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

The Lysinibacillus sphaericus proteins Tpp49Aa1 and Cry48Aa1 can together act as a toxin toward the mosquito Culex quinquefasciatus and have potential use in biocontrol. Given that proteins with sequence homology to the individual proteins can have activity alone against other insect species, the structure of Tpp49Aa1 was solved in order to understand this protein more fully and inform the design of improved biopesticides. Tpp49Aa1 is naturally expressed as a crystalline inclusion within the host bacterium, and MHz serial femtosecond crystallography using the novel nanofocus option at an X-ray free electron laser allowed rapid and high-quality data collection to determine the structure of Tpp49Aa1 at 1.62 Å resolution. This revealed the packing of Tpp49Aa1 within these natural nanocrystals as a homodimer with a large intermolecular interface. Complementary experiments conducted at varied pH also enabled investigation of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals-a crucial step in its mechanism of action. To better understand the cooperation between the two proteins, assays were performed on a range of different mosquito cell lines using both individual proteins and mixtures of the two. Finally, bioassays demonstrated Tpp49Aa1/Cry48Aa1 susceptibility of Anopheles stephensi, Aedes albopictus, and Culex tarsalis larvae-substantially increasing the potential use of this binary toxin in mosquito control.

Ectobacillus ponti sp. nov., a novel bacterium isolated from Pearl River Estuary.

A Gram-staining-positive, aerobic, motile, and rod-shaped strain, designated SYSU M60031T, was isolated from a Pearl River Estuary sediment sample, Guangzhou, Guangdong, China. The isolate could grow at pH 5.0-8.0 (optimum, pH 7.0), 25-37 °C (optimum, 28 °C) and in the presence of 0-1 % (w/v) NaCl (optimum, 0 %). The predominant respiratory menaquinone of SYSU M60031T was MK-7. The cellular polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, one unidentified aminophospholipid, and one unidentified aminolipid. The major fatty acids (>10 % of total) were iso-C14 : 0, iso-C15 : 0, anteiso-C15 : 0, iso-C16 : 0, and C16 : 0. The genomic DNA G+C content was 51.2 %. Phylogenetic analyses based on 16S rRNA gene sequences and core genes indicated that strain SYSU M60031T belonged to the genus Ectobacillus and showed the highest sequence similarity to Ectobacillus funiculus NAF001T (96.16%), followed by Ectobacillus antri SYSU K30001T (95.08 %). Based on the phenotypic, genotypic, and phylogenetic data, strain SYSU M60031T should be considered to represent a novel species of the genus Ectobacillus, for which the name Ectobacillus ponti sp. nov. is proposed. The type strain of the proposed novel isolate is SYSU M60031T (=CGMCC 1.19243T =NBRC 115614T).

Pelgipeptins, a Nonribosomal Lipopeptide Family, Show Larvicidal Activity against Vectors Transmitting Viruses.

Aedes aegypti and Culex quinquefasciatus are vectors of numerous diseases of worldwide public importance, such as arboviruses and filariasis. The main strategy for controlling these vectors is the use of chemicals, which can induce the appearance of resistant insects. The use of Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) with larvicidal activity against arboviral-transmitting insects has been successful in many studies. In contrast, the use and knowledge of peptides with insecticidal activity are so far scarce. In this work, 25 peptides and 5 strains of each bacterial species were prospected individually or together regarding their insecticidal activity. Initially, in vitro assays of cellular cytotoxicity of the peptides against SF21 cells of Spodoptera frugiperda were performed. The peptides Polybia-MPII and pelgipeptin caused 69 and 60% of cell mortality, respectively, at the concentration of 10 µM. Thus, they were evaluated in vivo against second-stage larvae of the two Culicidae. However, in the in vivo bioassays, only pelgipeptin showed larvicidal mortality against both larvae (LC50 6.40 µM against A. aegypti, and LC50 1.22 µM against C. quinquefasciatus). The toxin-producing bacterial strain that showed the lowest LC50 against A. aegypti was Bt S8 (LC50 = 0.71 ng/mL) and against C. quinquefasciatus, it was Ls S260 (LC50 = 2.32 ng/mL). So, the synergistic activity between the association of the bacterial toxins and pelgipeptin was evaluated. A synergic effect of pelgipeptin was observed with Ls strain S260 against C. quinquefasciatus. Our results demonstrate the possibility of synergistic or individual use of both biologically active larvicides against C. quinquefasciatus and A. aegypti.

