质粒RSF1010从大肠杆菌到稀有放线菌头状链轮丝菌和星状诺卡氏菌的？…

RSF1010 is a naturally occurring Escherichia coli broad host-range plasmid about 8.7 kb in size. It can be mobilized at high frequency between different gram-negative bacterial species when transfer functions are available in trans. Following the pioneering work of conjugational transfer of RSF1010 from E. coli to Streptomyces lividans and Mycobacterium smegmatis, the transfer of this plasmid by conjugation from E. coli S17.1 tp two gram-positive rare actinomycetes, Nocardia asteroides 3927 and Streptoverticillum caespitosus ATCC27422 was first time reported in this study. Southern blot analysis of the total DNA extracted from the actinomycetes' exconjugants proved that RSF1010 had been transferred from E. coli into the two new hosts and maintained staby in the exconjugants. Meanwhile, partial deletions of RSF1010 replicon loosing its antibiotics resistance makers were readily detected in E. coli. The implenmentation of this observation was discussed.     

质粒RSF1010从大肠杆菌到稀有放线菌头状链轮丝菌和星状诺卡氏菌的接合转移

１９８７年,ＴｒｉｅｎＣｕｏｔ等人［１］证明穿梭质粒可以在革兰氏阴性的大肠杆菌（Ｅｓｃｈｅｒｉｃｈｉａｃｏｌｉ）和多种革兰氏阳性细菌之间发生接合转移。在这种转移中质粒需具备大肠杆菌的复制起始位点,同时又具备革兰氏阳性细菌的广宿主范围复制起始位点。转…     

Gene fusion vehicles for the analysis of gene expression in Rhizobium meliloti.

A set of plasmid cloning vehicles was developed to facilitate the construction of gene or operon fusions in Rhizobium meliloti. The vehicles also contain a broad-host-range replicon and could be introduced into bacteria either by transformation or by transduction, using bacteriophage P2. Insertion of foreign DNA into a unique restriction endonuclease cleavage site promotes the synthesis of either the Escherichia coli lactose operon or the kanamycin phosphotransferase gene from transposon Tn5. Expression of the lactose operon could be detected by observing the color of Rhizobium colonies on medium that contained a chromogenic indicator. We also determined the growth conditions that make it possible to select either for or against the expression of the E. coli lactose operon in R. meliloti. Recombinant plasmids were constructed by inserting MboI restriction fragments of R. meliloti DNA into one of the vehicles, pMK353 . Expression of beta-galactosidase by a number of these recombinants was measured in both R. meliloti and E. coli.     

Induction of transposon Tn1 translocations as affected by different mutagens

Transposition of ampicillin Tn1 transposon is repressed under normal conditions and occurs with low frequency (10(-4) per cell). Treatment of Escherichia coli cells with 22 c+mytomycin C, nitrosoguanidine and UV light induced transposition of Tn1 into different replicons. The degree of induction depended upon the strain of bacteria and the replicon which the transposon was inserted into. Mutation recA did not influence spontaneous translocation of the transposon but fully repressed induction of transposition during the period of mutagen treatment. In the present paper, the connection of inducible transposition process with the recA and lexA inducible functions of E. coli is discussed.     

Conservation of bacterial protein synthesis machinery: initiation and elongation in Mycobacterium smegmatis

Most of our understanding of ribosome function is based on experiments utilizing translational components from Escherichia coli. It is not clear to which extent the details of translation mechanisms derived from this single organism are true for all bacteria. Here we investigate translation factor-dependent reactions of initiation and elongation in a reconstituted translation system from a Gram-positive bacterium Mycobacterium smegmatis. This organism was chosen because mutations in rRNA have very different phenotypes in E. coli and M. smegmatis, and the docking site for translational GTPases, the L12 stalk, is extended in the ribosomes from M. smegmatis compared to E. coli. M. smegmatis genes coding for IF1, IF2, IF3, EF-G, and EF-Tu were identified by sequence alignments; the respective recombinant proteins were prepared and studied in a variety of biochemical and biophysical assays with M. smegmatis ribosomes. We found that the activities of initiation and elongation factors and the rates of elemental reactions of initiation and elongation of protein synthesis are remarkably similar with M. smegmatis and E. coli components. The data suggest a very high degree of conservation of basic translation mechanisms, probably due to coevolution of the ribosome components and translation factors. This work establishes the reconstituted translation system from individual purified M. smegmatis components as an alternative to that from E. coli to study the mechanisms of translation and to test the action of antibiotics against Gram-positive bacteria.     

