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Anaerobic Glycerol-3-Phosphate Dehydrogenase and the Glpd

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Anaerobic Glycerol-3-Phosphate Dehydrogenase and the Glpd JOURNAL OF BACTERIOLOGY, Jan. 1990, p. 179-184 Vol. 172, No. 1 0021-9193/90/010179-06$02.00/0 Copyright © 1990, American Society for Microbiology Multiple Regulatory Elements for the glpA Operon Encoding Anaerobic Glycerol-3-Phosphate Dehydrogenase and the glpD Operon Encoding Aerobic Glycerol-3-Phosphate Dehydrogenase in Escherichia coli: Further Characterization of Respiratory Control S. IUCHI,1 S. T. COLE,2 AND E. C. C. LIN'* Department of Microbiology and Molecular Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115,1 and Laboratoire de Genetique Moleculaire Bacterienne, Institut Pasteur, 75724 Paris Cedex 15, France2 Received 31 May 1989/Accepted 15 October 1989 In Escherichia coli, sn-glycerol-3-phosphate can be oxidized by two different flavo-dehydrogenases, an anaerobic enzyme encoded by the glpACB operon and an aerobic enzyme encoded by the glpD operon. These two operons belong to the glp regulon specifying the utilization of glycerol, sn-glycerol-3-phosphate, and glycerophosphodiesters. In glpR mutant cells grown under conditions of low catabolite repression, the gipA operon is best expressed anaerobically with fumarate as the exogenous electron acceptor, whereas the glpD operon is best expressed aerobically. Increased anaerobic expression of gipA is dependent on the fnr product, a pleiotropic activator of genes involved in anaerobic respiration. In this study we found that the expression of a glpAl(Oxr) (oxygen-resistant) mutant operon, selected for increased aerobic expression, became less dependent on the FNR protein but more dependent on the cyclic AMP-catabolite gene activator protein complex mediating catabolite repression. Despite the increased aerobic expression of glpAl(Oxr), a twofold aerobic repressibility persisted. Moreover, anaerobic repression by nitrate respiration remained normal. Thus, there seems to exist a redox control apart from the FNR-mediated one. We also showed that the anaerobic repression of the glpD operon was fully relieved by mutations in either arcA (encoding a presumptive DNA recognition protein) or arcB (encoding a presumptive redox sensor protein). The arc system is known to mediate pleiotropic control of genes of aerobic function. Glycerol is utilized by Escherichia coli obligatorily expression of the gipA operon was dependent on the fnr through phosphorylation to glycerol-3-phosphate (G3P), product, a pleiotropic activator of genes involved in anaer- which can also be captured directly from the environment or obic respiration (for a review, see reference 26). In this study after periplasmic hydrolysis of glycerophosphodiesters (18, we further explored the respiratory control of glpA and glpD 19). G3P is oxidized by either the anaerobic dehydrogenase expression. encoded by the glpACB operon (min 49) or the aerobic dehydrogenase encoded by the glpD operon (min 75) (Fig. MATERIALS AND METHODS 1). These two operons, together with the gipFK encoding glycerol kinase and glycerol facilitator (min 88) and the Materials. Phenazine methosulfate, 3-(4,5-dimethylthiazol- glpTQ operons (min 49) encoding G3P permease and glyc- yl-2-)-2,5-diphenyl tetrazolium bromide (MTT), benzyl vi- erophosphodiesterase, are members of the glp regulon spe- ologen, o-nitrophenyl-3-galactopyranoside, and DL-dithio- cifically controlled by the gIpR-encoded repressor. The threitol were obtained from Sigma Chemical Co., St. Louis, glpACB and the glpTQ operons are adjacent to each other Mo.; 5-bromo-4-chloro-3-indolyl-p-galactopyranoside was but are divergently transcribed (3, 4, 14, 18-20; G. Sweet, obtained from Bachem, Inc., Torrance, Calif.; [U-14C]G3P, personal communication). Two structural genes of the same disodium salt, and Biofluor were obtained from Dupont, regulon but of unknown function, gipE and glpG, were found NEN Research Products, Boston, Mass.; MacConkey