Differentiation of Shigella by esterase electrophoretic polymorphism

The electrophoretic mobilities of four esterases (A, B, C, and I) of 182 strains of Shigella dysenteriae, S. flexneri, S. boydii and S. sonnei were compared to those of 636 strains of Escherichia coli from various origins, including the Alkalescens Dispar group and enteroinvasive strains. Discriminant analysis of the distribution of esterases among the strains revealed that Shigella could be distinguished from E. coli by differences in the distribution of allozymes of esterases C and I. Principal components analysis distinguished four major clusters of Shigella strains corresponding to the following: S. dysenteriae serotype 1; S. flexneri serotypes 1 to 5; S. flexneri serotype 6 and S. boydii serotypes 2 and 4; and S. sonnei. The last three were characterized by distinct electrophoretic variants of carboxylesterase B, as judged by the two-dimensional electrophoretic profile and titration curves. The distinct esterase pattern obtained for the strains of S. boydii serotype 13 substantiates the view that this serotype may constitute a new species.     

Ribosomal RNA Sequencing of Members of the Crypthecodinium cohnii (Dinophyceae) Species Complex; Comparison with Soluble Enzyme Studies

ABSTRACT. Sixty-five members of the Ctypthecodinium cohnii species complex were analyzed for sequence differences within the D2 region of the 23S ribosomal RNA molecule. On the basis of 46 sequence differences the strains fell into 19 distinct ribosets (strains of identical sequence), some with many members. Members of four of the seven major sibling species (widespread breeding groups) were each found within single ribosets. Members of three other major sibling species were each, however, divided into two ribosets by a single sequence difference correlated with geographic separation and with previously reported electrophoretic polymorphisms of soluble enzymes within the sibling species. In addition to members of major sibling species, some ribosets include many minor sibling species (each represented by only one strain). Of 38 minor sibling species, 22 shared sequence with a major sibling species. Of these 22, 14 were identical in soluble enzymes to their related major sibling species or differed by only one of three enzymes. Other minor sibling species appear to have diverged extensively from any others in both rRNA sequence and electrophoretic profile. As a group, major sibling species differ markedly in the number of minor sibling species associated with them, suggesting differences in frequency of sexually isolating events in their past histories. These findings are discussed in the context of the previously proposed model of sympatric speciation.     

Gel electrophoretic analysis of penicillin-binding proteins of coagulase negatibve staphylococci

We analyzed gel electrophoretic banding patterns of penicillin-binding proteins (PBPs) of 16 type strains of coagulase-negative staphylococci (CNS). S. epidermidis, S. haemolyticus, S. saprophyticus, S. hominis, S. xylosus, S. simulans, S. warneri, S. capitis, S. saccharolyticus, S. auricularis, S. caseolyticus, S. gallinarum, S. hycus subsp. hycus, S. cohnii, S. caprae, and S. sciuri subsp. sciuri. The PBP profile of each CNS species was found to be unique and was clearly distinguishable from those of the rest of the species. Together with the previous work of other researchers, this study substantiates the applicability of the PBP profile analysis to the identification of clinical CNS strains.     

Molecular characterisation of the species of the genus Zygosaccharomyces

The restriction fragments polymorphisms of the mitochondrial DNA and the PCR fragment that comprised the internal transcribes spacers and the 5.8S rRNA gene, together with the electrophoretic karyotypes of 40 strains from the 10 species of the genus Zygosaccharomyces, including the new species Z. lentus were examined. The RFLP's of the ITS-5.8S region showed a specific restriction pattern for each species, including the new species Z. lentus. The only exception were the species Z. cidri and Z. fermentati that produced identical restriction profiles. The electrophoretic chromosome patterns confirmed the differences between the species of this genus, including the phylogenetic closest species Z. cidri and Z. fermentati. They present few chromosomes ranging from 3 bands (4 or 5 chromosomes) for Z. florentinus to 7 bands (8 to 10 chromosomes) for Z. cidri and Z. fermentati. The strain level resolution power of RFLP's of mtDNA of this genus enabled the characterisation of strains from the same species, even where they are isolated from the same substrate. However, in the cases of Z. bailii and Z. lentus, electrophoretic karyotyping there was considerable variation.     

