Bacillus subtilis has a constitutive system for the uptake of alpha-keto-beta-methylvalerate, alpha-ketoisovalerate, and (probably) alpha-ketoisocaproate. A mutation, kauA1, which blocks the uptake of alpha-keto-beta-methylvalerate and alpha-ketoisovalerate, is located between metB and citK on the B. subtilis chromosome

Goldstein, Barry J.

Zahler, Stanley A.

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Uptake of branched-chain alpha-keto acids in \u3ci\u3eBacillus subtilis\u3c/i\u3e

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Thomas Jefferson UniversityJefferson Digital CommonsDepartment of Medicine Faculty Papers Department of MedicineJuly 1976Uptake of branched-chain alpha-keto acids inBacillus subtilisBarry J. GoldsteinThomas Jefferson University, Barry.Goldstein@jefferson.eduStanley A. ZahlerCornell UniversityLet us know how access to this document benefits youFollow this and additional works at: https://jdc.jefferson.edu/medfpPart of the Medical Genetics CommonsThis Article is brought to you for free and open access by the Jefferson Digital Commons. The Jefferson Digital Commons is a service of ThomasJefferson University's Center for Teaching and Learning (CTL). The Commons is a showcase for Jefferson books and journals, peer-reviewed scholarlypublications, unique historical collections from the University archives, and teaching tools. The Jefferson Digital Commons allows researchers andinterested readers anywhere in the world to learn about and keep up to date with Jefferson scholarship. This article has been accepted for inclusion inDepartment of Medicine Faculty Papers by an authorized administrator of the Jefferson Digital Commons. For more information, please contact:JeffersonDigitalCommons@jefferson.edu.Recommended CitationGoldstein, Barry J. and Zahler, Stanley A., "Uptake of branched-chain alpha-keto acids in Bacillussubtilis" (1976). Department of Medicine Faculty Papers. Paper 23.https://jdc.jefferson.edu/medfp/23JOURNAL OF BACTERIOLOGY, July 1976, p. 667-670Copyright 1976 American Society for MicrobiologyVol. 127, No. 1Printed in U.S.A.Uptake of Branched-Chain a-Keto Acids in Bacillus subtilisBARRY J. GOLDSTEIN AND STANLEY A. ZAHLER*Division ofBiological Sciences, Section ofGenetics, Development and Physiology, Cornell University, Ithaca,New York 14853Received for publication 1 March 1976Bacillus subtilis has a constitutive system for the uptake of a-keto-,8-methyl-valerate, a-ketoisovalerate, and (probably) a-ketoisocaproate. A mutation,kauAl, which blocks the uptake of a-keto-,B-methylvalerate and a-ketoisovaler-ate, is located between metB and citK on the B. subtilis chromosome.Bacillus subtilis strain CU906 is a derivativeof strain 168I- which carries an ilvB deletionmutation and a leuB mutation and, therefore,requires the branched-chain amino acids iso-leucine, valine, and leucine. It can also satisfythese growth requirements with the corre-sponding a-keto acids: a-keto-,3methylvalerate78 (18/1))87 (9,68)metB521 (Us4 L_ 19 (S.? /o)kauAl cItKlFIG. 1. Genetic map of the metB-citK region of thechromosome ofB. subtilis showing the position ofthekauAl marker. A three-point cross was carried outbetween two strains isogenic for an ilvB deletion(necessary for scoring kauAl, which is silent in theabsence of a requirement for isoleucine or valine).The donor in the phage PBS1 transduction wasstrain CU450 (trpC2, ilvBAl1, citKl). The recipientwas strain CU968 (trpC2, ilvBAl, metB5, kauAl).Selection was for Met+or for Kau+. Of81 Met+ trans-ductants tested, none were Kau and CitK- all otherrecombinant classes were found. This indicates theorder of genes shown. Data from a two-point crosslinking citKl and kauAl are included in the figure.Map distances are given as phage PBS1 transductionunits, calculated as (100 - percentage of co-transferof the two markers). The arrows point from the se-lected to the unselected markers. The numbers inparentheses indicate the actual number of co-trans-ductants found among the total colonies tested ineach cross. Genetic techniques used were previouslyreported (4).