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Bacteria of the genus Listeria (phylum Firmicutes ) include both human and animal pathogens, as well as saprophytic strains. A common component of Listeria spp. genomes are plasmids, i.e., extrachromosomal replicons that contribute to gene flux in bacteria. This study provides an in-depth insight into the structure, diversity and evolution of plasmids occurring in Listeria strains inhabiting various environments under different anthropogenic pressures. Apart from the components of the conserved plasmid backbone (providing replication, stable maintenance and conjugational transfer functions), these replicons contain numerous adaptive genes possibly involved in: (i) resistance to antibiotics, heavy metals, metalloids and sanitizers, and (ii) responses to heat, oxidative, acid and high salinity stressors. Their genomes are also enriched by numerous transposable elements, which have influenced the plasmid architecture. The plasmidome of Listeria is dominated by a group of related replicons encoding the RepA replication initiation protein. Detailed comparative analyses provide valuable data on the level of conservation of these replicons and their role in shaping the structure of the Listeria pangenome, as well as their relationship to plasmids of other genera of Firmicutes , which demonstrates the range and direction of flow of genetic information in this important group of bacteria.

Background: Gene overlapping is a frequent phenomenon in microbial genomes. Excluding so-called "trivial overlapping", there are significant implications of such genetic arrangements, including regulation of gene expression and modification of protein activity. It is also postulated that, besides gene duplication, the appearance of overlapping genes (OGs) is one of the most important factors promoting a genome's novelty and evolution. OGs coding for in-frame proteins with different functions are a particularly interesting case. In this study we identified and characterized two in-frame proteins encoded by OGs on plasmid pIGRK from Klebsiella pneumoniae, a representative of the newly distinguished pHW126 plasmid family., Results: A single repR locus located within the replication system of plasmid pIGRK encodes, in the same frame, two functional polypeptides: a full-length RepR protein and a RepR' protein (with N-terminal truncation) translated from an internal START codon. Both proteins form homodimers, and interact with diverse DNA regions within the plasmid replication origin and repR promoter operator. Interestingly, RepR and RepR' have opposing functions - RepR is crucial for initiation of pIGRK replication, while RepR' is a negative regulator of this process. Nevertheless, both proteins act cooperatively as negative transcriptional regulators of their own expression., Conclusions: Regulation of the initiation of pIGRK replication is a complex process in which a major role is played by two in-frame proteins with antagonistic functions. In-frame encoded Rep proteins are uncommon, having been described in only a few plasmids. This is the first description of such proteins in a plasmid of the pHW126 family.

Psychrobacter sp. DAB_AL32B, originating from Spitsbergen island (Arctic), carries the large plasmid pP32BP2 (54,438 bp). Analysis of the pP32BP2 nucleotide sequence revealed the presence of three predicted phenotypic modules that comprise nearly 30% of the plasmid genome. These modules appear to be involved in fimbriae synthesis via the chaperone-usher pathway (FIM module) and the aerobic and anaerobic metabolism of carnitine (CAR and CAI modules, respectively). The FIM module was found to be functional in diverse hosts since it facilitated the attachment of bacterial cells to abiotic surfaces, enhancing biofilm formation. The CAI module did not show measurable activity in any of the tested strains. Interestingly, the CAR module enabled the enzymatic breakdown of carnitine, but this led to the formation of the toxic by-product trimethylamine, which inhibited bacterial growth. Thus, on the one hand, pP32BP2 can enhance biofilm formation, a highly advantageous feature in cold environments, while on the other, it may prevent bacterial growth under certain environmental conditions. The detrimental effect of harboring pP32BP2 (and its CAR module) seems to be conditional, since this replicon may also confer the ability to use carnitine as an alternative carbon source, although a pathway to utilize trimethylamine is most probably necessary to make this beneficial. Therefore, the phenotype determined by this CAR-containing plasmid depends on the metabolic background of the host strain.

