Regulation of arginine and proline catabolism in Bacillus licheniformis

The enzymes in the arginine breakdown pathway (arginase, ornithine-delta-transaminase, and Delta'-pyrroline-5-carboxylate dehydrogenase) were found to be present in Bacillus licheniformis cells during exponential growth on glutamate. These enzymes could be coincidentally induced by arginine or ornithine to a very high level and their synthesis could be repressed by the addition of glucose, clearly demonstrating catabolite repression control of the arginine degradative pathway. The strongest catabolite repression control of arginase occurred when cells were grown on glucose and this control decreased when cells were grown on glycerol, acetate, pyruvate, or glutamate. The proline catabolite pathway was present in B. licheniformis during exponential growth on glutamate. The proline oxidation and the Delta'-pyrroline-5-carboxylate dehydrogenase in this breakdown pathway were induced by l-proline to a high level. The Delta'-pyrroline-5-carboxylate dehydrogenase was found to be under catabolite repression control. Arginase could be induced by proline and arginine addition induced proline oxidation, suggesting a common in vivo inducer for these convergent pathways.     

Secretion of a trypsin-like thiol protease by a new keratinolytic strain of Bacillus licheniformis

When cultured in feather-containing broth with a growth optimum of pH 7.0 and 47 degrees C, a Bacillus licheniformis strain exhibited a high chicken feather-degrading activity. A trypsin-like protease was isolated from its ferment broth and was partially characterized. The enzyme was constitutively secreted and was highly active towards N-benzoyl-Phe-Val-Arg-p-nitroanilide as chromogenic substrate. Its pH optimum was 8.5 and it exhibited the highest activity at 52 degrees C. Fractionation on Sephadex G-100 column revealed that its molecular mass was about 42 kDa. The enzyme, which is new for the genus Bacillus, is a thiol protease, as tosyl-L-phenylalanine chloromethyl ketone, tosyl-L-lysine chloromethyl ketone, phenylmethylsulfonyl fluoride and ethylenediamine tetraacetate did not inhibit it, while HgCl2 and para-chloromercuribenzoate lowered its activity.     

Re-identification of the halotolerant, biosurfactant-producing Bacillus licheniformis strain JF-2 as Bacillus mojavensis strain JF-2

Bacillus strain JF-2 (ATCC 39307) is a halotolerant, biosurfactant-producing bacterium that was initially described as a member of the species Bacillus licheniformis based on a limited set of phenotypic characteristics. Here, genetic and phenotypic analyses were employed to determine the relationship of Bacillus strain JF-2 to other Bacillus strains. The restriction patterns with AluI and analysis of gyrA and 16S rRNA gene sequences grouped Bacillus strain JF-2 with B. mojavensis^T and not with B. licheniformis^T. DNA–DNA similarity showed JF-2 was 75% similar to B. mojavensis^T and only 11% similar to B. licheniformis^T. Both strain JF-2 and B. mojavensis^T required DNA for anaerobic growth, but B. licheniformis^T did not. B. mojavensis^T and strain JF-2 did not grow anaerobically in thioglycollate medium or aerobically with propionate while B. licheniformis^T grew under these conditions. DNA–DNA similarity, gene sequence data and phenotypic characteristics all support the assignment of JF-2 as a member of the species B. mojavensis.     

