Multilocus sequence typing--what is resolved?

Nucleotide sequence-based methods for bacterial typing (multilocus sequence typing; MLST) allow rapid and global comparisons between results from different laboratories. Combining this advantage with the reduced cost of high throughput sequencing, increasing automation and the amenability of sequence data for evolutionary analysis, it seems inevitable that sequence-based typing will eventually predominate over gel-based methods such as pulsed-field gel electrophoresis (PFGE) for most bacterial species. The increasing availability of multiple genome sequences for single pathogenic species, and the recent development of many new MLST schemes, means that a re-examination of the utility of multilocus sequencing, and in particular the choice of gene loci, is now appropriate.     

Multilocus Sequence Analysis (MLSA) of ‘Rickettsiella agriotidis’, an Intracellular Bacterial Pathogen of Agriotes Wireworms

Wireworms, the polyphagous larvae of click beetles belonging to the genus Agriotes (Coleoptera: Elateridae) are severe and widespread agricultural pests that affect numerous crops globally. A new bacterial specimen identified in diseased wireworms had previously been shown by microscopy and 16S ribosomal RNA (rRNA) gene-based phylogenetic reconstruction to belong to the taxonomic genus Rickettsiella (Gammaproteobacteria) that comprises intracellular bacteria associated with and typically pathogenic for a wide range of arthropods. Going beyond these earlier results obtained from rRNA phylogenies, multilocus sequence analysis (MLSA) using a four marker scheme has been employed in the molecular taxonomic characterization of the new Rickettsiella pathotype, referred to as ‘Rickettsiella agriotidis’. In combination with likelihood-based significance testing, the MLSA approach demonstrated the close phylogenetic relationship of ‘R. agriotidis’ to the pathotypes ‘Rickettsiella melolonthae’ and ‘Rickettsiella tipulae’, i.e., subjective synonyms of the nomenclatural type species, Rickettsiella popilliae. ‘R. agriotidis’ forms, therefore, part of a Rickettsiella pathotype complex that most likely represents the species R. popilliae. As there are currently no genetic data available from the R. popilliae type strain, the respective assignment cannot be corroborated directly. However, an alternative taxonomic assignment to the species Rickettsiella grylli has been positively ruled out by significance testing. MLSA has been shown to provide a more powerful tool for taxonomic delineation within the genus Rickettsiella as compared to 16S rRNA phylogenetics. However, the limitations of the present MLSA scheme for the sub-species level classification of ‘R. agriotidis’ and further R. popilliae synonyms has been critically evaluated.     

Preparation of α and β anomers of various isomeric methyl O-d-galactopyranosyl-d-galactopyranosides. Standards for interpretation of 13C-n.m.r. spectra of d-galactopyranans

The four isomers of methyl O-β-d-galactopyranosyl-β-d-galactopyranoside were prepared by condensation of 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl bromide with appropriate, partially O-substituted derivatives of methyl β-d-galactopyranoside. Reaction of 3,4,6-tri-O-acetyl-1,2-O-(1-ethoxyethylidene)-α-d-galactopyranose with the same acceptors, in the presence of mercuric bromide, led to the formation of α and β linkages. Thus, it was possible to assign ¹³C-n.m.r. resonances of α and β anomers of methyl O-d-galactopyranosyl-β-d-galactopyranosides. In terms of application of these shift values and those of related d-galactobioses to the structural analysis of d-galactopyranans by shift comparisons, some generalizations can be made. For β-d-galactopyranans, the resonances of glycosyloxylated carbon atoms of methyl O-β-d-galactopyranosyl-β-d-galactopyranosides are sensitive to structure and appear to have typical values, whereas limited variation was observed with shifts of C-1′ signals. On the other hand, for assigning structures to d-galactopyranans containing α linkages, the C-1′ shifts (at higher field) of methyl O-α-d-galactopyranosyl-β-d-galactopyranosides are sensitive to linkage position, whereas those of glycosyloxylated carbon atoms vary only a little.     

Studies on the Constituents of Typha L.—IV. A Preliminary Study of Chemosystematics of Typha L.

From pollen grains of Typha davidiana, T. latifolia, T. angustata the same eight flavonoids have been isolated. They are identified as naringenin I, isorhamnetin II, quercetin III, isorhamnetin-3-O-(2^G-α-L-rhamnopyranosyl)-rutioside IV, quercetin-3-O-(2^G-α-L-rhamnopyranosyl)-rutinosida, V, isorhamnetio-3-O-rutinoside VI, isorhamnetino-3-O-neohesperidoside VII, kampferol-3-O-neohesperidoside VIII. Flavonoids of pollen grains of five species of Typha, including the above three species, were analysed by TLC with the result showing that the constituents in the pollen grains of the five species are very similar. The chemical comparison among Typha and Sparganium and 16 possibly related families shows that Typha is different from Pandanaceae or Pandanales and is similar to Restionaceae, Flagellariaceae, Juncaceae and Cyperaceae in some respects. Typha and Sparganium are very similar in many respects, and they could be treated in the same family, Typhaceae, which merit the rank of order, Typhales.     

