Diversity and role of plasmids in adaptation of bacteria inhabiting the Lubin copper mine in Poland, an environment rich in heavy metals

Dziewit L, Pyzik A, Szuplewska M, et al. Diversity and role of plasmids in adaptation of bacteria inhabiting the Lubin copper mine in Poland, an environment rich in heavy metals. Frontiers in Microbiology. 2015;6: 152.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 600m) 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

Insights into the Transposable Mobilome of Paracoccus spp. (Alphaproteobacteria)

Several trap plasmids (enabling positive selection of transposition events) were used to identify a pool of functional transposable elements (TEs) residing in bacteria of the genus Paracoccus (Alphaproteobacteria). Complex analysis of 25 strains representing 20 species of this genus led to the capture and characterization of (i) 37 insertion sequences (ISs) representing 9 IS families (IS3, IS5, IS6, IS21, IS66, IS256, IS1182, IS1380 and IS1634), (ii) a composite transposon Tn6097 generated by two copies of the ISPfe2 (IS1634 family) containing two predicted genetic modules, involved in the arginine deiminase pathway and daunorubicin/doxorubicin resistance, (iii) 3 non-composite transposons of the Tn3 family, including Tn5393 carrying streptomycin resistance and (iv) a transposable genomic island TnPpa1 (45 kb). Some of the elements (e.g. Tn5393, Tn6097 and ISs of the IS903 group of the IS5 family) were shown to contain strong promoters able to drive transcription of genes placed downstream of the target site of transposition. Through the application of trap plasmid pCM132TC, containing a promoterless tetracycline resistance reporter gene, we identified five ways in which transposition can supply promoters to transcriptionally silent genes. Besides highlighting the diversity and specific features of several TEs, the analyses performed in this study have provided novel and interesting information on (i) the dynamics of the process of transposition (e.g. the unusually high frequency of transposition of TnPpa1) and (ii) structural changes in DNA mediated by transposition (e.g. the generation of large deletions in the recipient molecule upon transposition of ISPve1 of the IS21 family). We also demonstrated the great potential of TEs and transposition in the generation of diverse phenotypes as well as in the natural amplification and dissemination of genetic information (of adaptative value) by horizontal gene transfer, which is considered the driving force of bacterial evolution

Mobility and Generation of Mosaic Non-Autonomous Transposons by Tn<i>3</i>-Derived Inverted-Repeat Miniature Elements (TIMEs)

<div><p>Functional transposable elements (TEs) of several <i>Pseudomonas</i> spp. strains isolated from black shale ore of Lubin mine and from post-flotation tailings of Zelazny Most in Poland, were identified using a positive selection trap plasmid strategy. This approach led to the capture and characterization of (i) 13 insertion sequences from 5 IS families (IS<i>3</i>, IS<i>5</i>, IS<i>L3</i>, IS<i>30</i> and IS<i>1380</i>), (ii) isoforms of two Tn<i>3</i>-family transposons – Tn<i>5563</i>a and Tn<i>4662</i>a (the latter contains a toxin-antitoxin system), as well as (iii) non-autonomous TEs of diverse structure, ranging in size from 262 to 3892 bp. The non-autonomous elements transposed into AT-rich DNA regions and generated 5- or 6-bp sequence duplications at the target site of transposition. Although these TEs lack a transposase gene, they contain homologous 38-bp-long terminal inverted repeat sequences (IRs), highly conserved in Tn<i>5563</i>a and many other Tn<i>3</i>-family transposons. The simplest elements of this type, designated TIMEs (Tn<i>3</i> family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of <i>Pseudomonas</i> spp. It was demonstrated that TIMEs are able to mobilize segments of plasmid DNA for transposition, which results in the generation of more complex non-autonomous elements, resembling IS-driven composite transposons in structure. Such transposon-like elements may contain different functional genetic modules in their core regions, including plasmid replication systems. Another non-autonomous element “captured” with a trap plasmid was a TIME derivative containing a predicted resolvase gene and a <i>res</i> site typical for many Tn<i>3</i>-family transposons. The identification of a portable site-specific recombination system is another intriguing example confirming the important role of non-autonomous TEs of the TIME family in shuffling genetic information in bacterial genomes. Transposition of such mosaic elements may have a significant impact on diversity and evolution, not only of transposons and plasmids, but also of other types of mobile genetic elements.</p></div

Possible mechanism for the generation of diverse non-autonomous and autonomous elements of the Tn<i>3</i> transposon family.

