Mechanistic basis of substrate-O2 coupling within a chitin-active lytic polysaccharide monooxygenase: An integrated NMR/EPR study.

Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x2-y2) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O2 activation in chitin-active AA10 LPMOs.

Stability and cytotoxicity of DPPH inhibitory peptides derived from biodegradation of chicken feather.

The antioxidant activity and cell viability of feather hydrolysates obtained with the Bacillus licheniformis were evaluated using an in-vitro model. The results indicate that feathers-derived peptides under 3 kDa have antioxidant activity with IC50 values of 5.03 ± 0.215 mg/mL by using DPPH antioxidant assay. Although the antioxidant activity of the peptides under 3 kDa preserved after applying diverse heating (from 20 to 100 °C), they lost their activity under strongly acidic or alkaline conditions. Antioxidant activity of the mixed feather bioactive peptides (MFBPs) obtained with partial purification of peptides under 3 kDa was with IC50 amount of 0.169 mg/mL ± 0.004 using DPPH radical scavenging assay. Also, MFBPs within an amount range of from 0.0048 to 5.0 mg/mL, illustrated no cytotoxicity to gingival fibroblast blood cell lines. In light of our results, the obtained value-added peptides could be useful in different food products as a future functional ingredient with antioxidant potency.

Stability of BSE infectivity towards heat treatment even after proteolytic removal of prion protein.

The unconventional infectious agents of transmissible spongiform encephalopathies (TSEs) are prions. Their infectivity co-appears with PrPSc, aberrant depositions of the host's cellular prion protein (PrPC). Successive heat treatment in the presence of detergent and proteolysis by a keratinase from Bacillus licheniformis PWD-1 was shown before to destroy PrPSc from bovine TSE (BSE) and sheep scrapie diseased brain, however data regarding expected reduction of infectivity were still lacking. Therefore, transgenic Tgbov XV mice which are highly BSE susceptible were used to quantify infectivity before and after the bovine brain treatment procedure. Also four immunochemical analyses were applied to compare the levels of PrPSc. After heating at 115 °C with or without subsequent proteolysis, the original BSE infectivity of 106.2-6.4 ID50 g-1 was reduced to a remaining infectivity of 104.6-5.7 ID50 g-1 while strain characteristics were unaltered, even after precipitation with methanol. Surprisingly, PrPSc depletion was 5-800 times higher than the loss of infectivity. Similar treatment was applied on other prion strains, which were CWD1 in bank voles, 263 K scrapie in hamsters and sheep PG127 scrapie in tg338 ovinized mice. In these strains however, infectivity was already destroyed by heat only. These findings show the unusual heat resistance of BSE and support a role for an additional factor in prion formation as suggested elsewhere when producing prions from PrPC. Leftover material in the remaining PrPSc depleted BSE preparation offers a unique substrate for searching additional elements for prion infectivity and improving our concept about the nature of prions.

Fructanogenic traits in halotolerant Bacillus licheniformis OK12 and their predicted functional significance.

AIMS: Isolating a novel bacterial source of fructan from a saltern and analysis of its genome to better understand the possible roles of fructans in hypersaline environments. METHODS AND RESULTS: Bacteria were isolated from crude salt samples originating from Çamalti Saltern in Western Turkey and screened for fructanogenic traits in high-salt and sucrose-rich selective medium. Exopolysaccharide accumulated in the presence of sucrose by isolate OK12 was purified and chemically characterized via HPLC, FT-IR and NMR, which revealed that it was a levan-type fructan (ß-2,6 linked homopolymer of fructose). The isolate was taxonomically classified as Bacillus licheniformis OK12 through 16S rRNA gene and whole-genome sequencing methods. Strain OK12 harbours one levansucrase and two different levanase genes, which altogether were predicted to significantly contribute to intracellular glucose and fructose pools. The isolate could withstand 15% NaCl, and thus classified as a halotolerant. CONCLUSIONS: Fructanogenic traits in halotolerant B. licheniformis OK12 are significant due to predicted influx of glucose and fructose as a result of levan biosynthesis and levan hydrolysis, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Fructans from the residents of hypersaline habitats are underexplored compounds and are expected to demonstrate physicochemical properties different from their non-halophilic counterparts. Revealing fructanogenic traits in the genome of a halotolerant bacterium brings up a new perspective in physiological roles of fructans.