The Use of the Expectation–Maximization (EM) Algorithm for Maximum Likelihood Estimation of Gametic Frequencies of Multilocus Polymorphic Codominant Systems Based on Sampled Population Data

Estimation of gametic frequencies in multilocus polymorphic systems based on the numerical distribution of multilocus genotypes in a population sample (analysis without pedigrees) is difficult because some gametes are not recognized in the data obtained. Even in the case of codominant systems, where all alleles can be recognized by genotypes, so that direct estimation of the frequencies of genes (alleles) is possible (complete data), estimation of the frequencies of multilocus gametes based on the data on multilocus genotypes is sometimes impossible, whether population data or even family data are used for studying genotypic segregation or analysis of linkage (incomplete data). Such incomplete data are analyzed based on the corresponding genetic models using the expectation–maximization (EM) algorithm. In this study, the EM algorithm based on the random-marriage model for a nonsubdivided population was used to estimate gametic frequencies. The EM algorithm used in the study does not set any limitations on the number of loci and the number of alleles of each locus. Locus and alleles are identified by numeration making possible to arrange loops. In each combination of alleles for a given combination of m out of L loci (L is the total number of loci studied), all alleles are assigned value 1, and the remaining alleles are assigned value 0. The sum of zeros and unities for each gamete is its gametic value (h), and the sum of the gametic values of the gametes that form a given genotype is the genotypic value (g) of this genotype. Then, gametes with the sameh are united into a single class, which reduces the number of the estimated parameters. In a general case of m loci, this procedure yields m + 1 classes of gametes and 2m + 1 classes of genotypes with genotypic valuesg = 0, 1, 2,... 2m. The unknown frequencies of them + 1 classes of gametes can be represented as functions of the gametic frequencies whose maximum likelihood estimations (MLEs) have been obtained in all previous EM procedures and the only unknown frequency (P _m(m)) that is to be estimated in the given EM procedure. At the expectation step, the expected frequencies (F _m(g) of the genotypes with genotypic valuesg are expressed in terms of the products of the frequencies of m + 1 classes of gametes. The data on genotypes are the numbers (n _g) of individuals with genotypic values g = 0, 1, 2, 3, ..., 2m. The maximization step is the maximization of the logarithm of the likelihood function (LLF) for n _g values. Thus, the EM algorithm is reduced, in each case, to solution of only one equation with one unknown parameter with the use of the n _g values, i.e., the numbers of individuals after the corresponding regrouping of the data on the individuals" genotypes. Treatment of the data obtained by Kurbatova on the MNSs and Rhesus systems with alleles C, C ^w , c, D, d, E, e with the use of Weir's EM algorithm and the EM algorithm suggested in this study yielded similar results. However, the MLEs of the parameters obtained with the use of either algorithm often converged to a wrong solution: the sum of the frequencies of all gametes (4 and 12 gametes for MNSs and Rhesus, respectively) was not equal to 1.0 even if the global maximum of LLF was reached for each of them (as it was for MNSs with the use of Weir's EM algorithm), with each parameter falling within admissible limits (e.g., 0, min(P _N, P _s) for P _Ns). The ² function is suggested to be used as a goodness-of-fit function for the distribution of genotypes in a sample in order to select acceptable solutions. However, the minimum of this function only guarantee the acceptability of solutions if all limitations on the parameters are met: the sum of estimations of gametic frequencies is 1.0, each frequency falls within the admissible limits, and the gametic algebra is complied with (none of the frequencies is negative).     

Phylogenenetic approach of the section Bulbocodii D.C. of Narcissus based on cpDNA. A case of taxonomic inflation?

In this paper, we analyzed the phylogeny of the section Bulbocodii (genus Narcissus; Amarillydaceae) using the matK and trnL-F fragments of cpDNA in order to review the validity of the recognized taxa. Our results indicate that Narcissus obesus should be considered a valid species, and that N. blancoi is a distinct taxon. In addition, seven previously recognized species, N. juressianus, N. subnivalis, N. graellsii, N. conspicuus, N. citrinus, N. nivalis, and N. quintanilhae, should be assigned to an infraspecific rank under N. bulbocodium, as they are not valid species. In addition, we analyzed the distribution of the three morphological characters widely used in the systematics of this section and found that their variation does not agree with the phylogenetic results, rendering these characters limited taxonomical utility. This result suggests that the section Bulbocodii shows high morphological lability, which can explain the proliferation of nominal species.     

Genetics of esterases in Drosophila. V. Characteristics of the “pupal” esterase in D. virilis

From analysis of the properties of the pupal esterase (p-esterase) in Drosophila virilis, it is concluded that it is heat stable, its electrophoretic detection depends on culture density, its expression is stage specific, and it is not a variant of esterase 2. It was also demonstrated that p-esterase, like esterase 6, is activated by injections of the juvenile hormone into larvae. Heat treatment of heat-resistant D. virilis stocks led to decreased activities of the juvenile hormone dependent esterases but did not affect those of the heat-sensitive stocks. It is suggested that heat resistance in D. virilis is related to some functional features of the system of modifier genes controlling the phenotypic expression of esterases.     

