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Applied and Environmental Microbiology, September 2008, p. 5686-5694, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.01235-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Construction of arsB and tetH Mutants of the Sulfur-Oxidizing Bacterium Acidithiobacillus caldus by Marker Exchange

Leonardo J. van Zyl, Jolanda M. van Munster, and Douglas E. Rawlings^*

Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa

Received 4 June 2008/ Accepted 15 July 2008

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Acidithiobacillus caldus is a moderately thermophilic, acidophilic bacterium that has been reported to be the dominant sulfur oxidizer in stirred-tank processes used to treat gold-bearing arsenopyrite ores. It is also widely distributed in heap reactors used for the extraction of metals from ores. Not only are these bacteria commercially important, they have an interesting physiology, the study of which has been restricted by the nonavailability of defined mutants. A recently reported conjugation system based on the broad-host-range IncW plasmids pSa and R388 was used to transfer mobilizable narrow-host-range suicide plasmid vectors containing inactivated and partially deleted chromosomal genes from Escherichia coli to A. caldus. Through the dual use of a selectable kanamycin resistance gene and a hybridization probe made from a deleted portion of the target chromosomal gene, single- and double-recombinant mutants of A. caldus were isolated. The functionality of the gene inactivation system was shown by the construction of A. caldus arsB and tetH mutants, and the effects of these mutations on cell growth in the presence of arsenic and by means of tetrathionate oxidation were demonstrated.

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The creation of null mutants (nonfunctional gene products) of any organism is pivotal to our understanding of individual gene function, as it provides a baseline of activity to which comparisons can be made. We have developed a method for generating such mutants in the acidophilic, moderately thermophilic (45°C) bacterium Acidithiobacillus caldus (8, 14) through allelic exchange (homologous recombination). Together with the iron-oxidizing bacterium Leptospirillum ferriphilum, A. caldus has been reported to dominate the microbial populations in commercial arsenopyrite concentrate bio-oxidation tanks that operate at 40°C and pH 1.6, as well as in pilot scale stirred-tank bioleaching operations at 45°C (5, 20, 24). A. caldus has also been found in heap reactors (12, 25), making the study of this organism of economic importance. These bacteria are also interesting to study from a fundamental biological point of view due to their unusual metabolism and physiological characteristics (8).

The development of genetic systems for members of the genus Acidithiobacillus and other acidophilic bacteria found in biomining environments has been particularly challenging. Nevertheless, plasmid transfer and the expression of heterologous genes have been reported for three species of acidithiobacilli, Acidithiobacillus ferrooxidans (16, 19, 22, 23), Acidithiobacillus thiooxidans (13, 30), and A. caldus (15, 18, 32). In spite of this, there has been only one report of the construction of a null mutant among these bacteria, a recA mutant of A. ferrooxidans (19). Attempts to create other mutants have so far been unsuccessful. Although the ability to express or overexpress a chosen gene(s) in a particular organism is useful for strain improvement, the lack of a more complete suite of genetic tools, such as the construction of knockout mutants, has hampered the study of the acidithiobacilli.

The genome sequence of A. caldus is not yet available; however, two sets of genes that are candidates for the construction of mutants have been reported. These are the genes conferring arsenic resistance (15) and the genes for tetrathionate utilization (26).

The chromosomally located arsenic resistance operon from A. caldus has been described previously (15); it consists of three genes: arsR, arsB, and arsC (Fig. 1). The product of the arsB gene is the arsenite-exporting membrane-located pump, while the arsR and arsC genes encode an arsenite-sensitive regulator and an arsenate reductase, respectively. Inactivation of arsB should therefore result in an arsenic-sensitive phenotype unless another resistance mechanism is present.

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FIG. 1. (A) Arrangement of the arsenic resistance genes and the tetrathionate hydrolase operon on the A. caldus genome. The solid boxes below the ORFs indicate the regions used in construction of the suicide vectors (parsB::Km and ptetH::Km). The fragments removed between the KpnI sites (in boldface) internal to arsB and between the BstEII sites (in boldface) in tetH were used as probes to distinguish between single- and double-crossover mutants. The positions of the primers used to amplify the 2.3-kb fragment (TetH For and TetH Rev) used in the construction of ptetH::Km are indicated. (B) The parsB::Km and ptetH::Km suicide vectors used to generate double-crossover mutants. The 427-bp KpnI fragment internal to arsB and the 659-bp BstEII fragment internal to the tetH gene were replaced with the kan gene.

