The Lubin underground mine, is one of three mining divisions in the Lubin-Glogow Copper District in Lower Silesia province (Poland). It is the source of polymetallic ore that is rich in copper, silver and several heavy metals. Black shale is also significantly enriched in fossil organic matter in the form of long-chain hydrocarbons, polycyclic aromatic hydrocarbons, organic acids, esters, thiophenes and metalloporphyrins. Biological analyses have revealed that this environment is inhabited by extremophilic bacteria and fungi. Kupfershiefer black shale and samples of water, bottom and mineral sediments from the underground (below 600 m) Lubin mine were taken and 20 bacterial strains were isolated and characterized. All exhibited multi-resistant and hypertolerant phenotypes to heavy metals. We analyzed the plasmidome of these strains in order to evaluate the diversity and role of mobile DNA in adaptation to the harsh conditions of the mine environment. Experimental and bioinformatic analyses of 11 extrachromosomal replicons were performed. Three plasmids, including a broad-host-range replicon containing a Tn3 family transposon, carried genes conferring resistance to arsenic, cadmium, cobalt, mercury and zinc. Functional analysis revealed that the resistance modules exhibit host specificity, i.e., they may increase or decrease tolerance to toxic ions depending on the host strain. The other identified replicons showed diverse features. Among them we identified a catabolic plasmid encoding enzymes involved in the utilization of histidine and vanillate, a putative plasmid-like prophage carrying genes responsible for NAD biosynthesis, and two repABC-type plasmids containing virulence-associated genes. These findings provide an unique molecular insight into the pool of extrachromosomal replicons and highlight their role in the biology and adaptation of extremophilic bacteria inhabiting terrestrial deep subsurface.

Two microbial mats found inside two old (gold and uranium) mines in Zloty Stok and Kowary located in SW Poland seem to form a natural barrier that traps heavy metals leaking from dewatering systems. We performed complex physiological and metagenomic analyses to determine which microorganisms are the main driving agents responsible for self-purification of the mine waters and identify metabolic processes responsible for the observed features. SEM and energy dispersive X-ray microanalysis showed accumulation of heavy metals on the mat surface, whereas, sorption experiments showed that neither microbial mats were completely saturated with heavy metals present in the mine waters, indicating that they have a large potential to absorb significant quantities of metal. The metagenomic analysis revealed that Methylococcaceae and Methylophilaceae families were the most abundant in both communities, moreover, it strongly suggest that backbones of both mats were formed by filamentous bacteria, such as Leptothrix, Thiothrix, and Beggiatoa. The Kowary bacterial community was enriched with the Helicobacteraceae family, whereas the Zloty Stok community consist mainly of Sphingomonadaceae, Rhodobacteraceae, and Caulobacteraceae families. Functional (culture-based) and metagenome (sequence-based) analyses showed that bacteria involved in immobilization of heavy metals, rather than those engaged in mobilization, were the main driving force within the analyzed communities. In turn, a comparison of functional genes revealed that the biofilm formation and heavy metal resistance (HMR) functions are more desirable in microorganisms engaged in water purification than the ability to utilize heavy metals in the respiratory process (oxidation-reduction). These findings provide insight on the activity of bacteria leading, from biofilm formation to self-purification, of mine waters contaminated with heavy metals.

Sinorhizobium sp. M14 is an As(III)-oxidizing, psychrotolerant strain, capable of growth in the presence of extremely high concentrations of arsenic and many other heavy metals. Metallotolerant abilities of the M14 strain depend upon the presence of two extrachromosomal replicons: pSinA (∼ 109 kb) and pSinB (∼ 300 kb). The latter was subjected to complex analysis. The performed analysis demonstrated that the plasmid pSinB is a narrow-host-range repABC-type replicon, which is fully stabilized by the phd-vapC-like toxin-antitoxin stabilizing system. In silico analysis showed that among the phenotypic gene clusters of the plasmid pSinB, eight modules are potentially involved in heavy metals resistance (HMR). These modules carry genes encoding efflux pumps, permeases, transporters and copper oxidases, which provide resistance to arsenic, cadmium, cobalt, copper, iron, mercury, nickel, silver and zinc. The functional analysis revealed that the HMR modules are active and have an effect on the minimal inhibitory concentration (MIC) values observed for the heterological host cells. The phenotype was manifested by an increase or decrease of the MICs of heavy metals and it was strain specific. The analysis of distribution of the heavy metal resistance genes, i.e. resistome, in Sinorhizobium spp. plasmids, revealed that the HMR modules are common in these replicons.

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