Stimulation of T7 DNA polymerase by a new phage-coded protein

Summary A bacteriophage-induced DNA-binding protein was purified from T7 infected E. coli. The protein has a molecular weight of about 25000, as judged by SDS-polyacrylamide gel electrophoresis. The purified protein binds to single-stranded but not to native T7 DNA. Like the T4 gene-32 protein and the 22000-dalton unwinding protein of E. coli, the T7 25000 protein lowers the melting temperature of poly d(A-T). Using partially single-stranded T7 DNA as template-primer, the protein stimulates in vitro DNA synthesis by T7 DNA polymerase about five-fold. It was also found that the DNA-unwinding protein of E. coli stimulates T7 DNA polymerase to approximately the same extent. However, neither of the unwinding proteins stimulate DNA polymerase I of E. coli.     

Development of an electro-transformation system for Escherichia coli DH10B

Summary Escherichia coli can be transformed to high efficiencies by subjecting a mixture of cells and DNA to a brief but intense electrical field. Factors that affect the transformation efficiency of E.coli strain DH10B were analysed. Optimal conditions gave an efficiency of 10⁸ to 10⁹ transformants/g DNA with E.coli strains K803 and DH10B, and plasmids pB1221.23 and pBSK+. The use of ligated DNA resulted in 10⁶ transformants/g DNA. Detailed protocols for these systems are given.     

Electroporation and stable maintenance of plasmid DNAs in a biocontrol strain of Pseudomonas syringae

Transformation efficiencies as high as 10⁷ transformants g^–1 DNA have been previously reported for pseudomonads using electroporation protocols established for E. coli with plasmid DNAs prepared from methylation proficient E. coli hosts. We report here a protocol for electroporation of plasmid DNAs into a biocontrol strain of Pseudomonas syringae which could not be electroporated by standard E. coli methods. Transformation efficiencies of 10⁷ or higher were obtained with DNA recovered from initial P. syringae transformation or with DNA prepared from methylation deficient E. coli. Both plasmids used in this study were stably maintained in the absence of selection for at least 50 generations.     

Isolation and characterization of DNA repetitions carrying the chromosomal beta-lactamase gene of Escherichia coli K-12.

Summary A ColE1 hybrid plasmid, pNU1, carrying the amp operon coding for chromsomal -lactamase was isolated from the Clarke and Carbon collection and physically mapped. The physical location of ampC within this plasmid was further deduced by in vitro cloning.By reciprocal recombination between pNU1 and chromosome of two unstable -lactamase hyperproducing E. coli K-12 mutants a large plasmid from each mutant was obtained. The respective plasmid was physically mapped and found to contain five and two repeated DNA segments. The repetitions within each plasmid were equal in size, 9,800 bp and 11,900 bp respectively and were organized in tandem. The end points of the repeats were different in the two plasmids but shared a DNA segment carrying the ampC gene. The chromosomal DNA of the -lactamase hyperproducing E. coli mutants were found to contain an amplified DNA segment equal in size to the repeated unit found in the respective plasmid. The data shows that up to 10 identical repeats organized in tandem can be generated by a normal mutation frequency in E. coli.     

Induction of DNA breakdown and death in Escherichia coli by phleomycin. Its association with dark-repair processes

Summary Phleomycin, at concentrations above 1 g/ml, induced breakdown of DNA and death in E. coli. Exponentially growing cultures were about 10 times more sensitive to phleomycin than were stationary cultures, and the effect was somewhat dependent on the medium.Excisionless (HCR) mutants of E. coli were insensitive to doses of phleomycin which killed over 99% of wild-type organisms within an hour, while EXR mutants were considerably more sensitive.Mutants of E. coli selected for phleomycin resistance were unable to reactivate U.V. irradiated Tl phage (HCR).It is concluded that the DNA breakdown, inhibition of DNA replication and cell death are a consequence of initial attack by an excision-endonuclease stimulated by the phleomycin.     

