A dye elution method for the quantification of insecticidal crystal proteins from Bacillus thuringiensis and its compatibility with the presence of agro-industrial raw materials and waste products.

The insecticidal crystal proteins produced by Bacillus thuringiensis during sporulation are active ingredients against lepidopteran, dipteran, and coleopteran insects. Several methods have been reported for their quantification, such as crystal counting, ELISA, and SDS-PAGE/densitometry. One of the major tasks in industrial processes is the analysis of raw material dependency and costs. Thus, the crystal protein quantification method is expected to be compatible with the presence of complex and inexpensive culture medium components. This work presents a revalidated elution-based method for the quantification of insecticidal crystal proteins produced by the native strain B. thuringiensis RT. To quantify proteins, a calibration curve was generated by varying the amount of BSA loaded into SDS-PAGE gels. First, SDS-PAGE was performed for quality control of the bioinsecticide. Then, the stained protein band was excised from 10% polyacrylamide gel and the protein-associated dye was eluted with an alcoholic solution of SDS (3% SDS in 50% isopropanol) during 45 min at 95°C. This protocol was a sensitive procedure to quantify proteins in the range of 2.0-10.0 µg. As proof of concept, proteins of samples obtained from a complex fermented broth were separated by SDS-PAGE. Then, Cry1 and Cry2 proteins were properly quantified.

Bacillus thuringiensis-Based Bioproduct: Characterization and Performance Against Spodoptera frugiperda Strains in Maize Under Different Environmental Temperatures.

Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) is an important pest in several regions being the use of Bacillus thuringiensis-based bioproducts an alternative for its control. Firstly, 3 L of an aqueous bioproduct suspension was produced and characterized. Its 50% lethal concentration against molecularly identified corn and rice S. frugiperda strains using an artificial diet were 77.01% (95% CL, 68.16-90.47) and 2.22% (95% CL, 0.01-6.68), respectively. The next objective of this work was to evaluate the performance of this bioproduct in maize against S. frugiperda strains under different simulated agrological regions mimicking their corresponding periodic day/night temperatures. Thus, the impact of environmental temperature on the bioproduct efficacy (E) was studied. It was observed that a warmer scenario (35 °C day/30 °C night) could favor the tolerance of corn S. frugiperda strain to the bioproduct (E = 56.36 ± 0.61%) maintaining a high efficacy (92.44 ± 6.55%) when it was tested against rice S. frugiperda strain. Conversely, under temperate conditions, efficacy values ranged from 84 to 95% for both S. frugiperda strains. On the other hand, based on a foliar feeding damage analysis, our bioproduct displayed a significant foliar protection in maize plants infested with either corn or rice S. frugiperda strains.

Industrial biotransformations catalyzed by microbial lipases: screening platform and commercial aspects.

The successfulness of a lipase-catalyzed industrial process depends on a proper lipase selection. In this work, an alternative screening platform for industrially important biotransformations catalyzed by microbial lipases was proposed. Thus, the reactivity of sixty lipase activities from spore-forming microorganisms towards hydrolytic and transesterification reactions by using p-nitrophenyl palmitate as a chromogenic acyl donor substrate was explored. Only three biocatalysts were capable of catalyzing all reactions tested. Fourteen biocatalysts did not show hydrolytic activity at all; however, they displayed transesterification activities using ethanol, starch, low-methoxyl (LM) pectin, high-methoxyl (HM) pectin, or vitamin C as acyl acceptors. Using heat-treated biocatalysts, hydrolytic activities were not highly correlated with the corresponding transesterification activities using ethanol (r = -0.058, p = 0.660), starch (r = 0.431, p = 0.001), LM pectin (r = -0.010, p = 0.938), HM pectin (r = 0.167, p = 0.202), and vitamin C (r = -0.048, p = 0.716) as acyl acceptor. In addition, to the best of our knowledge, several transesterification activities produced from microorganisms of the genus Bacillus, Brevibacillus, Lysinibacillus, Geobacillus, or Sporosarcina were reported for first time. Finally, the global lipase market was presented and segmented by date, application, geography and player highlighting the commercial contribution of microbial lipases.

Control of microbial biofilm formation as an approach for biomaterials synthesis.