Study of the Ca2+-dependent gene expression of EuPrt, an extracellular metalloprotease produced by the psychro-tolerant bacterium Exiguobacterium undae Su-1.

The effect of a Ca2+ ion on the gene expression of an on-demand type of metalloprotease from psychrotrophic Exiguobacterium undae Su-1 (EuPrt) was studied. We first established a modified m m9 medium for strain Su-1 to examine its effect in more detail. Then, when the strain was cultured in m m9 medium and 1.0 m m CaCl2 was added, we detected the mature EuPrt and its precursor proteins via Western blotting analysis and found the relative protease activity and its transcription increased by 50-fold and 7-fold, respectively, at the peak. Furthermore, the intracellular concentration of Ca2+ ions was analyzed using inductively coupled plasma atomic emission spectroscopy (ICP-AES) with other metal ions along the growth of strain Su-1. The intracellular concentration of Ca2+ ion was found to increase as much as 3-fold in response to the addition of an extracellular Ca2+ ions, indicating that euPrt gene expression is regulated by sensing its intracellular concentration.

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: This study aimed 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 it exhibited larvicidal activity against Aedes aegypti. 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.

Lysinibacillus agricola sp. nov., isolated from soil.

A gram-staining-positive, rod-shaped bacterium, designed strain FJAT-51161T was isolated from farmland soil collected from Fujian Province, China. Growth was observed at 25-40 °C (optimum 30 °C), pH 7.0-9.0 (optimum 7.0), and NaCl tolerance in the range of 0-7% (w/v), respectively. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that the strain FJAT-51161T belonged to the genus Lysinibacillus, and had the closest relationship with Lysinibacillus xylanilyticus XDB9T (99.0% 16S rRNA sequence similarity). The digital DNA-DNA hybridization (dDDH) and average nucleotide identity (ANI) values based on the genome sequence analysis between strain FJAT-51161T and the closest reference strain were 38.0% for dDDH and 88.7% for ANI, respectively, lower than the prokaryotic species delineation values. Further analysis showed that strain FJAT-51161T shared the fatty acid profiles such as iso-C15:0 (46.7%), iso-C16:0 (15.8%), C16:1 ω7c alcohol (14.0%), anteiso-C15:0 (6.9%) with other members of the genus Lysinibacillus. As the peptidoglycan contained the amino acids alanine, lysine, glycine and aspartic acid, the type A4α was deduced as found in the closest relatives of strain FJAT-51161T. The peptidoglycan of strain FJAT-51161T was L-Lys-D-Asp (type A4α). The main quinone was MK-7 and MK-6. The major polar lipids were diphosphatidylglycerol (DPG) and phosphatidylethanolamine (PE). The DNA G + C content is 36.6 mol%. Based on the phenotypic characters and taxono-genomics study, strain FJAT-51161T is considered to represent a novel Lysinibacillus species, for which the name Lysinibacillus agricola sp. nov. is proposed. The type strain is FJAT-51161T (GDMCC1.2350T = KCTC 43326T).

Statistical optimization of experimental parameters for extracellular synthesis of zinc oxide nanoparticles by a novel haloalaliphilic Alkalibacillus sp.W7.

