Sensing domain and extension rate of a family B-type DNA polymerase determine the stalling at a deaminated base.

The uracil-sensing domain in archaeal family B-type DNA polymerases recognizes pro-mutagenic uracils in the DNA template, leading to stalling of DNA polymerases. Here, we describe our new findings regarding the molecular mechanism underpinning the stalling of polymerases. We observed that two successive deaminated bases were required to stall TNA1 and KOD1 DNA polymerases, whereas a single deaminated base was enough for stalling Pfu DNA polymerase, in spite of the virtually identical uracil-sensing domains. TNA1 and KOD1 DNA polymerases have a much higher extension rate than Pfu DNA polymerase; decreasing the extension rate resulted in stalling by TNA1 and KOD1 DNA polymerases at a single deaminated base. These results strongly suggest that these polymerases require two factors to stop DNA polymerization at a single deaminated base: the presence of the uracil-sensing domain and a relatively slow extension rate.

Characterization of a dITPase from the hyperthermophilic archaeon Thermococcus onnurineus NA1 and its application in PCR amplification.

In this study, we found that deoxyinosine triphosphate (dITP) could inhibit polymerase chain reaction (PCR) amplification of various family B-type DNA polymerases, and 0.93% dITP was spontaneously generated from deoxyadenosine triphosphate during PCR amplification. Thus, it was hypothesized that the generated dITP might have negative effect on PCR amplification of family B-type DNA polymerases. To overcome the inhibitory effect of dITP during PCR amplification, a dITP pyrophosphatase (dITPase) from Thermococcus onnurineus NA1 was applied to PCR amplification. Genomic analysis of the hyperthermophilic archaeon T. onnurineus NA1 revealed the presence of a 555-bp open reading frame with 48% similarity to HAM1-like dITPase from Methanocaldococcus jannaschii DSM2661 (NP_247195). The dITPase-encoding gene was cloned and expressed in Escherichia coli. The purified protein hydrolyzed dITP, not deoxyuridine triphosphate. Addition of the purified protein to PCR reactions using DNA polymerases from T. onnurineus NA1 and Pyrococcus furiosus significantly increased product yield, overcoming the inhibitory effect of dITP. This study shows the first representation that removing dITP using a dITPase enhances the PCR amplification yield of family B-type DNA polymerase.

Differential stringent responses of Streptomyces coelicolor M600 to starvation of specific nutrients.

This study focused on the involvement of the unusual nucleotide (p)ppGpp, a stringent factor, during the morphological and physiological differentiation of Streptomyces coelicolor. Two genes, relA and rshA, were disrupted to demonstrate the roles of the stringent factor in the differentiation. The intracellular concentration of (p)ppGpp in the wild-type (M600) and disrupted mutants was measured in relation to the intentional starvation of a specific nutrient such as carbon, nitrogen, and phosphate or the in situ depletion of nutrients in a batch culture. As a result, it was found that the morphological characteristic of the deltarelA mutant was a bld phenotype forming condensed mycelia, whereas the deltarshA mutant grew fast-forming spores and straightforward mycelia. In both mutants, the production of actinorhodin (Act) was completely abolished, yet the undecylprodigiosin (Red) production was increased. Intracellular (p)ppGpp was detected in the deltarelA mutant in the case of limited phosphate, yet not with limited carbon or nitrogen sources. In contrast, (p)ppGpp was produced in the deltarshA mutant under limited carbon and nitrogen conditions. Therefore, (p)ppGpp in S. coelicolor was found to be selectively regulated by either the RelA or RshA protein, which was differentially expressed in response to the specific nutrient limitation. These results were also supported by the in situ ppGpp production during a batch culture. Furthermore, it is suggested that RelA and RshA are bifunctional proteins that possess the ability to both synthesize and hydrolyze (p)ppGpp.

Engineering of primary carbohydrate metabolism for increased production of actinorhodin in Streptomyces coelicolor.

The objectives of the current studies were to determine the roles of key enzymes in central carbon metabolism in the context of increased production of antibiotics in Streptomyces coelicolor. Genes for glucose-6-phosphate dehydrogenase and phosphoglucomutase (Pgm) were deleted and those for the acetyl coenzyme A carboxylase (ACCase) were overexpressed. Under the conditions tested, glucose-6-phosphate dehydrogenase encoded by zwf2 plays a more important role than that encoded by zwf1 in determining the carbon flux to actinorhodin (Act), while the function of Pgm encoded by SCO7443 is not clearly understood. The pgm-deleted mutant unexpectedly produced abundant glycogen but was impaired in Act production, the exact reverse of what had been anticipated. Overexpression of the ACCase resulted in more rapid utilization of glucose and sharply increased the efficiency of its conversion to Act. From the current experiments, it is concluded that carbon storage metabolism plays a significant role in precursor supply for Act production and that manipulation of central carbohydrate metabolism can lead to an increased production of Act in S. coelicolor.

Two relA/spoT homologous genes are involved in the morphological and physiological differentiation of Streptomyces clavuligerus.

This study is focused on the involvement of the unusual nucleotide (p)ppGpp during the morphological and physiological differentiation of Streptomyces clavuligerus. In particular, the functional and structural elements of two genes encoding the proteins RelA and Rsh were identified. The relA gene encodes an 843 aa protein (RelA), while the rsh gene encodes a 738 aa protein (Rsh). The relA and rsh genes were disrupted by the insertion of a hygromycin resistance gene and an apramycin resistance gene, respectively. The synthesis of ppGpp in the relA gene-disrupted mutant was completely eliminated under conditions of starvation for amino acids, whereas synthesis persisted, but was greatly reduced in the rsh gene-disrupted mutant. The relA gene-disrupted mutant had a bald appearance on agar plate cultures and retarded growth in submerged culture, while the rsh-disrupted mutant was unchanged in growth characteristics relative to the wild-type culture. The production of both clavulanic acid and cephamycin C were completely abolished in the relA-disrupted mutant. Thus, it is concluded that the relA gene rather than rsh is essential for morphological and physiological differentiation in S. clavuligerus and that RelA primarily governs the stringent response of S. clavuligerus to starvation for amino acids.