Interaction between uranium and the cytochrome <Emphasis Type="Italic">c</Emphasis><Subscript>3</Subscript> of <Emphasis Type="Italic">Desulfovibrio desulfuricans</Emphasis> strain G20

Cytochrome c(3) of Desulfovibrio desulfuricans strain G20 is an electron carrier for uranium (VI) reduction. When D. desulfuricans G20 was grown in medium containing a non-lethal concentration of uranyl acetate (1 mM), the rate at which the cells reduced U(VI) was decreased compared to cells grown in the absence of uranium. Western analysis did not detect cytochrome c(3) in periplasmic extracts from cells grown in the presence of uranium. The expression of this predominant tetraheme cytochrome was not detectably altered by uranium during growth of the cells as monitored through a translational fusion of the gene encoding cytochrome c(3) ( cycA) to lacZ. Instead, cytochrome c(3) protein was found tightly associated with insoluble U(IV), uraninite, after the periplasmic contents of cells were harvested by a pH shift. The association of cytochrome c(3) with U(IV) was interpreted to be non-specific, since pure cytochrome c(3) adsorbed to other insoluble metal oxides, including cupric oxide (CuO), ferric oxide (Fe(2)O(3)), and commercially available U(IV) oxide.     

Electrostatic attraction between cytochrome <Emphasis Type="Italic">bc</Emphasis><Subscript>1</Subscript> and cytochrome <Emphasis Type="Italic">c</Emphasis> affects kinetics of cytochrome <Emphasis Type="Italic">c</Emphasis> reduction

The kinetics of the ubiquinol-cytochrome c reductase reaction was examined using membrane fragments and purified bc(1) complexes derived from a wild-type (WT) and a newly constructed mutant (MUT) strains of Paracoccus denitrificans. The cytochrome c(1) of the WT samples possessed an additional stretch of acidic amino acids, which was lacking in the mutant. The reaction was followed with positively charged mitochondrial and negatively charged bacterial cytochromes c, and specific activities, apparent k(cat) values, and first-order rate constant values were compared. These values were distinctly lower for the MUT fractions using mitochondrial cytochrome c but differed only slightly with the bacterial species. The MUT preparations were less sensitive to changes of ionic strength of the reaction media and showed pure first-order kinetics with both samples of cytochrome c. The reaction of the WT enzyme was first order only with bacterial cytochrome c but proceeded with a non-linear profile with mitochondrial cytochrome c. The analysis of the reaction pattern revealed a rapid onset of the reaction with a successively declining rate. Experiments performed in the absence of an electron donor indicated that electrostatic attraction could directly participate in cytochrome c reduction.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Phylogenetic analyses of <Emphasis Type="Italic">Uromyces viciae-fabae</Emphasis> and its varieties on <Emphasis Type="Italic">Vicia</Emphasis>, <Emphasis Type="Italic">Lathyrus</Emphasis>, and <Emphasis Type="Italic">Pisum</Emphasis> in Japan

A pea rust fungus, Uromyces viciae-fabae, has been classified into two varieties, var. viciae-fabae and var. orobi, based on differences in urediniospore wall thickness and putative host specificity in Japan. In principal component analyses, morphological features of urediniospores and teliospores of 94 rust specimens from Vicia, Lathyrus, and Pisum did not show definite host-specific morphological groups. In molecular analyses, 23 Uromyces specimens from Vicia, Lathyrus, and Pisum formed a single genetic clade based on D1/D2 and ITS regions. Four isolates of U. viciae-fabae from V. cracca and V. unijuga could infect and sporulate on P. sativum. These results suggest that U. viciae-fabae populations on different host plants are not biologically differentiated into groups that can be recognized as varieties.Contribution no. 184, Laboratory of Plant Parasitic Mycology, Institute of Agriculture and Forestry, University of Tsukuba, Japan     

Molecular phylogenetic perspective on speciation in the genus <Emphasis Type="Italic">Sebastes</Emphasis> (Scorpaenidae) from the Northwest Pacific and the position of <Emphasis Type="Italic">Sebastes</Emphasis> within the subfamily Sebastinae

