Translocation in Bacillus subtilis: Irreversible Inactivation of Elongation Factor G and Viability of a Temperature-Sensitive Mutant

A temperature-sensitive mutant of Bacillus subtilis 168 lost its viability irreversibly when grown at temperatures higher than 50 C. It is suggested that this loss of viability is due to irreversible alteration of elongation factor G activity, which was shown in vitro by two different assay methods.     

Structural Changes in the Nucleoid of Bacillus subtilis at Low Temperature

The external shape of the nucleoid of Bacillus subtilis strain w23 was examined with a new electron microscopic technique, the rapid freezing and substitution fixation method. The nucleoid of the log and stationary phase cells was recognized as an area devoid of ribosomes and widely dispersed in the cytoplasm, which was different from that observed in OsO₄-fixed cells. If the bacteria were exposed to low temperatures (0 to 10 C), the nucleoid showed a highly concentrated shape in the middle of the cytoplasm. These structural changes were observed only when the bacteria were maintained in a high-salt buffer. The results are discussed in relation to the membrane fluidity at low temperature.     

Cloning, Sequencing, and Characterization of the recA Gene from Rhodopseudomonas viridis and Construction of a recA Strain

A recombination-deficient strain of the phototrophic bacterium Rhodopseudomonas viridis was constructed for the homologous expression of modified photosynthetic reaction center genes. The R. viridis recA gene was cloned and subsequently deleted from the R. viridis genome. The cloned R. viridis recA gene shows high identity to known recA genes and was able to complement the Rec⁻ phenotype of a Rhizobium meliloti recA strain. The constructed R. viridis recA strain showed the general Rec⁻ phenotype, i.e., increased sensitivity to DNA damage and severely impaired recombination ability. The latter property of this strain will be of advantage in particular for expression of modified, nonfunctional photosynthetic reaction centers which are not as yet available.     

Inactivation of ccmO in Synechococcus sp. Strain PCC 7942 Results in a Mutant Requiring High Levels of CO2

Inactivation of ccmO in Synechococcus sp. strain PCC 7942 resulted in a mutant which possesses aberrant carboxysomes and a normal inorganic carbon uptake capability but a reduced ability to photosynthetically utilize the internal inorganic carbon pool. Consequently, it exhibits low apparent photosynthetic affinity for extracellular inorganic carbon and demands high levels of CO₂ for growth.     

Thialysine-Resistant Mutant of SALMONELLA TYPHIMURIUM with a Lesion in the thrA Gene

A mutant of Salmonella typhimurium was selected for its spontaneous resistance to the lysine analog, thialysine (S-2-aminoethyl cysteine). This strain, JB585, exhibits a number of pleiotropic properties including a partial growth requirement for threonine, resistance to thiaisoleucine and azaleucine, excretion of lysine and valine, and inhibition of growth by methionine. Genetic studies show that these properties are caused by a single mutation in the thrA gene which encodes the threonine-controlled aspartokinase-homoserine dehydrogenase activities. Enzyme assays demonstrated that the aspartokinase activity is unstable and the threonine-controlled homoserine dehydrogenase activity absent in extracts prepared from the mutant. These results explain the growth inhibition by methionine because the remaining homoserine dehydrogenase isoenzyme would be repressed by methionine, causing a limitation for threonine. The partial growth requirement for threonine during growth in glucose minimal medium may also, by producing an isoleucine limitation, cause derepression of the isoleucine-valine enzymes and provide an explanation for both the valine excretion, and azaleucine and thiaisoleucine resistance. The overproduction of lysine may confer the thialysine resistance.     

Inactivation of the Flagellin Gene flaA in Magnetospirillum gryphiswaldense Results in Nonmagnetotactic Mutants Lacking Flagellar Filaments

Magnetotactic bacteria synthesize magnetosomes, which cause them to orient and migrate along magnetic field lines. The analysis of magnetotaxis and magnetosome biomineralization at the molecular level has been hindered by the unavailability of genetic methods, namely the lack of a means to introduce directed gene-specific mutations. Here we report a method for knockout mutagenesis by homologous recombination in Magnetospirillum gryphiswaldense. Multiple flagellin genes, which are unlinked in the genome, were identified in M. gryphiswaldense. The targeted disruption of the flagellin gene flaA was shown to eliminate flagella formation, motility, and magnetotaxis. The techniques described in this paper will make it possible to take full advantage of the forthcoming genome sequences of M. gryphiswaldense and other magnetotactic bacteria.     

