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We report a regulation system in Escherichia coli for independent regulation of two distinct reporter genes by application of Tet repressors with different specificities. One Tet repressor variant comprises wild-type tet operator (tetO) recognition and exclusive induction with the novel inducer 4-dedimethylamino-anhydrotetracycline. The other Tet repressor variant shows tetO-4C recognition and induction with tetracycline. We demonstrate that both variants are independently active in vivo and allow selective regulation of two genes in the same cell without any cross talk.

Tet(M) protein interacts with the protein biosynthetic machinery to render this process resistant to the tetracycline in vivo and in vitro (V. Burdett, J. Biol. Chem. 266:2872-2877, 1991). To understand this process more completely, a mutant of Escherichia coli which is altered in the ability of Tet(M) to confer resistance has been identified. This mutation maps to miaA and displays phenotypes characteristic of previously isolated miaA mutations. The miaA gene product modifies A37 adjacent to the anticodon of several tRNA species. Both the mutant isolated in this work and previously isolated miaA mutants confer tetracycline sensitivity in the presence of functional Tet(M), both share a slow growth phenotype, and in neither case is a wild-type phenotype restored in trans by F'112 carrying the 89- to 98-min region of the chromosome. These similar phenotypes further substantiate the assignment of the mutation described here to the miaA locus.

To make a comprehensive study of tetracycline resistance determinant distribution in the genus Shigella, a collection of 577 clinical isolates of Shigella spp. and enteroinvasive Escherichia coli (EIEC) from a variety of geographical locations was screened to identify tetracycline-resistant strains. The 459 tetracycline-resistant isolates identified were then screened by PCR analysis to determine the distribution in these strains of tetracycline efflux resistance determinants belonging to classes A to E, G, and H that have been identified in gram-negative bacteria. Only classes A to D were represented in these strains. Although Tet B was the predominant determinant in all geographical locations, there were geographical and species differences in the distribution of resistance determinants. An allele of tet(A), designated tet(A)-1, was identified and sequenced, and the 8.6-kb plasmid containing determinant Tet A-1, designated pSSTA-1, was found to have homologies to portions of a Salmonella enterica cryptic plasmid and the broad-host-range resistance plasmid RSF1010. This allele and pSSTA-1 were used as epidemiological markers to monitor clonal and horizontal transmission of determinant Tet A-1. An analysis of serotype, distribution of tetracycline resistance determinants, and resistance profiles indicated that both clonal spread and horizontal transfer had contributed to the spread of specific tetracycline resistance determinants in these populations and demonstrated the use of these parameters as an epidemiological tool to follow the transmission of determinants and strains.

Tet(M) protein interacts with the protein biosynthesis machinery to render this process resistant to tetracycline by a mechanism which involves release of the antibiotic from the ribosome in a reaction dependent on GTP hydrolysis. To clarify this resistance mechanism further, the interaction of Tet(M) with the ribosome has been examined by using a gel filtration assay with radioactively labelled Tet(M) protein. The presence of GTP and 5'-guanylyl imido diphosphate, but not GDP, promoted Tet(M)-ribosome complex formation. Furthermore, thiostrepton, which inhibits the activities of elongation factor G (EF-G) and EF-Tu by binding to the ribosome, blocks stable Tet(M)-ribosome complex formation. Direct competition experiments show that Tet(M) and EF-G bind to overlapping sites on the ribosome.

Effector triggered conformational changes of proteins such as regulators of transcription, receptors, or enzymes are the molecular basis for regulation in biology. Most proteins perform their biological functions intracellularly, in the presence of many potential interaction partners. Studies of conformational changes have mainly been performed in vitro using sophisticated physical and biochemical methods that usually require purified proteins. Here we describe the observation of conformational changes of Tet repressor in the cytoplasm of growing Escherichia coli cells, analyzed by ligand dependent disulfide crosslinking of cysteine residues substituted into mobile regions of the protein. The amount of protein undergoing the structural change is quantitatively linked to the concomitant induction of transcription of a reporter gene.

An interdomain hybrid Tet protein consisting of a class C alpha domain and a class B beta domain (Tet(C/B)) lacks detectable efflux ability and provides only minimal levels of resistance to tetracycline (Tc) (3 microg/ml) compared with intact class B (256 microg/ml) and class C (64 microg/ml). Twenty-one independently isolated mutants of the Tet(C/B) protein with increased Tc resistance were generated by random chemical mutagenesis. Nine mutants with a Glu substitution for Gly-152 in helix 5 of the class C alpha domain produced a resistance of 48 microg/ml, whereas another 9 with an Asp replacement of Gly-247 in helix 8 of the class B beta domain mediated resistance at 32 microg/ml. The third type of mutation, found in 3 mutants expressing 24 microg/ml resistance, was a S202F replacement in the putative interdomain cytoplasmic loop of Tet(C/B). The latter underscores a previously unappreciated function of the interdomain cytoplasmic loop. All three types of Tet(C/B) mutant proteins were expressed in amounts comparable with that of the original protein and demonstrated restored energy-dependent efflux of tetracycline. Site-directed mutational analysis demonstrated that a Gly-247 to Asn mutation could also facilitate Tc resistance by the Tet(C/B) hybrid, and a negatively charged side chain at position 152 was required for Tet(C/B) activity. These mutations appear to promote the necessary functional interactions between the interclass domains that do not occur in the Tet(C/B) hybrid protein and suggest a direct association between helix 5 and helix 8 in the function of Tet efflux proteins.