Green synthesis of zinc oxide nanoparticles (ZnO NPs) through simple, rapid, eco-friendly and an economical method with a new haloalkaliphilic bacterial strain (Alkalibacillus sp. W7) was investigated. Response surface methodology (RSM) based on Box-Behnken design (BP) was used to optimize the process parameters (ZnSO4.7H2O concentration, temperature, and pH) affecting the size of Alkalibacillus-ZnO NPs (Alk-ZnO NPs). The synthesized nanoparticles were characterized using UV-visible spectrum, X-ray diffraction (XRD), Scanning electron microscope-energy dispersive X-ray spectroscopy (SEM-EDX), Transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR) and Zeta potential. The UV-Vis spectrum of ZnO NPs revealed a characteristic surface plasmon resonance (SPR) peak at 310 nm. XRD pattern confirmed the hexagonal wurtzite structure of highly pure with a crystallite size 19.5 nm. TEM proved the quasi-spherical shape nanoparticles of size ranging from 1 to 30 nm. SEM-EDX showed spherical shaped and displayed a maximum elemental distribution of zinc and oxygen. FTIR provided an evidence that the biofunctional groups of metabolites in Alkalibacillus sp.W7 supernatant acted as viable reducing, capping and stabilizing agents.

Characterization of the major antifungal extrolite from rice endophyte Lysinibacillus sphaericus against Rhizoctonia solani.

Sheath blight of rice caused by Rhizoctonia solani is regarded as one of the most widely distributed diseases of rice, and is one of the major production constraints for rice in India and most rice-growing countries of Asia. Biological control of plant diseases using antagonistic bacteria is now considered as a promising alternative to the use of hazardous chemical fungicides or bactericides. Several bacterial endophytes have been reported to support growth and improve the health of the plants and therefore, may be important as biocontrol agents. In the present study, putative antifungal metabolites were extracted from rice foliage endophyte Lysinibacillus sphaericus KJ872548 by solvent extraction methods and purified using HPTLC techniques. Separated bands were subjected to assess the in vitro antagonistic activity toward rice sheath blight pathogen Rhizoctonia solani using a dual culture method. Partially purified active fraction B2 obtained from HPTLC analysis showed the highest percentage of inhibition (76.9%). GC MS and FTIR analyses of B2 revealed the compound as1, 2-Benzenedicarboxylic acid butyl 2-Ethylhexyl ester, a strong antifungal volatile organic compound. Light microscopic analysis of the fungal mycelium from the dual culture plate of both culture filtrate and 1, 2-Benzenedicarboxylic acid butyl 2-Ethylhexyl ester disclosed strong mycolytic activity as evident by mycelial distractions and shrinkage. This is the first report on antifungal production by endophytic Lysinibacillus sphaericus against R. solani, the rice sheath blight pathogen. The findings of this study biologically prospect the endophyte L. sphaericus as an inexpensive broad spectrum bioagent for eco-friendly, economic and sustainable agriculture.

Potential of silver nanoparticles synthesized using low active mosquitocidal Lysinibacillus sphaericus as novel antimicrobial agents.

Silver nanoparticles (AgNPs) were synthesized using extracellular filtrates of some Lysinibacillus sphaericus (Ls) strains under simple conditions. Ls synthesized AgNPs showed the optical absorption peaks at 388-412 nm as detected by UV-visible spectrophotometer. Transmission electron micrographs of bacterial synthesized AgNPs revealed that they were polycrystalline with spherical, hexagonal, cuboidal, rod and irregular shapes. The average diameter of the tested AgNPs were ranged from 14-21 nm and they were negatively charged as detected by DLS (-18.2 to -28.9). FTIR spectra showed the presence of nitrogenous biomolecules capping the synthesized AgNPs. The filtrates of tested Ls strains showed nitrate reductase activity (1.45-2.56 µmol/ml/min). Tested AgNPs showed bactericidal activity against Gram positive and Gram negative bacteria, fungicidal activity against yeast and filamentous fungi, and virucidal activity against rotavirus. In addition, it showed synergistic antimicrobial effect to cephradine and nizoarm against all tested microorganisms. Cytotoxicity test revealed the safety of the tested nanoparticles at tested concentrations.Finally, Ls strains represent microbial sources for ecofriendly, simple and economic biosynthesis of antimicrobial AgNPs. Also, this research may contribute to the medicinal chemistry and pharmaceutical industry for the development of new products used for the public health.

