Characterization of plasmids from Haemophilus parainfluenzae and Haemophilus haemolyticus.
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DNA, Bacterial↗
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Tn554: isolation and characterization of plasmid insertions.
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Bacteriophages↗
Physical characterization of plasmids from Streptomyces kasugaensis MB273.
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Centrifugation, Density Gradient↗
Characterization of plasmids from plant pathogenic pseudomonads.
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Base Sequence↗
Characterization of plasmids in aminocyclitol-resistant Staphylococcus aureus: electron microscopic and restriction endonuclease analysis.
Plasmid species isolated from aminocyclitol-resistant Staphylococcus aureus have been analyzed by restriction endonuclease digestion and electron microscopy. These plasmids can be divided into two interrelated groups; intergroup variability is due to the gain or loss of defined DNA sequences. Plasmids pSJ1 and pSJ24 are related to staphylococcal penicillinase plasmid pI524 which was first described over 20 years ago. Both pSJ1 and pSJ24 differ from pI524 by the acquisition of 8 and 4 kbp, respectively, and encode additional resistance to the antibiotics erythromycin and kanamycin. The gain of these resistance determinants suggests that the evolution of staphylococcal resistance plasmids parallels that observed for plasmids of gram-negative bacteria and has serious implications for the spread of antibiotic resistance among the staphylococci.
Aminoglycosides↗
Characterization of plasmids and plasmid-borne macrolide resistance from Lactobacillus sp. strain 100-33.
Lactobacillus sp. strain 100-33 is resistant to macrolides, lincosamides, and streptogramin B-type antibiotics (MLSR) and appears to contain several major and minor plasmids. One of these plasmids, pLAR33, is approximately 18 kbp in size. When cells of strain 100-33 were protoplasted and regenerated, an MLSS isolate was derived. The derivative, designated strain ES1, contained a unique plasmid complement in which it had apparently lost the major plasmids of the parental strain, including pLAR33, and retained only a minor plasmid seen in low concentrations in strain 100-33. The MLSR determinant was cloned from plasmid DNA of strain 100-33 on a 3-kbp EcoRV fragment into pBR322 and localized to pLAR33. The determinant expressed macrolide and lincosamide resistance in Escherichia coli HB101, was localized to approximately 1 kbp on the cloned sequence, and is apparently under the control of its own promoter. MLSR electroporants were derived from strain ES1 electroporated with plasmid DNA from strain 100-33; these MLSR isolates had acquired a plasmid complement similar to that of strain 100-33, including pLAR33. Endonuclease digestion and Southern analysis of plasmid DNA from both strains indicated that the major plasmids are multimeric and deleted forms of one archetypal extrachromosomal element.
Anti-Bacterial Agents↗
Characterization of plasmids from Listeria sp.
Out of 139 isolates of Listeria sp. (mainly L. monocytogenes) 107 (78%) contained extrachromosomal DNA. Plasmids from 51 of these isolates were investigated further and covered a range of 8 different-sized molecules as shown by agarose gel electrophoresis. Only one of the 107 isolates contained more than one plasmid. With the exception of one plasmid from an isolate of L. seeligeri, restriction analysis and hybridization experiments showed a high degree of homology between the different plasmids.
Animals↗
New shuttle vectors for Escherichia coli and Bacillus subtilis. I. Construction and characterization of plasmid pHY460 with twelve unique cloning sites.
We have constructed chimeric plasmid vectors, pHY460 and pHY310, from the streptococcal tetracycline resistance (TcR) plasmid pAM alpha 1 (9.2 kb) and the Escherichia coli vector pACYC177 (3.7 kb). These bifunctional plasmids can replicate and express the TcR gene in both E. coli and Bacillus subtilis. Plasmids pHY460 (7.0 kb) and pHY310 (4.8 kb) contain the TcR gene of pAM alpha 1 and the ampicillin resistance (ApR) gene of pACYC177. Both plasmids showed high transformation efficiency in both host cells. pHY460 was maintained stably in B. subtilis and, thus, is a useful shuttle vector functioning in E. coli and B. subtilis. The PvuI, PstI, BglI and BanI sites in the ApR gene and the HpaI, BalI and EcoRV sites in the TcR gene can be used for selection of recombinant plasmids by insertional inactivation. In addition, plasmid pHY460 has unique sites for SacII, BstEII, XbaI, AvaI and BamHI.