Although the ability of A. caldus to utilize reduced inorganic sulfur compounds (RISCs) as energy sources is well known, the exact mechanism by which it decomposes these compounds and derives energy from them remains elusive. Tetrathionate (S₄O₆^2–) can be used as its sole energy source, and the enzyme thought to be responsible for its breakdown is tetrathionate hydrolase (encoded by tetH) (6). A gene cluster from A. caldus KU^T containing a homolog of this gene was recently cloned and sequenced. Identification of the gene as encoding a tetrathionate hydrolase was made on the basis of sequence similarity to other known tetrathionate hydrolases, and the tetrathionate hydrolase activity was linked to this gene by N-terminal sequencing of the protein responsible (26).

Here, we report the inactivation of the chromosomally located arsenic resistance gene arsB in A. caldus KU^T through marker exchange mutagenesis to serve as a model system for the knockout of other genes of interest from the microorganism. We further demonstrated the usefulness of this technique by constructing a second A. caldus KU^T knockout mutant, that of the tetH gene.

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Media, bacterial strains, and plasmids.
The bacterial strains and plasmids used in this study are listed in Table 1. Escherichia coli strains were grown in Luria-Bertani (LB) broth medium (27), with ampicillin (200 µg/ml), kanamycin (50 µg/ml), naladixic acid (35 µg/ml), or trimethoprim (50 µg/ml) added as required. To culture A. caldus strains in liquid medium, a 10x 9K basal salts solution (29) was made, the pH was adjusted to 2.5 using concentrated H₂SO₄, and the solution was autoclaved. When Na₂S₂O₃·5H₂O was used as the sulfur source, the 10x stock was diluted to a 1x solution and the pH was adjusted to 4.7 using 10 N NaOH, to which other components were added to make the growth media. A 20% (wt/vol) stock solution of Na₂S₂O₃·5H₂O was made and filter sterilized, 500 µl of which was added to every 100 ml of 1x basal salts to give a final concentration of 0.1% (wt/vol). To this, 50 µl of 1,000x trace elements [ZnSO₄·7H₂O, 1 g; CuSO₄·5H₂O, 0.1 g; MnSO₄·4H₂O, 0.1 g; CoSO₄·7H₂O, 0.1 g; Cr₂(SO₄)₃·15H₂O, 0.05 g; Na₂B₄O₇·10H₂O, 0.05 g; Na₂MoO₄·2H₂O, 0.05 g; and NaVO₃ (optional), 0.01 g, dissolved in 100 ml distilled H₂O, with 53 µl H₂SO₄ added and the solution autoclaved] was added per 100 ml medium. The final pH of the medium was ±4.5. The cultures were incubated in shake flasks at 37°C with active aeration (100 rpm). When K₂S₄O₆ served as the sulfur source (K₂S₄O₆ is less acid labile than Na₂S₂O₃), the 10x basal salts stock was diluted to 1x and the pH was adjusted to 2.5 instead of 4.7 using concentrated H₂SO₄.

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TABLE 1. Bacteria and plasmids used in this study

Solid medium for culturing A. caldus was made by diluting the 10x basal salts stock to a 2x solution, and the pH was adjusted to 4.7 using 10 N NaOH and autoclaved. This made up half of the final volume of the final medium. The other half consisted of a 1.75% (wt/vol) Phytagel (Sigma-Aldrich) solution made up in distilled H₂O and autoclaved. The solutions were allowed to cool to 50°C. To the 2x basal salts solution was added Na₂S₂O₃·5H₂O to a final concentration of 0.2% (wt/vol) and 100 µl trace element solution for every 100 ml of the 2x basal salts solution. When it was used as a selective medium, kanamycin was added to a final concentration of 100 µg/ml. The basal salts and phytagel solutions were then mixed, and the plates were poured.

