Detailed mutational analysis examines the roles of individual residues of the Vga(A) linker in determining the antibiotic resistance phenotype. It defines a narrowed region of residues 212 to 220 whose composition determines the resistance specificity to lincosamides, pleuromutilins, and/or streptogramins A. From the analogy with the recently described function of the homologous ABC-F protein EttA as a translational factor, we infer that the Vga(A) linker interacts with the ribosome and directly or indirectly affects the binding of the respective antibiotic.T he ABC-F family of ABC transporters comprises soluble proteins with two nucleotide binding domains (NBD) (Pfam accession number PF00005) separated by a flexible linker of approximately 80 amino acid residues. In contrast to typical ABC transporters, ABC-F proteins do not have any transmembrane domain. They usually participate in nontransport cellular functions, including translation regulation and DNA excision, and are also involved in antibiotic resistance. Antibiotic resistance ABC-F proteins are collectively referred to as ARE (antibiotic resistance) proteins (1). The mechanism of resistance conferred by ARE proteins is not well understood.Vga(A) is one of the most-studied ARE proteins (2-4). Since the first report of Vga(A) as a streptogramin A (SgA) resistance determinant (5), several variants differing in their ability to confer resistance to SgA, lincosamides (L), and/or pleuromutilins (P) were reported ( Fig. 1) (4, 6-11). For clarity in this paper, we refer to unspecified Vga(A) variants as Vga(A)*, use Vga(A) only for the originally described Vga(A) protein (NCBI protein database accession number AAA26684) conferring resistance to SgA (5), and use Vga(A) LC (the LC subscript indicates resistance to lincomycin and clindamycin) for the variant with NCBI protein database accession number ABH10964 that confers resistance to both L and SgA (4).Comparison of the Vga(A) and Vga(A) LC proteins showed that the shift in resistance between SgA and L is determined by amino acid substitutions L212S, G219V, A220T, and G226S clustered in a sequence only 15 amino acids long within the interdomain linker, which is the main source of polymorphism for all Vga(A)* proteins ( Fig. 1) (4). Nothing is known about how these individual variable residues contribute to antibiotic specificity. We set out to decipher the relationship between the linker variability of Vga(A)* variants and the resistance phenotype using detailed mutational study of the Vga(A) and Vga(A) LC linkers. Implications of our results for the proposed resistance mechanism are discussed.We have mutated amino acid residues of the linker in positions 212, 219, 220, and 226 to create a set of mutant forms of Vga(A)* that contain all but one of the possible combinations of Vga(A)-and Vga(A) LC -specific residues in their respective positions. Sitedirected mutagenesis was performed using two complementary primers according to the QuikChange site-directed mutagenesis kit protocol (Stratagene). The construct pB...

Vga(A) protein variants confer different levels of resistance to lincosamides, streptogramins A and pleuromutilins (LSAP) by displacing antibiotics from the ribosome. Here we show that expression of vga(A) variants from Staphylococcus haemolyticus is regulated by cis-regulatory RNA in response to the LSAP antibiotics by the mechanism of ribosome-mediated attenuation. The specificity of induction depends on the Vga(A)-mediated resistance rather than on the sequence of the riboregulator. Fine-tuning between Vga(A) activity and its expression in response to the antibiotics may contribute to the selection of more potent Vga(A) variants because newly acquired mutation can be immediately phenotypically manifested.