(KMV), a-ketoisovalerate (KIV), and a-keto-isocaproate (KIC). We mutagenized strainCU906 with N-methyl-N'-nitro-N-nitrosoguan-idine and imposed a penicillin selection (4) formutants unable to use the a-keto acids forgrowth. A mutant strain, CU961, was isolatedthat could not use 0.3 mM DL-KMV or KIV tosatisfy its isoleucine and valine requirements,although it could still use 0.3 mM KIC to satisfyits leucine auxotrophy. We have named themutation responsible for this phenotype kauAl;its genetic map position is in the metB-citK re-TABLE 1. Rate ofKIV uptake in the presence ofvarious inhibitorsaRate of uptake (%)Inhibitorb 5 AM inhibi- 100 A±M inhibi-tor concn tor concnL,Leucine - 53L-Isoleucine - 63L-Valine - 74KIC 31 3.7DL-KMV 48 2.5KIV 60d -Pyruvate - 96a-Ketoglutarate - 85a-Ketobutyrate - 53a The rate of KIV uptake is given as a percentageof the rate ofuptake seen in this experiment with noinhibitor added. The [KIVI was 1.6 ,uM and the rateof uptake with no inhibitor present was observed tobe 1.7 nmol/min per mg of protein. Uptake rateswere measured as described in Fig. 3A.b Inhibitors were adjusted to pH 7 before use.c A dash indicates that the assay was not per-formed.d In this assay, enough unlabeled KIV was addedto bring the final [KIV] to 6.6 uM. This point maythus be directly compared to the other values in thiscolumn.gion (Fig. 1), unlinked to the ilvB and leuB mu-tations. We have isolated two other mutantswith similar defects; their mutations lie veryclose to kauAl and are probably in the samegene. The kauA mutations are silent in strainsthat do not carry ilv mutations; i.e., such67668 NOTESstrains are prototrophic. The aminotransferaseactivities of the mutant strain CU961 for iso-leucine and valine, measured in both the for-ward and reverse directions with a-ketoglu-tarate and L-glutamate by the method ofTaylorand Jenkins (3), did not differ significantlyfrom those of the parent strain CU906.Strain CU992 (trpC2 kauAl) and the iso-genic strain CU120 (trpC2) were tested for up-take of KIV; the results are shown in Fig. 2.The uptake of KIV was virtually absent in theJ. BACTERIOL.strain carrying the kauAl marker, whereas es-sentially linear uptake was observed for atleast 5 min in the Kau+ strain (Fig. 2, inset).Since similar activity was seen whether or notthe glucose-salts growth medium containedKIV, the uptake system for KIV appears tobe constitutive.Using strain CU120, the kinetics of the up-take system for KIV was studied. The resultsof a substrate saturation experiment are shownin Fig. 3A; a double-reciprocal plot of thesec 1Ee~~~~~~~TIMEELAPSED (seconds)ca~~~~~2 gTIME ELAPSED (sConds)FIG. 2. Uptake ofKIV versus time with strains CU992 (trpC2, kauAl) and CU120 (trpC2). Symbols: 0,CU120 grown in minimal medium; 0, CU120 grown with KIV; *, CU992 grown in minimal medium; U,CU992 grown with KIV. For each time point, two separate determinations were made; the line is drawnthrough their mean. Cells were grown for 5 h at 37 C with aeration in 70 ml of medium containing (inmicrograms per milliliter): L-glutamate, 100; L-tryptophan, 50; D-glucose, 5,000; and, where applicable, KIV,40; in BS. BS is a salts solution containing (in milligrams per milliliter): (NH4)2SO4, 2; K2HPO4, 14;KH2PO4, 6; sodium citrate 2H20, 1; and MgSO4 * 7H20, 0.2. After harvesting, cells were washed twice withBS, resuspended in about 5 ml ofBS, and kept on ice prior to their immediate use. Samples were removed,and total cell protein (usually about 300 pg/ml) was estimated by the method ofLowry et al. (1), using bovineserum albumin as standard. For the uptake assay, 0.2 ml ofcell suspension was equilibrated in 0.4 ml of0.2%D-glucose in BS at 30 C for 5 min. Then, 5 p,l of [14C]KIV (49 p.Cilpmol; 0.2 pmollml) (prepared by amodification of the method ofMeister [2] and adjusted to pH 7) was added and the tube was reincubated at30 C. The final concentration of KIV in this assay was thus 1.7 pM. Samples of 100 p1 were removed atappropriate times and filtered through 0.45-p,m pore size nitrocellulose filters (Matheson-Higgins Co.)presoaked with 0.15 MNaCl and immediately washed with 8 ml of0.15 MNaCl. Filters were dried for 12 minat 165 C and counted in a fluid