Nucleotide Sequence Of Plasmid Research Articles

Sequence Analysis of IncA/C and IncI1 Plasmids Isolated from Multidrug-Resistant Salmonella Newport Using Single-Molecule Real-Time Sequencing.

Multidrug-resistant (MDR) plasmids play an important role in disseminating antimicrobial resistance genes. To elucidate the antimicrobial resistance gene compositions in A/C incompatibility complex (IncA/C) plasmids carried by animal-derived MDR Salmonella Newport, and to investigate the spread mechanism of IncA/C plasmids, this study characterizes the complete nucleotide sequences of IncA/C plasmids by comparative analysis. Complete nucleotide sequencing of plasmids and chromosomes of six MDR Salmonella Newport strains was performed using PacBio RSII. Open reading frames were assigned using prokaryotic genome annotation pipeline (PGAP). To understand genomic diversity and evolutionary relationships among Salmonella Newport IncA/C plasmids, we included three complete IncA/C plasmid sequences with similar backbones from Salmonella Newport and Escherichia coli: pSN254, pAM04528, and peH4H, and additional 200 draft chromosomes. With the exception of canine isolate CVM22462, which contained an additional IncI1 plasmid, each of the six MDR Salmonella Newport strains contained only the IncA/C plasmid. These IncA/C plasmids (including references) ranged in size from 80.1 (pCVM21538) to 176.5 kb (pSN254) and carried various resistance genes. Resistance genes floR, tetA, tetR, strA, strB, sul, and mer were identified in all IncA/C plasmids. Additionally, blaCMY-2 and sugE were present in all IncA/C plasmids, excepting pCVM21538. Plasmid pCVM22462 was capable of being transferred by conjugation. The IncI1 plasmid pCVM22462b in CVM22462 carried blaCMY-2 and sugE. Our data showed that MDR Salmonella Newport strains carrying similar IncA/C plasmids clustered together in the phylogenetic tree using chromosome sequences and the IncA/C plasmids from animal-derived Salmonella Newport contained diverse resistance genes. In the current study, we analyzed genomic diversities and phylogenetic relationships among MDR Salmonella Newport using complete plasmids and chromosome sequences and provided possible spread mechanism of IncA/C plasmids in Salmonella Newport Lineage II.

Characterization of a blaNDM‑1‑harboring plasmid from a Salmonellaenterica clinical isolate in China.

The plasmid-mediated transmission of antibiotic resistance genes has been reported to be involved in the development of antibiotic resistance in bacteria, and poses a serious threat for the success of bacterial infection treatment and human health worldwide. The present study used a 454 GS-FLX pyrosequencing system to determine the ~140 kb nucleotide sequence of plasmid pHS36-NDM, which was identified in a Salmonella Stanley isolate from the stool sample of an 11-month-old girl at Lishui Central Hospital, China, and which contains a New Delhi metallo-β-lactamase-1 (NDM-1) carbapenem resistance gene (blaNDM-1). The 181 open reading frames encode proteins with functions including replication, stable inheritance, antibiotic resistance and mobile genetic elements. Both horizontal transfer and passage stability-related genes were identified in pHS36-NDM, including a conserved type 4 secretion system and stbA (stable plasmid inheritance protein A). Two multidrug resistance gene islands were identified: The ISEcp1-blaCMY transposition unit which contains a CMY-6 β-lactamase gene (blaCMY-6) and a quaternary ammonium compound resistance gene (sugE); and the intI1-ISCR27 accessory region, which contained a trimethoprim resistance gene (dfrA12), two aminoglycoside resistance genes (aadA2 and rmtC), a truncated quaternary ammonium compound resistance gene (qacE∆1), a sulfonamide resistance gene (sul1), the blaNDM-1 carbapenemase and a bleomycin resistance gene (bleMBL). pHS36-NDM shared high homology with other blaNDM-1-containing plasmids reported in Sweden, Italy and Japan. However, no previous international travel history was documented for the patient and her family, even to neighboring cities. Furthermore, pHS36-NDM is of a different incompatibility group to other published blaNDM-1-carrying plasmids reported in China, with low homology in the surrounding structure of blaNDM-1. The present study will facilitate the understanding of the underlying resistance and dispersal mechanism of pHS36-NDM, and will deepen our recognition of the ongoing spread of the blaNDM-1-containing plasmids.