Plasmids play an important role in the adaptation of bacteria to changeable environmental conditions. As the main vectors of horizontal gene transfer, they can spread genetic information among bacteria, sometimes even across taxonomic boundaries. Some plasmids carry genes involved in the utilization of particular carbon compounds, which can provide a competitive advantage to their hosts in particular ecological niches. Analysis of the multireplicon genome of the soil bacterium P. aminovorans JCM 7685 revealed the presence of an extrachromosomal replicon pAMV1 (185 kb) with a unique structure and properties. This lifestyle-determining plasmid carries genes facilitating the metabolism of many different carbon compounds including sugars, short-chain organic acids and C1 compounds. Plasmid pAMV1 contains a large methylotrophy island (MEI) that is essential not only for the utilization of particular C1 compounds but also for the central methylotrophic metabolism required for the assimilation of C1 units (serine cycle). We demonstrate that the expression of the main serine cycle genes is induced in the presence of C1 compounds by the transcriptional regulator ScyR. The extrachromosomal localization of the MEI and the distribution of related genes in Paracoccus spp. indicate that it could have been acquired by HGT by an ancestor of P. aminovorans., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The Svalbard archipelago (Spitsbergen Island) is the northernmost landmass in the European Arctic and has a variety of small- and medium-sized glaciers. The plasmidome of eleven psychrophilic strains of Variovorax spp. isolated from the ice surface of Hans and Werenskiold Glaciers of Spitsbergen Island, was defined. This analysis revealed the presence of six plasmids whose nucleotide sequences have been determined. Four of them, exhibiting high reciprocal sequence similarity, possess unique structures, since their genomes lack any recognized genes. These miniature replicons, not exceeding 1 kb in size, include pHW69V1 (746 bp), which is the smallest autonomous replicon so far identified in free-living bacteria. The miniature plasmids share no similarity with known sequences present in the databases. In silico and experimental analyses identified conserved DNA regions essential for the initiation of replication of these replicons., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Paracoccus kondratievae NCIMB 13773(T), isolated from the maize rhizosphere, carries a large (95,049 bp) plasmid pKON1, whose structure has been significantly influenced by transposition. Almost 30% of the plasmid genome is composed of complete or truncated insertion sequences (ISs), representing seven IS families. The ISs are accompanied by numerous genes and gene clusters commonly found in bacterial chromosomes, encoding, among others, (i) a putative type III secretion system of the Rhizobiales-T3SS family, (ii) a type I restriction-modification system associated with the anti-codon nuclease (ACNase) gene prrC and (iii) OstA and OstB proteins involved in trehalose synthesis. The backbone of pKON1 is composed of replication and partitioning modules conserved in several large alphaproteobacterial replicons, including secondary chromid pAMI6 of Paracoccus aminophilus JCM 7686 and chromosome 2 (chromid) of Rhodobacter sphaeroides 2.4.1. pKON1 also contains a toxin-antitoxin system of the hipAB family, whose presence precludes removal of the plasmid from bacterial cells. This system, unlike two other related hipAB-family loci originating from plasmid pAMI8 and the chromosome of Paracoccus aminophilus JCM 7686, is highly efficient and permits very stable maintenance of a heterologous replicon in various hosts., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Bacterial genomic information can be divided between various replicons, including chromosomes, plasmids, and chromids (essential plasmid-like replicons with properties of both chromosomes and plasmids). Comparative analyses of bacterial plasmids, including homology searches, phylogenetic and phylogenomic analyses, as well as network construction for the characterization of their relationships, are good starting points for the identification of chromids. Chromids possess several chromosome-like genetic features (e.g., codon usage, GC content), but most significantly, they carry housekeeping genes, which make them indispensable for cell viability. However, it is important to confirm in silico predictions experimentally. The essential nature of a predicted chromid is usually verified by the application of a target-oriented replicon curing technique, based on the incompatibility phenomenon. Further tests examining growth in various media are used to distinguish secondary chromids from plasmids, and mutational analysis (e.g., using the yeast FLP/FRT recombination system) is employed to identify essential genes carried by particular chromids.

Antibiotic resistance determinants are frequently associated with plasmids and other mobile genetic elements, which simplifies their horizontal transmission. Several groups of plasmids (including replicons of the IncL/M incompatibility group) were found to play an important role in the dissemination of resistance genes encoding β-lactamases. The IncL/M plasmids are large, broad host range, and self-transmissible replicons. We have identified and characterized two novel members of this group: pARM26 (isolated from bacteria inhabiting activated sludge from a wastewater treatment plant) and pIGT15 (originating from a clinical strain of Escherichia coli). This instigated a detailed comparative analysis of all available sequences of IncL/M plasmids encoding β-lactamases. The core genome of these plasmids is comprised of 20 genes with conserved synteny. Phylogenetic analyses of these core genes allowed clustering of the plasmids into four separate groups, which reflect their antibiotic resistance profiles. Examination of the biogeography of the IncL/M plasmids revealed that they are most frequently found in bacteria of the family Enterobacteriaceae originating from the Mediterranean region and Western Europe and that they are able to persist in various ecological niches even in the absence of direct antibiotic selection pressure.

Plasmids are components of many bacterial genomes. They enable the spread of a large pool of genetic information via lateral gene transfer. Many bacterial strains contain mega-sized replicons and these are particularly common in Alphaproteobacteria. Considerably less is known about smaller alphaproteobacterial plasmids. We analyzed the genomes of 14 such plasmids residing in 4 multireplicon carotenoid-producing strains of the genus Paracoccus (Alphaproteobacteria): P. aestuarii DSM 19484, P. haeundaensis LG P-21903, P. marcusii DSM 11574 and P. marcusii OS22. Comparative analyses revealed mosaic structures of the plasmids and recombinational shuffling of diverse genetic modules involved in (i) plasmid replication, (ii) stabilization (including toxin-antitoxin systems of the relBE/parDE, tad-ata, higBA, mazEF and toxBA families) and (iii) mobilization for conjugal transfer (encoding relaxases of the MobQ, MobP or MobV families). A common feature of the majority of the plasmids is the presence of AT-rich sequence islets (located downstream of exc1-like genes) containing genes, whose homologs are conserved in the chromosomes of many bacteria (encoding e.g. RelA/SpoT, SMC-like proteins and a retron-type reverse transcriptase). The results of this study have provided insight into the diversity and plasticity of plasmids of Paracoccus spp., and of the entire Alphaproteobacteria. Some of the identified plasmids contain replication systems not described previously in this class of bacteria. The composition of the plasmid genomes revealed frequent transfer of chromosomal genes into plasmids, which significantly enriches the pool of mobile DNA that can participate in lateral transfer. Many strains of Paracoccus spp. have great biotechnological potential, and the plasmid vectors constructed in this study will facilitate genetic studies of these bacteria.