Effects of Bacillus licheniformis on the growth, antioxidant capacity, intestinal barrier and disease resistance of grass carp (Ctenopharyngodon idella).

To study the effect of dietary supplementation of Bacillus licheniformis FA6 on the growth, survival and intestinal health of grass carp, we assessed the antioxidant capacity, intestinal barrier, expression levels of immune genes, and the resistance to Aeromonas hydrophila AH-1 infection. Experimental setup comprised three groups (90 specimens each; average initial weight = 16.5 g): the control group was fed the basal diet without B. licheniformis, the low-dose (LD) group was supplemented with B. licheniformis at the concentration of 1 × 105 cfu/g, and the high-dose (HD) group with 1 × 106 cfu/g. After 56 days of growth trial, the challenge test with A. hydrophila AH-1 was conducted for 14 days. The results revealed that the grass carp in LD group and HD group had significantly (p < 0.05) improved percent weight gain (PWG) and specific growth rate (SGR) parameters. Additionally, the antioxidant status was improved, which included increased superoxide dismutase (SOD) activity and decreased malondialdehyde (MDA) levels in the serum, and upregulated mRNA levels of antioxidant enzymes MnSOD and catalase (CAT) in the intestine. Meanwhile, B. licheniformis FA6 supplementation groups exhibited a decreased mRNA expression of proinflammatory cytokines (such as IL-1ß and TNF-α) and increased the expression of anti-inflammatory cytokine IL-10. Histological (villi length was increased) and gene expression (qPCR: upregulated ZO-1, occludin, and claudin-c) analyses suggested improved functioning of the intestinal barrier. Post-challenge mortality rates in LD and HD groups were significantly lower (56.6% and 70% respectively) than in the control group (100%). Overall, these results indicated that dietary supplementation of B. licheniformis FA6 can improve growth, antioxidant capacity, intestinal barrier functions and disease resistance of grass carp.

Effects of Bacillus lincheniformis feeding frequency on the growth, digestion and immunity of Haliotis discus hannai.

To study the effects of Bacillus lincheniformis feeding frequency on the survival and growth of Haliotis discus hannai abalone, we measured the expression levels of nonspecific immune genes and monitored the anti-Vibrio parahaemolyticus immune reaction. H. discus hannai (shell length: 32.75 ± 2.63 mm, body weight: 4.91 ± 0.34 g) was selected to perform a 70 d laboratory culture experiment including a 14 d V. parahaemolyticus artificial infection experiment. The control group (C) was fed normal commercial feed every day. The M1 experimental group was given experimental feed and basal feed on alternating days until the end of the experiment. The M2 experimental group was given experimental feed for 4 d and basal feed for 3 d, and this cycle was repeated every 7 d until the end of the experiment. The M3 experimental group was given experimental feed for 2 d and basal feed for 5 d, and this cycle was repeated every 7 d until the end of the experiment. The M4 group was continuously given experimental feed for the duration of the experiment. The concentration of added B. lincheniformis in each experimental group was 105 cfu/g (according to the quantity of viable bacteria). The specific growth rate (as measured by body weight) and the feed conversion efficiency of the abalone in M1 and M2 were significantly higher than those in M4 and C (P < 0.05). The cellulose and lipase activities of abalone in M1, M2 or M4 were significantly higher than those in M3 or C (P < 0.05). The acid phosphatase, superoxide dismutase, total haemocyte counts, O2- levels generated by respiratory bursts, and the expression levels of Mn-SOD, TPx, GSTs and GSTm in abalone in the M2 group were significantly higher than those in any other feeding frequency group (P < 0.05). At the end of the V. parahaemolyticus infection, the cumulative mortality of the abalone in M2 was significantly lower than that in any other group (P < 0.05). Consequently, given the growth advantages and the enhancement of immune function, the feeding plan in which B. lincheniformis was applied for 4 d per week, and basal feed was then applied for 3 d, did not lead to a high level of immune reaction, immune fatigue or waste of resources, but increased the growth rate of individuals and their resistance to V. parahaemolyticus infection.

Microbiological and real-time mechanical analysis of Bacillus licheniformis and Pseudomonas fluorescens dual-species biofilm.

