An enrichment selection for mutants resulting from oligonucleotide-directed mutagenesis of double-stranded DNA

We report here a simple and rapid procedure for enrichment and selection of mutants from oligonucleotide-directed mutagenesis on double-stranded plasmid DNA. Mutagenic oligonucleotides were designed to insert or delete a unique restriction site with silent codon changes. After mutagenesis, plasmid DNA from all resulting colonies was pooled, restricted with the appropriate endonuclease, and the resulting unique form of DNA (linear or circular) was isolated and used for transformation of competent E. coli. These procedures provided an enrichment of mutant plasmid from the 4% obtained by more conventional techniques to greater than 65%.     

Mouse infection and pathogenesis by Trypanosoma brucei motility mutants

The flagellum of Trypanosoma brucei is an essential and multifunctional organelle that drives parasite motility and is receiving increased attention as a potential drug target. In the mammalian host, parasite motility is suspected to contribute to infection and disease pathogenesis. However, it has not been possible to test this hypothesis owing to lack of motility mutants that are viable in the bloodstream life cycle stage that infects the mammalian host. We recently identified a bloodstream‐form motility mutant in 427‐derived T. brucei in which point mutations in the LC1 dynein subunit disrupt propulsive motility but do not affect viability. These mutants have an actively beating flagellum, but cannot translocate. Here we demonstrate that the LC1 point mutant fails to show enhanced cell motility upon increasing viscosity of the surrounding medium, which is a hallmark of wild type T. brucei, thus indicating that motility of the mutant is fundamentally altered compared with wild type cells. We next used the LC1 point mutant to assess the influence of trypanosome motility on infection in mice. Wesurprisingly found that disrupting parasite motility has no discernible effect on T. brucei bloodstream infection. Infection time‐course, maximum parasitaemia, number of waves of parasitaemia, clinical features and disease outcome are indistinguishable between motility mutant and control parasites. Our studies provide an important step toward understanding the contribution of parasite motility to infection and a foundation for future investigations of T. brucei interaction with the mammalian host.     

利用机器学习提高M. jannaschii酪氨酰tRNA合成酶底物特异性分子建模预测的准确度

设计结合不同化学结构底物的酶结合袋是一个巨大的挑战. 传统的湿实验要筛选成千上万甚至上百万个突变体来寻找对特定配体结合的突变体,此过程需要耗费大量的时间和资源. 为了加快筛选过程,我们提出了一种新的工作流程,将分子建模和数据驱动的机器学习方法相结合,生成具有高富集率的突变文库,用于高效筛选能识别特定底物的蛋白质突变体. M. jannaschii酪氨酰tRNA合成酶（Mj. TyrRS）能识别特定的非天然氨基酸并催化形成氨酰tRNA,其不同的突变体能够识别不同结构的非天然氨基酸,并且已经有了许多报道和数据的积累,因此我们使用TyrRS作为一个例子来进行此筛选流程的概念验证. 基于已知的多个Mj. TyrRS的晶体结构及分子建模的结果,我们发现D158G/P是影响残基158~163位α螺旋蛋白骨架变化的关键突变. 我们的模拟结果表明,在含有687个突变体的测试数据中,与随机突变相比,分子建模和打分函数计算排序可以将目标突变体的富集率提高2倍,而使用已知突变体和对应的非天然氨基酸数据训练的机器学习模型进行校准后,筛选富集率可提高11倍. 这种分子建模和机器学习相结合的计算和筛选流程非常有助于Mj.TyrRS的底物特异性设计,可以大大减少湿实验的时间和成本. 此外,这种新方法在蛋白质计算设计领域具有广泛的应用前景.     

Selection of spontaneous mutants ofYarrowia lipolytica by inositol-less death

Summary A procedure was developed for the selection of spontaneous mutants of the yeastYarrowia lipolytica. An inositol-requiring mutant of a wild-typeY. lipolytica, YB 3-122, was derived by mutagenesis and screening. The mutant had a reversion frequency of less than 6×10^–9. A mutant selection procedure based on inositolless death was then developed using this mutant strain. The selection procedure killed growingY. lipolytica cells and enriched for mutants yielding cultures that consisted of 60–98% spontaneous mutants after two rounds of inositol-less death. The procedure enriched for four classes of mutants, strains that were auxotrophic, metabolite analog sensitive, temperature sensitive, or unable to grow on citric acid as the sole carbon source. Since strain YB 3-122 is now available to yeast researchers, inositol-less death will be useful for the routine isolation of spontaneous mutants ofY. lipolytica.     