Epidemiological typing of Acinetobacter strains by esterase electrophoresis

Fifty-three strains of Acinetobacter, belonging to the species A baumannii, A. haemolyticus and A. johnsonii, were differentiated by electrophoretic typing of their esterases, on the basis of both the enzyme specific activity profiles and their electrophoretic mobilities. Each esterase was defined by its spectrum of hydrolytic activity toward five synthetic substrates and its sensitivity to di-isopropyl fluorophosphate. Since each enzyme was not detected in all strains of a given species, several zymotypes could be defined by the patterns of combinations of esterases. Thus, 24 zymotypes were defined in the 32 A. baumannii strains, 4 were defined in the 10 A. haemolyticus strains and 6 were defined in the 11 A. johnsonii strains. When the electrophoretic mobilities of the various esterases were included, each of the 53 strains of Acinetobacter (with the exception of three A. haemolyticus strains) showed a distinct electrotype.     

表型相似的热带假丝酵母和麦芽糖假丝酵母在脉冲电泳核型上的差异

热带假丝酵母Ｃａｎｄｉｄａ ｔｒｏｐｉｃａｌｉｓ （Ｃａｓｔｅｌｌａｎｉ ）Ｂｅｒｋｈｏｕｔ和麦芽糖假丝酵母Ｃ． ｍａｌｔｏｓａＫｏｍａｇａｔａ, Ｎａｋａｓｅ ＆ Ｋａｔｓｕｙａ是两种可利用烃类作为碳和能量来源的酵母菌,前者还是一种条件致病菌,可引起系统感染。这两种假丝酵母菌在形态和生理生化性状上非常相似,用常规分类方法不易准确地鉴别。本研究对Ｃ． Ｔｒｏｐｉｃａｌｉｓ和Ｃ ｍａｌｔｏｓａ的模式菌株以及中国普通微生物菌种保藏中心（ＣＧＭＣＣ）保藏的归于这两个种名下的其它菌株进行了脉冲电泳核型比较分析。发现这两个表型相似的种具有明显不同的染色体ＤＮＡ分子带型,而同一种内的不同菌株却具有相同或相似的分子核型。Ｃ．Ｔｒｏｐｉｃａｌｉｓ的特异染色体ＤＮＡ分子带谱为２条８．５—１．２ Ｍｂ的带, ４条２．３－３．４ Ｍｂ的带。 Ｃ ｍａｌｔｏｓａ的特异带谱为： ３～４条分子量在１．１－１．３Ｍｂ范围内的带, １条约为２．２Ｍｂ的带以及２－３条大小为３．２－３．５Ｍｂ的带。 Ｃ ｔｒｏｐｉｃａｌｉｓ与Ｃ ｍａｌｔｏｓａ在染色体ＤＮＡ分子带型上的差异与二者在可溶性淀粉的同化能力和４０℃下的生长能力上的差异具有明显的相关性…     

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.     

The taxonomic position of Saccharomyces boulardii as evaluated by sequence analysis of the D1/D2 domain of 26S rDNA, the ITS1-5.8S rDNA-ITS2 region and the mitochondrial cytochrome-c oxidase II gene

A taxonomic study was carried out on eight strains of Saccharomyces boulardii. Morphological and physiological characteristics were consistent with those of Saccharomyces cerevisiae. Sequences of the D1/D2 domain of the 26S rDNA were identical for all strains examined and had a similarity value of 100% compared to sequences of the type strain of S. cerevisiae (CBS 1171T) and strain S288c. For all S. boulardii isolates was found the exact same ITS1-5.8S rDNA-ITS2 sequence, which displayed a close resemblance with the sequences published for S288c (99.9%), CBS 1171(T) (99.3%) and other S. cerevisiae strains. Sequence analysis of the mitochondrial cytochrome-c oxidase II gene (COX2) also resulted in identical sequences for the S. boulardii isolates and comparisons with available nucleotide sequences revealed close relatedness to strains of S. cerevisiae including S288c (99.5%) and CBS 1171(T) (96.6%). The electrophoretic karyotypes of the S. boulardii strains appeared quite uniform and although very typical of S. cerevisiae, they formed a cluster separate from strains of this species. The results of the present study strongly indicate a close relatedness of S. boulardii to S. cerevisiae and thereby support the recognition of S. boulardii as a member of S. cerevisiae and not as a separate species.     

