Browsing School, Graduate by Subject "dCTP pyrophosphoydrolase"
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Characterization of nudix hydrolases from predatory Bdellovibrio and like-organismsThe Bdellovibrio and like organisms are motile gram negative bacteria that prey other gram negative bacteria by entering into their periplasm and feeding from the degraded host macromolecules of the prey. The Nudix hydrolases are a superfamily of phosphoanhydrases that catalyze the hydrolysis of a nucleoside diphosphate linked to different moieties, X (NUDIX). They have in common the signature sequence GX<sub>5</sub>EX<sub>7</sub>REUXEEXGU (U=I, L or V). Some examples of phenotypes associated with Nudix enzymes are dGTPases involved in preventing mutations by removing damaged nucleotides from the nucleotide pool (mutT gene) and diadenosine tetraphosphatases involved in host invasion. The Bd2220 and Bm2 ORFs from the predatory Bdellovibrio bacteriovorus HD100 and Bacteriovorax marinus SJ, respectively, have the Nudix consensus sequence. The hypothesis proposed is that these predatory microorganisms contain Nudix hydrolases in their genome that could be related to their predatory behavior. To address this hypothesis, Bd2220 and Bm2 gene, both annotated as mutT gene at the genbank, were tested through an antimutator complementation assay. Both genes were confirmed to be not orthologue to E. coli mut T gene. The Bd2220 gene was further characterized by cloning, protein purification, enzymatic assay, subcellular localization, and a genomic in-frame deletion of the Bd2220 ORF. The BD2220 enzyme, localized at the periplasmic space of the Bd cell, was found to be a dCTP pyrophosphatase with a K<sub>M</sub> of 0.199 mM. The in-frame deletion of Bd2220 gene was conducted using the suicide vector pSSK10 and counter selecting the knock out strains (∆Bd2220) with 5% sucrose. The ∆Bd2220 showed phenotypic differentiation compared with the wild type (wt) strain. The lack of BD2220 enzyme activity caused the formation of aggregations in the ∆Bd2220 strain liquid cultures during the development stage of the bdelloplast. These aggregates were dissolved with DNase I treatment. In addition, the bdelloplast progeny was reduced, plaques were smaller and viable prey cells were observed even after several days of incubation. The proposed model is that formation of aggregates is due by the leaking of unprocessed DNA from the bdelloplast, which acted as extracellular DNA, inducing aggregation. All these findings suggest that the BD2220 enzyme is involved in a mechanism to process DNA during the bdelloplast development of the predatory life cycle.