Isolation and Characterization of Mutants of Colicin Plasmids E1 and E2 After Mu Bacteriophage Infection

Cells colicinogenic for the colicin plasmids E1 or E2 (Col E1 and Col E2, respectively) were selected for a loss of colicin production after infection with bacteriophage Mu. Extrachromosomal deoxyribonucleic acid that was larger than the original colicin plasmids was found in such cells. A small insertion mutant in Col E1 deoxyribonucleic acid affecting active colicin production without affecting either expression of colicin immunity or Col E1 deoxyribonucleic acid replication was found. Cells carrying this Col E1 plasmid mutant do not exhibit the lethal event associated with colicin E1 induction, suggesting that synthesis of active colicin is required for killing during induction. The altered Col E2 plasmid, containing an insertion at least as large as phage Mu, was maintained unstably in the mutants examined.     

Lipopolysaccharide Defective Mutant of Escherichia coli with Decreased Sensitivity to Colicin E2

Several colicin-sensitivity mutants were isolated from Escherichia coli K-12. The mutants could not form colonies in the presence of colicin E2, but recovered their colony-forming ability on trypsin treatment even after prolonged incubation with the colicin. They showed increased sensitivity to hydrophobic antibiotics and detergents, as well as resistance against P1 and T4 phages, both of which seemed due to structural changes of lipopolysaccharide (LPS). Quantitative analysis by gas-liquid chromatography revealed that the mutant-LPS contained a different stereoisomer of heptose with decreased amounts of neutral sugars (rhamnose, glucose and galactose). LPS extracted from the parental colicin-sensitive strain could neutralize the killing activity of colicin E2 in vitro, but the mutant-LPS could not. The mutant strains retained functional receptor proteins for colicin E2. These observations suggest that LPS plays an important role in the early stage of the interaction of colicin E2 with E. coli cells.     

毕赤氏酵母醇氧化酶-2基因启动子突变体的分离和鉴定

巴斯毕赤我苯酵母表达系统已被广泛用于生产外源蛋白的寄主菌。利用该系统将外源基因整合交换到染色体上时,ＡＯＸ１基因被破力的甲醇利用缓慢,给发本报生产千古 定影响。在不改变现有表达系统前提下,从ＡＯＸＩ功能缺陷 株分离出Ｍｕｔ＾＋自发突变化突变体,通过突变体在甲醇培养基中生长曲线的测定,ＨＳＡ表达产物的聚丙烯酰胺凝胶电泳检测,证明突变体的甲醇利用能力和蛋白表达比原始菌株大大提高,突变体ＡＯＸ２基因上游     

Isolation and Characterization of a Competition-Defective Bradyrhizobium japonicum Mutant

Tn5 mutagenesis was coupled with a competition assay to isolate mutants of Bradyrhizobium japonicum defective in competitive nodulation. A double selection procedure was used, screening first for altered extracellular polysaccharide production (nonmucoid colony morphology) and then for decreased competitive ability. One mutant, which was examined in detail, was deficient in acidic polysaccharide and lipopolysaccharide production. The wild-type DNA region corresponding to the Tn5 insertion was isolated, mapped, and cloned. A 3.6-kb region, not identified previously as functioning in symbiosis, contained the gene(s) necessary for complementation of the mutation. The mutant was motile, grew normally on minimal medium, and formed nodules on soybean plants which fixed almost as much nitrogen as the wild type during symbiosis.     

Isolation and Characterization of a Phosphatidylethanolamine-Deficient Mutant of Bacillus subtilis

A mutant of Bacillus subtilis ATCC 6051 deficient in phosphatidylethanolamine, an important membrane lipid, was isolated by a combination of nitrosoguanidine mutagenesis and penicillin concentration of auxotrophs employing phosphatidylethanolamine as a supplement. The mutant was compared to the parent strain with regard to lipid composition, growth, osmotic fragility, and staining character and differed substantially in each category. In addition to scant amounts of phosphatidylethanolamine, the mutant contained phosphatidylglycerol, cardiolipin, lysyl phosphatidylglycerol, and diglucosyldiglyceride, though in amounts differing from those found in the parent strain. The mutant was unable to grow appreciably on synthetic media, had enhanced osmotic fragility of protoplasts, and resisted decolorization in staining.     

