Genome analysis of Bradyrhizobium japonicum serocluster 123 field isolates by using field inversion gel electrophoresis

The genomes of 11 Bradyrhizobium japonicum serocluster 123 field isolates were analyzed by using field inversion gel electrophoresis. Genomic fingerprints produced by digestion of intact genomic DNA in agarose plugs with the rare-cutting restriction enzymes AseI, DraI, SpeI, and XbaI showed that the isolates were genetically diverse. Few (30 to 50%) isolates exhibited the same fingerprint as the USDA serogroup strain with which they are antigenically related. Southern hybridization with a nifHD gene probe to the blotted field inversion electrophoresis gels provided further evidence of the relatedness between members of serogroups 123 and 127.     

A systematic study of field inversion gel electrophoresis.

The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field inversion gel electrophoresis (FIGE) were measured at different pulse times and electric fields. Also the ratios between forward and backward pulse times and/or field gradients were varied. The problem of 'band inversion' during FIGE, leading to an ambiguity in the mobility of large DNA fragments, was solved by using two dimensional gel electrophoresis with different parameters in the first and second dimension. The results are compared with those obtained with other pulsed electrophoresis systems and with a theoretical model.     

Pulsed field gel electrophoresis techniques for separating 1- to 50-kilobase DNA fragments

Conventional agarose gel electrophoresis separates DNA using a static electric field. The maximum size limit for separation of DNA by this method is about 20 kilobase pairs (kb). A number of new electrophoretic techniques which employ periodic reorientation of electric fields permit separation of DNA well beyond this size limit. We sought to determine whether the use of very fast (millisecond) field switching could improve separation of DNA in the size range of 1 to 50 kb. Additionally, we have compared the resolution obtained with each of the different field switching regimens for DNA in this size range. Switching intervals of from 0.2 to 900 ms were used with unidirectional pulsing of a single electric field, with pulsed field gels, and with field inversion gel electrophoresis. Plotting the mobility of DNA as a function of size demonstrates that under the conditions used, each of these techniques offers comparable resolution. We also have examined the separation obtained when field inversion gels are run with forward and reverse fields of equal voltage and different durations, versus using fields of equal duration and different voltages. Field inversion which uses forward and reverse fields of different voltages yields resolution which is superior to the other methods examined.     

The basis of high resolution separation of small DNAs by asymmetric-voltage field inversion electrophoresis and its application to DNA sequencing gels.

We have previously shown that asymmetric-voltage field inversion electrophoresis produces more uniform separation for fragments between 1 and 50 kilobases (kb) than other modes of pulsed field gel electrophoresis. We now report on the basis of this phenomenon. As in conventional electrophoresis, the pulsed field mobility of DNAs between 1 and 50 kb varies with voltage in a size dependent manner. The complex migration pattern obtained with asymmetric-voltage field inversion electrophoresis reflects the difference between the mobilities of each sized fragment under the conditions used for the forward and reverse fields. We have applied this technique to DNA sequencing gels and find improvement in resolution for single-stranded fragments in polyacrylamide gels.     

BBC microcomputer controlled field inversion gel electrophoresis

Agarose gel electrophoresis to separate DNA molecules is a widelyused technique in molecular biology but there is an upper limitto the sizes that can be resolved. Pulsed field techniques haveextended this limit but require expensive equipment. Here wedescribe a home-made control unit to interface conventionalelectrophoresis equipment to a BBC microcomputer for the purposesof field inversion gel electrophoresis. Received on October 6, 1987; accepted on November 10, 1987     

Clonal differences within M-types of the group A streptococcus revealed by pulsed field gel electrophoresis

Digestion of chromosomal DNA with the rare cutting restriction enzyme SfiI in association with pulsed field gel electrophoresis was used to observe restriction fragment length polymorphisms (RFLP) among isolates of group A Streptococcus. Streptococci examined included isolates belonging to the same M-type (epidemiologically related and unrelated), and isolates from other M-types. RFLP patterns were quite distinct between all serotypes tested. More importantly, isolates from within a serotype could be differentiated by this technique.     

