Mutations in multicopy Tn10 tet plasmids that confer resistance to inhibitory effects of inducers of tet gene expression.

Escherichia coli K-12 strains that carry the Tn10 tetracycline resistance determinant (tet) on multicopy plasmids are hypersensitive to 5a,6-anhydrotetracycline and heated chlortetracycline, two tetracycline derivatives that are relatively more effective as inducers of tet gene expression than as inhibitors of bacterial growth. Twenty spontaneous mutations that confer resistance to anhydrotetracycline (Atr) and resistance to heated chlortetracycline (Ctr) were isolated and characterized. All of these Atr mutations are located in the Tn10 tet region; the majority (18 of 20) have no effect on tetR repressor function. Atr mutations can increase, reduce, or eliminate the phenotypic expression of plasmid tetracycline resistance (Tcr). IS insertions that result in an Atr Tcs phenotype are clustered in a 150-base-pair promoter-proximal region of the tetA resistance gene. Some Atr mutations reduce expression of the tetA gene by altering either the tetR repressor or the tetA promoter. In addition, it appears that E. coli cannot tolerate constitutive expression of the wild-type tetA gene from a multicopy plasmid containing a tetR deletion. These observations support the proposal that high level expression of the 36-kilodalton tetA gene product inhibits the growth of E. coli. We speculate that this inhibition is related to the interaction of the tetA gene product with the cytoplasmic membrane.     

Promoter mutations affecting divergent transcription in the Tn10 tetracycline resistance determinant

The tetracycline resistance determinant in transposon Tn10 consists of two genes, the tetA resistance gene and the tetR repressor gene, that are transcribed from divergent overlapping promoters. We determined the levels of pulse-labeled tet messenger RNA in Escherichia coli strains with the Tn10 tet genes on a multicopy plasmid. Addition of the inducer 5a,6-anhydrotetracycline results in a 270- to 430-fold increase in tetA mRNA and a 35- to 65-fold increase in tetR mRNA. As judged by the relative molar amounts of tetA and tetR mRNA synthesized under maximally inducing conditions, the tetA promoter (tetPA) is 7 to 11 times more active than the two tetR promoters (tetPR1 and tetPR2) combined. We characterized ten mutations in tetPA, including nine single-base-pair substitutions and a 30-base-pair deletion. All of the single-base-pair changes reduce the agreement with the consensus sequence for promoters recognized by E. coli RNA polymerase. Mutations in highly conserved nucleotides result in a 200- to 600-fold reduction in tetPA activity in vivo. Unexpectedly, tetPA mutations reduce by two- to fourfold the combined activity in vivo of tetPR1 and tetPR2, in spite of their locations outside the -35 and -10 regions of tetPR1 and tetPR2. For two tetPA mutations, the negative effect on tetPR activity was also demonstrated in tetR- tetPR-lacZ operon fusion strains, thus eliminating the possibility that it is due to variations in either plasmid copy-number or induction efficiency. The pleiotropic effects of tetPA mutations are discussed in terms of the expectation that the overlapping tet promoters compete for RNA polymerase.     

Plasmid vectors based on Tn10 DNA: gene expression regulated by tetracycline

The regulatory region of the tetracycline resistance determinant from transposon Tn10 has been used to construct plasmid vectors for gene expression regulated by tetracycline. Plasmids pRS tetBam-8 and pRS tetBam-16 include the tet regulatory region, the segment coding for the first four amino acids of the tetracycline resistance protein (tetA protein), and a linker region with SalI, HpaII, and BamHI restriction sites for gene fusions. Plasmid pTB-1, a derivative of pRS tetBam-8 and of the beta-galactosidase gene-containing plasmid pMC1403, constitutively expresses a tetA fragment-beta-galactosidase fusion protein. If a multicopy runaway replication plasmid, pMOBglII-16 that includes a 2.7-kb BglII DNA fragment from Tnl10 that provides tetR protein is present along with pTB-1, the expression of beta-galactosidase is reduced eightfold. Tetracycline acts as an inducer of the system and restores the level of beta-galactosidase activity measured in transformants containing pTB-1 alone. Plasmid mutants unable to produce active tetR protein are ineffective in reducing expression. Escherichia coli carrying plasmids that express both tetA protein and tetR protein show an increase in the tetracycline resistance level after incubation with the drug. The observations are consistent with the previously proposed mechanism of regulation of tetracycline resistance in Tn10.     

