9) Differential effects of point mutations on phenotypes of the inner-membrane protein SbmA

CORBALÁN NATALIA S; MARÍA FERNANDA POMARES; ADLER CONRADO; DE CRISTOBAL, RICARDO; PAULA A. VINCENT

Salta

Congreso; XXXIX Annual Meeting of the Argentinian Biophysical Society and 3rd Latin American Protein Society Meeting; 2010

The E. coli sbmA gene encodes a 406-amino-acid inner membrane protein which was predicted to have seven transmembrane-spanning domains1. sbmA gene homologues were found in a variety of bacteria. However, the physiological role of this protein is, until now, unknown. SbmA catalyzes the internalization of antibiotics containing rings thiazoles as microcin B17 and the glycopeptide antibiotic bleomycin2. At first it was thought that the recognition motive for this transporter was the thiazole ring. Later, with the discovery of MccJ25 as a new substrate of SbmA, this assumption was rejected, since the latter antibiotic lacks this structural feature3. Recently, Mattiuzzo et al. showed that SbmA is required for the uptake of proline-rich antimicrobial peptides from eukaryotic origin4. In this work we carried out a genetic analysis to determine which amino acids in the protein are critical for this different protein function. We have created a set of 16 site-directed SbmA mutants in which selected individual conserved amino acids were replaced with glycine residues. The mutants were characterized to determine how the various amino acid changes affected the different phenotypes associated with SbmA function. We observed that the mutants V102G, F219G and E276G had null phenotypes for all functions of SbmA, while mutants W53G, F60G, S69G, N155G, R190, L233G, T255G, A344G and R385G were indistinguishable from wild-type strains. The remaining 5 site-directed mutants demonstrate mixed phenotypic characteristics. The study of specificity in the transport of SbmA could give some evidence about the nature of the natural substrate of this transporter protein.