) was inserted in vitro into each plasmid. This resulted in kanamycin-resistant plasmids that were capable of replication upon transformation into E. coli EC100D pir+ cells. A single insertion into each plasmid was confirmed by sequencing from the transposon using primers KAN-2 FP-1 (forward primer; 5'-ACCTACAACAAAGCTCTCATCAACC-3') and R6KAN-2 RP1 (reverse primer; 5'-CTACCCTGTGGAACACCTACATCT-3'). Plasmids were mapped for restriction endonuclease digestion sites. Both plasmids were sequenced on both strands by a process of subcloning and primer walking. Sequencing reactions were carried out on five of the cloned plasmids, using EZ::TN <R6Kori/KAN-2>-specific, outward-reading primers. One plasmid with a single insertion into one type of plasmid (named p49879.1) was identified, while four plasmids with single insertions into different sites were identified for the second type of plasmid (p49879.2). One of each type of plasmid was sequenced using an ABI 3100 genetic analyzer.
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Sequence analysis of plasmids p49879.1 and p49879.2.
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Plasmids p49879.1 and p49879.2 were found to be of similar sizes, i.e., 28,878 and 28,012 bp, and their overall G+C mol% ratios were 57.8 and 55.2%, respectively. Using a combination of the Glimmer 2 (4; www.tigr.org/softlab), DNAMAN (Lynnon BioSoft), and BLAST (1; http://www.ncbi.nlm.nih.gov) software programs, altogether a total of 67 potential open reading frames (ORFs) were identified in the two plasmids. The locations and orientations of these ORFs are shown in Fig. 2. Of the products of the 33 ORFs in p49879.1, 11 had high homology to proteins of known function, while 7 had homology to previously identified proteins of unknown function and 15 had no homologues in the NCBI database. Of the products of the 34 ORFs identified in plasmid p49879.2, 21 had significant homology to proteins of known function, 4 had homology to previously identified proteins of unknown function, and 9 had no homology to any proteins in the NCBI database. Therefore, approximately half of the ORFs present on the two plasmids had products related to proteins whose functions had been identified previously. The ORFs present in the plasmids have been arbitrarily numbered in the order in which they occur in the plasmids (Fig. 2), followed by a decimal point and either a 1 or 2, indicating that they originated from plasmid p49879.1 or p49879.2, respectively.
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Putative replicons.
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The ability to replicate is an essential requirement of all plasmids, and most plasmids encode at least one protein that is associated with plasmid replication. In the case of both p49879.1 and p49879.2, none of the products of the predicted ORFs were related to proteins previously associated with plasmid replication. Furthermore, there were no ORFs present on the two plasmids that had products that were suggestive of replication proteins. Many plasmid replicons contain iterons that serve as replication protein binding sites and affect the plasmid copy number and incompatibility. Both plasmids contain four 20-bp iterons with 70% nucleotide sequence identity (14 of 20 bp) and with similar spacing between the iterons (Fig. 3A). Based on our experience with other plasmids, the differences in nucleotide sequence of the iterons of the two plasmids would be sufficient for them to be compatible should these iterons be involved with plasmid replication (13). This prediction is consistent with the expectation that the origins of replication of the two plasmids should be compatible since they were isolated from the same host strain.
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FIG. 3. (A) Comparison of nucleotide sequences of the 20-bp iterons present on p49879.1 and p49879.2 and their comparative spatial arrangement. (B) DNA sequences in the oriT-like regions of p49879.1, p49879.2, pTF1, and the IncQ plasmid RSF1010. (C) Alignment of sequences of the long direct repeats present on p49879.1. A consensus sequence present in all of the repeats is shown below the alignment.
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No genetic system for any of the leptospirilli exists, and thus we were unable to test biologically for replicon function in the natural host. Since the R6K origin of replication inserted by in vitro transposition into the plasmids functions only in E. coli pir+ cells, an attempt was made to transform E. coli DH5
(pir) as well as Pseudomonas putida cells (previously shown to not support R6K ori replication [11]) with the p49879.1 and p49879.2 plasmids containing the R6K plasmid oriV and a kanamycin resistance gene. No transformants were obtained, suggesting that neither plasmid was capable of replication in either E. coli or P. putida or that the replicons were disrupted during insertion of the EZ::TN <R6Kori/KAN-2> transposon. Disruption of the replicon was unlikely for p49879.2, as the insertion was into the transposase gene of another transposon, whereas for p49879.1 the insertion was into ORF 6.1, for which no close relatives have been identified and which may be associated with replication.
