Isozyme Variation among Biological Species in the Gibberella fujikuroi Species Complex (Fusarium Section Liseola)

Isozyme phenotypes were determined for 101 strains of Gibberella fujikuroi and 2 strains of Gibberella nygamai that represent seven biological species (mating populations) isolated from a variety of plant hosts in dispersed geographic locations. Fourteen enzymes were resolved in one or more of three buffer systems. Two of the enzymes, arylesterase and acid phosphatase, were polymorphic within two or more biological species and are suitable for intraspecific studies of population variation. Six enzymes, alcohol dehydrogenase, aspartate aminotransferase, glucose-6-phosphate dehydrogenase, mannitol dehydrogenase, phosphoglucomutase, and phosphogluconate dehydrogenase, were monomorphic in all of the isolates examined. The remaining six enzymes, fumarase, glucose phosphate isomerase, glutamate dehydrogenase (NADP), isocitrate dehydrogenase (NADP), malate dehydrogenase, and triose-phosphate isomerase, could potentially be used to distinguish the different biological species. Mating populations C and D are the most similar, since the mating population C isolates examined had the same isozyme phenotype as did a subset of the isolates in mating population D. Mating population E is the least similar to the other taxa examined. Unique isozyme phenotypes are present but are composed of banding patterns shared among the biological species. This finding supports the hypothesis that these biological species, with the possible exception of mating populations C and D, are reproductively isolated from one another and that no significant gene flow is occurring between them. Isozyme analysis is a useful method to distinguish these closely related biological species. Examination of isozyme phenotypes is more rapid than the present technique, which is based on sexual crosses; can be applied to strains that are not sexually fertile; and is more sensitive than traditional morphological characters, which cannot distinguish more than three or four morphological groups among the seven biological species. While emphasizing the discreteness of the mating populations as biological entities, our isozyme data also reaffirm the close genetic relationship among these groups.     

Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing

Pernin P. 1984. Isoenzyme patterns of pathogenic and nonpathogenic thermophilic Naegleria strains by isoelectric focusing. International Journal for Parasitology14: 459–465. The isoenzymatic patterns of different strains of Naegleria were studied by isoelectric focusing (I.E.F.) on polyacrylamide gels for seven enzymatic activities (leucine amino peptidase; lactate dehydrogenase; glucose 6 phosphate dehydrogenase; propionyl esterase; glucose phosphate isomerase; malate dehydrogenase; acid phosphatase), two of which (lactate dehydrogenase and glucose 6 phosphate dehydrogenase) were being investigated for the first time. The three pathogenic N. fowleri strains share a common pattern for most of the enzymes tested except for glucose 6 phosphate dehydrogenase, and thus form a very homogeneous species, while thermophilic nonpathogenic strains show more heterogeneity particularly for leucine amino peptidase and glucose 6 phosphate dehydrogenase.I.E.F. must be considered as a supplementary and rapid method for the identification of N. fowleri and as a powerful tool to demonstrate the complexity of different genera of free-living amoebas.     

Enzyme electrophoretograms in the analysis of taxon relatedness of Micrococcus cryophilus, Branhamella catarrhalis and atypical Neisserias.

Extracts were prepared from Micrococcus cryophilus, several strains of Branhamella catarrhalis and Neisseria spp. Esterases, NADP-dependent isocitrate dehydrogenase and malate dehydrogenase activities were assayed after electrophoresis of extracts of polyacrylamide gels. Except for Neisseria perflava and N. sicca which resolved activity bands for the acetate-esterase only, the remaining bacteria exhibited species-specific esterase patterns also for the propionate and butyrate substrates. The multiple esterase patterns from B. catarrhalis ATCC25238 were qualitatively and quantitatively different from those of B. catarrhalis ATCC23246. This finding and other evidence supports a taxonomic shift of the latter to a species level of that genus. The atypical neisserias N. caviae and N. ovis appeared to exhibit an intrageneric specificity in their esterase patterns with those from B. catarrhalis but not to the other Neisseria spp. tested. The malate dehydrogenase patterns from the atypical neisserias and B. catarrhalis ATCC23246 were qualitatively similar; however, the patterns of isocitrate dehydrogenase activity were variable for these species. Micrococcus cryophilus was distinct in its esterase and dehydrogenase bands, strongly suggesting its taxon unrelatedness to the genus Branhammella or the atypical neisserias. Of the enzymes assayed, esterase proved to be the most reliable for taxonomic identifications.     

