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Biologia 66/1: 18—26, 2011 Section Cellular and Molecular Biology DOI: 10.2478/s11756-010-0145-0 The isolation of heavy-metal resistant culturable bacteria and resistance determinants from a heavy-metal-contaminated site Edita Karelová, Jana Harichová, Tatjana Stojnev, Domenico Pangallo & Peter Ferianc* Institute of Molecular Biology, Slovak Academy of Sciences, Dúbravská cesta 21, SK-84551 Bratislava, Slovakia; e-mail: [email protected] Abstract: In this study we performed a phylogenetic analysis of a culturable bacterial community isolated from heavymetal-contaminated soil from southwest Slovakia using 16S rRNA (16S rDNA) and heavy-metal resistance genes. The soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg), smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg), and a trace amount of cadmium (<0.25 mg/kg). A total of 100 isolates were grown on rich (Nutrient agar No. 2) or minimal (soil-extract agar medium) medium. The isolates were identified by phylogenetic analysis using partial sequences of their 16S rRNA (16S rDNA) genes. Representatives of two broad taxonomic groups, Firmicutes and Proteobacteria, were found on rich medium, whereas four taxonomic groups, Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria, were represented on minimal medium. Forty-two isolates grown on rich medium were assigned to 20 bacterial species, while 58 bacteria grown on minimal medium belonged to 49 species. Twenty-three isolates carried czcA- and/or nccA-like heavy-metal-resistance determinants. The heavy-metalresistance genes of nine isolates were identified by phylogenetic analysis of their protein sequences. Key words: bacterial community structure; cultivation-dependent approaches; isolation of until now uncultured bacteria; heavy-metal-contaminated soil; heavy-metal resistance genes. Abbreviations: 16S rRNA, 16S ribosomal RNA; 16S rDNA, 16S ribosomal DNA; czcA, heavy-metal-resistance determinant; MM, minimal medium; NCBI, National Center for Biotechnology Information; nccA, heavy-metal-resistance determinant; RM, rich medium. Introduction The use of microbial communities to assess the impact caused by anthropogenic stress in natural habitats is increasing; however, there is considerable debate as to which approach is the most useful (Chapman 1999). Culture-independent methods have received particular attention because it is commonly known that only a small proportion of the bacteria present in any given environment will form colonies on general laboratory media (Hugenholtz 2002), but on the other hand, the traditional microbiological methods directly provide live bacteria, and not merely a “molecular strain”. Heavy metals are highly persistent in the environment and are known to alter soil ecosystem diversity, structure and function (Sandaa et al. 2006; Ashraf & Ali 2007). While the acute effect of heavy metals on the microbial community appears to lead to a subsequent shift in the community toward a more metaltolerant or metal-resistant population (Ranjard et al. 2000; Sandaa et al. 2001), in chronically contaminated sites natural selection should have resulted in a pre- dominantly metal-tolerant community (Kandeler et al. 2000; Becker et al. 2006). Many studies have focused on the effects of heavy metals on bacterial community structure (Ranjard et al. 2006; Ogilvie & Grant 2008; Khan et al. 2010; Pechrada et al. 2010), and relatively many bacteria have already been isolated from different heavy-metal-contaminated environments and their metabolic pathways for pollutant detoxification have been studied in detail. These studies included the mercury-reducing bacteria Bacillus megaterium MB1 (Huang et al. 1999); the cadmium-accumulating bacteria Rhodospirillum rubrum (Smiejan et al. 2003); strains resistant to multiple heavy metals (Schmidt & Schlegel 1994; Taghavi et al. 1994; Mergeay et al. 2003); bacteria specific to HgCl2 -contaminated soils, such as Duganella violaceinigra, Lysobacter koreensis and Bacillus panaciterrae (Mera & Iwasaki 2007); cadmium-resistant bacteria, such as Alcaligenes xylosoxidans, Comamonas testosteroni, Klebsiella planticola, Pseudomonas fluorescens and Serratia liquefaciens (Chovanová et al. 2004); Ochrobactrum sp. (Pandey et al. 2010); a Ralstonia pickettii strain, highly * Corresponding author c 2011 Institute of Molecular Biology, Slovak Academy of Sciences Unauthenticated Download Date | 8/12/17 7:49 PM Isolation of culturable bacteria and resistance determinants resistant to cadmium, and a Sphingomonas sp. strain, highly resistant to zinc (Xie et al. 2010); Aeromonas spp. and Pseudomonas spp., both highly resistant to copper (Matyar et al. 2010); and Geobacter daltonii sp. nov., an Fe(III)- and uranium(VI)-reducing bacterium (Prakash et al. 2010). The most well-characterized operons conveying resistance against heavy metals in Gram-negative bacteria are the czc operon from Cupriavidus metallidurans CH34 (Mergeay et al. 2003) and the ncc system from Achromobacter xylosoxidans 31A (Schmidt & Schlegel 1994). In Gram-positive bacteria, the cad operon from Bacillus and Staphylococcus members has been well studied (Silver & Phung 1996). In both Grampositive and Gram-negative bacteria the ars operons from Escherichia coli (Mobley et al. 1983; Saltikov & Olson 2002) and Staphylococcus strains (Ji & Silver 1992; Rosenstein et al. 1992), and the mer systems from Escherichia coli (Nascimento & Chartone-Souza 2003) and Bacillus populations (Bogdanova et al. 1998) have been well characterized. In addition, the cyanobacterial smt locus from Synechococcus PCC 7942 also contains a well-characterized heavy metal resistance system (Erbe et al. 1995). In the present study, a phylogenetic analysis was performed to examine both the culturable bacterial community and the heavy-metal resistance genes carried by the bacteria in this community in an anthropogenically disturbed soil sample using DNA solutions extracted from independently grown bacterial isolates on two different culture media. We have also tried to isolate new and heretofore uncultured bacteria carrying previously unknown resistance determinants to toxic metals. Material and methods Field site, soil samples and heavy metal content measurement Soil samples, down to 10 cm depth, were collected from farmland near the town of Sereď (48◦ 16 59 N, 17◦ 43 35 E) in southwest Slovakia. The sampling site was situated near by a dump containing heavy-metal-contaminated waste. The soil sample contained high concentrations of nickel (2,109 mg/kg), cobalt (355 mg/kg) and zinc (177 mg/kg); smaller concentrations of iron (35.75 mg/kg) and copper (32.2 mg/kg); and also a trace amount of cadmium (<0.25 mg/kg). The content of these heavy metals in the soil sample was measured using an atomic absorption spectrometer (Perkin-Elmer model 403, USA). Preparation of soil-borne bacterial suspension A 10 g portion (wet weight) of the soil was mixed in a sterile 250 mL Erlenmeyer flask with 90 mL of a 0.85% (w/v) salt solution and incubated at 30 ◦C in a shaker incubator at 90 rpm for 2 h. The suspensions obtained were filtered through Whatman 1 filter paper (Merck, Germany) under sterile conditions, and these filtered bacterial suspensions were used for further work. 19 Isolation of bacteria Subsamples (1.0 mL) withdrawn from the soil-borne bacterial suspension were serially diluted (in range: 10−1 –10−6 ) and each dilution was plated in triplicate on either nutrient agar No. 2 (Rich Medium, RM; Biomark, India) or soilextract agar medium (Minimal Medium, MM) containing 500 mL/L soil extract and 15 g/L agar. The pH of the final medium was adjusted to 7.2. If appropriate, 50 mg/L of actinomycin D was added to the medium to preclude the growth of fungi (Hamaki et al. 2005). The soil extract was prepared by mixing 1,000 g of soil with 2 L of 50 mM NaOH and incubating overnight at room temperature. The mixture was filtrated and then centrifuged for 60 min at 18,000 rpm. The supernatant was sterilized repeatedly over a period of 3 days at 100 ◦C in steam. The plates were incubated aerobically at 30 ◦C for either 24–48 h or 1–2 weeks (the numbers of CFUs were repeatedly counted to insure that, at the time of isolation, the appearance of new colonies had leveled off). Independently growing colonies were randomly selected (on the basis of morphology) for further analysis. DNA extraction Bacterial DNA from soil isolates and from Cupriavidus metallidurans CH34 (BCCMT M /LMG 1195) cells was isolated using the DNeasy purification kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The resulting high-molecular-weight