Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens

Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.     

The Transposon-Like Structure of IS26-Tetracycllne,and Kanamycin Resistance Determinant Derived from Transferable R Plasmid of Fish Pathogen,Pasteurella piscicida

Tetracycline (pp-tet), and kanamycin (pp-kan) resistance genes were cloned from a transferable R plasmid of fish pathogen Pasteurella piscicida, and complete nucleotide sequences were determined. The pp-tet was a class D Tet determinant constructed with the tetA resistance gene of 1,182 bp encoding a protein with a deduced molecular mass of 41 kDa and the tetR repressor gene of 654 bp encoding a product of 24 kDa. The pp-tet was highly homologous to the tet(D) of plasmid RA1 isolated from Aeromonas hydrophila with two nucleotide differences in the tetR, and of plasmid pIP173 from Salmonella ordonez with two nucleotide differences in the tetA. The pp-kan contained 813 bp encoding a 31 kDa protein of 271 amino acids, and was classified into type aph-Ic. It was identical to the aphA7 in the IAB operon of pBWH77, in which was originally found an isolate of Klebsiella pneumoniae, in its nucleotide sequences and hybrid promoter construction. The genes were connected by an insertion sequence IS 26 of 820 bp, and were flanked by repeated copies in direct orientation at the 3′ flanking region of the pp-tetA and in inverted orientation at the 3′ flanking region of the pp-kan. The genetic elements are organized like a complex transposon by close linkage of the IS 26 and the pp-tet and -kan.     

Development of a Tetracycline-Inducible Gene Expression System for the Study of Helicobacter pylori Pathogenesis

Deletion mutants and animal models have been instrumental in the study of Helicobacter pylori pathogenesis. Conditional mutants, however, would enable the study of the temporal gene requirement during H. pylori colonization and chronic infection. To achieve this goal, we adapted the Escherichia coli Tn10-derived tetracycline-inducible expression system for use in H. pylori. The ureA promoter was modified by inserting one or two tet operators to generate tetracycline-responsive promoters, named uPtetO, and these promoters were then fused to the reporter gfpmut2 and inserted into different loci. The expression of the tetracycline repressor (tetR) was placed under the control of one of three promoters and inserted into the chromosome. Conditional expression of green fluorescent protein (GFP) in strains harboring tetR and uPtetO-GFP was characterized by measuring GFP activity and by immunoblotting. The two tet-responsive uPtetO promoters differ in strength, and induction of these promoters was inducer concentration and time dependent, with maximum expression achieved after induction for 8 to 16 h. Furthermore, the chromosomal location of the uPtetO-GFP construct and the nature of the promoter driving expression of tetR influenced the strength of the uPtetO promoters upon induction. Integration of uPtetO-GFP and tetR constructs at different genomic loci was stable in vivo and did not affect colonization. Finally, we demonstrate tetracycline-dependent induction of GFP expression in vivo during chronic infection. These results open new experimental avenues for dissecting H. pylori pathogenesis using animal models and for testing the roles of specific genes in colonization of, adaptation to, and persistence in the host.     

Nucleotide sequence of class D tetracycline resistance genes from Salmonella ordonez

Summary Plasmid pIP173, isolated from Salmonella ordonez strain BM2000, confers resistance to tetracycline and a number of other antibiotics. We determined the nucleotide sequence of the pIP173 tetR repressor and tetA resistance genes. The pIP173 tetR gene is essentially identical to the class D tetR gene from plasmid RA1. The pIP173 tet genes are flanked by directly repeated copies of the insertion sequence IS26. Interestingly, the 3 end of the tetR gene, encoding the C-terminal 16 amino acids of the TetR protein, extends into the flanking IS26 sequence. The relationships between the class A, B, C, and D TetA sequences parallel the relationships between the corresponding TetR sequences; class D is more closely related to class B than to either class A or C. Overall, the four TetA sequences show 38% identity and 57% similarity.     

Nucleotide sequence homology of the tetracycline-resistance determinant naturally maintained in Bacillus subtilis Marburg 168 chromosome and the tetracycline-resistance gene of B. subtilis plasmid pNS1981

The nucleotide sequence (1579 bp) of tetracycline-resistance determinant and flanking regions of the cloned 5.1 kb DNA fragment from Bacillus subtilis GSY908 chromosome (Sakaguchi, R. and Shishido, K. (1988) Biochim. Biophys. Acta 949, 49–57) were determined and compared with those of the B. subtilis tetracycline-resistance plasmid pNS1981. The tetracycline-resistance structural (tet) genes of the B. subtilis GSY908 chromosome (tet BS908) and pNS1981 (tet pNS1981) were found to be highly homologous (80% identical). Both tet genes were composed of 1374 bp and 458 amino-acid residues initiating from a GTG codon preceded by a ribosome-binding site (RBS-2). Upstream from tet BS908 there exists a short open reading frame (20 amino acids) initiating from a ATG codon preceded by its own RBS (RBS-1). This leader sequence was also highly homologous to that of tet pNS1981 except for a deletion of one bp between the RBS-1 and the ATG codon.     