containing 4.02 g of2,5-diphenyloxazole (PPO) and 0.10 g of 1,4-bis[2]-(4-methyl-5-phenyloxazolyl)benzene (dimethyl-POPOP) per liter of toluene in a Beckman LS-230 scintillationcounter for 10 min. Background counts for the assay were determined by using tubes equilibrated at 30 C for 5min and then chilled to 0 C; the assay was run as above but at 0 C. These counts were subtracted from theexperimental values obtained at 30 C to calculate the counts taken up by the cells after various elapsed times.The number of moles of KIV taken up was estimated from these data by counting a known quantity of['4C]KIV solution absorbed into filters, which were subsequently dried and counted as above. Data for theinset were obtained in a separate experiment from that shown in the figure.NOTES 66910.0.@ 80c 60-2.0-0 30 60 90 120[KIV] (pM)EI,04.3150 180[KIVJ-1 PM1FIG. 3. (A) Rate ofKIV uptake versus external concentration ofKIV. The uptake assay was performed asdescribed in Fig. 2. Cells were grown without KIV. Unlabeled KIV (adjusted to pH 7) was added to assaytubes to increase the external concentration ofKIV; the quantity of['4C]KIVpresent was the same as describedin Fig. 2. The rate of uptake was calculated by measuring KIV uptake in triplicate assay samples at 1 and 3min elapsed time and finding the KIV uptake per minute. (B) Double-reciprocal plot of the data presented in(A).data appears in Fig. 3B. The measured K,,, forKIV in this experiment was 14 ,uM; the V,,,(,,.of uptake was 9.6 nmol/min per mg of protein.To investigate the specificity of the KIV up-take system, the rate of KIV uptake was meas-ured for strain CU120, with various potentialinhibitors present at either 5 or 100 ,uM in theusual uptake assay (Table 1). Strong inhibitionwas seen with KMV and KIC. A lower butsignificant amount of inhibition occurred withthe branched-chain amino acids. It was not dueto contamination of the amino acid solutions bya-keto acids.We examined the inhibition of KIV uptakeby KMV and KIC in strain CU120 (Fig. 4). Theinhibition due to KMV and to KIC was foundto be competitive; the measured K, values foruptake were 3.5 and 4.1 u.M for racemic KMVand for KIC, respectively.The system affected by the kauAl mutationappears to function in the uptake of the threebranched-chain a-keto acids (KIV, KMV, andA/zXVOL. 127, 1976670 NOTES30'' 2.5SCL 2.0c1.5ii .E.1I.1.00.5[I. ~~~~+KMV0~~~~0/noInhibitor02 0.4CKIVJ1 (JM)40.6FIG. 4. Double-reciprocal plots of rate ofKIV uptake versus external concentration ofKIV in the absenceand presence of2O MM DL-KMV. This experiment was performed as described in Fig. 3A. For each data point,the average oftwo separate determinations is shown. In this experiment, the K,) for KIV was 9.7 uM and theVmar for uptake was 15 nmol/min per mg ofprotein. An analogous experiment was performed with 20 pMKIC, and similar results were obtained. In this latter experiment, the K,m forKIV was 20 pM and the V,,,l was24 nmol/min per mg ofprotein.KIC) in B. subtilis. Since the mutant strain,CU961, originally isolated from the penicillinselection was able to utilize externally suppliedKIC, there must be an alternate system for theuptake of KIC in B. subtilis that does not func-tion in the uptake of KIV or KMV.We wish to thank J. Calvo for helpful discussions andthe generous gift of ('4C]valine, R. Hallberg for the use ofhis scintillation counter, and J. Ludford for the details ofthe uptake assay.LITERATURE CITED1. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J.Randall. 1951. Protein measurement with the Folinphenol reagent. J. Biol. Chem. 193:265-275.2. Meister, A. 1951. Studies on d- and l-a-keto-f-methyl-valeric acids. J. Biol. Chem. 190:269-276.3. Taylor, R. T., and W. T. Jenkins. 1966. Leucine amino-transferase. I. Colorimetric assays. J. Biol. Chem.241:4391-4395.4. Ward, J. B., Jr., and S. A. Zahler. 1973. Genetic studiesof leucine biosynthesis in Bacillus subtilis. J. Bacte-riol. 116:719-726.J. BACTERIOL.i.I,,

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Uptake of branched-chain alpha-keto acids in \u3ci\u3eBacillus subtilis\u3c/i\u3e

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