Structure of the Lysinibacillus sphaericus Tpp49Aa1 pesticidal protein elucidated from natural crystals using MHz-SFX

Tpp49Aa1 from Lysinibacillus sphaericus is a Toxin_10 family protein that must interact with Cry48Aa1, a 3-domain crystal protein, to produce potent mosquitocidal activity, specifically against Culex quinquefasciatus mosquitoes. We use Culex cell lines to demonstrate for the first time transient detrimental effects of individual toxin components and widen the known target range of the proteins. MHz serial femtosecond crystallography at a nano-focused X-ray free electron laser allowed rapid and high-quality data collection to determine the Tpp49Aa1 structure at 2.2 Å resolution from the merged X-ray diffraction data. The structure revealed the packing of Cry49Aa1 within the natural nanocrystals isolated from sporulated bacteria, as a homodimer with a large intermolecular interface. We then modelled the potential interaction between Tpp49Aa1 and Cry48Aa1. The structure sheds light on natural crystallisation and, along with cell-based assays broadens our understanding of this two-component system.

Directional Changes in the Intestinal Bacterial Community in Black Soldier Fly (Hermetia illucens) Larvae

Black soldier fly (BSF) larvae, Hermetia illucens (Diptera: Stratiomyidae) have emerged as an efficient system for the bioconversion of organic waste. Intestinal microorganisms are involved in several insect functions, including the development, nutrition, and physiology of the host. In order to transform the intestinal bacterial community of BSF directionally, six different potential functional strains (Lysinibacillus sphaericus, Proteus mirabilis, Citrobacter freundii, Pseudocitrobacter faecalis, Pseudocitrobacter anthropi, and Enterococcus faecalis) were added to aseptic food waste, and aseptic food waste was used without inoculants as a blank control to evaluate the changes in the intestinal microbiota of BSF under artificial intervention conditions. These six strains (which were isolated from the larval intestinal tract in selective media and then identified and screened) may be considered responsible for the functional characteristics of larvae. The results imply that the increase in the abundance of Lysinibacillus in the experimental group that was exposed to Lysinibacillus sphaericus was significantly different to the other groups (p < 0.05). The results revealed that it is feasible to transform the intestinal microbiota of BSF directionally; there are differences in the proliferation of different strains in the intestine of BSF.

Genome analysis of Lysinibacillus sphaericus isolate 6.2 pathogenic to Culex quinquefasciatus Say, 1823 (Diptera: Culicidae)

Viersanova A, Purwanto H. 2021. Genome analysis of Lysinibacillus sphaericus isolate 6.2 pathogenic to Culex quinquefasciatus Say, 1823 (Diptera: Culicidae). Biodiversitas 22: 5211-5222. Lysinibacillus sphaericus is an entomopathogenic bacteria that is specific to vector mosquitoes, especially Culex spp., and Anopheles spp., so it has been widely used as a bioinsecticide. L. sphaericus has a wide variation of toxicity efficiencies, which have led to continuous exploration of new isolates with higher toxicity and a new toxin to deal with resistance problems. This study aimed to identify the genomic characteristics and toxin characteristics of isolate 6.2 based on whole genome analysis and analyze the identification of isolate 6.2. Isolate 6.2 was previously obtained from rhizosphere in Yogyakarta. To analyze the genome and toxins, the NGS technique was used and then the analysis was carried out using a couple of freely available bioinformatics tools. Molecular identification was carried out with the 16SrRNA gene and the relationship was analyzed by reconstructing the phylogenetic tree using Neighbours-Joining. The genomic analysis of isolate 6.2 showed good results with G+C content and genome size that matched the reference genome of L. sphaericus. The result of the 16SrRNA gene blasting showed that the closest related gene of isolate 6.2 is L. fusiformis (NR_042072.1). However, the reconstructed phylogenetic tree did not show the formation of clusters according to the species. Toxin analysis indicates that isolate 6.2 has Mtx, s-layer protein, hemolysin, and chitin-binding protein genes. All of which are known to be associated with the toxicity of L. sphaericus to binary toxin resistant population of Culex quinquefasciatus.

Bioconversion of lignin and methane production from Corn cobs (Zea mays) treated by lignin-degrading bacteria

Corn cobs are one of the potential feedstocks consisting of cellulose, hemicellulose and lignin, which provide potential lignocellulose biomass to be converted into renewable energy such as biogas through anaerobic digestion (AD). However, the recalcitrant structure of corn cobs lignocellulose makes it resistant to microbial access to the cell wall, and therefore the effective pre-treatment needs to be conducted. The biological pre-treatment using lignin-degrading bacteria is one of the promising bioconversion processes which will help to break down the lignocellulose structure. This study aims to analyse the ability of bacteria, Agrobacterium sp., Lysinibacillus sphaericus and Paenibacillus sp. in degrading lignin of corn cobs and therefore will enhance the methane released from AD. The ability of bacteria to degrade lignin was observed by analysis of total reducing sugar, total soluble phenols, lignin content, and weight loss, while the methane production was determined by the biochemical methane potential (BMP). The percentage of lignin content of untreated and pre-treated corn cobs with bacteria Agrobacterium sp., L. sphaericus and Paenibacillus sp. is 18.34%; 9.66%; 11.48% and 9.06%, respectively. The methane concentration (specific methane production) produced by using inoculum of Agrobacterium sp., L. sphaericus and Paenibacillus sp. with the addition of pre-treated corn cobs are 1.79%; 1.16% and 2.51%, respectively. These results were higher than the inoculum with the addition of untreated corn cobs.

