Characterization of Lactobacillus sp. strain 100-37 from the murine gastrointestinal tract: ecology, plasmid content, and antagonistic activity toward Clostridium ramosum H1

A gram-positive, nonsporulating, microaerophilic rod that had two colonial variants was obtained during a study in which anaerobic bacteria were isolated from murine gastrointestinal tracts and screened for cryptic plasmids. The rod (both colonial variants) was identified as a Lactobacillus sp. (strain 100-37) by selective media, gas chromatography, and biochemical tests. In monoassociated, ex-germfree mice, the bacterium colonized the gastrointestinal tract and formed a thick, continuous layer on the keratinized squamous epithelium of the nonsecreting portion of the stomach. When lysate preparations of both colonial variants were electrophoresed in agarose gels, two bands which stained with ethidium bromide were detected with each lysate. When the DNA preparations were exposed to UV light, the lower ethidium bromide band gradually disappeared while the top band became either broader or more intense. The approximate size of the lower band was 2.2 megadaltons, as determined by comparison with plasmid molecular weight standards. In a search for phenotypes which could be encoded by the cryptic 2.2-megadalton plasmid, we detected an antagonistic activity toward an obligate anaerobe isolated from mouse feces, Clostridium ramosum H1. The antagonistic factor was precipitated with (NH4)2SO4 (70% saturation) from supernatant solutions of broth cultures of strain 100-37. The factor was not inducible with mitomycin C or UV light, but was stable in flowing steam for up to 50 min, and in buffers of pHs over a range of 1.6 to 6.8. It was nondialyzable and inactivated by trypsin and papain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Copy number determination of different derivatives of the streptomycete mini-plasmid pSLG33

Copy numbers of the streptomycete plasmid vector pRS410 and five other recombinant plasmid derivatives of the original cryptic streptomycete plasmid pSLG33 were determined using calibrated laser densitometry. DNA preparations, electrophoretically separated on agarose gels, were stained with ethidium bromide, photographed and the negatives were subsequently scanned in a laser densitometer. The pSLG33 replicon is very stable, as no effect of the selective pressure was observed. It is a multicopy plasmid with up to 220 detected copies per chromosome. The use of deletion and/or insertion mutants allowed us to define two regions of the pSLG33 molecule involved in the control of plasmid replication.     

A novel phage genome integrated into a plasmid in Bacillus thuringiensis strain AF101

Bacillus thuringiensis strain AF101 possesses a single plasmid (pAF101) with a molecular size of 42 MDa (69 kb). During plasmid curing experiments in strain AF101, we found that a phage (J7W-1) was induced by ethidium bromide treatment. Moreover, the phage genome (48 kb) hybridized only with pAF101 on a Southern blot of the DNA of a cleared lysate prepared from strain AF101. Comparison of the restriction patterns of pAF101 and J7W-1 phage DNA revealed that pAF101 contains not only the entire phage DNA but also a plasmid-specific DNA region. These results indicate that the J7W-1 genome has been stably integrated into pAF101 in strain AF101. Integration of the J7W-1 genome into a plasmid was also observed after phage infection of the type strain of B. thuringiensis subsp. israelensis.     

Interaction of 2-chloro-N10-substituted phenoxazine with DNA and effect on DNA melting

Five N10-substituted phenoxazines having different R groups and -Cl substitution at C-2 were found to bind to calf -thymus DNA and plasmid DNA with high affinity as seen from by UV and CD spectroscopy. The effect of phenoxazines on DNA were studied using DNA-ethidium bromide complexes. Upon addition of phenoxazines, the ethidium bromide dissociated from the complex with DNA. The binding of phenoxazines to plasmid PUC18 reduced ethidium bromide binding as seen from the agarose gel electrophoresis. Butyl, and propyl substituted phenoxazines were able to release more ethidium bromide compared with that of acetyl substitution. Addition of phenoxazines also enhanced melting temperature of DNA.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. II. Membrane properties

Aspects of membrane stucture and functions were studied in ethidium bromide resistant cells. Submitochondrial particles were solubilized and electrophoresed. The gel patterns, representing mitochondiral membrane proteins, demonstrated qualitative and quantitative alterations in mitochondrial preparations derived from virus-transformed cells and ethidium bromide resistant cells as compared to the control cells. The plasma membrane glycoproteins were labelled by the sodium borohydride method. The glycoporteins were released with Triton X-100 and electrophoresed. Fluorograms of the gels demonstratred some marked differences between the ethidium bromide resistant cells and their parental strain. The observed alterations in the membrane glycoproteins did not result in altered glucose transport properties or in the elution patterns of plasma membrane glycopeptides as analyzed by Sephadex G-50 chromatography. Dye uptake and binding studies with intact parental and drug resistant cells and their isolated mitochondria demonstrated no alteration of the membrane permeability or the number of binding sites for ethidium bromide. Similar results were also obtained with a cyanine dye. This latter finding was significant in that it permitted one to exclude dye exclusion as a mechanism for ethidium bromide resistance.     