Conjugation.
A. caldus KU^T was cultured on the higher-pH (4.5) medium and at 37°C to preadapt it to the conditions of the conjugation experiments. Cells from late-stationary-phase cultures (5 days) served as recipients. E. coli HB101 bacteria to be used in the conjugation experiments were initially cultured in LB broth but were then inoculated into 50 ml of the pH 4.5 medium with thiosulfate and trace elements added to the same final concentration as for the A. caldus cultures, but also supplemented with 0.05% (wt/vol) yeast extract, and cultured overnight at 37°C. This was done to preadapt the donor to the mating medium. Cells from donor (50-ml) and recipient (500-ml) cultures were collected by centrifugation and washed twice in 2 ml of the 1x high-pH basal salts solution. Donor and recipient cells were then separately resuspended in 250 µl of the salts solution each and mixed in a 1:1 ratio. Of this 500-µl mixture, 100 µl was spread evenly onto a Supor 0.2-µm filter (PALL Gelman Laboratory), which was placed on the mating medium. Thus, five filters were used per mating to accommodate the whole mating mixture. The mating medium was the same as the solid medium for culturing A. caldus described above but supplemented with 0.05% (wt/vol) yeast extract and 0.5x 10^–4 M diaminopimelic acid (19). After 5 days of incubation at 37°C, the cells were harvested by scraping growth from the filters with a loop and washed twice in 2 ml of 1x high-pH basal salt solution. Following mating and to provide the opportunity for the generation of a mutant, the cells were collected, washed, and inoculated into several 500-ml high-pH liquid cultures with selection for kanamycin resistance (100 µg/ml). This was done for several reasons. First, to avoid inoculation with too many cells, thereby making it difficult to judge whether growth had taken place. Second, to negate the protective effect that cells appear to afford one another when too many cells are inoculated or plated for a given antibiotic concentration. Third, the cells were cultured with selection to raise the number of mutants (single or double crossover) to a detectable level, as the combined mating and crossover frequencies could be well below our detection limit (approximately 10^–7 transconjugants/recipient). The cultures were incubated at 37°C for 6 to 7 days in shake flasks with vigorous aeration.

In an attempt to determine the rate at which the suicide vector (pOTF101) was transferred from E. coli to A. caldus, the cells were collected after mating and washed, and a serial dilution was plated onto selective and nonselective plates. The plates were incubated for 5 to 6 days at 37°C. The frequencies of plasmid transfer were expressed as the "apparent transfer frequency," i.e., the number of transconjugant colonies that grew on selective medium per recipient divided by the number of colonies that grew on nonselective thiosulfate medium. Donor bacteria were counterselected by the absence of a carbon source in the selective medium.

PCR.
PCRs were performed using Biotaq DNA polymerase from Bioline according to the manufacturer's recommendations. In general, 50 ng of DNA was used in a 50-µl reaction volume containing 2 mM MgCl₂, 0.25 µM of each primer, 200 µM of each deoxynucleoside triphosphate, and 1 U TacI polymerase. Reactions were carried out in a Hybaid Sprint thermocycler, with an initial denaturation at 94°C for 60 s, followed by 25 cycles of denaturation (30 s at 94°C), an annealing step of 30 s, and a variable elongation step at 72°C. The annealing temperatures and elongation times were altered as required. The 557-bp origin of transfer from the IncW plasmid R388 was amplified using primers R388oriTF (5'-TATAGAATTCAGCTCGCCTTGCAAGTCG-3') and R388oriTR (5'-TCGCGAATTCAAGGTCGTTTGCCTGCAT-3'). This product was cloned using the pGEM cloning kit from Promega. Primers TetH Fwd (5'-TAGAACCAAGGACAGC-3') and TetH Rev (5'-AACATCGGCACAGAGA-3') were used to amplify the 2.3-kb fragment from the A. caldus chromosome that contains part of the tetrathionate hydrolase operon used in making the suicide vector (Fig. 1A). The fragment was then cloned using pGEM. Primers KmFor (5'-TTGCACGCAGGTTCTCC-3') and KmRev (5'-TCGGGAGCGGCGATACC-3') were used to amplify a 714-bp fragment internal to the kanamycin resistance gene from Tn5. Figure 2 shows how the oligonucleotide pair ORF1-RTRev (5'-GATCGCGCAGCCAGAGTT-3') and ORF5-RTRev (5'-GTTTGGCAGGGATTGCGG-3') allowed the insertion junction between open reading frame 1 (ORF1) and the interrupted arsB to be checked. Similarly, Fig. 2 indicates how the primer pair arsB-Crossover (5'-GTTTGATCGCTATGCCC-3') and ORF7-Crossover (5'-GTCTGCACGGACTGCAT-3') could be used to check the insertion junction between ORF7 and the interrupted arsB.

