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.

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.

Genome analysis of Rossellomorea sp. y25, a deep sea bacterium isolated from the sediments of South China Sea.

Rossellomorea sp. y25, a putative new species of yellow pigment-producing, aerobic and chemoheterotrophic bacterium belonging to the family Bacillaceae, was isolated from the sediments at the depth of 1829 m in the South China Sea. In this study, we present the complete genome sequences of strain y25, which consisted of only one circular chromosome with 4,633,006 bp and the content of G + C was 41.76%. A total of 4466 CDSs, 106 tRNA, 33 rRNA, and 101 sRNA genes were obtained. Genomic analysis of strain y25 showed that it has the ability to produce antioxidant carotenoids and a large number of heavy metal resistance genes, such as arsenic, cadmium and zinc. In addition, strain y25 contains a prophage that may contribute to host protection against lysis by related Bacillus-like phages. This is the first report of genome-wide information on a bacterium of the genus Rossellomorea isolated from the deep sea, providing insights into how microorganisms of this genus adapt to deep-sea environments.

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).

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.

Biosynthetic potential of the sediment microbial subcommunities of an unexplored karst ecosystem and its ecological implications.

Microbial communities from various environments have been studied in the quest for new natural products with a broad range of applications in medicine and biotechnology. We employed an enrichment method and genome mining tools to examine the biosynthetic potential of microbial communities in the sediments of a coastal sinkhole within the karst ecosystem of the Yucatán Peninsula, Mexico. Our investigation led to the detection of 203 biosynthetic gene clusters (BGCs) and 55 secondary metabolites (SMs) within 35 high-quality metagenome-assembled genomes (MAGs) derived from these subcommunities. The most abundant types of BGCs were Terpene, Nonribosomal peptide-synthetase, and Type III polyketide synthase. Some of the in silico identified BGCs and SMs have been previously reported to exhibit biological activities against pathogenic bacteria and fungi. Others could play significant roles in the sinkhole ecosystem, such as iron solubilization and osmotic stress protection. Interestingly, 75% of the BGCs showed no sequence homology with bacterial BGCs previously reported in the MiBIG database. This suggests that the microbial communities in this environment could be an untapped source of genes encoding novel specialized compounds. The majority of the BGCs were identified in pathways found in the genus Virgibacillus, followed by Sporosarcina, Siminovitchia, Rhodococcus, and Halomonas. The latter, along with Paraclostridium and Lysinibacillus, had the highest number of identified BGC types. This study offers fresh insights into the potential ecological role of SMs from sediment microbial communities in an unexplored environment, underscoring their value as a source of novel natural products.

Genomic characterization of a novel ureolytic bacteria, Lysinibacillus capsici TSBLM, and its application to the remediation of acidic heavy metal-contaminated soil.

Soil heavy metal contamination is an essential challenge in ecological and environmental management, especially for acidic soils. Microbially induced carbonate precipitation (MICP) is an effective and environmentally friendly remediation technology for heavy metal contaminated sites, and one of the key factors for its realization lies in the microorganisms. In this study, Lysinibacillus capsici TSBLM was isolated from heavy metal contaminated soil around a gold mine, and inferred to be a novel ureolytic bacteria after phylogenomic inference and genome characterization. The urease of L. capsici TSBLM was analyzed by genetic analysis and molecular docking, and further applied this bacteria to the remediation of Cu and Pb in solution and acidic soils to investigate its biomineralization mechanism and practical application. The results revealed L. capsici TSBLM possessed a comprehensive urease gene cluster ureABCEFGD, and the encoded urease docked with urea at the lowest binding energy site (ΔG = -3.43 kcal/mol) connected to three amino acids threonine, aspartic, and alanine. The urease of L. capsici TSBLM is synthesized intracellularly but mainly functions extracellularly. L. capsici TSBLM removes Cu/Pb from the solution by generating heavy metal carbonates or co-precipitating with CaCO3 vaterite. For acidic heavy metal-contaminated soil, the carbonate-bound states of Cu and Pb increased significantly from 7 % to 16 % and from 23 % to 35 % after 30 days by L. capsici TSBLM. Soil pH improved additionally. L. capsici TSBLM maintained the dominant status in the remediated soil after 30 days, demonstrating good environmental adaptability and curing persistence. The results provided new strain resources and practical application references for the remediation of acidic heavy metal contaminated soil based on MICP.