Prevalence of tet(B) and tet(M) genes among tetracycline-resistant Vibrio spp. in the aquatic environments of Korea

Of 24 tetracycline(Tc)-resistant Vibrio spp. isolated from different marine sources in Korea between 1993 and 2003, 23 were identified as carrying both tet(B) and tet(M), while 1 strain carried tet(B) only. In conjugation experiments, 3 strains appeared to be able to transfer both tet(B) and tet(M) to the recipient. Both discriminatory PCR and sequence analysis showed that tet(M) genes of Vibrio spp. appear to be a single allele containing a specific region of tet(M) in Tn1545. However, erm(B) and aphA3, known to be linked to Tn1545-like genes, were not detected in Tc-resistant Vibrio spp., even in 9 strains resistant to erythromycin. In analysis to examine the relative position of tet(B) and tet(M), it was shown that tet(M) was present at the 3'-end of the insertion sequence IS10 of Tn10 carrying tet(B). At the junctional region between Tn10 and tet(M), we found a 14 bp sequence of unknown function and the deletion of regulatory sequences reported to be needed for tet(M) expression in conjugative transposons. This is the first report of the simultaneous presence of tet(B) and tet(M), and of the tet(M) gene being linked to the 3'-end of Tn10 in Tc-resistant Vibrio spp. in Korea.     

Homology among tet determinants in conjugative elements of streptococci.

A mutation to tetracycline sensitivity in a resistant strain of Streptococcus pneumoniae was shown by several criteria to be due to a point mutation in the conjugative omega (cat-tet) element found in the chromosomes of strains derived from BM6001, a clinical strain resistant to tetracycline and chloramphenicol. Strains carrying the mutation were transformed back to tetracycline resistance with the high efficiency of a point marker by donor deoxyribonucleic acids from its ancestral strain and from nine other clinical isolates of pneumococcus and by deoxyribonucleic acids from group D Streptococcus faecalis and group B Streptococcus agalactiae strains that also carry conjugative tet elements in their chromosomes. It was not transformed to resistance by tet plasmid deoxyribonucleic acids from either gram-negative or gram-positive species, except for one that carried transposon Tn916, the conjugative tet element present in the chromosomes of some S. faecalis strains. The results showed that the tet determinants in conjugative elements of several streptococcal species share a high degree of deoxyribonucleic acid sequence homology and suggested that they differ from other tet genes.     

Constitutive expression of tetracycline resistance mediated by a Tn10-like element in Haemophilus parainfluenzae results from a mutation in the repressor gene.

The Tn10-like constitutively expressed tetracycline resistance determinant from a Haemophilus parainfluenzae strain was cloned in Escherichia coli. Toxicity resulting from expression on multicopy plasmids necessitated its being cloned on a low-copy plasmid vector or in cells containing the Tn10-encoded repressor. Constitutive expression of tetracycline resistance was found to result from the synthesis of a truncated inactive repressor molecule. Instead of the 23-kilodalton repressor found in other Tn10-containing strains, this determinant encoded a 14.5-kilodalton molecule. The DNA sequence of the 700-base-pair region spanning the repressor gene and promoter-operator regions of the Haemophilus determinant was identical to that of the same region of Tn10, except for the absence of a single T X A base pair in the repressor gene. This deletion leads to premature termination of the protein. Antisera to the repressor suggested that the repressor was also absent in a second independently isolated H. parainfluenzae strain bearing a Tn10-like constitutive tetracycline resistance determinant.     

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.     

Sphingobacterium sp. strain PM2-P1-29 harbours a functional tet(X) gene encoding for the degradation of tetracycline

Aims:  The tet (X) gene has previously been found in obligate anaerobic Bacteroides spp., which is curious because tet (X) encodes for a NADP-dependent monooxygenase that requires oxygen to degrade tetracycline. In this study, we characterized a tetracycline resistant, aerobic, Gram-negative Sphingobacterium sp. strain PM2-P1-29 that harbours a tet (X) gene.
Methods and Results:  Sphingobacterium sp. PM2-P1-29 demonstrated the ability to transform tetracycline compared with killed controls. The presence of the tet (X) gene was verified by PCR and nucleotide sequence analysis. Additional nucleotide sequence analysis of regions flanking the tet (X) gene revealed a mobilizable transposon-like element (Tn 6031 ) that shared organizational features and genes with the previously described Bacteroides conjugative transposon CTnDOT. A circular transposition intermediate of the tet (X) region, characteristic of mobilizable transposons, was detected. However, we could not demonstrate the conjugal transfer of the tet (X) gene using three different recipient strains and numerous experimental conditions.
Conclusions:  This study suggests that Sphingobacterium sp. PM2-P1-29 or a related bacterium may be an ancestral source of the tet (X) gene.
Significance and Impact of the Study:  This study demonstrates the importance of environmental bacteria and lateral gene transfer in the dissemination and proliferation of antibiotic resistance among bacteria.     

