Determination of chromosome size and number of rrn loci in Lactobacillus plantarum by pulsed-field gel electrophoresis

Abstract When genomic DNA fragments from Streptomyces venezuelae ISP5230 were probed at moderate stringency with recA from Mycobacterium tuberculosis a 2.0-kb Sma I fragment was identified. The fragment was isolated by cloning a Bam HI digest of S. venezuelae DNA in pHJL400 and screening the plasmids in Escherichia coli by Southern hybridization using a sib-selection technique. Sequencing the hybridizing region located an open reading frame encoding 377 amino acids. Its deduced amino acid sequence resembled that of recA genes from other bacteria. The cloned S. venezuelae gene conferred partial resistance to ethyl methanesulfonate when expressed in E. coli from the lacZ promoter.     

Estimation of circular DNA size using gamma-irradiation and pulsed-field gel electrophoresis

A method is described for estimating the size of large circular DNAs found within complex chromosomal DNA preparations. DNAs are treated with low levels of gamma-irradiation, sufficient to introduce a single double-stranded break per circle, and the resulting linear DNA is sized by pulsed-field electrophoresis and blot hybridization. The method is fast, reproducible, and very conveniently applied to the agarose-enclosed chromosomal DNA preparations commonly used in pulsed field electrophoresis.     

High molecular weight DNA from nucleated erythrocytes for use in pulsed-field gel electrophoresis (PFGE)

Nucleated erythrocytes of lower vertebrates provide a source of genomic DNA that can be used in pulsed-field gel electrophoresis (PFGE) studies. Difficulties reported in the preparation of chicken erythrocyte DNA for analysis by PFGE suggest that the presence of hemoglobin iron may result in iron-mediated DNA degradation. We report here modifications to the procedures established for isolation of high molecular weight DNA from mammalian cells. By increasing the volume of buffers and extending the incubation periods to allow for the removal of hemoglobin iron, we have successfully prepared channel catfish erythrocyte DNA suitable for analysis of PFGE.     

Determination of Wolbachia genome size by pulsed-field gel electrophoresis

Genome sizes of six different Wolbachia strains from insect and nematode hosts have been determined by pulsed-field gel electrophoresis of purified DNA both before and after digestion with rare-cutting restriction endonucleases. Enzymes SmaI, ApaI, AscI, and FseI cleaved the studied Wolbachia strains at a small number of sites and were used for the determination of the genome sizes of wMelPop, wMel, and wMelCS (each 1.36 Mb), wRi (1.66 Mb), wBma (1.1 Mb), and wDim (0.95 Mb). The Wolbachia genomes studied were all much smaller than the genomes of free-living bacteria such as Escherichia coli (4.7 Mb), as is typical for obligate intracellular bacteria. There was considerable genome size variability among Wolbachia strains, especially between the more parasitic A group Wolbachia infections of insects and the mutualistic C and D group infections of nematodes. The studies described here found no evidence for extrachromosomal plasmid DNA in any of the strains examined. They also indicated that the Wolbachia genome is circular.     

Genome size of Myxococcus xanthus determined by pulsed-field gel electrophoresis.

Genomic DNA of the myxobacterium Myxococcus xanthus was digested with the rare cutting restriction endonuclease AseI or SpeI, and the restriction products were separated by pulsed-field gel electrophoresis. Transposons Tn5-132 and Tn5 lac, which contain AseI restriction sites, were used to determine the number of restriction fragments in each band. The size of the genome was determined by adding the molecular sizes of the restriction products. The genomes of strains DK101, MD2, and DZF1 have identical restriction patterns and were estimated to be 9,454 +/- 101 kilobase pairs from the AseI digestions and 9,453 +/- 106 kilobase pairs from the SpeI digestions. DK1622, which was derived from DK101 by treatment with UV light, has suffered a 220- to 222-kilobase-pair deletion that removed an AseI and an SpeI restriction site. The deleted DNA may consist exclusively of Mx alpha-associated sequences.     

Construction of yeast artificial chromosome libraries by pulsed-field gel electrophoresis

Yeast artificial chromosome (YAC) libraries have been constructed from a variety of organisms using different approaches. This protocol outlines in detail the construction of YAC libraries with large inserts using size fractionation of partially digested DNA by pulsed-field gel electrophoresis.     

Circularity of the Caulobacter crescentus chromosome determined by pulsed-field gel electrophoresis.