Microbial metabolism of aromatic nitriles. Enzymology of C–N cleavage by Nocardia sp. (Rhodochrous group) N.C.I.B. 11216

1. An organism utilizing benzonitrile as sole carbon and nitrogen source was isolated by the enrichment-culture technique and identified as a Nocardia sp. of the rhodochrous group. 2. Respiration studies indicate that nitrile degradation proceeds through benzoic acid and catechol. 3. Cell-free extracts of benzonitrile-grown cells contain an enzyme that catalyses the conversion of benzonitrile directly into benzoic acid without intermediate formation of benzamide. 4. This nitrilase enzyme was purified by DEAE-cellulose chromatography and gel filtration on Sephadex G-100 in the presence and absence of substrate. The purity of the enzyme was confirmed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and isoelectric focusing on polyacrylamide gel. 5. The enzyme shows a time-dependent substrate-activation process in which the substrate catalyses the association of inactive subunits of mol.wt. 45000 to form the polymeric 12-unit active enzyme of mol.wt. 560000. The time required for complete association is highly dependent on the concentration of the enzyme, temperature and pH. 6. The associated enzyme has a pH optimum of 8.0 and K(m) with benzonitrile as substrate of 4mm. The activation energy of the reaction as deduced from the Arrhenius plot is 51.8kJ/mol. 7. Enzyme activity is inhibited by thiol-specific reagents and several metal ions. 8. Studies with different substrates indicate that the nitrilase is specific for nitrile groups directly attached to the benzene ring. Various substituents in the ring are compatible with activity, though ortho-substitution, except by fluorine, renders the nitrile invulnerable to attack. 9. The environmental implications of these findings and the possible significance of the enzyme in the regulation of metabolism are discussed.     

Use of protoplasts in the improvement of filamentous microorganisms. I. Action of N-methyl-N′-nitro-N-nitrosoguanidine on protoplasts of Streptomycetes

Summary The effects of N-methyl-N–nitro-N-nitroso-guanidine (NTG) on protoplasts of Streptomycetes are markedly different from its action on spores, showing high mutagenic activity even at concentrations having no marked effect on protoplast survival. Strain improvement, eg in chlorotetracycline-producing strains of S. aureofaciens, was most effective when protoplasts were subjected to prolonged treatment (2 h) with low concentrations of NTG (50 /ug/ml).     

Micropropagation of Calluna vulgaris cv. ‘H.E. Beale’

Axillary shoot producing cultures were obtained from microcuttings and shoot tips of Calluna vulgaris cv. H.E. Beale. For cultures derived from microcuttings the highest multiplication rate of 38 shoots (5 mm or longer) was obtained on a reduced salt medium with the addition of 0.5 mgl^-1 2-isopentenyladenine (2iP) during an 8 week subculture. For shoot tip derived cultures 0.2 mgl^-1 6-benzyladenine (BA) was the best cytokinin and led to a multiplication rate of 26 for a 6 week subculture. The addition of 1 g/l casein hydrolysate to a multiplication medium enhanced shoot proliferation in presence of 0.5 mgl^-1 BA.Despite various auxin treatments shoots formed no roots in vitro but rooted readily if transferred to a peat substrate ex vitro. A high rooting percentage (80%) was also obtained with shoots taken from the end of a multiplication phase and rooted directly. An additional subculture on low auxin containing media before transfer to peat substrate is recommended because the shoot condition can be improved in this way. A high number of rooted plantlets was produced, so the methods described will allow mass propagation.     

Reversed phase h.p.l.c. of fully protected,synthetic βh- endorphin peptide segments of extreme hydrophobicity

Reversed-phase high-pressure liquid chromatography (h.p.l.c.) using trifluoroethanol/water as eluent has been used successfully in the analysis of two fully protected peptide segments of extreme hydrophobicity, β_h-endorphin (17–26) and β_h-endorphin (18–26).     

Monte Carlo simulation of the α-amylolysis of amylopectin potato starch. 2. α-amylolysis of amylopectin

A model is presented that describes all the saccharides that are produced during the hydrolysis of starch by an -amylase. Potato amylopectin, the substrate of the hydrolysis reaction, was modeled in a computer matrix. The four different subsite maps presented in literature for -amylase originating from Bacillus amyloliquefaciens were used to describe the hydrolysis reaction in a Monte Carlo simulation. The saccharide composition predicted by the model was evaluated with experimental values. Overall, the model predictions were acceptable, but no single subsite map gave the best predictions for all saccharides produced. The influence of an (16) linkage on the rate of hydrolysis of nearby (14) linkages by the -amylase was evaluated using various inhibition constants. For all the subsites considered the use of inhibition constants led to an improvement in the predictions (a decrease of residual sum of squares), indicating the validity of inhibition constants as such. As without inhibition constants, no single subsite map gave the best fit for all saccharides. The possibility of generating a hypothetical subsite map by fitting was therefore investigated. With a genetic algorithm it was possible to construct hypothetical subsite maps (with inhibition constants) that gave further improvements in the average prediction for all saccharides. The advantage of this type of modeling over a regular fit is the additional information about all the saccharides produced during hydrolysis, including the ones that are difficult to measure experimentally.