Molecular identification and GC-MS analysis of a newly isolated novel bacterium (Lysinibacillus sp. VCRC B655) for mosquito control.

BACKGROUND: Mosquitoes are widespread globally and have contributed to transmitting pathogens to humans and the burden of vector-borne diseases. They are effectively controlled at their larval stages by biocontrol agents. Unravelling natural sources for microbial agents can lead us to novel potential candidates for managing mosquito-borne diseases. In the present study, an attempt was made to isolate a novel bacterium from the field-collected agricultural soil for larvicidal activity and promising bacterial metabolites for human healthcare. METHODS AND RESULTS: Field-collected soil samples from the Union territory of Puducherry, India, have been used as the source of bacteria. Isolate VCRC B655 belonging to the genus Lysinibacillus was identified by 16S rRNA gene sequencing and exhibited promising larvicidal activity against different mosquito species, including Culex (Cx.) quinquefasciatus, Anopheles (An.) stephensi, and Aedes (Ae.) aegypti. The lethal concentration (LC) of Lysinibacillus sp. VCRCB655 was observed to be high for Cx. quiquefasciatus: LC50 at 0.047 mg/l, LC90 at 0.086 mg/l, followed by An. stephensi and Ae. aegypti (LC50: 0.6952 mg/l and 0.795 mg/l) respectively. Additionally, metabolic profiling of the culture supernatant was carried out through Gas chromatography and Mass spectrophotometry (GC/MS) and identified 15 major secondary metabolites of different metabolic classes. Diketopiperazine (DKPs), notably pyro lo [1, 2-a] pyrazine1, 4-dione, are the abundant compounds reported for antioxidant activity, and an insecticide compound benzeneacetic acid was also identified. CONCLUSIONS: A new bacterial isolate, Lysinibacillus sp. VCRC B655 has been identified with significant larvicidal activity against mosquito larvae with no observed in non-target organisms. GC-MS analysis revealed diverse bioactive compounds with substantial biological applications. In conclusion, Lysinibacillus sp. VCRC B655 showed promise as an alternative biocontrol agent for mosquito vector control, with additional biological applications further enhancing its significance.

Fate, inducibility, and behavior of Latilactobacillus curvatus temperate phage TMW 1.591 P1 during sausage fermentation.

AIMS: Temperate phages insert their genome into the host's chromosome. As prophages, they remain latent in the genome until an induction event leads to lytic phage production. When this occurs in a starter culture that has been added to food fermentation, this can impair the fermentation success. This study aimed to analyze prophage inducibility in the Latilactobacillus curvatus TMW 1.591 strain during meat fermentation and investigate whether an induction signal before cryopreservation is maintained during storage and can lead to phage-induced lysis after culture activation. METHODS AND RESULTS: A prophage-free isogenic derivative of the model starter organism, L. curvatus TMW 1.591, was developed as a negative control (L. curvatus TMW 1.2406). Raw meat fermentation was performed with the wild-type (WT) and phage-cured strains. The WT strain produced high numbers of phages (5.2 ± 1.8 × 107 plaque-forming units g-1) in the meat batter. However, the prophage did not significantly affect the meat fermentation process. Induction experiments suggested an acidic environment as a potential trigger for prophage induction. Phage induction by ultraviolet light before strain cryopreservation remains functional for at least 10 weeks of storage. CONCLUSIONS: Intact prophages are active during meat fermentation. However, in this study, this has no measurable consequences for fermentation, suggesting a high resiliency of meat fermentation against phages. Inadequate handling of lysogenic starter strains, even before preservation, can lead to phage introduction into food fermentation and unintended host lysis.

The effect of extracellular polymeric substances on MICP solidifying rare earth slags and stabilizing Th and U.