Previous genetic analyses of the Caulobacter crescentus chromosome have resulted in the construction of a linear genetic map. To establish the circularity of the C. crescentus chromosome, restriction fragments generated by digestion with AseI and SpeI were analyzed by pulsed-field gel electrophoresis and Southern hybridization. The size of each fragment was calculated and used to demonstrate that C. crescentus has a genome size of approximately 4,000 kilobases. In addition, both enzymes gave rise to large DNA fragments which contained genes from both ends of the genetic map. Thus, there is physical linkage between the genes at the ends of the genetic map and the chromosome is circular. Since this region of the chromosome appears to contain the replication terminus, we propose that recombination occurs at a high frequency in the vicinity of the terminus. This high frequency of recombination would prevent genetic linkage from being observed between genes on opposite sides of the terminus. Additional experiments using insertions which introduced new AseI and DraI restriction sites into the genome allowed us to calculate the physical distance between genes located in the vicinity of the replication terminus.     

Estimation of ruminal bacteriophage numbers by pulsed-field gel electrophoresis and laser densitometry.

To investigate phage activity in the rumen, a method for quantifying phage has been developed. By differential centrifugation and ultrafiltration, phage particles were separated and concentrated from ruminal fluid. Linear double-stranded DNA from this fraction containing predominantly tailed phage was isolated and separated by size, using pulsed-field gel electrophoresis (PFGE). Laser densitometry of gel photographs allowed the numbers of phages with DNA in each size region to be calculated and, therefore, the total numbers per milliliter of ruminal fluid to be estimated. Phage numbers were estimated to be between 3 x 10(9) and 1.6 x 10(10) particles ml of ruminal fluid-1. The phage population, as gauged by the appearance of DNA on PFGE gels, had two major components. A broad region of DNA between 30 and 200 kb was always present on PFGE gels. It appears this region comprises DNA from a great many different phages and would include most of the temperate phages. In addition, discrete DNA bands ranging in size from 10 to 850 kb were frequently observed. DNA from one such band, of 12 kb in size, was shown to consist primarily of a single DNA type, suggesting that it originated from a specific phage. It is postulated that the discrete bands are due to epidemics or blooms of phage activity from specific, probably lytic, phages. The method that has been developed will greatly enhance future investigations into the interactions between the ruminal phage population, the ruminal bacterial population, and animal nutrition and growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nucleic acid sequence-based amplification (NASBA) detection of medically important Candida species.

Nucleic Acid Sequence Based Amplification (iNASBA), an isothermal amplification technique for nucleic acids, was evaluated for the identification of medically important Candida species using primers selected from 18S rRNA sequences conserved in fungi. An RNA fragment of 257 nucleotides was amplified for Candida albicans. Nineteen different fungi were tested for rRNA amplification with the NASBA. All were positive when analyzed on agarose gel, whereas human RNA was negative. For the identification of Candida species, NASBA amplification products were analyzed in an enzyme bead-based detection format, using species-specific biotinylated probes and a generic Candida HRPO probe or a membrane-based system using biotinylated probes and avidin-HPRO. Discrimination of the major human pathogenic Candida spp. was based on a panel of biotinylated probes for C. krusei, C. tropicalis, C. albicans, C. glabrata, and C. lusitaniae. Using rRNA dilutions obtained from pure cultures of C. albicans, the combination of NASBA and the enzymatic bead-based detection yielded a sensitivity equivalent to 0.01 CFU. In a model system using 1 ml of artificially contaminated blood as few as 1-10 CFU of C. albicans could be detected. Testing of 68 clinical blood samples from patients suspected of candidemia showed that eight samples were positive for C. albicans and one for C. glabrata. Testing of 13 clinical plasma samples from patients suspected of fungemia identified the presence of C. albicans in two specimens. The whole procedure of sample preparation, amplification and identification by hybridization can be performed in 1 day. This speed and the observed sensitivity of the assay make the NASBA a good alternative to PCR for the detection of candidemia.     

