Oral vaccination with envelope protein VP28 against white spot syndrome virus in Procambarus clarkii using Bacillus subtilis as delivery vehicles

Aims: To achieve high‐level expression and secretion of active VP28 directed by a processing‐efficient signal peptide in Bacillus subtilis WB600 and exploit the possibility of obtaining an oral vaccine against white spot syndrome virus (WSSV) using vegetative cells or spores as delivery vehicles. Methods and Results: The polymerase chain reaction (PCR)‐amplified vp28 gene was inserted into a shuttle expression vector with a novel signal peptide sequence. After electro‐transformation, time‐courses for recombinant VP28 (rVP28) secretion level in B. subtilis WB600 were analysed. Crayfish were divided into three groups subsequently challenged by 7‐h immersion at different time points after vaccination. Subgroups including 20 inter‐moult crayfish with an average weight of 15 g in triplicate were vaccinated by feeding coated food pellets with vegetative cells or spores for 20 days. Vaccination trials showed that rVP28 by spore delivery induced a higher resistance than using vegetative cells. Challenged at 14 days postvaccination, the relative per cent survival (RPS) values of groups of rVP28‐bv and rVP28‐bs was 51·7% and 78·3%, respectively. Conclusions: The recombinant B. subtilis strain with the ability of high‐level secretion of rVP28 can evoke protection of crayfish against WSSV by oral delivery. Significance and Impact of the Study: Oral vaccination by the B. subtilis vehicle containing VP28 opens a new way for designing practical vaccines to control WSSV.     

Inactivation of Alicyclobacillus acidoterrestris vegetative cells in model system,apple, orange and tomato juices by high hydrostatic pressure

The objective of this study is to determine the effect of high hydrostatic pressure (HHP) on inactivation of Alicyclobacillus acidoterrestris vegetative cells in a model system (BAM broth) and in orange, apple and tomato juices. The shelf-life stability of pressurized juices is also studied. In general the viability loss was enhanced significantly as the level of pressure and temperature were increased (P < 0.05). 4.70 log cycle reduction was obtained after pressurization at 350 MPa at 50 °C for 20 min in BAM broth whereas thermal treatment at 50 °C for 20 min caused only 1.13 log cycle inactivation showing the effectiveness of HHP treatment on inactivation. The D values for pressure (350 MPa at 50 °C) and temperature (50 °C) treatments were 4.37 and 18.86 min in BAM broth, respectively. All juices were inoculated with A. acidoterrestris cells to 10⁶ c.f.u./ml and were pressurized at 350 MPa at 50 °C for 20 min. More than 4 log cycle reduction was achieved in all juices studied immediately after pressurization. The pressurized juices were also stored up to 3 weeks at 30 °C and the viable cell numbers of A. acidoterrestris in orange, apple and tomato juices were 3.79, 2.59 and 2.27 log cycles, respectively after 3 weeks. This study has indicated that A. acidoterrestris vegetative cells can be killed by HHP at a predictable rate even at temperatures at which the microorganism would normally grow.     

Survival of Plasmid-Containing Bacillus subtilis Released into Mushroom Compost

Abstract The survival of a plasmid-containing Bacillus subtilis released into mushroom compost was investigated. The indigenous Bacillus population of mushroom compost exhibited an antibiotic-resistance profile that was distinguished by almost complete absence of chloramphenicol resistance. Bacillus subtilis containing the chloramphenicol-resistance plasmid pC194 was released into mushroom compost microcosms and populations were monitored at different incubation temperatures. The organism colonized both sterile and untreated compost at 37°C, and to a lesser extent at 50°C, but was eliminated after 30 d at 65°C. Although sporulation of the B. subtilis population occurred within compost, the population was maintained for up to 13 weeks at 50°C, largely as vegetative cells. Experiments in which the B. subtilis host strain, without plasmid, was released demonstrated that plasmid carriage had no effect on the ability of the bacterium to colonize and survive in compost. Furthermore, the size and composition of the indigenous bacterial population was unaffected by the presence of the introduced B. subtilis strain. Virtually no loss of plasmid pC194 from the B. subtilis population in compost was observed, and experiments at low growth rates in chemostats confirmed the stability of this host/vector system in the absence of positive selection pressure. Received: 9 July 1997; Accepted: 20 October 1997     

