Molecular epidemiology and antibiotic susceptibility of livestock Brucella melitensis isolates from Naryn Oblast, Kyrgyzstan.

The incidence of human brucellosis in Kyrgyzstan has been increasing in the last years and was identified as a priority disease needing most urgent control measures in the livestock population. The latest species identification of Brucella isolates in Kyrgyzstan was carried out in the 1960s and investigated the circulation of Brucella abortus, B. melitensis, B. ovis, and B. suis. However, supporting data and documentation of that experience are lacking. Therefore, typing of Brucella spp. and identification of the most important host species are necessary for the understanding of the main transmission routes and to adopt an effective brucellosis control policy in Kyrgyzstan. Overall, 17 B. melitensis strains from aborted fetuses of sheep and cattle isolated in the province of Naryn were studied. All strains were susceptible to trimethoprim-sulfamethoxazole, gentamicin, rifampin, ofloxacin, streptomycin, doxycycline, and ciprofloxacin. Multilocus variable number tandem repeat analysis showed low genetic diversity. Kyrgyz strains seem to be genetically associated with the Eastern Mediterranean group of the Brucella global phylogeny. We identified and confirmed transmission of B. melitensis to cattle and a close genetic relationship between B. melitensis strains isolated from sheep sharing the same pasture.

THEORETICAL STUDIES ON THE METHODS OF RECONSTRUCTING PHYLOGENETIC TREES FROM DNA SEQUENCE DATA (MOLECULAR EVOLUTION, HOMINOID EVOLUTION, COMPUTER SIMULATION)

(1) A mathematical theory for computing the probabilities of various nucleotide configurations is developed, and the probability of obtaining the correct phylogenetic tree (model tree) from sequence data is evaluated for six phylogenetic tree-making methods (UPGMA, distance Wagner method, transformed distance method, Fitch-Margoliash's method, maximum parsimony method, and compatibility method). The number of nucleotides (m*) necessary to obtain the correct tree with a probability of 95% is estimated with special reference to the human, chimpanzee, and gorilla divergence. m* is at least 4,200, but the availability of outgroup species greatly reduces m* for all methods except UPGMA. m* increases if transitions occur more frequently than transversions as in the case of mitochondrial DNA. (2) A new tree-making method called the neighbor-joining method is proposed. This method is applicable either for distance data or character state data. Computer simulation has shown that the neighbor-joining method is generally better than UPGMA, Farris' method, Li's method, and modified Farris method on recovering the true topology when distance data are used. A related method, the simultaneous partitioning method, is also discussed. (3) The maximum likelihood (ML) method for phylogeny reconstruction under the assumption of both constant and varying evolutionary rates is studied, and a new algorithm for obtaining the ML tree is presented. This method gives a tree similar to that obtained by UPGMA when constant evolutionary rate is assumed, whereas it gives a tree similar to that obtained by the maximum parsimony tree and the neighbor-joining method when varying evolutionary rate is assumed. ^

Molecular evidence for multiple origins of woodiness and a New World biogeographic connection of the Macaronesian Island endemic Pericallis (Asteraceae: Senecioneae)

The prevalence of woody species in oceanic islands has attracted the attention of evolutionary biologists for more than a century. We used a phylogeny based on sequences of the internal-transcribed spacer region of nuclear ribosomal DNA to trace the evolution of woodiness in Pericallis (Asteraceae: Senecioneae), a genus endemic to the Macaronesian archipelagos of the Azores, Madeira, and Canaries. Our results show that woodiness in Pericallis originated independently at least twice in these islands, further weakening some previous hypotheses concerning the value of this character for tracing the continental ancestry of island endemics. The same data suggest that the origin of woodiness is correlated with ecological shifts from open to species-rich habitats and that the ancestor of Pericallis was an herbaceous species adapted to marginal habitats of the laurel forest. Our results also support Pericallis as closely related to New World genera of the tribe Senecioneae.

The phylogeny of closely related species as revealed by the genealogy of a speciation gene, Odysseus

Molecular differentiation between races or closely related species is often incongruent with the reproductive divergence of the taxa of interest. Shared ancient polymorphism and/or introgression during secondary contact may be responsible for the incongruence. At loci contributing to speciation, these two complications should be minimized (1, 2); hence, their variation may more faithfully reflect the history of the species' reproductive differentiation. In this study, we analyzed DNA polymorphism at the Odysseus (OdsH) locus of hybrid sterility between Drosophila mauritiana and Drosophila simulans and were able to verify such a prediction. Interestingly, DNA variation only a short distance away (1.8 kb) appears not to be influenced by the forces that shape the recent evolution of the OdsH coding region. This locus thus may represent a test case of inferring phylogeny of very closely related species.

