A soil bacterial catabolic pathway on the move: Transfer of nicotine catabolic genes between Arthrobacter genus megaplasmids and invasion by mobile elements

The 165,137 bp plasmid pAO1 of Paenarthrobacter nicotinovorans carries the genes of a nicotine catabolicpathway. The genes are organized into several gene modules responsible for the catabolism of L- and D-nicotineto nicotine blue, a-ketoglutarate and succinate. Various modules of these genes have been shown to be presentin gram-positive (Gram?) soil bacteria. The presence of the identical pAO1 nic-genes on the 288,370 bpplasmid pZXY21 of Arthrobacter sp. ZXY2 (96% to 100% at the nucleotide level) permitted the identificationof the limits of this DNA fragment. At the 50 end of the nic-genes are located the ORFs of two predictedintegrases of the tyrosine recombinase family with conserved R, H, R and Y catalytic residues and that of asmall transposase with a predicted leucine zipper motive. They are related to Tn554A, Tn554B and Tn554C ofStaphylococcus aureus and suggest that the entire nic-genes DNA fragment represents a large catabolictransposon. Surprisingly the nic-genes on pZXY21 were found to be interspersed by mobile elements encodingtransposases of various IS families. Insertion of these IS elements disrupts nicotine degradation and divide thenic-genes DNA into potentially new transposons. This finding may illustrate how nicotine catabolic genes canbe mobilized and spread by horizontal gene transfer to other soil bacteria.

Foldback Intercoil DNA and the Mechanism of DNA Transposition

Foldback intercoil (FBI) DNA is formed by the folding back at one point of a non-helical parallel track of double-stranded DNA at as sharp as 180degrees and the intertwining of two double helixes within each other's major groove to form an intercoil with a diameter of 2.2 nm. FBI DNA has been suggested to mediate intra-molecular homologous recombination of a deletion and inversion. Inter-molecular homologous recombination, known as site-specific insertion, on the other hand, is mediated by the direct perpendicular approach of the FBI DNA tip, as the attP site, onto the target DNA, as the attB site. Transposition of DNA transposons involves the pairing of terminal inverted repeats and 5-7-bp tandem target duplication. FBI DNA configuration effectively explains simple as well as replicative transposition, along with the involvement of an enhancer element. The majority of diverse retrotransposable elements that employ a target site duplication mechanism is also suggested to follow the FBI DNA-mediated perpendicular insertion of the paired intercoil ends by non-homologous end-joining, together with gap filling. A genome-wide perspective of transposable elements in light of FBI DNA is discussed.

Transposable Elements and Genome Size Variations in Plants

Although the number of protein-coding genes is not highly variable between plant taxa, the DNA content in their genomes is highly variable, by as much as 2,056-fold from a 1C amount of 0.0648 pg to 132.5 pg. The mean 1C-value in plants is 2.4 pg, and genome size expansion/contraction is lineage-specific in plant taxonomy. Transposable element fractions in plant genomes are also variable, as low as ~3% in small genomes and as high as ~85% in large genomes, indicating that genome size is a linear function of transposable element content. Of the 2 classes of transposable elements, the dynamics of class 1 long terminal repeat (LTR) retrotransposons is a major contributor to the 1C value differences among plants. The activity of LTR retrotransposons is under the control of epigenetic suppressing mechanisms. Also, genome-purging mechanisms have been adopted to counter-balance the genome size amplification. With a wealth of information on whole-genome sequences in plant genomes, it was revealed that several genome-purging mechanisms have been employed, depending on plant taxa. Two genera, Lilium and Fritillaria, are known to have large genomes in angiosperms. There were twice times of concerted genome size evolutions in the family Liliaceae during the divergence of the current genera in Liliaceae. In addition to the LTR retrotransposons, non-LTR retrotransposons and satellite DNAs contributed to the huge genomes in the two genera by possible failure of genome counter-balancing mechanisms.

