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The recognition and characterization of gene-encoded nitric oxide synthase (NOS) from Exiguobacterium profundum are reported in this study. A new gene was sequenced and cloned from E. profundum and heterologously expressed in E. coli for functional identification, followed by protein purification using the His-tag. The stability and activity characteristics of the recombinant NOS were evaluated using different concentrations of IPTG at various time points. A band of approximately 42 kDa was observed by SDS-PAGE. The Km value of NOS, calculated based on the Michaelis-Menten equation, was 0.59 µmol/L. Additionally, homologous sequence alignment analysis indicated that the new NOS shared 80.48% similarity with the same protein from Bacillus subtilis and Umezawaea. The construction of the NOS expression vector and the purification of the recombinant protein provide a foundation for further functional research and inhibitor development.

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Several exogenous probiotics are applicable in fish culture; however, challenges in isolation and verification have hindered the full utilization of numerous host probiotics. Therefore, this study aimed to apply the host probiotic Exiguobacterium acetylicum G1-33 to hybrid grouper (Epinephelus fuscoguttatus ♀ × Epinephelus lanceolatus ♂) cultures and explore its mechanism of action. In total, 360 hybrid grouper were divided into four groups, which were fed the following for 60 days: three received commercial feed with varying concentrations of E. acetylicum G1-33 (106, 108, and 1010 CFU/g), while a control group received commercial feed. The results showed that supplementation with 106 and 108 CFU/g of E. acetylicum G1-33 enhanced gut morphology, upregulated growth-related genes (ghr1, igf-2, s6k1, tor), and promoted growth, with supplementation with 108 CFU/g resulting in the most notable enhancement. However, supplementation with 1010 CFU/g inhibited growth, possibly because of changes in intestinal morphology. Additionally, supplementation with E. acetylicum G1-33 upregulated the expression of immune-related genes (c3, myd88, Cu/Zn-sod, tlr3, and tnf2) in the liver and head kidney but led to an increase in malondialdehyde content, as well as a decrease in alkaline phosphatase and acid phosphatase activities, in the liver and serum, indicating increased oxidative stress. Moreover, supplementation with 106 and 108 CFU/g E. acetylicum G1-33 enhanced the widespread expression of immune-related genes in the head kidney and liver, respectively, and improved resistance to Vibrio harveyi, whereas supplementation with 1010 CFU/g weakened this resistance. In conclusion, E. acetylicum G1-33, particularly at 108 CFU/g, emerged as an effective probiotic, optimizing growth performance and immunity in hybrid grouper. This research is pioneering in its application of E. acetylicum in mariculture, potentially broadening the range of probiotic strategies in aquaculture.

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Antimicrobial resistance (AMR) is one of the most alarming global public health challenges of the 21st century. Over 3 million antimicrobial-resistant infections occur in the United States annually, with nearly 50,000 cases being fatal. Innovations in drug discovery methods and platforms are crucial to identify novel antibiotics to combat AMR. We present the isolation and characterization of potentially novel antibiotic lead compounds produced by the cross-feeding of two rhizosphere bacteria, Acinetobacter sp. RIT 592 and Exiguobacterium sp. RIT 594. We used solid-phase extraction (SPE) followed by liquid chromatography (LC) to enrich antibiotic extracts and subsequently mass spectrometry (MS) analysis of collected fractions for compound structure identification and characterization. The MS data were processed through the Global Natural Product Social Molecular Networking (GNPS) database. The supernatant from RIT 592 induced RIT 594 to produce a cocktail of antimicrobial compounds active against Gram-positive and negative bacteria. The GNPS analysis indicated compounds with known antimicrobial activity in the bioactive samples, including oligopeptides and their derivatives. This work emphasizes the utility of microbial community-based platforms to discover novel clinically relevant secondary metabolites. Future work includes further structural characterization and antibiotic activity evaluation of the individual compounds against pathogenic multidrug-resistant (MDR) bacteria.

