Transposon mutagenesis in preparing mutants of a strain--a producer of a new antibiotic batumin

Various plasmids carrying transposon Tn5 were used to generate insertion mutants synthesizing batumin, a unique antibiotic with a selective antistaphylococcal effect. One of the plasmids used provided a sufficient yield of the clones in question. An analysis of over 7000 clones allowed us to select the mutant clones with increased and decreased levels of batumin synthesis and the mutants that lost the ability to synthesize this antibiotic.     

Obtaining a high-activity strain capable of producing the antistaphylococcal antibiotic batumin

The use of chemical and UV-induced mutageneses allowed us to increase the biosynthetic activity of the strain capable of producing new antistaphylococcal antibiotic, batumin. The strain ofPseudomonas batumici N17 producing 87–100 mg batumin per liter culture liquid was selected. Its activity was 3.5-5 times higher than the activity of the most potent natural strain.P. batumici N17 was shown to be stable in relation to the synthesis of batumin.     

Isolation of highly active strain producing the antistaphylococcal antibiotic batumin

The use of chemical and UV-induced mutageneses allowed us to increase the biosynthetic activity of the strain capable of producing new antistaphylococcal antibiotic, batumin. The strain of Pseudomonas batumici N17 producing 87-100 mg batumin per liter culture liquid was selected. Its activity was 3.5-5 times higher than the activity of the most potent natural strain. P. batumici N17 was shown to be stable in relation to the synthesis of batumin.     

Novel motility mutants of synechocystis strain PCC 6803 generated by in vitro transposon mutagenesis

We screened for transposon-generated mutants of Synechocystis sp. strain PCC 6803 that exhibited aberrant phototactic movement. Of the 300 mutants generated, about 50 have been partially characterized; several contained transposons in genes encoding chemotaxis-related proteins, while others mapped to novel genes. These novel genes and their possible roles in motility are discussed.     

Pigmentation mutants produced by transposon mutagenesis in Antirrhinum majus

New pigmentation mutants were generated by transposon mutagenesis in Antirrhinum majus, in three previously described loci, nivea, delila and incolorata, and two new loci, daphne and olive. The wild-type olive gene is required for the production of dark-green leaves, and the daphne gene for the synthesis of flavones. Five out of the six mutants were both germinally and somatically unstable, indicating that they resulted from transposon insertions. Molecular analysis of the mutant at nivea (niv-600) showed that it was caused by insertion of a new transposon, Tam4. The sequence of Tam4 suggests that it is unable to transpose autonomously and that it is related to Tam1 and Tam2. All three of these transposons have identical inverted repeats, produce 3 bp target duplications, leave similar excision footprints and share at one end a 600-700 bp region containing many palindromic copies of a motif sequence, possibly required in cis for transposition. The somatic excision of Tam4 in niv-600 is at a very low rate compared to germinal excision but it can be activated by crossing to lines carrying derivative alleles of a Tam1 insertion at niv. Molecular analysis of four different pigmentation mutants has shown that insertions of Tam1, Tam2, Tam3 and Tam4 have been obtained, illustrating the potential of general transposon mutagenesis for trapping and isolating new transposons as well as for tagging genes.     

Isolation of OprM-deficient mutants of Pseudomonas aeruginosa by transposon insertion mutagenesis: Evidence of involvement in multiple antibiotic resistance

Abstract We have shown that azadirachtin, a compound from the neem tree, Azadirachta indica , and selected semi-synthetic derivatives, block the development of the motile male malarial gamete in vitro. Changes in the hemiacetal group at position C₁₁ in the molecule result in a loss of activity in this assay. The motility of fully formed male gametes, and other selected flagellated cells, is unaffected by azadirachtin in vitro. These findings raise the possibility of developing azadirachtin-based compounds as antimalarials with transmission-blocking potential, as well as permitting the further study of structure-activity relationships in these compounds.     

转座子随机插入鉴定肠炎沙门氏菌生物膜形成相关基因

[目的]生物膜在沙门氏菌的致病性和引起沙门氏菌食物中毒等方面起着重要作用,本研究为了鉴定影响沙门氏菌生物膜形成的基因.[方法]利用结晶紫染色定量法对74株鸡源的肠炎、鸡白痢和鸡伤寒沙门氏菌进行生物膜测定,选择生物膜生长较好的肠炎沙门氏菌C050041,采用转座子随机插入法构建突变株库.[结果]84%的鸡源沙门氏菌菌株可在塑料表面形成生物膜;通过转座子插入获得1924个突变株,筛选的生物膜降低突变株经生长曲线测定、测序和序列比对及Southern blot分析鉴定出15个插入基因,它们分别为metE、ompR、rpoS、,和G、rfaJ、rfaK、rfaP、rfbH、rhlE、spiA、steB、tpx、ybdN和2个未知功能的基因.[结论]我们鉴定出了多个影响生物膜形成的新基因,这些基因的发现为进一步研究沙门氏菌生物膜形成的调控机制,研制减毒沙门氏菌疫苗奠定了基础.     

