Visualizing group II intron catalysis through the stages of splicing.

Group II introns are self-splicing ribozymes that share a reaction mechanism and a common ancestor with the eukaryotic spliceosome, thereby providing a model system for understanding the chemistry of pre-mRNA splicing. Here we report 14 crystal structures of a group II intron at different stages of catalysis. We provide a detailed mechanism for the first step of splicing, we describe a reversible conformational change between the first and the second steps of splicing, and we present the ligand-free intron structure after splicing in an active state that corresponds to the retrotransposable form of the intron. During each reaction, the reactants are aligned and activated by a heteronuclear four-metal-ion center that contains a metal cluster and obligate monovalent cations, and they adopt a structural arrangement similar to that of protein endonucleases. Based on our data, we propose a model for the splicing cycle and show that it is applicable to the eukaryotic spliceosome.

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.

Application of mutational profiling: New functional analyses reveal the tRNA recognition mechanism of tRNA m1A22 methyltransferase.

Transfer RNAs undergo diverse posttranscriptional modifications to regulate a myriad of cellular events including translation, stress response, and viral replication. These posttranscriptional modifications are synthesized by site-specific modification enzymes. Recent RNA-seq techniques have revealed multiple features of tRNA such as tRNA abundance, tRNA modification, and tRNA structure. Here, we adapt a tRNA-sequencing technique and design a new functional analysis where we perform mutational profiling of tRNA modifications to gain mechanistic insights into how tRNA modification enzymes recognize substrate tRNA. Profiling of Geobacillus stearothermophilus tRNAs and protein orthology analysis predict the existence of natural modifications in 44 tRNA molecular species of G. stearothermophilus. We selected the 1-methyladenosine modification at position 22 (m1A22) and tRNA (m1A22) methyltransferase (TrmK) for further analysis. Relative quantification of m1A22 levels in 59 tRNA transcripts by mutational profiling reveals that TrmK selectively methylates a subset of tRNAs. Using 240 variants of tRNALeu transcripts, we demonstrate the conserved nucleosides including U8, A14, G15, G18, G19, U55, Purine57, and A58 are important for the methyl transfer reaction of TrmK. Additional biochemical experiments reveal that TrmK strictly recognizes U8, A14, G18, and U55 in tRNA. Furthermore, these findings from tRNALeu variants were crossvalidated using variants of three different tRNA species. Finally, a model of the TrmK-tRNA complex structure was constructed based on our findings and previous biochemical and structural studies by others. Collectively, our study expands functional analyses of tRNA modification enzyme in a high-throughput manner where our assay rapidly identifies substrates from a large pool of tRNAs.

Elucidating the biotechnological potential of the genera Parageobacillus and Saccharococcus through comparative genomic and pan-genome analysis.

BACKGROUND: The genus Geobacillus and its associated taxa have been the focal point of numerous thermophilic biotechnological investigations, both at the whole cell and enzyme level. By contrast, comparatively little research has been done on its recently delineated sister genus, Parageobacillus. Here we performed pan-genomic analyses on a subset of publicly available Parageobacillus and Saccharococcus genomes to elucidate their biotechnological potential. RESULTS: Phylogenomic analysis delineated the compared taxa into two distinct genera, Parageobacillus and Saccharococcus, with P. caldoxylosilyticus isolates clustering with S. thermophilus in the latter genus. Both genera present open pan-genomes, with the species P. toebii being characterized with the highest novel gene accrual. Diversification of the two genera is driven through the variable presence of plasmids, bacteriophages and transposable elements. Both genera present a range of potentially biotechnologically relevant features, including a source of novel antimicrobials, thermostable enzymes including DNA-active enzymes, carbohydrate active enzymes, proteases, lipases and carboxylesterases. Furthermore, they present a number of metabolic pathways pertinent to degradation of complex hydrocarbons and xenobiotics and for green energy production. CONCLUSIONS: Comparative genomic analyses of Parageobacillus and Saccharococcus suggest that taxa in both of these genera can serve as a rich source of biotechnologically and industrially relevant secondary metabolites, thermostable enzymes and metabolic pathways that warrant further investigation.

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.

Exploration of culturable bacterial associates of aphids and their interactions with entomopathogens.

