Purification and Characterization of a Nonylphenol （NP）-degrading Enzyme from Bacillus cereus.Frankland

An extracellular NP-degrading enzyme secreted by Bacillus cereus.Frankland was purified to homogeneity by a combination of ammonium sulfate precipitation,Phenyl-Sepharose hydrophobic-interaction chromatography and DEAE anion-exchange chromatography.On SDS（sodium dodecyl sulfate）-polyacrylamide gel electrophoresis analysis,the purified enzyme showed a relative molecular mass of 58.3 kDa.The depolymerzation of subunits was accompanied with the loss of NP-degrading enzyme activity,and removing denaturing factors by dialysis could restore the dimer structure and enzymatic activity.The enzyme had an isoelectric point of 5.5 and an optimal temperature of 60℃,and was the most active at pH 6.0.The enzymatic activity was stable at pH 4-8 and inhibited by Cu2＋.TenN-terminal amino acids were determined to be ASVNSIKIGY,demonstrating that the purified enzyme was a novel one.The hydrolysis pattern of the purified enzyme indicated that the NP-degrading enzyme was an endo NP-degrading enzyme.The extraordinary thermo-stability provided the enzyme with a good prospect to be used as a new tool for clean-production process for textile industry.

Liver failure after Bacillus cereus food poisoning,an underrecognized entity:A case report

BACKGROUND Bacillus cereus(B.cereus)is known to cause 2 types of foodborne diseases;the diarrheal and emetic syndromes.They are largely underreported due to their usually self-limiting course.Rare and sometimes fatal cases of liver failure,pulmonary hemorrhage and cerebral oedema have been reported mainly in children and young adults.We present here a case of liver failure associated with B.cereus food poisoning in a middle-aged patient.CASE SUMMARY A 48-year-old female patient presented to the emergency department for emesis,diarrhea,chills without fever,asthenia and diffuse abdominal cramps that started less than 30 minutes after eating a rice salad.Her past medical history was relevant for cholecystectomy and a cured Hashimoto’s disease.She did not take any medication,drugs and declared a consumption of one glass of wine per week.In the emergency department,she was treated with acetaminophen,metoclopramide,ondansetron,and an intravenous normal saline infusion.Blood gas analysis revealed a metabolic acidosis with hyperlactatemia,coagulation revealed a low prothrombin activity[32%;normal values(N):70-140]and a low Factor V activity(15%;N:>70).Transaminases were elevated with hyperbilirubinemia,elevated lipase and rhabdomyolysis.N-acetylcysteine treatment was introduced.Abdominal echography revealed no signs of chronic hepatopathy or hepatomegaly.Day after the admission,psychomotor activity improved,transaminases and lipase started decreasing.Rhabdomyolysis gradually worsened to peak on day 3.Screening tests for liver disease were negative for viral and autoimmune cause of liver failure.Stools cultures were positive for colonies of the B.cereus group which were also identified in the rice salad samples processed whereas blood cultures were negative.The patient’s condition improved gradually including her liver function parameters and psychomotor activity which allowed her discharged home on day 9.CONCLUSION We describe a rare case of hepatocellular dysfunction due to a foodborne B.cereus intoxication in an adult patient.Even if it is uncommon,the severity of liver dysfunction reported and mechanism of the cereulide toxin toxicity on liver suggest that acetaminophen should be avoided in case of a foodborne intoxication and n-acetylcysteine could be a potential therapy helping to prevent hepatocytes necrosis due to the oxidative stress induced by mitochondrial dysfunction.

Lactobacillus rhamnosus GR-1 attenuates foodborne Bacillus cereus-induced NLRP3 inflammasome activity in bovine mammary epithelial cells by protecting intercellular tight junctions