Characterization of Enterobacter cloacae and E. sakazakii by electrophoretic polymorphism of acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases

Acid phosphatase, esterases, and glutamate, lactate and malate dehydrogenases of 34 strains of Enterobacter cloacae and 22 strains of Enterobacter sakazakii were analysed by horizontal polyacrylamide agarose gel electrophoresis and by isoelectrofocusing in thin-layer polyacrylamide gel. The two species could be separated on the basis of distinct electrophoretic patterns of all enzymes analysed. Glutamate dehydrogenase and acid phosphatase were detected exclusively in E. cloacae, whereas esterase bands were more intensively stained in E. sakazakii. For each species, two zymotypes could be distinguished, on the basis of electrophoretic mobilities of malate dehydrogenase and banding patterns of esterase for E. cloacae, and by both isoelectric point and electrophoretic mobilities of an esterase and of lactate and malate dehydrogenases for E. sakazakii. The high degree of enzyme polymorphism within the two species permitted precise identification of strains. The variations in electrophoretic patterns might therefore provide useful epidemiological markers.     

Nitrogen Metabolizing Enzyme Activities In the Free-Living Soil Amoebae Acanthamoeba Castellanii, Acanthamoeba Polyphaga and Hartmannella Vermiformis

ABSTRACT. Free-living soil amoebae consume a wide variety of food, including algae, yeast, small protozoa and especially bacteria, which they digest to fulfil their nutritional requirements. Amoebae play an active role in the nitrogen mineralization in soils due to their nitrogen metabolizing capacities. However, little is known about nitrogen metabolizing enzyme activities in these free-living soil amoebae. In this study a number of key enzymes involved in the nitrogen metabolism of the axeaically cultivated free-living soil amoebae Acanthamoeba castellanii, Acanthamoeba polyphaga and two different strains of Hartmannella vermiformis were determined. the specific enzyme activities for exponential growth phase ceils were calculated and it appeared that the species tested possessed urate oxidase, glutamine synthetase, NADH-dependent glutamate dehydrogenase, glutamate oxaloacetate transaminase and glutamate pyruvate transaminase activity. Glutamate synthase activity could not be detected in any of these species. the levels of specific activities varied depending on the enzymes tested. For all species the highest activities were detected for the transaminase reactions yielding glutamate, and for glutamate dehydrogenase. A general conclusion is that the pathway of nitrogen assimilation in free-living soil amoebae is similar to the one observed for other eukaryotes. Differences in specific activities were detected between the species.     

Isozyme Variation among 40 Frankia Strains

Forty Frankia strains belonging to the Alnus and Elaeagnus host specificity groups and isolated from various plant species from different geographical areas were characterized by the electrophoretic separation of isozymes of eight enzymes. All the enzyme systems that were investigated showed large variation. Diaphorases and esterases gave multiple band patterns and confirmed the identification of specific Frankia strains. Less variability was observed with enzymes such as phosphoglucose isomerase, leucine aminopeptidase, and malate dehydrogenase, which allowed for the delineation of larger groups of Frankia strains. Cluster analysis, based on the pair-wise similarity coefficients calculated between strains, delineated three large, dissimilar groups of Frankia strains, although each of these groups contained a large amount of heterogeneity. However, numerous Frankia strains, mainly from the Alnus host specificity group, demonstrated a perfect homology for all the enzymes tested.     

Characterization of Staphylococcus species by ribosomal RNA gene restriction patterns

The rRNA gene restriction pattern sof 110 strains belonging to 12 staphylococcal species have been determined. The strains, isolated from various sources, were epidemiologically unrelated. Total DNA was cleaved with restriction enzymes HindIII and EcoRI, electrophoretically separated and probed with radiolabelled 16S rDNA from Bacillus subtilis inserted in a plasmid vector, pBR322. Fourty-four distinct HindIII patterns and 44 distinct EcoRI patterns were observed. Strains belonging to different species had different patterns. Although distinct patterns were also observed with some species, a core of common bands could be discerned within each species or subspecies. Analysis of the patterns revealed two taxa in Staphylococcus xylosus which were not evident using phenotypic characteristics. Of 18 strains which were difficult to identify using phenotypic schemes, 15 showed patterns typical of known species. The three remaining atypical strains showed unusual patterns and may belong either to a known species, not included in the study, or to a new species. Since various patterns were observed within some species (e.g.S.aureus and S. epidermidis), rRNA gene restriction patterns may have epidemiological, as well as taxonomic interest.     

