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PubMed2001

Molecular characterization of a cryptic plasmid from Campylobacter jejuni.

Campylobacter jejuni is a leading bacterial cause of human enterocolitis. Molecular genetic characterization of this pathogen has been hampered by the lack of genetic tools that are functional in this organism. Cloning vectors commonly used in other organisms usually do not replicate within C. jejuni. To develop a system for functional analysis of C. jejuni genes, a small plasmid (pCJ01) identified in a poultry isolate of C. jejuni was sequenced and characterized in this study. By using inverse PCR, the full sequence of pCJ01 was amplified and subsequently determined. Results indicate that pCJ01 is a circular molecule of 3212 bp, with a G + C content of 33.5%. A typical plasmid replication origin with iteron sequences is identified upstream of the DNA sequences encoding replication initiation proteins. Four open reading frames (ORFs) are present in pCJ01. ORF1 and ORF2 share high homology with the putative RepA and RepB proteins, respectively, of known C. coli plasmids. ORF3 and ORF4, of unknown function, do not exhibit homology with any sequences deposited in the GenBank database. Hydropathy analysis predicts that ORF3 and ORF4 contain multiple stretches of hydrophobic amino acids, suggesting that they may encode transmembrane proteins. Since pCJ01 is a small plasmid and can be readily prepared from C. jejuni, it may be modified for use in molecular characterization of C. jejuni virulence genes.

Base Sequence↗

PubMed2001

Genetic transformation system for a psychrotrophic deep-sea bacterium: isolation and characterization of a psychrotrophic plasmid.

A versatile system that permits genetic manipulation of a psychrotrophic deep-sea bacterium, Pseudoalteromonas sp. PS1M3, has been developed. A cryptic indigenous plasmid, pPS1M3, of 3.1 kb from the above strain was isolated and characterized. The nucleotide sequence analysis of plasmid pPS1M3 revealed the presence of one open reading frame, and its deduced amino acid sequence was identified as the essential protein for plasmid maintenance. Transformation with the pPS1M3 harboring antibiotic resistance genes by electroporation was fully successful using the pPS1M3-cured strain as a host. This plasmid was quite stable under nonselective culture conditions for about 100 generations at 4 degrees C. The copy number of this plasmid in the cell was about 5 copies per chromosome.

Journal Article↗

PubMed1994

Characterization of pPT23B, the plasmid involved in syringolide production by Pseudomonas syringae pv. tomato PT23.

Avirulence gene D (avrD) in strain PT23 of Pseudomonas syringae pv. tomato (Pst) specifies the production of syringolides, which are elicitors of plant defense reactions. An 83-kb indigenous plasmid (pPT23B) that carries avrD has been mapped and characterized and a putative par region was identified. pPT23B contains a large amount of DNA that is repeated in other native plasmids in PT23. A putative mobile insertion element that occurs on plasmid pPT23A as well as on the chromosome was also identified in strain PT23. New broad-host-range expression vectors that functioned in Pst were constructed for overexpression of the cloned avrD gene and high-level production of the syringolides. Introduction of an avrD overexpression plasmid into PT23 or plasmid-cured strains led to identical syringolide peaks on HPLC with no new peaks observed. These results suggested that neither pPT23B nor other indigenous plasmids in Pst carry additional genes required for syringolide production or metabolism. Pst strains lacking pPT23B were not impaired in virulence on tomato plants.

Bacterial Toxins↗

PubMed1986

Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204.