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This study aims to examine the effects of the mixture of Bacillus cereus G1-11 and Exiguobacterium acetylicum G1-33, isolated from the gut of hybrid groupers (Epinephelus fuscoguttatus ♀ × E. lanceolatus ♂), on the host. The hybrid groupers were divided into a control (C, without any probiotics), B. cereus (BC, 1010 cfu/g), E. acetylicum (EA, 108 cfu/g), compound (mix, a 1:1 mixture of B. cereus and E. acetylicum), and positive reference group (P, Lactobacillus acidophilus, 5 × 108 cfu/L). Each group had four replicates, with 30 fish per replicate (53.30 ± 0.50 g), and were fed for 60 days. The results showed that adding probiotics to the feed significantly improved the weight gain, weight growth rate, specific growth rate, and digestive enzyme activities of hybrid groupers compared to the C group. The compound group was the most significant. In addition, composite probiotics added to feed significantly upregulated the expression levels of several growth-related genes in the liver and muscles. The activities of alkaline phosphatase, catalase, glutathione peroxidase, glutathione transferase, lysozyme, and total antioxidant capacity in the serum and liver were significantly influenced through mixed probiotic feeding. Moreover, the expression levels of several immune-related genes in the liver, spleen, and head kidney were significantly enhanced by adding single and mixed probiotics to feed, with the synergy of mixed probiotics being the best. An analysis of the gut microbiota showed that adding composite bacteria enhanced the richness and diversity of the gut microbiota, significantly increasing the relative abundance of potential probiotics (Cetobacterium and Microbacterium) while decreasing the presence of potential pathogens (Mycoplasma). Overall, our findings highlighted the efficacy of mixed probiotics (B. cereus and E. acetylicum) in enhancing growth performance, nutritional value of hybrid grouper feed, antioxidant capacity, immune response, and intestinal health, in finding the best combination of functional feed additives.

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We purified a lytic bacteriophage from soil collected in Guasave, Sinaloa: phiExGM16. This bacteriophage was isolated using the host, Exiguobacterium acetilycum. Its 17.6 kb genome contains 33 putative genes and shows a cover of 64% with 76.37% of nucleotide identity to Microbacterium phage Noelani.

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Here, we report the draft genome sequence of Exiguobacterium sp. strain MMG028, isolated from Rose Creek, San Diego, CA, USA, assembled and analyzed by undergraduate students participating in a marine microbial genomics course. A genomic comparison suggests that MMG028 is a novel species, providing a resource for future microbiology and biotechnology investigations.

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Delineating cohesive ecological units and determining the genetic basis for their environmental adaptation are among the most important objectives in microbiology. In the last decade, many studies have been devoted to characterizing the genetic diversity in microbial populations to address these issues. However, the impact of extreme environmental conditions, such as temperature and salinity, on microbial ecology and evolution remains unclear so far. In order to better understand the mechanisms of adaptation, we studied the (pan)genome of Exiguobacterium, a poly-extremophile bacterium able to grow in a wide range of environments, from permafrost to hot springs. To have the genome for all known Exiguobacterium type strains, we first sequenced those that were not yet available. Using a reverse-ecology approach, we showed how the integration of phylogenomic information, genomic features, gene and pathway enrichment data, regulatory element analyses, protein amino acid composition, and protein structure analyses of the entire Exiguobacterium pangenome allows to sharply delineate ecological units consisting of mesophilic, psychrophilic, halophilic-mesophilic, and halophilic-thermophilic ecotypes. This in-depth study clarified the genetic basis of the defined ecotypes and identified some key mechanisms driving the environmental adaptation to extreme environments. Our study points the way to organizing the vast microbial diversity into meaningful ecologically units, which, in turn, provides insight into how microbial communities adapt and respond to different environmental conditions in a changing world.

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Halophiles are excellent sources of detergent proteases that are attributed to stability in alkaline pH, salts, surfactants, and hydrophobic solvents. The lower enzymatic yields and tedious downstream processes necessitate the search for newer halophilic sources. We have previously reported a halotolerant Exiguobacterium sp. TBG-PICH-001, which secretes solvent-tolerant alkaline protease/s. The present study describes the heterologous expression of two protease genes, namely, rsep metalloprotease (WP_195864791, 1.23 Kb) and tpa serine protease (WP_195864453, 0.879 Kb) genes. These were cloned into the pET 22b + plasmid vector and expressed in Escherichia coli BL21(DE3). The recombinant proteases rsep and tpa showed respective yields of 6.3 and 6.7 IU/mg, 11 and 12-fold higher than the crude native protease/s from TBG-PICH-001. These showed soluble expression at 46 and 32 KDa, respectively. These were purified to homogeneity through Ni-NTA-affinity chromatography. The purified proteases were characterized for properties like pH & temperature optima and stability, substrate specificity, kinetic parameters, and detergent attributes. They showed affinity towards various substrates with a respective Km of 392 and 301 µM towards casein. The recombinant proteases exhibited stability in the alkaline pH (7-10), surfactants, metal ions, detergents, and hydrophobic solvents, rendering their suitability as detergent additives.