The tigecycline resistance gene tet(X4) was widespread in various bacteria. However, limited information about the plasmid harboring the tet(X4) gene spread among the different species is available. Here, we investigated the transmission mechanisms of the tet(X4) gene spread among bacteria in a pig farm. The tet(X4) positive Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae and Enterobacter hormaeche were identified in the same farm. The whole genome sequencing (WGS) analysis showed that the K. pneumoniae belonged to ST727 (n = 11) and ST3830 (n = 1), E. cloacae and E. hormaeche belonged to ST524 (n = 1) and ST1862 (n = 1). All tet(X4) genes were located on the IncHI1 plasmids that could be conjugatively transferred into the recipient E. coli C600 at 30°C. Moreover, a fusion plasmid was identified that the IncHI1 plasmid recombined with the IncN plasmid mediated by ISCR2 during the conjugation from strains B12L to C600 (pB12L-EC-1). The fusion plasmid also has been discovered in a K. pneumoniae (K1L) that could provide more opportunities to spread antimicrobial resistance genes. The tet(X4) plasmids in these bacteria are derived from the same plasmid with a similar structure. Moreover, all the IncHI1 plasmids harboring the tet(X4) gene in GenBank belonged to the pST17, the newly defined pMLST. The antimicrobial susceptibility testing was performed by broth microdilution method showing the transconjugants acquired the most antimicrobial resistance from the donor strains. Taken together, this report provides evidence that IncHI1/pST17 is an important carrier for the tet(X4) spread in Enterobacteriaceae species, and these transmission mechanisms may perform in the environment. [ABSTRACT FROM AUTHOR]

N,N-dimethylglycylamido (DMG) derivatives of 6-demethyl-6-deoxytetracycline and doxycycline bind 5-fold more effectively than tetracycline to the tetracycline high-affinity binding site on the Escherichia coli 70S ribosome, which correlates with a 10-fold increase in potency for inhibition of E. coli cell-free translation. The potencies of DMG-doxycycline and DMG-6-demethyl-6-deoxytetracycline were unaffected by the ribosomal tetracycline resistance factors Tet(M) and Tet(O) in cell-free translation assays and whole-cell bioassays with a conditional Tet(M)-producing E. coli strain.

Biosafety of engineered bacteria as living therapeutics requires a tight regulation to control the specific delivery of protein effectors, maintaining minimum leakiness in the uninduced (OFF) state and efficient expression in the induced (ON) state. Here, we report a three repressors (3R) genetic circuit that tightly regulates the expression of multiple tac promoters (Ptac) integrated in the chromosome of E. coli and drives the expression of a complex type III secretion system injectisome for therapeutic protein delivery. The 3R genetic switch is based on the tetracycline repressor (TetR), the non-inducible lambda repressor cI (ind-) and a mutant lac repressor (LacI W220F ) with higher activity. The 3R switch was optimized with different protein translation and degradation signals that control the levels of LacI W220F . We demonstrate the ability of an optimized switch to fully repress the strong leakiness of the Ptac promoters in the OFF state while triggering their efficient activation in the ON state with anhydrotetracycline (aTc), an inducer suitable for in vivo use. The implementation of the optimized 3R switch in the engineered synthetic injector E. coli (SIEC) strain boosts expression of injectisomes upon aTc induction, while maintaining a silent OFF state that preserves normal growth in the absence of the inducer. Since Ptac is a commonly used promoter, the 3R switch may have multiple applications for tight control of protein expression in E. coli. In addition, the modularity of the 3R switch may enable its tuning for the control of Ptac promoters with different inducers., (© 2023 The Authors. Microbial Biotechnology published by Applied Microbiology International and John Wiley & Sons Ltd.)

The emergence and spread of carbapenemase genes, colistin resistance genes mcr-1, and tigecycline resistance gene tet(X) represent a significant threat to clinical therapy and public health. In this study, we investigated the presence of carbapenemase genes, mcr-1, and tet(X) in 298 Escherichia coli strains obtained froma teaching hospital inChina. In total, eight (2.68%), six (2.01%), and one (0.34%) E. coli isolates carried blaNDM, mcr-1, and tet(X4), respectively. The blaNDM gene was located on IncX3 (n = 4), F2:A-:B- (n = 3), and F2:A1:B1 (n = 1) plasmids, with high similarity to multiple plasmids belonging to the same incompatibility type from Enterobacteriaceae. Six MCR-producing strains contained mcr-1-carrying IncI2 plasmids, organized similarly to other mcr-1-bearing IncI2 plasmids from animals in China. The blaCTX--M--55/64/132/199 gene located within a typical transposition unit (ISEcp1-blaCTX--M-orf477Δ) was inserted near dnaJ to generate 5-bp direct repeats in four mcr-1-positive plasmids. The tet(X) and another four resistance genes [aadA2, tet(A), floR, and Δlnu(F)] were co-located on an IncX1 plasmid, highly similar to other tet(X4)-carrying IncX1 plasmids from Escherichia and Klebsiella of animal or food origin, except that the conjugative transfer region of IncX1 plasmids was absent in our plasmid. Although a low prevalence of blaNDM, mcr-1, and tet(X) was observed in E. coli from patients in this study, their dissemination associated with some successful pandemic plasmids is of great concern. The continued surveillance of these crucial resistance genes in patients should be strengthened. [ABSTRACT FROM AUTHOR]

The Escherichia coli Lac repressor (Lac system) and tetracycline responsive promoter (Tet system) systems have been used individually to regulate gene expression at the cellular as well as the organismal levels. In this study, these two systems were combined (designated Lac/Tet dual-inducible system) to regulate two inducible genes simultaneously in a single cell. The isopropyl-beta-D-thiogalactopyranoside (IPTG) and tetracycline (used for the operation of the Lac and the Tet systems) were non-cytotoxic to the cells when added together into the cells at around the optimal concentrations (IPTG: < or = 5 mM; tetracycline: < 1.5 micrograms). The rate and efficiency of induction and repression of two inducible genes regulated by the Lac/Tet dual-inducible system were similar to the results obtained when one inducible gene is regulated by one inducible system in a single cell. The Lac/Tet dual-inducible system could function in many cell lines, which was demonstrated by regulating the expression of beta-galactosidase and luciferase reporter genes in five tumor cell lines by transient transfection analysis. The feasibility of introducing a second inducible system into an already established inducible cell line was confirmed. Finally, we showed that the Lac/Tet dual-inducible system functions at translational and at functional levels in a stable cell line named 7-4-b, which contains the Ha-ras and bc1-2 inducible genes. In conclusion, this study extends the application of prokaryotic inducible systems from the regulation of a single gene to two genes and helps clarify the relationship between two genes and the effects of two genes on the cells.