Mesobacillus aurantius sp. nov., isolated from an orange-colored pond near a solar saltern.

An endospore producing, strict aerobic, Gram-stain-positive, orange-colored colony forming bacterium designated as strain JC1013T was isolated from an orange pond near a solar saltern of Tamil Nadu, India. Phylogenetic analysis of the 16S rRNA gene sequences indicated that strain was affiliated to the family Bacillaceae of the phylum Firmicutes. Strain showed highest 16S rRNA gene sequence identity of 98.7% with Mesobacillus selenatarsenatis SF-1 T and below 98.3% with other members of the genus Mesobacillus. Strain JC1013T produced carotenoid pigments and indole compounds. Major cellular fatty acids of strain JC1013T were iso-C15:0, anteiso-C15:0, C16:0 3-OH, iso-C17:0ω10c and summed feature 4 (iso-C17:1 I/ anteisoB). Polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylglycerol, two unidentified aminolipids and four unidentified phospholipids. Strain JC1013T constituted m-diaminopimelic acid as diagnostic cell wall amino acids. MK-7 is the predominant menaquinone of strain JC1013T. The genome size of strain JC1013T was 4.6 Mbp and its G + C content was 42.7 mol%. For the affirmation of strain's taxonomic status, a detailed phylogenomic study was done. Based on the phylogenetic analyses, low ANI (84.6%), AAI (88.5%) values, in-silico DDH (< 29%) value, morphological, physiological and chemo-taxonomical characteristics, strain JC1013T was clearly distinguished from the nearest phylogenetic neighbor, Mesobacillus selenatarsenatis SF-1T to conclude that it is a new species of the genus Mesobacillus. We propose the name as Mesobacillus aurantius with type strain JC1013T (= NBRC 114146T = KACC 21451 T).

Evaluation of biosurfactant production potential of Lysinibacillus fusiformis MK559526 isolated from automobile-mechanic-workshop soil.

BACKGROUND: Biosurfactants are amphipathic biological compounds with surface active potential and are produced by many microorganisms. Biosurfactant production by Lysinibacillus fusiformis MK559526 isolated from automobile-mechanic-shop soil was investigated with a view to assessing its potential for production and potential for optimization. MATERIALS AND METHODS: Effects of carbon and nitrogen sources, pH, temperature and incubation periods on biosurfactant production were evaluated with a view to optimizing the processes. Fourier Transform Infra-Red absorption peaks and Gas chromatography mass spectrometry were used to determine the functional groups of the chemical make-up and the chemical profile of the biosurfactant respectively. RESULTS: Lysinibacillus fusiformis surfactant had emulsification index of 65.15 ± 0.35 %, oil displacement of 2.7 ± 0.26 mm, zone of haemolysis of 7.3 ± 0.16 mm and a positive drop collapse test. Optimized culture conditions for biosurfactant production: temperature, 35 ºC; pH, 7.0; starch solution, 40 g/L and urea, 1.5 g/L showed a reduction in surface tension to 28.46 ± 1.11 mN/m and increased emulsification index to 93.80 ± 0.41 %. Maximum biosurfactant production of 2.92 ± 0.04 g/L was obtained after 72 h. The biosurfactant contained peptides and fatty acids. The predominant fatty acid was 9-Octadecenoic acid (80.80%). CONCLUSIONS: The above results showing high emulsification potential and remarkable reduction in the surface tension are good biosurfactant attributes. Consequently, Lysinibacillus fusiformis MK559526 is a good candidate for biosurfactant production.

Lysinibacillus Isolate MK212927: A Natural Producer of Allylamine Antifungal 'Terbinafine'.

