外源glnA基因干扰大肠杆菌的代谢

采用分子生物学的方法构建了含Bacillus subtilis glnA基因的重组菌株Escherichia coli DH5α(pMD19-glnA),用毛细管电泳和核磁共振对重组菌株的转化谷氨酸的产物进行定性鉴定,并进一步通过荧光定量RT-PCR测定谷氨酰胺合成酶基因(glnA)mRNA水平的相对表达量,最后用SDS-聚丙烯酰胺凝胶电泳对蛋白的相对表达情况进行了分析。结果表明重组菌株并没有增加谷氨酰胺的产量,而是明显增加了γ-氨基丁酸(GABA)的产量。实验表明重组菌株中的glnA基因可以正常转录,但是谷氨酰胺合成酶的蛋白表达量并没有增加。这种外源基因干扰大肠杆菌代谢的现象值得进一步研究。     

Reversible inhibition of mammalian glutamine synthetase by tyrosine nitration

The effect of tyrosine nitration on mammalian GS activity and stability was studied in vitro. Peroxynitrite at a concentration of 5 micro mol/l produced tyrosine nitration and inactivation of GS, whereas 50 micro mol/l peroxynitrite additionally increased S-nitrosylation and carbonylation and degradation of GS by the 20S proteasome. (-)Epicatechin completely prevented both, tyrosine nitration and inactivation of GS by peroxynitrite (5 micro mol/l). Further, a putative "denitrase" activity restored the activity of peroxynitrite (5 micro mol/l)-treated GS. The data point to a potential regulation of GS activity by a reversible tyrosine nitration. High levels of oxidative stress may irreversibly damage and predispose the enzyme to proteasomal degradation.     

Construction and characterization of Bacillus subtilis deletion mutants lacking the prophage 2-trnS region

During development of a novel method for constructing a series of deletions in Bacillus subtilis using an isogenic set of gene-disrupted mutants created by integration of pMutin, deletion of the trnS operon, consisting of seven tRNA genes, was found to affect cell growth, development of competence and spore formation. A suppressor (sts1) of the DeltatrnS mutant was isolated, sequenced and found to have undergone a single base change, CAG to GAG, in the first anticodon of tRNA(Leu), in the trnB operon.     

Deoxyribonucleoside-requiring mutants of Bacillus subtilis.

A number of deoxyribonucleoside-requiring mutants (dns) of Bacillus subtilis were isolated and their growth characteristics and ribonucleotide reductase activities were compared with those of the wild type and of a dna mutant (tsA13). Both tsA13 and dns mutants required the presence of a mixture of deoxyribonucleosides for growth at 45 degrees C but not at 25 degrees C. All the mutant strains tested contained ribonucleotide reductase activity which showed heat sensitivity similar to that of the enzyme from a wild-type strain. The reductase in B. subtilis seemed to reduce ribonucleoside triphosphates in a similar manner to the enzyme in Lactobacillus leichmannii.     

Recombination-deficient mutants of Bacillus subtilis.

Two mutant strains of Bacillus subtilis Marburg, NIG43 and NIG45, were isolated. They showed high sensitivities to gamma rays, ultraviolet light (UV), and chemicals. Deficiencies in genetic recombination of these two mutants were shown by the experiments on their capacity in transformation. SPO2 transfection, and PBS1 phage transduction, as well as on their radiation and drug sensitivities and their Hcr+ capacity for UV-exposed phage M2. Some of these characteristics were compared with those of the known strains possessing the recA1 or recB2 alleles. Mapping studies revealed that the mutation rec-43 of strain NIG43 lies in the region of chromosome replication origin. The order was purA dna-8132 rec-43. Another mutation, rec-45, of strain NIG45 was found to be tightly linked to recA1. The mutation rec-43 reduced mainly the frequency of PBS1 transduction. On the other hand, the mutation rec-45 reduced the frequency of recombination involved both in transformation and PBS1 transduction. The mutation rec-43 of strain NIG43 is conditional, but rec-45 of strain NIG45 is not. The UV impairment in cellular survival of strain NIG43 was gradually reverted at higher salt or sucrose concentrations, suggesting cellular possession of a mutated gene produce whose function is conditional. In contrast to several other recombination-deficient strains, SPO2 lysogens of strain NIG43 and NIG45 were not inducible, indicating involvement of rec-43+ or rec-45+ gene product in the development of SPO2 prophage to a vegetative form. The UV-induced deoxyribonucleic acid degradation in vegetative cells was higher in rec-43 and rec-45 strains.     

Kasugamycin-resistant mutants of Bacillus subtilis.

Kasugamycin-resistant mutants of Bacillus subtilis were isolated and classified into two groups, one of which had resistance to kasugamycin in in vitro protein synthesis and mapped in the ribosomal region. The other group had no resistance to kasugamycin in in vitro protein synthesis and had weak cross-resistance to gentamicin and kanamycin. Neither group could sporulate in the presence of kasugamycin.     