In the Northeast and the Northwest Pacific, 26 and 68 species of Sebastes occur, respectively, the similar appearance of some pairs of shallow coastal water species in the two regions having been interpreted as evidence either for migration of ancestral species or of morphological convergence. To distinguish between these competing hypotheses, a portion of the mitochondrial DNA (mtDNA) cytochrome b gene was sequenced from 23 species of Sebastes from the Northwest Pacific and phylogenetic trees constructed so as to include the existing data from Northeastern species of Sebastes. The results clearly indicated separate origins for external morphology in Northeast and Northwest Pacific species. The shallow coastal water species in the Northwest Pacific are thought to have diverged from about 9.8MYA. Furthermore, the phylogenetic position of Sebastes was inferred among the subfamily Sebastinae, Helicolenus, Hozukius, Sebastes, and Sebastiscus, the results suggesting that Helicolenus and Hozukius are more closely related to Sebastes than Sebastiscus.     

Mediated catalysis of <Emphasis Type="Italic">Paracoccus pantotrophus</Emphasis> cytochrome <Emphasis Type="Italic">c</Emphasis> peroxidase by <Emphasis Type="Italic">P. pantotrophus</Emphasis> pseudoazurin: kinetics of intermolecular electron transfer

This work reports the direct electrochemistry of Paracoccus pantotrophus pseudoazurin and the mediated catalysis of cytochrome c peroxidase from the same organism. The voltammetric behaviour was examined at a gold membrane electrode, and the studies were performed in the presence of calcium to enable the peroxidase activation. A formal reduction potential, E ⁰′, of 230 ± 5 mV was determined for pseudoazurin at pH 7.0. Its voltammetric signal presented a pH dependence, defined by pK values of 6.5 and 10.5 in the oxidised state and 7.2 in the reduced state, and was constant up to 1 M NaCl. This small copper protein was shown to be competent as an electron donor to cytochrome c peroxidase and the kinetics of intermolecular electron transfer was analysed. A second-order rate constant of 1.4 ± 0.2 × 10⁵ M⁻¹ s⁻¹ was determined at 0 M NaCl. This parameter has a maximum at 0.3 M NaCl and is pH-independent between pH 5 and 9.     

Molecular phylogeography of two sibling species of <Emphasis Type="Italic">Eurema</Emphasis> butterflies

The common yellow butterfly Eurema hecabe is widely distributed in East Asia, and is one of the most burdensome species for taxonomists due to the numerous geographic and seasonal wing colour patterns. Moreover, within this species, individuals with a yellow wing fringe that occur in temperate regions of Japan (Y type) proved to be biologically different from others that occur widely in subtropical regions of Japan and all over East Asia (B type). To unveil the genetic variation within and between the two types, a total of 50 butterflies collected at 18 geographic localities in East Asia were examined for nucleotide sequence variation of three mitochondrial regions: cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit III (COIII) and NADH dehydrogenase subunit 5 (ND5). In addition, they were also examined for infection status with the endosymbiotic bacteria Wolbachia. The three mitochondrial sequences consistently showed that (i) Y type and B type were highly divergent, (ii) nucleotide variation within B type was very small although sampled from a geographically wide range, and (iii) a weak association existed between mitochondrial DNA haplotypes and Wolbachia infection status.     

Genetic variation in populations of <Emphasis Type="Italic">Allothrombium pulvinum</Emphasis> (Acari: Trombidiidae) from Northern Iran revealed by mitochondrial <Emphasis Type="Italic">cox</Emphasis>I and nuclear rDNA <Emphasis Type="Italic">ITS</Emphasis>2 sequences

Allothrombium pulvinum Ewing is a common natural enemy of aphids and some other arthropods. So far, there are no studies that have addressed genetic variation of this predatory mite. We investigated genetic variation of A. pulvinum across its whole known range in Iran. A 410 bp portion of the mitochondrial cytochrome c oxidase subunit I gene (coxI) and 797–802 bp portion of the internal transcribed spacer 2 of rDNA (ITS2) were sequenced for 55 individuals from 11 populations, resulting in 12 and 26 haplotypes, respectively. In the coxI region, haplotype and nucleotide diversities varied among populations from 0.00 to 0.90 and from 0.0000 to 0.0110, respectively. In the ITS2 region they varied from 0.20 to 0.91 and from 0.0006 to 0.0023, respectively. For both gene regions the highest haplotype and nucleotide diversities were detected in population Mahmoud Abad from northern Iran. Statistically significant population differentiation (F _ST) was detected in most pair-wise population comparisons. The results of population differentiation for both gene regions were generally congruent indicating that A. pulvinum from Iran consists of genetically different populations. This suggests that A. pulvinum comprises at least two geographically distinct populations or even more than one species. This study is an initial step towards understanding genetic variation of A. pulvinum, a taxon for which little molecular information is available. More intensive sampling and analysis of additional DNA regions are necessary for more detailed classification of this taxon.     