Construction of an Obligate Photoheterotrophic Mutant of the Cyanobacterium Synechocystis 6803 : Inactivation of the psbA Gene Family

psbA in Synechocystis 6803 was found to belong to a small multigene family with three copies. The psbA gene family was inactivated in vitro by insertation of bacterial drug resistance markers. Inactivation of all three genes resulted in a transformant that is unable to grow photosynthetically but can be cultured photoheterotrophically. This mutant lacks oxygen evolving capacity but retains photosystem I activity. Room temperature measurements of chlorophyll a fluorescence induction demonstrated that the transformant exhibits a high fluorescence yield with little or no variable fluorescence. Immunoblot analyses showed complete loss of the psbA gene product (the DI polypeptide) from thylakoid membranes in the transformant. However, the extrinsic 33 kilodalton polypeptide of the water-splitting complex of photosystem II, is still present. The results indicate that assembly of a partial photosystem II complex may occur even in the absence of the intrinsic D1 polypeptide, a protein implicated as a crucial component of the photosystem II reaction center.     

利用CRISPR-Cas9技术失活黑曲霉中果胶酶基因及突变株性能评价*

我国果胶酶制剂使用广泛但专一性不高,高效、专一的果胶酶制剂在市场上仍然匮乏。利用基因工程技术改造果胶酶生产菌株——黑曲霉来生产单一成分的果胶酶成为解决果胶酶应用需求的一种有效方案。构建一种高效的CRISPR-Cas9基因编辑技术,可为构建高产单一性果胶酶的黑曲霉底盘菌株提供有效的基因编辑工具。首先敲除产果胶酶黑曲霉基因组上的pyrG基因构建尿嘧啶营养缺陷型菌株AnΔpyrG,并在AnΔpyrG菌株的pyrG基因位点定点整合Cas9基因表达盒和pyrG基因表达盒,构建组成型表达Cas9基因的黑曲霉菌株AnCas9,再构建含有gpdA启动子、锤头结构核酶、HDV核酶的稳定性表达sgRNA的pLM2-sgRNA质粒,建立CRISPR-Cas9基因编辑体系。利用该技术失活AnCas9菌株中的2个聚半乳糖醛酸酶基因4978020和4983861来检测构建的CRISPR-Cas9基因编辑效率并检测4978020基因功能缺失菌株的表型变化和产酶变化,结果表明果胶酶基因编辑效率大于50%,AnΔ4978020的表型和果胶酶酶活性与出发菌株均无明显变化。在黑曲霉中成功构建了高效的Cas9基因编辑技术,4978020基因功能缺失也不影响菌株表型,为构建高产单一性果胶酶黑曲霉底盘菌株奠定基础。     

Inactivation of ccmO in Synechococcus sp. Strain PCC 7942 Results in a Mutant Requiring High Levels of CO(2)

Inactivation of ccmO in Synechococcus sp. strain PCC 7942 resulted in a mutant which possesses aberrant carboxysomes and a normal inorganic carbon uptake capability but a reduced ability to photosynthetically utilize the internal inorganic carbon pool. Consequently, it exhibits low apparent photosynthetic affinity for extracellular inorganic carbon and demands high levels of CO(2) for growth.     

Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele

Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities. Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264→Glu) and recA665 (Tyr264→His) bearing mutations at this site. As expected both mutations affected all RecA activities in vivo. Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways. Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.     

Detection of Mutant Uromodulin in Transgenic Mouse Harboring a Mutant Human UMOD Gene

Uromodulin is the most abundant protein secreted in urine, and the mutated form of the uromodulin gene is associated with uromodulin-associated kidney disease (UAKD). Although uromodulin accumulates in the kidney of UAKD patients, it is unclear whether this is the wildtype or mutant form. In this study, we established a liquid chromatography (LC)-mass spectrometry/mass spectrometry (MS/MS)-based method for the detection of uromodulin mutants, using the C148W mutant as a target molecule. Membrane and cytosolic fractions of kidney samples from transgenic (Tg) mice expressing the C148W uromodulin mutant were shown to contain human uromodulin by western blotting, and mutant uromodulin with the C148W mutant sequence was observed by proteomic and selected reaction monitoring analyses. Our LC-MS/MS-based method is therefore useful for detection of mutant uromodulin that is undetectable by western blotting alone.     

