Proteomic Analysis and Promoter Modification of Bacillus thuringiensis to Improve Insecticidal Vip3A Protein Production.

Insecticidal protein Vip3A secreted from B. thuringiensis is a potential biocontrol agent for control of lepidopteran pests. Under laboratory conditions, high albeit variable Vip3A production from the local isolate Bt294 was only obtained from a much enriched TB culture medium. Proteomic analysis and strain improvement were therefore performed to improve Vip3A production. Studies indicated that the buffer capacity, carbon source, and nitrogen source are critical to efficiently produce Vip3A. Medium with lower amounts of peptone and yeast extract (compared to TB), with an additional carbon source and phosphate buffer (LB*G medium) was found to give reasonable yields of Vip3A. Proteomic analysis revealed higher expression of proteins involved in glutamate and histidine biosynthesis in cells cultured in TB compared to LB about 58 and 33 times, respectively. Experiments confirmed that glutamate supplementation could increase Vip3A production. In addition, promoter substitution with that of cry3A increased Vip3A yields by about 20-30%. Overall, very high yields of Vip3A could be obtained by culturing Bt294 (Pcry3A-vip3Aa64) in LB*G medium with glutamate supplementation.

Identification and Functional Characterization of Two Homologous SpoVS Proteins Involved in Sporulation of Bacillus thuringiensis.

Sporulation is an important part of the life cycle of Bacillus thuringiensis and the basis for the production of parasporal crystals. This study identifies and characterizes two homologous spoVS genes (spoVS1 and spoVS2) in B. thuringiensis, both of whose expression is dependent on the σH factor. The disruption of spoVS1 and spoVS2 resulted in defective B. thuringiensis sporulation. Similar to Bacillus subtilis, B. thuringiensis strain HD(ΔspoVS1) mutants showed delayed formation of the polar septa, decreased sporulation efficiency, and blocked spore release. Different from B. subtilis, B. thuringiensis HD(ΔspoVS1) mutants had disporic septa and failed to complete engulfment in some cells. Moreover, HD(ΔspoVS2) mutants had delayed spore release. The effect of spoVS1 deletion on polar septum delay and sporulation efficiency could be compensated by spoVS2. ß-Galactosidase activity analysis showed that the expression of pro-sigE and spoIIE decreased to different degrees in the HD(ΔspoVS1) and HD(ΔspoVS2) mutants. The different effects of the two mutations on the expression of sporulation genes led to decreases in Cry1Ac production of different levels. IMPORTANCE There is only one spoVS gene in B. subtilis, and its effects on sporulation have been reported. In this study, two homologous spoVS genes were found and identified in B. thuringiensis. The different effects on sporulation and parasporal crystal protein production in B. thuringiensis and their relationship were investigated. We found that these two homologous spoVS genes are highly conserved in the Bacillus cereus group, and therefore, the functional characterization of SpoVS is helpful to better understand the sporulation processes of members of the Bacillus cereus group.

vB_BcM_Sam46 and vB_BcM_Sam112, members of a new bacteriophage genus with unusual small terminase structure.

One of the serious public health concerns is food contaminated with pathogens and their vital activity products such as toxins. Bacillus cereus group of bacteria includes well-known pathogenic species such as B. anthracis, B. cereus sensu stricto (ss), B. cytotoxicus and B. thuringiensis. In this report, we describe the Bacillus phages vB_BcM_Sam46 and vB_BcM_Sam112 infecting species of this group. Electron microscopic analyses indicated that phages Sam46 and Sam112 have the myovirus morphotype. The genomes of Sam46 and Sam112 comprise double-stranded DNA of 45,419 bp and 45,037 bp in length, respectively, and have the same GC-content. The genome identity of Sam46 and Sam112 is 96.0%, indicating that they belong to the same phage species. According to the phylogenetic analysis, these phages form a distinct clade and may be members of a new phage genus, for which we propose the name 'Samaravirus'. In addition, an interesting feature of the Sam46 and Sam112 phages is the unusual structure of their small terminase subunit containing N-terminal FtsK_gamma domain.