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Mobilization genes.
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Two ORFs with products that have high identity to proteins associated with plasmid mobilization (Fig. 2) as well as an oriT-like region (Fig. 3B) were identified on each plasmid. The products of ORFs 19.1 (94 amino acids [aa]) and 27.2 (110 aa) had related sequences (72% aa identity) and had the highest amino acid sequence identity to the MobC (90 aa) (CAB75593.1) protein encoded by the broad-host-range IncQ-family plasmid pIE1130 (42% aa identity) and to MobS (98 aa) (CAA36926.1), encoded by Acidithiobacillus ferrooxidans plasmid pTF1 (49% aa identity), respectively. The products of the adjacent ORFs 18.1 (237 aa) and 26.2 (504 aa) were also related to each other (76% aa identity), and both had the highest aa sequence identity (40 to 41%) to the N-terminal portion of the TraA protein (1,277 aa) encoded by a conjugative plasmid from Agrobacterium tumefaciens C58 (accession no. NP_355808.1 and NP_535333.1). They were slightly less related (39 and 32% identity, respectively) to the N terminus of the MobA/RepB protein (714 aa) encoded by a cryptic plasmid from Xylella fastidiosa (AF3229081.1) and the N terminus of the MobL protein (378 aa) encoded by plasmid pTF1 from A. ferrooxidans (CAA36927.1). The TraA, MobA/RepB, and MobL proteins are considerably larger than the products of ORFs 18.1 and 26.2, with the closest in size being the MobL protein encoded by plasmid pTF1 (5). It has been shown that the N-terminal portions of this family of proteins have a relaxase activity involved in the specific nicking of plasmid DNA at the origin of transfer (oriT) prior to plasmid mobilization (16, 20). Between these pairs of divergently transcribed ORFs (19.1/18.1 and 27.2/26.2), a region of DNA with features typical of the oriT regions of pTF1 and the IncQ plasmids (Fig. 3B) was present. The four oriT regions have clear structural (bilateral symmetry) and sequence similarity. Plasmid pTF1 and the IncQ family of plasmids, such as RSF1010 and pIE1130, are efficiently mobilized by IncP plasmids such as RP4 and R751 (18). The region containing the cloned mobilization genes and oriT of p49789.1 and p49879.2 was tested for mobilization by using E. coli S17.1 (26), which has a derivative of the conjugative plasmid RP4 integrated on its chromosome, as a donor, while E. coli strain CSH56 served as the recipient. The method used was as previously described (28). No plasmid mobilization was detected with either test plasmid, although mobilization occurred at a high frequency with a control plasmid. The reason for this lack of mobilization is unknown. Possibly, the ORFs may not be expressed in E. coli or the IncP plasmids may be unable to mobilize p49879.1 and p49879.2.
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Plasmid stability proteins.
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Both plasmids p49879.1 and p49879.2 contain ORFs (11.1 and 21.2, respectively) that encode proteins with high amino acid sequence identity to ParA-like ATPase proteins. These are proteins that are responsible for providing the energy required for the active partitioning of plasmids into progeny cells upon cell division (7). The products of these ORFs have very high aa sequence identity (in excess of 80%) to different ParA-like proteins, but when aligned with each other, the aa sequences of the products of ORFs 11.1 and 21.2 are only 29% identical. The products of the ParA-like ORFs on plasmids p49879.1 (AAD19678.1) and p49879.2 (AE008924.1) therefore belong to different families of ParA-like proteins. Although they are different, both are Walker box (ATP-binding site)-containing proteins (7). The lack of sequence homology between the two ParA-like proteins is not unexpected, as the possession of similar partition systems is usually associated with plasmid incompatibility, and one would expect the two partitioning systems to function independently for both plasmids to be stable in the same host cell (15). ORF 21.2 appears to be transcriptionally linked to ORF 20.2, as the stop codon of one ORF overlaps with the start codon of the next. Furthermore, the product of ORF 20.2 was closely related to the products of ORFs downstream of those for the ParA-like proteins of Xanthomonas axonopodis and the ParA-like protein encoded by the 48-kb plasmid pRSB101-37 (accession no. NC_006385). It is therefore likely that ORF 20.2 is also involved in plasmid partitioning. Plasmid p49879.1 does not have a second ORF of this type downstream of ORF 11.1. Plasmid stability experiments were carried out by cloning the par-containing regions of p49879.1 and p49879.2 into the unstable mini-R1 test plasmid pOU82, which segregates from cells at a frequency of 1% per cell per generation (6). A 1,471-bp EcoRV-EcoRI region containing the putative par gene (ORF 11.1) from p49879.1 was cloned into the pUCBM21 vector, and from there into pOU82, using BamHI and EcoRI sites, to give pOU82.1. A 2,660-bp BamHI-BamHI fragment from p49879.2 containing the putative par gene (ORF 21.2) was cloned directly into pOU82, giving pOU82.2. E. coli(pOU82.1) and E. coli(pOU82.2) were grown in Luria broth at 37°C without selection. Samples taken at 20-generation intervals were diluted in saline and plated onto Luria agar plates containing 40 µg of 5-bromo-4-chloro-3-indolyl-D-galactopyranoside (X-Gal)/ml (3). Blue and white colonies were scored as plasmid-containing and non-plasmid-containing colonies, respectively. After 100 generations in the absence of selection, plasmid pOU82.1 (par of p49879.1) was present in approximately 18% of cells, the same proportion as the pOU82 control. In contrast, after 80 generations, pOU82.2 (par of p49879.2) was present in over 54% of cells. Stability experiments were repeated three times with similar results, and therefore the par system of p49879.2, but not that of p49879.1, appeared to be functional in E. coli.
