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PubMed1975

Effect of plasmid carriage on the virulence of staphylococcus aureus.

The possession of any of eight different plasmids by Staphylococcus aureus strain 649--either singly or simultaneously (in no. 649MR)--caused changes in growth kinetics. Six of the plasmids caused an increase in exponential doubling time (by 8-25%), and most also altered the duration of the lag period. Strain 649MR was significantly less virulent for 10-day chick embryos than the corresponding plasmid-negative culture (no. 649N). The avirulence persisted even after loss of the plasmids from no. 649MR. The presence of a single plasmid specifying tetracycline resistance produced a moderate reduction in virulence, but chromosomal tetracycline resistance had an insignificant effect on it. The decrease in virulence could not be attributed to reduced formation of soluble products. It probably resulted from alterations in the cell surface, but membrane-polypeptide profiles of virulent and avirulent cells lacking plasmids were similar. Survival of strains 649MR and 649N on glass was identical. Therefore, reduction in the incidence of staphylococcal sepsis may be due in part to loss of virulence that has resulted from plasmid carriage.

Animals↗
PubMed1975

Factors that may prevent transfer of anti-biotic resistance between gram-negative bacteria in the gut.

Antibiotic-resistance transfer between populations of donor and recipient strains of Escherichia coli was completely inhibited in broth by dense suspensions of Bacteroides fragilis. Comparable amounts of inert bacterial matter (for-molised suspensions of E. coli or B. fragilis), or smaller numbers of viable B. fragilis, Streptococcus faecalis, Staphylococcus albus, Neisseria catarrhalis, or solutions of sodium taurocholate or glycocholate were only moderately inhibitory. Anaeroboisis had no effect upon plasmid transfer. Population densities of enteric organisms in these studies were similar to those found in faeces. The presence of dense cultures of B. fragilis provide a satisfactory explanation for almost total inhibition of conjugation in the human gut. Other factors inhibiting conjugation to a lesser degree may reinforce the effect of B. fragilis in vivo. As well as selecting for resistant organisms, antibiotics may also indirectly increase populations of R factor-bearing organisms in the gut by interfering with the anaerobic flora and so permit an increase in the frequency of conjugation.

Anaerobiosis↗
PubMed1976

Genetic and molecular characterisation of resistance determinants in methicillin-resistant Staphylococcus-aureus.

A genetic analysis of resistance to antibiotics in methicillin-resistant Staphylococcus aureus was performed. Demonstration of plasmid-specific DNA either in transductants that had received antibiotic-resistance markers from multiply-resistant strains, or in segregants of methicillin-resistant strains that had lost unstable determinants except the one under study, indicated that markers of resistance to penicillin, chloramphenicol and neomycin are present on separate, mutually compatible plasmids. Absence of covalently closed circular DNA was demonstrated in transductants that were resistant to methicillin, tetracycline, erythromycin and streptomycin, as well as in segregants that had lost the penicillinase, chloramphenicol and neomycin plasmid, but were still resistant to methicillin, tetracycline, erythromycin, streptomycin and the sulphonamides. Analysis of plasmid DNA either in a 5-20% neutral sucrose gradient or by electron microscopy revealed the presence of three readily distinguishable plasmids. The molecular weights of these plasmids were estimated by comparing the sedimentation rate constants with those of known reference plasmids and by contour-length measurements. The molecular weight of the penicillinase plasmid was estimated to be 20 X 10(6) daltons, that of the chloramphenicol plasmid 3 X 10(6) daltons and that of the plasmid carrying the neomycin resistance marker 37 X 10(6) daltons.

Anti-Bacterial Agents↗
PubMed1976

A transmissible plasmid determining lactose fermentation and multiple antibiotic resistance in a strain of Klebsiella pneumoniae.

In a wild strain of Klebsiella pneumoniae the plasmid that determined lactose fermentation also determined resistance to chloramphenicol, ampicillin, tetracyclines, streptomycin, spectinomycin, and sulphonamides. The plasmid transferred at a very low rate to Escherichia coli K12 and Salmonella typhi. By implanting other transfer factors in the strain the rate of transfer and the recipient range were increased. Plasmid transfer from the modified strain to Salm. typhimurium and Salm. gallinarum was detected in the alimentary tract of experimentally infected chicks fed diets containing ampicillin.

