Bacterial Chromosomal DNA Research Articles

Comparison of Arbitrarily Primed Polymerase Chain Reaction and Ribotyping for Subtyping Actinobacillus actinomycetemcomitans

This study investigated the compatibility of arbitrarily primed polymerase chain reaction (AP-PCR) and ribotyping in the characterization of Actinobacillus actinomycetemcomitans , a major pathogen in the mixed anaerobic microflora of human periodontitis. AP-PCR was performed directly on lysed bacterial colonies using a random-sequence 10-base oligonucleotide primer. Ribotyping was carried out by using purified bacterial chromosomal DNA digested with BglI. DNA fragments were separated electrophoretically, blotted onto a nylon membrane and hybridized with the plasmid pKK3535 containing the rRNA operon of Escherichia coli. The two genetic methods were evaluated on isolates from single individuals and from family members. Twelve AP-PCR types and 47 ribotypes were distinguished among 76 A. actinomycetemcomitans isolates of different serotypes. AP-PCR typing and ribotyping gave compatible results in 18 of 20 comparisons. Although AP-PCR detected less genetic heterogeneity in A. actinomycetemcomitans than ribotyping, the rapid and relatively simple AP-PCR technique seems to be sufficiently discriminative to be used in large scale epidemiological studies which preclude the application of the more laborious ribotyping technique.

Preelectrophoresis of Agarose Plugs Containing Bacterial Chromosomal DNA Prepared for Analysis by Pulsed Field Gel Electrophoresis Can Improve the Clarity of Restriction Patterns

Pulsed field gel electrophoresis has indicated that chromosomal DNA isolated from stationary phase Pseudomonas fluorescens, Pseudomonas putida, and Escherichia coli cells immobilized in agarose can be fragmented during its release. For P. fluorescens it was demonstrated that the entire chromosome is affected and that there are no specifically fragile sites. The extent of the damage increased both during storage of the DNA and also when magnesium ions were provided, suggesting that nucleases may be involved. Chromosomal DNA isolated from exponentially growing cultures was subjected to significantly less damage during release and storage. Removal of damaged DNA by electrophoresis prior to digestion was shown to result in clearer restriction patterns of chromosomal DNA.

Analysis of the amino acid sequence of the Pseudomonas aeruginosa galactophilic PA-I lectin

Based on the NH2-terminal 30-amino acid sequence of Pseudomonas aeruginosa galactophilic PA-I lectin, two degenerate primer oligonucleotides were synthesized and used in polymerase chain reaction with the bacterial chromosomal DNA as a template. A predominant DNA fragment of the appropriate size was radiolabeled and used as a probe for screening a P. aeruginosa genomic lambda gt11 library. One positive clone carrying an insert of about 630 base pairs encompassing the entire PA-I lectin gene was isolated and found to contain a 369-base pair open reading frame between an initiation codon (19 base pairs downstream from the insertion site, subsequent to a Shine-Dalgarno sequence) and two consecutive stop codons, followed by an oligo (seven) A sequence, in a partial dyad symmetry. The deduced amino acid sequence shows excellent agreement with the quantitative amino acid analysis and a perfect match with the NH2-terminal amino acid sequence of the purified lectin. It reveals that the PA-I lectin subunit contains 121 amino acids (M(r) 12,754; pI 4.94) with a predominant central hydrophilic core between two hydrophobic domains. Secondary structure algorithms predict that it is rich in beta sheets and contains several highly antigenic epitopes, but no signal peptide. In the carboxyl region a potential glycosylation site (Asn-Asn-Ser) was identified. Comparative analyses of this lectin sequence with those of lectins from other sources, reported in the protein and gene data banks, did not reveal any extensive homology.

An improved method for the purification of high molecular weight bacterial chromosomal DNA.

An improved method for the purification of high molecular weight bacterial chromosomal DNA.