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Tn3 family transposons.
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A feature of both plasmids is the presence of a Tn3/Tn21-related transposon. Plasmid p49879.1 has ORF 12.1, with a product that is 75% identical to the resolvase of Tn2501 from E. coli (AAA27426.1), while plasmid p49879.2 has ORF12.2, whose product is 73% identical to the resolvase of a transposon from Erwinia amylovora (NP_981999.1). Similarly, the product of ORF 13.1 of plasmid p49879.1 is 72% identical to the transposase of Tn3926 of E. coli (CAA32453.1), while that of ORF 11.2 is 73% identical to the same transposase. When aligned with each other, the resolvases and transposases encoded by p49879.1 and p49879.2 are 99% and 97% identical, respectively (Table 1). Although they are not identical, both transposons have tnpA (transposase)- and tnpR (resolvase)-like genes that are divergently transcribed in the same manner as those of Tn3, but different from those of Tn21, where the genes are transcribed in the same direction (24). On either side of the tnpA and tnpR genes are two long inverted repeat sequences (IR), of 65 bp in the case of p49879.1 and 67 bp in the case of p49879.2. The sequences of the 65-bp inverted repeats are identical between both ends of each individual transposon and also between the ends of the two different transposons. The outermost 38 bp of the IR sequence are closely related to the 38-bp inverted repeats of Tn21 (24). Outside the IR sequences, plasmid p49879.1 has two identical 5-bp target sequence duplications (AATCA), while p49879.2 has 7-bp target sequence duplications (CTTGCTC). Unlike many transposons of the Tn3-like family that carry either antibiotic or metal resistance genes (12, 24), the two Tn3 family transposons on plasmids p49879.1 and p49879.2 carry no markers. The presence of such highly related sequences on two plasmids suggests that the plasmids could undergo homologous recombination and form a plasmid cointegrate. Furthermore, should the transposon also be present on the chromosome, this would allow for the plasmids to insert into the chromosome. Whether the transposons are also present in the chromosomes of any of the L. ferrooxidans strains used in this study has not been tested.
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Additional sequence-related ORFs of plasmids p49879.1 and 49879.2.
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Besides the mobilization-like ORFs and Tn3/Tn21-related transposase- and resolvase-like ORFs, plasmids p49879.1 and p49879.2 had four other ORFs whose products shared >45% aa sequence identity (Table 1). These included ORFs 7.1 and 19.2, which were 78% related to each other and had 70% aa identity to protein Lfe211p1 expressed by L. ferrooxidans strain L3.2 (AY204453.1). This partially sequenced protein was upregulated 1.9-fold upon ammonia starvation (17; V. Parro, personal communication), but its function is unknown. The products of ORFs 6.1 (YP_065905.1) and 18.2 (ZP_00329799.1) are of a similar size (98 aa), are closely related to each other (93% aa sequence identity), and are related to bacterial nucleoid HU DNA-binding proteins. The products of ORFs 4.1 (87 aa) (NP_779305.1) and 23.2 (86 aa) (AAF05107.1) are 48% identical to each other, and both are highly related to DinJ-like DNA damage-inducible proteins of similar size. Both ORFs 4.1 and 23.2 are situated adjacent to ORFs 3.1 (YP_025380.1) and 22.2 (AAF05106.1), and their products have high sequence similarity to DinJ2-like proteins, although the products of ORFs 4.1 and 23.2 have only 28% aa sequence identity. ORF 3.1 has a frame shift and is unlikely to be functional. The products of two other ORFs of p49879.1 and p49879.2 (ORF 2.1 and 13.2) are of the same length (187 aa) and have aa sequences that are 89% identical to each other. These are closely related to uncharacterized proteins of unknown function that are conserved among the cyanobacteria (ZP_00329379.1).
