Characterization of a plasmid mutation affecting maintenance, transfer and elimination by novobiocin

Summary We have isolated a mutant H group plasmid temperature-sensitive for plasmid maintenance. Unlike the wild type plasmid (pSD114), the mutant (pDT4) was eliminated at 37° C and also at 30° C after novobiocin treatment. The mutant plasmid interfered with host cell growth at the non-permissive temperature. Conjugative transfer of the mutant was reduced at 30° C compared to the wild-type plasmid. Introduction of a coumermycin-novobiocin resistance DNA gyrase (cou) mutation into Escherichia coli prevented pDT4 elimination by novobiocin but did not affect the temperature-sensitive phenotype. The evidence indicates that the mutant plasmid used bacterial DNA gyrase for replication. Models to account for the behaviour of this unusual mutant are discussed.     

A new runaway type episomal vector for mammalian cells based on a temperature-sensitive simian virus 40 and inducible erythropoietin production

A runaway vector for mammalian cells was constructed from the simian virus 40 (SV40) genome with a temperature-sensitive mutation of the large T antigen and bacterial neo ^r gene. Replication of this plasmid was repressed above 39°C and vigorous DNA propagation was observed below 33°C in simian CV-1 cells. The human erythropoietin gene was inserted downstream of the SV40 late promoter of the plasmid and the recombinant plasmid was introduced into CV-1 cells. By a temperature shift from 37 to 33°C, the plasmid copy number increased from 5 × 10² to 5 × 10³ copies per cell and the specific production rate of erythropoietin increased more than ten-fold. The bacterial-derived sequences such as the neo ^r gene and vector pUC sequences were prone to delete but the main body of the recombinant plasmid such as SV40 and the erythropoietin-coding sequences were stably maintained at either 33 or 37°C.     

Assessment of the tolerance of Lactococcus lactis cells at elevated temperatures

When Lactococcus lactis strains were exposed directly to the lethal temperature of 50 C for 30 ;min, 0.1–31% of the cells survived. However, when pre-exposed to 40 °C, prior to exposure at 50 °C, 4–61% of the cells survived. A plasmid carrying a unique heat shock gene from the thermophile Streptococcus thermophilus was cloned into L. ;lactis. When the transformed cells were cultivated at 30 °C the introduction of the plasmid had no obvious effect on the growth of L. ;lactis. However, when the temperature was abruptly shifted from 30 °C to 42 °C at mid-growth phase the growth decreased by 50%.     

High Frequency Conjugative Mobilization Accomplished by a Natural Bacillus subtilisStrain Carrying a Large Plasmid

Conjugative properties of the strain Bacillus subtiliscarrying a large plasmid approximately 95 kb in size and isolated in Belarus from forest soil were described. The staphylococcal plasmid pUB110 that had previously been introduced into this strain was transferred to recipient cells of the Bacillus subtilis168 strain with a frequency of approximately 10^–2. The transfer occurred with approximately the same frequency both upon donor and recipient cell contact on the surface of membranes and in a liquid medium. The latter fact makes this system suitable as a model for studying conjugative mobilization in bacilli. A large plasmid cannot be transferred to recipients. An optimal temperature for conjugation of donor and recipient cells was 37°C, but conjugation also proceeded at lower temperatures, up to 21°C.     

Increased efficiency of transformation ofBacillus stearothermophilus by a plasmid carrying a thermostable kanamycin resistance marker

Recombinant plasmids carrying either the wildtype kanamycin nucleotidyltransferase gene encoded originally by the mesophilic plasmid pUB110 or the gene encoding the thermostable TK101 mutant were constructed and introduced intoBacillus stearothermophilus by a protoplast transformation procedure. When kanamycin-resistant transformants were selected at 47°C, the transformation efficiency of the plasmid bearing the TK101 gene was nine times higher than that of the plasmid encoding the wildtype enzyme. The difference in transformation efficiencies between the two plasmids was increased when transformants were selected at higher temperatures, reflecting the difference in thermostabilities of the respective kanamycin nucleotidyltransferases. We conclude that, even though the pUB110 enzyme is sufficiently active at 47°C to confer kanamycin resistance toB. stearothermophilus, the additional stability of the TK101 mutant is advantageous in transformation ofB. stearothermophilus. The TK101 gene may also have broad utility as a marker for cloning vectors in other thermophiles.     

