Synthesis and Behavior of DNA Oligomers Containing the Ambiguous Z-Nucleobase 5-Aminoimidazole-4-carboxamide.

5-Amino-1-ß-D-ribofuranosylimidazole-4-carboxamide 5'-monophosphate (ZMP) is a central intermediate in de novo purine nucleotide biosynthesis. Its nucleobase moiety, 5-aminoimidazole-4-carboxamide (Z-base), is considered an ambiguous base that can pair with any canonical base owing to the rotatable nature of its 5-carboxamide group. This idea of ambiguous base pairing due to free rotation of the carboxamide has been applied to designing mutagenic antiviral nucleosides, such as ribavirin and T-705. However, the ambiguous base-pairing ability of Z-base has not been elucidated, because the synthesis of Z-base-containing oligomers is problematic. Herein, we propose a practical method for the synthesis of Z-base-containing DNA oligomers based on the ring-opening reaction of an N1-dinitrophenylhypoxanthine (HxaDNP) base. Thermal denaturation studies of the resulting oligomers revealed that the Z-base behaves physiologically as an A-like nucleobase, preferentially forming pairs with T. We tested the behavior of Z-base-containing DNA oligomers in enzyme-catalyzed reactions: in single nucleotide insertion, Klenow fragment DNA polymerase recognized Z-base as an A-like analog and incorporated dTTP as a complementary nucleotide to Z-base in the DNA template; in PCR amplification, Taq DNA polymerase similarly incorporated dTTP as a complementary nucleotide to Z-base. Our findings will contribute to the development of new mutagenic antiviral nucleoside analogs.

Genetic Basis Underlying the Hyperhemolytic Phenotype of Streptococcus agalactiae Strain CNCTC10/84.

Streptococcus agalactiae (group B streptococcus [GBS]) is a major cause of infections in newborns, pregnant women, and immunocompromised patients. GBS strain CNCTC10/84 is a clinical isolate that has high virulence in animal models of infection and has been used extensively to study GBS pathogenesis. Two unusual features of this strain are hyperhemolytic activity and hypo-CAMP factor activity. These two phenotypes are typical of GBS strains that are functionally deficient in the CovR-CovS two-component regulatory system. A previous whole-genome sequencing study found that strain CNCTC10/84 has intact covR and covS regulatory genes. We investigated CovR-CovS regulation in CNCTC10/84 and discovered that a single-nucleotide insertion in a homopolymeric tract in the covR promoter region underlies the strong hemolytic activity and weak CAMP activity of this strain. Using isogenic mutant strains, we demonstrate that this single-nucleotide insertion confers significantly decreased expression of covR and covS and altered expression of CovR-CovS-regulated genes, including that of genes encoding ß-hemolysin and CAMP factor. This single-nucleotide insertion also confers significantly increased GBS survival in human whole blood ex vivoIMPORTANCE Group B streptococcus (GBS) is the leading cause of neonatal sepsis, pneumonia, and meningitis. GBS strain CNCTC10/84 is a highly virulent blood isolate that has been used extensively to study GBS pathogenesis for over 20 years. Strain CNCTC10/84 has an unusually strong hemolytic activity, but the genetic basis is unknown. In this study, we discovered that a single-nucleotide insertion in an intergenic homopolymeric tract is responsible for the elevated hemolytic activity of CNCTC10/84.