Characterization of tetracycline modifying enzymes using a sensitive in vivo reporter system

Abstract Background Increasing our understanding of antibiotic resistance mechanisms is critical. To enable progress in this area, methods to rapidly identify and characterize antibiotic resistance conferring enzymes are required. Results We have constructed a sensitive reporter system in Escherichia coli that can be used to detect and characterize the activity of enzymes that act upon the antibiotic, tetracycline and its derivatives. In this system, expression of the lux operon is regulated by the tetracycline repressor, TetR, which is expressed from the same plasmid under the control of an arabinose-inducible promoter. Addition of very low concentrations of tetracycline derivatives, well below growth inhibitory concentrations, resulted in luminescence production as a result of expression of the lux genes carried by the reporter plasmid. Introduction of another plasmid into this system expressing TetX, a tetracycline-inactivating enzyme, caused a marked loss in luminescence due to enzyme-mediated reduction in the intracellular Tc concentration. Data generated for the TetX enzyme using the reporter system could be effectively fit with the known Km and kcat values, demonstrating the usefulness of this system for quantitative analyses. Conclusion Since members of the TetR family of repressors regulate enzymes and pumps acting upon almost every known antibiotic and a wide range of other small molecules, reporter systems with the same design as presented here, but employing heterologous TetR-related proteins, could be developed to measure enzymatic activities against a wide range of antibiotics and other compounds. Thus, the assay described here has far-reaching applicability and could be adapted for high-throughput applications

The induction of folding cooperativity by ligand binding drives the allosteric response of tetracycline repressor

Tetracycline (Tc) repressor (TetR) undergoes an allosteric transition upon interaction with the antibiotic, Tc, that abrogates its ability to specifically bind its operator DNA. In this work, by performing equilibrium protein unfolding experiments on wild-type TetR and mutants displaying altered allosteric responses, we have delineated a model to explain TetR allostery. In the absence of Tc, we show that the DNA-binding domains of this homodimeric protein are relatively flexible and unfold independently of the Tc binding/dimerization (TBD) domains. Once Tc is bound, however, the unfolding of the DNA binding domains becomes coupled to the TBD domains, leading to a large increase in DNA-binding domain stability. Noninducible TetR mutants display considerably less interdomain folding cooperativity upon binding to Tc. We conclude that the thermodynamic coupling of the TetR domains caused by Tc binding and the resulting rigidification of the DNA-binding domains into a conformation that is incompatible with DNA binding are the fundamental factors leading to the allosteric response in TetR. This allosteric mechanism can account for properties of the whole TetR family of repressors and may explain the functioning and evolution of other allosteric systems. Our model contrasts with the prevalent view that TetR populates two distinct conformations and that Tc causes a switch between these defined conformations

Polymorphisms in the human tropoelastin gene modify in vitro self-assembly and mechanical properties of elastin-like polypeptides.

Elastin is a major structural component of elastic fibres that provide properties of stretch and recoil to tissues such as arteries, lung and skin. Remarkably, after initial deposition of elastin there is normally no subsequent turnover of this protein over the course of a lifetime. Consequently, elastic fibres must be extremely durable, able to withstand, for example in the human thoracic aorta, billions of cycles of stretch and recoil without mechanical failure. Major defects in the elastin gene (ELN) are associated with a number of disorders including Supravalvular aortic stenosis (SVAS), Williams-Beuren syndrome (WBS) and autosomal dominant cutis laxa (ADCL). Given the low turnover of elastin and the requirement for the long term durability of elastic fibres, we examined the possibility for more subtle polymorphisms in the human elastin gene to impact the assembly and long-term durability of the elastic matrix. Surveys of genetic variation resources identified 118 mutations in human ELN, 17 being non-synonymous. Introduction of two of these variants, G422S and K463R, in elastin-like polypeptides as well as full-length tropoelastin, resulted in changes in both their assembly and mechanical properties. Most notably G422S, which occurs in up to 40% of European populations, was found to enhance some elastomeric properties. These studies reveal that even apparently minor polymorphisms in human ELN can impact the assembly and mechanical properties of the elastic matrix, effects that over the course of a lifetime could result in altered susceptibility to cardiovascular disease

Mechanical properties of elastin-like peptides (ELPs) containing G to S substitutions.

<p>The four bar graphs indicate the means and standard errors for tensile mechanical properties of sheets of materials constructed with reference ELP, EP20–24–24, and ELPs containing the single G to S substitution. Materials constructed from ELPs containing the triple G to S substitution were too fragile to generate meaningful values. The number of replicates for each experiment (n) is indicated. ** indicates a significant difference between the two materials (ANOVA with Bonferoni correction, p<0.01).</p

Single nucleotide polymorphisms causing non-synonymous changes in tropoelastin.

<p>Tropoelastin protein sequence corresponds to RefSeq, variant 1 (NM_000501). This variant does not include exons 22 or 26a (an extension of exon 26). Exons are boxed, with exon numbers above. The positions of mutations and substituted amino acids are indicated within the shaded boxes, minor allele is indicated second.</p

Mechanical properties of elastin-like peptides (ELPs) containing K to R substitutions.

<p>The four bar graphs indicate the means and standard errors for tensile mechanical properties of sheets of materials constructed with reference ELP, EP20–24–24, and ELPs containing the double K to R substitution. The number of replicates for each experiment (n) is indicated. *** indicates a significant difference between the two materials (ANOVA with Bonferoni correction, p<0.005).</p

Single nucleotide polymorphisms identified in human tropelastin.

<p>1. SNPs identified through dbSNP are indicated with an appropriate SNP reference. EST indicates that the polymorphism was identified through EST libraries.</p><p>2. Tropoelastin RefSeq variant 1 (NM_000501) was used for numbering mRNA and amino acid positions, counting from the initiator methionine. Exons 22 and 26a (an extension of exon 26) are not present in this variant and are not included in the position count.</p><p>3. T/C and Leu/Pro designate Major/Minor allele respectively e.g. from T to C or Leu to Pro, etc.</p><p>4. Minor allele amino acid is indicated in square brackets</p><p>5. For SNPs detected through ESTs, minor allele frequency (MAF) indicates the proportion of ESTs sequences coding the minor allele. For SNPs obtained from dbSNP, MAF indicates the range across populations provided through dbSNP.</p><p>6. Because of phase 1 intron/exon borders, although the mutation site is in the last base of exon 11 the mutated amino acid is the first amino acid coded by exon 12.</p