Research advances in the identification of regulatory mechanisms of surfactin production by Bacillus: a review.

Surfactin is a cyclic hexalipopeptide compound, nonribosomal synthesized by representatives of the Bacillus subtilis species complex which includes B. subtilis group and its closely related species, such as B. subtilis subsp subtilis, B. subtilis subsp spizizenii, B. subtilis subsp inaquosorum, B. atrophaeus, B. amyloliquefaciens, B. velezensis (Steinke mSystems 6: e00057, 2021) It functions as a biosurfactant and signaling molecule and has antibacterial, antiviral, antitumor, and plant disease resistance properties. The Bacillus lipopeptides play an important role in agriculture, oil recovery, cosmetics, food processing and pharmaceuticals, but the natural yield of surfactin synthesized by Bacillus is low. This paper reviews the regulatory pathways and mechanisms that affect surfactin synthesis and release, highlighting the regulatory genes involved in the transcription of the srfAA-AD operon. The several ways to enhance surfactin production, such as governing expression of the genes involved in synthesis and regulation of surfactin synthesis and transport, removal of competitive pathways, optimization of media, and fermentation conditions were commented. This review will provide a theoretical platform for the systematic genetic modification of high-yielding strains of surfactin.

Evaluation of Antifungal Activity of Endophytic Bacillus spp. and Identification of Secondary Metabolites Produced Against the Phytopathogenic Fungi.

Endophytic bacteria serve as a rich source of diverse antimicrobial compounds. Recently, there has been a growing interest in utilizing endophytic Bacillus spp. as biological agents against phytogenic fungi, owing to their potential to produce a wide range of antimicrobial substances. The objective of this research was to investigate the protective abilities of 15 endophytic Bacillus spp. isolated from previous study from wheat plant, against the phytopathogenic fungi, Fusarium graminearum and Macrophomina phaseolina. A dual culture plate assay was conducted as a preliminary analysis, revealing that 7 out of 15 endophytic Bacillus spp. demonstrated inhibition against one or both of the phytopathogenic fungi used in this study. All seven endophytes were further assessed for the presence of diffusible antifungal metabolites. The cultures were grown in potato dextrose broth for 120 h, and the cell-free supernatant was extracted and analyzed using the cup plate method. The methanolic extract yielded similar results to the dual culture plate analysis, except for WL2-15. Additionally, deformities in the mycelial structure were examined under the light microscope upon exposure to methanolic extract. Furthermore, the analysis and identification of metabolites were carried out via gas chromatography-mass spectrometry of methanolic extract from selected seven endophytic Bacillus spp. The chromatogram revealed the presence of some major peaks such as tridecanoic acid, methyl ester, hydroperoxide, 1-methylbutyl, 9-octadecenamide, (z)-, hexane-1,3,4-triol, 3,5-dimethyl- tetradecanoic acid. To the best of our knowledge, this is the first report of these biocontrol agents in endophytic Bacillus spp. Interestingly, volatile organic compound production was also seen in all the isolates against the phytopathogenic fungi.

Comparative negation of amphiphile production using nutrition factors: Amyloids versus biosurfactants.

Microbial amphiphiles play an important role in environmental activities such as microbial signaling, bioremediation, and biofilm formation. Microorganisms rely on their unique characteristics of interfaces to carry out critical biological functions, which are helped by amphipathic biomolecules known as amphiphiles. Bacillus amyloids aid in cell adhesion and biofilm formation. Pseudomonas sp. are essential in biofilm development and are a vital survival strategy for many bacteria. Furthermore, Pseudomonas and Bacillus are well-known for their ability to produce biosurfactants with a range of applications, including bioremediation and removing biological pollutants from different environments. The study employed 31 different media types and a range of analytical techniques to assess the presence of amyloid proteins and the absence of biosurfactants in Bacillus licheniformis K125 (GQ850525.1) and Pseudomonas fluorescens CHA0. The presence of amyloid proteins was confirmed through Congo red and thioflavin T staining. The carefully constructed medium also efficiently inhibited the synthesis of biosurfactants by these bacteria. Additionally, surface tension measurements, emulsification index, thin-layer chromatography, and high-performance thin-layer chromatography analyses indicated the absence of biosurfactants in the tested media.