Use of pulsed field gel electrophoresis to size the chromosome of the bacterial fish pathogen Yersinia ruckeri

Field inversion gel electrophoresis (FIGE) and contour-clamped homogeneous field (CHEF) electrophoresis were used to analyse the chromosome of Yersinia ruckeri. The 8 base-pair recognition endonucleases, NotI and SfiI, generated less than 47 DNA fragments whose size and distribution were appropriate for pulsed field separation. Each isolate displayed a characteristic restriction pattern, with about 20% of bands in common. Depending on the strain used, the estimated genome size for this bacterial fish pathogen ranged from 4460 to 4770 kilobase pairs.     

Chromosomal heterogeneity of various Mycoplasma hyopneumoniae field strains.

Restriction enzyme digestion and field inversion gel electrophoresis were used to analyze the chromosomes of strains of Mycoplasma hyopneumoniae and the related organism Mycoplasma flocculare. The chromosome size for the M. hyopneumoniae type strain was calculated from individual fragments to be 1,011.3 +/- 32.9 kbp. The chromosomes of M. hyopneumoniae field strains were approximately the same size. The restriction patterns obtained for the chromosomes of phenotypically similar M. hyopneumoniae strains were quite different. Therefore, the species M. hyopneumoniae seems to be very heterogeneous. A field inversion gel electrophoresis analysis of the entire chromosomes allowed us to distinguish M. hyopneumoniae strains easily and hence to characterize further the species M. hyopneumoniae. The chromosome size for M. flocculare was calculated to be 988.3 +/- 39.5 kbp. Restriction enzyme XhoI, which statistically should cut the M. hyopneumoniae chromosome frequently, did not cut the DNA of any of the M. hyopneumoniae strains but did digest M. flocculare DNA, indicating that there is a site-specific modification at CTCGAG which probably belongs to a restriction modification system in M. hyopneumoniae and is absent in M. flocculare.     

Mapping genomic organization by field inversion and two-dimensional gel electrophoresis: application to the murine T-cell receptor gamma gene family.

A new two-dimensional gel electrophoresis technique has been developed for the mapping of multigene families. Resolution in the first dimension is based on the generation of large size DNA fragments by infrequently-cutting restriction enzymes, and separation of these fragments by field inversion gel (FIG) electrophoresis. A second restriction enzyme digestion is then carried out with the separated DNA fragments in the agarose gel. Standard gel electrophoresis in the second dimension allows one to estimate the number of hybridizing genes contained in each large DNA fragment. We have also developed a novel method to increase the separation, resolution and hybridization signal in the second dimension by condensing the bands from the first dimension into spots. As an example, we have applied these techniques to determine the organization of the murine T-cell receptor gamma locus. The murine gamma gene family was found to be contained on two DNA fragments encompassing 195 kilobases of DNA. The two-dimensional gel electrophoresis method is particularly useful in the analysis of the organization of multigenic families where single copy probes are not readily available, and should extend the potential usefulness of field inversion gel electrophoresis in gene mapping.     

Nodulation and Competition for Nodulation of Selected Soybean Genotypes among Bradyrhizobium japonicum Serogroup 123 Isolates

Twenty recently obtained field isolates of Bradyrhizobium japonicum serogroup 123 were tested for their nodule mass production on the standard commercial soybean (Glycine max (L.) Merr. cv. Williams) and on two soybean plant introduction (PI) genotypes previously determined to restrict nodulation by strain USDA 123. Four of the field isolates showed similar restricted nodulation on the two genotypes, while all 20 isolates produced a normal amount of nodules on G. max cv. Williams. Serological analyses with adsorbed fluorescent antibodies showed that members of the 123 serotype ranked low in nodulation of the two PIs, in contrast to members of serotypes 127 and 129. Competition studies on the PIs indicated that isolates which were restricted were not competitive for nodule occupancy against strain USDA 110. However, unrestricted isolates of serogroup 123 were very competitive against USDA 110. On G. max cv. Williams, all serogroup 123 isolates tested were very competitive against USDA 110.     

Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis

A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.     

