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The number of viable spores is the most important quality index of Bacillus-based microbial pesticides. This study aimed to establish a novel pre-treatment method of samples for improving the traditional plate counting method thereby detecting the number of viable spores accurately and reproducibly in Paenibacillus polymyxa-based microbial pesticides. Firstly, three pre-treatment methods of Shaker shaking (SSP), ultrasonic oscillation (UOP), and ultrasonic crushing (UCP) were optimised and compared. The results showed that only the UCP-based plate counting method could detect accurately the viable spore counts (5.2 × 109 CFU/g) in the 5 × 109 CFU/g P. polymyxa original powder, and were repeated well with a coefficient of variation (CV) of 5.0%, compared to SSP (3.46 × 109 CFU/g, CV was 23.4%) and UOP (3.5 × 109 CFU/g, CV was 26.0%). The optimal conditions of UCP were ultrasonic power of 200 W, ultrasonic/intermittent time of 3 s/3 s, and total time of 3 mins (30 cycles). Furthermore, the scanning electron microscope observed that spores were completely desorbed from the carrier by the UCP method. and the average particle size of the carrier pellet was about 2–5 μm, which is significantly smaller than the average particle size of the carrier pellet without pre-treatment (about 15–20 μm) and by the SSP (about 7–12 μm). The plate counting method based on UCP could desorb all spores from the carrier, and the detection results of viable spore counts in P. polymyxa microbial pesticides were accurate and reproducible. [ABSTRACT FROM AUTHOR]

Cell physiology relies on metalloenzymes and can be easily disrupted by imbalances in metal ion pools. Bacillus subtilis requires manganese for growth and has highly regulated mechanisms for import and efflux that help maintain homeostasis. Cells defective for manganese (Mn) efflux are highly sensitive to intoxication, but the processes impaired by Mn excess are often unknown. Here, we employed a forward genetics approach to identify pathways affected by manganese intoxication. Our results highlight a central role for the membrane‐localized electron transport chain in metal intoxication during aerobic growth. In the presence of elevated manganese, there is an increased generation of reactive radical species associated with dysfunction of the major terminal oxidase, the cytochrome aa3 heme‐copper menaquinol oxidase (QoxABCD). Intoxication is suppressed by diversion of menaquinol to alternative oxidases or by a mutation affecting heme A synthesis that is known to convert QoxABCD from an aa3 to a bo3‐type oxidase. Manganese sensitivity is also reduced by derepression of the MhqR regulon, which protects cells against reactive quinones. These results suggest that dysfunction of the cytochrome aa3‐type quinol oxidase contributes to metal‐induced intoxication. [ABSTRACT FROM AUTHOR]

Paenibacillus polymyxa (formerly known as Bacillus polymyxa) has been extensively studied for agricultural applications as a plant-growth-promoting rhizobacterium and is also an important biocontrol agent. Our team has developed the P. polymyxa strain HY96-2 from the tomato rhizosphere as the first microbial biopesticide based on P. polymyxa for controlling plant diseases around the world, leading to the commercialization of this microbial biopesticide in China. However, further research is essential for understanding its precise biocontrol mechanisms. In this paper, we report the complete genome sequence of HY96-2 and the results of a comparative genomic analysis between different P. polymyxa strains. The complete genome size of HY96-2 was found to be 5.75 Mb and 5207 coding sequences were predicted. HY96-2 was compared with seven other P. polymyxa strains for which complete genome sequences have been published, using phylogenetic tree, pan-genome, and nucleic acid co-linearity analysis. In addition, the genes and gene clusters involved in biofilm formation, antibiotic synthesis, and systemic resistance inducer production were compared between strain HY96-2 and two other strains, namely, SC2 and E681. The results revealed that all three of the P. polymyxa strains have the ability to control plant diseases via the mechanisms of colonization (biofilm formation), antagonism (antibiotic production), and induced resistance (systemic resistance inducer production). However, the variation of the corresponding genes or gene clusters between the three strains may lead to different antimicrobial spectra and biocontrol efficacies. Two possible pathways of biofilm formation in P. polymyxa were reported for the first time after searching the KEGG database. This study provides a scientific basis for the further optimization of the field applications and quality standards of industrial microbial biopesticides based on HY96-2. It may also serve as a reference for studying the differences in antimicrobial spectra and biocontrol capability between different biocontrol agents. [ABSTRACT FROM AUTHOR]