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.

Short communication: Investigating optimal laboratory growth conditions of Gracilibacillus halotolerans in media supplemented with salt.

Gracilibacillus halotolerans, a new and relatively unstudied extremophile, extracted from the Great Salt Lake USA, survives in an extreme saline environment. Uncovering optimal laboratory growth conditions can be useful to improve treatment strategies against antibiotic resistance and biofilm formation. In the current study, G. halotolerans growth optimization was tested to determine the ideal saline concentration. In addition, a variety of G. halotolerans'-derived survival strategies were reviewed. The major findings of the current study includes the optimal laboratory growth condition for G. halotolerans that requires the supplement of 5% NaCl. In addition, optimal growth was observed up to 72 h in Luria Bertani (LB) broth. Identifying the optimal laboratory growth conditions for G. halotolerans will standardize growth methods, reduce laboratory cost, and can improve future investigations of extremophile bacteria as model organisms to combat antibiotic resistance, biofilm, and other persister cell characteristics that negatively affect research and clinical settings.

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.

Genes encoding a novel thermostable bacteriocin in the thermophilic bacterium Aeribacillus pallidus PI8.

AIM: Aeribacillus pallidus PI8 is a Gram-positive thermophilic bacterium that produces thermostable antimicrobial substances against several bacterial species, including Geobacillus kaustophilus HTA426. In the present study, we sought to identify genes of PI8 with antibacterial activity. METHODS AND RESULTS: We isolated, cloned, and characterized a thermostable bacteriocin from A. pallidus PI8 and named it pallidocyclin. Mass spectrometric analyses of pallidocyclin revealed that it had a circular peptide structure, and its precursor was encoded by pcynA in the PI8 genome. pcynA is the second gene within the pcynBACDEF operon. Expression of the full-length pcynBACDEF operon in Bacillus subtilis produced intact pallidocyclin, whereas expression of pcynF in G. kaustophilus HTA426 conferred resistance to pallidocyclin. CONCLUSION: Aeribacillus pallidus PI8 possesses the pcynBACDEF operon to produce pallidocyclin. pcynA encodes the pallidocyclin precursor, and pcynF acts as an antagonist of pallidocyclin.

Biochemical characterisation of a novel broad pH spectrum subtilisin from Fictibacillus arsenicus DSM 15822T.

Subtilisins from microbial sources, especially from the Bacillaceae family, are of particular interest for biotechnological applications and serve the currently growing enzyme market as efficient and novel biocatalysts. Biotechnological applications include use in detergents, cosmetics, leather processing, wastewater treatment and pharmaceuticals. To identify a possible candidate for the enzyme market, here we cloned the gene of the subtilisin SPFA from Fictibacillus arsenicus DSM 15822T (obtained through a data mining-based search) and expressed it in Bacillus subtilis DB104. After production and purification, the protease showed a molecular mass of 27.57 kDa and a pI of 5.8. SPFA displayed hydrolytic activity at a temperature optimum of 80 °C and a very broad pH optimum between 8.5 and 11.5, with high activity up to pH 12.5. SPFA displayed no NaCl dependence but a high NaCl tolerance, with decreasing activity up to concentrations of 5 m NaCl. The stability enhanced with increasing NaCl concentration. Based on its substrate preference for 10 synthetic peptide 4-nitroanilide substrates with three or four amino acids and its phylogenetic classification, SPFA can be assigned to the subgroup of true subtilisins. Moreover, SPFA exhibited high tolerance to 5% (w/v) SDS and 5% H2 O2 (v/v). The biochemical properties of SPFA, especially its tolerance of remarkably high pH, SDS and H2 O2 , suggest it has potential for biotechnological applications.

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.

Genome-based reclassification of Bacillus acidicola, Bacillus pervagus and the genera Heyndrickxia, Margalitia and Weizmannia.