Microbially induced carbonate precipitation (MICP) has been used to cure rare earth slags (RES) containing radionuclides (e.g. Th and U) and heavy metals with favorable results. However, the role of microbial extracellular polymeric substances (EPS) in MICP curing RES remains unclear. In this study, the EPS of Lysinibacillus sphaericus K-1 was extracted for the experiments of adsorption, inducing calcium carbonate (CaCO3) precipitation and curing of RES. The role of EPS in in MICP curing RES and stabilizing radionuclides and heavy metals was analyzed by evaluating the concentration and morphological distribution of radionuclides and heavy metals, and the compressive strength of the cured body. The results indicate that the adsorption efficiencies of EPS for Th (IV), U (VI), Cu2+, Pb2+, Zn2+, and Cd2+ were 44.83%, 45.83%, 53.7%, 61.3%, 42.1%, and 77.85%, respectively. The addition of EPS solution resulted in the formation of nanoscale spherical particles on the microorganism surface, which could act as an accumulating skeleton to facilitate the formation of CaCO3. After adding 20 mL of EPS solution during the curing process (Treat group), the maximum unconfined compressive strength (UCS) of the cured body reached 1.922 MPa, which was 12.13% higher than the CK group. The contents of exchangeable Th (IV) and U (VI) in the cured bodies of the Treat group decreased by 3.35% and 4.93%, respectively, compared with the CK group. Therefore, EPS enhances the effect of MICP curing RES and reduces the potential environmental problems that may be caused by radionuclides and heavy metals during the long-term sequestration of RES.

Ethanologenic fermentation by Parageobacillus thermoglucosidasius with continuous hot microbubble gas-stripping.

The increased use of biofuels in place of fossil fuels is one strategy to support the transition to net-zero carbon emissions, particularly in transport applications. However, expansion of the use of 1st generation crops as feedstocks is unsustainable due to the conflict with food use. The use of the lignocellulosic fractions from plants and/or co-products from food production including food wastes could satisfy the demand for biofuels without affecting the use of land and the availability of food, but organisms which can readily ferment all the carbohydrates present in these feedstocks often suffer from more severe bioethanol inhibition effects than yeast. This paper demonstrates the potential of hot gas microbubbles to strip ethanol from a thermophilic fermentation process using Parageobacillus thermoglucosidasius TM333, thereby reducing product inhibition and allowing production to continue beyond the nominal toxic ethanol concentrations of ≤ 2% v/v. Using an experimental rig in which cells were grown in fed-batch cultures on sugars derived from waste bread, and the broth continuously cycled through a purpose-built microbubble stripping unit, it was shown that non/low-inhibitory dissolved ethanol concentrations could be maintained throughout, despite reaching productivities equivalent to 4.7% v/v dissolved ethanol. Ethanol recovered in the condensate was at a concentration appropriate for dewatering to be cost effective and not prohibitively energy intensive. This suggests that hot microbubble stripping could be a valuable technology for the continuous production of bioethanol from fermentation processes which suffer from product inhibition before reaching economically viable titres, which is typical of most thermophilic ethanologenic bacteria.

Comparative genomics reveals insights into the potential of Lysinibacillus irui as a plant growth promoter.

Members of the genus Lysinibacillus attract attention for their mosquitocidal, bioremediation, and plant growth-promoting abilities. Despite this interest, comprehensive studies focusing on genomic traits governing plant growth and stress resilience in this genus using whole-genome sequencing are still scarce. Therefore, we sequenced and compared the genomes of three endophytic Lysinibacillus irui strains isolated from Canary Island date palms with the ex-type strain IRB4-01. Overall, the genomes of these strains consist of a circular chromosome with an average size of 4.6 Mb and a GC content of 37.2%. Comparative analysis identified conserved gene clusters within the core genome involved in iron acquisition, phosphate solubilization, indole-3-acetic acid biosynthesis, and volatile compounds. In addition, genome analysis revealed the presence of genes encoding carbohydrate-active enzymes, and proteins that confer resistance to oxidative, osmotic, and salinity stresses. Furthermore, pathways of putative novel bacteriocins were identified in all genomes. This illustrates possible common plant growth-promoting traits shared among all strains of L. irui. Our findings highlight a rich repertoire of genes associated with plant lifestyles, suggesting significant potential for developing inoculants to enhance plant growth and resilience. This study is the first to provide insights into the overall genomic signatures and mechanisms of plant growth promotion and biocontrol in the genus Lysinibacillus. KEY POINTS: • Pioneer study in elucidating plant growth promoting in L. irui through comparative genomics. • Genome mining identified biosynthetic pathways of putative novel bacteriocins. • Future research directions to develop L. irui-based biofertilizers for sustainable agriculture.