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We report the draft genome sequence of a novel species, Exiguobacterium sp., isolated from a freshly harvested and untreated cantaloupe in North Carolina. The strain Exiguobacterium wild type exhibited inhibitory activity against the foodborne pathogen Listeria monocytogenes, including strains of diverse serotypes and genotypes, both on agar media and in biofilms.

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This study focused on the isolation and identification of a novel alkaline protease-producing strain from Lake Van, the largest soda lake on Earth. The objective was to purify, characterize, and investigate the potential application of protease in the detergent industry. Through a combination of classical and molecular methods, the most potent protease producer was identified as Exiguobacterium alkaliphilum VLP1. The purification process, involving ammonium sulfate precipitation, ultrafiltration, and anion exchange chromatography, resulted in a 45-fold purification with a yield of 6.4% and specific activity of 1169 U mg-1 protein. The enzyme exhibited a molecular weight of 69 kDa, a Km value of 0.4 mm, and a maximal velocity (Vmax ) value of 2000 U mg-1 . The optimum activity was observed at 40°C and potential of hydrogen (pH) 9, while the enzyme also exhibited remarkable stability in the ranges of 30-60°C and pH 9-12. Notably, this study represents the first report of an alkaline protease isolated and characterized from E. alkaliphilum. This study also highlighted the potential of the enzyme as a detergent additive, as it showed compatibility with commercial detergents and effectively removed blood and chocolate stains from fabrics.

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Breast cancer (BC) continues to be one of the major causes of cancer deaths in women. Progress has been made in targeting hormone and growth factor receptor-positive BCs with clinical efficacy and success. However, little progress has been made to develop a clinically viable treatment for the triple-negative BC cases (TNBCs). The current study aims to identify potent agents that can target TNBCs. Extracts from microbial sources have been reported to contain pharmacological agents that can selectively inhibit cancer cell growth. We have screened and identified pigmented microbial extracts (PMBs) that can inhibit BC cell proliferation by targeting legumain (LGMN). LGMN is an oncogenic protein expressed not only in malignant cells but also in tumor microenvironment cells, including tumor-associated macrophages. An LGMN inhibition assay was performed, and microbial extracts were evaluated for in vitro anticancer activity in BC cell lines, angiogenesis assay with chick chorioallantoic membrane (CAM), and tumor xenograft models in Swiss albino mice. We have identified that PMB from the Exiguobacterium (PMB1), inhibits BC growth more potently than PMB2, from the Bacillus subtilis strain. The analysis of PMB1 by GC-MS showed the presence of a variety of fatty acids and fatty-acid derivatives, small molecule phenolics, and aldehydes. PMB1 inhibited the activity of oncogenic legumain in BC cells and induced cell cycle arrest and apoptosis. PMB1 reduced the angiogenesis and inhibited BC cell migration. In mice, intraperitoneal administration of PMB1 retarded the growth of xenografted Ehrlich ascites mammary tumors and mitigated the proliferation of tumor cells in the peritoneal cavity in vivo. In summary, our findings demonstrate the high antitumor potential of PMB1.

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IMPORTANCE: In this work, we characterized the composition, structure, and functional potential for biofilm formation of Exiguobacterium strains isolated from the Salar de Huasco in Chile in the presence of arsenic, an abundant metalloid in the Salar that exists in different oxidation states. Our results showed that the Exiguobacterium strains tested exhibit a significant capacity to form biofilms when exposed to arsenic, which would contribute to their resistance to the metalloid. The results highlight the importance of biofilm formation and the presence of specific resistance mechanisms in the ability of microorganisms to survive and thrive under adverse conditions.