Pseudomonas sp. GLE121 (a psychrophilic Antarctic strain) carries three plasmids: pGLE121P1 (6899 bp), pGLE121P2 (8330 bp) and pGLE121P3 (39,583 bp). Plasmids pGLE121P1 and pGLE121P2 show significant sequence similarity to members of the IncP-9 and IncP-7 incompatibility groups, respectively, while the largest replicon, pGLE121P3, is highly related to plasmid pNCPPB880-40 of Pseudomonas syringae pathovar tomato NCPPB880. All three plasmids have a narrow host range, limited to members of the genus Pseudomonas. Plasmid pGLE121P3 encodes a conjugal transfer system, while pGLE121P1 carries only a putative MOB module, conserved in many mobilizable plasmids. Plasmid pGLE121P3 contains an additional load of genetic information, including a pair of genes with homology to the rulAB operon, responsible for ultraviolet radiation (UVR) tolerance. Given the increasing UV exposure in Antarctic regions, the expression of these genes is likely to be an important adaptive response., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Six strains of Psychrobacter spp. isolated from guano of little auks collected on Spitsbergen island (Arctic) carried nine plasmids that were fully sequenced. These replicons (ranging in size from 2917 to 14924 bp) contained either repA (ColE2-type) or repB (iteron-type) replication systems of a relatively narrow host range, limited to Psychrobacter spp. All but one of the plasmids carried predicted mobilization for conjugal transfer systems, encoding relaxases of the MOBQ, MOBV or MOBP families. The plasmids also contained diverse additional genetic load, including a type II restriction-modification system and a gene encoding a putative subunit C of alkyl hydroperoxide reductase (AhpC)-an antioxidant enzyme and major scavenger of reactive oxygen species. Detailed comparative sequence analyses, extended to all plasmids identified so far in psychrophilic bacteria, distinguished groups of the most ubiquitous replicons, which play a key role in horizontal gene transfer in cold environments.

Klebsiella pneumoniae 287-w carries three small narrow host range (NHR) plasmids (pIGMS31, pIGMS32, and pIGRK), which could be maintained in several closely related species of Gammaproteobacteria, but not in Alphaproteobacteria. The plasmids contain different mobilization systems (MOB), whose activity in Escherichia coli was demonstrated in the presence of the helper transfer system originating from plasmid RK2. The MOBs of pIGMS31 and pIGMS32 are highly conserved in many bacterial plasmids (members of the MOB family), while the predicted MOB of pIGRK has a unique structure, encoding a protein similar to phage-related integrases. The MOBs of pIGMS31 and pIGMS32 enabled the transfer of heterologous replicons from E. coli into both gammaproteobacterial and alphaproteobacterial hosts, which suggests that these NHR plasmids contain broad host range MOB systems. Such plasmids therefore represent efficient carrier molecules, which may act as natural suicide vectors promoting the spread of diverse genetic information (including other types of mobile elements, e.g. resistance transposons) among evolutionarily distinct bacterial species. Thus, mobilizable NHR plasmids may play a much more important role in horizontal gene transfer than previously thought., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

Plasmid pAMI7 of the methylotrophic bacterium Paracoccus aminophilus JCM 7686 (Alphaproteobacteria) encodes a functional type II restriction-modification (R-M) system designated PamI. Homologous systems were identified in the genomes of distinct taxonomic groups of Bacteria and Archaea, which provides evidence that horizontal gene transfer has contributed to the wide dissemination of R-M modules - even between domains. Analysis of the cleavage specificity of the R.PamI endonuclease revealed that this protein is an isoschizomer of restriction enzyme NcoI. Interestingly, bioinformatic analyses suggest that R.PamI and NcoI are accompanied by methyltransferases of different methylation specificities (C5-methylcytosine and N4-methylcytosine methyltransferases, respectively), which possibly exemplifies recombinational shuffling of genes coding for individual components of R-M systems. The PamI system can stabilize plasmid pAMI7 in a bacterial population, most probably at the postsegregational level. Therefore, it functions in an analogous manner to plasmid-encoded toxin-antitoxin (TA) systems. Since the TA system of pAMI7 is nonfunctional, it is highly probable that this lack is compensated by the stabilizing activity of PamI. This indicates the crucial role of the analyzed R-M system in the stable maintenance of pAMI7, which is, to our knowledge, the first report of 'symbiosis' between a R-M system and a plasmid in the Alphaproteobacteria., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

We have developed a DIY (Do It Yourself) series of genetic cassettes, which facilitate construction of novel versatile vectors for Alphaproteobacteria. All the cassettes are based on defined genetic modules derived from three natural plasmids of Paracoccus aminophilus JCM 7686. We have constructed over 50 DIY cassettes, which differ in structure and specific features. All of them are functional in eight strains representing three orders of Alphaproteobacteria: Rhodobacterales, Rhizobiales and Caulobacterales. Besides various replication and stabilization systems, many of the cassettes also contain selective markers appropriate for Alphaproteobacteria (40 cassettes) and genetic modules responsible for mobilization for conjugal transfer (24 cassettes). All the DIY cassettes are bordered by different types of polylinkers, which facilitate vector construction. Using these DIY cassettes, we have created a set of compatible Escherichia coli-Alphaproteobacteria mobilizable shuttle vectors (high or low copy number in E. coli), which will greatly assist the genetic manipulation of Alphaproteobacteria., (Copyright © 2011 Elsevier B.V. All rights reserved.)

