Effect of salt stress on physiological and antioxidative responses in two species of <Emphasis Type="Italic">Salicornia</Emphasis> (<Emphasis Type="Italic">S. persica</Emphasis> and <Emphasis Type="Italic">S. europaea</Emphasis>)

The effects of salt stress on growth parameters, free proline content, ion accumulation, lipid peroxidation, and several antioxidative enzymes activities were investigated in S. persica and S. europaea. The seedlings were grown for 2 months in half-strength Hoagland solution and treated with different concentrations of NaCl (0, 85, 170, 340, and 510 mM) for 21 days. The fresh and dry weights of both species increased significantly at 85 and 170 mM NaCl and decreased at higher concentrations. Salinity increased proline content in both the species as compared to that of control. Sodium (Na⁺) content in roots and shoots increased, whereas K⁺ and P_i content in both organs decreased. At all NaCl concentrations, the total amounts of Na⁺ and K⁺ were higher in shoots than in roots. Malondialdehyde (MDA) content declined at moderate NaCl concentrations (85 and 170 mM) and increased at higher levels. With increased salinity, superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPX) activities also increased gradually in both species. In addition, it seems that GPX, CAT, and SOD activities play an essential protective role in the scavenging reactive oxygen species (ROS) in both species. Native polyacrylamide gel electrophoresis (PAGE) indicated different isoform profiles between S. persica and S. europaea concerning antioxidant enzymes. These results showed that S. persica exhibits a better protection mechanism against oxidative damage and it is more salt-tolerant than S. europaea possibly by maintaining and/or increasing growth parameters, ion accumulation, and antioxidant enzyme activities.     

<Emphasis Type="Italic">Wolbachia</Emphasis> Infections of the Whitefly <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

We report the first systematic survey for the presence of Wolbachia endosymbionts in aphids and whiteflies, particularly different populations and biotypes of Bemisia tabaci. Additional agriculturally important species included were predator species, leafhoppers, and lepidopterans. We used a polymerase chain reaction (PCR)-based detection assay with ribosomal 16S rDNA and Wolbachia cell surface protein (wsp) gene primers. Wolbachia were detected in a number of whitefly populations and species, whitefly predators, and one leafhopper species; however, none of the aphid species tested were found infected. Single, double, and triple infections were detected in some of the B. tabaci populations. PCR and phylogenetic analysis of wsp gene sequences indicated that all Wolbachia strains found belong to group B. Topologies of the optimal tree derived by maximum likelihood (ML) and a ML tree in which Wolbachia sequences from B. tabaci are constrained to be monophyletic are significantly different. Our results indicate that there have been at least four independent Wolbachia infection events in B. tabaci. The importance of the presence of Wolbachia infections in B. tabaci is discussed. RID= ID= <E5>Correspondence to: </E5>K. Bourtzis; <E5>email:</E5> kbourtz&commat;cc.uoi.gr Received: 9 September 2002 / Accepted: 25 September 2002     

Molecular analysis of the genus <Emphasis Type="Italic">Anoxybacillus</Emphasis> based on sequence similarity of the genes <Emphasis Type="Italic">recN</Emphasis>, <Emphasis Type="Italic">flaA</Emphasis>, and <Emphasis Type="Italic">ftsY</Emphasis>