Background:Bacillus cereus is an important pathogen that causes human food poisoning,specifically diarrhea and vomiting.B.cereus can also induce mastitis in dairy cows and has a strong survival ability in milk,as it cannot be inactivated by high-temperature short-time pasteurization.Therefore,B.cereus can enter the market through pasteurized milk and other dairy products,imposing enormous hidden dangers on food safety and human health.Results:In this study,B.cereus 2101(BC)was isolated from milk samples of cows with mastitis.BC grew rapidly with strong hemolysis,making it difficult to prevent mastitis and ensure food security.MAC-T cells were treated with BC and/or Lactobacillus rhamnosus GR-1(LGR-1).Pretreatment with LGR-1 protected the integrity of tight junctions and the expression of zonula occludens-1(ZO-1)and occludin destroyed by BC.Furthermore,LGR-1 pretreatment reduced the expression of NOD-like receptor family member pyrin domain-containing protein 3(NLRP3),caspase recruitment and activation domain(ASC),Caspase-1 p20,gasdermin D(GSDMD)p30,inflammatory factors(interleukin(IL)-1βand IL-18),and cell death induced by BC.Moreover,LGR-1 pretreatment reduced NLRP3 inflammasome activity and increased expressions of ZO-1 and occludin induced by lipopolysaccharides(LPS)+ATP stimulation.MAC-T cells were transfected with NLRP3 si RNA or MCC950 and/or treated with BC and/or LGR-1.NLRP3-si RNA transfection and MCC950 attenuated BC-induced NLRP3 inflammasome activity.Expression of inflammatory cytokines and cell death suggested that the inflammatory pathway might play an important role in the induction of the NLRP3 inflammasome by BC and the protection of LGR-1.Conclusions:These results suggest that LGR-1 might be a probiotic alternative to antibiotics and could be administered to prevent mastitis in dairy cows,thus ensuring food security.

Biofilm formation under high temperature causes the commensal bacteria Bacillus cereus WPySW2 to shift from friend to foe in Neoporphyra haitanensis in vitro model

Although biofilm formation may promote growth,biofilms are not always beneficial to their hosts.The biofilm formation characteristics of Bacillus cereus WPySW2 and its changes at different temperatures were studied.Results show that B.cereus WPySW2 promoted the growth of Neoporphyra haitanensis(an economically cultivated seaweed)at 20℃ but accelerated algal rot at 28℃.Thicker B.cereus WPySW2 biofilms covered the surface of N.haitanensis thalli at 28℃,which hindered material exchange between the algae and surrounding environment,inhibited algal photosynthesis and respiration,and accelerated algal decay.Compared with planktonic bacteria,mature biofilm cells had lower energy consumption and metabolic levels.The biofilm metabolic characteristics of B.cereus WPySW2 changed significantly with temperature.High temperature accelerated biofilm maturation,which made it thicker and more stable,allowing the bacteria to easily adapt to environmental changes and obtain greater benefits from their host.High temperature did not affect the production or increased the abundance of toxic metabolites,indicating that the negative effects of B.cereus WPySW2 on algae were not caused by toxins.This study shows that increased temperature can transform a harmless bacterium into a detrimental one,demonstrating that temperature may change the ecological function of phycospheric bacteria by affecting their morphology and metabolism.

六价铬还原细菌Bacillus cereus S5.4的筛选鉴定及还原特性研究

六价铬生物毒性极大，是造成环境污染的主要重金属之一，其生物治理策略已引起了广泛关注。已经发现许多微生物具有六价铬抗性和还原性，但能工业应用的还十分有限。从宝钢电镀污泥中分离得到一系列高六价铬抗性菌株，其中一株S5．4显示出高六价铬还原性，经形态和生理生化特征及16s rDNA序列比对，鉴定为Bacillus cereus。该菌株好氧生长，在固体LB培养基上培养48h能耐受40mmol／L Cr^6＋，并对Mn^2＋、Ba^2＋和Mo^6＋也显出高抗性；在液体LB培养基中培养72h完全还原2mmol／L Cr^6＋，并能在补充培养基和六价铬的条件下连续还原。该菌株还原六价铬时，最适浓度为2mmol／L Cr^6＋，最适温度范围30～37℃，最适pH7～9。

Control Efficacy and Antifungal Mechanism of Bacillus Cereus Strain JK14 against Wheat Take-all Disease

[Objective] Study on control efficacy and inhibitory effect of Bacillus cereus strain JK14^·against wheat take-all disease, investigating its antifungal mechanism. [Method] B. cereus JK14^· was isolated from soil in the rhizosphere of wheat, inhibitory effects of whose nutrient solution form against Gaeumannomyces graminis var tritici strains 9862 and 9812 were measured in laboratory and then for its antifungal mechanism. The strain JK14^· with rifampicin and wheat take-all disease resistance was screened by increasing concentration of the two substrates, and colonization of JK14^·was studied. [Result] In pot experiment, the control effects of JK14^·, against 9862 and 9812 are 63% and 59%, respectively, which are higher than that of chemical fungicides, with 55% and 51% , respectively. JK14^· could deform mycelium and causes degradation of cell wall. And there are also dynamic change of JK14^· in root system. JK14^· on seed could extend to root along with seed germination and rooting, but per unit tissue mycelium number decreased gradually. [Conclusion] The results indicate some control efficacy of B. cereus strain JK14^· against wheat take-all disease.