Plasmid Incidence in Bacteria from Deep Subsurface Sediments

Bacteria were isolated from deep terrestrial subsurface sediments underlying the coastal plain of South Carolina. A total of 163 isolates from deep sediments, surface soil, and return drill muds were examined for plasmid DNA content and resistance to the antibiotics penicillin, ampicillin, carbenicillin, streptomycin, kanamycin, and tetracycline. MICs of Cu, Cr, and Hg for each isolate were also determined. The overall frequency of plasmid occurrence in the subsurface bacteria was 33%. Resistance was most frequent to penicillin (70% of all isolates), ampicillin (49%), and carbenicillin (32%) and was concluded to be related to the concentrations of the individual antibiotics in the disks used for assaying resistance and to the production of low levels of beta-lactamase. The frequencies of resistance to penicillin and ampicillin were significantly greater for isolates bearing plasmids than for plasmidless isolates; however, resistance was not transferable to penicillin-sensitive Escherichia coli. Hybridization of subsurface bacterial plasmids and chromosomal DNA with a whole-TOL-plasmid (pWWO) probe revealed some homology of subsurface bacterial plasmid and chromosomal DNAs, indicating a potential for those bacteria to harbor catabolic genes on plasmids or chromosomes. The incidences of antibiotic resistance and MICs of metals for subsurface bacteria were significantly different from those for drill mud bacteria, ruling out the possibility that bacteria from sediments were derived from drill muds.

A one-step procedure for the purification of high molecular weight bacterial chromosomal DNA.

A one-step procedure for the purification of high molecular weight bacterial chromosomal DNA.

Rapid small scale preparation of bacterial genomic DNA, suitable for cloning and hybridization analysis

We describe a rapid procedure for extracting purified chromosomal DNA from Pseudomonas putida, and some minor modifications needed for use in other organisms, including Gram‐positive strains. The technique is rapid and generally produces between 1 and 5 μg of DNA from 1 ml of liquid culture. The DNA is highly purified and can be readily cut with small quantities of restriction endo‐nucleases, cloned into plasmid vectors and used as a substrate for hybridization with labelled DNA probes. Genetical analysis and cloning of genomic DNA from environmentally important organisms like Ps. putida has become increasingly important. Bacterial chromosomal DNA is usually prepared from large volumes of liquid culture, and involves time‐consuming steps necessary to purify the DNA sufficiently for use in cloning experiments (e. g. Marmur 1961). We have developed a method for preparing small quantities of genomic DNA, which involves whole cell lysis and purification by precipitation and centrifugation. Loss of DNA occurs by shearing, but the yield of DNA is sufficient, and the quality is high. The method has been used to prepare DNA from a variety of organisms and is particularly applicable where the preparation and analysis of DNA from a large number of isolates is required.

A method for rapid extraction of Providencia chromosomal DNA and restriction enzyme digestion in agarose pellets

A simple and fast (2–3 d) method is described for the extraction and restriction endonuclease digestion of bacterial chromosomal DNA in low melting point agarose pellets. The technique minimized the random mechanical shearing of DNA caused by conventional preparative methods and improved the resolution of electrophoretic band patterns, particularly for the smaller (< 10 kb) fragments. The method was tested on 14 strains of Providencia stuartii and Prov. rustigianii.

Isolation and characterization of plasmid DNA fromRuminococcus

A method has been developed, utilizing mutanolysin and proteinase K, for the rapid lysis of strains of the rumen cellulolytic bacteriumRuminococcus. This has enabled bacterial chromosomal and plasmid DNA to be isolated. A small cryptic plasmid has been identified inRuminococcus flavefaciens strain 186. It is 5.2 kb long, contains a singleBamHI site, and two sites forBglII,EcoRI, andHindIII. This plasmid has potential in the development of genetic vectors for rumen bacteria.

Characterization of nonattaching mutants of Agrobacterium tumefaciens.

The first step in tumor formation by Agrobacterium tumefaciens is the site-specific binding of the bacteria to plant host cells. Transposon mutants of the bacteria which fail to attach to carrot suspension culture cells were isolated. These mutants showed no significant attachment to carrot cells with either microscopic or viable cell count assays of bacterial binding. The nonattaching mutants were all avirulent. When revertants of the mutants were obtained by enriching for bacteria which do bind to carrot cells, the bacteria were found to have regained the ability to bind to carrot cells and virulence simultaneously. These results suggest that the ability of the bacteria to bind to plant cells is required for virulence. Like the parent strain, all of the nonattaching mutants synthesized cellulose, but unlike the parent strain, they failed to aggregate carrot suspension culture cells. The transposon Tn5, which was used to obtain the mutants, was located on a 12-kilobase EcoRI fragment of the bacterial chromosomal DNA in all of the nonattaching mutants from strain C58. That the mutant phenotype was due to the Tn5 insertion was shown by cloning the Tn5-containing DNA fragment from the mutant bacteria and using it to replace the wild-type fragment in the parent strain by marker exchange. The resulting bacteria had the same mutant phenotype as the original Tn5 mutants; they did not attach to carrot cells, they did not cause the aggregation of carrot cells, and they were avirulent. No difference was seen between the parent strain and the nonattaching mutants in hydrophobicity, motility, flagella, fimbriae, beta-2-glucan content, size of lipopolysaccharide, or ability of the lipopolysaccharide to inhibit bacterial attachment to tissue culture cells. Differences were seen between the parent strain and the nonattaching mutants in the polypeptides removed from the bacteria during the preparation of spheroplasts. Three of the mutants were lacking a polypeptide of about 34 kilodaltons (kDa). One mutant was lacking the 34-kDa polypeptide and another polypeptide of about 38 kDa. The fifth mutant was lacking a polypeptide slightly smaller than the 34-kDa polypeptide missing in the other four mutants. These missing polypeptides all reappeared in the revertants of the mutants. Thus, bacterial binding to plant cells appears to require the presence of these polypeptides.