A general method for plasmid isolation in lactobacilli

A simple procedure for rapid isolation and detection of plasmid DNA fromLactobacillus species is described. Using an alkaline-detergent lysis method, plasmid DNA was released and characterized from cells treated with either mutanolysin or lysozyme for 1 h at 0°C. Treatment of cells with either enzyme at 37°C for 1h was detrimental to plasmid isolation and charaterization in someLactobacillus species. The procedure was effective with small volumes of cells and allowed rapid characterization of plasmid DNA inLactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus helveticus, andLactobacillus bulgaricus strains.     

Tn1 insertion mutagenesis in Escherichia coli K-12 using a temperature-sensitive mutant of plasmid RP4

Summary A method for Tn1 insertion mutagenesis in Escherichia coli has been developed using pTH10, a mutant plasmid of RP4 temperature-sensitive for maintenance. The mutagenesis involves three steps. Firstly, from strains carrying pTH10 showing resistance to the antibiotics kanamycin, tetracycline, and ampicillin at 30° C but not at 42° C, clones are isolated resistant to kanamycin at 42° C. Such temperature-independent, drug resistant clones probably carry pTH10 integrated into the host chromosome. Secondly, they are cultivated at 30° C. At this temperature segregants carrying pTH10, which has been excised from the host chromosome, accumulate. Thirdly, to cure such segregants of autonomous pTH10, they are cultivated at 42° C. By these procedures, clones free of pTH10, but carrying Tn1 insertions on the host chromosome, were obtained.About 3% of the clones carrying Tn1 insertions were auxotrophic. Distribution of auxotrophic mutations was not random, indicating the existence of preferential integration sites of Tn1 on the host chromosome. The frequency of precise excision of Tn1 was less than 10^-10.The pTH10 plasmid has a wide host range among Gram-negative bacteria and thus may serve as a excellent vector for insertion mutagenesis of Tn₁ in many Gram-negative bacterial species.     

Initial studies on aBacillus subtilis mutant lacking the dnaK-homologue protein

A sequence of 412bp, spanning the terminal half of thegrpE and the proximal portion of thednaK-homologues inBacillus subtilis, was amplified with PCR technology. This fragment was cloned into pJH101, anEscherichia coli plasmid, and transformed intoB. subtilis strain YB886. Several chloramphenicol-resistant colonies were obtained from this transformation. The integration of the plasmid into theB. subtilis chromosome was verified by restriction endonuclease analysis and Southern hybridization. Strain BUL101, a chloramphenicol-resistant transformant, lacked the DnaK-homologue as demonstrated by two-dimensional polyacrylamide gel electrophoresis and Western blot analysis. BUL101 grew at slower rates than parental cells at both 37°C and 48°C, produced abnormal cell shapes at 48°C, and was unable to grow at 51°C. The 412bp fragment did not exhibit detectable promoter activity.     

Development of Chainia as a host for xylanase gene cloning : Evidence for occurrence of a restriction-modification system

Summary Conditions for genetic transformation of the xylanase-negative (X^–) strain of Chainia with pIJ 702 were optimized. The growth of Chainia at 30°C for 36 – 40 h and addition of geletin (1%) to the medium resulted in the maximum yield of protoplasts and regeneration efficiency. Poor transformation efficiency of Chainia (X^–) protoplasts by native pIJ 702 versus improved efficiency (16 transformants ug^–1 of plasmid DNA) by prior heating of protoplasts at 42°C for 10 min suggests the occurrence of a restriction system in Chainia. Increased transformation efficiency by passage of the plasmid through Chainia together with the altered methylation status of the transformant plasmid presents evidence for the existence of an operative modification system in Chainia. Development of thiostrepton resistance and formation of me1amin pigment in Chainia (X^–) by transformation with pIJ 702 reveal that genes from Streptomyces can be functionally expressed by Chainia (X^–).(NCL Communication No. 6207)     

The extracellular nuclease of Serratia marcescens: studies on the activity in vitro and effect on transforming DNA in a groundwater aquifer microcosm