In natural habitats, bacterial species often coexist in biofilms. They interact in synergetic or antagonistic ways and their interactions can influence the biofilm development and properties. Still, very little is known about how the coexistence of multiple organisms impact the multispecies biofilm properties. In this study, we examined the behaviour of a dual-species biofilm at the air-liquid interface composed by two environmental bacteria: Bacillus licheniformis and a phenazine mutant of Pseudomonas fluorescens. Study of the planktonic and biofilm growths for each species revealed that P. fluorescens grew faster than B. licheniformis and no bactericidal effect from P. fluorescens was detected, suggesting that the growth kinetics could be the main factor in the dual-species biofilm composition. To validate this hypothesis, the single- and dual-species biofilm were characterized by biomass quantification, microscopy and rheology. Bacterial counts and microscale architecture analysis showed that both bacterial populations coexist in the mature pellicle, with a dominance of P. fluorescens. Real-time measurement of the dual-species biofilms' viscoelastic (i.e. mechanical) properties using interfacial rheology confirmed that P. fluorescens was the main contributor of the biofilm properties. Evaluation of the dual-species pellicle viscoelasticity at longer time revealed that the biofilm, after reaching a first equilibrium, created a stronger and more cohesive network. Interfacial rheology proves to be a unique quantitative technique, which combined with microscale imaging, contributes to the understanding of the time-dependent properties within a polymicrobial community at various stages of biofilm development. This work demonstrates the importance of growth kinetics in the bacteria competition for the interface in a model dual-species biofilm.

Bacillus licheniformis peptidoglycan characterization by CZE-MS: Assessment with the benchmark RP-HPLC-MS method.

Peptidoglycan or murein is an essential polymer found in bacterial cell wall. It is a dynamic structure that is continuously remodeled or modified during bacterial cell growth or in presence of cell wall stresses. These modifications are still poorly understood mainly due to the peptidoglycan, which is rather non-soluble, and the difficulties to separate the hydrophilic glycopeptides (muropeptides) by reversed phase liquid chromatography, generated by the enzymatic digestion using mutanolysin, an N-acetyl-muramidase, cleaving the ß1→4 bound between N-acetylglucosamine and N-acetylmuramic acid. Here, we report the use of CZE-MS for an easy and fast screening of muropeptides generated by the action of muramidase on the Bacillus licheniformis cell wall. Electron transfer and CID-MS were also used to unambiguously identify and localize the presence or the absence of amidation and acetylation moieties on muropeptide variants. The reference method to analyse muropeptides by reversed phase chromatography was also tested and the advantages and disadvantages of both methods were evaluated.

Structure-guided design combined with evolutionary diversity led to the discovery of the xylose-releasing exo-xylanase activity in the glycoside hydrolase family 43.

Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure-guided design of a xylose-releasing exo-ß-1,4-xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys247 ) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose-releasing exo-ß-1,4-xylanase reported in the GH43 family. This enzyme presents a unique active-site topology when compared with closely related ß-xylosidases, which is the absence of a hydrophobic barrier at the positive-subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the ß-propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate-active enzyme family abundant in most plant cell-wall-degrading microorganisms.

Bacillus pumilus probiotic feed supplementation mitigates Lawsonia intracellularis shedding and lesions.

The causative agent of ileitis, Lawsonia intracellularis, is commonly associated with diarrhea and reduced weight gain in growing pigs. The effect of in-feed probiotics on L. intracellularis infection dynamics was evaluated. In brief, 70 2.5-week-old-pigs were randomly divided into six groups with 10-20 pigs each. All pigs were fed an age appropriate base ration for the duration of the study, which was supplemented with one of three Bacillus strains including B. amyloliquefaciens (T01), B. licheniformis (T02) and B. pumilus (T03). Another group was orally vaccinated with a commercial live L. intracellularis vaccine (VAC) at 3 weeks of age. At 7 weeks of age, T01-LAW, T02-LAW, T03-LAW, VAC-LAW and the POS-CONTROL groups were challenged with L. intracellularis while the NEG-CONTROL pigs were not challenged. All pigs were necropsied 16 days later. By the time of inoculation, all VAC-LAW pigs had seroconverted and at necropsy 10-65% of the pigs in all other challenged groups were also seropositive. The results indicate a successful L. intracellularis challenge with highest bacterial DNA levels in POS-CONTROL pigs, VAC-LAW pigs and T01-LAW pigs. There was a delay in onset of shedding in T02-LAW and T03-LAW groups, which was reflected in less severe macroscopic and microscopic lesions, reduced intralesional L. intracellularis antigen levels and a lower area under the curve for bacterial shedding. Under the study conditions, two of the probiotics tested suppressed L. intracellularis infection. The obtained findings show the potential of probiotics in achieving antibiotic-free control of L. intracellularis.