Isoenzymes in two micromycetes species: taxonomic aspects

Strains of different origins belonging to the two species of fungi imperfecti Cylindrocarpon ianothele and Calcarisporium arbuscula exhibit difference in their morphological or physiological appearances. We have attempted to determine if these variabilities could also be observed at the level of other phenotypic markers, the enzymes glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, octanol dehydrogenase and malate dehydrogenase. Isozymes were separated by polyacrylamide gel electrophoresis and demonstrated isozyme variability as a function of growth and physiological states of a given strain. At the same state of development, the different strains exhibited highly heterogeneous isozyme groups which appear difficult to use for taxonomic purposes.     

Production of bacteriolytic enzymes as a tool for characterizing enterococci

Bacteriolytic enzymes secreted by log-phase cultures of enterococci ( Enterococcus faecalis, Ent. faecium, Ent. durans, Ent. hirae, Ent. casseliflavus, Ent. avium, Ent. mundtii ) were analysed by means of a zymogram technique to resolve activities according to the size of their polypeptide component and their specificities towards different substrates. Heterogeneous patterns of lytic activity were observed with different species. For each test substrate, homogeneous patterns of lytic activities were observed with strains of the same species, except for Ent. faecalis strains, which showed heterogeneous lytic patterns even towards the same substrate, and could be divided into at least four different groups according to their lytic pattern. No lytic activity was common to all strains tested. Results of zymogram analysis of Enterococcus bacteriolytic enzymes were consistent with current knowledge on enterococcal taxonomy, indicating that this analytical approach may be a useful tool for fine-tuned characterization of different enterococcal strains.     

Glucose-6-phosphate dehydrogenase and malate dehydrogenase enzyme electrophoretic patterns amongst strains of Bacteroides fragilis

Fifty-two strains of Bacteroides fragilis were examined for their enzyme electrophoretic patterns of glucose-6-phosphate dehydrogenase (G6PDH) and malate dehydrogenase (MDH). All strains tested possessed high levels of both enzymes but the G6PDH reduced NADP whereas MDH was NAD-dependent. Twenty-seven strains produced single bands of both G6PDH and MDH. In all cases G6PDH migrated faster than MDH. Strains clustered by a single linkage algorithm were recovered in eight clusters at the 77% similarity level. The remaining 25 strains produced multiple bands of one or both enzymes. These were recovered in six clusters at the 72% similarity level using the same algorithm. The results of this study revealed considerable heterogeneity of enzyme patterns within B. fragilis.     

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.     

The Species Problem in a Ciliate with a High Multiple Mating Type System, Euplotes crassus

ABSTRACT. One hundred twenty non-autogamous wild-type strains of Euplotes crassus , collected over seven years, mainly from the Mediterranean coasts, were investigated for their mating interactions. The strains were mixed pair-wise and data from mating reactions were evaluated and organized by means of a specially constructed computer program. The program identified 38 strains with distinctive mating patterns which could be clustered in nine clumps, all of which were connected either directly or indirectly. Thus, all these strains appeared to be components of the same gene pool, even though direct genetic exchange between strains was not possible in every combination. Subsequently, the 38 strains were subjected to cytometric analysis and scored for zymic variations resulting from electrophoretic patterns of five enzyme systems (acid phosphatases, amylases, malic dehydrogenase, malic enzyme, and tetrazolium oxidases) of proved diagnostic value in the identification of Euplotes species. No significant discontinuities correlated with mating patterns was apparent from these analyses. It was concluded that the E. crassus strains analyzed are not properly divided in sibling species and it was consistently suggested to avoid a genetic partitioning of ciliate species endowed with high multiple mating type systems, in which the sets of wild strains brought under investigation with difficulty represent the natural dimensions of the species.     

Localization in cardiac muscle of some enzymes related to glutamate metabolism.

A tetrazolium staining medium incorporated in a gel has been used in a histochemical study of enzymes in thin sections of heart muscle. Formazan distribution patterns given by mitochondrial enzymes were inconsistent with the location of these enzymes revealed by the extraction of whole tissue. Similar stain distributions were given by lactate dehydrogenase, glutamate oxaloacetate transaminase and glutamate dehydrogenase. The distribution given by succinate dehydrogenase was not the same as that given by cytochrome oxidase stained by a different technique. Alcohol dehydrogenase added to the tissue assumed a distribution which suggested some adsorption of the enzyme to the tissue. But experiments suggested that this enzyme was not firmly bound to muscle proteins in the manner of some glycolytic enzymes.     

