Detection of penicillinase and bacteriocinogenicity plasmids in Staphylococcus aureus strain No. 580]

Staphylococcus aureus No. 580 strain contains simultaneously two plasmids: bacteriocinogenicity plasmid and penicillinase plasmid. Both plasmids are lost spontaneously with a high frequency, and also under the effect of acridine orange at temperatures of 37 degrees C and 44 degrees C, and in cultivation at a temperature of 44 degrees C for 5 days. Loss of one of the plasmids failed to lead to stabilization of another plasmid, and it was eliminated spontaneously with the same frequency as in the population of the initial strain. Plasmid loss did not lead to the changes in biochemical and pathogenic properties and also of the phagovar and bacteriogenovar. At the same time in elimination of one or both plasmids lag-phase diminished from 220 to 120 min.     

Effects of some curing agents on phenotypic stability in Pseudomonas putida degrading ε-caprolactam

A strain of Pseudomonas putida MCM B-408 capable of utilizing ε-caprolactam (monomer of nylon-6) as the sole source of carbon and nitrogen was found to harbour a single 32-kb plasmid with the same electrophoretic mobility as that of pARI180, a reference plasmid. Acridine orange, ethidium bromide, mitomycin C and SDS failed to cure the plasmid and the phenotype. Elevated temperature alone (40°C) was found to be ineffective in curing. Phenotype, but not the plasmid, was cured at a frequency of 2.63% when acridine orange and elevated temperature (40°C) were used together. The studies therefore indicated that the phenotypic expression of caprolactam degradative genes is quite stable and that Pseudomonas putida MCM B-408 may degrade ε-caprolactam from waste-water satisfactorily without spontaneous loss of the property under adverse environmental conditions.     

Curing of symbiotic plasmid of Mesorhizobium huakuii subsp. rengei isolated from Astragalus sinicus

Astragalus sinicus (Chinese Milk vetch), a green manure leguminous plant, harbors Mesorhizobium huakuii subsp. rengei strain B3 in the root nodules. The visualization of symbiotic plasmid of strain B3 showed the presence of one sym plasmid of about 425 kbp. Curing of sym plasmid by temperature and acrydine orange was studied. Growing rhizobial cells at high temperature (37 degrees C) or treating the cells with acrydine orange at 50 mg/l eliminated sym plasmid of M. huakuii strain B3, which was confirmed by sym plasmid visualization and plant infection test of cured strains.     

Effects of some curing agents on phenotypic stability in Pseudomonas putida degrading ε-caprolactam

A strain of Pseudomonas putida MCM B-408 capable of utilizing -caprolactam (monomer of nylon-6) as the sole source of carbon and nitrogen was found to harbour a single 32-kb plasmid with the same electrophoretic mobility as that of pARI180, a reference plasmid. Acridine orange, ethidium bromide, mitomycin C and SDS failed to cure the plasmid and the phenotype. Elevated temperature alone (40°C) was found to be ineffective in curing. Phenotype, but not the plasmid, was cured at a frequency of 2.63% when acridine orange and elevated temperature (40°C) were used together. The studies therefore indicated that the phenotypic expression of caprolactam degradative genes is quite stable and that Pseudomonas putida MCM B-408 may degrade -caprolactam from waste-water satisfactorily without spontaneous loss of the property under adverse environmental conditions.     

Mechanism of conjugation and recombination in bacteria. XXI. Role of F factor genes in post-conjugational recombination in Escherichia coli K-12

Temperature sensitive dnaA recipient crossed at the restrictive temperature with HfrH, free from contaminating F+ cells, forms recombinants almost as proficiently as at the permissive temperature. The merozygotes are able to synthesize DNA at 42 degrees C, although the recipient and donor cells do not incorporate 3H-thymine. A substantial fraction of Lac+ recombinants, irrespective of the mating temperature, is temperature resistant (42 C-R); 15% from among those mated at 35 C and 30% from those mated at 42 C. The presence of dnaA mutation in these Lac+ 42 C-R recombinants was ascertained by co-transduction with ilv. Cell division at 42 C is inhibited in the Lac 42 C-R recombinants by acridine orange. The presence of F factor DNA in these recombinants was demonstrated directly by DNA: DNA hybridization. Suppression of dnaA mutation in Lac+ 42 degrees C-R recombinants and their sensitivity to acridine orange at 42 degrees C suggests that at least part of the F factor is integrated into the recombinant chromosome. A large fraction of the Lac+ 42 degrees C-R recombinants (up to 80%) is sensitive to male phage R17 and fertile. In crosses with HfrC there is a marked decrease of recombination frequency at 42 degrees C in the dnaA recipient. The fraction of Lac+ 42 degrees C-R recombinants is low (up to 10%) and the 42 degrees C-R recombinants are neither sensitive to male phage nor fertile. The results are discussed on the basis of the previously proposed model of post-conjugational recombination.     