Deletion of valR,a homolog of Vibrio harveyis´ luxR generates an intermediate colony phenotype between opaque/rugose and translucent/smooth in Vibrio alginolyticus

A previous study has shown that Vibrio alginolyticus ZJ-51 undergoes colony phase variation between opaque/rugose (Op) and translucent/smooth (Tr). The AI-2 quorum-sensing master regulator ValR, a homolog to V. harveyi LuxR, was suggested to be involved in the transition. To investigate the role of ValR in the variation and in biofilm formation, an in-frame deletion of valR in both Op and Tr backgrounds was carried out. The mutants in both backgrounds showed an intermediate colony morphotype, where the colonies were less opaque/rugose but not fully translucent/smooth either. They also showed an intermediate level of motility. However, biofilm formation was severely decreased in both mutants and polar flagella were depleted also. Quantitative PCR showed that most of the genes related to flagellar and polysaccharide biosynthesis were upregulated in the mutant of Op background (ΔvalR/Op) but downregulated in the mutant of Tr background (ΔvalR/Tr) compared with their parental wild-type strains. This suggests that ValR may control biofilm formation by regulating flagellar biosynthesis and affect the expression of the genes involved in colony phase variation in V. alginolyticus.     

Isolation of Spontaneously Derived Mutants of CAULOBACTER CRESCENTUS

Caulobacter crescentus has a penicillinase which precludes the use of penicillin for mutant enrichment. However, two other antibiotics, fosfomycin and D-cycloserine, can be enrich for C. crescentus mutants. In enrichment procedures for C. crescentus auxotrophs, spontaneously derived mutants occur at a frequency of 5-10% among the survivors of an enrichment procedure. Consequently, large numbers of mutants are readily obtained without any need for mutagenesis. These mutants are heterogeneous both with regard to the type of mutation and to the nutritional requirement. A similar procedure has been used to isolate temperature-sensitive mutants.     

Procedure for the isolation of mutants of Bacillus subtilis with defective cytoplasmic membranes

A procedure has been devised to isolate mutants of Bacillus subtilis with structurally defective membranes. The procedure used to screen for the mutants involved comparison of the stability of protoplasts of the mutant with those of the wild type in a medium of sufficient osmotic strength to stabilize wild-type protoplasts. Mutagenized cells were grown as clones on agar plates, and then replicated onto plates containing 0.5 m lactose, which is sufficient to stabilize wild-type protoplasts. The colonies on the lactose-containing plates were then treated with lysozyme to convert the cells to protoplasts. Colonies of wild-type protoplasts remained opaque; however, colonies of mutant protoplasts lysed and became clear. Twenty-nine osmotically fragile mutants were isolated in this manner; the membranes of several mutants were found to contain alterations in the composition of their proteins or lipids.     

An easy method for screening and isolating rod mutants of<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

A convenient and rapid method for screening and identifying rod mutants of Bacillus subtilis is described. At the restrictive temperature (45 °C), all rod mutants of B. subtilis screened lost their ability to sporulate. The morphology and colour of mutant colonies grown on sporulation agar plates differed from those of rod⁺ cells, which were able to sporulate even at elevated temperature. These characteristics provide an alternative approach for the identification of rod mutants in B. subtilis culture by streaking the cells onto a minimal glucose agar plate and incubating at the restrictive temperature. After 30 h of incubation at this temperature, rod mutants are easily identified. This method will facilitate the screening and isolation of rod mutants of B. subtilis.     

Isolation and characterization of respiration-deficient mutants inAspergillus ochraceus

Two respiration-deficient mutants (rd) were isolated from the acetate-nonutilizing mutants (acu) induced by N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) inAspergillus ochraceus. A complementation analysis of the tword mutants indicated that MNNG had caused a mutation at a single locus. The diameter of the tword mutant colonies in glucose medium was found to be small in comparison to that of the wild type and the otheracu mutants; the diameter of the isolated mutant colonies in acetate medium was very small. The grown zone ofrd mutants remained colorless up to 20 h incubation in 2,3,5-triphenyltetrazolium-overlaid solid Czapek-Dox medium and it turned pink after prolonged incubation, whereas the wild type and the otheracu mutants became pink within 30 min in the same medium. Therd mutants were further characterized by measuring the respiratory activities of intact mycelia in the presence of glucose.     