A rapid biochemical method for purifying high molecular weight bacterial chromosomal DNA for restriction enzyme analysis

A rapid biochemical method for purifying high molecular weight bacterial chromosomal DNA for restriction enzyme analysis

Action of citrinin on bacterial chromosomal and plasmid DNA in vivo and in vitro.

Citrinin, a mycotoxin of Penicillium citrinum and other species of the genera Penicillium and Aspergillus, caused the following effects at different concentrations in Escherichia coli. In vivo at 100 micrograms/ml single-strand breaks were caused in the chromosomal DNA. In the presence of 100 micrograms/ml, UV (254 nm)-induced DNA damage was repaired in the bacterial cells without need for a complete growth medium. At 300 micrograms/ml lambda ts prophage was induced in a lysogenic E. coli strain. In an E. coli strain carrying a F' lac plasmid, 4.7% of the cells displayed the Lac- phenotype after treatment with 200 micrograms of citrinin per ml, suggesting elimination of the F' factor. In vitro, DNA repair synthesis was observed at 5 micrograms of citrinin per ml in permeabilized cells, and replicative DNA synthesis was inhibited at 200 micrograms/ml. In these systems synthesis of stable RNAs was slightly diminished at 300 micrograms/ml, and protein synthesis was not affected at concentrations up to 450 micrograms/ml. Lambda and ColE1 plasmid DNA were cleaved in vitro when small amounts of copper ions were present. This DNA-attacking activity was prevented by NADPH, catalase, and superoxide dismutase and by higher concentrations of hydroxyl radical scavengers, suggesting the involvement of free radicals in the mechanism of action of citrinin on DNA.

Higher order structural organization of the nucleoid in the thiobacteriumThiosphaera pantotropha

Thiosphaera pantotropha cells treated with mitomycin C produced bacteriophages and showed cell lysis. Upon occurrence of cell lysis, samples were mounted for electron microscopy by negative staining. During mounting, the cell contents were spread at the surface of the support film. Besides polysomes, strands interpreted as DNA could be seen, most of them complexed with particles interpreted as DNA-binding proteins. Single and twisted strands were revealed, and complex structures with diameters around 35 nm were common. They exhibited an ordered arrangement of the proteins. Our findings suggest that bacterial chromosomal DNA complexed with DNA-binding proteins may be organized in higher order, similar to the compactation of nucleosome strands in eukaryotic chromosomal DNA.

Colony probing as an alternative to standard sequencing as a means of direct analysis of chromosomal DNA to determine the spectrum of single-base changes in regions of known sequence.

We demonstrate here the means to directly analyze bacterial chromosomal DNA for all classes of single-base mutations in a specific codon in any region of known sequence through the use of DNA probing as a powerful substitute for standard sequencing techniques. With this method, chromosomal DNA from hundreds of mutants can be examined for single-base changes without any DNA cloning. This method can conveniently provide a large data base for the assessment of all classes of single-base mutations occurring spontaneously or induced by a known or suspected mutagen. The method is demonstrated for the analysis of histidine-independent (His+) revertants of hisG46, a missense mutation of Salmonella typhimurium that can revert in six or seven ways.

Efficiency of Escherichia coli repair processes on uv-damaged transforming plasmid DNA

A comparison has been made of the efficiencies with which the dark repair processes of Escherichia coli act on ultraviolet irradiated bacterial chromosomal DNA and ultraviolet damaged transforming plasmid DNA. It is shown that postreplicational repair pathways act very inefficiently on transforming plasmid DNA, and that the majority of repair is carried out by excision repair pathways. However, even excision repair pathways act less efficiently on damaged plasmid DNA than they do on chromosomal DNA. The large effect of mutations in recB on plasmid survival suggests that the product of this gene may be essential for the excision repair pathways which act on plasmid DNA, but not for those which act on chromosomal DNA.

Polyethyleneglycol-induced transformation of Bacillus subtilis protoplasts by bacterial chromosomal DNA.