Plasmid curing of Oenococcus oeni

Two strains of Oenococcus oeni, RS1 (which carries the plasmid pRS1) and RS2 (which carries the plasmids pRS2 and pRS3), were grown in the presence of different curing agents and at different temperatures. Sublethal temperature together with acriflavine generated all possible types of cured strains, i.e., lacking pRS1 (from strain RS1), and lacking pRS2, pRS3, or both (from strain RS2). Sublethal temperature together with acridine orange only generated cured strains lacking pRS3. These results suggest that acriflavine is a better curing agent than acridine orange for O. oeni, and that pRS3 is the most sensitive to these curing agents. We also observed spontaneous loss of pRS2 or both pRS2 and pRS3 by electroporation. The ability to cure O. oeni strains of plasmids provides a critical new tool for the genetic analysis and engineering of this commercially important bacterium.     

The elimination of urease activity in Streptococcus faecium as evidence for plasmid-coded urease.

A strain of Streptococcus faecium from the sheep rumen showed spontaneous loss of urease activity when subcultured at the normal rumen temperature of 38 degrees C, although in mixed cultures in vivo or in vitro loss of urease was not apparent. The rate of loss of urease in pure cultures was increased at incubation temperatures above 38 degrees C, but loss was never complete. However, at temperatures below 38 degrees C loss was greater, and at 22 or 18 degrees C the urease was completely eliminated. Incubation with sodium dodecyl sulphate (0-002%) or ethidium bromide (2-5 X 10(-5)M) caused complete loss of urease activity. The urease activity was also eliminated when the streptococcus was grown aerobically, and this loss of activity was irreversible. It is suggested that the urease activity is controlled by a plasmid gene and that aeration, low growth temperature and chemical agents 'cure' the streptococcus of the plasmid. Attempts to demonstrate the presence of covalently closed circular extrachromosomal DNA by caesium chloride-ethidium bromide equilibrium density-gradient centrifugation were unsuccessful.     

Mutation and identification of the F plasmid locus determining resistance to acridine orange curing

A region in plasmid F responsible for resistance to acridine orange curing has been identified. Insertion of the transposable element for ampicillin resistance Tn3 at the 45.8- or 46.35-kilobase coordinate in mini-F plasmids results in resistance to curing by acridine orange and also results in increased plasmid copy numbers. In contrast, copy number mutants of mini-F induced by chemical mutagenesis are sensitive to acridine-induced loss of plasmid. Hence, the Tn3-induced acridine orange resistance does not represent a suppression of sensitivity because of elevated plasmid copy numbers. General hypotheses to explain our results are presented.     

High-frequency induction of nodulation and nitrogen fixation mutants of Rhizobium japonicum.

More than 50 symbiotic mutants of Rhizobium japonicum were isolated by purported plasmid-curing techniques. Wild-type R. japonicum strains were grown in liquid culture at 28 or 36 degrees C in different concentrations of acridine orange, ethidium bromide, or sodium dodecyl sulfate for selection of mutants. The symbiotic traits of 133 isolates from nine treatment groups were determined. Forty-two isolates were Nod- Nif+, seven were Nod+ Nif-, and two were Nod- Nif-. The nifDH genes were deleted in three mutants and consequently showed no hybridization to a nifDH probe. None of these mutants showed any detectable loss of plasmid DNA.     