A Pseudomonas putida strain designated RE204, able to utilize isopropylbenzene as the sole carbon and energy source, was isolated. Tn5 transposon mutagenesis by means of the suicide transposon donor plasmid pLG221 yielded mutant derivatives defective in isopropylbenzene metabolism. These were characterized by the identification of the products which they accumulated when grown in the presence of isopropylbenzene and by the assay of enzyme activities in cell extracts. Based on the results obtained, the following metabolic pathway is proposed: isopropylbenzene----2,3-dihydro -2,3-dihydroxyisopropylbenzene----3-isopropylcatechol----2 -hydroxy-6-oxo-7-methylocta-2,4-dienoate----isobutyrate + 2-oxopent-4-enoate----amphibolic intermediates. Plasmid DNA was isolated from strain RE204 and mutant derivatives and characterized by restriction enzyme cleavage analysis. Isopropylbenzene-negative isolates carried a Tn5 insert within a 15-kilobase region of a 105-kilobase plasmid designated pRE4. DNA fragments of pRE4 carrying genes encoding isopropylbenzene catabolic enzymes were cloned in Escherichia coli with various plasmid vectors; clones were identified by (i) selection for Tn5-encoded kanamycin resistance in the case of Tn5 mutant plasmids, (ii) screening for isopropylbenzene dioxygenase-catalyzed oxidation of indole to indigo, and (iii) use of a Tn5-carrying restriction fragment, derived from a pRE4::Tn5 mutant plasmid, as a probe for clones carrying wild-type restriction fragments. These clones were subsequently used to generate a transposon insertion and restriction enzyme cleavage map of the isopropylbenzene metabolic region of pRE4.

Benzene Derivatives↗

PubMed2003

Characterization of the nodulation plasmid encoded chemoreceptor gene mcpG from Rhizobium leguminosarum.

BACKGROUND: In general, chemotaxis in Rhizobium has not been well characterized. Methyl accepting chemotaxis proteins are sensory proteins important in chemotaxis of numerous bacteria, but their involvement in Rhizobium chemotaxis is unclear and merits further investigation. RESULTS: A putative methyl accepting chemotaxis protein gene (mcpG) of Rhizobium leguminosarum VF39SM was isolated and characterized. The gene was found to reside on the nodulation plasmid, pRleVF39d. The predicted mcpG ORF displayed motifs common to known methyl-accepting chemotaxis proteins, such as two transmembrane domains and high homology to the conserved methylation and signaling domains of well-characterized MCPs. Phenotypic analysis of mcpG mutants using swarm plates did not identify ligands for this putative receptor. Additionally, gene knockouts of mcpG did not affect a mutant strain's ability to compete for nodulation with the wild type. Notably, mcpG was found to be plasmid-encoded in all strains of R. leguminosarum and R. etli examined, though it was found on the nodulation plasmid only in a minority of strains. CONCLUSIONS: Based on sequence homology R. leguminosarum mcpG gene codes for a methyl accepting chemotaxis protein. The gene is plasmid localized in numerous Rhizobium spp. Although localized to the sym plasmid of VF39SM mcpG does not appear to participate in early nodulation events. A ligand for McpG remains to be found. Apparent McpG orthologs appear in a diverse range of proteobacteria. Identification and characterization of mcpG adds to the family of mcp genes already identified in this organism.

Amino Acid Sequence↗

PubMed1986

Characterization of the Streptomyces plasmid pVE1.

pVE1 (11.0 kb) was isolated from Streptomyces venezuelae ATCC 14585 and characterized. pVE1 has a broad host range and an apparent copy number of about 100 during exponential growth, rising to 1500 during stationary phase. It is a pock-forming, self-transmissible fertility factor which promotes chromosome recombination in S. lividans at frequencies of about 0.1% per total parents. A detailed restriction map for 15 enzymes was determined. Genes for ThioR (thiostrepton resistance), NeoR (neomycin resistance), and pBR322 derivatives were inserted into pVE1 and the resulting plasmids were analyzed for self-transmissibility and stability. The plasmid has an essential region of approximately 2.5 kb and a region of 1.0-3.6 kb required for conjugation. NeoR and ThioR vectors were constructed with unique HindIII, PvuI, BamHI, EcoRI, BglII, and EcoRV sites available for insertion of foreign DNA.

Chromosome Mapping↗

PubMed1987

Plasmid-mediated trimethoprim-resistance in Staphylococcus aureus. Characterization of the first gram-positive plasmid dihydrofolate reductase (type S1).

