Screening of high-yielding biocontrol bacterium Bs<Emphasis Type="Italic">-</Emphasis>916 mutant by ion implantation

Bacillus subtilis 916 was an effective biocontrol agent in control rice sheath blight caused by Rhizoctonia solani. To further improve its antagonistic ability, low-energy ion implantation was applied in Bs-916. We studied the effects of different doses of N⁺ implantation. The optimum dose of ion implantation for the Bs-916 was from 15 × 2.6 × 10¹⁴ N⁺/cm² to 25 × 2.6 × 10¹⁴ N⁺/cm². The mutant strain designated as Bs-H74 was obtained, which showed higher inhibition activity in the screening plate. Its inhibition zone against the indicator organism increased by 30.7% compared to the parental strain. The control effect of rice sheath blight was improved by 14.6% over that of Bs-916. Thin-layer chromatography and high-performance liquid chromatography analysis indicated that lipopeptides produced by Bs-916 and the mutant strains belonged to the surfactin family. Bs-H74 produced approximately 3.0-fold surfactin compared to that of Bs-916. To determine the role of surfactin in biocontrol by Bs-916, we tested another mutant strain, Bs-M49, which produced lower levels of surfactin significantly, and found that Bs-M49 had no obvious effects against R. solani. These results suggested that the surfactin produced by Bs-916 plays an important role in the suppression of sheath blight. These observations also showed that the Bs-H74 mutant strain is a better biocontrol agent than the parental strain.     

The effects of caffeine, ultraviolet and x-irradiation on DNA breakdown in E. coli B and some of its dark repair mutants

Summary Caffeine (4 and 8 mM) increased the rate of spontaneous DNA breakdown which occurred during normal growth of a culture of E. coli Bs-11, but neither affected breakdown in the EXR strain Bs-2 nor the parental strain B. It slightly impaired the rate of DNA breakdown which follows UV-irradiation of E. coli B, but did not depress the final fraction degraded. On the other hand even 12 mM caffeine had no detectable effect on the (excessive) rate of DNA breakdown of U. V.-irradiated cultures of the EXR strain Bs-2. The effects of X-irradiation on DNA breakdown in E. coli B and a number of its U. V.-sensitive and resistant mutants are described. Caffeine had no detectable effect on the kinetics of X ray-induced DNA breakdown of E. coli B.     

Caffeine-death in Escherichia coli

Summary Growth of a culture of E. coli strain B or 15 in medium containing caffeine resulted in the accumulation of inviable cells in the population. A caffeine concentration of 8 mM caused the death of between 30% and 50% of the cells in 12 independent populations grown for 15 generations or more. The thymine dimer excision-defective strains Bs-1, Bs-8 and Bs-12 and the exr ^– mutant Bs-2 were resistant to this lethal effect. The reckless, hcr ⁺ mutant Bs-11 was more sensitive than the parental B strain. Although 100mM caffeine did not impair DNA synthesis in vitro, concentrations of the drug 8 mM caused a significant decline in DNA synthesis in vivo in E. coli B cells. From the fit of an experimental growth curve to an algebraic model of growth in which a proportion of cells are inactivated at each replication it is suggested that caffeine does not affect the replication rate of the viable cells. The observed impairment of DNA synthesis in vivo is equated with this cell death (caffeine-death). For E. coli 15 or B, 8 mM caffeine induced caffeine-death at a rate of 18% per cell generation. Caffeine-resistant mutants of E. coli B and E. coli 15 were isolated. Of those studied in detail a substantial proportion proved to be U.V. and X-ray sensitive and excision-defective. Others were more U.V. and X-ray resistant than strain B. Yet another class proved highly unstable. A chromosome breakage model of caffeine-death implicating enzymes of the excision-repair process is discussed.     