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The identification and applicability of bacteria are inconclusive until comprehended with genomic repositories. Our isolate, Exiguobacterium sp. TBG-PICH-001 exhibited excellent halo- and organic solvent tolerance with simultaneous production of alkaline protease/s (0.512 IU/mL). The crude protease (1 IU) showed a 43.57% degradation of whey protein. The bulk proteins in the whey were hydrolyzed to smaller peptides which were evident in the SDS-PAGE profile. With such characteristics, the isolate became interesting for its genomic studies. The TBG-PICH-001 genome was found to be 3.14 Mb in size with 17 contigs and 47.33% GC content. The genome showed 3176 coding genes, and 2699 genes were characterized for their functionality. The Next-Generation-Sequencing of the genome identified only the isolate's genus; hence we attempted to delineate its species position. The genomes of the isolate and other representative Exiguobacterium spp. were compared based on orthologous genes (Orthovenn2 server). A pan-genomic analysis revealed the match of TBG-PICH-001 with 15 uncharacterized Exiguobacterium genomes at the species level. All these collectively matched with Exiguobacterium indicum, and the results were reconfirmed through phylogenetic studies. Further, the Exiguobacterium indicum genomes were engaged for homology studies rendering 11 classes of protease genes. Two putative proteases (Zinc metalloprotease and Serine protease) obtained from homology were checked for PCR amplification using genomic DNA of TBG-PICH-001 and other Exiguobacterium genomes. The results showed amplification only in the Exiguobacterium indicum genome. These protease genes, after sequencing, were matched with the TBG-PICH-001 genome. Their presence in its whole genome experimentally validated the study. Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03796-5.

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As the ecological niche most closely associated with polymers, microorganisms in the 'plastisphere' have great potential for plastics degradation. Microorganisms isolated from the 'plastisphere' could colonize and degrade commercial plastics containing different additives, but the observed weight loss and surface changes were most likely caused by releasing the additives rather than actual degradation of the plastics itself. Unlike commercial plastics that contain additives, whether marine microorganisms in the 'plastisphere' have adapted to additive-free plastics as a surface to colonize and potentially degrade is not yet known. Herein, a novel marine bacterium, Exiguobacterium marinum a-1, was successfully isolated from mature 'plastisphere' that had been deployed in situ for up to 20 months. Strain a-1 could use additive-free polypropylene (PP) films as its primary energy and carbon source. After strain a-1 was incubated with additive-free PP films for 80 days, the weight of films decreased by 9.2%. The ability of strain a-1 to rapidly form biofilms and effectively colonize the surface of additive-free PP films was confirmed by Scanning Electron Microscopy (SEM), as reflected by the increase in roughness and visible craters on the surface of additive-free PP films. Additionally, the functional groups of -CO, -C-H, and -OH were identified on the treated additive-free PP films according to Fourier Transform Infrared (FTIR). Genomic data from strain a-1 revealed a suite of key genes involved in biosurfactant synthesis, flagellar assembly, and cellular chemotaxis, contributing to its rapid biofilm formation on hydrophobic polymer surfaces. In particular, key enzymes that may be responsible for the degradation of additive-free PP films, such as glutathione peroxidase, cytochrome p450 and esterase were also recognized. This study highlights the potential of microorganisms present in the 'plastisphere' to metabolize plastic polymers and points to the intrinsic importance of the new strain a-1 in the mitigation of plastic pollution.

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Microbial reduction is an effective way to deal with hexavalent chromium [Cr(VI)] contamination in the environment, which can significantly mitigate the biotoxicity and migration of this pollutant. The present study investigated the influence of environmental factors on aqueous Cr(VI) removal by a newly isolated facultative anaerobic bacterium, Exiguobacterium sp. PY14, and revealed the reduction mechanism. This strain with a minimum inhibitory concentration of 400 mg/L showed the strongest Cr(VI) removal capacity at pH 8.0 because of its basophilic nature, which was obviously depressed by increasing the Cr(VI) initial concentration under both aerobic and anaerobic conditions. In contrast, the removal rate constant for 50 mg/L of Cr(VI) under anaerobic conditions (1.82 × 10-2 h-1) was 3.3 times that under aerobic conditions. The co-existence of Fe(III) and Cu(II) significantly promoted the removal of Cr(VI), while Ag(I), Pb(II), Zn(II), and Cd(II) inhibited it. Electron-shuttling organics such as riboflavin, humic acid, and anthraquinone-2,6-disulfonate promoted the Cr(VI) removal to varying degrees, and the enhancement was more significant under anaerobic conditions. The removal of aqueous Cr(VI) by strain PY14 was demonstrated to be due to cytoplasmic rather than extracellular reduction by analyzing the contributions of different cell components, and the end products existed in the aqueous solution in the form of organo-Cr(III) complexes. Several possible genes involved in Cr(VI) metabolism, including chrR and chrA that encode well-known Chr family proteins responsible for chromate reduction and transport, respectively, were identified in the genome of PY14, which further clarified the Cr(VI) reduction pathway of this strain. The research progress in the influence of crucial environmental factors and biological reduction mechanisms will help promote the potential application of Exiguobacterium sp. PY14 with high adaptability to environmental stress in Cr(VI) removal in the actual environment.

