Transductional evidence for plasmid linkage of lactose metabolism in streptococcus lactis C2.

A lactose-negative (Lac-), proteinase-negative (Prt-) mutant, designated C145 was isolated from Streptococcus lactis C2 after treatment with nitrosoguanidine and ultraviolet irradiation. The mutant appeared to be cured of the prophage(s) present in S. lactis C2 based on non-inducibility by ultraviolet irradiation or mitomycin C. When cleared lysate material from C145 was subjected, to cesium chloride-ethidum bromide (EB) density gradient centrifugation, no plasmid peak was observed, suggesting that C145 was cured of plasmid deoxyribonucleic and (DNA). A histogram showing distribution of contour lengths of circular molecules of DNA from C145, however, revealed the presence of a greatly diminished number of DNA molecules as compared with the parent culture and indicated the absence of the 30 x 10(6) plasmid. Cesium chloride-ethidium bromide gradient profiles from Lac+, Prt- and Lac+ Prt+ transductants of C145 revealed no plasmid peak, but electron microscopy of the fractions normally possessing the satellite band of DNA showed the presence of a new plasmid species having a molecular weight from 20 x 10(6) to 22 x 10(6). This plasmid was lost when the transductants became Lac-. Examination of a plasmid histogram from a spontaneous Lac- Prt- mutants of S. lactis C2 resembled that of C145, with the absence of the 30 x 10(6) plasmid and the presence of the 22 x 10(6) plasmid in Lac+ Prt+ transductants. The results suggest that lactose metabolism is mediated through the 30 x 10(6) plasmid in S. lactis C2 and that the transducing bacteriophage, which is too small to accommodate the entire plasmid, is transferring about two-thirds of the original plasmid through a process termed transductional shortening.     

Plasmid deoxyribonucleic acid in Rhizobium trifolii.

In deoxyribonucleic acid of Rhizobium trifolii centrifuged in cesium chloride-ethidium bromide equilibrium was found a sattelite peak containing covalently closed circular deoxyribonucleic acid. The plasmid had a molecular weight of about 64 x 10(6) shown by sedimentation in sucrose gradients and electron microscopy.     

Plasmids in Streptococcus lactis: evidence that lactose metabolism and proteinase activity are plasmid linked.

Populations of lactose positive (Lac+) and proteinase positive (Prt+) cells from Streptococcus lactis M18, C10, and ML3 grown at 39 degrees C gave rise to increasing proportions of Lac- Prt- clones. The deficiencies did not appear until after a number of generations at the elevated temperature, and the rate depended on the strain.Lac- Prt+ and Lac+ Prt- mutants were isolated after treatment with ethidium bromide. Plasmid deoxyribonucleic acid was isolated by cesium chloride-ethidium bromide equilibrium density gradient centrifugation from the parent cultures as well as from their Lac- Prt-, Lac- Prt+, and Lac+ Prt- mutants. Five distinct plasmid sizes of approximate molecular weights of 2,4, 8, 21, and 27 million were found in S. lactis C10, whereas the Lac- Prt- derivative lacked the 8- and 21-million-dalton plasmids, but the 8-million-dalton plasmid was present in the Lac-Att mutant. In S. lactis m18 five plasmids possessing molecular weights of about 2, 4, 10, 18 and 27 million were observed. The 10- and 18-million-dalton plasmids were not detected in the Lac- Prt- mutants, whereas the Lac- Prt+ derivative lacked only the 18-million-dalton plasmid and the Lac+ Prt- mutant lacked only the 10-million-dalton plasmid. In S. lactis ML3 five distinct plasmids, with approximate molecular weights of 2, 4, 8, 22, and 30 million, were present. The 8- and 22-million-dalton plasmids were not detected in the Lac- Prt- derivative, but the 8-million-dalton plasmid was present in the Lac- Prt+ mutant. The evidence suggests that lactose-fermenting ability and proteinase activity in these organisms are mediated through two distinct plasmids having molecular weights of 8 x 10(6) to 10 x 10(6) for proteinase activity and 18 x 10(6) to 22 x 10(6) for lactose metabolism.     

Isolation and characterization of four plasmids from Bacillus subtilis.

