Characterization of Bacillus amyloliquefaciens PM415 as a potential bio-preserving probiotic.

Animal feed is vulnerable to fungal infections, and the use of bio-preserving probiotics has received increasing attention. In contrast to Lactobacillus and Bifidobacteria spp., fewer Bacillus spp. have been recognized as antifungal probiotics. Therefore, our objective was to screen antifungal strains and provide more Bacillus candidates to bridge this gap. Here, we screened 56 bacterial strains for cyclic lipopeptide genes and conducted an antifungal assay with Aspergillus niger as a representative fungus. We found that a Bacillus strain Bacillus amyloliquefaciens PM415, isolated from pigeon manure, exhibited the highest fungal inhibition activity as demonstrated by the confrontation assay and morphological observation under scanning electron microscope (SEM). Preliminary safety assessment and probiotic characterization revealed its non-pathogenic feature and stress tolerance capability. Whole genome sequencing of Bacillus amyloliquefaciens PM415 revealed a genome size of 4.16 Mbp and 84 housekeeping genes thereof were used for phylogenetic analysis showing that it is most closely related to Bacillus amyloliquefaciens LFB112. The in silico analysis further supported its non-pathogenic feature at the genomic level and revealed potential biosynthetic gene clusters responsible for its antifungal property. RNA-seq analysis revealed genome-wide changes in transportation, amino acid metabolism, non-ribosomal peptides (NRPs) biosynthesis and glycan degradation during fungal antagonism. Our results suggest that Bacillus amyloliquefaciens PM415 is a safe and effective probiotic strain that can prevent fungal growth in animal feeds.

Effect of Bacillus amyloliquefaciens LSG2-8 on the intestinal barrier function of Amur minnow (Rhynchocypris lagowskii).

Probiotics are widely used in aquaculture. This article aims to study the effect of Bacillus amyloliquefaciens LSG2-8 on the intestinal barrier function of Rhynchocypris lagowskii. B. amyloliquefaciens LSG2-8 were added to R. lagowskii basal diets (CK) as additives at four concentrations: 1.0 × 106 (D-6), 1.0 × 107 (D-7), 1.0 × 108 (D-8) and 1.0 × 109 (D-9) CFU g-1 by dry weight of basal diet. After a 56-day feeding experiment, the activities of intestinal digestive enzymes and immunity-related enzymes of R. lagowskii on group D-6, D-7, D-8 and D-9 diet were significantly higher than the control (P < 0.05). In molecular experiments, the authors found that the levels of TGF-ß mRNA, IL-10 mRNA, ZO-1 mRNA and claudin-3 mRNA in group D-8 R. lagowskii were significantly higher (P < 0.05) than those of the control and other groups. Furthermore, the levels of IL-1ß and IL-8 mRNA of R. lagowskii on group D-6, D-7, D-8 and D-9 diet were significantly lower than those of the control (P < 0.05). In addition, the authors found that B. amyloliquefaciens LSG2-8 can regulate the intestinal flora balance and improve the intestinal structure of R. lagowskii. In conclusion, B. amyloliquefaciens LSG2-8 can improve the intestinal barrier function of R. lagowskii and can be used as a feed additive in aquaculture.

Transcriptome Analysis of Bacillus amyloliquefaciens Reveals Fructose Addition Effects on Fengycin Synthesis.

Fengycin is a lipopeptide produced by Bacillus that has a strong inhibitory effect on filamentous fungi; however, its use is restricted due to poor production and low yield. Previous studies have shown that fengycin biosynthesis in B. amyloliquefaciens was found to be significantly increased after fructose addition. This study investigated the effect of fructose on fengycin production and its regulation mechanism in B. amyloliquefaciens by transcriptome sequencing. According to the RNA sequencing data, 458 genes were upregulated and 879 genes were downregulated. Transcriptome analysis results showed that fructose changed the transcription of amino acid synthesis, fatty acid metabolism, and energy metabolism; alterations in these metabolic pathways contribute to the synthesis of fengycin. In an MLF medium (modified Landy medium with fructose), the expression level of the fengycin operon was two-times higher than in an ML medium (modified Landy medium). After fructose was added to B. amyloliquefaciens, the fengycin-synthesis-associated genes were activated in the process of fengycin synthesis.

