Expression of mel gene improves the UV resistance of Bacillus thuringiensis

Aims:  To improve ultraviolet (UV) resistance of Bacillus thuringiensis for increasing the duration of the Bt product applied in the field, a genetically engineered strain Bt TD841 that produced both melanin and Cry1A protein was constructed, and its UV resistance was evaluated in the laboratory.
Methods and Results:  Melanin quantitative analysis revealed that the recombinant strain Bt TD841 could synthesize 0.15 mg melanin ml⁻¹ sporulated culture. Atomic force microscopy confirmed the production of diamond crystal and SDS-PAGE results showed the expression of the 130 kDa Cry1A protein. Bioassay results demonstrated that the LC₅₀ value of Bt TD841 was 3.69 μl ml⁻¹ against Helicoverpa armigera and the UV resistance of this recombinant was enhanced 9.7-fold compared to its parental strain Bt HC42 after 4-h UV irradiation.
Conclusion:  Expression of the mel gene can significantly increase UV resistance of B. thuringiensis.
Significance and Impact of the Study:  This is the first report on genetically engineered Bt strain with co-expression of melanin and the insecticidal crystal proteins gene, and the results may offer a practical solution for improving the photoprotection of Bt products in field application.     

胞苷生产菌的选育

目的：筛选得到胞苷发酵单位较高的菌株,并对发酵过程作初步研究。方法：以胞苷脱氨酶缺失枯草芽孢杆菌DOS7为出发菌株,对其进行紫外诱变、5-氟胞苷（5FCR＋）和2-杂氮尿嘧啶抗性（2AU＋）抗性筛选。结果：通过紫外诱变和抗性筛选得到突变株DOS7-2-1000-15,抗5-氟胞苷和2-杂氮尿嘧啶的临界浓度分别为800mg/L和1 000mg/L。同时检测了抗5-氟胞苷突变株中CTP合成酶的活性,比原始菌株提高了12.4%,突变株DOS7-2-1000-15发酵过程结果为：36℃发酵72h能积累胞苷最高为3.5g/L。结论：筛选得到的突变株DOS7-2-1000-15的遗传稳定性较好,可稳定发酵。     

苏云金杆菌酪氨酸酶基因转化子黑色素的研究

苏云金杆菌(Bacillusthuringiensis,Bt)是使用广泛的杀虫细菌。将构建的含有嗜麦芽假单胞菌酪氨酸酶基因的重组质粒导入苏云金杆菌体内,使后者获得了稳定产生黑色素的能力。所产黑色素是无定形物质,在大多数有机和无机溶剂中溶解度低或不溶解,在碱性溶液中有较好的溶解性,在中性及弱酸性溶液中溶解度低,pH<4时沉淀;可用过氧化氢或次氯酸钠氧化,也能被银离子或硫化氢还原;含有天然物质中少见的自由基,并能清除由紫外线诱导产生的自由基;能吸收所有波长的光线,在紫外波长下吸收值最大,能有效地保护紫外辐射对蛋白质及DNA等生物大分子物质的损害,具有很强的抗辐射作用。使转化子抗辐射能力显著增强,杀虫活性稳定保持;能抵抗病毒的侵袭,生长旺盛;对畜禽安全。     

Transformation and Transduction in Recombination-defective Mutants of Bacillus subtilis

The effects on transformation and transduction of an ultraviolet sensitivity (uvr(-)) and two ultraviolet sensitivity-recombination deficiency (rec-1(-) and rec-2(-)) mutations in isogenic strains of Bacillus subtilis were investigated. Transformation frequency in the rec-1(-) and rec-2(-) strains was reduced to approximately 5 and 25%, respectively, of the parental strains. Normal kinetics of deoxyribonucleic acid dose response in transformation were found for the rec-1(+) and rec-2(-) strains. Biphasic curves were obtained with the rec-1(-) strains. Transduction frequency with bacteriophage SP-10 decreased parallel to transformation frequency in the rec-1(-) and rec-2(-) strains. This result suggests that transformation and SP-10 transduction share a common mechanism for genetic recombination. It also indicates that the reduction in transformation frequency of these strains was not due to altered competence. Transduction frequency with bacteriophage PBS-1 or 3NT, on the contrary, was not diminished in rec-1(-) strains. This frequency was reduced in rec-2(-) strains but not as severely as that of transformation or SP-10 transduction. Several hypotheses to interpret these differences are presented. Recombination frequency between linked markers was reduced more than 50% in transformation by the presence of the rec-1(-) mutation. Linkage was unaffected in the rec-2(-) strains. Neither the rec-1(-) nor the rec-2(-) mutation had an effect on linkage in PBS-1 or 3NT transduction. The uvr(-) strains were transformed at a frequency equal to or greater than that of the parental strains. These strains were transduced by all bacteriophage systems at frequencies about twofold higher than those of parental strains.     

