Functional characterization of SbmA, a bacterial inner membrane transporter required for importing the antimicrobial peptide Bac7(1-35).
SbmA is an inner membrane protein of Gram-negative bacteria that is involved in the internalization of glycopeptides and prokaryotic and eukaryotic antimicrobial peptides, as well as of peptide nucleic acid (PNA) oligomers. The SbmA homolog BacA is required for the development of Sinorhizobium meliloti bacteroids within plant cells and favors chronic infections with Brucella abortus and Mycobacterium tuberculosis in mice. Here, we investigated functional features of SbmA/BacA using the proline-rich antimicrobial peptide Bac7(1-35) as a substrate. Circular dichroism and affinity chromatography studies were used to investigate the ability of SbmA to bind the peptide, and a whole-cell transport assay with fluorescently labeled peptide allowed the determination of transport kinetic parameters with a calculated Km value of 6.95 ± 0.89 μM peptide and a Vmax of 53.91 ± 3.17 nmol/min/mg SbmA. Use of a bacterial two-hybrid system coupled to SEC-MALLS (size exclusion chromatography coupled with multiangle laser light scattering) analyses established that SbmA is a homodimer in the membrane, and treatment of the cells with arsenate or ionophores indicated that the peptide transport mediated by SbmA is driven by the electrochemical gradient. Overall, these results shed light on the SbmA-mediated internalization of peptide substrates and suggest that the transport of an unknown substrate(s) represents the function of this protein.
Department of Life Sciences, University of Trieste, Trieste, Italy.
Giulia
G
Runti
Maria del Carmen
Mdel C
Lopez Ruiz
Tatiana
T
Stoilova
Rohanah
R
Hussain
Matthew
M
Jennions
Hassanul G
HG
Choudhury
Monica
M
Benincasa
Renato
R
Gennaro
Konstantinos
K
Beis
Marco
M
Scocchi
D000255:Adenosine Triphosphate / Q000378:metabolism; D023181:Antimicrobial Cationic Peptides / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011522:Protons; D011993:Recombinant Fusion Proteins
D023181:Antimicrobial Cationic Peptides; C481640:Bac7(1-35) peptide; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; C557704:PTD4-apoptin protein; D011522:Protons; D011993:Recombinant Fusion Proteins; C529573:SbmA protein, E coli; D000255:Adenosine Triphosphate; C488524:BacA protein, E coli; D010744:Phosphoric Monoester Hydrolases