H. S. Levinson, A. L. Sonenshein, and D. J. Tipper (ed.),. Sporulation and germination. American Society for Microbiolo- gy, Washington, D.C.. 12. Henner, D. J.
JOURNAL
OF
BACTERIOLOGY, Feb. 1984, p. 454-459
Vol. 157, No. 2
0021-9193/84/020454-06$02.00/0 Copyright © 1984, American Society for Microbiology
Genetics of Leucine Biosynthesis in Bacillus megaterium QM B1551 JAMES C. GARBE, GERARD F. HESS, MARGARET A. FRANZEN, AND PATRICIA S. VARY* Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115 Received 22 September 1983/Accepted 12 November 1983
Genes involved in the biosynthesis of leucine have been mapped in Bacillus megaterium QM B1551, using transducing phage MP13. Mutations were designated leuA, leuB, or leuC on the basis of enzyme assays. Two mutant strains were deficient in the enzyme activities of leuA (a-isopropylmalate synthase) and leuC (,-isopropylmalate dehydrogenase) and so may contain polar mutations. Fine-structure transduction mapping established the gene order leuC-leuB-leuA-ilv-hem-phe. The orientation of the leu genes to the ilv gene is the same as in Bacillus subtilis, but the relationship in respect to two other linked markers, hem and phe, differs.
Bacillus megaterium has been used extensively in studies of bacterial sporulation and germination because of its ability to sporulate efficiently and germinate synchronously. It has also been used industrially to produce amino acids and vitamins and to modify antibiotic molecules and hormones (9). A number of mutants are available for study, including strains temperature sensitive for germination (23, 28) and some altered in ATPase activity (8). However, until recently there has been no method of genetic analysis available in B. megaterium. We have previously reported the isolation and characterization of bacteriophage MP13, which mediates generalized transduction in B. megaterium (29, 30), and have used this phage for initial genetic studies on the trp-his region (5) of this species. Genetic analysis of the leucine genes has been initiated since leucine biosynthesis has been extensively studied in Salmonella typhimurium (4, 20, 26) and B. subtilis (31, 32) and the enzymes and intermediates have been identified. Leucine biosynthesis involves only three specific enzymes, making biochemical and genetic characterization much simpler. Finally, the leucine genes in both B. subtilis and Salmonella spp. are linked (20, 31), suggesting that a similar linkage might exist in B. megaterium. Indeed, preliminary experiments suggested that these genes are clustered in this species (11). In this paper, we report the results of our studies on the leucine biosynthetic enzymes in B. megaterium and the fine mapping of the leu region, using MP13mediated transduction.
Mutant isolation. Late logarithmic cells of strain QM B1551 were mutagenized by treatment with ethyl methanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine, using the methods of Vary (27). Methicillin enrichment was also used in some experiments. Mutagenesis with 6-chloro-9 ((3((2-chloroethyl)amino)propyl)amino)-2-methoxy acridine (ICR-191) was performed as follows: a cell suspension was diluted to a concentration of 107 CFU/ml, and ICR-191 was added to a final concentration of 10 ,g/ml. The treated suspension was incubated with aeration for 12 to 18 h, after which the cells were washed twice in minimal medium, resuspended in supplemented nutrient broth, and plated on minimal medium. Presumptive auxotrophs, appearing as tiny colonies, were picked to supplemented nutrient broth plates and screened for auxotrophic requirements. Spontaneous hemin-requiring mutants were isolated by spreading spores of JV85 on supplemented nutrient broth plates plus 10 ,ug of neomycin per ml. Tiny neomycinresistant colonies were tested for growth on supplemented nutrient broth, with and without 1.5 ,ug of hemin per ml. Colonies which grew on supplemented nutrient broth plus hemin but not on supplemented nutrient broth alone were saved as hem auxotrophs. Mutants were further tested for growth on 8-aminolevulinic acid since all but hemA mutants fail to grow on this intermediate (2). Enzyme assays. Cells were grown from lyophilized spores which were heat activated at 60°C for 10 min in 1 ml of sterile distilled water, added to 25 ml of prewarmed MC broth plus 20 ,ug of L-leucine per ml to mid-log phase, and diluted 1:4 into 100 ml of MC plus 20 p,g of leucine per ml. Cells were harvested by centrifugation for 15 to 45 min after leaving log phase, washed once in 10 ml of MC, and then resuspended in 3.0 ml of lysing buffer prewarmed to 37°C. Lysing buffer wasmixed immediately before use and contained 1.0 