Interaction of 2‐Chloro‐N10‐Substituted Phenoxazine with DNA and Effect on DNA Melting

Five N¹⁰‐substituted phenoxazines having different R groups and –Cl substitution at C‐2 were found to bind to calf –thymus DNA and plasmid DNA with high affinity as seen from by UV and CD spectroscopy. The effect of phenoxazines on DNA were studied using DNA‐ethidium bromide complexes. Upon addition of phenoxazines, the ethidium bromide dissociated from the complex with DNA. The binding of phenoxazines to plasmid _PUC18 reduced ethidium bromide binding as seen from the agarose gel electrophoresis. Butyl, and propyl substituted phenoxazines were able to release more ethidium bromide compared with that of acetyl substitution. Addition of phenoxazines also enhanced melting temperature of DNA.     

Plasmid Profiles of Lactose-Negative and Proteinase-Deficient Mutants of Streptococcus lactis C10, ML(3), and M18

Lactose- and proteinase-negative (Lac Prt) mutants of Streptococcus lactis C10, ML3, and M18 were isolated after treatment with ethidium bromide. The Lac Prt mutants of C10 were missing a 40-megadalton plasmid. A 33-megadalton plasmid was absent in the ML3 mutants, and the M18 variants lacked a 45-megadalton plasmid. The results suggest a linkage of these metabolic traits to the respective plasmids. The possible complexity of the interrelationship between lactose metabolism and proteinase activity is presented.     

Translocation of the chloramphenicol-resistance determinant in Staphylococcus aureus

pC658: a plasmid determining resistance to chloramphenicol in the hospital strain of Staphylococcus aureus JM658 was transduced after irradiation of phage lysate with high doses of UV. The localization of determinants causing resistance to chloramphenicol in the obtained transductants was investigated by modified Arber's method and variants resistant to chloramphenicol, but suspected of absence of chloramphenicolase plasmid were selected. Additionally the absence of plasmid DNA was demonstrated in the selected strains. The possibility of plasmid and chromosomal localization of the same gene indicates its translocable nature. The obtained results suggests transposomal character of the genes determining resistance to chloramphenicol in the pC658 plasmid, occurring in the hospital strain of S. aureus.     

Method for obtaining more-accurate covalently closed circular plasmid-to-chromosome ratios from bacterial lysates by dye-buoyant density centrifugation.

A method that gives high recovery of deoxyribonucleic acid (DNA) from crude bacterial lysates using ethidium bromide-cesium chloride density gradient centrifugation is presented. After Pronase digestion and shearing of the lysate, essentially 100% recovery of chromosomal DNA and a reproducible recovery of covalently closed circular (CCC) plasmid DNA is obtained for a specific plasmid in a given strain. This method should be useful for comparing the CCC plasmid/chromosome ratio of various plasmid-host combinations.     

Evidence for a chromosomal location of the genes coding for chloramphenicol production in Streptomyces venezuelae.

Of seven chloramphenicol-producing actinomycetes examined, only Streptomyces venezuelae strain 13s contained extrachromosomal DNA detectable by agarose gel electrophoresis and cesium chloride-ethidium bromide density gradient centrifugation. The single 17-megadalton plasmid present in this strain was indistinguishable from plasmid pUC3 previously isolated from mutagenized cultures. Strains selected for their inability to produce chloramphenicol after treatment with acriflavine or ethidium bromide still contained a plasmid that had the same electrophoretic mobility as plasmid pUC3 and yielded similar fragments when digested with restriction endonucleases. By regenerating protoplasts of strain 13s and screening for isolates lacking extrachromosomal DNA, strain PC51-5 was obtained. The absence of plasmid pUC3 sequences in this strain was confirmed by Southern hybridization using 32P-labeled plasmid as a probe. Since the plasmidless strain produced as much chloramphenicol as did the parent strain, pUC3 contains neither structural nor regulatory genes for antibiotic production. Evidence from electrophoretic analysis of BamHI digests of total cellular DNA from wild-type and dye-treated nonproducing progeny indicated that acriflavine caused structural changes in the chromosome.     

Rapid micropreps and minipreps of Ti plasmids and binary vectors fromAgrobacterium tumefaciens

A simple and versatile procedure has been developed for the isolation of both large helper/Ti plasmids and binary vectors fromAgrobacterium tumefaciens. Using a slightly modified alkaline lysis protocol, intact plasmid can be recovered from cultures grown in standard micro-centrifuge tubes or culture tubes in sufficient yield and purity to allow for restriction analysis on ethidium bromide stained gels of the >200 kb Ti plasmid DNA. Contamination by chromosomal DNA is minimal and there is thus no need for isopycnic gradient purification. This same procedure can be combined with a high temperature treatment (37°C) and antibiotic selection to generate preparations containing binary vector DNA that are virtually free of interfering Ti plasmid DNA. Restriction patterns produced from these binary vector DNA preparations are unambiguous and therefore preliminary screening by Southern hybridization can be eliminated.     