The possibility of gene suppression by the expression of anti-sense sequences has been tested for tet gene of pBR322 plasmid. Anti-tet gene has been inserted into lac-promoter regulated site of M13mp 10 single-stranded high copy phage vector. To achieve that, HihdIII-BamHI fragment of pBR322 carrying part of the tet gene was inserted into poly-linker of mp 10. The influence of the anti-tet gene expression on growth parameters of cells with or without tetracycline in the growth media was monitored for JM103 cells. The results indicate that in this system the detectable suppression of the tet gene by anti-tet expression was not manifested.

Previous studies [Wasylewski et al. (1996), J. Protein Chem. 15, 45-58] have shown that the W43 residue localized within the helix-turn-helix structure domain of Tet repressor can exist in the ground state in two conformational states. In this paper we investigate the fluorescence properties of W43 of TetR upon binding of tetracycline inducer and its chemical analogs such as anhydro- and epitetracycline. Binding of the drug inducer to the protein indicates that the W43 residue still exists in two conformational states; however, its environment changes drastically, as can be judged by the changes in fluorescence parameters. The FQRS (fluorescence-quenching-resolved spectra) method was used to decompose the total emission spectrum. The resolved spectra exhibit maxima of fluorescence at 346 and 332 nm and the component quenchable by KI (346 nm) is shifted 9 nm toward the blue side of the spectrum upon inducer binding. The observed shift does not result from the changes in the exposure of W43, since the bimolecular quenching rate constant remains the same and is equal to about 2.7 x 10(9) M-1 sec-1. The binding of tetracycline leads to drastic decrease of the W43 fluorescence intensity and increase of the tetracycline intensity as well as the decrease of fluorescence lifetime, especially of the W43 component characterized by the emission at 332 nm. The observed energy transfer from W43 to tetracycline is more efficient for the state characterized by the fluorescence emission at 332 nm (88%) than for the component quenchable by iodide (53%). Tetracycline and several of its derivatives were also used to observe how chemical modifications of the hydrophilic groups in tetracycline influence the mechanism of binding of the antibiotic to Tet repressor. By use of pulsed-laser photoacoustic spectroscopy it is shown that the binding of tetracyclines to Tet repressor leads to significant increase of tetracycline fluorescence quantum yields. Steady-state fluorescence quenching of tetracycline analogs in complexes with Tet repressor using potassium iodide as a quencher allowed us to determine the dependence of the exposure of bound antibiotic on the modifications of hydrophilic substituents of tetracycline. Circular dichroism studies of the TetR-[Mg.tc]+ complex do not indicate dramatic changes in the secondary structure of the protein; however, the observed small decrease in the TetR helicity may occur due to partial unfolding of the DNA recognition helix of the protein. The observed changes may play an important role in the process of induction in which tetracycline binding results in the loss of specific DNA binding.

Steady-state fluorescence quenching and time-resolved measurements have been performed to resolve the fluorescence contributions of the two tryptophan residues, W43 and W75, in the subunit of the homodimer of the Tet repressor from Escherichia coli. The W43 residue is localized within the helix-turn-helix structural domain, which is responsible for sequence-specific binding of the Tet repressor to the tet operator. The W75 residue is in the protein matrix near the tetracycline-binding site. The assignment of the two residues has been confirmed by use of single-tryptophan mutants carrying either W43 or W75. The FQRS (fluorescence-quenching-resolved-spectra) method has been used to decompose the total emission spectrum of the wild-type protein into spectral components. The resolved spectra have maxima of fluorescence at 349 and 324 nm for the W43 and W75 residues, respectively. The maxima of the resolved spectra are in excellent agreement with those found using single-tryptophan-containing mutants. The fluorescence decay properties of the wild type as well as of both mutants of Tet repressor have been characterized by carrying out a multitemperature study. The decays of the wild-type Tet repressor and W43-containing mutant can be described as being of double-exponential type. The W75 mutant decay can be described by a Gaussian continuous distribution centered at 5.0 nsec with a bandwidth equal to 1.34 nsec. The quenching experiments have shown the presence of two classes of W43 emission. One of the components, exposed to solvent, has a maximum of fluorescence emission at 355 nm, with the second one at about 334 nm. The red-emitting component can be characterized by bimolecular-quenching rate constant, kq equal to 2.6 x 10(9), 2.8 x 10(9), and 2.0 x 10(9) M-1 sec-1 for acrylamide, iodide, and succinimide, respectively. The bluer component is unquenchable by any of the quenchers used. The W75 residue of the Tet repressor has quenching rate constant equal to 0.85 x 10(9) and 0.28 x 10(9) M-1 sec-1 for acrylamide and succinimide, respectively. These values indicate that the W75 is not deeply buried within the protein matrix. Our results indicate that the Tet repressor can exist in its ground state in two distinct conformational states which differ in the microenvironment of the W43 residue.