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FIG. 2. Construction of single- and double-crossover recombination mutants. On introduction of the nonreplicating vector parsB::Km into A. caldus KUT, a single recombination event results in one of two possible single-crossover strains, Single A or Single B. A second recombination event can result in either restoration of the wild-type gene or a mutant double-crossover strain. The wild type should not be observed due to selection with kanamycin. The annealing sites for primers used to characterize the various recombinants are indicated. The solid boxes indicate where the arsB probe hybridizes, while the cross-hatched boxes indicate the hybridization positions of the kanamycin probe.

Construction of the A. caldus arsB and tetH mutants.
The origin of transfer was excised from pGEM using the EcoRI restriction endonuclease and cloned into the EcoRI site on pUC19 to give pOT. A 2.2-kb PstI-PvuII fragment from pAtcars4 (Table 1) containing part of the chromosomally encoded arsenic resistance operon was cloned into the PstI and blunted SacI sites of pOT to give pOTars (Fig. 1A). This construct was digested with KpnI, which removed two ±200-bp fragments from the center of the arsB gene, which were later used as a probe for detecting single- and double-crossover mutants, and the ends were filled in using the T4 DNA polymerase (Fermentas, Vilnius, Lithuania). The ±1.45-kb XbaI-XhoI fragment from pSKM2 carrying the kanamycin resistance cassette from Tn5 was also blunt-end cloned into the blunted KpnI sites of arsB to give parsB::Km (Fig. 1B). The 2.3-kb DraI-EcoRI fragment containing part of the tetrathionate hydrolase operon was excised from pGEM. The EcoRI site was blunted, and the fragment was cloned into the SmaI site of pOT to give pOTtet (Table 1). This construct was digested with BstEII, which removed 659 bp from the middle of tetH, and the ends were filled in using T4 polymerase. The ±1.45-kb NotI-SalI fragment from pSKM2 carrying the kanamycin resistance cassette from Tn5 was also blunt-end cloned into the blunted BstEII sites of tetH to give ptetH::Km (Fig. 1B).

Plasmids parsB::Km and ptetH::Km were each transformed into E. coli HB101 cells that contained pR388 by using kanamycin and trimethoprim resistance selection. The abilities of both of these suicide vectors to be mobilized were checked by transferring them by conjugation on Luria agar from E. coli HB101-R388 to E. coli CSH56 with selection for nalidixic acid and kanamycin resistance. The mobilizable suicide vectors were then transferred from E. coli HB101-R388 to A. caldus KU^T under the conditions described above.

DNA manipulations and sequencing.
Plasmid preparation, restriction endonuclease digestion, gel electrophoresis, ligation, and Southern/colony blot hybridization were performed using standard methods or the manufacturers' recommendations (27). Ultrapure plasmid DNA was obtained using the Wizard Plus SV miniprep DNA purification system from Promega. Total DNA from A. caldus was prepared as previously described (15). Large-scale plasmid preparations were made using the Nucleobond AX kit from Machery-Nagel. The sequence of the origin of transfer amplification product, cloned into pGEM, was determined using an ABI Prism 377 automated DNA sequencer. The sequence was analyzed using the PC-based DNAMAN (version 4.1) package from Lynnon BioSoft.

Arsenic resistance assay of the wild-type A. caldus KU^T strain versus the arsB double-crossover mutant strain.
To test for growth of A. caldus in the presence of arsenite, cells were cultured in the pH 4.5 thiosulfate medium containing 0.0, 0.25, 0.50, 0.75, and 1.0 mM arsenite. Early-stationary-phase cultures were diluted 2,000-fold into fresh medium containing As(III) and incubated for 12 days, and the cell density was determined by measurement of the optical density at 600 nm. Growth in the presence of arsenate was not tested, as the phosphate in the growth medium would contribute to apparent arsenate resistance (28).