Nineteen Bacillus subtilis isolates obtained from type culture collections were examined for the presence of covalently closed circular duplex deoxyribonucleic acid molecules by the technique of cesium chloride-ethidium bromide density gradient centrifugation. Four of the 19 strains tested carried covalently closed circular molecules. Two of these strains (IFO3022, IFO3215) harbored a similar plasmid with a molecular weight of 5.4 X 10(6). The other two strains (IAM1232, IAM1261) carried 4.9 C 10(6)-and 5.3 X 10(6)-dalton plasmids, respectively. These plasmid-harboring strains did not show phenotypic traits such as antibiotic resistance orbacteriocin production. The plasmid deoxyribonucleic acids were digested by three restriction endonucleases, EcoRI, HindIII, and BamNI, and were classified into three different types from their electrophoretic patterns in agarose gels.     

Loss of Lactose Metabolism in Lactic Streptococci

Lactose-negative mutants occurred spontaneously in broth cultures of Streptococcus lactis C(2)F. Instability of lactose metabolism was noted in other strains of S. lactis, in strains of S. cremoris, and in S. diacetilactis. Colonies of S. lactis C(2)F grown with lactose as the carbohydrate source also possessed lac(-) cells. Treatment of lactic streptococci with the mutagen acriflavine (AF) increased the number of non-lactose-fermenting variants. The effect of AF on growth and on loss of lactose-fermenting ability in S. lactis C(2)F was consequently further examined. The presence of AF appears to favor competitively the growth of spontaneously occurring lactose-negative cells and appears to act in the conversion of lactose-positive to non-lactose-fermenting cells. The lactose-negative mutants partially revert to lactose-positive variants which remain defective in lactose metabolism and remain unable to coagulate milk. The lactose-negative cells become dominant in continuous culture growth and provide evidence that alterations in the characteristics of starter strains can be produced by continuous culture, in this case, the complete loss in ability to ferment lactose.     

Isolation and partial characterization of four plasmids from antibiotic-resistant thermophilic bacilli.

Twenty-nine antibiotic-resistant isolates of thermophilic bacilli were examined for the presence of covalently closed circular duplex DNA molecules by agarose-gel electrophoresis and caesium chloride-ethidium bromide density gradient centrifugation. Five of the 29 strains tested contained covalently closed circular molecules. Two of the streptomycin-resistant strains contained the same two plasmids: pAB118A of molecular weight 4.9 X 10(6) (7.0 kilobases) and pAB118B of molecular weight 3.0 X 10(6) (4.3 kilobases). Two of the tetracycline-resistant strains each contained a plasmid (pAB124) of molecular weight 2.9 X 10(6) (4.14 kilobases), while a third harboured a small plasmid (pAB128) of molecular weight 2.5 X 10(6) (3.57 kilobases). These plasmids were digested with 19 different restriction endonucleases and the numbers of cleavage sites were determined. Transformation of Bacillus subtilis (168 (Trp-) with purified plasmid DNA indicated that pAB124 conferred tetracycline resistance on the host.     

Isolation of plasmid deoxyribonucleic acid from two strains of Bacteroides.

Two clinical isolates of Bacteroides contained covalently closed circular deoxyribonucleic acid (DNA) as shown by sedimentation in an alkaline sucrose gradient, CsCl ethidium bromide equilibrium centrifugation, and electron microscopy. Bacteriodes fragilis N1175 contained a homogeneous species of plasmid DNA with a molecular weight of 25 x 10(6). Bacteroides ochraceus 2228 contained two distinct, covalently closed circular DNA elements. The larger cosedimented with the covalently closed circular DNA form of the R plasmid, R100, corresponding to a molecular weight of 70 x 10(6); the smaller sedimented as a 58S molecule with a calculated molecular weight of 25 x 10(6). The roles of these plasmids are unknown. Neither strain transferred antibiotic resistance to plasmid-negative Bacteroides or Escherichia coli, and neither produced bacteriocins active against other Bacteroides or sensitive indicator strains of E. coli.     