N,N-Dimethylformamide (DMF), a toxic solvent used in the chemical industry, is frequently present in industrial wastes. Plasmid pAMI2 (18.6 kb) of Paracoccus aminophilus JCM 7686 carries genetic information which is crucial for methylotrophic growth of this bacterium, using DMF as the sole source of carbon and energy. Besides a conserved backbone related to pAgK84 of Agrobacterium radiobacter K84, pAMI2 carries a three-gene cluster coding for the protein DmfR, which has sequence similarities to members of the LuxR family of transcription regulators, and two subunits (DmfA1 and DmfA2) of N,N-dimethylformamidase, an enzyme of high substrate specificity that catalyzes the first step in the degradation of DMF. Genetic analysis revealed that these genes, which are all placed in the same orientation, constitute an inducible operon whose expression is activated in the presence of DMF by the positive transcription regulator DmfR. This operon was used to construct a strain able to degrade DMF at high concentrations that might be used in the biotreatment of DMF-containing industrial wastewaters. To our knowledge, this is the first study to provide insights into the genetic organization and regulation as well as the dissemination in bacteria of genes involved in the enzymatic breakdown of DMF.

We demonstrated that a single copy of insertion sequence ISPme1 can mobilize adjacent segments of genomic DNA of Paracoccus methylutens DM12, which leads to the generation of diverse transposable elements of various size and DNA contents. All elements (named transposable modules [TMos]) contain ISPme1 (placed at the 5' ends of the elements) and have variable 3'-end regions of between 0.5 and 5 kb. ISPme1 was shown to encode an outwardly oriented promoter, which may activate the transcription of genes transposed within TMos in evolutionarily distinct hosts. TMos may therefore be considered to be natural systems enabling gene capture, expression, and spread. However, unless these elements have been inserted into a highly conserved genetic context to enable a precise definition of their termini, it is extremely difficult or even impossible to identify them in bacterial genomes by in silico sequence analysis. We showed that TMos are present in the chromosome and plasmids of strain DM12. Sequence analysis of plasmid pMTH1 (32 kb) revealed that four TMos, previously identified with a trap vector, pMEC1, comprise 87% of its genome. Repeated TMos within pMTH1 may stimulate other structural rearrangements resulting from homologous recombination between long repeat sequences. This illustrates that TMos may play a significant role in shaping the structure of natural plasmids, which consequently may have a great impact on the evolution of plasmid genomes.

A group of proteic toxin-antitoxin (TA) cassettes whose representatives are widely distributed among bacterial genomes has been identified. These cassettes occur in chromosomes, plasmids, bacteriophages, and noncomposite transposons, as well as in the SXT conjugative element of Vibrio cholerae. The following four homologous loci were subjected to detailed comparative studies: (i) tad-ata from plasmid pAMI2 of Paracoccus aminophilus (the prototype of this group), (ii) gp49-gp48 from the linear bacteriophage N15 of Escherichia coli, (iii) s045-s044 from SXT, and (iv) Z3230-Z3231 from the genomic island of enterohemorrhagic Escherichia coli O157:H7 strain EDL933. Functional analysis revealed that all but one of these loci (Z3230-Z3231) are able to stabilize heterologous replicons, although the host ranges varied. The TA cassettes analyzed have the following common features: (i) the toxins are encoded by the first gene of each operon; (ii) the antitoxins contain a predicted helix-turn-helix motif of the XRE family; and (iii) the cassettes have two promoters that are different strengths, one which is located upstream of the toxin gene and one which is located upstream of the antitoxin gene. All four toxins tested are functional in E. coli; overexpression of the toxins (in the absence of antitoxin) results in a bacteriostatic effect manifested by elongation of bacterial cells and growth arrest. The toxins have various effects on cell viability, which suggests that they may recognize different intracellular targets. Preliminary data suggest that different cellular proteases are involved in degradation of antitoxins encoded by the loci analyzed.

The replication system of plasmid pMTH4 (22 kb) of dichloromethane-degrading Paracoccus methylutens DM12 (Alphaproteobacteria) has been cloned within a mini-replicon pMTH100 (4.7 kb) and preliminarily characterized. Functional analysis, performed with a series of mutated plasmid mini-derivatives, showed that the replicator region consists of three elements: (i) gene repA coding for a replication initiation protein RepA, (ii) origin of replication (oriV), placed in the promoter region of repA and containing a set of imperfect directly repeated sequences (iterons) together with putative DnaA and IHF-binding DNA sequences as well as (iii) an enhancer (0.65 kb) upstream of oriV. We showed that the enhancer was not crucial for plasmid replication, however, it was necessary to assure the proper plasmid copy number. Additionally its presence has increased the strength of a determinant of incompatibility (located within the oriV region) as well as the level of transcription carried from the repA promoter. The enhancer region was shown not to encode any proteins or promoter sequences. We speculate that this region might constitute a site of binding of plasmid or host-encoded proteins that are able to interact with the origin, which positively regulates the initiation of replication.