Draft genome sequence of a lipopeptide-producing Bacillus amyloliquefaciens strain isolated from wheat field soil with antagonistic activity against azole-resistant Fusarium graminearum.

OBJECTIVES: Lipopeptides have been revealed as good potential biocontrol agents against various pathogenic microbes. In the present work, we report the draft genome sequence of a lipopeptide-producing strain of Bacillus amyloliquefaciens (7D3) that showed good antifungal activity against the azole-resistant pathogenic fungus Fusarium graminearum. METHODS: Whole-genome sequencing of strain 7D3 was performed on an Illumina MiSeq 300 platform. Raw data were cleaned using Trim Galore v.0.4.0 and were checked for quality using FastQC. De novo assembly was performed using the SOAPdenovo2 package. Genes responsible for the biosynthesis of secondary metabolites were identified using antiSMASH. RESULTS: Bacillus amyloliquefaciens 7D3 genome assembly resulted in a total genome size of 3 913 220 bp with a G+C content of 46.13%. There were 3998 predicted genes with 72 tRNAs and 9 rRNAs. A total of ten gene clusters were found to be related to secondary metabolite biosynthesis, of which five were identified as lipopeptide synthesis clusters. CONCLUSION: This study presents the genome sequence of B. amyloliquefaciens 7D3, which exhibited intense antagonistic activity against azole-resistant fungi. The whole genome sequence will help in the search for novel antifungal peptides against drug-resistant pathogens.

A Thermotolerant Marine Bacillus amyloliquefaciens S185 Producing Iturin A5 for Antifungal Activity against Fusarium oxysporum f. sp. cubense.

Fusarium wilt of banana (also known as Panama disease), is a severe fungal disease caused by soil-borne Fusarium oxysporum f. sp. cubense (Foc). In recent years, biocontrol strategies using antifungal microorganisms from various niches and their related bioactive compounds have been used to prevent and control Panama disease. Here, a thermotolerant marine strain S185 was identified as Bacillus amyloliquefaciens, displaying strong antifungal activity against Foc. The strain S185 possesses multiple plant growth-promoting (PGP) and biocontrol utility properties, such as producing indole acetic acid (IAA) and ammonia, assimilating various carbon sources, tolerating pH of 4 to 9, temperature of 20 to 50 °C, and salt stress of 1 to 5%. Inoculation of S185 colonized the banana plants effectively and was mainly located in leaf and root tissues. To further investigate the antifungal components, compounds were extracted, fractionated, and purified. One compound, inhibiting Foc with minimum inhibitory concentrations (MICs) of 25 µg/disk, was identified as iturin A5 by high-resolution electrospray ionization mass spectrometry (HR-ESI-MS) and nuclear magnetic resonance (NMR). The isolated iturin, A5, resulted in severe morphological changes during spore germination and hyphae growth of Foc. These results specify that B. amyloliquefaciens S185 plays a key role in preventing the Foc pathogen by producing the antifungal compound iturin A5, and possesses potential as a cost-effective and sustainable biocontrol strain for Panama disease in the future. This is the first report of isolation of the antifungal compound iturin A5 from thermotolerant marine B. amyloliquefaciens S185.

Direct Antibiotic Activity of Bacillibactin Broadens the Biocontrol Range of Bacillus amyloliquefaciens MBI600.