培养条件对Bacilluscereus 357生长及产生拮抗物质的影响

为了了解Bacillus cereus357菌株生长及产生拮抗物质的影响因子,对Bacillus cereus357菌株在KMB、PDA、LB、Davis、SMA和淡薄培养基等6种培养基上的生长及产生的拮抗物质的生物活性进行检测和比较.结果发现在这六种培养基中,KMB培养基最适合该菌株的生长,所产生的拮抗物质生物活性最大;但相同菌量相应的拮抗物质生物活性却以Davis培养基为最高;同时研究发现利用液体培养基培养与固体培养基培养对拮抗物质的产生没有影响.另外,在不同温度、不同pH值等培养条件下对B.cereus357菌株的生长及产生的拮抗物质活性进行测定,结果表明在37℃和pH 7.5的培养条件下,该菌株产生的拮抗物质抑菌活性最大.生长曲线分析表明该菌株在对数生长终期达到最大生物活性.为今后大量制备Bacillus cereus357产生的拮抗物质和进行生长条件的深入分析提供了实验基础.

耐冷菌Bacillus cereus SYP-A3-2低温蛋白酶的发酵条件

研究了营养因子及环境条件对中国冰川1号耐冷菌BacilluscereusSYP-A3-2产低温蛋白酶的影响。结果表明对该菌产酶影响较大的因素分别是温度、酪蛋白浓度和pH,在酪蛋白浓度0·8%、pH7、15℃下摇瓶发酵48h产酶水平可达3,800U/mL。

蜡样杆菌(Bacillus cereus)M22 Mn-SOD cDNA片断的克隆

分析不同种细菌Mn SOD氨基酸序列的保守区域 ,设计一对简并引物进行RT PCR扩增 ,产物经T A载体连接转化大肠杆菌JM 1 0 9,筛选阳性克隆、测序并进行同源性分析 ,得到 4 36bpMn SODcDNA片段。根据此片段设计 5′端特异性引物 ,然后进行 5′RACE扩增 ,获得Mn SOD 5′端 387bpcDNA片断。将 2段序列进行拼接获得 5 94bpcDNA片断 ,编码 1 72个氨基酸。对推定氨基酸序列进行BLAST分析 ,结果表明其与炭疽芽孢杆菌 (Bacillusanthracis)同源性为 95 % ,且Gly77,Aly78,Phe86,Gln1 50 和Asp1 51 在已报道的Mn SOD中均存在 ,构成Mn SOD的活性中心。表明该序列为蜡样芽孢杆菌Mn

耐有机溶剂蛋白酶产生菌Bacillus cereus WQ9-2发酵条件及酶学性质

以蜡状芽孢杆菌Bacillus cereus WQ9-2为产耐有机溶剂蛋白酶的出发菌株,对其产酶条件进行优化并初步研究了其酶学性质。在单因素实验基础上,通过中心复合实验确定了产酶的最佳发酵条件为酵母粉8 g/L,葡萄糖17 g/L,KH2PO40.5 g/L,无水MgSO40.3 g/L,CaCl20.5 g/L,NaCl 1.0 g/L;pH 7.5。实验中发现采用低温发酵能大大缩短菌体产酶周期;通过优化发酵时间由最初84 h缩短到48 h,最高比酶活为3 921 U/mL。金属离子螯合剂1,10-菲罗啉(1,10-phenanthroline)、乙二胺四乙酸(EDTA)对该酶有较强的抑制作用,表明该酶可能为金属蛋白酶;Ca2+对该酶的活力及热稳定性有显著提高作用。

B.cereus MBL13-U产牛骨胶原蛋白酶工艺优化

为了获得能够高效降解牛骨胶原蛋白的新型胶原蛋白酶,以蜡样芽孢杆菌(Bacillus cereus MBL13-U)为出发菌种,以胶原蛋白酶活力为检测指标,对影响发酵的4个因素(菌种接种量、发酵温度、p H、发酵时间)进行单因素试验。通过四元二次正交旋转组合试验,确定了B.cereus MBL13-U菌株发酵制备胶原蛋白酶的最佳工艺。当菌种接种量4%(体积分数)、发酵温度35℃、初始p H6.4、发酵时间46 h时,胶原蛋白酶活力达(92.31±1.13)U/m L。将其作用于牛骨胶原蛋白,通过扫描电子显微镜对酶解过程中的牛骨胶原蛋白进行结构分析。分析结果表明:酶解破坏了牛骨胶原蛋白原本完整的表面结构,使其所含的Ⅰ型胶原蛋白更多地暴露在表面,加快水解。