The trimethoprim-resistance gene located on plasmid pSK1, originally identified in a multi-resistant Staphylococcus aureus from Australia, encodes the production of a dihydrofolate reductase (type S1), which confers a high degree of resistance to its host and is quite unlike any plasmid-encoded dihydrofolate reductase hitherto described. It has a low Mr (19,700) and has a higher specific activity than the constitutive Gram-negative plasmid dihydrofolate reductases. The type S1 enzyme is heat-stable and has a relatively low affinity for the substrate, dihydrofolate (Km 10.8 microM). It is moderately resistant to trimethoprim, and is competitively inhibited by this drug with an inhibitor-binding constant of 11.6 microM. This is the first identification and characterization of a plasmid-encoded trimethoprim-resistant dihydrofolate reductase derived from a Gram-positive species.

Drug Resistance, Microbial↗

PubMed1997

Antibiotic-resistant Salmonella typhi from two outbreaks: few ribotypes and IS200 types harbor Inc HI1 plasmids.

To investigate factors that could be involved in the emergence of antibiotic resistant S. typhi, we characterized R plasmids and antibiotic resistant S. typhi strains from two outbreaks of typhoid in Peru and Chile. Differences in the Inc HI1 plasmids of Peruvian and Chilean strains were identified by conjugation and incompatibility studies and plasmid DNA characterization. Antibiotic-resistant S. typhi harboring Inc HI1 plasmids belonged to a reduced number of Pst1 and Cla1 ribotypes and IS200 types, in contrast to the high genetic diversity found among epidemic antibiotic-susceptible S. typhi. The low diversity of antibiotic-resistant S. typhi suggests that they may express properties that are related to both their ability to harbor Inc HI1 R plasmids and to disseminate.

Bacteriophage Typing↗

PubMed1984

Construction and characterization of autonomously replicating plasmids in the green unicellular alga Chlamydomonas reinhardii.

Plasmids that replicate autonomously in Chlamydomonas reinhardii were constructed by inserting random DNA fragments from this alga into a plasmid containing the yeast ARG4 locus. Arginine prototrophy was used as a selective marker. The presence of free plasmids in the DNA of the transformants was demonstrated by hybridization with a specific plasmid probe and by recovering these plasmids in E. coli after transformation. Four of them were characterized. Their inserts of 415, 257, 153, and 102 bp all hybridize to chloroplast DNA and were localized on the physical map of the chloroplast genome. One of these plasmids also promotes autonomous replication in yeast. Sequence analysis of the inserts of the plasmids reveals several short direct and inverted repeats and two semiconserved AT-rich elements of 19 and 12 bp that may play a role in promoting autonomous replication in C. reinhardii.

Base Sequence↗

PubMed1992

Rapid plasmid DNA isolation from mucoid gram-negative bacteria.

Exopolysaccharides interfere with the isolation and characterization of plasmid DNA from gram-negative bacteria. To repress capsular polysaccharide production, bacteria were cultured in medium containing bismuth nitrate and sodium salicylate. Rapid removal of other contaminating bacterial surface components was achieved by mild acidic zwitterionic detergent extraction. After treatment, bacterial cells were more readily lysed in alkaline detergents. The resulting plasmid preparations contained virtually no capsular polysaccharide and relatively small quantities of lipopolysaccharide and protein, yet they produced yields of nucleic acids similar to those of conventional plasmid preparations. Conventional preparations from encapsulated organisms were largely insoluble and appeared as smears following agarose gel electrophoresis, with indefinite plasmid banding. Plasmids prepared by the new method were highly soluble in conventional buffers and exhibited high-resolution plasmid banding patterns in agarose gels. Plasmids as large as 180 kbp could be isolated and visualized, without apparent nicking, and were readily digested by restriction endonuclease enzymes. The method proved effective with encapsulated or mucoid strains of Klebsiella pneumoniae, Escherichia coli, Acinetobacter anitratus, Salmonella typhimurium, and Enterobacter species. The complete method for plasmid isolation was not suitable for Pseudomonas aeruginosa because of the inhibitory effects of bismuth. Thus, removal of contaminating bacterial surface structures enabled the rapid isolation and characterization of plasmids from mucoid clinical isolates, without the use of organic solvents, CsCl gradients, or expensive, disposable columns.

