pANKS2, a New Plasmid of Salmonella typhimurium and the Pathway of Plasmid Evolution for Antibiotic Resistance

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Buse TÜREGÜN ATASOY1, Mehmet TAŞPINAR2, Devran GERÇEKER1,A. Derya AYSEV3, Birsel ERDEM1, Fikret ŞAHİN1
1Department of Microbiology, Ankara University, School of Medicine, Ankara, Turkey
2Department of Medical Biology, Van Yüzüncü Yıl University, School of Medicine, Van, Turkey
3Department of Pediatrics, Ankara University, School of Medicine, Cebeci, Ankara, Turkey
Article Type
Orginal Article
Plasmid, Salmonella Typhimurium, pAnkS2, transposons, antibiotic resistance


Information of antibiotic resistance is an important tool in epidemiologic control and treatment of infections. The mobile genetic elements such as plasmidsand transposons responsible for developing resistance against mostly used antibiotics.Previously we isolated characterized and named a plasmid pAnkS from Salmonella Typhimurium (S. Typhimurium). Plasmid pAnkS is one of the few completely sequencedplasmids from S. Typhimurium and is made of transposition of Tn3 transposons of 4950bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, ampicillin resistance blaTEM, and the right terminal repeat into thep4821. In here another plasmid isolated from S. Typhimurium and named pAnkS2.The plasmid was analyzed partially for potential reading frames and structural featuresindicative of transposons and transposon relics. The overlapping restriction fragmentsof pAnkS2 were cloned into E. coli plasmid vectors, sequenced and analyzed with theBLAST programs. Plasmid pAnkS2 consist of EHEC plasmid p4821 as a core regionand also complete Tn3 and Tn5-like transposons. pAnkS2 showed strong homologywith plasmids described previously, pAnkS, and pNTP16 for sequences belong top4821. P4821 is made the core region of pAnkS2 as in pAnkS, and pNTP16. pAnkS2also carries Tn5-like transposon consisting of aminoglycoside 3’-phosphotransferasetype II, bleomycin resistant genes and ORF sequence. Therefore, pAnkS2 was madeup from the transpositions of the Tn5 transposon into the pAnkS which consist of transposition of Tn3 into the p4821. pAnkS2 shows multiresistant phenotype due to carryingaminoglycoside 3’-phosphotransferase type II, bleomycin and ampicillin sequences.The analysis of p4821, pNTP16, pAnkS and partial analysis of pAnkS2 revealed thatpAnkS is a simple form of plasmid found in the salmonella and might be the precursorof the most of the salmonella plasmids carrying multiple resistance genes includingpNTP16, and pAnkS2.


Salmonellosis is an important public health problem throughout the world, resulting in hospitalizations and deaths (1). During the past decades,Salmonella enterica serovar Typhimurium infections have increased in many parts of the world(1). In general, the primary approach to the treatment and control of Salmonella infections is theuse of antimicrobial agents. However, bacteria arebecoming increasingly resistant to multiple antibiotics in many areas of the world (2). In a number ofbacteria, resistance is acquired from mobile genetic elements including bacteriophages, transposons,and plasmids, and is subjected to frequent rearrangements (3). The presence of antibiotic resistance genes on transposable elements is a matterof concern with regard to the dissemination of theresistance genes via horizontal gene transfer (4).In particular, genes coding for resistances to ß-lactams or tetracyclines in Gram-negative bacteriahave been associated with transposons. Genes coding for ß-lactamases of the TEM-type are amongthe most prevalent ß-lactam resistance genes inGram-negative bacteria. The blaTEM-1 gene hasbeen detected on transposon Tn3 which is also located either on conjugative and non-conjugativeplasmids or on the chromosome. Tn3 and Tn1721are members of the Tn3 family of transposons andexhibit similar mechanisms of replicative transposition (4). Moreover, both transposons have inverted terminal repeats of 35–38 bp and produce characteristic 5 bp direct repeats at their integrationsites in the genome. This suggests that these extraresistance genes may be located in other chromosomal regions or on plasmids (4). Some of theseresistance plasmids from various bacterial originshave been partially or completely sequenced (4,5).We previously identified and characterized an ampicillin resistant gene carrying plasmid from clinically isolated and described as Salmonella entericaserovar Typhimurium that conferred the ampicillin, chloramphenicol, streptomycin, sulfonamides and tetracycline (ACSSuT) resistance phenotype.The plasmid we named as pAnkS is 8271 bp size.The complete analysis of pAnkS revealed that itwas made of the transposition of the Tn3 transposon into the enterohemorrhagic Escherichia coli(EHEC) O157:H7 plasmid p4821 which containthe basic requirements of a plasmid such as replication, stability and mobilization genes (6). Further analysis of pAnkS showed that it is precursorform of another S. Typhimurium plasmid pNTP16which carries kanamycin resistance gene in addition to ampicillin gene (5). In this study, we isolated and described a new plasmid named pAnkS2(plasmid Ankara Salmonella 2). pAnkS2 showsmultiresistant phenotype due to carrying aminoglycoside 3’-phosphotransferase type II, bleomycinand ampicillin sequences and was made up fromthe transpositions of the Tn5 transposon carryingaminoglycoside 3’-phosphotransferase type II, andbleomycin into the pAnkS which consist of transposition of Tn3 into the p4821.