Both domains, alpha and beta, of the cytoplasmic membrane-localized Tet proteins encoded by the tet gene family (classes A through E) are required for resistance to tetracycline (Tcr) in gram-negative bacteria. Two inactive proteins, each containing a mutation in the opposite domain, are capable of complementation to produce Tcr. Similarly, inactive hybrid proteins expressed by interdomain gene hybrids constructed between tet(B) and tet(C) [tet(B) alpha/(C) beta and tet(C) alpha/(B) beta] together produce significant Tcr via trans complementation (R.A. Rubin and S. B. Levy, J. Bacteriol. 172:2303-2312, 1990). A derivative of tet(B) was constructed to express the two domains of Tet(B) as separate polypeptides, neither containing intact the central, hydrophilic interdomain region. Cells harboring this tet(B) mutant expressed Tcr at about 20% the level conferred by intact tet(B). As expected, no detectable amount of a full-length Tet protein was expressed. A polypeptide corresponding to the alpha domain was observed. Interdomain hybrids between tet(B) and tet(C) containing a frameshift at the fusion junction, designed to result in expression of each of the four domains on separate polypeptides, showed trans complementation without production of detectable full-length proteins. Levels of Tcr were greater than or equal to those previously observed in complementations using full-length hybrid proteins. These results strongly suggest that polypeptides harboring individual alpha and beta domains, lacking an intact interdomain region, can interact productively in the cell to confer Tcr.

We have constructed a genetic assay which selects positively for a functional interaction between Tet repressor and its cognate operator in Escherichia coli. In this strain Tet repressor blocks expression of lacI and lacZ. This leads to derepression of a lacPO controlled galK gene. The strain can be selected by growth on galactose as the sole carbon source and screened for the beta-galactosidase phenotype. These features allow the identification of one candidate among 10(8) false clones on a single plate. The assay was applied to select mutants with a ts DNA binding phenotype and to screen oligonucleotide generated Tet repressor mutants. Analysis of these mutations revealed that they affect DNA and inducer binding and possibly the dimerization domains. These mutations are located at residues 21, 48, 49, 89 and at the C terminus of the protein (193), respectively.

Inner membrane Tet proteins encoded by tet genes in gram-negative bacteria mediate resistance to tetracycline (Tcr) by directing its export. Total sequences for class A, B, and C tet genes demonstrate that their products have a common ancestor, with Tet(A) and Tet(C) being more closely related (78% identical) than either is to Tet(B) (45% identical). The N- and C-terminal halves of Tet(B) and Tet(C) appear to comprise separate domains, and trans-complementation observed between tetracycline sensitive mutants in either domain of Tet(B) suggests separate but interactive functions for these domains. In this present study, interdomain hybrid genes were constructed to express hybrid tet products whose N- and C-terminal halves were derived from different family members [Tet(A/C), Tet(B/C), and Tet(C/B)]. Tet(A/C) specified a level of Tcr comparable to wild-type Tet(C) and 60% that of Tet(A), indicating that domains from these closely related tet products can function in cis. Although neither Tet(B/C) nor Tet(C/B) hybrids conferred significant Tcr, cells producing both of these types of hybrid proteins expressed substantial Tcr, indicating that productive interactions can occur in trans between Tet(B/C) and Tet(C/B). Taken together, these results suggest that highly specific interactions between the N- and C-terminal domains are necessary for Tcr and do not occur in individual hybrids derived from the more distant relatives, Tet(B) and Tet(C). This requirement for specific interactions suggests that N- and C-terminal domains have coevolved in each member of the Tet family.

The aspartate 15 residue within the first predicted intramembrane helix of the tetracycline efflux protein Tet has been conserved in four tetracycline resistance determinants from gram-negative bacteria. Its replacement in class B Tet by tyrosine, histidine, or asparagine resulted in a 60 to 85% loss of tetracycline resistance and a similar loss of tetracycline-proton antiport. The tyrosine and histidine substitutions lowered the Vmax of the efflux system by some 90% but did not alter the Km. The asparagine substitution raised the Km over 13-fold, while the Vmax was equal to or greater than that of the wild type. Therefore, although the nature of its role is unclear, aspartate 15 is important for normal Tet function.

Atypical tetracyclines were active against Escherichia coli and Staphylococcus aureus strains containing determinants that mediate resistance to typical tetracyclines by efflux (Tet B and Tet K) or ribosomal protection (Tet M) mechanisms. The results support recently published data that tetracyclines are divisible into at least two classes on the basis of their modes of action.

The transmission of antibiotic resistance genes (ARGs) in pathogenic bacteria affects culture animal health, endangers food safety, and thus gravely threatens public health. However, information about the effect of disinfectants - triclosan (TCS) on ARGs dissemination of bacterial pathogens in aquatic animals is still limited. One Citrobacter freundii (C. freundii) strain harboring tet (X4)-resistant plasmid was isolated from farmed grass carp guts, and subsequently conjugative transfer frequency from C. freundii to Escherichia coli C600 (E. coli C600) was analyzed under different mating time, temperature, and ratio. The effect of different concentrations of TCS (0.02, 0.2, 2, 20, 200 and 2000 μg/L) on the conjugative transfer was detected. The optimum conditions for conjugative transfer were at 37 °C for 8h with mating ratio of 2:1 or 1:1 (C. freundii : E. coli C600). The conjugative transfer frequency was significantly promoted under TCS treatment and reached the maximum value under 2.00 μg/L TCS with 18.39 times that of the control group. Reactive oxygen species (ROS), superoxide dismutase (SOD) and catalase (CAT) activities, cell membrane permeability of C. freundii and E. coli C600 were obviously increased under TCS stress. Scanning electron microscope showed that the cell membrane surface of the conjugative strains was wrinkled and pitted, even broken at 2.00 μg/L TCS, while lysed or even ruptured at 200.00 μg/L TCS. In addition, TCS up-regulated expression levels of oxidative stress genes (kat E, hem F, bcp , hem A, kat G, ahp F, and ahp C) and cell membrane-related genes (fim C, bam E and omp A) of donor and recipient bacteria. Gene Ontology (GO) enrichment demonstrated significant changes in categories relevant to pilus, porin activity, transmembrane transporter activity, transferase activity, hydrolase activity, material transport and metabolism. Taken together, a tet (X4)-resistant plasmid could horizontal transmission among different pathogens, while TCS can promote the propagation of the resistant plasmid. [Display omitted] • A Citrobacter freundii carrying tet (X4) gene was isolated from fish gut. • The tet (X4) resistance plasmids can transfer between bacterial intergeneric. • Triclosan can remarkably increase conjugative transfer frequency of the plasmids. • Triclosan damages the structure and permeability of the cell membrane. • Triclosan aggravated oxidative stress of the donor and recipient bacteria. [ABSTRACT FROM AUTHOR]

