Extremophile Metal Resistance: Plasmid-Encoded Functions in Streptomyces mirabilis

Abstract

The extreme metal tolerance of up to 130 mM NiSO₄ in Streptomyces mirabilis P16B-1 was investigated. Genome sequencing revealed the presence of a large linear plasmid, pI. To identify plasmid-encoded determinants of metal resistance, a newly established transformation system was used to characterize the predicted plasmid-encoded loci nreB, hoxN, and copYZ. Reintroduction into the plasmid-cured S. mirabilis ΔpI confirmed that the predicted metal transporter gene nreB constitutes a nickel resistance factor, which was further supported by its heterologous expression in Escherichia coli. In contrast, the predicted nickel exporter gene hoxN decreased nickel tolerance, while copper tolerance was enhanced. The predicted copper-dependent transcriptional regulator gene copY did not induce tolerance toward either metal. Since genes for transfer were identified on the plasmid, its conjugational transfer to the metal-sensitive Streptomyces lividans TK24 was checked. This resulted in acquired tolerance toward 30 mM nickel and additionally increased the tolerance toward copper and cobalt, while oxidative stress tolerance remained unchanged. Intracellular nickel concentrations decreased in the transconjugant strain. The high extracellular nickel concentrations allowed for biomineralization. Plasmid transfer could also be confirmed into the co-occurring actinomycete Kribbella spp. in soil microcosms. IMPORTANCE Living in extremely metal-rich environments requires specific adaptations, and often, specific metal tolerance genes are encoded on a transferable plasmid. Here, Streptomyces mirabilis P16B-1, isolated from a former mining area and able to grow with up to 130 mM NiSO₄, was investigated. The bacterial chromosome, as well as a giant plasmid, was sequenced. The plasmid-borne gene nreB was confirmed to confer metal resistance. A newly established transformation system allowed us to construct a plasmid-cured S. mirabilis as well as an nreB-rescued strain in addition to confirming nreB encoding nickel resistance if heterologously expressed in E. coli. The potential of intra- and interspecific plasmid transfer, together with the presence of metal resistance factors on that plasmid, underlines the importance of plasmids for transfer of resistance factors within a bacterial soil community.

Keywords: Streptomyces; cross-kingdom transformation; genome sequence; heavy metal resistance; metal efflux; soil.

FIG 1

Examples for trench plate tests (TSB; n > 3) showing increased tolerance toward Ni²⁺ caused by transfer of plasmid. The first four lines compared three S. lividans transconjugants (T1 to T3) carrying the plasmid to the recipient S. lividans TK24 wild type (WT). For control, nickel tolerance of the donor S. mirabilis P16B-1 (fifth lane) is shown (left). The growth of two cured strains of the donor was added and compared to the S. mirabilis P16B-1 wild type (right). Growth toward a trench containing NiSO₄ at the top that produced a metal concentration gradient starting from the trench was tested; sensitivity is seen by lack of growth toward the trench. The strong effect on nickel tolerance led to further investigations of nickel resistance.

FIG 2

Formation of biominerals below and near colonies of S. lividans TK24 carrying the heterologous plasmid, cultivated on TSB amended with NiSO₄ at 10 to 20 mM concentrations (for comparison to other strains, see Table S1 in the supplemental material).

FIG 3

Drop plate test for metal sensitivity of selected strains. (A) GYM medium with 2.5 mM NiSO₄ (left) or TSB with 8 mM CuSO₄ (right) to evaluate the potential resistance determinants carried on three cosmids carrying either nreB and kmtR, copYZ, or hoxN reintroduced into the cured S. mirabilis ΔpI (for constructs, see Fig. S4 in the supplemental material). (B) TSB with 5 mM NiSO₄ (left) or 4 mM CoSO₄ (right) for checking the effect of nreB cloned with its native S. mirabilis promoter only, compared to the empty vector control (evc), transfected into S. lividans TK24. The spore counts applied with each drop had been derived from plates with the same agar, but lacking the metal, are given at the top.

FIG 4

Growth on metal-containing media of E. coli overexpressing nreB. The cells carried either nreB cloned in-frame (black squares or triangles) or the empty vector control pTrc99a (open squares or triangles). E. coli was grown in LB medium supplemented with NiSO₄ at 2 mM (squares) and 2.5 mM (triangles) concentrations (A), NiCl₂ at 1.5 mM (squares) and 2 mM (triangles) (B), CoSO₄ at 0.5 mM (squares) and 1 mM (triangles) concentrations (C), and CuSO₄ at 2.5 mM (squares) and 3 mM (triangles) concentrations (D); bars indicate standard deviation of six replicates. The growth rates μ [h] are indicated in each diagram for every growth curve.

FIG 5

Genomic region for potential plasmid transfer functions. Plasmid loci SMIR_40565 to SMIR_40600 encoding putative plasmid transfer proteins vtrA, tcpC, tcpE, and virE components are shown. ORFs without predicted function are shaded in gray. Accession numbers are given for gene identification.

FIG 6

Detection of plasmid transfer from S. mirabilis P16B-1 to Kribbella spp. Potential Kribbella transconjugant DNA was checked by PCR (K) and compared to S. mirabilis P16B-1 DNA (C) as a positive control. For detection of plasmid transfer, primers were designed that target plasmid sequences and do not match sequences elsewhere in known genomes (compare in Table 3). Negative controls for Kribbella before plasmid transfer were performed (data not shown).