地衣芽胞杆菌FLP/FRT基因编辑系统的构建及验证

地衣芽胞杆菌有效的基因编辑工具有限,为了拓展和丰富其基因编辑手段,在地衣芽胞杆菌中构建一个抗性标记可重复使用的FLP/FRT基因编辑系统,并通过敲除α-淀粉酶基因amyL、蛋白酶基因aprE及敲入外源透明颤菌血红蛋白基因vgb对该系统进行初步验证。首先以温敏质粒pNZT1为载体分别构建amyL和aprE基因敲除质粒pNZTT-AFKF和pNZTT-EFKF,两个敲除质粒各自包含针对目标基因的同源臂、抗性基因及同向的FRT位点;将敲除质粒转化地衣芽胞杆菌并经过两次同源交换过程实现目标基因的敲除;最后导入一个FLP重组酶表达质粒通过FLP/FRT系统的重组作用介导抗性基因的回收。为进一步验证本系统的实用性及编辑效率,构建了包含透明颤菌血红蛋白编码基因vgb表达盒及基因组丙酮酸甲酸裂解酶编码基因pflB敲除盒的重组质粒pNZTK-PFTF-vgb,并以此进行外源基因vgb在基因组上的定向敲入。结果显示,成功敲除amyL及aprE并回收了抗性标记卡那霉素基因,敲除后淀粉酶活和蛋白酶活分别减少95.3%和80.4%;vgb基因成功整合入基因组pflB位点并回收了抗性标记四环素基因,并利用荧光定量PCR技术检测到vgb的整合表达。文中首次建立了一个适用于地衣芽胞杆菌的、抗性标记可重复使用的FLP/FRT基因编辑系统,并进行了基因敲除及基因敲入验证,为地衣芽胞杆菌遗传改造提供了良好的方法参考。     

Cloning and characterization of the glutamate dehydrogenase gene in Bacillus licheniformis

The gdhA genes of IRC-3 GDH-strain and IRC-8 GDH+ strain were cloned,and they both successfully complemented the nutritional lesion of an E.coli glutamate auxotroph,Q100 GDH-.However,the gdhA gene from the mutant IRC-8 GDH+ strain failed to complement the glutamate deficiency of the wild type strain IRC-3.The gdhA genes of the wild type and mutant origin were sequenced separately.No nucleotide difference was detected between them.Further investigations indicated that the gdhA genes were actively expressed in both the wild type and the mutant.Additionally,no GDH inhibitor was found in the wild type strain IRC-3.It is thus proposed that the inactivity of GDH in wild type is the result of the deficiency at the post-translational level of the gdhA expression.Examination of the deduced amino acid sequence of Bacillus licheniformis GDH revealed the presence of the motifs characteristic of the familyⅠ-type hexameric protein,while the GDH of Bacillus subtilis belongs to family II.     

地衣芽孢杆菌治疗新生儿母乳性黄疸的疗效观察

目的了解地衣芽孢杆菌活菌胶囊(整肠生)治疗母乳性黄疸的疗效。方法选取2012年1月至2015年4月我院门诊治疗的新生儿母乳性黄疸69例,随机分为两组,对照组给予口服茵栀黄颗粒,观察组给予茵栀黄颗粒及整肠生治疗,观察治疗效果及两组血清胆红素值及黄疸消退时间、大便次数。结果 (1)治疗后观察组总有效率97.14%,对照组治疗总有效率82.35%,差异具有统计学意义(P0.05)。(2)治疗第5天观察组血清总胆红素值明显低于对照组,黄疸消退时间短于对照组,平均大便次数多于对照组,差异具有统计学意义(P0.01)。(3)两组患儿均无过敏及脱水等不良反应。结论整肠生治疗母乳性黄疸可缩短黄疸持续时间,显著降低血清总胆红素水平,提高治疗有效率。     

Expression in Escherichia coli of the carboxy terminal domain of the BLAR sensory-transducer protein of Bacillus licheniformis as a water-soluble Mr 26 000 penicillin-binding protein

A cloning vector has been constructed which allows production and export by Escherichia coli of the Met346-Arg601 carboxy terminal domain of the 601 amino acid BLAR sensory-transducer involved in beta-lactamase inducibility in Bacillus licheniformis. The polypeptide, referred to as BLAR-CTD, accumulates in the periplasm of E. coli in the form of a water-soluble, Mr 26,000 penicillin-binding protein. These data and homology searches suggest that BLAR has a membrane topology similar to that of other sensory-transducers involved in chemotaxis.     

Destruction of Bacillus licheniformis spores by microwave irradiation

Aims:  To investigate the sporicidal mechanisms of microwave irradiation on Bacillus licheniformis spores.
Methods and Results:  We measured spore viability and the release of DNA and proteins, and performed transmission electron microscopy (TEM). A microwave oven (0·5 kW) was modified to output power at 2·0 kW, which allowed a shorter sterilization cycle. A 2·0 kW microwave treatment at the boiling temperature for 1 min did not kill all spores, but killed most spores. The spore inactivation rate was faster than that of boiling and 0·5 kW microwave oven. In contrast to boiling and 0·5 kW microwave treatments, the 2·0 kW microwave resulted in significant leakage of proteins and DNA from spores due to injury to the spore structure. TEM revealed that 2·0 kW microwave irradiation affected spore cortex hydrolysis and swelling, and ruptured the spore coat and inner membrane.
Conclusions:  These results suggest that 2·0 kW microwave irradiation ruptures the spore coat and inner membrane, and is significantly different from boiling.
Significance and Impact of the Study:  This study provides information on the sporicidal mechanisms of microwave irradiation on B. licheniformis spores.     