Isolation and Characterization of a Photorepair-Deficient Mutant in Drosophila melanogaster

A mutation abolishing photorepair has been localized to map position 56.8 centimorgans on the second chromosome of Drosophila melanogaster. Strains homozygous for the phr allele are totally devoid of photorepair and partially deficient in excision repair. Both defects map to the chromosomal region between pr and c. Since a homozygous phr stock exhibits reduced photoreactivation, the corresponding wild-type allele plays a significant role in UV resistance.     

Isolation and Characterization of a Temperature-sensitive Sporulation Mutant of Bacillus subtilis

Temperature-sensitive sporulation mutants were isolated spontaneously from Bacillus subtilis 168 TT by a sequential transfer method. A representative mutant strain, ts32, was characterized in detail. The mutant grew normally at 30°C and 42°C, but did not sporulate at 42°C. Electron microscopic observation and physiological analysis showed that the mutant was blocked at stage 0-1 of sporulation. Genetic analysis suggested that the mutation was located at the spo0B locus on the B. subtilis chromosome. Temperature-shift experiments clearly showed that the spo0B gene product functions only at the beginning of sporulation.     

拟南芥根生长缺陷突变体rei1的分离和鉴定

植物体根发育是一个复杂的过程,尽管对其研究颇多,但对其中的分子机制尚缺乏足够认识。以模式植物拟南芥（Arabidopsis thaliana）为研究材料,在T-DNA突变体库中分离到一个拟南芥根生长缺陷突变体rei1（root elongationinhi-bited1）。通过表型分析发现,rei1在生长发育方面与野生型存在明显的差异,突变体的根较野生型短,且角果较小,花出现部分的败育。对突变体进行显微结构分析,发现突变体的根在内部结构上表现为表皮及皮层细胞形态不规则,排列疏松且横向膨大。遗传学分析表明,rei1是单基因隐性突变且与一个T-DNA插入共分离,通过图位克隆的方法成功分离了缺失的候选基因。以上研究结果表明,REI1对植物的根发育具有非常重要的调节作用。     

Characterization of Colicin Ia and Colicin Ib: Antigenic Homology

Immunological techniques have been employed to determine antigenic homology between colicin Ia and colicin Ib. Results of these experiments demonstrate that the two colicins are antigenically similar.     

Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on beta-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being beta-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.     

Isolation and Characterization of Temperature-Sensitive, High-CO2 Requiring Mutant of Anacystis nidulans R2

Temperature-sensitive (ts), high-CO₂ requiring mutants of Anacystisnidulans R2 were isolated by N-methyl-N'-nitro-N-nitrosoguanidine(MNNG) mutagenesis and ampicillin enrichment. One of these mutantswas able to grow under ordinary air enriched with 5% CO₂, butnot under ordinary air at 40?C. At 40?C, the concentration ofCO₂ at which the rate of oxygen evolution reached half the maximumvelocity (apparent Km(CO₂) in photosynthesis) was 1,000 timeshigher in mutant cells than in wild type cells, whereas therewas no significant difference in the maximum rate of photosynthesis. When wild type and mutant cells were incubated with 55 µmNaHC0₃ under illumination at 40?C, the initial rate of inorganiccarbon (IC) transport from the medium into the cells and themaximum internal IC accumulation were significantly higher inwild type cells than in mutant cells. These results indicate that the isolated high-CO₂ requiringmutant lacks the ability in transporting IC into the cells at40?C. Furthermore, the finding that the mutant cells which aredefective in IC transport cannot grow under ordinary air suggeststhe importance of IC concentrating system(s) in photosynthesisof cyanobacteria. Present address: Department of Molecular Biology, The Universityof Wyoming, Laramie, Wyoming 82071, U.S.A. To whom reprints should be requested. (Received April 28, 1988; Accepted September 14, 1988)     

Isolation and Characterization of a Phosphonomycin-Resistant Mutant of Escherichia coli K-12