Bacillus cereus SZ-4产蛋白酶脱毛蓝湿革机械性能研究

将蜡状芽孢杆菌(Bacillus cereus)SZ-4摇瓶发酵后所产脱毛蛋白酶粗酶液应用于猪皮、牛皮和绵羊皮的脱毛后,对经软化工序与未经软化工序制备成的蓝湿革进行比较。绵羊皮二者之间,在抗张强度、撕裂强度检测结果上未表现出明显的差别;而对于猪皮、牛皮而言,因为其皮张较羊皮厚且脱毛作用时间比较短,导致脱毛酶只能作用至毛囊附近而未能进一步对皮胶原纤维间质等进行水解,未能完全达到软化皮张的作用。

海洋细菌Bacillus cereus OPF-0031产普鲁兰酶的发酵条件优化研究

利用响应面法对海洋细菌Bacillus cereus OPF-0031产普鲁兰酶发酵条件进行优化。在单因素实验基础上,首先通过两水平的PB(Plackett-Burman design)设计筛选出影响普鲁兰酶发酵的四个显著因素:硫酸铵、硫酸镁、硫酸铁、糯米粉;通过4因素4水平的响应面二次回归中心组合实验(central composite design,CCD)对上述影响因素进行优化。实验结果运用Design-Expert软件进行回归分析,最终确定最佳的培养条件:糯米粉15.53 g/L,硫酸铵9 g/L,硫酸镁0.9 g/L,硫酸铁0.018 g/L,蛋白胨10 g/L,磷酸二氢钾0.5 g/L反应温度为30℃,转速为180 r/min,最终酶活可达到2.7742 U/mL。

脱毛蛋白酶生产菌株Bacillus cereus SZ-4的筛选及遗传稳定性检测

采用平皿菌落分离法、透明圈法及摇瓶发酵法从盐腌皮上分离、筛选得到脱毛蛋白酶生产菌株Bacillus cereus SZ-4,该菌株经发酵后得到的粗酶液酶活力为72/mL;对其遗传稳定性检测结果表明,Bacillus cereus SZ-4遗传稳定性较差,在1～4代菌种之间差异不是很显著,可用于发酵生产接种。

Isolation and characterization of glacier VMY22, a novel lytic cold-active bacteriophage of Bacillus cereus

As a unique ecological system with low temperature and low nutrient levels, glaciers are considered a "living fossil" for the research of evolution. In this work, a lytic cold-active bacteriophage designated VMY22 against Bacillus cereus MYB41-22 was isolated from Mingyong Glacier in China, and its characteristics were studied. Electron microscopy revealed that VMY22 has an icosahedral head(59.2 nm in length, 31.9 nm in width) and a tail(43.2 nm in length). Bacteriophage VMY22 was classified as a Podoviridae with an approximate genome size of 18 to 20 kb. A one-step growth curve revealed that the latent and the burst periods were 70 and 70 min, respectively, with an average burst size of 78 bacteriophage particles per infected cell. The pH and thermal stability of bacteriophage VMY22 were also investigated. The maximum stability of the bacteriophage was observed to be at pH 8.0 and it was comparatively stable at p H 5.0–9.0. As VMY22 is a cold-active bacteriophage with low production temperature, its characterization and the relationship between MYB41-22 and Bacillus cereus bacteriophage deserve further study.

Anaerobic benzene biodegradation by a pure bacterial culture of Bacillus cereus under nitrate reducing conditions

A pure culture using benzene as sole carbon and energy sources was isolated by screening procedure from gasoline contaminated soil.The analysis of the 16S rDNA gene sequence,morphological and physiological characteristics showed that the isolated strain was a member of genus Bacillus cereus.The biodegradation performance of benzene by B.cereus was evaluated,and the results showed that benzene could be efficiently biodegraded when the initial benzene concentration was below 150 mg/L.The metabolites of anaerobic nitrate-dependent benzene oxidation by strain B.cereus were identified as phenol and benzoate.The results of substrate interaction between binary combinations for benzene,phenol and benzoate showed that the simultaneous presence of benzene stimulated the degradation of benzoate,whereas the addition of benzene inhibited the degradation of phenol.Benzene degradation by B.cereus was enhanced by the addition of phenol and benzoate,the enhanced effects were more pronounced at higher concentration.To our knowledge,this is the first report that the isolated bacterial culture of B.cereus can efficiently degraded benzene under nitrate reducing conditions.