Growth curve of the wild-type A. caldus KU^T strain versus the tetH double-mutant crossover strain on tetrathionate.
To determine the ability of the mutant to grow in tetrathionate-containing medium, cells from both the mutant and wild type were initially cultured in the pH 4.5 thiosulfate-containing medium until early stationary phase. The cells were then diluted 2,000-fold in fresh tetrathionate medium (pH 2.5), and the growth was monitored by measuring the change in optical density at 600 nm over 5 days. The growth of the wild type was compared to that of the double-crossover mutant.

Screening for single- or double-crossover mutants.
Screening of transconjugants to determine whether they were single or double crossovers was done by colony blot Southern hybridization. After a serial dilution of the 500-ml culture (inoculated with cells directly after mating) was plated on selective plates, Km^r colonies were picked and streaked on fresh selective plates, which were incubated at 37°C for 5 days. The colony blot was performed using standard methods. The membrane was first probed against a 714-bp internal fragment of the kanamycin resistance cassette under stringent conditions. This indicated whether enough cell mass had transferred to the membrane and whether proper lysis of cells had taken place to give a strong signal. The blot was stripped of the kanamycin probe and reprobed, this time using either the two ±200-bp arsB KpnI fragments or the 659-bp tetH BstEII internal fragment under stringent conditions. The colonies that gave a strong signal with both probes were considered to be single-crossover mutants, while those that gave a strong signal for the kanamycin probe but gave no signal with the arsB probe were possible double-crossover mutants (Fig. 2; also see Fig. 4).

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FIG. 4. PCR analyses of selected single- and double-crossover mutants of A. caldus KUT (KUT) and pAtcars4 using primer pairs ORF1-ORF5 and arsB-ORF7 (Fig. 2). See the text for details.

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Construction of the A. caldus KU^T arsB and tetH mutants.
To test the procedure for the construction of a null mutant, we chose the arsB gene from the chromosomally encoded arsenic resistance operon found in A. caldus (Fig. 1A). The arsenic resistance operon was selected because it was one of the few areas of the A. caldus genome sequence that was publicly available (15). Since the A. caldus ars operon has been well characterized with respect to which other ORFs are in its vicinity and how these genes are transcribed, we had confidence that the inactivation of the arsB gene would not result in a lethal mutation (15). Furthermore, inactivation of the gene should be easily confirmed by a simple growth assay in the presence of arsenite.

To make the A. caldus arsB mutant, a mobilizable suicide vector was constructed, parsB::Km (see Materials and Methods). The suicide vector was based on pUC19, as that plasmid has a narrow-host-range ColE1-type replicon. The suicide vector carried a copy of the arsB gene with an internal fragment replaced by the kanamycin resistance cassette from the E. coli transposon Tn5 (Fig. 1B). The conjugative plasmid pR388 was used to mobilize the suicide vector from E. coli HB101 to A. caldus KU^T as described above. After several unsuccessful attempts to detect mutants directly after mating by plating them on selective medium, it was decided to inoculate cells directly after mating into fresh liquid medium with selection to try to amplify the number of mutants to a detectable level. After 6 to 7 days of incubation at 37°C, growth was observed; the cells were harvested and washed, and serial dilutions were plated on kanamycin selective plates.

Of the colonies that grew on selective medium, 140 were picked and streaked onto fresh solid selective medium. Colony blot Southern hybridization was used to screen the transconjugants to determine which of them were possibly single- or double-crossover mutants. All 140 colonies probed using a 714-bp PCR fragment internal to the kanamycin resistance gene gave a signal, suggesting that all were transconjugants. When the blot was then stripped of the kanamycin probe and reprobed with the two ±200-bp KpnI fragments internal to the arsB gene, six colonies did not give a positive hybridization signal using the second probe. As the probe fragments should not be present on the chromosomes of double-crossover mutants, those colonies that gave a signal with the kanamycin gene but not the internal arsB fragment were considered to be potential double-crossover mutants (Fig. 2). The ratio of single- to potential double-crossover mutants was therefore approximately 1 to 23.

A second suicide vector targeting the tetH gene was constructed (ptetH::Km) similarly to the vector used in disruption of the arsB gene (Fig. 1B). This vector carried a copy of tetH interrupted by the kanamycin resistance cassette from the E. coli transposon Tn5 and could be mobilized by the IncW plasmid R388. Since it had been determined that an "enrichment" step was required to amplify mutants to a detectable level, cells were collected after conjugation and inoculated into 500 ml liquid culture with selection and incubated at 37°C for 6 to 7 days, after which growth was observed. The cells were harvested and washed, and a serial dilution was plated on selective medium.