Chestnut bur-shaped aggregates of chrysotile particles enable inoculation of <Emphasis Type="Italic">Escherichia coli</Emphasis> cells with plasmid DNA

In the present study, Escherichia coli cells exhibited antibiotic resistance after transformation with exogenous plasmid DNA adsorbed onto chrysotile particles during agar-exposure. We previously demonstrated penetration of E. coli by chrysotile particles during agar-exposure. To further investigate the mechanism by which transformation of E. coli is achieved through the use of chrysotile fibers, the interaction between E. coli cells and chrysotile was examined during agar-exposure. Dispersion of chrysotile particles within the chrysotile solution was analyzed by flow cytometry. A suspension containing E. coli cells expressing blue fluorescence protein and chrysotile particles was exposed to agar using stirring apparatus, which allowed a constant vertical reaction force to be applied to the surface of the gel. Fluorescence microscopy was then used to illustrate the adsorption of fluorescein isothiocyanate-conjugated DNA oligomers to chrysotile. Larger aggregates were observed when increasing concentrations of chrysotile were added to the solution. With prolonged exposure, during which surface moisture diffused into the agar gel, greater concentrations of chrysotile were observed on the agar surface. In addition, chrysotile aggregates exceeding 50 m developed on the agar surface. They were shaped like a chestnut bur. The chrysotile aggregates penetrated the cell membranes of adherent E. coli cells during agar-exposure due to sliding friction forces generated at the interface of the agar and the stirring stick. E. coli cells thus acquired plasmid DNA and antibiotic resistance, since the plasmid DNA had been adsorbed onto the chrysotile particles. The inoculation of plasmid DNA into E. coli cells demonstrates the usefulness of chrysotile for E. coli transformation.     

Electroporation of Bradyrhizobium japonicum

Summary Electroporation offers a fast, efficient and reproducible way to introduce DNA into bacteria. We have successfully used this technique to transform two commercially important strains of Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont. Initially, electroporation conditions were optimized using plasmid DNA which had been prepared from the same B. japonicum strain into which the{imDNA was to b}e transformed. Efficiencies of 10⁵-10⁶ transformants/g DNA were obtained for strains USDA 110 and 61A152 with ready-to-use frozen cells. Successful electroporation of B. japonicum with plasmid DNA prepared from Escherichia coli varied with the E. coli strain from which the plasmid was purified. The highest transformation efficiencies (10⁴ transformants/g DNA) were obtained using DNA prepared from a dcm ^– dam ^– strain of E. coli. This suggests that routine isolation of DNA from an E. coli strain incapable of DNA modification should help in increasing transformation efficiencies for other strains of bacteria where DNA restriction appears to be a significant obstacle to successful transformation. We have also monitored the rate of spontaneous mutation in electroporated cells and saw no significant difference in the frequency of streptomycin resistance for electroporated cells compared to control cells.     

Cloning of mglB, the structural gene for the galactose-binding protein of Salmonella typhimurium and Escherichia coli

Summary From libraries of EcoRI fragments of Salmonella thyphimurium and Escherichia coli DNA in gt7, phages could be isolated that carry mglB, the structural gene of the galactose-binding protein as well as other mgl genes. Lysogenization of an E. coli mutant carrying a defective galactose-binding protein with gt7 mglB (Salmonella) restores full galactose transport and galactose chemotaxis. Both the E. coli mutant protein as well as the wild-type Salmonella galactose-binding protein are synthesized in this strain. The EcoR1 fragments of both organisms carrying the mgl genes were 6 Kb long. They were subcloned into the multicopy plasmid pACUC184. The hybrid plasmid containing the Salmonella mgl DNA gives rise to the synthesis of large amounts of galactose-binding protein in the periplasm of E. coli. The protein can be precipitated by antibodies against the E. coli binding protein and is identical to the fully processed protein isolated from Salmonella typhimurium LT2. In vitro protein synthesis (Zubay-system) with either gt7 mgl phages as well as the hybrid plasmid as DNA matrix produces the galactose-binding protein mainly in precursor form that is precipitable by specific antibodies.     

