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Appl Environ Microbiol. Feb 2002; 68(2): 838–845.
PMCID: PMC126727

Molecular Relationship between Two Groups of the Genus Leptospirillum and the Finding that Leptospirillum ferriphilum sp. nov. Dominates South African Commercial Biooxidation Tanks That Operate at 40°C

Abstract

Iron-oxidizing bacteria belonging to the genus Leptospirillum are of great importance in continuous-flow commercial biooxidation reactors, used for extracting metals from minerals, that operate at 40°C or less. They also form part of the microbial community responsible for the generation of acid mine drainage. More than 16 isolates of leptospirilla were included in this study, and they were clearly divisible into two major groups. Group I leptospirilla had G+C moles percent ratios within the range 49 to 52% and had three copies of rrn genes, and based on 16S rRNA sequence data, these isolates clustered together with the Leptospirillum ferrooxidans type strain (DSM2705 or L15). Group II leptospirilla had G+C moles percent ratios of 55 to 58% and had two copies of rrn genes, and based on 16S rRNA sequence data, they form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present among the leptospirilla tested, with two DNA-DNA hybridization similarity subgroups found within group I. The two groups could also be distinguished based on the sizes of their 16S-23S rRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a separate species with the name Leptospirillum ferriphilum sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and by carrying out restriction enzyme digests of the products. Several, but not all, isolates of the group II leptospirilla, but none from group I (L. ferrooxidans), were capable of growth at 45°C. All the leptospirilla isolated from commercial biooxidation tanks in South Africa were from group II.

Bioleaching and biooxidation of minerals are industrial processes which involve a consortium of acidophilic iron- and/or sulfur-oxidizing bacteria (21). Acidithiobacillus ferrooxidans (previously Thiobacillus ferrooxidans) was the first microorganism isolated from an acidic leaching environment, and subsequently, microbial research in this field has centered around the elucidation of the properties of this chemolithoautotrophic bacterium. Although A. ferrooxidans was considered to be the primary biological catalyst in biomining processes, leptospirilla have been found to be the dominant iron-oxidizing bacteria in industrial continuous-flow biooxidation tanks, such as those used for the treatment of gold-bearing arsenopyrite concentrates (19, 20). There are several possible reasons for this, probably the most important being that the high ferric-ferrous iron ratio present in biooxidation tanks is less inhibitory to leptospirilla than it is to A. ferrooxidans (22). In many environmental samples, Leptospirillum has also been shown to outnumber Acidithiobacillus at a ratio of 2:1 under appropriate conditions (25). Temperatures above 40°C and pH values below 1.0 are two other conditions more suitable to the growth of leptospirilla than acidithiobacilli. Under these conditions, leptospirilla have been reported to be important contributors to the generation of acid mine drainage and its associated environmental problems (26). Together, these findings have suggested that leptospirilla are more important to both uncontrolled (natural) and deliberate mineral bioleaching and biooxidation processes than has been generally recognized.

Bacteria belonging to the genus Leptospirillum are small, gram-negative, vibrio- or spiral-shaped cells (14, 16). They are obligately chemolithotrophic organisms, fixing carbon by the Benson-Calvin cycle, using ferrous iron as their sole electron donor and oxygen as their electron acceptor (11, 14). These obligately acidophilic bacteria grow optimally in inorganic media within the pH range 1.3 to 2.0. Since they use only ferrous iron as an electron donor, they are among the most metabolically restricted organisms known. Possibly as a result of this substrate specificity, they have a high affinity for ferrous iron (Km = 0.25 mM) relative to A. ferrooxidans (Km = 1.34 mM) (17). Optimum leaching efficiency is obtained at lower substrate concentrations than have been reported for A. ferrooxidans (25).