DNA was stored at −20 ◦C and was used as a template in appropriate PCR experiments. Detection of 16S rRNA (16S rDNA), czcA- and nccA-like genes DNA extracted from soil bacterial isolates and C. metallidurans CH34 (BCCMT M /LMG 1195) was used in PCR either with universal 16S rRNA gene primers or with non-specific degenerated czcA and nccA primer sets (Table 1). Each 50 µL reaction mixture contained 2 µL of the DNA template, 5 µL 10×Taq buffer (Qiagen, Hilden, Germany), 2.5 U Taq DNA polymerase (Hot-Star; Qiagen, Hilden, Germany), 1.5 mM MgCl2 , 200 µM deoxynucleotide triphosphates and 0.5 µM of each primer. PCRs were performed in a T1 thermal cycler (Biometra, Goettingen, Germany) with the following cycling conditions: 2 min of denaturation at 94 ◦C, 25 cycles of 1 min at 53 ◦C(16S rRNA), 50 ◦C (czcA) or 59 ◦C (nccA), 1.5 min (16S rRNA), 2 min (czcA) or 1 min (nccA) at 72 ◦C, 1 min at 94 ◦C, and a final cycle of extension at 72 ◦C for 5 min. PCR products were separated by electrophoresis in a 1% (w/v) agarose gel (Merck, Germany) and stained with Gold View Nucleic Acid Stain (SBS Genetech Co., Ltd., China). DNA bands, approximately 696, 822 or 581 bp in size for the 16S rRNA, czcA and nccA genes, respectively, were excised and purified using the QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. After PCR amplification, PCR products were prepared using a BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, USA) in accordance with the manufacturer’s protocol and then sequenced using an ABI 3130 XL DNA Analyser (Appied Biosystems, Foster City, USA). The sequenced PCR products of the czcA and nccA genes from C. metallidurans CH34 were used as positive controls in PCR-detection of czcA- and nccA-like genes. Bacterial strain identification and phylogenetic analysis Specific 16S rRNA (16S rDNA) sequences were edited with Chromas Lite software (version 2.01) for further DNA analysis. A BLAST search (Altschul et al. 1990) at the NCBI Unauthenticated Download Date | 8/12/17 7:49 PM 20 genome database server (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) was conducted to identify the nearest neighbors. Multiple alignments were generated with the CLUSTALW2 program (Thompson et al. 1994) from the EMBL Nucleotide Sequence Database at the EBI server (http://www.ebi.ac. uk/Tools/clustalw2/), and phylogenetic trees were displayed with the TreeView software (Page 1996) on the basis of the evolutionary distances calculated by the neighborjoining method (Saitou & Nei 1987) using Kimura’s twoparameter model (Kimura 1980). Identification of czcA- and nccA-like gene products and phylogenetic analysis After trimming of specific czcA- and nccA-like sequences with Chromas Lite software (version 2.01), nucleotide sequences were translated to protein sequences by using the ExPASy proteomics server (http://www.expasy.ch/tools/ dna.html). A BLAST search of the NCBI protein databases was conducted to identify the nearest czcA- and nccA-like gene products. Multiple alignments were generated with the CLUSTALW2 program from the EMBL Protein Sequence Databases (Swiss-Prot and TrEMBL) of the EBI server, and phylogenetic trees were displayed with the TreeView software on the basis of the evolutionary distances calculated by the neighbour-joining method (Saitou & Nei 1987) using Kimura’s two-parameter model (Kimura 1980). Nucleotide sequence accession numbers The sequences generated in this study have been deposited with the GenBank database (Benson et al. 2010) under the accession numbers GU935266–GU935334 for bacterial 16S rRNA (16S rDNA) genes and GU935257–GU935265 for czcA- and nccA-like genes. Results and discussion Structure of culturable bacterial community A phylogenetic analysis was performed to determine the structure of the culturable bacteria using partial sequences of the 16S rRNA (16S rDNA) genes. Bacteria were isolated from heavy-metal-contaminated farmland soil in southwest Slovakia. The specific 16S rDNA PCR products derived from a total of 100 bacterial isolates grown either on a rich (RM) or a minimal (MM) medium were subsequently sequenced. The sequences of bacteria growing on RM fell into two broad taxonomic groups (Figs 1, 2). Most bacteria (17 species) were classified as Proteobacteria, but three species were classified as Firmicutes. The