<Emphasis Type="Italic">Toxoplasma gondii</Emphasis> glycosylphosphatidylinositols are not involved in <Emphasis Type="Italic">T. gondii</Emphasis>-induced host cell survival

Toxoplasma gondii is an intracellular parasite able to both promote and inhibit apoptosis. T. gondii renders infected cells resistant to programmed cell death induced by multiple apoptotic triggers. On the other hand, increased apoptosis of immune cells after in vivo infection with T. gondii may suppress the immune response to the parasite. Glycosylphosphatidylinositol (GPI)-anchored proteins dominate the surface of T. gondii tachyzoites and GPIs are involved in the pathogenicity of protozoan parasites. In this report, we determine if GPIs are responsible for inhibition or induction of host cell apoptosis. We show here that T. gondii GPIs fail to block apoptosis that was triggered in human-derived cells via extrinsic or intrinsic apoptotic pathways. Furthermore, characteristics of apoptosis, e.g. caspase-3/7 activity, phosphatidylserine exposition at the cell surface or DNA strand breaks, were not observed in the presence of T. gondii GPIs. These results indicate that T. gondii GPIs are not involved in survival or in apoptosis of host cells. This absence of effect on apoptosis could be a feature common to GPIs of other parasites.     

Phylogeny of Chinese <Emphasis Type="Italic">Polystichum</Emphasis> (Dryopteridaceae) based on chloroplast DNA sequence data (<Emphasis Type="Italic">trnL-F</Emphasis> and <Emphasis Type="Italic">rps4-trnS</Emphasis>)

Polystichum is one of the largest and most taxonomically complex fern genera in China. The evolutionary relationships of Chinese Polystichum and related genera, and the relationship between our Polystichum phylogeny and ecogeographic distribution, were tested by the use of DNA sequence data. Fifty-one species of Polystichum and 21 species in allied genera were sequenced for the plastid intergenic spacers rps4-trnS and trnL-F. Maximum parsimony and Bayesian phylogenetic analyses of both individual and combined data sets showed that Chinese Polystichum as commonly recognized was paraphyletic: one clade (the CCPC clade) included Cyrtomidictyum lepidocaulon, two Cyrtogonellum species, three Cyrtomium species, and a small number of Polystichum species usually occurring on limestone. A second clade, Polystichum sensu stricto, included the remainder of the Polystichum species; these often occur on non-limestone substrates. The remaining Cyrtomium species formed the third clade. Three subclades resolved within Polystichum sensu stricto (s.s.) clade do not correspond with recent sectional classifications, and we outline the issues relevant to a new classification for the genus. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Cytochromes <Emphasis Type="Italic">c</Emphasis> of the anaerobic methacrylate reducer <Emphasis Type="Italic">Geobacter sulfurreducens</Emphasis> AM-1

Cytochromes c were found in the cells of the bacterium Geobacter sulfurreducens AM-1 grown on acetate and methacrylate. The periplasmic extract of G. sulfurreducens AM-1 contained about 88% of the total content of cytochromes c of intact cells. The analysis of cytochromes c from the native cells of G. sulfurreducens AM-1, from the periplasmic extract and from the cells treated by an alkaline solution showed the presence of nine proteins containing heme c. The molecular masses of cytochromes c from G. sulfurreducens AM-1 were 12.5, 15.5, 25.7, 29.5, 34.7, 41.7, 50.1, 63.1, and 67.6 kDa; localization of each cytochrome c was determined. Three heme-containing proteins (15.5 kDa, 25.7 kDa, and 29.5 kDa with the most intensive staining) were present mainly in the periplasm of the bacterium. The other two (50.1 and 67.6 kDa) were supposedly localized in the cell membrane. Cytochromes c with the molecular masses of 12.5, 15.5, and 67.6 kDa are considered as possible components of the methacrylate redox system of G. sulfurreducens AM-1.     

