Epsilon Open Archive

Abstract

Mycoplasmas (class mollicutes) are the smallest self-replicating bacteria. They are obligate parasites in a wide range of organisms, including humans, plants, and animals. This thesis describes studies related to Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), the etiological agent of contagious bovine pleuropneumoniae, Mycoplasma capricolum (Mcap), causing caprine pneumonia, Mycoplasma hyopneumoniae (Mhp), the etiological agent of porcine enzootic pneumonia, Ureaplasma urealyticum (Uu), a commensal of human urogenital tract causing nongonococcal urethritis, Mycoplasma genitalium (Mg), a causative agent of nongonococcal, chlamydia-negative urethritis, and Mycoplasma pneumoniae (Mpn) causing atypical pneumonia and tracheobronchitis. The first part concerns deoxynucleoside metabolism in Uu and growth inhibition of Uu and Mpn by nucleoside analogs. The uptake and metabolism of the deoxynucleosides, thymidine (dThd), deoxyuridine, deoxycytidine were studied and all these deoxynucleosides were readily taken up by Uu and dThd showed the highest rate of uptake. Pyrimidine nucleosides e.g., fluoropyrimidines gave complete inhibition of Uu growth. The addition of dUrd and dThd to the growth medium abolished inhibitory effect. These results suggested a new pathway of thymidylate synthesis in Uu. The second part deals with identification of a new family of flavin-dependent thymidylate synthases (FDTS) in mycoplasmas. Thymidylate synthases (TS) are essential enzymes in the de novo synthesis of thymidylate and are coded either by thyA or thyX genes. Analysis of the fully sequenced Mycoplasma genomes revealed that there were no TS genes annotated in the genomes of Uu, MmmSC, Mhp, and Mcap. Still, TS activities were detected in Uu and MmmSC extracts. By using a bioinformatics approach together with biochemical methods putative genes for (FDTS) were identified in Uu, MmmSC, Mhp and Mcap genomes and functional verified. New members of this family were also discovered in Mg and Mpn, which also have the thyA genes. These mycoplasma FDTS have similar catalytic activity to the ThyX proteins, but share very little sequence homology to the known bacterial ThyX. The sequence homologies within Mycoplasma FDTS were also very low. Since Mycoplasma FDTS share no sequence or structural homology to ThyA or ThyX this makes them promising targets for future design of new antibiotics.