phylum Firmicutes included only six representatives, while the phylum Proteobacteria included up to 36 members. In addition, three, one, and thirteen bacterial species from Proteobacteria could be classified as belonging to the three subgroups α-, β- and γ-Proteobacteria, respectively. The sequences of bacteria grown on MM were distributed into four broad taxonomic groups (Figs 1, 2). The majority of them were classified as Proteobacteria or Actinobacteria; from a total of 49 identifiable species, the former phylum included 17 species and 22 representatives and the latter, 15 species and 18 representatives. Nine species were classified as Firmicutes and the remaining eight as Bacteroidetes. Both of these phyla were represented by nine isolates. Representatives E. Karelová et al. of the phylum Proteobacteria were assigned to three subgroups, α-, β- and γ-Proteobacteria which were represented by two, eight and seven species, respectively. The most abundant subgroup on both cultivation media was γ-Proteobacteria followed by β-Proteobacteria and α-Proteobacteria. The predominance of Proteobacteria generally and the γ-Proteobacteria in particular is not surprising. The low occurrence of α- and β-Proteobacteria, and the absence of δ-Proteobacteria are common phenomena in plated communities, probably because the members of these groups are generally slow-growing (Mitsui et al. 1997), or may have specific physiological requirements and therefore may have been lost during sub-culturing (Ellis et al. 2003). The choice of culture medium appears to be very important for a survey of the structure and diversity of the culturable part of the bacterial community. This fact is underlined mainly by the significant differences observed in the occurrence of the growing isolates on both culture media and their distribution into broad taxonomic groups. It seems evident that MM generates more appropriate conditions for the growth of a wide variety of natural bacterial isolates than RM does, suggesting that MM offers conditions more similar to those found in the environmental sample than is offered by RM. Furthermore, these results also suggest that the use of both types of culture media provided a better picture of the structure and diversity of the culturable part of the microbial communities, because this approach allows the cultivation of bacterial strains with different growth requirements, enabling a wide variety of natural bacteria to be isolated than would be possible using only one type of medium. However, the importance of a nutritionally poor medium for the cultivation and isolation of unculturable microorganisms must be emphasized. Conventional culture media are nutritionally rich and their use for the cultivation and isolation of microorganisms from environmental samples renders the majority of microorganisms unculturable. By using nutritionally poor media, 93 (27%) of 350 isolated microorganisms belonging to 20 as yet unnamed family level groupings have been successfully cultivated (Joseph et al. 2003). Identification of bacterial isolates All isolated bacteria were identified based on their nearest bacterial relatives using phylogenetic analysis. From our soil sample, 68 isolates were assigned to known species or genera and 18 isolates were identified as belonging to known but unculturable bacteria with sequence similarity ranging between 79–99%. Both the new cultivable bacterial isolates and the known cultivable strains with low similarity to their close relatives were considered to belong to new species or genera (Fig. 1). Only nine of the actinomycete strains from 18 isolates could be assigned to an already known species or genus; the remaining nine individuals are thought to belong to a new species or genus (Fig. 1). More specifically, isolates EK-I73, EK-I74, EK-I76, EKUnauthenticated Download Date | 8/12/17 7:49 PM Isolation of culturable bacteria and resistance determinants 21 EK-I2 [GU935267], RM Pseudomonas jessenii, strain PS06 [AY206685], 99% EK-I1 [GU935266], RM Uncultured gamma proteobacterium clone C-CU62 [AY622234], 99% EK-I3-6 [GU935268], RM EK-I7 [GU935269], RM Bacillus pumilus, strain BPT-18 [EF523475], 100% Bacillus licheniformis, strain BPT-18 [X68416], 100% EK-I43 [GU935286], MM Bacillus sp., strain LMG-19415 [AJ276809], 97% EK-I65 [GU935301], MM