Aphids shelter several bacteria that benefit them in various ways. The associates having an obligatory relationship are non-culturable, while a few of facultative associates are culturable in insect cell lines, axenic media or standard microbiology media. In the present investigation, isolation, and characterization of the culturable bacterial associates of various aphid species, viz., Rhopalosiphum maidis, Rhopalosiphum padi, Sitobion avenae, Schizaphis graminum, and Lipaphis erysimi pseudobrassicae were carried out. A total of 42 isolates were isolated using different growth media, followed by their morphological, biochemical, and molecular characterization. The isolated culturable bacterial associates were found to belong to the genera Acinetobacter, Bacillus, Brevundimonas, Cytobacillus, Fictibacillus, Planococcus, Priestia, Pseudomonas, Staphylococcus, Sutcliffiella, and Tumebacillus which were grouped under seven families of four different orders of phyla Bacillota (Firmicutes) and Pseudomonata (Proteobacteria). Symbiont-entomopathogen interaction study was also conducted, in which the quantification of colony forming units of culturable bacterial associates of entomopathogenic fungal-treated aphids led us to the assumption that the bacterial load in aphid body can be altered by the application of entomopathogens. Whereas, the mycelial growth of entomopathogens Akanthomyces lecanii and Metarhizium anisopliae was found uninhibited by the bacterial associates obtained from Sitobion avenae and Rhopalosiphum padi. Analyzing persistent aphid microflora and their interactions with entomopathogens enhances our understanding of aphid resistance. It also fosters the development of innovative solutions for agricultural pest management, highlighting the intricate dynamics of symbiotic relationships in pest management strategies.

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).

Characterization of individual spores of two biological insecticides, Bacillus thuringiensis and Lysinibacillus sphaericus, in response to glutaraldehyde using single-cell optical approaches.

Bacillus thuringiensis (Bt) and Lysinibacillus sphaericus (Ls) are the most widely used microbial insecticides. Both encounter unfavorable environmental factors and pesticides in the field. Here, the responses of Bt and Ls spores to glutaraldehyde were characterized using Raman spectroscopy and differential interference contrast imaging at the single-cell level. Bt spores were more sensitive to glutaraldehyde than Ls spores under prolonged exposure: <1.0% of Bt spores were viable after 10 min of 0.5% (v/v) glutaraldehyde treatment, compared to ~ 20% of Ls spores. The Raman spectra of glutaraldehyde-treated Bt and Ls spores were almost identical to those of untreated spores; however, the germination process of individual spores was significantly altered. The time to onset of germination, the period of rapid Ca2+-2,6-pyridinedicarboxylic acid (CaDPA) release, and the period of cortex hydrolysis of treated Bt spores were significantly longer than those of untreated spores, with dodecylamine germination being particularly affected. Similarly, the germination of treated Ls spores was significantly prolonged, although the prolongation was less than that of Bt spores. Although the interiors of Bt and Ls spores were undamaged and CaDPA did not leak, proteins and structures involved in spore germination could be severely damaged, resulting in slower and significantly prolonged germination. This study provides insights into the impact of glutaraldehyde on bacterial spores at the single cell level and the variability in spore response to glutaraldehyde across species and populations.

A novel type IIb L-asparaginase from Latilactobacillus sakei LK-145: characterization and application.

We succeeded in homogeneously expressing and purifying L-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using L-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called "Type IIb" novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.

Tepidibacillus marianensis sp. nov., a novel heterotrophic iron-reducing bacterium isolated from Mariana Trench sediment.

