JOUtNAL OF BACTERIOLOGY, Mar. 1980, p. 1055-1063 Vol. 141, No. 3 0021-9193/80/03-1055/09$02.00/0 Purification and Properties of a Binding Protein for Branched- Chain Amino Acids in Pseudomonas aeruginosa TOSHIMITSU HOSHINO* AND MAKOTO KAGEYAMA Mitsubishi-Kasei Institute ofLife Sciences, Machida-shi, Tokyo 194, Japan A binding protein for branched-chain amino acids was purified to a homoge- neous state from shock fluid of Pseudomonas aeruginosa PML14. It was a monomeric protein with an apparent molecular weight of4.3 x 10' or 4.0 x 10' by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration, re- spectively. The isoelectric point was determined to be pH 4.1 by electrofocusing. Amino acid analysis of the protein showed that aspartic acid, glutamic acid, glycine, and alanine were major components and that the protein contained only one residue each of tryptophan and cysteine per molecule. The binding protein contained no sugar. The binding activity of the protein was specific for the branched-chain amino acids. The protein also bound alanine and threonine with lower affinity. The dissociation constants of this protein for leucine, isoleucine, and valine were found to be 0.4, 0.3, and 0.5 !M, respectively. Mutants defective in the production of the binding protein were identified among the mutants deficient in a transport system for branched-chain amino acids (LIV-I). The revertants from these mutants to LIV-I-positive phenotype simultaneously re- covered normal levels of the binding protein. These findings suggest strongly the association of the binding protein with the LIV-I transport system. In some bacterial transport systems, peri- preferential decrease in the activity of the LIV- plasmic binding proteins are found to associate I system, which was lost in membrane vesicles with the transport of amino acids, sugars, and (17). Leucine-binding activity was found in the ions (10, 24). Transport by such systems is sen- shock fluid of P. aeruginosa. The binding activ- sitive to osmotic shock (9) and is not operative ity of the shock fluid was smilar to the activity in membrane vesicles (31). The mechanism of of the LIV-I transport system in affinity for energy coupling ofsuch systems appears to differ leucine and in substrate specificity (16). How- from that in shock-resistant transport systems ever, no conclusive evidence has been obtained (11, 18, 31). However, such an association of for the association ofsuch a binding protein with binding proteins with transport systems in Pseu- the LIV-I transport system of P. aeruginosa. domonas aeruginosa has not been well estab- In this report, we describe the purification and lished, although Stinson et al. (27, 28) have properties ofthe leucine-binding protein from P. recently reported that the glucose-binding pro- aeruginosa cells. We also describe the isolation tein (GBP) is associated with glucose transport and characterization of mutants defective in the by the organism. LIV-I system with normal LIV-II activity and P. aeruginosa accumulates the branched- their revertants to LIV-I-positive phenotype. chain amino acids by at least two systems with The results convince us of the association of the different substrate specificities, LIV-I and LIV- binding protein with the LIV-I transport system. II (16, 17). The LIV-I system, which has a high affinity, is operative in the absence of Na+ and may be specific for alanine and threonine in MATERIALS AND METHODS addition to branched-chain amino acids. The Bacterial strains and growth conditions. The LIV-I system, which has a lower affinity, is bacterial strains used were PML14 (prototroph) and operative only when Na+ is present and is spe- its derivatives. Bacteria were grown aerobically at cific only for branched-chain amino acids. Trans- 370C in a synthetic medium (D-medium) described port activities for branched-chain amino acids previously (16) with 0.5% D-glucose as an energy source unless otherwise indicated. by the two systems can be assayed separately Mutagenesis. Mutagenesis with N-methyl-N'-ni- with whole-cell suspensions. Our previous study tro-N-nitrosoguanidine (NTG) was done according to (16) also suggested that a binding protein(s) the procedure of Fargie and Holloway (13) with mod- might be required for the LIV-I transport sys- ifications. Cells were harvested at the midexponential tem. Osmotic shock treatment of cells caused a phase, suspended to 2 x 108 cells per ml in 50 mM 1055 1056 HOSHINO AND KAGEYAMA J. BACTERIOL. potassium phosphate buffer, pH 7, containing 20 ug of onto 8% polyacrylamide gel columns (0.5 by 7.5 cm) NTG per ml, and incubated at 370C for 60 min. Cell containing 0.5% SDS and 8 M urea and developed at survival under the above conditions was approxi- 4 mA per gel. The gels were fixed with 20% (wt/wt) mately 5%. After mutagenesis, cells were centrifuged, sulfosalicylic acid and stained with Coomassie brilliant suspended in nutrient broth and cultured overnight at blue R250 according to the method of Maizel (21). 