Resistance to antifungal agents represents a major clinical challenge, leading to high morbidity and mortality rates, especially in immunocompromised patients. In this study, we screened soil bacterial isolates for the capability of producing metabolites with antifungal activities via the cross-streak and agar cup-plate methods. One isolate, coded S6, showed observable antifungal activity against Candida (C.) albicans ATCC 10231 and Aspergillus (A.) niger clinical isolate. This strain was identified using a combined approach of phenotypic and molecular techniques as Lysinibacillus sp. MK212927. The purified metabolite displayed fungicidal activity, reserved its activity in a relatively wide range of temperatures (up to 60 °C) and pH values (6-7.8) and was stable in the presence of various enzymes and detergents. As compared to fluconazole, miconazole and Lamisil, the minimum inhibitory concentration of the metabolite that showed 90% inhibition of the growth (MIC90) was equivalent to that of Lamisil, half of miconazole and one fourth of fluconazole. Using different spectroscopic techniques such as FTIR, UV spectroscopy, 1D NMR and 2D NMR techniques, the purified metabolite was identified as terbinafine, an allylamine antifungal agent. It is deemed necessary to note that this is the first report of terbinafine production by Lysinibacillus sp. MK212927, a fast-growing microbial source, with relatively high yield and that is subject to potential optimization for industrial production capabilities.

Cross Resistance in S-Methoprene-Resistant Culex quinquefasciatus (Diptera: Culicidae).

The juvenile hormone analog S-methoprene is the only synthetic biopesticide that is registered with the United States Environmental Protection Agency to control arthropods of economic importance in public health, livestock, pets, urban, and stored products. The high activity, relative target specificity, and benign environmental profile of S-methoprene have been well documented. While the risk of resistance in mosquitoes to S-methoprene is generally low, there is a lack of information regarding cross resistance in S-methoprene-resistant mosquitoes to other pesticides. In this paper, a population of the southern house mosquito Culex quinquefasciatus Say from southern California acquired low levels of resistance to S-methoprene in the field, where the resistance ratios ranged 7.0- to 8.8-fold as compared with a laboratory reference colony. After 30 generations of laboratory selections by S-methoprene when resistance was elevated to 57.4- to 168.3-fold relative to an unselected population, various levels of cross resistance to other commonly used pesticides were revealed in the selected population. Cross resistance to the microbial mosquito larvicide Lysinibacillus sphaericus (Meyer & Neide) (Bacillales: Bacillaceae) was the most profound, amounting to 77.50- to 220.50-fold. The mechanism and potential management tactics toward cross resistance are discussed to preserve the unique value of this synthetic biopesticide.

Genetic and biochemical characterization of thermophilic ß-cyclodextrin glucanotransferase from Gracilibacillus alcaliphilus SK51.001.

BACKGROUND: Gracilibacillus alcaliphilus SK51.001, a strain that produces ß-CGTase (ß-cyclodextrin glucanotransferase) (EC 2.4.1.19), was screened and isolated from Sudanese soil. The objective of this study was to sequence and characterize the ß-CGTase gene from G. alcaliphilus SK51.001. RESULTS: According to 16S rRNA analysis of the strain and its morphological shape, it was identified as G. alcaliphilus. The ß-CGTase gene was successfully cloned, sequenced, and expressed in Escherichia coli BL21. This gene showed 706 amino acid residues including 33 amino acids as a signal peptide. The active site residues of G. alcaliphilus SK51.001CGTase were described using enzyme modeling and docking with the products. The estimated molecular mass of G. alcaliphilus SK51.001CGTase was approximately 74 kDa as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the evaluation of the gel filtration showed approximately 85 kDa, which means G. alcaliphilus SK51.001CGTase is a monomer. The optimum temperature and pH of G. alcaliphilus SK51.001CGTase were 60 °C and 7.0 respectively. Gracilibacillus alcaliphilus SK51.001CGTase was comparatively stable at a pH levels between 6.0 and 9.0 and temperatures of 30-50 °C. The activity of G. alcaliphilus SK51.001CGTase was increased by Ni2+ , and Co2+ but inhibited by Al3+ and Fe3+ . The kinetic parameters of Km and Vmax were 2068.52 µg mL-1 and 0.13 µmol mL-1  min-1 , respectively. CONCLUSION: Gracilibacillus alcaliphilus SK51.001CGTase could hydrolyze soluble starch into α-, ß-, and γ-cyclodextrin in a ratio of 2: 83: 15% respectively. This high ratio production of ß-CD could allow the enzyme to be used in ß-CD production. © 2020 Society of Chemical Industry.

