The Murein Hydrolase of the Bacteriophage φ3626 Dual Lysis System Is Active against All Tested Clostridium perfringens Strains

Clostridium perfringens commonly occurs in food and feed, can produce an enterotoxin frequently implicated in food-borne disease, and has a substantial negative impact on the poultry industry. As a step towards new approaches for control of this organism, we investigated the cell wall lysis system of C. perfringens bacteriophage 3626, whose dual lysis gene cassette consists of a holin gene and an endolysin gene. Hol3626 has two membrane-spanning domains (MSDs) and is a group II holin. A positively charged beta turn between the two MSDs suggests that both the amino terminus and the carboxy terminus of Hol3626 might be located outside the cell membrane, a very unusual holin topology. Holin function was experimentally demonstrated by using the ability of the holin to complement a deletion of the heterologous phage λ S holin in λΔSthf. The endolysin gene ply3626 was cloned in Escherichia coli. However, protein synthesis occurred only when bacteria were supplemented with rare tRNA^Arg and tRNA^Ile genes. Formation of inclusion bodies could be avoided by drastically lowering the expression level. Amino-terminal modification by a six-histidine tag did not affect enzyme activity and enabled purification by metal chelate affinity chromatography. Ply3626 has an N-terminal amidase domain and a unique C-terminal portion, which might be responsible for the specific lytic range of the enzyme. All 48 tested strains of C. perfringens were sensitive to the murein hydrolase, whereas other clostridia and bacteria belonging to other genera were generally not affected. This highly specific activity towards C. perfringens might be useful for novel biocontrol measures in food, feed, and complex microbial communities.     

Morphing of the phylogeographic lineages of the Balkan alpine newts (Ichthyosaura alpestris, Caudata, Salamandridae): in situ morphological diversification

Numerous alpine newt (Ichthyosaura alpestris) populations from the Balkans, representing all the previously established phylogeographic lineages, were studied for variations in various morphological characteristics (body size and shape, skull qualitative traits and number of trunk vertebrae). Here, we present a decoupling of morphological and mtDNA phylogeographic substructuring in the alpine newt on the Balkan Peninsula. In sharp contrast to other European newts (Triturus spp., Lissotriton spp.), the vast majority of morphological variation in the alpine newt is concentrated at the population level indicating an in situ morphological diversification. We found that the rate of morphological change is similar to the rate of mtDNA change. We hypothesize that the alpine newts are characterized by non-adaptive morphological evolution.     

Heavy metal accumulation of Danube river aquatic plants — indication of chemical contamination

In this paper, the ecological status of a section of the Danube River flowing through Serbia from Bezdan to Djerdap was evalutated. Using the chemical composition of water, sediment samples from the littoral zone and dominant aquatic macrophytes, the level of chemical pollution was ascertained. Chemical analyses of the water and sediment indicated that the tributaries flowing into the Danube significantly influenced the chemical load of the water and as a direct consequence, the sediment. The concentration of heavy metals including Cu, Mn and Cd found in plants of the Potamogeton genus, further indicated significant chemical pollution, establishing a clear link between the chemical composition of plant tissues and the chemical composition of water and sediment. This paper therefore describes how the chemical composition of aquatic plants can be used as a reliable indicator for heavy metal pollution of aquatic ecosystems.     

Alien invasive neophytes of the Southeastern part of the Pannonian Plain

Floristic diversity in the Pannonian Plain is the result of mosaic habitat composition, characterized by steppes, saline meadows, and lowland forests along major rivers. In the last few centuries, the flora has become enriched by species introduced by human activities, such as colonization and industrialization. Presently it consists of nearly 1800 species. As a result of 30 years of field research as well as herbarium and literature searches, a list of invasive alien neophytes has been compiled and is presented here. The list includes 152 species, classified as either naturalized, casual, or unknown. One of the species is listed in Red Book of Serbian Flora. Eighty species are naturalized, and 61 are casual species. The majority of the introduced flora originates from North America (66) and the Old World (43). Most alien invasive species of the Southeastern part of the Pannonian Plain, were introduced accidentally by seed material or deliberately by nursery saplings. The annual and perennial herbaceous plants, which flower in the summer and summer-autumn periods, are represented in the greatest numbers. The alien flora was further analyzed with respect to taxonomy, life forms, pollination, and seed dispersal mode. The greatest number of alien species appears in anthropogenic and semi-natural habitats (59). In natural habitats, 45 invasive taxa were recorded, representing a conservation threat to biodiversity.     

The evolution of the adult body form of the crested newt (Triturus cristatus superspecies,Caudata, Salamandridae)

We characterized the adult body form of the crested newt (Triturus cristatus superspecies) and explored its evolution. From seven morphometric traits, we determined that body size, interlimb distance and head width define the body form. None of the morphometric traits showed a phylogenetic signal. Three body‐shape morphotypes (Triturus dobrogicus + T. cristatus, Triturus carnifex + Triturus macedonicus and Triturus karelinii + Triturus arntzeni) and three body‐size morphotypes (T. dobrogicus, T. cristatus and all other crested newts) could be recognized. The ancestral phenotype (a large body with a short trunk and a wide head) characterized T. karelinii and T. arntzeni. Triturus carnifex and T. macedonicus had a somewhat different phenotype (large body and wide head, accompanied by mild body elongation). The most derived phenotype included body size reduction and more pronounced body elongation in T. cristatus and, especially, in T. dobrogicus. Body elongation occurred by trunk lengthening but not head and tail lengthening. Additionally, contrary to other tetrapods, evolutionary axis elongation in crested newts was followed by a decrease in body size. We advocate the hypothesis that ecology drives the evolution of body form in crested newts.     