Characterisation of persistent and sporadic Listeria monocytogenes strains by pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP)

This study was set up to evaluate the genetic similarity or dissimilarity of persistent and sporadic Listeria monocytogenes strains existing in eleven food processing facilities, including fish, dairy, meat and poultry processing plants. In each plant persistent and sporadic strains were selected on the basis of PFGE typing results. A total of 17 strains representing persistent strains and 38 sporadic strains originating from eleven food processing plants were included in the study. PFGE macrorestriction patterns of persistent and sporadic strains from different processing plants were compared and the strains were further studied by amplified fragment length polymorphism (AFLP), being a characterisation method giving more whole genome based information. The 17 persistent and 38 sporadic strains showed 14 and 35 pulsotypes, 14 and 28 AFLP types, respectively. The combination of PFGE and AFLP typing results yielded a total of 48 genotypes. Thirteen of 15 genotypes presented by persistent strains were only associated with persistent strains and similarly 94% (33/35) of genotypes showed by sporadic strains were recovered among sporadic strains only. Our results showed that L. monocytogenes strains causing persistent contamination differ from sporadic strains. In AFLP analysis persistent strains did not, however, form any specific clusters and neither was there any difference between the known two genomic groups. These results indicate that even though persistent strains differ from sporadic strains there seems not to be any specific evolutional lineage of persistent strains.     

Karyotyping of Candida albicans and Candida glabrata isolates from recurrent vaginal infections by pulsed-field gel electrophoresis

In the present study, 16 women with recurrent vulvovaginal candidiasis (RVVC) due to Candida albicans and Candida (Torulopsis) glabrata were followed for a period of 4 to 12 months, and 36 vaginal isolates were evaluated by pulsed-field gel electrophoresis (PFGE). Eleven women were infected by C. albicans and 5 by C. glabrata. Three electrophoretic karyotypes of C. albicans and 3 of C. glabrata were identified throughout the follow-up. All patients but one was infected with the same karyotype of C. albicans or C. glabrata during the follow-up period. Two different karyotypes of C. glabrata were identified in one patient in the course of 12 months. The results confirmed the diversity of the karyotypes of C. albicans and C. glabrata causing vulvovaginitis, and demonstrated the persistence of colonization with the same strain over different periods of time despite therapy (15/16 women).     

Comparison of the separation of Candida albicans chromosome-sized DNA by pulsed-field gel electrophoresis techniques.

Pulsed-field gel electrophoresis techniques were used to study chromosome-sized DNA molecules of C. albicans. Chromosome-sized DNA of two strains of Candida albicans has been resolved into 8 bands by orthogonal-field-alternation gel electrophoresis (OFAGE). Six bands were observed in chromosomal preparations of C. albicans using field-inversion gel electrophoresis (FIGE). Differences in the electrophoretic mobilities of bands of the strains of C. albicans examined suggests that chromosome-length polymorphisms exist and make it difficult to correlate the banding patterns among strains. These correlations were facilitated, however, by assignment of C. albicans chromosomes by hybridization using a collection of cloned DNA probes specific for each of the 8 observed bands. Southern blotting showed that the 6 FIGE bands consisted of 4 singlets and 2 comigrating doublets, accounting for the 8 bands observed by OFAGE analysis. The agreement between OFAGE and FIGE analysis suggests that the C. albicans haploid genome contains a minimum of 8 chromosomes.     

Molecular typing of Salmonella weltevreden and Salmonella chincol by pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR)

Genomic DNA of Salmonella weltevreden (10 isolates from poultry, two isolates each from raw vegetables and river water) and S. chincol (15 isolates from poultry) were characterized by pulsed-field gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus-polymerase chain reaction (ERIC-PCR) analysis. These isolates originated from a single location in Kajang, Selangor. The results of the PFGE and ERIC-PCR were analysed and comparisons were made using GelCompar software. ERIC-PCR with primers ERIC1R and ERIC2 discriminated the S. weltevreden into five clusters and two single isolates and S. chincol into two clusters and two single isolates at a similarity level of 80%, respectively. PFGE produced a single cluster and eight single isolates for S. weltevreden, and one cluster and 11 single isolates for S. chincol at a similarity level of 80% after digestion with the restriction enzyme XbaI, respectively. These results demonstrate that both PFGE and ERIC-PCR are suitable tools for molecular typing of the isolates examined. This revised version was published online in July 2006 with corrections to the Cover Date.     

Legionella pneumophila in cooling towers: fluctuations in counts, determination of genetic variability by pulsed-field gel electrophoresis (PFGE), and persistence of PFGE patterns

The concentrations of Legionella pneumophila in cooling towers may vary considerably over short periods of time, producing significant fluctuations throughout the year. Despite genetic variability, in small geographical areas the same indistinguishable pulsed-field gel electrophoresis patterns may be shared among different cooling towers and persist over time.     

