Conjugative Plasmid pPPUT-Tik1-1 from a Permafrost Pseudomonas putida Strain and Its Present-Day Counterparts Inhabiting Environments and Clinics.

A novel group of conjugative plasmids of Pseudomonas is characterized. The prototype plasmid pPPUT-Tik1-1 (153,663 bp), isolated from a permafrost strain of P. putida Tik1, carries a defective mercury transposon, Tn501, and a streptomycin resistance transposon, Tn5393. Ten plasmids and 34 contigs with backbone regions closely related to pPPUT-Tik1-1 have been found in GenBank. Two of these plasmids from clinical strains of P. putida and P. fulva are almost identical to the ancient plasmid. A characteristic feature of this group of plasmids is the presence of two genes encoding the initiators of replication (repA1 and repA2). None of these genes have high similarity with plasmid replication genes belonging to known incompatibility groups. It has been demonstrated that while pPPUT-Tik1-1-like plasmids have homologous backbone regions, they significantly differ by the molecular structure and the predicted functions of their accessory regions. Some of the pPPUT-Tik1-1-related plasmids carry determinants of antibiotic resistance and/or heavy metal salts. Some plasmids are characterized by the ability to degrade xenobiotics. Plasmids related to pPPUT-Tik1-1 are characterized by a narrow host range and are found in various species of the Pseudomonas genus. Interestingly, we also found shorter plasmid variants containing the same replication module, but lacking conjugation genes and containing other structural changes that strongly distinguish them from plasmids related to pPPUT-Tik1-1, indicating that the structure of the replication module cannot be used as the sole criterion for classifying plasmids. Overall, the results suggest that the plasmids of the novel group can be spread using conjugation in environmental and clinical strains of Pseudomonas and may play diverse adaptive functions due to the presence of various accessory regions.

Plasmids as Key Players in Acinetobacter Adaptation.

This review briefly summarizes the data on the mechanisms of development of the adaptability of Acinetobacters to various living conditions in the environment and in the clinic. A comparative analysis of the genomes of free-living and clinical strains of A. lwoffii, as well as the genomes of A. lwoffii and A. baumannii, has been carried out. It has been shown that plasmids, both large and small, play a key role in the formation of the adaptability of Acinetobacter to their living conditions. In particular, it has been demonstrated that the plasmids of various strains of Acinetobacter differ from each other in their structure and gene composition depending on the lifestyle of their host bacteria. Plasmids of modern strains are enriched with antibiotic-resistant genes, while the content of genes involved in resistance to heavy metals and arsenic is comparable to plasmids from modern and ancient strains. It is concluded that Acinetobacter plasmids may ensure the survival of host bacteria under conditions of various types of environmental and clinical stresses. A brief overview of the main mechanisms of horizontal gene transfer on plasmids inherent in Acinetobacter strains is also given.

Ubiquitous Conjugative Mega-Plasmids of Acinetobacter Species and Their Role in Horizontal Transfer of Multi-Drug Resistance.

Conjugative mega-plasmids play a special role in adaptation since they carry a huge number of accessory genes, often allowing the host to develop in new niches. In addition, due to conjugation they are able to effectively spread themselves and participate in the transfer of small mobilizable plasmids. In this work, we present a detailed characterization of a recently discovered family of multiple-drug resistance mega-plasmids of Acinetobacter species, termed group III-4a. We describe the structure of the plasmid backbone region, identify the rep gene and the origin of plasmid replication, and show that plasmids from this group are able not only to move between different Acinetobacter species but also to efficiently mobilize small plasmids containing different mob genes. Furthermore, we show that the population of natural Acinetobacter strains contains a significant number of mega-plasmids and reveal a clear correlation between the living conditions of Acinetobacter strains and the structure of their mega-plasmids. In particular, comparison of the plasmids from environmental and clinical strains shows that the genes for resistance to heavy metals were eliminated in the latter, with the simultaneous accumulation of antibiotic resistance genes by incorporation of transposons and integrons carrying these genes. The results demonstrate that this group of mega-plasmids plays a key role in the dissemination of multi-drug resistance among Acinetobacter species.

Acinetobacter Plasmids: Diversity and Development of Classification Strategies.