Characterization of Plasmid Deoxyribonucleic Acid in Streptococcus lactis subsp. diacetylactis: Evidence for Plasmid-Linked Citrate Utilization

The use of Streptococcus diacetylactis as a flavor producer in dairy fermentations is dependent upon its ability to produce diacetyl from citrate. Treatment of S. diacetylactis strains 18-16 and DRC1 with acridine orange resulted in the conversion of approximately 2% of the DRC1 population and 20% of the 18-16 population to citrate negative, which is indicative of the involvement of plasmid deoxyribonucleic acid (DNA). Growth in the presence of acridine orange also resulted in the appearance of 2% lactose-negative derivatives in S. diacetylactis 18-16 and 99% lactose-defective, proteinase-negative derivatives in S. diacetylactis DRC1. Cesium chloride-ethidium bromide equilibrium density gradients of cleared lysate material from each strain revealed the presence of covalently closed circular DNA. Samples of this covalently closed circular DNA were subjected to agarose gel electrophoresis to determine the plasmid composition of each strain. S. diacetylactis 18-16 was found to possess six plasmids, of approximately 41, 28, 6.4, 5.5, 3.4, and 3.0 megadaltons (Mdal). S. diacetylactis DRC1 contained six plasmids, of approximately 41, 31, 18, 5.5, 4.5, and 3.7 Mdal. Variants of S. diacetylactis 18-16 which failed to produce acetoin plus diacetyl from citrate (citrate negative) were missing a 5.5-Mdal plasmid. Lactose-negative mutants of the same strain were devoid of a 41-Mdal plasmid. Lactose-defective, proteinase-negative mutants of S. diacetylactis DRC1 were missing a 31-Mdal plasmid. The citrate-negative mutants of S. diacetylactis DRC1 isolated in this study did not possess a 5.5-Mdal plasmid. Thus, we have evidence that there is a correlation between the ability to utilize citrate and the presence of a 5.5-Mdal plasmid. A relationship was also noted between lactose fermentation and proteinase activity and plasmid DNA in S. diacetylactis.     

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.     

R6K Plasmid Replication: Influence of Chromosomal Genotype in Minicell-Producing Strains of Escherichia coli K-12

Alkaline sucrose velocity sedimentation and cesium chloride-ethidium bromide equilibrium centrifugation have been used to determine the number of copies per chromosomal equivalent of the relaxedly replicating R6K plasmid (a conjugative plasmid conferring ampicillin and streptomycin resistance) in two minicell-producing strains of Escherichia coli K-12. In one strain, the average number of covalently closed circular R6K molecules per chromosomal equivalent is 13 in log-phase and 35 in stationary-phase cells. In the other strain, there is an average of six covalently closed circular R6K molecules per chromosomal equivalent in both log- and stationary-phase cells. Selection from this strain of spontaneously occurring mutants resistant to high concentrations of ampicillin has been accomplished and such mutants show a two- to threefold increase in the number of R6K copies per chromosomal equivalent. Relative to the parental strain, mutants display the following properties: (i) elevated streptomycin resistance, (ii) a 10-fold increase in R6K conjugal transfer, (iii) a 10-fold increase in the amount of R6K plasmid deoxyribonucleic acid segregated into minicells, and (iv) a two- to threefold increase in R6K-specified beta-lactamase. The mutation(s) responsible for the increase in the number of R6K molecules per chromosomal equivalent is located on the bacterial chromosome. No R6K-linked mutations conferring the above phenotypes have been obtained. The mutations are presumed to be in chromosomal genes which play a role in the regulation of R6K replication in this strain.     

Simultaneous Loss of Proteinase- and Lactose-Utilizing Enzyme Activities in Streptococcus lactis and Reversal of Loss by Transduction

During studies on spontaneous loss of lactose metabolism in Streptococcus lactis C2, it was found that the lactose-negative (lac(-)) mutants were also proteinase negative (prt(-)). This pleiotropic effect was observed in S. diacetilactis 18-16, but not in S. cremoris B1. The lac(-)prt(-) mutants from S. lactis C2 were able to grow in milk, but no pH change or measurable protein breakdown occurred. When the milk was supplemented with glucose, a slow decline in pH occurred. Addition of a protein hydrolysate to milk did not stimulate acid production. When both supplements were added to milk, normal growth and pH change were obtained. When the lac(-)prt(-) mutant of S. lactis C2 was transduced with the temperate phage from the lac(+)prt(+) parent culture, approximately equal numbers of lac(+)prt(-) and lac(+)prt(+) transductants were obtained. When the spontaneous lac(+)prt(-) strain of S. lactis C2 was converted to a lac(-)prt(-) derivative and transduced, similar results were obtained. The co-transduction of the lactose and proteinase markers suggest they are closely associated. The findings indicate that the transducing phage from S. lactis C2 can be used to examine the causes of instability in both the lactose and proteinase enzyme systems of this organism.     