Using cointegrate formation, we constructed a basic replicon of the megaplasmid/mini-chromosome pTAV3 of Paracoccus versutus UW1. It is composed of two adjacent modules, responsible for plasmid replication (rep) and partitioning (par). Functional analysis of the par region identified a determinant of incompatibility (inc2), whose presence is crucial for proper partitioning (the partitioning site). Database searches revealed that the only known replicon with significant homology to that of pTAV3 is encoded by the chromosome cII of Rhodobacter sphaeroides 2.4.1. Incompatibility studies showed that closely related basic replicons are also encoded by megaplasmids (above 400 kb) harbored by four strains of P. pantotrophus. Basic replicons of the pTAV3-type are able to maintain large bacterial genomes, therefore they appear to be good candidates for the construction of vectors specific for Alphaproteobacteria.

Two functional regions within the basic replicon of plasmid pMTH4 of Paracoccus methylutens DM12 have been distinguished that are responsible for the replication of the plasmid (REP) and its stabilization (STA). In the REP region, a gene encoding the putative replication initiation protein RepA has been identified, with the highest similarity to the replication protein of plasmid pALC1 (Paracoccus alcaliphilus). The potential origin of replication (oriV), consisting of five long repeated sequences (iterons) as well as putative DnaA and IHF boxes, has been localized in the promoter region of the gene repA. The STA region was found to ensure stability for heterogeneous plasmid pABW3 that is unstable itself in paracocci. The mini-STA region (850 bp) contains two short open reading frames, one of which shows similarity to the RelB protein of Escherichia coli. Our investigations suggest that the stabilizing system of pMTH4 is based on the toxin and antidote principle.

The repABC replicons have an unusual structure, since they carry genes coding for partitioning (repA, repB) and replication (repC) proteins, which are organized in an operon. So far, the presence of these compact bi-functional modules has been reported only in the megaplasmids of the Rhizobiaceae and within the plasmid pTAV1 (107kb) of Paracoccus versutus. We studied the distribution of repABC-type replicons within bacteria belonging to the genus Paracoccus. We found that repABC replicons occur only in the group of pTAV1-like plasmids: pKLW1, pHG16-a, pWKS2, and pPAN1, harbored by different strains of Paracoccus pantotrophus. A partial sequencing approach followed by phylogenetic analysis revealed that these replicons constitute a distinct evolutionary branch of repABC replicons. Incompatibility studies showed that they represent two incompatibility groups designated IncABC1 (pTAV1, pKLW1, and pHG16-a) and IncABC2 (pPAN1). Sequence comparison using available databases allowed the identification, within plasmid pRS241d of Rhodobacter sphaeroides 2.4.1, of an additional sequence highly homologous to the paracoccal repABC replicons, which has been included in comparative analyses.

The complete nucleotide sequence of the small, cryptic plasmid pWKS1 (2697 bp) of Paracoccus pantotrophus DSM 11072 was determined. The G+C content of the sequence of this plasmid was 62 mol%. Analysis revealed that over 80% of the plasmid genome was covered by two ORFs, ORF1 and ORF2, which were capable of encoding putative peptides of 44.1 and 37.8 kDa, respectively. Mutational analysis showed that ORF2 was crucial for plasmid replication. The translational product of ORF2 shared local homologies with replication proteins of several theta-replicating lactococcal plasmids, as well as with the Rep proteins of plasmids residing in Gram-negative hosts. An A+T-rich region, located upstream of the rep gene and containing three tandemly repeated 21 bp long iteron-like sequences, served as the origin of replication (oriV). ORF1 encoded a putative mobilization protein with similarities to mobilization proteins (Mob) from the broad-host-range plasmid pBBR1 and plasmids of Gram-positive bacteria. A plasmid bearing the MOB module of pWKS1 (the mob gene and the oriT sequence) could be mobilized for transfer (by IncP RP4 transfer apparatus) at low frequency between different strains of Escherichia coli. MOB modules of pWKS1 and pBBR1 were functionally complementary to each other. Hybridization analysis revealed that only plasmid pSOV1 (6.5 kb), among all of the paracoccal plasmids identified so far, carries sequences related to pWKS1. Plasmid pWKS1 could replicate in 10 species of Paracoccus and in Agrobacterium tumefaciens, Rhizobium leguminosarum and Rhodobacter sphaeroides, but it could not replicate in E. coli.