EK-I66 [GU935302], MM Bacillus fusiformis, strain SW-B9 [AY907676], 98% Lysinibacillus fusiformis, strain X-25 [EU187498], 98% EK-I67 [GU935303], MM Bacillus cereus, strain IBL01058 [EU168407], 99% EK-I93 [GU935328], MM Bacillus niabensis isolate BRL02-13 [DQ339647], 99% EK-I94 [GU935329], MM Uncultured bacterium clone SBK_d10_1_1 [EU778785], 97% EK-I88 [GU935323], MM Staphylococcus sp. Ver2 [AM778685], 98% EK-I15 [GU935272], RM Staphylococcus epidermidis, strain RP62A [CP000029], 98% EK-I53 [GU935293], MM Carnobacterium sp. H126a [EF204312], 94% EK-I90 [GU935325], MM Uncultured bacterium clone THM-4 [AY147274], 95% EK-I81 [GU935317], MM EK-I44-47 [GU935287], MM EK-I97 [GU935332], MM Arthrobacter sp. 1663 [EU086792], 98% Arthrobacter sp. ZY007 [EU652876], 99% EK-I89 [GU935324], MM Streptomyces sp. LS12 [EU721604], 99% EK-I92 [GU935327], MM Streptomyces sp. M1019 [EU876684], 99% EK-I50 [GU935290], MM Actinomycetales bacterium HPA19 [DQ144224], 99% EK-I91 [GU935326], MM Uncultured bacterium clone AKAU3875 [DQ339647], 99% EK-I79 [GU935315], MM EK-I82 [GU935318], MM Uncultured bacterium clone CZ52H03 [EF507108], 79% Uncultured bacterium clone FFCH10433 [EU132625], 94% Pseudonocardia halophobica, strain IMSNU 21327T [AJ252827], 94% EK-I48 [GU935288], MM Uncultured bacterium clone CZ51F07 [EF507054], 98% EK-I76 [GU935312], MM EK-I73 [GU935309], MM EK-I80 [GU935316], MM Glycomyces sp. IM-0594 [AF131369], 88%, 93% Glycomyces algeriensis [AY462044], 96%, 99% Glycomyces illinoisensis [AY462043], 95% EK-I74 [GU935310], MM EK-I86 [GU935321], MM EK-I16-17 [GU935273], RM Acinetobacter baumannii N2S4 [EU221350], 99% EK-I78 [GU935314], MM EK-I32 [GU935280], RM Pseudomonas alcaligenes [AF094721], 99% EK-I68 [GU935304], MM Haloanella gallinarum, strain 020-3.3-CV-A-02 [EF010560], 85% Uncultured Chryseobacterium sp. clone ChsS5 [AY621824], 82% Uncultured Bacteroidetes bacterium JL-ETNP-Z65 [AY726972], 79% EK-I77 [GU935313], MM EK-I71 [GU935307], MM EK-I72 [GU935308],MM EK-I70 [GU935306], MM Bacterium EK-I70 [GU935306], 100% EK-I51 [GU935291], MM Sphingobacterium sp. P-17 [AM411963], 98% EK-I83-84 [GU935319], MM Uncultured bacterium, clone 17RHF23 [AJ863366], 97% EK-I85 [GU935320], MM EK-I30 [GU935278], RM EK-I24 [GU935275], RM Agrobacterium tumefaciens, strain A2P3 [EU221409], 99% Ochrobactrum anthropi, type strain LMG 3331T [AM114398], 99% Sphingomonas sp. 47 [EF016512], 86% Sphingomonas sp. SMCC B0618 [EU442226], 93% EK-I33-35 [GU935281], RM Sphingomonas paucimobilis, strain 20006FA [DQ400860], 98% EK-I69 [GU935305], MM EK-I75 [GU935311], MM EK-I18-23 [GU935274], RM Burkholderia cepacia, strain AN 2.9 [EU156336], 99% EK-I54 [GU935294], MM EK-I62 [GU935298], MM EK-I49 [GU935289], MM Duganella violaceusniger, isolate CP177-4 [AJ871470], 98% Uncultured organism clone M8907D07 [AY898078], 99% Beta proteobacterium 8c-1 [AY561542], 98% EK-I61 [GU935289], MM Oxalobacteraceae bacterium EK-I61 [GU935297], 100% EK-I98 [GU935333], MM EK-I95 [GU935330], MM EK-I96 [GU935331], MM EK-I99-100 [GU935334], MM Variovorax sp. isolate PBD-H1 [AM502919], 85% Uncultured beta proteobacterium clone Cl-13-TB4-II [AY599735], 83% Variovorax sp. VC-YC6672 [EU734636], 87% Variovorax paradoxus strain A11 [AM882682], 97% EK-I8 [GU935270], RM Acinetobacter rhizosphaerae, strain BIHB 723 [DQ536511], 99% EK-I87 [GU935322], MM Uncultured Acinetobacter sp. clone 2P-4-1-C01 [EU705405], 92% EK-I38 [GU935283], RM Moraxella lacunata [AF005170], 98% EK-I26-29 [GU935277], RM Moraxella caprae [DQ156148], 99% EK-I36-I37 [GU935282], RM Moraxella lincolnii, strain CCUG 9405T [AJ417490], 99% EK-I39 [GU935284], RM Psychrobacter immobilis, strain DSM 7229T [AJ309942], 99% EK-I25 [GU935276], RM EK-I31 [GU935279], RM Aeromonas salmonicida ssp. masoucida, strain Fin 3 [AM296506], 98% Aeromonas sobria, strain NCIMB 12065 [X60412], 99% EK-I52 [GU935292], MM Pseudomonas sp. P97.26 [AY456707], 98% EK-I63 [GU935299], MM EK-I58-60 [GU935296], MM EK-I55-57 [GU935295], MM Pseudomonas sp. STATEMENT 368 [AJ968715], 82%, 91%, EK-I64 [GU935300], MM Pseudomonas sp. EK-I64 [GU935300], 100% EK-I9-14 [GU935271], RM Pseudomonas luteola, strain ATCC 43330 [D84003], 99% EK-I40-42 [GU935285], RM Pseudomonas fluorescens, isolate TC222 [DQ402053], 99% 0.1 Fig. 1. Neighbor-joining tree showing phylogeny of 16S rRNA (16S rDNA) gene sequences