Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “plasmid characterization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 199 records · Page 11Linked to original sources
PubMed2026

Global lessons from antibiotic resistance: Metformin-hydrolysing genes in transposable elements, a new threat for type II diabetic patients?

OBJECTIVES: To investigate the evolutionary origin, genomic mobility, and potential dissemination of metformin-hydrolysing genes (mfmAB), and to assess whether environmental selection by metformin pollution may drive the emergence of transferable pharmaceutical-degrading traits analogous to antibiotic resistance. METHODS: Large-scale comparative genomics was performed using publicly available bacterial genomes carrying mfmAB homologs. Phylogenomic reconstruction, average nucleotide identity analysis, genomic context comparison, plasmid characterization, and insertion sequence mapping were used to infer evolutionary history and identify mechanisms of horizontal gene transfer. RESULTS: mfmAB homologs were identified in twelve Aminobacter and three Pseudomonas genomes within a conserved ∼8.2 kb gene cluster. Phylogenomic analyses showed that metformin-degrading capacity emerged independently in multiple Aminobacter lineages across distinct continents, consistent with convergent evolution under anthropogenic selective pressure. Genomic comparisons indicated a chromosomal origin of mfmAB, followed by mobilization onto conjugative plasmids through IS1182-mediated transposition. In Pseudomonas, additional IS3/IS6-mediated transposition events integrated mfmAB into diverse plasmid backbones, frequently within composite transposons also encoding guanylurea and biguanide degradation pathways (guuH, bguH). These findings reveal a dynamic modular assembly of metabolic functions facilitating adaptation to pharmaceutical pollutants. CONCLUSIONS: Metformin pollution appears to promote the emergence and mobilization of pharmaceutical-degrading genes through mechanisms paralleling antibiotic resistance evolution. Although no clinical impact has yet been demonstrated, the potential spread of such genes into human-associated microbiomes and their possible co-selection with antibiotic resistance determinants represent an emerging One Health concern. Environmental surveillance of pharmaceutical-degrading genes is warranted to anticipate future threats to drug efficacy.

Convergent evolution↗
PubMed1996

Multi-drug resistant non-typhi salmonellae in Kenya.

Two methods of plasmid characterization, restriction digest patterns and incompatibility grouping, were used to study self-transmissible multi-drug resistance among non-typhi salmonellae (NTS). Resistance to ampicillin and other commonly applied beta-lactams was evaluated by iso-electric focusing and disc inactivation. Of the NTS isolated from blood, 75% were Salmonella typhimurium but those included several different phage types. Over 47% of isolates were resistant to three or more of the readily available drugs including ampicillin, cefuroxime, chloramphenicol, co-trimoxazole, streptomycin and tetracycline. Self-transferable resistance plasmids (c. 100 kb) were essentially of incompatibility group incFIIA, but their restriction fragment patterns revealed a diversity in relatedness. More than half of parent strains and their transconjugants produced beta-lactamases which co-electrophoresed with TEM-1 and OXA-1. This study has observed a disturbingly high prevalence of transmissible multi-drug resistance among NTS which are an important cause of morbidity in HIV-1 seropositive individuals.

Ampicillin Resistance↗
PubMed2002

Characterization of chimeric ADPglucose pyrophosphorylases of Escherichia coli and Agrobacterium tumefaciens. Importance of the C-terminus on the selectivity for allosteric regulators.