A novel chemoheterotrophic iron-reducing micro-organism, designated as strain LSZ-M11000T, was isolated from sediment of the Marianas Trench. Phylogenetic analysis based on the 16S rRNA gene revealed that strain LSZ-M11000T belonged to genus Tepidibacillus, with 97 % identity to that of Tepidibacillus fermentans STGHT, a mesophilic bacterium isolated from the Severo-Stavropolskoye underground gas storage facility in Russia. The polar lipid profile of strain LSZ-M11000T consisted of diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, as well as other unidentified phospholipids and lipids. The major fatty acids were C16 : 0 (28.4 %), C18 : 0 (15.8 %), iso-C15 : 0 (12.9 %), and anteiso-C15 : 0 (12.0 %). Strain LSZ-M11000T had no menaquinone. Genome sequencing revealed that the genome size of strain LSZ-M11000T was 2.97 Mb and the DNA G+C content was 37.9 mol%. The average nucleotide identity values between strain LSZ-M11000T and its close phylogenetic relatives, Tepidibacillus fermentans STGHT and Tepidibacillus decaturensis Z9T, were 76.4 and 72.6 %, respectively. The corresponding DNA-DNA hybridization estimates were 20.9 and 23.4 %, respectively. Cells of strain LSZ-M11000T were rod-shaped (1.0-1.5×0.3-0.5 µm). Using pyruvate as an electron donor, it was capable of reducing KMnO4, MnO2, As(V), NaNO3, NaNO2, Na2SO4, Na2S2O3, and K2Cr2O7. Based on phenotypic, genotypic, and phylogenetic evidence, strain LSZ-M11000T is proposed to be a novel strain of the genus Tepidibacillus, for which the name Tepdibacillus marianensis is proposed. The type strain is LSZ-M11000T (=CCAM 1008T=JCM 39431T).

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.

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.

Harnessing the power of Neobacillus niacini AUMC-B524 for silver oxide nanoparticle synthesis: optimization, characterization, and bioactivity exploration.

BACKGROUND: Biotechnology provides a cost-effective way to produce nanomaterials such as silver oxide nanoparticles (Ag2ONPs), which have emerged as versatile entities with diverse applications. This study investigated the ability of endophytic bacteria to biosynthesize Ag2ONPs. RESULTS: A novel endophytic bacterial strain, Neobacillus niacini AUMC-B524, was isolated from Lycium shawii Roem. & Schult leaves and used to synthesize Ag2ONPS extracellularly. Plackett-Burman design and response surface approach was carried out to optimize the biosynthesis of Ag2ONPs (Bio-Ag2ONPs). Comprehensive characterization techniques, including UV-vis spectral analysis, Fourier transform infrared spectroscopy, transmission electron microscopy, X-ray diffraction, dynamic light scattering analysis, Raman microscopy, and energy dispersive X-ray analysis, confirmed the precise composition of the Ag2ONPS. Bio-Ag2ONPs were effective against multidrug-resistant wound pathogens, with minimum inhibitory concentrations (1-25 µg mL-1). Notably, Bio-Ag2ONPs demonstrated no cytotoxic effects on human skin fibroblasts (HSF) in vitro, while effectively suppressing the proliferation of human epidermoid skin carcinoma (A-431) cells, inducing apoptosis and modulating the key apoptotic genes including Bcl-2 associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), Caspase-3 (Cas-3), and guardian of the genome (P53). CONCLUSIONS: These findings highlight the therapeutic potential of Bio-Ag2ONPs synthesized by endophytic N. niacini AUMC-B524, underscoring their antibacterial efficacy, anticancer activity, and biocompatibility, paving the way for novel therapeutic strategies.

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.

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.

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.

Lysinibacillus pinottii sp. nov., a novel species with anti-mosquito and anti-mollusk activity.

An isolate of a Gram-positive, strictly aerobic, motile, rod-shaped, endospore forming bacterium was originally isolated from soil when screening and bioprospecting for plant beneficial microorganisms. Phylogenetic analysis of the 16S rRNA gene sequences indicated that this strain was closely related to Lysinibacillus fusiformis NRRL NRS-350T (99.7%) and Lysinibacillus sphaericus NRRL B-23268T (99.2%). In phenotypic characterization, the novel strain was found to grow between 10 and 45 °C and tolerate up to 8% (w/v) NaCl. Furthermore, the strain grew in media with pH 5 to 10 (optimal growth at pH 7.0). The predominant cellular fatty acids were observed to be iso-C15: 0 (52.3%), anteiso-C15: 0 (14.8%), C16:1ω7C alcohol (11.2%), and C16: 0 (9.5%). The cell-wall peptidoglycan contained lysine-aspartic acid, the same as congeners. A draft genome was assembled and the DNA G+C content was determined to be 37.1% (mol content). A phylogenomic analysis on the core genome of the new strain and 5 closest type strains of Lysinibacillus revealed this strain formed a distinct monophyletic clade with the nearest neighbor being Lysinibacillus fusiformis. DNA-DNA relatedness studies using in silico DNA-DNA hybridizations (DDH) showed this species was below the species threshold of 70%. Based upon the consensus of phylogenetic and phenotypic analyses, we conclude that this strain represents a novel species within the genus Lysinibacillus, for which the name Lysinibacillus pinottii sp. nov. is proposed, with type strain PB211T (= NRRL B-65672T, = CCUG 77181T).