Bacillus subtilis↗
Construction and characterization of plasmid and lambda phage vector systems for study of transcriptional control in Escherichia coli.
We constructed a family of lambda phage and plasmid vectors which facilitate cloning and quantitative analysis of transcriptional regulator in both single and multiple copies. Their expression system was modified from the ara-trp-lac fusion operon of plasmid pMC81 [Casadaban and Cohen, J. Mol. Biol. 138 (1980) 179-207], which is designed to assay both promoters and terminators with a single vehicle. To eliminate transcriptional and translational polar effects liable to occur in the original fusion operon upon insertion of a foreign nucleotide sequence, intracistronic Rho-dependent terminators, that are present within the trpB gene and distal to the cloning site were deleted, and DNA spacers containing stop codons were introduced immediately before and after the cloning site. In analysis of the cloned trp regulatory region, the lambda phage system faithfully reproduced the tight regulation by tryptophan characteristic to the natural trp operon on the E. coli chromosome, whereas the plasmid counterpart exhibited a substantially relaxed response. Comparative studies on the relative strengths of various promoters and terminators have further demonstrated that the lambda phage vector system permits accurate assays of exceptionally strong promoters like Ptrp and lambda pL without disturbing the bacterial growth, while being sensitive enough for detecting low-level transcription under the control of weak promoters or potent terminators. Cloning with the lambda phage vector can be greatly facilitated by transferring the target regulatory site precloned with the plasmid onto the phage genome through in vivo recombination.
Bacteriophage lambda↗
Sequence analysis and characterization of plasmid pSFD10 from Salmonella choleraesuis.
The nucleotide sequence of a small plasmid, designated pSFD10, is isolated from the vaccine strain Salmonella choleraesuis C500 in China, has been determined. This plasmid is 4091 bp long with a total G+C content of 51.4%, which is in the range of Salmonella genomic DNA. Analysis of the complete nucleotide sequence reveals that pSFD10 has a high degree of similarity to ColE1-type plasmid, having the possible cer and rom genes, and a putative mobilization origin of ColE1-type. Plasmid pSFD10 possesses six main open reading frames (ORFs), five of which have a very high degree of amino acid identity to ColE1-type plasmid gene products involved in mobilization and copy number control. The other ORF (ORF6) encodes a putative protein, which has 49% homology to the invasion plasmid antigen J protein (IpaJ) secreted by the type III secretion apparatus of Shigella flexneri. In addition, pSFD10 belongs to a different incompatibility group than ColE1-type and pMB1-type to which it is related. Plasmid pSFD10 can be mobilized by the plasmid RP4 in E. coli.
China↗
Identification of the origin of replications and partial characterization of plasmid pRK100.
In search for the evolutionary origin of the conjugative F-like plasmid pRK100, the plasmid's functional replication regions were identified. Additionally targeted genetic analysis was used to investigate origins of other regions of the plasmid. Construction of minireplicons via ligation of Tn1725 with plasmid fragments and targeted cloning of putative replication regions, followed by sequence analysis indicated two functional replication regions, a F plasmid related RepFIB and a R1 plasmid related RepFIIA replication region. Partial nucleotide sequencing of regions of the plasmid revealed genes that encode a putative enterochelin iron uptake system previously associated with an Escherichia coli pathogenicity island, PAI III536, and the pColV-like aerobactin genes. In addition, a homologue of the R100 plasmid related rmoA gene was found that exhibits strong similarity to hha/ymoA encoding the Hha/YmoA class of modulators of gene expression. PCR and hybridization experiments further demonstrated that pRK100 harbors multiple IS2 and IS3 insertion sequences that may have facilitated in the acquisition of elements from other DNA molecules. These data together with the previous identification of a F-like tra region and a pColIa-like colicin Ia, indicate that pRK100 has a highly mosaic structure with elements derived from many different known large natural plasmids.
Base Sequence↗
Sequence analysis and characterization of plasmids from Streptococcus thermophilus.