Genetic footprinting in bacteria

In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage lambda transposon delivery system.     

New thermosensitive plasmid for gram-positive bacteria.

We isolated a replication-thermosensitive mutant of the broad-host-range replicon pWV01. The mutant pVE6002 is fully thermosensitive above 35 degrees C in both gram-negative and gram-positive bacteria. Four clustered mutations were identified in the gene encoding the replication protein of pVE6002. The thermosensitive derivative of the related plasmid pE194 carries a mutation in the analogous region but not in the same position. Derivatives of the thermosensitive plasmid convenient for cloning purposes have been constructed. The low shut-off temperature of pVE6002 makes it a useful suicide vector for bacteria which are limited in their own temperature growth range. Using pVE6002 as the delivery vector for a transposon Tn10 derivative in Bacillus subtilis, we observed transposition frequencies of about 1%.     

A new theta-type thermosensitive replicon from Lactoccocus lactis as an integration vector for Enterococcus faecalis

We isolated a replication thermosensitive mutant of the theta-type lactococcal pUCL22 replicon. An improved version of this thermosensitive replicon was obtained by fusioning the replication repA gene with the downstream repB gene. The resulting plasmid was named pUCB3522Ts. It is highly instable at 42°C in Enterococcus faecalis. Integration into the chromosome via homologous recombination was monitored using the npr gene of E. faecalis JH2-2 as a target. A 513 bp PCR amplification product from an internal region of this npr gene was cloned into pUCB3522Ts. Integration of this construction into the JH2-2 npr gene was selected by shift temperature, from 30°C to 42°C. 85% of the analysed clones showed integration into the npr gene, demonstrating the practicality of this thermosensitive replicon as a genetic integrative tool for E. faecalis.     

Transformation of Mycobacterium smegmatis with Escherichia coii plasmids carrying a selectable resistance marker

One limiting factor in studies of tuberculosis and leprosy is the difficulty of genetic analysis and manipulation of mycobacteria. Two approaches were adopted for the construction of vectors, based on different Escherichia coli plasmids and using Mycobacterium smegmatis as the host. In both cases we found that the original E. coli plasmid is capable of being replicated in M. smegmatis, yielding chloramphenicol-resistant colonies. One such plasmid has been recovered from a M. smegmatis transformant and used to re-transform both M. smegmatis and E. coli to chloramphenicol resistance. This plasmid is indistinguishable from the original plasmid by restriction analysis, and can be used as a shuttle vector for the genetic manipulation of mycobacterial species.     

Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum.

In a two-step mating experiment with recipient strains of Mycobacterium smegmatis, the Mycobacterium fortuitum cryptic plasmid pJAZ38 was isolated. Plasmid pJAZ38 was genetically labeled by cointegration formation mediated by the kanamycin-resistant mycobacterial transposon Tn611. The region responsible for replication of pJAZ38 was located and sequenced. This region showed homology with the Mycobacterium avium plasmid pLR7 and the Mycobacterium scrofulaceum plasmid pMSC262, a family of plasmids which have been found to be widespread throughout the mycobacteria. Further experiments showed pJAZ38 to be stably inherited in the absence of selection pressure and compatible with the most commonly used mycobacterial replicon, pAL5000. In contrast to pLR7 and pMSC262, pJAZ38 was able to replicate in M. smegmatis mc(2)155, making it a useful tool for mycobacterial genetics.     

Saturation mutagenesis in Escherichia coli of a cloned Xanthomonas campestris DNA fragment with the lux transposon Tn4431 using the delivery plasmid pDS1, thermosensitive in replication

A system allowing transposon mutagenesis of cloned DNA fragments in Escherichia coli with Tn4431, which carries the promotorless luciferase (lux) operon of Vibrio fischeri, has been developed. The transposon delivery plasmid, pDS1, based on an IncF replicon, is thermosensitive in replication and mobilizable to many Gram-negative bacteria. We used pDS1 for Tn4431-saturation mutagenesis of a 10-kb DNA fragment of Xanthomonas campestris pv. campestris (X.c.c.) in E. coli and showed that the expression of the lux operon was dependent on orientation and location of the transposon. Transfer of a specific Tn4431 insertion to X.c.c. allowed the determination of the bioluminescence phenotype in planta. Correspondence to: U. B. Priefer     

Integration of temperature-sensitive nonconjugative plasmid carrying kanamycin gene into conjugative R plasmids.