mtDNA Diversity and genetic lineages of eighteen cattle breeds from <Emphasis Type="Italic">Bos taurus </Emphasis>and<Emphasis Type="Italic"> Bos indicus</Emphasis> in China

In order to clarify the origin and genetic diversity of indigenous cattle breeds in China, we carried out phylogenetic analysis of representatives of those breeds by employing mitochondrial gene polymorphism. Complete cyt b gene sequences, 1140 bp in length, were determined for a total of 136 individuals from 18 different breeds and these sequences were clustered into two distinct genetic lineages: taurine (Bos taurus) and zebu (Bos indicus). In analysis of the cyt b gene diversity, Chinese cattle showed higher nucleotide (0.00923) and haplotype diversity (0.848) than the reports from other studies, and the animals from the taurine lineage indicated higher nucleotide diversity (0.00330) and haplotype diversity (0.746) than the ones from the zebu lineage (0.00136; 0.661). The zebu mtDNA dominated in the southern breeds (63.3–100%), while the taurine dominated in the northern breeds (81.8–100%). Six cattle breeds from the central area of China exhibited intermediate frequencies of zebu mtDNA (25–71.4%). This polymorphism revealed a declining south-to-north gradient of female zebu introgression and a geographical hybrid zone of Bos taurus and Bos indicus in China.     

Phylogenetic differentiation between Atlantic <Emphasis Type="Italic">Scomber colias</Emphasis> and Pacific <Emphasis Type="Italic">Scomber japonicus</Emphasis> based on nuclear DNA sequences

In the classical taxonomy, three Scomber species are distinguished: S. scombrus, S. australasicus, and S. japonicus. Yet, some fish taxonomists have recently recognized Scomber colias, inhabiting the Atlantic Ocean, as a separate species from S. japonicus, distributed in the Pacific Ocean. Such proposal was based on significant mitochondrial DNA divergence as well as great phenotypic variation among individuals from these two ocean basins. However, in the absence of nuclear DNA data this issue remains still controversial. In this study, a phylogenetic analysis of nuclear 5S rDNA sequences was performed. A total of 30 individuals of S. colias collected in the Atlantic and 34 specimens of S. japonicus from the Pacific were characterized. Moreover, nine individuals of Pacific S. australasicus and eight of Atlantic S. scombrus were included. Maximum likelihood, maximum parsimony, and neighbor-joining analyses revealed the presence of two well-supported distinct clades corresponding to S. colias and S. japonicus, respectively. Altogether, morphologic and genetic data are in agreement with the recognition of two different species, S. colias in the Atlantic, and S. japonicus in the Pacific.     

Floral development and systematic position of <Emphasis Type="Italic">Arillastrum</Emphasis>, <Emphasis Type="Italic">Allosyncarpia</Emphasis>, <Emphasis Type="Italic">Stockwellia</Emphasis> and <Emphasis Type="Italic">Eucalyptopsis</Emphasis> (Myrtaceae)

We have investigated the floral ontogeny of Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis (of the eucalypt group, Myrtaceae) using scanning electron microscopy and light microscopy. Several critical characters for establishing relationships between these genera and to the eucalypts have been determined. The absence of compound petaline primordia in Arillastrum, Allosyncarpia, Stockwellia and Eucalyptopsis excludes these taxa from the eucalypt clade. Post-anthesis circumscissile abscission of the hypanthium above the ovary in Stockwellia, Eucalyptopsis and Allosyncarpia is evidence that these three taxa form a monophyletic group; undifferentiated perianth parts and elongated fusiform buds are characters that unite Stockwellia and Eucalyptopsis as sister taxa. No floral characters clearly associate Arillastrum with either the eucalypt clade or the clade of Stockwellia, Eucalyptopsis and Allosyncarpia.We gratefully acknowledge Clyde Dunlop and Bob Harwood (Northern Territory Herbarium) for collecting specimens of Allosyncarpia, and Bruce Gray (Atherton) for collecting specimens of Stockwellia. The Australian National Herbarium (CANB) kindly lent herbarium specimens of Eucalyptopsis for examination. This research was supported by a University of Melbourne Research Development Grant to Andrew Drinnan.     

