Further Electrophoretic Characterization of Strains of Tetrahymena pyriformis*

Mobility patterns of 5 isozymes in strains of Tetrahymena pyriformis were demonstrated using polyacrylamide disc gel electrophoresis. Six stock strains were compared in these patterns to 4 strains representative of each of the previously described 4 major “phenotypic sets.” Stock strains segregated into predicted “phenosets,” and essentially confirmed validity and reproducibility of such a discrimination method. The proposal that new strain designations be assigned on a basis of “phenoset” conformity is reaffirmed.     

DNA restriction endonuclease cleavage patterns,DNA sequence similarity and phenotypical characteristics in some strains of Lactobacillus helveticus and Lactobacillus jugurti

Physiological characteristics, deoxyribonucleic acid (DNA) base composition (% guanine + cytosine; % GC), DNA sequence similarity (% DNA-DNA hybridization) and DNA restriction endonuclease cleavage patterns of two strains of Lactobacillus helveticus and four strains of Lactobacillus jugurti were examined.All the strains investigated were closely related genetically, having DNA-DNA hybridization values ranging from 89–100%. Nevertheless, these strains can be differentiated from one another on the basis of the digestion of their DNA by specific restriction endonucleases, such as Bam HI, Eco RI and Hind III. The DNA of these strains shows clear, reproducible and distinct cleavage patterns. Cleavage patterns of DNA from strains L. jugurti S.35.19 and S.36.2 were found to be similar. These findings suggest that fingerprinting of DNA by restriction endonuclease cleavage might provide, in addition to the conventional methods, a useful tool for the characterization of closely related microorganisms at the strain level.     

Phylogenetic relatedness among Spirulina and related cyanobacterial genera

Molecular polymorphisms in a selected set of Spirulina and related genera using random primers based on repetitive sequences along with biochemical parameters, led to the unambiguous differentiation of the strains and understanding of their phylogenetic relationships. A combination of 10 sets of dual primers generated 100% distinct polymorphic bands ranging from 150 to 5,000 bp. Total number of fragments ranged from 68 to 159 whereas polymorphic bands ranged from 13 to 32 for different Random Amplified Polymorphic DNA (RAPD) reactions. Spirulina platensis strains, Sp-2 and Sp-3, possessed quite comparable chlorophyll and protein content besides having maximum similarity coefficient (0.88) between them on the basis of RAPD reactions, thus proved to be closely related. Sp-8 (Spirulina from Loktak Lake) having the highest protein content and protein: chlorophyll ratio, showed close similarity with the mutant of Spirulina platensis (Sp-7) on the basis of RAPD analysis. Duncan’s Multiple Range Test (DMRT) ranking for the biochemical parameters were quite closer for the strains of Spirulina and Arthrospira. This is also supported by the cluster analysis based on RAPD data, as the strains of Spirulina and Arthrospira are placed together in the same subcluster in the dendrogram. The comparative closeness among the strains of Lyngbya, Oscillatoria and Phormidium is reflected by the low content of protein and protein: chlorophyll ratio, which is also supported by the dendrogram based upon RAPD; thus, exhibiting the usefulness of multiplex RAPD along with biochemical parameters for the phylogenetic analysis of Spirulina and related genera.     

The mushroom habitat as an ecological arena for global exchange of Wolbachia

Wolbachia infect a variety of arthropod and nematode hosts, but in arthropods, host phylogenetic relationships are usually poor predictors of strain similarity. This suggests that new infections are often established by horizontal transmission. To gain insight into the factors affecting the probability of horizontal transmission among host species, we ask how host phylogeny, geographical distribution and ecology affect patterns of Wolbachia strain similarity. We used multilocus sequence typing (MLST) to characterize Wolbachia strain similarity among dipteran hosts associated with fleshy mushrooms. Wolbachia Supergroup A was more common than Supergroup B in Diptera, and also more common in mycophagous than non‐mycophagous Diptera. Within Supergroup A, host family within Diptera had no effect on strain similarity, and there was no tendency for Wolbachia strains from sympatric host species to be more similar to one another than to strains from hosts in different biogeographical realms. Supergroup A strains differed between mycophagous and non‐mycophagous Diptera more than expected by chance, suggesting that ecological associations can facilitate horizontal transmission of Wolbachia within mycophagous fly communities. For Supergroup B, there were no significant associations between strain similarity and host phylogeny, biogeography, or ecology. We identified only two cases in which closely related hosts carried closely related Wolbachia strains, evidence that Wolbachia‐host co‐speciation or early introgression can occur but may not be a major contributor to overall strain diversity. Our results suggest that horizontal transmission of Wolbachia can be influenced by host ecology, thus leading to partial restriction of Wolbachia strains or strain groups to particular guilds of insects.     