枯草芽孢杆菌对杨树水泡溃疡病菌菌丝生长的抑制作用

测定了枯草芽孢杆菌BS-2菌株对杨树水泡溃疡病菌菌丝生长的影响,发现BS-2菌株能够抑制病菌菌丝的生长,并导致菌丝形态畸变,表现为菌丝伸长生长受抑制、扭曲、原生质浓缩、局部膨大成球形或椭圆形以及菌丝断裂和菌丝细胞变成空泡等。BS-2发酵液对病菌菌丝生长也有明显的抑制作用,培养时间越长,其培养液的抑菌作用越强;在不同培养基中获得的发酵液对病菌的抑制效果不同。     

Initiation and Termination Mutants of Bacillus subtilis Bacteriophage SPO1

Mutants affected in cistrons 21 and 32 of bacteriophage SPO1 are defective specifically in the initiation of DNA replication. Mutations in cistron 32 also specifically affect the termination of replication.     

滨海白首乌提取物对酪氨酸酶活力的影响

以l-dopa为底物,研究了白首乌提取物的酪氨酸酶活性影响作用。结果表明白首乌提取物对酪氨酸酶抑制作用显著,可使酶促反应进程出现较长迟滞期,稳态酶活力缓慢下降。白首乌提取物终浓度80.0μg/mL时,迟滞时间为626 s,稳态酶活力相对维生素C抑制率为56.25%。白首乌提取物对酪氨酸酶的抑制机理为可逆的竞争性抑制。     

Hyperproduction of Orotic Acid and Orotidine by Mutants of Bacillus subtilis

The fatty acid composition of lipids in various organs and in the stomach contents of two species of frigate mackerel, Auxis rocheri and Auxis thazard, related to the tuna species was determined. Docosahexaenoic acid was the dominant unsaturated fatty acid accounting for 20% or more of the total fatty acids in all organs of the two frigate mackerel species (mean ±S.D.: 22.6 ± 6.0% for rocheri, 28.0 ± 4.3% for A. thazard), while the fatty acids in lipids from their stomach contents were comparatively low (1.5–13.0% for A. rocheri, 15.4–16.5% for A. thazard). It is suggested that the high content of DHA in the lipids of tuna species is a general characteristic.     

Isolation and Characterization of Tricarboxylic Acid Cycle Mutants of Bacillus subtilis

A technique was developed for the detection, on agar, of mutants of Bacillus subtilis that lacked a functional tricarboxylic acid cycle. Mutants devoid of detectable levels of aconitase, isocitric dehydrogenase, alpha-ketoglutarate dehydrogenase, succinic dehydrogenase, fumarase, and malate dehydrogenase have been isolated and characterized. Several mutants with conditionally expressible lesions, including a mutant with a heat-sensitive citrate synthase, have also been isolated. All of the mutants examined express all the biochemical markers normally absent in early-stage sporulation mutants except elastase, and some of these mutants sporulated nearly as well as the prototroph.     

Isolation and Characterization of Thymineless Mutants from Ultraviolet-Sensitive Bacillus subtilis Strains

Thymineless mutants of Bacillus subtilis 168 ind, hcr-9 were isolated by using trimethoprim. These and other Thy⁻ strains differed drastically from Thy⁺ ones in their patterns of [³H]thymidine uptake and growth in trimethoprim-containing medium. Transformation was negligible between most mutants derived from the ultraviolet-sensitive strain 168 ind, hcr-9 but significant between 168 ind, thy and these mutants. The latter and these new mutants all grow in the presence of trimethoprim plus thymidine or thymine and fail to grow if thymine or thymidine is omitted.     