In the present study, the taxonomic positions of Bacillus acidicola, Bacillus pervagus and members of the genera Heyndrickxia, Margalitia and Weizmannia were evaluated. The 16S rRNA gene sequence similarity between Bacillus acidicola DSM 14745T, Bacillus pervagus DSM 23947T and members of the genera Heyndrickxia and Margalitia were above the cut-off level (>95 %) for genus delineation. Amino acid identity (AAI) values and the results of phylogenomic analysis suggested that B. acidicola and the members of the genera Heyndrickxia, Margalitia and Weizmannia belong to the same genus. Furthermore, the AAI and phylogenomic results also differentiate B. pervagus from B. acidicola and the members of the genera Heyndrickxia, Margalitia and Weizmannia. Based on the results, we propose to transfer Bacillus acidicola, Margalitia and Weizmannia to the genus Heyndrickxia. We also propose the reclassification of B. pervagus into a new genus Oikeobacillus gen. nov., with the type species Oikeobacillus pervagus comb. nov.

Proposal to transfer Bacillus dafuensis and Bacillus massiliigabonensis to the genus Cytobacillus as Cytobacillus dafuensis comb. nov., and Cytobacillus massiliigabonensis comb. nov., respectively.

In the present study, the taxonomic position of Bacillus dafuensis and Bacillus massiliigabonensis was evaluated using genome-based comparison. The 16S rRNA gene sequence obtained from the Bacillus dafuensis FJAT-25496T genome showed 99.7% similarity with the type strain of Cytobacillus citreus, while Bacillus massiliigabonensis Marseille-P2639T showed 98.7% similarity with the type species of Cytobacillus solani. The 16S rRNA gene sequence similarity of Bacillus dafuensis FJAT-25496T and Bacillus massiliigabonensis Marseille-P2639T with Cytobacillus members was above the threshold (94.5%) for genus delineation. In phylogenetic (based on 16S rRNA gene sequences) and phylogenomic (based on 71 bacterial single-copy genes) trees, Bacillus dafuensis and Bacillus massiliigabonensis clustered with Cytobacillus members. The 16S rRNA gene sequence, amino acid identity and percentage of conserved proteins analysis indicated Bacillus dafuensis FJAT-25496T and Bacillus massiliigabonensis Marseille-P2639T as a member of the genus Cytobacillus. The digital DNA-DNA hybridization and the average nucleotide identity values of Bacillus dafuensis FJAT-25496T and Bacillus massiliigabonensis Marseille-P2639T with Cytobacillus members was below the cut-off value (70/94%-95%) for species delineation. Based on the results we propose to transfer Bacillus dafuensis and Bacillus massiliigabonensis to the genus Cytobacillus as Cytobacillus dafuensis comb. nov., and Cytobacillus massiliigabonensis comb. nov., respectively.

Cytobacillus spongiae sp. nov. isolated from sponge Diacarnus spinipoculum.

A novel Gram-stain-positive, aerobic and motile bacterium, designated strain CY-GT, was isolated from a sponge (Diacarnus spinipoculum) collected from the Red Sea. The strain grew at 13-43 °C (optimum 30 °C), pH 5.5-10.0 (optimum pH 9.0) and with 0-8.0 % (w/v) (0-1.37 M) NaCl (optimum 0 %). The results of phylogenetic analysis based on the 16S rRNA gene sequences indicated that CY-GT represents a member of the genus Cytobacillus, with the highest sequence identity to Cytobacillus oceanisediminis H2T (97.05 %), followed by Cytobacillus firmus IAM 12464T (96.76 %). The major cellular fatty acids (>5 % of the total) of CY-GT were C15 : 0iso, C16 : 0iso, C16 : 1ω7c alcohol, C16 : 0, C17 : 1iso ω10c and C17 : 0iso. The major polar lipids were glycolipid, diphosphatidylglycerol, phosphatidylethanolamine and phosphatidylglycerol. The major respiratory quinone is menaquinone-7 (MK-7). The cell-wall peptidoglycan contains meso-diaminopimelic acid. The total genome size of CY-GT is 4 789 051 bp. The DNA G+C content is 38.83 mol%. The average nucleotide identity and DNA-DNA hybridization among CY-GT and type strains of other species of the genus Cytobacillus were 76.79-78.97 % and 20.10-24.90 %, respectively. On the basis of the results of phylogenetic analysis, physiological and biochemical characterization, strain CY-GT represents a novel species of the genus Cytobacillus, for which the name Cytobacillus spongiae sp. nov. is proposed. The type strain is CY-GT (=MCCC 1K06383T=KCTC 43348T).

Lysinibacillus capsici 38,328 isolated from agricultural soils as a promising probiotic candidate for intestinal health.