mg of lysozyme and 5.0 ,g of DNase per ml in 0.1 M Tris, pH 8.0. Lysis was checked by phase-contrast microscopy. If cells were not completely lysed after 10 min, they were briefly sonicated. Additional DNase (10 ,ug/ml) was added to viscous lysates. This crude extract was used to assay for Pisopropylmalate (IPM) dehydrogenase. For the other enzymes, lysed cell suspensions were centrifuged at 10,000 x g for 10 min to pellet debris, and the supematant fractions were stored in ice while assays were performed. The assay for a-IPM synthase was performed by the method of Kohlaw and Leary (15) with two modifications: the amount of acetyl-coenzyme A was doubled, and samples were centrifuged after addition of ethanol to remove precip-
MATERIALS AND METHODS Organisms. All strains used in this study were derived from B. megaterium QM B1551 (ATCC 12872) (Table 1). Bacteriophage MP13 (29) was the vector for all transductions. Media and cultural conditions. The medium used for routine growth of bacteria and for lysate production was supplemented nutrient broth (25). Screening of mutants and transductants was done on minimal glucose salts medium (16), and transductions were plated on MCT medium (30). Conditions for cell growth and transductions were as previously described (30). All incubations were at 30°C with aeration at 240 rpm unless otherwise indicated. * Corresponding author. t Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
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TABLE 2. Specific activities of leucine biosynthetic enzymes Sp act (p.mol/min per mg of protein)' Mutant IPM-S IPM-I IPM-DH LDH 0.030 13.99 0.036 Wild type 0.052 0.000 0.015 36.84 PV36 0.046 20.54 0.016 0.032 PV37 0.004 0.003 27.02 0.026 PV39 0.045 21.08 0.005 0.019 0.041 PV40 33.69 0.029 0.000 PV41 0.042 0.029 0.019 29.20 PV45 0.012 25.80 0.026 0.028 0.000 JV75 0.06 0.006 0.041 JV76 0.004 62.30 0.024 JV78 0.039 a IPM-S, a-IPM synthase; IPM-I, IPM isomerase; IPM-DH, IPM dehydrogenase; LDH, leucine dehydrogenase.
TABLE 1. B. megaterium strains Strain' Genotype Mutagenesisb QM B1551 Prototrophic NTG PV5 cys-20 NTG PV30 pheA20 ICR-191 PV36 leuB20 ICR-191 PV37 1euA21 ICR-191 PV39 leuC22 ICR-191 PV40 leu-23 PV41 NTG leuC24 PV43 NTG cysC21 PV45 NTG leuA25 NTG of JV78 PV93 leuC4 ilv-22 str-3 Spontaneous from JV85 PV146 ilv4 hem-I str-3 Spontaneous from JV85 PV147 ilv4 hem-2 str-3 Transduction PV159 ilv4 leuBi str-3 Transduction PV160 ilv4 leuA25 str-3 Transduction PV162 ilv4 leuBI PV169 Transduction ilv4 leuC24 Transduction PV172 ilv-4 leu-23 Transduction PV173 ilv-4 leu-2 NTG PV194 argA32 NTG JV71 argO2 NTG JV75 leuBI NTG JV76 leu-2 EMS JV78 leuC4 str-3 NTG JV82 ilvAl NTG JV85 ilv4 str-3 EMS JV86 ilv-5 str-3 EMS JV98 glu-l str-3 JV100 EMS ser-2 str-3 a Strains with a JV prefix were kindly supplied by James C. Vary, University of Illinois Medical Center, Chicago, and have not previously been reported in the literature except for JV71, JV78, and JV100 (26). All other strains were isolated or constructed for this study. b NTG, N-Methyl-N'-nitro-N-nitrosoguanidine; ICR-191, 6chloro-9((3 -((2-chloroethyl)amino)propyl)amino)-2- methoxy acridine; EMS, ethyl methanesulfonate.
except that the P-IPM and NAD+ concentrations were increased to 1 and 2 mM, respectively. Crude extract was assayed 4 min after the addition of lysing buffer before centrifugation. The extract was concentrated 1:2 (by using 2 ml instead of 4 ml of NaCO3) after the toluene extraction. Standards of ketoisocaproic acid (0 to 0.2 ,umol in increments of 0.05 ,umol) were treated in parallel with the samples. L-Leucine dehydrogenase activity was determined as a control for enzyme activity in the extracts and was assayed by the method of Sanwal and Zink (24). Protein concentrations were determined by the method of Lowry et al. (18). A Perkin-Elmer A3 UV-visible double-beam spectrophotometer was used for all absorbancy measurements. Chemicals and reagents. NAD+ and acetyl-coenzyme A were purchased from Boehringer Mannheim Biochemicals (Indianapolis, Ind.). ,B-IPM was a gift from H. E. Umbarger at Purdue University (Lafayette, Ind.). All other chemicals were of reagent grade and were obtained from Sigma Chemical Co. (St. Louis, Mo.).
RESULTS
itated DNA. Dithionitrobenzoate was then added to the supernatant fraction. IPM isomerase was assayed by the method of Cho-Chang et al. (7). IPM dehydrogenase was assayed by the colorimetric method of Ward and Zahler (31)
Nine leu mutants were assayed for activity of the leucine biosynthetic enzymes (Table 2). None of the mutants were defective in leucine aminotransferase as defined by the
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