转座子Tn917在短小芽孢杆菌中的转座作用

Transposition Tn917 was introduced into Bacillus pumilus 289 by protoplast transformation with plasmid pTV32. The temperature-sensitive replication property of pTV32 was maintained in B. pumilus. Tn917 was transposed efficiently in B. pumilus with 4.8 x 10(-4) transposition rate. The yield of auxotrophs was about 0.65% in all insertional mutants. It indicated a prospects for the use of Tn917 as a tool for insertional mutagenesis and genetic manipulation in B. pumilus.     

Transposition of the gram-positive transposon Tn917 in Escherichia coli.

The streptococcal transposon Tn917 was demonstrated to transpose in Escherichia coli from the Bacillus subtilis-E. coli shuttle plasmid pHK1207 into an F' plasmid derivative. Subsequently, a second round of transposition from the F' plasmid into pACYC184 could be readily demonstrated. These results represent the initial demonstration of the transposition of a gram-positive transposon in a gram-negative bacterium at a relatively high frequency.     

Chromosomal insertions of Tn917 in Bacillus subtilis.

We describe 46 insertions of the Streptococcus faecalis transposon Tn917 into the chromosome of Bacillus subtilis. These insertion mutations were mapped genetically. Some caused auxotrophic requirements, and others were cryptic. These insertions were scattered around the B. subtilis chromosome. The mutant strains were useful in several ways for mapping and cloning B. subtilis genes and were added to the Bacillus Genetic Stock Center collection. Among the auxotrophic markers were a new serine auxotrophy and deletion-insertions that caused auxotrophy in one case for homoserine and threonine, in another case for uracil and either cysteine or methionine, and in a third case for leucine, isoleucine, and valine.     

Mutagenesis using Tn917 in Bacillus licheniformis

Abstract Two plasmid vectors containing Tn 917 have been transformed into protoplasts of Bacillus licheniformis . Replication of the vectors was inhibited at high temperature. The transporon integrated efficiently into the chromosome and auxotroph formation was high (9% of transposed colonies). Most of these were argG or argH indicating a hot-spot region for integration of Tn 917 into the chromosome. However, about 0.5% of the mutants had other auxotrophies.     

Isolation of Gln- mutants through transposon, Tn917, mutagenesis in Bacillus brevis.

Transposon, Tn917, carried on pTV1 plasmid has been used successfully to mutagenise Bacillus brevis. The transposon showed preference for insertion at an "aro" site. A second insertional event after elimination of the preferred site with ethidium bromide/acridine orange treatment has permitted isolation of Gln- mutants in B. brevis.     

Isolation and characterization of Tn917lac-generated competence mutants of Bacillus subtilis

We isolated 28 mutants of Bacillus subtilis deficient in the development of competence by using the transposon Tn917lacZ as a mutagen. The mutant strains were poorly transformable with plasmid and chromosomal DNAs but were normally transducible and exhibited wild-type resistance to DNA-damaging agents. The mutations were genetically mapped, and the mutants were characterized with respect to their abilities to bind and take up radiolabeled DNA. All were defective in uptake, and some failed to bind significant amounts of DNA. The abilities of the mutant strains to resolve into two buoyant density classes on Renografin gradients were studied. Most resolved normally, but several banded in Renografin only at the buoyant density of noncompetent cells. The genetic mapping studies and the other analyses suggested that the mutations define a minimum of seven distinct com genes.     

Isolation and characterization of Tn917-generated bacitracin deficient mutants of Bacillus licheniformis

Abstract Two Tn917-generated bacitracin deficient mutants of Bacillus licheniformis were isolated. Southern blot analysis of chromosomal DNA extracted from both insertional mutants showed that Tn917 inserted in the vicinity of the gene coding for the enzyme BA2 of the bacitracin synthetase enzyme complex. Measurements of bacitracin synthetase activity in cell-free extracts and positive hybridization signals in the vicinity of the BA2 gene indicate that in both bacitracin deficient mutants Tn917 could be inserted in the BA1 gene or in segments involved in regulation. Thus, it could be possible that the genes for bacitracin synthetase are clustered in the B. licheniformis genome.     

转座子Tn917诱变的炭疽杆菌芽孢形成缺陷株的筛选

目的:诱导转座子Tn917随机插入炭疽杆菌染色体,产生在不同位点突变的突变体库,从中筛选芽孢形成缺陷型突变株。方法:用含转座子Tn917的质粒pLTV3转化炭疽杆菌,以低浓度红霉素诱导转座因发生转座,产生大量的突变株。进而用氯化三苯基四氮唑染色法和复红美蓝染色法从突变体库中筛选芽孢形成缺陷株;用Southern杂交法对芽孢形成缺陷株进行验证。结果:对2000个突变体进行了筛选,共得到6株芽孢形成缺陷株,在LB培养基中培养5d后,镜下仍未见有芽孢形成,呈现明显的芽孢形成缺陷特征。Southern杂交表明野生株无杂交带,突变株均有且只有1条杂交带,且杂交带的位置不尽相同。结论:转座子Tn917可以单拷贝随机诱变炭疽杆菌野生株,产生在不同位点突变的突变株。     

Properties of erythromycin-inducible transposon Tn917 in Streptococcus faecalis.