Valorization of mutant Bacillus licheniformis M09 supernatant for green synthesis of silver nanoparticles: photocatalytic dye degradation, antibacterial activity, and cytotoxicity.

The present study reports the optimization of a green method for the synthesis of silver nanoparticles (AgNPs) via reduction of Ag+ ions using cell-free supernatant of mutant Bacillus licheniformis M09. UV-Visible spectroscopy showing an absorption peak at ~ 430 nm confirmed the synthesis of AgNPs. Transmission electron microscope (TEM) analysis exhibited spherical AgNPs within the size range of 10-30 nm. Fourier transform infrared (FTIR) measurements assured the presence of effective functional molecules which could be responsible for stabilizing the AgNPs. X-ray diffraction (XRD) pattern verified the crystalline nature of AgNPs. Furthermore, the synthesized AgNPs showed an excellent photocatalytic degradation of methylene blue dye in less than 3 h under visible light proving their potential as a catalytic agent for bioremediation for next-generation dye degradation in effluent treatment. The AgNPs demonstrated antimicrobial activity against Gram-positive and Gram-negative foodborne pathogens which endorsed its suitability as agents to extend shelf-life in food packaging and food safety applications. The results also revealed a strong concentration-dependent cytotoxicity of AgNPs against human breast adenocarcinoma cells (MCF-7), while 15.07 µg/mL of IC50 was attained. The outcome suggests the possible application of these AgNPs in nanomedicine formulations. Thus, these findings propose promising ways for the valorization of the waste fermentation supernatant left after cell harvesting and desired metabolite extraction.

Production and Application of Biosurfactant Produced by Bacillus Licheniformis Ali5 in Enhanced Oil Recovery and Motor Oil Removal from Contaminated Sand.

The present study describes the production of biosurfactant from isolate B. licheniformis Ali5. Seven different, previously-reported minimal media were screened for biosurfactant production, and two selected media were further optimized for carbon source. Further, various fermentation conditions such as (pH 2-12, temperature 20-50 °C, agitation speed 100-300 rpm, NaCl (0-30 g·L-1) were investigated. The partially purified biosurfactant was characterized by Fourier transform infrared spectroscopy (FTIR) and matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF MS) and found a lipopeptide mixture, similar to lichenysin-A. Biosurfactant reduced surface tension from 72.0 to 26.21 ± 0.3 and interfacial tension by 0.26 ± 0.1 mN.m-1 respectively, biosurfactant yield under optimized conditions was 1 g·L-1, with critical micelle concentration (CMC) of 21 mg·L-1 with high emulsification activity of (E24) 66.4 ± 1.4% against crude oil. Biosurfactant was found to be stable over extreme conditions. It also altered the wettability of hydrophobic surface by changing the contact angle from 49.76° to 16.97°. Biosurfactant efficiently removed (70-79%) motor oil from sand, with an efficiency of more than 2 fold as compared without biosurfactant (36-38%). It gave 32% additional oil recovery over residual oil saturation upon application to a sand-packed column. These results are indicative of potential application of biosurfactant in wettability alteration and ex-situ microbial enhanced oil recovery.

Characterization and mode of action of a potent bio-preservative from food-grade Bacillus licheniformis MCC 2016.

The antibacterial peptide of Bacillus licheniformis MCC 2016 have potential biopreservative efficacy. Here, we report the purification process, properties, and mode of action of this antibacterial peptide for its potential application in the food industry. The antibacterial peptide from the cell-free supernatant was purified using a sequence of purification steps. The purified antibacterial peptide showed a specific activity of 68817 AU mg-1 and 0.4% yield. Liquid chromatography-mass spectroscopy analysis showed an mz-1 value of 279.28 for the active peptide. The SDS-PAGE analysis confirmed the antibacterial peptide is low-molecular weight and the size is between 3.0 and 3.5 kDa. Scanning electron microscopy, Fourier transform infrared spectroscopy, ß-gal induction assay and release of UV-absorbing materials indicated that the antibacterial peptide targets the cell wall of pathogens. Minimum inhibitory concentration of the antibacterial peptide against Listeria monocytogenes Scott A and others (Kocuria rhizophila ATCC 9341, Staphylococcus aureus FRI 722 and Salmonella typhimurium MTCC 1251) was found to be 1600 and 800 AU mL-1, respectively. The antibacterial peptide is temperature and pH stable, proteolytic-enzyme-sensitive, low-molecular weight, cell wall active class I bacteriocin and exhibits remarkable antibacterial activity against pathogens, suggesting its application as a potential biopreservative in the food industry.