Bacteria of the genus Acinetobacter, with their numerous species common in various habitats, play a significant role as pathogens. Their ability to adapt to different living conditions is largely due to the presence of numerous plasmids containing the necessary adaptive genes. At the same time the diversity of Acinetobacter plasmids and their evolutionary dynamics have not been sufficiently studied. Here, we characterized 44 plasmids isolated from five permafrost Acinetobacter lwoffii strains, examined their relationship with plasmids of modern Acinetobacter strains and identified groups of related plasmids. For this purpose, we have developed a combined approach for classifying all known Acinetobacter plasmids. The classification took into account the size of plasmids, the presence and structure of the rep and mob genes, as well as the structure of their backbone and accessory regions. Based on the analysis, 19 major groups (lineages) of plasmids were identified, of which more than half were small plasmids. The plasmids of each group have common features of the organization of the backbone region with a DNA identity level of at least 80%. In addition, plasmids of the same group have similarities in the organization of accessory regions. We also described a number of plasmids with a unique structure. The presence of plasmids in clinical strains that are closely related to those of environmental permafrost strains provides evidence of the origin of the former from the latter.

Adaptive dif Modules in Permafrost Strains of Acinetobacter lwoffii and Their Distribution and Abundance Among Present Day Acinetobacter Strains.

The dif/Xer system of site-specific recombination allows resolution of chromosomal dimers during bacterial DNA replication. Recently, it was also shown to be involved in horizontal transfer of a few known Xer-dependent mobile elements. Here, we show that plasmids of various Acinetobacter species, including clinically important strains, often contain multiple pdif sites that are mainly located within their accessory regions. Chromosomes of Acinetobacter strains may also contain additional dif sites, and their similarity with plasmid pdif sites is higher than with the main chromosomal site dif1. We further identify putative mobile genetic elements containing pdif sites on both flanks of adaptive genes and analyze their distribution in Acinetobacter species. In total, we describe seven mobile elements containing genes with various adaptive functions from permafrost strains of A. lwoffii group. All of them are also spread in modern plasmids of different Acinetobacter species including A. baumannii. We could not detect pdif sites and corresponding mobile elements in closely related bacterial genera, including Psychrobacter and Moraxella. Thus, the widespread distribution of dif modules is a characteristic feature of Acinetobacter species and may contribute to their high adaptability both in the environment and in the clinic.

Chromium resistance genetic element flanked by XerC/XerD recombination sites and its distribution in environmental and clinical Acinetobacter strains.

A novel mobile genetic element has been identified in small plasmids isolated from permafrost strains of Acinetobacter lwoffii. This element, designated the chrAB dif module, contains the chromium resistance genes chrA and chrB, functionally active both in the original host strains and after transfer into Acinetobacter baylyi. The 3011 bp chrAB dif module is flanked by XerC/XerD recombination sites highly homologous to those of the site-specific recombination system dif/Xer. Analysis of public databases revealed almost identical variants of the chrAB dif module in different plasmids in strains of various Acinetobacter species predominantly inhabiting the environment (A. lwoffii, Acinetobacter indicus, Acinetobacter idrijaensis, Acinetobacter shindleri and Acinetobacter nosocomialis). Together with previously described Acinetobacter antibiotic resistance elements, the chrAB dif module defines a new group of mobile elements that rely on the dif/Xer system for their mobility. Our observations suggest an ancient origin of the mobile elements flanked by dif sites and their participation in the mobilization of plasmid genes bearing adaptive functions.

Resistance of Permafrost and Modern Acinetobacter lwoffii Strains to Heavy Metals and Arsenic Revealed by Genome Analysis.