The long-term survival of Escherichia coli in river water

Escherichia coli introduced into autoclaved filtered river water survived for up to 260 d at temperatures from 4 degrees to 25 degrees C with no loss of viability. Survival times were less in water which was only filtered through either a Whatman filter paper or a 0.45 micron Millipore filter or in untreated water, suggesting that competition with the natural microbial flora of the water was the primary factor in the disappearance of the introduced bacteria. Survival was also dependent upon temperature with survival at 4 degrees C greater than 15 degrees C greater than 25 degrees C greater than 37 degrees C for any water sample. Direct counts showed that bacterial cells did not disappear as the viable count decreased. The possession of the antibiotic resistance plasmids, R1drd-19 or R144-3, did not enhance survival nor cause a faster rate of decay, indicating that the metabolic burden imposed by a plasmid was not a factor in survival under starvation conditions. There was no evidence of transfer of either plasmid at 15 degrees C or of loss of plasmid function during starvation.     

Studies on the role of polyamines associated with the ribosomes from Bacillus stearothermophilus

1. Spermine and spermidine were the main polyamines detectable in Bacillus stearothermophilus. 2. When grown at 65 degrees B. stearothermophilus contained lower concentrations of polyamines per mg. of RNA than when grown at 45 degrees or at 55 degrees . 3. Ribosomes isolated from B. stearothermophilus in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride contained sufficient polyamines to neutralize between 4% and 9% of their RNA phosphorus. 4. Removal of polyamines from the ribosomes by dialysis against m-potassium chloride did not appreciably alter the hypochromicity or thermal denaturation profiles of the ribosomes when measured in 0.01m-tris-hydrochloric acid buffer (pH7.4)-0.01m-magnesium chloride, though it did cause a loss of ribosome particles sedimenting at greater than 78s. 5. When ribosomes were dialysed against acridine orange solutions acridine orange bound to the ribosomes and did not displace spermine, but when a mixture of ribosomal RNA and spermine was dialysed against acridine orange the acridine orange displaced the spermine. It is concluded that polyamines in the ribosomes are less accessible for displacement by acridine orange than when polyamines are bound to ribosomal RNA.     

Evidence of a dormant but infective state of the fish pathogen Pasteurella piscicida in seawater and sediment.

The stability of Pasteurella piscicida strains in seawater and sediment microcosms at different temperatures (6 and 20 degrees C) was investigated during a 1-month period. Three strains of P. piscicida showed similar survival kinetics. By a standard plate count method they survived in water and sediment for only 6 to 12 days, depending on the strain and type of microcosm. During this starvation period, the metabolic activity of the cells was reduced by more than 80%. Culturable cells of each P. piscicida strain persisted better in sediment than in water, as well as at 20 degrees C compared to 6 degrees C. However, in all the microcosms, the acridine orange direct counts remained at about 10(5) cells per ml during the experimental period, which demonstrated that P. piscicida possesses a capacity to enter a viable but not culturable state. Moreover, dormant cells were always resuscitated by the addition of fresh medium to the microcosms, since we recovered numbers of culturable cells similar to the acridine orange direct counts. These resuscitated cells exhibited the same respiration rate as that seen prior to the start of the experiments. Although the biochemical, physiological, and serological characteristics; lipopolysaccharides; membrane proteins; and plasmid content of P. piscicida strains were unaffected during the starvation conditions, the dormant cells were smaller (dwarf cells) and had increased surface hydrophobicity. The starved cells maintained their infectivity and pathogenic potential for fish, with 50% lethal doses similar to those of the original strains.     

Cloning of genes affecting capsule expression in Staphylococcus aureus strain M

Staphylococcus aureus strain M produces large amounts of capsular polysaccharide. It produces a non-encapsulated variant at a frequency of 0.01% at 37 degrees C. At high temperature (43 degrees C), the frequency of capsule loss was shown to be 1-38%. A 19 kb plasmid and a prophage were found to be carried by the M strain, but curing of these elements did not affect capsular production. To clone the capsular (cap) genes, a plasmid library of S. aureus M was constructed directly in S. aureus RN4200. The library was then infected with phage 80 alpha. After transduction of the phage lysates to a Cap- mutant derived from M strain, a recombinant plasmid was obtained which complemented the mutant to a Cap+ phenotype. Chromosomal walking experiments were used to clone additional nearby cap genes. Complementation tests using a collection of Cap- mutants showed that most of the mutants were complemented by a 19.4 kb DNA fragment, suggesting that the majority of the cap genes affecting capsule production are clustered together.     

