An analysis of the genes encoding the 51.4- and 41.9-kDa toxins of Bacillus sphaericus 2297 by deletion mutagenesis: the construction of fusion proteins

A series of deletion mutants have been constructed, in which varying numbers of amino acids have been deleted from both the N- and C-termini of both the 51.4- and 41.9-kDa toxins of Bacillus sphaericus. The results show that between 34-39 and 52-54 amino acids respectively at the N- and C-termini of the 51.4-kDa protein, are not essential for toxicity. In the case of the 41.9-kDa protein, the removal of only 7 amino acids from the C-terminus abolishes toxicity whilst at least 17 amino acids can be deleted from the N-terminus without loss of toxicity. A fusion protein with the 51.4-kDa derived sequence N-terminal to the 41.9-kDa sequence yielded a protein of Mr 87 kDa which was not toxic by itself. When supplemented with cells expressing only the 51.4-kDa protein, toxicity was restored. In contrast, another fusion protein, in which the gene order was reversed, was shown to be fully active in toxicity assays.     

Isolation of the RAD18 gene of Saccharomyces cerevisiae and construction of rad18 deletion mutants

Summary The RAD18 gene of Saccharomyces cerevisiae is involved in mutagenic DNA repair. We describe its isolation from a yeast library introduced into the centromeric YCp50 vector, a low copy number plasmid. The insert was sublconed into YCp50 and into the multicopy YRp7 plasmid. RAD18 is not toxic when present in multiple copies but the UV survival response indicates an heterogeneity in the cell population, a fraction of it being more sensitive. A DNA segment, close to RAD18, is toxic on the multicopy plasmid and may correspond to the tRAN sup61 known to be tightly linked to RAD18. Chromosomal deletions of RAD18 were constructed. The gene is not essential and the deleted strains have the properties of single site mutants. Thus, RAD18 appears to be essentially involved in DNA repair metabolism.     

自絮凝工业酒精酵母营养缺陷型的筛选和鉴定

运用紫外诱变方法成功获得了自絮凝酵母的营养缺陷型突变体,并且优化了诱变方法,证明了通过紫外诱变也可获得自絮凝酵母的营养缺陷型突变体。实验证明,较低的致死率更容易获得突变体,利用制霉菌素的富集可明显减少非缺陷型背景。本实验获得了组氨酸和尿嘧啶营养缺陷型各一株,其中组氨酸缺陷型茵株失去絮凝特性,而尿嘧啶缺陷型保持了良好的絮凝特性。继代实验表明,二株突变体均可以稳定遗传。并利用交配型PCR方法证明了絮凝酵母及其两株突变体与其酿酒酵母亲本类似,均为交配型杂合体。     

Isolation and characterisation of mutants from the halotolerant yeast Pichia sorbitophila defective in H+/glycerol symport activity

Abstract Pichia sorbitophila , a yeast species that is highly resistant to osmotic stress in general and to salt stress in particular, was subjected to a mutagenesis strategy in order to obtain mutants deficient in the glycerol active uptake previously described. Density centrifugation was used for enrichment of NaCl sensitive mutants in either glucose or glycerol media. Several phenotypic classes of mutants were identified, to which physiological tests were applied concerning the activity of the symporter, its accumulation capacity and the detection of the activity of glycerol pathway specific enzymes. From these, two mutant strains were selected, presenting a clearly deficient phenotype on H⁺/glycerol symport activity.     

Computational modeling and prediction of deletion mutants

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Properties of the menaquinol oxidase (Qox) and of qox deletion mutants of Bacillus subtilis

Menaquinol oxidase isolated from the membrane of Bacillus subtilis W23 was found to consist of four polypeptides (QoxA, B, C, and D) that were predicted by the sequence of the qox operon of B. subtilis 168 (Santana et al. 1992). The preparation contained 7 mol cytochrome aa ₃ per g protein, which corresponds to 2mol heme A per mol enzyme of 144 kDa molecular mass. Respiration with dimethylnaphthoquinol catalyzed by the enzyme was ten times faster than that with menadiol. Activities with more electropositive quinols were negligible. The activity of the enzyme was inhibited by equimolar amounts of HQNO, while antimycin, myxothiazol, and stigmatellin were more than tenfold less effective. When cells of both strains of B. subtilis (W23 and 168) were grown with glucose, quinol respiration was an order of magnitude more active than respiration with N,N,N,N-tetramethyl-1,4-phenylenediamine plus ascorbate. Surprisingly, the same result was obtained with mutant strains lacking qoxB. As cytochromes a and d were virtually absent, a second quinol oxidase, possibly of the cytochrome o-type, was apparently formed by the mutants.Abbreviations cat Chloramphenicol resistance gene - cta Cytochrome oxidase genes - DMN 2,3-Dimethyl-1,4-naphthoquinone - DMNH ₂ Reduced DMN - HQNO 2-(n-Heptyl)-4-hydroxyquinoline-N-oxide - qox Quinol oxidase genes - TMPD N,N,N,N-tetramethyl-1,4-phenylenediamine     