Delineating thermophilic xylanase from Bacillus licheniformis DM5 towards its potential application in xylooligosaccharides production.

In this present study novel endoxylanase producing Bacillus licheniformis DM5 isolated, identified based on 16S rDNA from Garampani hotspring, Assam, India and enzyme was purified. RNA secondary structure predicted the similarity of B. licheniformis DM5 with B. licheniformis ATCC14580. Highest production of xylanase from B. licheniformis DM5 was achieved in the TY medium with cell densities 12 g/l and extracellular protein concentration containing xylanase 400 mg/l. Partially purified extracellular xylanase displayed optimum pH 6.5 and temperature 50 °C. Thermostability of the xylanase at the elevated temperature showed stability between 50 and 60 °C retaining its 99% activity. Kinetic parameters of thermophilic xylanase revealed Km 1.5 ± 0.2 mg/ml, Vmax 2.7 ± 0.2 U/ml and and Kcat 1.8 ± 0.2 s-1 against beechwood xylan but ruled out any exo-acting activity against synthetic pNP-xylopyranoside substrate. Time dependent enzymatic hydrolysis of beechwood xylan and preprocessed agrowaste corncob exhibited the release of xylotriose and xylobiose oligosaccharide (XOS) significantly high. Xylobiose and xylotriose exhibited higher binding affinities with BIAXP transporter protein of probiotic bacteria explaining their easy uptake by the cells. Mixed oligosaccharides also exhibited better prebiotic activity by promoting growth of Bifidobacterium infantis and Lactobacillus delbrueckii. Mixed XOS when tested for their cytotoxicity on Hela cell lines in in vitro MTT assay displayed significant lowering of cell viability after 48 h and 24 h at 100 µg/ml to 60% and 50%, respectively. In contrast, cytotoxicity wasn't observed against normal cervical cell line (VK2/E6E7-ATCC-CRL-2616). Therefore, thermophilic endoxylanase from B. licheniformis DM5 could be attributed for the production of prebiotic and anti-inflammatory XOS from agrowaste.

Effects of a commercial probiotic BS containing Bacillus subtilis and Bacillus licheniformis on growth, immune response and disease resistance in Nile tilapia, Oreochromis niloticus.

The present study evaluated a commercial probiotic designated as BS (a mix of B. subtilis and B. licheniformis) to ascertain its efficacy and the dose necessary to improve growth, immune response, and disease resistance in tilapia, Oreochromis niloticus. Fish (53.01 ±â€¯1.0 g) were fed with a basal diet supplemented with 0 g kg−1 (CT), 3 g kg−1(BS3), 5 g kg−1 (BS5), 7 g kg−1 (BS7), and 10 g kg−1 (BS10) [corrected] of the probiotic BS for 4 weeks. At the end of the feeding trial, the weight gain, specific growth rate, and feed conversion ration were enhanced in all probiotic BS enriched groups but with better (P < 0.05) improvement in the BS10 group. The lysozyme, protease, anti-protease, superoxide dismutase activities, and immunoglobulin M level were significantly (P < 0.05) highest in the BS10 group in both serum and skin mucus. Enhanced (P > 0.05) catalse activity in all treated groups in the serum and myeloperoxidase activity in the B10 group in both serum and skin mucus were observed. The expression of C-lysozyme, heat shock protein 70, ß-defensin, transforming growth factor beta, and small body size decapentaplegic homolog 3, genes in the mid-intestines and the head-kidney were up-regulated in all treated groups with the BS10 group provoking the highest up-regulation (P < 0.05). After challenge with Streptococcus agalactiae, cumulative mortality was 80 %, 47.5 %, 42.8 %, 30 %, and 20 % [corrected] for fish fed with CT, BS3, BS5, BS7, BS10 groups respectively. In conclusion, probiotic BS application at 10 g kg−1(BS10) [corrected] can be considered to improve growth and immunological status in tilapia farming.