Genome predictions based on selected genes would be a very welcome approach for taxonomic studies. We analyzed three genes, recN, flaA, and ftsY, for determining if these genes are useful tools for systematic analyses in the genus Anoxybacillus. The genes encoding a DNA repair and genetic recombination protein (recN), the flagellin protein (flaA), and GTPase signal docking protein (ftsY) were sequenced for ten Anoxybacillus species. The sequence comparisons revealed that recN sequence similarities range between 61% and 99% in the genus Anoxybacillus. Comparisons to other bacterial recN genes indicated that levels of similarity did not differ from the levels within genus Anoxybacillus. These data showed that recN is not a useful marker for the genus Anoxybacillus. A 550–600-bp region of the flagellin gene was amplified for all Anoxybacillus strains except for Anoxybacillus contaminans. The sequence similarity of flaA gene varies between 61% and 76%. Comparisons to other bacterial flagellin genes obtained from GenBank (Bacillus, Pectinatus, Proteus, and Vibrio) indicated that the levels of similarity were lower (3–42%). Based on these data, we concluded that the variability in this single gene makes it a particularly useful marker. Another housekeeping gene ftsY suggested to reflect the G+C (mol/mol) content of whole genome was analyzed for Anoxybacillus strains. A mean difference of 1.4% was observed between the G+C content of the gene ftsY and the G+C content of the whole genome. These results showed that the gene ftsY can be used to represent whole G+C content of the Anoxybacillus species.     

A new methanol assimilating yeast, <Emphasis Type="Italic">Ogataea</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>, the ascosporic state of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">parapolymorpha</Emphasis>

Ogataea parapolymorpha sp. n. (NRRL YB-1982, CBS 12304, type strain), the ascosporic state of Candida parapolymorpha, is described. The species appears homothallic, assimilates methanol as is typical of most Ogataea species and forms hat-shaped ascospores in asci that become deliquescent. O. parapolymorpha is closely related to Ogataea angusta and Ogataea polymorpha. The three species can be resolved from gene sequence analyses but are unresolved from fermentation and growth reactions that are typically used for yeast identification. On the basis of multiple isolates, O. angusta is known only from California, USA, in association with Drosophila and Aulacigaster flies, O. parapolymorpha is predominantly associated with insect frass from trees in the eastern USA but O. polymorpha has been isolated from various substrates in the USA, Brazil, Spain and Costa Rica.     

<Emphasis Type="Italic">Drosophila</Emphasis><Emphasis Type="Italic">P</Emphasis> transposons of the urochordata <Emphasis Type="Italic">Ciona intestinalis</Emphasis>

P transposons belong to the eukaryotic DNA transposons, which are transposed by a cut and paste mechanism using a P-element-coded transposase. They have been detected in Drosophila, and reside as single copies and stable homologous sequences in many vertebrate species. We present the P elements Pcin1, Pcin2 and Pcin3 from Ciona intestinalis, a species of the most primitive chordates, and compare them with those from Ciona savignyi. They showed typical DNA transposon structures, namely terminal inverted repeats and target site duplications. The coding region of Pcin1 consisted of 13 small exons that could be translated into a P-transposon-homologous protein. C. intestinalis and C. savignyi displayed nearly the same phenotype. However, their P elements were highly divergent and the assumed P transposase from C. intestinalis was more closely related to the transposase from Drosophila melanogaster than to the transposase of C. savignyi. The present study showed that P elements with typical features of transposable DNA elements may be found already at the base of the chordate lineage. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Association Between the <Emphasis Type="Italic">cfx</Emphasis>A Gene and Transposon Tn<Emphasis Type="Italic">4555</Emphasis> in <Emphasis Type="Italic">Bacteroides distasonis</Emphasis> Strains and Other <Emphasis Type="Italic">Bacteroides</Emphasis> Species

The Bacteroides genus, the most prevalent anaerobic bacteria of the intestinal tract, carries a plethora of the mobile elements, such as plasmids and conjugative and mobilizable transposons, which are probably responsible for the spreading of resistance genes. Production of β-lactamases is the most important resistance mechanism including cephalosporin resistance to β-lactam agents in species of the Bacteroides fragilis group. In our previous study, the cfxA gene was detected in B. distasonis species, which encodes a clinically significant broad-spectrum β-lactamase responsible for widespread resistance to cefoxitin and other β-lactams. Such gene has been associated with the mobilizable transposon Tn4555. Therefore, the aim of this study was to detect the association between the cfxA gene and the presence of transposon Tn4555 in 53 Bacteroides strains isolated in Rio de Janeiro, Brazil, by PCR assay. The cfxA gene was detected in 11 strains and the Tn4555 in 15. The transposon sequence revealed similarities of approximately 96% with the B. vulgatus sequence which has been deposited in GenBank. Hybridization assay was performed in attempt to detect the cfxA gene in the transposon. It was possible to associate the cfxA gene in 11 of 15 strains that harbored Tn4555. Among such strains, 9 presented the cfxA gene as well as Tn4555, but in 2 strains the cfxA gene was not detected by PCR assay. Our results confirm the involvement of Tn4555 in spreading the cfxA gene in Bacteroides species.     

