Recent invasion and low level of divergence between diploid and triploid forms of <Emphasis Type="Italic">Carassius auratus</Emphasis> complex in Croatia

Carassius auratus is an invasive species in European waters, comprising a complex of diploid and polyploid forms with different modes of reproduction. However, the evolutionary history and relationships between the diploids and polyploids are still unresolved. In this study, 51.5% diploids and 48.5% triploids, including four triploid males, were discovered among the 363 individuals sampled in Croatia. We used eight microsatellite loci and mitochondrial displacement loop sequences to analyze the structure and origin of populations; and to attempt to infer the evolutionary history of the two different forms in Croatia. Microsatellite analyses revealed high allelic and clonal diversity, corroborating that high propagule vectors can compensate for the negative effects of genetic bottlenecks in successful invasive species. The absence of significant population structuring confirmed recent origin and rapid spreading of populations. No evidence was found for the existence of native European populations. Distances between individuals using both nuclear and mtDNA markers revealed the absence of substantial clustering on the ploidy level, while the split between the different ploidies on population level was only partial, suggesting that the reproductive isolation between the two forms is either of a very recent origin, or that there exists uni-, or bidirectional gene flow between the diploid and triploid forms.     

Isolation and characterization of <Emphasis Type="Italic">Clostridium difficile</Emphasis> from shellfish and marine environments

This pilot study was carried out to evaluate the occurrence of Clostridium difficile in marine environments and in edible shellfish. Samples of seawater, sediment, and zooplankton were collected at five sampling stations in the Gulf of Naples. Six samples of edible shellfish, furthermore, were obtained: two from mussel farms and four from wholesalers. The isolation and the characterization of C. difficile strains were carried out using selective media and molecular techniques, respectively. C. difficile was isolated from nine of the 21 samples investigated. Shellfish and zooplankton showed the highest prevalence of positive samples. No C. difficile was detected in marine sediment. Majority of the C. difficile isolates were toxin A/B positive. Six known different PCR ribotypes (003, 005, 009, 010, 056, and 066) were identified, whereas one strain may represent a new PCR ribotype. C. difficile may be present in the marine environment in Southern Italy, including shellfish and zooplankton. This study is reporting the isolation of C. difficile from zooplankton, clams, and mussels and pointing out a new possible route to exposure to C. difficile of healthy individuals in the community.     

<Emphasis Type="Italic">allB</Emphasis>, allantoin utilisation and <Emphasis Type="Italic">Salmonella enterica</Emphasis> serovar Enteritidis and Typhimurium colonisation of poultry and mice

Natural variation in the presence or the absence of STM0517-0529 genes allowing allantoin utilisation has been described in field isolates of the multidrug resistant Salmonella enterica serovar Typhimurium belonging to the phage type DT104. Interestingly, S. enterica subspecies enterica serovar Typhimurium DT104 is quite frequent in pigs and cattle, but rarely present in egg-laying hens. Taking into account the different mode of allantoin metabolism in birds and mammals, we were interested in whether the absence of STM0517-0529 genes may disable this clone in poultry colonisation. We have therefore constructed the allB (also designated as STM0523) mutants in S. enterica subspecies enterica serovar Typhimurium and S. enterica subspecies enterica serovar Enteritidis, and with these, we infected mice, newly hatched chickens and adult egg-laying hens to show that the defect in allantoin utilisation does not influence S. enterica virulence for mice or adult hens, but slightly decreases virulence of S. enterica for chickens. The decrease in virulence of the allB mutant was relatively minor as it could be observed only after a mixed infection model, consistent with a lower prevalence, but not a total absence of such clones in poultry flocks.     

Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula

Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C₄ intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.Sulfolobales (Crenarchaeota) comprise extreme thermoacidophiles from volcanic areas that grow best at a pH of around 2 and a temperature of 60 to 90°C (32, 33). Most Sulfolobales can grow chemoautotrophically on sulfur, pyrite, or H₂ under microaerobic conditions, which also applies to Metallosphaera sedula (31), the organism studied here. Its genome has been sequenced (2). Some species of the Sulfolobales secondarily returned to a facultative anaerobic or even strictly anaerobic life style (33), and some laboratory strains appear to have lost their ability to grow autotrophically (8). Autotrophic representatives of the Sulfolobales use a 3-hydroxypropionate/4-hydroxybutyrate cycle (in short, hydroxypropionate/hydroxybutyrate cycle) for autotrophic carbon fixation (Fig. ​(Fig.1)1) (6-8, 38). The enzymes of this cycle are oxygen tolerant, which predestines the cycle for the lifestyle of the aerobic Crenarchaeota (8). The presence of genes coding for key enzymes of the hydroxypropionate/hydroxybutyrate cycle in the mesophilic aerobic “marine group I” Crenarchaeota suggests that these abundant marine archaea use a similar autotrophic carbon fixation mechanism (6, 24, 68) (for a review of autotrophic carbon fixation in Archaea, see reference 7).Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle functioning in autotrophic carbon fixation in Sulfolobales and its relation to the central carbon metabolism, as studied in this work for Metallosphaera sedula. The situation may be similar in other Sulfolobales and possibly in autotrophic marine Crenarchaeota. Enzymes: 1, acetyl-CoA/propionyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonic semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, acetyl-CoA/propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14 and 15, crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase; 17, succinyl-CoA synthetase (ADP forming); 18, succinic semialdehyde dehydrogenase; 19, succinate dehydrogenase (natural electron acceptor unknown); 20, fumarate hydratase; 21, malate dehydrogenase; 22, malic enzyme; 23, PEP carboxykinase (GTP); 24, pyruvate:water dikinase (ATP); 25, enolase; 26, phosphoglycerate mutase; 27, phosphoglycerate kinase; 28, glyceraldehyde 3-phosphate dehydrogenase; 29, triosephosphate isomerase; 30, fructose 1,6-bisphosphate aldolase/phosphatase; 31, (si)-citrate synthase; 32, aconitase; 33, isocitrate dehydrogenase.In the cycle, one molecule of acetyl-coenzyme A (CoA) is formed from two molecules of bicarbonate. The key carboxylating enzyme is a bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase (10, 11, 36, 38, 48, 49). In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step in fatty acid biosynthesis. However, archaea do not contain fatty acids, and therefore acetyl-CoA carboxylase obviously plays a different metabolic role. The hydroxypropionate/hydroxybutyrate cycle can be divided into two parts. The first transforms acetyl-CoA and two bicarbonate molecules via 3-hydroxypropionate to succinyl-CoA, and the second converts succinyl-CoA via 4-hydroxybutyrate to two acetyl-CoA molecules. In brief, the product of the acetyl-CoA carboxylase reaction, malonyl-CoA, is reduced via malonic semialdehyde to 3-hydroxypropionate, which is further reductively converted to propionyl-CoA. Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by the same carboxylase as that that carboxylates acetyl-CoA (11, 36). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Succinyl-CoA then is converted into two molecules of acetyl-CoA via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxyacetyl-CoA, and acetoacetyl-CoA. This reaction sequence apparently is common to the autotrophic Crenarchaeota, as it also is used by autotrophic Crenarchaeota of the orders Thermoproteales and Desulfurococcales, which use a dicarboxylate/4-hydroxybutyrate cycle for autotrophic carbon fixation (8, 34, 55, 56) (also see the accompanying work [57]).From the list of intermediates of the hydroxypropionate/hydroxybutyrate cycle, acetyl-CoA and succinyl-CoA are the only intermediates considered common to the central carbon metabolism. In this work, we addressed the question of which intermediate of the cycle most biosynthetic routes branch off, and we came to the conclusion that succinyl-CoA serves as the main precursor for cellular carbon. This requires one turn of the cycle to regenerate the CO₂ acceptor and to generate one extra molecule of acetyl-CoA from two molecules of bicarbonate. Acetyl-CoA plus another two bicarbonate molecules are converted by an additional half turn of the cycle to succinyl-CoA. This strategy differs from that of the anaerobic pathways, in which acetyl-CoA is reductively carboxylated to pyruvate, and from there the other precursors for building blocks ultimately are derived (discussed in reference 7).     

Preparation of D-galactofuranosyl nitromethanes: a revision and a new approach

Sodium methoxide-promoted methanolysis of 7-deoxy-7-nitro-L-glycero-L-galacto-heptitol peracetate rapidly and nearly quantitatively accumulates 7-deoxy-6-O-methyl-7-nitro-L-glycero-L-galacto-heptitol. The prolonged treatment then provides 76% of D-galactofuranosyl nitromethanes and finally results in the equilibrium of 77% of β-D-galactopyranosyl nitromethane and 7-9% of three other tautomeric D-galactosyl nitromethanes. Thermal treatment of 7-deoxy-7-nitro-L-glycero-L-galacto-heptitol in boiling water peaks at a 58% content of D-galactofuranosyl nitromethanes and ends in a similar equilibrium mixture of four D-galactosyl tautomers. The relevant kinetic parameters of the latter transformation are determined by a curve fitting using the nonlinear least-squares Marquardt-Levenberg algorithm.     