Analysis of the reduction in expression of tetracycline resistance determined by transposon Tn10 in the multicopy state

The tet genes of transposon Tn10 have been mapped in a 2,200 bp DNA sequence by analysing deletion and Tn5 insertion mutations. When the tet genes were present on multi-copy plasmids the level of resistance expressed was about ten-fold lower than that determined by a single copy of Tn10 in the E. coli chromosome. The 36K tet protein known to be encoded by R100 in E. coli minicells was not detected when they harboured a multicopy tet plasmid. However, normal high levels of resistance were expressed when the tet genes were recombined into the host chromosome as part of a lambda lysogen, showing that the multicopy effect was phenotypic. Most of the Tn5 insertions and deletions in tet which caused Tc^s mutations also prevented expression of high level Tc^r from a chromosomal Tn10 element present in the same cell. Only those insertions in the promoter-proximal 90–130 bp of a 1,275 bp HindII fragment known to carry the gene encoding the 36K tet protein did not reduce the single copy Tn10 resistance level.A gene fusion system that results in the constitutive synthesis of -galactosidase from a tet promoter has been used to assay tet repressor activity. The basal (uninduced) -galactosidase level in cells carrying multicopy tet plasmids was 10–20 fold lower than those carrying a single copy. The tet:: Tn5 mutants defective in the trans-dominant multicopy effect still made normal amounts of tet repressor showing that repressor overproduction was not responsible for this effect. In addition a repressor-defective constitutive mutant did not exhibit a higher resistance level when located on a multicopy plasmid vector. We postulate that a regulatory mechanism recognises the amino-terminus of the tet structural gene product when attempts are being made to overproduce the protein and prevents further translation.     

The application of Tet repressor in prokaryotic gene regulation and expression

Inducible gene expression based upon Tet repressor (tet regulation) is a broadly applied tool in molecular genetics. In its original environment, Tet repressor (TetR) negatively controls tetracycline (tc) resistance in bacteria. In the presence of tc, TetR is induced and detaches from its cognate DNA sequence tetO, so that a tc antiporter protein is expressed. In this article, we provide a comprehensive overview about tet regulation in bacteria and illustrate the parameters of different regulatory architectures. While some of these set‐ups rely on natural tet‐control regions like those found on transposon Tn10, highly efficient variations of this system have recently been adapted to different Gram‐negative and Gram‐positive bacteria. Novel tet‐controllable artificial or hybrid promoters were employed for target gene expression. They are controlled by regulators expressed at different levels either in a constitutive or in an autoregulated manner. The resulting tet systems have been used for various purposes. We discuss integrative elements vested with tc‐sensitive promoters, as well as tet regulation in Gram‐negative and Gram‐positive bacteria for analytical purposes and for protein overproduction. Also the use of TetR as an in vivo biosensor for tetracyclines or as a regulatory device in synthetic biology constructs is outlined. Technical specifications underlying different regulatory set‐ups are highlighted, and finally recent developments concerning variations of TetR are presented, which may expand the use of prokaryotic tet systems in the future.     

The inactivation of tet genes on a plasmid by the duplication of one inverted repeat of a transposon-like structure which itself mediates tetracycline resistance

Derivatives of a naturally occurring IncFII tetracycline resistance plasmid (pUB889) which are unable to confer resistance to tetracycline, and which were isolated from both partners of a married couple, have been examined. The tet genes are part of a transposon-like structure which is closely related to, but distinct from, Tn10. The lesions in the two plasmids examined are identical and involve the insertion of a nucleotide sequence of 0.9 × 10⁶ within the region encoding tetracycline resistance, expression of which is lost as a consequence. The extra DNA is homologous with the nucleotide sequence, a pair of which form the inverted repeats of the Tn10-like structure. We conclude that this nucleotide sequence comprises an IS element. The epidemiological implications arising from the origin of these plasmids are discussed.     