Bacillus amyloliquefaciens is considered the most successful biological control agent due to its ability to colonize the plant rhizosphere and phyllosphere where it outgrows plant pathogens by competition, antibiosis, and inducing plant defense. Its antimicrobial function is thought to depend on a diverse spectrum of secondary metabolites, including peptides, cyclic lipopeptides, and polyketides, which have been shown to target mostly fungal pathogens. In this study, we isolated and characterized the catecholate siderophore bacillibactin by B. amyloliquefaciens MBI600 under iron-limiting conditions and we further identified its potential antibiotic activity against plant pathogens. Our data show that bacillibactin production restrained in vitro and in planta growth of the nonsusceptible (to MBI600) pathogen Pseudomonas syringae pv. tomato. Notably, it was also related to increased antifungal activity of MBI600. In addition to bacillibactin biosynthesis, iron starvation led to upregulation of specific genes involved in microbial fitness and competition. IMPORTANCE Siderophores have mostly been studied concerning their contribution to the fitness and virulence of bacterial pathogens. In the present work, we isolated and characterized for the first time the siderophore bacillibactin from a commercial bacterial biocontrol agent. We proved that its presence in the culture broth has significant biocontrol activity against nonsusceptible bacterial and fungal phytopathogens. In addition, we suggest that its activity is due to a new mechanism of action, that of direct antibiosis, rather than by competition through iron scavenging. Furthermore, we showed that bacillibactin biosynthesis is coregulated with the transcription of antimicrobial metabolite synthases and fitness regulatory genes that maximize competition capability. Finally, this work highlights that the efficiency and range of existing bacterial biocontrol agents can be improved and broadened via the rational modification of the growth conditions of biocontrol organisms.

Improving the catalytic characteristics of phenolic acid decarboxylase from Bacillus amyloliquefaciens by the engineering of N-terminus and C-terminus.

BACKGROUND: 4-vinylphenols produced by phenolic acid degradation catalyzed by phenolic acid decarboxylase can be used in food additives as well as flavor and fragrance industry. Improving the catalytic characters of phenolic acid decarboxylase is of great significance to enhance its practical application. RESULTS: A phenolic acid decarboxylase (P-WT) was created from Bacillus amyloliquefaciens ZJH-01. Mutants such as P-C, P-N, P-m1, P-m2, P-Nm1, and P-Nm2 were constructed by site-directed mutagenesis of P-WT. P-C showed better substrate affinities and higher turnover rates than P-WT for p-coumaric acid, ferulic acid, and sinapic acid; however, P-N had reduced affinity toward p-coumaric acid. The extension of the C-terminus increased its acid resistance, whereas the extension of the N-terminus contributed to the alkali resistance and heat resistance. The affinity of P-m1 to four substrates and that of P-m2 to p-coumaric acid and ferulic acid were greatly improved. However, the affinity of P-Nm2 to four phenolic acids was greatly reduced. The residual enzyme activities of P-Nm1 and P-Nm2 considerably improved compared with those of P-m1 and P-m2 after incubation at 50 °C for 60 min. CONCLUSIONS: The extension of the N-terminus may be more conducive to the combination of the binding cavity with the substrate in an alkaline environment and may make its structure more stable.

New Class of Chitosanase from Bacillus amyloliquefaciens for the Generation of Chitooligosaccharides.

Chitooligosaccharides (COS) generated from either chitin (chitin oligosaccharides) or chitosan (chitosan oligosaccharides) have a wide range of applications in agriculture, medicine, and other fields. Here, we report the characterization of a chitosanase from Bacillus amyloliquefaciens (BamCsn) and the importance of a tryptophan (Trp), W204, for BamCsn activity. BamCsn hydrolyzed the chitosan polymer by an endo mode. It also hydrolyzed chitin oligosaccharides and interestingly exhibited transglycosylation activity on chitotetraose and chitopentaose. Mutation of W204, a nonconserved amino acid in chitosanases, to W204A abolished the hydrolytic activity of BamCsn, with a change in the structure that resulted in a decreased affinity for the substrate and impaired the catalytic ability. Phylogenetic analysis revealed that BamCsn could belong to a new class of chitosanases that showed unique properties like transglycosylation, cleavage of chitin oligosaccharides, and the presence of W204 residues, which is important for activity. Chitosanases belonging to the BamCsn class showed a high potential to generate COS from chitinous substrates.