Molecular phylogenetic analysis of evolutionary trends in stonefly wing structure and locomotor behavior

Insects in the order Plecoptera (stoneflies) use a form of two-dimensional aerodynamic locomotion called surface skimming to move across water surfaces. Because their weight is supported by water, skimmers can achieve effective aerodynamic locomotion even with small wings and weak flight muscles. These mechanical features stimulated the hypothesis that surface skimming may have been an intermediate stage in the evolution of insect flight, which has perhaps been retained in certain modern stoneflies. Here we present a phylogeny of Plecoptera based on nucleotide sequence data from the small subunit rRNA (18S) gene. By mapping locomotor behavior and wing structural data onto the phylogeny, we distinguish between the competing hypotheses that skimming is a retained ancestral trait or, alternatively, a relatively recent loss of flight. Our results show that basal stoneflies are surface skimmers, and that various forms of surface skimming are distributed widely across the plecopteran phylogeny. Stonefly wings show evolutionary trends in the number of cross veins and the thickness of the cuticle of the longitudinal veins that are consistent with elaboration and diversification of flight-related traits. These data support the hypothesis that the first stoneflies were surface skimmers, and that wing structures important for aerial flight have become elaborated and more diverse during the radiation of modern stoneflies.

Phylogeny of genes for secretion NTPases: Identification of the widespread tadA subfamily and development of a diagnostic key for gene classification

Macromolecular transport systems in bacteria currently are classified by function and sequence comparisons into five basic types. In this classification system, type II and type IV secretion systems both possess members of a superfamily of genes for putative NTP hydrolase (NTPase) proteins that are strikingly similar in structure, function, and sequence. These include VirB11, TrbB, TraG, GspE, PilB, PilT, and ComG1. The predicted protein product of tadA, a recently discovered gene required for tenacious adherence of Actinobacillus actinomycetemcomitans, also has significant sequence similarity to members of this superfamily and to several unclassified and uncharacterized gene products of both Archaea and Bacteria. To understand the relationship of tadA and tadA-like genes to those encoding the putative NTPases of type II/IV secretion, we used a phylogenetic approach to obtain a genealogy of 148 NTPase genes and reconstruct a scenario of gene superfamily evolution. In this phylogeny, clear distinctions can be made between type II and type IV families and their constituent subfamilies. In addition, the subgroup containing tadA constitutes a novel and extremely widespread subfamily of the family encompassing all putative NTPases of type IV secretion systems. We report diagnostic amino acid residue positions for each major monophyletic family and subfamily in the phylogenetic tree, and we propose an easy method for precisely classifying and naming putative NTPase genes based on phylogeny. This molecular key-based method can be applied to other gene superfamilies and represents a valuable tool for genome analysis.

Molecular cloning of the first metazoan beta-1,3 glucanase from eggs of the sea urchin Strongylocentrotus purpuratus.

We report the molecular cloning of the first beta-1,3 glucanase from animal tissue. Three peptide sequences were obtained from beta-1,3 glucanase that had been purified from eggs of the sea urchin Strongylocentrotus purpuratus and the gene was cloned by PCR using oligonucleotides deduced from the peptide sequences. The full-length cDNA shows a predicted enzyme structure of 499 aa with a hydrophobic signal sequence. A 3.2-kb message is present in eggs, during early embryogenesis, and in adult gut tissue. A polyclonal antibody to the native 68-kDa enzyme recognizes a single band during early embryogenesis that reappears in the adult gut, and recognizes a 57-kDa fusion protein made from a full-length cDNA clone for beta-1,3 glucanase. The identity of this molecule as beta-1,3 glucanase is confirmed by sequence homology, by the presence of all three peptide sequences in the deduced amino acid sequence, and by the recognition of the bacterial fusion protein by the antibody directed against the native enzyme. Data base searches show significant homology at the amino acid level to beta-1,3 glucanases from two species of bacteria and a clotting factor from the horseshoe crab. The homology with the bacteria is centered in a 304-aa region in which there are seven scattered regions of high homology between the four divergent species. These four species were also found to have two homologous regions in common with more distantly related plant, fungal, and bacterial proteins. A global phylogeny based on these regions strongly suggests that the glucanases are a very ancient family of genes. In particular, there is an especially deep split within genes taken from the bacterial genus Bacillus.