Genomic and functional evaluation of exopolysaccharide produced by Liquorilactobacillus mali t6-52: technological implications.

BACKGROUND: This study explores the biosynthesis, characteristics, and functional properties of exopolysaccharide produced by the strain Liquorilactobacillus mali T6-52. The strain demonstrated significant EPS production with a non-ropy phenotype. RESULTS: The genomic analysis unveiled genes associated with EPS biosynthesis, shedding light on the mechanism behind EPS production. These genes suggest a robust EPS production mechanism, providing insights into the strain's adaptability and ecological niche. Chemical composition analysis identified the EPS as a homopolysaccharide primarily composed of glucose, confirming its dextran nature. Furthermore, it demonstrated notable functional properties, including antioxidant activity, fat absorption capacity, and emulsifying activity. Moreover, the EPS displayed promising cryoprotective activities, showing notable performance comparable to standard cryoprotective agents. The EPS concentration also demonstrated significant freeze-drying protective effects, presenting it as a potential alternative cryoprotectant for bacterial storage. CONCLUSIONS: The functional properties of L. mali T6-52 EPS reveal promising opportunities across various industrial domains. The strain's safety profile, antioxidant prowess, and exceptional cryoprotective and freeze-drying characteristics position it as an asset in food processing and pharmaceuticals.

Seed endophytic bacterium Lysinibacillus sp. (ZM1) from maize (Zea mays L.) shapes its root architecture through modulation of auxin biosynthesis and nitrogen metabolism.

Seed endophytic bacteria have been shown to promote the growth and development of numerous plants. However, the underlying mechanism still needs to be better understood. The present study aims to investigate the role of a seed endophytic bacterium Lysinibacillus sp. (ZM1) in promoting plant growth and shaping the root architecture of maize seedlings. The study explores how bacteria-mediated auxin biosynthesis and nitrogen metabolism affect plant growth promotion and shape the root architecture of maize seedlings. The results demonstrate that ZM1 inoculation significantly enhances root length, root biomass, and the number of seminal roots in maize seedlings. Additionally, the treated seedlings exhibit increased shoot biomass and higher levels of photosynthetic pigments. Confocal laser scanning microscopy (CLSM) analysis revealed extensive colonization of ZM1 on root hairs, as well as in the cortical and stellar regions of the root. Furthermore, LC-MS analysis demonstrated elevated auxin content in the roots of the ZM1 treated maize seedlings compared to the uninoculated control. Inoculation with ZM1 significantly increased the levels of endogenous ammonium content, GS, and GOGAT enzyme activities in the roots of treated maize seedlings compared to the control, indicating enhanced nitrogen metabolism. Furthermore, inoculation of bacteria under nitrogen-deficient conditions enhanced plant growth, as evidenced by increased root shoot length, fresh and dry weights, average number of seminal roots, and content of photosynthetic pigments. Transcript analysis indicated upregulation of auxin biosynthetic genes, along with genes involved in nitrogen metabolism at different time points in roots of ZM1-treated maize seedlings. Collectively, our findings highlight the positive impact of Lysinibacillus sp. ZM1 inoculation on maize seeds by improving root architecture through modulation of auxin biosynthesis and affecting various nitrogen metabolism related parameters. These findings provide valuable insights into the potential utilization of seed endophytic bacteria as biofertilizers to enhance plant growth and yield in nutrient deficient soils.

Enhancing spinach (Spinacia oleracea L.) resilience in pesticide-contaminated soil: Role of pesticide-tolerant Ciceribacter azotifigens and Serratia marcescens in root architecture, leaf gas exchange attributes and antioxidant response restoration.