ADPglucose pyrophosphorylase catalyzes the regulatory step in the pathway for bacterial glycogen synthesis. The enzymes from different organisms exhibit distinctive regulatory properties related to the main carbon metabolic pathway. Escherichia coli ADPglucose pyrophosphorylase is mainly activated by fructose 1,6-bisphosphate (FBP), whereas the Agrobacterium tumefaciens enzyme is activated by fructose 6-phosphate (F6P) and pyruvate. Little is known about the regions determining the specificity for the allosteric regulator. To study the function of different domains, two chimeric enzymes were constructed. "AE" contains the N-terminus (271 amino acids) of the A. tumefaciens ADPglucose pyrophosphorylase and the C-terminus (153 residues) of the E. coli enzyme, and "EA", the inverse construction. Expression of the recombinant wild-type and chimeric enzymes was performed using derivatives of the pET24a plasmid. Characterization of the purified chimeric enzymes showed that the C-terminus of the E. coli enzyme is relevant for the selectivity by FBP. However, this region seems to be less important for the specificity by F6P in the A. tumefaciens enzyme. The chimeric enzyme AE is activated by both FBP and F6P, neither of which affect EA. Pyruvate activates EA with higher apparent affinity than AE, suggesting that the C-terminus of the A. tumefaciens enzyme plays a role in the binding of this effector. The allosteric inhibitor site is apparently disrupted, as a marked desensitization toward AMP was observed in the chimeric enzymes.

Agrobacterium tumefaciens↗
PubMed1992

Resistance of enterococci to aminoglycosides and glycopeptides.

High-level resistance to aminoglycosides in enterococci often is mediated by aminoglycoside-modifying enzymes, and the corresponding genes generally are located on self-transferable plasmids. These enzymes are similar to those in staphylococci but differ from the modifying enzymes of gram-negative bacteria. Three classes of enzymes are distinguished, depending upon the reaction catalyzed. All but amikacin and netilmicin confer high-level resistance to the antibiotics that are modified in vitro. However, the synergistic activity of these last two antibiotics in combination with beta-lactam agents can be suppressed, as has always been found in relation to high-level resistance to the aminoglycosides. Acquisition of glycopeptide resistance by enterococci recently was reported. Strains of two phenotypes have been distinguished: those that are resistant to high levels of vancomycin and teicoplanin and those that are inducibly resistant to low levels of vancomycin and susceptible to teicoplanin. In strains of Enterococcus faecium highly resistant to glycopeptides, we have characterized plasmids ranging from 34 to 40 kilobases that are often self-transferable to other gram-positive organisms. The resistance gene vanA has been cloned, and its nucleotide sequence has been determined. Hybridization experiments showed that this resistance determinant is present in all of our enterococcal strains that are highly resistant to glycopeptides. The vanA gene is part of a cluster of plasmid genes responsible for synthesis of peptidoglycan precursors containing a depsipeptide instead of the usual D-alanyl-D-alanine terminus. Reduced affinity of glycopeptides to these precursors confers resistance to the antibiotics.

Aminoglycosides↗
PubMed1985

Molecular cloning of cDNAs cognate to genes sensitive to hormonal control in rat liver.

Poly(A)-RNAs were prepared from livers of rats treated with hydrocortisone and cycloheximide, then enriched for large mRNAs by successive sucrose gradients and gel electrophoresis. The size-selected RNAs were used as templates for synthesis of double-stranded cDNAs that were cloned in Escherichia coli using the pBR322 plasmid vector. Recombinant plasmids characterized as carrying inserts of potential interest were further analyzed by differential hybridization to mRNAs from untreated and hydrocortisone-treated rats. Seven of the cloned cDNAs were identified as complementary to mRNAs whose content in liver is sensitive to modulation by the steroid. Further screening for hormonal responsiveness revealed that two of the cloned cDNAs hybridize to a 3.4-kilobase mRNA that is rapidly induced in liver by treatment with insulin or cAMP as well as by hydrocortisone; restriction enzyme analysis demonstrated that the cDNAs from these two clones are derived from the same mRNA. In isolated nuclei, the rate of transcription of this mRNA is increased by each of the inducing hormones. This unusually regulated mRNA codes for a protein of 53 kDa on denaturing gels, undergoes rapid intracellular degradation that is prevented by cycloheximide, and appears to be the product of a single copy gene.

Animals↗
PubMed1995

Mobile genetic elements of Fusobacterium nucleatum.