生防菌Bs-916 合成脂肽类化合物Bac操纵子突变株构建及功能

摘要：生防枯草芽孢杆菌Bs-916对水稻纹枯病有很好的防治效果。【目的】明确其合成脂肽类化合物操纵子Bac功能,为遗传改良提高Bs-916防效奠定基础。【方法】采用同源重组方法成功更换Bac内源启动子为组成性表达强启动子和插入失活Bac启动子,分别得到突变株BGG104和BGG105。突变株生物学活性测定结果表明：BGG104上清液相对Bs-916上清液对几种病原真菌毒力,血红细胞的溶血能力都显著提高;而BGG105则显著降低。采用6 mol/L盐酸沉淀及甲醇抽提方法从Bs-916和突变株培养液中制备脂肽     

Decadent sporulation mutants of Bacillus subtilis.

In decadent sporulation mutants, sporulating populations are heterogeneous: the cells reach successive chemical and physical resistances with progressively decreasing frequencies. Each decadent mutant can be characterized by the shape and slope of the curve describing the frequency of cells resistant to various agents ('the resistance spectrum'). In some mutants the resistance spectrum decreases progressively from xylene resistance to heat resistance; in other mutants it decreases rapidly between octanol resistance and chloroform resistance. Electron microscopy showed that in two mutants the majority of the cells are blocked at stages III and IV; the number of cells that develop further to reach successive morphological stages falls off progressively. In two other mutants most cells reach stage V. Cortexless spores are also frequent. One of the decadent mutations, SpoL1, was localized between aroD and acf. The phenotype of decadent mutants is discussed in terms of sequential gene activation.     

Alcohol-resistant sporulation mutants of Bacillus subtilis.

About 80% of Bacillus subtilis cells form spores when grown in nutrient broth. In medium containing various short-chain aliphatic alcohols, the frequency of sporulation was reduced to 0.5%. Mutants sporulated in the presence of alcohols at a frequency of 30 to 40%. Sporulation in the wild-type cells was sensitive to alcohol at the beginning of sporulation (stage zero). Sensitivity to alcohol in the mutants was also at stage zero, even though the sensitivity was considerably reduced. This sensitivity of sporulation to alcohol is the phenotypic expression of a genetic locus designated ssa. Mutations at this locus lead to a decreased sensitivity of sporulation to alcohol without modifying the sensitivity of growth. Genetic analysis by transduction was bacteriophage PBS1 revealed that ssa mutations are near the previously described spo0A locus. ssa mutants also differ from wild-type cells in the composition of membrane phospholipids. The relative amount of phosphatidylglycerol increased, whereas the relative amount of phosphatidylethanolamine and lysylphosphatidylglycerol decreased relative to the proportions in the wild type. The distribution of fatty acids in membrane lipids is the same as in the wild type. No differential sensitivity of phospholipid metabolism to alcohol could be detected in the mutant. This work therefore reveals that the extensive, pleiotropic changes in the membranes of ssa mutants are the phenotypic reflection of alterations at a specific gene locus.     

Catabolite repression-resistant mutants of Bacillus subtilis.

Mutants of Bacillus subtilis that are able to sporulate under the condition of catabolite repression were isolated by a simple selection technique. The mutants used in the present study were able to grow normally on minimal medium with ammonium sulphate as the nitrogen source and glucose as the carbon source. Studies carried out with these mutants show that there is no close relation between catabolite repression of an inducible enzyme, acetoin dehydrogenase, and that of sporulation. Certain mutants are able to sporulate in the presence of all the carbon sources tested but some mutants are resistant only to the carbon source used in isolation. It is suggested that several metabolic steps may be affected in catabolite repression of sporulation.     

枯草芽孢杆菌核黄素操纵子rib operon的克隆与表达

目的:构建产核黄素的枯草芽孢杆菌基因工程菌.方法:以穿梭载体pEB03构建核黄素操纵子的表达质粒载体pGJB13和pGJB14,与质粒pMX45分别转化产核黄素的枯草芽孢杆菌GJ07,并通过发酵摇瓶实验检测核黄素的产量.结果:得到产核黄素的工程菌GJ13 、GJ14和GJ08,在以蔗糖为碳源的发酵条件下,GJ08可产核黄素820mg/L,提高了约55%.结论:得到了产核黄素的高产菌种G J08.     

Glutamine synthetase gene of Bacillus subtilis

The glutamine synthetase gene (glnA) of Bacillus subtilis was purified from a library of B. subtilis DNA cloned in phage lambda. By mapping the locations of previously identified mutations in the glnA locus it was possible to correlate the genetic and physical maps. Mutations known to affect expression of the glnA gene and other genes were mapped within the coding region for glutamine synthetase, as determined by measuring the sizes of truncated, immunologically cross-reacting polypeptides coded for by various sub-cloned regions of the glnA gene. When the entire B. subtilis glnA gene was present on a plasmid it was capable of directing synthesis in Escherichia coli of B. subtilis glutamine synthetase as judged by enzymatic activity, antigenicity, and ability to allow growth of a glutamine auxotroph. By use of the cloned B. subtilis glnA gene as a hybridization probe, it was shown that the known variability of glutamine synthetase specific activity during growth in various nitrogen sources is fully accounted for by changes in glnA mRNA levels.     