The nucleotide sequences of eight plasmids isolated from seven Streptococcus thermophilus strains have been determined. Plasmids pSt04, pER1-1, and pJ34 are related and replicate via a rolling circle mechanism. Plasmid pJ34 encodes for a replication initiation protein (RepA) and a small polypeptide with unknown function. Plasmids pSt04 and pER1-1 carry in addition to repA genes coding for small heat shock proteins (sHsp). Expression of these proteins is induced at elevated temperatures or low pH and increases the thermo- and acid resistance. Plasmids pER1-2 and pSt22-2 show identical sequences with five putative open reading frames (ORFs). The gene products of ORF1 and ORF4 reveal some similarities to transposon encoded proteins of Bacillus subtilis and Tn916. ORF1 of plasmid pSt106 encodes a protein similar to resolvases of different Gram-positive bacteria. Integrity of ORF2 and 3, encoding a putative DNA primase and a replication protein, is essential for replication. ORF1 to 3 of plasmid pSt08, which are organized in a tricistronic operon, encode a RepA protein, an adenosine-specific methyltransferase, and a type II restriction endonuclease. Another type II restriction-modification (R/M) system is encoded on plasmid pSt0 which is highly similar to those encoded on lactococcal plasmid pHW393 and B. subtilis plasmid pXH13. Plasmid-free derivatives of strains St0 and St08 show increased phage sensitivity, indicating that in the wild-type strains the R/M systems are functionally expressed. Recombinant plasmids based on the replicons of plasmids pSt04, pJ34, pSt106, pSt08, and pSt0, are able to replicate in Lactococcus lactis and B. subtilis, respectively, whereas constructs carrying pER1-2 only replicate in S. thermophilus.
Bacillus subtilis↗
Detection of Escherichia coli O157:H7 by multiplex PCR and their characterization by plasmid profiling, antimicrobial resistance, RAPD and PFGE analyses.
Twenty-five and three strains of Escherichia coli O157:H7 were identified from 25 tenderloin beef and three chicken meat burger samples, respectively. The bacteria were recovered using the immunomagnetic separation procedure followed by selective plating on sorbitol MacConkey agar and were identified as E. coli serotype O157:H7 with three primer pairs that amplified fragments of the SLT-I, SLT-II and H7 genes in PCR assays. Susceptibility testing to 14 antibiotics showed that all were resistant to two or more antibiotics tested. Although all 28 strains contained plasmid, there was very little variation in the plasmid sizes observed. The most common plasmid of 60 MDa was detected in all strains. We used DNA fingerprinting by randomly amplified polymorphic DNA (RAPD) and pulsed-field gel electrophoresis (PFGE) to compare the 28 E. coli O157:H7 strains. At a similarity level of 90%, the results of PFGE after restriction with XbaI separated the E. coli O157:H7 strains into 28 single isolates, whereas RAPD using a single 10-mer oligonucleotides separated the E. coli O157:H7 strains into two clusters and 22 single isolates. These typing methods should aid in the epidemiological clarification of the E. coli O157:H7 in the study area.
Animals↗
Characterization of plasmids from Listeria monocytogenes and Listeria innocua strains isolated from short-ripened cheeses.
The plasmid content of 30 isolates of Listeria monocytogenes and 18 isolates of Listeria innocua obtained from short-ripened cheeses was analysed. The isolates of L. monocytogenes serogroup 1 harboured a single plasmid, pLM33 (33.2 kbp), whereas the serogroup 4 isolates did not contain plasmids. One group of L. innocua strains harboured the plasmid pLI71 (71 kbp) and another one contained two plasmids: pLI59 (59.5 kbp) and pLI56 (56.5 kbp). These plasmid groups were in accordance with clusters previously defined by pulsed-field gel electrophoresis analysis of the chromosomal DNA of Listeria isolates. Plasmids pLM33, pLI71 and pLI59 shared homology regions of at least 20 kbp. Plasmid pLI56 did not encode genes for any known character (such as carbohydrate fermentation, resistance to antibiotics, heavy metals or disinfectants, growth at low pH, NaCl tolerance or thermal inactivation by pasteurisation) and displayed different characteristics to the other three plasmids. It was also the only one cured from the parent strain and the sole plasmid not digested by the restriction enzyme PstI. In addition, its lack of homology with pLM33, pLI71 and pLI59 enhanced the possibility of a different origin for plasmid pLI56.
