Functional and Structural Study of the Dimeric Inner Membrane Protein SbmA

Abstract

g SbmA protein has been proposed as a dimeric secondary transporter. The protein is involved in the transport of microcins B17 and J25, bleomycin, proline-rich antimicrobial peptides, antisense peptide phosphorodiamidate morpholino oligomers, and peptide nucleic acids into the Escherichia coli cytoplasm. The sbmA homologue is found in a variety of bacteria, though the physiological role of the protein is hitherto unknown. In this work, we carried out a functional and structural analysis to determine which amino acids are critical for the transport properties of SbmA. We created a set of 15 site-directed sbmA mutants in which single conserved amino acids were replaced by glycine residues. Our work demonstrated that strains carrying the site-directed mutants V102G, F219G, and E276G had a null phenotype for SbmA transport functions. In contrast, strains carrying the single point mutants W19G, W53G, F60G, S69G, N155G, R190, L233G, A344G, T255G, N308G, and R385G showed transport capacities indistinguishable from those of strains harboring a wild-type sbmA. The strain carrying the Y116G mutant exhibited mixed phenotypic characteristics. We also demonstrated that those sbmA mutants with severely impaired transport capacity showed a dominant negative phenotype. Electron microscopy data and in silico three-dimensional (3D) homology modeling support the idea that SbmA forms a homodimeric complex, closely resembling the membrane-spanning region of the ATP-binding cassette transporter family. Direct mapping of the sbmA single point mutants on the protein surface allowed us to explain the observed phenotypic differences in transport ability.

Functional Characterization of SbmA, a Bacterial Inner Membrane Transporter Required for Importing the Antimicrobial Peptide Bac7(1-35)

“…Given the constraints of the two-hybrid system, which can functionally reconstitute the two subunits of the adenylate cyclase only on the cytoplasmic side of the bacterial membrane, in this study we provide indirect evidence on the localization of the C terminus of SbmA inside the bacterial cell, as the two subunits fused to the C termini gave a detectable interaction. In addition, when the T25 and T18 subunits were cloned at the N terminus of SbmA, the two monomers still continued to interact, suggesting that their N terminus ends are also on the cytoplasmic side, as detailed in the accompanying article by Corbalan et al (32). These data are in agreement with a previous study on the global topology of the E. coli inner membrane proteome (33), indicating that the C terminus of this protein is in the cytoplasm.…”

Mechanism of Escherichia coli Resistance to Pyrrhocoricin

“…In order to elucidate genetic determinants that confer resistance to pyrrhocoricin, we deter- mined and compared the entire genome sequences of the parent strain and one of its resistant mutants, Mut2. This analysis revealed only one difference: a chromosomal deletion of a 5,465-bp region in Mut2, removing (fully or partially) the following five genes: yaiU, encoding a hypothetical outer membrane protein (26); yaiV and yaiW, encoding putative DNA-binding transcriptional regulators; ampH, encoding a bifunctional DD-endopeptidase/DD-carboxypeptidase (29,30); and sbmA, encoding a dimeric transporter (31)(32)(33) (Fig. 2A).…”

“…SbmA is dimeric and shares homology with the transmembrane domain of ABC transporters; however, it lacks the nucleotide binding domain, and its function requires an electrochemical gradient across the inner membrane rather than ATP hydrolysis (32,33). Although the sbmA gene is found in many bacteria, in particular in most Enterobacteriaceae, its product has been shown not to be essential under laboratory conditions (31,35).…”

The C-Terminal Part of Microcin B Is Crucial for DNA Gyrase Inhibition and Antibiotic Uptake by Sensitive Cells

“…It is noteworthy that I69K and I69E were even less active than McB . We next tested the mutants on lawns of sensitive cells overproducing SbmA, an inner membrane transporter responsible for McB uptake (24,25). SbmA-overproducing cells were 10 times more sensitive to wild-type McB, indicating that the sensitivity of wild-type E. coli is limited by McB transport.…”