We performed whole-genome sequencing of five permafrost strains of Acinetobacter lwoffii (frozen for 15-3000 thousand years) and analyzed their resistance genes found in plasmids and chromosomes. Four strains contained multiple plasmids (8-12), which varied significantly in size (from 4,135 to 287,630 bp) and genetic structure; the fifth strain contained only two plasmids. All large plasmids and some medium-size and small plasmids contained genes encoding resistance to various heavy metals, including mercury, cobalt, zinc, cadmium, copper, chromium, and arsenic compounds. Most resistance genes found in the ancient strains of A. lwoffii had their closely related counterparts in modern clinical A. lwoffii strains that were also located on plasmids. The vast majority of the chromosomal resistance determinants did not possess complete sets of the resistance genes or contained truncated genes. Comparative analysis of various A. lwoffii and of A. baumannii strains discovered a number of differences between them: (i) chromosome sizes in A. baumannii exceeded those in A. lwoffii by about 20%; (ii) on the contrary, the number of plasmids in A. lwoffii and their total size were much higher than those in A. baumannii; (iii) heavy metal resistance genes in the environmental A. lwoffii strains surpassed those in A. baumannii strains in the number and diversity and were predominantly located on plasmids. Possible reasons for these differences are discussed.

The ancient small mobilizable plasmid pALWED1.8 harboring a new variant of the non-cassette streptomycin/spectinomycin resistance gene aadA27.

The small mobilizable plasmid pALWED1.8 containing a novel variant of the streptomycin/spectinomycin resistance gene aadA27 was isolated from the permafrost strains of Acinetobacter lwoffii. The 4135bp plasmid carries mobА and mobC genes that mediate its mobilization by conjugative plasmids. The nucleotide sequences of mobА and mobC are similar to those of mobilization genes of the modern plasmid pRAY* and its variants, which contain aadB gene, and are widespread among the pathogenic strains of Acinetobacter baumannii. Almost identical pALWED1.8 variants were detected in modern environmental Аcinetobacter strains. A highly similar plasmid was revealed in a strain of Acinetobacter parvus isolated from mouse intestine. Furthermore, we discovered six previously unidentified variants of plasmids related to pALWED1.8 and pRAY* in public databases. In contrast to most known variants of aadA which are cassette genes associated with integrons, the aadA27 variant harbored by pALWED1.8 is a non-cassette, autonomously transcribed gene. Non-cassette aadA genes with 96% sequence identity to aadA27 were detected in the chromosomes of Acinetobacter gyllenbergii and several uncharacterized strains of Аcinetobacter sp. Moreover, we revealed that the autonomous aadA-like genes are present in the chromosomes of many gram-positive and gram-negative bacteria. The phylogenetic analysis of amino acid sequences of all identified AadA proteins showed the following: (i) cassette aadA genes form a separate monophyletic group and mainly reside on plasmids and (ii) chromosomal non-cassette aadA genes are extremely diverse and can be inherited both vertical and via horizontal gene transfer.

Genetic structure and biological properties of the first ancient multiresistance plasmid pKLH80 isolated from a permafrost bacterium.

A novel multidrug-resistance plasmid, pKLH80, previously isolated from Psychrobacter maritimus MR29-12 found in ancient permafrost, was completely sequenced and analysed. In our previous studies, we focused on the pKLH80 plasmid region containing streptomycin and tetracycline resistance genes, and their mobilization with an upstream-located ISPpy1 insertion sequence (IS) element. Here, we present the complete sequence of pKLH80 and analysis of its backbone genetic structure, including previously unknown features of the plasmid's accessory region, notably a novel variant of the ß-lactamase gene blaRTG-6. Plasmid pKLH80 was found to be a circular 14 835 bp molecule that has an overall G+C content of 40.3 mol% and encodes 20 putative ORFs. There are two distinctive functional modules within the plasmid backbone sequence: (i) the replication module consisting of repB and the oriV region; and (ii) the mobilization module consisting of mobA, mobC and oriT. All of the aforementioned genes share sequence identities with corresponding genes of different species of Psychrobacter. The plasmid accessory region contains antibiotic resistance genes and IS elements (ISPsma1 of the IS982 family, and ISPpy1 and ISAba14 of the IS3 family) found in environmental and clinical bacterial strains of different taxa. We revealed that the sequences flanking blaRTG-6 and closely related genes from clinical bacteria are nearly identical. This fact suggests that blaRTG-6 from the environmental strain of Psychrobacter is a progenitor of blaRTG genes of clinical bacteria. We also showed that pKLH80 can replicate in different strains of Acinetobacter and Psychrobacter genera. The roles of IS elements in the horizontal transfer of antibiotic resistance genes are examined and discussed.