This study unveils the detoxification potential of insecticide-tolerant plant beneficial bacteria (PBB), i.e., Ciceribacter azotifigens SF1 and Serratia marcescens SRB1, in spinach treated with fipronil (FIP), profenofos (PF) and chlorantraniliprole (CLP) insecticides. Increasing insecticide doses (25-400 µg kg-1 soil) significantly curtailed germination attributes and growth of spinach cultivated at both bench-scale and in greenhouse experiments. Profenofos at 400 µg kg-1 exhibited maximum inhibitory effects and reduced germination by 55%; root and shoot length by 78% and 81%, respectively; dry matter accumulation in roots and shoots by 79% and 62%, respectively; leaf number by 87% and leaf area by 56%. Insecticide application caused morphological distortion in root tips/surfaces, increased levels of oxidative stress, and cell death in spinach. Application of insecticide-tolerant SF1 and SRB1 strains relieved insecticide pressure resulting in overall improvement in growth and physiology of spinach grown under insecticide stress. Ciceribacter azotifigens improved germination rate (10%); root biomass (53%); shoot biomass (25%); leaf area (10%); Chl-a (45%), Chl-b (36%) and carotenoid (48%) contents of spinach at 25 µg CLP kg-1 soil. PBB inoculation reinvigorated the stressed spinach and modulated the synthesis of phytochemicals, proline, malondialdehyde (MDA), superoxide anions (O2•-), and hydrogen peroxide (H2O2). Scanning electron microscopy (SEM) revealed recovery in root tip morphology and stomatal openings on abaxial leaf surfaces of PBB-inoculated spinach grown with insecticides. Ciceribacter azotifigens inoculation significantly increased intrinsic water use efficiency, transpiration rate, vapor pressure deficit, intracellular CO2 concentration, photosynthetic rate, and stomatal conductance in spinach exposed to 25 µg FIP kg-1. Also, C. azotifigens and S. marcescens modulated the antioxidant defense systems of insecticide-treated spinach. Bacterial strains were strongly colonized to root surfaces of insecticide-stressed spinach seedlings as revealed under SEM. The identification of insecticide-tolerant PBBs such as C. azotifigens and S. marcescens hold the potential for alleviating abiotic stress to spinach, thereby fostering enhanced and safe production within polluted agroecosystems.

Oceanobacillus picturae alleviates cadmium stress and promotes growth in soybean seedlings.

Cadmium (Cd) is a heavy metal that significantly impacts human health and the environment. Microorganisms play a crucial role in reducing heavy metal stress in plants; however, the mechanisms by which microorganisms enhance plant tolerance to Cd stress and the interplay between plants and microorganisms under such stress remain unclear. In this study, Oceanobacillus picturae (O. picturae) was isolated for interaction with soybean seedlings under Cd stress. Results indicated that Cd treatment alone markedly inhibited soybean seedling growth. Conversely, inoculation with O. picturae significantly improved growth indices such as plant height, root length, and fresh weight, while also promoting recovery in soil physiological indicators and pH. Metabolomic and transcriptomic analyses identified 157 genes related to aspartic acid, cysteine, and flavonoid biosynthesis pathways. Sixty-three microbial species were significantly associated with metabolites in these pathways, including pathogenic, adversity-resistant, and bioconductive bacteria. This research experimentally demonstrates, for the first time, the growth-promoting effect of the O. picturae strain on soybean seedlings under non-stress conditions. It also highlights its role in enhancing root growth and reducing Cd accumulation in the roots under Cd stress. Additionally, through the utilization of untargeted metabolomics, metagenomics, and transcriptomics for a multi-omics analysis, we investigated the impact of O. picturae on the soil microbiome and its correlation with differential gene expression in plants. This innovative approach unveils the molecular mechanisms underlying O. picturae's promotion of root growth and adaptation to Cd stress.

Ureibacillus aquaedulcis sp. nov., isolated from freshwater well and reclassification of Lysinibacillus yapensis and Lysinibacillus antri as Ureibacillus yapensis comb. nov. and Ureibacillus antri comb. Nov.