Production of tannase by the immobilized cells of Bacillus licheniformis KBR6 in Ca-alginate beads

AIMS: The present study was aimed at finding the optimal conditions for immobilization of Bacillus licheniformis KBR6 cells in calcium-alginate (Ca-alginate) beads and determining the operational stability during the production of tannin-acyl-hydrolase (tannase) under semicontinous cultivation. METHODS AND RESULTS: The active cells of B. licheniformis KBR6 were immobilized in Ca-alginate and used for the production of tannase. The influence of alginate concentration (5, 10, 20 and 30 g l(-1)) and initial cell loading on enzyme production were studied. The production of tannase increased significantly with increasing alginate concentration and reached a maximum enzyme yield of 0.56 +/- 0.03 U ml(-1) at 20 g l(-1). This was about 1.70-fold higher than that obtained by free cells. The immobilized cells produced tannase consistently over 13 repeated cycles and reached a maximum level at the third cycle. Scanning electron microscope study indicated that the cells in Ca-alginate beads remain in normal shape. CONCLUSIONS: The Ca-alginate entrapment is a promising immobilization method of B. licheniformis KBR6 for repeated tannase production. Tannase production by immobilized cells is superior to that of free cells because it leads to higher volumetric activities within the same period of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of tannase production from immobilized bacterial cells. The bacterium under study can produce higher amounts of tannase with respect to other fungal strains within a short cultivation period.     

Biochemical characteristics of chitosanase from the Indonesian Bacillus licheniformis MB-2

Bacillus licheniformis MB-2, isolated from a hot spring water in Manado, Indonesia, secreted a unique chitosanase. Media consisted of 0.24% chitosan, 0.25% casiton, 1% MgSO₄, 1.4% K₂HPO₄, 0.02% CaCl₂·2H₂O, 0.002% FeSO₄·7H₂O (w/v) was used for enzyme production. Purification of the enzyme through the hydrophobic interaction chromatography system (butyl Sepharose 4 FF) resulted in two major active fractions; the F₂ fraction was shown as a single band at both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram analysis with apparent molecular mass of 75 kDa. The enzyme worked best at 70°C and pH between 6.0 and 7.0. When incubated at 70, 80, and 90°C, the t_1/2 values were 26.56, 18.44, and 16.74 min, respectively with the k constant being at 0.026, 0.037, and 0.04/min. When heated at 90°C, the enzyme retained its activity up to 8 h in the presence of 1mM MnCl₂. The enzyme's activity was unaffected by the presence of 1 M NaCl and 6 M urea but was decreased by 2 M of guanidine hydrochloride. Albeit the enzyme did not degrade colloidal and glycol chitin, it hydrolyzed glycol chitosan up to 0.8% and colloidal chitosan up to 11%. The 85% deacetylated (DDA) soluble chitosan was the most susceptible to this enzyme, followed by 90% and 100% DDA chitosan. The K _{m app} values of the 85, 90, and 100% DDA soluble chitosans were found as 0.23, 0.24, and 0.58 mg/mL, whereas the V_max values were 843, 668, and 261 U/mg, respectively. The hydrolysis products of F₂ chitosanase at 24 h incubation (70°C) were pentasaccharide (GlcN)₅ and hexasaccharide (GlcN)₆. The prelimiaary test showed inhibitory effect of chitooligosaccharides resulted from enzymatic degradation toward Pseudomonas aeruginosa, Salmonella typhimurium. Listeria monocytogenes, Bacillus cereus, Escherichia coli, and Staphylococcus aureus.     