Regulated expression of heterologous genes inBacillus subtilis using the Tn10 encodedtet regulatory elements

Summary TheEscherichia coli-derivedtet regulatory elements from Tn10 have been used to construct vectors allowing the regulated, inducible, high-level expression of foreign genes inBacillus subtilis. While the wild-typetet promoters are inactive inB. subtilis, a synthetic mutanttet sequence with improved promoter consensus sequences and upstream poly A blocks shows activity inB. subtilis. The expression of an indicatorcat gene is inducible by sublethal amounts of tetracycline, indicating that the Tet repressor protein and thetet operator sequences are functional. However, the inducibility and maximal expression are not sufficient in this construct. To improve these properties atet operator sequence was placed between the —35 and —10 boxes of theB. subtilis-derived very strongxyl promoter. In the presence of atetR gene this construct is about 100-fold inducible and has high promoter strength, but some basal expression. This is avoided by placing a secondtet operator downstream resulting in no detectable basal expression at the expense of reduced inducibility. Using the system with a singletet operator inducible expression of glucose dehydrogenase fromB. megaterium was obtained at a very high level, and inducible expression of human single-chain urokinase-like plasminogen activator was achieved at the same level as inE. coli. Unlike inE. coli, the product was not degraded up to 4 h after induction inB. subtilis. These results demonstrate that the regulated expression vector described here should be very useful for production of foreign gene products fromB. subtilis cultures.     

A Potential New Gene (tccA) on IncP Plasmid RK2 and Transposon Tn1721:Relationship of Its Product to the TrwC Relaxase/Helicase of IncW Plasmid R388

Computational analysis of the fully sequenced 60-kb genome of broad-host-range IncPα plasmid RK2 revealed a previously unreported potential protein-coding sequence, an 80-codon open reading frame (tccA), located in the region between the vegetative origin of replication (oriV) and thetetRgene of the tetracycline resistance determinant. The coding region is also present in the transposon Tn1721 tetregion, which is nearly identical to thetetregion of RK2. Remarkably, the predicted polypeptide product of the coding region displays 56% identity and 72% similarity with the C-terminal domain of the TrwC relaxase/helicase protein of IncW plasmid R388.     

Phenotypic and genotypic characterization of tetracycline and minocycline resistance in Clostridium perfringens

The aim of this study was to determine the incidence of tetracycline resistance and the prevalence of tetracycline-resistance genes in strains of Clostridium perfringens isolated from different sources between 1994 and 2005. Susceptibility to tetracycline and minocycline in strains from humans (35 isolates), chickens (15 isolates), food (21 isolates), soil (16 isolates) and veterinary sources (6 isolates) was determined, and tetracycline-resistance genes were detected. Resistance was most common in strains isolated from chickens, followed by those from soils, clinical samples and foods. The most highly resistant strains were found among clinical and food isolates. tetA(P) was the most common resistance gene, and along with tetB(P) was found in all resistant strains and some sensitive strains. One tetracycline-resistant food isolate had an intact tet(M) gene. However, PCR fragments of 0.4 or 0.8 kb with high degrees of identity to parts of the tet(M) sequences of other bacteria were found, mainly in clinical isolates, and often in isolates with tetB(P). No correlation between level of sensitivity to tetracycline or minocycline and the presence of tetA(P), tetB(P) or part of tet(M) was found. The presence of part of tet(M) in some strains of C. perfringens containing tetB(P) may have occurred by recent gene transfer.     

Molecular Characterization of tet(M) Genes in Lactobacillus Isolates from Different Types of Fermented Dry Sausage

The likelihood that products prepared from raw meat and milk may act as vehicles for antibiotic-resistant bacteria is currently of great concern in food safety issues. In this study, a collection of 94 tetracycline-resistant (Tc^r) lactic acid bacteria recovered from nine different fermented dry sausage types were subjected to a polyphasic molecular study with the aim of characterizing the host organisms and the tet genes, conferring tetracycline resistance, that they carry. With the (GTG)₅-PCR DNA fingerprinting technique, the Tc^r lactic acid bacterial isolates were identified as Lactobacillus plantarum, L. sakei subsp. carnosus, L. sakei subsp. sakei, L. curvatus, and L. alimentarius and typed to the intraspecies level. For a selection of 24 Tc^r lactic acid bacterial isolates displaying unique (GTG)₅-PCR fingerprints, tet genes were determined by means of PCR, and only tet(M) was detected. Restriction enzyme analysis with AccI and ScaI revealed two different tet(M) allele types. This grouping was confirmed by partial sequencing of the tet(M) open reading frame, which indicated that the two allele types displayed high sequence similarities (>99.6%) with tet(M) genes previously reported in Staphylococcus aureus MRSA 101 and in Neisseria meningitidis, respectively. Southern hybridization with plasmid profiles revealed that the isolates contained tet(M)-carrying plasmids. In addition to the tet(M) gene, one isolate also contained an erm(B) gene on a different plasmid from the one encoding the tetracycline resistance. Furthermore, it was also shown by PCR that the tet(M) genes were not located on transposons of the Tn916/Tn1545 family. To our knowledge, this is the first detailed molecular study demonstrating that taxonomically and genotypically diverse Lactobacillus strains from different types of fermented meat products can be a host for plasmid-borne tet genes.     