生防菌Bs-916突变菌株M49芽胞形成和感受态形成能力

摘要：生防枯草芽胞杆菌Bs-916（Bacillus subtilis）在水稻纹枯病的防治上效果显著。应用离子注入突变对Bs-916进行了突变,获得了一系列的突变菌株。其中突变菌株M49,其表面活性素Surfactin分泌量比出发菌株Bs-916大大降低并导致其防效降低。【目的】为了确认影响该菌株防效降低的影响因子,对其表型和相关基因表达水平进行了研究。【方法】应用生孢培养基,通过芽胞形成能力评测方法比较该菌株和野生菌株Bs-916的芽胞形成能力;通过转化质粒的实验评测突变菌株M49和野生型Bs-916的     

油桐尺蠖卵巢细胞系BS-484克隆株特性的研究

通过有限稀释法由Bs—484细胞系中成功地分离出四个克隆株(Bs—484B,E,F,G)。克隆细胞侏的生长特性不同于原细胞株Bs—484,各克隆株之间的形态特征、细胞倍增时间、以及在维持油桐尺蠖核型多角体病毒的复制能力等方面均有差异。用三种同工酶(乳酸脱氢酶、苹果酸脱氢酶和酯酶)比较了各克隆株与原细胞株之间的异同。     

Extensive and equivalent repair in both radiation-resistant and radiation-sensitive E. coli determined by a DNA-unwinding technique.

The extent of strand breakage and repair in irradiated E. coli B/r and Bs-1 was studied using a DNA-unwinding technique in denaturing conditions of weak alkali. Although these two strains show widely different responses to the lethal effects of ionizing radiation, they both have an equal capacity to repair radiation-induced breaks in DNA. Oxygen enhancement ratios for the killing of B/r and Bs-1 were respectively 4 and 2; but after repair in non-nutrient or nutrient post-irradiation conditions, the oxygen enhancement values for the residual strand breaks were always the same for the two strains. The equal abilities of E. coli B/r and E. coli Bs-1 to remove the strand breaks measured by this weak-alkali technique leads us to suggest that some other type of damage to either DNA or another macromolecule may play a major role in determining whether or not the cells survive to proliferate.     

转鞭毛蛋白基因水稻细菌性条斑病抗性研究

该研究从生防菌枯草芽胞杆菌Bs-916中克隆了鞭毛蛋白基因,利用转基因载体pCAMBIA1300转入水稻,筛选得到98株阳性转基因植株。分子检测结果表明,有12个转基因株系可检测到目的基因的表达。随后抗病性鉴定表明,有3个转基因株系对水稻细菌性条斑病具有较高的抗性。该研究为目前水稻抗细菌性条斑病转基因研究拓宽了可应用基因资源的范围。     

Characterization of S-Sulfokeratein from Pig Hair and Its Use as a Modifier of Type I Collagen Gel

S-sulfokeratein is prepared through S-sulfonation after the cleavage of disulfide bonds in keratin using ditiothreitol in urea. S-sulfokeratein is composed of two fractions, matrix and microfibril components, and S-sulfokeratein from the matrix component (Bs) can regenerate disulfide bonds. In this study, the effects of Bs and partially reduced Bs on type I collagen self-assembly and properties of reconstructed Bs- or partially reduced Bs-collagen gel were investigated. It was proved that collagen self-assembly was accelerated by the increased amount of added Bs, but partially reduced Bs with 10 mg DTT/100 mg Bs (Bs-10) did not affect the ratio of collagen self-assembly. The mechanical strength of Bs-collagen gel proved to be lower than control, but that of Bs-10-collagen gel was times higher than that of control.     

Effects of puromycin aminonucleoside on excision repair and recombination repair inescherichia coli

Ultraviolet (UV) lethality was increased when puromycin aminonucleoside (PAN) (3.0 mM) was added to the postirradiation medium ofEscherichia coli strains. The extent of repair inhibition differed greatly for strains WP-2hcr ⁺, B/r()hcr ⁺, WP-2hcr ^–, and Bs-1hcr ^–. The interaction between PAN and UV was synergistic in thehcr ⁺ strains. PAN enhanced UV lethality in strain B/r () to a greater degree than in WP-2hcr ⁺. There was no UV lethality enhancement by PAN (3.0 mM) in thehcr ^– strains, but the interaction of PAN (8.0 mM) with UV was synergistic. PAN decreased plaque formation of T1 UV-irradiated phage plated onE. coli Bhcr ⁺ but had no effect on phage plated on Bs-1 or WP-2hcr ^– strains. These results suggest that PAN interferes with thehcr function in UV-irradiated bacteria.     

