Comparisons of the transferability of plasmids pCAR1, pB10, R388, and NAH7 among Pseudomonas putida at different cell densities

The transferability of plasmids pCAR1, pB10, R388, and NAH7 was compared using the same donor-recipient system at different cell density combinations in liquid or on a solid surface. pCAR1 was efficiently transferred in liquid, whereas the other plasmids were preferentially transferred on a solid surface. Difference of liquid or solid affected the transfer frequency especially at lower cell densities.     

Evidence for a life span-prolonging effect of a linear plasmid in a longevity mutant of Podospora anserina

The linear mitochondrial plasmid pAL2-1 of the long-lived mutant AL2 of Podospora anserina was demonstrated to be able to integrate into the high molecular weight mitochondrial DNA (mtDNA). Hybridization analysis and densitometric evaluation of the mitochondrial genome isolated from cultures of different ages revealed that the mtDNA is highly stable during the whole life span of the mutant. In addition, and in sharp contrast to the situation in certain senescence-prone Neurospora strains, the mutated P. anserina mtDNA molecules containing integrated plasmid copies are not suppressive to wild-type genomes. As demonstrated by hybridization and polymerase chain reaction (PCR) analysis, the proportion of mtDNA molecules affected by the integration of pAL2-1 fluctuates between 10% and 50%. Comparative sequence analysis of free and integrated plasmid copies revealed four differences within the terminal inverted repeats (TIRs). These point mutations are not caused by the integration event since they occur subsequent to integration and at various ages. Interestingly, both repeats contain identical sequences indicating that the mechanism involved in the maintenance of perfect TIRs is active on both free and integrated plasmid copies. Finally, in reciprocal crosses between AL2 and the wild-type strain A, some abnormal progeny were obtained. One group of strains did not contain detectable amounts of plasmid pAL2-1, although the mtDNA was clearly of the type found in the long-lived mutant AL2. These strains exhibited a short-lived phenotype. In contrast, one strain was selected that was found to contain wild-type A-specific mitochondrial genomes and traces of pAL2-1. This strain was characterized by an increased life span. Altogether these data suggest that the linear plasmid pAL2-1 is involved in the expression of longevity in mutant AL2.     

Survey of plasmid profiles of <Emphasis Type="Italic">Shigella</Emphasis> species isolated in Malaysia during 1994–2000

Summary Plasmid profiling was used to characterize 219 strains of Shigellaspecies isolated from sporadic cases of shigellosis in Malaysia during the period 1994–2000. Heterogeneous plasmid patterns were observed in all Shigella spp. There was a correlation between plasmid patterns and serotypes of S. flexneri, S. dysenteriaeand S. sonnei. Five common small plasmids (>20.0 kb) were observed in S. flexneri1b and 2a, whereas six common small plasmids were found in serotype 3a. Some of these plasmids appeared to maintain their existence stably in each individual serotype. Plasmids of size 11.40 and 4.20 kb were present only in S. flexneri2a isolates, whereas the 4.40 kb plasmid was unique for serotype 3a. Large (>150 kb) or mid-range plasmid (20.0–150 kb) was not observed from any S. flexneri1b isolates. Eighty-nine percent of S. flexneriof various serotypes harboured the plasmid of 3.20 kb. All S. dysenteriaetype 2 isolates harboured the 9.00 kb plasmid, while four common small plasmids were found in S. sonneiisolates. The 2.10 kb plasmid was only seen in S. sonnei. Streptomycin resistance in S. dysenteriaetype 2 and multi-drug resistance in S. sonneimay be associated with the 9.00 and 14.8 kb plasmids, respectively. Plasmid profiling provided a further discrimination beyond serotyping and a useful alternative genotypic marker for differentiation of Shigellaspecies. To the best of our knowledge, this is the first report on the plasmid prevalence of the Malaysian Shigellaspecies.     