Strain variation in Campylobacter pylori detected by numerical analysis of one-dimensional electrophoretic protein patterns

A total of 21 clinical isolates of Campylobacter pylori from Peru and the United Kingdom and two reference strains (from Australia), including the type strain (NCTC 11637^T), were characterized by high resolution one-dimensional SDS-polyacrylamide gel electrophoresis of cellular proteins. The protein patterns contained more than 40 discrete bands and the approximate molecular weights of the major bands were 22, 27, 46, 57, 60, 65 and 93 kD. The total patterns were used as the basis of numerical analysis. Most strains were clustered in four phenons at 91% similarity with the exception of six ungrouped strains. Overall similarity was high with all strains linked in the phenogram at 81%. Variation among strains was attributable principally to qualitative and quantitative band differences in the 47 to 56 kD (hypervariable) region of the C. pylori protein profile. From the analysis, ten different electropherotypes (EP-types) were identified. We demonstrated that differences were detectable among isolates from widely separated geographical locations as well as from the same location, although multiple isolates from two Peruvian patients had the same electropherotype. Our results indicate that determination of protein profiles provides the basis of a reproducible method for characterization of C. pylori isolates.     

Surface‐based protein binding pocket similarity

Protein similarity comparisons may be made on a local or global basis and may consider sequence information or differing levels of structural information. We present a local three‐dimensional method that compares protein binding site surfaces in full atomic detail. The approach is based on the morphological similarity method which has been widely applied for global comparison of small molecules. We apply the method to all‐by‐all comparisons two sets of human protein kinases, a very diverse set of ATP‐bound proteins from multiple species, and three heterogeneous benchmark protein binding site data sets. Cases of disagreement between sequence‐based similarity and binding site similarity yield informative examples. Where sequence similarity is very low, high pocket similarity can reliably identify important binding motifs. Where sequence similarity is very high, significant differences in pocket similarity are related to ligand binding specificity and similarity. Local protein binding pocket similarity provides qualitatively complementary information to other approaches, and it can yield quantitative information in support of functional annotation. Proteins 2011; © 2011 Wiley‐Liss, Inc.     

Genetic diversity analysis of <Emphasis Type="Italic">Monascus</Emphasis> strains using SRAP and ISSR markers

In this study, sequence-related amplification polymorphism (SRAP) and inter-simple sequence repeat (ISSR) were analyzed for accessing the genetic diversity of 37 Monascus isolates and 14 control strains. According to the dendrogram produced by SRAP data, all the tested strains were grouped into four clusters at a 78% similarity level. Comparatively, 51 tested strains were divided into four major groups at a similarity level of 74% based on the dendrogram generated via ISSR marker analysis. Based on the two sets of dendrograms, Monascus aurantiacus, M. purpureus, M. serorubescens, M. anka, and M. ruber were clustered in the same clade; M. albidus, M. fuliginosus, and M. barkeri were clustered with M. pilosus in a second clade; and M. lunisporas and M. argentinensis occurred together in a third cluster distinct from the other Monascus species. The cluster result produced by SRAP data shared great similarity with that by ISSR data with minor differences in the subgroups, which is basically in agreement with morphological observations. In general, SRAP and ISSR are more simple, rapid, and efficient, which may provide alternative molecular approaches to studying genetic diversity, classification, and identification of Monascus strains.     

Mechanisms of prey selection by predaceous stoneflies: roles of prey morphology,behavior and predator hunger

Summary Laboratory feeding experiments using Hesperoperla pacifica (Banks), Perlidae, and Megarcys signata (Hagen), Perlodidae, as predators and Baetis tricaudatus Dodds and Ephemerella altana Allen as prey indicate a strong effect of prey morphology and mobility and predator hunger on prey selection by stoneflies. Knowledge of both dietary composition and feeding behavior was necessary to fully understand prey selection by these stoneflies.Fasted stoneflies presented with live prey ate more E. altana while satiated stoneflies ate approximately equal numbers of the two mayfly species. This pattern of dietary composition was the result of a reduction of attack frequency on the slower swimming E. altana with predator satiation and a continued high attack rate on B. tricaudatus regardless of recent feeding history. In contrast, fasted H. pacifica fed fresh frozen mayflies ate more B. tricaudatus indicating the importance of differences in prey mobility in controlling dietary composition.The high degree of similarity in patterns of feeding and mechanisms underlying those patterns for H. pacifica and M. signata suggest that they may be using similar rules for choosing mayfly prey and we suggest that mayfly prey are ranked by stoneflies on the basis of handling times. A general mechanistic model for stoneflies feeding on mayflies is presented and discussed.     