IS CR1 asociado con genes blaCTX-M-1 y bla CTX-M-2 en plásmidos IncN e IncFIIA aislados en Klebsiella pneumoniae de origen hospitalario en Mérida, Venezuela / IS CR1 associated with bla CTX-M-1 y bla CTX-M-2 genes in IncN and IncFIIA plasmids isolated from Klebsiella pneumoniae of nosocomial origin in Mérida, Venezuela

Introducción. Las secuencias de inserción tales como IS CR1 promueven la captura, transposición y expresión de los genes bla CTX-M, facilitando, de esta manera, su diseminación rápida en la población bacteriana. Objetivo. Se determinó la presencia del elemento IS CR1 y su asociación con genes bla CTX-M-1 y bla CTX-M-2 en plásmidos de diferentes grupos de incompatibilidad en Klebsiella pneumoniae de origen hospitalario. Materiales y métodos. Se aislaron tres cepas de K. pneumoniae con sensibilidad disminuida a cefalosporinas de amplio espectro, de neonatos con septicemia hospitalaria. La presencia de β -lactamasas de espectro expandido (BLEE) fue determinada fenotípicamente. Los plásmidos se aislaron y clasificaron según grupos de incompatibilidad por tipificación del replicón por reacción en cadena de la polimerasa (PCR). Los genes bla BLEE y su asociación a IS CR1 se determinaron por PCR y secuenciación directa, usando varios juegos de iniciadores. Resultados. Todas las cepas demostraron un perfil fenotípico indicativo de producción de BLEE, transferibles por conjugación. Los ensayos de PCR para para cefotaximasas (CTX-M) y el análisis de la secuenciación, revelaron que las cepas portaban genes bla CTX-M-1 y bla CTX-M-2. Estos genes se encontraron en plásmidos conjugados de 150 kb, aproximadamente, relacionados con los grupos IncN e IncFIIA, respectivamente. IS CR1 se encontró ´aguas arriba´ ( upstream ) y asociado con los genes bla CTX-M-1 y bla CTX-M-2. Conclusión. Este es el primer reporte realizado en Venezuela donde la presencia de IS CR1 está estrechamente asociada con la movilización de los genes bla CTX-M-1 y bla CTX-M-2 en plásmidos conjugativos IncN y IncFIIA en cepas de K. pneumoniae que circulan en una Unidad de Alto Riesgo Neonatal.

Introduction: Insertion sequences such as IS CR1 promote capture, transposition and expression of bla CTX-M genes. Thus, gene dissemination in bacterial populations occurs rapidly. Objective: To determine the presence of IS CR1 sequence genes and their association with bla CTX-M-1 and bla CTX-M-2 on plasmids IncN and IncFIIA from K. pneumoniae of nosocomial origin, was determined. Materials and methods: Three strains of K. pneumoniae with reduced susceptibility to extendedspectrum cephalosporins were isolated from neonatal sepsis cases of nosocomial origin. Phenotypic tests showed the presence of ESBLs. Plasmids were isolated and classified according to incompatibility groups by PCR replicon typing. Detection and association of IS CR1 with bla CTX-M genes were determined by PCR and direct sequencing through the use of several sets of PCR primers. Results: All strains showed phenotypic profile consistent with ESBL-producing transferred by conjugation. PCR amplification assay for CTX-M together with sequencing analysis revealed that strains carrying bla CTX-M-1 y bla CTX-M-2 genes were localized in plasmids of approximately 150 kb related to IncN and IncFIIA groups, respectively. IS CR1 was found upstream and associated with bla CTX-M-1 y bla CTX-M-2 genes. Conclusion. Thus far, this is the first Venezuelan report, in which IS CR1 presence is closely related to bla CTX-M-1 y bla CTX-M-2 gene mobilization in IncN and IncFIIA conjugative plasmids located in K. pneumonaiae strains circulating at a neonatal high risk unit.

Transposable Elements: No More 'Junk DNA'

Since the advent of whole-genome sequencing, transposable elements (TEs), just thought to be 'junk' DNA, have been noticed because of their numerous copies in various eukaryotic genomes. Many studies about TEs have been conducted to discover their functions in their host genomes. Based on the results of those studies, it has been generally accepted that they have a function to cause genomic and genetic variations. However, their infinite functions are not fully elucidated. Through various mechanisms, including de novo TE insertions, TE insertion-mediated deletions, and recombination events, they manipulate their host genomes. In this review, we focus on Alu, L1, human endogenous retrovirus, and short interspersed element/variable number of tandem repeats/Alu (SVA) elements and discuss how they have affected primate genomes, especially the human and chimpanzee genomes, since their divergence.

