Isolation of Higher Molecular Weight DNA from Bacillus Cereus T Using Proteinase K

A method is described for the isolation of deoxyribonucleic acid (DNA) from Bacillus cereus T. The DNA is released from the cells autolytically in the presence of proteinase K which prevents degradation by nucleases. The method is simple and eliminates many of the previously required steps which cause mechanical shear. The DNA is of high molecular weight and can be efficiently trapped in agar to give DNA agar which is suitable for hybridization studies.     

琼脂固体培养基培养蜜环菌菌索总DNA的提取

野外采集的蜜环菌[Armillaria mellea(Vahl.ex Fr.)Quel]在提取DNA前需要分离获得纯化的菌丝体。常规液体培养获得菌丝团的方法感杂率较高,采集固体培养基表面cellophane膜上形成的菌丝则难以获得足量的DNA提取材料。蜜环菌细胞内含有大量多醣类物质,也使得蜜环菌高质量DNA的提取存在一定的困难。本研究通过改进试验,提供一个直接从琼脂固体培养基培养的蜜环菌菌索中提取高质量DNA的方法。其中样品的预先冻融处理方法可以促使蜜环菌菌索与琼脂分离;而在裂解提取缓冲液裂解材料细胞后加入1.25 mol/L KAc溶液,则有利于除去蜜环菌细胞内的多醣类物质以及残留的少量琼脂。通过琼脂糖电泳、紫外分光光度计对DNA浓度及OD值的测定、ISSR引物的PCR扩增以及酶切产物的PCR扩增等方法的检测,结果均表明该方法提取的DNA质量较好,符合进一步进行分子生物学研究的要求。     

Isolation and partial characterization of Bacillus subtilis mutants impaired in DNA entry

Transformation-deficient mutants of Bacillus subtilis have been identified either by screening for a nuclease-deficient phenotype on methyl green-DNA agar or for nontransformability on transforming DNA-containing agar. After purification of the mutations causing a reduction in the entry of DNA, a set of isogenic entry-deficient strains was obtained. In addition to being entry deficient to various extents, the strains usually were less capable of association with DNA than the entry-proficient parent. Likewise, the specific transforming activity in the purified mutant strains continued to be less than that in the wild type. With the possible exception of one strain, no evidence was obtained that the mutant strains were impaired in recombination. Since the breakdown of transforming DNA to acid-soluble products correlated fairly well with the residual capacity of the strains to take up DNA, nucleolytic activity is likely to be involved in the entry of DNA in B. subtilis.     

Inhibition of polynucleotide kinase by agar, dextran sulfate and other polysaccharide sulfates

In certain preparations of lambda DNA, the phosphorylation of their 5′-ends as catalyzed by polynucleotide kinase was inhibited. The inhibitor appeared to be due to traces of agar left in the DNA preparation. Of the several polysaccharides and their sulfuric acid esters tested for their inhibitory activity, all the polysaccharide sulfates are found to be strong inhibitors of the polynucleotide kinase whereas the non-sulfated polysaccharides are non-inhibitory. Since agar is sometimes used in growing the phages and dextran sulfate is often used in purifying the phages or DNA preparations, these potential inhibitors must be carefully removed from the final DNA preparations before a polynucleotide kinase reaction can be successfully carried out. However, the effect of small amounts of these inhibitors on the kinase can be counteracted by the addition of either polylysine or spermine.     

Unstable transformation of mouse 3T3 cells by transfection with DNA from normal human lymphocytes.

DNA fragments (0.5-4.5 kb) of normal human lymphocytes induced pre-neoplastic mouse NIH/3T3 cells after transfection to grow in soft agar medium at low efficiency (0.0007 colonies/micrograms DNA/10(6) cells). In secondary transfections high mol. wt. DNA (greater than 20 kb) of cells transformed by DNA fragments induced neoplastic transformation with high efficiency (0.16-1.1 soft agar colonies/micrograms DNA/10(6) cells). These results confirm previous data obtained by others with chicken and mouse donor DNA. We describe here that independent secondary transformants harbored human Alu repetitive DNA sequences on similar restriction fragments and formed progressively growing tumors in BALB/c mice or nude mice. The corresponding primary transformants were not tumorigenic, however, and the ability to proliferate in semi-solid agar medium was gradually lost when the cells were grown as non-confluent monolayers. Furthermore, in contrast to secondary transformants, DNA from primary transformants showed only relatively weak hybridization to a human Alu repetitive DNA probe. We conclude that in primary transformants the transformed phenotype is expressed in an unstable fashion whereas secondary transformants appear to be stably transformed.     