Genetic characterization of three strains of Hymenolepis diminuta at 39 enzyme loci

The technique of allozyme electrophoresis was applied to three strains of Hymenolepis diminuta to distinguish between three hypotheses [(1) multiple species, (2) genetically distinct founder stocks and (3) response to differential selection among similar stocks] proposed to account for metabolic differences among strains. There was no evidence from the 39 enzyme loci established that the three strains represented more than one species. In the absence of knowledge of the population structure of H. diminuta in the wild, electrophoretic data herein could not distinguish between the latter hypotheses. Nevertheless, all three strains were distinguishable on electrophoretic profiles and allelic similarities between strains question the view of their proposed origins.     

脉冲电泳核型分析在酿酒酵母菌分类学研究中的应用

根据酵母属（ Ｓａｃｃｈａｒｏｍｙｃｅｓ Ｍｅｙｅｎ ｅｘ Ｒｅｅｓｓ） 分类学研究最新进展,核实并更新了保藏于中国普通微生物菌种保藏中心的该属菌株的种类归属。在形态和生理生化性状,包括对６ 种糖的发酵能力、对１８ 种碳源和３ 种氮源化合物的同化能力、在无维生素培养基中和３７ ℃下的生长情况、对放线菌素酮的抗性等常规分类学研究的基础上,对部分疑难菌株进行了脉冲电泳核型比较分析。酿酒酵母（ Ｓａｃｃｈａｒｏｍｙｃｅｓｃｅｒｅｖｉｓｉａｅ） 、贝酵母（ Ｓ．Ｂａｙａｎｕｓ） 和巴氏酵母（ Ｓ．Ｐａｓｔｏｒｉａｎｕｓ） 三者与少孢酵母（ Ｓ．Ｅｘｉｇｕｕｓ） 在电泳核型上具有明显的差异,主要表现在前三者染色体ＤＮＡ 分子的大小范围均为２２５ ～２２００ ｋｂ ,而Ｓ．Ｅｘｉｇｕｕｓ 缺少小于３６５ ｋｂ 的染色体ＤＮＡ 分子。Ｓ．Ｃｅｒｅｖｉｓｉａｅ 的模式和权威菌株具有１２ ～１４ 条染色体ＤＮＡ 带;Ｓ．Ｂａｙａｎｕｓ 和Ｓ．Ｐａｓｔｏｒｉａｎｕｓ 的模式菌株均有１７ 条带,但在带型上存在一定差异。原归于Ｓ．Ｃｅｒｅｖｉｓｉａｅ 的株菌ＡＳ２－１００ 具有１６ 条带,与Ｓ．Ｃｅｒｅｖｉｓｉａｅ 区别明显而与Ｓ．…     

Variations in mRNA transcript levels of cell wall-associated genes of Saccharomyces cerevisiae following spheroplasting

A natural subgroup (that we refer to as Saccharomyces uvarum) was identified, within the heterogeneous species Saccharomyces bayanus. The typical electrophoretic karyotype, interfertility of hybrids between strains, distinctive sugar fermentation pattern, and uniform fermentation characteristics in must, indicated that this subgroup was not only highly homogeneous, but also clearly distinguishable from other species within the Saccharomyces sensu stricto group. Investigation of the S. bayanus type strain and other strains that have been classified as S. bayanus, confirmed the apparent lack of homogeneity and, in some cases, supported the hypothesis that they are natural hybrids.     

Isoenzyme patterns: a valuable molecular tool for the differentiation of Zygosaccharomyces species and detection of misidentified isolates

Electrophoretic analysis of esterase, acid phosphatase, lactate dehydrogenase, glucose-6-phosphate dehydrogenase and alcohol dehydrogenase isoenzymes was performed in 39 strains classified into six species of the yeast genus Zygosaccharomyces. The electrophoretic profiles obtained allowed the clear separation of Z. bailii, Z. bisporus, Z. florentinus, Z. lentus, Z. mellis and Z. rouxii, strains of the latter species clustering into two subgroups. Furthermore, this methodology enabled the detection of misidentified strains, as subsequently confirmed by DNA-DNA reassociation and sequencing of the D1/D2 domain of the 26S rRNA gene. Cluster analysis of the global electrophoretic data and those obtained using only two of the isoenzyme systems, esterase and lactate dehydrogenase, yielded similar grouping of the strains examined, indicating that these enzymes are good markers for the differentiation of Zygosaccharomyces species.     

