Functional reconstitution of a maltose ATP-binding cassette transporter from the thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius

Abstract

The thermoacidophilic gram-positive bacterium Alicyclobacillus acidocaldarius grows at 60 degrees C and pH 2-3. The organism can utilize maltose and maltodextrins as energy source that are taken up by an ATP-binding cassette (ABC) import system. Genes encoding a maltose binding protein, MalE, and two membrane-integral subunits, MalF and MalG, are clustered on the chromosome but a malK gene translating into a cognate ATPase subunit is lacking. Here we report the cloning of malK from genomic DNA by using the msiK gene of Streptomyces lividans as a probe. Purified MalK exhibited a spontaneous ATPase activity with a Vmax of 0.13 micromol Pi/min/mg and a Km of 330 microM that was optimal at the growth temperature of the organism. Coexpression of malK, malF and malG in Escherichia coli resulted in the formation of a complex that could be coeluted from an affinity matrix after solubilization of membranes with dodecylmaltoside. Proteoliposomes prepared from the MalFGK complex and preformed phospholipid vesicles of A. acidocaldarius displayed a low intrinsic ATPase activity that was stimulated sevenfold by maltose-loaded MalE, thereby indicating coupling of ATP hydrolysis to substrate translocation. These results provide evidence for MalK being the physiological ATPase subunit of the A. acidocaldarius maltose transporter. Moreover, to our knowledge, this is the first report on the functional reconstitution of an ABC transport system from a thermophilic microorganism.

Maltodextrin Utilization Plays a Key Role in the Ability of Group A Streptococcus To Colonize the Oropharynx

“…The spc cassette replacement of malE removes an XmnI-cut site, thereby increasing the fragment size by ϳ2500 bps. (39,45). Upstream of malE and divergently transcribed are malR, which encodes a putative transcriptional regulator, and malP and malQ, which encode enzymes putatively involved in generating various forms of glucose from maltodextrins (Fig.…”

Identification of a gene cluster encoding an arginine ATP-binding-cassette transporter in the genome of the thermophilic Gram-positive bacterium Geobacillus stearothermophilus strain DSMZ 13240

“…The ArtMP complex was incorporated into liposomes essentially as described previously (Scheffel et al, 2004). Lipids (20 mg) were dried under a stream of nitrogen, and slowly redissolved in 50 mM MOPS/KOH, pH 7?5, containing 1 % octyl b-D-glucopyranoside.…”

“…ABC transporter functions of ArtJMP were subsequently studied in proteoliposomes by assaying the following wellestablished criteria: (i) stimulation of ATPase activity of the complex by the cognate binding protein (Davidson et al, 1992;Landmesser et al, 2002;Scheffel et al, 2004), and (ii) sensitivity of this activity to the specific inhibitor orthovanadate (Landmesser et al, 2002;Sharma & Davidson, 2000). To this end, proteoliposomes were prepared from purified ArtMP and a crude total lipid extract of G. stearothermophilus (mainly composed of phosphatidylethanolamine, diphosphatidylglycerol and phosphatidylglycerol; Jurado et al, 1991) in the presence of L-arginine (1 mM) and/or ArtJ.…”

Structure, Function, and Evolution of Bacterial ATP-Binding Cassette Systems

“…Structures of BPD importers. The desire to obtain highresolution structural data has fueled an interest in archaeal ABC transporters, as thermostable proteins may stand up better to the rigors of crystallization than their bacterial counterparts do (177,423,508). Several recent reviews discuss the unique properties of the archaeal transporters (4,74,270).…”