Multilocus enzyme electrophoresis: a practical guide

Multilocus enzyme electrophoresis (MEE) uses the relative electrophoretic mobilities of intracellular enzymes to characterize and differentiate organisms by generating an electromorph type (ET). This article presents the chemical conditions that may be useful, a guide to the successful practice of the electrophoretic technique, and analysis of the results.     

Multilocus enzyme electrophoresis analysis of Trypanosoma cruzi isolates from a geographically restricted endemic area for Chagas' disease in Argentina

A set of 65 Trypanosoma cruzi stocks from dogs, opossums, insect vectors and humans was isolated in a geographically restricted endemic area for Chagas' disease in Argentina and was analysed by multilocus enzyme electrophoresis for 15 loci. The results show that at least five multilocus genotypes (clonets) circulate in the study area, one belonging to T. cruzi IIe, one to T. cruzi IId and three clonets belonging to T. cruzi I; and they confirm the presence of these lineages in the country. The three clonets attributed to T. cruzi I were identical to each other for all loci except for Sod-2, where three different patterns were identified. These patterns suggest the presence of two homozygous genotypes and one heterozygous genotype. Our results also suggest association of clonet IIe with dogs, clonet IId with humans and the three T. cruzi I clonets with Didelphis albiventris. On the other hand, there was no significant association between Triatoma infestans and any particular clonet circulating in the area. These findings are consistent with the hypothesis of natural selection, from mixed populations of T. cruzi in vectors, toward more restricted populations in mammals. The epidemiological implications of the possible selection of different clonets by different mammal hosts and the significance of two homozygous genotypes and one heterozygous genotype for the Sod-2 locus are discussed.     

Multilocus sequence analysis of homologous recombination and diversity in Arthrobacter sensu lato named species and glacier-inhabiting strains

Members of the bacterial genus Arthrobacter sensu lato are Gram-positive actinomycetes distributed worldwide and found in numerous environments including soil, water, glacier ice, and sewage. Homologous recombination is an important driving force in bacterial evolution, but its impact on Arthrobacter sensu lato evolution is poorly understood. We evaluated homologous recombination among 41 Arthrobacter sensu lato named species, using multilocus sequence analysis (MLSA). A high level of recombination was found, associated with strong diversification and a reticulate evolutionary pattern of Arthrobacter sensu lato. We also collected a total of 31 cold-adapted Arthrobacter sensu lato strains from two cold glaciers located in northwest China and two temperate glaciers in southwest China, and evaluated their diversity and population structure by MLSA. The glacier strains displayed high diversity, but rates of recombination among the four glacier groups were quite low, indicating that barriers to homologous recombination formed in the past among the populations on different glaciers. Our findings indicate that historical glaciation events shaped the contemporary distributions, taxonomic relationships, and phylogeographic patterns of Arthrobacter sensu lato species on glaciers.     

Clonal relationship among invasive and non-invasive strains of enteroinvasive Escherichia coli serogroups

The genetic relatedness among 96 invasive Escherichia coli belonging to several serogroups and 13 non-invasive of several serotypes that share the same O antigen was investigated by multilocus enzyme electrophoresis analysis. The invasive strains were isolated in different parts of the world and most of them recovered from dysentery. Twenty-nine electrophoretic types were distinguished and the most invasive strains were found to belong to two major lineages. These results suggested that the invasive ability in these strains has evolved in divergent chromosomal backgrounds, presumably through the horizontal spread of plasmid-borne invasion genes. The maintenance of invasive phenotypes in separate lineages suggests that this ability confers a selective advantage to invasive strains.     

Multilocus phylogeography and phylogenetics using sequence-based markers

We review recent trends in phylogeography and phylogenetics and argue that these two fields stand to be reunited by the common yardstick provided by sequence and SNP data and by new multilocus methods for phylogenetic analysis. Whereas the modern incarnation of both fields was spawned by PCR approaches applied to mitochondrial DNA in the late 1980s, the two fields diverged during the 1990s largely due to the adoption by phylogeographers of microsatellites, in contrast to the adoption of nuclear sequence data by phylogeneticists. Sequence-based markers possess a number of advantages over microsatellites, even on the recent time scales that are the purview of phylogeography. Using examples primarily from vertebrates, we trace the maturation of nuclear gene phylogeography and phylogenetics and suggest that the abundant instances of gene tree heterogeneity beckon a new generation of phylogenetic methods that focus on estimating species trees as distinct from gene trees. Whole genomes provide a powerful common yardstick on which both phylogeography and phylogenetics can assume their proper place as ends of a continuum.     