Cloning of bacterial DNA replication genes in bacteriophage lambda

Summary Recombinant lambda phages containing the genes for dnaZ protein (the subunit of DNA polymerse III holoenzyme), primase (dnaG protein) and dnaC protein from Escherichi coli and Salmonella typhimurium were isolated. Each gene cloned from S. typhimurium has extensive DNA sequence homology to the corresponding E. coli gene. Clones selected by complementation of a dnaA temperature-sensitive mutant appear similar to other isolated suppressors of dnaA (Projan and Wechsler 1981). Derivatives of each cloned fragment suitable for overproduction of the protein were constructed. Of those tested, only the phage containing the E. coli dnaZ gene resulted in significant overproduction.Abbreviations DTT dithiothreitol - Ec Escherichia coli - EDTA ethylene diamine tetra acetic acid - kb kilobase 1,000 bases or base-pairs - moi multiplicity of infection - pol I E. coli DNA polymerase I - pol III holoenzyme E. coli DNA polymerase III holoenzyme - pri dnaG, primase-coding gene - SSB single-strand binding protein - St Salmonella typhimurium - sup gene coding for suppressor - ts temperature-sensitive     

High-frequency electroporation and maintenance of pUC- and pBR-based cloning vectors inPseudomonas stutzeri

A number ofEscherichia coli cloning vectors, based on ColE1-like replicons, were shown to be maintained inPseudomonas stutzeri ATCC 17588. A restrictionless mutant ofP. stutzeri was isolated, and this strain was used to develop an efficient electroporation system. With theE. coli cloning vector pHSG298, transformation frequencies of up to 2×10⁷ transformants/g DNA were achieved. This frequency is comparable to that obtained for CaCl₂-mediated transformation ofE. coli; thus, direct cloning of DNA intoP. stutzeri is feasible. As will be discussed, this may prove useful for cloning DNA from high mol% G+C genera in cases in whichE. coli is not a suitable heterologous cloning host.     

Molecular cloning of a gene which regulates the adaptive response to alkylating agents in Escherichia coli

Summary The ada ⁺ gene of E. coli is a regulatory gene of the adaptive response to simple alkylating agents. ada mutants are sensitive to both the mutagenicity and toxicity of alkylating agents, and are unable to induce O⁶-methylguanine DNA methyltransferase and 3-methyladenine DNA glycosylase II. The ada ⁺ gene was cloned from wild type E. coli B by ligating bacterial DNA partially digested with Sau3A into the cosmid vector pJB8. The hybrid cosmid, pCS33, conveyed N-methyl-N-nitro-N-nitrosoguanidine resistance to ada mutants of E. coli B and E. coli K12, and resulted in the constitutive synthesis of the two DNA repair enzymes at high levels. An alk mutation, which results in a deficiency of only the DNA glycosylase, was not complemented by this cosmid. It was concluded that the product of the ada ⁺ gene is a positive regulator of the adaptive response. The cosmid insert DNA was subcloned into the plasmid vector pAT153, and the ada ⁺ plasmids pCS42 and pCS58 selected. The ada ⁺ gene located in PCS58 by transposon mutagenesis and subcloning. Two polypeptides of Mr 37,000 and 27,000, were identified in maxicells as products of the ada ⁺ gene(s). It is as yet unclear whether they represent different forms of the same gene product, or are encoded by separate ada ⁺ genes within the same operon.     

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

Summary A convenient procedure for the isolation of specificEcoRI-fragments ofE. coli genome and their amplification on Km-resistance plasmid vector CK 11 is described. The hybrid molecules were constructedin vitro usingEcoRI-digestion, followed by ligation. Then appropriatedE. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK 11-arg ⁺, CK 11-his ⁺, CK 11-thr ⁺ and CK 11-leu ⁺ containing loci ofE. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of oneEcoRI-fragment of initial plasmid CK 11 and one respective specific portion ofE. coli genome.     