Biogenesis of antibacterial silver nanoparticles using the endophytic bacterium Bacillus cereus isolated from Garcinia xanthochymus

Objective:To synthesize the ecofriendly nanoparticles,which is viewed as an alternative to the chemical method which initiated the use of microbes like bacteria and fungi in their synthesis.Methods:The current study uses the endophytic bacterium Bacillus cereus isolated from the Garcinia xanthochymus to synthesize the silver nanoparticles(AgNPs).The AgNPs were synthesized by reduction of silver nitrate solution by the endophytic bacterium after incubation for 3-5 d at room temperature.The synthesis was initially observed by colour change from pale white to brown which was confirmed by UV-Vis spectroscopy.The AgNPs were further characterized using FTIR,SEM-EDX and TEM analyses.Results:The synthesized nanoparticles were found to he spherical with the size in the range of 20-40 nm which showed a slight aggregation.The energy-dispersive spectra of the nanoparticle dispersion confirmed the presence of elemental silver.The AgNPs were found to have antibacterial activity against a few pathogenic bacteria like Escherichia coli,Pseudomonas aeruginosa,Staphylococcus aureus,Salmonella typhi and Klebsiella pneumoniae.Conclusions:The endophytic bacteria identified as Bacillus cereus was able to synthesize silver nanoparticles with potential antibacterial activity.

Surface reaction of Bacillus cereus biomass and its biosorption for lead and copper ions

In this study, the surface chemical functional groups of Bacillus cereus biomass were identified by Fourier transform infrared （FTIR） analytical technique. It had been shown that the B. cereus cells mainly contained carboxyl, hydroxyl, phosphate, amino and amide functional groups. The potentiometric titration was conducted to explain the surface acid-base properties of aqueous B. cereus biomass. The computer program FITEQL 4.0 was used to perform the model calculations. The optimization results indicated that three sitesthree pKas model, which assumed the cell surface to have three distinct types of surface organic functional groups based on the IR analysis results, simulated the experimental results very well. Moreover, batch adsorption experiments were performed to investigate biosorption behavior of Cu（Ⅱ） and Pb（Ⅱ） ions onto the biomass. Obviously, the adsorption equilibrium data for the two ions were reasonably described by typical Langmuir isotherm.

Detection and sequencing of plasmid encoded tetracycline resistance determinants(tetA and tetB) from food-borne Bacillus cereus isolates

Objective:To investigate the detection and sequencing of plasmid encoded tetracycline resistance genes(tet A and tet B) from food-borne and standard strains of Bacillus cereus(B. cereus).Methods:PCR was carried out to detect the tetracycline resistance genes(tet A and tet B) in food-borne B.cereus strains and the amplified products were sequenced.Results:The phenotypic resistance against tetracycline was observed in 39 of the 118 food-borne isolates and two reference strains(MTCC 430 and MTCC 1307) of B.cereus.Among the phenotypically resistant isolates,tet A was detected in 36 food-borne isolates and two reference strains(MTCC 430 and MTCC 1307).whereas,tel B was detected in 12 food-bome isolates and MTCC 1307 strain. Conclusions:A close association was therefore found between phenotypic resistance against tetracycline and presence of tetracycline resistance genes.The tet A and tet B gene fragments were amplified,purified and sequenced.The gene sequences of the isolates studied herein were found similar to tetA and tetB gene sequences of other bacteria available in NCBI.The occurrence of tetA and tetB genes in B.cereus indicate the horizontal transfer of antibiotic resistance determinants from other bacteria into B.cereus.The transfer of these resistant determinants to other potentially pathogenic bacteria may be a matter of great concern.

Colonization of Bacillus cereus NJSZ-13,a species with nematicidal activity in Masson pine(Pinus massoniana Lamb.)

Bacillus cereus NJSZ-13,an endophytic bacterium with nematicidal activity,was isolated from stems of healthy Pinus elliottii Engelm.Colonization of P.massoniana Lamb.by endophytic B.cereus was studied using scanning electron microscopy and confocal laser scanning microscopy.After the plasmid p GFP78 containing the green fluorescent protein(GFP)gene was transformed into the NJSZ-13 strain,the NJSZ-13:gfp showed the same nematicidal activity and growth curve as the wild-type strain,and the plasmid p GFP78 was stably maintained in strain NJSZ-13 for at least 96 h of bacterial cultivation on medium without antibiotics.After inoculation into Masson pine roots,colonization of the NJSZ-13:gfp strain in plant roots and stems was visualized using confocal laser scanning and the strain was enumerated in inoculated roots and stems.These results suggest that NJSZ-13:gfp is an efficient colonizer of Masson pine and can transfer vertically from roots to stems.