紫外诱变原生质体选育D-核糖生产菌株

以D-核糖产生菌枯草芽孢杆(Bacillus subtilis)B941为出发菌株,采用紫外诱变原生质体的方法,获得了4株可以在含有6．0％D-核糖的培养基上生长的D-核糖高产菌株,其摇瓶发酵产糖达55．0g／L左右。通过摇瓶发酵试验,研究了D-核糖高产菌株Buvp-24的遗传稳定性。研究结果表明,经多次传代,菌株Buvp-24的发酵产糖能力及对发酵产物D-核糖的耐受性没有改变,有望应用于工业生产中。     

Applications of transposition mutagenesis in antibiotic producing streptomycetes

Several transposons have been developed from the streptomycete insertion sequence IS493. They have broad host specificity in Streptomyces species and insert relatively randomly into a consensus target sequence of gNCaNTgNNy. Collectively, they have specialized features that facilitate the following: cloning of DNA flanking insertions; physical mapping of insertions; construction of highly stable mutants; and efficient construction of mutant libraries. All of the transposons can be introduced into streptomycetes by conjugation from E. coli, and can be delivered by curing the temperature sensitive delivery plasmid. Tn5099 was used to physically map genes involved in daptomycin and red pigment production in Streptomyces roseosporus, and to clone daptomycin biosynthetic genes. Tn5099 was also used in Streptomyces fradiae to identify and clone a neutral genomic site for the insertion of a second copy of the tylF gene. Recombinants containing two copies of the tylF gene carried out the no rmally rate limiting conversion of macrocin to tylosin very efficiently, thus causing substantial increases in tylosin yield.     

Genome-wide transposon mutagenesis of Borrelia burgdorferi for identification of phenotypic mutants

The spirochete Borrelia burgdorferi is the causative agent of Lyme disease, the leading vector-borne illness in the United States. Many of the genetic factors affecting spirochete morphology and physiology are unknown due to the limited genetic tools available and the large number of open reading frames with unknown functions. By adapting a mariner transposon to function in B. burgdorferi, we have developed a random mutagenesis system that tags the mutated locus for rapid identification. Transposition occurs at saturating levels in B. burgdorferi and appears to be random, targeting both linear and circular replicons. By combining the transposon system with a screen for factors affecting growth rate, mutations were readily identified in genes putatively involved in cell division and chemotaxis and a hypothetical open reading frame involved in outer membrane integrity. The successful adaptation of a mariner transposon to function in B. burgdorferi should aid in identifying virulence factors and novel gene products related to spirochete physiology.     

Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis

Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.     

Isolation of Citrobacter sp. mutants defective in decolorization of brilliant green by transposon mutagenesis

To identify genes involved in the decolorization of brilliant green, we isolated random mutants generated by transposon insertion in brilliant green-decolorizing bacterium, Citrobacter sp. The resulting mutant bank yielded 19 mutants with a complete defect in terms of the brilliant green color removing ability. Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants and these mutants appeared to have insertions at different sites of the chromosome. Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined. By comparing these with a sequence database, putative protein products encoded by bg genes were identified as follows: bg 3 as a LysR-type regulatory protein; bg 11 as a MalG protein in the maltose transport system; bg 14 as an oxidoreductase; and bg 17 as an ABC transporter. The sequences deduced from the three bg genes, bg 2, bg 7 and bg 16, showed no significant similarity to any protein with a known function, suggesting that these three bg genes may encode unidentified proteins responsible for the decolorization of brilliant green.     

原生质体激光诱变选育少根根霉脂肪酶高产菌株

对少根根霉BUCT-11原生质体制备、再生条件及激光诱变育种进行了研究.结果显示,少根根霉BUCT-11原生质体形成及再生最佳条件为:菌龄24 h,混合酶由27 mg/mL的蜗牛酶和53 mg/mL的纤维素酶组成,酶解时间1.5 h,酶解温度30 ℃,渗透压稳定剂为0.6 mol/L NH4Cl、0.02 mol/L ...     

紫外诱变原生质体选育低温碱性脂肪酶高产菌株

以产低温碱性脂肪酶约氏不动杆菌(Acinetobacter johnsonii)LP28为出发菌株,采用EDTA和溶菌酶处理制备原生质体.确定其最佳处理条件为37℃的水浴下,以终浓度为0.15 mg/mL的溶菌酶处理45 min,最终可获得90%的原生质体形成率及0.9%左右的再生率.采用紫外诱变原生质体的方法,筛选得...     