Chlorella pyrenoidosa is an extensively investigated species. This species has been used as a commercial microalgal feedstock of proteins, lipids, and chlorophyll for nourishment and aquaculture. As such, reliable and easy genetic modification procedures should be developed to improve this industrial microalga. In this study, a pGreen 0029 vector containing an eGFP gene under the control of a Ubiquitin promoter and Npt II, which is a selective marker gene, was constructed to explore and optimize the electroporation method of C. pyrenoidosa . The optimal transformation efficiency was approximately 101 ± 7 transformants per μg plasmid and was obtained under the following conditions: 5 × 10 6 /plate cell density in logarithmic phase; 30 μg/mL plasmid; 660 V pulse voltage; 3.5 ms pulse width; and 30 μg/mL G418. The Npt II and eGFP genes were identified successfully at the DNA level after transformants were screened and purified. High-intensity green fluorescence was observed in the transformants by using fluorescence and laser confocal microscope. Results showed that foreign genes in the transformants were integrated stably and could be expressed. Hence, the efficient transformation system of C. pyrenoidosa can be used to biotechnologically improve this important industrial microalgal species and to facilitate systematic functional genomic studies. [ABSTRACT FROM AUTHOR]

Spore survival and moisture content are two important properties of biopesticides, and both are related to field biocontrol efficacy and storage shelf life. In this study,Paenibacillus polymyxa(HY96-2) was spray-dried on both pilot plant and production scales, and the effects of inlet and outlet temperatures on spore survival and moisture content were investigated. The results showed that inlet temperatures ranging from 170 to 230 °C (at an outlet temperature of 80 °C) had no obvious effect on the two properties during pilot scale processing, although an inlet temperature of 230 °C resulted in higher feed speed. When the outlet temperature on the pilot scale was reduced from 100 to 80 °C, no obvious variations in spore survival and moisture content were found, while a further reduction from 80 to 65 °C resulted in a decline in spore survival from 81.0 to 67.0% and an increase in moisture content from 2.3 to 31.7%. These results indicate that both outlet temperature and moisture content have an effect on spore survival. Optimum inlet and outlet temperatures forP. polymyxaprocessing were 230 °C and 85–90 °C on a production scale. Under these conditions, spore survival and moisture content were 83.5–86.6% and 2.73––4.12%, respectively. [ABSTRACT FROM AUTHOR]

Marine-derived Bacillus velezensis B-9987 is an important biocontrol bacterium with a broad-spectrum antibacterial effect. The traditional plate counting method is widely used for quantitative detection of viable bacteria and spores but has some disadvantages such as being laborious and time-consuming (at least 24–48 h). This study aimed to develop a new PMA-qPCR method for rapid and accurate detection of viable bacteria and spores of B-9987. The specific primers were designed for qPCR amplification based on the conserved region of the bmmA gene (encoding a malonyl CoA-ACP transacylase) of B-9987. According to the characteristic that propidium monoazide (PMA) dye can distinguish viable and dead bacteria, the optimal PMA concentration of 10 μg/ml and optimal exposure time of 10 min were achieved under PMA treatment conditions. The B-9987 spores' genomic DNA was successfully extracted after the spore coat was removed and spore germination was induced. The quantification limits of the PMA-qPCR method were determined for viable B-9987 bacteria, spores in pure culture, and spores in marine Bacillus wettable powder (marine Bacillus WP) and were 1.5 × 103 CFU/ml, 6.5 × 102 CFU/ml, and 103 CFU/ml, respectively. Compared with the qPCR method, the PMA-qPCR method could sensitively detect viable bacteria in the viable/dead bacterial mixture. In this study, the developed PMA-qPCR method was found to have excellent sensitivity and specificity in the context of a pure culture of B-9987 strain, which could accurately and rapidly detect viable B-9987 bacteria within 3–4 h and viable B-9987 spores in marine Bacillus WP within 4–6 h. • The PMA-qPCR method could detect viable B. velezensis bacteria within 3–4 h. • The PMA-qPCR method could detect viable B. velezensis spores within 4–6 h. • Genomic DNA of B. velezensis spores was successfully extracted. [ABSTRACT FROM AUTHOR]

Microbes encounter a myriad of stresses during their life cycle. Dysregulation of metal ion homeostasis is increasingly recognized as a key factor in host-microbe interactions. Bacterial metal ion homeostasis is tightly regulated by dedicated metalloregulators that control uptake, sequestration, trafficking, and efflux. Here, we demonstrate that deletion of the Bacillus subtilis yqgC-sodA (YS) complex operon, but not deletion of the individual genes, causes hypersensitivity to manganese (Mn). YqgC is an integral membrane protein of unknown function, and SodA is a Mn-dependent superoxide dismutase (MnSOD). The YS strain has reduced expression of two Mn efflux proteins, MneP and MneS, consistent with the observed Mn sensitivity. The YS strain accumulated high levels of Mn, had increased reactive radical species (RRS), and had broad metabolic alterations that can be partially explained by the inhibition of Mg-dependent enzymes. Although the YS operon deletion strain and an efflux-deficient mneP mneS double mutant both accumulate Mn and have similar metabolic perturbations, they also display phenotypic differences. Several mutations that suppressed Mn intoxication of the mneP mneS efflux mutant did not benefit the YS mutant. Further, Mn intoxication in the YS mutant, but not the mneP mneS strain, was alleviated by expression of Mg-dependent, chorismate-utilizing enzymes of the m enaquinone, s iderophore, and t ryptophan (MST) family. Therefore, despite their phenotypic similarities, the Mn sensitivity in the mneP mneS and the YS deletion mutants results from distinct enzymatic vulnerabilities.IMPORTANCEBacteria require multiple trace metal ions for survival. Metal homeostasis relies on the tightly regulated expression of metal uptake, storage, and efflux proteins. Metal intoxication occurs when metal homeostasis is perturbed and often results from enzyme mis-metalation. In Bacillus subtilis , Mn-dependent superoxide dismutase (MnSOD) is the most abundant Mn-containing protein and is important for oxidative stress resistance. Here, we report novel roles for MnSOD and a co-regulated membrane protein, YqgC, in Mn homeostasis. Loss of both MnSOD and YqgC (but not the individual proteins) prevents the efficient expression of Mn efflux proteins and leads to a large-scale perturbation of the metabolome due to inhibition of Mg-dependent enzymes, including key chorismate-utilizing MST (menaquinone, siderophore, and tryptophan) family enzymes., Competing Interests: The authors declare no conflict of interest.