The replicon of the pTAV3 megaplasmid (approx. 400 kb) of Paracoccus versutus has been localized to a 4center dot3 kb EcoRI restriction fragment and its entire nucleotide sequence determined. The G+C content of the entire sequence is 66 mol%, which is within the range (62-66 mol%) previously determined for P. versutus total DNA. ORF1 encodes a replication initiation protein Rep (47.2 kDa), which shares substantial similarity with putative proteins of the Coxiella burnetii plasmids QpH1 and QpDV, and the replication protein of Pseudomonas syringae plasmid pPS10. ORF2, located in the opposite transcriptional orientation to ORF1, encodes a putative protein that shares similarity to a subfamily of ATPases involved in plasmid partitioning. The highest similarity was observed with homologous proteins (RepA) encoded by the repABC family of replicons found in several plasmids of Agrobacterium, Rhizobium and Paracoccus spp. The predicted product of ORF3 was similar to AcoR, Nif and NtrC transcriptional activators. A strong incompatibility determinant (inc) was localized between ORF1 (rep) and ORF2 (parA). The origin of replication of pTAV400 contains a short A+T-rich region and several imperfect palindromic sequences. Curing experiments demonstrated that the megaplasmid bears genes required for growth in minimal media and can therefore be referred to as a mini-chromosome. Megaplasmids pTAV3 of P. versutus UW1 and pKLW2 of Paracoccus pantotrophus DSM 11073 were found to carry closely related, incompatible replicons. It has been shown that plasmid pORI6 (containing oriV of pTAV3 cloned into plasmid pABW1, which does not replicate in Paracoccus spp.) can be trans activated not only by pTAV3, but also by pKLW2. Using pORI6, it was demonstrated that replication systems related to pTAV3 are also present in the replicons of Paracoccus alcaliphilus JCM 7364, Paracoccus thiocyanatus IAM 12816 and Paracoccus methylutens DM 12.

The second replicator region of the native plasmid pTAV1 of Paracoccus versutus has been identified thus proving the composite nature of this replicon. The minimal replicon designated pTAV320 (4.3 kb) was cloned and sequenced. pTAV320 encodes three putative proteins--RepA, RepB and RepC. This replicator region shows strong structural and functional similarity to repABC-type replicons found in several Agrobacterium and Rhizobium plasmids. The origin of replication appears to be localized within the coding sequence of the repC gene. RepC was shown to be essential for replication. RepA and RepB were necessary for stable maintenance of the plasmid, which implies a role in active partitioning. The presence of the complete sequence of pTAV320 (in its non-replicative form) could stabilize in cis pTAV202, a mini-replicon derived from the other pTAV1 replicator region. Deletions introduced into the repC gene abolished the 'stabilizing' activity of pTAV320, suggesting that the centromere-like sequence, necessary for partitioning, might be localized within this gene. The two replicator regions of pTAV1 (pTAV320 and pTAV202) expressed incompatibility towards the parental plasmid but were compatible in trans in P. versutus cells. The pTAV320 replicon can be maintained in several Paracoccus, Agrobacterium, Rhizobium and Rhodobacter strains in addition to P. versutus.

The cold-adapted Psychrobacter sp. strain DAB_AL62B, isolated from ornithogenic deposits on the Arctic island of Spitsbergen, harbors a 34.5 kb plasmid, pP62BP1, which carries a genetic SLF module predicted to enable the host bacterium to metabolize alkyl sulfates including sodium dodecyl sulfate (SDS), a common anionic surfactant. In this work, we experimentally confirmed that the pP62BP1-harboring strain is capable of SDS degradation. The slfCHSL genes were shown to form an operon whose main promoter, PslfC, is negatively regulated by the product of the slfR gene in the absence of potential substrates. We showed that lauryl aldehyde acts as an inducer of the operon. The analysis of the draft genome sequence of the DAB_AL62B strain revealed that the crucial enzyme of the SDS degradation pathway—an alkyl sulfatase—is encoded only within the plasmid. The SLF module is flanked by two restriction–modification systems, which were shown to exhibit the same sequence specificity. We hypothesize that the maintenance of pP62BP1 may be dependent on this unique genetic organization. [ABSTRACT FROM AUTHOR]

Bacteria of the genus Paracoccus are common components of the microbiomes of many naturally- and anthropogenically shaped environments. One species, Paracoccus yeei , is unique within the genus because it is associated with opportunistic human infections. Therefore, strains of P. yeei may serve as an interesting model to study the transition from a saprophytic to a pathogenic lifestyle in environmental bacteria. Unfortunately, knowledge concerning the biology, genetics and genomic content of P. yeei is fragmentary; also the mechanisms of pathogenicity of this bacterium remain unclear. In this study we provide the first insight into the genome composition and metabolic potential of a clinical isolate, P. yeei CCUG 32053. This strain has a multipartite genome (4,632,079 bp) composed of a circular chromosome plus eight extrachromosomal replicons pYEE1–8: 3 chromids and 5 plasmids, with a total size of 1,247,173 bp. The genome has been significantly shaped by the acquisition of genomic islands, prophages (Myoviridae and Siphoviridae phage families) and numerous insertion sequences (ISs) representing seven IS families. Detailed comparative analysis with other complete genomic sequences of Paracoccus spp. (including P. yeei FDAARGOS_252 and TT13, as well as non-pathogenic strains of other species in this genus) enabled us to identify P. yeei species-specific genes and to predict putative determinants of virulence. This is the first attempt to identify pathoadaptive genetic information of P. yeei and to estimate the role of the mobilome in the evolution of pathogenicity in this species. [ABSTRACT FROM AUTHOR]