A quantitative endonuclease assay, which relies on the introduction of single and double strand breaks into supercoiled plasmid DNA, was used to study the activity of the extracellular nuclease of Serratia marcescens SM6 in buffer and in groundwater. The parallel enzyme concentration-dependent production of relaxed and linear plasmid molecules suggests that the nuclease produces single and double strand breaks in duplex DNA. Bovine serum albumin stimulated the nuclease activity towards DNA and RNA and increased the stability of the enzyme against thermal inactivation. The DNase activity at 4 °C and 50 °C was almost half of that at the optimum temperature (37 °C). The nuclease was active in groundwater, although the specific activity was lower than in buffer. In a groundwater aquifer microcosm, mineral-adsorbed transforming DNA was substantially less accessible to the nuclease than was dissolved DNA. The data suggest that the extracellular nuclease of Serratia marcescens may contribute to DNA turnover in the environment and that adsorption of DNA to minerals provides protection against the nuclease.Abbreviations GW groundwater GWA groundwater aquifer     

DNA degradation at elevated temperatures after plasmid amplification in amino acid-starved Escherichia coli cells

Summary Amino acid starvation of Escherichia coli relA mutants may be used as a method for efficient plasmid DNA amplification. Here we demonstrate DNA degradation which occurs at elevated temperatures (42–43°C) after plasmid amplification in amino acid-starved bacteria. These results may explain the previously described low efficiency of plasmid DNA amplification at elevated temperatures.     

Effect of temperature on plasmid stability and expression of cloned cellulase gene in a recombinantBacillus megaterium

Summary The expression of carboxymethyl cellulase gene inBacillus megaterium (pCK108) was investigated with respect to temperature in batch culture. The suboptimal temperature supporting maximal cell growth rate was determined to be 30 °C at which stability of the plasmid pCK108 could be maintained stable. However, cellular plasmid contents, production rate of cellulase of the cell, and efficiency of the gene expression increased significantly with increase of the temperature from 30 °C to 44 °C, even though the plasmid stability decreased up to 60% level at the end of the culture.     

Plasmid instability inLactobacillus plantarum strain caTC2

The stability of plasmids inLactobacillus plantarum was investigated by extended incubation of bacterial cells in the presence of different carbohydrates. Strain caTC2, carrying a plasmid-encoded chloramphenicol-resistant (Cm^r) phenotype, was grown overnight (16–18 h) in MRS, MRS-L, and MRS-M broths containing 2% glucose, lactose, and maltose respectively at 30°C. The cultures were subsequently held at 30°C and room temperature (21±1°C) for an extended period (7 days). The total viable cell counts were assayed on MRS agar plates and tested for sensitivity to 30 g chloramphenicol/ml by replica plating. The plasmid profiles of the chloramphenicol-sensitive strains showed that there was a loss of the 8.5-kb plasmid, but not the 10.6 or 6.5 kb plasmids. Concomitant loss of the chloramphenicol resistance phenotype and plasmid at high frequency, particularly by using MRS-L at 21°C method, suggests that this would be a simple and efficient method for curing selected plasmids in lactobacilli.Contribution No. 2039 from the Centre for Food and Animal Research.     

DNA supercoiling in a thermotolerant mutant ofEscherichia coli

A spontaneously occurring, nalidixic acid-resistant (Nal^R), thermotolerant (T/r) mutant ofEscherichia coli was isolated. Bacteriophage P1-mediated transduction showed that Nal^R mapped at or neargyr A, one of the two genes encoding DNA gyrase. Expression ofgyrA ⁺ from a plasmid rendered the mutant sensitive to nalidixic acid and to high temperature, the result expected for alleles mapping ingyrA. Plasmid linking number measurements, made with DNA from cells grown at 37° C or shifted to 48° C, revealed that supercoiling was about 12% less negative in the T/r mutant than in the parental strain. Each strain preferentially expressed two different proteins at 48° C. The genetic and supercoiling data indicate that thermo-tolerance can arise from an alteration in DNA gyrase that lowers supercoiling. This eubacterial study, when. coupled with those of archaebacteria, suggests that DNA relaxation is a general aspect of thermotolerance.     