Filtering across Spatial Scales: Phylogeny,Biogeography and Community Structure in Bumble Bees

Despite the expansion of phylogenetic community analysis to understand community assembly, few studies have used these methods on mobile organisms and it has been suggested the local scales that are typically considered may be too small to represent the community as perceived by organisms with high mobility. Mobility is believed to allow species to mediate competitive interactions quickly and thus highly mobile species may appear randomly assembled in local communities. At larger scales, however, biogeographical processes could cause communities to be either phylogenetically clustered or even. Using phylogenetic community analysis we examined patterns of relatedness and trait similarity in communities of bumble bees (Bombus) across spatial scales comparing: local communities to regional pools, regional communities to continental pools and the continental community to a global species pool. Species composition and data on tongue lengths, a key foraging trait, were used to test patterns of relatedness and trait similarity across scales. Although expected to exhibit limiting similarity, local communities were clustered both phenotypically and phylogenetically. Larger spatial scales were also found to have more phylogenetic clustering but less trait clustering. While patterns of relatedness in mobile species have previously been suggested to exhibit less structure in local communities and to be less clustered than immobile species, we suggest that mobility may actually allow communities to have more similar species that can simply limit direct competition through mobility.     

Biodiversity of populations of phosphate solubilizing rhizobia that nodulates chickpea in different Spanish soils

Within rhizobia, two species nodulating chickpea, Mesorhizobium ciceri and Mesorhizobium mediterraneum, are known as good phosphate solubilizers. For this reason, we have analysed the ability to solubilize phosphate of a wide number of strains isolated from Cicer arietinum growing in several soils in Spain. The aim of this work was to analyse microbial populations nodulating chickpea, that are able to solubilize phosphates, using molecular techniques. In the present work we analyzed 19 strains isolated from effective nodules of C. arietinum growing in three soils from the North of Spain. Nineteen strains showed ability to solubilize phosphate in YED-P medium. These strains were separated into 4 groups according to the results obtained by 879F-RAPD fingerprinting. The 16S rDNA sequencing of a representative strain from each group allowed the identification of strains as belonging to the genus Mesorhizobium. Strains from groups I and II showed a 99.4% and 99.2% similarity with M. mediterraneum UPM-CA142^T, respectively. The strains from group III were related to M. tianshanense USDA 3592^T at a 99.4% similarity level. Finally, the strain from group IV was related to M. ciceri USDA 3383^T with a 99.3% similarity. The LMW RNA profiles confirmed these results. Strains from groups I and II showed an identical LMW RNA profile to that of M. mediterraneum UPM-CA142^T; the profile of strains from group III was identical to that of M.␣tianshanense USDA 3592^T and the profile of strains from group IV was identical to that of M. ciceri USDA 3383^T. Different 879F-RAPD patterns were obtained for strains of the group I, group II and the M.␣mediterraneum type strain (UPM-CA142^T). The 879-RAPD patterns obtained for group III also differed from the pattern shown by M. tianshanense USDA 3592^T. Finally, the patterns between group IV and M. ciceri USDA 3383^T were also different. These results suggest that groups I and II may be subspecies of M. mediterraneum, group III a subspecies of M. tianshanense and group IV a subspecies of M. ciceri. Nevertheless, more studies are needed to establish the taxonomic status of strains isolated in this study.     

Novel Endophytes of Rice form a Taxonomically Distinct Subgroup of Serratia marcescens

Six endophytic strains isolated from surface-sterilized rice roots and stems of different rice varieties grown in the Philippines were characterized. They were analyzed by physiological and biochemical tests, SDS-PAGE of whole-cell protein patterns, DNA-DNA hybridization and 16S rDNA sequencing. SDS-PAGE of whole-cell patterns showed that the six isolates fell into two subgroups which were similar but not identical in protein patterns to S. marcescens. The phylogenetic analysis of 16S rDNA sequences of two representative strains IRBG 500 and IRBG 501 indicated that they were closely related to S. marcescens(more than 99% identity). Physiological and biochemical tests corroborated that the isolates were highly related to each other and to S. marcescens. In cluster analysis, all six isolates were clustered together at 93% similarity level and grouped closely with Serratia marcescens at 86% similarity level. DNA-DNA hybridization studies revealed that the isolates shared high similarity levels with S. marcescens(≥86% DNA-DNA binding), indicating they belong to the same species. However, the isolates differed in several biochemical characteristics from the type strain. They produce urease and utilize urea and L(+) sorbose as a substrate, which is different from all known Serratia reference strains. These results suggest that the six endophytic isolates represent a novel, non-pigmented subgroup of S. marcescens.     