同步辐射软X射线对枯草杆菌的诱变效应

采用同步辐射软X射线对枯草芽孢杆菌(Bacillus subtilis)1831菌株进行辐照处理,研究了不同剂量下3．1nm的软X射线对其芽孢的失活和诱变作用。结果表明：同步辐射软X射线对枯草杆菌芽孢的剂量存活曲线表现为典型的“肩型”,对芽孢的失活作用属于“单靶多击”方式,失活击中数等于3。根据脱脂牛奶平板上蛋白酶活力大小的测量统计,以变异系数作为诱变效应指标,软X射线对芽孢具有一定的诱变作用。     

Mechanism of Anucleate Cell Production in the oriC-Deleted Mutants of Bacillus subtilis

We constructed oriC-deleted mutants of Bacillus subtilis byintegrating the minimal replication region of plasmid pLS32into the proA (115°), spoIIIJ (360°) and thrS (256°)loci of the chromosome, respectively. All three mutants producedanucleate cells and the DNA/protein ratio was lower than thatof the wild-type strain when grown in nutrient broth. However,when grown in minimal-glucose medium, the frequency of anucleatecells was reduced in all of them and the DNA/protein ratio wasrestored to normal. Especially, the oriC-deleted mutant in whichthe plasmid was integrated near oriC produced almost no anucleatecell. These results indicate that initiation frequency of chromosomereplication from the integrated plasmid origin were reduceddisproportionately to cell mass increase in rich medium, whichin turn disrupted coordination between DNA replication cycleand cell division cycle. The locations of the plasmid originrelative to the natural oriC locus affected the production ofanucleate cell remarkably, suggesting that partition mechanismof chromosome was also impaired by the translocation of itsreplication origin.     

Production of Guanosine by Psicofuranine and Decoyinine Resistant Mutants of Bacillus subtilis

Growth of Bacillus subtilis AG169 that produced large amounts of xanthosine and guanosine was inhibited by psicofuranine. When AG169 was mutated to resistance against psicofuranine, a mutant, GP–1, which yielded more guanosine was obtained. Psicofuranine did not inhibit growth of GP–1 any more. The guanosine 5′-monophosphate (GMP) synthetase activities were then assayed. In GP–1, the specific activity decreased about half, the complete loss of repression by GMP was found, and the inhibition by GMP was slightly loosed, when compared with those of AG169.

Furthermore, as growth of GP–1 was strongly inhibited by decoyinine, decoyinine resistant mutants were derived from GP–1. Of these mutants, two strains, MG–1 and MG–4, were resistant to decoyinine completely and showed the exclusive accumulation of guanosine in high yields, i.e. 16.0 and 15.5 g of guanosine per liter with weight yields of 20.0 and 19.4% of consumed sugar, respectively. GMP synthetase activity of MG–1 increased remarkably in comparison with that of GP–1 or AG169, and the inhibitions by GMP, psicofuranine and decoyinine were completely released in MG–1. Namely, the psicofuranine and decoyinine resistances seemed to cause mainly variations of GMP synthetase, and as results, the conversion of xanthosine 5′-monophosphate (XMP) to GMP proceeded more smoothly, and a larger amount of guanosine was accumulated.     

Genetic Changes in Regulation Occurring in the Tryptophan Biosynthetic Pathway of l-Tryptophan Producing Mutants Derived from Bacillus subtilis K

The regulatory mechanism for l-tryptophan (l-Trp) synthesis was compared between the wild type strain and l-Trp producing mutants of B. subtilis K. In the wild type strain, indolmycin (IM) repressed the synthesis of anthranilate synthetase (AS) more strongly than 5-fluorotryptophan ? (5FT), which repressed AS to the same extent as l-Trp did. 5FT inhibited the activity of AS as strongly as l-Trp did, while IM had no inhibitory effect. In the 5FT resistant strains, the syntheses of AS and tryptophan synthetase (TS-B) were markedly increased by genetic derepression, while AS remained still sensitive to the feedback inhibition by l-Trp. The facts that IM repressed the syntheses of AS and TS-B in the strain which was 5FT^r and IM^S, and did not repress those in the IM-resistant mutant suggested that IM acts as a co-repressor in a different way from 5FT.     

Production of L-Tryptophan by 5-Fluorotryptophan Resistant Mutants of Bacillus subtilis

The growth of Bacillus subtilis TR–44, a prototrophic transductant from one of inosine producers, was completely inhibited by 200 µg/ml of 5-fiuorotryptophan, a tryptophan analogue, and the inhibition was reversed by the addition of L-tryptophan.