Bacteria, Plasmid and Culture Conditions
Plasmid pAnkS2 was found in a Salmonella enterica subsp. enterica serovar Typhimurium isolated from patient has gastroenteritis in Ankara.Plasmid DNA was prepared by alkaline lysis andsubsequent purification. For preparation of theplasmid DNA bacteria were grown in L-broth (1%w/v NaCl, 1% w/v tryptone, 0.5% yeast extract, andpH7.5). For maintenance of recombinant plasmid,culture media supplemented with ampicillin to afinal concentration of 100ug/ml.

Antibiotic Susceptibility Testing
After pAnkS2 transformed into the E. coli DH5α,antibiotic susceptibility testing was performed.The disk-diffusion assay was used to determinethe antibiotic resistance profile of the strains usingMueller–Hinton agar (Difco Laboratories, Detroit,USA) following the National Committee for Clinical Laboratory Standards (NCCLS) guidelines(NCCLS 2002). The antibiotic disks used were:ampicillin, ampicillin-sulbactam, carbenicillin,cephalothin, chloramphenicol, ciprofloxacin, ceftriaxone, cefuroxime, erythromycin, gentamicin,kanamycin, Polymyxin B, streptomycin, sulfisoxazole, tetracycline, trimethoprim, and trimethoprim-sulfamethoxazole. All antibiotic disks werepurchased either from Difco or BBL (Becton–Dickinson, Sparks, MD).

General Recombinantion Techniques
After restriction mapping with enzymes, BamH1,EcoRI, EcoRV HinfIII, HindIII, Not I, and XmnI.BamHI fragments of pAnkS2 were cloned separately into pBSK (+) (Invitrogen, Groningen, TheNetherlands) and transformed into E. coli DH5.Restriction endonuclease digestion was carriedout according to the supplier’s instructions (Fermentas- Life science technologies Lithuania). Purification of DNA fragments from agarose gels wasperformed with Gene-clean kit (GeneMark-Hopegen Biotechnology-China). Ligation and transformation experiments were carried out accordingto standard methods. Initial sequence analyseswere conducted with the M13 reverse and forwardprimers for pUC/M13 vectors as described (7). Fordetermination of the complete sequence, primerwalking was carried out with oligonucleotide primers (IDT USA) designed from sequences previouslyobtained with the M13 reverse and forward primers.Nucleotide Sequencing and Analysis of theSequences

Nucleotide sequencing was performed with BigDye
Terminator (PE Applied Biosystems) sequencingchemistry on an automatic DNA sequencer ABI 377(Applied Biosystems) with universal and reverseprimers for pBSK/M13 vectors and customizedprimer chosen during primer walking. Sequenceanalysis was carried out with the BLAST programsblastn and blastp ( BLAST/; last accessed 24 September 2016) as wellas with the ORF Finder program (; last accessed 2016).