Comparative analysis of genetic variation among <Emphasis Type="Italic">Xanthomonas axonopodis pv manihotis</Emphasis> isolated from the western states of Nigeria using RAPD and AFLP

Xanthomonas axonopodis pv manihotis is the causal agent of cassava bacterial blight (CBB) worldwide. CBB disease is a major constraint to cassava cultivation, and losses can be extremely severe in regions where highly susceptible cultivars are grown. To develop an efficient disease management policy, the genetic diversity of the pathogens population must be known. There is dearth of information on the genetic diversity of X. axonopodis pv manihotis population in Nigeria. We used RAPD (random amplified polymorphic DNA) and AFLP (amplified fragment length polymorphism), a PCR-based technique, to characterize the X. axonopodis pv manihotis isolates from the western States of Nigeria. Thirteen strains Xam and 2 reference strains were tested with eight primers combination of AFLP and 4 RAPD primers. RAPD amplified DNA fragment data showed four major clusters at 80 % similarity coefficient level and two strains were not clustered by this analysis. Strains Kwa76A and Ond48A were also separated in the principal component analysis of the same data. Numerical analysis differentiated the AFLP patterns into four distinct clusters and grouped two strains separately at 66 % similarity. PCA assembly grouped the bacterial strains into 4 and one of the strains was singled out from the others. The two DNA analyses techniques seem to be complimentary to one another and informative on the genomic structure of Xam population in Western Nigeria. The genetic analysis presented here contributes to understanding of the Xam population structure in Western Nigeria.     

Protein profile and biochemical properties of Bacillus circulans isolated from intestines of small free-living animals in Poland

Forty-seven strains ofBacillus circulans isolated from the intestinal tract of free-living small mammals from theNarvia and Biebrza National Park (NE Poland) were compared with the electrophoretic whole-cell protein patterns on the basis of SDS-PAGE and biochemical characteristic using API tests (50 CHB and 20E). The strains were grouped into two clusters (I and II) at the similarity of protein pattern of 78% using the simple matching coefficient and clustering on unweighted pair group arithmetic average algorithm (S_SM/UPGMA). Each of the clusters comprised reference strains. The rods included in the cluster I are characterized by higher biochemical activity in comparison with the bacilli II.     

Genetic diversity in populations of a freshwater ciliate

RAPD fingerprinting with nine different primers revealed that all of 18 E. aediculatus isolates from nine ponds and streams in western Germany, France and the U.S.A. were genetically different. The extent of genetic similarity between genotypes from different waters did not show a significant relationship with the geographical distance among habitats, although genotypes isolated from the same habitat showed a higher genetic similarity than genotypes isolated from different habitats. Phylogenetic analyses of RAPD patterns indicate a separation of E. aediculatus strains into subgroups within one species, but all strains were genetically more similar to one another than to strains from two other Euplotes species. Crossings of the different E. aediculatus strains revealed they belonged to seven mating types of one gene pool. The high genetic diversity observed is explained by a frequent occurrence of conjugation in the studied populations.     

Pattern of transposable elements distribution on <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> polytene chromosomes before and after quantitative trait selection

The effect of selecting the length of a radial vein on the distribution of hybridization sites for P and hobo transposons and mdg1 and mdg2 retrotransposons on polytene chromosomes of Drosophila melanogaster salivary glands was studied. The pattern of the transposable element (TEs) distribution was polymorphic in both parental and selected strains. The similarity in mdg1 and mdg2 distribution between strains selected in one direction was closer than between strains selected in the opposite direction, but the selected strains were closer to each other than to the parental strain, regardless of direction of selection. No new mdg2 hybridization sites that would be absent from the control were found in the selected strains compared to the control. The number of mdg1 and hobo hybridization sites was more selected in strains in the (+) direction than in the (−) direction. The mobility of hobo copies in the strains correlated with the presence of its full-sized copy in the genome. The polymorphism of all TEs studied except for mdg1 was higher in strains selected in the (+) direction than in the (−) direction. These results suggest that some TEs migrate over the genome independently of selection, whereas the other are markers of evolutionary events rather than their causes.     

Molecular assessment of diversity among endophytic diazotrophs isolated from subtropical Indian sugarcane

Twenty-two endophytic bacterial isolates from the roots of sugarcane were compared morphologically, biochemically and genetically. Gram staining, colony pigment, texture and other cultural characteristics were taken for morphological characterization. Oxidation-fermentation tests for D-glucose and D-sucrose, production of acid and hydrogen from different carbon source, oxidase activity, antibiotic and drug resistance patterns were chosen as the biochemical and physiological criteria. Twelve random decamer primers were used to analyze and compare these isolates through RAPD among themselves as well as with known standard diazotrophic strains. The isolates were compared through dendrograms constructed on the basis of similarity patterns obtained from biochemical and RAPD analysis. The estimated diversity through RAPD analysis was more evident than the diversity on the basis of morphological and biochemical characters. Within Acetobacter group, the isolates showed substantial genetic diversity for future exploitation as PGPRs and diazotrophic associative endophytes.     