Bacterial Typing Techniques↗

PubMed2004

Functional characterization of a catabolic plasmid from polychlorinated- biphenyl-degrading Rhodococcus sp. strain RHA1.

Rhodococcus sp. strain RHA1, a potent polychlorinated-biphenyl (PCB)-degrading strain, contains three linear plasmids ranging in size from 330 to 1,100 kb. As part of a genome sequencing project, we report here the complete sequence and characterization of the smallest and least-well-characterized of the RHA1 plasmids, pRHL3. The plasmid is an actinomycete invertron, containing large terminal inverted repeats with a tightly associated protein and a predicted open reading frame (ORF) that is similar to that of a mycobacterial rep gene. The pRHL3 plasmid has 300 putative genes, almost 21% of which are predicted to have a catabolic function. Most of these are organized into three clusters. One of the catabolic clusters was predicted to include limonene degradation genes. Consistent with this prediction, RHA1 grew on limonene, carveol, or carvone as the sole carbon source. The plasmid carries three cytochrome P450-encoding (CYP) genes, a finding consistent with the high number of CYP genes found in other actinomycetes. Two of the CYP genes appear to belong to novel families; the third belongs to CYP family 116 but appears to belong to a novel class based on the predicted domain structure of its reductase. Analyses indicate that pRHL3 also contains four putative "genomic islands" (likely to have been acquired by horizontal transfer), insertion sequence elements, 19 transposase genes, and a duplication that spans two ORFs. One of the genomic islands appears to encode resistance to heavy metals. The plasmid does not appear to contain any housekeeping genes. However, each of the three catabolic clusters contains related genes that appear to be involved in glucose metabolism.

Amino Acid Sequence↗

PubMed1986

Characterization and incompatibility properties of ROM- derivatives of pBR322-based plasmids.

We have characterized a copy number mutant of the pBR322-based plasmid pWT111. A single nucleotide transversion in loop II' of RNAI results in an eightfold increase in plasmid copy number. Removal of the rom coding region from pWT111 cop results in a further sixfold increase in copy number. We present evidence that ROM is involved in the strong incompatibility effect seen between pMB1 and ColE1 type plasmids.

Base Sequence↗

PubMedDate not supplied

Characterization of non-virulence plasmids with homology to the virulence plasmid of Salmonella dublin.

Six wild-type (wt) strains of Salmonella typhimurium, one wt strain of S. heidelberg and 12 wt strains of Escherichia coli were isolated based on both hybridization to a 6-kb HindIII fragment of the non-virulence coding part of the S. dublin serovar-specific virulence plasmid and the absence of hybridization to the virulence genes (spv genes) of the same plasmid. Such hybridization was shown to be caused by resident plasmids in all strains and to involve the same region of 30 to 37 kb of consecutive HindIII fragments on the S. dublin virulence plasmid, suggesting a common origin of this plasmid DNA. Nine of the plasmids were selected for detailed characterization and were shown not to be of the same plasmid species. They varied in size between 44 and 88 kb, they showed incompatibility with the plasmid K-MP10, or belonged to incompatibility group X, and with the exception of five plasmids from E. coli, they showed different HindIII restriction profile patterns.

DNA, Bacterial↗

PubMed2003

Characterization of the cryptic plasmid pBGR1 from Bartonella grahamii and construction of a versatile Escherichia coli-Bartonella spp. shuttle cloning vector.