Bacillus subtilis protoplasts, which in the presence of polyethyleneglycol (PEG) are transformed by plasmid DNA (Chang and Cohen 1979) can also be transformed under these conditions by chromosomal DNA. Transformation in this case occurs at a much lower frequency, not fully accounted for by the heterogeneity of this DNA. Another unexpected feature of the transformation studied, which may explain why it previously went unnoticed, is that DNA concentrations higher than 1--2 microgram/ml decrease the yield of transformants, without showing signs of general toxicity. PEG-induced protoplasts (PIP) transformation for chromosomal markers operates normally with protoplasts prepared from a non-transformable bacterial mutant. The evidence indicates that both native linear and plasmid DNAs must somehow be forced into the cells as a result of PEG action. Denatured chromosomal DNA however is almost inactive in PIP transformation. No competition between chromosomal and plasmid DNAs could be detected, when the DNA tested as inhibitor was in tenfold excess.

Communication through fimbriae during conjugation in a fungus.

LONG fine hairs, termed fimbriae1 or pili2, have been observed in many bacteria and in fungi3–5. Bacterial sex pili transfer chromosomal and plasmid DNA during conjugation6. This paper describes evidence that the fimbriae of the smut fungus Ustilago violacea also transfer sex-specific molecules between the two conjugants.

Determination of the molecular complexity of double-stranded phage genome DNA from initial renaturation rates. The effect of DNA base composition.

The molecular complexity of double-stranded phage genome DNA was determined by an adaptation of the initial renaturation rate method (Gillis et al., 1970) . The exact conditions, the reproducibility, the reliability and the effects of salt concentration, optimal temperature, renaturation time, DNA concentration and base composition were determined. The method is sensitive: a sample size of 20 determinations yields 4% confidence limits on the sample mean. The molecular complexity and length of genome DNA from several phages were determined. Our results agree perfectly with the most reliable values proposed by Freifelder (1970) . Our method appears to offer advantages by its rapidity, reproducibility and reliability. A statistical treatment shows that the available literature data on the molecular weight of bacterial and phage chromosome DNA, and on the length of phage chromosome DNA, of the same organisms are so widely scattered that an eventual effect of the DNA base composition on the renaturation rate is smaller than the experimental scatter. When the most reliable literature data on phage T5, T7 and λ+ genome sizes (Freifelder, 1970) are used in conjunction with our k′ measurements, the effect of the % (G+C) on the renaturation rate is negligible. We propose to disregard this effect until more precise reference data become available.

Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli: VII. Mapping the ribosomal RNA genes of plasmid F14

Escherichia coli episome F14 contains two 16 S + 23 S ribosomal RNA gene sets. Their positions and structure have been studied by electron microscope heteroduplex methods. The A gene set maps from 81.4 to 86.7 kilobases from the counterclockwise junction of F DNA with bacterial chromosomal DNA of F14; the B gene set maps very close to the clockwise junction, that is, at map position 204.4 to 209.7 kilobases from the counterclockwise junction. The structure of the rRNA-rDNA hybrids indicates that each gene set consists of one 16 S gene (r16) of length 1.28 ± 0.12 kilobases, a spacer (rsp) of length 0.57 ± 0.12 kilobase and a 23 S gene (r23) of length 2.60 ± 0.20 kilobases. We propose the genetic notation rrn for the entire gene set. The structure of the DNA/DNA duplexes between the rrnA set and the rrnB set shows that there is a region of non-homology of length 0.29 ± 0.06 kilobase within the otherwise homologous rrnA and rrnB sequences. This non-homology region is probably part, but not all, of the spacer between the 16 S and 23 S genes.

Isolation of circular DNA molecules from whole cellular DNA by use of ATP-dependent deoxyribonuclease.

A technique is described for isolation of plasmid DNA in closed and open circular double-stranded forms from bacterial cells, by use of ATP-dependent deoxyribonuclease purified from Micrococcus luteus. This DNase, acting only upon linear DNA molecules, degrades all bacterial chromosomal DNA extracted in the linear form. Circular plasmid DNAs are left intact, and are then separated by sedimentation through a sucrose gradient. Unlike previous techniques for analysis of plasmid DNA, this technique can be used to isolate not only closed circular DNA but also open circular DNA. Several plasmids, such as those from phage (lambdadv1 and lambdadv21), a colicinogenic factor (Col E2), a sex factor (F(8)' gal), and "minicircles" in Escherichia coli 15, in both the open and closed circular forms, were well separated from chromosomal DNA by this technique.