Defining a similarity threshold for a functional protein sequence pattern: The signal peptide cleavage site

When preparing data sets of amino acid or nucleotide sequences it is necessary to exclude redundant or homologous sequences in order to avoid overestimating the predictive performance of an algorithm. For some time methods for doing this have been available in the area of protein structure prediction. We have developed a similar procedure based on pair-wise alignments for sequences with functional sites. We show how a correlation coefficient between sequence similarity and functional homology can be used to compare the efficiency of different similarity measures and choose a nonarbitrary threshold value for excluding redundant sequences. The impact of the choice of scoring matrix used in the alignments is examined. We demonstrate that the parameter determining the quality of the correlation is the relative entropy of the matrix, rather than the assumed (PAM or identity) substitution model. Results are presented for the case of prediction of cleavage sites in signal peptides. By inspection of the false positives, several errors in the database were found. The procedure presented may be used as a general outline for finding a problem-specific similarity measure and threshold value for analysis of other functional amino acid or nucleotide sequence patterns.     

Interpreting the SDS-PAGE protein patterns with self-organizing maps: application for the characterization of mosquito-pathogenic Bacillus strains

Aims: To present the pairwise comparison of potential mosquito‐pathogenic Bacillus strains based on their SDS‐PAGE protein patterns and to evaluate their characteristic toxicity patterns. Methods and Results: In this work, 20 Bacillus strains were subjected to qualitative toxicity tests against Aedes aegypti and Culex quinquefasciatus larvae. The selected strains were then characterized by SDS‐PAGE protein profiles. The highly heterogeneous multiple protein components of protein patterns were analysed using self‐organizing map (SOM), a ‘visualization and clustering’ tool. Members of mosquitocidal Bacillus species were classified in four distinct clusters, and then toxicity patterns were examined. Cluster (1, 1) comprised of three highly toxic strains of Bacillus sphaericus: SPH88, 1593 and KSD‐4; cluster (1, 2) consisted of two B. sphaericus strains: SSII‐1 and Bsp‐R that showed weak larvicidal activity; cluster (2, 1) constituted two B. sphaericus strains: WHO2297 and ISPC‐5 that possessed moderate toxicity; and cluster (2, 2) contained four B. thuringiensis ssp. israelensis strains: ONR‐60A, HD500, IPS70 and IPS82 belonging to serotype H14 but exhibited moderate to high mosquito larvicidal toxicity. Conclusions: SOM served as a colour‐coded alternate for easy visualization of similarities or dissimilarities between the strains even at the infra subspecies level. Furthermore, characteristic toxicity patterns of Bacillus strains of different clusters were determined. Significance and Impact of the Study: Analysis of electrophoretic protein patterns using SOM provides a better insight into the inter‐relationships of bacterial strains through similarity‐based clustering and pairwise comparison of two strains.     

Subfamily specific conservation profiles for proteins based on n-gram patterns

Background  

A new algorithm has been developed for generating conservation profiles that reflect the evolutionary history of the subfamily associated with a query sequence. It is based on n-gram patterns (NP{n,m}) which are sets of n residues and m wildcards in windows of size n+m. The generation of conservation profiles is treated as a signal-to-noise problem where the signal is the count of n-gram patterns in target sequences that are similar to the query sequence and the noise is the count over all target sequences. The signal is differentiated from the noise by applying singular value decomposition to sets of target sequences rank ordered by similarity with respect to the query.     

Pyrolysis-gas-liquid chromatography of fungi: Observations on variability among nine Penicillium species of the section asymmetrica,subsection fasciculata

Mathematical expressions of variability or similarity based on data from pyrolysis-gas-liquid chromatograms were calculated for each combination among three strains of each of nine fasciculatePenicillium spp. The mean similarity for eight of these species clustered around 41 but the similarity forP. cyclopium was 58. Within each of the nine species, exceptP. urticae, at least one combination of strains had a similarity of 51 or greater. Similarities were also calculated for 576 possible combinations among 27 strains used in this study. These data indicate that certain strains of these nine penicillia may be more closely related to strains of other fasciculate species than they are to strains within their own species. This agrees with previous observations on variability and intergradation in the subsection Fasciculata.