Evaluation of UV-Induced Mutants of Chlorella vulgaris for Single Cell Protein I. The Screening Effect of Cycloheximide and 6-Methylpurine

Chlorella vulgaris was irradiated with UV doses allowing a 0.1 per cent survival. Dark recovery and photoreactivation were carried out either in the presence or in the absence of an inhibitor of protein synthesis. Faster growing and faster greening colonies were selected, and the amino acid composition of the mutant strains were evaluated. Higher growth and photosynthetic rates, higher chlorophyll content and lower respiration rate were shown by all mutants irrespective of the selection procedure. Selection against cycloheximide led to higher protein and RNA, but lower carotenoid content, whereas mutants selected against 6-methylpurine showed, in addition to a protein and carotenoid increase, a more favourable protein: RNA ratio.     

Effect of gacS and gacA mutations on colony architecture, surface motility, biofilm formation and chemical toxicity in Pseudomonas sp. KL28

GacS and GacA proteins form a two component signal transduction system in bacteria. Here, Tn5 transposon gacS and gacA (Gac) mutants of Pseudomonas sp. KL28, an alkylphenol degrader, were isolated by selecting for smooth colonies of strain KL28. The mutants exhibited reduced ability to migrate on a solid surface. This surface motility does not require the action of flagella unlike the well-studied swarming motility of other Pseudomonas sp. The Gac mutants also showed reduced levels of biofilm and pellicle formation in liquid culture. In addition, compared to the wild type KL28 strain, these mutants were more resistant to high concentrations of m-cresol but were more sensitive to H2O2, which are characteristics that they share with an rpoS mutant. These results indicate that the Gac regulatory cascade in strain KL28 positively controls wrinkling morphology, biofilm formation, surface translocation and H2O2 resistance, which are important traits for its capacity to survive in particular niches.     

Temporal sequence of the recovery of traits during phenotypic curing of a Cytophaga johnsonae motility mutant.

The lack of cell translocation and the resulting formation of nonspreading colonies of mutants of the gram-negative gliding bacterium Cytophaga johnsonae have been correlated with the loss of cell surface features of the organism. These cell surface traits include the ability to move polystyrene-latex beads over the cell surface and the ability to be infected by bacteriophages that infect the parent strain. In order to assess whether these traits reflect structures or functions that actually play a role in gliding, we studied a mutant (21A2I) selected for its inability to form spreading colonies; it is deficient in sulfonolipid, lacks bead movement ability, and is resistant to at least one bacteriophage. The provision of cysteate (a specific sulfonolipid precursor) restores lipid content and gliding to the mutant; hence, the lipids are necessary for motility. Growth with cysteate also restores bead movement and phage sensitivity. In order to determine the temporal relationship of these traits, we undertook a kinetic study of the appearance of them after addition of cysteate to the mutant. One predicts that appearance of a trait essential for cell translocation will either precede or accompany the appearance of this ability, while a nonessential trait need not do so. Sulfonolipid synthesis was the only trait that appeared before gliding; this is consistent with its established importance for motility. Bead movement and phage sensitivity first appeared only after gliding started, suggesting that the machinery involved in those processes is not necessary, at least for the initiation of gliding.     

Cloning and characterization of the Flavobacterium johnsoniae gliding-motility genes gldB and gldC

The mechanism of bacterial gliding motility (active movement over surfaces without the aid of flagella) is not known. A large number of mutants of the gliding bacterium Flavobacterium johnsoniae (Cytophaga johnsonae) with defects in gliding motility have been previously isolated, and genetic techniques to analyze these mutants have recently been developed. We complemented a nongliding mutant of F. johnsoniae (UW102-99) with a library of wild-type DNA by using the shuttle cosmid pCP26. The complementing plasmid (pCP200) contained an insert of 26 kb and restored gliding motility to 4 of 50 independently isolated nongliding mutants. A 1.9-kb fragment which encompassed two genes, gldB and gldC, complemented all four mutants. An insertion mutation in gldB was polar on gldC, suggesting that the two genes form an operon. Disruption of the chromosomal copy of gldB in wild-type F. johnsoniae UW101 eliminated gliding motility. Introduction of the gldBC operon, or gldB alone, restored motility. gldB appears to be essential for F. johnsoniae gliding motility. It codes for a membrane protein that does not exhibit strong sequence similarity to other proteins in the databases. gldC is not absolutely required for gliding motility, but cells that do not produce GldC form colonies that spread less well than those of the wild type. GldC is a soluble protein and has weak sequence similarity to the fungal lectin AOL.     

