The lack of a complete genome sequence for V tapetis and, theref

The lack of a complete genome sequence for V. tapetis and, therefore, BMS-354825 cost the unavailability of an appropriate database is reflected in our study, where only 27 of the 60 proteins sequenced by MS were identified, and indicates the necessity for further studies to characterize the proteome of this pathogen. In comparison with proteomics, genetic procedures such as MLSA have the advantage that the information is fairly consistent; the procedure is unaffected

by the growth conditions of bacteria and can generate highly reproducible and portable data, which enables the comparison of results between laboratories using the public online databases. MLSA has been demonstrated to be a powerful, both intra- and interspecific, discriminative tool within the Vibrio genus (Thompson et al., 2004, 2005, 2007, 2009; Pascual et al., 2010). The choice of the protein encoding genes for the MLSA is the most important aspect in a correct MLSA analysis. This choice is particularly difficult in the case of a set of strains

belonging to the same species or to closely related taxa, due for the need for genes that are able to measure such low variability. In our case, each selected gene has been used previously for Vibrio species (Thompson et al., 2004, 2005, 2007) and the results obtained were in agreement with those reached using genotyping methods (Castro et al., 1996, 1997; Romalde et al., 2002; Rodríguez et al., 2006). Both methods, 2D-PAGE and MLSA, rendered trees

with similar topology, the clam isolates appearing to be more closely related than those from fish. In addition, the relative branching order is clearly in agreement with the three genetic groups previously described on the basis of typing methods (Romalde et al., 2002; Rodríguez et al., 2006). The congruence between the results obtained in the phylogenetic study of housekeeping genes (conservative approach) and the analysis of the whole proteome of the isolates (dynamic approach) provide an inter-validation of the techniques. In conclusion, the proteomic approach using 2D-PAGE can be a useful complementary tool NADPH-cytochrome-c2 reductase for the study of the intraspecific variability of V. tapetis. In addition, the method does not require prior information about the genome sequence and possesses the added value of describing gene expression at protein level, which can furnish helpful information on host–pathogen interaction and pathogenic processes. This work was partially supported by Grants AGL2006-13208-C02-01 and AGL2010-18438 from the Ministerio de Ciencia e Innovación (MICINN) (Spain). The kind donation of strains by Drs J.J. Borrego (University of Málaga, Spain) and T.H. Birkbeck (University of Glasgow, UK) is gratefully acknowledged. S.B. and J.B.C.

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