A Gram-stain-positive aerobic, rod-shaped, spore-producing bacterium forming colonies with convex elevation and a smooth, intact margin was isolated from a freshwater sample collected from a well situated in an agricultural field. The 16S rRNA gene sequence of the isolated strain BA0131T showed the highest sequence similarity to Lysinibacillus yapensis ylb-03T (99.25%) followed by Ureibacillus chungkukjangi 2RL3-2T (98.91%) and U. sinduriensis BLB-1T (98.65%). The strain BA0131T was oxidase and catalase positive and urease negative. It also tested positive for esculin hydrolysis and reduction of potassium nitrate, unlike its phylogenetically closest relatives. The predominant fatty acids in strain BA0131T included were anteiso-C15:0, iso-C16:0, iso-C15:0, iso-C14:0 and the major polar lipids comprised were phosphatidylglycerol, diphosphatidylglycerol and phosphatidylethanolamine. The respiratory quinones identified in strain BA0131T were MK8 (H2) (major) and MK8 (minor). The strain BA0131T shared the lowest dDDH values with L. yapensis ylb-03T (21%) followed by U. chungkukjangi 2RL3-2T (24.2%) and U. sinduriensis BLB-1T (26.4%) suggesting a closer genetic relationship U. sinduriensis BLB-1T. The ANI percentage supported the close relatedness with U. sinduriensis BLB-1T (83.61%) followed by U. chungkukjangi 2RL3-2T (82.03%) and U. yapensis ylb-03T (79.57%). The core genome-based phylogeny constructed using over 13,704 amino acid positions and 92 core genes revealed the distinct phylogenetic position of strain BA0131T among the genus Ureibacillus. The distinct physiological, biochemical characteristics and genotypic relatedness data indicate the strain BA0131T represents a novel species of the genus Ureibacillus for which the name Ureibacillus aquaedulcis sp. nov. (Type strain, BA0131T = MCC 5284 = JCM 36475) is proposed. Additionally, based on extensive genomic and phylogenetic analyses, we propose reclassification of two species, L. yapensis and L. antri, as U. yapensis comb. nov. (Type strain, ylb-03T = JCM 32871T = MCCC 1A12698T) and U. antri (Type strain, SYSU K30002T = CGMCC 1.13504T = KCTC 33955T).

Overcoming the inhibitory effects of urea to improve the kinetics of microbial-induced calcium carbonate precipitation (MICCP) by Lysinibacillus sphaericus strain MB284.

Among different microbial-induced calcium carbonate precipitation (MICCP) mechanisms utilized for biomineralization, ureolysis leads to the greatest yields of calcium carbonate. Unfortunately, it is reported that urea-induced growth inhibition can delay urea hydrolysis but it is not clear how this affects MICCP kinetics. This study investigated the impact of urea addition on the MICCP performance of Lysinibacillus sphaericus MB284 not previously grown on urea (thereafter named bio-agents), compared with those previously cultured in urea-rich media (20 g/L) (hereafter named bio-agents+ or bio-agents-plus). While it was discovered that initial urea concentrations exceeding 3 g/L temporarily hindered cell growth and MICCP reactions for bio-agents, employing bio-agents+ accelerated the initiation of bacterial growth by 33% and led to a 1.46-fold increase in the initial yield of calcium carbonate in media containing 20 g/L of urea. The improved tolerance of bio-agents+ to urea is attributed to the presence of pre-produced endogenous urease, which serves to reduce the initial urea concentration, alleviate growth inhibition, and expedite biomineralization. Notably, elevating the initial concentration of bio-agents+ from OD600 of 0.01 to 1, housing a higher content of endogenous urease, accelerated the initiation of MICCP reactions and boosted the ultimate yield of biomineralization by 2.6 times while the media was supplemented with 20 g/L of urea. These results elucidate the advantages of employing bio-agents+ with higher initial cell concentrations to successfully mitigate the temporary inhibitory effects of urea on biomineralization kinetics, offering a promising strategy for accelerating the production of calcium carbonate for applications like bio self-healing of concrete.

Conversion of metal-enriched magnetite mine tailings into suitable soil for vegetation by phytoremediation process with Bougainvillaea glabra under the influence of Thiobacillus ferroxidance.

