Extremophile Metal Resistance: Plasmid-Encoded Functions in Streptomyces mirabilis.

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.

Changes in element availability induced by sterilization in heavy metal contaminated substrates: A comprehensive study.

Microbiome analyses of soils and microcosm experiments depend on conditions that include sterilization in order to perform experimental manipulation of microbial communities. Still, they should represent conditions close to nature. When using metal contaminated soils, sterilization methods might alter metal availability. Here, four typical metal contaminated substrates were analyzed, representing different contamination histories and soil types. They included two very poor substrates, as they are often found at metal contaminated sites. The low contents in organic carbon and nitrogen as well as two substrates with slightly higher nutrient availability were used to perform a comprehesive study for element availability changes induced by sterilization. Autoclaving, dry heat or gamma raγ sterilization were applied and compared to a non-treated control. The sterile substrates were analyzed using sequential extraction to account for different associations of the elements. Metals forming specific (hydro)oxide layers were specifically analyzed since they in turn may also impact other metals or ions. In addition, (heavy) metals and (micro)nutrients were analyzed for changes in speciation. The effects of autoclaving (wet heat) was found acceptable, while γ-ray irradiation did show unexpected changes in metal associations, especially for one substrate. Dry heat changed metal availability to the highest degree.