Clostridium thermocellum与Bacillus licheniformis共培养分解纤维素的性质

【目的】采取人工构建复合菌系的方法探索微生物协同降解纤维素的机理及菌间关系。【方法】从一组高温发酵木质纤维素原料产沼气的菌群中分离获得若干菌株,其中一株细菌经16S rRNA基因全序列测序比对后鉴定为地衣芽孢杆菌(Bacillus licheniformis),将该菌株与厌氧纤维素分解菌Clostridium thermocellum CTL-6进行共培养,菌株组合表现出很强的滤纸纤维素分解能力。【结果】两菌共培养9 d,累计滤纸分解量为484.6 mg,滤纸相对分解率高达93.2%;pH变化呈先下降后逐步回升,培养3 d后pH由初始时的7.00降到最低值6.57,第9天升至7.73;菌株组合能同时产生纤维素酶和半纤维素酶,培养过程中两种酶活性大小均呈不断上升趋势,最大值分别为0.32 U/m L和0.57 U/m L。利用HPLC监测了乳酸、甲酸、乙酸、丙酸和丁酸5种有机酸含量的变化,其中丁酸、丙酸代谢量最高,分别为1 477.3 mg/L和1 068.8 mg/L;除丙酸外,其他4种有机酸含量变化趋势与滤纸降解的变化均无明显相关性。5种有机酸总含量的变化与p H的变化趋势一致,表明对pH变化起决定性作用的很可能是某种未检测的酸性较强的物质含量变化。【结论】Bacillus licheniformis能有效促进Clostridium thermocellum CTL-6的纤维素分解活性,且该菌株组合可作为后期进一步构建纤维素甲烷转化复合菌系的基础。     

Nucleotide Sequence and Features of the Bacillus licheniformis gnt Operon

Bacillus licheniformis was able to utilize gluconate as thesole carbon source as efficiently as Bacillus subtilis did.Southern analysis indicated that B. licheniformis likely possessesonly one gnt determinant. The nucleotide sequence (6278 bp)of the B. licheniformis DNA containing the gnt operon was determined,revealing the five complete open reading frames (ORF; genes).The putative product of the first gene, oug, did not show anysignificant homology to known proteins, but those of the secondto fifth genes exhibited striking homology to the gntRKPZ genesof B. subtilis, respectively, indicating that they are the correspondinggnt genes of B. licheniformis. Not only is the organizationof the gnt genes of these two Bacilli highly conserved, butso are the cis regulatory elements of their gnt operon. Sequenceanalysis of the upstream regions of these two gnt operons impliedthat a chromosome rearrangement in B. subtilis might have occurredimmediately upstream of the gnt operon during evolution, causingit to diverge from a common ancestor into B. licheniformis andB. subtilis.     

Amplification of bacitracin transporter genes in the bacitracin producing Bacillus licheniformis

We have amplified the previously cloned and sequenced genes of the bacitracin exporter (bcr), a member of the ATP-binding transport protein family, within the chromosome of the bacitracin producing Bacillus licheniformis. Amplification of the transporter genes was followed by greatly increased bacitracin resistance. Antibiotic production was enhanced at a low level of bcr genes amplification. An enlarged increase in the copy number of the bcr genes negatively affects the overall growth of bacteria.     

强化底物利用酶系表达,提升地衣芽孢杆菌生产碱性蛋白酶性能

地衣芽孢杆菌2709由于易于培养、GRAS状态和完善的蛋白质分泌能力,是已经投入工业生产碱性蛋白酶的菌株.为改善该菌株的发酵生产性能,提高菌体对培养基成分的利用和碱性蛋白酶产量,对菌株的胞外分泌酶系进行完善.利用同源重组机制,在基因组复制起始位点附近引入了来源于短小芽孢杆菌的木聚糖酶基因xynA和在复制起始位点中心对称...     