Limited phylogenetic studies of a relatively small number of members of the genus Leptospirillum have been reported. Harrison and Norris (10) obtained evidence to suggest that there was considerable variation among isolates belonging to the genus Leptospirillum. One group of isolates had a moles percent G+C content of ca. 51%, and another group had a G+C content of 55 to 56%. This result was further supported by DNA-DNA hybridization studies, in which two isolates had DNA sequence similarity of 71 to 73% while all other isolates had >6 to 31% DNA-DNA similarity. Unfortunately, all but the L. ferrooxidans type strain (DSM2705) from this early study were lost (P. R. Norris, personal communication). Hallmann et al. (8) carried out DNA-DNA hybridization studies with six isolates of leptospirilla. Two pairs of strains were 100% related to each other, and there was 38 to 50% relatedness between these pairs and 31 to 50% relatedness among all other isolates. A moderately thermophilic Leptospirillum isolate with an optimum temperature of 45 to 50°C (maximum, 55 to 60°C), a moles percent G+C of 56%, and a DNA similarity of 27% with a mesophilic strain was reported (5). This strain was named Leptospirillum thermoferrooxidans, but it has also been lost and so is unavailable for comparative studies (14). The genus name Leptospirillum and the species names ferrooxidans and thermoferrooxidans have recently been validated (11). Also recently, 16S ribosomal DNA (rDNA) belonging to a third group of leptospirilla was amplified from DNA isolated directly from slime streamers of an acid mine drainage site; however, bacteria belonging to the third group have not been isolated (2).

Members of the genus Leptospirillum have a limited range of physiological characteristics that can be used in their identification (14). One objective of the present study, therefore, was to determine the diversity of Leptospirillum isolates from different geographical locations using a variety of molecular techniques to establish whether there were sufficient differences to warrant subdivision at a species level. These studies provide an extended description of a number of characteristics that can be used in the identification of the more commonly encountered leptospirilla. A second aim was to determine which Leptospirillum type dominated industrial biooxidation tanks. This would help identify which species should be the focus of long-term molecular biology research. From these findings, we propose that two distinct Leptospirillum species are represented among these isolates.

MATERIALS AND METHODS

Bacterial strains, media, and growth.

The strains used in this study are listed in Table Table1.1. Strains were routinely grown at 30°C in 800 ml of basal medium [(NH4)2SO4, 0.2% (wt/vol); K2HPO4, 0.05% (wt/vol); MgSO4 · 7H2O, 0.05% (wt/vol); KCl, 0.01% (wt/vol); and Ca(NO3)2, 0.001% (wt/vol)] supplemented with FeSO4 · 7H2O (500 mM) and adjusted to pH 1.6 with concentrated H2SO4. Strain purity was checked using the overlay technique of Johnson (13). Experiments at 45°C were carried out using the same medium. The ferrous iron concentration was determined by volumetric titration with potassium dichromate using diphenylamine 4-sulfonic acid indicator (28).

TABLE 1.
Strains of Leptospirillum

DNA preparation.

Bacterial cells were harvested by centrifugation at 15,000 × g for 35 min and washed with acid water (pH 1.2) to remove ferric iron precipitate. The cells were either used immediately or stored frozen at −20°C in SET buffer (25% sucrose, 2 mM EDTA, 50 mM Tris; pH 8.0). Prior to lysis, the cells were treated with proteinase K (20 ng/μl) at 37°C for 30 min. Cell lysis was achieved by the addition of 10% sodium dodecyl sulfate. DNA was extracted via spooling and resuspended in Tris-EDTA buffer by overnight shaking at 30°C.

DNA techniques and Southern hybridization.

Standard methods as described by Sambrook et al. (24) were used for restriction enzyme digestions and gel electrophoresis. Restriction enzymes and buffers were obtained from Roche Biochemicals and used in accordance with the manufacturer's specifications. For Southern hybridization used in ribotyping, 5 μg of chromosomal DNA was digested with BamHI, and the restriction nuclease fragments were separated by agarose gel electrophoresis. The DNA was denatured in 0.25 M HCl, neutralized in 0.4 M NaOH, and transferred to a nylon Hybond N+ membrane (Amersham) by capillary blotting overnight. The 1.5-kb 16S rDNA PCR product of isolate P3a (chosen randomly from the 15 isolates) was labeled with digoxigenin using the DIG oligonucleotide 3′-end labeling and detection kit (Roche Biochemicals) and used as the hybridization probe. The hybridization temperature was 40°C. Washing was done for 20 min at room temperature, followed by 20 min at 65°C. Membrane detection was performed in accordance with the manufacturer's instructions (Roche Biochemicals).