Dietary supplementation of probiotic Bacillus licheniformis Dahb1 improves growth performance, mucus and serum immune parameters, antioxidant enzyme activity as well as resistance against Aeromonas hydrophila in tilapia Oreochromis mossambicus.

The present study evaluated the dietary supplementation of probiotic Bacillus licheniformis Dahb1 on the growth performance, immune parameters and antioxidant enzymes activities in serum and mucus as well as resistance against Aeromonas hydrophila in Mozambique tilapia Oreochromis mossambicus. Fish (24 ±â€¯2.5 g) were fed separately with three diets, 1) commercial diet (control), 2) diet containing probiotic at 105 cfu g-1 (D1) and 3) diet containing probiotic at 107 cfu g-1 (D2) for 4 weeks. Growth performance in term of final weight (FW) specific growth rate (SGR) and feed conversion ratio (FCR), immune parameters of total protein (TP), alkaline phosphatase (ALP), myeloperoxidase (MPO), lysozyme (LYZ), reactive oxygen species (ROS), reactive nitrogen species (RNS) and antioxidant parameters of superoxide dismutase (SOD) and glutathione peroxidase (GPx) in serum and mucus were evaluated after 2nd and 4th weeks. The FW, SGR, and FCR of fish fed with D1 and D2 significantly improved (p < 0.05). The activities of ALP, LYZ and MPO in the mucus were significantly higher (p < 0.05) in fish that fed D1 and D2. The TP, ROS, RNS, SOD and GPx in the serum were significantly higher (p < 0.05) in fish that fed D1 and D2. In addition, the challenge test showed that fish fed D1 and D2 enhanced significantly (p < 0.05) the resistance against A. hydrophila (1 × 107 cells ml-1). In conclusion, probiotic B. licheniformis Dahb1 can be applied in diet at 107 cfu g-1 to improve healthy status and resistance against A. hydrophila in tilapia farming.

Effects of a probiotic (Bacillus licheniformis) on the growth, immunity, and disease resistance of Haliotis discus hannai Ino.

To study the effects of a probiotic (Bacillus lincheniformis) on the survival and growth of Haliotis discus hannai Ino, the expression levels of nonspecific immune genes and the resistance to Vibrio parahaemolyticus infection were assessed. Abalones (shell length: 27.64 ±â€¯1.59 mm, body weight: 4.17 ±â€¯0.32 g) were selected for use in an 8-week culture experiment and a 2-week V. parahaemolyticus artificial infection experiment. In both experiments, the control group (C) was fed with a basal feed and the experimental groups were fed with experimental food prepared by spraying the probiotic on the basal feed at different concentrations: 103 (B1), 105 (B2), and 107 (B3) cfu/mL. The survival rate, total number of blood lymphocytes, activity of acid phosphatase, and expression level of heat shock protein 70 were significantly higher in B1, B2, and B3 than in C (P < 0.05). The specific growth rate of shell length, food intake, food conversion rate, phagocytic activity of blood lymphocytes, activities of myeloperoxidase and catalase (CAT), and expression levels of CAT and thioredoxin peroxidase of abalones in B2 were significantly higher than those in B1 and C (P < 0.05). Although the level of O2- produced by the respiratory burst of blood lymphocytes in B2 was not significantly different from those in B1 and B3, they were significantly higher than that in C (P < 0.05). The activity of superoxide dismutase (SOD), the nitric oxide levels produced by the respiratory burst of blood lymphocytes, and the expression levels of Mn-SOD in B1 and B3 were significantly higher than those in C but significantly lower than those in B2 (P < 0.05). Fourteen days after infection with V. parahaemolyticus, the cumulative mortality of abalones in B2 was significantly lower than those in B1 and C (P < 0.05). These results indicate that the food containing 105 cfu/mL Bacillus licheniformis promoted food intake and growth of abalones and also improved their resistance to V. parahaemolyticus infection. Thus, B. licheniformis is a good potential probiotic.

A novel antifungal property for the Bacillus licheniformis ComX pheromone and its possible role in inter-kingdom cross-talk.