Characterization of S-sulfokeratein from pig hair and its use as a modifier of type I collagen gel

S-sulfokeratein is prepared through S-sulfonation after the cleavage of disulfide bonds in keratin using ditiothreitol in urea. S-sulfokeratein is composed of two fractions, matrix and microfibril components, and S-sulfokeratein from the matrix component (Bs) can regenerate disulfide bonds. In this study, the effects of Bs and partially reduced Bs on type I collagen self-assembly and properties of reconstructed Bs- or partially reduced Bs-collagen gel were investigated. It was proved that collagen self-assembly was accelerated by the increased amount of added Bs, but partially reduced Bs with 10 mg DTT/100 mg Bs (Bs-10) did not affect the ratio of collagen self-assembly. The mechanical strength of Bs-collagen gel proved to be lower than control, but that of Bs-10-collagen gel was times higher than that of control.     

Further analysis of gene-for-gene disease resistance specificity in flax

The flax rust resistance gene L , a nucleotide binding site, leucine-rich repeat (NBS-LRR) class of plant resistance gene, has 12 characterized alleles with different gene-for-gene resistance specificities. Here the specificities of presumptive L1 , L5, L8 and L11 genomic clones are confirmed by transgenic expression. L6 and L11 differ by 33 amino acids, 32 in the LRR region and one in the C-terminal non-LRR region, and recognize unrelated avirulence proteins, AvrL567 and AvrL11, respectively. To analyse the specificity differences, 13 L6L11 recombinant genes were constructed in vitro and tested in transgenic flax for resistance to F2 progeny of rust strain CH5, in which the unlinked avirulence genes AvrL567 and AvrL11 segregate. The data show that the single C-terminal non-LRR region polymorphism is not involved in L6–L11 specificity differences, that polymorphisms necessary for specificity are spread throughout the LRR region and that some polymorphisms essential for L11 are not essential for L6. Seven 'null' recombinants expressed no resistance when tested with CH5-derived rusts. These were tested for new resistance specificities by inoculation with a strain of rust, Bs-1, which is distantly related to CH5 and which potentially carries a different range of avirulence specificities. The 'null' recombinant L6L11RV , which differs from L6 and L11 by its susceptibility to CH5, was resistant to strain Bs-1. The specificity difference is due to a reduction in the number of AvrL567 variants recognized by L6L11RV compared with L6 and not due to recognition of an unrelated Avr gene product in strain Bs-1.     

The response of E. coli Bs-1 to tritium-beta particles under aerated and anoxic conditions.

E. coli Bs-1 cells were exposed to acute doses of tritium-beta particles by suspension in tritiated water for known lengths of time. The resulting survival rate was compared with that obtained for external irradiation with 7 MeV electrons. The o.e.r. measured for tritium-beta s was not significantly different from the value of 2.15 measured for 7 MeV electrons. The r.b.e. of the tritium beta s relative to 7 MeV electrons was 1.21 in both air and nitrogen. These results were compared with existing data for low voltage electron irradiations and with track segment studies of the effect of varying LET on the radiosensitivity of E. coli Bs-1.     

Survival and repair of single-stranded DNA breaks after gamma-irradiation of Escherichia coli cells depending on the presence of plasmids pKN101 and COIIB-P9

Plasmids, pKM101 and ColIb-P9, present in an autonomous state in E. coli AB 1157, JC5519, and P3478 cells at the stationary and logarithmic phases of growth, somewhat sensitize the cells to the lethal effect of gamma-radiation and do not influence the radiosensitivity of B/r, Bs-1 gamma R, Bs-1, and W3110 cells. The efficiency of repair of gamma-ray-induced DNA single-strand breaks in AB1157 and P3478 cells containing plasmids is somewhat lower than that in the same non-plasmid strains.     

油桐尺蠖核型多角体病毒的空斑测定

在油桐尺蠖卵巢细胞系上对油桐尺蠖核型多角体病毒(BsNPV)进行了空斑测定。用此方法测定了BsNPV的感染力,并将所得的结果与其TCID_(50)进行比较,结果显示这两种方法在测定病毒感染力时敏感性相似。     

The synthesis of DNA by DNA-membrane complexes from irradiated E. coli B/r and Bs-1: the role of the membrane.