Seaweed-associated heterotrophic bacteria: new paradigm of prospective anti-infective and anticancer agents.

Ever since the development of the first antibiotic compound with anticancer potential, researchers focused on isolation and characterization of prospective microbial natural products with potential anti-infective and anticancer activities. The present work describes the production of bioactive metabolites by heterotrophic bacteria associated with intertidal seaweeds with potential anti-infective and anticancer activities. The bacteria were isolated in a culture-dependent method and were identified as Shewanella algae MTCC 12715 (KX272635) and Bacillus amyloliquefaciens MTCC 12716 (KX272634) based on combined phenotypic and genotypic methods. Further, the bacteria were screened for their ability to inhibit drug-resistant infectious pathogens and prevent cell proliferation of human liver carcinoma (HepG2) and breast cancer (MCF7) cell lines, without affecting the normal cells. Significant anti-infective activity was observed with bacterial cells and their organic extracts against broad-spectrum multidrug-resistant pathogens, such as vancomycin-resistant Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia and Pseudomonas aeruginosa with minimum inhibitory concentration ≤ 3.0 µg mL-1 as compared to the antibiotic agents' chloramphenicol and ampicillin, which were active at ≥ 6.25 mg mL-1. The extracts also exhibited anticancer activity in a dose-responsive pattern against HepG2 (with IC50, half maximal inhibitory concentration ~ 78-83 µg mL-1) and MCF7 (IC50 ~ 45-48 µg mL-1) on tetrazolium bromide screening assay with lesser cytotoxic effects on normal fibroblast (L929) cell lines (IC50 > 100 µg mL-1). The results revealed that seaweed-associated heterotrophic bacteria could occupy a predominant role for a paradigm shift towards the development of prospective anti-infective and anticancer agents.

Prebiotic, Probiotic, Antimicrobial, and Functional Food Applications of Bacillus amyloliquefaciens.

Bacillus amyloliquefaciens belongs to the genus Bacillus and family Baciliaceae. It is ubiquitously found in food, plants, animals, soil, and in different environments. In this review, the application of B. amyloliquefaciens in probiotic and prebiotic microbes in fermentation, synthesis, and hydrolysis of food compounds is discussed as well as further insights into its potential application and gaps. B. amyloliquefaciens is also a potential microbe in the synthesis of bioactive compounds including peptides and exopolysaccharides. In addition, it can synthesize antimicrobial compounds (e.g., Fengycin, and Bacillomycin Lb), which makes its novelty in the food sector greater. Moreover, it imparts and improves the functional, sensory, and shelf life of the end products. The hydrolysis of complex compounds including insoluble proteins, carbohydrates, fibers, hemicellulose, and lignans also shows that B. amyloliquefaciens is a multifunctional and potential microbe which can be applied in the food industry and in functional food processing.

Comparative transcriptome analysis of Sclerotinia sclerotiorum revealed its response mechanisms to the biological control agent, Bacillus amyloliquefaciens.

Biological control mechanisms of plant diseases have been intensively studied. However, how plant pathogens respond to and resist or alleviate biocontrol agents remains largely unknown. In this study, a comparative transcriptome analysis was performed to elucidate how the pathogen of sclerotinia stem rot, Sclerotinia sclerotiorum, responds and resists to the biocontrol agent, Bacillus amyloliquefaciens. Results revealed that a total of 2,373 genes were differentially expressed in S. sclerotiorum samples treated with B. amyloliquefaciens fermentation broth (TS) when compared to control samples (CS). Among these genes, 2,017 were upregulated and 356 were downregulated. Further analyses indicated that various genes related to fungal cell wall and cell membrane synthesis, antioxidants, and the autophagy pathway were significantly upregulated, including glucan synthesis, ergosterol biosynthesis pathway, fatty acid synthase, heme-binding peroxidase related to oxidative stress, glutathione S-transferase, ABC transporter, and autophagy-related genes. These results suggest that S. sclerotiorum recruits numerous genes to respond to or resist the biocontrol of B. amyloliquefaciens. Thus, this study serves as a valuable resource regarding the mechanisms of fungal pathogen resistance to biocontrol agents.