HP17, a new pigment-like antibiotic produced by a new strain of Spirillospora

Spirillospora strain 719 produces several antibiotics. On solid and liquid media, a deep red pigment is formed and diffuses throughout the culture. It was extracted with methanol from the mycelium cake and from the fermentation broth after precipitation at pH 2 and purified using TLC and HPLC. Its u.v. absorption spectrum and its physicochemical characteristics place this antibiotic in the 3.3.2.2.8 of the Berdy et al. classification. In most respects, it resembles proteinaceous pigment from Spirillospora 1655 and 1309-b that was studied and named spirillomycin. However, HP17 differs from spirillomycin principally in molecular weight and chemical nature.     

Generalized transposon shuttle mutagenesis in Neisseria gonorrhoeae: a method for isolating epithelial cell invasion-defective mutants

Hybrid genes were constructed to express bifunctional hybrid proteins in which staphyloccal nuclease A with or without an amino-terminai OmpA signal sequence was fused with TEM β-lactamase (at the carboxyl terminal side) using the signal peptide of the major outer membrane lipoprotein of Escherichia coli as an internal linker. The hybrid proteins were found to be inserted in the membrane. Orientation of the hybrid protein with the OmpA signal peptide showed that the nuclease was translocated into the periplasm and the β-lactamase remained in the cytoplasm. This indicates that the cleavable OmpA signal peptide served as a secretory signal for nuclease and the internal lipoprotein signal served as the transmembrane anchor, in the absence of the OmpA signal sequence the topology of the hybrid protein was reversed indicating that the internal lipoprotein signal peptide initially served as the signal peptide for the secretion of the carboxy terminal β-lactamase domain across the membrane and subsequently as a membrane anchoring signal. The role of charged amino acids in the translocation and transmembrane orientation of membrane proteins was also analysed by introducing charged amino acids to either or both sides of the internal lipoprotein signal sequence in the bifunctional hybrid proteins in the absence of the amino-terminal signal sequence. Introduction of two lysine residues at the carboxy-terminal side of the internal signal sequence reversed the topology of the transmembrane protein by translocating the aminoterminal nuclease domain across the membrane, leaving the carboxyl terminal β-actamase domain in the cytoplasm. When three more lysine residues were added to the amino-terminal side of the internal signal sequence of the same construct the membrane topology flipped back to the original orientation. A similar reversion of the topology could be obtained by introducing negatively charged residues at the amino-terminal side of the internal signal sequence. Present results demonstrate for the first time that a bifunctional transmembrane protein can be engineered to assume either of the two opposite orientations and that charge balance around the transmembrane domain is a major factor in controlling the topology of a transmembrane protein.     

Aflagellated mutants of Helicobacter pylori generated by genetic transformation of naturally competent strains using transposon shuttle mutagenesis

Three out of 10 Helicobacter pylori clinical isolates were found to be naturally competent for genetic transformation to streptomycin resistance by chromosomal DNA extracted from a spontaneous streptomycin-resistant H. pylori mutant. The frequency of transformation varied between 5 × 10^?4 and 4 × 10^?6, depending on the H. pylori isolate used. Transposon shuttle mutagenesis based on this natural competence was established using the flagellin gene flaA as the target. The cloned flaA gene was interrupted by insertion of TnMax1, a mini-Tn1721 transposon carrying a modified chloramphenicol-acetyltransferase gene, the cat_GC cassette. Natural transformation of competent H. pylori strains with plasmid constructs harbouring a cat_GC-inactivated flaA gene resulted in chloramphenicol-resistant transformants at an average frequency of 4 × 10^?5. Southern hybridization experiments confirmed the replacement of the chromosomal H. pylori flaA gene by the cat-inactivated cloned gene copy via homologous recombination resulting in allelic exchange. Phenotypic characterization of the mutants demonstrated the absence of flagella under the electron microscope and the loss of bacterial motility. Immunoblots of cell lysates of the H. pylori mutants with an antiserum raised against the C-terminal portion of recombinant H. pylori major flagellin (FlaA) confirmed the absence of the 54kDa FlaA protein. This efficient transposon shuttle mutagenesis procedure for H. pylori based on natural competence opens up new possibilities for the genetic assessment of putative H. pylori virulence determinants.     

Epitope mapping of recombinant antigens by transposon mutagenesis

We describe a method for generating a plasmid library expressing random truncations of a recombinant protein and for epitope mapping by screening the library with monoclonal antibodies. The key step is the random introduction of the transposon, Tn1000, which carries stop codons in all three reading frames, into a bacterial expression plasmid by using a simple bacterial mating procedure. Antibody-positive clones are then selected and the point of protein truncation is determined by sequencing the plasmid DNA at the point of transposon insertion. One advantage of the method is that no subcloning or in vitro manipulation of DNA is necessary.