Evidence for plasmid-associated lactose metabolism inLactobacillus casei subsp.casei

Lac variants ofLactobacillus casei subsp.casei DR1002 (formerly 64H) have been produced using acriflavin, ethidium bromide, mitomycin C, or combinations of these agents. Two successive transfers in the presence of acriflavin and mitomycin C or ethidium bromide and mitomycin C resulted in nearly a 100% loss of lactose fermentation. Cesium chloride-ethidium bromide isopycnic gradient ultracentrifugal analysis of purified lysates demonstrated that the 23-mdal plasmid (pDR101) found inL. casei DR1002 was consistently absent in Lac⁻ clones. We concluded that, as in lactic streptococci, lactose metablism is a plasmid-mediated train inL. casei DR1002.     

Isolation of extrachromosomal deoxyribonucleic acid for exfoliative toxin production from phage group II Staphylococcus aureus.

The ability of phage group II staphylococcal strain UT 0101 to produce exfoliative toxin and bacteriocin could be eliminated at a high frequency after growth at high temperatures or in the presence of ethidium bromide or sodium dodecyl sulfate. Extrachromosomal deoxyribonucleic acid, associated with the genes for exfoliative toxin and bacteriocin production, was isolated from strain UT 0101 but was absent from an ethidium bromide-cured substrain. The molecular weight of the exfoliative toxin plasmid, determined by co-sedimentation with the penicillinase plasmid, PI258, was 3.3 times 10-7. The 56S covalently closed circular form of the exfoliative toxin plasmid converted to a 38S open circular form after storage or exposure to sodium dodecyl sulfate. Plasmid deoxyribonucleic acid associated with penicillin resistance could not be identified in the penicillin-resistance Tox+ strains, UT 0007 and UT 0001.     

Ethidium Bromide Resistance, a New Marker on the Staphylococcal Penicillinase Plasmid

A strain of Staphylococcus aureus resistant to ethidium bromide is described. The genetic determinants for the resistance are present on the same plasmid as the penicillinase genes.     

Rapid isolation of plasmid DNA by LiCl-ethidium bromide treatment and gel filtration.

We established a simple and rapid plasmid DNA purification method. Crude plasmid DNA preparations are treated with 4 M LiCl in the presence of 0.6 mg/ml ethidium bromide to precipitate RNA and proteins contained in the DNA preparations. After removal of RNA and protein precipitates, the supernatant is filtered through a Sepharose CL6B column to remove low-molecular-weight contaminants. This procedure takes only 30 min and provides pure plasmid DNA preparations that consist mainly of covalently closed circular plasmid DNA but have no detectable RNA and protein. The purified DNA preparations are susceptible to various six- and four-base-recognition restriction endonucleases, T4 DNA ligase, the Klenow fragment of DNA polymerase I, and T7 and Taq DNA polymerase. Since no special equipment is needed for this purification method, 20 or more samples of microgram to milligram levels can be treated in parallel.     

Plasmid chromosome isolation: An improved batch procedure for large plasmids

A one-step, batch fractionation procedure for the isolation of Escherichia coli plasmids with molecular weights up to at least 1.3 × 10⁸ has been devised. A 15-ml lysate of 5 × 10¹⁰ bacteria is gently prepared and underlain with 15 ml of CsCl-ethidium bromide solution in a 30-ml centrifuge bottle. During an 18- to 24-h 100,000g centrifugation in an anglehead rotor, an aggregate of dense cellular debris including almost all of the host DNA pellets. Plasmid chromosomes are banded in the supernatant isopycnic gradient and are detected by fluorescence of their intercalated ethidium bromide. Plasmid recoveries of 40–70% are achieved. The preparations are pure enough to be used for plasmid circumference measurements by electron microscopy.     

Densitometric quantification of DNA topoisomers in ethidium bromide-stained agarose gels and chemiluminescence-detected X ray films

This study investigates the contribution of deformational strain imposed by topological interconversions of DNA in ethidium bromide-binding on agarose gels. Closed-circular plasmid DNAs were nicked using UV exposure and the DNA bands were quantified by densitometry. The results show that the closed circular DNA binds the same amount of the dye as its nicked counterpart. The relationship between the band intensity on X-ray films of chemiluminescence-detected Southern blots and DNA concentration was shown to be linear.     

Involvement of plasmid in degradation of pentachlorophenol by Pseudomonas sp. from a chemostat

Pseudomonas sp. strain IST103 obtained from a stable bacterial consortium was capable of utilizing pentachlorophenol (PCP) as sole carbon and energy source. The consortium was developed by continuous enrichment in a chemostat. The degradation of PCP by bacterial strain proceeded through an oxidative route as indicated by accumulation of tetrachloro-p-hydroquinone and chlorohydroquinone determined by high performance liquid chromatography (HPLC), and chloride molecules released in culture medium. Two different molecular size plasmids, of approximately 80 and 4 kilobase, were found to be responsible for carrying genes for degradation of PCP. This was evidenced by mutants produced by curing of plasmid by treatment of ethidium bromide. The derivatives were not able to utilize PCP, however, transformation of low molecular size plasmid of Pseudomonas sp. strain 103 into E. coli JM109 utilized PCP, indicated a possible involvement of plasmid in degradation of pentachlorophenol.