Multilocus selection in subdivided populations II. Maintenance of polymorphism under weak or strong migration

The potential of maintaining multilocus polymorphism by migration-selection balance is studied. A large population of diploid individuals is distributed over finitely many demes connected by migration. Generations are discrete and nonoverlapping, selection may vary across demes, and loci are multiallelic. It is shown that if migration and recombination are strong relative to selection, then with weak or no epistasis and intermediate dominance at every locus and in every deme, arbitrarily many alleles can be maintained at arbitrarily many loci at a stable equilibrium. If migration is weak relative to selection and recombination, then with weak or no epistasis and intermediate dominance at every locus and in every deme, as many alleles as there are demes can be maintained at arbitrarily many loci at equilibrium. In both cases open sets of such parameter combinations are constructed, thus the results are robust with respect to small, but arbitrary, perturbations in the parameters. For weak migration, the number of demes is, in fact, a generic upper bound to the number of alleles that can be maintained at any locus. Thus, several scenarios are identified under which multilocus polymorphism can be maintained by migration-selection balance when this is impossible in a panmictic population.      

Genetic diversity analysis of Rhodothermus reflects geographical origin of the isolates

The genetic diversity and relationships of 81 Rhodothermus isolates from different geothermal environments in Iceland were examined by analysis of electrophoretically demonstrable allelic variation of 13 genes encoding enzymes. All the enzymes were polymorphic. A total of 71 distinctive multilocus genotypes (electrophoretic types, ETs) were identified. The mean genetic diversity per locus (H _t ) was 0.586. The relatively high genetic variance observed within Rhodothermus isolates from different locations is most likely the result of genetic changes occurring independently in the locations studied. A high G _st value (0.284) indicates that a considerable part of the variance observed is due to differences between locations. Cluster analysis revealed two major groups of ET clusters diverging at a genetic distance of 0.75, reflecting strongly the geographic origin of isolates. Estimation of the association index (I _A) indicates that Rhodothermus marinus is a clonal species in which recombination events occur rarely. Partial or whole sequencing of the 16S rRNA genes of Rhodothermus isolates grouping at genetic distance of 0.40 confirmed that all the isolates belonged to the species Rhodothermus marinus. The results of this study confirm that, despite phylogenetic and phenotypic similarity, genetic diversity within Rhodothermus marinus is quite high. Received: January 21, 2000 / Accepted: April 27, 2000     

Genetic analysis of four sympatric morphs of Arctic charr,Salvelinus alpinus,from Thingvallavatn,Iceland

Synopsis The degree of genetic differentiation among four morphs of Arctic charr (small benthivorous, large benthivorous, piscivorous, and planktivorous) from Thingvallavatn, Iceland, was determined electrophoretically. Five of 36 enzyme loci were found to be polymorphic (Est2, Gpi3, Ldh4, Mdh4, 5 and Pgm2). However, only Est2 and Mdh4,5 showed enough variability to permit statistical analysis of divergence among morphs. All four morphs are very closely related; the values of Nei's (D) range from 0.00004 to 0.00126. These morphs are conspecific and do not represent different evolutionary lineages. There is significant genetic differentiation between the small benthivorous charr and the other three morphs. The relative relatedness of morphs based on gene frequency data is only partially concordant with that based on morphology and ecological specialization. The biological significance of this result is unclear because of the limited number of polymorphic loci upon which the genetic analysis is based and the high degree of relatedness among morphs.     

Genetic diversity and methylation polymorphism analysis of Chrysanthemum nankingense

The diploid species Chrysanthemum nankingense (Anthemideae, Asteraceae) is closely related to the commercially important hexaploid ornamental species Chrysanthemum morifolium and is well adapted to poor environments. In this study, phenotypic variants of C. nankingense were first identified by morphological traits. Using EST-SSR (simple sequence repeat) analysis, we detected some absent EST-SSRs. The percentage of AFLP (amplified fragment length polymorphism) polymorphic fragments was 78.2%, indicating high genetic diversity. To evaluate the genome methylation level and methylation polymorphism, we used the MSAP (methylation-sensitive amplification polymorphism) technique to analyze the 30 C. nankingense lines. The total DNA methylation level ranged from 54.6% to 62.6%. Most of the MSAP-methylated fragments (97%) were polymorphic in the lines. The U-values associated with hemi-methylation were larger than those associated with full methylation in four of the 30 lines, and six individual values were statistically significant (U > 1.96). The high genomic diversity as well as the high methylation polymorphism may be responsible for the morphological polymorphism. There was no significant correlation between the phenotypic and genetic diversity among the lines.     