Streptococcus faecalis strain DS16 harbors two plasmids, a conjugative plasmid, pAD1, which encodes hemolysin and bacteriocin activities, and a nonconjugative plasmid, pAD2, encoding resistance to streptomycin, kanamycin, and erythromycin, the latter of which is inducible. The erythromycin resistance determinant is located on a 3.3-megadalton transposable element designated Tn917, which could be transposed to pAD1 as well as to two other plasmids, pAm gamma 1 and pAM alpha 1. When strain DS16 was exposed to low (inducing) concentrations of erythromycin for a few hours, the frequency of Tn917 transposition from pAD2 to pAD1 increased by an order of magnitude. This induction paralleled induction of erythromycin resistance and was prevented by exposing the cells to inhibitors of deoxyribonucleic acid, ribonucleic acid or protein synthesis. The exposure of strain DS16 to inducing concentrations of erythromycin also enhanced the frequency of erythromycin-resistant transconjugants appearing during mating. Initially, cointegrate molecules, whose molecular weights were approximately the sum of pAD1 and pAD2, accounted for these transconjugants; however, as the induction time increased, pAD1::Tn917 became increasingly prominent.     

Transposition of Tn4351 in Porphyromonas gingivalis.

Genetic analysis of Porphyromonas gingivalis, an obligately anaerobic gram-negative bacterium, has been hindered by the apparent lack of naturally occurring bacteriophages, transposable elements, and plasmids. Plasmid R751::*omega 4 has previously been used as a suicide vector to demonstrate transposition of Tn4351 in B. uniformis. The erythromycin resistance gene on Tn4351 functions in Bacteroides and Porphyromonas. Erythromycin-resistant transconjugants were obtained at a mean frequency of 1.6 x 10(-7) from matings between Escherichia coli HB101 containing R751::*omega 4 and P. gingivalis 33277. Southern blot hybridization analysis indicated that about half of the erythromycin-resistant P. gingivalis transconjugants contained simple insertions of Tn4351 and half contained both Tn4351 and R751 sequences. The presence of R751 sequences in some P. gingivalis transconjugants most likely occurred from Tn4351-mediated cointegration of R751, since we were unable to detect autonomous plasmid in these P. gingivalis transconjugants. The P. gingivalis-Tn4351 DNA junction fragments from different transconjugants varied in size. These results are consistent with transposition of Tn4351 and with insertion at several different locations in the P. gingivalis chromosome. Tn4351 may be useful as a mutagen to isolate well-defined mutants of P. gingivalis.     

Transduction in Bacillus megaterium.

A bacteriophage, MP13, isolated from the soil on $\text{B. megaterium}$ QM B1551 has been found to transduce several auxotrophic markers. Transduction required inactivation of the phage to approximately 0.01% survival with UV light and it was enhanced by the absence of salts that are probably necessary for phage readsorption.     

Transposition of Tn1000: in vivo properties.

Transposition mediated by the Tn1000 transposase was investigated by using transposon variants carrying synthetic or wild-type termini but no intact Tn1000 genes. Transposon Tn1001, whose only homologies to Tn1000 are in its 38-base-pair terminal inverted repeats, transposed at the same rate as Tn1005, an artificial construct carrying wild-type Tn1000 termini and approximately 1 kilobase of flanking Tn1000 DNA at each end, when transposase was supplied in trans. The majority of the transpositions into pOX38 gave rise to cointegrates, but approximately 10% of the products expressed phenotypes of direct transpositions. The expression and temperature dependence of the tnpA gene product were examined by studying transposition of Tn1001 to bacteriophage lambda. The temperature optimum for transposition was 37 degrees C, and the transposase was stable for up to 2 h at this temperature.     

Phosphatidyltransferase activity in Bacillus megaterium.

Phosphatidyl transfer between phosphatidylethanolamine, phosphatidylglycerol or phosphatidylserine as donors and primary hydroxyl acceptors including ethanolamine, glycerol, serine and Triton X-100 has been shown to be catalysed by membrane particles derived from Bacillus megaterium strains ATCC 13632 and ATCC 14581. The rate of cardiolipin synthesis from phosphatidylglycerol in the presence of ethanolamine was an order of magnitude greater than that of phosphatidylethanolamine formation. Cardiolipin synthesis from phosphatidylethanolamine in the presence of glycerol was also observed, and was 1.5-fold greater than the formation of phosphatidylglycerol. Similar heat lability, effects of pH and of Triton X-100 for phosphatidyl transfer and cardiolipin synthesis indicate that both reactions were catalysed by cardiolipin synthase.     

Tryptophan catabolism in Bacillus megaterium.

Bacillus megaterium grows in a medium containing L-tryptophan as the sole carbon, nitrogen, and energy source. Kynurenine, anthranilic acid, and catechol are metabolic intermediates, suggesting that this organism used the anthranilic acid pathway for tryptophan degradation. Cells that grow on L-tryptophan oxidize kynurenine, alanine, and anthranilic acid and the presence of tryptophan oxygenase (EC 1.13.1.12), kynureninase (EC 3.7.1.3), and catechol oxygenase (EC 1.13.1.1) in cell extracts provide additional evidence for the degradative pathway in B. megaterium. Tryptophan oxygenase is inhibited by sodium azide, potassium cyanide, and hydroxylamine, indicating that the enzyme has a functional heme group. D-Tryptophan is not a substrate for tryptophan oxygenase, and the D-isomer does not inhibit this enzyme. Formamidase (EC 3.5.1.9) and anthranilate hydroxylase are not detectable in extracts. Tryptophan catabolism is inducible in B megaterium and is subject to catabolite repression by glucose and glutamate. Arginine does not cause repression, and kynurenine induces both tryptophan oxygenase and kynureninase.