RESUMO
We have identified and partially characterized a putative HD domain hydrolase, LMOf2365_2464, which is highly expressed during listerial intracellular replication. LMOf2365_2464 is annotated as a putative HD domain-containing hydrolase. The ability of an isogenic mutant strain, F2365Δ2464, to adhere, invade and replicate in intestinal epithelial cells (Caco-2) was significantly lower than parent strain F2365. Colonization of mouse liver and spleen by L. monocytogenes F2365 was significantly higher than it was for the mutant. The recombinant protein showed phosphodiesterase activity in the presence of divalent metal ions, indicating its role in nucleotide metabolism. It has activity against several cyclic nucleotides and cyclic dinucleotides, but its strongest activity is against cyclic di-AMP and cyclic AMP. Based on this enzymatic activity, we designated LMOf2365_2464 phosphodiesterase E (PdeE).
Assuntos
Hidrólise , Listeria monocytogenes/enzimologia , Listeria monocytogenes/patogenicidade , Nucleotídeos/metabolismo , Diester Fosfórico Hidrolases/metabolismo , Virulência , Animais , Aderência Bacteriana , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Células CACO-2 , AMP Cíclico/metabolismo , DNA Bacteriano , Modelos Animais de Doenças , Ensaios Enzimáticos , Feminino , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Humanos , Concentração de Íons de Hidrogênio , Listeria monocytogenes/genética , Listeria monocytogenes/crescimento & desenvolvimento , Listeriose/microbiologia , Fígado/microbiologia , Manganês/metabolismo , Camundongos , Mutagênese , Mutação , Diester Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas Recombinantes , Baço/microbiologia , Temperatura , Virulência/genéticaRESUMO
The structure of Archaeal chromatin or nucleoid is believed to have characteristics similar to that found in both eukaryotes and bacteria. Recent comparative studies have suggested that DNA compaction in Archaea requires a bridging protein (e.g., Alba) along with either a wrapping protein (e.g., a histone) or a bending protein such as Sac7d. While X-ray crystal structures demonstrate that Sac7d binds as a monomer to create a significant kink in duplex DNA, the structure of a multiprotein-DNA complex has not been established. Using cross-linked dimers of Sac7d with a defined orientation, we present evidence that indicates that Sac7d is able to largely coat duplex DNA in vivo by binding in alternating head-to-head and tail-to-tail orientations. Although each Sac7d monomer promotes a significant kink of nearly 70°, coated DNA is expected to be largely extended because of compensation of repetitive kinks with helical symmetry.