A new high molecular weight immunoglobulin class from the carcharhine shark: implications for the properties of the primordial immunoglobulin.

All immunoglobulins and T-cell receptors throughout phylogeny share regions of highly conserved amino acid sequence. To identify possible primitive immunoglobulins and immunoglobulin-like molecules, we utilized 3' RACE (rapid amplification of cDNA ends) and a highly conserved constant region consensus amino acid sequence to isolate a new immunoglobulin class from the sandbar shark Carcharhinus plumbeus. The immunoglobulin, termed IgW, in its secreted form consists of 782 amino acids and is expressed in both the thymus and the spleen. The molecule overall most closely resembles mu chains of the skate and human and a new putative antigen binding molecule isolated from the nurse shark (NAR). The full-length IgW chain has a variable region resembling human and shark heavy-chain (VH) sequences and a novel joining segment containing the WGXGT motif characteristic of H chains. However, unlike any other H-chain-type molecule, it contains six constant (C) domains. The first C domain contains the cysteine residue characteristic of C mu1 that would allow dimerization with a light (L) chain. The fourth and sixth domains also contain comparable cysteines that would enable dimerization with other H chains or homodimerization. Comparison of the sequences of IgW V and C domains shows homology greater than that found in comparisons among VH and C mu or VL, or CL thereby suggesting that IgW may retain features of the primordial immunoglobulin in evolution.

Population dynamics of flaviviruses revealed by molecular phylogenies.

The phylogeny of 123 complete envelope gene sequences was reconstructed in order to understand the evolution of tick- and mosquito-borne flaviviruses. An analysis of phylogenetic tree structure reveals a continual and asymmetric branching process in the tick-borne flaviviruses, compared with an explosive radiation in the last 200 years in viruses transmitted by mosquitoes. The distinction between these two viral groups probably reflects differences in modes of dispersal, propagation, and changes in the size of host populations. The most serious implication of this work is that growing human populations are being exposed to an expanding range of increasingly diverse viral strains.

Australian microhylid frogs (Cophixalus and Austrochaperina): phylogeny, taxonomy, calls, distributions and breeding biology

Despite a considerable surge in herpetological research in Australia over the last couple of decades the Australian microhylid frogs (Cophixalus and Austrochaperina) remain relatively poorly known. Herein I present the results of extensive fieldwork and molecular, morphological and call analysis with the aim of resolving taxonomy, call variation and distributions, and increasing our understanding of breeding biology. Analysis of 943 base pairs of mitochondrial 16S rRNA and 12S rRNA provides a well supported phylogeny that is largely consistent with current taxonomy. Levels of divergence between species are substantial and significant phylogeographic structuring is evident in C. ornatus, C. neglectus and C. aenigma, sp. nov. The description of C. concinnus was based on a mixed collection of two species from Thornton Peak and a new species is described to resolve this. C. aenigma, sp. nov., is described from high-elevation (>750 m) rainforest across the Carbine, Thornton, Finnigan and Bakers Blue Mountain uplands, north-east Queensland. C. concinnus is redescribed as a highly distinct species restricted to rainforest and boulder fields at the summit of Thornton Peak (>1100 m). Despite protection in Daintree National Park in the Wet Tropics World Heritage Area, predictions of the impact of global warming suggest C. concinnus to be of very high conservation concern ( Critically Endangered, IUCN criteria). The mating call of two species ( C. mcdonaldi and C. exiguus) is described for the first time and high levels of call variation within C. ornatus, C. neglectus, C. hosmeri, C. aenigma and Austrochaperina fryi are presented. Such variation is often attributable to genetically divergent lineages, altitudinal variation and courtship; however, in some instances ( particularly within C. hosmeri) the source or function of highly distinct calls at a site remains obscure. Molecular, morphological and call analyses allow the clarification of species distributions, especially in the northern mountains of the Wet Tropics. Notes are presented on the breeding biology of C. aenigma, C. bombiens, C. concinnus, C. exiguus, C. infacetus, C. mcdonaldi, C. monticola, C. neglectus, C. ornatus and C. saxatilis, which are largely consistent with previous accounts: small terrestrial clutches usually attended by a male. Courtship behaviour in C. ornatus is described and the first records of multiple clutching in Australian microhylids are presented (for C. ornatus and C. infacetus).

