Bacterial communities are known to reflect their microenvironmental conditions by readily responding at extremely fast rates to environmental and pollution changes (Bell
In our previous work, a few of hardly cultivable and previously uncultured bacterial isolates from toxic-metal contaminated soil were cultivated, partly identified and characterised by using a diffusion chamber approach. Obtained results showed that all these isolates were resistant to nickel, cobalt, zinc, copper and cadmium ions and that this resistance in majority of β- or γ-Proteobacteria was mediated via a system of transmembrane metal pumps carried by these bacteria (Remenár
In detail, CBA transporters are three-component protein complexes that span the whole cell wall of Gram-negative bacteria. The most important component of the transporter is an RND protein that is located in the inner membrane. It mediates the active part of the transport process, determines the substrate specificity and is involved in the assembly of the trans-envelope protein complex. The RND protein family was first described as a related group of bacterial transport proteins involved in heavy metal resistance (
The best-characterized CBA transporter is the CzcCBA complex from
Thus, in our studies, we wanted to accurately identify a newly isolated bacterium tentatively assigned to uncultured betaproteobacteria and its heavy-metal-resistance gene product because we expected that such bacterium isolated from extreme environment could serve as a specific soil bacterial strain carrying heavy-metal-resistance gene product that facilitates the cells to survive in soil contaminated with nickel and also containing other metals, such as cobalt, zinc, iron, copper and cadmium.
Bacterium MR-CH-I2 was cultivated on LB (Luria-Bertani) agar plates aerobically at 30°C for 24 h and independently growing colonies were used for further analysis.
Primer sets used in this study.
Probes | Sequence | Descriptiona |
---|---|---|
27F | 5’ AGAGTTTGATCCTGGCTCAG 3’ | 16S rDNA universal primers, positions 8–27 and 704–685 and 1512–1492, resp. in the |
1492R | 5’ ACGGCTACCTTGTTACGACTT 3’ | |
2555nccAF | 5’ AGCCG (C,G) GA (C,G) AACGG CAAGCG 3’ | 2536–2555 and 3136–3117 degenerative nccA primers, positions on plazmid p9 in the |
3117nccAR | 5’ CCGATCACCACCGT (T,C) GC CAG 3’ | |
nccA9F | 5’ ACGTATCATTAGTTTCGCCA 3’ | 1861923–1861904 and 1859057–1859076 |
nccA2875R | 5’ ATCGGATAAACGACAGCATC 3’ | |
nccA1244F | 5’ GCTCTCGAAAGAGGAAGGCA 3’ | 1862989–1862970 and 1861746–1861765 |
nccA1244R | 5’ TTCGGTTTCGAGCGGTGAAT 3’ | |
nccA642F | 5’ GCTAGTCTTCACGGGCATT 3’ | 1859211–1859193 and 1858570–1858589 |
nccA642R | 5’ GCTCTTCGTCATGACACCAC 3’ | |
nccA923F | 5’ GGTCGCTTCCATTAACCG 3’ | 1860996–1860979 and 1860074–1860091 |
nccA923R | 5’ GATCGGATGCAATCTCCG 3’ | |
nccA-F | 5’ GTCGCCTTGTTCATCGG 3’ | 1860425–1860409 and 1860301–1860319 |
nccA-R | 5 GCAAACGTCAATACAACGG 3’ | |
gdhA-F | 5’ CGTACTCAATGAACGAAGGC 3’ | 388722–388741 and 388866–388850 |
gdhA-R | 5’ TCGATGCCGAGATTGCG 3’ | |
UP1 | 5’ GAAGTCATCATGACCGTTCTG CA(TC)GC(TCAG)GG(TCAG)GG (TCAG)AA(AG)TT(TC)GA 3’ | |
UP2r | 5’ AGCAGGGTACGGATGTGCGAG CC(AG)TC(TCAG)AC(AG)TC(TC AG)GC(AG)TC(TCAG)GTCAT 3’ | |
UP-1S | 5’ GAAGTCATCATGACCGTTCT GCA 3’ | |
UP-2Sr | 5’ AGCAGGGTACGGATGTGCG AGCC 3’ |
Numbers in parenthesis indicate the GenBank accession number.
amplification of beginning of amplification of terminal part of amplification of middle part of
Each 50 μl reaction mixture contained 1 μl (10 ng) of the DNA template, 5.0 μl 10
Unrooted phylogenetic tree obtained by the maximum likelihood method with 100 bootstrap replications showing phylogeny of 16S rRNA (16S rDNA) gene sequences of MR-CH-I2 isolate (in bold) and members of the genera
Unrooted phylogenetic tree obtained by the maximum likelihood method with 100 bootstrap replications showing phylogeny of partial
In addition, pulsed-field gel electrophoresis (PFGE) showed that the bacterium MR-CH-I2 carried a unique high molecular weight plasmid of about 50 kb in size (Fig. 3). It is known that there are two separate strains of
Pulsed-field gel electrophoresis analysis of high molecular plasmids from bacterium MR-CH-I2.
Legends: Lane 1 = mass standard (Lambda Ladder PFGE Marker); lane 2 = plasmid from isolate MR-CH-I2; lane 3 = control sample (without plasmid); lane 4 = mass standard (1 Kb DNA Ladder). The arrow indicates the band of about 50 kb in size.
The whole MR-CH-I2-nccA [KR476581] gene sequencing strategy of MR-CH-I2 isolate (cf. Detection of complete
Legends: Numbers in bold indicate positions of the MR-CH-I2-nccA gene (dark-skinned grey arrow) and its neighbourhood areas (light grey arrow) on chromosome in the
Unrooted phylogenetic tree obtained by the maximum likelihood method with 100 bootstrap replications showing phylogeny of whole
Expression of MR-CH-I2-nccA [KR476581] gene after heavy metal additions to the medium.
Time (h) | Nickela | Cadmiuma | Cobalta | Coppera | Zinca | |||||
---|---|---|---|---|---|---|---|---|---|---|
ΔΔCt | RQ | ΔΔCt | RQ | ΔΔCt | RQ | ΔΔCt | RQ | ΔΔCt | RQ | |
0 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 | 1.00 | 0.00 | 1.00 |
2 | –4.08 | 16.91 | 0.53 | 0.69 | 3.23 | 0.11 | 5.50 | 0.02 | 1.81 | 0.29 |
4 | –0.17 | 1.13 | 6.32 | 0.01 | 5.18 | 0.03 | 6.61 | 0.01 | 3.17 | 0.11 |
6 | –0.51 | 1.42 | 5.82 | 0.02 | 5.22 | 0.03 | 9.38 | 0.002 | 3.65 | 0.08 |
8 | 0.67 | 0.63 | 6.13 | 0.01 | 7.61 | 0.005 | 7.52 | 0.005 | 2.81 | 0.14 |
Standardization of gene expression according to the house-keeping gene after heavy metal additions;
Ct – threshold cycle;
ΔΔCt = ΔCt1 (Ct
RQ = 2–ΔΔCt;