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We report the complete genome sequence of Exiguobacterium profundum TSS-3, a strain isolated from the sediment of an extremely saline-alkaline spring located in Ixtapa, Chiapas-México (16° 47´ LN and 92° 54´ LO). Its genome is composed of a 2.8-Mb chromosome and a small 4.6-Kb plasmid.

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Hexavalent chromium (Cr(VI)) is a cytotoxic heavy metal pollutant that adversely affects all life forms. Interestingly, the crustacean Procambarus clarkii exhibits a relatively high tolerance to heavy metals. The underlying mechanisms remain unclear. In this study, we investigated the role of symbiotic bacteria in P. clarkii in alleviating Cr(VI)-induced damage and explored their potential mechanisms of action. Through transcriptomic analysis, we observed that Cr(VI) activated P. clarkii's antimicrobial immune responses and altered the bacterial composition in the hemolymph. After antibiotic treatment to reduce bacterial populations, Cr(VI)-induced intestinal and liver damage worsened, and crayfish exhibited lower levels of GSH/CAT/SOD activity. The Exiguobacterium, the symbiotic bacteria in the hemolymph of P. clarkii, were proved to be primary contributor to Cr(VI) tolerance. Further investigation suggested that it resists Cr(VI) through the activation of the ABC transporter system and the reduction of Cr(VI) via the reductase gene nfsA. To validate the role of Exiguobacterium in Cr(VI) tolerance, crayfish treated with antibiotics then supplemented with Exiguobacterium H6 and recombinant E. coli (with the nfsA gene), reduced Cr(VI)-induced ovarian damage. Overall, this study revealed that the symbiotic bacteria Exiguobacterium can absorb and reduce hexavalent chromium, mitigating Cr(VI)-induced damage in P. clarkii. These findings provide new insights into hexavalent chromium tolerance mechanisms in crustaceans.

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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.

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The microalgae Chlorella saccharophila UTEX247 was co-cultured with its symbiotic indigenous isolated bacterial strain, Exiguobacterium sp., to determine the possible effects of bacteria on microalgae growth and lutein productivity. Under optimal conditions, the lutein productivity of co-culture was 298.97 µg L-1 d-1, which was nearly 1.45-fold higher compared to monocultures i.e., 103.3 µg L-1 d-1. The highest lutein productivities were obtained in co-cultures, accompanied by a significant increase in cell biomass up to 0.84-fold. These conditions were analyzed using an untargeted metabolomics approach to identify metabolites enhancing valuable renewables, i.e., lutein, without compromising growth. Our qualitative metabolomic analysis identified nearly 30 (microalgae alone), 41 (bacteria alone), and 75 (co-cultures) metabolites, respectively. Among these, 46 metabolites were unique in the co-culture alone. The co-culture interactions significantly altered the role of metabolites such as thiamine precursors, reactive sugar anomers like furanose and branched-chain amino acids (BCAA). Nevertheless, the central metabolism cycle upregulation depicted increased availability of carbon skeletons, leading to increased cell biomass and pigments. In conclusion, the co-cultures induce the production of relevant metabolites which regulate growth and lutein simultaneously in C. saccharophila UTEX247, which paves the way for a new perspective in microalgal biorefineries.

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The roles of plant growth-promoting rhizobacteria in promoting plant growth and soil health, including alteration in plant metabolism and production of phytohormones such as indole-3-acetic acid (IAA) and the enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminase, are indisputable. This study aimed to isolate and characterize beneficial bacteria isolated from the rhizosphere of pineapple from distinct stress-inducing habitats, including water excess-, herbicide-over-treated-, and pathogen-infected areas at PT Great Giant Foods located in Lampung, Indonesia. The isolated bacteria were screened based on IAA production and ACC deaminase activities. Six selected isolates produced IAA with concentrations of up to 36.93 mgL-1. The highest value belongs to Bacillus sp. NCTB5I, followed by Brevundimonas sp. CHTB 2C (13.13 mgL-1) and Pseudomonas sp. CHTB 5B (6.65 mgL-1). All isolates were detected with ACC deaminase activities with Brevundimonas sp. CHTJ 5H consuming 88% of ACC over 24 h, the highest among all. Brevundimonas sp. CHTB 2C was detected with the highest ACC deaminase activity with the value of 13,370 nm α-ketobutyrate mg-1h-1. In another experiment, it was revealed that all selected isolates promote soybean growth. These bacteria are potential to be developed in the future as bioagents to promote plant growth, especially under stressful environmental conditions.