Complete sequence of Enterococcus faecium pVEF3 and the detection of an omega-epsilon-zeta toxin-antitoxin module and an ABC transporter.

Glycopeptide resistant Enterococcus faecium (GREF) persists on Norwegian poultry farms despite the ban on the growth promoter avoparcin. The biological basis for long-term persistence of avoparcin resistance is not fully understood. This study presents the complete DNA sequence of the E. faecium R-plasmid pVEF3 and functional studies of some plasmid-encoded traits (a toxin-antitoxin (TA) system and an ABC transporter) that may be of importance for plasmid persistence. The pVEF3 (63.1 kbp), isolated from an E. faecium strain of poultry origin sampled in Norway in 1999, has 71 coding sequences including the vanA avoparcin/vancomycin resistance encoding gene cluster. pVEF3 encodes the TA system omega-epsilon-zeta, and plasmid stability tests and transcription analysis show that omega-epsilon-zeta is functional in Enterococcus faecalis OGIX, although with decreasing effect over time. The predicted ABC transporter was not found to confer reduced susceptibility to any of the 28 substances tested. The TA system identified in the pVEF-type plasmids may contribute to vanA plasmid persistence on Norwegian poultry farms. However, size and compositional heterogeneity among E. faecium vanA plasmids suggest that additional plasmid maintenance systems in combination with host specific factors and frequent horizontal gene transfer and rearrangement causes the observed plasmid composition and distribution patterns.

Environment factors can influence mitochondrial inheritance in the fungus Cryptococcus neoformans.

Cryptococcus neoformans is a model basidiomycete yeast. Strains of this species belong to one of two mating types: mating type a (MATa) or mating type alpha (MATalpha). In typical crosses between MATa and MATalpha strains, the progeny inherit mitochondria from the MATa parent. However, the underlying mechanisms remain largely unknown. To help elucidate the molecular mechanisms, we examined the effects of four environmental factors on the patterns of mtDNA inheritance. These factors are temperature, UV irradiation, and the addition of either the methylation inhibitor 5-aza-2'-deoxycytidine (5-adc) or the ubiquitination inhibitor ammonium chloride. Except temperature, the other three factors have been shown to influence organelle inheritance during sexual mating in other eukaryotes. Our results indicate that while the application of 5-adc or ammonium chloride did not influence mtDNA inheritance in C. neoformans, both UV irradiation and high temperature treatments did. Progeny from a cross involving a high temperature-sensitive mutant with the calcineurin subunit A gene deleted showed biparental mtDNA inheritance in all examined temperatures, consistent with a role of calcineurin and temperature in mtDNA inheritance. Furthermore, the zygote progeny population from a cross performed at a high-temperature environment had a greater variability in their vegetative fitness than that from the same cross conducted at a low temperature. Our results indicate a potentially adaptive role of biparental mtDNA inheritance and mtDNA recombination in certain environments in C. neoformans.

Damage to mitochondrial DNA induced by the quinolone Bay y 3118 in embryonic turkey liver.