The host specificity system inEscherichia coli SK

E. coli SK has its own enzyme system providing DNA host specificity which differs from the known types of specificity inE. coli K12 andE. coli B. Modification and restriction are observed when the PBVI or PBV3 phages are transferred fromE. coli SK toE. coli B or K12 (and back).A methylase has been isolated fromE. coli SK cells and partly purified. This methylase catalyzesin vitro transfer of the labelled methyl groups from S-adenosylmethionine (SAM) to DNA of both phage and tissue origin which gives rise to 5-methylcytosine (5MC) and 6-methylaminopurine (6MAP). The methylase preparations isolated from the cells at the stationary growth have proved to be 1.5–1.7 times as active as the enzyme from the cells at the logarithmic growth stage. The extract ofE. coli SK cells infected with the phage S_D cannot methylate DNAin vitro. This fact is due tode novo synthesis of the enzyme which disintegrates SAM down to 5-methylthioadenosine (5MTA) and homoserine (HS). This enzyme is not found in the cells infected with the S_D phage in the presence of chloroamphenicole. The activity of the enzyme which disintegrates SAM is the highest between the 4th and the 5th minutes of infection. Thus it may be assumed that this enzyme, most probably, is an early virus specific protein and preventsin vivo methylation of the phage DNA.     

Restriction enzyme cleavage of DNA resulting from gently lysed Escherichia coli.

Summary When E. coli or infected E. coli are gently lysed the DNA is released as a very fast sedimenting species that is presumably bound to membrane material. If this complex is now subjected to restriction enzyme cleavage, only a minor fraction of the fast sedimenting DNA remains and this is found, after purification, to be enriched for branched molecules.     

A bacteriocinogenic factor of Enterobacter cloacae

Summary Enterobacter cloacae strain DF13 produces a bacteriocin which is able to kill other strains of Enterobacter and Klebsiella. This property can be transmitted to Enterobacter cloacae strain O 2 (up to 90% of the acceptor population became bacteriocinogenic), to E. coli K12F^- and E. coli K 12 Hfr. Transfer of chromosome material was never observed, suggesting that the production of the bacteriocin is determined by a plasmid. However all attempts to eliminate this plasmid failed. The plasmid F trp cys Col B Col V could be transferred from E. coli into Ent. cloacae DF13 and subsequently it could be eliminated by acridine orange treatment. Ent. cloacae DF13 harbours in addition two independently transferable R-factors, one determining resistance against streptomycin and sulfanilamide and the other resistance against penicillin.Most but not all Ent. cloacae O2 recombinants which have received only the bacteriocinogenic factor upon conjugation with Ent. cloacae DF 13, can transfer this property to Ent. cloacae O2 but not to E. coli. E. coli F^- recombinants, which have received only the bacteriocinogenic factor cannot transfer this property. The results suggest that the bacteriocinogenic factor cannot mediate its own transfer, but can be transferred when another transmissible plasmid is present. This may be the R(str sul) factor, the F-factor in E. coli Hfr or a transfer factor () in Ent. cloacae O2.Closed circular DNA molecules were selectively isolated from these strains and investigated by electron microscopy and velocity sedimentation. Ent. cloacae DF13 harbours small closed circular DNA molecules ranging from 0.5 to 3.2 in contour length, 98% of which corresponds to a size class of 0.7±0.1 . Ent. cloacae O2 also harbours closed circular DNA ranging from 0.8 to 3.0 in contour length, with major size classes of 0.9 and 1.4 respectively. Circular DNA of a contour length of 3.0±0.2 (S_20,w=26 S) corresponding to a molecular weight of 6.0×10⁶ daltons was transferred to E. coli and Ent. cloacae O 2 concomitantly with the ability to produce the bacteriocin. A significant difference was observed in the number of copies of the plasmid between Ent. cloacae and E. coli.     

Moving pictures of DNA released upon lysis from bacteria,chloroplasts, and mitochondria

Summary Cells and organelles suspended in gelled agarose agarose were lysed with detergent and protease, stained with ethidium bromide and their DNA was observed by fluorescence microscopy. The migration of individual DNA molecules during electrophoresis on a microscope slide was recorded on video tape so that moving pictures could be analyzed. The DNA from lysed bacteria (Escherichia coli andAgrobacterium tumefaciens) appeared as a rosette of at least twenty loops of varying size, whereas that from bacterial spheroplasts (E. coli andPseudomonas aeruginosa) appeared as circular forms or rods with many fine filaments of RNA extending toward the anode. The DNA from chloroplasts of watermelon (Citrullus vulgaris) and pea (Pisum sativum) did not appear as a rosette of loops. Many or most of the chloroplast DNA molecules per lysed chloroplast were immobile in the electric field, as if in circular form hooked on agarose fibers. The amount of DNA-fluorescence per watermelon mitochondrial particle was much less than that found for either chloroplasts or bacteria. The appearance of the mitochondrial DNA during electrophoresis was that of linear molecules, no obviously circular forms were evident and no rosette structures were observed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole - kb kilobase pairs - mtDNA mitochondrial DNA - PFGE pulsed-field gel electrophoresis     