Cold-active bacteria of the genus Polaromonas (class Betaproteobacteria) are important components of glacial microbiomes. In this study, extrachromosomal replicons of 26 psychrotolerant Polaromonas strains, isolated from Arctic and Antarctic glaciers, were identified, sequenced, and characterized. The plasmidome of these strains consists of 13 replicons, ranging in size from 3,378 to 101,077 bp. In silico sequence analyses identified the conserved backbones of these plasmids, composed of genes required for plasmid replication, stable maintenance, and conjugal transfer. Host range analysis revealed that all of the identified plasmids are narrow-host-range replicons, only able to replicate in bacteria of closely related genera (Polaromonas and Variovorax) of the Comamonadaceae family. Special attention was paid to the identification of plasmid auxiliary genetic information, which may contribute to the adaptation of bacteria to environmental conditions occurring in glaciers. Detailed analysis revealed the presence of genes encoding proteins potentially involved in (i) protection against reactive oxygen species, ultraviolet radiation, and low temperatures; (ii) transport and metabolism of organic compounds; (iii) transport of metal ions; and (iv) resistance to heavy metals. Some of the plasmids also carry genes required for the molecular assembly of iron–sulfur [Fe-S] clusters. Functional analysis of the predicted heavy metal resistance determinants demonstrated that their activity varies, depending on the host strain. This study provides the first molecular insight into the mobile DNA of Polaromonas spp. inhabiting polar glaciers. It has generated valuable data on the structure and properties of a pool of plasmids and highlighted their role in the biology of psychrotolerant Polaromonas strains and their adaptation to the environmental conditions of Arctic and Antarctic glaciers. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Functional transposable elements (TEs) of several Pseudomonas 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 (IS3, IS5, ISL3, IS30 and IS1380), (ii) isoforms of two Tn3-family transposons – Tn5563a and Tn4662a (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 Tn5563a and many other Tn3-family transposons. The simplest elements of this type, designated TIMEs (Tn3 family-derived Inverted-repeat Miniature Elements) (262 bp), were identified within two natural plasmids (pZM1P1 and pLM8P2) of Pseudomonas 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 res site typical for many Tn3-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. [ABSTRACT FROM AUTHOR]

Background Paracoccus aminophilus JCM 7686 is a methylotrophic α-Proteobacterium capable of utilizing reduced one-carbon compounds as sole carbon and energy source for growth, including toxic N,N-dimethylformamide, formamide, methanol, and methylamines, investigate P. aminophilus which are widely used in the industry. P. aminophilus JCM 7686, as many other Paracoccus spp., possesses a genome representing a multipartite structure, in which the genomic information is split between various replicons, including chromids, essential plasmid-like replicons, with properties of both chromosomes and plasmids. In this study, whole-genome sequencing and functional genomics approaches were applied to investigate P. aminophilus genome information. Results The P. aminophilus JCM 7686 genome has a multipartite structure, composed of a single circular chromosome and eight additional replicons ranging in size between 5.6 and 438.1 kb. Functional analyses revealed that two of the replicons, pAMI5 and pAMI6, are essential for host viability, therefore they should be considered as chromids. Both replicons carry housekeeping genes, e.g. responsible for de novo NAD biosynthesis and ammonium transport. Other mobile genetic elements have also been identified, including 20 insertion sequences, 4 transposons and 10 prophage regions, one of which represents a novel, functional serine recombinase-encoding bacteriophage, Pam-6. Moreover, in silico analyses allowed us to predict the transcription regulatory network of the JCM 7686 strain, as well as components of the stress response, recombination, repair and methylation machineries. Finally, comparative genomic analyses revealed that P. aminophilus JCM 7686 has a relatively distant relationship to other representatives of the genus Paracoccus. Conclusions P. aminophilus genome exploration provided insights into the overall structure and functions of the genome, with a special focus on the chromids. Based on the obtained results we propose the classification of bacterial chromids into two types: "primary" chromids, which are indispensable for host viability and "secondary" chromids, which are essential, but only under some environmental conditions and which were probably formed quite recently in the course of evolution. Detailed genome investigation and its functional analysis, makes P. aminophilus JCM 7686 a suitable reference strain for the genus Paracoccus. Moreover, this study has increased knowledge on overall genome structure and composition of members within the class Alphaproteobacteria. [ABSTRACT FROM AUTHOR]

The complete nucleotide sequence of plasmid pP62BP1 (34,467 bp), isolated from Arctic Psychrobacter sp. DAB_AL62B, was determined and annotated. The conserved plasmid backbone is composed of several genetic modules, including a replication system (REP) with similarities to the REP region of the iteron-containing plasmid pPS10 of Pseudomonas syringae. The additional genetic load of pP62BP1 includes two highly related type II restriction-modification systems and a set of genes ( slfRCHSL) encoding enzymes engaged in the metabolism of organic sulfates, plus a putative transcriptional regulator (SlfR) of the AraC family. The pP62BP1 slf locus has a compact and unique structure. It is predicted that the enzymes SlfC, SlfH, SlfS and SlfL carry out a chain of reactions leading to the transformation of alkyl sulfates into acyl-CoA, with dodecyl sulfate (SDS) as a possible starting substrate. Comparative analysis of the nucleotide sequences of pP62BP1 and other Psychrobacter spp. plasmids revealed their structural diversity. However, the presence of a few highly conserved DNA segments in pP62BP1, plasmid 1 of P. cryohalolentis K5 and pRWF-101 of Psychrobacter sp. PRwf-1 is indicative of recombinational shuffling of genetic information, and is evidence of lateral gene transfer in the Arctic environment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A sacB-based trap plasmid was used to clone a mosaic transposable element (TE) in the carotenoid producer Paracoccus marcusii OS22. This element is composed of an insertion sequence (IS) IS Pmar4 (IS As1 family), which contains an inserted functional transposable module (TMo) generated by a copy of IS 1247a (of the IS 1380 family). Besides IS 1247a sequences, the TMo also contains the 3′-end region of a putative α/β hydrolase gene, whose expression might be activated from the P2 promoter of IS 1247a. The identification of this novel TE provides evidence that transposition of TMos may change an IS into a more complex element resembling noncomposite transposons. [ABSTRACT FROM AUTHOR]