Quinolones are a class of antibiotics that induce damage to and loss of DNA from bacteria. The structural organization of bacterial DNA is more similar to eukaryotic mitochondrial DNA (mtDNA) than to eukaryotic chromosomal or nuclear DNA (nDNA). Antibiotics affecting the bacterial genome may therefore preferentially damage mtDNA rather than nDNA. We investigated the effect of a quinolone on mtDNA in avian embryonic hepatocytes in ovo. The quinolone Bay y 3118 (1-cyclopropyl-7-(2,8-diazabicyclo[4.3.0]non-8-yl) 6-fluoro-8-chloro-1,4-dihydro-4-oxo-3-quinolinecarboxylic acid hydrochloride, chemical structure see Bremm et al. [K.D. Bremm, U. Petersen, K.G. Metzger, R. Endermann, In vitro evaluation of Bay-y 3118, a new full-spectrum fluoroquinolone, Chemotherapy 38 (1992) 376-387] was injected into fertilized turkey eggs 8 days before hatching at doses of 1, 3, 10 and 30 mg per egg. The embryos were removed from the eggs after 4 days and liver samples were shock frozen. Mitochondrial DNA was purified from samples of the embryonic liver. The integrity of mtDNA was investigated by electrophoresis on agarose gels with native mtDNA and with ribonuclease-treated mtDNA. Fluorescent staining of the electrophoresis gels allows the densitometric quantification of the mtDNA of the regular band at 16 kilobases (kb) and the amount of DNA fragments of irregular size (smear). The genotoxic nitrosamine nitrosodiethylamine (NDEA) has previously been shown to reduce the content of mtDNA of the regular size of 16 kb and to induce the occurrence of smaller fragments of mtDNA [H. Enzmann, C. Kühlem, E. Löser, P. Bannasch, Damage to mitochondrial DNA induced by the hepatocarcinogen, diethylnitrosamine in ovo, Mutation Res. 329 (1995) 113-120]. After exposure to 10 and 30 mg Bay y 3118, a dose-dependent induction of damage to the mtDNA was found, whereas exposure to 3 and 1 mg showed no effect. NDEA (25 mg) was used as positive control. Testing chemical compounds in the in ovo model is a simple and rapid approach for investigations on chemically induced alterations of mtDNA.

[The effect of sodium butyrate and luminol on reciprocal exchanges and gene conversion during extrachromosomal DNA recombination in cultured animal cells]. / Issledovanie vliianiia butirata natriia i liuminola na retsiproknye obmeny i gennuiu konversiiu pri ékstrakhromosomnoi rekombinatsii DNK v kul'tiviruemykh kletkakh zhivotnykh.

For determination of the extrachromosomal homologous DNA recombination efficiency, somatic cells of various lines have been transformed with plasmid DNAs which contain copies of neo-gene with non-overlapping deletions. Reconstruction of the neo-gene functional activity, which imparts a geneticin-resistant phenotype to cells, indicates that recombination has occurred. If dP1 and dR copies of the neo-gene are used, a single (reciprocal) exchange is necessary for reconstruction of the neo-gene by homologous DNA recombination, but a double exchange (gene conversion) is needed in the case of dP1 and dS copies. It is shown that in human cells of line HeLa and in mouse cells of line LMtk-, in contrast to the Chinese hamster cells of line A238, the frequency of double exchanges is comparable to that of the single DNA exchanges which is an evidence of participation in DNA recombination of gene conversion in addition to a single exchange mechanism. The treatment of cells with sodium butyrate and luminol exerts different influences on the rate of the single DNA exchanges and on that of gene conversion (double exchanges) in cells of lines LMtk- and HeLa, respectively. Essential distinctions in correlation of the single DNA exchange frequency and the gene conversion frequency in cells of the studied lines, and the possibility to distinguish between these mechanisms of recombination, under the treatment by sodium butyrate and luminol, may suggest the existence of two mechanisms of homologous DNA recombination in cultured animal cells, which function independently of one another, to a considerable extent.

[The participation of endonuclease in the formation of extrachromosomal DNA and the possible mechanisms of the occurrence of gene amplification]. / Uchastie éndonukleazy v orbazovanii ékstrakhromosomnoi DNK i vozmozhnye mekhanizmy vozniknoveniia amplifikatsii genov.

We examined the extrachromosomal DNA (exDNA, Hirt fraction) in ethidium bromide sensitive and resistant cells of line L929. The exDNA amount is greater in the latter. The amount of exDNA in L929 cells makes 0.19% of the total cellular DNA; the exDNA amounts in cells, resistant to 5 and 50 micrograms/ml ethidium bromide are 0.22 and 0.33%, resp. Using labelling by BudR, it is shown that approximately 16% exDNA in L cells constituted amplified sequences to be excreting to the culture medium. The Zn-independent endogenous nuclease is activated in the resistant cells. The treatment with cycloheximide (50 micrograms/ml) resulted in the increase in the exDNA amount and in the activation of Zn-independent endonuclease. The data obtained suggested that the activation of Zn-independent endonuclease may lead to the increase in the exDNA amount and determine presumably a high rate of cell adaptability to environmental conditions.