Hemagglutinating activity and motility of the bacterium <Emphasis Type="Italic">Azospirillum brasilense</Emphasis> in the presence of various nitrogen sources

Hemagglutinating activity of the Azospirillum brasilense strain Sp245 grown in liquid media and the swarming motility of those bacteria grown in semisolid media vary significantly depending on the nitrogen source. In media with nitrate or nitrite, an increase in the hemagglutinating activity and a decrease in the swarming circles’ diameter of Sp245 were observed, compared to bacteria grown in the presence of ammonium or N₂. A ∼67-kDa hemagglutinin exhibiting affinity to the O-specific polysaccharide, an acidic D-rhamnan (OPS-I), was isolated from the surface of Sp245 cells. Introduction of the hemagglutinin into the media resulted in a decrease in the Sp245 cell motility while not affecting its mutants lacking the acidic D-rhamnan or the Sp245.5 mutant with a different OPS structure. Cells of strain Sp245.5 demonstrated hemagglutinating activity two times higher than that of the parent Sp245 strain and formed “diffuse” colonies, rather than distinct swarming circles Sp245 formed when grown in a semisolid medium. The data obtained demonstrate that intercellular contacts mediated by the interaction between the surface hemagglutinin and OPS-I, which is sensitive to environmental factors, affect the collective motility of cells.     

Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): Genes controlling movement of single cells

Summary M. xanthus is a gliding bacterium whose motility is subject to intercellular control. Strain DK101 of M. xanthus gives rise to 6 distinct types of nonmotile mutants and transduction of motility between mutants, mediated by the generalized transducing phage Mx8, identifies the gene loci that underlie the six types. Five of the types, B, C, D, E, and F, are contitional mutants that can be stimulated to move by wild-type cells or by cells of a different mutant type. Mutants of each stimulation type lie in separate and distinct loci, cglB, cglC, cglD, cglE and cglF. The sixth mutant type can stimulate any of the five other types to move, never moves itself, and is produced by mutations in at least 17 loci.     

Identification of genes involved in <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> biofilm formation by <Emphasis Type="Italic">mariner</Emphasis>-based transposon mutagenesis

Listeria monocytogenes is a ubiquitous food-borne pathogen, whose distribution and survival in food-processing environments are associated with the ability to form biofilms. The process of biofilm formation is complex and its molecular mechanism is relatively poorly understood in L. monocytogenes. To better understand the genetics of this process, a mariner-based transposon mutagenesis strategy was used to identify genes involved in biofilm formation of L. monocytogenes. A library of 6,500 mutant colonies was screened for reduced biofilm formation using a microtiter plate biofilm assay. Forty biofilm-deficient mutants of L. monocytogenes were identified based on DNA sequences of the transposon-flanking regions and Southern hybridization with a transposon-based probe. The insertions harbored by these mutants led to the identification of 24 distinct loci, 18 of which, to our knowledge, have not been previously reported to function in the biofilm formation in L. monocytogenes. Genetic complementation confirmed the importance of lmo1386, a gene encoding a putative DNA translocase, for biofilm formation. Molecular analyses of mutants indicated that the majority of the 24 identified genes are related to flagella motility, gene regulation, and cell surface structures.     

Isolation of motile and non-motile insertional mutants of Campylobacter jejuni: the role of motility in adherence and invasion of eukaryotic cells

A method of insertional mutagenesis for naturally transformable organisms has been adapted from Haemophilus influenzae and applied to the study of the pathogenesis of Campylobacter jejuni. A series of kanamycin-resistant Insertional mutants of C. jejuni 81–176 has been generated and screened for loss of ability to invade INT407 cells. Eight noninvasive mutants were identified which showed 18-200-fold reductions in the level of invasion compared with the parent. Three of these eight show defects in motility, and five are fully motile. The three mutants with motility defects were further characterized to evaluate the method. One mutant, K2–32, which is non-adherent and non-invasive, has an insertion of the kanamycin-resistance cassette into the flaA flagellin gene and has greatly reduced motility and a truncated flagellar filament typical of flaA mutants. The adherent non-invasive mutants K2–37 and K2–55 are phenotypically paralysed, i.e. they have a full-length flagellar filament but are non-motile. All three mutants show an aberration in flagellar structure at the point at which the filament attaches to the cell. Mutants K2–37 and K2–55 represent overlapping deletions affecting the same gene, termed pflA (paralysed flagella). This gene encodes a predicted protein of 788 amino acid residues and a molecular weight of 90 977 with no significant homology to known proteins. Site-specific insertional mutants into this open reading frame result in the same paralysed flagellar phenotype and the same invasion defects as the original mutants.