Tetracycline resistance in the Enterobacteriaceae is mediated by a number of genetically related, usually plasmid-borne, determinants which specify an efflux system involving an inner membrane protein, Tet. Attempts to overproduce the Tn10 (Class B)-encoded Tet in Escherichia coli by cloning the structural gene tet downstream of the lambda PL promoter under regulation by temperature-sensitive lambda repressor cI857 were unsuccessful; induction at 42 degrees C resulted in filamentous, non-viable cells containing little detectable overproduction of the protein. However, cells containing tet fused to lacZ were resistant to tetracycline at 30 degrees C and synthesized modest amounts of a large fusion protein when induced at 42 degrees C. Fusion of the N-terminal half or the first 38 amino acids of tet to lacZ did lead to increased production of fusion proteins. Fusions could be purified by size or by LacZ immunoaffinity or substrate-affinity chromatography. In the latter method, selected detergents were required to counteract nonspecific binding of Tet to the adsorbant. Amino acid sequencing of the N-terminus of Tet-LacZ fusion proteins indicated that most molecules were blocked at this terminus. The sequence of an unblocked subpopulation was consistent with that expected from the nucleotide sequence. A collagen peptide linker, genetically placed between tet and lacZ, allowed recovery of purified Tet protein after collagenase treatment of the purified fusion protein.

The major in vitro transcripts from the tet promoter of pBR322 derivatives pTA22 and pTA33 have heterogeneous 5' ends consisting of variable lengths of oligo(A). Their structure is 5'pppAnU..., where n ranges from 1 to greater than 12, but the template strand can encode at most four A residues at the site of transcription initiation. The abundance of additional A residues at the 5' end of the pTA22 and pTA33 tet transcripts could be reduced by elevating the concentration of UTP, but even at high concentrations (greater than 1 mM) non-cognate A residues were still observed. Aberrant initiation was not artifactual since the major and minor transcripts of the pBR322 tet promoter region, and other transcripts arising from minor promoters on pTA22 or pTA33 DNA all had unique 5' termini. Mixing experiments showed that RNA polymerase did not utilize pppA2-4-OH produced by abortive initiation as primers. The data suggest that the initial nascent RNA chain 'slips' in the 5' direction during elongation opposite T4 on the template strand causing RNA polymerase to reiteratively add A residues to the 5' end of the transcript. The generality and possible significance of this mechanism is discussed.

The mechanism of resistance to tetracycline in Escherichia coli mediated by the Campylobacter jejuni-derived resistance determinant Tet(O) was investigated. The cloned Tet(O) protein had no detectable effect on the intracellular accumulation of tetracycline. The presence of Tet(O) markedly diminished the inhibitory effect of tetracycline on protein synthesis both in vivo and in vitro. Ribosomes prepared from tetracycline-resistant and susceptible E. coli cells bound almost identical amounts of radiolabeled tetracycline. Thus, a reduction in the binding of the antibiotic to its target site on the ribosome is not the primary mechanism of resistance. Poly(U)-directed polyphenylalanine synthesis revealed that an S-100 fraction prepared from tetracycline-resistant cells made the ribosomes prepared from susceptible cells considerably more resistant to the inhibitory action of tetracycline. The N-terminal portion (1 to 150 residues) of Tet(O) is highly homologous to the GTP-binding domain of elongation factor Tu and to elongation factor G, indicating that the Tet(O) protein has the potential to bind GTP. These data suggest that the Tet(O) protein could function either as a tetracycline-resistant analog of this elongation factor(s) or by modifying the target sites on the ribosomes in a catalytic fashion.

Background and Aims: Escherichia coli is a natural microflora of the human heart and all the warm animals. Mastitis is a disease caused by various pathogens. One of the important factors in the production of mastitis in the livestock is E. coli and is known as peripheral mastitis. The aim of this study was to isolate sulfonamide, integron and tetracycline resistance genes in E.coli isolated from mastitis with clinical symptoms using Multiplex PCR. Materials and Methods: 50 milk isolates with mastitis symptoms were collected from livestock farms in west of Tehran. Biochemical and microbial tests were performed and antibiotic susceptibility test using disc diffusion method was performed according to CLSI 2016 and E-test with antibiotics of different groups. After isolating the bacteria and extracting DNA, the presence of the genes was investigated by Multiplex PCR. Results: Based on the findings of this study, out of a total of 50 samples, 1 sample (2%) had Sul2 gene and 32 samples (64%) had the tetA gene and 11 samples (36%) of the tetc gene. Also Int gene was detected in 1 sample of 2% of the samples and the highest antibiotic resistance to the antibiotic sultrim and cotrimoxazole were 68% and 50%, respectively, and the highest sensitivity to tetracycline was 96%. Conclusion: Comparison of this study with other studies has shown that the resistance gene in E.coli isolated from mastitis is high. The difference between the results of the M-PCR method in this study and the results of studies by other researchers in different parts of the world may be due to the source of the sample, while in studies of other researchers, most cases have been studied on samples of blood diarrhea. Another reason for the difference is the difference in geographical areas.