Genomic characterization and integrative properties of phiSMA6 and phiSMA7, two novel filamentous bacteriophages of Stenotrophomonas maltophilia.

Two novel filamentous phages, phiSMA6 and phiSMA7, were isolated from Stenotrophomonas maltophilia environmental strain Khak84. We identified and annotated 11 potential open reading frames in each phage. While the overall layout of the functional gene groups of both phages was similar to that of the known filamentous phages, they differed from them in their molecular structure. The genome of phiSMA6 is a mosaic that evolved by acquiring genes from at least three different filamentous S. maltophilia phages and one Xanthomonas campestris phage related to Cf1. In the phiSMA6 genome, a gene similar to the bacterial gene encoding the mating pair formation protein trbP was also found. We showed that phiSMA6 possesses lysogenic properties and upon induction produces high-titer lysates. The genome of phiSMA7 possesses a unique structure and was found to be closely related to a prophage present in the chromosome of the completely sequenced S. maltophilia clinical strain D457. We suggest that the other three filamentous phages of S. maltophilia described previously also have the capacity to integrate into the genome of their bacterial host.

A new subgroup of the IS3 family and properties of its representative member ISPpy1.

Recently, we described a novel insertion element, ISPpy1, isolated from a permafrost strain of Psychrobacter maritimus. In this work, we demonstrated that ISPpy1 is a member of a novel subgroup of the IS3 family of insertion sequences (ISs) that was not identified and characterized previously. IS elements of this subgroup termed the ISPpy1 subgroup are broadly distributed among different taxa of Eubacteria, including Geobacteraceae, Chlorobiaceae, Desulfobacteraceae, Methylobacteriaceae, Nitrosomonadaceae and Cyanobacteria. While displaying characteristic features of the IS3-family elements, ISPpy1 subgroup elements exhibit some unusual features. In particular, most of them have longer terminal repeats with unconventional ends and frameshifting box with an atypical organization, and, unlike many other IS3-family elements, do not exhibit any distinct IS specificity. We studied the transposition and mutagenic properties of a representative member of this subgroup, ISPpy1 and showed that in contrast to the original P. maritimus host, in a heterologous host, Escherichia coli K-12, it is able to translocate with extremely high efficiency into the chromosome, either by itself or as a part of a composite transposon containing two ISPpy1 copies. The majority of transposants carry multiple chromosomal copies (up to 12) of ISPpy1. It was discovered that ISPpy1 is characterized by a marked mutagenic activity in E. coli: its chromosomal insertions generate various types of mutations, including auxotrophic, pleiotropic and rifampicin-resistance mutations. The distribution of IS elements of the novel subgroup among different bacteria, their role in the formation of composite transposons and the horizontal transfer of genes are examined and discussed.

A novel gene, ardD, determines antirestriction activity of the non-conjugative transposon Tn5053 and is located antisense within the tniA gene.

The mercury-resistance transposon Tn5053 inhibits restriction activity of the type I restriction-modification endonuclease EcoKI in Escherichia coli K12 cells. This is the first report of antirestriction activity of a non-conjugative transposon. The gene (ardD) coding for the antirestriction protein has been cloned. The ardD gene is located within the tniA gene, coding for transposase, on the complementary strand. The direction of transcription is opposite to transcription of the tniA gene.

Tn5045, a novel integron-containing antibiotic and chromate resistance transposon isolated from a permafrost bacterium.

A novel antibiotic and chromate resistance transposon, Tn5045, was isolated from a permafrost strain of Pseudomonas sp. Tn5045 is a compound transposon composed of three distinct genetic elements. The backbone element is a Tn1013-like Tn3 family transposon, termed Tn1013∗, that contains the tnpA and the tnpR genes, encoding the transposase and resolvase, respectively, the res-site and four genes (orfA, B, C, D) related to different house-keeping genes. The second element is class 1 integron, termed InC∗, which is inserted into the Tn1013∗ res-region and contains 5'-CS-located integrase, 3'-CS-located qacE∆1 and sulfonamide resistance sulI genes, and a single cassette encoding the streptomycin resistance aadA2-gene. The third element is a TnOtChr-like Tn3 family transposon termed TnOtChr∗, which is inserted into the transposition module of the integron and contains genes of chromate resistance (chrB, A, C, F). Tn5045 is the first example of an ancient integron-containing mobile element and also the first characterized compound transposon coding for both antibiotic and chromate, resistance. Our data demonstrate that antibiotic and chromate resistance genes were distributed in environmental bacteria independently of human activities and provide important insights into the origin and evolution of antibiotic resistance integrons.