DNA-membrane complexes were isolated from lysed E. coli B/r and Bs-1, either by low g forces from a low salt solution, or by high g forces through a discontinuous sucrose gradient. The latter method was more gentle. Irradiation of the intact bacteria had no effect on the membrane macromolecules or on RNA components of these complexes. DNA loss was not significant after irradiation under anoxic conditions but complexes isolated from from Bs-1 irradiated in air showed an appreciable decrease in DNA content. In the presence of the appropriate nucleotide mixture, both 'free' DNA, found in the supernatant fractions, and rapidly sedimented membrane-associated DNA were able to synthesize DNA in the absence of added polymerase. DNA synthesis associated with 'free' DNA was more sensitive to radiation than that associated with DNA bound to the membrane, which appeared to moderate the effects of radiation on new DNA synthesis. It is concluded that the depression of DNA synthesis is primarily a result of irradiation-induced changes on genome-DNA. The interpretation of earlier work from our laboratories that DNA-membrane complexes contained the macromolecular structure which responded to radiation with a high o.e.r. is not supported by the evidence in this work.     

Deoxyribonucleic acid strand breaks during freeze-drying and their repair in Escherichia coli.

Freeze-drying of Escherichia coli cells caused strand breaks of deoxyribonucleic acid (DNA) in both radiation-sensitive and -resistant strains. However, in the radiation-resistant strain E. coli B/r the damaged DNA was repaired after rehydration, whereas in the radiation-sensitive strain E. coli Bs-1 the damaged DNA was not repaired and the DNA was degraded. Repeated freeze-drying did not break the damaged DNA into smaller pieces.     

Dark- and K-reactivation in UV irradiatedEscherichia coli

Summary UV survival of E. coli strains with different sensitivities has been measured under several post-irradiation conditions, which have been found to influence only the efficiency of two known reactivation mechanisms.Strains BB, B and Bs-1 (equivalent to fil⁺ and probably lon^–) cannot use a reactivation mechanism which is active in K 12 (K-reactivation). Only a regulatory component is lacking, which can be substituted for by pantoyl lactone or heat treatment. B or BB cells in the growing state perform incomplete dark-reactivation; liquid holding or growth to the stationary phase restore it. K-reactivation also allows completion of darkreactivation.     

Some features of the reaction of intracellular bacteriophages T1 to UV irradiation.

The reaction of complexes pf phage T1-cells of E. coli B or E. coli Bs-1 to UV irradiation was investigated. The complexes were irradiated at various stage of infection, and their survival, extent of Hcr and Phr, were evaluated. It was found that the UV resistance of phage DNA in the second half of the latent period fluctuates. Hcr after UV exposure at these stages of infection operates in a small volume. The ability of intracellular phage to photoreactivate when cells of E. coli B were infected is constant after irradiation at many stages of infection, except the early ones. In the complexes of phage T1-bacteria of E. coli Bs-1 this ability declines while infection is promoted. The daughter phage particles released from UV irradiated complexes undergo Phr and Hcr only after irradiation at the late stages of infection. This was not the cases when complexes of phage-bacteria were irradiated during the first half of the latent period. A possible tole of UV-damaged phage DNA in propagation of infection and in maturation of phage particles is discussed.     

Survival and the repair of single-stranded DNA breaks in gamma-irradiated Escherichia coli cells adapted to methylmethane sulfonate]

The survival and repair of single-strand breaks of DNA in gamma-ray-irradiated E. coli adapted to MMS (20 mkg/ml during 3 hours) have been investigated. It is shown that the survival of adapted bacteria of radioresistant strains B/r, H/r30, AB1157 and W3110 pol+ increases with DMF (dose modification factor) ranging within 1.4-1.8 and in radiosensitive strains Bs-1, AB1157 recA13 and AB1157 lexA3 with DMF ranging within 1.3-1.4, and does not change in strains with mutation in polA gene P3478 polA1 and 016 res-3. There is no increase in radioresistance during the adaptation to MMS under the action of the protein synthesis inhibitor chloramphenicol. The increase in radioresistance during the adaptation to MMS correlates with the acceleration of repair of gamma-ray-induced single-strand breaks in the radioresistant strains B/r and W3110 pol+ and with the appearance of the ability to repair some part of DNA single-strand breaks in the mutant Bs-1, which beyond the adaptation to MMS does not repair these damages. The incomplete reparability of DNA single-strand breaks in P3478 polA1 strain cells, both adapted and non-adapted to MMS, is equal.