We report herein the isolation and molecular characterization of pBGR1, the first native plasmid isolated from the genus Bartonella. Cloning and sequencing revealed a 2725-base pair (bp) cryptic plasmid comprising two open reading frames of considerable length, which were designated rep and mob. The regions containing rep and mob are separated by 140-bp inverted repeat sequences and display a difference in G + C content from one another. A 1435-bp SacI-BclI fragment containing the rep gene is sufficient to mediate replication in the species Bartonella henselae and Bartonella tribocorum, while this replicon does not appear to be functional in Escherichia coli. The Rep protein of 190 amino acids (aa) shares homology to putative replication proteins of cryptic plasmids of Gram-negative origin, which form a subgroup of the rolling-circle replication proteins of the pSN2 plasmid superfamily of Gram-positive bacteria. The Mob protein of 333 aa is related to mobilization proteins of several cryptic plasmids and is associated with a conserved recombination site A. The tra functions of RP4 can mobilize pBGR1 derivatives in a mob-dependent manner. Mobilizable pBGR1-based E. coli-Bartonella spp. shuttle vectors were constructed and were shown to be maintained in B. tribocorum during in vivo passage in a rat model in the absence of antibiotic selection. The small size and stability of these shuttle cloning vectors should render them particularly valuable for genetic studies in Bartonella spp.

Adenosine Triphosphatases↗

PubMed1984

Saccharomyces cerevisiae RAD2 gene: isolation, subcloning, and partial characterization.

A plasmid (pNF2000) containing a 9.7-kilobase pair DNA insert that complements the UV sensitivity of rad2-1, rad2-2, and rad2-4 mutants of Saccharomyces cerevisiae has been isolated from a yeast genomic library. Genetic analysis of strains derived by transformation of rad2 mutants with an integrating plasmid containing a 9.3-kilobase pair fragment from pNF2000 shows that the fragment integrates exclusively at the chromosomal rad2 gene. We therefore conclude that this plasmid contains the RAD2 gene. The 9.3-kilobase pair fragment was partially digested with Sau3A and cloned into a multicopy yeast vector designed for easy retrieval of Sau3A inserts. The smallest subclone that retains the RAD2 gene is 4.5 kilobase pairs. This fragment was partially digested with Sau3A and cloned into an integrating plasmid. These plasmids were isolated and integrated into a heterozygous rad2/RAD2 strain. Plasmids containing internal fragments of the RAD2 gene were identified because they yielded UV-sensitive transformants due to disruption of the RAD2 gene. Sporulation of diploids transformed with integrating plasmids containing internal fragments of RAD2 gave rise to four viable haploids per tetrad, indicating that unlike the RAD3 gene of S. cerevisiae, the RAD2 gene is not essential for the viability of haploid cells under normal growth conditions. Measurements of the RNA transcript by RNA-DNA hybridization with the internal fragment as the probe indicate a size of approximately 3.2 kilobases.

Cloning, Molecular↗

PubMed1993

Direct expression of adrenodoxin reductase in Escherichia coli and the functional characterization.

A plasmid for direct expression in Escherichia coli of the mature form bovine adrenodoxin reductase was constructed from the full-size cDNA for the enzyme [Y. Sagara, Y. Takata, T. Miyata, T. Hara, and T. Horiuchi, J. Biochem. (Tokyo), 102, 1333 (1987)] and an expression vector pCWori+. The recombinant adrenodoxin reductase was purified from the transformed E. coli cell lysates using adrenodoxin-Sepharose affinity chromatography [T. Sugiyama and T. Yamano, FEBS Lett., 52, 145 (1975)] with a yield of 2.5 mg/l of culture. The purified recombinant enzyme showed a single band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and migration was identical with that of the authentic enzyme purified from bovine adrenal cortex mitochondria. The recombinant enzyme had Ser at its amino-terminus and the sequence of the amino terminal 9 residues was identical with that of the authentic bovine enzyme. The absorption spectrum of the recombinant enzyme showed peaks at 270, 376, and 450 nm and shoulders at 425 and 475 nm. Flavin content of the recombinant enzyme was 0.8 mol FAD/mol. The apparent Km value for bovine adrenodoxin in NADPH-cytochrome c reductase activity using a reconstitution system was 16 nM, a value comparable with that of the authentic bovine enzyme (17 nM). The cholesterol side chain cleavage activity with a reconstitution system was about 75% of that obtained when the authentic enzyme was used.

Adrenal Cortex↗

PubMed2007

Characterization of a cryptic plasmid from Bacillus sphaericus strain LP1-G.