The presence of the tet gene from cloning vectors impairs Salmonella survival in macrophages

Cloning, mutagenesis and complementation of virulence factors are key steps to understand the mechanisms of bacterial pathogenesis and cloning vectors are routinely utilized for these processes. We have investigated the effect of the presence of commonly used cloning vectors on the survival of the intracellular bacterial pathogen Salmonella during macrophage infection. We demonstrate that the presence of the pSC101 derived tetracycline resistance gene on plasmids causes a lower survival rate of Salmonella in macrophages. The decrease in survival caused by the presence of the tet gene was not due to a higher susceptibility to gentamicin, a growth defect, or to increased sensitivity to acid. Higher susceptibility to hydrogen peroxide was observed in vitro for strain containing plasmid with the tet gene when the strains were grown at high densities but not when they were grown at low densities. Our findings demonstrate that the use of the tet gene for mutation or complementation can have deleterious effects and should thus be carefully considered.     

Tryptophan in alpha-helix 3 of Tet repressor forms a sequence-specific contact with tet operator in solution

The Tn10-encoded Tet repressor contains two tryptophan residues at positions 43 and 75. The typical tryptophan fluorescence is decreased upon binding of tet operator. The Tet repressor gene was engineered to replace either or both of the Trp codons by Phe codons. The resulting single tryptophan mutants are called F43 and F75 and the double mutant F43F75. The mutant proteins were purified to homogeneity. They recognize tet operator DNA only in the absence of the inducer tetracycline, indicating an intact tertiary structure of the engineered proteins. F75 and wild-type bind tet operator with the same association constant. The association constants of F43 and F43F75 with tet operator are about 3 orders of magnitude smaller. This indicates that Trp43 is important for tet operator recognition. Trp43 fluorescence is completely quenched in the complex with tet operator DNA while Trp75 remains unaffected. Binding to nonspecific DNA leads only to a 40% decrease of Trp43 fluorescence. This is interpreted as the contribution of the changed environment while the complete quench reflects a tight sequence-specific contact of tryptophan 43 to tet operator DNA. Trp43 is solvent-exposed, while Trp75 is buried in the hydrophobic interior of the protein. These results are discussed in light of the alpha-helix turn-alpha-helix DNA binding motif deduced from homology to other repressor proteins.     

Analysis of tetracycline resistance encoded by transposon Tn10: deletion mapping of tetracycline-sensitive point mutations and identification of two structural genes

Deletions in the tet genes derived from Tn10 were formed from different tet::Tn5 insertion mutations by removing DNA sequences located between a HindIII site in Tn5 and a HindIII site adjacent to the tet genes. Tetracycline-sensitive point mutations were mapped in recombination tests with the deletions and were thus aligned with the genetic and physical map of the tet region. Plasmids carrying point mutations were tested for complementation with derivatives of pDU938, a plasmid carrying cloned tet genes derived from Tn10 which had been inactivated by Tn5 insertions. Complementation occurred between promoter-proximal tet point mutations and distal tet::Tn5 insertions, suggesting the existence of two structural genes, tetA and tetB. These results, together with the analysis of polypeptides in minicells harboring pDU938tet::Tn5 mutants, suggested that tetA and tetB are expressed coordinately in an operon. The tetB gene encodes the previously characterized 36,000-dalton cytoplasmic membrane TET protein, but the product of tetA was not identified. Point mutations in either tetA or tetB led to the defective expression of the resistance mechanism involving tetracycline efflux. It is suggested that the tetA and tetB products interact cooperatively in the membrane to express resistance.     

Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida

Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc(r)) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried [tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tc(r) plasmids to an Escherichia coli recipient at 15 degrees C. Five of the isolates, with genetically related Tc(r) plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7x10(-4) to 2.8x10(-6) per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 degrees C is the lowest temperature that has been documented in bacteria.