PCR amplification of DNA sequence related to the hrpD gene of Xanthomonas cucurbitae in leaf spot disease of pumpkin and their antagonism by soil bacteria

A sensitive and specific assay was developed to detect and biological control of bacterial leaf spot of pumpkin, Xanthomonas cucurbitae was identified on the basis of the morphological, biochemical and molecular assay. The antibiotic sensitivity of the isolate showed that, Carbenicillin revealed highest antibacterial activity with 29 ± 0.00 mm zone of inhibition against isolated bacterial strain. Isolated bacterial strains from soil were also identified by biochemical and molecular characterisation. By analysing morphological and biochemical characteristics and 16S rDNA of three bacterial strains isolated from soil was matched 96% with Bacillus subtilis, 98% with Bacillus brevis and 97% with Pseudomonas fluorescens strain. They were subjected to the antagonistic activity against Xanthomonas cucurbitae by disc diffusion method. Among them, B. subtilis showed significant positive antagonistic activity with 17.0 ± 0.28 mm zone of inhibition against Xanthomonas cucurbitae. The presence of DNA sequence related to the hrpD gene successfully amplified in some isolates of Xanthomonas cucurbitae.     

Surface display of recombinant protein on the cell surface of Bacillus subtilis by the CotB anchor protein

We developed a novel surface display system based on the CotB anchoring motif in order to express foreign protein on the surface of vegetative Bacillus subtilis cells. CotB is a protein in the B. subtilis spore coat. In this system, three repeats of the immunodominant ovalbumin T-cell epitope (OVA_323–339) were linked with the cholera toxin B subunit (CTB) to construct a fusion protein, CTB-OVA epi, which was then fused to the C-terminal of the CotB protein so that CTB-OVA epi was expressed in vegetatively-growing B. subtilis. The expression and localization of the CTB-OVA epi protein was confirmed by western blotting, immunofluorescence microscopy, and flow cytometry. The results indicated that a CotB-based surface display system was successfully used to express the CTB-OVA epi protein on the surface of vegetative B. subtilis cells.     

BIOETHANOL PRODUCTION FROM LEAFY BIOMASS OF MANGO (Mangifera indica) INVOLVING NATURALLY ISOLATED AND RECOMBINANT ENZYMES

The present study describes the usage of dried leafy biomass of mango (Mangifera indica) containing 26.3% (w/w) cellulose, 54.4% (w/w) hemicellulose, and 16.9% (w/w) lignin, as a substrate for bioethanol production from Zymomonas mobilis and Candida shehatae. The substrate was subjected to two different pretreatment strategies, namely, wet oxidation and an organosolv process. An ethanol concentration (1.21 g/L) was obtained with Z. mobilis in a shake-flask simultaneous saccharification and fermentation (SSF) trial using 1% (w/v) wet oxidation pretreated mango leaves along with mixed enzymatic consortium of Bacillus subtilis cellulase and recombinant hemicellulase (GH43), whereas C. shehatae gave a slightly higher (8%) ethanol titer of 1.31 g/L. Employing 1% (w/v) organosolv pretreated mango leaves and using Z. mobilis and C. shehatae separately in the SSF, the ethanol titers of 1.33 g/L and 1.52 g/L, respectively, were obtained. The SSF experiments performed with 5% (w/v) organosolv-pretreated substrate along with C. shehatae as fermentative organism gave a significantly enhanced ethanol titer value of 8.11 g/L using the shake flask and 12.33 g/L at the bioreactor level. From the bioreactor, 94.4% (v/v) ethanol was recovered by rotary evaporator with 21% purification efficiency.     