Evaluation of Antibiotic Biodegradation by a Versatile and Highly Active Recombinant Laccase from the Thermoalkaliphilic Bacterium Bacillus sp. FNT.

Laccases are industrially relevant enzymes that have gained great biotechnological importance. To date, most are of fungal and mesophilic origin; however, enzymes from extremophiles possess an even greater potential to withstand industrial conditions. In this study, we evaluate the potential of a recombinant spore-coat laccase from the thermoalkaliphilic bacterium Bacillus sp. FNT (FNTL) to biodegrade antibiotics from the tetracycline, ß-lactams, and fluoroquinolone families. This extremozyme was previously characterized as being thermostable and highly active in a wide range of temperatures (20-90 °C) and very versatile towards several structurally different substrates, including recalcitrant environmental pollutants such as PAHs and synthetic dyes. First, molecular docking analyses were employed for initial ligand affinity screening in the modeled active site of FNTL. Then, the in silico findings were experimentally tested with four highly consumed antibiotics, representatives of each family: tetracycline, oxytetracycline, amoxicillin, and ciprofloxacin. HPLC results indicate that FNTL with help of the natural redox mediator acetosyringone, can efficiently biodegrade 91, 90, and 82% of tetracycline (0.5 mg mL-1) in 24 h at 40, 30, and 20 °C, respectively, with no apparent ecotoxicity of the products on E. coli and B. subtilis. These results complement our previous studies, highlighting the potential of this extremozyme for application in wastewater bioremediation.

Microbial degradation of low-density polyethylene, polyethylene terephthalate, and polystyrene by novel isolates from plastic-polluted environment.

Biodegradation is an eco-friendly measure to address plastic pollution. This study screened four bacterial isolates that were capable of degrading recalcitrant polymers, i.e., low-density polyethylene, polyethylene terephthalate, and polystyrene. The unique bacterial isolates were obtained from plastic polluted environment. Dermacoccus sp. MR5 (accession no. OP592184) and Corynebacterium sp. MR10 (accession no. OP536169) from Malaysian mangroves and Bacillus sp. BS5 (accession no. OP536168) and Priestia sp. TL1 (accession no. OP536170) from a sanitary landfill. The four isolates showed a gradual increase in the microbial count and the production of laccase and esterase enzymes after 4 weeks of incubation with the polymers (independent experiment set). Bacillus sp. BS5 produced the highest laccase 15.35 ± 0.19 U/mL and showed the highest weight loss i.e., 4.84 ± 0.6% for PS. Fourier transform infrared spectroscopy analysis confirmed the formation of carbonyl and hydroxyl groups as a result of oxidation reactions by enzymes. Liquid chromatography-mass spectrometry analysis showed the oxidation of the polymers to small molecules (alcohol, ethers, and acids) assimilated by the microbes during the degradation. Field emission scanning electron microscopy showed bacterial colonization, biofilm formation, and surface erosion on the polymer surface. The result provided significant insight into enzyme activities and the potential of isolates to target more than one type of polymer for degradation.

The impact of orphan histidine kinases and phosphotransfer proteins on the regulation of clostridial sporulation initiation.

Sporulation is an important feature of the clostridial life cycle, facilitating survival of these bacteria in harsh environments, contributing to disease transmission for pathogenic species, and sharing common early steps that are also involved in regulating industrially important solvent production by some non-pathogenic species. Initial genomics studies suggested that Clostridia lack the classical phosphorelay that phosphorylates Spo0A and initiates sporulation in Bacillus, leading to the hypothesis that sporulation in Clostridia universally begins when Spo0A is phosphorylated by orphan histidine kinases (OHKs). However, components of the classical Bacillus phosphorelay were recently identified in some Clostridia. Similar Bacillus phosphorelay components have not yet been found in the pathogenic Clostridia or the solventogenic Clostridia of industrial importance. For some of those Clostridia lacking a classical phosphorelay, the involvement of OHKs in sporulation initiation has received support from genetic studies demonstrating the involvement of several apparent OHKs in their sporulation. In addition, several clostridial OHKs directly phosphorylate Spo0A in vitro. Interestingly, there is considerable protein domain diversity among the sporulation-associated OHKs in Clostridia. Further adding to the emergent complexity of sporulation initiation in Clostridia, several candidate OHK phosphotransfer proteins that were OHK candidates were shown to function as phosphatases that reduce sporulation in some Clostridia. The mounting evidence indicates that no single pathway explains sporulation initiation in all Clostridia and supports the need for further study to fully understand the unexpected and biologically fascinating mechanistic diversity of this important process among these medically and industrially important bacteria.