Dominant egg surface bacteria of Holotrichia oblita (Coleoptera: Scarabaeidae) inhibit the multiplication of Bacillus thuringiensis and Beauveria bassiana.

Holotrichia oblita (Coleoptera: Scarabaeidae) and some other scarab beetles are the main soil-dwelling pests in China. Bacillus thuringiensis (Bt) and Beauveria bassiana (Bb) are entomopathogens that have been used as biocontrol agents of various pests. However, scarab larvae especially H. oblita exhibited strong adaptability to these pathogens. Compared to other scarabs, H. oblita could form a specific soil egg case (SEC) structure surrounding its eggs, and young larvae complete the initial development process inside this structure. In this study, we investigated the role of SEC structure and microorganisms from SEC and egg surface in pathogen adaptability. 16S rRNA gene analysis revealed low bacterial richness and high community unevenness in egg surface, with Proteobacteria, Firmicutes, Bacteroidetes and Fusobacteria dominating. In terms of OTUs composition analysis, the data show that the egg surface contains a large number of unique bacteria, indicating that the egg bacterial community may be derived from maternal transmission. Furthermore, we found that all culturable bacteria isolated from egg surface possessed antimicrobial activity against both Bt and Bb. The Pseudomonas bacteria with a significantly higher abundance in egg surface showed strong Bt- and Bb antagonistic ability. In conclusion, this study demonstrated a unique and antimicrobial bacterial community of H. oblita egg surface, which may contribute to its adaptability. Furthermore, the specific SEC structure surrounding the H. oblita eggs will provide a stable microenvironment for the eggs and egg surface bacteria, which probably provides more advantages for H. oblita adaptation ability.

Compatibility of Bt biopesticides and adjuvants for Spodoptera frugiperda control.

Spodoptera frugiperda is a pest of economic importance for several crops with resistance reports to Bt crops and pesticides. Eco-friendly Bt biopesticides may be an alternative to chemical insecticides due to their selectivity and specificity. However, the efficacy of Bt biopesticides may be influenced by the association with other chemicals, such as adjuvants. This study evaluated the compatibility and toxicity of Bt biopesticides mixed with adjuvants for the control of S. frugiperda. The treatments included the association of Dipel SC and Dipel PM with adjuvants. Compatibility tests were used to evaluate the Bt mixture. Bt suspensions obtained from mixtures of Bt and adjuvants at 106 and 3 × 108 spores/mL-1 were used to evaluate S. frugiperda mortality and distilled water was used as the control. The addition of the adjuvant LI increased growth and sporulation, indicating compatibility with Bt biopesticides. The other adjuvants were toxic to reducing Bt growth and sporulation. Only the mixture of Bt with LI and Bt alone was effective to S. frugiperda. The addition of adjuvants to Bt biopesticide affect the Bt sporulation, growth and mortality.

A selected bacterial strain for the self-healing process in cementitious specimens without cell immobilization steps.

The use of microorganisms capable of mediating the bioprecipitation process can be an important application in the self-healing processes of cement specimens. Thus, the present study identified and evaluated five Bacillus strains for potential application in the protocol of self-healing via bioprecipitation. Cell growth, enzyme production, and kinetic parameters conditions were evaluated during the fermentation process. Based on the analysis of 16S rDNA in conjunction with biochemical testing, results demonstrate that the strains are either Bacillus cereus or Bacillus thuringiensis. Strategically it was found that the addition of glycerol to fermentative medium was essential to increase the bacterial concentration (≈ 4.2 × 107 cells mL-1) and production of the enzyme urease (≈ 3.623,2 U.mL-1). The addition of this medium after 40 days of fermentation promoted the self-healing of cracks and increased compressive strength in ≈ 14.2% of the cementitious specimens; therefore, increasing the sustainability and engineering properties of cement-based materials.

Interpretation of the bacterial growth process based on the analysis of the speckle field generated by calibrated scattering media.