Moving away from traditional antibiotic treatment: can macrocyclic lactones from marine macroalga-associated heterotroph be the alternatives?

Intertidal red algae Hypnea valentiae associated Bacillus amyloliquefaciens MTCC 12716 revealed potential inhibitory effects on the growth of drug-resistant pathogens. In the genome of B. amyloliquefaciens MTCC 12716, biosynthetic gene clusters encoding antibacterial metabolites were predicted, which might be expressed and contributed to the broad-spectrum anti-infective activity. Three homologue members of the 24-membered macrocyclic lactone family, named as bacvalactones 1-3 bearing 13-O-ethyl (1); 15-O-furanyl-13-O-isobutyl-7-O-propyl-propanoate (2); and 15-O-furanyl-13-O-isobutyl-7-O-propyl-propanoate-7,24-dimethyl (3) functionalities, were acquired through bioactivity-guided purification. The macrocyclic lactones displayed bactericidal activity against opportunistic pathogens causing nosocomial infections, for instance, methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis (VREfs), and multidrug-resistant strains of Pseudomonas aeruginosa and Klebsiella pneumonia with MIC ≤ 3.0 µg/mL, whereas standard antibiotics ampicillin and chloramphenicol were active only at concentrations of ≥ 6.25 mg/mL. The biosynthetic pathway of macrocyclic lactones that are generated by trans-AT polyketide synthases through stepwise extension of an acetyl starter unit by eleven sequential Claisen condensations with malonyl-CoA was established, and the structures were correlated with the gene organization of the mln operon, which encompasses nine genes mln A-I (approximately 47 kb in size). The best binding poses for each compounds (1-3) with Staphylococcus aureus peptide deformylase (SaPDF) unveiled docking scores (≥ 9.70 kcal/mol) greater than that of natural peptide deformylase inhibitors, macrolactin N and actinonin (9.14 and 6.96 kcal/mol, respectively), which supported their potential in vitro bioactivities. Thus, the present work demonstrated the potential of macrocyclic lactone for biotechnological and pharmaceutical applications against emerging multidrug-resistant pathogens. Key Points •Three antibacterial bacvalactones were identified from the symbiotic bacterium. •The symbiotic bacterial genome was explored to identify the biosynthetic gene clusters. •Trans-AT pks-assisted mln biosynthetic pathway of the macrocyclic lactone was proposed. •In silicomolecular interactions of the bacvalactones with S. aureus PDF were analyzed.

Plant-bacterial endophyte secondary metabolite matching: a case study.

Some studies focused on metabolic relationships between plants and their endophytic bacterial associates, and more research is required to generate critical evidence for these relationships. In the current interest, we tried to confirm the relationship between the traditional medicinal plant, Calotropis procera (Aiton) W.T. Aiton, and its associated endophytes, Bacillus siamensis and Bacillus amyloliquefaciens, as the first matching study regarding the production of bioactive secondary metabolites from the plant vis-a-vis its bacterial endophytes.Secondary metabolites of both the plant and its endophytic bacteria were extracted using different solvents, e.g., water, methanol, and ethyl acetate. All extracts exhibited high quantities of phenolics, flavonoids, tannins, and saponins. In addition, they showed significant antioxidant capacity which was found to be positively correlated with total phenolic contents. The highest total antioxidant capacity (99.28 ± 0.0 mg AA equivalent/g extract) was measured for the aqueous extract of B. siamensis.Antibacterial activity of the different extracts was evaluated against certain pathogenic bacteria, i.e., Escherichia coli, Klebsiella pneumoniae, Streptococcus agalactiae, Salmonella typhi, Serratia marcescens, and Staphylococcus aureus. It was strikingly found that the broadest antibacterial spectrum was revealed by extracts of both C. procera and its endophytic B. siamensis. Interestingly, antibacterial activity was significantly correlated to phenolic and flavonoid contents.

