Occurrence of Horizontal Gene Transfer of PIB-type ATPase Genes among Bacteria Isolated from the Uranium Rich Deposit of Domiasiat in North East India

Uranium (U) tolerant aerobic heterotrophs were isolated from the subsurface soils of one of the pre-mined U-rich deposits at Domiasiat located in the north-eastern part of India. On screening of genomic DNA from 62 isolates exhibiting superior U and heavy metal tolerance, 32 isolates were found to be positive for PIB-type ATPase genes. Phylogenetic incongruence and anomalous DNA base compositions revealed the acquisition of PIB-type ATPase genes by six isolates through horizontal gene transfer (HGT). Three of these instances of HGT appeared to have occurred at inter-phylum level and the other three instances indicated to have taken place at intra-phylum level. This study provides an insight into one of the possible survival strategies that bacteria might employ to adapt to environments rich in uranium and heavy metals.


Introduction
Domiasiat (25u 309 N 91u 309 E) represents one of the premined uranium rich areas located in Meghalaya in the northeastern part of India. Exploration by the Atomic Mineral Division (AMD) of India in 1986 led to the discovery of sandstone type uranium deposits occurring at shallow depth. As is the case for other U-ore bearing sites, Domiasiat co-hosts additional heavy metals including zinc, copper, cadmium and lead [1]. Metals may influence the growth and occurrence of organisms, either as limiting factors or through toxicity. Bacteria existing in metal rich/ contaminated soils are known to have evolved strategies to negate the toxic effects of metals. One such mechanism relates to the maintenance of metal homeostasis within the cell, usually accomplished by the active efflux of metal ions [2]. P IB -type ATPases are transmembrane proteins that are known to govern the transport of heavy metals such as Cu, Zn, Pb and Cd in bacteria [3,4]. These transport mechanisms use energy from ATP hydrolysis to pump out metal ions from the cytoplasm towards the periplasm [3,4]. Although P IB -type ATPases are mostly chromosomally encoded, some studies have reported the presence of PIBtype ATPase genes on mobile genetic elements (i.e. plasmids and transposons) in both gram-positive bacteria [5][6][7][8] and gramnegative bacteria [9,10]. Based on substrate specificity, members of the P-type ATPase proteins are classified into distinguishable families [11]. Furthermore, the P IB -type sub-family tends to separate into two distinct groups based on the cation they transport, i.e. (a) those specific to monovalent cations like Cu + or Ag + and (b) those transporting divalent cations like Zn 2+ , Cd 2+ , Co 2+ and Pb 2+ [3,11]. So far, no signature motif has been identified that suggests the substrate specificity in these proteins. Further biochemical characterization is therefore required to delineate the metal specificity in these proteins.
Horizontal gene transfer (HGT) is considered to be a key factor in providing adaptive features to microbes for their survival and proliferation by defining the structure and function of microbial communities in their natural habitats [12,13]. It has been shown to be prevalent in the prokaryotic genome with low frequencies of recombination and is considered to be responsible for generating diversity and adaptability among microorganisms [14]. While the role of HGT has been well documented for the transfer of antibiotic resistance and pathogenic genes in bacteria [15], its contribution to the transfer of metal transporting genes is being recently explored. Genome wide studies by Coombs and Barkay [16] showed that HGT of metal-transporting P IB -type ATPase encoding genes can occur albeit at a low frequency. Molecular evidence for HGT was observed among lead-resistant bacteria isolated from deep subsurface environments contaminated with radionuclides and heavy metals [17,18].
In the present study, we isolated uranium tolerant aerobic heterotrophs from subsurface soils of one of the uranium rich deposits located at Domiasiat in north eastern part of India. We examined the presence of P IB -type ATPase genes in these uranium tolerant bacterial isolates. Six isolates belonging to Bacteroidetes, Firmicutes, and Proteobacteria phyla, among the population of P IB -type ATPase positive isolates, revealed the acquisition of zntA/ cadA/pbrA-like genes through HGT.

Experimental site and soil samples
Soil samples were collected from the proposed mining site of one of the U ore deposits at Domiasiat (25u 309 N 91u 309 E) in North-East India. This site formed a part of a Cretaceous Mahadek basin covering roughly an area of 13,000 km 2 containing 9.22 million metric tonnes of ore reserves with an average ore grade of circa 0.1% U 3 O 8 [19]. The soil samples contained variable concentrations of U ranging from 40 mM to 5 mM (unpublished data). The pH of the soil was acidic within the range of 4.3 to 6.3 [20].