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.     

Demonstration of IS<Emphasis Type="Italic">711</Emphasis> transposition in <Emphasis Type="Italic">Brucella ovis</Emphasis> and <Emphasis Type="Italic">Brucella pinnipedialis</Emphasis>

Background  

The Brucella genome contains an insertion sequence (IS) element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose.     

Gene gain and loss events in <Emphasis Type="Italic">Rickettsia</Emphasis> and <Emphasis Type="Italic">Orientia</Emphasis>species

Background  

Genome degradation is an ongoing process in all members of the Rickettsiales order, which makes these bacterial species an excellent model for studying reductive evolution through interspecies variation in genome size and gene content. In this study, we evaluated the degree to which gene loss shaped the content of some Rickettsiales genomes. We shed light on the role played by horizontal gene transfers in the genome evolution of Rickettsiales.     

Evolution of nematode-resistant <Emphasis Type="Italic">Mi</Emphasis>-<Emphasis Type="Italic">1</Emphasis> gene homologs in three species of <Emphasis Type="Italic">Solanum</Emphasis>

Plants have evolved several defense mechanisms, including resistance genes. Resistance to the root-knot nematode Meloidogyne incognita has been found in wild plant species. The molecular basis for this resistance has been best studied in the wild tomato Solanum peruvianum and it is based on a single dominant gene, Mi-1.2, which is found in a cluster of seven genes. This nematode attacks fiercely several crops, including potatoes. The genomic arrangement, number of copies, function and evolution of Mi-1 homologs in potatoes remain unknown. In this study, we analyzed partial genome sequences of the cultivated potato species S. tuberosum and S. phureja and identified 59 Mi-1 homologs. Mi-1 homologs in S. tuberosum seem to be arranged in clusters and located on chromosome 6 of the potato genome. Previous studies have suggested that Mi-1 genes in tomato evolved rapidly by frequent sequence exchanges among gene copies within the same cluster, losing orthologous relationships. In contrast, Mi-1 homologs from cultivated potato species (S. tuberosum and S. phureja) seem to have evolved by a birth-and-death process, in which genes evolve mostly by mutations and interallelic recombinations in addition to sequence exchanges.     

Molecular evolution of the clustered <Emphasis Type="Italic">MIC</Emphasis>-<Emphasis Type="Italic">3</Emphasis> multigene family of <Emphasis Type="Italic">Gossypium</Emphasis> species

The Gossypium MIC-3 (Meloidogyne Induced Cotton-3) gene family is of great interest for molecular evolutionary studies because of its uniqueness to Gossypium species, multi-gene content, clustered localization, and root-knot nematode resistance-associated features. Molecular evolution of the MIC-3 gene family was studied in 15 tetraploid and diploid Gossypium genotypes that collectively represent seven phylogenetically distinct genomes. Synonymous (d_S) and non-synonymous (d_N) nucleotide substitution rates suggest that the second of the two exons of the MIC-3 genes has been under strong positive selection pressure, while the first exon has been under strong purifying selection to preserve function. Based on nucleotide substitution rates, we conclude that MIC-3 genes are evolving by a birth-and-death process and that a ‘gene amplification’ mechanism has helped to retain all duplicate copies, which best fits with the “bait and switch” model of R-gene evolution. The data indicate MIC-3 gene duplication events occurred at various rates, once per 1 million years (MY) in the allotetraploids, once per ~2 MY in the A/F genome clade, and once per ~8 MY in the D-genome clade. Variations in the MIC-3 gene family seem to reflect evolutionary selection for increased functional stability, while also expanding the capacity to develop novel “switch” pockets for responding to diverse pests and pathogens. Such evolutionary roles are congruent with the hypothesis that members of this unique resistance gene family provide fitness advantages in Gossypium.     