Electrophoretic Variants of Phosphoglucomutase in Saccharomyces Species

Strains of Saccharomyces cerevisiae and species with which S. cerevisiae is interfertile display a characteristic pattern of electrophoretic variants of phosphoglucomutase (PGM) consisting of a major component and one or two minor components, all of which migrate toward the cathode. The patterns are consistent with an earlier finding that two unlinked genes, one of which has two known alleles, determine the synthesis of PGM in S. cerevisiae. The PGM patterns of strains of S. fragilis, S. lactis, and S. marxianus, species thought to be closely related to each other and only distantly related to S. cerevisiae, also displayed a characteristic pattern of PGM variants, but it was quite different from that of S. cerevisiae. In these species five or six electrophoretic variants could be detected, all of which migrated toward the anode. We interpret the differences in the PGM variants of the two groups of species as a reflection of differences in genetic composition which have arisen in two phylogenetically distinct groups that have become sexually isolated from each other.     

Characterization of enterobacteria by esterase specific-activity profiles

The spectrum of specific activities and the electrophoretic mobilities of esterases produced by 550 strains of Enterobacteriaceae belonging to 36 species and subclassified into six groups (group 1, Escherichia coli, Shigella and Escherichia hermanii; group 2, genus Salmonella and genus Citrobacter; group 3, genus Klebsiella and genus Enterobacter; group 4, genus Serratia and Serratia fonticola; group 5, genus Proteus, genus Providencia and genus Morganella; and group 6, genus Yersinia) were analysed by acrylamide/agarose gel electrophoresis using standardized methods for staining and mobility comparisons. Nineteen types of esterase were defined by their respective esterase specific-activity profile (ESAP). A multiple correspondence analysis (MCA) of the ESAP data enabled 82% of the strains in the 36 species to be correctly classified. In each group, the species were clearly delineated after MCA on both ESAP and electrophoretic mobility data. In addition, the smallest number of characters providing species identification of Yersinia strains by esterase polymorphism was identified by means of a binary segmentation tree technique.     

Polyacrylamide gel electrophoresis analysis of ribosomal protein: a new approach for actinomycete taxonomy.

The ribosomal (r)-proteins from eleven Streptomyces strains representing various numerical taxonomic clusters were compared by two-dimensional polyacrylamide-gel electrophoresis (2D-PAGE). The protein patterns were specific for each species. An attempt was made to identify one strain of Streptomyces by both traditional taxonomic methods and 2D-PAGE analysis of the r-protein patterns. Both methods identified the strain as Streptomyces lavendulae, and protein pattern analysis also showed that S. griseolavendus was a variant of S. lavendulae. Actinomycete r-protein AT-L30 exhibited electrophoretic mobility that is specific for each genus. On the basis of this observation, we analyzed AT-L30 r-proteins from numerous strains of species belonging to the genera Actinomadura, Microtetraspora, Streptosporangium, Nocardia, Rhodococcus and mycolate-less wall chemotype-IV actinomycetes. The results strongly supported the conclusions of previous work and thus proved the efficacy of r-protein analysis as a novel approach for taxonomy of actinomycetes.     

Precise molecular weight determination of PCR products of the rRNA intergenic spacer region using electrospray quadrupole mass spectrometry for differentiation of B. subtilis and B. atrophaeus, closely related species of bacilli

Assessment of 16S–23S rRNA intergenic spacer region (ISR) sequence variability is an important supplement to 16S rRNA sequencing for differentiating closely related bacterial species. Species differentiation can also be achieved by determination of approximate size of PCR (polymerase chain reaction) products of ISRs, based on their relative electrophoretic mobility on agarose gels. Closely-related species can have ISR PCR products that are similar in size. More precise molecular weight (M.W.) determination of these products might allow improved discrimination of such species. Electrospray quadrupole mass spectrometry (ESI-Q-MS) has the potential to provide such precision. For ESI-Q-MS analysis, size limitation of PCR products is currently limited to around 130 base pairs (bp). Bacillus subtilis and Bacillus atrophaeus are two closely related species with few distinguishing phenotypic characteristics. B. subtilis has recently been sub-divided into two subgroups, W23 (type strain, W23) and 168 (type strain, 168). PCR products amplified from the ISR including the 5′ terminal end of the 23S rRNA and a conserved portion of the ISR were analyzed by ESI-Q-MS. A 119 or 120 bp PCR product was produced for B. atrophaeus strains. However, strains of B. subtilis subgroups W23 and 168 each produced 114 bp products. In summary, a mass spectrometry method was developed for differentiation of B. subtilis and B. atrophaeus. Also, the genetic similarity of B. subtilis subgroups W23 and 168 was confirmed. Accurate determination of the molecular weight of PCR products from the 16S–23S rRNA intergenic spacer region using electrospray quadrupole mass spectrometry has great potential as a general technique for characterizing closely related bacterial species.     