Detection of P IB -type ATPase genes
The bacterial isolates were screened for the presence of metal transporting P IB-type ATPase (e.g., zntA/cadA/pbrA-like) genes. Genomic DNA was isolated from the respective isolates using genomic DNA extraction kit (HiMedia, India) and amplified with PCR primers specific for P IB -type ATPase encoding genes [17,18]. PCR mixtures (25 mL) contained approximately 30 ng of template DNA, 2 mM forward primer (81JC or 133JC), 2 mM reverse primer (84JC), Taq DNA Polymerase buffer with 15 mM MgCl 2 , deoxynucleoside triphosphates (250 mM each of dATP, dCTP, dGTP and dTTP) and 1.0 U of Taq DNA polymerase. DNA amplification was carried out in a GeneAmpH PCR system 9700 (Applied Biosystems, USA) with an initial denaturation step of 94uC for 5 min, followed by 30 cycles consisting of denaturation at 94uC for 1 min, annealing at 49uC for 1 min, and extension at 72uC for 1.5 min followed by a final extension step of 72uC for 5 min. This PCR procedure yielded amplicons of the expected size (,750 bp).
Phylogenetic analyses of zntA/cadA/pbrA-like gene products and 16S rRNA genes Amplicons of P IB -type ATPase genes (zntA/cadA pbrA-like) of the representative 16 isolates from different phyla were purified using the QIAquick Gel Extraction Spin Kit (QIAGEN, Germany) and sequenced using the Big Dye Terminator cycle sequencing kit v.3.1 (Applied Biosystems, USA) deploying the standard protocol and an automated Genetic Analyzer ABI 3130XL (Applied Biosystems, USA). The Basic Local Alignment Search Tool (BLAST, sub-program BLASTX) [26] was used to determine the phylogenetic neighbours of P IB -type ATPase genes against the GenBank database (National Center for Biotechnology Information, Bethesda, USA). Molecular Evolutionary Genetics Analysis software (MEGA v4) was used for phylogenetic analyses [27]. The sequences of identified phylogenetic neighbours were aligned with the sequences using ClustalW of MEGA4. The Escherichia coli kdpB gene was used as outlier. For phylogenetic tree construction we used the neighbor joining method with 1000 bootstrap replications for nodal support. Phylogenetic analyses based on the maximum likelihood and maximum parsimony of P IB -type ATPase amino acid sequences were in agreement with the data generated by the above described neighbour joining method. On average, 600 nucleotides were included in the phylogenetic analyses of the P IBtype ATPase sequences. The 16S rRNA gene sequences of the representative bacteria were amplified and sequenced as previously described [28]. Phylogenetic neighbours were obtained against the database of type strains with validly published prokaryotic names (available online http://eztaxon-e.ezbiocloud. net/) [29]. Phylogenetic analyses by MEGA4 were performed using an average of 1200 nucleotides of 16S rRNA encoding DNA sequence [27]. The 16S rRNA gene sequence of Deinococcus radiodurans M21413 was taken as an outlier. The phylogenetic tree was constructed using the scale bars of 0.05 change per nucleotide position for the 16S rRNA gene and 0.1 change per amino acid position for PIB-type ATPase.
The G+C content of each zntA/cadA/pbrA-like amplicon was calculated by using Oligo Calculator available at http://mcb. berkeley.edu/labs and were compared to the G+C contents of all other organisms belonging to the same genus.

Nucleotide sequence accession numbers
Nucleotide sequences of 16S rRNA and P IB -type ATPase encoding genes have been deposited in NCBI with accession numbers as shown in Figure 1A and 1B, respectively.

Results
Tolerance to uranium and heavy metals among natural bacterial isolates and analysis of P IB -type ATPase genes One hundred and thirty uranium tolerant isolates were obtained from LPM plates enriched with uranium. Out of those, 62 isolates were selected for further studies based on amplified ribosomal DNA restriction analysis (ARDRA) grouping (data not shown). The natural isolates from Domiasiat were able to tolerate significant concentrations of U (2mM) and other heavy metals (data not shown). These metal-tolerant isolates were screened for the presence of PIB-type ATPase (zntA/cadA/pbrA-like) encoding genes using primers reported in earlier investigation [18]. Out of the 62 isolates analysed for the presence of P IB -type ATPase encoding genes, 32 isolates tested positive and yielded gene products of the expected size of ,750 bp. Based on the superior metal tolerance potential, 16 out of 32 PCR positive isolates (representing each genus), were selected for further sequence analysis. Fourteen of these isolates showed PCR amplifiable zntA/ cadA/pbrA loci whereas the other two isolates displayed copA-like loci. The amino acid sequences deduced from the PIB-type ATPase gene sequences of seven isolates, i.e. WAKH2, NONG2, LONG1, KMSDS4, RSBA2, PMSZPI and KMSII3 showed $80% similarity with the PIB-type ATPase signature sequences of the expected genus in the GenBank database ( Table 1). The deduced PIB-type amino acid sequences of the other nine isolates either showed close similarity with another genus or displayed poor similarity (,70%) with the corresponding genus. The first closest match revealed by phylogenetic analysis of PIB-type ATPases for the isolates PKSW3 and RSBA2 indicated cation transporting ATPases while the second match corresponded to Cd and Cu transporting genes, respectively (Table 1). Neighbour joining analyses of gene sequences of natural isolates were performed with the nearest homologous zntA/cadA/copA-like loci from complete genomes available at NCBI GenBank. The combined phylogenetic clustering with the PCR amplified gene sequences for PIB-type ATPases obtained from Domiasiat isolates corresponded to ZntA-like, CopA-like, and CadA-like major clusters ( Figure 1B). The phylogenetic tree created using the CopA-like loci of OT6 and RSBA2 along with the corresponding sequences from complete genomes from NCBI (as identified by sequence similarity using BLASTX) resulted in CopA1-like cluster ( Figure 1B, Figure S1). This is in agreement with previous reports indicating the coherence between the substrate specificity and phylogeny of P-type ATPases [11]. The MICs of U and other heavy metals like Cd, Cu and Pb for the 16 representative bacteria are shown in Table 1. All the isolates exhibited a MIC of 4 mM for U against 1-2 mM shown by type strains. Half (50%) of the bacterial isolates showed a MIC of 8 mM for Cu against 2-4 mM demonstrated by the type strains. For Cd, 56% of the isolates displayed a MIC of 2 mM while 44% showed a MIC of 1 mM as compared to a MIC of 60-500 mM for Cd by the type strains. While one half of the population exhibited a MIC of 2 mM for Pb, the other half showed a MIC of 1 mM against 500 mM shown by the tested type strains (Table 1). These results confirmed the superior metal tolerance phenotypes of the natural isolates of Domiasiat harbouring PIB-type ATPases.

Horizontal transfer of P IB -type ATPase genes
Phylogenetic incongruence between a gene of interest and a marker gene such as the 16S rRNA encoding gene has been used in earlier studies to predict HGT during gene evolution [30,31]. BLASTX analysis was performed using zntA/cadA/pbrA-like sequences obtained from our isolates to find out the closest relative from the NCBI database. The similarity percentage of some of the zntA/cadA/pbrA-like sequences was found to be lower (,70%) as compared to the corresponding gene from the same genus reported in the NCBI database ( Table 1). Six of our isolates appeared to have acquired zntA/cadA/pbrA-like genes by HGT ( Figure 1, Table S1). The P IB -type ATPase gene sequences from Sphingobacterium sp. isolates, KMSDrP1, KMSDrP2, and KMSDrP3, exhibited circa 66% similarity with the zntA gene of Sphingobacterium spiritivorum ATCC33861 (as derived from its complete genome sequence; NCBI reference sequence, NZ_ACHA02000002.1 (Table 1). However, these isolates showed 100% similarity with the zntA like sequence of Paenibacillus pabuli PSS2 isolated from Domiasiat site and clustered with the zntA-like gene cluster of Firmicutes and Bacteroidetes unlike the clusters obtained using 16S rRNA gene sequence analysis ( Figure 1A, B). Initial analysis suggested the occurrence of HGT from Bacteroidetes (Sphingobacterium isolates) to the Firmicutes (Paenibacillus isolate). However, on inclusion of another important criterion for implicating HGT, i.e., moles percent (mol%) of G+C content (atypical sequence composition) [32], HGT appeared to have taken place from Firmicutes to Bacteroidetes. The G+C content of the zntA-like gene sequences from three Sphingobacterium isolates (47.3 mol%) was clearly higher than the DNA G+C content reported so far for Sphingobacterium species (i.e., 37.3 to 44.2 mol%) [33]. Furthermore, the G+C contents of zntA like gene sequences of the Sphingobacterium isolates were found to be almost similar to that of PSS2 identified as Paenibacillus pabuli by 16S rRNA gene phylogeny. The G+C content of the Paenibacillus pabuli genome is reported to be 45.3 mol% [34] which compares well to that of the zntA sequences of the isolates KMSDrP1(47.4% mol G+C), KMSDrP2(47.3% mol G+C), KMSDrP3 (47.3% mol G+C), and PSS2 (47.5% mol G+C), indicating HGT from Firmicutes to Bacteroidetes. In addition to a resemblance in mol% G+C contents, an incongruence in the phylogenetic tree was clearly observed when zntA like sequences of other Bacteroidetes isolates, i.e. PMSZPI and KMSZPIII, were excluded ( Figure S2). However, on inclusion of representatives of all phyla, we found that the Sphingobacterium isolates (KMSDrP1, KMSDrP2, and KMSDrP3) and Firmicutes isolate (PSS2) clustered with both Bacteroidetes and Firmicutes with a strong bootstrap support of 100% ( Figure 1B). As the mol% G+C content of PSS2, KMSDrP1, KMSDrP2, and KMSDrP3 corresponded well to that of Firmicutes, we have assigned these as Firmicutes rather than Bacteroidetes ( Figure 1B). The possibility of HGT of zntA like sequence for other two Bacteroidetes, Chryseobacterium culicis PMSZPI and Sphingobacterium siyangense KMSZPIII, was not taken into consideration as these showed a closest match with a zinc exporting ATPase gene of the complete genome sequence of Chryseobacterium gleum ATCC35910 with maximum identity of 92% and 76% respectively. Unlike the Sphingobacterium isolates, the G+C contents observed in these isolates matched well with those of the respective genera.
Our study showed intra-phylum evidence for HGT in three other instances. The isolate KMSZP5 was identified as Bacillus thuringiensis based on its 16S rRNA gene sequence ( Figure 1A) whereas when analysed for its zntA/cadA/pbrA-like gene sequences, it exhibited good similarity (94%) with the cadmium-transporting ATPase gene sequence of Lysinibacillus sphaericus C3-41 (Table 1) and clustered with Lysinibacillus sp. (Figure 1B). The proposition of HGT in the present analysis was established with incongruence between 16S rRNA and ATPase phylogenies. Additionally, the G+C content of the KMSZP5 P IB -type ATPase gene was higher (39.8 mol%) than the genomic G+C content of Bacillus thuringiensis i.e. 34-35.5 mol% (available at www.ncbi.nlm.nih.gov/genomes/ MICROBES). The isolate LONG2 clustered with Bacillus halmaplus based on 16S rRNA gene phylogeny ( Figure 1A) but the same isolate branched with Geobacillus kaustophilus HTA426 using zntA/cadA/pbrA-like sequences with 100% bootstrapping ( Figure 1B). The phylogenetic incongruence (Figure 1) and the higher G+C content at 45 mol% (considerably higher than the expected 38.6 mol% G+C content of Bacillus halmapalus), provided strong evidence for the acquisition of the P IB -type ATPase gene through HGT. We confirmed our observations by comparing the mol% G+C content for both species of Bacillus as the range for genomic mol% G+C content for the genus Bacillus is known to be very wide (32-66%) [35]. The isolate OT6, identified by us as Pseudomonas koreensis by 16S rRNA phylogeny, possessed zntA/cadA/pbrA-like gene sequences that showed good similarity (92%) with the copper-transporting P IB -type ATPase gene of the complete genome of Providencia rettgeri (NCBI reference sequence, ZP_06125459.2) with a 100% bootstrap support ( Figure 1B). This observation was unaltered even on inclusion of another copper transporting ATPase gene of Pseudomonas. The observed mol% G+C content for the isolate OT6 was found to be much lower (48.1%) than is expected for Pseudomonas sp. (58-70 mol%) [36]. Thus, in the present investigation, the occurrence of HGT could be demonstrated in six instances (3 inter-phylum and 3 intra-phyla) which were supported by both phylogenetic incongruence and unusual mol% G+C contents.

Discussion
Bacterial isolates from Domiasiat site displayed remarkable tolerance towards U and other co-occurring heavy metals. The MICs of the isolates towards the tested metals were found to be 2-4 fold higher than those of the respective type strains and as reported for E.coli [37]. Among the tested metals, Cd and Pb were found to be the most toxic metals in our study, which is in agreement with earlier findings [37][38][39][40]. The ability of our subsurface natural isolates of Domiasiat to tolerate and survive against toxic concentrations of U and other heavy metals made us to explore the presence of P IB -type ATPases. Further, we analysed the possible dissemination of P IB -type ATPase genes through HGT among the natural isolates as HGT was found to be a common occurrence in previously explored metal contaminated sites [17,18].
In the present study, 32 natural isolates showed a positive signal for a P IB -type ATPases gene sequence using gene-specific PCR primers [16]. We obtained zntA/cadA/pbrA-like loci from Bacteroidetes (Sphingobacterium sp. and Chryseobacterium sp.) and Firmicutes (Lysinibacillus sp. and Paenibacillus sp.) which deviated from the earlier reported genera [17,18]. In the case of Lysinibacillus sp. and Paenibacillus sp., this might be explained by the fact that the amplification primers were designed to target P IB -type ATPase genes of Bacillus sp. (another genus of Firmicutes). Further, it was observed that PCR amplification of P IB -type ATPase genes in Arthrobacter sp. (RSBA2) and Pseudomonas sp. (OT6) showed copAlike loci instead of the expected zntA/cadA/pbrA-like loci as reported earlier [18]. Possibly, these deviations might be due to the limited number of control strains used earlier for designing the initial primer sets for P IB -type zntA/cadA/pbrA-specific ATPases [16]. In our study, phylogenetic incongruence was observed in six isolates which was also supported by the aberrant G+C contents of their amplified genes. In three instances, acquisition of zntA/cadA/ pbrA-like loci by Bacteroidetes (KMSDrP1, KMSDrP1, and KMSDrP1) from Firmicutes (PSS2) was observed indicating an inter-phylum transfer. The other three isolates KMSZP5, LONG2 and OT6 showed evidences of intra-phylum gene transfer. It is proposed that phylogenetically close microbes were more likely to exchange their genetic material [14] with less likelihood of unveiling the molecular footprint of this transfer in their genomes [41]. Similar results were obtained in our studies where closely related phylogenetic groups were isolated using conditioned enrichment based cultivation method targeting uranium tolerant populations. We might have underestimated the magnitude of HGT of the zntA/cadA/pbrA-like loci among the natural isolates as the diversity at the genus level in our study was low. Similar observations were shown by Coombs and Barkay [17] who suggested the use of variety of selection and enrichments methods to obtain diverse isolates for achieving better clarity of HGT in a given environment. With the advent of metagenome and metaproteome applications in microbial ecology and the creation of databases relating 16S rRNA genes and functional genes in uncultured microbes [42][43][44][45], investigating HGT events among uncultured members of the microbial community should be more feasible in the near future.
Although it is not clear whether HGT events reported in this study occurred before or after U ore mineralization in the selected area, our results clearly indicate the evolution of an ecologically important phenotype suggesting the dissemination of horizontally acquired P IB -type ATPase genes among Firmicutes, Bacteroidetes, and Proteobacteria phyla. This study provides an important insight into HGT of P IB -type ATPase genes among natural isolates of the U rich deposit located in Domiasiat in North East India. Figure S1 Phylogenetic analysis of CopA-like cluster of isolates OT6 and RSBA2. Neighbor-joining cluster analyses were performed for the PCR amplified gene sequences of CopAlike loci from the isolates OT6 and RSBA2 with the gene sequences from complete genomes identified by their gene sequence similarity from NCBI GenBank using BLASTX. Phylogenetic clustering showed that CopA-like loci from OT6 and RSBA2 belonged to the CopA1-gene cluster. The scale bars indicate 0.1 change per amino acid position for P IB -type ATPase phylogeny. (PDF)