Pulsed-field gel electrophoresis for genotypic comparison of Rhizobium bacteria that nodulate leguminous trees

Abstract Pulsed-field gel electrophoresis (PFGE) was applied to characterize Rhizobium bacteria isolated from the root nodules of Acacia senegal and Prosopis chilensis trees growing in Sudan and Keya. For the electrophoresis, the total DNA of 42 isolates, embedded in agarose, was digested by a rare-cutting restriction endonuclease, Xba I. The PFGE run resulted in good resolution of the DNA fragments and gave the strains distinctive fingerprint patterns. The patterns were analysed visually and using automated clustering analysis, which divided the strains into groups resembling the results generated by numerical taxonomy. However, several strains had unique banding patterns, which indicates that these strains are genetically very diverse.     

A simple and rapid method for the differentiation and identification of thermophilic bacteria

In total, 170 strains of thermophilic bacteria were isolated from deep-sea hydrothermal fields in the Pacific Ocean and a hot spring in Xiamen of China. To facilitate the identification of thermophilic strains, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole-cell proteins of these strains was first performed. The results showed that there exist four different protein patterns, indicating that the 170 strains might belong to four species or genera. The RAPD (random amplified polymorphic DNA) profiles of nine representative strains were consistent with those of SDS-PAGE. To further identify the species of the nine strains, their 16S rDNA sequences were analyzed. The results showed that the nine strains fell into four species of three genera, which was the same as revealed by SDS-PAGE. Therefore, SDS-PAGE of whole-cell proteins could be used as a rapid and simple method for the discrimination of thermophilic bacteria as the first step of species identification.     

Distribution of repetitive DNA sequences in eubacteria and application to fingerprinting of bacterial genomes.

Dispersed repetitive DNA sequences have been described recently in eubacteria. To assess the distribution and evolutionary conservation of two distinct prokaryotic repetitive elements, consensus oligonucleotides were used in polymerase chain reaction [PCR] amplification and slot blot hybridization experiments with genomic DNA from diverse eubacterial species. Oligonucleotides matching Repetitive Extragenic Palindromic [REP] elements and Enterobacterial Repetitive Intergenic Consensus [ERIC] sequences were synthesized and tested as opposing PCR primers in the amplification of eubacterial genomic DNA. REP and ERIC consensus oligonucleotides produced clearly resolvable bands by agarose gel electrophoresis following PCR amplification. These band patterns provided unambiguous DNA fingerprints of different eubacterial species and strains. Both REP and ERIC probes hybridized preferentially to genomic DNA from Gram-negative enteric bacteria and related species. Widespread distribution of these repetitive DNA elements in the genomes of various microorganisms should enable rapid identification of bacterial species and strains, and be useful for the analysis of prokaryotic genomes.     

Comparison of ribotyping, pulsed-field gel electrophoresis and random amplified polymorphic DNA for typing Clostridium difficile strains

Abstract Clostridium difficile is a Gram-positive sporulating anaerobic bacillus which causes pseudomembranous colitis. Nosocomial acquisition of this bacteria has proved frequent, and epidemiological markers are needed to recognize and control common-source outbreaks. We therefore compared the results of pulsed-field gel electrophoresis (PFGE) after restriction with Sma I or Nru I, random-amplified polymorphic DNA (RAPD) using 3 10-mer oligonucleotides, and ribotyping to differentiate between 30 unrelated strains of C. difficile belonging to 8 serotypes. The strains were separated into 26 different types by PFGE, 25 by RAPD, but into only 18 types by ribotyping. Median percentages of similarity between strains ranged from 27 in the PFGE assay to 90 in the ribotyping assay, but there was good agreement between the 3 methods for the clustering of strains. PFGE was more time-consuming than RAPD but its patterns were easier to analyze.     

Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR

On the basis of 23S rRNA gene sequences, 1 universal forward and 4 taxon (species/biovar)-specific reverse primers were designed for multiplex PCR to aid in identification and differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2. In reactions with DNA of 119 bacterial strains belonging to: Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Phyllobacterium, as well as phytopathogenic bacteria representing various genera, the primers developed for identification of A. vitis, A. rubi or Agrobacterium biovar 1 amplified only DNA of strains belonging to these taxa, producing fragments of the expected sizes: 478, 1006 and 184bp, respectively. However, in the case of the primer developed for identification of Agrobacterium biovar 2, the characteristic 1066bp PCR product was obtained not only with DNA of this biovar, but also with DNA of 3 atypical biovar 1 strains and some rhizobial strains. Differentiation between Agrobacterium biovar 2 and the other strains was possible using the restriction analysis of this product with endonuclease Alw26I. The method developed is an excellent tool for rapid classification of these 4 taxa of Agrobacterium.     

Identification and characterization of Streptococcus thermophilus strains by pulsed-field gel electrophoresis

The chromosomal DNA of a number of strains of the lactic acid bacterium Streptococcus thermophilus was analysed with the aim of rapidly differentiating and assessing the characteristics of each strain. Pulsed-field gel electrophoresis was used to separate large DNA fragments formed by the restriction enzymes Sma I, Sfi I or Apa I. Hybridization with a non-radioactive DNA probe confirmed the identification of strains as Strep. thermophilus and analysis of the electrophoretic patterns differentiated some strains from others. A more extensive study of the pulsed-field electrophoresis restriction patterns of new isolates of Strep. thermophilus may facilitate assessment of their technological properties by comparison of their restriction patterns with those of reference strains.     

Esterase electrophoretic polymorphism of human and animal strains of Clostridium perfringens.

Esterase electrophoretic polymorphism in human and animal strains of Clostridium perfringens was studied by using polyacrylamide-agarose gel electrophoresis. Five types of esterases, designated E-I to E-V and defined by their hydrolytic specificities toward five synthetic substrates, were found in protein extracts of bacteria grown without glucose (glucose-containing media allowed only the expression of esterase E-I). Mobility variants of esterase E-I, which hydrolyzes alpha- and beta-naphthyl acetates and butyrates, were used as a basis for the distribution of strains into 11 zymogroups. When all five types of esterases and their electrophoretic variants were considered, 77 electrophoretic types (ETs) could be described for the 89 strains tested. Animal strains did not constitute a distinctive subpopulation, as revealed by their distribution in the zymogroups and by clustering analysis. Statistical analysis also emphasized the importance of esterase E-IV (which hydrolyzes only naphthyl acetates) and esterase E-V (which hydrolyzes only alpha-naphthyl acetate) in clustering by the relatedness of the ETs. ETs allowed the epidemiological characterization of stool isolates recovered from elderly inpatient residents and from adolescent chronic-care psychiatric patients. These results indicate that esterase electrophoretic typing may be a marker for epidemiological and ecological analyses.     

Application of agarose gel electrophoresis to the characterization of plasmid DNA in drug-resistant enterobacteria.

A simple gel electrophoresis method has been described for the detection of plasmid DNA in bacteria (Meyers et al., 1976). We investigated further the problems encountered in using this method for the analysis of plasmids in wild enterobacterial strains. The migration of open circular and linear plasmid DNA was examined, since these forms sometimes caused difficulty in the interpretation of the plasmid content of uncharacterized strains. Electrophoresis at different agarose concentrations was employed to resolve clearly plasmid DNA from the chromosomal DNA fragments in the crude preparations. Dissociation of some plasmids occurs in Salmonella typhimurium, and this was detected by electrophoresis. The technique was applied to the study of drug-resistant strains of S. typhimurium phage type 208 from several Middle Eastern countries. The cultures carry a drug resistance plasmid of the FIme compatibility group, and at least two other plasmids which were detected and identified by gel electrophoresis. The studies supported and extended the genetic findings and provided information on the distribution of particular plasmids.     

