Double-strand DNA end-binding and sliding of the toroidal CRISPR-associated protein Csn2

The adaptive immunity of bacteria against foreign nucleic acids, mediated by CRISPR (clustered regularly interspaced short palindromic repeats), relies on the specific incorporation of short pieces of the invading foreign DNA into a special genomic locus, termed CRISPR array. The stored sequences (spacers) are subsequently used in the form of small RNAs (crRNAs) to interfere with the target nucleic acid. We explored the DNA-binding mechanism of the immunization protein Csn2 from the human pathogen Streptococcus agalactiae using different biochemical techniques, atomic force microscopic imaging and molecular dynamics simulations. The results demonstrate that the ring-shaped Csn2 tetramer binds DNA ends through its central hole and slides inward, likely by a screw motion along the helical path of the enclosed DNA. The presented data indicate an accessory function of Csn2 during integration of exogenous DNA by end-joining.     

A highly pathogenic strain of Bacillus thuringiensis serovar kurstaki in lepidopteran pests

In order to detect and identify the most toxic Bacillus thuringiensis strains against pests, we isolated a B. thuringiensis strain (Bn1) from Balaninus nucum (Coleoptera: Curculionidae), the most damaging hazelnut pest. Bn1 was characterized via morphological, biochemical, and molecular techniques. The isolate was serotyped, and the results showed that Bn1 was the B. thuringiensis serovar, kurstaki (H3abc). The scanning electron microscopy indicated that Bn1 has crystals with cubic and bipyramidal shapes. The Polymerase Chain Reactions (PCRs) revealed the presence of the cry1 and cry2 genes. The presence of Cry1 and Cry2 proteins in the Bn1 isolate was confirmed via SDS-PAGE, at approximately 130 kDa and 65 kDa, respectively. The bioassays conducted to determine the insecticidal activity of the Bn1 isolate were conducted with four distinct insects, using spore-crystal mixtures. We noted that Bn1 has higher toxicity as compared with the standard B. thuringiensis subsp. kurstaki (HD-1). The highest observed mortality was 90% against Malacosoma neustria and Lymantria dispar larvae. Our results show that the B. thuringiensis isolate (Bn1) may prove valuable as a significant microbial control agent against lepidopteran pests.     

The crystal structure of the CRISPR-associated protein Csn2 from Streptococcus agalactiae

The prokaryotic immune system, CRISPR, confers an adaptive and inheritable defense mechanism against invasion by mobile genetic elements. Guided by small CRISPR RNAs (crRNAs), a diverse family of CRISPR-associated (Cas) proteins mediates the targeting and inactivation of foreign DNA. Here, we demonstrate that Csn2, a Cas protein likely involved in spacer integration, forms a tetramer in solution and structurally possesses a ring-like structure. Furthermore, co-purified Ca(2+) was found important for the DNA binding property of Csn2, which contains a helicase fold, with highly conserved DxD and RR motifs found throughout Csn2 proteins. We could verify that Csn2 binds ds-DNA. In addition molecular dynamics simulations suggested a Csn2 conformation that can "sit" on the DNA helix and binds DNA in a groove on the outside of the ring.     

A new biopesticide from a local <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> var. <Emphasis Type="Italic">tenebrionis</Emphasis> (Xd3) against alder leaf beetle (Coleoptera: Chrysomelidae)

Use of chemical pesticides in agriculture harms humans, non-target organisms and environments, and causes increase resistance against chemicals. In order to develop an effective bio-pesticide against coleopterans, particularly against Agelastica alni (Coleoptera: Chrysomelidae) which is one of the serious pests of alder leaf and hazelnut, we tested the insecticidal effect of 21 Bacillus isolates against the larvae and adults of the pest. Bacillus thuringiensis var. tenebrionis-Xd3 (Btt-Xd3) showed the highest insecticidal effect based on screening tests. For toxin protein production and high sporulation of Xd3, the most suitable medium, pH and temperature conditions were determined as nutrient broth medium enriched with salts, pH 7 and 30?°C, respectively. Sporulated Btt-Xd3 in nutrient broth medium enriched with salts transferred to fermentation medium containing soybean flour, glucose and salts. After fermentation, the mixture was dried in a spray dryer, and spore count of the powder product was determined as 1.6?×?10¹⁰ c.f.u. g^?1. Moisture content, suspensibility and wettability of the formulation were determined as 8.3, 86% and 21 s, respectively. Lethal concentrations (LC₅₀) of formulated Btt-Xd3 were determined as 0.15?×?10⁵ c.f.u. ml^?1 for larvae at laboratory conditions. LC₅₀ values were also determined as 0.45?×?10⁶ c.f.u. ml^?1 at the field condition on larval stage. Our results showed that a new bio-pesticide developed from B. thuringiensis tenebrionis (Xd3) (Btt-Xd3) may be valuable as a biological control agent for coleopteran pests.     

