Assaying Paenibacillus alvei CsaB-Catalysed Ketalpyruvyltransfer to Saccharides by Measurement of Phosphate Release

Ketalpyruvyltransferases belong to a widespread but little investigated class of enzymes, which utilise phosphoenolpyruvate (PEP) for the pyruvylation of saccharides. Pyruvylated saccharides play pivotal biological roles, ranging from protein binding to virulence. Limiting factors for the characterisation of ketalpyruvyltransferases are the availability of cognate acceptor substrates and a straightforward enzyme assay. We report on a fast ketalpyruvyltransferase assay based on the colorimetric detection of phosphate released during pyruvyltransfer from PEP onto the acceptor via complexation with Malachite Green and molybdate. To optimise the assay for the model 4,6-ketalpyruvyl::ManNAc-transferase CsaB from Paenibacillus alvei, a β-d-ManNAc-α-d-GlcNAc-diphosphoryl-11-phenoxyundecyl acceptor mimicking an intermediate of the bacterium’s cell wall glycopolymer biosynthesis pathway, upon which CsaB is naturally active, was produced chemo-enzymatically and used together with recombinant CsaB. Optimal assay conditions were 5 min reaction time at 37 °C and pH 7.5, followed by colour development for 1 h at 37 °C and measurement of absorbance at 620 nm. The structure of the generated pyruvylated product was confirmed by NMR spectroscopy. Using the established assay, the first kinetic constants of a 4,6-ketalpyuvyl::ManNAc-transferase could be determined; upon variation of the acceptor and PEP concentrations, a KM, PEP of 19.50 ± 3.50 µM and kcat, PEP of 0.21 ± 0.01 s−1 as well as a KM, Acceptor of 258 ± 38 µM and a kcat, Acceptor of 0.15 ± 0.01 s−1 were revealed. P. alvei CsaB was inactive on synthetic pNP-β-d-ManNAc and β-d-ManNAc-β-d-GlcNAc-1-OMe, supporting the necessity of a complex acceptor substrate.

Biocontrol of Fusarium oxysporum f. sp. vasinfectum With Bacillus spp. Strains

The wilt caused by Fusarium oxysporum f.sp. vasinfectum in cotton is one of the main diseases of this crop. The pathogen is difficult to control because it produces chlamydospores, which allows it to survive in the soil for long periods without the presence of its main host. The control of this disease is based on varieties with some resistance, certified seeds, and fungicides used to treat seeds. Thus, the development of control alternatives is needed to increase the number of resources to manage this disease in the field. Bacillus are bioregulators and antagonists of several important phytopathogens. Therefore, the objective of this work was to select strains of Bacillus (Paenibacillus alvei, Bacillus thuringiensis, B. megaterium, and B. lentus), whose metabolites are toxic to F. oxysporum in laboratory and greenhouse conditions, in addition to adjusting in vitro methodologies to perform and evaluate selection tests. This work determined the best culture medium for the direct confrontation assays, proposed a grading scale to facilitate decisions about selection with the dual tests, identified among the strains selected in the confrontation assays those that produce volatile and non-volatile metabolites toxic to F. oxysporum, and tested these selected strains for disease control during seed germination in greenhouse trials. In last test, some strains of Bacillus had a potential protective effect during the germination of cotton seeds.

Revisiting Substrate Specificity Concept in Microbial Screening Methodologies for Fish Waste Management

Microbial and enzymatic processing is an attractive area for production of valuable byproducts from fish waste. Functional screening methodologies for the purpose are still based on activities in non-specific substrates, and concept of substrate specificity is not yet validated. Therefore, reliability of using non-specific substrate for the purpose was checked. Results revealed the existence of a limited number of mutually inclusive positive isolates in non-specific and specific substrate based assays (13% for fish proteolysis and 22% for fish lipolysis), with no significant positive correlations (P>0.05). Further, using non-specific substrates in screening assays missed 57.1% and 53.33% of fish proteolytic and fish lipolytic microbes respectively, signifying the use of same substrates. Beyond methodological perspectives, the paper forms the first report on fish proteolytic activity of Bacillus tropicus, Bacillus vallismortis, Paenibacillus alvei, Staphylococcus epidermidis and Staphylococcus hominis. Similarly, fish oil hydrolyzing capacities of B. tropicus, Cronobacter sakazakii, P. alvei, Paenibacillus pinisoli, Pantoea stewartii, S. hominis and Staphylococcus caprae are recorded for the first time. Further, the paper points out 6 and 3 potential microbial species producing > 1 protease units/ml and >1 enzymatic index for fish proteolytic and lipolytic activities, without any optimization, warranting future use in fish waste management.