Lymphocyte chromatin changes in Down's disease detected by heat denaturation]

By using fluorescent microscopy and acridine orange staining it was shown in the studies on short-term culture of human cells that the melting patterns of chromatin DNA of intact lymphocytes of healthy individuals represented the curves with 6 maxima (F530) at the temperature ranges of 45 degrees C, 65 degrees C, 85 degrees C, and 92 degrees C (P less than 0.01). The melting patterns of lymphocytes from patients with Down's disease represented curves with 4 maxima at the temperature ranges of 65 degrees C, 85 degrees C, 88 degrees C, 92 degrees C (P less than 0.01). No decline in the fluorescence intensity at the temperature intervals of 78 degrees C-85 degrees C was apparently due to a greater degree of condensation of definite regions of the trisomal cell chromatin complex. Possible mechanisms accounting for structural readjustments of the interphasic human lymphocyte chromatin occurring under thermal effects are discussed.     

Regulation of toxin biosynthesis by plasmids in Vibrio cholerae

Vibrio cholerae strain 569B Inaba harbouring P plasmid produced less toxin than the parent strain. To examine the effect of plasmid loss on toxin production, temperature-sensitive (ts) mutants of P, unable to replicate at 42 degrees C, were isolated. One ts plasmid was unstable at 42 degrees C and its loss yielded a cured strain that resumed a normal level of toxin biosynthesis characteristic of the plasmid-free parent strain. Toxin production was again suppressed in the cured strain after reacquisition of P plasmid. This suggested a role for plasmid-borne genes in the regulation of toxin biosynthesis. A mutant of strain 569B Inaba that produced mutant toxin was isolated by transfer of P and V plasmids. The mutant toxin was similar to choleragenoid because it did not give rise to symptoms of cholera but induced antitoxin immunity in rabbits.     

Escherichia coli dnaK null mutants are inviable at high temperature.

DnaK, a major Escherichia coli heat shock protein, is homologous to major heat shock proteins (Hsp70s) of Drosophila melanogaster and humans. Null mutations of the dnaK gene, both insertions and a deletion, were constructed in vitro and substituted for dnaK+ in the E. coli genome by homologous recombination in a recB recC sbcB strain. Cells carrying these dnaK null mutations grew slowly at low temperatures (30 and 37 degrees C) and could not form colonies at a high temperature (42 degrees C); furthermore, they also formed long filaments at 42 degrees C. The shift of the mutants to a high temperature evidently resulted in a loss of cell viability rather than simply an inhibition of growth since cells that had been incubated at 42 degrees C for 2 h were no longer capable of forming colonies at 30 degrees C. The introduction of a plasmid carrying the dnaK+ gene into these mutants restored normal cell growth and cell division at 42 degrees C. These null mutants showed a high basal level of synthesis of heat shock proteins except for DnaK, which was completely absent. In addition, the synthesis of heat shock proteins after induction in these dnaK null mutants was prolonged compared with that in a dnaK+ strain. The well-characterized dnaK756 mutation causes similar phenotypes, suggesting that they are caused by a loss rather than an alteration of DnaK function. The filamentation observed when dnaK mutations were incubated at a high temperature was not suppressed by sulA or sulB mutations, which suppress SOS-induced filamentation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conjugal Transfer of Plasmid-Borne Multiple Antibiotic Resistance in Streptococcus faecalis var. zymogenes

A strain of Streptococcus faecalis var. zymogenes, designated JH1, had high-level resistance to the antibiotics streptomycin, kanamycin, neomycin, erythromycin, and tetracycline. These resistances were lost en bloc from approximately 0.1% of cells grown in nutrient broth at 45 C. The frequency of resistance loss was not increased by growth in the presence of the "curing" agents acriflavine or acridine orange, but after prolonged storage in nutrient agar 17% of cells became antibiotic sensitive. Covalently closed circular deoxyribonucleic acid (DNA) molecules were isolated from the parental strain and from antibiotic-sensitive segregants by using cesium chloride-ethidium bromide gradients. DNA molecular species were identified by using neutral sucrose gradients. Strain JH1 contained two covalently closed circular DNA species of molecular weights 50 x 10(6) and 38 x 10(6). An antibiotic-sensitive segregant, strain JH1-9, had lost the larger molecular species. A second sensitive segregant, strain JH1-5, had also lost the larger molecular species but a new molecular species of approximate molecular weight 6 x 10(6) was present. The antibiotic resistances that were curable from the parental strain were transferred to antibiotic-sensitive strains of S. faecalis and to strain JH1-9, during mixed incubation in nutrient broth at 37 C. Data to be described are interpreted to suggest that the transfer is by a conjugal mechanism. Analysis of the plasmid species in recipient clones showed that all had received the plasmid of molecular weight 50 x 10(6). Strain JH1-5 was not a good recipient. Analysis of one successful recipient clone of JH1-5 revealed that it had gained the 50 x 10(6) molecular weight plasmid but lost the 6 x 10(6) molecular weight species. These data are interpreted to mean that the multiple antibiotic resistance is borne by a transferable plasmid of 50 x 10(6) molecular weight, and that in clone JH1-5 this plasmid suffered a large deletion leaving only a 6 x 10(6) remnant which was incompatible with the complete replicon.     