The gram-negative anaerobic bacterium, Fusobacterium nucleatum, is a predominant member of the human oral flora. As a major component of subgingival plaque, this bacterium has a significant impact on the ecology of the oral cavity due to its ability to adhere to many different microbial species. The objective of this study was to identify and characterize plasmids and transposons that may have the potential to be developed into tools for cloning, genetic transformation, and mutagenesis of oral isolates of F. nucleatum. Analysis of a collection of laboratory strains resulted in the identification of a homologous family of small cryptic plasmids. Plasmids within this family ranged in size from 6.0 to 6.6 kb. Eighteen percent of all strains examined (n = 74) contained DNA sequences related to the plasmids. Homologous plasmid sequences were found in strains belonging to 2 of the 3 subspecies of the bacterium. The 2 smallest plasmid species were cloned in Escherichia coli to facilitate endonuclease restriction mapping. Among the strains examined for plasmids, 5 exhibited resistance to at least 10 micrograms/ml of tetracycline. These strains, all members of the subsp. polymorphum, contained a tetracycline resistance determinant (TetM) as part of a Tn916-like integrated transposon sequence. The Tn916-like element and 1 of the plasmid species co-resided in a single strain of the bacterium. Hybridization patterns of the Tn916-like sequences were identical in all 5 tetracycline-resistant strains. However, these strains appeared to be clonally distinct based on genomic fingerprinting.

Base Sequence↗
PubMed1988

Regulation of proline utilization in Salmonella typhimurium: molecular characterization of the put operon, and DNA sequence of the put control region.

The two genes required for proline utilization (put) in Salmonella typhimurium form a divergent operon. Extensive genetic evidence suggests that transcription of the put operon is autoregulated by the putA gene product, a membrane-associated dehydrogenase. In order to understand the mechanism of regulation, we characterized plasmid clones of the put operon. A 7.5 kb clone contains both of the put structural genes and regulatory sites. This clone only expressed two unique proteins corresponding to the putA and putP gene products. By comparing the physical and genetic maps of the put operon, the position of the put regulatory region was defined and the DNA sequence of this region was determined. Analysis of the DNA sequence indicated several potential regulatory sites for the put genes. Based on genetic and physical mapping studies, the most likely regulatory sites are two convergent promoters approximately 30 bp apart. A 27 bp palindrome located between the two promoters may be the operator for autoregulation by the PutA protein. The putA translational start site is 40 bp downstream of its putative mRNA start site. The putP promoter and its translational start site are separated by a 400 bp untranslated region.

Base Sequence↗
PubMed2006

Improvement of enterocin P purification process.

Purification and heterologous expression of enterocin P (EntP), a sec-dependent bacteriocin produced by Enterococcus faecium, in Escherichia coli is described. PCR-amplified product of the enterocin P structural gene entP was cloned into plasmid pET-32b under the control of the inducible T7lac promoter. The neo-synthesized EntP was genetically modified by an addition of 3 extra amino acids, leading to recombinant EntRP. Active EntRP was recovered from the cytoplasmic soluble fraction of E. coli harboring appropriate recombinant plasmid, characterized by ELISA and Western-blot analysis and purified by immunoaffinity chromatography. The use of E. coli as heterologous host and pET-32b as expressing vector offers promising tools for heterologous production of class IIa bacteriocin.

Bacteriocins↗
PubMed2005

Construction of highly efficient E. coli expression systems containing low oxygen induced promoter and partition region.

A series of high-copy-number Escherichia coli expression vectors equipped with an oxygen-sensitive promoter P(vgb) of Vitreoscilla hemoglobin (encoded by the vgb gene) were constructed and characterized. Plasmid pKVp containing P(vgb) was inducible by low oxygen tension, while plasmid pKVpP containing a partition (par) region from plasmid pSC101 ligated to P(vgb) provided inheritable stability for the vectors in the absence of ampicillin. Plasmid pKVpV had the Vitreoscilla hemoglobin operon vgb ligated to P(vgb), while a construct containing P(vgb), the vgb operon and a par region constituted plasmid pKVpPV. Shake-flask studies demonstrated that plasmids pKVpV and pKVpPV expressed higher levels of Vitreoscilla hemoglobin under low aeration condition (5% air saturation in water) compared with the levels observed under strong aeration (20% air saturation in water). Introduction of either the enhanced green fluorescent protein (eGFP) gene egfp or the toluene dioxygenase (TDO) gene tod into either pKVpV (P(vgb), vgb operon) or pKVpPV (P(vgb), vgb operon, par) slightly attenuated (approximately 30%) the strong expression of VHb under low aeration. However, all displayed approximately a three-fold increase versus that observed for strong aeration. Recombinant E. coli harboring either pKVp-E (P(vgb), egfp) or pKVpP-E (P(vgb), par, egfp) displayed at least a two-fold increase in eGFP expression under conditions of low aeration and absence of antibiotic, compared with that under strong aeration after 24 h of cultivation. Strong expression of TDO was also observed using low aeration in recombinant E. coli harboring pKVpPV-T (P(vgb), vgb operon, par, tod) or pKVpP-T (P(vgb), par, tod). Plasmids containing the par region were stable over 100 generations. These results indicate that the novel expression system combining plasmid stability over the cell growth phase and a promoter inducible by low oxygen tension will be very useful for high-density production of foreign proteins.