Paenibacillus alvei K165 and Fusarium oxysporum F2: Potential Biocontrol Agents against Phaeomoniella chlamydospora in Grapevines

In the last two decades grapevine trunk diseases (GTDs) have emerged as the most significant threat for grapevine sustainability worldwide. The tracheomycotic fungus Phaeomoniella chlamydospora (Pch) is the predominant GTD-associated species and cannot be controlled with available chemicals. In the present study, we evaluated the effectiveness of two microbial strains (Paenibacillus alvei K165 and Fusarium oxysporum F2) against Pch in grapevine. In vitro bioassays, performed in a growth culture medium simulating the xylem environment, indicated that F2 decreased Pch growth and sporulation, whereas K165 did not have any effect on Pch growth. In planta experiments revealed that root-drench and stem-puncture application of K165 and F2 reduced the endophytic relative DNA amount of Pch by 90% and 82%, respectively, compared to controls. However, wood discoloration, the typical symptom of Pch infection, was not reduced in the F2 treated grapevines. Nevertheless, the F2 treated grapevines harbored higher lignin levels compared to mocks, as it was also done by K165. Therefore, F2 and K165 have the potential to be used as biocontrol agents against Pch in grapevines.

TUYỂN CHỌN VÀ XÂY DỰNG BỘ SƯU TẬP VI KHUẨN FRUCTOPHILIC LACTIC ACID (FLAB) TỪ HỆ TIÊU HÓA ONG MẬT

Nghiên cứu này bao gồm: phân lập, định danh và tuyển chọn vi khuẩn FLAB từ hệ tiêu hóa ong mật với khả năng kháng vi khuẩn gây bệnh thối ấu trùng ở ong mật, định hướng sử dụng probiotic cho ong, định hướng cho giải pháp thay thế hoặc phối hợp với việc sử dụng kháng sinh trong điều trị bệnh ở ong hiện nay. Kết quả tuyển chọn được 4 chủng vi khuẩn FLAB có khả năng kháng cao với vi khuẩn gây bệnh thối ấu trùng trên ong mật gồm Melissococcus pluton, Paenibacillus larvae và Paenibacillus alvei với đường kính vòng kháng khuẩn từ 8,67±0,58 mm đến 15,33±0,58 mm, có khả năng bám dính khá cao và tồn tại được trong điều kiện pH thấp của hệ tiêu hóa ong mật. Bên cạnh đó, các vi khuẩn đều không nhạy cảm với 2 loại kháng sinh thường sử dụng trong điều trị bệnh ở ong là streptomycin và kanamycin để có thể phối hợp sử dụng trong giới hạn cho phép, mà không làm ảnh hưởng đến hệ vi sinh vật có lợi. Các vi khuẩn được khảo sát có khả năng tồn tại ở nhiệt độ đến 60oC trong định hướng cho việc tạo chế phẩm bằng phương pháp sấy phun. Kết quả định danh theo trình tự 16s rRNA xác định cả 4 chủng đều là Lactobacillus kunkeei với độ tương đồng là 99,24%-99,44%. Nổi bật là chủng L. kunkeei M8, chủng FLAB bản địa với các hoạt tính probiotic cao để có thể phát triển tạo chế phẩm probiotic cho ong mật.

Thermostable cellulase biosynthesis from Paenibacillus alvei and its utilization in lactic acid production by simultaneous saccharification and fermentation

Cellulosic date palm wastes may have beneficial biotechnological applications for eco-friendly utilization. This study reports the isolation of thermophilic cellulase-producing bacteria and their application in lactic acid production using date palm leaves. The promising isolate was identified as Paenibacillus alvei by 16S rRNA gene sequencing. Maximum cellulase production was acquired using alkaline treated date palm leaves (ATDPL) at 48 h and yielded 4.50 U.mL-1 FPase, 8.11 U.mL-1 CMCase, and 2.74 U.mL-1 β-glucosidase. The cellulase activity was optimal at pH 5.0 and 50°C with good stability at a wide temperature (40-70°C) and pH (4.0-7.0) range, demonstrating its suitability in simultaneous saccharification and fermentation. Lactic acid fermentation was optimized at 4 days, pH 5.0, 50°C, 6.0% cellulose of ATDPL, 30 FPU/ g cellulose, 1.0 g. L-1 Tween 80, and 5.0 g. L-l yeast extract using Lactobacillus delbrueckii. The conversion efficiency of lactic acid from the cellulose of ATDPL was 98.71%, and the lactic acid productivity was 0.719 g. L-1 h-1. Alkaline treatment exhibited a valuable effect on the production of cellulases and lactic acid by reducing the lignin content and cellulose crystallinity. The results of this study offer a credible procedure for using date palm leaves for microbial industrial applications.