Magnetite mining is a significant contributor to land deterioration as well as HM-based soil contamination. The characteristics of magnetite mine tailing were examined in the present study, in addition to the positive and sustainable restoration strategy with Bougainvillaea glabra under the influence of Thiobacillus ferroxidance. The traits of test soil analysis findings demonstrated that the majority of the parameters exceeded the allowable limits (For instance: HMs such as Cr, Cu, Zn, Pb, Fe, and Co were found to be 208 ± 2.3, 131.43 ± 1.6, 185.41 ± 3.3, 312 ± 5.11, 956 ± 5.3, and 26.89 ± 2.43 mg kg-1 respectively). T. ferroxidance exhibited impressive HMs tolerance for as much as 800 g mL-1 concentrations of Cr, Cu, Zn, Pb, Fe, and Co. To prevent HMs toxic effects, the HMs contents in test soil were decreased by diluting with normal soil in the ratios of Ex-3 and Ex-2. A typical greenhouse study was carried out to assess the phytoremediation ability of B. glabra across six setups for experiments (Ex-1 to Ex-6). According to the findings of this research, the HMs tolerant T. ferroxidance from Ex-3 and Ex-2 had an outstanding impact on the growth, biomolecules level (such as chlorophylls: 65.84 & 41.1 mg g-1, proteins: 165.1 & 151.1 mg g-1, as well as carbohydrates: 227.4 & 159.3 mg g-1) as well as phytoremediation potential of B. glabra on magnetite mine soil. These findings indicated that a mixture of B. glabra as well as T. ferroxidance might serve as a valuable sustainable agent for removing HMs from contaminated soil.

Parageobacillus thermoglucosidasius as an emerging thermophilic cell factory.

Parageobacillus thermoglucosidasius is a thermophilic and facultatively anaerobic microbe, which is emerging as one of the most promising thermophilic model organisms for metabolic engineering. The use of thermophilic microorganisms for industrial bioprocesses provides the advantages of increased reaction rates and reduced cooling costs for bioreactors compared to their mesophilic counterparts. Moreover, it enables starch or lignocellulose degradation and fermentation to occur at the same temperature in a Simultaneous Saccharification and Fermentation (SSF) or Consolidated Bioprocessing (CBP) approach. Its natural hemicellulolytic capabilities and its ability to convert CO to metabolic energy make P. thermoglucosidasius a potentially attractive host for bio-based processes. It can effectively degrade hemicellulose due to a number of hydrolytic enzymes, carbohydrate transporters, and regulatory elements coded from a genomic cluster named Hemicellulose Utilization (HUS) locus. The growing availability of effective genetic engineering tools in P. thermoglucosidasius further starts to open up its potential as a versatile thermophilic cell factory. A number of strain engineering examples showcasing the potential of P. thermoglucosidasius as a microbial chassis for the production of bulk and fine chemicals are presented along with current research bottlenecks. Ultimately, this review provides a holistic overview of the distinct metabolic characteristics of P. thermoglucosidasius and discusses research focused on expanding the native metabolic boundaries for the development of industrially relevant strains.

Genomic insights of Fictibacillus terranigra sp. nov., a versatile metabolic bacterium from Amazonian Dark Earths.

The Amazon rainforest, a hotspot for biodiversity, is a crucial research area for scientists seeking novel microorganisms with ecological and biotechnological significance. A key region within the Amazon rainforest is the Amazonian Dark Earths (ADE), noted for supporting diverse plant and microbial communities, and its potential as a blueprint for sustainable agriculture. This study delineates the isolation, morphological traits, carbon source utilization, and genomic features of Fictibacillus terranigra CENA-BCM004, a candidate novel species of the Fictibacillus genus isolated from ADE. The genome of Fictibacillus terranigra was sequenced, resulting in 16 assembled contigs, a total length of 4,967,627 bp, and a GC content of 43.65%. Genome annotation uncovered 3315 predicted genes, encompassing a wide range of genes linked to various metabolic pathways. Phylogenetic analysis indicated that CENA-BCM004 is a putative new species, closely affiliated with other unidentified Fictibacillus species and Bacillus sp. WQ 8-8. Moreover, this strain showcased a multifaceted metabolic profile, revealing its potential for diverse biotechnological applications. It exhibited capabilities to antagonize pathogens, metabolize multiple sugars, mineralize organic matter compounds, and solubilize several minerals. These insights substantially augment our comprehension of microbial diversity in ADE and underscore the potential of Fictibacillus terranigra as a precious resource for biotechnological endeavors. The genomic data generated from this study will serve as a foundational resource for subsequent research and exploration of the biotechnological capabilities of this newly identified species.