Radiocarbon based assessment of soil organic matter contribution to soil respiration in a pine stand of the Campine region, Belgium

The contribution of decomposing soil organic carbon (SOC) to total annual soil respiration (SR) was evaluated by radiocarbon measurements at a Scots pine stand growing on a plaggen soil in the Belgian Campine region. Two approaches were used to estimate the contribution of different C pools to SR. In the first approach, the variations in ¹⁴C content of soil CO₂ efflux were monitored during one year (2003) and compared to the atmospheric and SOC ¹⁴C signatures to determine the contribution of ??fast?? (root respiration and fast decomposing SOC) and ??slow?? cycling C pools to total SR. In the second approach an estimate of the total heterotrophic soil respiration (Rh), comprising the slow cycling C and the heterotrophic part of the fast-cycling C pools, was derived applying a box model based on the amount of the bulk SOC pool and its ¹⁴C-derived mean residence time (MRT). The quantification of the Rh and the decomposition rate of the slow-cycling SOC allows to indirectly determining the contribution of the heterotrophic C that decompose within a year. Measurements of total SR performed in the field allowed assessing the contribution of the different C pools to total soil C efflux. On an annual basis, the fast-cycling C was the main contributor to SR, about 85%, while the contribution of the slow-cycling C (with MRT >1 yr) to total SR was 15%. Total annual Rh was 36% of total SR, which is in the lower range reported for temperate coniferous forests. The comparison of Rh with other estimates for the same site (47?C50% of total SR) suggest a possible underestimation of the C flux from the mineral soil. In fact, the ??very old?? C contained in the plaggen horizon strongly affects the signature of the mostly young C leaving the soil. In conclusion, our results indicate that the contribution of SOC decomposition to total soil CO₂ flux in this forest is less than 40%, and at least half of it comes from organic compounds less than 1 year old.     

Late Quaternary (Weichselian) alluvial history and neotectonic control on fluvial landscape development in the southern Körös plain,Hungary

Four drill cores and a clay pit section have been examined in the southern part of the Körös plain to understand the history and controls on alluvial sedimentation for the last ~ 40 ka. Four facies groups were identified, such as channel, channel margin, floodplain and floodbasin with seven distinctive facies. Magnetic susceptibility and mineralogy have further characterized the sedimentary facies indicating shifts in humidity conditions, variations in sediment flux and pedogenesis. Detailed pollen analysis of a 7.5 m thick clayey succession indicated climatic variability within the MIS 3 period. The spatial distribution of the different facies allowed outlining alluvial architecture of the study area. Three depositional units composed of various facies were identified based on OSL and radiocarbon data. These packages correspond to three major phases of channel activity: (F-I) pre-LGM period (> 30 ka to 24 ka), (F-II) post-LGM interstadial (18–16 ka), and (F-III) Late Glacial < 15 ka to ~ 10 ka). The pre-LGM and post-LGM “interstadial” phases are characterized by meandering river patterns, while the Late Glacial fluvial activity is characterized by a braided system in the area. Higher sediment supply feeding this braided river was probably caused by neotectonic uplift of the southern margin of the basin, documented by a significant stratigraphic gap between 25 and 14 ka.     

Azido analogs of neuroactive steroids

Analogs of pregnanolone (3α-hydroxy-5β-pregnan-20-one), modified in position 17 were prepared. Compounds with 20-keto pregnane side chain replaced completely by azide (17α- and 17β-azido-5β-androstan-3α-ol), compounds with its part replaced (20-azido-21-nor-5β-pregnan-3α-ol), and compounds with keto group only replaced ((20R)- and (20S)-20-azido-5β-pregnan-3α-ol) were synthesized using tosylate displacements with sodium azide or Mitsunobu reaction with azoimide. All five azido steroids were converted into corresponding sulfates. Subsequent tests for inhibition of glutamate induced response on NMDA receptors revealed that modification of pregnanolone sulfate side chain with azide did not disturb the activity and some of sulfates tested were more active than parent compound.     

Isolation and characterization of <Emphasis Type="Italic">Arabidopsis</Emphasis> mutants with enhanced tolerance to oxidative stress

We have previously reported a method for isolation of mutants with enhanced tolerance to the fungal AAL toxin and given a detailed characterization of atr1 (AAL toxin resistant, Gechev et al. in Biochem Biophys Res Commun 375:639–644, 2008). Herewith, we report eight more mutants with enhanced tolerance to the AAL toxin. Phenotypic analysis showed that six of the mutants were reduced in size compared with their original background loh2. Furthermore, atr2 showed delayed flowering and senescence. The mutants were also evaluated for oxidative stress tolerance by growing them on ROS-inducing media supplemented with either aminotriazole or paraquat, generating, respectively, H₂O₂ or superoxide radicals. Oxidative stress, confirmed by induction of the marker genes, HIGH AFFINITY NITRATE TRANSPORTER At1G08090 and HEAT SHOCK PROTEIN 17 At3G46230, inhibited growth of all lines. However, while the original background loh2 developed necrotic lesions and died rapidly on ROS-inducing plant growth media, atr1, atr2, atr7 and atr9 remained green and viable. The tolerance against oxidative stress-induced cell death was confirmed by fresh weight and chlorophyll measurements. Real-time PCR analysis revealed that the expression of the EXTENSIN gene At5G46890, previously shown to be downregulated by aminotriazole in atr1, was repressed in all lines, consistent with the growth inhibition induced by oxidative stress. Taken together, the data indicate a complex link between growth, development and oxidative stress tolerance and indicates that growth inhibition can be uncoupled from oxidative stress-induced cell death.