Metabolomics and Molecular Networking-Guided Screening of Bacillus-Derived Bioactive Compounds Against a Highly Lethal Vibrio Species.

Microorganisms are important sources of bioactive natural products. However, the complexity of microbial metabolites and the low abundance of active compounds render the isolation and purification process laborious and inefficient. During our search for active substances capable of inhibiting the newly discovered highly lethal Vibrio strain vp-HL, we found that the fermentation broth of multiple Bacillus strains exhibited antibacterial activity. However, the substances responsible for the activity remained unclear. Metabolomics, molecular networking (MN), and the Structural similarity Network Annotation Platform for Mass Spectrometry (SNAP-MS) were employed in conjunction with bioactivity screening to predict the antibacterial compounds from Bacillus strains. The analysis of fractions, and their isolation, NMR-based annotation, and bioactivity evaluation of an amicoumacin compound partially confirmed the prediction from these statistical analyses. This work presents the potential of marine Bacillus in producing active substances against Vibrio species. Additionally, it highlighted the significance and feasibility of metabolomics and MN in the dereplication of compounds and the determination of isolation targets.

Advances in recombinant protease production: current state and perspectives.

Proteases, enzymes that catalyze the hydrolysis of peptide bonds in proteins, are important in the food industry, biotechnology, and medical fields. With increasing demand for proteases, there is a growing emphasis on enhancing their expression and production through microbial systems. However, proteases' native hosts often fall short in high-level expression and compatibility with downstream applications. As a result, the recombinant production of proteases has become a significant focus, offering a solution to these challenges. This review presents an overview of the current state of protease production in prokaryotic and eukaryotic expression systems, highlighting key findings and trends. In prokaryotic systems, the Bacillus spp. is the predominant host for proteinase expression. Yeasts are commonly used in eukaryotic systems. Recent advancements in protease engineering over the past five years, including rational design and directed evolution, are also highlighted. By exploring the progress in both expression systems and engineering techniques, this review provides a detailed understanding of the current landscape of recombinant protease research and its prospects for future advancements.

Potential of the quorum-quenching and plant-growth promoting halotolerant Bacillus toyonensis AA1EC1 as biocontrol agent.

The use of fertilizers and pesticides to control plant diseases is widespread in intensive farming causing adverse effects together with the development of antimicrobial resistance pathogens. As the virulence of many Gram-negative phytopathogens is controlled by N-acyl-homoserine lactones (AHLs), the enzymatic disruption of this type of quorum-sensing (QS) signal molecules, mechanism known as quorum quenching (QQ), has been proposed as a promising alternative antivirulence therapy. In this study, a novel strain of Bacillus toyonensis isolated from the halophyte plant Arthrocaulon sp. exhibited numerous traits associated with plant growth promotion (PGP) and degraded a broad range of AHLs. Three lactonases and an acylase enzymes were identified in the bacterial genome and verified in vitro. The AHL-degrading activity of strain AA1EC1 significantly attenuated the virulence of relevant phytopathogens causing reduction of soft rot symptoms on potato and carrots. In vivo assays showed that strain AA1EC1 significantly increased plant length, stem width, root and aerial dry weights and total weight of tomato and protected plants against Pseudomonas syringae pv. tomato. To our knowledge, this is the first report to demonstrate PGP and QQ activities in the species B. toyonensis that make this strain as a promising phytostimulant and biocontrol agent.