The speckle imaging technique has been proven to be a reliable and effective method for real-time monitoring of the growth kinetics of any bacterium in suspension. To understand the interaction between the light and the bacterial density, a simulation of the bacterial growth of Bacillus thuringiensis was performed using calibrated microspheres of different concentrations and sizes. Results show that the decrease of speckle grain size with the increase of the medium scattering coefficient reveals the two essential phases of the bacterial growth: the exponential phase where the number of the bacteria increases and the stationary phase where sporulation and cell lysis occur.

Respiration, predatory behavior and prey consumption by Podisus nigrispinus (Heteroptera: Pentatomidae) nymphs exposed to some insecticides.

Podisus nigrispinus Dallas (Heteroptera: Pentatomidae) preys on insect pests in eucalyptus plantations where it can be exposed to insecticides used in pest control. The effect of insecticides on non-target natural enemies requires further study. The objective of the present study was to evaluate the side-effects of Bacillus thuringiensis (Bt), permethrin, tebufenozide and thiamethoxam on third instar nymphs of the predator P. nigrispinus in the laboratory. The toxicity of insecticides for this insect was determined by estimating their lethal concentrations. Podisus nigrispinus behavior after exposure to insecticides was analyzed using a video tracking system and the respiratory rate with a respirometer. Prey/nymph consumption was assessed after 24 h of starvation. The preference of P. nigrispinus nymphs, for prey treated or not with the insecticides, was evaluated in free choice tests. The insecticides Bt [LC50 = 1.10(0.83-1.46) mg mL-1], permethrin [LC50 = 0.25(0.17-0.34) mg mL-1], tebufenozide [LC50 = 5.71(4.17-7.57) mg mL-1] and thiamethoxam [LC50 = 0.04(0.02-0.06) mg mL-1] are toxic to P. nigrispinus nymphs. Bt and the insecticides tebufenozide, permethrin and thiamethoxam reduced the respiratory rate of P. nigrispinus. The insecticides permethrin, tebufenozide and thiamethoxam affect the locomotion of this insect's nymphs. Prey treated with Bt, permethrin and thiamethoxam are less preferred by P. nigrispinus. The survival of the nymphs of this predator was 93.3%, 66.7%, 56.6%, 0% and 0% in the control, tebufenozide, Bt, permethrin and thiamethoxam treatments, respectively. In addition, the reduction of prey consumption, treated with neurotoxic insecticides, reduces the predatory potential of this natural enemy. Bt and tefubenozide present low toxicity for P. nigrispinus, but the neurotoxic products have low compatibility with this natural enemy and, therefore, are not recommended, with this predator in the management of forest insect pests.

Temperature-Dependent Growth Characteristics of Bacillus thuringiensis in a Ratatouille Food Model.

ABSTRACT: In contrast to Bacillus cereus, the role of Bacillus thuringiensis in foodborne illness has been controversially discussed. As B. thuringiensis-based biopesticides containing a mixture of crystal toxins and viable spores are widely used, a current European Food Safety Authority opinion underlines the need for additional data to enable risk assessment. However, it is currently poorly understood if B. thuringiensis is able to multiply in food, which is crucial to sound risk assessment. Therefore, the aim of this study was to investigate growth of selected B. thuringiensis strains from food and insecticides in a ratatouille food model. To this end, the growth parameters of three B. thuringiensis strains were determined: insecticide strain ABTS-351 (CH_119, B. thuringiensis serovar kurstaki), insecticide strain ABTS-1857 (CH_121, B. thuringiensis serovar aizawai), and CH_48 (wild-type B. thuringiensis isolated from rosemary), producing extremely high levels of enterotoxins. After an initial drop in colony counts, we observed a statistically significant growth for the tested B. thuringiensis strains between 6 and 24 h at 22, 30, and 37°C, conditions mimicking prolonged holding times. We were also able to show that the enterotoxin overproducer CH_48 can grow up to 108 CFU/g in the ratatouille matrix within 24 h at 37°C. The two midlevel enterotoxin formers ABTS-351 (CH_119) and ABTS-1857 (CH_121) isolated from biopesticides exhibited growth between 6 and 24 h, with one of the strains growing to 107 CFU/g. To our knowledge, this is the first study providing evidence of B. thuringiensis growth in a food model with intact competitive flora. Our findings suggest strain-specific variation and stress the complexity of assessing the risk related to B. thuringiensis in food, indicating that some strains can represent a risk to consumer health when vegetable-based foods are stored under conditions of prolonged temperature abuse.