All of the 249 colonies picked gave a signal following colony blot Southern hybridization using the 714-bp PCR fragment internal to the kanamycin resistance gene as a probe, suggesting that all were transconjugants. The blot was then stripped of the kanamycin probe and reprobed with the 659-bp BstEII fragment internal to the tetH gene. Only one colony did not give a positive hybridization signal with the second probe, indicating that it was a possible double-crossover mutant. The ratio of single- to potential double-crossover mutants was therefore 1 in 249.

To determine the frequency at which the suicide vector was mobilized from E. coli to A. caldus, the vector pOTF101 was constructed. The vector consisted of the pTC-F14 IncQ replicon and kanamycin resistance cassette cloned into pOT instead of the 2.2-kb PstI-PvuII fragment from the A. caldus chromosomally located arsenic operon (Table 1). As plasmid pTC-F14 originated from A. caldus (7), this would allow the plasmid to replicate in A. caldus and permit us to determine the frequency at which the IncW oriT is transferred from E. coli to A. caldus. As with the mutant construction matings, plasmid R388 was used as the conjugative plasmid to transfer pOTF101 from E. coli HB101 to A. caldus. Mating frequencies varied considerably within the range 10^–5 to 10^–7 transconjugants per recipient.

Genetic characterization of arsB and tetH mutants.
To demonstrate that the potential double- and single-crossover mutants identified by colony blot Southern hybridization had a gene layout that would be predicted after homologous recombination (Fig. 2), genomic DNA was prepared from randomly selected clones. They were compared by Southern blot hybridization and PCR analysis to DNA from the wild-type strain.

A Southern blot was prepared using genomic DNA from wild-type A. caldus, as well as putative double- and single-crossover arsB mutants digested with the restriction endonucleases BstEII, SalI, and PvuI (Fig. 3). When an internal fragment of the arsB gene was used as the probe (Fig. 3A), the chromosomal DNA from the putative double-crossover mutant did not give a signal, indicating the absence of these fragments in double-crossover mutants and confirming the colony blot Southern hybridization result. When wild-type DNA was the target, this probe gave 2.6-kb BstEII, ±7-kb SalI, and 3.5-kb PvuI signals, whereas 5.4-kb BstEII, 9.2-kb SalI, and 3.5-kb PvuI signals were observed for the putative single-crossover mutant (Fig. 2 and 3A). When an internal fragment of the kanamycin gene was used as a probe, the wild-type DNA did not give a signal, as expected. Genomic DNA from the putative double-crossover mutant gave signals of 3.5 kb, 3.8 kb, and 4.4 kb for BstEII-, SalI-, and PvuI-digested DNA, respectively (Fig. 2 and 3B). In contrast, signals of 3.5 kb, 9.2 kb, and 4.4 kb were obtained for BstEII, SalI, and PvuI digests, respectively, when genomic DNA from the putative single-crossover mutants was used. From these data, it was concluded that the single crossover took place according to option B in Fig. 2.

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FIG. 3. Southern blot analysis of BstEII-, SalI-, and PvuI-digested DNA isolated from A. caldus KUT (KU-WT), a double-crossover arsB mutant, and a single-crossover arsB mutant. The blots were probed with either a fragment internal to the arsB gene (A) or a fragment internal to the kanamycin resistance gene (B). The numbers between the panels indicate the sizes (in kilobases) of some fragments from the PstI molecular size marker.

Using PCR primers internal to ORF1 and ORF5, which flank the arsBRC operon, the A. caldus wild type and a clone of the wild-type genes gave similar-size (±2.7-kb) PCR fragments, as predicted (Fig. 2 and 4). The putative single- and double crossover mutants gave similar-size, but larger, PCR fragments (±3.7 kb) due to the replacement of a small piece of the arsB gene with a larger fragment containing the kanamycin resistance gene. This also indicated that the single-crossover mutant was option "Single B" rather than option "Single A" (Fig. 2 and 4). Although there are two places where the ORF5 and arsB-Crossover primers can anneal, the elongation times for the PCRs were chosen to give fragments in the <5.0-kb size range only. When PCR primers internal to arsB and ORF7 were used, similar-size (±2.4-kb) fragments were obtained for the wild-type and cloned wild-type DNA, as expected (Fig. 4). An identical-size fragment was also obtained for the single crossover (Fig. 4), as predicted from the single-crossover option "Single B" (Fig. 2). A larger, ±3.2-kb fragment (Fig. 4) was obtained for the double-crossover mutant, as predicted (Fig. 2).