ABSTRACTThe recent emergence of plasmid-mediated tigecycline resistance genes, tet(X3) and tet(X4), in animals and humans in China would pose a foreseeable threat to public health. To illustrate this paradigm shift in tigecycline resistance, here, covering the period 2008-2018, we retrospectively analysed a national strain collection of Escherichia coli (n = 2254), obtained from chickens and pigs, in six representative provinces of China. The gene tet(X4) was identified in five pig isolates collected in 2016 and 2018 from the provinces of Sichuan (3/15, 2018), Henan (1/25, 2018) and Guangdong (1/28, 2016), but not in the isolates prior to 2016. None of the isolates was detected harbouring tet(X3). All tet(X4)-positive E. coli exhibited high levels of tigecycline resistance (MICs, 16-64 mg/L), and two were confirmed as colistin resistant, harbouring chromosome-borne mcr-1 gene. The gene tet(X4) was detected on a plasmid in all five isolates, whereas a co-location of tet(X4) on the chromosome of one isolate was observed. Diverse host strains and novel plasmids related to the tet(X4) gene were observed. Our timely findings of the recent emergence of tet(X4) gene in food animal support the rapid surveillance and eradication of this gene before it is established.

tet(X4) is the critical resistance gene for tigecycline degradation that has been continually reported in recent years. In particular, pathogenic bacteria carrying tet(X4) are a severe threat to human health. However, information describing Escherichia coli coharboring tet(X4) with virulence genes is limited. Here, we isolated an E. coli strain coharboring tet(X4) and the heat-stable toxin gene astA from a dead piglet. The strain named 812A1-131 belongs to ST10. The genome was sequenced using the Nanopore and Illumina platforms. The virulence genes astA and tet(X4) are located on the chromosome and in the IncHI1-type plasmid p812A1-tetX4-193K, respectively. The plasmid could be conjugatively transferred to recipient E. coli J53 with high frequency. In vivo experiments showed that strain 812A1-131 is pathogenic to Galleria mellonella and could colonize the intestines of mice. In summary, pathogenic E. coli could receive a plasmid harboring the tet(X4) gene, which can increase the difficulty of treatment. The prevalence and transmission mechanisms of pathogenic bacteria coharboring the tet(X4) gene need more attention. [ABSTRACT FROM AUTHOR]

Escherichia coli , a rod-shaped Gram-negative bacterium, is a significant causative agent of severe clinical bacterial infections. This study aimed to analyze the epidemiology of extended-spectrum β-lactamase (ESBL)-producing mcr-1 -positive E. coli in Shandong, China. We collected 668 non-duplicate ESBL-producing E. coli strains from clinical samples at Shandong Provincial Hospital between January and December 2018, and estimated their minimum inhibitory concentrations (MICs) using a VITEK ® 2 compact system and broth microdilution. Next-generation sequencing and bioinformatic analyses identified the mcr-1 gene and other resistance genes in the polymyxin B-resistant strains. The conjugation experiment assessed the horizontal transfer capacity of the mcr- 1 gene. Of the strains collected, 24 polymyxin B-resistant strains were isolated with a positivity rate of 3.59% and among the 668 strains, 19 clinical strains carried the mobile colistin resistance gene mcr-1 , with a positivity rate of approximately 2.8%. All 19 clinical strains were resistant to ampicillin, cefazolin, ceftriaxone, ciprofloxacin, levofloxacin, and polymyxin B. Seventeen strains successfully transferred the mcr-1 gene into E. coli J53. All transconjugants were resistant to polymyxin B, and carried the drug resistance gene mcr-1 . The 19 clinical strains had 14 sequence types (STs), with ST155 (n = 4) being the most common. The whole-genome sequencing results of pECO-POL-29&#95;mcr1 revealed that no IS Apl1 insertion sequences were found on either side of the mcr-1 gene. Our study uncovered the molecular epidemiology of mcr-1 -carrying ESBL-producing E. coli in the region and suggested horizontal transmission mediated by plasmids as the main mode of mcr-1 transmission., (© 2024 Yue Liu et al., published by Sciendo.)

The emergence of the plasmid-mediated tigecycline resistance gene tetX family in pig farms has attracted worldwide attention. The use of tetracycline antibiotics in pig farms has a facilitating effect on the prevalence of the tetX family, but the relationship among its presence, expression, and resistance phenotype in resistant bacteria is unknown. In this study, the presence and expression characteristics of tetracycline resistance genes (TRGs) in 89 strains of doxycycline-resistant E. coli (DRE) isolated from pig manure samples from 20 pig farms under low concentrations of doxycycline stress (2 μg/mL) were analyzed. The detection rate of tetO was 96.63%, which is higher than those of other TRGs, such as tetA (94.38%), tetX (76.40%), tetB (73.03%), and tet(X4) (69.66%). At least three TRG types were present in DRE strains, which thus showed extensive resistance to tetracycline antibiotics, and 37% of these strains were resistant to tigecycline. In the presence of a low concentration of doxycycline, tetA played an important role, and the expression and existence ratio of TRGs indicated low expression of TRGs. Furthermore, the doxycycline resistance of DRE was jointly determined by the total absolute abundance of TRGs, and the absolute abundance of tetX and tet(X4) was significantly positively associated with tigecycline resistance in DRE (P < 0.05). Overall, DRE isolated from swine manure is an important reservoir of the tetX family, which suggests that DRE in swine manure has a high risk of tigecycline resistance, poses a potential threat to human health, and should be of public concern., (© 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

In a collection of Escherichia coli isolates, we discovered a new mechanism leading to frequent and high-level tigecycline resistance involving tandem gene amplifications of an efflux pump encoded by the tet(A) determinant. Some isolates, despite carrying a functional tet(A), could not evolve high-level tigecycline resistance by amplification due to the presence of a deletion in the TetR(A) repressor. This mutation impaired induction of tetA(A) (encoding the TetA(A) efflux pump) in presence of tetracyclines, with the strongest effect observed for tigecycline, subsequently preventing the development of tet(A) amplification-dependent high-level tigecycline resistance. We found that this mutated tet(A) determinant was common among tet(A)-carrying E. coli isolates and analysed possible explanations for this high frequency. First, while the mutated tet(A) was found in several ST-groups, we found evidence of clonal spread among ST131 isolates, which increases its frequency within E. coli databases. Second, evolution and competition experiments revealed that the mutation in tetR(A) could be positively selected over the wild-type allele at sub-inhibitory concentrations of tetracyclines. Our work demonstrates how low concentrations of tetracyclines, such as those found in contaminated environments, can enrich and select for a mutation that generates an evolutionary dead-end that precludes the evolution towards high-level, clinically relevant tigecycline resistance. A study on evolution of antimicrobial resistance reveals how sub-inhibitory concentrations of antibiotics enrich and select for a mutated allele that prevents evolution towards clinically significant levels of antibiotic resistance. [ABSTRACT FROM AUTHOR]