Screening antibacterial activity of entomopathogenic bacteria isolated from pests of hazelnut

Thirty-seven entomopathogenic bacteria isolated from six common pests of hazelnut in the Black Sea Region of Turkey have been screened for their potential of antibacterial substance production against indicator bacteria by the agar spot assay and well diffusion assay. Results indicated that 13.5% of entomopathogenic bacteria, Pseudomonas fluorescens (Pf-Xd1), Bacillus polymyxa (Bp-Ar2), Bacillus thuringiensis (Bt-Bn1), Serratia marcescens (Sm-Mm3) and Pseudomonas flourescens (Pf-Aa4) isolated from pests of Xyleborus dispar, Anoplus roboris, Balaninus nucum, Melolontha melolontha and Agelastica alni, respectively, showed significant levels of inhibitor activities against indicator bacteria. Well diffusion assay showed that supernatants of Bp-Ar2, Bt-Bn1 and Sm-Mm3 have antibacterial activity, whereas Pf-Xd1 and Pf-Aa4 did not show any activity. Furthermore, the antibacterial activity of the substance produced by the Bp-Ar2 has a narrow spectrum, whereas those of Bt-Bn1 and Sm-Mm3 exhibit broad spectrum. The production of these antibacterial substances were similarly determined at early logarithmic phase in the growth cycle of three bacteria and continued until the beginning of the stationary phase as primer metabolite. In addition, optimal pH (at 7–9 forBt-Bn1 and 5–9 forSm-Mm3), medium (Muller Hinton broth forBt-Bn1 and Luria Bertani broth forSm-Mm3), temperature (25°C for Bt-Bn1 and Sm-Mm3) and production time (24h forBt-Bn1 and 72h forSm-Mm3) of these substances were determined. Our results demonstrate that entomopathogenic bacteria are a potential source of antibacterial substances.     

The biology of Chilo iridescent virus

Chilo iridescent virus (CIV) is the type species for genus Iridovirus, and belongs to the family Iridoviridae. Since the discovery of CIV in 1966, many attempts were made to elucidate the viral genome structure. The virions contain a single linear ds DNA molecule that is circularly permuted and terminally redundant. The genome of CIV has been entirely sequenced. The CIV virion consists of an unusual three-layer structure containing an outer proteinaceous capsid, an intermediate lipid membrane, and a core DNA-protein complex containing the genome. CIV has a broad host spectrum and has, in general, a limited mortality effect on its hosts. Up to now there have been several studies about CIV describing its structure, ecology, and molecular biology. In this review study we present all these studies together to describe the CIV.     

Isolation and characterization of entomopathogenic fungi from hazelnut-growing region of Turkey

Although Turkey is the first among all hazelnut-producing countries, yield per unit area of this crop is low in comparison to other countries, mainly because many insect species seriously damage hazelnut trees and their fruit. To find effective and safe biocontrol agents, we conducted a survey study to isolate entomopathogenic fungi from the hazelnut-growing region of Turkey and characterized the isolated strains in detail. In addition, we determined the effectiveness of seven selected strains from this region against Melolontha melolontha (Coleoptera: Scarabaeidae) which is one of the most serious pests of hazelnut. In 2006 and 2007, 301 soil samples were collected randomly and analyzed for presence of entomopathogenic fungi using the Galleria bait method. Entomopathogenic fungi were found to occur in 20.59% of the soil samples studied. Based on morphology, ITS sequence and partial sequencing of the 18S (SSU rDNA) and EF1-α genes, the isolates were identified as Metarhizium anisopliae var. anisopliae, Metarhizium sp., Beauveria bassiana, Beauveria cf. bassiana, Isaria fumosorosea and Evlachovaea sp. Metarhizium anisopliae var. anisopliae was isolated from 34 sites and was the most frequent and abundant entomopathogenic species recovered. All the isolates tested were pathogenic to M. melolontha. M. anisopliae var. anisopliae KTU-27 and Evlachovaea sp. KTU-36 produced the highest insecticidal activity (86.6%) within 15 days after inoculation. Our results suggest that entomopathogenic fungi could be good biocontrol agents against M. melolontha, and are discussed with respect to ecology of fungi in relation to habitat in order to evaluate biocontrol potential of these isolates. This is the first study of the distribution of entomopathogenic fungi in the hazelnut-growing region of Turkey and of their pathogenicities against M. melolontha.     