Plant defense approach of Bacillus subtilis (BERA 71) against Macrophomina phaseolina (Tassi) Goid in mung bean

This study was aimed to elucidate the mitigation mechanism of an endophytic bacterium, Bacillus subtilis (BERA 71) against Macrophomina phaseolina (Tassi) Goid disease in mung bean. M. phaseolina reduced the plant growth by inducing disease, hydrogen peroxide (H₂O₂) and lipid peroxidation. The inoculation of B. subtilis to diseased plants increased chlorophyll, ascorbic acids, and superoxide dismutase, catalase, peroxidase, ascorbate peroxidase and glutathione reductase activities, and while inhibited H₂O₂ and lipid peroxidation for enhancing plant growth. In addition, B. subtilis association in plants mitigated the M. phaseolina infection due to increase of indole acetic acids and indole butyric acid, and also a decrease of abscisic acid. However, the nutrients (N, K, Ca, Mg, Zn, Cu, Mn and Fe) were increased, except Na in M. phaseolina diseased plants treated with B. subtilis. The result of this study suggests that B. subtilis interaction with plants can modulate the metabolism of pigments, hormones, antioxidants and nutrients against M. phaseolina to induce disease resistance in mung bean.     

Extraction of ginsenosides from fresh ginseng roots (Panax ginseng C.A. Meyer) using commercial enzymes and high hydrostatic pressure

A combination of high hydrostatic pressure (HHP) and enzymatic hydrolysis (HHP-EH) was applied for the extraction of ginsenosides from fresh ginseng roots (Panax ginseng C.A. Myer). The highest yield of ginsenosides was obtained by using a mixture of three enzymes (Celluclast + Termamyl + Viscozyme) along with HHP (100 MPa, at 50 °C for 12 h) in comparison to control samples (no enzymes, atmosphere pressure, P < 0.05). Total ginsenosides increased by 184 % while Rg1 + Rb1 increased by 273 %. Application of these conditions significantly increased total ginsenosides by 49 % and Rg1 + Rb1 by 103 % compared to HHP treatment alone (P < 0.05). The effect of HHP on increased yield of ginsenosides is likely due in part, to acceleration of enzyme activity. Thus HHP-EH significantly improves the extraction of ginsenosides from fresh ginseng roots.     

Potency of Bacillus thuringiensis and Bacillus subtilis against the cotton leafworm,Spodoptera littoralis (Bosid.) Larvae

The biological activities of two species of bacteria isolated from soil of cotton fields identified as Bacillus subtilis strain NRC313 (BS NRC313) and Bacillus thuringiensis strain NRC335 (BT NRC335) were evaluated against the third larval instar of the cotton leafworm, Spodoptera littoralis (Boisd.). The different entomopathogenic bacteria of BS NRC313and BT NRC335 contained 10 × 10⁸ cell/ml, and caused mortality of 100 and 97.3% for the above mentioned strains, respectively. Concentrations of 2.5 × 10⁸ to 10 × 10⁸ cell/ml of strains BS NRC313 and BT NRC335 were applied to the larvae: LC₅₀ were 3.3 × 10⁸ and 3.9 × 10⁸ cell/ml respectively. The influence of exposure to toxin concentrations manifested in terms of decreasing the adult emergence and prolongation of the generation period. The percentage of larvae that survived and succeeded to pupate increased by decreasing the concentration. The longevity of adult emergence that resulted from larvae treated with Bacillus subtilis were 6.0 ± 0.51 and 9.0 ± 0.63 days at 5 × 10⁸ and 2.5 × 10⁸ cell/ml, respectively compared with 9.8 ± 0.47 in control. The results indicated that Bacillus subtilis was more potent than Bacillus thuringiensis. Field applications of B. thuringiensis, B. subtilis and Reldan achieved 55.6, 67.4 and 89.4% reduction of the cotton leafworm larvae Spodoptera littoralis in clover plants under field conditions.     

