Download The isolation of heavy-metal resistant culturable

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.
Acknowledgements
This work was supported by VEGA Grant No. 2/7022/7, by
The Centre of Excelence for the Protection and Exploitation
of Landscape and Biodiversity under project code ITMS26240120014 and by New microbial isolates containing the
genes of catabolic and detoxification pathways and their use
in biotechnology under project code ITMS-26240220010.
References
Altschul S.F., Gish W., Miller W., Myers E.W. & Lipman D.J.
1990. Basic local alignment search tool. J Mol. Biol. 215:
403–410.
Ashraf R. & Ali T.A. 2007. Effect of heavy metals on soil microbial community and mung beans seed germination. Pak. J.
Bot. 39: 629–636.
Becker J.M., Parkin T., Nakatsu C.H., Wilbur J.D. & Konopka A.
2006. Bacterial activity, community structure, and centimetre-scale spatial heterogeneity in contaminated soil. Microb.
Ecol. 51: 220–231.
Benson D.A., Karsch-Mizrachi I., Lipman D.J., Ostell J. & Sayers E.W. 2010. GenBank. Nucleic Acids Res. 38 (Database
issue): D46–D51.
Bogdanova E.S., Bass I.A., Minakhin L.S., Petrova M.A., Mindlin
S.Z., Volodin A.A., Kalyaeva E.S., Tiedje J.M., Hobman J.L.,
Brown N.L. & Nikiforov V.G. 1998. Horizontal spread of mer
operons among Gram-positive bacteria in natural environments. Microbiology 144: 609–620.
Chapman P.M. 1999. The role of soil microbial tests in ecological
risk assessment. Hum. Ecol. Risk Assess. 5: 657–660.
Chovanová K., Sládeková D., Kmeť V., Prokšová M., Harichová
J., Puškárová A., Polek B. & Ferianc P. 2004. Identification
and characterization of eight cadmium resistant bacterial isolates from a cadmium-contaminated sewage sludge. Biologia
59: 817–827.
Ellis R.J., Morgan P., Weightman A.J. & Fry J.C. 2003.
Cultivation-dependent and -independent approaches for determining bacterial diversity in heavy-metal-contaminated
soil. Appl. Environ. Microbiol. 69: 3223–3230.
Erbe J.L., Taylor K.B. & Hall L.M. 1995. Metalloregulation of
the cyanobacterial smt locus: identification of SmtB binding
sites and direct interaction with metals. Nucleic Acids Res.
23: 2472–2478.
Hamaki T., Suzuki M., Fudou R., Jojima Y., Kajiura T., Tabuchi
A., Sen K. & Shibai H. 2005. Isolation of novel bacteria and
actinomycetes using soil-extract agar medium. J. Biosci. Bioeng. 99: 485–492.
Huang C.C., Narita M., Yamagata T., Itoh Y. & Endo G. 1999.
Structure analysis of a class II transposon encoding the mercury resistance of the gram-positive bacterium Bacillus megaterium MB1, a strain isolated from Minamata Bay, Japan.
Gene 234: 361–369.
Hugenholtz P. 2002. Exploring prokaryotic diversity in the genomic era. Genome Biol. 3: reviews0003–reviews0003.8.
Ji G. & Silver S. 1992. Regulation and expression of the arsenic
resistance operon from Staphylococcus aureus plasmid pI258.
J. Bacteriol. 174: 3684–3694.
25
Joseph S.J., Hugenholtz P., Sangwan P., Osborne C.A. & Janssen
P.H. 2003. Laboratory cultivation of widespread and previously unculturable soil bacteria. Appl. Environ. Microbiol.
69: 7210–7215.
Kandeler E., Tscherko D., Bruce K.D., Stemmer M., Hobbs P.J.,
Bardgett R.D. & Amelung W. 2000. Structure and function
of the soil microbial community in microhabitats of a heavy
metal polluted soil. Biol. Fertil. Soils 32: 390–400.
Khan S., Hesham A.E-L., Qiao M., Rehman S. & He J.Z. 2010.
Effect of Cd and Pb on soil microbial community structure
and activities. Environ. Sci. Polut. Res. 17: 288–296.
Kimura M. 1980. A simple method for estimating evolutionary
rates of base substitutions through comparative studies of
nucleotide sequences. J. Mol. Evol. 16: 111–120.
Lane D.J. 1991. 16S/23S rRNA sequencing, pp. 115–148. In:
Stackebrandt E. & Goodfellow M. (eds), Nucleic Acid Techniques in Bacterial Systematics, John Wiley & Sons, New
York.
Matyar F., Akkan T., Uçak Y. & Eraslan B. 2010. Aeromonas and
Pseudomonas: antibiotic and heavy metal resistance species
from Iskenderun Bay, Turkey (northeast Mediterranean Sea).
Environ. Monit. Assess. 167: 309–320.
Mera N. & Iwasaki K. 2007. Use of plate-wash samples
to monitor the fates of culturable bacteria in mercuryand trichloroethylene-contaminated soils. Appl. Microbiol.
Biotechnol. 77: 437–445.
Mergeay M., Monchy S., Vallaeys T., Auquier V., Benotmane A.,
Bertin P., Taghavi S., Dunn J., Van Der Lelie D. & Wattiez R. 2003. Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metalresponsive genes. FEMS Microbiol. Rev. 27: 385–410.