<Emphasis Type="Italic">Anthocyaninless1</Emphasis> gene of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> encodes a UDP-glucose:flavonoid-3-<Emphasis Type="Italic">O</Emphasis>-glucosyltransferase

We isolated several mutants of Arabidopsis thaliana (L.) Heynh. that accumulated less anthocyanin in the plant tissues, but had seeds with a brown color similar to the wild-type. These mutants were allelic with the anthocyaninless1 (anl1) mutant that has been mapped at 15.0 cM of chromosome 5. We performed fine mapping of the anl1 locus and determined that ANL1 is located between the nga106 marker and a marker corresponding to the MKP11 clone. About 70 genes are located between these two markers, including three UDP-glucose:flavonoid-3-O-glucosyltransferase-like genes and a glutathione transferase gene (TT19). A mutant of one of the glucosyltransferase genes (At5g17050) was unable to complement the anl1 phenotype, showing that the ANL1 gene encodes UDP-glucose:flavonoid-3-O-glucosyltransferase. ANL1 was expressed in all tissues examined, including rosette leaves, stems, flower buds and roots. ANL1 was not regulated by TTG1.     

Identification and characterisation of <Emphasis Type="Italic">ssrA</Emphasis> in members of the <Emphasis Type="Italic">Helicobacter</Emphasis> genus

Bacterial ssrA encodes tmRNA that functions both as a tRNA and an mRNA to rescue the stalled ribosome on defective mRNAs. In this study, ssrA was identified in four gastric species of Helicobacters and four enterohepatic species of Helicobacters. The tag peptide of 14 amino acids encoded by ssrA showed a pattern of Val(1)Ala(13) in gastric species, a pattern of Ala(1)Val(13 )in enterohepatic species, in contrast to the pattern of Ala(1)Ala(13) in W. succinogenes and C. jejuni, which are closely related to helicobacters. Phylogenetic analysis and the patterns of the tag peptide suggest that the Helicobacter genus could be separated into two genera. High conservation of ssrA in H. pylori was observed. The annotated ORF HP0784 in H. pylori, which largely overlaps ssrA, is unlikely to be functional. H. pylori ssrA interestingly expressed a large and a small tmRNA molecule.     

Molecular phylogeny of horsetails (<Emphasis Type="Italic">Equisetum</Emphasis>) including chloroplast <Emphasis Type="Italic">atpB</Emphasis> sequences

Equisetum is a genus of 15 extant species that are the sole surviving representatives of the class Sphenopsida. The generally accepted taxonomy of Equisetum recognizes two subgenera: Equisetum and Hippochaete. Two recent phylogenetical studies have independently questioned the monophyly of subgenus Equisetum. Here, I use original (atpB) and published (rbcL, trnL-trnF, rps4) sequence data to investigate the phylogeny of the genus. Analyses of atpB sequences give an unusual topology, with E. bogotense branching within Hippochaete. A Bayesian analysis based on all available sequences yields a tree with increased resolution, favoring the sister relationships of E. bogotense with subgenus Hippochaete.     

The Evolutionary History of <Emphasis Type="Italic">Shigella</Emphasis> and Enteroinvasive <Emphasis Type="Italic">Escherichia coli</Emphasis> Revised

In Shigella and enteroinvasive Escherichia coli (EIEC), the etiologic agents of shigellosis in humans, the determinants responsible for entry of bacteria into and dissemination within epithelial cells are encoded by a virulence plasmid. To understand the evolution of the association between the virulence plasmid and the chromosome, we performed a phylogenetic analysis using the sequences of four chromosomal genes (trpA, trpB, pabB, and putP) and three virulence plasmid genes (ipaB, ipaD, and icsA) of a collection of 51 Shigella and EIEC strains. The phylogenetic tree derived from chromosomal genes showed a typical star phylogeny, indicating a fast diversification of Shigella and EIEC groups. Phylogenetic groups obtained from the chromosomal and plasmidic genes were similar, suggesting that the virulence plasmid and the chromosome share similar evolutionary histories. The few incongruences between the trees could be attributed to exchanges of fragments of different plasmids and not to the transfer of an entire plasmid. This indicates that the virulence plasmid was not transferred between the different Shigella and EIEC groups. These data support a model of evolution in which the acquisition of the virulence plasmid in an ancestral E. coli strain preceded the diversification by radiation of all Shigella and EIEC groups, which led to highly diversified but highly specialized pathogenic groups.