Elimination of extrachromosomally amplified MYC genes from human tumor cells reduces their tumorigenicity.

Oncogene amplification has been observed in a broad spectrum of human tumors and has been associated with a poor prognosis for patients with several different types of malignancies. Importantly, at biopsy, the amplified genes localize to acentric extrachromosomal elements such as double-minute chromosomes (DMs) in the vast majority of cases. We show here that treatment of several human tumor cell lines with low concentrations of hydroxyurea accelerates the loss of their extrachromosomally amplified oncogenes. The decreases in MYC copy number in a human tumor cell line correlated with a dramatic reduction in cloning efficiency in soft agar and tumorigenicity in nude mice. No effect on gene copy number or tumorigenicity was observed for a closely related cell line containing the same number of chromosomally amplified MYC genes. One step involved in the accelerated loss of extrachromosomal elements is shown to involve their preferential entrapment of DMs within micronuclei. The data suggest that agents that accelerate the loss of extrachromosomally amplified genes could provide valuable tools for moderating the growth of a large number of human neoplasms.

Analysis of antibiotic susceptibility and extrachromosomal DNA content of Ruminococcus albus and Ruminococcus flavefaciens.

Seventeen Ruminococcus albus and Ruminococcus flavefaciens strains have been screened for naturally occurring antibiotic resistance, as determined by zones of inhibition from antibiotic disks. These strains were also examined for extrachromosomal DNA content. All strains screened are resistant to low levels (10-200 micrograms/mL) of streptomycin. In contrast to the previously reported data, we have found that R. flavefaciens C-94 is now susceptible to both kanamycin and tetracycline. However, R. flavefaciens FD-1 is not susceptible to kanamycin (minimum inhibitory concentration (MIC) = 40 micrograms/mL). Furthermore, R. albus 8 is resistant to tetracycline (MIC = 40 micrograms/mL), and erythromycin (MIC = 100 micrograms/mL). Six freshly isolated strains showed resistance to tetracycline (35-70 micrograms/mL), and all tetracycline-resistant strains also showed resistance to minocycline. None of these Ruminococcus determinants share homology with the streptococcal tetL, tetM, or tetN determinants. All 17 strains were screened for extrachromosomal DNA content. Nine different techniques for the detection and isolation of extrachromosomal DNA were tested. However, owing to difficulties in demonstrating or isolating plasmid DNA, it has not been possible to determine if these antibiotic resistance genes are plasmid borne. Evidence is presented to suggest that the presence of oxygen may affect the quality of the DNA obtained from Ruminococcus.

[Toxic action of the polyene antibiotic levorin on the superproducer Streptomyces levoris]. / Tokischeskoe deistvie polienovogo antibiotika levorina na superprodutsent Streptomyces levoris.

Along with an increase in the activity of a superproductive strain of Str. levoris there was observed in its population an increase in the number of low active cells sensitive to high concentrations of the own antibiotic. Levorin had an especially high toxic action on viability and antibiotic activity of the low productive strain 78/19 as compared to the highly productive strain 78/72. Resistance of the levorin-producing organism to its own antibiotic was associated with composition and properties of the cytoplasmic membrane. Acridine orange had no effect on survival and morphological variation of the strains studied. It had either no effect on the antibiotic production by the low active strain 78/19. However, under the effect of the dye there was induced marked variation in the highly productive strain 78/72 with respect to the antibiotic production property: decreasing of the population activity due to elimination of highly productive variants and accumulation of low productive variants. The data suggested the presence of plasmid genes in the levorin-producing organism determining biosynthesis of the antibiotic and resistance to it.

[Role of multiple drug resistance in the biology of pathogenic and opportunistic bacteria]. / Rol' mnozhestvennoi lekarstvennoi ustoichivosti v biologii patogennykh i uslovno-patogennykh bakterii.