Interaction of silicon and cadmium in <Emphasis Type="Italic">Brassica juncea</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

The objective of this study was to determine the effect of silicon (Si) and cadmium (Cd) on root and shoot growth and Cd uptake in two hydroponically cultivated Brassica species (B. juncea (L.) Czern. cv. Vitasso and B. napus L. cv. Atlantic). Both species are potentially usable for phytoextraction. Inhibitory effects of Cd on root elongation were diminished by the impact of Si. Primary roots elongation in the presence of Cd + Si compared with Cd was stronger and the number of lateral roots was lower in B. juncea than in B. napus. Cd content per plant was higher in B. napus roots and shoots compared with B. juncea. Suberin lamellae were formed closer to the root apex in Cd + Si than in Cd treated plants and this effect was stronger in B. napus than in B. juncea. Accelerated maturation of endodermis was associated with reduced Cd uptake. Cd decreased the content of chlorophylls and carotenoids in both species, but Si addition positively influenced the content of photosynthetic pigments which was higher in B. napus than in B. juncea. Si enhanced more substantially translocation of Cd into the shoot of B. napus than of B. juncea. Based on our results B. napus seems to be more suitable for Cd phytoextraction than B. juncea because these plants produce more biomass and accumulate higher amount of Cd. The protective effect of Si on Cd treated Brassica plants could be attributed to more extensive development of suberin lamellae in endodermis.     

Expression of a lipid-inducible,self-regulating form of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> lipase <Emphasis Type="Italic">LIP2</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Saccharomyces cerevisiae is frequently used as a bioreactor for conversion of exogenously acquired metabolites into value-added products, but has not been utilized for bioconversion of low-cost lipids such as triacylglycerols (TAGs) because the cells are typically unable to acquire these lipid substrates from the growth media. To help circumvent this limitation, the Yarrowia lipolytica lipase 2 (LIP2) gene was cloned into S. cerevisiae expression vectors and used to generate S. cerevisiae strains that secrete active Lip2 lipase (Lip2p) enzyme into the growth media. Specifically, LIP2 expression was driven by the S. cerevisiae PEX11 promoter, which maintains basal transgene expression levels in the presence of sugars in the culture medium but is rapidly upregulated by fatty acids. Northern blotting, lipase enzyme activity assays, and gas chromatographic measurements of cellular fatty acid composition after lipid feeding all confirmed that cells transformed with the PEX11 promoter–LIP2 construct were responsive to lipids in the media, i.e., cells expressing LIP2 responded rapidly to either free fatty acids or TAGs and accumulated high levels of the corresponding fatty acids in intracellular lipids. These data provided evidence of the creation of a self-regulating positive control feedback loop that allows the cells to upregulate Lip2p production only when lipids are present in the media. Regulated, autonomous production of extracellular lipase activity is a necessary step towards the generation of yeast strains that can serve as biocatalysts for conversion of low-value lipids to value-added TAGs and other novel lipid products.     