Mitsui H., Gorlach K., LEE H.J., Hattori R. & Hattori T. 1997.
Incubation time and media requirements of culturable bacteria from different phylogenetic groups. J. Microbiol. Methods
30: 103–110.
Mobley H.L., Chen C.M., Silver S. & Rosen B.P. 1983. Cloning
and expression of R-factor mediated arsenate resistance in
Escherichia coli. Mol. Gen. Genet. 191: 421–426.
Nascimento A.M.A. & Chartone-Souza E. 2003. Operon mer:
bacterial resistance to mercury and potential for bioremediation of contaminated environments. Genet. Mol. Res. 2:
92–101.
Ogilvie L.A. & Grant A. 2008. Linking pollution induced community tolerance (PICT) and microbial community structure
in chronically metal polluted estuarine sediments. Mar. Environ. Res. 65: 187–198.
Page R.D. 1996. TreeView: an application to display phylogenetic
trees on personal computers. Comput. Appl. Biosci. 12: 357–
358.
Pandey S., Saha P., Barai P.K. & Maiti T.K. 2010. Characterization of a Cd2+ -resistant strain of Ochrobactrum sp. isolated
from slag disposal site of an iron and steel factory. Curr. Microbiol. 61: 106–111.
Pechrada J., Sajjaphan K. & Sadowsky M.J. 2010. Structure and
diversity of arsenic-resistant bacteria in an old tin mine area
of Thailand. J. Microbiol. Biotechnol. 20: 169–178.
Prakash O., Gihring T.M., Dalton D.D., Chin K.-J., Green S.J.,
Akob D.M., Wanger G. & Kostka J.E. 2010. Geobacter daltonii sp. nov., an Fe(III)- and uranium(VI)-reducing bacterium isolated from a shallow subsurface exposed to mixed
heavy metal and hydrocarbon contamination. Int. J. Syst.
Evol. Microbiol. 60: 546–553.
Ranjard L., Brothier E. & Nazaret S. 2000. Sequencing bands of
ribosomal intergenic spacer analysis fingerprints for characterization and microscale distribution of soil bacterium populations responding to mercury spiking. Appl. Environ. Microbiol. 66: 5334–5339.
Ranjard L., Lignier L. & Chaussod R. 2006. Cumulative effect
of short-term polymetal contamination on soil bacterial community structure. Appl. Environ. Microbiol. 72: 1684–1687.
Rosenstein R., Peschel A., Wieland B. & Götz F. 1992. Expression and regulation of the antimonite, arsenite, and arsenate
resistance operon of Staphylococcus xylosus plasmid pSX267.
J. Bacteriol. 174: 3676–3683.
Unauthenticated
Download Date | 8/12/17 7:49 PM
26
Saitou N. & Nei M. 1987. The neighbour-joining method: a new
method for reconstructing phylogenetic trees. Mol. Biol. Evol.
4: 406–425.
Saltikov C.W. & Olson B.H. 2002. Homology of Escherichia coli
R773 arsA, arsB, and arsC genes in arsenic-resistant bacteria
isolated from raw sewage and arsenic-enriched creek waters.
Appl. Environ. Microbiol. 68: 280–288.
Sandaa R.A., Torsvik V. & Enger Ø. 2001. Influence of long-term
heavy-metal contamination on microbial communities in soil.
Soil Biol. Biochem. 33: 287–295.
Sandaa R.A., Torsvik V., Enger Ø., Daae F.L., Castberg T. &
Hahn D. 2006. Analysis of bacterial communities in heavy
metal-contaminated soils at different levels of resolution.
FEMS Microbiol. Ecol. 30: 237–251.
Schmidt T. & Schlegel H.G. 1994. Combined nickel-cobaltcadmium resistance encoded by the ncc locus of Alcaligenes
xylosoxidans 31A. J. Bacteriol. 176: 7045–7054.
E. Karelová et al.
Silver S. & Phung L.T. 1996. Bacterial heavy metal resistance: New surprises. Ann. Rev. Microbiol. 50: 753–789.
doi:10.1146/annurev.micro.50.1.753.
Smiejan A., Wilkinson K.J. & Rossier C. 2003. Cd bioaccumulation by a freshwater bacterium, Rhodospirillum rubrum. Environ. Sci. Technol. 37: 701–706.
Taghavi S., Van Der Lelie D. & Mergeay M. 1994. Electroporation
of Alcaligenes eutrophus with (mega) plasmids and genomic
DNA fragments. Appl. Environ. Microbiol. 60: 3585–3591.
Thompson J.D., Higgins D.G. & Gibson T.J. 1994. CLUSTAL
W: improving the sensitivity of progressive multiple sequence
alignment through sequence weighting, position specific gap
penalties and weight matrix choice. Nucleic Acids Res. 22:
4673–4680.
Xie X., Fu J., Wang H. & Liu J. 2010. Heavy metal resistance
by two bacteria strains isolated from a copper mine tailing in
China. African J. Biotechnol. 9: 4056–4066.
Received June 25, 2010
Accepted October 26, 2010
Unauthenticated
Download Date | 8/12/17 7:49 PM