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Other ORFs present on plasmids p49879.1 and p49879.2.
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Other ORFs identified on p49879.1 that had sequence-related proteins present in the GenBank/NCBI protein database were ORF 30.1 (related to a guanylate cyclase-like protein; NP_840191.1), ORF 5.1 (related to a site-specific recombinase-like protein; NP_840910.1), and ORFs 1.1, 6.1, and 29.1 (related to different types of DNA-binding proteins; NP_518246.1, YP_065905.1, NP_926370.1). ORFs 21.1 (ZP_00086243.1) and 20.1 (ZP_00086242.1) are interesting in that they are related to two adjacent ORFs encoding hypothetical proteins of similar size from Pseudomonas fluorescens. ORF 10.1's product is related to a hypothetical protein of similar size from Caulobacter crescentus (AE005760.1). There were 15 ORFs encoding potential proteins that had no clear sequence relatedness to any proteins in the GenBank/NCBI database.
Plasmid p49879.2 contained several other ORFs with products that were related to previously identified proteins. These were ORF 1.2 (related to a possible phosphate-binding protein; NP_519650.1), ORF 3.2 (related to a redox protein regulator-like protein; NP_953832.1), ORF 4.2 (related to a cytochrome c biogenesis-like protein; ZP_00208849.1), ORF 6.2 (related to a cysteine desulfurase-like protein; YP_074470.1), ORF 7.2 (related to an S-adenosyl-methionine-dependent methyl transferase-like protein; ZP_00329389.1), ORF 8.2 (related to a TniR resolvase-like protein; NP_361078.1), ORF 9.2 (related to permeases of the drug/metabolite transporter superfamily; ZP_00355899.1), ORF 10.2 (related to a 77-aa fragment of a truncated transposase; ZP_00148816.1), ORF 17.2 (related to a resolvase-like protein; NP_569373.1), ORF 28.2 (related to a transcriptional regulator of the Cro/Ci family, but with a frame shift; NP_778602.1), ORF 30.2 (related to a hypothetical protein of similar size from Geobacter metallireducens; ZP_00298389.1), ORF 32.2 (related to a major facilitator superfamily-like permease; ZP_00334637.1), ORF 33.2 (related to a transcriptional regulator-like protein; NP_487783.1), and ORF 34.2 (related to a Zn-dependent hydrolase-like protein; ZP_00334638.1). In addition, nine ORFs (2.2, 5.2, 14.2, 15.2, 16.2, 24.2, 25.2, 29.2, and 31.2) were identified that had products with no clear sequence relationship to proteins in the NCBI database.
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Nucleotide sequence repeats.
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Besides the 20-bp iterons referred to earlier, both plasmids had some other unusual sequence features. One of the most notable of these was a series of five and one-half direct repeats of 78 to 87 bp that was present on plasmid p49879.1. Although the nucleotide sequence of each repeat was not perfectly conserved, there was a striking degree of conservation between them (Fig. 3C). These long direct repeats were located within ORF 25.1, a region that encodes a putative protein with no sequence relationship to any protein in the NCBI database. Plasmid p49879.1 has another region containing two identical 18-bp tandem repeats followed by two 23-bp tandem repeats containing 17 bp of the 18-bp nucleotide sequence (Fig. 4A). There is no nucleotide spacing between the 18-bp repeats, but there is a 6-bp space to the first 23-bp repeat and a 73-bp space to the second 23-bp repeat. Also in this region is another pair of 23-bp direct repeat sequences, 8 bp of which are identical to the 18-bp and other 23-bp repeat sequences. These repeats are situated in the region between ORFs 33.1 and 32.1, extending into the C-terminal region of ORF 33.1. The function of these repeat DNA sequences is unclear.