Nuclear and mitochondrial phylogeography of the European fire-bellied toads Bombina bombina and Bombina variegata supports their independent histories

Exact location and number of glacial refugia still remain unclear for many European cold‐blooded terrestrial vertebrates. We performed a fine‐scaled multilocus phylogeographic analysis of two Bombina species combining mitochondrial variation of 950 toads from 385 sites and nuclear genes (Rag‐1, Ncx‐1) from a subset of samples to reconstruct their colonization and contemporary variation patterns. We identified the lowlands northwest of the Black Sea and the Carpathians to be important refugial areas for B. bombina and B. variegata, respectively. This result emphasizes the importance of Central European refugia for ectothermic terrestrial species, far north of the Mediterranean areas regarded as exclusive glacial refugia for the animals. Additional refugia for B. variegata have been located in the southern Apennines and Balkans. In contrast, no evidence for the importance of other east European plains as refugial regions has been found. The distribution of mtDNA and Ncx‐1 variation suggests the presence of local refugia near the Black Sea for B. bombina; however, coalescent simulations did not allow to distinguish whether one or two refugia were present in the region. Strong genetic drift apparently accompanied postglacial expansions reducing diversity in the colonization areas. Extended sampling, coupled with the multilocus isolation with migration analysis, revealed a limited and geographically restricted gene flow from the Balkan to Carpathian populations of B. variegata. However, despite proximity of inferred B. bombina and B. variegata refugia, gene exchange between them was not detected.     

Monitoring of aphid flight activities in?seed?potato?crops?in Serbia

Aphid flight activities in seed potato fields have been studied by the yellow water traps. It is a good method for monitoring aphids as vectors of viruses, but this study also showed it is a suitable method for insect-diversity research. During the four-year studies, over 11.500 specimens were collected and a total of 107 different taxa of aphids were identified. The most abundant species were polyphagous species, such as: Acyrthosiphon pisum (Haris), Aphis fabae Scopoli, Aphis gossypii Gloverand Brachycaudus helichrysi (Kaltenbach). The results of the studies show that diversity of aphids in different regions of Serbia is similar regardless of the altitude and the diversity of terrain. At most sites it ranged from 2 to 3. The highest value was recorded in Begeč, locality in northern part of Serbia, in year 2008, and it was 2.92. The maximum values of the Shannon-Weaver diversity index at all sites were recorded in the first weeks of the monitoring of aphid flight activities. Morisita-Horn similarity index shows no significant differences between sites regardless of altitudes. The sites are grouped by year, not by similarity of relief. In spite of these results, the Chi-square analysis showed highly significant difference in vector frequencies among seasons and sites with more pronounced differences for PVY. As a consequence of differences in vector frequencies, the vector pressure index in some regions was different also. The number of vectors and vector pressure index vary depending on the altitude of localities. At localities at altitudes under 1000 m, they were high. The highest index was at Kotraža, locality in central part of Serbia, in 2007, when PVY index exceeded the value of 180, while for PLRV it was 60. At high altitudes on mountain Golija, above 1100 m, the number of aphids was low, as well as the vector pressure index which indicates that these regions are suitable for producing virus-free seed potato.     

Identification and partial characterization of the nonribosomal peptide synthetase gene responsible for cereulide production in emetic Bacillus cereus

Cereulide, a depsipeptide structurally related to valinomycin, is responsible for the emetic type of gastrointestinal disease caused by Bacillus cereus. Due to its chemical structure, (D-O-Leu-D-Ala-L-O-Val-L-Val)(3), cereulide might be synthesized nonribosomally. Therefore, degenerate PCR primers targeted to conserved sequence motifs of known nonribosomal peptide synthetase (NRPS) genes were used to amplify gene fragments from a cereulide-producing B. cereus strain. Sequence analysis of one of the amplicons revealed a DNA fragment whose putative gene product showed significant homology to valine activation NRPS modules. The sequences of the flanking regions of this DNA fragment revealed a complete module that is predicted to activate valine, as well as a putative carboxyl-terminal thioesterase domain of the NRPS gene. Disruption of the peptide synthetase gene by insertion of a kanamycin cassette through homologous recombination produced cereulide-deficient mutants. The valine-activating module was highly conserved when sequences from nine emetic B. cereus strains isolated from diverse geographical locations were compared. Primers were designed based on the NRPS sequence, and the resulting PCR assay, targeting the ces gene, was tested by using a panel of 143 B. cereus group strains and 40 strains of other bacterial species showing PCR bands specific for only the cereulide-producing B. cereus strains.     

Influenza vaccine and learning in C57BL mice with an acute experimental autoimmune encephalomyelitis

Literature data suggest possible link between influenza vaccination and development of autoimmune processes. Therefore, the aim of the study was to investigate the effect of influenza vaccination on spatial learning in mice with experimental autoimmune encephalomyelitis (EAE). EAE was induced in eight-week-old C57BL/6J female mice by subcutaneous immunization (MOG_35–55 in complete Freund’s adjuvant) and Pertussis vaccine injected intraperitoneally. Mice were vaccinated with influenza vaccine three days before MOG immunization. The hippocampal-dependent spatial learning test, Morris Water Maze test (MWM), was performed before and after EAE induction. Significant difference (P < 0.05) in the time for completing the Morris Water Maze task was found between mice with mild clinical signs of EAE when compared to other mice. However no significant difference was observed between mice with EAE and mice with EAE that were vaccinated with influenza vaccine. Hippocampal tissue lesions in EAE mice are in correlation with memory impairment. Study shows no influence of influenza vaccine on progression of clinical signs of EAE, spatial learning and memory impairment.