Filamentation promotes F'lac loss in Escherichia coli K12.

The stability of plasmid F'lac in Escherichia coli strain SP45 (a temperature conditional mutant which grows as spherical cells at 42 degrees C and as a rod at 30 degrees C) was studied. F'lac elimination was demonstrated when bacteria exposed to subinhibitory concentrations of various chemicals were induced to form filaments. No plasmid loss was found when spherical cells were subjected to the same treatments. Plasmid loss was also observed in dnaA46 and lexA41 mutants when cell filamentation was induced at 42 degrees C, but not when they were cultured at 30 degrees C. Nalidixic acid promoted F'lac elimination at 0.25 micrograms ml-1 in a recA13 mutant and at 1.5 micrograms ml-1 in the recA+ counterpart. A marked difference was found in the rate of F'lac elimination from thermosensitive DNA gyrase mutants [gyrA43(Ts) and gyrB41(Ts)] between rods and their spherical (rodA51) derivatives growing at semipermissive temperature (36.5 degrees C). Plasmids carrying the ccd segment of F in DNA gyrase mutants were lost after 2.5 generations from rods and after 6 generation from spherical cells. Plasmid segregation into non-viable minicell-like elements was found after induction of filaments. These data suggest that plasmid stability is correlated with cell shape and that curing is more easily achieved when bacteria can elongate normally.     

Transfer of the drug-resistance transposon Tn5 toErwinia herbicola and the induction of insertion mutations

Translocatable drug-resistance element (transposon) Tn5 was transferred through conjugation from its carrier suicidal plasmid pJB4JI, harbored byEscherichia coli, toErwinia herbicola. The frequencies of transfer ranged from 0.4×10^–8 to 26×10^–8 per recipient cell. Membrane filter mating yielded more transconjugants than the spread plate technique. Several insertion mutations resulting in loss of bacteriocinogenicity were detected. The location of Tn5 in the mutant genomes was determined by Southern blot hybridization using Tn5-containing pRZ102 as the³²P-labelled probe. The resulting autoradiogram showed specific hybridization with a 96 megadalton plasmid in nine mutants ofErwinia herbicola out of ten tested. However, in one mutant, the 96 megadalton plasmid was missing; instead a larger plasmid containing Tn5 was apparent which was not present in the original strain. This plasmid may have arisen by dimerization of the 96 megadalton plasmid as a result of Tn5 insertion. Using this method, we show that the insertion of Tn5 in that plasmid may have caused the loss of bacteriocinogenicity. The potential usefulness of this technique in genetic analyses of otherErwinia species and other phytobacteria is discussed.     

洋葱伯克霍尔德菌T1828质粒消除方法及条件

采用十二烷基硫酸钠和提高生长温度结合法、紫外线涂布平板法、冻融法、吖啶橙法和黄芩甙提取液消除法等方法消除洋葱伯克霍尔德菌T1828菌株质粒,探讨适合洋葱伯克霍尔德菌质粒消除的方法,并研究最佳质粒消除条件。结果表明十二烷基硫酸钠和提高生长温度结合法最适合于洋葱伯克霍尔德菌T1828质粒消除,同时最佳消除条件为:在T1828培养16 h后加入SDS使其终浓度0.1%,36°C处理18 h,消除率达到49%。筛选到的无质粒突变株可以用于进一步的研究。