Cytochrome c(551) from Starkeya novella - Characterization, spectroscopic properties, and phylogeny of a diheme protein of the SoxAX family

Cytochromes from the SoxAX family have a major role in thiosulfate oxidation via the thiosulfate-oxidizing multi-enzyme system (TOMES). Previously characterized SoxAX proteins from Rhodovulum sulficlophilum and Paracoccus pantotrophus contain three heme c groups, two of which are located on the SoxA subunit. In contrast, the SoxAX protein purified from Starkeya novella was found to contain only two heme groups. Mass spectrometry showed that a disulfide bond replaced the second heme group found in the diheme SoxA subunits. Apparent molecular masses of 27,229 +/- 10.3 Da and 20,258.6 +/- 1 Da were determined for SoxA and SoxX with an overall mass of 49.7 kDa, indicating a heterodimeric structure. Optical redox potentiometry found that the two heme cofactors are reduced at similar potentials (versus NHE) that are as follows: + 133 mV (pH 6.0); + 104 mV (pH 7.0); +49 (pH 7.9) and +10 mV (pH 8.7). EPR spectroscopy revealed that both ferric heme groups are in the low spin state, and the spectra were consistent with one heme having a His/Cys axial ligation and the other having a His/Met axial ligation. The His/Cys ligated heme is present in different conformational states and gives rise to three distinct signals. Amino acid sequencing was used to unambiguously assign the protein to the encoding genes, soxAX, which are part of a complete sox gene cluster found in S. novella. Phylogenetic analysis of soxA- and soxX-related gene sequences indicates a parallel development of SoxA and SoxY, with the diheme and monoheme SoxA sequences located on clearly separated branches of a phylogenetic tree.

Molecular characterisation, pathogenesis and fungicide sensitivity of Pythium spp. from table beet (Beta vulgaris var. vulgaris) grown in the Lockyer Valley, Queensland

Table beet production in the Lockyer Valley of south-eastern Queensland is known to be adversely affected by soilborne root disease from infection by Pythium spp. However, little is known regarding the species or genotypes that are the causal agents of both pre- and post-emergence damping off. Based on RFLP analysis with HhaI, HinfI and MboI of the PCR amplified ITS region DNA from soil and diseased plant samples, the majority of 130 Pythium isolates could be grouped into three genotypes, designated LVP A, LVP B and LVP C. These groups comprised 43, 41 and 7% of all isolates, respectively. Deoxyribonucleic acid sequence analysis of the ITS region indicated that LVP A was a strain of Pythium aphanidermatum, with greater than 99% similarity to the corresponding P. aphanidermatum sequences from the publicly accessible databases. The DNA sequences from LVP B and LVP C were most closely related to P. ultimum and P. dissotocum, respectively. Lower frequencies of other distinct isolates with unique RFLP patterns were also obtained with high levels of similarity (> 97%) to P. heterothallicum, P. periplocum and genotypes of P. ultimum other than LVP B. Inoculation trials of 1- and 4-week-old beet seedlings indicated that compared with isolates of the LVP B genotype, a higher frequency of LVP A isolates caused disease. Isolates with the LVP A, LVP B and LVP C genotypes were highly sensitive to the fungicide Ridomil MZ, which suppressed radial growth on V8 agar between approximately four and thirty fold at 5 mu g/mL metalaxyl and 40 mu g/mL mancozeb, a concentration far lower than the recommended field application rate.

Molecular characterization of Thelastomatoidea (Nematoda : Oxyurida) from cockroaches in Australia

A molecular approach was used to genetically characterize 5 species (Aoruroides queenslandensis. Blattophila sphaerolaima, Cordonicola gibsoni, Desmicola ornato and Leidynemella fusiformis) belonging to the superfamily. Thelastomatoidea fi (Nematoda: Oxyurida), a group of pinworms that parasitizes terrestrial arthropods. The D3 domain of the large subunit Of nuclear ribosomal RNA (LSU) was sequenced for individual specimens, and the analysis of the sequence data allowed the genetic relationships of the 5 species to be studied dagger. The sequence variation in the D3 domain within individual species (0-1-8%) was significantly less than the differences among species (4(.)3-12(.)4%). Phylogenetic analyses, Using maximum parsimony, maximum likelihood, and neighbour-joining, tree-building methods, established relationships among the 5 species of Thelastomatoidea and Oxyuris equi (a species of the order Oxyurida). The molecular approach employed provides the prospect for developing DNA tools for the specific identification of the Thelastomatoidea, irrespective of developmental stage and sex, as a basis for systematic, ecological and/or population genetic investigations of members within this superfamily.