N-terminal-capturing screening system for the isolation of Brucella abortus genes encoding surface exposed and secreted proteins.
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. 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.
Marchesini
María Inés
MI
Instituto de Investigaciones Biotecnológicas, Universidad Nacional de General San Martín (IIB-UNSAM), Consejo Nacional de Investigaciones Científicas, Provincia de Buenos Aires, Argentina.
Ugalde
Juan Esteban
JE
Czibener
Cecilia
C
Comerci
Diego José
DJ
Ugalde
Rodolfo Augusto
RA
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D002003:Brucella abortus / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism / Q000472:pathogenicity; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism*; D003001:Cloning, Molecular; D005822:Genetic Vectors; D016254:Mutagenesis, Insertional; D014774:Virulence; D037521:Virulence Factors / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D037521:Virulence Factors; D015500:Chloramphenicol O-Acetyltransferase