A Southern blot was also prepared using genomic DNA from wild-type A. caldus, as well as a putative tetH double-crossover mutant digested with the restriction endonuclease KspI and a DraI-SalI double digest (Fig. 5). When a fragment internal to tetH was used as the probe, the chromosomal DNA from the putative double-crossover mutant did not give a signal (Fig. 5A). When wild-type DNA was the target, this probe gave 3.4-kb KspI and 4.2-kb DraI-SalI signals (Fig. 5A). When an internal fragment of the kanamycin gene was used as a probe, the wild-type DNA did not give a signal, as expected. Genomic DNA from the putative double-crossover mutant gave signals of 4.2-kb KspI- and 5-kb DraI-SalI-digested DNA, respectively (Fig. 5B).

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FIG. 5. Southern blot analysis of KspI- and DraI-SalI-digested DNA isolated from A. caldus KUT (KU-WT) and a double-crossover mutant. The blots were probed with either a fragment internal to the tetH gene (A) or a fragment internal to the kanamycin resistance gene (B). The numbers between the panels indicate the sizes (in kilobases) of some fragments from the PstI molecular size marker.

Taken together, these results suggest that the selected arsB potential single-crossover clone was a single-crossover mutant and had a gene arrangement similar to "Single B," as shown in Fig. 2. It also shows that the selected potential double-crossover clone is indeed a double-crossover mutant and that in this mutant, the arsB gene has been interrupted by the kanamycin resistance cassette. Similarly, the potential tetH double-crossover mutant was confirmed, and tetH had been disrupted by the kanamycin resistance cassette in this clone.

Arsenite resistance in the A. caldus KU^T wild type and its arsB disrupted mutant.
To compare the ability of the wild type and the arsB double-crossover mutant to cope with As(III) toxicity, we compared their growth after 12 days in liquid medium at a range of As(III) concentrations (Fig. 6A). The arsB double-crossover mutant displayed a greatly reduced capacity to deal with As(III) compared to the wild type and grew poorly, or not at all, in >0 mM As(III) (Fig. 6A).

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FIG. 6. (A) Growth of A. caldus KUT (KU-WT) and KU-arsB::Km after 12 days in the presence of various concentrations of arsenite. Cell densities were determined (optical density at 600 nm[OD600]) and are represented as percentages of growth in the absence of arsenite. (B) Growth of A. caldus KU-WT and KU-tetH::Km on tetrathionate-containing media over 5 days. Each data point represents duplicate results of at least two experiments. The error bars indicate standard deviations.

Growth of the A. caldus KU^T wild type and its tetH disrupted mutant on tetrathionate.
To determine whether the proposed tetH is responsible for the decomposition of tetrathionate to other sulfur intermediates, allowing the bacterium to utilize it as a sole energy source, the wild type and double-crossover mutants were initially cultured on thiosulfate medium and then switched to medium containing only tetrathionate as a sulfur source. Figure 6B shows that whereas the wild-type cells grew relatively well in tetrathionate medium, the mutant did not grow to a level that was detectable using optical-density measurements. To determine if any mutant growth had taken place, the CFU of both wild-type and mutant cultures were determined on day 5. The CFU count for the mutant (tetH::Km) was 1.3 x 10⁶ ± 0.8 x 10⁶ CFU/ml, while the CFU count for the wild type was 1.6 x 10⁸ ± 0.4 x 10⁸ CFU/ml. As the CFU count of the cultures after inoculation was ±8 x 10⁴ CFU/ml, growth of the mutant had taken place, although approximately 100-fold reduced compared with that of wild-type cells.