Protoplast transformation of recalcitrant alkaliphilic Bacillus sp. with methylated plasmid DNA and a developed hard agar regeneration medium

Among the diverse alkaliphilic Bacillus strains, only a little have been reported to be genetically transformed. In this study, an efficient protoplast transformation procedure was developed for recalcitrant alkaliphilic Bacillus sp. N16-5. The procedure involved polyethylene glycol-induced DNA uptake by the protoplasts and subsequent protoplast regeneration with a developed hard agar regeneration medium. An in vivo methylation strategy was introduced to methylate the exogenous plasmid DNA for improving the transformation efficiency. The transformation efficiency reached to 1.1×10(5) transformants per μg plasmid DNA with methylated plasmid pHCMC04 and the developed hard agar regeneration medium. This procedure might also be applicable to the genetic transformation of other Bacillus strains.     

An Agar Diffusion Method for the Determination Of Antibodies Against Staphylococcus Aureus Deoxyribonuclease

On the addition of small concentrations of deoxyribonuclease, produced by Staphylococcus aureus, to Toluidine Blue DNA agar, a medium is produced on which antibodies against S. aureus deoxyribonuclease may be detected. When samples of milk, or blood serum, containing antibodies against S. aureus are applied into wells in the agar, the deoxyribonuclease activity is inhibited by the antibodies diffusing into the agar. As a result of this inhibition, blue zones are produced around the wells in the otherwise bluish-red agar. The diameters of the zones correspond to the concentrations of antibodies, and the method may consequently be used for qualitative and quantitative examinations of antibodies against S. aureus deoxyribonuclease in milk and serum. The procedure and certain limitations of the method are described.     

Development of an agar lift-DNA/DNA hybridization technique for use in visualization of the spatial distribution of Eubacteria on soil surfaces.

While microbial growth is well-understood in pure culture systems, less is known about growth in intact soil systems. The objective of this work was to develop a technique to allow visualization of the two-dimensional spatial distribution of bacterial growth on a homogenous soil surface. This technique is a two-step process wherein an agar lift is taken and analyzed using a universal gene probe. An agar lift is comprised of a thin layer of soil that is removed from a soil surface using an agar slab. The agar is incubated to allow for microbial growth, after which, colonies are transferred to a membrane for conventional bacterial colony DNA/DNA hybridization analysis. In this study, a eubacterial specific probe was used to demonstrate that growing bacterial populations on soil surfaces could be visualized. Results show that microbial growth and distribution was nonuniform across the soil surface. Spot supplementation of the soil with benzoate or glucose resulted in a localized microbial growth response. Since only growing colonies are detected, this technique should facilitate a greater understanding of the microbial distribution and its response to substrate addition in more heterogenous soil systems.     

Hemolytic activity reevaluation of putative nonpathogenic Listeria monocytogenes strains.

Identification of 12 strains originally characterized as nonpathogenic Listeria monocytogenes was reassured following the evaluation of their hemolytic capability with a newly developed horse blood agar plate. Seven of the strains were observed consistently to be hemolytic and confirmed as L. monocytogenes with the use of two commercial systems: the Gene-Trak L. monocytogenes-specific colorimetric DNA hybridization assay and the API Listeria system. Except for one strain that formed typical smooth colonies, these hemolytic strains formed rough colonies on a selective medium, lithium chloride-ceftazidime agar. The rest of the strains were nonhemolytic and did not hybridize with the DNA probe; they were identified as Listeria innocua on the basis of their API Listeria system biochemical profile. All but one of these nonhemolytic strains formed smooth colonies on lithium chloride-ceftazidime agar.     