Bacterial Proteins↗
PubMed1986

Construction of recombinant vaccinia virus strains using single-stranded DNA insertion vectors.

The ability of single-stranded (ss) DNA, isolated from recombinant M13 bacteriophage, to direct the insertion of foreign genetic elements into the vaccinia virus (VV) genome was examined. An identical chimeric transcriptional unit [VV promoter/chloramphenicol acetyl transferase (CAT) gene embedded in DNA sequences encoding vaccinia virus thymidine kinase (TK)] was inserted into either the previously characterized plasmid insertion vector, pGS20, or into M13mp18. It was found that the ss vector (M13mp18:TK/CAT) was four times more efficient than the plasmid vector (pGS20:CAT) in catalyzing homologous recombination of the cat gene by marker transfer into the VV genome. Furthermore, Southern blot analyses and CAT enzymatic activity assays confirmed that the structure of the M13-derived recombinant genomes were as expected and that the chimeric genes were fully active. Although the precise mechanism responsible for the ss DNA-catalyzed insertion event is not known, these results are discussed with respect to the advantages of using M13-based vectors with which to manipulate and insert genetic information into infectious VV recombinants.

Cloning, Molecular↗
PubMed1998

Suspected Brazilian purpuric fever in a toddler with overwhelming Epstein-Barr virus infection.

We describe a toddler from Connecticut who developed purulent conjunctivitis, fever, and a morbilliform rash. Blood cultures were positive for Haemophilus influenzae biogroup aegyptius; further investigation was performed to assess the possibility that the illness was consistent with Brazilian purpuric fever, which, to our knowledge, has not been reported in the United States. This isolate shared morphological and some biochemical characteristics with previously studied H. influenzae biogroup aegyptius strains but differed according to slide agglutination testing, plasmid characterization, and ribotyping. Blood and tissue samples obtained during his hospitalization were also positive for Epstein-Barr virus. The child died 8 days after hospitalization. Fifty other cases of invasive H. influenzae infection were identified by active surveillance studies. Of the 49 viable surveillance isolates, 10 were biotype III (two of which had the same ribotype as the strain from our case.

Bacteremia↗
PubMed1990

Genetic and molecular analysis of a long-lived strain of Podospora anserina.

A genetic and molecular analysis of a long-lived strain of Podospora anserina, Mn19, was undertaken to detect mutations in genes responsible for senescence. In crosses between Mn19 and wild type about 15% of the progeny were long-lived, regardless of the female parent. Molecular analysis of the long-lived progeny showed that none of the strains inherited a mtDNA rearrangement characteristic of the Mn19 parent. Instead, all long-lived strains initially inherited wild-type mtDNA. Over time the mtDNA of most long-lived strains underwent rearrangements, deletions and amplifications. The change over time in the presence of two previously characterized plasmids associated with either senescence or longevity was monitored. Crosses between Mn19 and its long-lived progeny also yielded only a small percent of individuals recovering from senescence. Analysis of mtDNA from crosses suggests that wild-type mtDNA from the paternal parent can be selected over mtDNA from the maternal parent. The life span phenotypes of progeny were not consistent with the hypothesis that mutations in a few nuclear genes were responsible for longevity.