Unleashing Bacillus species as versatile antagonists: Harnessing the biocontrol potentials of the plant growth-promoting rhizobacteria to combat Macrophomina phaseolina infection in Gloriosa superba.

Charcoal rot caused by Macrophomina phaseolina is one of the most devastating diseases that cause severe yield loss in Gloriosa superba cultivation. Plant growth-promoting rhizobacteria (PGPR) are extensively harnessed as biocontrol agents due to their effectiveness in combating a wide array of plant pathogens through a multifaceted approach. The present study delved into the mechanisms underlying its ability to inhibit root rot pathogen and its capacity to promote plant growth in G. superba, commonly known as glory lily. PGPR isolated from the rhizosphere of glory lily were subjected to in vitro assessments using the dual plate technique. The isolated Bacillus subtilis BGS-10 and B. velezensis BGS-21 showed higher mycelial inhibition (61%) against M. phaseolina. These strains also promote plant growth by producing indole-3-acetic acid, siderophore, ammonia, amylase, cellulase, pectinase, xylanase, and lipase chemicals. Genome screening of BGS-10 and BGS-21 revealed the presence of antimicrobial peptide genes such as Iturin (ituD gene), surfactin (srfA and sfp genes) along with the mycolytic enzyme ß-1,3-glucanase. Further, the presence of secondary metabolites in the bacterial secretome was identified through gas chromatography-mass spectrometry (GC/MS) analysis. Notably, pyrrolo[1,2-a] pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl), 9 H-pyrido[3,4-b] indole and L-leucyl-D-leucine exhibited the highest docking score against enzymes responsible for pathogen growth and plant cell wall degradation. Under glasshouse conditions, tuber treatment and soil application of talc-based formulation of B. subtilis BGS-10 and B. velezensis BGS-21 suppress the root rot incidence with a minimal disease incidence of 27.78% over untreated control. Concurrently, there was a notable induction of defense-related enzymes, including peroxidase (PO), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL), in glory lily. Therefore, it can be concluded that plant growth-promoting Bacillus strains play a significant role in fortifying the plant's defense mechanisms against the root rot pathogen.

Investigation of non-classical secretion of oxalate decarboxylase in Bacillus mojavensis XH1 mediated by exopeptide YydF: Mechanism and application.

Non-classical secretory proteins are widely found in bacteria and have been extensively studied due to their important physiological roles. However, the relevant non-classical secretory mechanisms remain unclear. In this study, we found that oxalate decarboxylase (Bacm OxDC) from Bacillus mojavensis XH1 belongs to non-classical secretory proteins. Its N-terminus showed high hydrophilicity, which was different from the conventional signal peptide. The truncation test revealed that the deletion of the N-terminus affects the structure resulting in its inability to cross the cell membrane. Further studies verified that the exported peptide YydF played an important role in the secretion process of Bacm OxDC. Experimental results on the secretion mechanism indicated that Bacm OxDC bound to the exported peptide YydF and they are translocated to the cell membrane together, after which Bacm OxDC caused cell membrane relaxation for transmembrane secretion. Thereafter, three recombinant proteins were successfully secreted with certain enzymatic activity by fusing Bacm OxDC as a guide protein with various target proteins. To the best of our knowledge, this was the first time that non-classical secretion mechanism in bacteria has been analyzed. The novel discovery may provide a reference and broaden the horizons of the secretion pathway and expression regulation of proteins.

One-step synthesis of ferrous disulfide and iron nitride modified hydrochar for enhanced adsorption and reduction of hexavalent chromium in Bacillus LD513 by promoting electron transfer and microbial metabolism.

