Ivan Gelbič

Bacillus thuringiensis (Bt) is efficient, strongly specific, and avirulent to humans, making it one of the most popular biopesticides in the world. Bt LLP29 is a mosquitocidal strain that was first isolated from Magnolia denudata. To understand its molecular mechanism against mosquitoes, the genome of Bt LLP29 was sequenced and annotated in this study. The LLP29 genome was found to have a total length of 5.99 Mb, with an average G + C content of 35.21%. A total of 6107 coding sequences were also detected, together with 42 rRNAs and 124 tRNAs and 135 other RNAs. With the help of annotation databases, including GO, COG, KEGG, Nr and Swiss-Prot, most unigene functions were identified. At the same time, a collinear analysis was performed on the genome of LLP29. There were also some virulence genes detected, including cry, chitinase, zwittermicin and vip.

Pectinase is widely used in numerous industrial fields, including the food, wine, and paper industries. In this work, seven bacteria were isolated from orange peel and their pectinase production activity was assayed. One bacterium (OR-B2) identified as a Bacillus sp. showed the highest enzyme activity towards others. A gene encoding a pectate lyase designed as PelB-B2 in this work was amplified and heterogeneous expressed in E.coli. PelB-B2 was defined as a member of the PelB pectate lyase family after phylogenic tree analysis. 3D model of PelB-B2 was constructed by SWISS-MODEL and PelB-B2 showed conserved para-β structure. After inducing culture and purified by Ni-affinity chromatography, the properties of the purified PelB-B2 were assayed. Optimal pH and temperature for PelB-B2 was pH 8.0 and 50 °C, respectively. PelB-B2 showed excellent pH stability and thermostability. It was stable within pH range 3.0–11.0 and retained more than 51% activity after incubation at 40 °C, 50 °C, or 60 °C for 1 h. Furthermore, we determined that PelB-B2 was a Ca²⁺-dependent pectinase and the pectin extracted from citrus was the benefit substrate for PelB-B2. The K_m and V_max of PelB-B2 were 1.64 g/L and 232.56 mol/(L min), respectively. The OR-B2 can be a new resource for pectinase production and the PelB-B2 has potential for industrial application. Graphic abstract 7 bacteria were isolated from orange peel, namely OR-B1 to OR-B7 and their pectinase activities were assayed. One pectate lyase belongs to PelB family was cloned from OR-B2 and heterogeneous expressed in E. coli. Purified PelB-B2 was further studied with its properties. Effects of pH, temperature, chemicals, substrate on the enzyme activity were assayed and the enzyme kinetic was also measured.

Thuricin 4AJ1, produced by Bacillus thuringiensis strain 4AJ1, showed inhibition activity against Bacillus cereus 0938 and ATCC 10987. It began to appear in the stationary phase and reached its maximum activity level of 209.958 U at 18 h against B. cereus 0938 and 285.689 U at 24 h against B. cereus ATCC 10987. Tricine–SDS-PAGE results showed that the partly purified thuricin 4AJ1 was about 6.5 kDa. The molecular weights of the known B. thuringiensis bacteriocins and the ones obtained by the two mainstream websites for predicting bacteriocins were inconsistent with the size of the thuricin 4AJ1, indicating that the bacteriocin obtained in this study may have a novel structure. Based on the biochemical properties, the thuricin 4AJ1 activities increased after treatment with proteinase K and lipase II, and were not affected by a-amylase, catalase, α-chymotrypsin VII and α-chymotrypsin II. It was heat tolerant, being active up to 90º C. In the pH 3–10 range, it maintained most of its activity. Finally, the sensitivity of the strain 4AJ1 to commonly used antibiotics was tested. In view of its stability and antibacterial activity, thuricin 4AJ1 may be applied as a food biopreservative.

A novel Bacillus thuringiensis (Bt) bacteriocin BtCspB, active against a food-borne pathogen Bacillus cereus, was identified and purified by a traditional four-step chromatographic process with low yield (44.5 µg/L) in our lab previously. The aim of this study was to dramatically increase its yield by heterologous expression of BtCspB. The BtCspB gene from Bt BRC-ZYR2 was successfully heterologously expressed in Escherichia coli BL21 (DE3). Affinity chromatography was used to obtain the pure BtCspB up to 20 mg/L. The purified BtCspB showed a MIC value of 12.5 µg/mL and a MBC value of 50.0 µg/mL against Bacillus cereus ATCC 10987. The bacteriocin activity of BtCspB against B. cereus ATCC 10987 was further directly detected in a gel-overlay assay. The anti-B. cereus activity, however, was lower than the bacteriocin purified by the traditional four-step chromatographic process probably because of structural modifications. Compared with the traditional method, the yield of the bacteriocin by heterologous expression increased by 449 times, and the purification step was dramatically simplified, which laying a foundation for the industrial production of this novel cold-shock protein-like bacteriocin BtCspB active against B. cereus.

Ectropis oblique Prout (Lepidoptera: Geometridae) is one of the main pests that damages the tea crop in Southeast Asia. To understand the molecular mechanisms of its feeding biology, transcriptomes of the alimentary tract (AT) and of the body minus the AT of E. oblique were successfully sequenced and analyzed in this study. A total of 36,950 unigenes from de novo sequences were assembled. After analysis using six annotation databases (e.g., Gene Ontology, Kyoto Encyclopedia of Genes and Genome, and NCBI nr), a series of putative genes were found for this insect species that were related to digestion, detoxification, the immune system, and Bacillus thuringiensis (Bt) receptors. From this series of genes, 21 were randomly selected to verify the relative expression levels of transcripts using quantitative real-time polymerase chain reaction. These results will provide an invaluable genomic resource for future studies on the molecular mechanisms of E. oblique, which will be useful in developing biological control strategies for this pest.

The presence of the thermophilic species Anopheles hyrcanus in the lower Dyje River basin has been regularly monitored since 2008. Mosquito trapping has been conducted mainly in summer using EVS traps with CO₂ as attractant. Anopheles hyrcanus and Culex modestus were studied in a group of ponds near the Czech–Austrian border. At the Sedlec location, occurrence of An. hyrcanus was low during 2008–2011, with around 1–9 individuals/trap/collection day. At the Lednice location on the edge of Mlýnský Pond, the first capture of An. hyrcanus occurred in 2010. In 2013, there was a sudden increase in the An. hyrcanus mosquito at all monitored locations. The largest presence was recorded at Sedlec and reached 524.4 individuals/trap/collection day. Culex modestus was one of the predominant species throughout the period. At Sedlec, the smallest finding was in 2009 (at 9.6 individuals) while the greatest was in 2010 (at 988.6 individuals/trap/collection day). At Lednice, the smallest finding was in 2011 (at 56.3 individuals) while the largest was in 2013 (at 3850 individuals/trap/collection day).

In addition to being used increasingly as a model system in modern molecular biology studies, the free-living nematode Caenorhabditis elegans (Maupas, 1900) is