Dietary Bacillus amyloliquefaciens enhance survival of white spot syndrome virus infected crayfish.

Bacillus amyloliquefaciens, which is closely related to Bacillus subtilis, produces a series of metabolites that can inhibit the growth of fungi and bacteria. Here, we investigated the effect of B. amyloliquefaciens used as a probiotic on the innate immunity of the crayfish Procambarus clarkii when challenged with white spot syndrome virus (WSSV). Dietary B. amyloliquefaciens supplement significantly reduced the mortality of WSSV-challenged crayfish and reduced copy numbers of WSSV. The quantitative reverse transcription-polymerase chain reaction results showed that B. amyloliquefaciens supplement increased the expression of several immune-related genes, including Toll-like receptor, NF-κB and C-type-lectin. Further analysis showed that B. amyloliquefaciens supplement also had an effect on three immune parameters, including total hemocyte count, phenoloxidase activity and superoxide dismutase activity. In both infected and uninfected crayfish, B. amyloliquefaciens supplement significantly decreased hemocyte apoptosis. Our results showed that B. amyloliquefaciens can regulate innate immunity of crayfish and reduce the mortality following WSSV challenge. This study provides a novel insight into the potential for therapeutic or prophylactic intervention with B. amyloliquefaciens to regulate crayfish immunity and protect against WSSV infection, and also provides a theoretical basis for the use of probiotics as aquatic feed additives.

Preparation of phosphatidylcholine nanovesicles containing bacteriocin CAMT2 and their anti-listerial activity.

A novel bacteriocin CAMT2, produced by Bacillus amyloliquefaciens ZJHD3-06, has potential as a natural biopreservative for the control of food-borne spoilage and pathogenic bacteria. To avoid interaction of CAMT2 with components of food that may adversely impact its antibacterial activity, CAMT2 was encapsulated into nanovesicles prepared from soybean phosphatidylcholine. The encapsulation of CAMT2 exhibited a limited impact on functional structure and crystallinity of bacteriocin CAMT2, but a high anti-listerial activity in agar, and increase its stability in food at refrigeration temperature (4 °C). The results also showed that both encapsulated and free CAMT2 had good anti-listerial effect in skim milk at refrigeration temperature. However, encapsulated CAMT2 performed better than free CAMT2 against Listeria in whole milk. These results showed that nano-encapsulation is an effective method of protecting bacteriocin from fat in milk and retaining its antimicrobial efficacy.

Potential Probiotics Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895 Co-Aggregate with Clinical Isolates of Proteus mirabilis and Prevent Biofilm Formation.