The coordinated action of glucuronoyl esterase and α-glucuronidase promotes the disassembly of lignin-carbohydrate complexes.

Glucuronoxylans represent a significant fraction of woody biomass, and its decomposition is complicated by the presence of lignin-carbohydrate complexes (LCCs). Herein, LCCs from birchwood were used to investigate the potential coordinated action of a glucuronoyl esterase (TtCE15A) and two α-glucuronidases (SdeAgu115A and AxyAgu115A). When supplementing α-glucuronidase with equimolar quantities of TtCE15A, total MeGlcpA released after 72 h by SdeAgu115A and AxyAgu115A increased from 52% to 67%, and 61% to 95%, respectively. Based on the combined TtCE15A and AxyAgu115A activities, ~ 34% of MeGlcpA in the extracted birchwood glucuronoxylan was occupied as LCCs. Notably, insoluble LCC fractions reduced soluble α-glucuronidase concentrations by up to 70%, whereas reduction in soluble TtCE15A was less than 30%, indicating different tendencies to adsorb onto the LCC substrate.

Cannibalism in mosquito larvae during microbial larvicide potency tests.

We observed instances of cannibalism (intraspecific predation) among intra-instar larvae of Culex pipiens Linnaeus, 1758 while performing a bioassay of Lysinibacillus sphaericus (formerly named Bacillus sphaericus) larvicide, when the larvae were exposed to the larvicide for 48 h in the absence of food. Larvae without symptoms of poisoning attacked and devoured those visibly affected. Cannibalism was more prevalent in 1st-2nd instar larvae than in 3rd-4th instar. This phenomenon should be taken into account when interpreting the results of larvicide bioassays, especially when the exposure lasts over 24 h. The necessity of creating optimal conditions for organisms tested is emphasised.

Interaction of curium(III) with surface-layer proteins from Lysinibacillus sphaericus JG-A12.

Trivalent actinides such as Cm(III) are able to occupy natural Ca(II) binding sites in biological systems. For this investigation, we studied the formation of aqueous Cm(III) complexes with S-layer proteins by time-resolved laser-induced fluorescence spectroscopy (TRLFS). S-layer proteins serve as protective biointerfaces in bacteria and archaea against the surrounding solution. Experimental assays were performed at a fixed total concentration of Cm(III) (0.88 µM) using an S-layer protein (5 g/L / 39.6 µM) at varying pH levels (2.0-9.0), as well as several types of S-layer proteins of L. sphaericus JG-A12. Based on resulting luminescence spectra and lifetime data, specific and unspecific binding sites could be distinguished. Notably, specific Cm(III) binding to S-layer proteins was confirmed by the appearance of a sharp emission band at 602.5 nm, combined with a long lifetime of 310 µs. The high affinity of these specific binding sites was also verified using competing EDTA, wherein only a high EDTA concentration (40 µM) could efficiently remove Cm(III) from S-layer proteins.

The Cry48Aa N-terminal Domain is Responsible for Cry48Aa-Cry49Aa Interaction in Lysinibacillus sphaericus Toxin.

The Cry48Aa/Cry49Aa binary toxin from Lysinibacillus sphaericus is composed of a three-domain Cry-like toxin (Cry48Aa) and a binary-like protein (Cry49Aa) that work together to kill Culex quinquefasciatus mosquito larvae through a novel interaction between its two components. The aim of this study was to identify the functional regions of Cry48Aa that were involved in the interaction with Cry49Aa. Eight Cry48Aa truncated fragments were constructed from both N- and C-termini and expressed in Escherichia coli. Only the individual or combined N69K truncated fragment, a Cry48Aa N-terminal derivative consisting of three domains, showed larvicidal activity against C. quinquefasciatus larvae, while the other fragments exhibited significant loss of biological activity. Far-Western dot blot analysis showed that Cry48Aa N-terminal regions had the ability to bind to Cry49Aa protein. These results demonstrate that the N-terminal domain of Cry48Aa plays a crucial role in responsible for the full virulence to mosquito larvae and the interaction with Cry49Aa as a binary toxin.