Comparison of Mycoplasma agalactiae isolates by pulsed field gel electrophoresis, SDS-PAGE and immunoblotting

Abstract We have analyzed 81 isolates of Mycoplasma agalactiae from four different regions of Italy between 1990 and 1995 in order to identify antigenic differences through SDS-PAGE and Western blotting and chromosomal DNA restriction endonuclease cleavage pattern differences. Antigenic variability in M. agalactiae isolates was investigated analyzing hydrophobic membrane protein fractions by immunoblotting using pooled sheep antiserum from naturally infected sheep. Large restriction fragments obtained cleaving genomic DNAs with Sma I, Nru I, Sal I, Xho I, Bss HII and Kpn I were analyzed by pulsed field gel electrophoresis. Genetic analysis indicates that isolates are all similar without intraspecific differences. This homogeneity was confirmed by immunoblotting: 80 and 50 kDa antigens are present in all strains analyzed.     

Field inversion gel electrophoresis with different pulse time ramps.

The influence of different pulse time ramps on the separation of yeast chromosomes with field inversion gel electrophoresis (FIGE) was investigated by the means of two dimensional gel electrophoresis. The problem of band inversion, which makes it difficult to distinguish DNA molecules of different size, has been solved by using double randomized pulse times. A major disadvantage of the field inversion technique is thereby overcome, making this system comparable to other pulsed field techniques.     

Cystic fibrosis: screening for a DNA deletion by field inversion gel electrophoresis

Summary We analyzed DNA of ten cystic fibrosis (CF) patients representing DNA of 19 different CF chromosomes and screened for deletions by means of field inversion gel electrophoresis (FIGE). No differences were detected after digestion of the DNA samples with the restriction enzymes Not I and Sfi I and hybridization with the probes MetH, MetD, J3.11, and 7C22. Thus the percentage of deletions occurring within the CF region and detectable with FIGE is less than 15.2% (95% confidence interval, N=19)     

Diversity among Field Populations of Bradyrhizobium japonicum in Poland

Genetic structure in field populations of Bradyrhizobium japonicum isolated in Poland was determined by using several complementary techniques. Of the 10 field sites examined, only 4 contained populations of indigenous B. japonicum strains. The Polish bradyrhizobia were divided into at least two major groups on the basis of protein profiles on polyacrylamide gels, serological reaction with polyclonal antisera, repetitive extragenic palindromic PCR fingerprints of genomic DNA, and Southern hybridization analyses with nif and nod gene probes. Serological analyses indicated that 87.5% of the Polish B. japonicum isolates tested were in serogroups 123 and 129, while seven (12.5%) of the isolates tested belonged to their own unique serogroup. These seven strains also could be grouped together on the basis of repetitive extragenic palindromic PCR fingerprints, protein profiles, and Southern hybridization analyses. Cluster analyses indicated that the seven serologically undefined isolates were genetically dissimilar from the majority of the Polish B. japonicum strains. Moreover, immuno-cross-adsorption studies indicated that although the Polish B. japonicum strains reacted with polyclonal antisera prepared against strain USDA123, the majority failed to react with serogroup 123- and 129-specific antisera, suggesting that Polish bradyrhizobia comprise a unique group of root nodule bacteria which have only a few antigens in common with strains USDA123 and USDA129. Nodulation studies indicated that members of the serologically distinct group were very competitive for nodulation of Glycine max cv. Nawiko. None of the Polish serogroup 123 or 129 isolates were restricted for nodulation by USDA123- and USDA129-restricting soybean plant introduction genotypes. Taken together, our results indicate that while genetically diverse B. japonicum strains were isolated from some Polish soils, the majority of field sites contained no soybean-nodulating bacteria. In addition, despite the lack of long-term soybean production in Poland, field populations of unique B. japonicum strains are present in some Polish soils and these strains are very competitive for nodulation of currently used Polish soybean varieties.     