A complete sequence of the pGA1611 binary vector

We report the nucleotide sequence of the binary vector pGA1611, which is used for the transformation of foreign DNA into rice. This vector is 13,476 bp long. The 5577- bp T- DNA region consists of a 1987- bp ubiquitine promoter region, 45 bp for the multiple cloning site, a 253- bpnos terminator region, and the 2045- bpCaMV35S- hph- T7 chimaeric gene. The vector backbone (7004 bp) carriesoriT,traJ,trfA,tetA,tetR, andoriV. An 892- bp RB region and the 489- bp LB region are also present The T- DNA possesses 15 unique sites, six of which are at the multiple cloning site. This information will be valuable for cloning foreign DNA and modifying the vector.     

Specificity of Transposon Tn5 Insertion

Genetic mapping studies had shown that the bacterial transposon Tn5 can insert into many sites in a gene, but that some sites are preferred. To begin understanding Tn5's insertion specificity at the molecular level, we selected transpositions of Tn5 from the Escherichia coli chromosome to the plasmid pBR322 and analyzed the resultant pBR322::Tn5 plasmids by restriction endonuclease digestion and DNA sequencing. Seventy-five insertions in the tet gene were found at 28 sites including one major hotspot (with 21 insertions) and four lesser hotspots (with four to ten insertions each). All five hotspots are within the first 300 of the 1250-base pair (bp) tet gene. In contrast, 31 independent insertions in the amp gene were found in at least 27 distinct sites.—Tn5 generates 9 bp target sequence duplications when it transposes. Such transposon-induced duplications are generally taken to indicate that cleavages of complementary target DNA strands are made 9 bp apart during transposition. DNA sequence analysis indicated that GC base pairs occupy positions 1 and 9 in the duplications at each of the five hotspots examined, suggesting a GC-cutting preference during Tn5 transposition.     

Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing

A series of promoter hybrids has been constructed by exchanging the ? 35 and ? 10 regions of lacUV5, tet, and trp promoters. These three promoters and the six hybrid promoters constructed from them have been inserted into a pKO plasmid which places galactokinase expression under the control of the inserted promoter. Additionally, promoter mutants were prepared which had altered the spacing between the ? 35 and ? 10 regions of the promoter. Derivatives of the tet promoter with one or two extra base pairs in this spacer region and constructions of the lac:: tet hybrid promoter with two different spacings have been inserted into the galactokinase expression plasmid. Measurements of galactokinase levels in strains harboring these plasmids permited the comparison of in vivo activities of the promoters. The strongest of the hybrid promoters (order: ? 35, ? 10) were trp:: lac and trp:: tet suggesting a high efficiency for the ? 35 region of the trp promoter. The weakest promoters were tet:: trp, lac:: trp and lac::tet indicating a weak ? 10 region for the trp promoter and the importance of ? 35 to ? 10 spacing. Analysis of activity of related promoters with differences in spacing indicated that a distance of 19 bp yields a very weak promoter, and that 18 bp is less active than the 17-bp spacing, which is the most frequently found spacing in promoters.     

Tetracycline-Dependent Conditional Gene Knockout in Bacillus subtilis

Reversible tetracycline-dependent gene regulation allows induction of expression with the tetracycline repressor (TetR) or gene silencing with the newly developed reverse mutant revTetR. We report here the implementation of both approaches with full regulatory range in gram-positive bacteria as exemplified in Bacillus subtilis. A chromosomally located gene is controlled by one or two tet operators. The precise adjustment of regulatory windows is accomplished by adjusting tetR or revtetR expression via different promoters. The most efficient induction was 300-fold in the presence of 0.4 μM anhydrotetracycline obtained with a Pr-xylA-tetR fusion. Reversible 500-fold gene knockouts were obtained in B. subtilis after adjusting expression of revTetR by synthetically designed promoters. We anticipate that these tools will also be useful in many other gram-positive bacteria.