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FIG. 4. (A) Region of p49879.1 showing nucleotide sequence and spatial arrangement of the direct repeats present in the terminal region of ORF 1.1. (B) Region of p49879.2 showing nucleotide sequence and spatial arrangement of the direct and inverted repeats present in the terminal region of ORF 20.2. Arrows with solid heads indicate direct repeats, while arrows with open heads indicate inverted repeats. The 15-bp sequence shown is present as both direct and inverted repeats.
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Plasmid p49879.2 also had a region with a complex set of direct and indirect repeats (Fig. 4B). This region was situated immediately downstream of ORF 21.2, the ParA-like protein-encoding ORF, and the apparently translationally linked ORF 20.2, whose product is possibly involved in plasmid partitioning. Proteins involved in partitioning systems frequently act at sites of repeated sequences and secondary structure, but whether these sequences function in that role has still to be determined.
Plasmids p49879.1 and p49879.2 are the first two plasmids to have been isolated and sequenced from L. ferrooxidans. No clear candidates for replication proteins were identified. Both plasmids encode DNA-binding proteins and nitrogen-regulated Lfe211p1-like proteins (Table 1) previously identified in another strain of L. ferrooxidans (17) on either side of 20-bp iterons (Fig. 3A). The location of these ORFs suggests that they could be candidates for putative replication proteins. There is no obvious reason why a potential replication protein like Lfe211p1 should be nitrogen regulated, but frequently small shifts in gene expression data obtained by microarray hybridization assays are spurious and require confirmation using other techniques. An origin of replication must lie somewhere on both plasmids, but this speculation on the location of the potential replicon requires confirmation. Since there is no genetic system available for this iron-oxidizing, obligately autotrophic acidophile, it is difficult to study the expression and function of the plasmids in their host organism. Since the two plasmids occur in the same host strain, it is of interest to determine whether the plasmids are likely to have exchanged DNA fragments or are likely to recombine and form cointegrates. As described earlier, both plasmids contain several ORFs whose products contain aa sequences that are related. The DNA sequences of many of these ORFs are also highly related (Table 1). The region of the two plasmids that had very high DNA identity (99.92%) was the 3,761-bp region containing the transposon of the Tn3 family. The DNA sequence over most of this region was identical, except that p49879.2 had an 84-bp deletion within ORF 11.2 (transposase) that maintained the reading frame. There were four other regions with DNA sequence identities of 84 to 96% (Table 1). This suggests that with the exception of the Tn3-type transposon, the plasmids probably have not recently exchanged DNA. The high identity with the transposon could provide a site of plasmid cointegration, although how frequently this occurs is unknown.
Now that the first two medium-sized plasmids isolated from L. ferrooxidans have been analyzed, an obvious question to be asked is as follows: of what value are the plasmids and the potential genes that they carry to their host? It is not yet possible to answer this question. Taking into account all 67 identified ORFs on both plasmids, about half had products that did not have aa sequences that were related to proteins in the databases. Many of the other ORFs appear to encode an eclectic collection of proteins whose functions seem to be unrelated. The reason for their presence on a plasmid is therefore not obvious. Two interesting exceptions to this are found on plasmid 49879.2. ORFs 3.2 (encodes an unidentified possible redox protein regulator), 4.2 (encodes a cytochrome c biogenesis-like protein), 5.2 (encodes an unknown protein), 6.2 (encodes a cysteine desulfurase-like protein), and 7.2 (encodes a methyl transferase-like protein) have a spatial orientation and intergenic distances that suggest that they may be expressed as an operon that might have a role in the synthesis of an electron carrier. The second exception is ORFs 31.2 (no match), 32.2 (encodes a permease-like protein), 33.2 (encodes a regulator-like protien), and 34.2 (encodes a Zn-dependent hydrolase-like protein), which may fulfill an as yet unidentified export function. Whether these potential genes are coordinately expressed and what their function is remain to be tested.
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Nucleotide sequence accession numbers.
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The annotated sequences of p49879.1 and p49879.2 are available under GenBank/EMBL accession numbers AY941098 and AY941099, respectively.
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ACKNOWLEDGMENTS
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We thank Wolfgang Sand for the gift of L. ferrooxidans ATCC 49879.
We acknowledge the financial support of BHP-Billiton, the EU framework 6 BioMinE project, the University of Stellenbosch, and the National Research Foundation (Pretoria).
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FOOTNOTES
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* Corresponding author. Mailing address: Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa. Phone: 27-21-808 5848. Fax: 27-21-808 5846. E-mail: der{at}sun.ac.za. 
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Abstract
Introduction