Multilocus models in the infinite island model of population structure

Different methods have been developed to consider the effects of statistical associations among genes that arise in population genetics models: kin selection models deal with associations among genes present in different interacting individuals, while multilocus models deal with associations among genes at different loci. It was pointed out recently that these two types of models are very similar in essence. In this paper, we present a method to construct multilocus models in the infinite island model of population structure (where deme size may be arbitrarily small). This method allows one to compute recursions on allele frequencies, and different types of genetic associations (including associations between different individuals from the same deme), and incorporates selection. Recursions can be simplified using quasi-equilibrium approximations; however, we show that quasi-equilibrium calculations for associations that are different from zero under neutrality must include a term that has not been previously considered. The method is illustrated using simple examples.     

Identification of metolachlor mineralizing bacteria in aerobic and anaerobic soils using DNA-stable isotope probing

The influence of soil environmental factors such as aeration on the ecology of microorganisms involved in the mineralization and degradation of the popular soil-applied pre-emergent herbicide, metolachlor is unknown. To address this knowledge gap, we utilized DNA-based stable isotope probing (SIP) where soil microcosms were incubated aerobically or anaerobically and received herbicide treatments with unlabeled metolachlor or ¹³C-metolachlor. Mineralization of metolachlor was confirmed as noted from the evolution of ¹⁴CO₂ from ¹⁴C-metolachlor-treated microcosms and clearly demonstrated the efficient utilization of the herbicide as a carbon source. Terminal restriction fragment length polymorphisms (T-RFLP) bacterial community profiling performed on soil DNA extracts indicated that fragment 307 bp from aerobic soil and 212 bp from anaerobic soil were detected only in the herbicide-treated (both unlabeled metolachlor and ¹³C-metolachlor) soils when compared to the untreated control microcosms. T-RFLP profiles from the ultracentrifugation fractions illustrated that these individual fragments experienced an increase in relative abundance at a higher buoyant density (BD) in the labeled fractions when compared to the unlabeled herbicide amendment fractions. The shift in BD of individual T-RFLP fragments in the density-resolved fractions suggested the incorporation of ¹³C from labeled herbicide into the bacterial DNA and enabled the identification of organisms responsible for metolachlor uptake from the soil. Subsequent cloning and 16S rRNA gene sequencing of the ¹³C-enriched fractions implicated the role of organisms closely related to Bacillus spp. in aerobic mineralization and members of Acidobacteria phylum in anaerobic mineralization of metolachlor in soil.     

Simultaneous carriage of Candida albicans strains from HIV-infected patients with oral candidiasis: multilocus enzyme electrophoresis analysis

Abstract Genetic diversity of 160 Candida albicans isolates from the oral cavity of 16 HIV-infected adults prior to antifungal treatment was assessed using multilocus enzyme electrophoresis (10 C. albicans colonies were randomly chosen from each specimen culture). 20 electrophoretic types were distinguished from the analysis of 21 enzyme loci (10 were polymorphic). Five patients (31%) were found to be colonized by 2 or 3 genetically distinct strains. Nevertheless, in these five cases, one strain predominated (from 7 to 9 of the 10 colonies). Some HIV + patients with oral candidiasis appear to be simultaneously infected with several genetically different C. albicans strains before antifungal treatment.     

Relationship between enzyme heterozygosity and quaternary structure

The need for proteins to maintain particular quaternary structures constrains variability in amino acid sequence. Monomeric enzymes are then expected to be more variable than dimeric forms, which in turn are expected to be more variable than tetrameric forms. These predictions are confirmed by analysis of available data on enzyme variation. Theories relating enzyme heterozygosity to metabolic function are discussed in the light of these findings.Financial support for part of the work described in this article was derived from NERC Grant GR3/1558 to J. A. Beardmore.     

厌氧菌和需氧菌性阴道混合感染发病调查

目的对妇女厌氧菌和需氧菌性阴道混合感染发病进行调查分析。方法分别采用Donders高倍镜湿片镜检法和细菌预成酶快速检测法对257例患者进行联合检测。结果257例患者中共检出单纯的需氧菌性阴道感染患者52例,占总人数的20．2％,检出单纯的厌氧菌阴道感染患者125例,占总人数的48．6％,检出厌氧菌和需氧菌性阴道混合感染患者67例,占总人数的26．1％,检出其他感染患者13例,占总人数的5％,经统计学分析,患者的厌氧菌和需氧菌性阴道混合感染的检出率差异无统计学意义（P〉0．05）。结论厌氧菌和需氧菌性阴道混合感染,可以给临床医生应采取合理有效的措施进行及时治疗,防止阴道上行感染的发生。     

Differentiation of intestinal spirochaetes by multilocus enzyme electrophoresis analysis and 16S rRNA sequence comparisons

Abstract Multilocus enzyme electrophoresis (MEE) analysis and comparisons of nearly complete 16S rRNA gene sequences (1416 nucleotide positions) were used to evaluate phylogenetic relationships among Serpulina hyodysenteriae strain B78^T, S. innocens strain B256^T, Brachyspira aalborgi strain 513A^T, and eight uncharacterised strains of swine, avian, and human intestinal spirochaetes. From MEE analysis, nine strains could be assigned to five groups containing other intestinal spirochaetes ( genetic distances between groups = 0.6–0.9). Chicken spirochaete strain C1 and B. aalborgi 513A^T represented unique electrophoretic types and formed their own MEE groups. Despite MEE differences, the 11 strains had highly similar (96.3–99.9%) 16S rRNA sequences. These findings point out limitations of both MEE analysis and 16S rRNA sequence comparisons when used as solitary techniques for classifying intestinal spirochaetes related to Brachyspira/ Serpulina species.     