The Complete Sequence and Comparative Analysis of a Multidrug-Resistance and Virulence Multireplicon IncFII Plasmid pEC302/04 from an Extraintestinal Pathogenic Escherichia coli EC302/04 Indicate Extensive Diversity of IncFII Plasmids.

Extraintestinal pathogenic Escherichia coli (ExPEC) that causes extraintestinal infections often harbor plasmids encoding fitness traits such as resistance and virulence determinants that are of clinical importance. We determined the complete nucleotide sequence of plasmid pEC302/04 from a multidrug-resistant E. coli EC302/04 which was isolated from the tracheal aspirate of a patient in Malaysia. In addition, we also performed comparative sequence analyses of 18 related IncFIIA plasmids to determine the phylogenetic relationship and diversity of these plasmids. The 140,232 bp pEC302/04 is a multireplicon plasmid that bears three replication systems (FII, FIA, and FIB) with subtype of F2:A1:B1. The plasmid is self-transmissible with a complete transfer region. pEC302/04 also carries antibiotic resistance genes such as blaTEM−1 and a class I integron containing sul1, cml and aadA resistance genes, conferring multidrug resistance (MDR) to its host, E. coli EC302/04. Besides, two iron acquisition systems (SitABCD and IutA-IucABCD) which are the conserved virulence determinants of ExPEC-colicin V or B and M (ColV/ColBM)-producing plasmids were identified in pEC302/04. Multiple toxin-antitoxin (TA)-based addiction systems (i.e., PemI/PemK, VagC/VagD, CcdA/CcdB, and Hok/Sok) and a plasmid partitioning system, ParAB, and PsiAB, which are important for plasmid maintenance were also found. Comparative plasmid analysis revealed only one conserved gene, the repA1 as the core genome, showing that there is an extensive diversity among the IncFIIA plasmids. The phylogenetic relationship of 18 IncF plasmids based on the core regions revealed that ColV/ColBM-plasmids and non-ColV/ColBM plasmids were separated into two distinct groups. These plasmids, which carry highly diverse genetic contents, are also mosaic in nature. The atypical combination of genetic materials, i.e., the MDR- and ColV/ColBM-plasmid-virulence encoding regions in a single ExPEC plasmid is rare but of clinical importance. Such phenomenon is bothersome when the plasmids are transmissible, facilitating the spread of virulence and resistance plasmids among pathogenic bacteria. Notably, certain TA systems are more commonly found in particular ExPEC plasmid types, indicating the possible relationships between certain TA systems and ExPEC pathogenesis.

Molecular characterization of plasmid pMoma1of Moraxella macacae, a newly described bacterial pathogen of macaques.

We report the complete nucleotide sequence and characterization of a small cryptic plasmid of Moraxella macacae 0408225, a newly described bacterial species within the family Moraxellaceae and a causative agent of epistaxis in macaques. The complete nucleotide sequence of the plasmid pMoma1 was determined and found to be 5,375 bp in size with a GC content of 37.4 %. Computer analysis of the sequence data revealed five open reading frames encoding putative proteins of 54.4 kDa (ORF1), 17.6 kDa (ORF2), 13.3 kDa (ORF3), 51.6 kDa (ORF4), and 25.0 kDa (ORF5). ORF1, ORF2, and ORF3 encode putative proteins with high identity (72, 42, and 55 %, respectively) to mobilization proteins of plasmids found in other Moraxella species. ORF3 encodes a putative protein with similarity (about 40 %) to several plasmid replicase (RepA) proteins. The fifth open reading frames (ORF) was most similar to hypothetical proteins with unknown functions, although domain analysis of this sequence suggests it belongs to the Abi-like protein family. Upstream of the repA gene, a 470-bp intergenic region, was identified that contained an AT-rich section and two sets of tandem direct and indirect repeats, consistent with a putative origin of replication site. In contrast to other plasmids of Moraxella, the occurrence of pMoma1 in M. macacae isolates appears to be common as PCR testing of 14 clinical isolates from two different research institutions all contained the plasmid.

Analysis of plasmids encoding the tyrosine decarboxylase gene in Tetragenococcus halophilus isolated from fish sauce

In order to elucidate mechanisms of tyramine accumulation during fish sauce production, two tyramine-producing bacterial strains, referred to as TyrA and TyrB, were isolated from fish sauce mash accumulating over 141 mg of tyramine per 100 g of sample. Both strains were identified as Tetragenococcus halophilus based on phenotypic characterization and a 16S rRNA gene sequence analysis. Molecular analysis of the tyramine-producing gene in the two strains confirmed the presence of a ~30-kb plasmid encoding a single copy of the pyridoxal phosphate-dependent tyrosine decarboxylase gene (tdcA) along with three other genes related to tyramine synthesis (tdc cluster). The complete nucleotide sequences of plasmids extracted from the two strains indicated that both plasmids were almost identical, except for a 1.6-kb transposon sequence in the plasmid from the strain TyrB. Both plasmids had a replication region, a plasmid maintenance region, and two putative mobile genetic elements located upstream and downstream of the tdc cluster. This structure was identical to that of tetragenococcal plasmids encoding histidine decarboxylase (hdcA), which were sequenced previously. These results suggest a common origin for plasmids encoding hdcA and tdcA. In addition, the genes for both these biogenic amines are distributed among tetragenococcal species via this plasmid.

Single origin of three plasmids bearing blaCTX-M-15 from different Klebsiella pneumoniae clones.

To determine and compare the complete nucleotide sequences of plasmids carrying blaCTX-M-15 from three different Klebsiella pneumoniae clones. IncFII-type plasmids pKP02022, pKP09085 and pKP007 were extracted from three K. pneumoniae strains. These strains belong to sequence types (STs) ST15, ST48 and ST23, respectively, and were isolated in Korea. Plasmids were sequenced using the 454 Genome Sequencer FLX system. The three plasmids, pKP02022 (203577 bp), pKP09085 (213019 bp) and pKP007 (246 176 bp), all exhibited a very similar structure, with a pKPN3-like backbone and a resistance region including blaOXA-1, aac(6')-Ib-cr and cat genes as well as blaCTX-M-15. They were also very similar to pUUH239.2, previously isolated in Sweden. Iron (III) uptake-related genes were found in pKP007 from the ST23 strain, which has been reported to be associated with liver abscesses. The resistance region contained several insertion sequences, such as IS26, which may play an important role in structural rearrangements of plasmids. The very similar structure of the three plasmids, extracted from different clones, suggests that the spread of CTX-M-producing K. pneumoniae isolates might result from the horizontal transfer of plasmids and subsequent integration and recombination.

Mosaic Structure of p1658/97, a 125-Kilobase Plasmid Harboring an Active Amplicon with the Extended-Spectrum β-Lactamase Gene bla SHV-5

Escherichia coli isolates recovered from patients during a clonal outbreak in a Warsaw, Poland, hospital in 1997 produced different levels of an extended-spectrum beta-lactamase (ESBL) of the SHV type. The beta-lactamase hyperproduction correlated with the multiplication of ESBL gene copies within a plasmid. Here, we present the complete nucleotide sequence of plasmid p1658/97 carried by the isolates recovered during the outbreak. The plasmid is 125,491 bp and shows a mosaic structure in which all modules constituting the plasmid core are homologous to those found in plasmids F and R100 and are separated by segments of homology to other known regions (plasmid R64, Providencia rettgeri genomic island R391, Vibrio cholerae STX transposon, Klebsiella pneumoniae or E. coli chromosomes). Plasmid p1658/97 bears two replication systems, IncFII and IncFIB; we demonstrated that both are active in E. coli. The presence of an active partition system (sopABC locus) and two postsegregational killing systems (pemIK and hok/sok) indicates that the plasmid should be stably maintained in E. coli populations. The conjugative transfer is ensured by the operons of the tra and trb genes. We also demonstrate that the plasmidic segment undergoing amplification contains the blaSHV-5 gene and is homologous to a 7.9-kb fragment of the K. pneumoniae chromosome. The amplicon displays the structure of a composite transposon of type I.

What is, mutatis mutandis, the sequence of plasmid DNAs used in gene therapy?