A mutant was isolated from Escherichia coli K-12 which showed increased resistance towards phosphonomycin, a new bactericidal antibiotic recently isolated from strains of Streptomyces. Evidence is presented which suggests that this mutant is resistant to lysis by phosphonomycin because of a lower affinity of phosphoenolpyruvate: uridine diphospho-N-acetylglucosamine enolpyruvyl transferase for this antibiotic. This mutant was also found to be temperature-sensitive in growth. At 42 C mutant cells grew poorly, and the rate of incorporation of (3)H-diaminopimelic acid into trichloroacetic acid-insoluble material was also greatly reduced. Genetic studies indicate that the increased resistance toward phosphonomycin and temperature sensitivity in growth of this mutant are probably the consequences of a single mutation.     

Isolation and Characterization of a Mutant of Arabidopsis thaliana Resistant to alpha-Methyltryptophan

Mutants of Arabidopsis thaliana have been selected for resistance to growth inhibition at the seedling stage by α-methyltryptophan (aMT). One mutant, amt-1 has been characterized in detail. The appearance and growth rate of the mutant in the absence of the inhibitor are similar to wild type, both as plants and callus. However, mutant plant growth is unaffected by 25 micromolar aMT and mutant callus growth by 50 micromolar aMT, concentrations that completely inhibit the growth of wild-type plants and callus, respectively. Tryptophan levels in mutant and wild-type plants are 24.3 ± 2.7 and 4.7 ± 1.2 micrograms per gram fresh weight, respectively, and in the corresponding callus 64.0 ± 2.6 and 31.8 ± 8.4 micrograms per gram fresh weight, respectively. Anthranilate synthase (AS) activity levels in crude extracts from whole plants are 3.09 ± 0.54 nanomoles per milligram protein per hour in amt-1 and 1.32 ± 0.21 nanomoles per milligram protein per hour in wild-type plants. In crude extracts from callus, anthranilate synthase levels are 11.54 ± 2.05 nanomoles per milligram protein per hour and 7.74 ± 1.58 in amt-1 and wild type, respectively. Enzyme extracts are inhibited by l-tryptophan; the concentrations required for 50% inhibition (I₅₀) are 3.9 and 1.9 micromolar for amt-1 and for wild type, respectively. The mutation segregates as a single nuclear allele and shows incomplete dominance. The concomitant increases in both AS activity and its I₅₀ for tryptophan suggest that the mutation amt-1 either resides in one of the AS structural genes or causes increased expression of an AS isoform with an I₅₀ greater than the average for the entire extract.     

Isolation and Characterization of a Temperature-Sensitive Circadian Clock Mutant of Neurospora Crassa

A new circadian clock mutant has been isolated in Neurospora crassa. This new mutation, called period-6 (prd-6), has two features novel to known clock mutations. First, the mutation is temperature sensitive. At restrictive temperatures (above 21°) the mutation shortens circadian period length from a wild-type value of 21.5 hr to 18 hr. At permissive temperatures (below 21°) the mutant has a 20.5-hr period length close to that of the wild-type strain. Second, the prd-6 mutation is epistatic to the previously isolated clock mutation period-2 (prd-2). This epistasis is unusual in that the prd-2 prd-6 double mutant strain has an 18-hr period length at both the restrictive and permissive temperatures. That is, the temperature-sensitive aspect of the phenotype of the prd-6 strain is lost in the prd-2 prd-6 double mutant strain. This suggests that the gene products of the prd-2 and prd-6 loci may interact physically and that the presence of a normal prd-2(+) protein is required for low temperature to ``rescue' the prd-6 mutant phenotype. These results, combined with our recent finding that prd-2 and some alleles of the frq gene show genetic synergy, suggest that it may be possible to establish a more comprehensive model of the Neurospora circadian clock.     

Isolation and Characterization of a Bacillus cereus Mutant Strain Hyperproductive of Exo-beta-Amylase

Starting with a strain of Bacillus cereus excreting about 40-fold more beta-amylase than does the original wild-type strain, we isolated, after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, a strain designated BQ10-S1 SpoIII which showed under optimal conditions a further 5.5-fold increase in beta-amylase activity. The amylase production of this strain was observed to increase in the presence of 0.5% glucose or 1% maltose and, more markedly, in the presence of 2% soluble starch in the culture medium. The enzyme produced by this strain was immunologically identical to the wild-type enzyme, suggesting that either the copy number of the gene or the efficiency of enzyme synthesis from it, or both, are altered in this strain.     