Isolation and characterization of respiration-deficient mutants from the pathogenic yeastCandida albicans

The isolation of several respiration deficient mutants of the pathogenic yeastCandida albicans is described. These show greatly reduced respiration rates, loss of cytochromesaa ₃ andb, and reduced growth rates. All of the mutants had lost the ability to assimilate a wide range of carbon sources. Ultrastructural studies showed reduced development of mitochondrial cristae in the mutants. The mutants can be divided into three classes depending on their respiration responses to the addition of cyanide.     

Isolation,characterization and mapping of temperature-sensitive mutants of mycobacteriophage I3

Eighteen temperature-sensitive mutants of mycobacteriophage I3 have been isolated and partially characterized. All the mutants were defective in vegetative replication. Based on temperature shift experiments with the temperature sensitive mutants, the thermosensitive phase of the phage development period has been characterized for each mutant. The genes have been mapped by recombination analysis. The early, continuous and middle genes seem to cluster on the genetic map     

Efficacy of non-toxic deletion mutants of protective antigen from Bacillus anthracis

Current human anthrax vaccines available in the United States and Europe consist of alum-precipitated supernatant material from cultures of a toxigenic, nonencapsulated strain of Bacillus anthracis. The major component of human anthrax vaccine that confers protection is protective antigen (PA). A second-generation human vaccine using the recombinant PA (rPA) is being developed. In this study, to prevent the toxicity and the degradation of the native rPA by proteases, we constructed two PA variants, delPA (163-168) and delPA (313-314), that lack trypsin (S(163)-R(164)-K(165)-K(166)-R(167)-S(168)) or chymotrypsin cleavage sequence (F(313)-F(314)), respectively. These proteins were expressed in Bacillus brevis 47-5Q. The delPAs were fractionated from the culture supernatant of B. brevis by ammonium sulfate at 70% saturation, followed by anion exchange chromatography on a Hitrap Q, Hiload 16/60 superdex 200 gel filtration column and phenyl sepharose hydrophobic interaction column. In accordance with previous reports, both delPA proteins combined with lethal factor protein did not show any cytotoxicity on J774A.1 cells. The delPA (163-168) and delPA (313-314) formulated either in Rehydragel HPA or MPL-TDM-CWS (Ribi-Trimix), elicited a comparable amount of anti-PA and neutralizing antibodies to those of native rPA in guinea pigs, and confers full protection of guinea pigs from 50xLD50 of fully virulent B. anthracis spore challenges. Ribi-Trimix was significantly more effective in inducing anti-PA and neutralizing antibodies than Rehydragel HPA. These results indicate the possibility of delPA (163-168) and delPA (313-314) proteins being developed into nontoxic, effective and stable recombinant vaccine candidates.     

Two naturally occurring deletion mutants of 12S seed storage proteins in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Two naturally occurring Arabidopsis mutants, Cape Verde Islands and Monte (Mr-0), with aberrant 12S seed storage protein (SSP) profiles have been identified by SDS-PAGE. In both mutants, one of the 12S globulin bands is missing while a new band of lower molecular mass is present. Tandem mass spectrometry-mass spectrometry (MS/MS) analyses of the mutant peptides have revealed that both are shorter variants of 12S globulin with deletion sites detected within the -subunits of 12S globulin cruciferin B (CRB) and C (CRC), respectively. Sequence analyses of the genomic DNA flanking the deletion sites have demonstrated that both deletions occurred at the genomic level. These two mutants are referred to as CRB12 and CRC13 with the delta sign indicating a deletion and the number indicating amino acids deleted. Alignment of these two mutant sequences with that of soybean A3B4 subunit, for which the crystal structure was determined recently, have revealed that the CRC13 deletion is located in a hypervariable/disordered region, and will probably not affect the structure of the hexameric globulin. The CRB12 deletion, however, is located in a binding region that is thought to be important for the hexamer formation. However, CRB12 appears to accumulate normally as judged by its band intensity relative to the other SSP subunits on the protein gels. Thus it seems that the seed can, to a certain extent, tolerate some mutations in its storage proteins.     