DNA gel electrophoresis: effect of field intensity and agarose concentration on band inversion

We study the effect of electric field intensity and agarose gel concentration on the anomalous electrophoretic mobility recently predicted by the biased reptation model and experimentally observed for linear DNA fragments electrophoresed in continuous electric fields. We show that high fields and low agarose concentrations eliminate the physical mechanism responsible for anomalous DNA mobility and band inversion, in good agreement with theory, thus restoring the monotonic mobility-size relationship necessary for unambiguous interpretation of the results of DNA gel electrophoresis.     

Genotypic characterization of Ureaplasma species by pulsed field gel electrophoresis

We describe the first use of pulsed field gel electrophoresis to genotype human Ureaplasma species. This technique can distinguish between U. urealyticum and U. parvum, differentiate most of the 14 serovars from one another, and identify differences among clinical isolates of the same serovar.     

Genetic Diversity in Bradyrhizobium japonicum Serogroup 123 and Its Relation to Genotype-Specific Nodulation of Soybean

The genetic diversity among 20 field isolates of Bradyrhizobium japonicum serogroup 123 was examined by using restriction endonuclease digestions, one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis of total cell proteins, Southern hybridization analysis of nif and nod genes, and intrinsic antibiotic resistance profiles. All of the isolates were previously separated into three broad nodulation classes (low, medium, and high) based on their ability to form symbioses with specific soybean genotypes. Results of our studies indicate that there is a relationship between these three genotype-specific nodulation classes and groupings that have been made based on genomic DNA digestion patterns, sodium dodecyl sulfate-protein profiles, and Southern hybridizations to a nifHD gene probe. Intrinsic antibiotic resistance profiles and nodAB gene hybridizations were not useful in determining interrelationships between isolates and nodulation classes. Southern hybridizations revealed that two of the isolates had reiterated nod genes; however, there was no correlation between the presence of extra nodAB genes and the nodulation classes or symbiotic performance on permissive soybean genotypes. Hybridizations with the nif gene probe indicated that there is a relationship among serogroup, nodulation class, and the physical organization of the genome.     

Genome analysis of Pseudomonas aeruginosa by field inversion gel electrophoresis

Abstract The genome of Pseudomonas aeruginosa was analysed by digestion with rare-cutting restriction endonucleases and subsequent field inversion gel electrophoresis (FIGE). P. aeruginosa strain PAO and the 17 IATS strains were investigated. Each strain displayed a unique pattern of restriction fragments. Digestion with Dra I and Ssp I yielded, respectively 7–11 and 2–5 fragments of more than 130 kb in size, indicating the non-random occurrence of AT-rich sequences in the P. aeruginosa genome. The genome size of P. aeruginosa PAO was estimated to be (2.2 ± 0.3) × 10⁶ bp. The applications of DNA fingerprinting for gene cloning, construction of a physical chromosome map, and epidemiological studies, are discussed.     

Phylloplane bacteria increase seedling emergence, growth and yield of field-grown groundnut (Arachis hypogaea L.)

AIM: To isolate and characterize groundnut-associated bacterial isolates for growth promotion of groundnut in field. METHODS AND RESULTS: Three hundred and ninety-three groundnut-associated bacteria, representing the geocarposphere, phylloplane and rhizosphere, and endophytes were applied as seed treatment in greenhouse. Maximum increase in plant biomass (up to 26%) was observed following treatment with a rhizosphere isolate identified as Bacillus firmis GRS 123, and two phylloplane isolates Bacillus megaterium GPS 55 and Pseudomonas aeruginosa GPS 21. There was no correlation between the production of L-tryptophan-derived auxins and growth promotion by the test isolates. Actively growing cells and peat formulations of GRS 123 and GPS 55, and actively growing cells of GPS 21, significantly increased the plant growth and pod yield (up to 19%) in field. Rifampicin-resistant mutants of GRS 123 and GPS 21 colonized the ecto- and endorhizospheres of groundnut, respectively, up to 100 days after sowing (DAS), whereas GPS 55 was recovered from both the habitats at 100 DAS. CONCLUSION: Seed bacterization with phylloplane isolates promoted groundnut growth indicating the possibility of isolating rhizosphere beneficial bacteria from different habitats. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of phylloplane bacteria as effective plant growth-promoting rhizobacteria (PGPR) broadens the spectrum of PGPR available for field application.