Detection of root mat associated Agrobacterium strains from plant material and other sample types by post-enrichment TaqMan PCR

AIMS: The development of a fluorogenic, 5' nuclease, TaqMan PCR assay for the detection of Ri-plasmids from root mat inducing Agrobacterium biovar 1 strains. METHODS AND RESULTS: A TaqMan probe and primer set were designed within the T-DNA sequence of a known root mat inducing Agrobacterium strain. One hundred and ten Agrobacterium and closely related bacteria were tested using this novel PCR and compared with results from a conventional PCR which detects Ti and Ri-plasmids. The Agrobacterium selective media, Medium 1A was modified into broth form for use as an enrichment of the pathogen from samples prior to the TaqMan PCR. CONCLUSIONS: The root mat pathogen was detected successfully from a range of sample types using the enriched fluorogenic PCR assay, negating the need for complex DNA extraction procedures and post-PCR processing techniques such as gel electrophoresis. The technique is therefore a rapid and cost-effective detection method. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first known report of a fluorogenic, 5' nuclease, TaqMan assay designed to detect an Agrobacterium plant pathogen. The method can be used as a model system for the detection of other Agrobacterium pathogens.     

Estimation of ribosomal RNA operon (rrn) copy number in Acinetobacter isolates and potential of patterns of rrn operon-containing fragments for typing strains of members of this genus

The copy number of the rrn operon in 70 strains of Acinetobacter including the type strains of almost all the genomic species with validated names was estimated after digestion of their genomic DNA by the restriction enzymes BglII and PstI, and Southern blotting. Copy number estimates varied between and among species, with between 3 and 7 rrn operon copies detected. Copy number estimates obtained from the same strain with the two enzymes sometimes varied. BglII generated RFLP patterns of the rrn containing fragments obtained from Southern blots after agarose gel electrophoresis were examined for their value in identifying Acinetobacter isolates. This method was very reproducible with the same fragment pattern always generated from the same isolate on repeated analysis. Often multiple strains of the same genomic species gave identical or very similar patterns (e.g. Acinetobacter baylyi), clustering closest together on the dendrogram generated after numerical analysis of these patterns. However, with some, like BG5 and BG8, the patterns derived from the different strains, some of which had been placed in this genomic species from DNA:DNA hybridization data, varied considerably to each other and to the type strain. Little similarity was seen when relationships between these strains based on these patterns were compared to those using DNA:DNA hybridization data. Often these patterns could be used to question earlier identification of strains using phenotypic characters. Thus, strain AB82 thought to belong to genomic species 5 gave an identical pattern to A. bouvetii(T) (DSM 14964). In some cases this pattern analysis suggested that novel species of Acinetobacter might exist among the strains examined.     

AUTOMATED 35S-LABELLED PROTEIN ELECTROPHORESIS FOR IDENTIFICATION OF STRAINS OF RHIZOBIUM LEGUMINOSARUM bv. TRIFOLII

A technique for strain-level identification within a Rhizobium biovar is described, based on automated sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of ³⁵S-labelled proteins, offering substantial improvements on existing SDS-PAGE methods particularly in the areas of standardization of electrophoresis conditions and rapidity of positive identification. Gels were analysed with a β-scanner, the beta particle emission data being directly relayed to an IBM PC/AT computer for subsequent manipulation. Analysis of the total protein profiles obtained by this method revealed regions of variability between strains of R. leguminosarum bv. trifolii. Automated comparison of these regions enabled identification of strains. The method was successfully used for identifying root nodule isolates obtained from competition studies between known pairs of R. leguminosarum bv. trifolii strains inoculated onto Trifolium repens.     

黑木耳菌株酯酶同工酶酶谱多样性研究

目的：为了探索黑木耳菌株之间的遗传距离,构建出供试菌株酯酶同工酶鉴别图。方法：采用垂直板聚丙烯酰胺凝胶电泳对21个黑木耳菌株酯酶同工酶的酶谱多样性进行了研究。结果：酯酶同工酶电泳各个菌株分别具有2~7条酶带,其中在Rf值为0.344处,21个菌株都有谱带出现。21个菌株之间的遗传相似系数在0.167-1.000之间,应用NTSYS软件进行聚类分析,当相似水平为0.73时,可将供试的21个黑木耳菌株分为6个不同的类群。结论：酯酶同工酶可以有效快捷的对黑木耳菌株进行菌种鉴定,是黑木耳菌株遗传多样性研究的理想手段。     