The significance of cloning of pathogenic and opportunistic multiple drug-resistant bacteria is analysed from the present viewpoints of medicine and veterinary. Distribution of drug-resistant strains in men, animals and environment and occurrence of new type hospital and nosocomial infections are described. The controversial points of the antibiotics role in the formation of multiple drug resistance plasmids, occurrence of hospital resistant strains and the part of R plasmids in changing the biological characteristics of pathogenic and opportunistic bacteria are analysed. It is shown in relation to Salmonella that formation of definite clones of multiple drug resistant bacteria results in significant changing of epidemiology of the diseases caused by these organisms. The data on the identity of the biological and genetic characteristics of the Salmonella strains of some phage vars isolated in hospitals and outside the hospitals, as well as the data on the effect of R plasmids on the Salmonella virulence are presented. The thesis of a higher infective capacity of the strains with multiple drug resistance associated with their increased survival levels and a higher colonization ability is accentuated. The danger of occurrence of new type causative agents on formation of complex plasmids with additional determinants connected with the bacteria pathogenicity is discussed. The tasks of the further investigation of these aspects are formulated and the possible means for the control of this phenomenon are presented.

Mutagenesis of extrachromosomal genetic determinants for exfoliative toxin B and bacteriocin R1 synthesis in Staphylococcus aureus after plasmid transfer by protoplast fusion.

In previous studies, we have shown that a 27-megadalton plasmid (pRW002) in Staphylococcus aureus contains genetic determinants for exfoliative toxin B (ET B) and bacteriocin (Bac R1) synthesis and Bac R1 resistance. Attempts to transform or transduce this plasmid to S. aureus or Bacillus subtilis recipients were not successful. However, genetic transfer of the plasmid was possible after polyethylene glycol-induced fusion of S. aureus protoplasts containing pRW002 and S. aureus protoplasts lacking this plasmid. Some of the resulting fusants lost the ability to make ET B, Bac R1, or both products. Fusants that were Bac R1-, Bac R1s, ET B- all lacked the 27-megadalton pRW002 plasmid. The largest class of fusants was Bac R1+, Bac R1r, ET B-. Immunodiffusion analyses of ET B extracts from 28 fusants showed that four ET B+ strains were cross-reacting mutants that produced ET B protein that was serologically related to, but not identical to, the wild-type toxin. Results indicated that genetic transfer of pRW002 after protoplast fusion induced molecular rearrangements that resulted in mutation of the genetic determinants for ET B and Bac R1 synthesis. Recombination of chromosomal genes was enhanced after CaCl2 was added to the protoplast-fusion mixture.

5-bromodeoxyuridine-induced amplification of prolactin gene in GH cells is an extrachromosomal event.

Treatment of a 5-bromodeoxyuridine-resistant (brdUrdr) and prolactin-nonproducing (Prl-) subclone of GH cells with this drug led to amplification of the prolactin (Prl) gene and induced Prl synthesis. Withdrawal of the drug treatment reversed both of these processes. In normal rats, the increased Prl synthesis observed during late pregnancy and lactation does not seem to be mediated via amplification of the gene. Amplification of the Prl gene and induction of Prl synthesis can also be observed in the Prl-, brdUrd-sensitive (brdUrds) GH cell strain. Prl gene amplification thus does not seem to be associated with the mechanism that confers the brdUrdr phenotype to these cells. brdUrd-induced amplification of the Prl gene can be identified with the low molecular weight, extrachromosomal, supernatant DNA fraction, isolated by Hirt's method. Southern blot analysis of Hirt's supernatant DNA (undigested) from brdUrd-treated cells generated a distinct band following hybridization with [32P]pDNAPrl-insert. The size of this band is greater than 23 kb but smaller than chromosomal DNA. Growth hormone (Gh) and albumin (Alb) gene sequences can be detected in the chromosomal DNA preparation but are absent in the extrachromosomal DNA prepared from Hirt's supernatant. The levels of Gh and Alb sequences are unaffected by brdUrd treatment of these cells. Results presented here suggest that in rat pituitary glands as well as in GH cells, hormonally controlled increased Prl synthesis is not caused by gene amplification. However, the brdUrd-induced expression of the Prl gene seems to be linked to the mechanism of drug-induced amplification of the Prl gene, mediated via an extrachromosomal event.