Isolation and identification of bacteria from <Emphasis Type="Italic">Thaumetopoea pityocampa</Emphasis> Den. and Schiff. (Lep., Thaumetopoeidae) and determination of their biocontrol potential

The pine processionary moth Thaumetopoea pityocampa (Den. and Schiff.) is one of the most harmful insect pest for pine species in Mediterranean countries including Turkey. The objective of the present study is to find a more effective and safe biological control agent against T. pityocampa. Thus, we investigated the bacterial flora of the pest insect, collected from the Middle Black Sea Region of Turkey from 2003 to 2004. Based on morphological, physiological, biochemical and molecular methods, 14 different bacterial isolates were determined. The identified bacterial flora of T. pityocampa consisted of bacteria belonging to the Enterobacteriaceae (Tp1), Arthrobacter sp. (Tp2), Staphylococcus spp. (Tp3 and 10), Bacillus subtilis (Tp4), Serratia liquefaciens (Tp5), Bacillus thuringiensis subsp. morrisoni (Tp6 and 14), an acrystalliferous form Bacillus thuringiensis (Tp7), Staphylococcus cohnii (Tp8), Bacillus licheniformis (Tp9), Bacillus pumilus (Tp11), Brevibacterium sp. (Tp12) and Bacillus simplex (Tp13). After analysing the conclusions of conventional and molecular tests Tp1 (Enterobacteriaceae), Tp2 (Arthrobacter sp.) and Tp12 (Brevibacterium sp.) were assigned as novel bacterial species. Isolate Tp5 had a promising insecticidal effect on third instar larvae of T. pityocampa (up to 70% mortality within 10 days).     

Characterisation of three Alphabaculovirus isolates from the gypsy moth,Lymantria dispar dispar (Lepidoptera: Erebidae), in Turkey

In this study, Lymantria dispar dispar larvae, collected from three different localities in Turkey, were examined for the presence of inclusion bodies under phase contrast and electron microscopes. Inclusion bodies from infected larvae were subjected to polymerase chain reaction using the conserved primers for polyhedrin (polh), late expression factor 8 (lef-8) and late expression factor 9 (lef-9) genes. Sequence analysis confirmed that larvae collected from the three different localities contained multiple nucleopolyhedrosis viruses (MNPVs). These isolates were designated LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3. Phylogenetic analyses of these isolates were performed using target genes polh, lef-8 and lef-9. Restriction endonuclease analysis of the three geographic isolates with EcoRI and PstI enzymes demonstrated some differences existed among the isolates. According to the EcoRI profile, the mean estimated size for the complete genome of each isolate (LdMNPV-T1, LdMNPV-T2 and LdMNPV-T3) was calculated to be approximately 170, 153 and 170?kb, respectively. Insecticidal activities of each isolate were tested on L. d. dispar larvae using four different viral concentrations between 10³ and 10⁶?OBs/ml. Results showed that the mortalities for LdMNPV-T1, -T2 and -T3 ranged between 13–53%, 47–100% and 46–93%, respectively. The LC₅₀ and LC₉₅ values of LdMNPV-T2 were not significantly different from the respective corresponding values of the other two isolates. However, isolate LdMNPV-T2 killed larvae with a LC₅₀ value that was lower than the other two isolates. Our results suggested there are promising LdMNPV isolates in Turkey that can be used for microbial control of L. d. dispar larvae.