Rapid lactic acid bacteria identification in dairy products by high-resolution melt analysis of DGGE bands

Aim: To investigate the application of high‐resolution melt (HRM) analysis for rapid species‐level identification of lactic acid bacteria (LAB) communities in dairy products, as well as for bacterial community profiling and monitoring. Methods and Results: First, comparisons of HRM profiles of known reference strains of LAB and their denaturing gradient gel electrophoresis (DGGE) bands showed very good agreement, allowing species recognition and identification from DGGE bands by HRM. Second, samples of cheese, kefir grains and kefir were characterized by PCR‐DGGE, and melting profiles of DGGE bands were compared with known reference strains. Of the 13 DGGE bands, ten were identified by HRM by comparison with the reference strains and only three required sequencing for identification. Use of HRM profiling for comparison and monitoring of total LAB communities from dairy products or starter cultures was also evaluated, and good agreement was found when comparing clustering of DGGE band profiles with clustering of HRM melting profiles. Conclusion: Identification of DGGE bands is possible by comparison of HRM melting profiles with known reference strains. Significance and Impact of the Study: HRM profiling is suggested as an additional approach for identification of DGGE bands.     

Universal PCR primers for detection of phytopathogenic Agrobacterium strains.

Two PCR primer pairs, based on the virD2 and ipt genes, detected a wide variety of pathogenic Agrobacterium strains. The endonuclease domain of VirD2 protein, which cleaves transferred DNA (T-DNA) border sequences, is highly conserved; primer oligonucleotides specific for the endonuclease portion of virD2 detected all pathogenic strains of Agrobacterium tested. PCR primers corresponding to conserved sequences in ipt, the T-DNA-borne cytokinin synthesis gene, detected only Agrobacterium tumefaciens and distinguished it from Agrobacterium rhizogenes. The virD2 and ipt primer pairs did not interfere with each other when included in the same PCR amplification, and this permitted simultaneous detection of both genes in a single reaction. One nonpathogenic Agrobacterium radiobacter strain contained virD2 but not ipt; we speculate that this strain arose from a pathogenic progenitor through a deletion in the T-DNA. The virD2 primer pair appears to be universal for all pathogenic Agrobacterium species; used together, the primer sets reported here should allow unambiguous identification of Ti plasmid DNA in bacteria isolated from soil and plants.     

Differentiation of Citrobacter strains using chromosomal DNA restriction patterns

The aim of this study was checking of the usefulness of chromosomal DNA restriction patterns in differentiation of Citrobacter strains. Molecular characterization of total 56 isolates of Citrobacter from Poland and Czech Republic, was performed by pulsed-field gel electrophoresis after digestion of chromosomal DNA with restriction endonuclease Xba I (5'-TCTAGA-3'). Chromosomal DNA of all tested Citrobacter strains gave after electrophoresis 12 to 21 bands and patterns consisting of 12 to 21 fragments ranging in size from 790 kb to 48.5 kb and smaller, which where not distinguishable. Pulsed-field gel electrophoresis patterns were useful for comparing Citrobacter strains. Identical restriction patterns generated by PFGE were observed in the case of selected strains e.g. strains C. sedlakii studied in this study, coming from an outbreak, having the some phenotype. In addition, PFGE patterns can be used to evaluate the clonal relatedness among bacterial isolates. PFGE can be helpful for assessing genetic relatedness among strains epidemiologicaly unrelated e.g. C. werkmanii strains tested in this study. The sum of DNA fragments after Xba I digestion indicates the genome size of Citrobacter strains. This suggests that PFGE should be useful for epidemiological investigations of Citrobacter strains.     