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.

Cr(VI) reduction by Agrobacterium sp. Cr-1 and Lysinibacillus sp. Cr-2, novel Cr(VI)-reducing strains isolated from chromium plant soil.

The bioremediation of Cr(VI)-contaminated soil is a promising strategy; however, the performance of Cr(VI)-reducing bacteria is limited by the toxicity of Cr(VI). In this study, two novel Cr(VI)-reducing bacteria were isolated from a Cr salt plant and identified as Agrobacterium sp. and Lysinibacillus sp. The Cr(VI) reduction conditions of the two strains were optimized. At a Cr(VI) concentration of 500 mg/L, Agrobacterium sp. Cr-1 reduced Cr(VI) with a removal rate of 96.91%, while that for Lysinibacillus sp. Cr-2 was 92.82%. First-order reaction kinetic equations simulated the positive relationship between time and Cr(VI) concentration during Cr(VI) reduction in these two strains. Agrobacterium sp. Cr-1 was further studied, and the effects of different cell components on Cr(VI) reduction were detected. The extracellular extracts of Agrobacterium sp. Cr-1 played a major role in Cr(VI) reduction, followed by intracellular extracts and cell membranes. The scanning electron microscope-energy dispersive spectrometer (SEM-EDS) images show that the precipitation was Cr. The high Cr(VI) reducing ability of Agrobacterium sp. Cr-1 suggests that this strain is promising for the remediation of Cr(VI)-contaminated sites.

Structural and Biochemical Insights into Bis(2-hydroxyethyl) Terephthalate Degrading Carboxylesterase Isolated from Psychrotrophic Bacterium Exiguobacterium antarcticum.

This study aimed to elucidate the crystal structure and biochemically characterize the carboxylesterase EaEst2, a thermotolerant biocatalyst derived from Exiguobacterium antarcticum, a psychrotrophic bacterium. Sequence and phylogenetic analyses showed that EaEst2 belongs to the Family XIII group of carboxylesterases. EaEst2 has a broad range of substrate specificities for short-chain p-nitrophenyl (pNP) esters, 1-naphthyl acetate (1-NA), and 1-naphthyl butyrate (1-NB). Its optimal pH is 7.0, losing its enzymatic activity at temperatures above 50 °C. EaEst2 showed degradation activity toward bis(2-hydroxyethyl) terephthalate (BHET), a polyethylene terephthalate degradation intermediate. We determined the crystal structure of EaEst2 at a 1.74 Å resolution in the ligand-free form to investigate BHET degradation at a molecular level. Finally, the biochemical stability and immobilization of a crosslinked enzyme aggregate (CLEA) were assessed to examine its potential for industrial application. Overall, the structural and biochemical characterization of EaEst2 demonstrates its industrial potency as a biocatalyst.

Identification and characterization of a novel bacteriocin gene cluster in Lysinibacillus boronitolerans.