A urinary tract infection (UTI) is a multi-factorial disease including cystitis, pyelonephritis, and pyelitis. After Escherichia coli, Proteus mirabilis is the most common UTI-associated opportunistic pathogen. Antibiotic resistance of bacteria and infection recurrence can be connected to biofilm formation by P. mirabilis. In this study, human and sheep isolates of P. mirabilis were investigated for antibiotic sensitivity using an antibiotic disk test. Co-aggregation of the tested potential probiotic bacilli, Bacillus amyloliquefaciens B-1895 and Bacillus subtilis KATMIRA1933, with the isolated pathogen was also evaluated. Then, the anti-biofilm activity of naturally derived metabolites, such as subtilin and subtilosin, in the bacilli-free supernatants was assessed against biofilms of P. mirabilis isolates. The isolated pathogens were sensitive to 30 µg of amikacin and 5 µg of ciprofloxacin but resistant to other tested antibiotics. After 24 h, auto-aggregation of B. amyloliquefaciens B-1895 was at 89.5% and higher than auto-aggregation of B. subtilis KATMIRA1933 (59.5%). B. amyloliquefaciens B-1895 strongly co-aggregated with P. mirabilis isolates from human UTIs. Cell-free supernatants of B. amyloliquefaciens B-1895 and B. subtilis KATMIRA1933 showed higher antimicrobial activity against biofilms of P. mirabilis isolated from humans as compared with biofilms of sheep isolates. According to our knowledge, this is the first report evaluating the anti-biofilm activity of probiotic spore-forming bacilli against clinical and animal UTI isolates of P. mirabilis. Further studies are recommended to investigate the anti-biofilm activity and the mode of action for the antimicrobial substances produced by these bacilli, subtilosin and subtilin.

In silico identification and characterization of antineoplastic asparaginase enzyme from endophytic bacteria.

It is generally accepted that L-asparagine is an important amino acid required for the fast growth of cells. Cancerous cells receive this amino acid from extracellular sources. The depletion of L-asparagine from its surrounding environments by asparaginase enzyme can be used as a therapeutic strategy in cancer patients. This therapeutic enzyme is produced commercially mainly from bacteria such as Escherichia coli and Erwinia chrysanthemi. The side effects of such drugs have persuaded scientists to find new enzyme sources. In this study, in silico approach was applied to investigate L-asparaginase producing endophytic bacteria that produce more compatible enzymes within the body. Protein-protein basic local alignment search tool with E. coli and E. chrysanthemi asparaginase enzyme sequences against 262 endophytic bacteria were performed. The results with identity more than 35%, coverage more than 80%, and E-value less than 10-4 were selected. Then, some of bioinformatics tools were used to characterize them. A total of nine sequences consisting of seven known and two hypothetical proteins were identified in six bacterial species. The results showed that some of the asparaginase enzymes produced by endophytic bacteria possess more suitable immunological indices compared with asparaginase enzymes of E. coli and E. chrysanthemi. Herbaspirillum rubrisubalbicans was predicted to produce a nonallergen and nonantigen asparaginase enzyme. The number of antigenic determinants was predicted to be lower in asparaginase enzymes produced by Bacillus amyloliquefaciens, H. rubrisubalbicans, and H. seropedicae. Moreover, the number of high-scored B-cell epitopes was lower in enzyme sequences related to the mentioned bacteria and Paenibacillus polymyxa. The number of discontinuous epitopes and the number of T-cell epitopes were lower in B. amyloliquefaciens produced enzymes. Therefore, the therapeutic use of these enzymes is possible.

Oxygenated elansolid-type of polyketide spanned macrolides from a marine heterotrophic Bacillus as prospective antimicrobial agents against multidrug-resistant pathogens.

Three homologous oxygenated elansolid-type of polyketide spanned macrolides were isolated from a heterotrophic marine bacterium, Bacillus amyloliquefaciens MTCC 12716, associated with an intertidal red alga Hypnea valentiae. The complete genome of the bacterium was sequenced and all detectable natural product gene clusters were analysed. The B. amyloliquefaciens MTCC 12716 genome features polyketide synthase (pks) systems of every known formally classified family, nonribosomal peptide synthetases and hybrid clusters. Comprehensive spectroscopic studies revealed the compounds to possess isobenzofuranyl benzoate and 1H-furopyrano[2,3-c]oxacyclononadecine-6-carboxylate moieties. The identified compounds displayed broad-spectrum bactericidal activity against methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecalis, and drug-resistant strains of Pseudomonas aeruginosa and Klebsiella pneumoniae with minimum inhibitory concentrations (MICs) of ≤1.0 µg/mL, whereas the standard antibiotics ampicillin and chloramphenicol were active only at concentrations of ≥6.25 µg/mL. The plausible mechanism of elansolid-type macrolide biosynthesis by trans-AT polyketide synthases through the pks starter unit para-hydroxybenzoic acid was hypothesised, and the structures were correlated with the gene organisation, with the predicted gene cluster comprising 16 genes (~81 kb in size). The best binding poses for each compound with the peptide deformylase (PDF) protein of S. aureus revealed docking scores (>11.30 kcal/mol) greater than actinonin (6.96 kcal/mol), a natural PDF inhibitor. The higher electronic values along with optimum lipophilic parameters support the potential anti-infective properties of the studied macrolides. These antibacterial elansolid-type of polyketide spanned macrolides in marine symbiotic B. amyloliquefaciens could be potential leads for biotechnological and pharmaceutical applications against emerging multidrug-resistant pathogens.