The signaling peptide PapR is required for the activity of the quorum-sensor PlcRa in Bacillus thuringiensis.

The transcriptional regulator PlcR, its cognate cell-cell signaling heptapeptide PapR7, and the oligopeptide permease OppABCDF, required for PapR7 import, form a quorum-sensing system that controls the expression of virulence factors in Bacillus cereus and Bacillus thuringiensis species. In B. cereus strain ATCC 14579, the transcriptional regulator PlcRa activates the expression of abrB2 gene, which encodes an AbrB-like transcriptional regulator involved in cysteine biosynthesis. PlcRa is a structural homolog of PlcR: in particular, its C-terminal TPR peptide-binding domain could be similarly arranged as in PlcR. The signaling peptide of PlcRa is not known. As PlcRa is a PlcR-like protein, the cognate PapR7 peptide (ADLPFEF) is a relevant candidate to act as a signaling peptide for PlcRa activation. Also, the putative PapRa7 peptide (CSIPYEY), encoded by the papRa gene adjacent to the plcRa gene, is a relevant candidate as addition of synthetic PapRa7 induces a dose-dependent increase of abrB2 expression. To address the issue of peptide selectivity of PlcRa, the role of PapR and PapRa peptides in PlcRa activity was investigated in B. thuringiensis 407 strain, by genetic and functional complementation analyses. A transcriptional fusion between the promoter of abrB2 and lacZ was used to monitor the PlcRa activity in various genetic backgrounds. We demonstrated that PapR was necessary and sufficient for PlcRa activity. We showed that synthetic PapRs from pherogroups II, III and IV and synthetic PapRa7 were able to trigger abrB2 expression, suggesting that PlcRa is less selective than PlcR. Lastly, the mode of binding of PlcRa was addressed using an in silico approach. Overall, we report a new role for PapR as a signaling peptide for PlcRa activity and show a functional link between PlcR and PlcRa regulons in B. thuringiensis.

Real-time monitoring of bacterial growth kinetics in suspensions using laser speckle imaging.

In microbiology, monitoring the growth of any microorganism in culture is important for studying and optimizing the growth kinetics, the biomass and the metabolite production. In this work, we show that laser speckle imaging is a reliable technique that can be used to perform real-time monitoring of bacteria growth kinetic in liquid culture media. Speckle parameters, specifically speckle grain size and the spatial contrast of the speckle images, and standard analytical parameters (optical density, pH and colony forming units) were measured during the culture of different strains of Bacillus thuringiensis. Our results show that both speckle grain size and spatial contrast decrease with bacterial growth. Furthermore, speckle parameters are sensitive to the fermentation conditions. Statistical analysis revealed a relatively high correlation between speckle and analytical parameters.

Rapid Assessment of Water Toxicity by Plasmonic Nanomechanical Sensing.

The ability to rapidly and accurately detect water toxicity is crucial for monitoring water quality and assessing toxic risk, but such detection remains a great challenge. Here, we present a plasmonic nanomechanical sensing (PNMS) system for the rapid assessment of water toxicity. This technique is based on the plasmonic sensing of the nanomechanical movement of single bacterial cells, which could be inhibited upon exposure to potential toxicants. By correlating the amplitude of nanomechanical movement with bacterial activity, we detected a variety of toxic substances in water. The direct readout of bacterial activity via PNMS allowed for a high sensitivity to toxicants in water, thereby enabling us to evaluate the acute toxicological effect of chemical compounds rapidly. The PNMS method is promising for online alerts of water quality safety and for assessing chemical hazards. We anticipate that PNMS is also suitable for a wide range of other applications, including bacterial detection and high-throughput screening of antibacterial materials.