Rapid construction of Campylobacter jejuni deletion mutants

AIMS: To develop a novel method for rapid construction of Campylobacter jejuni deletion mutants. METHODS AND RESULTS: We used overlapping extension PCR protocol to amplify a target sequence region of Camp. jejuni genomic DNA in which an internal fragment, Cj0618 coding sequence, was replaced by a chloramphenicol resistance cassette. After the resulting PCR product was introduced into electrocompetent Camp. jejuni 81-176, chloramphenicol-resistant mutants in which the wild type allele has been replaced by the deletion cassette were selected. DNA sequencing confirmed precise deletion in the Cj0618 gene. As expected from the previously reported role of Cj0618 in chick colonization, the resulting deletion mutant showed a caecal colonization defect in chick infection. CONCLUSIONS: This method can be used for rapid construction of Camp. jejuni deletion mutants. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of this method should facilitate functional characterization of various Camp. jejuni genes.     

Isolation and preliminary characterisation of twenty-five temperature-sensitive mutants of mouse cytomegalovirus

Abstract To study the pathogenicity of mouse cytomegalovirus (MCMV) and to identify virulence determinants, we have isolated and phenotypically characterised 25 temperature-sensitive ( ts ) mutants. Six of these ( tsm 9, tsm 13, tsm 20, tsm 22, tsm 28 and tsm 30) failed to replicate in mice and were avirulent. Five mutants ( tsm 14, tsm 18, tsm 19, tsm 25 and tsm27 ) were to similar virulence to the parenthal wild-type ( wt ) virus, five ( tsm 7, tsm 15, tsm 24, tsm31 ) were 12–100 fold less virulent, five ( tsm 8, tsm 12, tsm 16, tsm 23 and tsm 29) were 150–1500 fold less virulent and four ( tsm 10, tsm 11, tsm 17 and tsm 21) were between 2,000 and 85,000 fold less virulent than wt . One mutant ( tsm 28) did not plaque or replicate at 39°C while 5 other mutants ( tsm 7, tsm 9, tsm 23, tsm 24 and tsm 27) also failed to plaque at 39°C but only failed to replicate or replicated poorly at 40°C. A further two mutants ( tsm 10 and tsm 13) were able to plaque and replicate at 39°C but not 40°C. Six other mutants ( tsm 14, tsm 15, tsm 16, tsm21 , tsm 22 and tsm 30) failed to form plaques at 40°C and were severely restricted in their replication at 40°C. The remaining 11 mutants exhibited varying degrees of restriction in ability to plaque and/or replicate at non-permissive temperatures. These 25 mutants, together with 6 isolated previously, comprise at least 24 complementation groups.     

A complete collection of single‐gene deletion mutants of Acinetobacter baylyi ADP1

We have constructed a collection of single‐gene deletion mutants for all dispensable genes of the soil bacterium Acinetobacter baylyi ADP1. A total of 2594 deletion mutants were obtained, whereas 499 (16%) were not, and are therefore candidate essential genes for life on minimal medium. This essentiality data set is 88% consistent with the Escherichia coli data set inferred from the Keio mutant collection profiled for growth on minimal medium, while 80% of the orthologous genes described as essential in Pseudomonas aeruginosa are also essential in ADP1. Several strategies were undertaken to investigate ADP1 metabolism by (1) searching for discrepancies between our essentiality data and current metabolic knowledge, (2) comparing this essentiality data set to those from other organisms, (3) systematic phenotyping of the mutant collection on a variety of carbon sources (quinate, 2‐3 butanediol, glucose, etc.). This collection provides a new resource for the study of gene function by forward and reverse genetic approaches and constitutes a robust experimental data source for systems biology approaches.     