Characterization of New Fengycin Cyclic Lipopeptide Variants Produced by Bacillus amyloliquefaciens (ET) Originating from a Salt Lake of Eastern Algeria.

Fengycin antibiotic displays a strong antifungal activity and inhibits the growth of a wide range of plant pathogens especially filamentous fungi. The main objective of the present study is to characterize fengycin variants produced by B. amyloliquefaciens strain (ET). LC-MS analysis of fengycin extracts has shown several molecular ion peaks corresponding to conventional fengycin homologues (MH + : m/z 1463.9; 1491.9; 1506) and some new ones (MH + : m/z 1433; 1447; 1461; and 1477). Further characterization of these precursor ions was carried out by LC-MS.MS analysis. Reporter fragment ions were observed (named A and B), they correspond to the cleavage of Orn2-Tyr3 (A), Glu1-Orn2 (B), and used for identifying fengycin variants. The reporter fragment couple ions [A/B] at [m/z 966.5/1080.5] and [m/z 994.4 /1108.5] represent fengycin A and B, respectively. The diagnostic ions at ([m/z 980/1094]) may correspond to fengycin C3, D, S or B2. Interestingly, unknown diagnostic product ions at [m/z 951/1065] and [m/z 979/1093] were detected for the first time in this study which prove that they correspond to new fengycin variants, named fengycin X and fengycin Y, respectively. The fengycin X results from a substitution of the glutamine amino acid (Q), at position 8 of the fengycin A peptide part, by an isoleucine (I) or a leucine (L) residue. This mutation should be the same in fengycin Y but compared to fengycin B.

Synthesis and evaluation of a novel nanoparticle carrying urokinase used in targeted thrombolysis.

Acute thrombosis, which can lead to vessel occlusion and ischemic complication, is a major cause of death and disability from myocardial infarction and stroke in recent years. Traditional drugs used in clinical thrombolytic therapy have many disadvantages, such as, higher drug dosage, shorter halftime. Thus, an efficient, safe, noninvasive, and easy thrombolysis method is urgently needed. A novel nanometer targeted carrier of a thrombolytic drug was developed in this study to improve the efficacy of thrombolytic therapy. To prepare the carrier, the carbodiimide-mediated amide was adopted to link urokinase (UK) and Arg-Gly-Asp-Ser tetrapeptide (RGDS) to the surface of the treated Bacillus amyloliquefaciens spores (BA) to synthesize UK-BA-RGDS nanoparticles. The thrombolysis rate of UK-BA-RGDS was 87%, which was nearly fivefold higher than that of commercial UK. Additionally, the tail bleeding time of UK-BA-RGDS was decreased compared with commercial UK (601 vs. 1,145 s). Furthermore, the blood flow velocity at the thrombus was recovered to ~80% of the initial state in 80 min after injection of UK-BA-RGDS. These results might indicate that the UK-BA-RGDS nanoparticles may be an effective targeted drug for the treatment of thromboembolism without hemorrhagic complication. Besides, the BA nanoparticle is a potential carrier of other drugs a wide range of possible applications.