Ampicillin↗
PubMed2005

Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.

DNA, Intergenic↗
PubMed2003

Transcription-dependent recombination and the role of fork collision in yeast rDNA.

It is speculated that the function of the replication fork barrier (RFB) site is to avoid collision between the 35S rDNA transcription machinery and the DNA replication fork, because the RFB site is located near the 3'-end of the gene and inhibits progression of the replication fork moving in the opposite direction to the transcription machinery. However, the collision has never been observed in a blockless (fob1) mutant with 150 copies of rDNA. The gene FOB1 was shown previously to be required for replication fork blocking activity at the RFB site, and also for the rDNA copy number variation through unequal sister-chromatid recombination. This study documents the detection of fork collision in an fob1 derivative with reduced rDNA copy number (approximately 20) using two-dimensional agarose gel electrophoresis. This suggests that most of these reduced copies are actively transcribed. The collision was dependent on the transcription by RNA polymerase I. In addition, the transcription stimulated rDNA copy number variation, and the production of the extrachromosomal rDNA circles (ERCs), whose accumulation is thought to be a cause of aging. These results suggest that such a transcription-dependent fork collision induces recombination, and may function as a general recombination trigger for multiplication of highly transcribed single-copy genes.

DNA Replication↗
PubMed2003

Extrachromosomal circular DNA of tandemly repeated genomic sequences in Drosophila.

One characteristic of genomic plasticity is the presence of extrachromosomal circular DNA (eccDNA). This DNA is found in various eukaryotes from yeast to humans, and its levels are elevated by exposure to carcinogens. eccDNA is heterogeneous in size and composed of chromosomal sequences. In this study we used two-dimensional gel electrophoresis to detect and characterize eccDNA in Drosophila. We found eccDNA throughout the fly's life cycle. These molecules comprise up to 10% of the total repetitive DNA content, and their size ranges from <1 kb to >20 kb. The eccDNA population contains circular multimers of tandemly repeated genes such as histones, rDNA, Stellate, and the Suppressor of Stellate. Multimers of centromeric heterochromatin sequences are included in eccDNA as well. Our findings are consistent with the hypothesis that intramolecular homologous recombination between direct tandem repeats is a favorite mechanism for eccDNA formation. The level of eccDNA increased following MMS treatment of wild-type larvae, consistent with phenomena observed in cultured mammalian cells. This shows mutagen-induced eccDNA formation in the context of the whole organism for the first time. Mutations in the genes okra, mus309, and mei41 did not affect eccDNA under normal conditions or following mutagen treatment, implying that eccDNA formation is different from known pathways of DNA repair.

Animals↗
PubMed1975

Plasmid DNA as a possible state of Epstein-Barr virus genomes in nonproductive cells.

It has been shown that the EBV DNA in Raji cells is located in chromosomes and that most of this EBV DNA can be separated from high molecular weight cell DNA both in alkaline and neutral glycerol gradients. The data indicate that the latent EBV DNA may exist possibly as a plasmid DNA. As 50 genomes of EBV DNA are equal to 0.1% of the total cell DNA, it may be quite conceivable that such an amount of DNA should remain in a non-integrated form. Chromosomal location may be a requisite for any DNA to persist in the nucleus of cells and to be divided into daughter cells evenly. As the present studies measured only the status of the major portion of EBV genomes in Raji cells, a possibility still remains that a few genomes of EBV may be linearly integrated into cell DNA and these may be responsible for cellular transformation. Another possibility that a small piece of cellular DNA might be attached to EBV DNA in Raji cells, which might serve as a control signal for replication and/or transcription of the latent virust DNA, has also not been ruled out. Even though EBV DNA is not integrated covalently into cell DNA, the association of the virus genomes with cells is very stable and the number of EBV genomes remains unchanged throughout many cell generations. In synchronized cells, all EBV genomes replicate simultaneously at an early S phase before maximum synthesis of cell DNA replication occurs (Hamper et al. 1974). This indicates that EBV DNA in a plasmid state is strictly under a cellular control mechanism. How this latent virus DNA is controlled in cells will be examined in future studies.

Cell Line↗