Mechanisms of lactose utilization by lactic acid streptococci: enzymatic and genetic analyses

The apparent instability of beta-galactosidase in toluene-treated cells or cell-free extracts of lactic streptococci is explained by the fact that these organisms do not contain the expected enzyme. Instead, various strains of Streptococcus lactis, S. cremoris, and S. diacetilactis were shown to hydrolyze o-nitrophenyl-beta-d-galactoside-6-phosphate (ONPG-6-P), indicating the presence of a different enzyme. In addition, lactose metabolism in S. lactis C(2)F was found to involve enzyme I (EI), enzyme II (EII), factor III (FIII), and a heat-stable protein (HPr) of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system analogous to that of Staphylococcus aureus. Mutants of S. lactis C(2)F, defective in lactose metabolism, possessed the phenotype lac(-) gal(-). These strains were unable to accumulate (14)C-thiomethyl-beta-d-galactoside, to hydrolyze ONPG, or to utilize lactose when grown in lactose or galactose broth. In addition, these mutants contained EI and HPr, but lacked EII, FIII, and the ability to hydrolyze ONPG-6-P. This suggested that the defect was in the phosphorylation step. Lactose-negative mutants of S. lactis 7962, a strain containing beta-galactosidase, could be separated into several classes, which indicated that this organism is not dependent upon the PEP-phosphotransferase system for lactose metabolism.     

Lysogeny in Lactic Streptococci Producing and Not Producing Nisin

Eighty-seven strains of lactic streptococci (46 of Streptococcus lactis, 24 of S. diacetilactis, and 17 of S. cremoris) were tested for lysogeny; 12 S. lactis strains produced nisin. Lysogeny was found in five S. lactis strains (two of them were nisin producers) and in two S. diacetilactis strains. Four S. lactis and two S. diacetilactis lysogens liberated phages both spontaneously and after ultraviolet treatment, and one S. lactis strain liberated phages spontaneously only. No lysogens were found among the S. cremoris strains tested. An initial characterization of the lysogens and their phages was made. The lytic spectrum of some of the examined phages was very narrow (homospecific), whereas that of others was wide, including strains of the three investigated species.     

Replication of kinetoplast DNA of Crithidia acanthocephali. I. Density shift experiments using deuterium oxide

The protozoan Crithidia acanthocephali contains, within a modified region of a mitochondrion, a mass of DNA known as kinetoplast DNA (kDNA). This DNA consists mainly of an association of approximately 27,000 covalently closed 0.8-mum circular molecules which are apparently held together in a definite ordered manner by topological interlocking. After culturing of C. acanthocephali cells for 25 generations in medium containing 75% deuterium oxide, both nuclear DNA (rhonative, nondeuterated=1.717 g/cm3) and kDNA (rhonative, nondeuterated=1.702 g/cm3) increased in buoyant density by 0.012 g/cm3. The replication of the two DNAs was studied by cesium chloride buoyant density analysis of DNAs from exponentially growing cells taken at 1.0, 1.4, 2.0, 3.0, and 4.0 cell doublings after transfer of cells from D2O- containing medium into medium containing only normal water. The results obtained from analysis of both native and denatured nuclear DNAs indicate that this DNA replicates semiconservatively. From an analysis of intact associations of kDNA, it appears that this DNA doubles once per generation and that the newly synthesized DNA does not segregate from parental DNA. Fractions of covalently closed single circular molecules and of open circular and unit length linear molecules were obtained from associations of kDNA by sonication, sucrose sedimentation, and cesium chloride-ethidium bromide equilibrium gradient centrifugation. Buoyant density profiles obtained from these fractions indicate that: (a) doubling of the kDNA results from the replication of each circular molecule rather than from repeated replication of a small fraction of the circular molecules; (b) replication of kDNA is semiconservative rather than conservative, but there is recombination between the circles at an undefined time during the cell cycle.     

A simple method for the preparation of the covalently closed circular form of plasmid DNA

We describe a simple, rapid, inexpensive method for isolation of covalently closed circular plasmid DNA. The method involves the electrophoresis of crude DNA preparations in an agarose gel, electrotransfer onto a dialysis membrane and elution of the highly purified circular covalently closed plasmid DNA. Native and recombinant plasmid DNA have been purified by this method and shown to be suitable for restriction enzyme digestion and transformation of bacteria. The yield of this rapid purification procedure makes it a good alternative method to standard centrifugation in cesium chloride ethidium bromide gradients.     