Microbiology is a key basic professional course for all the students specializing in biology, biotechnology and related majors. To date, microbiology is mainly taught in Chinese within colleges and universities in China. Development of a microbiology course that is taught in English may satisfy the diversified learning needs of the students and promote the "Double First-Class" initiative. We started to teach the microbiology course in English at the East China University of Science and Technology since 2016. This practice was associated with reform and innovation in the teaching methods and contents. The microbiology course taught in English greatly attracted the interest of the attending students and helped improve their professional English learning as well as scientific research. This course provided important support for fostering innovative professional first-class undergraduates under the context of the "Double First-Class" initiative.

Microbes are involved in every aspect of human life. Microbiology is a mandatory subject at the undergraduate level covering majors including life sciences, pharmacy, medicine, agriculture, forestry and food. Along with internationalization and development of the first-class disciplines, teaching microbiology courses in English is highly valued. Here we discuss how to conduct curriculum reform of microbiology teaching in English, and what are the advantages and challenges when teaching in English. The teaching system can be advanced by enhancing interdisciplinary communication so as to promote study and research for students and teachers. We take this practical exploration as an example to communicate with relevant teachers.

Paenibacillus polymyxa (formerly known as Bacillus polymyxa ) has been extensively studied for agricultural applications as a plant-growth-promoting rhizobacterium and is also an important biocontrol agent. Our team has developed the P. polymyxa strain HY96-2 from the tomato rhizosphere as the first microbial biopesticide based on P. polymyxa for controlling plant diseases around the world, leading to the commercialization of this microbial biopesticide in China. However, further research is essential for understanding its precise biocontrol mechanisms. In this paper, we report the complete genome sequence of HY96-2 and the results of a comparative genomic analysis between different P. polymyxa strains. The complete genome size of HY96-2 was found to be 5.75 Mb and 5207 coding sequences were predicted. HY96-2 was compared with seven other P. polymyxa strains for which complete genome sequences have been published, using phylogenetic tree, pan-genome, and nucleic acid co-linearity analysis. In addition, the genes and gene clusters involved in biofilm formation, antibiotic synthesis, and systemic resistance inducer production were compared between strain HY96-2 and two other strains, namely, SC2 and E681. The results revealed that all three of the P. polymyxa strains have the ability to control plant diseases via the mechanisms of colonization (biofilm formation), antagonism (antibiotic production), and induced resistance (systemic resistance inducer production). However, the variation of the corresponding genes or gene clusters between the three strains may lead to different antimicrobial spectra and biocontrol efficacies. Two possible pathways of biofilm formation in P. polymyxa were reported for the first time after searching the KEGG database. This study provides a scientific basis for the further optimization of the field applications and quality standards of industrial microbial biopesticides based on HY96-2. It may also serve as a reference for studying the differences in antimicrobial spectra and biocontrol capability between different biocontrol agents.

This research was aimed at establishing the pilot-scale purification technology of lipopeptide from marine-derived Bacillus marinus. We studied lipopeptide surfactivity interferences on scale-up unit technologies including acid precipitation, methanol extraction, solvent precipitation, salting out, extraction, silica gel column chromatography and HZ806 macroporous absorption resin column chromatography. Then, the unit technologies were combined in a certain order, to remove the impurities gradually, and to gain purified lipopeptide finally, with high recovery rate throughout the whole process. The novel pilot-scale purification technology could effectively isolate and purify lipopeptide with 87.51% to 100% purity in hectograms from 1 ton of Bacillus marinus B-9987 fermentation broth with more than 81.73% recovery rate. The first practical hectogram production of highly purified lipopeptide derived from Bacillus marinus was achieved. With this new purification method, using complex media became possible in fermentation process to reduce the fermentation cost and scale-up the purification for lipopeptide production. For practicability and economy, foaming problem resulting from massive water evaporation was avoided in this technology.