The search for new biomaterials with superior mechanical properties is the focus in the area of materials science. A promising pathway is drawing inspiration from nature to design and develop materials with enhanced properties. In this work, a novel strategy to produce functionalized supramolecular bionanomaterials from the microbial biofilm is reported. Tuneable biofilms with specific characteristics were obtained by controlling the culture condition of the microorganism. When the exopolysaccharide (EPS) production was desired the tryptone was the best nutritional component for the EPS production into the biofilm. However, for the expression of a high amount of amyloid protein the combination of peptone and glucose was the best nutritional choice. Each biofilm obtained showed its owner rheology properties. These properties were altered by the addition of extracellular DNA, which increased the viscosity of the biofilm and induced a viscoelastic hydrogel behavior. Besides, as a proof of concept of bionanomaterial, a novel supramolecular polymeric hybrid EPS-Amyloid protein (EPAP) was obtained from the biofilm and it was tested as a new natural functionalized support for enzyme immobilization. The results suggest that this technology could be used as a new concept to obtain biomaterials from biofilms by controlling the nutritional conditions of a microorganism. Understanding environmental factors affecting biofilm formation will help the development of methods for controlling biofilm production and therefore obtaining new biomaterials.

Insecticidal crystal proteins from native Bacillus thuringiensis: numerical analysis and biological activity against Spodoptera frugiperda.

Fourteen strains of Bacillus thuringiensis collected from both larvae showing disease symptoms and soil samples in northwest Argentina were characterized by insecticidal activity against Spodoptera frugiperda. First instar larvae and protein profile SDS-PAGE analysis of whole cell proteins not only allowed the differentiation of native Bacillus thuringiensis but also revealed the possibility of applying protein profile analysis in classification of toxicity patterns. Cluster analysis showed that there were two main groups. Interestingly, one of them only contained the most pathogenic native strains. The biomass-bound protease activity of native pathogenic isolates and the reference strain Bt 4D1 is also reported.

Functional characterization of LotP from Liberibacter asiaticus.

Liberibacter asiaticus is an unculturable parasitic bacterium of the alphaproteobacteria group hosted by both citrus plants and a psyllid insect vector (Diaphorina citri). In the citrus tree, the bacteria thrive only inside the phloem, causing a systemically incurable and deadly plant disease named citrus greening or Huanglongbing. Currently, all commercial citrus cultivars in production are susceptible to L. asiaticus, representing a serious threat to the citrus industry worldwide. The technical inability to isolate and culture L. asiaticus has hindered progress in understanding the biology of this bacterium directly. Consequently, a deep understanding of the biological pathways involved in the regulation of host-pathogen interactions becomes critical to rationally design future and necessary strategies of control. In this work, we used surrogate strains to evaluate the biochemical characteristics and biological significance of CLIBASIA_03135. This gene, highly induced during early stages of plant infection, encodes a 23 kDa protein and was renamed in this work as LotP. This protein belongs to an uncharacterized family of proteins with an overall structure resembling the LON protease N-terminus. Co-immunoprecipitation assays allowed us to identify the Liberibacter chaperonin GroEL as the main LotP-interacting protein. The specific interaction between LotP and GroEL was reconstructed and confirmed using a two-hybrid system in Escherichia coli. Furthermore, it was demonstrated that LotP has a native molecular weight of 44 kDa, corresponding to a dimer in solution with ATPase activity in vitro. In Liberibacter crescens, LotP is strongly induced in response to conditions with high osmolarity but repressed at high temperatures. Electrophoretic mobility shift assay (EMSA) results suggest that LotP is a member of the LdtR regulon and could play an important role in tolerance to osmotic stress.

The Escherichia coli yjfP Gene Encodes a Carboxylesterase Involved in Sugar Utilization during Diauxie.

BACKGROUND: Acetylation and efflux of carbohydrates during cellular metabolism is a well-described phenomenon associated with a detoxification process to prevent metabolic congestion. It is still unclear why cells discard important metabolizable energy sources in the form of acetylated compounds. METHODS: We describe the purification and characterization of an approximately 28-kDa intracellular carboxylesterase (YjfP) and the analysis of gene and protein expression by qRT-PCR and Western blot. RESULTS: qRT-PCR and Western blot, respectively, showed that yjfP is upregulated during the diauxic lag in cells growing with a mixture of glucose and lactose. The ß-galactosidase activity in the ΔyjfP strain was both delayed and half the magnitude of that of the wild-type strain. YjfP-hyperproducing strains displayed a long lag phase when cultured with glucose and then challenged to grow with lactose or galactose as the sole carbon source. CONCLUSION: Our results suggest that YjfP controls the intracellular concentration of acetyl sugars by redirecting them to the main metabolic circuits. Instead of detoxification, we propose that sugar acetylation is utilized by the cell for protection and to prevent the metabolism of a necessary minimal intracellular sugar pool. Those sugars can eventually be exported as a side effect of these mechanisms.