Isolation of extrachromosomal deoxyribonucleic acids from extremely thermophilic bacteria

Eight strains of thermophilic bacteria were examined for the presence of covalently closed circular deoxyribonucleic acid molecules by caesium chloride-ethidium bromide density gradient centrifugation. Four of the eight strains tested, Thermus flavus BS1, AT61, AT62 and Thermus thermophilus HB8 carried covalently closed circular DNA molecules. Thermus flavus BS1 haboured two species of plasmids with molecular weights of 6.1 X 10(6) and 17.0 X 10(6) as determined by electron microscopy. Thermus thermophilus HB8, T. flavus AT61 and T. flavus AT62 carried plasmids with molecular weights of 6.2 X 10(6), 6.6 X 10(6) and 6.6 X 10(6), respectively. Plasmids from T. flavus AT61 and AT62 were indistinguishable in their electrophoretic patterns in agarose or acrylamide gel after digestion with various restriction endonucleases. This is the first evidence for the presence of plasmids in extremely thermophilic bacteria, though their functions are unknown.     

KINETOPLAST DEOXYRIBONUCLEIC ACID OF THE HEMOFLAGELLATE TRYPANOSOMA LEWISI

Cesium chloride centrifugation of DNA extracted from cells of blood strain Trypanosoma lewisi revealed a main band, ρ = 1.707, a light satellite, ρ = 1.699, and a heavy satellite, ρ = 1.721. Culture strain T. lewisi DNA comprised only a main band, ρ = 1.711, and a light satellite, ρ = 1.699. DNA isolated from DNase-treated kinetoplast fractions of both the blood and culture strains consisted of only the light satellite DNA. Electron microscope examination of rotary shadowed preparations of lysates revealed that DNA from kinetoplast fractions was mainly in the form of single 0.4 µ circular molecules and large masses of 0.4 µ interlocked circles with which longer, often noncircular molecules were associated. The 0.4 µ circular molecules were mainly in the covalently closed form: they showed a high degree of resistance to thermal denaturation which was lost following sonication; and they banded at a greater density than linear DNA in cesium chloride-ethidium bromide gradients. Interpretation of the large masses of DNA as comprising interlocked covalently closed 0.4 µ circles was supported by the findings that they banded with single circular molecules in cesium chloride-ethidium bromide gradients, and following breakage of some circles by mild sonication, they disappeared and were replaced by molecules made up of low numbers of apparently interlocked 0.4 µ circles. When culture strain cells were grown in the presence of either ethidium bromide or acriflavin, there was a loss of stainable kinetoplast DNA in cytological preparations. There was a parallel loss of light satellite and of circular molecules from DNA extracted from these cells.     

Isolation of covalently closed circular DNA of high molecular weight from bacteria.

A simple method is described in detail for the efficient isolation of high molecular weight covalently closed circular DNA (ccc-DNA) from Agrobacterium. Although this method was developed for isolating ccc-DNA of molecular weights greater than 10⁸ daltons in Agrobacterium, the technique also proves to be useful in isolating ccc-DNA of varying sizes from a variety of other bacteria. The technique involves the shearing and alkali denaturation of the chromosomal DNA, followed by the preferential removal of the single-stranded DNA by phenol extraction. The DNA which remains is largely ccc-DNA. The DNA is then concentrated, and the ccc-DNA is separated from the chromosomal DNA by centrifugation in a cesium chloride-ethidium bromide density gradient. By this technique, ccc-DNA of varying sizes has also been isolated from species of Escherichia, Rhizobium, Citrobacter, and Lactobacillus.     

Identification of lactose fermentation plasmids of streptococcal dairy starter strains by Southern hybridization

Twenty-two strains of Streptococcus cremoris , seven strains of Streptococcus lactis and three strains of Streptococcus lactis subsp. diacetilactis, each with a different plasmid complement, were isolated from a starter culture used in a Finnish dairy plant. By using DNA-DNA hybridization, with cloned 6-P-ß-galactosidase gene of the Strep, lactis plasmid pLP712 as a probe, the lactose fermentation genes were located, in each strain, in the large ( 30 MD) plasmid.     

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.