INVESTIGADORES
COMERCI Diego Jose
artículos
Título:
N-terminal-capturing screening system for the isolation of Brucella abortus genes encoding surface exposed and secreted proteins.
Autor/es:
MARCHESINI, M. I.; UGALDE, J. E.; CZIBENER, C.; DIEGO JOSE COMERCI; UGALDE, R. A.
Revista:
MICROBIAL PATHOGENESIS
Editorial:
Elsevier
Referencias:
Año: 2004 vol. 37 p. 95 - 105
ISSN:
0882-4010
Resumen:
Secreted as well as surface exposed proteins are assumed to play major roles in bacterial virulence. In this report we describe the
construction of an N-terminal protein-capturing system and its use for the isolation of Brucella abortus S2308 genes coding for putative
surface exposed or secreted proteins. For this purpose, a cloning vector that generates gene fusions to a ribosome binding site and start codon
deficient Chloramphenicol Acetyl Transferase (CAT) reporter gene was constructed and the resulting library introduced into B. abortusBrucella abortus S2308 genes coding for putative
surface exposed or secreted proteins. For this purpose, a cloning vector that generates gene fusions to a ribosome binding site and start codon
deficient Chloramphenicol Acetyl Transferase (CAT) reporter gene was constructed and the resulting library introduced into B. abortusB. abortus
S2308 and virB mutant strains. Secreted translational fusions were identified by determining CAT activity in culture supernatants. Secretion
was confirmed by Western Blot using a polyclonal anti-CAT antibody. A total of 864 clones were screened and 10 genes encoding putative
secreted/surface exposed proteins were identified. Seven are Brucella proteins with an assigned function, whereas three are hypothetical
proteins. The number of amino acid residues that promotes CAT secretion varies from 5 to 386 and no conserved motifs were detected.
Secretion in a virB mutant background of some of the isolated fusion proteins was also determined. Interestingly, some hybrid proteins
seemed to require a full VirB system for their secretion.virB mutant strains. Secreted translational fusions were identified by determining CAT activity in culture supernatants. Secretion
was confirmed by Western Blot using a polyclonal anti-CAT antibody. A total of 864 clones were screened and 10 genes encoding putative
secreted/surface exposed proteins were identified. Seven are Brucella proteins with an assigned function, whereas three are hypothetical
proteins. The number of amino acid residues that promotes CAT secretion varies from 5 to 386 and no conserved motifs were detected.
Secretion in a virB mutant background of some of the isolated fusion proteins was also determined. Interestingly, some hybrid proteins
seemed to require a full VirB system for their secretion.Brucella proteins with an assigned function, whereas three are hypothetical
proteins. The number of amino acid residues that promotes CAT secretion varies from 5 to 386 and no conserved motifs were detected.
Secretion in a virB mutant background of some of the isolated fusion proteins was also determined. Interestingly, some hybrid proteins
seemed to require a full VirB system for their secretion.virB mutant background of some of the isolated fusion proteins was also determined. Interestingly, some hybrid proteins
seemed to require a full VirB system for their secretion.