Long-term and stable expression of transgene mediated by piggyBac transposon in gynecological malignant tumor cells / 中华妇产科杂志

Objective To investigate the expression of exogenous gene transferred by piggyBac (PB) transposon in various gynecological malignant cell lines and reveal its potential application of gene therapy in gynecological cancer.Methods Amplified herpes simplex virus thymidine kinase (HSV-tk) gene coding region by PCR and integrated it into PB expression vector, PB[Act-RFP]DS, for reconstructing PB[Act-RFP, HSV-tk]DS (pPB/TK).By using different transfection reagents: FuGENE HD, jetPEI, lipofectamine 2000, pPB/TK together with helper plasmid Act-PBase were cotransfected into four mostly common gynecological malignant tumor cell lines HeLa, JEG-3, SKOV3 and HEC-1B.The mRFP1 report gene expressions was observed and detected by fluorescence microscope and flow cytometry to analyze transfection efficiency.The expressions of HSV-tk and mRFP1 gene were detected by reverse transcription PCR (RT-PCR).The cytotoxic effect of various concentration of pro-drug ganciclovir (GCV) for transfected cells was detected by methyl thiazole tetrazolium assay.The transfected cells were positive sorted by flow cytometry and limiting diluted to obtain the stable transfected cell line.The insertion sites of foreign gene tranferred by PB transposon in genome were analyzed by inverse PCR.Results (1) Double digests analysis and sequences test demonstrated that pPB/TK vector was reconstructed successfully.(2) Using three different transfective reagents, PB trausposon transferred HSV-tk gene and mRFP1 gene into HeLa, HEC-1 B, SKOV3 and JEG-3 cell efficiently, and the transfection efficiency of pPB/TK for the same cell was different by using different transfective reagents; in Hela cell, the transfection efficiency of FuGENE HD [(78.7 ± 9.2) %]was higher than that of lipofectamine 2000 [(54.1 ± 11.4) %]and jetPEI [(46.5 ± 7.4) %, all P < 0.05] ; using the same transfective reagent, the transfection efficiency of pPB/TK was also different on various cell lines, using FuGENE HD, the transfeetion efficiency of pPB/TK on HeLa, JEG-3 and SKOV3 cell was (78.7 ± 9.2) %, (74.4 ± 8.9) % and (83.2 ± 9.7) % respectively, which all were higher than that on HEC-1B [(39.5 ± 8.7) %, P < 0.05] .(3) RT-PCR showed that there were the mRNA expression of HSV-tk and mRFP1 in all cell lines.(4) 50% inhibitory concentration of GCV for transfected cells, HeLa, JEG-3, SKOV3 and HEC-1B, was 1.29, 3.35, 0.09 and 13.28 μg/ml respectively.Inhibitory effect of GCV (10 μg/ml) on SKOV3 transfected with pPB/TK was (86 ± 9) %, which was superior to that transfected with pORF-HSVtk alone [(52 ± 12)%, P < 0.05] .(5) The insertion sites of PB transposon in the target cells genome were located at TTAA sites, mRFP1 expression still could be detected in three months after transfected.Conclusions PB transposon could transfer exogenous gene into various gynecological malignant cells, which could integrated into genome and obtain a long-term and stable expression.It is expected that PB transposon may supply a more efficient and safer transgene technology platform for gene therapy in gynecological cancer.

Locus-specific analysis of IS100 insertion within Yersinia pestis genome / 中国地方病学杂志

Objective To study the genotyping distribution of the Yersinia pestis(Y.pestis)strains by characterizing the diversity of the insertion sequence IS100 within the Y.pestis genome.Methods Derived fromthe known sequence of oriental strain CO92,5 pairs of locus-specific primers originating from both sides of the adjacent region of IS100 copies were designed,and two other complementary primers inside the IS100 sequence were designed to correspond with the outer primers.Then,91 Y.pestis strains and l pseudotubebculosis strain were tested by the specific PCR method using the primers described above and the PCR products were conformed by the sequence analysis,then further analysis WaS performed after the IS100 status was marked on the map of the plague focus type of china.Results The 91 Y.pestis strains had different IS100 status in their genome on tested loci.some possessed IS100 insertion,some didn't,and others changed their genome constitution.The IS100 possession on the 5 loci also suggested a distribution of regionality.Conclusion The analysis of some IS100 insertion element loci reveals that the IS100 genotyping distribution is consistent with the plague focus of type of China.And IS100genotyping pattern of the Y.pestis stains well reflects its genome constitution and the high flowability in its natural evolution.

Structures of resistance transposons and multilocus sequencing typing in vancomycin-resistant enterococci strains isolated from five hospitals in Hangzhou / 中华检验医学杂志

Objective To determine the structures of resistance transposons and muhilocus sequencing typing(MLST)in the vancomycin resistant enterococcus(VRE).Methods Twenty-one VRE strains were isolated from five hospitals in Hangzhou.The resistance to antimicrobial agents was determined by Etest.Polymerase chain reaction(PCR),conjugation,plasmid extract,transposon structures,pulse field gel electrophoresis(PFGE),muhilocus sequencing typing(MLST),and multiple-locus variable-number tandem repeat analysis(MLVA)were carried out.Results All of the 21 VRE strains harbored the vanA gene.These strains were divided into 10 PFGE types,7 sequence types(STs)and 5 MLVA types.All of these VRE strains were susceptible to linezolid and tigecycline.The vanA genes in two VRE strains were located in transposon Tnl546,and those in the other 19 VRE strains were located in transpeson Tnl546- like,with ISl485 inserted in vanXY.Vancomycin resistance of 1 8 VRE isolates was transferred by filter mating. All of these conjugants had a plasmid containing a molecular size of about 54 000 bo.Conclusions These 21 VRE strains were all caused by the vanA gene and divided into 7 MIST types.A novel trasnposon was detected.Most of these VRE isolates belonged to the clonal complex(CC17)by MIST,which was the hospital-adapted and pandemic VRE clonal complex.

Bacteria resistance surveillance on Enterococci Isolated from pedlatric hospitals and distribution of resistance genes ermB,mefA,tetM and the integrase gene intTn of Tn1545 in Enterococci / 中华检验医学杂志

Objective To determine the drug-resistance rate of Enterococci isolated from patients of 5 padiatric hospitals located at different areas in China,and to investigate the distribution of resistance genes ermB,mefA,tetM and the integrase gene intTn of Tn1545 in Enterococci.Methods The antimicrobial susceptibility to 8 antibiotics of 2 216 Enteroeocei isolates was determined.PCR was used to detect the macrolide resistance genes ermB and mefA,tetracycline resistance genes tetM,and the integrase gene int-Tn of Tn1545.Results The resistance rates to erythromycin,ampicillin,gentamicin and teicoplanin were 86.5%,48.0%,60.5% and 0.7%,respectively.All isolated Enterococci straim were found sensitive to vancomycin.Of the detected 225 strains,70.7% of the 225 detected strains carried ermB gene while 75.1% of them carried tetracycline resistance gene tetM:only one strain had mefA.The presence of ermB gene in erythromycin MIC＞256 mg/L straim group(95.7%)strains was higher than those in erythromycin MIC＜256 mg/L group(2.5%).The int-Tn gene was detected in 40.9%(92/225)of the 225 test strains.The presence of ermB gene in int-Tn positive group strains was higher(84.8%)than those in int-Tn negative strains group(60.9%).So did the tetM in int-Tn positive group(83.7%)compared with those in int-Tn negative group(70.0%).Conclusions Enterococci sbowed a high resistance rate to the antibiotics we monitored,especially to erythromycin;but still very senstive to glycopeptide antibiotics. Resistance to macrolide in Enterococci collected from clinical in five Children's Hospital was generally mediated by methylation of 23S rRNA via ermB methylase. Enterococci resistance to tetracycline was predominantly due to ribosomal protection encoded by tetM. There was a strong relationship of the ermB and tetM genes with Tn1545-related elements.