The Ability of a Recombinant Escherichia coli Strain to Synthesize 2-C-Methyl-D-Erythritol-2,4-Cyclopyrophosphate Correlates with Its Tolerance to In Vitro Induced Oxidative Stress and to the Bactericidal Action of Murine Peritoneal Macrophages Olga D. Ogrel, 1 Konstantin V. Fegeding, 2, * Elena F. Kharatian, 1 Andrew B. Sudarikov, 2 Dmitry N. Ostrovsky 1 1 Bakh Institute of Biochemistry, RussianAcademy of Sciences, Leninsky Prospect 33, 117071 Moscow, Russia 2 Hematology Research Center, RussianAcademy of Medical Sciences, Novozykovsky per.4a, 125167 Moscow, Russia Abstract. A number of bacteria are able to synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophos- phate (BOSS) in response to oxidative stress. Here we show that the ability to synthesize BOSS can be genetically transferred from Corynebacterium ammoniagenes to Escherichia coli. A total DNA library from C. ammoniagenes ATCC 6872 established in the pBluescript SKII1 vector backbone was transfected into E. coli XL-1 blue. Recombinant clone 2–31, which was resistant to redox- cycling agents, was selected. NMR studies showed that this clone was able to synthesize BOSS. We also studied the resistance of clone 2–31 to the bactericidal action of macrophages. Clone 2–31 cells had better survival within murine peritoneal macrophages than parental E. coli XL-1-blue cells. Since the ability to synthesize BOSS correlates with increased survival of bacteria within macro- phages, we suggest that the pathogenicity of Corynebacteria could be mediated through the synthesis of BOSS. Corynebacterium ammoniagenes ATCC 6872 cells synthesize 2-C-methyl-D-erythritol-2,4-cyclopyrophos- phate (BOSS) in response to the action of a number of redox-cycling agents [3]. The chemical structure of BOSS has been established and confirmed by the contrary synthesis [6, 7, 10]. The ability to synthesize BOSS was found in different microorganisms includ- ing Mycobacterium smegmatis and M. phlei [4]. On the other hand, a group of novel nonproteinaceous superantigens responsible for g/d T-cell activation and proliferation have been detected recently in M. tuber- culosis lysates [1, 8, 13]. The chemical structure of the superantigens is not established yet, but it has been proposed that the substances are of low molecular weight carbohydrate pyrophosphate-like nature, and one of them may be isopentenyl pyrophosphate [13]. However, nothing is known about the genes involved in biosynthesis of novel nonproteinaceous superanti- gens and their relation to the pathogenicity of bacteria. Here we report the cloning of an 8-kb segment of the C. ammoniagenes genome that confers the ability to synthesize BOSS to E. coli cells. The data suggesting a possible relationship between BOSS and Corynebacte- ria pathogenicity are also presented. Materials and Methods Reagents. Benzylviologen dichloride (BV), menadione-SO 3 , ampi- cillin sodium salt, and gentamicin sulfate were purchased from Sigma Chemical Company (St. Louis, Missouri, USA). N,N8-bis-(2- carbamoylethyl)-4,48-dipyridilium hydrochloride (AV) was the gift of Y. Zelihover (Moscow). Strains. Corynebacterium ammoniagenes ATCC 6872 was pur- chased from the American Type Culture Collection. E. coli XL1-blue and pBluescript SKII1 plasmid DNA were from Strata- Gene. 31 P-NMR. The preparation of samples for NMR was described previously [4]. A ‘‘Gemini 300’’ spectrometer was used for the 31 P-NMR experiments. Bacteria were grown in LB broth with 50 μg/ml ampicillin overnight and diluted 1:50 with fresh medium. Indicated amounts * Present address: Department of Experimental Pathology, Divi- sion of Pathology, Walter Reed Army Institute of Research, 14th and Dahlia Sts. N.W., Washington, DC 20307-5100, USA Correspondence to: K.V. Fegeding CURRENT MICROBIOLOGY Vol. 32 (1996), pp. 225–228 An International Journal R Springer-Verlag New York Inc. 1996