Isozyme variability and differentiation between Rhodnius prolixus, R.robustus and R.pictipes, vectors of Chagas disease in Venezuela

Enzyme polymorphism in triatomine bugs of the genus Rhodnius (Hemiptera: Reduviidae), vectors of Chagas disease, is analysed using both starch and polyacrylamide gel electrophoresis. Out of forty-five enzymes assayed, the electromorphs of seventeen of them: AO, CA, DIA, ES, ES-A, FH, GPD, G6PD, GPI, MDH, ME, 6PGD, PGM, ACON, ACPH, LAP and SOD, involving twenty-two putative structural loci, were scorable. These gene-enzyme systems were therefore selected for routine characterization of R.prolixus Stal adults from different strains. The first thirteen enzymes, involving sixteen structural loci, were also analysed in first instar nymphs of the three species, R.prolixus, R.robustus Larrousse and R.pictipes Stal. Allelic frequencies were calculated for three R.prolixus strains: three to five loci appeared to be polymorphic. The proportion of polymorphic loci (22%) and the average heterozygosity (0.06) indicated low genetic variability, with significant differences between the strains at individual loci. Rhodnius prolixus and R.robustus were found to have identical isozymic patterns. R.pictipes was genetically well differentiated, with twelve diagnostic loci.     

New developments in oxidative fermentation

Oxidative fermentations have been well established for a long time, especially in vinegar and in L-sorbose production. Recently, information on the enzyme systems involved in these oxidative fermentations has accumulated and new developments are possible based on these findings. We have recently isolated several thermotolerant acetic acid bacteria, which also seem to be useful for new developments in oxidative fermentation. Two different types of membrane-bound enzymes, quinoproteins and flavoproteins, are involved in oxidative fermentation, and sometimes work with the same substrate but produce different oxidation products. Recently, there have been new developments in two different oxidative fermentations, D-gluconate and D-sorbitol oxidations. Flavoproteins, D-gluconate dehydrogenase, and D-sorbitol dehydrogenase were isolated almost 2 decades ago, while the enzyme involved in the same oxidation reaction for D-gluconate and D-sorbitol has been recently isolated and shown to be a quinoprotein. Thus, these flavoproteins and a quinoprotein have been re-assessed for the oxidation reaction. Flavoprotein D-gluconate dehydrogenase and D-sorbitol dehydrogenase were shown to produce 2-keto- D-gluconate and D-fructose, respectively, whereas the quinoprotein was shown to produce 5-keto- D-gluconate and L-sorbose from D-gluconate and D-sorbitol, respectively. In addition to the quinoproteins described above, a new quinoprotein for quinate oxidation has been recently isolated from Gluconobacter strains. The quinate dehydrogenase is also a membrane-bound quinoprotein that produces 3-dehydroquinate. This enzyme can be useful for the production of shikimate, which is a convenient salvage synthesis system for many antibiotics, herbicides, and aromatic amino acids synthesis. In order to reduce energy costs of oxidative fermentation in industry, several thermotolerant acetic acid bacteria that can grow up to 40 degrees C have been isolated. Of such isolated strains, some thermotolerant Acetobacter species were found to be useful for vinegar fermentation at a high temperature such 38-40 degrees C, where mesophilic strains showed no growth. They oxidized higher concentrations of ethanol up to 9% without any appreciable lag time, while alcohol oxidation with mesophilic strains was delayed or became almost impossible under such conditions. Several useful Gluconobacter species of thermotolerant acetic acid bacteria are also found, especially L-erythrulose-producing strains and cyclic alcohol-oxidizing strains. Gluconobacter frateurii CHM 43 is able to rapidly oxidize meso-erythritol at 37 degrees C leading to the accumulation of L-erythrulose, which may replace dihydroxyacetone in cosmetics. G. frateuriiCHM 9 is able to oxidize cyclic alcohols to their corresponding cyclic ketones or aliphatic ketones, which are known to be useful for preparing many different physiologically active compounds such as oxidized steroids or oxidized bicyclic ketones. The enzymes involved in these meso-erythritol and cyclic alcohol oxidations have been purified and shown to be a similar type of membrane-bound quinoproteins, consisting of a high molecular weight single peptide. This is completely different from another quinoprotein, alcohol dehydrogenase of acetic acid bacteria, which consists of three subunits including hemoproteins.     