PCR amplification for restriction enzyme mapping.

PCR amplifications of the 16S rRNA gene were routinely carried out to generate a 1.5-kb band on electrophoresis using the primers pfDD2 (5"-CCGGATCCGTCGACAGAGTTTGATCITGGCTCAG-3"), which contains BamHI and SalI cloning sites towards the 5" end, and primer prDD2 (5"-CCAAGCTTCTAGACGGITACCTTGTTACGACTT-3"), which has HindIII and XbaI cloning sites. Approximately 100 ng of chromosomal DNA was subjected to amplification in a total volume of 50 μl containing 20 mM (NH4)2SO4, 75 mM Tris-HCl (pH 8.8 at 25°C), 0.1% (vol/vol) Tween 20, 3 mM MgCl2, 2.5 μM each deoxyribonucleotide (dATP, dCTP, dGTP, and dTTP), 0.2 μM each primer, and 2 U of Redhot polymerase (Advanced Biotechnologies). Denaturation was performed at 94°C for 60 s followed by 25 amplification cycles of 30 s at 94°C, 30 s at 52°C, and 90 s at 72°C. An additional 120 s at 72°C and a cooling step at 4°C for 60 s completed the reaction. The reactions were carried out in a Biometra Personal Cycler. PCR product restriction enzyme analysis was performed using EcoRV, StuI, KpnI, AvaI, SmaI, AgeI, MroI, NcoI, AvrII, BfrI, SspI, SacII, and HindIII in order to generate a discriminatory banding pattern on gel electrophoresis.

PCR of 16S rDNA for sequencing.

Three different sets of prokaryotic specific primers targeting internal regions of the 16S rRNA gene were used. Forward and reverse sequencing primers from conserved 16S rRNA gene regions were made based on nucleotides 8 to 27, 517 to 536, and 1053 to 1074 in the forward direction and nucleotides 1512 to 1492, 1074 to 1053, and 536 to 515 in the reverse direction (Escherichia coli numbering). A maximum of 50 ng of template DNA was used per reaction in a 50-μl volume combined with 20 mM (NH4)2SO4, 75 mM Tris-HCl (pH 8.8 at 25°C), 0.1% (vol/vol) Tween 20, 0.5 mM MgCl2, 2.5 μM each deoxyribonucleotide (dATP, dCTP, dGTP, and dTTP), 10 μM each primer, and 2.5 U of Redhot polymerase. The amplification protocol was as follows: one cycle of 2 min at 96°C, followed by 25 cycles of 45 s at 96°C, 30 s at 51°C, and 90 s at 72°C, and finally one cycle of 45 s at 96°C, 30 s at 51°C, and 3 min at 72°C. The PCR products were purified using the QIAquick PCR purification kit (Qiagen), following the manufacturer's recommendations. Concentrations were determined by reading at 260 nm in a UV spectrophotometer.

Sequencing and analysis of the 16S rRNA gene.

The 16S rDNA was sequenced using the dideoxy chain termination method. Cycle-sequencing reactions (with a maximum of 40 ng of template DNA), using fluorescently labeled Cy5-Far Red primers, were performed with a Thermosequenase cycle-sequencing kit (Amersham Pharmacia Biotech United Kingdom Ltd.). The sequencing reactions were run on an Alfexpress automated DNA sequencer (Pharmacia Biotech, Uppsala, Sweden). Each isolate was sequenced in both the forward and reverse directions. PILEUP and CLUSTALW were used for multiple sequence alignments, and phylogenetic dendrogram construction (see Fig. Fig.2)2) was done with the DNAMAN for Windows program version 4.13. A secondary-structure model of the 16S rRNA molecule transcribed from the primary sequence of isolate Fairview was constructed by Robin Gutell (7), and the file was interpreted using Aladdin Ghostscript version 5.1 graphical interface software.

FIG. 2.
Map of 6-bp restriction endonuclease cutting sites within the 16S rRNA genes of L. ferrooxidans and L. ferriphilum. Sites which enable L. ferrooxidans to be distinguished from L. ferriphilum and which were consistent among all isolates used in this study ...

PCR amplification and analysis of the 16S-23S intergenic region (IR).

The conditions used for 16S-23S amplification were the same as those used for 16S rRNA gene amplification, except the annealing step took place at 45°C. The primers used in amplification were G1.2 (5"-GTCGTAACAAGGTAICCG-3") and L1.2 (5"-GCCIAGGCATCCACC-3"), modeled on primers designed by Jensen et al. (12).

Moles percent G+C content.

Genomic DNA was treated with RNase A at a final concentration of 50 μg/ml for 30 min at 37°C. The DNA was then phenol extracted, followed by ethanol precipitation. The purified DNA was dissolved in 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate, pH 7) at concentrations between 10 and 40 μg/ml and dialyzed against 0.1× SSC overnight. The DNA solutions were stored in 0.1× SSC at 4°C. The G+C content of the DNA was determined as described by Harrison (9).

DNA-DNA hybridization.

Genomic DNA was prepared as for moles percent G+C content determinations, with the exception of 0.1× SSC dialysis. DNA was resuspended in Tris-EDTA buffer. Three twofold dilutions, 125-ng starting concentration, of all genomic DNAs were prepared in a denaturing solution (final concentration, 0.4 M NaOH-10 mM EDTA). Samples were boiled for 10 min, flash cooled, and loaded onto a positively charged nylon membrane using a slot blot manifold as described by Sambrook et al. (24). The membrane was rinsed briefly in 2× SSC and air dried. Genomic DNA probes were sonicated for seven 10-s periods with a Biosonik III instrument (Bronwill Scientific Inc., Rochester, N.Y.) at an energy setting of 60% before being labeled with digoxigenin using the DIG oligonucleotide 3′-end labeling and detection kit. Hybridization was in DIG-Easyhyb at 40°C, followed by washing in 1× SSC at 25°C and a second washing in 0.1× SSC at 65°C. Quantification of hybridization signals was carried out on a Uvidoc gel documentation system using Alphaimager 2000 software.

Nucleotide sequence accession numbers.

The Leptospirillum sequences determined in this study were assigned the GenBank accession numbers listed in Table Table11.

RESULTS

Number of rrn genes and ribotyping.

Genomic DNA from 16 different Leptospirillum isolates was analyzed in Southern hybridization experiments using 16S rDNA from strain P3a as a probe. Each band represented a single copy of an rrn operon, as genomic DNA was digested with BamHI and it had been established that none of the Leptospirillum-derived 16S rDNA PCR products had an internal BamHI cleavage site. Two main groups of leptospirilla could be distinguished from each other, one with two rrn operon copies and the other with three rrn copies. This result was confirmed by digestion of Leptospirillum genomic DNA with SalI, which also has no internal 16S rDNA cleavage site (results not shown). A further subdivision of the two main groups into ribotype subgroups can be made from a comparison of hybridization fragment sizes (Table (Table2).2). These subgroups provide an indication of the positioning of BamHI restriction endonuclease sites flanking the 16S rRNA genes. Four subgroups within each rrn group were identified. Interestingly, some members that belonged to the same subgroup were isolated from very different geographical locations. For example the group with three rrn gene copies has a 5.08-, 2.8-, 2.1-kb ribotype subgroup containing leptospirilla isolated from Romania, Montana, and England, while the 5.0-, 4.5-, and 2.7-kb ribotype subgroup has leptospirilla isolated from Wales, Idaho, and Chile.

TABLE 2.
Some molecular characteristics of the leptospirilla in this study

Sequence analysis of the 16S rDNA PCR products.

The 16S rRNA genes of 10 of the 16 Leptospirillum isolates were sequenced directly from the PCR-amplified products in both forward and reverse directions. A homology matrix (not shown) between these sequences and five other Leptospirillum sequences previously deposited in GenBank, EMBL, and Ribosomal Database Project databases was constructed. Isolates within the group with two rrn gene copies had 16S rDNA sequences which were 97.2 to 100% identical, whereas those within the group with three rrn gene copies were 98.2 to 99.9% identical. Sequence identity between the members of the two groups was 91.0 to 93.4%. A dendrogram of all strains of Leptospirillum for which sequences are available illustrates the clustering of the two rrn groups (Fig. (Fig.1).1). With the assistance of Robin Gutell (7), a secondary-structure diagram of the Leptospirillum strain Fairview 16S rRNA was drawn (not shown). Although variations in sequence between groups with two and three rrn gene copies occurred in many regions of the 16S rRNA, most variation occurred within variable regions 3 and 6 (not shown). There have been reports of polymorphisms within multiple copies of 16S rRNA genes within the same organism. For example, Mycoplasma capripneumoniae subsp. capripneumoniae has two copies of 16S rRNA genes, and between 11 and 24 differences in nucleotide sequence between the copies were found in 20 isolates examined (18). The sequencing of the 16S rRNA genes of the leptospirilla in this study was carried out directly from the PCR-amplified products. Assuming that all copies of the 16S rRNA genes were amplified with equal efficiency, polymorphisms between gene copies would have resulted in a mixed population of nonidentical amplification products and ambiguous sequence data in certain positions. No positions with sequence ambiguity were found, and all copies of 16S rRNA genes therefore appeared to be identical.

FIG. 1.
Evolutionary-distance dendrogram of leptospirilla based on approximately 1,450 bp of 16S rDNA sequence. Branch points supported by bootstrap values of >75% are shown by solid circles, and those supported by bootstrap values between 50 and 75% ...

PCR amplification and restriction enzyme mapping of 16S rDNA.

We have routinely used restriction enzyme mapping of amplified 16S rDNA as a convenient method for rapidly identifying isolates of previously isolated iron- and sulfur-oxidizing microorganisms present in biooxidation tanks (19, 20). We wished to determine whether this simple technique could be used as a quick screening method to distinguish between the major groups of Leptospirillum. Comparison of the 16S rDNA sequence data from this study and those from previously sequenced leptospirilla deposited in the GenBank and Ribosomal Database Project databases enabled us to identify several 6-bp recognition sequence restriction endonucleases which would give different digestion patterns that could be used for this purpose. Based on the view that the presence of a cutting site has more value than the absence of a site, four endonucleases (AgeI, MroI, NcoI, and SmaI) were identified that allow for specific identification of the group of leptospirilla with two rrn gene copies and six endonucleases (AgeI, AvrII, BfrI, EcoRV, SspI, and StuI) were identified for specific identification of the group with three rrn gene copies (Fig. (Fig.2).2). The AgeI cutting site was present with the 16S rDNAs of both groups but in sufficiently different positions to allow specific identification. Although ApaI, HindIII, KpnI, and SacII cannot be used to distinguish among leptospirilla, these restriction enzymes can be used as diagnostic tools in distinguishing between Leptospirillum, Acidithiobacillus caldus, A. ferrooxidans, and Acidithiobacillus thiooxidans. To confirm the usefulness of this approach, 16S rDNAs of Leptospirillum strains for which the 16S rDNA had not been sequenced but for which the number of copies of rrn had been determined were amplified by PCR. Restriction enzyme digests for MroI, NcoI, SmaI, BfrI, EcoRV, SspI, and StuI were carried out, and in each case the Leptospirillum isolate could be correctly placed in the group with two or three rrn gene copies based on the restriction enzyme digests.

Amplification product profiles of the 16S-23S IRs.

The IRs between the 16S and 23S rRNA genes were amplified in all 16 Leptospirillum isolates. Both single and multiple banding patterns ranging in size from 3.0 to 0.47 kb were obtained (Table (Table2).2). PCR product profiles consisted of both intense, highly reproducible fragments (primary products) and weaker fragments, the presence of which varied depending on amplification purposes (secondary products). As secondary products are not used for classification purposes, they were ignored. A single 0.5-kb IR spacer was amplified from leptospirilla of the group with two rrn gene copies, whereas IR spacers of a variety of sizes were amplified from leptospirilla of the group with three rrn gene copies. Isolates P3a, N25, DSM2705, ATCC 49879, and Crys13 produced three different primary IR products, presumably a different-size product from each of the three rrn gene copies. These results are in agreement with existing evidence that multiple IRs of various sizes may be present within a single species (6).

DNA-DNA hybridization.

Although sequence analysis of 16S rRNA is a valuable tool in investigating phylogenetic relationships, it has been shown in several cases that almost identical 16S rRNA sequences have yielded DNA-DNA hybridization values of less than 70%, indicating separate species (27). For this reason, DNA-DNA hybridization was used in conjunction with 16S rRNA sequence analysis. DNA-DNA hybridization percentages were obtained for 16 isolates using genomic DNAs from 13 leptospirilla as hybridization probes. The results are given in Table Table3.3. The group I leptospirilla could be divided into two DNA-DNA hybridization subgroups with 94 to 100% and 93 to 100% similarity within a subgroup and 60 to 79% similarity between the two subgroups. We have named the subgroups I.1 and I.2. Group II leptospirilla formed a single DNA-DNA hybridization subgroup with 81 to 100% similarity. However, there was only <5 to 11% similarity between subgroups I.1 and I.2 of the group I leptospirilla and the group II leptospirilla.

TABLE 3.
DNA-DNA hybridization values between Leptospirillum groups I and II and between subgroupsa

Leptospirilla capable of growth at 45°C.

One of the few physiological differences reported among leptospirilla is the fact that some isolates are capable of growth at temperatures of >40°C (5, 26). We have previously investigated the bacteria present in pilot plants operating at 45°C and found that large numbers of leptospirilla were present (20). Furthermore, there is a report of a Leptospirillum isolate that is capable of iron oxidation at 55°C and that is considered to constitute a separate species, L. thermoferrooxidans. We wished to determine to which group the leptospirilla adapted in pilot plants to grow at 45°C belonged and whether any of the nonadapted Leptospirillum isolates were also capable of growth at 45°C. Each of the 16 original isolates was tested for the ability to oxidize ferrous iron at 30 and 45°C. In addition, three new leptospirilla (Adapt, BN Mod, and 617) isolated from bioreactors operating at 45 to 55°C were introduced into the study at this stage. Several members of the Leptospirillum group with two copies of rrn genes, including those not previously exposed to bioreactors operating at 40°C or above, were able to oxidize iron at 45°C (Table (Table2).2). However, the rate of iron oxidation was lower than at 30 or 40°C, and no leptospirilla from the group with three rrn gene copies were able to oxidize iron at 45°C.

Lack of marked physiological or physical differences between the two groups of leptospirilla.