Transfer RNAs as genotypic fingerprints of eubacteria

A new method was developed for rapid genotypic identification and classification of bacteria. The method is based on high resolution gel electrophoresis of the stable, low molecular weight (LMW) RNA fraction of single bacterial strains. This fraction comprises the total transfer RNA pool and the 5S ribosomal RNA. On a one-dimensional gel, every eubacterial strain exhibited a distinct LMW RNA profile, a set of bands belonging to three different size classes: 5S rRNAs (110–131 nt), class 2 tRNAs (82–96 nt) and class 1 tRNAs (72–79 nt). LMW RNA profiles of members of five of the ten major eubacterial groups, previously defined by 16S rRNA sequence analysis, were highly diverse. For some major groups, like flavobacteria and planctomyces, the distinctive sizes of their 5S rRNAs allowed the assignment of strains to these groups. More specific taxonomic information was gained from analysis of the tRNA part of the profile. Strains could be grouped as species and genera due to species- and genus-specific tRNA bands. From an evolutionary point of view, this order found in the total tRNA pool of eubacteria could indicate that cytoplasmic tRNA evolution reflects ribosomal RNA evolution. Given the universality of tRNAs, it is to be expected that their electrophoretic mobility profiles may serve as a convenient RNA fingerprint for defining bacterial species operationally and for identifying new genotypes by differing patterns.     

Genetic relationships among the oral streptococci.

Genetic relationships and species limits among the oral streptococci were determined by an analysis of electrophoretically demonstrable variation in 16 metabolic enzymes. Fifty isolates represented 40 electrophoretic types, among which the mean genetic diversity per locus was 0.857. Mannitol-1-phosphate dehydrogenase was not detected in isolates of the sanguis species complex, and glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were absent in species of the mutans complex. Clustering from a matrix of Gower's coefficient of genetic similarity placed the 40 electrophoretic types in 10 well-defined groups corresponding to the Streptococcus species S. mutans, S. sobrinus, S. cricetus, S. rattus, S. ferus, S. oralis (mitior), two distinct assemblages of S. sanguis strains, and two subdivisions of "S. milleri." The assignments of isolates to these groups were the same as those indicated by DNA hybridization experiments, and the coefficient of correlation between genetic distance estimated by multilocus enzyme electrophoresis and genetic similarity indexed by DNA hybridization was -0.897 (P less than 0.001) for 50 pairwise combinations of isolates. S. ferus, which is widely believed to be a member of the mutans complex, was shown to be phylogenetically closer to species of the sanguis complex.     

Mycoplasma taxonomy studiedy electrophoresis of cell proteins

The electrophoretic patterns of cell proteins in polyacrylamide gels were used for the study of several taxonomic problems in the Mycoplasmatales. The patterns of five Mycoplasma hominis strains showed marked differences that corresponded with their known serological and nucleic acid heterogeneity. The patterns of three M. mycoides var. mycoides strains isolated in different countries were essentially identical. The electrophoretic patterns of several caprine strains resembled those of M. mycoides var. mycoides, supporting their classification as M. mycoides var. capri. Strain B3, a swine isolate, accordingly was tentatively identified as M. mycoides var. capri. The bovine mastitis strain M. agalactiae var. bovis possessed a pattern basically similar to that of the goat mastitis strain M. agalactiae, supporting the inclusion of both strains in one species. Three M. pulmonis strains isolated from rats or tissue cultures showed nearly identical patterns. The pattern of the toxigenic M. neurolyticum (Sabin A) strain resembled but was not identical with that of the nontoxigenic PG28 strain. The avian Mycoplasma species, M. gallisepticum, M. meleagridis, M. synoviae, M. gallinarum, and M. iners showed easily distinguishable and specific patterns, supporting their present classification in different species. Several improvements in the electrophoretic technique are described, and its advantages and limitations as a taxonomic tool are discussed.