from heavy-metal-contaminated soil isolates growing either on rich (RM) or minimal (MM) medium (in bold). Numbers in square brackets indicate the GenBank accession number and similarity to closest relative is shown after the clone designation. Sequences were aligned with ClustalW. Bar indicates 10% sequence divergence. Aligned sequences were 696 bp in length. Unauthenticated Download Date | 8/12/17 7:49 PM E. Karelová et al. 22 TCB RM MM α β γ TCB RM MM 60 60 50 Mean abundance, % Mean abundance, % 50 40 30 20 40 30 20 10 10 0 0 Proteobacteria Taxonomic groups Fig. 2. Mean relative abundance distribution of broad taxonomic groups (right) and Proteobacteria (left) within the collection of isolates (n = 100), from heavy-metal-contaminated soil sample, growing either on rich (RM) or minimal (MM) medium. TCB indicates total cultivable bacteria. I82, and EK-I86 exhibited low similarity (88–96%) to their closest relatives AY462044, AY462043, AF131369, AJ252827 and AF131369, respectively. Similarly, isolates EK-I81 and EK-I79 also showed low similarity (79–94%) to their closest relatives EF507108 and EU132625. The final isolates EK-I48 and EK-I91 exhibited high similarity (98 and 99%) to the uncultured bacterium clones EF507054 and DQ125761, respectively. The taxonomic group Bacteroidetes was represented by 9 individuals, but only one isolate, EK-I51 could be assigned to a known genus, Sphingobacterium sp. (98% AM411963). The remaining eight individuals are thought to form a new species or genus (Fig. 1). Bacteria belonging to the large taxonomic groups Firmicutes and Proteobacteria were isolated from both culture media. The Firmicutes isolates EK-I90 and EK-I94 are probably members of new genus, because they displayed only low similarity (95–97%) to two uncultured bacterium clones AY147274 and EU778785. Thirty-six bacterial strains belonging to Proteobacteria, which were isolated on RM, were assigned to 17 species belonging to 9 genera and 1 uncultured bacterium clone. Twenty-one individuals recovered on MM were assigned to 17 species belonging to 6 genera and 4 uncultured bacteria clones (Fig. 1). However, great discrepancies were found among bacteria grown on both the media types used here and assigned to the subgroups of α-, β- and γ-Proteobacteria. Our results are similar to the findings of Mera & Iwasaki (2007). They isolated representatives of Actinobacteria – Arthrobacter globiformis, Arthrobacter nicotinovorans and Streptomyces avellaneus from HgCl2 -contaminated soil, as well as one representative of Bacteroidetes (Chryseobacterium soldanellicola), a few representatives of Bacillus spp., one species of β- Proteobacteria (Duganella violaceusnigra), and also one species of γ- (Lysobacter koreensis). One difference is that our isolate EK-I71, which was classified as uncultured Chryseobacterium sp. AY621824, will most likely belong to a new species, since it exhibits only low similarity (82%) to its closest relative. Occurrence and sequence analysis of czcA- and nccAlike genes in bacterial isolates At present, the effects of nickel, cobalt and zinc on the majority of the isolates identified in the present study is unknown, and only partly known for iron, copper and cadmium. In this situation, our results can give new information about bacterial resistance toward these heavy metals. All isolates from both media types were able to grow and survive sub-culturing in the presence of cobalt, nickel and cadmium. The isolates were screened for the presence of two genes coding for the heavy-metal-resistance determinants, czcA and nccA, by PCR-based methods using degenerative czcA and nccA primer sets (Table 1). The degenerative primer sets were designed using conserved gene fragments of Gram-negative bacteria: fragments from Ralstonia metallidurans CH34, pMOL30 [NC 006466] were used for czcA, whereas those from Alcaligenes xylosoxidans 31A [L31363] for nccA. The primers czcA or nccA amplified PCR-products of about 822 and 581 bp, respectively. Only 23 isolates from a total of 100 isolated bacteria carried at least one of the PCR bands of the expected size for the heavy-metal-resistance genes (Table 2). The PCR band corresponding to the nccA-like gene was the more abundant one; it was present alone in 11 isolates compared to the PCR band corresponding to the czcA-like gene, which was present alone in Unauthenticated Download Date | 8/12/17 7:49 PM Isolation of culturable bacteria and resistance determinants 23 Table 1. Primer sets used in this study. a Probes Sequence Descriptiona 27F 5’-AGAGTTTGATCCTGGCTCAG-3’ 16S rDNA universal primers, positions 8–27 and 704–685 in the Escherichia coli K12 [NC 000913] numbering system; (Lane 1991). 685R 1226czcAF 5’-TCTACGCATTTCACCGCTAC-3’ 5’-GACTTCGGCATCATC(A,G)TCGA(T,C)GG-3’ 2026czcAR 2555nccAF 5’-CGTTGAA(G,C)CGCA(G,A)CTGGATCGG-3’ 5’-AGCCG(C,G)GA(C,G)AACGGCAAGCG-3’ 3117nccAR 5’-CCGATCACCACCGT(T,C)GCCAG-3’ 1204–1226 and 2047–2026 degenerative czcA primer positions on plasmid pMOL30 in the Cupriavidus metallidurans CH34 [NC 006466] numbering system; this work. 2536–2555 and 3136–3117 degenerative nccA primer positions on plazmid p9 in the Achromobacter xylosoxidans 31A [L31363] numbering system; this work. Numbers in parenthesis indicate the GenBank accession number. Table 2. Presence of heavy-metal-resistance determinants in bacterial isolates and assignment of some bacterial nccA-like soil systems to the closest identified match in the GenBank database. PCR bandsc Soil isolates Acc. No.a EK-I1–hmr GU935257 EK-I2-hmr GU935258 EK-I3 EK-I4 EK-I5 EK-I6 EK-I8 EK-I9 EK-I15 EK-I16 EK-I17 EK-I18 EK-I19 EK-I45–hmr GU935259 EK-I46-hmr GU935260 EK-I49 EK-I51-hmr GU935261 EK-I52-hmr GU935262 EK-I59-hmr GU935263 EK-I61-hmr EK-I64-hmr GU935264 GU935265 EK-I99 EK-I100 Affiliation of nccA-like soil systemsb Protein of unknown function DUF6, transmembrane, Pseudomonas mendocina ymp, [YP001187314], 40% Transcriptional regulator, LysR family, Pseudomonas fluorescens PfO-1, [YP346150], 93% Hypothetical protein GL50803 6582, Giardia lamblia ATCC 50803, [XP 001709060], 42% NLP/P60 protein, Arthrobacter chlorophenolicus A6, [ZP 02837928], 55% Hypothetical protein ECA0431, Erwinia carotovora subsp. atroseptica SCRI1043, [YP 048549], 63% Probable cation efflux system transmembrane protein, Ralstonia solanacearum GMI1000, [NP 522054], 76% Heavy metal efflux pump CzcA, Alteromonas macleodii ‘Deep ecotype’, [ZP 01109838], 53% Transporter, Agrobacterium tumefaciens str. C58, [AAK88967], 67% Heavy metal efflux pump CzcA, Azotobacter vinelandii AvOP, [ZP 00415898], 80% czcAd nccAe + + + + + + – + + + – + + + – – – + + + + – + + + + + + – + – – + + – + – + – – + + + + – – a Accession number of identified nccA-like genes. Clone assignment of bacterial nccA-like gene products to the closest identified match in the GenBank database. Numbers in square brackets indicate the GenBank accession numbers and the level of identity is shown after the clone designation. c Presence of PCR bands of expected size for czcA- and nccA-like genes. All isolates were tested for the presence of PCR bands of expected size for both, czcA- and nccA-like genes. Only strains positive in PCR band of expected size for any resistance gene are listed in the table. (+) presence or (–) absence of PCR bands for investigated heavy-metal-resistance determinants. d DNA extracted from Cupriavidus metallidurans CH34 [NC 006466] was used as template for czcA-labeled amplicon by using nonspecific degenerated czcA primer set. e DNA extracted from Cupriavidus metallidurans CH34 [NC 006466] was used as template for nccA-labeled amplicon by using nonspecific degenerated nccA primer set. b Unauthenticated Download Date | 8/12/17 7:49 PM E. Karelová et al. 24 NccA-like [EK-I45] NccA-like [EK-I1] Protein of unknown function DUF6, transmembrane [Pseudomonas mendocina ymp] [YP001187314], 40% NccA-like [EK-I59] Heavy metal efflux pump CzcA [Alteromonas macleodii ‘Deep ecotype’] [ZP01109838], 53% NccA-like [EK-I52] Probable cation efflux system transmembrane protein [Ralstonia solanacearum GMI1000] [NP522054], 76% NccA-like [EK-I64] Heavy metal efflux pump CzcA [Azotobacter vinelandii AvOP] [ZP00415898], 80% CzcA-like [EK-I2] Transcriptional regulator, LysR family [Pseudomonas fluorescens PfO-1] [YP346150], 93% Hypothetical protein GL50803_6582 [Giardia lamblia ATCC 50803] [XP001709060], 42% NccA-like [EK-I46] NLP/P60 protein [Arthrobacter chlorophenolicus A6] [ZP02837928],55% NccA-like [EK-I61] Transporter [Agrobacterium tumefaciens str. C58] [AAK88967], 67% 0.1 NccA-like [EK-I51] Hypothetical protein ECA0431 [Erwinia carotovora subsp. atroseptica SCRI1043] [YP048549], 63% Fig. 3. Neighbor-joining tree showing phylogeny of partial either czcA-like sequences based on 274 derived amino acid sites or nccA-like sequences based on 193 derived amino acid sites cloned directly from heavy-metal-contaminated soil isolate DNA (in bold). Numbers in square brackets indicate the GenBank accession number and similarity to closest relative is shown after the clone designation. Sequences were aligned with ClustalW. Bar indicates 10% sequence divergence. only 4 bacteria. The simultaneous presence of PCR bands corresponding to both of these genes was found in 8 bacterial isolates. The strains harboring heavymetal-resistance elements were grown either on RM (13 isolates) or on MM (8 individuals). The distribution of heavy-metal-resistance genes varied by taxonomic group and respective bacterial species; these genes were present mainly in the γ-Proteobacteria isolates. The fact that the presence of at least one of the PCR bands corresponding to one of the resistance determinants could be confirmed only in 23% (23 isolates) of all analyzed bacteria (Table 2) may be partly because the degenerative primer sets used were designed only for conserved gene fragments from Gramnegative bacteria. On the other hand, both primer sets were designed to be degenerative, suggesting that they should have the ability to hybridize also with other, similar genes. For example, the primers for czcA also gave positive results with czcA from Ralstonia sp. CH34 [X98451], Alcaligenes sp. [D67044], Ralstonia solanacearum GMI1000, megaplasmid [AL646079], Pseudomonas aeruginosa PAO1 [AE004679], Pseudomonas putida KT2440 [AE016774, AE016783], czrA from Pseudomonas aeruginosa [Y14018], Pseudomonas putida strain P111 [AY026914], and cztA from Pseudomonas fluorescens ATCC 13525 [AY007258]. Similarly, the primers for nccA also gave positive results for nccA from Ralstonia metallidurans CH34, pMOL30 [X71400], czcA from Pseudomonas aeruginosa PAO1 [AE004679], czrA from Pseudomonas aeruginosa [Y14018] and Pseudomonas putida strain P111 [AY026914], and cztA from Pseudomonas fluorescens ATCC 13525 [AY007258]. It is therefore not surprising that the genes examined were found mainly in bacterial species belonging to the γ-Proteobacteria, in the Pseudomonas isolates especially, and that the presence of heavy-metal-resistance determinants could not be confirmed in the majority of Gram-positive bacteria. To identify the heavy-metal-resistance determinants in those bacterial isolates that harbored at least one of these two heavy-metal-resistance genes, the nearest relatives of the protein products of the czcA- and nccA-like genes were investigated using phylogenetic analysis (Fig. 3). The PCR products of czcA and nccA derived from bacterial isolates were subsequently sequenced. By sequencing it was possible to identify only 9 of the 23 heavy-metal-resistance determinants detected, however. This fact may partly be explained by the rather poor primers used: the primers only amplified a subset of the resistance genes, and were also insufficiently specific. A BLAST search of the GenBank revealed that while one sequence (EK-I2) showed relatively high level of similarity (93%) to the LysR transcriptional regulator family, eight remaining sequences showed too low levels of similarity (40–80%) to known proteins encoded by czcA and nccA genes (Fig. 3, Table 2). Therefore, all eight gene products are thought to form new heavymetal-resistance proteins. Although these gene products exhibited low similarity to their closest relatives, those carried by representatives of the Proteobacteria, the Pseudomonas isolates (EK-I1, EK-I52, EK-I59, EKI64) in particular, and one carried by one isolate belonging to the β-Proteobacteria (EK-I61), showed cerUnauthenticated Download Date | 8/12/17 7:49 PM Isolation of culturable bacteria and resistance determinants tain relatedness either to the heavy metal efflux pump CzcA, or to some types of transmembrane proteins (Table 2), suggesting that these genes could be involved in active protection against heavy metals. Thus, because all bacterial isolates which carried either czcA- or nccA-like genes were culturable, they could make a practical contribution to some fields of applied microbiology. 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