Escherichia coli is a universal bacterium causing infections in humans and animal and serves as a major pathogen of urinary tract infections (UTI) and Extraintestinal infection. The present study was conducted for current antibiotic resistance pattern of E. coli and molecular detection of resistance related gene in clinical isolates of E. coli. The study was a hospital based, prospective study which was done for a period of twelve months. This study was done by using the standard culture techniques for urine, pus, semen and sputum samples, Maximum number was from samples of urine 73 followed by pus 23 semen 2 and sputum 2. Hundred pathogenic E. coli isolates was further identified by standard microbiology techniques such as colony morphology, Gram staining and biochemical testing methods. Drug resistance was evaluated by disc diffusion method and relevant drug resistance gene detection done by Multiplex PCR. Out of 130 clinical samples total (n=100) isolates were identified as E. coli and their susceptibility patterns for different antibiotics were determined. Results showed that Gentamicin among aminoglycosides and Colistin sulfate among polymyxin were showed relatively less resistance in E. coli. Bacitracin, Ampicillin, trimethoprim, Erythromycin, Tetracycline, Ciprofloxacin, Amoxicillin and Piperacillin were found more resistant. Imepenum, Meropenum among ß lactam were most effective drug. PCR was employed to identify resistance causing gene. Among 100 pathogenic E. coli isolates 87% shown ampicillin resistance encoded by Tem B lactamase gene and 86 % shown tetracycline resistance causing by Tet A gene. Highest level of drug-resistance was observed against ampicillin and tetracycline (AMP-TET) among clinical isolates of pathogenic E. coli collected from hospitalized patients. [ABSTRACT FROM AUTHOR]

Objectives: Carbapenems, colistin, and tigecycline are critically important antibiotics in clinics. After the global appearance of bla NDM and mcr mediating the resistance to carbapenems and colistin, respectively, tigecycline becomes the last-resort drug against severe human infections caused by multidrug-resistant bacteria. Recently, a mobile tigecycline resistance gene tet (X4) has been identified in Escherichia coli , Klebsiella pneumoniae , and Acinetobacter baumannii that causes high resistance to tigecycline and other tetracyclines. In this study, the prevalence of tet (X4) in E. coli isolates from duck and goose farms in Southeast China was identified and characterized. Methods: Feces, soil, sewage, and dust samples were collected from duck and goose farms along with the southeast coast provinces of China. Antimicrobial susceptibility testing and polymerase chain reaction screening were performed to investigate the phenotype and genotype of tigecycline resistance. Conjugation, S1 pulsed-field gel electrophoresis (PFGE), and whole-genome sequencing were used to determine the transferability, genetic location, and the genomic characteristics of tet (X4). Results: In total, 1,716 samples were collected, and 16 isolates (0.9%) recovered from Guangdong, Shandong, and Jiangsu were positive for tet (X4) gene with tigecycline minimum inhibitory concentrations ≥16 mg/L. Notably, among these tet (X4)-positive E. coil isolates, seven of them were from the environment samples (soil and sewage). PFGE and multilocus sequence typing demonstrated that ST3997 was the most prevalent sequence type (eight isolates, 50%) in Jiangsu province. By conjugation assays, 11 isolates were able to transfer tet (X4) plasmid to E. coli C600 recipient, and these plasmids belonged to IncHI1 and IncX1 detected by sequence analysis. tet (X4) was found adjacent to an insertion sequence IS CR2 downstream and a cat D gene upstream for all isolates. In addition, multiple-drug resistance to tigecycline, chlortetracycline, ampicillin, florfenicol, ciprofloxacin, gentamicin, trimethoprim/sulfamethoxazole, and fosfomycin was profiled in most of the tet (X4)-positive isolates. Conclusion: The identification of tet (X4) harboring E. coli strains in duck farms and their surrounding environment enlarges our knowledge of the variety and prevalence of tigecycline resistance. The prevalence of tet (X4) raises concern for the use of tetracyclines in animal farming, and the tet (X4) gene should be listed as primary gene for resistance surveillance. [ABSTRACT FROM AUTHOR]

Carbapenem-resistant Klebsiella pneumoniae (CRKP), a superbug that can be difficult or impossible to treat, has become a worldwide problem. This study presents the first report of a CRKP strain carrying a plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A) and the subsequent analysis of its genomic features. Isolation and identification of bacteria, antimicrobial susceptibility test, whole genome sequencing, and conjugation experiments assay were conducted in clinical epidemiological investigations and plasmid genetic characterisation analysis. A total of 116 strains of bacteria were isolated from patients with bloodstream infections (BSI) between 2018 and 2023. A total of 89.66% of the isolates were carbapenem-resistant Enterobacteriaceae (CRE), with the majority (75/116) being CRKP. Among these, a novel plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A) simultaneously was found in CRKP46, and the three genes mediated conjugation by IS 26 , IS Aba125 , and IS 26 , respectively. This plasmid conferred carbapenem resistance to E. coli J53 after conjugative transfer, which was 2 times greater than that of CRKP46. The present study identified the occurrence of a rare plasmid co-harbouring bla NDM-1 , bla KPC-2 , and tet(A) , and the spread of these genes was mediated by the corresponding mobile elements. The increased carbapenem resistance created by this novel plasmid challenges public health security and poses a potential threat to human health; therefore, it deserves attention. [ABSTRACT FROM AUTHOR]