Microbial immobilization technology is effective in improving bioremediation efficiency and heavy metal pollution. Herein, Bacillus LD513 with hexavalent chromium (Cr(VI)) tolerance was isolated and immobilized on a novel ferrous disulfide (FeS2)/iron nitride (FeN) modified hydrochar (Fe3-SNHC) prepared from waste straws. The prepared Fe3-SNHC-based LD513 (FeLD) significantly improves Cr(VI) adsorption and reduction by 31.4 % and 15.7 %, respectively, compared to LD513 alone. Furthermore, the FeLD composite system demonstrates efficient Cr(VI) removal efficiency and good environmental adaptability under different culture conditions. Microbial metabolism and electrochemical analysis indicate that Fe3-SNHC is an ideal carrier for protecting LD513 activity, promoting extracellular polymer secretion, and reducing oxidative stress. Additionally, the carrier serves as an electron shuttle that accelerates electron transfer and promotes Cr(VI) reduction. Overall, FeLD is an environmentally friendly biocomposite that shows good promise for reducing Cr(VI) contamination in wastewater treatment.

Optimization of degradation conditions and analysis of degradation mechanism for nitrite by Bacillus aryabhattai 47.

Excessive nitrite levels cause significant damage to aquaculture, making it crucial to explore green and reliable nitrite removal technologies. In this study, A Bacillus aryabhattai (designated as the strain 47) isolated from aquaculture wastewater was used as the experimental strain. The nitrite degradation conditions of the strain 47 were optimized, and the optimal conditions are: glucose was 12.74 g/L, fermented special soybean meal was 21.27 g/L, MgCl2 369 mg/L, pH 7.0, incubated at 30 °C with the inoculum size of 2 % and the rotation speed of 170 rpm. Under the optimal conditions, the nitrite concentration of the culture solution was 200 mg/L, and the nitrite removal rate reached 91.4 %. Meanwhile, the mechanism by which Mg2+ enhanced the nitrite degradation ability of the strain 47 was investigated by transcriptomics. An operon structure directed cellular trafficking of Mg2+, and then, the Mg2+-mediated catalytic reaction of multiple enzymes enhanced and improved cellular metabolic processes (e.g. the transport and metabolism of nitrite, central carbohydrate metabolism oxidative phosphorylation). At the same time, with the progress of cell metabolism, cells secreted a series of enzymes related to nitrite transport and metabolism to promote the metabolism of nitrite. And the process of the assimilated nitrate reduction pathway of nitrite degradation in the strain 47 was elaborated at the transcriptome level. This study provided a new insight into nitrite treatment mediated by microbial organisms.

Precursor-Directed Biosynthesis of Antialgal Fluorinated Bacillamide Derivatives in Bacillus atrophaeus.

The bacillamides are a class of indole alkaloids produced by the Bacillus genus that possess significant antialgal activity. Incorporation of fluorine into the bacillamides was carried out using a precursor-directed biosynthesis approach, with 4-, 5-, and 6-fluorotryptophan added to growing cultures of Bacillus atrophaeus IMG-11. This yielded the corresponding fluorinated analogues of bacillamides A and C, in addition to new derivatives of the related metabolite N-acetyltryptamine, thus demonstrating a degree of plasticity in the bacillamide biosynthetic pathway. The bacillamide derivatives were tested for activity against bloom-forming algae, which revealed that fluorination could improve the antialgal activity of these compounds in a site-specific manner, with fluorination at the 6-position consistently resulting in improved activity.

Effects of Bacillus-based inoculum on odor emissions co-regulation, nutrient element transformations and microbial community tropological structures during chicken manure and sawdust composting.

This study aims to evaluate whether different doses of Bacillus-based inoculum inoculated in chicken manure and sawdust composting will provide distinct effects on the co-regulation of ammonia (NH3) and hydrogen sulfide (H2S), nutrient conversions and microbial topological structures. Results indicate that the Bacillus-based inoculum inhibits NH3 emissions mainly by regulating bacterial communities, while promotes H2S emissions by regulating both bacterial and fungal communities. The inoculum only has a little effect on total organic carbon (TOC) and inhibits total sulfur (TS) and total phosphorus (TP) accumulations. Low dose inoculation inhibits total potassium (TK) accumulation, while high dose inoculation promotes TK accumulation and the opposite is true for total nitrogen (TN). The inoculation slightly affects the bacterial compositions, significantly alters the fungal compositions and increases the microbial cooperation, thus influencing the compost substances transformations. The microbial communities promote ammonium nitrogen (NH4+-N), TN, available phosphorus (AP), total potassium (TK) and TS, but inhibit nitrate nitrogen (NO3--N), TP and TK. Additionally, the bacterial communities promote, while the fungal communities inhibit the nitrite nitrogen (NO2--N) production. The core bacterial and fungal genera regulate NH3 and H2S emissions through the secretions of metabolic enzymes and the promoting or inhibiting effects on NH3 and H2S emissions are always opposite. Hence, Bacillus-based inoculum cannot regulate the NH3 and H2S emissions simultaneously.