Transformation of plant protoplasts with DNA: cotransformation of non-selected calf thymus carrier DNA and meiotic segregation of transforming DNA sequences

Summary With the DNA transformation procedure developed in our laboratory (13) several transformed tobacco SR1 tissues were obtained which, apart from selected and non-selected pTi sequences (T⁺), also had acquired non-selected calf thymus carrier DNA sequences (C⁺), being integrated in their nuclear genomes. From one such tissue (cNT4), with a shooty crown gall phenotype and expressing mannopine synthesis activity (Mas⁺), shoots were grafted and mature, flowering plants (gNT4) were obtained. After cross pollination with wild type SR1 tobacco pollen 49% of the seedlings obtained, had the maternal NT4-like crown gall phenotype and 51% showed wild type (SR1) features. The mannopine locus segregated independently from the locus determining the crown gall phenotype. When screened for integrated (transforming) foreign DNA sequences 97% of the NT4-like seedlings turned out to be C⁺T⁺. Most of the SR1-like seedlings, having a wild type tobacco morphology, proved to be transformed as well: roughly a 1:1:1:1 ratio as found for C⁺T⁺:C^-T⁺: C⁺T:C T SR1-like seedlings. Based on the segregation of transforming sequences during meiosis a model is presented showing the integration of these sequences in three different host chromosomes.     

Host strain influences on supercoiled plasmid DNA production in Escherichia coli: Implications for efficient design of large-scale processes

We set out to investigate if E. coli genotype plays a significant role in host strain selection for optimal processing of plasmid DNA based on both quality and quantity of supercoiling. Firstly 17 E. coli commercial and non-commercial strains were selected and their available genetic backgrounds were researched in the open literature. Growth characteristics of all the strains were considered and made impartial by using a common medium and growth condition platform. By keeping the growth conditions constant for each strain/plasmid combination, we are only looking at one variable which is the host strain. The second step was to attempt to correlate the findings with common genotype characteristics (e.g. mutations such as endA or recA). We found that one can screen the number of strains which are likely to give good productivity early on, before any further optimisation and verification is performed, both for small and large plasmids. Also, it is worth noting that complex plasmid interactions with each strain prevent the use of genotype alone in making an intelligent choice for supercoiling optimisation. This leads to a third optimisation step selecting a few of the potentially high performing strains based on high DNA yield and supercoiling, with a view to identify the factors which need improvement in strain design and bioreactor optimisation. We found that high specific growth rates of some strains did not affect the level of DNA supercoiling but did influence the total plasmid yield, potentially an important aspect in the design of fermentation strategy. Interestingly, a few host/plasmid combinations result in what appears to be runaway plasmid replication.     

High-level resistance to gentamicin: genetic transfer between Enterococcus faecalis isolated from food of animal origin and human microbiota

Aims: To investigate the in vivo gene transfer of high‐level gentamicin resistance (HLRG) from Enterococcus faecalis isolated from the food of animal origin to a human isolate, using a mouse model of intestinally colonized human microbiota. Methods and Results: In vitro study: The presence of plasmids involved in HLRG coding was investigated. After the conjugation experiment, the recipient strain, Ent. faecalis JH2‐SS, acquired a plasmid responsible for HLRG [minimal inhibitory concentration (MIC) >800 μg ml^?1], in a similar position to the donor cells. In vivo study: Seven BALB/c mice were dosed with ceftriaxone (400 mg kg^?1) and then inoculated with a dilution of 1/100 of human faeces (HFc). After 72 h, Ent. faecalis JH2‐SS (recipient) was inoculated and then, after a further 72 h, the animals were given Ent. faecalis CS19, isolated from the food of animal origin, involved in HLRG (donor). The presence of transconjugant strains in HFc was subsequently recorded on a daily basis until the end of the experiment. The clonal relationship between Ent. faecalis and Escherichia coli in faeces was assessed by RAPD‐PCR. Both the in vitro and in vivo studies showed that the receptor strain acquired a plasmid responsible for HLRG (MICs >800 μg ml^?1), which migrated with a similar relative mobility value. Transconjugant strains were detected from 24 h after the donor strain inoculation and persisted until the end of the experiment. Conclusions: The in vivo gene transfer of HLRG from Ent. faecalis strains, isolated from the food of animal origin, to human microbiota has been demonstrated in a mouse model. Significance and Impact of the Study: The complexity found on the therapeutic responses of invasive infectious diseases caused by Ent. faecalis facilitates the assessment of food of animal origin as a resistant pathogen reservoir. In addition, this study may contribute to the understanding of antimicrobials’ resistance gene transfer between Ent. faecalis strains from food and human GI tract.     