Fructose-bisphosphatase-deficient mutants of the yeast Schizosaccharomyces pombe

We showed that in the yeast Schizosaccharomyces pombe, fructose-bisphosphatase is not subject to catabolite inactivation as it was observed in Saccharomyces cerevisiae. However, this enzyme activity is sensitive to catabolite repression in both yeasts. Two mutants lacking completely fructose-bisphosphatase activity were found. They were unable to grow on glycerol medium. They were still respiratory competent and exhibited the ability to derepress partially malate dehydrogenase activity. In glucose exponential phase culture, the parental strain lacks completely the fructosebisphosphatase activity due to catabolite repression. In these conditions, the growth is slowed down only in the mutants eventhough both mutants and their parental strain lack this enzyme activity. Normal sporulation and poor spore germination were observed for one mutant whereas, only in the presence of glucose, normal sporulation and normal spore germination were observed for the second mutant. Mendelian segregation of glycerol growth was found for the well germinating mutant. It is of nuclear heredity. The two mutations appeared to be closely linked.Abbreviations FBPase Fructose-1,6-bisphosphatase - fbp ^- genetic symbol for FBPase deficiency - glr ^- symbol for inability to grow on glycerol A. M. Colson is Research Associate au Fonds National de la Recherche Scientifique     

布鲁氏菌M5-90疫苗株virB2基因缺失株的构建及鉴定

【目的】构建布鲁氏菌M5-90疫苗株virB2基因缺失株。【方法】利用常规分子生物学技术构建自杀载体pGEM-7zf-ΔvirB2-sacB,通过同源重组的方法,将电转化后的布鲁氏菌分别经100 mg/L氨苄抗性筛选和5%蔗糖敏感性筛选,获得基因缺失株。对获得的基因缺失株进行PCR鉴定和稳定性检测。【结果】成功构建M5-90ΔvirB2基因缺失株,并且该缺失株在10代以内未发生回复突变。【结论】为研发新型布鲁氏菌弱毒基因缺失活苗奠定基础。     

Isolation of mutants of Clostridium pasteurianum

Twenty-nine independent mutants of Clostridium pasteurianum ATCC 6013, including several auxotrophs and UV resistants, have been isolated and characterized. The protoplast formation and regeneration procedure of Minton and Morris (1983) has also been successfully tried with some of these newly obtained mutants. The availability of these mutants together with the possibility of protoplast formation and regeneration will be useful for the development of a genetic exchange system in this species.Abbreviations CFU colony forming unit - DCCP dicyclohexyl carbodiamide - EMS ethylmethane sulfonate - IB isotonic buffer - MNNG N-methyl-N-nitro-N-nitrosoguanidine - PEG polyethylene glycol - UV ultraviolet     

Isolation and characterization of Anacystis nidulans R2 mutants affected in nitrate assimilation: Establishment of two new mutant types

Summary Eighteen mutant strains of the unicellular cyanobacterium Anacystis nidulans R2 that are unable to assimilate nitrate have been isolated after transposon Tn901 mutagenesis. Characterization of phenotypes and transformation tests have allowed the distinction of five different mutant types. The mutants exhibiting a nitrate reductase-less phenotype were identified as being affected in previously defined loci, as they could be transformed to the wild type by one of the plasmids pNR12, pNR63 or pNR193, which contain cloned genes of A. nidulans R2 involved in nitrate reduction. The mutations in strains FM2 and FM16 appear to affect two other genes involved in nitrate assimilation. Strain FM2 apparently bears a single mutation which results in both lack of nitrite reductase activity and loss of ammonium-promoted repression of nitrate reductase synthesis. FM16 has a low but significant level of nitrate reductase that is also freed from repression by ammonium, and an increased level of nitrite reductase activity. FM16 exhibited properties which indicate that this mutant strain might also be affected in the transport of nitrate into the cell.Abbreviations EDTA ethylenediamine-tetraacetic acid - MTA mixed alkyltrimethylammonium bromide - TES N-tris (hydroxymethyl)methyl-2-aminoethane sulfonic acid - Tricine N-[2-hydroxy-1,1-bis (hydroxymethyl)ethyl]-glycine - Tris Tris(hydroxymethyl)aminomethane     

Development of gene transfer systems in Methylobacillus flagellatum KT: Isolation of auxotrophic mutants

A collection of polyauxotrophic mutants of the obligate methylotroph Methylobacillus flagellatum KT was obtained. On the first step two stable auxotrophic mutants with a high requirement for amino acids supplements were isolated by treatment with nitrosoguanidine and selection on complete medium. Spontaneous variants of these mutants with a low requirement for nutrient supplements were the base for obtaining polyauxotrophic strains. It was shown, that the growth of mutants of M. flagellatum KT is inhibited by complete medium. Some amino acids and nucleotides are the inhibitor components of complete media. An approach for selection of auxotrophic mutants of individual genes was worked out on minimal medium. The optimal conditions for nitrosoguanidine mutagenesis of M. flagellatum KT were developed. The possible mechanisms of action of some of the nutrient supplements on the growth of M. flagellatum KT are discussed.