Fecundity and feeding of <Emphasis Type="Italic">Galerucella calmariensis</Emphasis> and <Emphasis Type="Italic">G. pusilla</Emphasis> on <Emphasis Type="Italic">Lythrum salicaria</Emphasis>

Fecundity and feeding of two introduced sibling biological control species, Galerucella calmariensis and G. pusilla (Coleoptera: Chrysomelidae) on purple loosestrife, Lythrum salicaria L. (Lythraceae) were compared at constant temperatures of 12.5, 15, 20, 25, and 27.5 °C. Larval feeding was also carried out at 30 °C, but at this temperature, larvae developed only to the L2 stage and none pupated. Thus, data for this temperature were not used in the analysis. There were significant species × temperature interactions in fecundity. Of the two species, Galerucella pusilla laid more eggs. Although egg production of both species was lowest at 12.5 °C and increased to 20 °C, at higher temperatures, the two species reacted differently. From 25 to 27.5 °C, egg production decreased for G. pusilla, but G. calmariensis fecundity peaked at 27.5 °C. Significant temperature × species × life-stage interactions were also observed in feeding. For each species, the amount of feeding varied with temperature and stage of development. Galerucella pusilla adults consumed more foliage at 15, 20, and 27.5 °C. However, at 12.5 °C G. calmariensis adults fed more than G. pusilla. G. pusilla larvae consumed an average of 25% less foliage than G. calmariensis. The lower larval consumption of G. pusilla suggests that when food is limited, G. pusilla larvae may have a higher survival rate because of its ability to complete larval development with less food and produce more progeny due to its greater fecundity. When food is not limited neither species would have a competitive advantage and both species could coexist temporally and spatially. However, since G. calmariensis larvae consumed more leaf material, the larval stage of this species would have a greater impact on purple loosestrife than G. pusilla.     

Detection of <Emphasis Type="Italic">Candida</Emphasis><Emphasis Type="Italic">dubliniensis</Emphasis> in Venezuela

Over the past decades there has been a significant increase in fungal infections caused by Candida species, and continues to be common in immunocompromised individuals infected with the human immunodeficiency virus (HIV). Although Candida albicans remains the fungal species most frequently isolated as an opportunistic oral pathogen, other non-albicans are often identified in this cohort of patients, including C. dubliniensis. This yeast is closely related to and shares many phenotypic characteristics with C. albicans. Colonies of these two species appear morphologically identical when not grown on special media. The shared phenotypic characteristics of C. dubliniensis and C. albicans suggest that many C. dubliniensis isolates may have been misidentified as C. albicans in the past. The present studies aim is to recover and identify C. dubliniensis, and presumptive clinical C. albicans, from the oral cavities of HIV-seropositive individuals, comparing conventional media to obtain a simple, low-cost and reliable identification system for C. dubliniensis. A total of 16 isolates (3,98%) had been obtained from 402 HIV infected individuals with recurrent oropharyngitis and were identified as C. dubliniensis. Out of these C. dubliniensis isolates 19% were resistant, with MICs above 64 μg/ml to fluconazole. This constitutes, to the authors knowledge the first recovery of this organism in Venezuela.     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

The karyology of the cyprinid genera <Emphasis Type="Italic">Scardinius</Emphasis> and <Emphasis Type="Italic">Rutilus</Emphasis> in southern Europe

The chromosomes of eight species of Rutilus and Scardinius, mostly endemic to the Italian and the Balkan peninsula, were analyzed by conventional and other banding techniques. Parallel analyses were conducted also on two leuciscine species, Alburnus albidus, for which we provide the first karyological analysis, and Leuciscus cephalus. All species examined displayed the same karyotype (2n=50 chromosomes, 8 metacentric+13 submetacentric+4 subtelo/acrocentric pairs) with nucleolus organizer regions (NORs) on the ends of the shorter arms of a medium-sized submetacentric pair. In contrast, interspecific variation was observed in the distribution of constitutive heterochromatin. The variation observed in this genomic material proved to be systematically and phylogenetically informative. Indeed, a peritelomeric C-band on the first telocentric pair characterizes species of Rutilus and Scardinius. In both genera heterochromatin differentiation appears to be directed to a centromere–telomere direction, particularly evident along the metacentric elements of their karyotypes.An erratum to this article can be found at