Restriction Enzyme Mapping
Restriction enzymes were tested for their ability to cleave plasmid pAnkS2. The enzymes BamHI, EcoRI, EcoRV HinfIII, Not I, and XmnI were tested. BamHI and EcoRI gave three bands individually and EcoRV, HindIII and XmnI gave two bands and NotI cut once the plasmid (Figure 1). Three BamHI fragments of pAnkS2 were cloned into the pBSK (+) cloning vector.

DNA Sequence
The nucleotide sequence of these fragment determined with an automized sequencer. For determination of the complete sequence, primer walkingwas carried out with oligonucleotide primers (IDTUSA) designed from sequences previously obtainedwith the M13 reverse and forward primers.

Structure and Organization of PlasmidpAnkS2
In vitro susceptibility testing of E. coli DH5α:pAnkS2 transformants revealed that this plasmidmediates resistance to ampicillin, kanamycin, butsusceptible to ampicillin-sulbactam, carbenicillin,cephalothin, chloramphenicol, ciprofloxacin, ceftriaxone, cefuroxime, erythromycin, polymyxinB, streptomycin, sulfisoxazole, tetracycline, trimethoprim, and trimethoprim-sulfamethoxazole.The partial nucleotide sequence analysis revealedthat Plasmid pAnkS2 GC content is 48%. The determined nucleotide sequence analysis of pAnkS2was used to search homology search with theBLAST programs. Analysis of pAnkS2`s core complex showed that this region harbored sequencesessential for replication and stable maintenanceof plasmid as also described by Haarmann et all(6) (Figures 2,3,5). Analysis of core complex revealed five open reading frames (Figures 2,3). Homology searches in the GenBank database library revealed close to 100% identity to the mobA geneand other ORFs of the S. Typhimurium plasmidpAnkS, NTP16 and of p4821. All functionally important features of cer are maintained in pAnkS2and are also present in pAnkS, NTP16 and p4821.The sequence of pAnkS2 coordinates origin oftransfer (oriT) is identical to the oriT of pAnkS,and pNTP16 and showed strong homology to theoriT of p4821 (Figure 3)

pAnkS2’s core region is flanked by the Tn3 transposon (Figure 1) as in pAnkS. The sequenced Tn3transposon part comprised the blaTEM gene codingfor a ß-lactamase, the genes tnpA and tnpR whoseproducts are essential for transposition of Tn3 aswell as the left and right terminal inverted repeatof Tn3. However, the partial analysis of the Tn3transposons revealed that the Tn3 transposon ofpAnkS2 separated by the tn5 transposon containing aminoglycoside 3’-phosphotransferase type IIand bleomycin resistant genes and ORF sequence(Figure 4). The aminoglycoside 3’-phosphotransferase type II in pAnkS2 showed 100% identity tothe complete sequence of the Salmonella enterica subsp. enterica serovar Dublin plasmid pMAK2DNA (GeneBank-accession no: AB366441), andSalmonella enterica subsp. enterica serovar Indiana strain D90 plasmid pD90-3, (GeneBank-accession no: CP022453) and bleomycine was identicalto the complete sequence of the pMAK2 DNA


Plasmid pAnkS2 is a new S. Typhimurium plasmid contains Tn3 transposon contain parts of the tn5 transposon carrying aminoglycoside 3’-phosphotransferase type II and bleomycin resistantgenes and ORF sequence and plasmid p4821 isolated from the EHEC 0157:H7. Plasmid p4821 is asimple form of plasmid contains the only information necessary for its replication, stability, and mobilization (6). Analysis of plasmid p4821 patternsof EHEC strains showed that plasmid occurred athigh frequencies (6). Presence of p4821 in the S. Typhimurium related plasmid pNTP1 and pNTP16,pAnkS and pAnkS2 suggest that p4821 is a phylogenetically long established plasmid; presumablydistributed various enterobacteria and genetictransfer must have occurred among the bacteria.P4821 is made the core region of pAnkS2 as inpAnkS and pNTP16. This core region was flankedby Tn3 in pAnkS2 as in pAnkS and pNTP16.Transposons of the Tn3 family do not show extended insertion site specificity, but appear to preferA + T-rich sequences (4). When integrating into anew vector molecule, these transposons producecharacteristic 5 bp direct repeats at the insertionsite. The 5 bp direct repeats might be variable indifferent plasmids and genomes including TAAAAin pFPTB1 (GenBank accession no: AJ634602),TATTA in pLEW517 (GenBank accession no:DQ390454), TTATT in pINF5 (GenBank accession no: AM234722), TATAA in Salmonella Enteritidis gene (GenBank accession no: AB103092).In pAnkS the core complex and the transposonwere separated on both sites by short direct repeats with the sequence TTCTT (7). This sequencealso occurred in p4821; however, here it occurredonly at one position and its obvious duplication onpAnkS at the boundaries to the Tn3 transposon ledto suggest that it may function as a target site forthe integration of transposon and segment mighthave integrated into the pAnkS basic replicon asone unit. When considering strong homology between pAnkS and p4821 and short direct repeatswith the sequence TTCTT in both plasmid led tosuggest that pAnkS is made of transposition of Tn3into the p4821 plasmid at the TTCTT site (7). As in pAnkS and pNTP16, pAnkS2 has the left andright inverted repeat of the Tn3 transposon. WhilepAnkS contain whole Tnp3 transposon, pNTP16contain terminal 400 of 3000 of tnpA and terminal part of tnpR, ampR gene and inverted repeatregions in both sites. PNTP16 contain Tn4351transposon carrying a protein identical to the aminoglycoside 3’-phosphotransferase of Tn903, in themissing portion of Tn3 (8).Analysis of Tn4352 flanking sequences in Tn3transposon of the pNTP16 provides evidence thatpNTP16 is a derivative of pAnkS since they arestrongly homologous to the terminal parts of tnpAand tnpR and ampR genes of Tn3 in addition tosharing the core region (Figure 6). Furthermore,it provides evidence that, subsequent to insertionwithin the resident Tn3 transposon, Tn4352 mediates an adjacent deletion of 2701 bps since thereis no target duplication flanking the element. It ishighly possible therefore that subsequent to theinitial tn4352 transposition event into the pAnkSin either the tnpA or tnpR site a deletion has occurred, presumably promoted ISI171 and initiatedat the one of the ends of Tn4352, resulting in theloss of 2701 bp of DNA (based on the Tn3 sequence)which contains most of sequence for tnpA and someof the sequence for tnpR (8).Analysis of Tn5 flanking sequences in Tn3 transposon of the pAnkS2 provides evidence pAnkS2contain parts of the tn5 transposon containingaminoglycoside 3’-phosphotransferase type II andbleomycin resistant genes and ORF sequences. Inaddition, they are strongly homologous to terminalparts of tnpA and tnpR and ampR genes of Tn3 inaddition to sharing the core region. The analysisprovides evidences that insertion sites of aminoglycoside 3’-phosphotransferase type II and bleomycin resistant genes and ORF sequences in Tn3transposon are different than the insertion side ofthe Tn4352 in Tn3 transposon. While ISI76 dividethe tnpA and tnpR in pNTP16, aminoglycoside3’-phosphotransferase type II and bleomycin resistant genes inserted in the tnpA side of the Tn3Transposon in pAnkS2 (Figure 6). Previous studies had shown that the bacterial transposon Tn5could insert into many sites in a gene (9). Sincethe sequence analysis is incomplate we couldn’t determined the exact side of instertion of neomycin-kanamycin and bleomycin in Tn3 transposon.The blaTEM gene of pAnkS codes for 286 aminoacid protein which identical the TEM extended-spectrum beta-lactamase (GenBank accession no. DQ221256). The same as pAnkS2 comprisedthe blaTEM gene coding for a ß-lactamase.Although p4821 plasmid originally found in England, the other Salmonella plasmids pNTP1,pNTP16, pAnkS, and pAnkS2 found in the closeregions (Middle East countries) (10) and p4821might be more universally available plasmid foundin Enterobacteriaceae family. Previously, it was suggested that pNTP16 must be evolved from aplasmid containing only ampicillin resistance gene(5) (8). Therefore, pAnkS is the most likely plasmidwhich the precursor form of pNTP16, pAnkS2 andpMAK2 considering the evidences described above(Figure 6). We believe that description of pAnkSand pAnkS2 may help to understand the plasmidevolution better.


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