Homoploid hybrid speciation in Doronicum L. (Asteraceae)? Morphological,karyological and molecular evidences

Abstract

The first unambiguous documentation of hybridism in the genus Doronicum (Senecioneae – Asteraceae) is reported. All our morphological, karyological and molecular data concur to indicate that Doronicum × minutilloi Peruzzi hybr. nov. (2n = 60) is a hybrid growing in Monti Aurunci (Central Italy), originated from the spontaneous crossing D. orientale Hoffm. (2n = 60) × D. columnae Ten. (2n = 60). This new hybrid shows a slightly higher morphological, karyotypic and ribotypic affinity with D. columnae, but shares a trnL-trnF IGS haplotype with D. orientale, and co-occurs with the latter species only; it has reduced fertility and a high potential for vegetative propagation through rhizome fragmentation. Our results led us to suspect in fieri homoploid hybrid speciation.     

Isolation and Identification of Polycyclic Aromatic Hydrocarbon–Degrading Bacteria from Crude Oil Exploration Bore Well Sludge

Two bacterial strains capable of degrading polycyclic aromatic hydrocarbons were isolated from the crude oil exploration bore well sludge and identified by 16s rRNA gene sequencing as Pseudomonas stutzeri and Bacillus subtilis. The bacterial strains Pseudomonas stutzeri and Bacillus subtilis were able to degrade 95.1% and 99.4% of naphthalene (100 mg L^?1) and 99.5% and 94.6% of anthracene (50 mg L^?1), respectively, as a sole carbon and energy source in the liquid phase within a period of 6 days. The specific growth rate was determined for both the species and found to be 0.169 and 0.124 day^?1.     

Development of a radio frequency heating system for sterilization of vacuum-packed fish in water

We developed equipment that quickly and uniformly heats packed whole fish in circulating tap water using radio frequency (RF) heating. Four vacuumed plastic-packed Pacific sauries in tap water were set in a radial arrangement between coaxial cylindrical electrodes in a closed vessel. For sterilization testing, Bacillus subtilis spores added in the center of the sauries were counted after treatment. For quality assurance, meat color and backbone hardness were measured after treatment. The temperature at the center of the sauries was increased up to 130 °C for 19 min using 9 kW RF heating, and up to 119 °C for 45 min using conventional heating (CH) at 120 °C. B. subtilis spores were decreased by five logarithmic orders using RF heating and by four logarithmic orders using CH. The RF-treated meat was brighter than the CH-treated meat, and the RF-treated backbone was softer than CH-treated one.     

Biological control of Botrytis mali on apple fruit by use of Bacillus bacteria,isolated from the rhizosphere of wheat

Bacillus pumilus (B19), B. subtilis (1J), B. crerus (B16), B. subtilis (B11) and B. cereus (B17), isolated from the rhizosphere of wheat, were tested for control of gray mold of apple caused by Botrytis mali. Dual culture, cell free metabolite and volatile tests showed that all five isolates of Bacillus spp. inhibited growth of the pathogen. Inhibition varied from 13.6 to 74% in the dual culture tests; 12.3 to 87% in the cell free metabolite tests; and 11 to 53% in the volatile tests. Five isolates of Bacillus spp. reduced B. mali lesion diameter from 7.2 to 32.2 mm compared with 41.6 to 51.4 mm in controls at 4°C. At 20°C the lesion diameters were from 7 to 24.9 mm for antagonistic treatments and from 46.2 to 46.6 mm for the control treatment after 15 days.     

Glycolaldehyde Dehydrogenase,Its Involvement in Vitamin B6 Biosynthetic Pathway of Escherichia coli B

The deoxyribonucleic acid (DNA) polymerases were partially purified from spores and vegetative cells of Bacillus subtilis. Some biochemical properties of the enzymes from the spores were studied in comparison with those from the vegetative cells. The spores and vegetative cells had at least three species of DNA polymerases (DNA polymerase I, II and III). These DNA polymerases in spores could not be distinguished from those in vegetative cells, respectively, with regard to the reresponses to ionic strength, the sensitivity to thiol-blocking agents, the template specificity, pH and temperature optima in assay, and the sedimentation behavior. It is inferred that DNA polymerases from spores was essentially identical to those from vegetative cells.