沙坡头地区根瘤菌DNA同源性及16SrDNA全序列

数值分类和多位点酶电泳分析表明,分离自宁夏沙坡头 地区的12株根瘤菌构成一个独立的表观群。对这一菌群进行了DNA同源性和群内中心菌株1 6SrDNA全序列分析。12个菌株的G+C mol%在56.4～62.2范围内;群内DNA同源性为72.3% ～9.5%,大于70%,属种内水平;中心株N220的16SrDNA全序列与参比菌株的序列比较,从 模拟系统发育树看出,它与三株土壤杆菌、三株根瘤菌的16SrDNA序列同源性在94.8%～99 .2%的相似性水平上构成一个分支,看来沙坡头地区这群根瘤菌是一个独立的新种群。     

Development of specific PCR primers for a rapid and accurate identification of Staphylococcus xylosus, a species used in food fermentation

Twenty-seven Staphylococcus strains isolated from food and food environments were assigned to Staphylococcus xylosus by API-Staph system. But only seven isolates had similar patterns to this species when compared to the pulse-field gel electrophoresis patterns of 12 S. xylosus strains. To perform a rapid identification of the S. xylosus species, a random amplified polymorphic DNA product of 539-bp shared by all of the S. xylosus strains was used to design a pair of primers. These primers were species-specific for S. xylosus when tested by PCR on 21 staphylococci species. This specific PCR assay confirms the identification of the seven isolates identified by PFGE to S. xylosus. In conclusion, we developed specific PCR primers for a rapid and accurate identification of the S. xylosus species.     

Adaptive changes of chemolithoautotrophic acidophilic sulfur-oxidizing bacteria during growth in sewage sludge

A chemolithoauthotrophic, acidophilic, sulfur-oxidizing strain was isolated from sewage sludge and identified as Acidithiobacillus thiooxidans. The morphology and physiology of the isolate grown in mineral medium or sterilized sewage sludge were investigated. Morphological and ultrastructural differences between cells grown in mineral medium and sewage sludge were clearly visible. Sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed some changes in the protein expression profiles in the periplasmic fraction as well as a lower level of cytochromes. Adaptation of A. thiooxidans to sewage sludge was not only a physiological process but also included genetic changes. Restriction fragment length polymorphism analysis using pulsed field gel electrophoresis showed structural changes in chromosomal DNA of such bacteria. Most of the restriction fragments were highly conserved and shared by strains grown under different conditions. Cultivation in mineral medium did, however, lead to the appearance of an additional restriction fragment. In combination, the obtained results provide evidence of adaptive responses by A. thioxidans during growth in sewage sludge and confirm that this bacteria can be useful in biotechnologies of heavy metal bioleaching from different environments polluted with hazardous compounds.     

Raw cow milk bacterial population shifts attributable to refrigeration

We monitored the dynamic changes in the bacterial population in milk associated with refrigeration. Direct analyses of DNA by using temporal temperature gel electrophoresis (TTGE) and denaturing gradient gel electrophoresis (DGGE) allowed us to make accurate species assignments for bacteria with low-GC-content (low-GC%) (<55%) and medium- or high-GC% (>55%) genomes, respectively. We examined raw milk samples before and after 24-h conservation at 4 degrees C. Bacterial identification was facilitated by comparison with an extensive bacterial reference database ( approximately 150 species) that we established with DNA fragments of pure bacterial strains. Cloning and sequencing of fragments missing from the database were used to achieve complete species identification. Considerable evolution of bacterial populations occurred during conservation at 4 degrees C. TTGE and DGGE are shown to be a powerful tool for identifying the main bacterial species of the raw milk samples and for monitoring changes in bacterial populations during conservation at 4 degrees C. The emergence of psychrotrophic bacteria such as Listeria spp. or Aeromonas hydrophila is demonstrated.     

Recognition of individual strains of fast-growing rhizobia by using profiles of membrane proteins and lipopolysaccharides.

Membrane protein and lipopolysaccharide profiles of Rhizobium leguminosarum (biovars viciae, trifolii, and phaseoli), R. meliloti, and Agrobacterium tumefaciens strains were analyzed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Differences in one or both profiles allowed us to distinguish all 18 R. leguminosarum strains tested in this study from each other.