Comparison of two selective media for the detection and enumeration of Lactobacilli in human faeces

The enumeration of faecal bacteria is an important requirement for many studies of bowel health. One approach is the use of selective culture media for the culture and identification of genera or species from faeces. This study compares the culture of Lactobacilli from dilution series of faecal samples from six healthy human volunteers on two commonly used media, LAMVAB and Rogosa agar. Colonies were counted after a 72-h anaerobic incubation at 37 degrees C, and colony morphology recorded by a single observer. DNA was isolated from a representative number of colonies and genus-specific PCR, single-stranded conformation polymorphism (SSCP) and DNA sequencing performed. Total colony counts ranged from <3.00 to 7.48 log(10) cfu/g of faeces for LAMVAB and 5.09 to 7.66 log(10) cfu/g for Rogosa. For each subject, the total colony count was higher on Rogosa than that obtained with LAMVAB agar. SSCP analysis and DNA sequencing indicated that colony morphology was not an accurate predictor of genus identity. Growth of two species, Lactobacillus acidophilus and Lactobacillus gasseri, was not supported on LAMVAB medium. Rogosa agar was more likely to support growth of non-Lactobacillus species. Therefore, neither medium gave a fully accurate representation of the Lactobacilli species present in human faecal samples.     

Densitometric determination of nucleic acids in gels after electrophoresis

A technique for the quantitative determining of nucleic acids after electrophoresis by double-wave densitometry in UV spectrum by scanning agar gel along both axes was developed. Optical characteristics of agar and acrylamide-agar gels are given. The range of quantities measured is 6 divided by 30 micrograms of DNA per gel; the standard deviation adjusted to 1 microgram is less than +/- 3%.     

Discolored Red Seaweed <Emphasis Type="Italic">Pyropia yezoensis</Emphasis> with Low Commercial Value Is a Novel Resource for Production of Agar Polysaccharides

The red seaweed Pyropia yezoensis has been demonstrated to be a novel resource for the production of high-quality agar. P. yezoensis is grown for the food industry in large-scale Japanese mariculture operations. However, discolored P. yezoensis is mostly discarded as an industrial waste, although it has some kind of utility values. Here, we evaluated the utility of discolored P. yezoensis as a resource for agar production. The quality of agar from the discolored seaweed was comparable to that from normal seaweed. In addition, as a distinguishing characteristic, agar yield was higher from discolored seaweeds than from normal types. Moreover, we successfully used agar from discolored P. yezoensis for bacterial plate media and DNA electrophoresis gels without agarose purification. Thus, our results demonstrate that discolored P. yezoensis is suitable for agar production and use in life science research. Diverting discolored P. yezoensis from disposal to agar production provides a solution to the current industrial waste problem in mariculture, as well as a secure source of agar for research purposes.     

The cell cycle of Bacillus subtilis as studied by electron microscopy

Bacillus subtilis strain Marburg was grown exponentially with a doubling time of 65 min. To follow the time course of various cell cycle events, cells were collected by agar filtration and were then classified according to length. The DNA replication cycle was determined by a quantitative analysis of radioautograms of tritiated thymidine pulse labeled cells. The DNA replication period was found to be 45 min. This period is preceded and followed by periods without DNA synthesis of about 10 min.The morphology and segregation of nucleoplasmic bodies was studied in thin sections. B. subtilis contains two sets of genomes. DNA replication and DNA segregation seem to go hand in hand and DNA segregation is completed shortly after termination of DNA replication.Cell division and cell separation were investigated in whole mount preparations (agar filtration) and in thin sections. Cell division starts about 20 min after cell birth; cell separation starts at about 45 min and before completion of the septum.     

DNA degradation in isolated rat liver nuclei

Purified rat liver nuclei were incubated at 4°C. After different intervals of incubation aliquots of the nuclear suspension were taken and DNA was extracted by a common SDS-phenol-chloroform procedure. The fractionation of DNA by agar gel electrophoresis revealed large DNA fragments. It was shown that the well-known DNA degradation to monomers and its multiples is preceeded by an earlier breakdown of DNA into characteristic large fragments.     

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.     