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
This is the first report of the construction of a null mutant of the bacterium A. caldus using marker exchange mutagenesis. Inactivation of the arsenite pump arsB gene was used as a model to demonstrate how this system could be used to study other genes of interest from the organism. Although plasmid transfer to A. caldus has been reported (15, 18, 32), the ability to generate mutants has not been demonstrated previously, possibly due to the difficulty in detecting these low-frequency events. Interruption of the A. caldus KU^T arsB gene with the kanamycin resistance cassette was confirmed using molecular analysis and resulted in a marked increase in sensitivity to arsenite displayed by the arsB double mutant compared to the wild-type strain. This also demonstrated that there are no other detectable mechanisms of arsenite resistance in A. caldus KU^T and showed the benefit of being able to create null mutants.

The transfer frequency of the pOTF101 plasmid varied widely between 10^–5 and 10^–7 transconjugants per recipient. As the protocol for all conjugation experiments was standardized, this result shows that, under these mating conditions, even slight variations can have a significant effect on the frequency of plasmid transfer and therefore the probable success rate of constructing and selecting mutants by homologous recombination. For E. coli, the frequency at which homologous recombination occurs has been found to be approximately 10^–3 to 10^–4 less than the transformation frequency (10). If one speculates that this same proportion holds for A. caldus, at a transformation efficiency of 10^–5 transconjugants per recipient, the best-case scenario would result in a single-crossover recombinant occurring at a rate of 10^–8 per recipient. The occurrence of double-crossover recombinants would be even lower, with only 1 in 23 of the crossovers being double crossovers in the case of the arsB mutant. This frequency could be much lower, however, in the worst-case scenario (10^–7 transconjugants per recipient and 10^–4 recombination efficiency), which then makes it exceedingly difficult to isolate a double-crossover mutant and probably shows why the "enrichment" step after conjugation was required to isolate mutants. The ratios of 1 in 23 for arsB and 1 in 249 for tetH reported in this study are not accurate estimates. The numbers of double crossovers were too small to allow statistical validity and could be further skewed, as this ratio was not measured directly after mating but only once the cells had been cultured long enough to bring the recombinants into detection range. If either the single- or double-crossover mutant has a growth rate slightly different from that of the other, the ratio would be affected.

The fact that there are more double-crossover mutants for arsB than for tetH could be partly due to the smaller total area available for recombination on the tetH vector (1,807 bp for arsB and 1,622 bp for tetH), as well as the relative sizes of the areas on either side of the kanamycin gene for the two different vectors (802 bp to 1,005 bp for arsB and 929 bp to 693 bp for tetH) (1, 10, 11). In addition, it has been reported that, depending on the locus being targeted, the frequency of recombination can vary by several orders of magnitude (21).

Efforts have been made to study the metabolism of this organism with respect to how it oxidizes RISCs and how it generates energy, in the form of ATP, from this process (2, 4, 6, 9, 26). However, the ability to knock out genes involved in these processes has been lacking, making it difficult to confirm their involvement or to identify other pathways involved. We have therefore constructed a null mutant for the chromosomally located tetrathionate hydrolase gene, tetH, which is thought to be responsible for the hydrolysis of tetrathionate to thiosulfate, sulfur, and sulfate, which A. caldus uses as energy sources (2, 26). The observation, using cell counts, that the tetH mutants were able to grow on tetrathionate media, although very poorly, could be due to one of two reasons. There may be an additional, less efficient mechanism of tetrathionate hydrolysis, or, alternatively, the tetrathionate in the medium may have been unstable so that there was a natural conversion to other RISCs, allowing growth to take place. These possibilities have yet to be investigated, and work is under way to characterize this mutant physiologically.

The addition of this technique to the suite of tools already available for A. caldus can be considered a significant milestone toward our ability to study the organism.

 
This work was funded by grants from the National Research Foundation (Pretoria), BHP-Billiton Johannesburg Technology Centre, and BioMinE project 500329 of EU framework 6.

We thank Mark Dopson and Olena Rzhepishevska (University of Umeå) for providing us with the sequence data used for the construction of the tetH mutant.

 
* Corresponding author. Mailing address: Department of Microbiology, University of Stellenbosch, Private Bag X1, Matieland 7602, South Africa. Phone: 27-21-808 3071. Fax: 27-21-808 3680. E-mail: der{at}sun.ac.za

Published ahead of print on 25 July 2008.

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Applied and Environmental Microbiology, September 2008, p. 5686-5694, Vol. 74, No. 18
0099-2240/08/$08.00+0     doi:10.1128/AEM.01235-08
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