Chemical biology

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in chemical biology.     

On the mechanism of action of tetracycline antibiotics

Oxytetracycline-sensitive and resistant strains of Staphylococci and ofListeria monocytogenes andPasteurella multocida were tested for differences in the reduction of triphenyltetrazolium (TTC) in the presence of glucose, acetate, lactate, pyruvate, glycerol, succinate, formate, malate, citrate, serine, glycine and asparagine. The sensitive staphylococci reduced TTC more actively than the resistant ones in the presence of glucose, acetate and serine. The resistant strains reduced TTC more actively in the presence of succinate, formate, glycine, pyruvate and malate. Oxytetracycline itself only inhibited the reducing activity greatly in the sensitive staphylococci. In the resistant ones, oxytetracycline only slightly decreased the TTC reduction. The insensitivity of the reducing activity of resistant staphylococci toward the effect of oxytetracycline indicates that this activity may be one of the sites of attack by this antibiotic. Ascorbic acid contained in the injection preparation of oxytetracycline interfered with the inhibitory effect of this antibiotic on the TTC reduction by staphylococci and actually increased the activity of reduction.     

Chemical biology

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in chemical biology.     

On the mechanism of action of tetracycline antibiotics

It was found that low oxytetracycline (OTC) concentrations inhibited malic dehydrogenase (MDH) and lactic dehydrogenase (LDH) inStaphylococcus aureus andEscherichia coli (1–5 μg/ml for MDH and 10 μg/ml for LDH). Inhibition of these enzymes occurred almost instantaneously and could be demonstrated after 3–4 minutes. No MDH activity was found in OTC-resistant variants of these microorganisms, but LDH activity was not lowered. The inhibitory effect of OTC is specific for bacterial MDH and LDH. The same enzymes of mammalian origin are not inhibitedin vitro even by high OTC concentrations (100 μg/ml).     

Spores of microorganisms X. Interference of tetracycline antibiotics with sporogenesis of Bacilli

Спорообразующие клетки бацилл, характеризующиеся чрезвычайно сильным накоплением кальция, служили моделью для изучения сущности действия тетрациклиновых антибиотиков на цельные клетки. В период образования преспор наблюдается усиление связывания клетками кальция. В тот же период резко повышается способность клеток Bacillus cereus и Bacillus megaterium связывать хлортетрациклин. Повидимому, как кальций, так и тетрациклин адсорбируются в этот период на поверхность клеток и могут быть отмыты различными методами промывания. Культивирование спорообразующих клеток с лимонной кислотой в значительной степени усиливает связывание хлортетрациклина с клетками. Хлортетрациклин, окситетрациклин и тетрациклин (в концентрации 3 µг/мл), блокируя поверхность клетки, по-видимому, резко тормозят внедрение кальция в клетки и синтез дипиколиновой кислоты в спорангиях, влияя так на морфологию оюразуюшихся спор и на автолиз спорангиев. Это их действие в значительной степени зависит от стадии развития культуры в момент прибавления к ней антибиотика. Избыток кальция в среде несколько повыwает образование дипиколиновой кислоты как в присутствии хлортетрациклина, так и в контроле. Избыток магния, марганци илп глюкозы не меняет торможения внедрения кальция. — Действие тетрациклиновых антибиотиков на спорообразование и на автолиз спорангиев сравнивается с действием этилендиаминтетрау ксусной кислоты. Полученные результаты обсуждаются с точки зрения теории блокады макромолекул и структур тетрациклиновыми антибиотиками через посредство металлов.     

Amplifiable resistance to tetracycline, chloramphenicol, and other antibiotics in Escherichia coli: involvement of a non-plasmid-determined efflux of tetracycline.

Increasing levels of resistance to tetracycline and to a number of other unrelated antibiotics, including chloramphenicol, beta-lactams, puromycin, and nalidixic acid, occurred in Escherichia coli after 50 to 200 generations of growth in the presence of subinhibitory concentrations of tetracycline or chloramphenicol. In the absence of selective pressure, resistances fell to low levels within 100 generations of growth. This amplification of resistance was observed in laboratory and naturally occurring E. coli strains as well as in polA and recA strains. With the exception of previously identified cmlA and cmlB mutations, tetracycline or chloramphenicol resistances were not P1 transducible. Coincident with the emergence of resistance was the appearance of a previously cryptic energy-dependent efflux system for tetracycline. The expression of resistance phenotypes and the tetracycline efflux system were temperature sensitive at 42 degrees C.     

Chemical biology of abscisic acid

Chemical biology is a discipline that utilizes chemicals to elucidate biological mechanisms and physiological functions. Various abscisic acid (ABA) derivatives have revealed the structural requirement for the perception by ABA receptors while biotin or caged derivatives of ABA have disclosed the localization of several ABA-binding proteins. Recently, selective ABA agonist has been used to identify ABA receptors. Furthermore, ABA biosynthesis and catabolic inhibitors have contributed to the identification of new ABA functions in plant growth and development. The physiological function of ABA in non-plant organisms has gradually been revealed. In this review, we discuss the development of small bioactive chemicals and their significance in ABA research.     

TetX is a flavin-dependent monooxygenase conferring resistance to tetracycline antibiotics

The tetracycline antibiotics block microbial translation and constitute an important group of antimicrobial agents that find broad clinical utility. Resistance to this class of antibiotics is primarily the result of active efflux or ribosomal protection; however, a novel mechanism of resistance has been reported to be oxygen-dependent destruction of the drugs catalyzed by the enzyme TetX. Paradoxically, the tetX genes have been identified on transposable elements found in anaerobic bacteria of the genus Bacteroides. Overexpression of recombinant TetX in Escherichia coli followed by protein purification revealed a stoichiometric complex with flavin adenine dinucleotide. Reconstitution of in vitro enzyme activity demonstrated a broad tetracycline antibiotic spectrum and a requirement for molecular oxygen and NADPH in antibiotic degradation. The tetracycline products of TetX activity were unstable at neutral pH, but mass spectral and NMR characterization under acidic conditions supported initial monohydroxylation at position 11a followed by intramolecular cyclization and non-enzymatic breakdown to other undefined products. TetX is therefore a FAD-dependent monooxygenase. The enzyme not only catalyzed efficient degradation of a broad range of tetracycline analogues but also conferred resistance to these antibiotics in vivo. This is the first molecular characterization of an antibiotic-inactivating monooxygenase, the origins of which may lie in environmental bacteria.