Several mutants resistant to 5FT* produced L-tryptophan in the growing cultures. The best producer, strain FT–39, which was selected on a medium containing 1500 µg/ml of 5FT, produced 2 g/liter of L-tryptophan, when cultured in a medium containing 8% of glucose but without any tryptophan precursors. In this mutant, anthranilate synthetase, a key enzyme of the tryptophan biosynthesis, had increased over 280-fold, presumably owing to a genetic derepression. From FT–39, mutants resistant to 7000 µg/ml of 5FT were derived. Among them, strain FF–25 produced 4 g/liter of L-tryptophan, twice as much as did the parental strain. Since this strain produced large amount of L-phenylalanine as well as L-tryptophan, the genetic alteration seemed to be involved in some metabolic regulation of common part of the aromatic amino acid biosynthetic pathway.

Further, some auxotrophs derived from these 5FT resistant mutants produced more L-tryptophan than did the parental strains.

Relationships between the accumulation of L-tryptophan and the regulation mechanisms of the L-tryptophan biosynthesis were discussed.     

Isolation and Properties of Suppressor-Sensitive Mutants of Bacillus subtilis Bacteriophage SPO2

Suppressor-sensitive mutants of Bacillus subtilis phage SPO2 were isolated and classified into 18 cistrons. A linear genetic map was constructed. The genes controlling early functions of phage development were clustered near the CI gene which may produce phage repressor.     

Sporulation and Antibiotic Production by Bacillus subtilis Mutants Deficient in Intracellular Proteases

Erythropoietin (Ep) was isolated from the urine of patients with aplastic anemia [Yanagawa et al., J. Biol. Chem., 259, 2707 (1984)] and burst-promoting activity (BPA) was extensively purified from the residue obtained after removal of Ep. These erythropoietic factors were studied for their effcects on erythroid burst-colony formation of human peripheral blood mononuclear cells in methylcellulose cultures. Reddish bursts were formed with the addition of Ep alone. Addition of BPA not only elevated the number of bursts but also greatly reduced the amount of Ep required for burst formation. The presence of BPA alone in cultures did not permit bursts to form but did permit the growth of small colonies that did not contain hemoglobin (Hb). Addition of Ep to these small colonies led to the formation of erythroid bursts. Administration of Ep to the cultures could be delayed for 6 days without decreasing the number of bursts if the cultures were initiated in the presence of BPA; in the absence of BPA, the erythroid precursors (BUF-E) were rapidly lost if Ep was not provided at the start of the cultures. BPA produced larger bursts than those formed in the presence of Ep alone. Microassays of Hb in the bursts indicated that BPA increased the amonut of Hb per burst. This increase could not be entirely explained by the augumentation in cell number per burst but was partly ascribable to the increased amount of Hb per cell.     

Regioselective synthesis of kojic acid esters by Bacillus subtilis protease

The lipophilicity of kojic acid [5-hydroxy-2-(hydroxymethyl)-4H-pyran-4-one] was improved by esterifying kojic acid with either divinyl adipate, vinyl hexanoate, vinyl octanoate or vinyl decanoate using protease from Bacillus subtilis for 7 d. ¹H-NMR and ¹³C-NMR showed that the primary hydroxyl group at the C-7 position of kojic acid was regioselectively esterified to afford 7-O-vinyl adipoyl kojic acid, 7-O-hexanoyl kojic acid, 7-O-octanoyl kojic acid and 7-O-decanoyl kojic acid (13–27% yield). The kojic acid esters had radical scavenging activities, inhibited tyrosinase activity and was biodegradable.     

Effect of Microwaves on Escherichia coli and Bacillus subtilis

Suspensions of Escherichia coli and Bacillus subtilis spores were exposed to conventional thermal and microwave energy at 2,450 MHz. The degrees of inactivation by the different energy sources were compared quantitatively. During the transient heating period by microwave energy, approximately a 6 log cycle reduction in viability was encountered for E. coli. This reduction was nearly identical to what is expected for the same time-temperature exposure to conventional heating. Heating of B. subtilis spores by conventional and microwave energy was also carried out at 100 C, in ice and for transient heating. The degree of inactivation by microwave energy was again identical to that by conventional heating. In conclusion, inactivation of E. coli and B. subtilis by exposure to microwaves is solely due to the thermal energy, and there is no per se effect of microwaves.