酱香型白酒发酵中地衣芽孢杆菌与酿酒酵母的相互作用

【目的】为解析酱香型白酒酿造群体微生物的发酵过程, 研究了酱香型白酒酿造中重要微生物地衣芽孢杆菌与酿酒酵母之间的相互作用, 并对它们之间的作用机制进行初步探讨。【方法】通过地衣芽孢杆菌与酿酒酵母共培养体系的构建, 认识了两者的相互作用, 初步分析了酿酒酵母产生抑制物的分子量, 耐热性及对蛋白酶敏感性等特性。【结果】研究表明, 酿酒酵母发酵造成的酸性环境以及某些代谢物质能够抑制地衣芽孢杆菌的生长, 这些物质分子量大于10 kD, 对热和蛋白酶敏感。【结论】白酒酿造中酿酒酵母通过产酸以及大分子的蛋白质类物质对地衣芽孢杆菌生长形成抑制, 该研究促进了对白酒酿造群体微生物发酵过程的解析。     

Calcite-forming Bacillus licheniformis Thriving on Underwater Speleothems of a Hydrothermal Cave

The underwater environment of Grotta Giusti (Monsummano Terme, Italy) is a suggestive setting with different types of speleothems including “leafy” and “cauliflower” concretions along the walls and roof, and conical pseudo-stalagmites on the floor. Very high calcium and dissolved CO₂ levels, and massive calcium carbonate precipitation characterize this cave environment. Yet, life thrives on the leafy concretion surfaces with loads of cultivable heterotrophic microorganisms around 10⁵ colony-forming units per cm². Bacillus licheniformis appeared to be the prevalent cultivable microorganism on a low-nutrient medium that was used for screening. 16S rRNA gene-based polymerase chain reaction–single strand conformation polymorphism profiling indicated that Group VI Bacillaceae species was well represented in the bacterial community of underwater speleothems. Interpretation of X-ray diffraction spectra and Raman spectroscopy data indicated that the B. licheniformis isolate produced in vitro abundant calcite microcrystals that were also characterized by scanning electron microscopy coupled with energy dispersive X-ray spectroscopy. Production of calcite microcrystals was analyzed in different media (Christensen’s urea agar and B4 calcium carbonate precipitation medium) and incubation conditions, and it was found to be enhanced by nitrate supplement in B4 medium under low-oxygen conditions. B4 and B4-nitrate media also stimulated antibiotic production by the B. licheniformis isolate, which was analyzed by microbiological assays.     

Introducing Bacillus licheniformis as the causal agent of pistachio dieback in Iran

During 2004 and 2006 growing seasons some pistachio trees in Kerman province of Iran showed dieback symptoms. Initial symptoms were observed at the beginning of the growing season and they developed during two weeks after green tip stage, as shoot tips turned black and dieback occurred. During the growing season, the symptoms developed and vessel destruction was observed. If these stems were not pruned during winter, dieback developed in the spring. During the growing season affected pistachio samples were collected and surface disinfected with 0.01% mercury chloride. Pieces of affected vessels were grown on nutrient agar (NA) and incubated at 25°C for three-to-four days. Bacterial colonies with Bacillus characteristics were isolated and 15 representative strains were selected for further characterisation. The pathogenicity of selected strains was verified on 2–3 year-old pistachio seedlings using injection of bacterial suspension (10⁷ cfu p/ml) and control plants inoculated with distilled water; vessel destruction developed after 20 days, and bacterial causal agent was isolated from seedlings. No symptoms were observed in control plants. The strains were Gram positive, motile, with central spores and caused a hypersensitivity reaction (HR) on tobacco and geranium; they were positive for anaerobic growth, nitrate reduction, utilisation of citrate, VP test, urease, catalase, growth at pH 5.7, 7% NaCl and 45°C, acid production from arabinose, xylose, glucose and mannitol, and anaerobic fermentation of glucose. They could hydrolyze starch, aesculin, Tween 80 and gelatin but indole production was negative. Based on the characteristics of the isolated strains, they were identified as Bacillus licheniformis. This is the first report of Bacillus licheniformis as a causal agent of pistachio dieback.     

Cloning of a thermostable α-amylase gene from Bacillus licheniformis and its expression in Escherichia coli and Bacillus subtilis

Abstract The gene coding for the thermostable α-amylase Bacillus licheniformis has been isolated from a direct shotgun in Escherichia coli using the bacteriophage lambda as a vector. The fragment containing the α-amylase gene has been sub-cloned in pBR322 and its restriction map determined. The α-amylase produced by the E. coli clones retained the thermostability of the B. licheniformis enzyme. Expression and properties of the gene product in E. coli and Bacillus subtilis have been examined.