Stabilization of Colicin E2 by Bovine Serum Albumin

Colicin E2 was partially purified from Escherichia coli W3110. This preparation was remarkably stabilized by bovine serum albumin in a solution at neutral pH, as shown by dilution experiments and tests on heat stability of colicin. One killing unit of colicin E2 was estimated to correspond to one molecule of colicin E2, on the assumption of a molecular weight of 60,000.     

Isolation and Characterization of Carotenosomes from a Bacteriochlorophyll c-less Mutant ofChlorobium tepidum

Chlorosomes are the light-harvesting organelles in photosynthetic green bacteria and typically contain large amounts of bacteriochlorophyll (BChl) c in addition to smaller amounts of BChl a, carotenoids, and several protein species. We have isolated vestigial chlorosomes, denoted carotenosomes, from a BChl c-less, bchK mutant of the green sulfur bacterium Chlorobium tepidum. The physical shape of the carotenosomes (86 ± 17 nm × 66 ± 13 nm × 4.3 ± 0.8 nm on average) was reminiscent of a flattened chlorosome. The carotenosomes contained carotenoids, BChl a, and the proteins CsmA and CsmD in ratios to each other comparable to their ratios in wild-type chlorosomes, but all other chlorosome proteins normally found in wild-type chlorosomes were found only in trace amounts or were not detected. Similar to wild-type chlorosomes, the CsmA protein in the carotenosomes formed oligomers at least up to homo-octamers as shown by chemical cross-linking and immunoblotting. The absorption spectrum of BChl a in the carotenosomes was also indistinguishable from that in wild-type chlorosomes. Energy transfer from the bulk carotenoids to BChl a in carotenosomes was poor. The results indicate that the carotenosomes have an intact baseplate made of remarkably stable oligomeric CsmA–BChl a complexes but are flattened in structure due to the absence of BChl c. Carotenosomes thus provide a valuable material for studying the biogenesis, structure, and function of the photosynthetic antennae in green bacteria.     

Isolation and Characterization of a Bacillus cereus Mutant Strain Hyperproductive of Exo-β-Amylase

Starting with a strain of Bacillus cereus excreting about 40-fold more β-amylase than does the original wild-type strain, we isolated, after mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine, a strain designated BQ10-S1 SpoIII which showed under optimal conditions a further 5.5-fold increase in β-amylase activity. The amylase production of this strain was observed to increase in the presence of 0.5% glucose or 1% maltose and, more markedly, in the presence of 2% soluble starch in the culture medium. The enzyme produced by this strain was immunologically identical to the wild-type enzyme, suggesting that either the copy number of the gene or the efficiency of enzyme synthesis from it, or both, are altered in this strain.     

Purification and Characterization of Colicin D

Colicin D-CA23, obtained by sonic treatment of mitomycin C-induced cells of Escherichia coli K-12 W1485 (colD), was purified by ammonium sulfate precipitation, gel filtration on Sephadex G200, ion-exchange chromatography on diethylaminoethyl cellulose, and isoelectrofocusing. Polyacrylamide-gel electrophoresis, sedimentation velocity analysis, and antigenic analysis indicated that the preparation was homogeneous. Colicin D is composed entirely of amino acids and hence is a simple protein uncomplexed with lipid or lipopolysaccharide. It contains six residues of cysteine per molecule. The molecular weight of colicin D is approximately 92,000, as determined by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis and gel filtration on Sephadex G200. Its sedimentation coefficient is 4.41S. The behavior of colicin D in solutions of sodium dodecyl sulfate and 2-mercaptoethanol indicates that it does not consist of subunits and exists as a single polypeptide chain. Its high molecular weight and presence of six cysteine residues per molecule distinguish colicin D from all colicins previously described. Although colicins D and E3 have similar modes of action, their gross molecular properties are entirely different.