Degradation of Escherichia coli DNA: evidence for limitation in vivo by protein X, the recA gene product.

Summary DNA is more extensively degraded after it is damaged in recA mutants of E. coli than in wild type cells. All data presented here are consistent with the recA gene product, protein X, being an inhibitor of nalidixic acid induced degradation of the bulk DNA (but not of newly replicated DNA). Production of protein X also is correlated with appearance of various S.O.S. repair functions. Evidence was obtained by comparing the rates of protein X synthesis and solubilization of uniformly-labeled DNA in intact cells, incubated in the presence of nalidixic acid. A set of mutants at the lexA locus produced protein X at different rates and degraded their DNA at rates which were inversely correlated to their rates of protein X production. A low concentration of rifampicin quite specifically inhibited protein X production by wild type E. coli, and allowed more rapid DNA degradation. After the DNA was damaged by the incubation of cells in the presence of nalidixic acid, cells preloaded with protein X degraded their DNA more slowly. We propose that protein X could protect DNA against degradation by binding to singlestranded regions, thereby inhibiting nuclease action.     

Sequence structure and expression of a cloned beta-glucosidase gene from an extreme thermophile

Summary The gene for a -glucosidase from the extremely thermophilic bacterium Caldocellum saccharolyticum has been isolated from a genomic library and sequenced. An open reading frame identified by computer analysis of the sequence could encode a protein of M_r 54400, which is close to the size of the polypeptide experimentally determined using maxicells. Analysis of the amino-terminal residues of the protein produced in Escherichia coli suggests that it is processed by a methionine aminopeptidase. A sequence within C. saccharolyticum DNA upstream of the -glucosidase gene was found to act as a promoter for expression of the thermophile gene in E. coli. The protein has been overproduced in E. coli and Bacillus subtilis where it retains its enzymatic activity and heat stability. There appears to be a single copy of the gene in Caldocellum DNA.     

RNA-linked nascent DNA pieces in T7 phage-infected Escherichia coli cells. I. Role of gene 6 exonuclease in removal of the linked RNA.

Summary The presence of RNA-linked nascent DNA pieces in T7 phage-infectedEscherichia coli cells has been shown by the selective degradation of the 5-hydroxyl-terminated nascent DNA, produced by alkali or RNase treatment, with spleen exonuclease. At 43°C, the proportion of RNA-linked DNA pieces in nascent short DNA is 50 to 60% in T7ts136 (ts mutant of gene 6) phage-infectedE. coli, whereas that in T7 wild-type phage-infected cells is less than 6%. Joining of the nascent pieces is greatly retarded in T7ts136-infectedE. coli temperature sensitivepolA mutants at 43° C. These results suggest that gene 6 exonuclease plays a role in removal of the linked RNA during the discontinuous replication of T7 DNA.     

Nucleotide sequence of a promoter recognized by Bacillus subtilis RNA polymerase

Summary We report the nucleotide sequence of a promoter recognized by RNA polymerase from the gram-positive bacterium Bacillus subtilis. This promoter, which was isolated from B. subtilis phage SP01 DNA, is homologous to promoters for Escherichia coli RNA polymerase; the sequences of the -35 region and the Pribnow box were 5TTGACT and 5CATAAT, respectively (T is the thymine analog 5-hydroxymethyluracil in SP01 DNA). These sequences each differed by only a single base pair from the preferred sequences for E. coli promoters. Not surprisingly, the SP01 promoter was actively transcribed in vitro by E. coli RNA polymerase as well as by B. subtilis RNA polymerase.