2-Methylcitrate cycle: a well-regulated controller of Bacillus sporulation.

Bacillus thuringiensis is the most widely used eco-friendly biopesticide, containing two primary determinants of biocontrol, endospore and insecticidal crystal proteins (ICPs). The 2-methylcitrate cycle is a widespread carbon metabolic pathway playing a crucial role in channelling propionyl-CoA, but with poorly understood metabolic regulatory mechanisms. Here, we dissect the transcriptional regulation of the 2-methylcitrate cycle operon prpCDB and report its unprecedented role in controlling the sporulation process of B. thuringiensis. We found that the transcriptional activity of the prp operon encoding the three critical enzymes PrpC, PrpD, and PrpB in the 2-methylcitrate cycle was negatively regulated by the two global transcription factors CcpA and AbrB, while positively regulated by the LysR family regulator CcpC, which jointly account for the fact that the 2-methylcitrate cycle is specifically and highly active in the stationary phase of growth. We also found that the prpD mutant accumulated 2-methylcitrate, the intermediate metabolite of the 2-methylcitrate cycle, which delayed and inhibited sporulation at the early stage. Thus, our results not only revealed sophisticated transcriptional regulatory mechanisms for the metabolic 2-methylcitrate cycle but also identified 2-methylcitrate as a novel regulator of sporulation in B. thuringiensis.

Comparative Evaluation of Selective Media for the Detection of Bacillus cereus.

The commercially available 3 types of selective media in Japan were compared for the detection of Bacillus cereus. When assessed inclusivity using 25 B. cereus strains, MYP agar, NGKG agar, and chromogenic X-BC agar demonstrated excellent inclusivity. For exclusivity study using 50 non-B. cereus strains, MYP, NGKG, and X-BC allowed to grow 11, 7, and 3 strains, respectively. Of the grown bacteria on each strains tested, only 2 strains of B. thuringiensis formed typical B. cereus colonies on all selective media tested. The NGKG and X-BC were compared with MYP as a reference using artificially contaminated food (fried rice, plain rice, fried noodle, and potato salad ), since MYP is recommended in ISO 7932: 2004. The both correlation coefficients between NGKG and MYP, and X-BC and MYP were 0.999. Therefore, we demonstrated that NGKG and X-BC can be adapted to ISO 7932: 2004 method for selected food as well as MYP.

Mass production of a S-layer protein of Bacillus thuringiensis and its toxicity to the cattle tick Rhipicephalus microplus.

The most commonly used biopesticides to control agricultural, forest and insect vectors of human diseases are derived from the bacterium Bacillus thuringiensis, which begins to produce Cry and Cyt insecticidal proteins during the onset of the sporulation phase. Some B. thuringiensis strains also produce S-layer proteins that are toxic to certain pests. S-layer proteins are the most abundant proteins in bacteria and archaea. This proteins' key trait to design high performace processes for mass production is their continuous expression during the vegetative phase, unlike Cry and Cyt, which are restricted to the sporulation phase. In this work, a S-layer protein expressed by the GP543 strain of B. thuringiensis that is toxic to the cattle tick Rhipicephalus microplus was mass produced using the batch culture fermentation technique. In addition, the spore-protein complex showed a mortality rate of 75% with a dose of 300 µg·mL-1 on adult females of R. microplus after fourteen days. The lethal concentration 50 was 69.7 µg·mL-1. The treatment also caused a decrease of 13% in the weight of the mass of oviposited eggs with 200 µg·mL-1 of the spore-protein complex and inhibition of the hatching of eggs from 80 to 92%. Therefore, this could be a good option for controlling this parasite. The advantages of S-layer protein synthesis are focused on the production of a new generation of proteins in pest control. This is the first report on the mass production of an S-layer protein that is responsible for toxicity.