A cryptic plasmid from Bacillus sphaericus strain LP1-G, designated as pLG, was sequenced and characterized. It was an 11,066bp circular molecule, with G+C content of 37%. The plasmid pLG was predicted to encode 23 putative ORFs, and ORF 21 shared the highest identity with Rep of pGI1 and pBMB9741, members of rolling-circle replication (RCR) pC194-family. Sequence analysis revealed a pC194-type double strand origin (dso) and a single strand origin (sso) like sequence located upstream and downstream of ORF 21, respectively. Moreover, Mung bean nuclease analysis and Southern hybridization confirmed the existence of single stranded DNA (ssDNA) intermediates, indicating that pLG belongs to the RCR pC194-family. Accumulation of multiple ssDNA intermediates in native strain LP1-G and decline of ssDNA and supercoiled DNA in rifampicin-treated strain implied that a special mechanism might be employed by pLG. Furthermore, the copy number of pLG in its original host was determined and about 58 copies of the plasmid exist in each cell. Subcloning and transformation experiments proved that the minimal replicon of pLG was within a 1.6-kb fragment, which was composed of rep gene and dso. These data are a good basis for the understanding of replication mechanisms and genetics of this B. sphaericus plasmid.

Bacillus↗

PubMed2026

Genomic characterization of KPC-2 and NDM coproducing carbapenem-resistant Klebsiella pneumoniae in a hospital: discovery of ST1869 clone and a novel hybrid plasmid.

UNLABELLED: To characterize the plasmid architecture and molecular background of KPC-NDM coproducing carbapenem-resistant Klebsiella pneumoniae (KN-CRKP) in a South China hospital. Five KN-CRKP isolates were collected, including three from one patient. All underwent Illumina sequencing; two (ST11 and ST1869) additionally had Nanopore sequencing. Antimicrobial susceptibility testing strain sequence types, conjugation assays, resistance gene profiling, plasmid typing, genetic structure comparison, core-genome single nucleotide polymorphisms (SNPs) analysis, and plasmid clustering were performed. All isolates exhibited an imipenem minimum inhibitory concentration (MIC) of ≥128 µg/mL and harbored multiple resistance genes. One isolate (1/5) belonged to ST1869 and co-harbored blaKPC-2 and blaNDM-5. The blaNDM-5-carrying plasmid was a novel IncI1/X3 fusion plasmid that also carried blaCMY-42. Unlike several IncX3 plasmids carrying blaNDM in publicly available KN-CRKP genomes from South China, this IncI1/X3 hybrid lacked a complete conjugative transfer system. ST11 was the predominant clone (4/5), co-harboring blaKPC-2 and blaNDM-1. A rare genetic structure, ΔISKpn6-blaKPC-2-ISKpn28, was identified on IncFII plasmids carrying blaKPC-2. Plasmid clustering analysis of 126 comparative KN-CRKP genomes showed diverse sequence types and plasmid backgrounds associated with the KPC/NDM co-production pattern. The observed plasmid diversity and structural variation in KN-CRKP support continued genomic surveillance, with particular attention to the ST1869 clone, the novel IncI1/X3 hybrid plasmid harboring blaNDM-5 and blaCMY-42, and the rare "ΔISKpn6-blaKPC-2-ISKpn28" genetic structure. Expanded genomic data on KN-CRKP are needed to further elucidate its resistance mechanisms and plasmid evolutionary trajectories. IMPORTANCE: The co-production of KPC and NDM carbapenemases in Klebsiella pneumoniae poses a formidable threat to clinical antimicrobial therapy, as these enzymes confer resistance to virtually all β-lactam agents, including carbapenems. Here, we report novel genomic features of KN-CRKP in South China, including the emergence of the ST1869 clone, a unique IncI1/X3 hybrid plasmid harboring blaNDM-5 and blaCMY-42, and the rare ΔISKpn6-blaKPC-2-ISKpn28 genetic structure. These findings substantially expand current understanding of plasmid evolution and resistance gene dissemination in this region. The identification of diverse resistance mechanisms and clonal backgrounds supports enhanced genomic surveillance and infection-control awareness for pan-resistant Enterobacterales.

Plasmids↗