Molecular structure and translocation of a multiple antibiotic resistance region of a Psychrobacter psychrophilus permafrost strain.

A Psychrobacter psychrophilus strain resistant to tetracycline and streptomycin was isolated from a 15,000-35,000-year-old permafrost subsoil sediment sampled from the coast of the Eastern-Siberian Sea. The genes conferring antibiotic resistance were localized on an c. 30-kb pKLH80 plasmid. It was shown that the antibiotic resistance region of this plasmid has a mosaic structure and contains closely linked streptomycin resistance (strA-strB) and tetracycline resistance [tetR-tet(H)] genes, followed by a novel IS element (ISPpy1) belonging to the IS3 family. Both the strA-strB and tetR-tet(H) genes of pKLH80 were highly similar to those found in modern clinical bacterial isolates. It was shown that the ISPpy1 element of pKLH80 can direct translocation of the adjacent antibiotic resistance genes to different target plasmids, either by one-ended transposition or by formation of a composite transposon resulting from the insertion of the ISPpy1 second copy at the other side of the antibiotic resistance region. Thus, our data demonstrate that clinically important antibiotic resistance genes originated long before the introduction of antibiotics into clinical practice and confirm an important role of horizontal gene transfer in the distribution of these genes in natural bacterial populations.

Present-day mercury resistance transposons are common in bacteria preserved in permafrost grounds since the Upper Pleistocene.

Transposons closely related to mercury resistance transposons Tn5041, Tn5053, and Tn5056, which have been previously described in present-day bacteria, were detected in a survey of 12 mercury-resistant Pseudomonas strains isolated from permafrost samples aged 15-40 thousand years. In addition, Tn5042, a novel type of mercury resistance transposon, was revealed in the permafrost strain collection and its variants found to be common among present-day bacteria. The results reveal that no drastic changes in the distribution mode of the different types of mercury resistance transposons among environmental bacteria have taken place in the last 15-40 thousand years.

Translocation of transposition-deficient (TndPKLH2-like) transposons in the natural environment: mechanistic insights from the study of adjacent DNA sequences.

A family of plasmid-borne DNA fragments of different length, apparently inherited from an ancient plasmid, has been identified in the world population of environmental Acinetobacter strains. These fragments, named PPFs (parental plasmid DNA fragments), were >/=99.8 % identical to each other in the common regions, and contained in their central region a variant of an aberrant mercury-resistance transposon (Tn(d)PKLH2) that has lost its transposition genes. As a rule, recombinogenic elements were found at the breakpoints of identity between the different PPFs. Of these recombinogenic elements, a newly identified IS6 family element, a transposon, or a resolvase gene interrupted one end of the PPFs. At the opposite end, the breakpoint of some PPFs was mapped to the recombination point within, in each case, a different variant of a res site (RS2), whilst in other PPFs, this end was eroded by insertion of a newly identified IS6 family element. On the basis of DNA sequence data, possible mechanisms of translocation of defective Tn(d)PKLH2-like elements via recombination events implicating the nearby res (resolution) site and IS element are proposed.

Tn5060 from the Siberian permafrost is most closely related to the ancestor of Tn21 prior to integron acquisition.

A Tn21-related mercury resistance transposon, Tn5060, has been isolated from Pseudomonas sp. strain A19-1 from a 8,000-10,000-year-old Siberian permafrost sample, and sequenced. Like Tn21, the element transposes to different plasmids at a frequency of 10(-2)-10(-3) per target plasmid transfer. Comparison of the complete Tn5060 DNA sequence (8,667 bp) with that of Tn21 (19,672 bp) shows that Tn5060 does not contain integron In2 and deviates from Tn21 in four nucleotide positions. These and other comparative data demonstrate that Tn5060 is the most closely related of the characterized mercury resistances to the as yet hypothetical immediate ancestor of Tn21, TnX.