Induction of DNA methyltransferase genes in Helicoverpa armigera following injection of pathogenic bacteria modulates expression of antimicrobial peptides and affects bacterial proliferation.

Following pathogen attack in a host, widespread changes are induced in the host's gene expression, in particular those involved in the immune system, growth and survival. Epigenetic mechanisms have been suggested to be involved in the regulation of these changes through a number of mechanisms. DNA methylation is one of the important epigenetic processes that is carried out by DNA (cytosine-5) methyltransferase (DNMT) and alters expression of target genes. Here, we identified two putative sequences of DNMT (i.e. DNMT1 and DNMT2) from the transcriptome dataset of Helicoverpa armigera that showed high similarity to the homologous sequences in Bombyx mori. Domain architectures of DNMT1 and DNMT2 exhibit the unique pattern of DNMTs that highlights conserved function of these genes in different insects. To see if these genes play any role in bacterial infection, we challenged the fifth instar larvae of H. armigera by injecting Bacillus thuringiensis and Serratia marcescens cells into the hemolymph. Transcript levels of the DNMTs were analyzed by RT-qPCR. The results showed that the expression levels of DNMT1 and DNMT2 increased in the bacteria-injected larvae. Injection of the heat-killed bacteria also induced the expression of the DNMTs, but lower than that of the live bacteria. To determine whether these genes function during bacterial infection, we injected the inhibitor of DNMTs, 5-azacytidine (5-AZA), into the larvae and 24 h later, the bacterial cells were also injected into the larvae. Bacterial replication and larval mortality were analyzed in the treated and control insects. We found that 5-AZA reduced bacterial replication and also mortality of the bacterial-injected larvae regardless of the pathogenic bacterial species. Interestingly, the expression levels of antimicrobial peptides (AMPs) were also modulated following 5-AZA treatment. In conclusion, we showed that upregulation of the DNMTs in H. armigera following bacterial infections modulates AMPs and thereby affects the insect-bacteria interactions.

Rapid nondestructive measurement of bacterial cultures with 3D interferometric imaging.

The agar culture plate has played a crucial role in bacteriology since the origins of the discipline and is a staple bioanalytical method for efforts ranging from research to standard clinical diagnostic tests. However, plating, inoculating, and waiting for microbes to develop colonies that are visible is time-consuming. In this work, we demonstrate white-light interferometry (WLI) as a practical tool for accelerated and improved measurement of bacterial cultures. High resolution WLI surface profile imaging was used for nondestructive characterization and counting of bacterial colonies on agar before they became visible to the naked eye. The three-dimensional (3D) morphology of Gram-negative (Pseudomonas fluorescens) and Gram-positive (Bacillus thuringiensis) bacterial species were monitored with WLI over time by collecting surface profiles of colonies on agar plates with high vertical resolution (3-5 nanometers) and large field of view (3-5 mm). This unique combination of sensitive vertical resolution and large field of view uniquely provided by WLI enables measurement of colony morphologies and nondestructive monitoring of hundreds of microcolonies. Individual bacteria were imaged within the first few hours after plating and colonies were accurately counted with results comparing favorably to counts made by traditional methods that require much longer wait times. Nondestructive imaging was used to track single cells multiplying into small colonies and the volume changes over time in these colonies were used to measure their growth rates. Based on the results herein, bioimaging with WLI was demonstrated as a novel rapid bacterial culture assay with several advantageous capabilities. Fast nondestructive counting of colony-forming units in a culture and simultaneous measurement of bacterial growth rates and colony morphology with this method may be beneficial in research and clinical applications where current methods are either too slow or are destructive.

Solid-state fermentation of Bacillus thuringiensis var kurstaki HD-73 maintains higher biomass and spore yields as compared to submerged fermentation using the same media.