<p>The elements of the Tn3 family originate from progenitor insertion sequences (IS). Tns – diverse autonomous non-composite or composite transposons generated by acquisition of foreign DNA and mobilization for transposition of genomic DNA results. TIME - non-autonomous elements resulting from a reduction in the number of transposon-encoded genes, able to form mosaic elements resembling non-composite and composite Tns in structure. Elements representative of the different types are shown as examples: IS<i>1071</i> (accession no. M65135), Tn<i>3434</i> (accession no. AY232820), Tn<i>5393</i> (accession no. M96392), Tn<i>Ppa1</i> (accession no. DQ149577) and Tn<i>5271</i> (accession no. U18133).</p

Differential Localization and Functional Specialization of parS Centromere-Like Sites in repABC Replicons of Alphaproteobacteria

International audienceThe repABC replication/partitioning loci are widespread in extrachromosomal replicons of Alphaproteobacteria . They are evolutionarily diverse, subject to multi-layer self-regulation, and are responsible for the maintenance of different types of replicons, such as plasmids (e.g., Agrobacterium pTi and pRi tumorigenic and rhizogenic plasmids), megaplasmids (e.g., Sinorhizobium pSymA and pSymB) and essential chromids (e.g., secondary chromosomes of Agrobacterium , Brucella and Rhodobacter )

Characteristics of transposable elements identified in this study.

a<p>The length of the IRs/the number of identical residues. nd - not determined.</p

Predicted secondary structures of TIMEs at the RNA level.

<p>RNA secondary structures predicted by <i>in silico</i> folding using Mfold software for TIME1 (class 1), two TIMEs representing class 3 and 4 elements shown in Fig. 3 (accession nos. AF020724 and AE016855, respectively), and the TIME-like elements ARM<i>phe</i> and IS<i>101</i> (see Discussion for details). The minimum folding energy (ΔG) of the predicted secondary structures was calculated by Mfold.</p

Genetic structure of <i>Pseudomonas</i> spp. plasmids containing non-autonomous TEs of the Tn<i>3</i> transposon family.

<p>Circular plasmids pZM1P1 (A) and pLM8P2 (B) originate from <i>Pseudomonas</i> spp. strains ZM1 and LM8, respectively. Predicted genetic modules involved in plasmid replication (REP), mobilization for conjugal transfer (MOB) and toluene resistance (TtgGHI efflux pump - truncated operon) are boxed and appropriately labeled. Predicted coding regions are represented by thick arrows indicating the direction of transcription. Broken arrows indicated truncated genes (<i>ttgH</i> and <i>orf5</i> of pLM8P2). Shaded areas connect DNA regions of pZM1P1 included in non-autonomous TIME-COMP transposons. Note that TIMEs are not drawn to scale. Thin black arrows above TIMEs indicate the orientation of the elements. A plot of the G+C content of pZM1P1 is shown above the structure diagram and the average G+C value is given to the right.</p

Non-autonomous and autonomous transposable elements of the Tn<i>3</i> transposon family identified in <i>Pseudomonas</i> spp. strains.

<p>A. The genetic organization of TEs identified with trap plasmid pMAT1 in strains ZM1 (TIME, TIME-COMP1, TIME-COMP2), LM8 (TIME, TIME-RES), LM10 and LM13 (Tn<i>4662</i>a), as well as LM7 (Tn<i>5563</i>a). Inverted repeats (IRs) flanking the elements are marked by red triangles. Predicted coding regions are represented by thick arrows indicating the direction of transcription. Thin black arrows above TIMEs indicate the orientation of the elements. Shaded areas connect homologous DNA regions. <i>res</i> indicates the cointegrate resolution site of the site-specific recombination systems. The predicted toxin-antitoxin system is boxed and denoted by TA. Note that TIMEs are not drawn to scale. B. Alignment of the terminal inverted repeat nucleotide sequences (IRL – left IR; IRR – right IR) of TIMEs and two autonomous Tn<i>3</i>-family transposons, Tn<i>5563</i>a and Tn<i>5044</i>. Identical residues are indicated by gray shading. C. Nucleotide sequences of direct repeats (DRs) generated by TIME elements during transposition into the selective cartridge of pMAT1.</p