Although the antibiotics inhibit or kill pathogens, the abuse leads to the resistance formation and even "Super Bacteria." Therefore, it is urgent to explore the natural and safe alternatives such as bacteriocin. In this study, an uncharacterized bacteriocin gene cluster for Lysinibacillus boronitolerans was first predicted by genome sequencing and bioinformatics analysis, of which including two biosynthetic genes, a regulatory gene, a transport-related gene, and six other genes. Subsequently, the 10.24-kb gene cluster was expressed in Escherichia coli BL21, and the lysate effectively inhibited the growths of pathogenic bacteria containing Bacillus pumilus, Bacillus velezensis, Pseudomonas syringae pv. tomato DC3000, and Xanthomonas axonopodis pv. manihotis. The antibacterial substance was purified by 70% ammonium sulfate precipitation and further identified by liquid chromatography-tandem mass spectrometry. The results showed that the antibacterial substance consisted of 44 amino acids and had 24.1% sequence identity with the cyanobacterin Piricyclamide 7005 E4 PirE4, a bacteriocin analogue. The minimal set of genes required for the biosynthesis of the antibacterial substance was determined by site-directed mutagenesis, suggesting both a transcriptional repressor and a phosphohydroxythreonine transaminase were essential. Subsequently, the evolution and conservation of the two proteins were analyzed among 22 Lysinibacillus species. Among them, the residues responsible for functions were identified. Collectively, our results set a solid foundation for investigation of the biosynthesis and application of bacteriocin.

Unveiling the genomic potential of a novel thermostable glycoside hydrolases producing Neobacillus sedimentimangrovi UE25.

Genetic and enzymatic potential of Neobacillus sedimentimangrovi has not been assembled to date. Here, we report a high-quality genome assembly of thermophilic bacterium Neobacillus sedimentimangrovi UE25 using Illumina Hi-seq 2500. The strain was isolated from a crocodile pond Manghopir, Karachi, Pakistan. QUAST quality parameters showed 37.75% GC content and exhibited the genome into 110 contigs, with a total size of 3,230,777 bases. Genome of N. sedimentimangrovi UE25 harbors phage mediated DNA through horizontal gene exchange from the phages, symbiotic and pathogenic bacteria. Most of the phage genome encodes for hypothetical proteins, protease, and phage assembly proteins. Gene clusters encoding the intrinsic resistance to glycopeptides, isoniazid, rifamycin, elfamycin, macrolide, aminoglycosides, tetracycline and fluoroquinolone were identified into the genome. Since, the strain has been reported for the production of many industrially important thermostable enzymes, therefore, the genomic data of thermostable enzymes might be helpful to employ this species in commercial sectors. Probing genes of multiple thermostable glycoside hydrolase enzymes especially xylanases of N. sedimentimangrovi UE25 showed genetic diversity among the genes and confer the industrial importance of this microorganism. Furthermore, the genome of N. sedimentimangrovi will greatly improve our understanding of its genetics and evolution.

Lysinibacillus spp.: an IAA-producing endospore forming-bacteria that promotes plant growth.

Lysinibacillus is a bacterial genus that has generated recent interest for its biotechnological potential in agriculture. Strains belonging to this group are recognized for their mosquitocidal and bioremediation activity. However, in recent years some reports indicate its importance as plant growth promoting rhizobacteria (PGPR). This research sought to provide evidence of the PGP activity of Lysinibacillus spp. and the role of the indole-3-acetic acid (IAA) production associated with this activity. Twelve Lysinibacillus spp. strains were evaluated under greenhouse conditions, six of which increased the biomass and root architecture of corn plants. In most cases, growth stimulation was evident at 108 CFU/mL inoculum concentration. All strains produced IAA with high variation between them (20-70 µg/mL). The bioinformatic identification of predicted genes associated with IAA production allowed the detection of the indole pyruvic acid pathway to synthesize IAA in all strains; additionally, genes for a tryptamine pathway were detected in two strains. Extracellular filtrates from all strain's cultures increased the corn coleoptile length in an IAA-similar concentration pattern, which demonstrates the filtrates had an auxin-like effect on plant tissue. Five of the six strains that previously showed PGPR activity in corn also promoted the growth of Arabidopsis thaliana (col 0). These strains induced changes in root architecture of Arabidopsis mutant plants (aux1-7/axr4-2), the partial reversion of mutant phenotype indicated the role of IAA on plant growth. This work provided solid evidence of the association of Lysinibacillus spp. IAA production with their PGP activity, which constitutes a new approach for this genus. These elements contribute to the biotechnological exploration of this bacterial genus for agricultural biotechnology.