A nonconjugative R plasmid, rMS3, whose molecular weight was 2.4 X 10(7) daltons, possessed a kanamycin resistance gene and was thermosensitive in its maintenance in Escherichia coli strains. We mobilized rMS3 with a conjugative R plasmid, R100 or T-tet, and obtained cointegrates carrying all the parental resistance markers. Various markers of the cointegrates were frequently deleted by P1 transduction and the deletion patterns among the different cointegrates were differed from each other. The cointegrates were thermoresistant, but the thermosensitive replicon could be segregated from the thermoresistant cointegrate by deletion. Some cointegrates between rMS3 and T-tet showed a derepressed state of transferability because of the integration of rMS3 and T-tet showed a derepressed state of transferability because of the integration of rMS3 into the regulator gene of the transfer loci. The genome size of the cointegrate so far tested was the sum of the sizes of the parental plasmids, indicating that the whole genome of rMS3 could integrate into various sites of the conjugative plasmids R100 and T-tet.     

Cloning and nucleotide sequence of luxR, a regulatory gene controlling bioluminescence in Vibrio harveyi.

Mutagenesis with transposon mini-Mulac was used previously to identify a regulatory locus necessary for expression of bioluminescence genes, lux, in Vibrio harveyi (M. Martin, R. Showalter, and M. Silverman, J. Bacteriol. 171:2406-2414, 1989). Mutants with transposon insertions in this regulatory locus were used to construct a hybridization probe which was used in this study to detect recombinants in a cosmid library containing the homologous DNA. Recombinant cosmids with this DNA stimulated expression of the genes encoding enzymes for luminescence, i.e., the luxCDABE operon, which were positioned in trans on a compatible replicon in Escherichia coli. Transposon mutagenesis and analysis of the DNA sequence of the cloned DNA indicated that regulatory function resided in a single gene of about 0.6-kilobases named luxR. Expression of bioluminescence in V. harveyi and in the fish light-organ symbiont Vibrio fischeri is controlled by density-sensing mechanisms involving the accumulation of small signal molecules called autoinducers, but similarity of the two luminescence systems at the molecular level was not apparent in this study. The amino acid sequence of the LuxR product of V. harveyi, which indicates a structural relationship to some DNA-binding proteins, is not similar to the sequence of the protein that regulates expression of luminescence in V. fischeri. In addition, reconstitution of autoinducer-controlled luminescence in recombinant E. coli, already achieved with lux genes cloned from V. fischeri, was not accomplished with the isolation of luxR from V. harveyi, suggesting a requirement for an additional regulatory component.     

Identification of clones carrying an E. coli tRNAPhe gene by suppression of phenylalanyl-tRNA synthetase thermosensitive mutants

Two libraries of cloned E. coli DNA were screened for plasmids which complemented thermosensitive phenylalanyl-tRNA synthetase mutants. Four plasmids were isolated which complemented pheS and pheT thermosensitive mutations but which do not carry pheS or pheT, the structural genes for phenylalanyl-tRNA synthetase. All these plasmids increased the intracellular tRNAPhe concentration. Three plasmids were shown to carry the structural gene for tRNAPhe which we call pheU. By restriction enzyme analysis, DNA blotting and DNA:tRNA hybridization, pheU was localised to a 280 bp fragment within a 5.6 kb PstI restriction fragment of E.coli DNA.     

Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes

Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn 5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1 D-myo-inosityl 2- L-cysteinylamido-2-deoxy-alpha-D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene.     

Two-step method for curing Escherichia coli of ColE1-derived plasmids

To cure Escherichia coli for plasmids derived from the ColE1 replicon advantage is taken of the fact that maintenance of this replicon requires a wild-type allele of polA, encoding DNA polymerase I. Curing is achieved by cotransduction of a mutant polA allele with metE::Tn10, fadAB::Tn10 or other transposon insertions near polA. Reciprocal transduction to Met(+) Pol(+) or to Fad(+) Pol(+) ensures reestablishment of the original genotype except for loss of the plasmid. A set of useful bacterial strains is provided.