RESUMEN
Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) whole-cell fingerprinting was used for characterization of 66 reference strains of Gallibacterium. The 4 recognised Gallibacterium species and Gallibacterium genomospecies 1 yielded reproducible and unique mass spectrum profiles, which were confirmed with Bruker Biotyper reference database version 3. The reproducibility of MALDI-TOF MS results were evaluated varying the age and storage of the cultures investigated. Reliable species identification was possible for up to 8 days of storage at 4°C and less reliable if the bacteria were stored at room temperature (20°C). However, if the strains were grown longer than 48h at 37°C under microaerobic atmosphere, poor identification results were obtained, due to changes in protein profile. The MALDI-TOF MS results of all 66 strains demonstrated 87.9% concordance with results based upon biochemical/physiological characterization. In addition, diversities outlined by MALDI-TOF MS were verified by sequencing the rpoB (n=43), 16S rRNA (n=28), infB (n=14), and recN (n=14) genes (multilocus sequence analysis, MLSA). In addition, discrepancies were observed between some of the genes sequenced. Results obtained demonstrated that MALDI-TOF MS fingerprinting represents a fast and reliable method for identification and differentiation of the 4 recognised Gallibacterium species and possible a fifth species Gallibacterium genomospecies 1, with applications in clinical diagnostics.
Asunto(s)
Tipificación de Secuencias Multilocus/métodos , Pasteurellaceae/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Enzimas de Restricción del ADN/genética , Enzimas de Restricción del ADN/aislamiento & purificación , Bases de Datos Genéticas , Biblioteca de Genes , Pasteurellaceae/clasificación , Fenotipo , Factor 2 Procariótico de Iniciación/genética , Factor 2 Procariótico de Iniciación/aislamiento & purificación , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/aislamiento & purificación , Programas InformáticosRESUMEN
The structure of the intact heterotrimeric translation initiation factor 2 (e/aIF2) is of great interest due to its key role in the initiator tRNA delivery to the ribosome and in translation initiation regulation in eukaryotes and archaea. We have chosen aIF2 from the hyperthermophilic archaeobacterium Sulfolobus solfataricus (SsoIF2) as an object for crystallization and structural investigations. Genes of the SsoIF2 subunits alpha, beta, and gamma were cloned and superexpressed. A method for heterotrimer SsoIF2alphabetagamma purification was elaborated with at least 95% purity. Highly ordered crystals of the full-sized SsoIF2, reflecting X-rays at the resolution up to 2.8 A, were obtained for the first time.
Asunto(s)
Proteínas Arqueales/química , Proteínas Arqueales/aislamiento & purificación , Factor 2 Procariótico de Iniciación/química , Factor 2 Procariótico de Iniciación/aislamiento & purificación , Sulfolobus solfataricus/química , Proteínas Arqueales/genética , Cristalización , Factor 2 Procariótico de Iniciación/genética , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Eukaryotic and archaeal translation initiation factors 2, heterotrimers that consist of alpha-, beta-, and gamma-subunits, deliver methionylated initiator tRNA to a small ribosomal subunit in a manner that depends on GTP. To evaluate correlation of the function and association of the subunits, we used isothermal titration calorimetry to analyze the thermodynamics of the interactions between the alpha- and gamma-subunits in the presence or absence of a nonhydrolyzable GTP analog or GDP. The alpha-subunits bound to the gamma-subunit with large heat capacity change (DeltaC(p)) values. The DeltaH and DeltaC(p) values for the interaction between the alpha- and gamma-subunits varied in the presence of the GTP analog but not in the presence of GDP. These results suggest that the binding of both the alpha-subunit and GTP changes the conformation of the switch region of the gamma-subunit and increases the affinity of the gamma-subunit for tRNA.
Asunto(s)
Proteínas Arqueales/química , Guanosina Trifosfato/química , Factor 2 Procariótico de Iniciación/química , Termodinámica , Proteínas Arqueales/genética , Cristalografía , Escherichia coli/genética , Guanosina Difosfato/química , Guanosina Trifosfato/análogos & derivados , Factor 2 Procariótico de Iniciación/genética , Factor 2 Procariótico de Iniciación/aislamiento & purificación , Conformación Proteica , Subunidades de Proteína/química , Subunidades de Proteína/genética , Sulfolobus solfataricusRESUMEN
Bacterial translation initiation factor IF2 is a multidomain protein that is an essential component of a system for ensuring that protein synthesis begins at the correct codon within a messenger RNA. Full-length IF2 from Escherichia coli and seven fragments of the protein were expressed, purified, and characterized using nuclear magnetic resonance (NMR) and circular dichroism (CD) methods. Interestingly, resonances of the 6 kD IF2N domain located at the extreme N terminus of IF2 can be clearly identified within the NMR spectra of the full-length 97-kD protein. (15)N NMR relaxation rate data indicate that (1) the IF2N domain is internally well ordered and tumbles in solution in a manner that is independent of the other domains of the IF2 protein, and (2) the IF2N domain is connected to the C-terminal regions of IF2 by a flexible linker. Chemical shifts of resonances within the isolated IF2N domain do not significantly differ from those of the corresponding residues within the context of the full-length 97-kD protein, indicating that IF2N is a structurally independent unit that does not strongly interact with other regions of IF2. CD and NMR data together provide evidence that Domains I-III of IF2 have unstructured and flexible regions as well as substantial helical content; CD data indicate that the helical content of these regions decreases significantly at temperatures above 35 degrees C. The features of structurally well-ordered N- and C-terminal domains connected by a flexible linker with significant helical content are reminiscent of another translation initiation factor, IF3.