There is an increasing interest in the use of spore-forming Bacillus spp. as probiotic ingredients on the market. However, probiotics Bacillus species are insufficient, and more safe Bacillus species were required. In the study, traditional fermented foods and soil samples were collected from more than ten provinces in China, and 506 Bacillus were selected from 109 samples. Using the optimized procedure, we screened nine strains, which successfully passed the acid, alkali, bile salt, and trypsin resistance test. Drug sensitivity test results showed that three Bacillus out of the nine isolates exhibited antibiotic sensitivity to more than 29 antibiotics. The three strains sensitive to antibiotics were identified by 16S ribosomal RNA, recA, and gyrB gene analysis, two isolates (38,327 and 38,328) belong to the species Lysinibacillus capsici and one isolate (37,326) belong to Bacillus halotolerans. Moreover, the three strains were confirmed safe through animal experiments. Finally, L. capsici 38,327 and 38,328 showed protections in the Salmonella typhimurium infection mouse model, which slowed down weight loss, reduced bacterial load, and improved antioxidant capacity. Altogether, our data demonstrated that selected L. capsici strains can be used as novel probiotics for intestinal health.

New robust subtilisins from halotolerant and halophilic Bacillaceae.

The aim of the present study was the characterisation of three true subtilisins and one phylogenetically intermediate subtilisin from halotolerant and halophilic microorganisms. Considering the currently growing enzyme market for efficient and novel biocatalysts, data mining is a promising source for novel, as yet uncharacterised enzymes, especially from halophilic or halotolerant Bacillaceae, which offer great potential to meet industrial needs. Both halophilic bacteria Pontibacillus marinus DSM 16465T and Alkalibacillus haloalkaliphilus DSM 5271T and both halotolerant bacteria Metabacillus indicus DSM 16189 and Litchfieldia alkalitelluris DSM 16976T served as a source for the four new subtilisins SPPM, SPAH, SPMI and SPLA. The protease genes were cloned and expressed in Bacillus subtilis DB104. Purification to apparent homogeneity was achieved by ethanol precipitation, desalting and ion-exchange chromatography. Enzyme activity could be observed between pH 5.0-12.0 with an optimum for SPPM, SPMI and SPLA around pH 9.0 and for SPAH at pH 10.0. The optimal temperature for SPMI and SPLA was 70 °C and for SPPM and SPAH 55 °C and 50 °C, respectively. All proteases showed high stability towards 5% (w/v) SDS and were active even at NaCl concentrations of 5 M. The four proteases demonstrate potential for future biotechnological applications. KEY POINTS: • Halophilic and halotolerant Bacillaceae are a valuable source of new subtilisins. • Four new subtilisins were biochemically characterised in detail. • The four proteases show potential for future biotechnological applications.

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.

Effect of Lysinibacillus isolated from environment on probiotic properties and gut microbiota in mice.

Soil microorganisms (SM) are primarily involved in organism degradation, plant nitrogen nutrient immobilization, host microorganisms and oxidation. However, research on the effect of soil-derived Lysinibacillus on the intestinal microbiota spatial disparity of mice is lacking. To test the probiotic properties of Lysinibacillus and the spatial disparity on mice intestinal microorganisms, hemolysis test, molecular phylogenetic analysis, antibiotic sensitivity testing, serum biochemical assays and 16S rRNA profiling were applied. The results showed that Lysinibacillus (LZS1 and LZS2) was resistant to two common antibiotics, Tetracyclines and Rifampin, and sensitive to other antibiotics among the 12 antibiotics tested and negative for hemolysis. In addition, the body weight of group L (treatment of Lysinibacillus, 1.0 × 108 CFU/d for 21days) mice was significantly greater than that of the control group; serum biochemical tests showed that the TG and UREA were significantly lower in group L. The spatial disparity of intestinal microorganisms in mice was significant, treatment of Lysinibacillus (1.0 × 108 CFU/d for 21days) reduced the intestinal microbial diversity and decreased the richness of Proteobacteria, Cyanobacteria and Bacteroidetes in mice. Furthermore, Lysinibacillus treatment enhanced Lactobacillus and Lachnospiraceae richness and significantly reduced 6 bacterial genera in jejunum community, reduced 8 bacterial genera, but increased bacteria at the 4 genera level in cecum microorganisms. In conclusion, this study demonstrated spatial disparity of intestinal microorganisms in mice and probiotic potential of Lysinibacillus isolated from soil.