Dimethyl sulfoxide affects the amount of extrachromosomal spleen focus-forming virus DNA in murine erythroleukemia cells.

We used Southern blot hybridization to titrate and map restriction enzyme cleavage sites of a 6.3-kilobase-pair species of extrachromosomal viral DNA found in derivatives of the 745A line of murine erythroleukemia cells, which vary in their ability to be induced to differentiate by dimethyl sulfoxide (DMSO). Greater than an eightfold variation was observed in the amount of this DNA, with the largest amounts being found in cells that were resistant to the induction of differentiation by DMSO. This increase in the level of extrachromosomal viral DNA was found to be dependent upon the continued presence of DMSO in the culture medium. The increase was shown not to be due to an immediate stimulatory effect of this agent on the synthesis or maintenance of this DNA, since cell lines sensitive to the differentiation-inducing effects of DMSO were shown to undergo a transient reduction in the amount of extrachromosomal viral DNA after the addition of DMSO to the culture medium. In addition to the 6.3-kilobase-pair linear form found in the cytoplasm, in some preparations two hybridizing bands were observed that migrated in agarose gels in the position expected of covalently closed circular species of viral DNA. Restriction enzyme mapping of the cytoplasmic linear form indicated a close relationship of this DNA to two polycythemic strains of spleen focus-forming virus that have been molecularly cloned by other workers. No obvious change in the number or arrangement of chromosomal viral sequences could be detected after treating cells with DMSO. Thus, the exposure of murine erythroleukemia cells to DMSO caused an obvious change in the amount of extrachromosomal spleen focus-forming virus DNA but no obvious change in the integration of the provirus.

Extent of BrdUrd-induced prolactin gene amplification in GH cells.

The thymidine analog, 5-bromodeoxyuridine (BrdUrd), induces prolactin (Prl) synthesis and Prl gene amplification in a subclone of GH cells (rat pituitary tumor cells in culture). Withdrawal of drug treatment reverses both processes. Our previous results show that Prl gene amplification is associated with an event involving extrachromosomal DNA. The results presented in this report show that a 20-kb length of DNA, including all the coding sequences of the Prl gene, is amplified following BrdUrd treatment of this strain of GH cells. Amplification of DNA sequences extends about 3 kb upstream from the 5' end and about 7 kb downstream from the 3' end of the Prl gene. The DNA sequences immediately adjacent to these 5'-and 3'-flanking regions of the Prl gene are not affected by BrdUrd treatment. These results suggest that BrdUrd-induced amplification of Prl gene coding and neighboring sequences is restricted to within 3 kb of the 5' end and within 7 kb of the 3' end of the structural gene Analysis of low-molecular-weight extrachromosomal DNA isolated by Hirt's procedure from drug-treated cells reveals a similar pattern of amplification of the Prl gene and its flanking sequences.

[Episome stability in Escherichia coli K-12]. / Izuchenie stabil'nosti épisom v shtammakh Escherichia coli K-12.

Arg+ and trp+ carrying episomes which have markedly different spontaneous efficiency of elimination, were obtained in experiments with AB673 and KL99 Escherichia coli donor strains. The level of episome maintenance is the result of mutation (deletion, insertion) that occurred in episomes during their formation. Differences in the efficiency of spontaneous elimination are independent of the origin of the donor strain used. The rate of loss of stably maintained episomes is not enhanced significantly by exposure of cells to AO. According to molecular characteristics, the episome demonstrating stable maintenance has a lower molecular mass than that of unstable maintenance.

Relationship of amplified dihydrofolate reductase genes to double minute chromosomes in unstably resistant mouse fibroblast cell lines.