Ascomycota↗
PubMed1983

A hospital outbreak of multiresistant Salmonella typhimurium belonging to phage type 193.

An outbreak of nosocomial infection was caused by strains of Salmonella typhimurium phage type 193 that were resistant to ampicillin, chloramphenicol, neomycin-kanamycin, streptomycin, spectinomycin, sulfonamides, tetracyclines, trimethoprim, and nalidixic acid. Resistances to drugs other than nalidixic acid were specified by plasmids, and, on the basis of phage typing and plasmid characterization studies, the multiresistant phage type 193 strains were determined to be clonal. In a two-year period, 488 patients infected with these bacteria were identified. An investigation in a pediatric surgical ward, where the outbreak was particularly severe, showed that patients exposed to antibiotics were more likely to be colonized with the epidemic strain and that young debilitated patients were more liable to show clinical signs of infection. Epidemiologic studies suggested that cross infection via the hands of the ward staff was the likely means of propagation of the epidemic.

Anti-Bacterial Agents↗
PubMed1985

Trimethoprim resistance in urinary coliforms from patients in the community: plasmids and R-transfer.

A study of urinary coliform isolates, from patients attending their general practitioner, between 1981 and 1983 showed that 10.2% were resistant to trimethoprim. Forty-four trimethoprim-resistant Escherichia coli were collected for further study and the results compared with those obtained from hospital isolates collected during 1979-80 and 1982. Thirty-two (73%) were highly resistant to trimethoprim (MIC greater than 1024 mg/l) a similar proportion to that in the hospital isolates. The highly resistant isolates bore a close resemblance to the hospital isolates in that 66% carried multiple plasmids, 97% were multiply drug-resistant and all were highly resistant to sulphamethoxazole. The frequency of antimicrobial resistance transfer (59%) was similar to that from the hospital isolates. More than half of the trimethoprim resistance plasmids characterized were indistinguishable in terms of mol.wt and resistance pattern from those found in isolates from the hospital collections. Our results suggest that, whereas trimethoprim-resistant E. coli are less commonly isolated from patients in the community than in hospitals, many of the community isolates may have originated from a hospital source.

Conjugation, Genetic↗
PubMed1986

Comparative activities of the beta-lactamase inhibitors YTR 830, clavulanate, and sulbactam combined with ampicillin and broad-spectrum penicillins against defined beta-lactamase-producing aerobic gram-negative bacilli.

The in vitro synergistic activities of the beta-lactamase inhibitors YTR 830, clavulanate, and sulbactam, combined with ampicillin, ticarcillin, mezlocillin, azlocillin, piperacillin, and apalcillin, were determined against 34 strains of members of the Enterobacteriaceae family, Pseudomonas aeruginosa, Aeromonas hydrophila, and Haemophilus influenzae with characterized plasmid or chromosomal beta-lactamases or both. Strains were tested against fixed concentrations of beta-lactamase inhibitors (8 micrograms/ml) combined with doubling dilutions of beta-lactams. Synergy was defined as a fourfold or greater decrease in the MIC of the beta-lactam. Against Enterobacteriaceae producing Richmond and Sykes class III and V plasmid-mediated beta-lactamases, synergy was obtained against most strains with YTR 830- and clavulanate-beta-lactam combinations, with sulbactam being less effective. Against Enterobacteriaceae producing class I chromosomal beta-lactamases, combinations containing YTR 830 or sulbactam were more synergistic than combinations containing clavulanate. Against strains producing class V PSE enzymes, all three inhibitors were synergistic with piperacillin and apalcillin against strains producing PSE-1, -3, and -4 enzymes, while the PSE-2-producing strain was resistant to all inhibitors. YTR 830-beta-lactam combinations were also synergistic against strains producing the novel beta-lactamases OHIO-1, TLE-1, AER-1, and ROB-1. Overall, YTR 830 with piperacillin or apalcillin was the most effective combination.

Ampicillin↗
PubMed1990

recB recC-dependent processing of heteroduplex DNA stimulates recombination of an adjacent gene in Escherichia coli.