300C. The nutrient broth culture was diluted in a 100- SDS slab-gel electrophoresis using a discontinuous fold volume ofD-medium, cultured at 30°C for several buffer system was conducted according to the method hours, and then plated. of Laemmli (19) with modifications by Mizuno and Elimination ofNa' from agar. Agar powder from Kageyama (22). Gels were fixed and stained with 10% a commercial source (Iwai Kagaku Co., Tokyo) was acetic acid, 25% isopropanol, and 0.1% Coomassie bril- found to contain about 0.2% Na+ as determined by liant blue R250 at 370C for 1 h. Cytochrome c (12,400), atomic absorption spectroscopy. Ifagar plates (usually ,8-lactalbumin (18,400), a-chymotrypsinogen A containing 1.5% agar powder) are prepared with this (25,000), hen egg albumin (45,000), and bovine serum agar powder, about 1.3 mM Na+ contaminates the albumin (68,000) were used as molecular weight stand- plates, which is enough for the LIV-II system of P. ards. aeruginosa to operate (16). For preparing Na+-free Polyacrylamide gel isoelectric focusing was per- agar plates, Nae was eliminated from agar powder as formed by the procedure of Wrigley (32) with modifi- follows. Agar powder was washed 10 times on a glass cations. A purified sample (5 ,ug) suspended in a solu- filter with 1 liter of 1 M KCl per 100 g of powder, tion of 30% glycerol and 2% ampholite was layered on followed by washing with distilled water. The washed a 7.5% polyacrylamide gel column (0.5 by 7 cm) con- powder was dried in a warm air stream. The agar taining 2% ampholite of desired pH ranges. A solution powder thus washed contained only 0.004% Na+, giving of 15% glycerol and 2% ampholite was overlaid onto a concentration of only 0.03 mM Na+ in the agar the sample solution. Ampholite used was a mixture of plates. Agar plates used in this study were all prepared ampholine with pH ranges 3.5 to 5, 5 to 7, and 3.5 to with this washed powder. When needed, NaCl was 10 (LKB Instruments, Inc.) in the ratio of 4:4:2. The added to plates to make the final concentration 20 samples were developed at 200 V for 3 h at 40C. The mM. gels were removed and sliced into 2-mm widths. After Extraction of leucine-binding protein from the pH was determined with a flat-type combination cells. Extraction of periplasmic proteins was per- pH electrode model 6210-05T (Horiba, Kyoto), each formed basically by the same method as described slice was fixed overnight with 15% trichloroacetic acid, previously (16). However, the osmotic shock stage stained for 2 h at 370C with 0.25% Coomassie brilliant after the MgCl2 extraction was omitted, for almost all blue G250, and destained with 7.5% acetic acid. the binding activity was recovered in the MgCl2 ex- Amino acid analysis. Samples were hydrolyzed in tract. Cells harvested at the midexponential phase twice-distilled 6 N HCI containing 0.02% phenol at were suspended in 50 mM Trs-hydrochloride, pH 7.3, 1050C for 24, 48, and 72 h in sealed evacuated tubes. containing 0.2 M MgCl6 at a concentration of 0.1 to To determine sulfur-containing amino acids, samples 0.2 g (wet weight) of cells per ml. After incubating for were oxidized with performic acid according to the 10 min at 300C, the cell suspension was rapidly chilled procedure of Moore (23) and hydrolyzed for 18 h. in an ice bath and was left standing for 15 min. The Amino acid analyses were performed with a Durrum temperature shift was repeated once more. The sus- type D-500 automatic amino acid analyzer. Trypto- pension was centrifged twice at 8,000 x g for 10 min phan content was determined spectrophotometrically at 40C to remove cells. The supernatant was recentri- with the unhydrolyzed sample according to the fuged at 20,000 x g for 10 min at 40C. The resulting method of Goodwin and Morton (15). fluid was concentrated to about 4 mg ofprotein per ml Other methods. Hexose content was determined by ultrafiltration (Diaflo PM10 filter, Amicon Corp.). by the phenol-sulfuric acid method of Dubois et al. This concentrated fluid was stored at -70°C until (12), with D-galactose as a standard. Protein content used. was determined by the method of Lowry et al. (20), Binding assays Binding activities were measured with bovine serum albumin as a standard. by the equilibrium dialysis method, using dialysis bags Chemicals. '4C-labeled L-amino acids and 3H-la- (7, 16). A dialysis bag filled with 0.3 ml of a sample beled L-leucine were all purchased from Radiochemi- was dialyzed for 20 h at 40C against 10 mM Tris- cal Centre.