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Aglow for antibiotics Antibacterial compounds used for veterinary therapeutic purposes have improved the health and productivity of farm animals. However, the use of these compounds is not without its downside. Overuse can lead to increased bacterial resistance to antibiotics. One way to reduce the negative effects is to monitor drug residues and make sure that only the needed amount of drug is used. In this issue (p. 4457), Matti T. Karp and colleagues at the University of Turku (Finland) describe a biosensor for the detection of one class of such antibacterial drugs the tetracyclines. The biosensor uses a genetically engineered E. coli strain that contains bacterial luciferase genes from P. luminescens and d regulatory unit that represses or promotes the production of luciferase, depending on the presence of tetracycline. The repressor protein (tetR) has a recognition site for tetracycline. When the tetracycline binds to the tetR, the protein changes conformation and dissociates from the promoter. The luciferase gene is turned on, and the cell luminesces. The sensor response depends on the pH and the external Mg2+ concentration. Increases in the Mg2+ concentration shifts the luminescence response curve toward higher amounts of tetracycline. The sensitivity of the sensor can be manipulated by adjusting the Mg2+ concentration. At sufficiently high concentrations of tetracycline, the bioluminescence begins to decrease, which the au-
Binding with mass spec As combinatorial libraries grow, so does the pressure on analytical scientists to find quick and effective ways to assay the many compounds for useful biological activity as drug candidates. Albert Heck working at the University of Warwick (United Kingdom) (now at Utrecht University, The Netherlands) has collaborated with Thomas Jorgensen and Peter Roepstorff, two molecular biologists at Odense University (Denmark), to find a new way of coping wiih such libraries. The team developed an electrospray ionization (ESI) MS technique for evaluating
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thors attribute to the toxicity of tetracycline to the E. coli cells. "The tetracycline starts ts inhibit the protein synthesis machinery of the sensor bacteria," Karp says. Although more than 1000 tetracycline derivatives are known, only seven have been used extensively for clinical or veterinary applications. The sensor responds effectively to all of the clinically relevant tetracyclines, which actually showed a higher affinity (about 1000-fold) for the repressor protein than for ribosomes (their usual targets). Although 90 min were required for optimal sensitivity of luminescence, a detectable signal was produced within a few minutes. The detection limit was 2 ng/sample, which is in the picomole range. "We have focused on rapid diagnostics of antibiotic residues.
The diffusion rate of tetracycline in the cells controls the speed of the assay," Karp says. 'With very long incubation times of 10-20 h, sensitivity would be even better." The sensor is specific to tetracyclines. Other antibiotics, such as rifampicin, kanamycin, nalidixic acid, chloramphenicol, streptomycin, and erythromycin, do not induce luciferase production in the sensor cells—even at high concentrations over a wide range. Karp says, "No antibiotics other than tetracyclines will cause an increase in luminescence over the background signal." Potential areas of application for this sensor include the detection of tetracyclines in different food matrixes and serum. According to Karp, the next step is to develop an assay that is suitable for routine work. Celia Henry
Mode of action of the sensor on the molecular level.
vancomycin analogues as new antibiotics. forming between peptidoglycans and kills The approach correlates peaks in their the bacterium. spectra directly with the binding of antiWorryingly, resistance, even to vancomybiotic candidates to bacterial cell wall pepcin, is becomming more comon, as bacteria tides and so avoids the complex and timehave swapped the D-alanine in their growing consuming spectroscopic titration techbacterial overcoat for a D--actate or D--erine. niques typical in such studies. This change repels vancomycin rather than hooking it—removing its inhibitory effects Bacterial drug resistance is a major and allowing the bacterium to mature. problem, and natural products such as the glycopeptide vancomycin are now used by In searching for effective vancomycin hospitals as last-resort drugs against killer analogues to bring up the rearguard, medicibugs such as Staphylococcus aureus. Vanco-nal chemists have discovered that the solumycin, and its chemical cousin ristocetin, tion association binding constant (K^ bework by hooking on to the D-Ala-D-Ala setween the antibiotic and the bacterial peptide quence of peptidoglycan precursors cell wall is an accurate indicator of potential present in the growing bacterial cell wall. activity as a drug. Now, Heck and his colThis stops stabilizing cross-links from leagues have found that they can determine
Analytical Chemistry News & Features, November 1, 1998