Characterization of nitrogen-fixing Paenibacillus species by polymerase chain reaction-restriction fragment length polymorphism analysis of part of genes encoding 16S rRNA and 23S rRNA and by multilocus enzyme electrophoresis

Forty-two strains representing the eight recognized nitrogen-fixing Paenibacillus species and 12 non-identified strains were examined by restriction fragment length polymorphism (RFLP) analysis of part of 16S and 23S rRNA genes amplified by polymerase chain reaction (PCR). Eleven different 16S rDNA genotypes were obtained from the combined data of RFLP analysis with four endonucleases and they were in agreement with the established taxonomic classification. Only one group of unclassified strains (Group I) was assigned in a separate genotype, suggesting they belong to a new species. Using the 23S PCR-RFLP method only six genotypes were detected, showing that this method is less discriminative than the 16S PCR-RFLP. Using the multilocus enzyme electrophoresis (MLEE) assay, the 48 strains tested could be classified into 35 zymovars. The seven enzymatic loci tested were polymorphic and the different profiles obtained among strains allowed the grouping of strains into 10 clusters. The PCR-RFLP methods together with the MLEE assay provide a rapid tool for the characterization and the establishment of the taxonomic position of isolates belonging to this nitrogen-fixing group, which shows a great potentiality in promoting plant growth.     

Scaling of enzyme activity in larval herring and plaice: effects of temperature and individual growth rate on aerobic and anaerobic capacity

Herring Clupea harengus and plaice Pleuronectes platessa were reared at 8 and 12° C from the fertilized egg to a larval age of up to 600 degree-days. Soluble protein as well as the activities of both citrate synthetase (CS) and lactate dehydrogenase (LDH) were measured in homogenate supernatants of individual larvae at 10° C. Scaling factors were calculated using the expression y = ax^b where y is the enzyme activity, x the protein content of the larva a is a constant and b the scaling factor. All scaling factors showed significant differences between the species. Within species, the scaling factors for CS activity were either small or not significantly different between the two rearing temperatures, but the scaling factors for the LDH activities were significantly different at the two temperatures for both species. Herring larvae, which had higher LDH activities when newly hatched, showed smaller scaling factors for LDH ( b =1·42 at 8°C and b =1·07 at 12° C) than plaice ( b =2·11 at 8°C and b =1·45 at 12° C). Activities converged as the larvae grew. The results of the current study together with reanalysis of data from the literature indicate an increasing aerobic and anaerobic capacity during the larval stage of fishes (positive allometry).     

Evaluation of pyrimidine- and hydantoin-degrading enzyme activities in aerobic bacteria

Abstract The enzyme activities responsible for the reductive pyrimidine base degradation by aerobic bacteria, which produce hydantoin-degrading enzymes, were investigated. Pseudomonas putida IFO 12996, which is a d-stereospecific hydantoinase producer, has dihydropyrimidinase activity, and Comamonas sp. E222c and Blastobacter sp. A17p-4, which are N-carbamoyl-D-amino acid amidohydrolase producers, have β-ureidopropionase activity. Blastobacter sp. also possesses both d-stereospecific hydantoinase and dihydropyrimidinase activities. Thus, two amide ring-opening activities and/or two N -carbamoyl amino acid-hydrolyzing activities coexist in these bacteria. However, the differences of the induction levels of each enzyme activities for the several pyrimidine- and hydantoin-related compounds suggest that these corresponding amide ring-opening or N -carbamoyl amino acid-hydrolyzing activities are not always catalyzed by the same enzymes.     

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S_SM). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S_SM^MLEE × S_SM^EK × S_SM^SSRs). Clustering analyses showed a mean of 9 ± 12.4 isolates per cluster (3.8 ± 8 isolates/taxon) for MLEE, 6.2 ± 4.9 isolates per cluster (4 ± 4.5 isolates/taxon) for SSRs, and 4.1 ± 2.3 isolates per cluster (2.6 ± 2.3 isolates/taxon) for EK. A total of 45 (13%), 39 (11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (S_J) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships.