Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9.

In vitro recombination via restriction endonucleases and the in vivo genetic translocation of the Ap resistance (Apr) gene resulted in the construction of a new cloning vehicle, the plasmid pBR313. This vector was derived from a ColE1-like plasmid and, while it does not produce colicon E1, it still retains colicin E1 immunity. The Apr and tetracycline resistance (Tcr) markers carried in pBR313 were derived from the ampicillin transposon (TnA) of pRSF2124 and pSC101 respectively. During the construction of pBR313, the TnA component was altered and the Apr gene in pBR313 can no longer be translocated. This plasmid has a molecular weight of 5.8 Mdalton and has been characterized using thirteen restriction enzymes, six of which (EcoRI, SmaI, HpaI, HindIII, BamHI and SalI) cleave the plasmid at unique restriction sites. This allows the molecular cloning of DNA fragments generated by these six enzymes. The restriction sites for the latter three enzymes, HindIII, BamHI and SalI, are located in the Tcr gene(s). Cloning DNA fragments into these sites alters the expression of the Tcr mechanisms thus providing a selection for cells carrying recombinant plasmid molecules. An enrichment method for AprTcS cells carrying recombinant plasmid molecules is described.     

Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique Eco RI sites for selection of Eco RI generated recombinant DNA molecules.

In vitro recombinant DNA techniques were used to construct two new cloning vehicles, pBR324 and pBR235. These vectors, derived from plasmid pBR322, are relaxed replicating elements. Plasmid pBR324 carries the genes from pBR322 coding for resistance to the antibiotics ampicillin (Apr) and tetracycline (Tcr) and the colicin E1 structural and immunity genes derived from plasmid pMBI. Plasmid pBR325 carries the Apr and Tcr genes from pBR322 and the cloramphenicol resistance gene (Cmr) from phage P1Cm. In these plasmids the unique EcoRI restriction site present in the DNA molecule is located either in the colicin E1 structural gene (pBR324) or in the Cmr gene (pBR325). These vectors were constructed in order to have a single EcoRI site located in the middle of a structural gene which when inactivated would allow, for the easy selection of plasmid recombinant DNA molecules. These plasmids permit the molecular cloning and easy selection of EcoRI, BamHI, HindIII, PstI, HincII, SalI, (XamI), Smal, (XmaI), BglII and DpnII restriction generated DNA molecules.     

Characterization of a new type of Bacteroides conjugative transposon, Tcr Emr 7853.

Results of previous investigations suggested that the conjugative transposons found in human colonic Bacteroides species were all members of a closely related family of elements, exemplified by Tcr Emr DOT. We have now found a new type of conjugative transposon, Tcr Emr 7853, that does not belong to this family. Tcr Emr 7853 has approximately the same size as the Tcr Emr DOT-type elements (70 to 80 kbp) and also carries genes encoding resistance to tetracycline (Tcr) and erythromycin (Emr); however, it differs from previously described conjugative transposons in a number of ways. Its transfer is not regulated by tetracycline and its transfer genes are not controlled by the regulatory genes rteA and rteB, which are found on Tcr Emr DOT and related conjugative transposons. Its ends do not cross-hybridize with the ends of Tcr Emr DOT-type conjugative transposons, and the Emr gene it carries does not cross-hybridize with ermF, the Emr gene found on all previously studied Bacteroides conjugative transposons. There is only one region with high sequence similarity between Tcr Emr 7853 and previously characterized elements, the region that contains the Tcr gene, tetQ. This sequence similarity ends 145 bp upstream of the start codon and 288 bp downstream from the stop codon. A 2-kbp region upstream of tetQ on Tcr Emr 7853 cross-hybridized with four additional EcoRV fragments of Bacteroides thetaiotaomicron 7853 DNA other than the one that contained tetQ. These additional cross-hybridizing bands were not part of Tcr Emr 7853, but one of them cotransferred with Tcr Emr 7853 in some matings. Thus, at least one of the additional cross-hybridizing bands may be associated with another conjugative element or with an element that is mobilized by Tcr Emr 7853. DNA that cross-hybridized with the upstream region was found in one clinical isolate of Bacteroides ovatus and four Tcr isolates of Prevotella ruminicola.     

Genetic analysis of a tetracycline resistance element from Clostridium difficile and its conjugal transfer to and from Bacillus subtilis

A tetracycline resistance (Tcr) determinant from Clostridium difficile strain 630 was cloned into the Escherichia coli plasmid vector pUC13. The resulting plasmid pPPM20, containing an insert of 3.4 kbp, was mapped and a 1.1 kbp SacI-HindIII fragment wholly within the Tcr gene was identified. Dot-blot hybridization studies with the 1.1 kbp fragment showed that the Tcr gene belonged to hybridization class M. Tcr could be transferred between C. difficile strains and to Bacillus subtilis at a frequency of 10(-7) per donor cell. The element could be returned from B. subtilis to C. difficile at a frequency of 10(-8) per donor cell. This is the first demonstration of C. difficile acting as a recipient in intergeneric crosses. DNA from C. difficile transconjugants digested with EcoRV always has two hybridizing fragments of 9.5 and 11.0 kbp when probed with pPPM20. DNA from B. subtilis transconjugants digested with EcoRV produced one hybridizing band of variable size when probed with pPPM20. The behaviour of the element was reminiscent of the conjugative transposons. Therefore we compared the element to the conjugative transposon Tn916. The HincII restriction maps of the two elements differed and no hybridization was detected to oligonucleotides directed to the ends of Tn916. However, the elements do have some sequence homology, detected by hybridization analysis.     

Insertion and excision of Bacteroides conjugative chromosomal elements.

Many strains of Bacteroides harbor large chromosomal elements that can transfer themselves from the chromosome of the donor to the chromosome of the recipient. Most of them carry a tetracycline resistance (Tcr) gene and have thus been designated Tcr elements. In the present study, we have used transverse alternating field electrophoresis to show that all but one of the Tcr elements screened were approximately 70 to 80 kbp in size. The exception (Tcr Emr 12256) was 150 to 200 kbp in size and may be a hybrid element. All of the Tcr elements inserted in more than one site, but insertion was not random. The Tcr elements sometimes cotransfer unlinked chromosomal segments, or nonreplicating Bacteroides units (NBUs). Transverse alternating field electrophoresis analysis showed that insertion of NBUs was not random and that the NBUs did not insert near the Tcr element. Although attempts to clone one or both ends of a Tcr element have not been successful, ends of a cryptic element (XBU4422) were cloned previously and shown to be homologous to the ends of Tcr elements. We have obtained DNA sequences of junction regions between XBU4422 and its target from several different insertions. Comparison of junction sequences with target sequences showed that no target site duplication occurred during insertion and that XBU4422 carried 4 to 5 bp of adjacent chromosomal DNA when it excised from the chromosome and inserted in a plasmid. We identified a short region of sequence similarity between one of the ends of XBU4422 and its target site that may be important for insertion. This sequence contained an 8-bp segment that was identical to the recombinational hot spot sequence on Tn21. XBU4422 could exise itself from plasmids into which it inserted. In most cases, the excision left a single additional A behind in the target site, but precise excision was seen in one case.     

Cloning, expression in Escherichia coli and nucleotide sequence of a tetracycline-resistance gene from Streptomyces rimosus

Determinants of tetracycline resistance have been cloned from two different tetracycline-producing industrial strains of Streptomyces into Streptomyces lividans using the plasmid vector pUT206. Three plasmids, pUT250 and pUT260 with a 9.5 and a 7.5 kb insert respectively of Streptomyces rimosus DNA, and pUT270 with a 14.0 kb insert of Streptomyces aureofaciens DNA, conferring resistance to tetracycline, have been isolated. By in vitro sub-cloning, a similar fragment of 2.45 kb containing the tetracycline resistance gene (tet347) was further localized on these plasmids. The S. rimosus gene has been cloned into Escherichia coli and expressed under the control of lambda pL or Lpp promoters. Differential protein extraction of E. coli cells revealed the presence of an additional membrane-embedded protein in tetracycline-resistant cells. On the basis of available restriction endonuclease maps, the tet347 gene is probably identical to the tetB gene from S. rimosus recently identified by T. Ohnuki and co-workers as responsible for the reduced accumulation of tetracycline. The nucleotide sequence of a 2052 bp DNA fragment containing the TcR structural gene from S. rimosus has been determined. The amino acid sequence of the tet347 protein (Mr35818) deduced from the nucleotide sequence shows a limited but significant homology to other characterized tetracycline transport acting determinants from pathogenic bacteria.     

A site-specific DNA inversion in Bacteroides plasmid pBF4 is influenced by the presence of the conjugal tetracycline resistance element.

pBF4 is a 42-kb R plasmid from Bacteroides fragilis which transfers clindamycin resistance (Clr) independently of the chromosomal tetracycline resistance (Tcr) transfer element. We have found that this plasmid exists in two nonequimolar conformations, A and B. These forms differ by an inversion of approximately 11.5 kb which does not involve the repeated DNA sequences previously mapped on the plasmid. The presence of chromosomal tetracycline resistance conjugal elements influences the relative amounts of the two conformations: induction with tetracycline shifts the dominant form from B to A.     

Nucleotide sequence analysis of tetracycline resistance gene tetO from Streptococcus mutans DL5.

Streptococcus mutans DL5, isolated from the dental plaque of a pig, was resistant to high levels of streptomycin (Sm, 20 mg/ml), erythromycin (Em, 1 mg/ml), and tetracycline (Tc, greater than 100 micrograms/ml), but contained no detectable plasmid DNA. The Smr and Emr determinants were cloned from cellular DNA on the self-replicating 5-kilobase-pair (kbp) EcoRI fragment of pAM beta 1 and the 4.2-kbp cryptic plasmid pVA380-1, respectively, by transformation of Streptococcus sanguis Challis. Helper plasmid cloning, with a Challis host containing pVA380-1, was required to clone the Tcr determinant of strain DL5 on this vector. A single-colony isolate of the original Tcr clone contained a hybrid plasmid, pDL421, composed of 2.6 kbp of vector DNA and 11.4 kbp of S. mutans DNA. Plasmid pDL421 did not hybridize to plasmids containing the streptococcal Tcr determinants tetL, tetM, and tetN. A shortened derivative of this hybrid plasmid, pDL422, missing a 4.9-kbp HincII fragment from the S. mutans DNA but still encoding Tcr, was obtained by subcloning in S. sanguis Challis. The Tcr gene was located in a 1,917-base-pair open reading frame (ORF) corresponding to a 72-kilodalton protein. The ORF exhibited 99.4% sequence identity with the 1,917-base-pair tetO gene from a strain of Campylobacter coli (W. Sougakoff, B. Papadopoulou, P. Nordmann, and P. Courvalin, FEMS Microbiol. Lett. 44:153-160, 1987). A 1.67-kbp NdeI fragment, internal to the ORF from strain DL5, as well as pDL421 hybridized under stringent conditions to DNA from 10 of 10 Tcr strains of C. coli and Campylobacter jejuni from human and animal sources, but not to DNA from Tcs isolates of these two species.     

端粒、端粒酶结合因子hPinx1基因启动子的克隆及活性分析

目的：克隆端粒、端粒酶结合因子hPinx1基因的启动子,分析并鉴定其活性调控元件。方法：采用PCR技术从人肝癌细胞系HepG2基因组中扩增出hPinx1启动子,构建到萤光素酶报告基因载体pGL3-basic中,确定所扩增的DNA序列,在HepG2细胞中检测其活性。结果：克隆了hPinx1基因转录起始位点上游4661bp且序列正确;活性分析表明hPinx1启动子含有多个调控元件,其中核心序列位于530bp内,在1329-2174bp间存在正调控序列,在2174-4661 bp间存在负调控序列。结论：构建的hPinx1启动子具有活性,为hPinx1的功能研究提供了重要基础。     

Tetracycline resistance transposon Tn1721: recA-dependent gene amplification and expression of tetracycline resistance

The 7.1-megadalton transposon Tn1721 codes for inducible tetracycline resistance (Tcr). The transposable element consists of a "minor transposon" (3.6 megadaltons) encoding functions required for transposition and a "tet region" (3.5 megadaltons) encoding resistance. Multiple tandem repeats of the tet region can be generated by recA-dependent gene amplification. This feature of Tn1721 has been used to analyze the relationship between gene dosage and Tcr. Derivatives of plasmid R388:Tn1721 containing from one to nine copies of the tet region were isolated and separately transformed into recA host cells, where they are stably maintained. The results of the study of Tcr in these strains were as follows: (i) the uninduced, "basal" level of Tcr was linearly related to gene dosage between 4 and 36 copies of tet per chromosome equivalent; (ii) the underlying mechanism could not be attributed to reduced accumulation of the drug; and (iii) induction with tetracycline elicited a four- to fivefold reduction in drug accumulation, independent of the gene dosage.     

Novel aerobic tetracycline resistance gene that chemically modifies tetracycline.

A tetracycline resistance gene that was found originally on the Bacteroides plasmid pBF4 confers resistance on Escherichia coli but only when cells are growing aerobically. When E. coli EM24 carrying this aerobic tetracycline resistance (*Tcr) gene is grown in medium containing tetracycline, the resulting spent medium is no longer toxic to tetracycline-sensitive (Tcs) E. coli EM24 (B.S. Speer and A.A. Salyers, J. Bacteriol. 170: 1423-1429, 1988). To determine whether the *Tcr gene product modified tetracycline, we characterized the material resulting from incubation of E. coli (*Tcr) with tetracycline. When [7-3H(N)]tetracycline was added to cultures of E. coli (*Tcr), at least 90% of the label was recovered in the extracellular fluid. Therefore, tetracycline was not being sequestered by the cells. The labeled material behaved similarly to tetracycline with respect to solubility in various organic solvents. However, the UV-visible light spectrum had a single peak at 258 nm, whereas the tetracycline spectrum had a peak at 364 nm. The labeled material also had a faster migration rate than did tetracycline on thin-layer plates in a solvent system of butanol-methanol-10% citric acid (4:1:2, vol/vol/vol) and was separable from tetracycline by reverse-phase high-pressure liquid chromatography, using an acetronitrile-0.1% trifluoroacetic acid solvent system. These results demonstrate that the *Tcr gene product chemically modifies tetracycline. The *Tcr gene is the first example of a chemically modifying tetracycline resistance mechanism.     

Characterization of genetic transformation in Streptococcus mutans by using a novel high-efficiency plasmid marker rescue system.

We developed a marker rescue system for study of competence development and genetic transformation in Streptococcus mutans. The system involved the recombinational rescue of a tetracycline resistance (Tcr) determinant by a homologous, inactive locus (Tcs because of a small deletion). Streptococcal cells harboring this in vitro-prepared Tcs construct (pVA1208) were restored to Tcr when plasmid (pVA981) DNA was used as donor material. pVA981 contained the intact streptococcal Tcr locus and was unable to autonomously replicate in streptococci. Marker rescue with this system followed first-order kinetics and occurred at a frequency 8- or 160-fold higher than did transformation with homologous chromosomal or plasmid DNA, respectively. By using the rescue system, we were able to confirm that competence of S. mutans appeared to be inducible. This was indicated by a sequential increase and then decrease in Tcr transformation frequencies during growth in complex medium. Also, donor DNA binding was not sequence specific, since the recovery of Tcr transformants was reduced by increasing the concentrations of heterologous DNA. We investigated the fate of donor DNA and the kinetics of plasmid establishment in the transformation of S. mutans with plasmid DNA. Monomeric plasmid molecules transformed S. mutans as a second-order process, whereas multimeric plasmid DNA and chromosomal markers were recovered as a first-order process. Approximately 50% of the initially bound donor plasmid DNA was found to remain in a trichloroacetic acid-insoluble form. Our results suggested that molecular cloning in S. mutans would be conducted most efficiently by using helper plasmid systems or shuttle vectors and that gene transfer by transformation of S. mutans occurred in a manner similar to that observed in Streptococcus sanguis.