Quantitative selective PCR of 16S ribosomal DNA correlates well with selective agar plating in describing population dynamics of indigenous Pseudomonas spp. in soil hot spots

We used a quantitative PCR method targeting 16S ribosomal DNA using competitive PCR for specific detection of indigenous Pseudomonas DNA in soil hot spots. The amount of Pseudomonas DNA corresponded to the number of culturable Pseudomonas bacteria on Gould's S1 agar. This represents the first use of PCR for quantification of indigenous bacteria in more than one sample of soil.     

Deoxyribonucleic acid synthesis in a temperature-sensitive Escherichia coli dnaH mutant, strain HF4704S.

The dnaH mutant strain HF4704S, isolated by Sakai et al. (1974), was examined for its effect on phiX174 deoxyribonucleic acid (DNA) synthesis. It was found to carry two mutations affecting DNA synthesis. One mutation had no affect on phiX174 DNA synthesis, but did affect the ability of the mutant cells to form colonies on agar medium at 41 degrees C, and caused host DNA synthesis to cease after 1 h at 41 degrees C. The mutant marker cotransduced with ilvD at a frequency of about 9%. It seems likely that this mutation is in the dnaA gene. The second mutation affected the ability of the mutant cells to form colonies on agar medium supplemented with only 2 mug of thymine per ml, and affected both host and phiX174 DNA synthesis in medium supplemented with only 2 mug of thymine per ml. Both effects could be overcone by adding excess exogenous thymine. We were not able to unambiguously determine the map position of this mutant locus. Our data show that the DNA synthesis phenotype of the mutant strain HE4704S is governed by both these mutations, neither of which directly affects the replication of phiX174 DNA.     

Plasmid transformation of Streptococcus lactis protoplasts: optimization and use in molecular cloning.

The parameters affecting polyethylene glycol-induced plasmid transformation of Streptococcus lactis LM0230 protoplasts were examined to increase the transformation frequency. In contrast to spreading protoplasts over the surface of an agar medium, their incorporation into soft agar overlays enhanced regeneration of protoplasts and eliminated variability in transformation frequencies. Polyethylene glycol with a molecular weight of 3,350 at a final concentration of 22.5% yielded optimal transformation. A 20-min polyethylene glycol treatment of protoplasts in the presence of DNA was necessary for maximal transformation. The number of transformants recovered increased as the protoplast and DNA concentration increased over a range of 3.0 X 10(6) to 3.0 X 10(8) protoplasts and 0.25 to 4.0 micrograms of DNA per assay, respectively. With these parameters, transformation was increased to 5 X 10(3) to 4 X 10(4) transformants per microgram of DNA. Linear and recombinant plasmid DNA transformed, but at frequencies 10- to 100-fold lower than that of covalently closed circular DNA. Transformation of recombinant DNA molecules enabled the cloning of restriction endonuclease fragments coding for lactose metabolism into S. lactis LM0230 with the Streptococcus sanguis cloning vector, pGB301. These results demonstrated that the transformation frequency is sufficient to clone plasmid-coded genes which should prove useful for strain improvement of dairy starter cultures.     

Heterologous expression and characterization of a recombinant thermophilic arylsulfatase from Thermotoga maritima

Production of low sulfated agar or agarose from agar or agaropectins by enzymatic hydrolysis has advantages but a high melting temperature is needed. The arylsulfatase gene from thermophilic Thermotoga maritima was cloned and expressed in Escherichia coli W3110 with pCol-MICT as the vector. The gene was comprised of 1,782 bp and encoded a protein of 593 amino acids with a molecular weight of 65 kDa. The recombinant arylsulfatase was partially purified by heat treatment (70°C, 30 min) and characterized. The enzyme was prepared with a total protein content of 2.4 mg and a specific activity of 20.63 U/mg. Optimal temperature and pH of the enzyme were 80°C and 7.0, respectively, for hydrolysis of p-nitrophenyl sulfate and sulfate content of agar was diminished to 40% after a 12 h treatment at that condition. Enhanced electrophoretic movement of DNA was observed in enzymetreated agar gel compared to that in a non-treated agar gel. These results suggest that thermophilic arylsulfatase expressed in E. coli could be useful for producing a low sulfated agar and electrophoretic grade agarose.