Quorum sensing molecules (QSMs) regulate, through a chemical communication process, multiple complex systems in bacterial and some fungal populations on the basis of cell density. The bacterial QSMs involved in inter-kingdom cross-talk may exhibit antagonistic activity against fungi. This provides an important opportunity for biocontrol of fungal invasion in plants. It has been shown that cultures of Bacillus spp. inhibit fungal growth. Here, we explore the inhibitory potential of the industrial workhorse Bacillus licheniformis NCIMB-8874 and its QSM (ComX pheromone) on the growth of Aspergillus flavus, a cereal, legume, and nut crop pathogen. Our studies show that ComX filtered extracts from cultures of B. licheniformis can cause a significant reduction in the growth of A. flavus NRRL 3357 and ESP 15 at a concentration as low as 13 µg ml-1. This work evidences, for the first time, the inter-kingdom utility of the bacterial quorum sensing ComX pheromone indicating potential antifungal food security against A. flavus.

Enhanced production of heterologous proteins by Bacillus licheniformis with defective D-alanylation of lipoteichoic acid.

Heterologous expression is an efficient strategy for target protein production. Dlt operon plays the important role in the D-alanylation of lipoteichoic acid, which might affect the net negative charge of cell wall for protein secretion. In this study, dlt operon was deleted to improve the target protein production, and nattokinase, α-amylase and ß-mannanase with different isoelectric points (PIs) were served as the target proteins. Firstly, our results implied that deletions of dltA, dltB, dltC and dltD improved the net negative charge of cell wall for extracellular protein secretion respectively, and among which, the dltB deficient strain DW2ΔdltB showed the best performance, its nattokinase (PI: 8.60) activity was increased by 27.50% compared with that of DW2/pP43SacCNK. Then, the dltABCD mutant strain was constructed, and the net negative charge and nattokinase activity were increased by 55.57% and 37.13% respectively, due to the deficiency of dltABCD. Moreover, it was confirmed that the activities of α-amylase (PI: 6.26) and ß-mannanase (PI: 5.75) were enhanced by 44.53% and 53.06% in the dltABCD deficient strains, respectively. Collectively, this study provided a strategy that deletion of dlt operon improves the protein secretion in B. licheniformis, and which strategy was more conducive to the target protein with lower PI.

Oral Administration of a Select Mixture of Bacillus Probiotics Affects the Gut Microbiota and Goblet Cell Function following Escherichia coli Challenge in Newly Weaned Pigs of Genotype MUC4 That Are Supposed To Be Enterotoxigenic E. coli F4ab/ac Receptor Negative.

Structural disruption of the gut microbiota and impaired goblet cell function are collateral etiologic factors in enteric diseases. Low, moderate, or high doses of a Bacillus licheniformis-B. subtilis mixture (BLS mix) were orally administered to piglets of genotype MUC4 that are supposed to be F4-expressing enterotoxigenic Escherichia coli strain (F4+ ETEC) F4ab/ac receptor negative (i.e., MUC4-resistant piglets) for 1 week before F4+ ETEC challenge. The luminal contents were collected from the mucosa of the colon on day 8 after F4+ ETEC challenge. The BLS mix attenuated E. coli-induced expansion of Bacteroides uniformis, Eubacterium eligens, Acetanaerobacterium, and Sporobacter populations. Clostridium and Turicibacter populations increased following F4+ ETEC challenge in pigs pretreated with low-dose BLS mix. Lactobacillus gasseri and Lactobacillus salivarius populations increased after administration of BLS mix during E. coli infection. The beneficial effects of BLS mix were due in part to the expansion of certain Clostridium, Lactobacillus, and Turicibacter populations, with a corresponding increase in the number of goblet cells in the ileum via upregulated Atoh1 expression, in turn increasing MUC2 production and thus preserving the mucus barrier and enhancing host defenses against enteropathogenic bacteria. However, excessive BLS mix consumption may increase the risk for enteritis, partly through disruption of colonic microbial ecology, characterized by expansion of Proteobacteria and impaired goblet cell function in the ileum. Our findings suggest that oral administration of BLS mix reprograms the gut microbiota and enhances goblet cell function to ameliorate enteritis. IMPORTANCE: The present study is important for improving our understanding of the protective role of probiotics against Escherichia coli infection in piglets. Structural disruption of the gut microbiota and impaired goblet cell function are collateral etiologic factors in enteric diseases. In this study, low, moderate, or high doses of a Bacillus licheniformis-B. subtilis mixture (BLS mix) were orally administered to MUC4-resistant piglets for 1 week before the F4-expressing ETEC strain (F4+ ETEC) challenge. Our findings suggest that oral administration of BLS mix reprograms the gut microbiota and enhances goblet cell function to ameliorate enteritis.