Mating due to loss of surface exclusion as a cause for thermosensitive growth of bacteria containing the Rtsl plasmid

Summary At 25° C, Rtsl⁺ bacteria grow to about 5x10⁹ bacterial/ml before leveling off, whereas at 42° C they grow from 2.6x10⁸ bacteria/ml for only 2.3 generations after temperature shift before the growth is inhibited with a zig-zag pattern at the plateau. When diluted, Rtsl⁺ bacteria grow rapidly at 42° C, until the concentration reaches as high as the undiluted 42° C culture when growth measured by colony counts stops and the zig-zag pattern again appears. This density-dependent growth inhibition is not due to the presence of stable growth inhibitor(s). Mating experiments show that at 42° C, Rtsl⁺ bacteria retain good donor ability; at the same time, they become good recipients in mating with Hfr (Rtsl) bacteria. SDS-PAGE reveals that membranes are altered at 42° C. Examination of DNA synthesis indicates that chromosomal DNA is synthesized at both 25° C and 42° C at high bacterial concentration, but that of the Rtsl plasmid is slowed down at 42° C. The labeling experiments suggest that in 2 h there are 2 rounds of plasmid replication at 25° C, 3.5 rounds at 42° C when bacteria are diluted, and 0.6 rounds at 42° C when bacteria are not diluted. These results suggest that the growth inhibition of Rtsl⁺ bacteria at 42° C is probably the consequence of mating initiated due to loss of surface exclusion.     

Possible involvement of the ugpA gene product in the stable maintenance of mini-F plasmid in Escherichia coli

Summary The seg-3 mutant Escherichia coli does not support the maintenance of mini-F plasmid at 42° C. We cloned the chromosomal DNA segment of the wild-type strain W3110 that complements the Seg^– phenotype of this mutant. Cleavage mapping of this segment showed that it was derived from the 76-min region of the E. coli chromosome map. Complementation tests using plasmids carrying subcloned DNA segments suggested that the seg-3 mutant carried two mutations that additively affected the maintenance of mini-F plasmid; one was in the ugpA gene and the other was presumably in the rpoH gene. We generated a disrupted ugpA null mutant and found that the mini-F plasmid was unstable in this ugpA null mutant even at 30° C. This suggests that the ugpA gene product is required for the stable maintenance of mini-F plasmid.     

An unusual mutation in RepA increases the copy number of a stringently controlled plasmid (Rtsl derivative) by over one hundred fold

A copy number mutant of the Rtsl replicon (copy number 1–2 copies/cell) was obtained. A one-base substitution in the repA region results in a single amino acid change from histidine to asparagine at position 159. This mutation increased the plasmid copy number by up to 120-fold depending upon the growth conditions. At 42.5° C the plasmid with the wild type replicon was unstable while the mutated replicon was relatively stable.     

Some properties of arctic rhizobia

Forty-eight strains of Rhizobium isolated from the root nodules of three species of legumes indigenous to the high tundra (Astragalus alpinus, Oxytropis maydelliana andOxytropis arctobia) are phenotypically heterogenous with respect to intrinsic antibiotic resistance, expression of nitrogenase activityex planta and plasmid content. All of the strains possess a 250–300 kb plasmid and are homologous to each other on the genomic DNA level but have little DNA homology with selected reference strains of well characterized species of rhizobia. The arctic rhizobia have an optimum growth temperature of 23°C and can grow slowly at 5°C. The DNA from four of the isolates, which were selected for detailed investigation, have sequences homologous tonif andnod genes fromRhizobium trifolii.     

A thermostable glucose isomerase having a relatively low optimum pH: study of activity and molecular cloning of the corresponding gene

A thermostable glucose isomerase from a newly isolated thermophilic Streptomyces sp. SK strain, had a wide pH range with an optimum of 6 at 60°C and 6.4 at 90°C. It was optimally active at 95°C and completely stable at 80°C for at least 5.5 h with a half-life of 5 h at 90°C. Using E.coli as a host strain and an internal fragment of xylA of S. olivochromogenes as a probe, a 6.5 kb DNA fragment from Streptomyces sp. SK was cloned. This fragment carries the entire xylA gene since the recombinant plasmid complements the E.coli xyl-5 mutant strain HB101. © Rapid Science Ltd. 1998