Involvement of the virulence gene products of Yersinia enterocolitica in the immune response of infected mice

The virulence of Yersinia enterocolitica is known to be highly dependent on its virulence plasmid. However, it remains unclear whether the virulence plasmid is engaged also in the induction of cell-mediated immunity that is essential for protective immunity in the host. In this study, we have compared the induction of type 1 helper T cell immunity against Y. enterocolitica using a virulent strain (P+) harboring the pYV plasmid and an avirulent strain (P-) harboring no pYV. Spleen cells from both groups of mice immunized with 1/10 LD50 of P+ strain and those with 1/10 LD50 of P- strain produced a high level of gamma interferon (IFN-gamma) upon stimulation with heat-killed bacteria, and CD4+ T cells were exclusively responsible for IFN-gamma production. When crude Yersinia outer proteins (Yops) were used for antigenic stimulation, IFN-gamma response of immune spleen cells against crude Yops was observed only in mice immunized with P+ strain. Flowcytometric analysis revealed a significant level of increase in IFN-gamma-producing CD8+ T cells as well as the increase in IFN-gamma-producing CD4+ T cells against crude Yops. These results suggest that the virulence plasmid of Y. enterocolitica is involved in the induction of Th1-type of possibly protective T cells in infected mice.     

The N-terminal domain of the replication initiator protein RepE is a dimerization domain forming a stable dimer

The initiator protein RepE of the mini-F plasmid in Escherichia coli plays an essential role in DNA replication, which is regulated by the molecular chaperone-dependent oligomeric state (monomer or dimer). Crosslinking, ultracentrifugation, and gel filtration analyses showed that the solely expressed N-terminal domain (residues 1-144 or 1-152) exists in the dimeric state as in the wild-type RepE protein. This result indicates that the N-terminal domain functions as a dimerization domain of RepE and might be important for the interaction with the molecular chaperones. The N-terminal domain dimer has been crystallized in order to obtain structural insight into the regulation of the monomer/dimer conversion of RepE.     

Electrotransfer of human IL-1Ra into skeletal muscles reduces the incidence of murine collagen-induced arthritis

BACKGROUND: It has previously been demonstrated that high levels of gene expression in skeletal muscles can be achieved after direct in vivo electrotransfer of naked plasmid DNA. The purpose of this study is to examine the potential of in vivo electroporation of plasmid DNA encoding human IL-1Ra for the prevention of murine collagen-induced arthritis (CIA). METHODS: DBA/1 mice were injected in gastrocnemius muscles with plasmid DNA followed by in vivo electroporation. To uncover the optimum conditions of gene transfer, various electric field strengths and different amounts of plasmid DNA were applied. Calf muscles around the injected areas were investigated with histological methods for damage to muscle tissue. The levels of human IL-1Ra expression in the injected area and also in the serum were determined with ELISA for human IL-1Ra. Based on these data, the effects of electrotransfer of plasmid DNA were tested using the murine CIA model. DBA/1 mice were immunized with bovine collagen type II at the base of the tail. On day 21, mice were given a booster injection with the same antigen. Mice were divided into two groups on day 26. One group of mice received plasmid containing the IL-1Ra cDNA sequence, while control mice were given plasmid lacking the IL-1Ra coding sequence. The incidence of arthritis was evaluated by macroscopic analysis, histological analysis, and the levels of inflammatory cytokines. RESULTS: IL-1Ra expression increased as a function of the electrical field strength and the amount of DNA. 200 V/cm (eight pulses; 20 ms per pulse; 1 Hz) and 15 microg of plasmid DNA per mouse were found to be optimum for gene transfer. After in vivo electroporation, gene expression in both muscle and serum increased gradually, reaching a peak value on day 10. Significant levels of human IL-1Ra expression were maintained for 20 days. Macroscopic analysis showed that the onset of CIA was significantly inhibited by direct electrotransfer of plasmid DNA encoding human IL-1Ra. Histological analysis of knee joints showed that the incidence of arthritis in knee joints was also prevented. The levels of mouse IL-1beta and IL-12 in paws were significantly lower in the group treated with IL-1Ra than those in the control group. CONCLUSIONS: These results demonstrate that direct electrotransfer of plasmid containing the human IL-1Ra cDNA sequence to skeletal muscle can reduce the incidence of CIA in mice.