Blotting, Southern↗
Characterization of plasmid MIP233 (IncHI3) of the H complex.
Plasmid MIP233, the unique member of the IncHI3 subgroup of the H incompatibility complex, is a large self-transferable plasmid encoding for sucrose utilization, the PacB character and resistance to tellurite. The current study examined whether pMIP233 conferred other putatively advantageous plasmid-encoded functions. Heavy metal studies revealed pMIP233 phenotypes of resistance to cadmium, cobalt, lead, zinc and nickel. Sequencing of random clones from a pMIP233 library revealed extensive homology with IS elements and genes involved in the metabolic processes demonstrated. Interestingly, pMIP233 did not appear to code for resistance to a wide range of antibiotics. pMIP233 is considered to be a metabolic plasmid.
Bacteria↗
Characterization of plasmids conferring resistance to gentamicin and apramycin in strains of Salmonella typhimurium phage type 204c isolated in Britain.
In Salmonella typhimurium phage type 204c isolated in Britain, gentamicin resistance is specified by plasmids of the I1 compatibility group which also confer resistance to apramycin. These plasmids have been subdivided into three types within the I1 group on the basis of their antibiotic resistance specificity, their ability to produce colicin Ib and their restriction enzyme digest fragmentation patterns. All three have been identified in strains from cattle, but as yet only two types have been found in strains from humans. It is suggested that the use of apramycin in animal husbandry is responsible for the appearance of gentamicin resistance in multiresistant strains of phage type 204c, a phage type already epidemic in bovine animals and with an increasing incidence in humans.
Acetyltransferases↗
Partial characterization and plasmid linkage of a non-proteinaceous antimicrobial compound in a Lactobacillus casei strain of vegetable origin.
Lactobacillus casei IMPC LC34 of vegetable origin produces a non-proteinaceous inhibitory compound with a broad spectrum of activity towards Gram-positive and Gram-negative bacteria, including pathogens. The active substance, mainly produced in the stationary phase of growth, is insensitive to proteolytic enzymes, lipase and catalase, and is stable at 121 degrees C for 30 min. The inhibitory activity was detected either at 8 degrees C or at 37 degrees C. The active compound does not contain glucidic groups, is inactivated by Na-metaperiodate, and its molecular mass is between 2000 and 5000 Da. Plasmid curing experiments showed that both antimicrobial compound immunity and production determinants were encoded by an 8.8 kbp plasmid. The effectiveness of the active agent was verified on ready-to-use vegetables, using either the Lact. casei strain or its culture supernatant fluid as inoculant, compared with cured clone. The application potential of the Lact. casei strain or its culture supernatant fluid for assuring the microbiological safety of ready-to-use vegetables is discussed.
Anti-Bacterial Agents↗
Characterization of plasmids that encode streptomycin-resistance in bacterial epiphytes of apple.
Streptomycin resistance in strains of Pseudomonas syringae pv. papulans, Pantoea agglomerans and a yellow-pigmented, non-fluorescent Pseudomonas sp. (Py), isolated from apple orchards in New York and Washington states, is predominantly associated with strA-strB genes carried on conjugal plasmids (R plasmids). None of 128 resistant Erwinia amylovora strains from the eastern and western USA hybridized with a strA-strB probe, SMP3. Resistant Py strains transfered R plasmids to Ps. syringae pv. papulans and to Py in vitro at frequencies of 10(-1)-10(-2) per recipient cell whereas Ps. syringae pv. papulans transferred its plasmids at frequencies of 10(-2) to below detectable levels. Transfer of R plasmids to P. agglomerans was not detected and resistant P. agglomerans did not transfer their R plasmids to any recipients. R plasmids were found to be highly diverse as measured by DNA fingerprint analysis. Transfer-deficient transposon mutants of R plasmid pCPP519 were generated, and 3.9 kb EcoRI and 3.0 kb SmaI fragments that hybridized with a Tn5 probe were cloned and sequenced. The deduced amino acid sequences of the 3.9 kb fragment were similar to proteins involved in replication, nicking at oriT, and piliation in other bacteria.
Amino Acid Sequence↗