Genetic control of alcohol dehydrogenase levels in Drosophila

Among the progeny of Drosophila flies heterozygous for two noncomplementing Adh-negative alleles, two individuals were found that had recovered appreciable alcohol dehydrogenase activity, thereby surviving the ethanol medium used as a screen. The most likely explanation is that these Adh-positive flies are the product of intracistronic recombination within the Adh locus. Judging by the distribution of outside markers, one of the crossovers would have been a conventional reciprocal exchange while the other appears to have been an instance of nonreciprocal recombination. The enzymes produced in strains derived from the original survivors can be easily distinguished from wild-type enzymes ADH-S and ADH-F on the basis of their sensitivity to denaturing agents. None of various physical and catalytic properties tested revealed differences between the enzymes of the survivor strains except that in one of them the level of activity is 55–65% of the other. Quantitative immunological determinations of ADH gave estimates of enzyme protein which are proportional to the measured activity levels. These results are interpreted to indicate that different amounts of ADH protein are being accumulated in the two strains.This work was supported in part by NSF Grant PCM 76-19563.     

Origin of cytokinin-and auxin-autonomy and changes in specific proteins in tobacco callus tissue

On the basis of earlier data it was suggested that the induction of cytokinin autonomy might be accompanied by disorders in plastid function and a decrease in cytokinin utilization. In the work presented below the formation of chlorophyll and the isozyme patterns of nine enzymes, some of which are known to be localized in plastids, were compared in tobacco callus tissues differing in their hormonal requirements. Tissues either not requiring cytokinin or both auxin and cytokinin for their growth, contained a lower amount of chlorophyll than the cytokinin-and auxin-dependent strain. The number of isozymes of glucose-6-phosphate and NADP-malate dehydrogenase (i.e. enzymes which are known to be located in plastids) was reduced from four in the cytokinin-and auxin-dependent strain to two and one in the two cytokinin-autonomous strains, respectively. The fully habituated tissue contained an additional isozyme of NADP-malate dehydrogenase. The total number of isozymes of the remaining enzymes (NAD-malate dehydrogenase, peroxidase, esterase and a-and β-galactosidase) either was decreased or not changed in the cytokinin autonomous strains. The exception was an additional anodic peroxidase in one strain. The number of these isozymes in tissue habituated with respect to both auxin and cytokinin either remained the same or increased. Tobacco callus strains with altered requirements for growth regulators contained some new isozymes which were not present in any other strain and some isozymes present in other strains were absent. These differences are discussed in relation to the possible role of plastid function disorder associated with habituation.     

Extracellular enzymes of mycobacteria

Abstract Extracellular enzymes were studied in different mycobacteria using a plate substrate assay. All the pathogenic mycobacteria included in the study showed the presence of protease, while lipase, ribonuclease, mucinase and β-lactamase could also be detected in some strains. In contrast, no protease was detected in the 3 saprophytic mycobacteria studied. DNase was not detected in any of the species studied. Thus, the demonstration of extracellular enzymes, in particular of protease, in mycobacteria may be relevant in understanding their role in pathogenicity.     

Intraspecific diversity of Oenococcus oeni isolated during red wine-making in Japan

Using molecular and chemotaxonomic techniques, we studied the intraspecific diversity of Oenococcus oeni, a lactic acid bacterium isolated during red wine-making in Japan. The results confirmed high values of DNA-DNA relatedness and strong similarity among 16S rDNA sequences of the isolates with the O. oeni-type strain. Pulsed-field gel electrophoresis (PFGE) by NotI identified four patterns among the strains. Three different patterns of lactate dehydrogenase mobility were seen and there was a strong correlation between PFGE pattern and mobility. The present results suggest that the different strains of O. oeni comprise one species, and that variations in the genomic profiles of the different strains of O. oeni, including Japanese isolates are well correlated.     

Discrimination of Corynebacterium glutamicum, Brevibacterium flavum and Brevibacterium lactofermentum by restriction pattern analysis of DNA adjacent to the hom gene

Different strains of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum were analysed for restriction fragment length polymorphism using the homoserine dehydrogenase gene (hom) as a probe. The hybridization patterns obtained PvuII- or Asp700-restriction of chromosomal DNA were specific and distinguishable for each of the three species and identical for the different strains of each species. Thus, the method employed allows rapid distinction of Corynebacterium glutamicum, Brevibacterium flavum, and Brevibacterium lactofermentum. The former species could also be discriminated from the latter two by its resistance to 0.5 g/l of the methionine analog ethionine.