The effect of DNA mismatched repair on the genetic recombination of a gene adjacent to the mismatch site (MS) was tested by using four mismatch configurations. An MS was constructed in a well-characterized plasmid recombination substrate, and recombination with a resident compatible plasmid was measured after transformation of the mismatched plasmid into Escherichia coli. The mismatched plasmids were constructed such that one of the DNA strands was methylated by the DNA adenine methylase (Dam), while the other strand was unmethylated. The processing of a hemimethylated single-base-pair mismatch had no effect on the recombination of the adjacent gene, suggesting that the most efficient (Dam-instructed) mismatch repair process does not secondarily promote genetic recombination. However, mismatches that could form an ordered secondary structure resembling a cruciform increased the recombination of this adjacent gene at least 20-fold. An identical mismatch that could not form an ordered secondary structure had no effect in this system. The increased frequency of recombination observed was found to require the recB or recC gene product or both. Furthermore, the recombination appeared unidirectional, in that the cruciform-containing plasmid did not produce stable transformants. Our results support a model in which the cruciform-containing plasmid can participate in recombination with the resident plasmid but is unable to produce stable transformant progeny. A proposed role for the RecBCD enzyme (ExoV) in this process is discussed.

DNA Repair↗
PubMed1996

Detection by PCR of the nim genes encoding 5-nitroimidazole resistance in Bacteroides spp.

A PCR method was developed for detection of the nim genes encoding 5-nitrolmidazole resistance in Bacteroides spp. Two PCR primers specific for nim genes were designed. They allowed amplification of a 458-bp fragment from all characterized plasmid- and chromosome-borne metronidazole resistance genes. The specificity of the method was tested with DNA from metronidazole-sensitive Bacteroides spp. strains and from other strains of unrelated species. Each DNA preparation was analyzed with and without an internal positive control to verify that the absence of PCR amplification product was not due to inhibition of the Taq polymerase inhibitors. By this technique, two newly discovered metronidazole-resistant clinical strains of Bacteroides fragilis were shown to harbor resistance genes undetectable by Southern blotting. In spite of the sequence divergence of the nim genes, the PCR method is thus suitable for epidemiological investigations. The amplification method also revealed that nim-related resistance genes were not present in either Streptomyces strain S6670, a natural producer of 2-nitroimidazole, or in Enterococcus faecalis strains, which have been suggested to possess metronidazole-inactivating enzyme.

Bacterial Proteins↗
PubMed1997

Emergence of rifampin-resistant Rhodococcus equi in an infected foal.

To investigate the emergence of rifampin resistance in Rhodococcus equi strains isolated from foals and their environment in Japan, we compared the in vitro antimicrobial susceptibilities to rifampin of 640 isolates from 64 infected foals and 98 soil isolates from their horse-breeding farms. As a control, 39 human isolates from patients with and without AIDS were also tested for susceptibility to rifampin. All of the isolates showed rifampin sensitivity, except isolates from one infected foal and two patients with AIDS that showed rifampin resistance. To investigate the emergence of rifampin-resistant R. equi in the infected foal, which had received rifampin monotherapy for a month before euthanasia, 99 isolates of R. equi from the lesions and 20 isolates from the intestinal contents of the one foal with rifampin-resistant organisms were analyzed for rifampin susceptibilities, pathogenicities, and ribotypes. Of the 99 isolates from the lesions, all of which were virulent R. equi strains containing a virulence plasmid with a size of 85 or 90 kb, 90 (91%) isolates were rifampin resistant (MIC, > or = 12.5 microg/ml). On the other hand, of the 20 isolates from the intestinal contents, 11 (55%) isolates showed rifampin resistance (MIC, > or = 25 microg/ml), and 5 of them were avirulent R. equi strains. Among these 101 rifampin-resistant R. equi isolates with and without virulence plasmids characterized by ribotyping, 58 were type I, 20 were type II, 11 were type III, and 12 were type IV. These results demonstrated that at least eight different rifampin-resistant R. equi strains emerged concurrently and respectively from the different lesions and intestinal contents of the infected foal.

Actinomycetales Infections↗