Murine 3T6 selected in increasing concentrations of methotrexate were unstable with respect to dihydrofolate reductase overproduction and methotrexate resistance when they are cultured in the absence of methotrexate. An analysis of the karyotypes of these resistant cells revealed the presence of numerous double minute chromosomes. We observed essentially identical kinetics of loss of dihydrofolate reductase gene sequences in total deoxyribonucleic acid and in deoxyribonucleic acid from fractions enriched in double minute chromosomes and in the numbers of double minute chromosomes per cell during reversion to methotrexate sensitivity, and this suggested that unstably amplified gene sequences were localized on double minute chromosomes. This conclusion ws also supported by an analysis of cell populations sorted according to dihydrofolate reductase enzyme contents, in which relative gene amplification and double minute chromosome content were related proportionally.

Loss and stabilization of amplified dihydrofolate reductase genes in mouse sarcoma S-180 cell lines.

We studied the loss and stabilization of dihydrofolate reductase genes in clones of a methotrexate-resistant murine S-180 cell line. These cells contained multiple copies of the dihydrofolate reductase gene which were associated with double minute chromosomes. The growth rate of these cells in the absence of methotrexate was inversely related to the degree of gene amplification (number of double minute chromosomes). Cells could both gain and lose genes as a result of an unequal distribution of double minute chromosomes into daughter cells at mitosis. The loss of amplified dihydrofolate reductase genes during growth in the absence of methotrexate resulted from the continual generation of cells containing lower numbers of double minute chromosomes. Because of the growth advantage of these cells, they became dominant in the population. We also studied an unstably resistant S-180 cell line (clone) that, after 3 years of continuous growth in methotrexate, generated cells containing stably amplified dihydrofolate reductase genes. These genes were present on one or more chromosomes, and they were retained in a stable state.

[Action of acridine dyes on antibiotic, pigment and aerial mycelium formation in streptomycete producers of multicomponent antibiotics]. / Deistvie akridinovykh krasitelei na obrazovanie antibiotika, pigmenta i vozdushnogo mitseliia u streptomitsetov--prudutsentov mnogokomponentnykh antibiotikov.

The ability of 5 streptomycetous species synthesizing multicomponent antibiotic to produce the antibiotic and water-soluble pigment and to form the aerial mycelium in the presence of acridine dyes was studied. It was found that the character of the produced complex changed, when acridine dyes were added to the medium under conditions not affecting the culture growth and the temperature was elevated. Colonies deficient with respect to formation of the aerial mycelium and with changed pigment and antibiotic production were detected in the monospore cultures of the streptomycetes treated with acridine dyes, when the spore survival was equal to 100 percent, the frequency of the colonies being about 40 per cent.

Elimination of mitochondrial elements and improved viability in hybrid cells.

Experiments were carried out to determine whether the mitochondria-specific dye rhodamine-6G (R6G) can affect transmission of cytoplasmic determinants in mammalian cells. When one parental cell type was treated with R6G prior to fusion with an untreated partner, the subsequent hybridization frequencies in both intra- and interspecific crosses were not adversely affected, even though R6G was extremely toxic to the parental cells. In addition, cells lethally treated with R6G could be rescued by fusion with cytoplasm alone from untreated cells. When chloramphenicol (CAP) resistant cells were used as the R6G-treated parent, the expression of CAP resistance in hybrids and cybrids was greatly reduced. Thus R6G can be used to control the input of cytoplasmic determinants into fused cells. In the interspecific (Chinese hamster x mouse) crosses, it was also seen that the majority of hybrids which had not been R6G pretreated grew poorly or degenerated after a short time. In contrast, nearly all hybrids in crosses where the hamster parent was R6G pretreated grew vigorously. The concomitant elimination of inviability and loss of mitochondrial determinants in R6G-pretreated hybrids suggests that interactions involving mitochondrial gene products or components can influence growth characteristics in interspecific somatic cell hybrids.