• Mobile tet (X)-variant genes, notably tet (X4), were prevalent in municipal WWTPs. • EpicPCR-directed cultivation facilitated the targeted isolation of ARG bacterial hosts. • Enterobacteriaceae was identified as the predominant tet (X4) host using the epicPCR. • tet (X4)-positive E. coli in WWTP were genetically identical to animal and clinical origin. • Four tet (X4)-plasmid types were transmissible across species of different origin. Mobile tet (X)-variant genes confer resistance to a wide range of tetracyclines, including the antibiotic of last-resort, tigecycline, raising significant concerns regarding their potential spread cross-environmental dissemination. However, the bacterial hosts and environmental spread of these genes remain poorly understood. Herein, a retrospective study unveiled the prevalence of tet (X)-variant genes (ranging from tet (X3) to tet (X6)) in activated sludge samples from five municipal wastewater treatment plants (WWTPs) from 2013 to 2021. Among these variants, tet (X4) exhibited the highest detection frequency (100 %) and abundance [(2.48 ± 3.07) × 107 copies/g dry weight] with an increasing trend. An epicPCR-directed cultivation strategy was proposed to facilitate the targeted isolation of tet (X4)-carrying bacterial hosts in activated sludge. This strategy involves the identification of bacterial host profiles using epicPCR and subsequent selective isolating target bacteria. Enterobacteriaceae emerged as the primary bacterial host for tet (X4), alongside previously unreported genera like Providencia, Advenella , and Moheibacter. Subsequent selective isolation of the most abundant Enterobacteriaceae based on the epicPCR-informed host spectrum yielded 39 tet (X4)-carrying Escherichia coli strains from the WWTP. Whole genome sequencing of tet (X4)-positive strains revealed that plasmid-mediated horizontal gene transfer is the primary mechanism driving tet (X4) dissemination. Plasmids including IncFIA(HI1)/IncHI1A/IncHI1B(R27) and IncX1, commonly reported in various clinical and animal settings, were identified as the predominant carriers of tet (X4). E. coli strains harbouring tet (X4) in the WWTP showed substantial genetic similarity to strains from hospital and animal sources, underscoring concerns about the potential risk of across diverse sectors. This study provided the first glimpse of the presence of mobile tet (X)-variants in WWTPs, and highlighted the promise of the epicPCR-directed cultivation strategy for exploring bacterial hosts of clinically important ARGs in different habitats from a One Health perspective. [Display omitted] [ABSTRACT FROM AUTHOR]

Plasmid-mediated tigecycline resistance genes, notably the tet(X) and tmexCD-toprJ genes, have garnered considerable attention due to their transferability. This study aims to investigate the prevalence and resistance mechanisms associated with tet(X) and tmexCD-toprJ in flies, which are important reservoirs of antimicrobial resistance genes. A total of 52 tigecycline resistant bacterial isolates were collected, among which 40 (76.9 %) and 12 (23.1 %) were positive for tet(X) and tmexCD-toprJ, respectively. Tigecycline resistant bacteria were isolated from diverse geographical locations in China, with tet(X4)-positive Escherichia coli and tmexCD1-toprJ1-positive Klebsiella pneumoniae dominant among the isolates. The prevalence of tet(X) in rural area was significantly higher than that in urban area (2.7 % vs. 0.3 %; P 0.05). Most tet(X)-positive strains (n = 40, 100.0 %), and 11(91.7 %) of the tmexCD1-toprJ1-positive strains exhibited multi-drug resistance. The IncFIB(Mar)/IncHI1B hybrid plasmid carrying tmexCD1-toprJ1 was identified by whole-genome sequencing analysis, which dominated the transmission of tmexCD1-toprJ1 in K. pneumoniae. Genetic context analysis showed that tmexCD1-toprJ1 was related locally to IS26, and IS26 may exacerbate the spread of tmexCD1-toprJ1 in different bacteria. In addition, the genetic structure of tmexCD1-toprJ1 also contains several antimicrobial resistance genes, including aph(3′)-Ic, sul1, blaDHA-1, blaCTX-M−5, etc., conferring resistance to aminoglycosides, sulfonamides, and carbapenems. This study provides insights into the epidemiology and transmission dynamics of tigecycline resistance genes, informing targeted intervention strategies to mitigate antimicrobial resistance dissemination.

The discovery of antibiotics has noticeably promoted the development of human civilization; however, antibiotic resistance in bacteria caused by abusing and overusing greatly challenges human health and food safety. Considering the worsening situation, it is an urgent demand to develop emerging nontraditional technologies or methods to address this issue. With the expanding of synthetic biology, optogenetics exhibits a tempting prospect for precisely regulating gene expression in many fields. Consequently, it is attractive to employ optogenetics to reduce the risk of antibiotic resistance. Here, a blue light-controllable gene expression system was established in Escherichia coli based on a photosensitive DNA-binding protein (EL222). Further, this strategy was successfully applied to repress the expression of β-lactamase gene (bla) using blue light illumination, resulting a dramatic reduction of ampicillin resistance in engineered E. coli. Moreover, blue light was utilized to induce the expression of the mechanosensitive channel of large conductance (MscL), triumphantly leading to the increase of streptomycin susceptibility in engineered E. coli. Finally, the increased susceptibility of ampicillin and streptomycin was simultaneously induced by blue light in the same E. coli cell, revealing the excellent potential of this strategy in controlling multidrug-resistant (MDR) bacteria. As a proof of concept, our work demonstrates that light can be used as an alternative tool to prolong the use period of common antibiotics without developing new antibiotics. And this novel strategy based on optogenetics shows a promising foreground to combat antibiotic resistance in the future., (© 2024 Wiley‐VCH GmbH.)