Mutation is a fundamental biological process occurring in each living organism. Plasmid DNA which is used in gene therapy protocols or DNA vaccination passes through two different living cells which are, respectively, the producing cell (bacterial) and the target cell (eukaryotic). Hence, modifications in the nucleotide sequence of plasmids are likely to occur both in bacteria during the amplification step of plasmid DNA and in eukaryotic cells following gene transfer. In addition to these biological modifications resulting from the physical passage of the plasmid into two different living organisms, an additional source of sequence alteration resides in our mode of representation of the nucleotide sequence of plasmid DNA which uses a four letters code, whereas, bacterial DNA is made of six different nucleosides. Indeed, the therapeutic DNA paradigm seems to have neglected the qualitative importance of these DNA sequence alterations. In this review we discuss the importance and the role of these DNA sequence modifications in the context of non-viral gene therapy approaches.

Complete Nucleotide Sequence of a Cryptic Plasmid from the Marine Bacterium Vibrio splendidus and Identification of Open Reading Frames

A 2.3-kb replication-proficient fragment was previously obtained from a cryptic plasmid (pPS41) isolated from a marine Vibrio splendidus isolate (P. A. Sobecky, T. J. Mincer, M. C. Chang, A. Toukdarian, and D. R. Helinski, 1998, Appl. Environ. Microbiol. 64, 2822–2830). Analysis of the complete nucleotide sequence of plasmid pPS41 revealed two additional open reading frames (ORFs). Analysis of ORF-1 revealed that its translated product has 125 amino acids with a predicted MW of 16,978 and ORF-2 encodes a putative protein of 151 amino acids with a predicted MW of 19,802. The ORF-2 encoded protein showed 31 to 35% sequence homology to proteins identified to have a role in plasmid mobilization. These proteins are encoded on plasmids found in Escherichia coli and Pasteurella multocida. Plasmid pPS41 could be mobilized by a conjugative plasmid at frequencies of 1 × 10−2 to 2 × 10−2.

The Complete Nucleotide Sequence of a Small Cryptic Plasmid from a Rumen Bacterium of the Genus Butyrivibrio

The complete nucleotide sequence of a plasmid, designated pRJF1, isolated from a rumen bacterium of the genus Butyrivibrio, has been determined. pRJF1 is a small plasmid (2631 bp) which shows the high AT content (64%) typical of plasmids from gram-positive organisms. Computer analysis of sequence data revealed two major open reading frames encoded on the same strand but in different frames. The smaller, ORF1 (435 bp), is preceded by Shine Dalgarno (SD) and Escherichia coli -10, -35 sequences and is predicted to encode an acidic polypeptide of 145 aa. The putative protein shows little homology to known bacterial proteins except in a small region similar to part of the Pre protein (plasmid recombination enzyme) of Bacillus plasmid pTB913. Pre proteins of the pTB913 family, however, show conservation of several amino acids in the amino terminal region of the protein that may be related to function. This pattern of amino acid conservation is not observed in ORF1. The predicted product of ORF2 (849 bp) is a basic protein of 283 as that shows significant similarity to repA gene products of plasmids from gram-negative (but not gram-positive) bacteria. No typical Shine Dalgarno sequence or -10, -35 sequences precede this coding region. While sequence homologous to nick sites of plasmids from gram-positive organisms that replicate via ssDNA intermediates were also detected in pRJF1, the order of the three consecutive inverted repeats typical of nick sites is not conserved. pRJF1, however, does contain an extended region of directly and inversely repeated sequence motifs reminiscent of theta replication origins.

The molecular relatedness among α-hemolytic plasmids from various incompatibility groups

Deoxyribonucleic acid (DNA) reassociation studies among α-hemolytic ( Hly) plasmids from FVI and FIII–IV incompatibility groups showed a close similarity between the nucleotide sequences of plasmids from the same group. With respect to R plasmids from the F overgroup, they have 20–26 Mdal in common, an amount of DNA close to the amount involved in the traF operon. No more extensive sequence homology was found between pSU316 (IncFIII–IV) and the incompatible plasmids ColB-K98 (IncFIII) or R124 (IncFIV). The IncIα I2 plasmid pSU5 has only the α-hemolytic region (5 Mdal) in common with plasmid pSU316 but it is much more closely related to IncFVI plasmids where the DNA in common amounts to 22 Mdal. Finally, the genetically unrelated plasmid pSU233 shares 66% of its nucleotide sequences (40 Mdal) with the IncFVI plasmids and has 16–23 Mdal in common with various F-like plasmids.