We examined the type strain of L. ferrooxidans (DSM2705) and the proposed type strain of Leptospirillum ferriphilum (ATCC 49881) for physiological and physical differences besides temperature tolerance. Both species had properties similar to those reported for L. ferrooxidans (11, 14). They were of similar size (0.3 to 0.5 μm wide and 0.9 to 3.0 μm long), with L. ferriphilum at the narrower end of the width range. Both species were vibrio shaped in young cultures (up to 4 days), helical (two to five turns) in older cultures, and motile by means of a single polar flagellum. They oxidized iron at similar rates (at 37°C), with a doubling time of 12 to 15 h, and could grow autotrophically at the expense of pyrite mineral (data not shown). In addition, both grew optimally on ferrous iron medium within similar pH ranges (pH 1.4 to 1.8 for L. ferriphilum and pH 1.6 to 2.0 for L. ferrooxidans). Both leptospirilla were catalase negative and peroxidase positive. These physical and physiological observations are in close agreement with those reported for L. ferriphilum strain P3a (now called ATCC 49881), which was a gift from Wolfgang Sand (25).

DISCUSSION

Studies of mesophilic leptospirilla by several workers (3, 4, 8, 10, 15) have indicated that more than one species of Leptospirillum exists. Nevertheless, all mesophilic leptospirilla have been generally referred to as L. ferrooxidans or Leptospirillum-like bacteria, as there have been insufficient physiological grounds or molecular information to decide whether they represented more than one species. Criteria commonly used to identify two bacteria as belonging to the same species are G+C moles percent ratios that differ by 5% or less and genome DNA-DNA hybridization of about 70% or greater (27). Comparison of 16S rRNA sequence data has been reported to be a somewhat less reliable criterion for separation of organisms into species. As a result of the compilation of data carried out by Stackebrandt and Goebel (27), it was suggested that organisms with 16S rRNA sequence identities of less than 97% are unlikely to have DNA-DNA hybridization values above 60%. Similar comparisons have been carried out by Rosselló-Mora and Amann (23), and they suggested a slightly more relaxed interpretation, that genomes should have less than 50 to 70% DNA-DNA hybridization before being considered as belonging to different species.

We suggest that the mesophilic leptospirilla investigated in this study may be subdivided into two groups and that the differences between these groups are sufficient for them to be regarded as separate species. Differences in G+C moles percent ratios of 49 to 52 versus 55 to 58% and 16S rRNA sequence identities of 91 to 93% suggest that division into two species is warranted. In addition, the groups differ in that one group has two copies of rrn genes while the other group has three copies. The DNA-DNA hybridization results support separation into two species, as there was a low level of hybrizidation between the group I and group II leptospirilla. The differences in hybridization between subgroups I.1 and 1.2 fall within the suggested guidelines for organisms to be considered as a single species. The differences in size of the IRs between the 16S-23S rRNA genes support separation into two species. Based on the above evidence, we propose that the leptospirilla used in this study should be divided into two species, one of which consists of two distinct subgroups, or genomovars (23). The name L. ferrooxidans should be used for group I because the L. ferrooxidans type strain (DSM2705) belongs to this group, and we propose that a new species name is required for group II. In the absence of a distinguishing physiological property for all members of both species, we suggest that the name L. ferriphilum (ferri, iron; philum, loving) could be used for the group II leptospirilla. This name reflects a common property of all leptospirilla, which is that they use only ferrous iron as their electron donor, and it will have to be validated by the International Committee on Systematic Bacteriology.

PCR amplification of 16S rDNA genes followed by restriction enzyme digestion and separation of the fragments on an agarose gel is a relatively simple procedure compared with DNA-DNA hybridization, 16S rRNA sequencing, and Southern hybridization studies. Since the restriction enzyme digestion maps shown in Fig. Fig.22 were consistent between the two main species of leptospirilla identified in this study, this could be used as a routine identification method. Where identification is uncertain, more comprehensive tests should be carried out.

