RESUMEN
Relaxases of the MOBH family are often found on large plasmids, genetic islands and integrative conjugative elements. Many members of this family contain an N-terminal relaxase domain (TraI_2) followed by a disordered middle part and a C-terminal domain of unknown function (TraI_2_C). The TraI_2 domain contains two putative metal-binding motifs, an HD domain motif and an alternative 3H motif. TraI, encoded within the gonococcal genetic island of Neisseria gonorrhoeae, is the prototype of the MOBH family. SAXS experiments showed that TraI_2 and TraI_2_C form globular structures separated by an extended middle domain. The TraI_2 domain cleaves oriT-ssDNA in a site-specific Mn2+ or Co2+ dependent manner. The minimal oriT encompasses 50 nucleotides, requires an inverted repeat 3' of the nic-site and several nucleotides around nic for efficient cleavage. Surprisingly, no stable covalent relaxase-DNA intermediate was observed. Mutagenesis of conserved tyrosines showed that cleavage was abolished in the Y212A mutant, whereas the Y212F and Y212H mutants retained residual activity. The HD and the alternative 3H motifs were essential for cleavage and the HD domain residues D162 and D267 for metal ion binding. We propose that the active site binds two metal ions, one in a high-affinity and one in a low-affinity site.
Asunto(s)
Proteínas Bacterianas/genética , ADN Helicasas/genética , ADN Nucleotidiltransferasas/genética , ADN Bacteriano/genética , Islas Genómicas/genética , Neisseria gonorrhoeae/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión/genética , Dominio Catalítico , División del ADN , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Nucleotidiltransferasas/química , ADN Nucleotidiltransferasas/metabolismo , ADN Bacteriano/metabolismo , Metales/química , Metales/metabolismo , Neisseria gonorrhoeae/metabolismo , Unión Proteica , Dominios Proteicos , Homología de Secuencia de Ácido NucleicoRESUMEN
Neisseria gonorrhoeae is an obligate human pathogen that is responsible for the sexually-transmitted disease gonorrhea. N. gonorrhoeae encodes a T4SS within the Gonococcal Genetic Island (GGI), which secretes ssDNA directly into the external milieu. Type IV secretion systems (T4SSs) play a role in horizontal gene transfer and delivery of effector molecules into target cells. We demonstrate that GGI-like T4SSs are present in other ß-proteobacteria, as well as in α- and γ-proteobacteria. Sequence comparison of GGI-like T4SSs reveals that the GGI-like T4SSs form a highly conserved unit that can be found located both on chromosomes and on plasmids. To better understand the mechanism of DNA secretion by N. gonorrhoeae, we performed mutagenesis of all genes encoded within the GGI, and studied the effects of these mutations on DNA secretion. We show that genes required for DNA secretion are encoded within the yaa-atlA and parA-parB regions, while genes encoded in the yfeB-exp1 region could be deleted without any effect on DNA secretion. Genes essential for DNA secretion are encoded within at least four different operons.
Asunto(s)
Islas de CpG/genética , Neisseria gonorrhoeae/genética , Sistemas de Secreción Bacterianos/genética , Mapeo Cromosómico , Análisis Mutacional de ADN , ADN Bacteriano/genética , Gammaproteobacteria/genética , Genes Bacterianos , Humanos , Operón/genética , Plásmidos/metabolismo , Transcripción GenéticaRESUMEN
ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c(11) ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c(<11), c(12), c(13), c(14), and c(>14) rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c(12) instead of c(11), is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.