Functional Bacillus thuringiensis Cyt1Aa Is Necessary To Synergize Lysinibacillus sphaericus Binary Toxin (Bin) against Bin-Resistant and -Refractory Mosquito Species.

The binary (Bin) toxin from Lysinibacillus sphaericus is effective to mosquito larvae, but its utilization is threatened by the development of insect resistance. Bin toxin is composed of the BinB subunit required for binding to midgut receptors and the BinA subunit that causes toxicity after cell internalization, mediated by BinB. Culex quinquefasciatus resistance to this toxin is caused by mutations that prevent expression of Bin toxin receptors in the midgut. Previously, it was shown that the Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis restores Bin toxicity to Bin-resistant C. quinquefasciatus and to Aedes aegypti larvae, which are naturally devoid of functional Bin receptors. Our goal was to elucidate the mechanism involved in Cyt1Aa synergism with Bin in such larvae. In vivo assays showed that the mixture of Bin toxin, or its BinA subunit, with Cyt1Aa was effective to kill resistant larvae. However, no specific binding interaction between Cyt1Aa and the Bin toxin, or its subunits, was observed. The synergy between Cyt1Aa and Bin toxins is dependent on functional Cyt1Aa, as demonstrated by using the nontoxic Cyt1AaV122E mutant toxin affected in oligomerization and membrane insertion, which was unable to synergize Bin toxicity in resistant larvae. The synergism correlated with the internalization of Bin or BinA into anterior and medium midgut epithelial cells, which occurred only in larvae treated with wild-type Cyt1Aa toxin. This toxin is able to overcome failures in the binding step involving BinB receptor by allowing the internalization of Bin toxin, or its BinA subunit, into the midgut cells.IMPORTANCE One promising management strategy for mosquito control is the utilization of a mixture of L. sphaericus and B. thuringiensis subsp. israelensis insecticidal toxins. From this set, Bin and Cyt1Aa toxins synergize and display toxicity to resistant C. quinquefasciatus and to A. aegypti larvae, whose midgut cells lack Bin toxin receptors. Our data set provides evidence that functional Cyt1Aa is essential for internalization of Bin or its BinA subunit into such cells, but binding interaction between Bin and Cyt1Aa is not observed. Thus, this mechanism contrasts with that for the synergy between Cyt1Aa and the B. thuringiensis subsp. israelensis Cry toxins, where active Cyt1Aa is not necessary but a specific binding between Cry and Cyt1Aa is required. Our study established the initial molecular basis of the synergy between Bin and Cyt1Aa, and these findings enlarge our knowledge of their mode of action, which could help to develop improved strategies to cope with insect resistance.

Field Efficacy of VectoMax FG and VectoLex CG Biological Larvicides for Malaria Vector Control in Northwestern Brazil.

Despite historical and contemporary evidence of its effectiveness, larval source management with insecticides remains little used by most malaria control programs worldwide. Here we show that environmentally safe biological larvicides under field conditions can significantly reduce anopheline larval density in fish farming ponds that have became major larval habitats across the Amazon Basin. Importantly, the primary local malaria vector, Anopheles darlingi Root (Diptera: Culicidae), feeds and rests predominantly outdoors, being little affected by interventions such as long-lasting insecticidal bed net distribution and indoor residual spraying. We found >95% reduction in late-instar density up to 7 d after the first application of VectoMax FG or VectoLex CG (both from Valent BioSciences), and up to 21 d after larvicide reapplication in fish ponds (n = 20) situated in the main residual malaria pocket of Brazil, irrespective of the formulation or dosage (10 or 20 kg/ha) used. These results are consistent with a substantial residual effect upon retreatment and support the use of biological larvicides to reduce the density of anopheline larvae in this and similar settings across the Amazon where larval habitats are readily identified and accessible.