耐辐射奇球菌recA基因的克隆、表达及其对recA缺损大肠杆菌辐射抗性的影响

将耐辐射奇球菌(Deinococcus radiodurans)recA基因克隆到表达质粒pET15b中,并在Escherichia coli HMS中高效表达了可溶性的RecA重组蛋白。同时将recA基因通过穿梭质粒pRADZ3导入recA缺损E.coli TG2细胞中,Western印迹实验显示RecA蛋白能够在不需要诱导剂IPTG的条件下稳定表达。辐射抗性实验表明,D.radiodurans的recA基因在E.coli细胞中的表达能够完全补偿recA缺损E.coli辐射抗性能力。     

Co-Occlusion and Persistence of a Baculovirus Mutant Lacking the Polyhedrin Gene

A co-occlusion process was evaluated as a commercially and ecologically acceptable strategy for the development of genetically improved baculovirus insecticides. Coinfection of Spodoptera frugiperda (IPLB-SF-21) tissue culture cells with Autographa californica nuclear polyhedrosis virus (AcMNPV) and an AcMNPV mutant (Ac-E10) lacking the polyhedrin gene resulted in occlusion of both virus types within polyhedra. The amount of occluded Ac-E10 virions in progeny polyhedra populations during serial passage in Trichoplusia ni larvae was evaluated. Maintenance of the mutant in progeny polyhedra required polyhedra inocula containing equal numbers of the two virus types at a high dose. A significant reduction in occluded mutant nucleocapsids occurs with inoculum levels below a 100% lethal dose. At inoculum levels below a 30% lethal dose, the majority of fourth-instar larvae were infected with only one type of virus. The commercial application and ecological advantages of the co-occlusion process are discussed.     

Characterization and Genetic Analysis of a Mutant of Escherichia coli K-12 with Rounded Morphology

A morphological mutant of Escherichia coli K-12 that grows as round cells at 30, 37, or 42 C in a variety of complex and synthetic media has been isolated and characterized. The gene concerned, designated rodA, has been shown to be on the chromosome between the purE and pyrC loci and to be located at about minute 15. The rodA gene has been found to be co-transducible with the lip gene at a frequency of 95%. The rodA mutant showed an increased resistance to ultraviolet irradiation and a changed sensitivity to drugs. The resistance to ultraviolet irradiation and mitomycin C appears to be co-transducible with the rodA gene.     

一种新型无花瓣油菜突变不育系花器官的形态解剖研究

在自育的甘蓝型油菜(Brassica napus)无花瓣品系AP197中, 发现并育成由4对隐性核基因控制的无花瓣油菜突变不育系AMS971。AMS是一种雄蕊心皮化为不结实的假性雌蕊而引起的新的雄性不育类型, 不育性非常彻底, 其植株形态特征与AP197相同。比较花器官发现, AMS971除一个正常的雌蕊外,还有6个梭形的心皮化假性雌蕊。假心皮化雌蕊上部变成近似倒U形的柱头结构区, 下部是完全突变的半开裂心皮, 其上着生4~14个不等的裸露的幼小胚珠; 蜜腺比正常油菜小, 数目为0~4个, 这与AP197没有差异。AMS971的四轮花器官由于突变造成了大小和重量的重新分配。推断AMS为类似拟南芥属B功能缺失的突变体。     

一种新型无花瓣油菜突变不育系花器官的形态解剖研究

在自育的甘蓝型油菜(Brassica napus)无花瓣品系AP197中,发现并育成由4对隐性核基因控制的无花瓣油菜突变不育系AMS971.AMS是一种雄蕊心皮化为不结实的假性雌蕊而引起的新的雄性不育类型,不育性非常彻底,其植株形态特征与AP197相同.比较花器官发现,AMS971除一个正常的雌蕊外,还有6个梭形的心皮化假性雌蕊.假心皮化雌蕊上部变成近似倒U形的柱头结构区,下部是完全突变的半开裂心皮,其上着生4～14个不等的裸露的幼小胚珠;蜜腺比正常油菜小,数目为0～4个,这与AP197没有差异.AMS971的四轮花器官由于突变造成了大小和重量的重新分配.推断AMS为类似拟南芥属B功能缺失的突变体.