Microbial-based metabolites associated with degradation of imidacloprid and its impact on stress-responsive proteins.

Imidacloprid (IMD), a neonicotinoid insecticide, is intensively used in agricultural fields for effective protection against aphids, cane beetles, thrips, stink bugs, locusts, etc., is causing serious environmental concerns. In recent years, seed treatment with Imidacloprid is being practiced mainly to prevent sucking insect pests. In India, due to the increase in application of this insecticide residue has been proven to have an impact on the quality of soil and water. In view of this, the current investigation is focussed on sustainable approach to minimize the residual effect of IMD in agricultural fields. The present study reveals a most promising imidacloprid resistant bacterium Lysinibacillus fusiformis IMD-Bio5 strain isolated from insecticide-contaminated soil. The isolated bacterial strain upon tested for its biodegradation potential on mineral salt medium (MSM) showed a significant survival growth at 150 g/L of IMD achieved after 3 days, whereas immobilized cells on MSM amended with 200 g/L of IMD as the sole carbon source provided degradation of 188 and 180 g/L of IMD in silica beads and sponge matrices, respectively. The liquid chromatography mass spectrometry was performed to test the metabolite responsive for IMD biodegradation potential of L. fusiformis IMD-Bio5 which showed the induced activity of the metabolite 6-Chloronicotinic acid. Furthermore, as compared to the untreated control, the Lysinibacillus fusiformis IMD-Bio5 protein profile revealed a range of patterns showing the expression of stress enzymes. Thus, results provided a most effective bacterium enabling the removal of IMD-like hazardous contaminants from the environment, which contributes to better agricultural production and soil quality, while long-term environmental advantages are restored.

Efficient bioremediation of multiple steroid hormones by halotolerant 17ß-hydroxysteroid dehydrogenase derived from moderately halophilic Pontibacillus chungwhensis HN14.

Steroid hormones exhibit potent endocrine disrupting activity and have been shown to disrupt the equilibrium of aquatic ecosystems and pose a threat to public health through their persistent and carcinogenic effects. Pontibacillus chungwhensis HN14, a moderately halophilic bacterium with the capacity to effectively degrade various polycyclic aromatic hydrocarbons and other organic pollutants, was previously isolated. Additionally, the strain HN14 showed strong environmental adaptability under various environmental stress conditions. In this study, the steroid degradation by strain HN14 was studied for the first time. We demonstrated that strain HN14 could degrade estradiol (E2) to maintain the growth of the strain and could convert E2 to estrone. Additionally, the efficient substrate degradation efficiency of P. chungwhensis HN14 under high salinity and high substrate concentration conditions was demonstrated. Furthermore, a 17ß-hydroxysteroid dehydrogenase, 17ß-HSD(HN14), was identified in strain HN14. Comparative analysis reveals that 17ß-HSD(HN14) shares approximately 38% sequence identity with 17ß-HSDx from Rhodococcus sp. P14. In addition, 100 µg of purified 17ß-HSD(HN14) could effectively convert about 40% of 0.25 mM of E2 within 1 h period, with an enzyme activity of 17.5 U/mg, and catalyze the dehydrogenation of E2 and testosterone at the C-17 position. The characterization of purified enzyme properties reveals that 17ß-HSD(HN14) exhibits exceptional structural robustness and enzymatic efficacy even under high salinity conditions of up to 20%. Overall, this study enhances our comprehension of steroid biodegradation in strain HN14 and contributes novel ideas and theoretical underpinnings for advancing bioremediation technologies targeting steroid pollution in high-saline environments.