Probiotic characterization of Bacillus smithii: Research advances, concerns, and prospective trends.

Bacillus smithii is a thermophilic Bacillus that can be isolated from white wine, hot spring soil, high-temperature compost, and coffee grounds, with various biofunctions and wide applications. It is resistant to both gastric acid and high temperature, which makes it easier to perform probiotic effects than traditional commercial probiotics, so it can maintain good vitality during food processing and has great application prospects. This paper starts with the taxonomy and genetics and focuses on aspects, including genetic transformation, functional enzyme production, waste utilization, and application in the field of food science as a potential probiotic. According to available studies during the past 30 years, we considered that B. smithii is a novel class of microorganisms with a wide range of functional enzymes such as hydrolytic enzymes and hydrolases, as well as resistance to pathogenic bacteria. It is available in waste degradation, organic fertilizer production, the feed and chemical industries, the pharmaceutical sector, and food fortification. Moreover, B. smithii has great potentials for applications in the food industry, as it presents high resistance to the technological processes that guarantee its health benefits. It is also necessary to systematically evaluate the safety, flavor, and texture of B. smithii and explore its biological mechanism of action, which is of great value for further application in multiple fields, especially in food and medicine.

Complete genome sequence analysis of Bacillus velezensis A5, a promising biocontrol agent from the Pacific Ocean.

Tobacco bacterial wilt (TBW) caused by Ralstonia solanacearum is a serious soil-borne disease, which seriously damages the growth of tobacco crops. Bacillus velezensis A5 was isolated from 3000 m deep-sea sediments of the Pacific Ocean, and was found to be antagonistic to TBW. Here, we report the complete genome sequence of strain A5, which has a 4,000,699-bp single circular chromosome with 3827 genes and a G + C content of 46.44%, 87 tRNAs, and 27 rRNAs. A total of 12 gene clusters were identified in the genome of strain A5, which were responsible for the biosynthesis of antibacterial compounds, including surfactin, bacillaene, fengycin, difficidin, bacillibactin, and bacilysin. Additionally, strain A5 was found to contain a series of genes related to the biosynthesis of carbohydrate-active enzymes and secreted proteins. Our results indicate that strain A5 can be considered a promising biocontrol agent against TBW in agricultural fields.

Bacillus sp. G2112 Detoxifies Phenazine-1-carboxylic Acid by N5 Glucosylation.

Microbial symbionts of plants constitute promising sources of biocontrol organisms to fight plant pathogens. Bacillus sp. G2112 and Pseudomonas sp. G124 isolated from cucumber (Cucumis sativus) leaves inhibited the plant pathogens Erwinia and Fusarium. When Bacillus sp. G2112 and Pseudomonas sp. G124 were co-cultivated, a red halo appeared around Bacillus sp. G2112 colonies. Metabolite profiling using liquid chromatography coupled to UV and mass spectrometry revealed that the antibiotic phenazine-1-carboxylic acid (PCA) released by Pseudomonas sp. G124 was transformed by Bacillus sp. G2112 to red pigments. In the presence of PCA (>40 µg/mL), Bacillus sp. G2112 could not grow. However, already-grown Bacillus sp. G2112 (OD600 > 1.0) survived PCA treatment, converting it to red pigments. These pigments were purified by reverse-phase chromatography, and identified by high-resolution mass spectrometry, NMR, and chemical degradation as unprecedented 5N-glucosylated phenazine derivatives: 7-imino-5N-(1'ß-D-glucopyranosyl)-5,7-dihydrophenazine-1-carboxylic acid and 3-imino-5N-(1'ß-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid. 3-imino-5N-(1'ß-D-glucopyranosyl)-3,5-dihydrophenazine-1-carboxylic acid did not inhibit Bacillus sp. G2112, proving that the observed modification constitutes a resistance mechanism. The coexistence of microorganisms-especially under natural/field conditions-calls for such adaptations, such as PCA inactivation, but these can weaken the potential of the producing organism against pathogens and should be considered during the development of biocontrol strategies.