One aim of this study was to identify which type of Leptospirillum was present in samples taken directly from the commercial biooxidation tanks which operate at the Fairview mine (Barberton, South Africa). These tanks are used to oxidize gold-bearing arsenopyrite concentrates and operate at pH 1.6 and 40°C (21). The isolate from the commercial biooxidation tanks at the Fairview mine was from the group II leptospirilla (two rrn gene copies). Likewise, the 45°C-adapted BN Mod, Adapt, and 617 isolates belonged to the group II leptospirilla and are therefore of a different species than L. ferrooxidans. In a continuous culture study on a culture being prepared for a commercial cobaltiferous pyrite ore bioleaching operation, a Leptospirillum-like bacterium (strain L8) with an optimum pH of 1.3 to 1.6 and an optimum temperature of 37.5°C, but which could grow at 45°C, was isolated (1). This bacterium had a G+C ratio of about 55.6 mol%, which suggests that it was also a group II leptospirillum rather than L. ferrooxidans. It must be pointed out that the commercial processes in which we report that only group II leptospirilla were present operate at temperatures of 35 to 40°C or higher. It would be interesting to determine whether strains of the L. ferrooxidans group with three rrn gene copies are found in industrial heap leaching- or aeration tank-type processes that operate at temperatures lower than 35°C. Since none of the L. ferrooxidans strains that we examined were capable of growth at 45°C, it may be that these bacteria are noncompetitive at temperatures of 35 to 45°C but may well be important in industrial processes that operate at lower temperatures.

Although it was hoped that the selection of more than 16 Leptospirillum isolates from many geographical locations would give a broad representation of Leptospirillum diversity, there are clearly some types of leptospirilla that were not represented in this group. It is unlikely that any of the isolates in this study are the same species as the moderately thermophilic L. thermoferrooxidans. L. thermoferrooxidans was reported to have an optimum growth temperature of 45°C and was capable of iron oxidation at 55°C, which is considerably higher than any isolate in this study. Since the culture has been lost, it was not possible to compare the leptospirilla in this study with L. thermoferrooxidans. The 16S rRNA sequence of this moderate thermophile is also unreported.

During a recent investigation of the microorganisms present in a subaerial slime from the Iron Mountain acid mine drainage site in California, a 16S rRNA sequence for what is proposed to be a third type of leptospirillum was discovered (2). Sequences corresponding to this Leptospirillum group III represented the majority of the clones in a clone bank of 16S rDNA genes prepared from the Iron Mountain slime. There are no reports of leptospirilla belonging to group III having been isolated in pure culture. No leptospirilla belonging to group III were present in our studies based on the direct amplification of 16S rDNA from total DNA isolated from biooxidation tanks nor in our collection of cultured environmental samples. However, the existence of group III illustrates the diversity of leptospirilla, some of which may await discovery.

Description of Leptospirillum ferriphilum sp. nov.

Leptospirillum ferriphilum (ferri, iron; philum, loving). This description is based on this study and that reported by Sand et al. (25). Cells are small curved rods or spirilla, measuring 0.3 to 0.6 μm wide and 0.9 to 3.5 μm long. Young cells are vibrio shaped, but in cultures older than 4 days, cells are mostly spiral shaped with two to five turns. Cells are gram negative, spore forming, and motile by means of a single polar flagellum. Growth is aerobic and chemolithotrophic, with ferrous iron or pyrite but not sulfur serving as the energy source. Optimum pH is 1.4 to 1.8 and temperature 30 to 37°C, with some isolates having the ability to grow at 45°C. Cells are catalase negative and peroxidase positive. G+C content of the DNA is 55 to 58%, there are two copies of rrn genes, and based on 16S rRNA sequence analysis, the cells form a phylogenetic cluster which is separate from L. ferrooxidans. The size of the 16S-23S rRNA intergenic region is conserved among isolates at 500 bp. The type strain is strain ATCC 49881, which is the same as strain P3a provided by Sand and originally isolated in Peru (25).

Acknowledgments

We thank Wolfgang Sand, Peggy Arps, Frank Roberto, Paul Norris, and especially Barrie Johnson for providing us with many of the Leptospirillum isolates used in this study. We also thank Paul Norris for making available the spectrophotometer used to determine DNA melting temperatures.

This work was funded by grants from the National Research Foundation (Pretoria, South Africa), the University of Stellenbosch, and Billiton Process Research (Randburg, South Africa).

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