Bacterial genome mapping by two-dimensional pulsed-field gel electrophoresis (2D-PFGE)

Macrorestriction mapping is often the first step toward a thorough physical and genetic characterization of a bacterial genome. The problem of deducing the order of partially or completely digested macrorestriction fragments to yield a physical genome map may readily be solved by applying twodimensional pulsed-field gel electrophoresis (2D-PFGE) techniques. These powerful methods are quick and technically easy to perform; specifically, they are independent of DNA probes and should therefore be applicable to any bacterial species irrespective of its prior genetic characterization. In this article, detailed step-by-step protocols are given to set up, run, and evaluate 2D pulsed-field gels. Two basic methods are described: partial/complete 2D gels of one restriction enzyme and complete/complete 2D gels of two different restriction enzymes. Other topics include preparation of bacterial genomic DNA, screening for suitable rare-cutting restriction enzymes and determination of optimal running conditions. Accompanied by many notes, these protocols are meant to offer the novice a sound and rapid access to these important methods.     

Construction of yeast artificial chromosome libraries with large inserts using fractionation by pulsed-field gel electrophoresis.

A method for constructing yeast artificial chromosome (YAC) libraries with large insert sizes is reported. High molecular weight human DNA was partially digested with EcoRI and cloned in the vector pYAC4. When unfractionated DNA was used, the mean YAC size was 120kb. Fractionation by pulsed-field gel electrophoresis using a 'waltzer' apparatus to remove small DNA fragments increased the mean YAC size to congruent to 220kb or congruent to 370kb depending on the fractionation conditions. Ligated DNA prepared by this method was stable at 4 degrees C and routinely yielded transformation efficiencies of greater than 700 colonies/micrograms. It should be possible to extend the method to produce even larger inserts and to use high molecular weight DNA from any source.     

Differentiation of Erwinia amylovora strains by pulsed-field gel electrophoresis.

Erwinia amylovora strains, isolated from several host plants in various geographic regions during different years, were analyzed by pulsed-field gel electrophoresis (PFGE) after digestion of the DNA from lysed, agar-embedded cells with rare-cutting restriction enzymes. The banding patterns obtained with enzyme XbaI digests revealed significant differences among strains from different areas. North American strains E9 and Ea-Rb, a Rubus strain, were highly divergent from other E. amylovora strains. French strains were different from central European and English strains. E. amylovora strains from central Europe and New Zealand had identical PFGE patters, as had strains from Egypt, Greece, and Turkey. PFGE of genomic DNA from American and English strains gave rise to dissimilar patterns. Patterns of some American strains resembled those from strains isolated in other parts of the world. The restriction fragment length polymorphisms observed by PFGE analysis can be used to group strains and may give hints about the course of distribution of the plant disease. From the sizes of the restriction fragments obtained, a molecular mass of approximately 4.5 Mb was calculated for the genome of E. amylovora.     

Comparison of coagulase-negative staphylococci by pulsed-field gel electrophoresis

Five pathogenic strains each of Staphylococcus epidermidis, S. haemolyticus, S. lugdunensis and S. schleiferi were analysed by conventional electrophoresis and field inversion gel electrophoresis. For these coagulase-negative staphylococci, the restriction endonuclease SmaI emerged as the most suitable enzyme for pulsed-field electrophoresis by providing an adequate number of clearly separated DNA fragments. Field inversion gel electrophoresis confirmed the differences among strains already discriminated by conventional electrophoresis, and furthermore, differentiated strains which had previously appeared identical. Among the species that were studied, S. epidermidis showed great genomic diversity with a few common bands. On the contrary, S. haemolyticus, S. lugdunensis and S. schleiferi showed less diversity. Although these minor variations may be epidemiologically significant, this question has to be investigated on a larger number of strains.     

Quantitative hybridization to genomic DNA fractionated by pulsed-field gel electrophoresis.

Hybridization to genomic DNA fractionated by CHEF electrophoresis can vary >100-fold if the DNA is acid depurinated prior to Southern blotting. The level of hybridization is high or low depending on whether the molecule being analyzed migrates at a size coincident with or different from the size of the majority of genomic DNA in the sample, respectively. Techniques that avoid acid depurination including in-gel hybridizations and UV irradiation of DNA prior to blotting provide more accurate quantitative results. CHEF analysis of DNA molecules containing repetitive satellite sequences is particularly prone to this effect.