Examines the location of active partitioning site of the mini-replicon of the composite Paracoccus versutus Plasmid pTAV1. Association between repARC replicons and genera Rhizobium and Agrobacterium; Use of repABc for the localization of pTAV320; Identification of incompatibility determinants of pTAV320.

Listeria monocytogenes is a pathogen responsible for severe cases of food poisoning. Listeria spp. strains occurring in soil and water environments may serve as a reservoir of resistance determinants for pathogenic L. monocytogenes strains. A large collection of Listeria spp. strains (155) isolated from natural, agricultural, and urban areas was screened for resistance to heavy metals and metalloids, and the presence of resistance determinants and extrachromosomal replicons. Of the tested strains, 35% were resistant to cadmium and 17% to arsenic. Sequence analysis of resistance plasmids isolated from strains of Listeria seeligeri and Listeria ivanovii, and the chromosome of L. seeligeri strain Sr73, identified a novel variant of the cadAC cadmium resistance efflux system, cadA6, that was functional in L. monocytogenes cells. The cadA6 cassette was detected in four Listeria species, including strains of L. monocytogenes, isolated from various countries and sources—environmental, food-associated, and clinical samples. This resistance cassette is harbored by four novel composite or non-composite transposons, which increases its potential for horizontal transmission. Since some cadAC cassettes may influence virulence and biofilm formation, it is important to monitor their presence in Listeria spp. strains inhabiting different environments. [ABSTRACT FROM AUTHOR]

Abstract The sixty-seven nonpathogenic Listeria spp. strains isolated from food and food processing environments in Poland were examined for the presence of benzalkonium chloride (BC) resistance cassette (bcrABC) and four different variants of cadmium resistance determinants (cadA1-cadA4). All the strains were phenotypically resistant to cadmium and 22 among them were also resistant to BC. PCR-based analysis revealed that bcrABC cassette was harbored by 95.5% of the strains phenotypically resistant to BC. All of them harbored also either cadA1 or cadA2 genes (none carried cadA3 or cadA4), which corresponded to the presence of plasmids with two restriction patterns. The strains resistant to cadmium but susceptible to BC harbored only the cadA1 gene variant. DNA-DNA hybridization analysis showed that all the identified bcrABC , cadA 1 and cadA2 genes were located within plasmids, classified into 11 groups of RFLP profiles. Only one of the plasmids – pLIS1 of Listeria welshimeri (carrying bcrABC and cadA2) – was capable of efficient conjugal transfer from nonpathogenic Listeria isolates to a pathogenic Listeria monocytogenes strain. Analysis of the complete nucleotide sequence of pLIS1 (the first sequenced plasmid of L. welshimeri species) revealed the presence of genes involved in plasmid replication, stabilization and transfer as well as genes conferring resistance phenotypes. Comparative analysis showed that pLIS1 genome is highly similar to a group of plasmids originating from L. monocytogenes strains. A common feature of pLIS1 and its relatives, besides the presence of the resistance genes, is the presence of numerous transposable elements (TEs). The analysis revealed the important role of TEs in both promoting genetic rearrangements within Listeria spp. plasmids and the acquisition of resistance determinants. Highlights • Nonpathogenic cadmium resistant Listeria spp. carry either cadA1 or cadA2 genes • Majority of strains resistant to benzalkonium chloride carry bcrABC genes • The resistance determinants are located within plasmids of 11 RFLP patterns • Plasmid pLIS1 of L. welshimeri is capable of efficient conjugal transfer • pLIS1 contains bcrABC and cadA2 as well as numerous transposable elements [ABSTRACT FROM AUTHOR]

Psychrobacter sp. DAB_AL43B, isolated from ornithogenic soil collected on the Arctic island of Spitsbergen, is a newly sequenced psychrophilic strain susceptible to conjugation and electrotransformation. Its genome consists of a circular chromosome (3.3 Mb) and four plasmids (4.4–6.4 kb). In silico genome mining and microarray-based phenotypic analysis were performed to describe the metabolic potential of this strain and identify possible biotechnological applications. Metabolic reconstruction indicated that DAB_AL43B prefers low-molecular-weight carboxylates and amino acids as carbon and energy sources. Genetic determinants of heavy-metal resistance, anthracene degradation and possible aerobic denitrification were also identified. Comparative analyses revealed a relatively close relationship between DAB_AL43B and other sequenced Psychrobacter species. In addition, the plasmids of this strain were used as the basis for the construction of Escherichia coli – Psychrobacter spp. shuttle vectors. Taken together, the results of this work suggest that DAB_AL43B is a promising candidate as a new model strain for studies on Psychrobacter spp. [ABSTRACT FROM AUTHOR]