Bacitracin resistance (bacitracin MIC, >256 g ml ؊1 ) has been reported in Enterococcus faecalis, and in the present study we report on the genetic basis for this resistance. Mutagenesis was carried out with transposon Tn917 to select for E. faecalis mutants with decreased resistance to bacitracin. Two bacitracin-sensitive mutants (MICs, 32 g ml ؊1 ) were obtained and Tn917 insertions were mapped to genes designated bcrA and bcrB. The amino acid sequences of BcrA (ATP-binding domain) and BrcB (membrane-spanning domain) are predicted to constitute a homodimeric ATP-binding cassette (ABC) transporter, the function of which is essential for bacitracin resistance in E. faecalis. The bcrA and bcrB genes were organized in an operon with a third gene, bcrD, that had homology to undecaprenol kinases. Northern analysis demonstrated that bcrA, bcrB, and bcrD were transcribed as a polycistronic message that was induced by increasing concentrations of bacitracin but not by other cell wall-active antimicrobials (e.g., vancomycin). Upstream of the bcrABD operon was a putative regulatory gene, bcrR. The bcrR gene was expressed constitutively, and deletion of bcrR resulted in a bacitracin-sensitive phenotype. No bcrABD expression was observed in a bcrR mutant, suggesting that BcrR is an activator of genes essential for bacitracin resistance (i.e., bcrABD). The bacitracin resistance genes were found to be located on a plasmid that transferred at a high frequency to E. faecalis strain JH2-2. This report represents the first description of genes that are essential for acquired bacitracin resistance in E. faecalis.

NOTE

We describe here purification and biochemical characterization of the F 1 F o -ATP synthase from the thermoalkaliphilic organism Bacillus sp. strain TA2.A1. The purified enzyme produced the typical subunit pattern of an F 1 F o -ATP synthase on a sodium dodecyl sulfate-polyacrylamide gel, with F 1 subunits ␣, ␤, ␥, ␦, and and F o subunits a, b, and c. The subunits were identified by N-terminal protein sequencing and mass spectroscopy. A notable feature of the ATP synthase from strain TA2.A1 was its specific blockage in ATP hydrolysis activity. ATPase activity was unmasked by using the detergent lauryldimethylamine oxide (LDAO), which activated ATP hydrolysis >15-fold. This activation was the same for either the F 1 F o holoenzyme or the isolated F 1 moiety, and therefore latent ATP hydrolysis activity is an intrinsic property of F 1 . After reconstitution into proteoliposomes, the enzyme catalyzed ATP synthesis driven by an artificially induced transmembrane electrical potential (⌬). A transmembrane proton gradient or sodium ion gradient in the absence of ⌬ was not sufficient to drive ATP synthesis. ATP synthesis was eliminated by the electrogenic protonophore carbonyl cyanide m-chlorophenylhydrazone, while the electroneutral Na ؉ /H ؉ antiporter monensin had no effect. Neither ATP synthesis nor ATP hydrolysis was stimulated by Na ؉ ions, suggesting that protons are the coupling ions of the ATP synthase from strain TA2.A1, as documented previously for mesophilic alkaliphilic Bacillus species. The ATP synthase was specifically modified at its c subunits by N,N-dicyclohexylcarbodiimide, and this modification inhibited ATP synthesis.

SummaryDespite the thermodynamic problem imposed on alkaliphilic bacteria of synthesizing adenosine triphosphate (ATP) against a large inverted pH gradient and consequently a low electrochemical proton potential, these bacteria still utilize a proton-coupled F1Fo-ATP synthase to synthesize ATP. One potential solution to this apparent thermodynamic problem would be the operation of a larger oligomeric c ring, which would raise the ion to ATP ratio, thus facilitating the conversion of a low electrochemical potential into a significant phosphorylation potential. To address this hypothesis, we have purified the oligomeric c ring from the thermoalkaliphilic bacterium Bacillus sp. strain TA2.A1 and determined the number of c-subunits using a novel mass spectrometry method, termed 'laser-induced liquid bead ion desorption' (LILBID). This technique allows the mass determination of non-covalently assembled, detergent-solubilized membrane protein complexes, and hence enables an accurate determination of c ring stoichiometries. We show that the Bacillus sp. strain TA2.A1 ATP synthase harbours a tridecameric c ring. The operation of a c ring with 13 subunits renders the thermodynamic problem of ATP synthesis at alkaline pH less severe and may represent a strategy for ATP synthesis at low electrochemical potential.

Bacillus sp. strain TA2.A1, this activity is intrinsic to the F 1 moiety. To study the mechanism of ATPase inhibition, we developed a heterologous expression system in Escherichia coli to produce TA2F 1 complexes from this thermoalkaliphile. Like the native F 1 F o -ATP synthase, the recombinant TA2F 1 was blocked in ATP hydrolysis activity, and this activity was stimulated by the detergent lauryldimethylamine oxide. To determine if the C-terminal domain of the subunit acts as an inhibitor of ATPase activity and if an electrostatic interaction plays a role, a TA2F 1 mutant with either a truncated subunit [i.e., TA2F 1 ( ⌬C )] or substitution of basic residues in the second ␣-helix of with nonpolar alanines [i.e., TA2F 1 ( 6A )] was constructed. Both mutants showed ATP hydrolysis activity at low and high concentrations of ATP. Treatment of the purified F 1 F o -ATP synthase and TA2F 1 ( WT ) complex with proteases revealed that the subunit was resistant to proteolytic digestion. In contrast, the subunit of TA2F 1 ( 6A ) was completely degraded by trypsin, indicating that the C-terminal arm was in a conformation where it was no longer protected from proteolytic digestion. In addition, ATPase activity was not further activated by protease treatment when compared to the untreated control, supporting the observation that was responsible for inhibition of ATPase activity. To study the effect of the alanine substitutions in the subunit in the entire holoenzyme, we reconstituted recombinant TA2F 1 complexes with F 1 -stripped native membranes of strain TA2.A1. The reconstituted TA2F o F 1 ( WT ) was blocked in ATP hydrolysis and exhibited low levels of ATP-driven proton pumping consistent with the F 1 F o -ATP synthase in native membranes. Reconstituted TA2F o F 1 ( 6A ) exhibited ATPase activity that correlated with increased ATP-driven proton pumping, confirming that the subunit also inhibits ATPase activity of TA2F o F 1 .