Diversity of midgut bacteria in larvae and females of Aedes aegypti and Aedes albopictus from Gampaha District, Sri Lanka

Background The midgut microbiota of mosquitoes maintain basal immune activity and immune priming. In recent years, scientists have focused on the use of microbial communities for vector control interventions. In the present study, the midgut bacteria of larvae and adults of Aedes aegypti and Ae. albopictus were assessed using both field-collected and laboratory-reared mosquitoes from Sri Lanka. Methods Adults and larvae of Ae. aegypti and Ae. albopictus were collected from three selected areas in Gampaha Medical Officer of Health area, Gampaha District, Western Province, Sri Lanka. Bacterial colonies isolated from mosquito midgut dissections were identified by PCR amplification and sequencing of partial 16S rRNA gene fragments. Results Adults and larvae of Ae. aegypti and Ae. albopictus harbored 25 bacterial species. Bacillus endophyticus and Pantoea dispersa were found more frequently in field-collected Ae. aegypti and Ae. albopictus adults, respectively. The midgut bacteria of Ae. aegypti and Ae. albopictus adults (X2 = 556.167, df = 72, P < 0.001) and larvae (X2 = 633.11, df = 66, P < 0.001) were significantly different. There was a significant difference among the bacterial communities between field-collected adults (X2 = 48.974, df = 10, P < 0.001) and larvae (X2 = 84.981, df = 10, P < 0.001). Lysinibacillus sphaericus was a common species in adults and larvae of laboratory-reared Ae. aegypti. Only P. dispersa occurred in the field-collected adults of Ae. aegypti and Ae. albopictus. Species belonging to genera Terribacillus, Lysinibacillus, Agromyces and Kocuria were recorded from Aedes mosquitoes, in accordance with previously reported results. Conclusions This study generated a comprehensive database on the culturable bacterial community found in the midgut of field-collected (Ae. aegypti and Ae. albopictus) and laboratory-reared (Ae. aegypti) mosquito larvae and adults from Sri Lanka. Data confirm that the midgut bacterial diversity in the studied mosquitoes varies according to species, developmental stage and strain (field vs laboratory). Graphical abstract

Bacterial Toxins Active against Mosquitoes: Mode of Action and Resistance

Larvicides based on the bacteria Bacillus thuringiensis svar. israelensis (Bti) and Lysinibacillus sphaericus are effective and environmentally safe compounds for the control of dipteran insects of medical importance. They produce crystals that display specific and potent insecticidal activity against larvae. Bti crystals are composed of multiple protoxins: three from the three-domain Cry type family, which bind to different cell receptors in the midgut, and one cytolytic (Cyt1Aa) protoxin that can insert itself into the cell membrane and act as surrogate receptor of the Cry toxins. Together, those toxins display a complex mode of action that shows a low risk of resistance selection. L. sphaericus crystals contain one major binary toxin that display an outstanding persistence in field conditions, which is superior to Bti. However, the action of the Bin toxin based on its interaction with a single receptor is vulnerable for resistance selection in insects. In this review we present the most recent data on the mode of action and synergism of these toxins, resistance issues, and examples of their use worldwide. Data reported in recent years improved our understanding of the mechanism of action of these toxins, showed that their combined use can enhance their activity and counteract resistance, and reinforced their relevance for mosquito control programs in the future years.

Production of Lysinibacillus sphaericus Mosquitocidal Protein Mtx2 from Bacillus subtilis as a Secretory Protein

Background: Mtx2 is a mosquitocidal toxin produced during the vegetative growth of Lysinibacillus sphaericus. The protein shows synergism with other toxins against mosquito larvae; hence it could be used in mosquito control formulations. The protein expression system is needed for Mtx2 development as a biocontrol agent. Objective: The objective of the study was to set up a Bacillus subtilis system to produce Mtx2 as a secreted protein since the protein contains a putative signal peptide. Methods: Initially, four different promoters (P43, Pspac, PxylA, and PyxiE) were compared for their strength using GFP as a reporter in B. subtilis. Subsequently, six different signal peptides (SacB, Epr, AmyE, AprE, LipA, and Vip3A)were tested in conjunction with the selected promoter and mtx2 to evaluate levels of Mtx2 secreted by B. subtilis WB800, an extracellular protease-deficient strain. Results: The promoter PyxiE showed the highest GFP intensity and was selected for further study. Mtx2 was successfully produced as a secreted protein from signal peptides LipA and AmyE, and exhibited larvicidal activity against Aedesaegypti. Conclusion: B. subtilis was successfully developed as a host for the production of secreted Mtx2 and the protein retained its larvicidal activity. Although the Mtx2 production level still needs improvement, the constructed plasmids could be used to produce other soluble proteins.