Biofilm-defective mutants of Bacillus subtilis

Many bacteria can adopt organized, sessile, communal lifestyles. The gram-positive bacterium, Bacillus subtilis,forms biofilms on solid surfaces and at air-liquid interfaces, and biofilm development is dependent on environmental conditions. We demonstrate that biofilm formation by B. subtilis strain JH642 can be either activated or repressed by glucose, depending on the growth medium used, and that these glucose effects are at least in part mediated by the catabolite control protein, CcpA. Starting with a chromosomal Tn917-LTV3 insertional library, we isolated mutants that are defective for biofilm formation. The biofilm defects of these mutants were observable in both rich and minimal media, and both on polyvinylchloride abiotic surfaces and in borosilicate tubes. Two mutants were defective in flagellar synthesis. Chemotaxis was shown to be less important for biofilm formation than was flagellar-driven motility. Although motility is known to be required for biofilm formation in other bacteria, this had not previously been demonstrated for B. subtilis. In addition, our study suggests roles for glutamate synthase, GltAB, and an aminopeptidase, AmpS. The loss of these enzymes did not decrease growth or cellular motility but had dramatic effects on biofilm formation under all conditions assayed. The effect of the gltAB defect on biofilm formation could not be due to a decrease in poly-gamma-glutamate synthesis since this polymer proved to be nonessential for robust biofilm formation. High exogenous concentrations of glutamate, aspartate, glutamine or proline did not override the glutamate synthase requirement. This is the first report showing that glutamate synthase and a cytoplasmic aminopeptidase play roles in bacterial biofilm formation. Possible mechanistic implications and potential roles of biofilm formation in other developmental processes are discussed.     

Porphyrin and corrinoid mutants of Bacillus subtilis.

Porphyrin auxotrophs of Bacillus subtilis can be divided into two groups. Strains belonging to the first group (hemA, hemB, or hemC) are not able to synthesize or metabolize porphobilinogen. These strains require cysteine, cystine, and methionine, respectively. Traces of aminolevulinic acid, in a hemin-containing medium, can replace the cysteine requirement in a mutant lacking aminolevulinic acid synthetase. In bacteria belonging to the second group (hemE, hemF, or hemG), porphyrin biosynthesis is blocked at later steps, and the amino acids mentioned above are not required. It is of interest that both the activity of ribonucleotide reductase and the amount of vitamin B12 were significantly lower in the first group. The addition of vitamin B12 to the medium did not promote the growth of strains examined. We assume that porphobilinogen deaminase is essential for the synthesis of corrinoids.     

Autolytic enzyme-deficient mutants of Bacillus subtilis 168.

Mutants of Bacillus subtilis strain 168 have been isolated that are at least 90 to 95% deficient in the autolytic enzymes N-acetylmuramyl-L-alanine amidase and endo-beta-N-acetylglucosaminidase. These mutants grow at normal rates as very long chains of unseparated cells. The length of the chains is directly related to the growth rates. They are nonmotile and have no flagella, but otherwise appear to have normal cell morphology. Their walls are fully sysceptible to enzymes formed by the wild type and have the same chemical composition as the latter. Cell wall preparations from the mutants lyse at about 10% of the rate of those from the isogenic wild type, with the correspondingly small liberation of both the amino groups of alanine at pH 8.0 and of reducing groups at pH 5.6. Likewise, Microcococcus luteus walls at pH 5.6 and B. subtilis walls at pH 8 are lysed only very slowly by LiCl extracts made from the mutants as compared with rates obtained with wild-type extracts. Thus, the activity of both autolytic enzymes in the mutants is depressed. The frequencies of transformation, the isolation of revertants, and observations with a temperature-sensitive mutant all point to the likelihood that the pleiotropic, phenotypic properties of the strains are due to a single mutation. The mutants did not produce more protease or amylase than did the wild type. They sporulate and the spores germinate normally. The addition of antibiotics to exponentially growing cultures prevents wall synthesis but leads to less lysis than is obtained with the wild type. The bacteriophage PBSX can be induced in the mutants by treatment with mitomycin C.     

5-Bromouracil-tolerant mutants of Bacillus subtilis

5-Bromouracil (BU)-tolerant mutants of Bacillus subtilis 23 (thy his) have been isolated. Several classes of tolerant mutants were obtained by a sequential selection procedure. The classes can be distinguished by their relative BU tolerance as well as several other phenotypic characteristics. The mutants can grow for an extended period of time in minimal medium supplemented with amino acids and BU, in which the sensitive parental strain (Bu(+)) undergoes rapid cell death. Both mutants But-1 and But-1310 have a greater rate of deoxyribonucleic acid (DNA) synthesis by a factor of two in the presence of BU than Bu(+), But-1 being somewhat faster than But-1310. The preferential incorporation of thymine to BU of But-1 is about half that of the Bu(+) strain during DNA replication in minimal medium supplemented with 10 mug of BU/ml and 1 mug of thymine/ml. It is not known at what step or steps this reduction in selectivity occurs.