The DNA polymerase activity decreased rapidly in the course of sporulation, and only about 20% is recovered in the spores, suggesting that an extentive inactivation mechanism of the enzymes would be involved during sporulation.     

Oxalic acid production by Fusarium oxysporum Schlecht and Botryodiplodia theobromae Pat., post-harvest fungal pathogens of yams (Dioscorea rotundata L.) and detoxification by Bacillus subtilis CM1 isolated from culturable cowdung microflora

Abstract

Fusarium oxysporum Schlecht and Botryodiplodia theobromae Pat., two important post-harvest pathogens of yam (Dioscorea rotundata L.) tubers in storage were found to produce oxalic acid (OA) in vitro and in vivo. The rate of OA accumulation was proportional to fungal growth (cell mass) in Potato Dextrose liquid medium during 10 days incubation period. Further, simultaneous co-culturing of either of the fungi with Bacillus subtilis CM1 isolated from cowdung culturable microflora resulted in 92% reduction in OA accumulation compared with that in the culture of the individual fungus. The effect was more prominent in pH 5 – 6 than in pH 7 – 8. B. subtilis CM1 was capable of detoxifying OA and several proteins were detected in the culture filtrates when it was grown in peptone-mineral salt medium containing OA. SDS-PAGE analysis of 70% ammonium sulphate fraction of the culture filtrate exhibited the presence of a predominant 97 kDa protein.     

Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon

clpC ofBacillus subtilis is part of an operon containing six genes. Northern blot analysis suggested that all genes are co-transcribed and encode stress-inducible proteins. Two promoters (P_A and P_B) were mapped upstream of the first gene. P_A resembles promoters recognized by the vegetative RNA polymerase Eσ^A. The other promoter (P_B) was shown to be dependent on σ^B, the general stress σ factor in B. subtilis, suggesting that clpC, a potential chaperone, is expressed in a σ^B-dependent manner. This is the first evidence that σ^B in B, subtilis is involved in controlling the expression of a gene whose counterpart, clpB, is subject to regulation by σ³² in Escherichia coli, indicating a new function of σ^B-dependent general stress proteins. P_B deviated from the consensus sequence of σ^B promoters and was only slightly induced by starvation conditions. Nevertheless, strong induction by heat, ethanol, and salt stress occurred at the σ^B-dependent promoter, whereas the vegetative promoter was only weakly induced under these conditions. However, in a sigB mutant, the σ^A-like promoter became inducible by heat and ethanol stress, completely compensating for sigB deficiency. Only the downstream σ^A-like promoter was induced by certain stress conditions such as hydrogen peroxide or puromycin. These results suggest that novel stress-induction mechanisms are acting at a vegetative promoter. Involvement of additional elements in this mode of induction are discussed.     

Synergistic growth of Bacillus and Pseudomonas and its degradation potential on pretreated polypropylene

This study investigated biodegradation of physically pretreated polypropylene (PP) by using two different combinations of microorganisms, namely, Bacillus flexus + Pseudomonas azotoformans(B1) and B. flexus + B. subtilis(B2), for a period of 12 months. The growth rate of (B1) was found to be high throughout the study period, and reached a maximum of 1 × 10¹⁴ colony-forming units (CFU)/mL. At the end of the experiment, the polymers become hydrophilic. Carbonyl indices showed that ultraviolet (UV)-treated polymers started degrading faster than the thermally treated PP. The thermogravimetric analysis also revealed that UV-treated PP exposed to the B. flexus + P. azotoformans combination for 1 year exhibited maximum degradation (22.7%). The gravimetric weight loss method showed 1.95% weight loss followed by 1.45% with B. flexus + B. subtilis. The changes in the carbonyl indices of the polymer through Fourier-transform infrared (FTIR) analysis also support the degradation.