To determine the growth kinetic parameters, substrate consumption and spore yields for Bacillus thuringiensis, liquid fermentation (SmF) and solid-state fermentation (SSF), on polyurethane foam (PUF), were analysed comprising strictly the same media. The analysis included three medium concentrations, maintaining the same C/N ratio, with initial glucose at 12.5, 25, and 50 g L-1 (1X, 2X and 4X, respectively). SSF at 2X and 4X produced higher amounts of total biomass, vegetative growth and even early sporulation. Notably, at all glucose concentrations, sporulation was not inhibited in SSF as seen partially in SmF at 2X, and totally at 4X. Micrographs from PUF cultures showed thin layers of bacteria forming large horizontal aggregates, associated with the higher biomass yields and the early cell differentiation. This is the first work showing that SSF improves spore yields of B. thuringiensis in media with high substrate concentrations, using PUF as a research tool for comparative analysis with application in new production systems including biofilm-forming microorganisms.

Effect of the spoIIID mutation on mother cell lysis in Bacillus thuringiensis.

SpoIIID is a small, sequence-specific DNA-binding protein which can direct many genes' transcription and has an effect on spore formation in Bacillus subtilis. We investigated the role of SpoIIID in mother cell lysis in Bacillus thuringiensis. A ß-galactosidase assay based on the promoter fusions with lacZ indicated that the sigK gene was positively regulated by SpoIIID and σK negatively regulated the expression of sigE. The spoIIID mutant strain exhibited no mother cell lysis in Schaeffer's sporulation medium (SSM) but did in ½ Luria-Bertani (LB) medium. cwlC is an essential hydrolase gene for mother cell lysis. Moreover, the expression of a PcwlC-lacZ fusion in spoIIID mutant was proved to be higher in ½ LB medium than in SSM. HD (ΔspoIIID)(ΔcwlC) mutant was obtained by knocking out the cwlC gene in HD(ΔspoIIID) and displayed no mother cell lysis in both SSM and ½ LB mediums. The deletion of spoIIID decreased the crystal protein production in HD73. The expression of Porf1cry8E and P5014 promoter fusions with lacZ gene in the acrystalliferous HD-(ΔspoIIID) mutant showed similar activity to that in the acrystalliferous HD73- strain before T7 and slightly higher than that in the acrystalliferous HD73- after T7. Sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Cry1Ac production in HD-(ΔspoIIID) directed by the Porf1cry8E and P5014 promoters was at a similar level as that in HD73 wild strain. Altogether, these results suggested that the spoIIID mutant with Porf1cry8E or P5014 promoters could be an alternative delivery system for cry gene expression with no mature spore formation and medium-dependent mother cell lysis.

A single point mutation in hmgA leads to melanin accumulation in Bacillus thuringiensis BMB181.

Bacillus thuringiensis BMB181 (Bt BMB181), with high melanin production, is an ideal candidate for industrial scale production of light-stable insecticides. However, its melanogenic pathways remain unclear. In the present study, we demonstrated that Bt BMB181 failed to produce melanin after treatment with mesotrione, an inhibitor of 4-hydroxyphenylpyruvate dioxygenase in the homogentisic acid pathway of melanin synthesis. Heterologous expression experiments suggested that homogentisate-1,2-dioxygenase (HmgA) in Bt BMB171 functions normally, yet HmgA in Bt BMB181 had lost its activity, at least partly. Using the CRISPR-Cas9 system, the hmgA gene in Bt BMB171 was knocked out and the mutant strain gained the ability to produce melanin. Furthermore, the complemented strain reverted to the wild-type phenotype. In addition, overexpression of its own hmgA gene in Bt BMB181 also resulted in failure to produce the pigment. BLAST results indicated that the amino acid alteration (G272E) in HmgA of Bt BMB181 was caused by a single point mutation (815G→ A). The enzyme activity of purified HmgA171 was more than 10-fold higher than that of HmgA181. Finally, we determined that the mutation in hmgA was responsible for melanin accumulation in Bt BMB181. Our results provided new insights into the synthesis and regulation of melanin production in B.thuringiensis and will promote its future industrial application.