Production of Pumilarin and a Novel Circular Bacteriocin, Altitudin A, by Bacillus altitudinis ECC22, a Soil-Derived Bacteriocin Producer.

The rise of antimicrobial resistance poses a significant global health threat, necessitating urgent efforts to identify novel antimicrobial agents. In this study, we undertook a thorough screening of soil-derived bacterial isolates to identify candidates showing antimicrobial activity against Gram-positive bacteria. A highly active antagonistic isolate was initially identified as Bacillus altitudinis ECC22, being further subjected to whole genome sequencing. A bioinformatic analysis of the B. altitudinis ECC22 genome revealed the presence of two gene clusters responsible for synthesizing two circular bacteriocins: pumilarin and a novel circular bacteriocin named altitudin A, alongside a closticin 574-like bacteriocin (CLB) structural gene. The synthesis and antimicrobial activity of the bacteriocins, pumilarin and altitudin A, were evaluated and validated using an in vitro cell-free protein synthesis (IV-CFPS) protocol coupled to a split-intein-mediated ligation procedure, as well as through their in vivo production by recombinant E. coli cells. However, the IV-CFPS of CLB showed no antimicrobial activity against the bacterial indicators tested. The purification of the bacteriocins produced by B. altitudinis ECC22, and their evaluation by MALDI-TOF MS analysis and LC-MS/MS-derived targeted proteomics identification combined with massive peptide analysis, confirmed the production and circular conformation of pumilarin and altitudin A. Both bacteriocins exhibited a spectrum of activity primarily directed against other Bacillus spp. strains. Structural three-dimensional predictions revealed that pumilarin and altitudin A may adopt a circular conformation with five- and four-α-helices, respectively.

2,4,6-Triphenyl-1-hexene, an Anti-Melanogenic Compound from Marine-Derived Bacillus sp. APmarine135.

Although melanin protects against ultraviolet radiation, its overproduction causes freckles and senile lentigines. Recently, various biological effects of metabolites derived from marine microorganisms have been highlighted due to their potential for biological and pharmacological applications. In this study, we discovered the anti-melanogenic effect of Bacillus sp. APmarine135 and verified the skin-whitening effect. Fractions of APmarine135 showed the melanin synthesis inhibition effect in B16 melanoma cells, and 2,4,6-triphenyl-1-hexene was identified as an active compound. The melanogenic capacity of 2,4,6-triphenyl-1-hexene (1) was investigated by assessing the intracellular melanin content in B16 cells. Treatment with 5 ppm of 2,4,6-triphenyl-1-hexene (1) for 72 h suppressed the α-melanocyte-stimulating hormone (α-MSH)-induced intracellular melanin increase to the same level as in the untreated control group. Additionally, 2,4,6-triphenyl-1-hexene (1) treatment suppressed the activity of tyrosinase, the rate-limiting enzyme for melanogenesis. Moreover, 2,4,6-triphenyl-1-hexene (1) treatment downregulated tyrosinase, Tyrp-1, and Tyrp-2 expression by inhibiting the microphthalmia-associated transcription factor (MITF). Furthermore, 2,4,6-triphenyl-1-hexene (1) treatment decreased the melanin content in the three-dimensional (3D) human-pigmented epidermis model MelanoDerm and exerted skin-whitening effects. Mechanistically, 2,4,6-triphenyl-1-hexene (1) exerted anti-melanogenic effects by suppressing tyrosinase, Tyrp-1, and Tyrp-2 expression and activities via inhibition of the MITF. Collectively, these findings suggest that 2,4,6-triphenyl-1-hexene (1) is a promising anti-melanogenic agent in the cosmetic industry.