me- between glpD and glpR (22). dium, tryptone, and yeast extract were obtained from Difco With a gIpR deletion mutant, Freedberg and Lin (5) Laboratories, Detroit, Mich.; and vitamin-free casein acid observed the highest activity level of the glpD enzyme after hydrolysate was obtained from ICN Nutritional Biochemi- aerobic growth on casein acid hydrolysate and the highest cals, Cleveland, Ohio. All other reagents used were com- activity level of the gipA enzyme after anaerobic growth on mercial products of the highest grade available. casein hydrolysate plus fumarate. Submaximal activity lev- Bacterial and phage strains. All strains used were deriva- els of both enzymes were observed when nitrate served as tives of E. coli K-12. Their origins and genotypes are given the exogenous electron acceptor. Since catabolite repression in Table 1. P1 vir phage were used for transductions. The could not account for the pattern of these variations, a D[glpAJ(Oxr)-lac] locus (Oxr, oxygen resistant) was trans- respiratory control mechanism was invoked (5). Kuritzkes et duced from strain ECL503 to strain ECL514 by selection for al. (16) subsequently discovered that the increased anaerobic aerobic growth on lactose mineral agar. The glpR mutation was transduced from strain ECL72 to strains ECL392 and ECL519 by selecting for the closely linked glpD+ marker * Corresponding author. which conferred aerobic growth ability on glycerol (20 mM) 179 180 IUCHI ET AL. J. BACTERIOL. Glycerol Focilit;-G~lycerolIotor .---EnzyEnzyme 111Glc Feedback Inhibition Fructose-1,6-P2 Kinase (g/pK) +ATP Aerobic dehydrogenase (9/pD) G3P Perm G3P Anaerobicdehydrogenose DHAP Glyceraldehyde-3-P (gIpACB) ROd -It \ G3P Oxidoreductose (gpsA) P-diesterrose] (g/p Biosynthesis G3P-OR FIG. 1. The dissimilatory system for glycerol, G3P, and glycerophosphodiesters in E. coli encoded by the glp regulon. The genetic symbols are in parentheses. Dashed arrows indicate feedback inhibitors of glycerol kinase activity: enzyme 111GIc, the protein III of the phosphoenolpyruvate phosphotransferase system for the vectorial phosphorylation of glucose, and fructose-1,6-P2, fructose-1,6-bisphos- phate. The dotted arrow indicates the pathway for G3P biosynthesis. Positions of the glp genes and operons (arrows over genetic symbols show the directions of transcriptions) are indicated on the circular figure (for a review, see reference 19). mineral agar. The fnr mutation in strains ECL538 and with the linked TnJO by selection on tetracycline-LB agar. ECL539, the chlE mutation in strain ECL566, the arcA The narH200::TnJO in strain ECL392 was selected for tetra- mutation in strain ECL597, and the arcB mutation in strain cycline resistance. All transductants were purified on agar ECL598 were acquired as nonselective markers together with the same composition as that used for the selection, and TABLE 1. E. coli strains used in this study Strain Genotype' Derivation Source or RK5263 narH200: :Tn10 28 ECL72 glpR 5 ECL323 fnr-1 zci::TnlO 16 ECL389 F- C(glpA-lac) AglpD102 recA srl::TnlO araD139 A(argF-lac) 8 U169 deoCI galflb-5301 ptsF relAl rpsL150 sdh-9 ECL392 F- 4(glpA-lac) AglpD102 araDJ39 A(argF-lac)U169 deoCI gal 8 flb-5301 ptsF relAl rpsL150 sdh-9 ECL503 1§[glpAJ(Oxr)-lac] frd-l(Con) 8 ECL514 F- 4(glpA-lacZ Y) ,AglpDI02 araD139 A(argF-lac)U169 deoCI 9 galflb-5301 ptsF relAl rpsL150 sdh-9 ECL519 'F[glpAJ(Oxr)-lac) AglpDI02 P1(ECL503) x ECL514 This study ECL526 Fb(glpA-lac) glpR P1(ECL72) x ECL392 This study ECL527 4?[glpAl(Oxr)-lac] glpR P1(ECL72) x ECL519 This study ECL538 ('(glpA-lac) glpR fnr-1 zci::TnJO P1(ECL323) x ECL526 This study ECL539 4f[glpAJ(Oxr)-lac] glpRfnr-1 zci::TnJO P1(ECL323) x ECL527 This study ECL560 4'(glpA-lac) narH200::TnlO AglpDl02 P1(RK5253) x ECL392 This study ECL562 chlEJ03 zbi-624: :TnlO &glpDlO2 10 ECL565 '1(glpA-lac) narL2J5::TnlO AglpDJ02 11 ECL566 'F(glpA-lac) chlE103 zbi-624::TnJO AglpDJ02 P1(ECL562) x ECL392 This study ECL585 arcAl 41::TnJO 12 ECL594 arcBI zgi::TnlO 7 ECL597 ¢(glpA-lac) glpR arcAl zy::TnJO P1(ECL585) x ECL526 This study ECL598 't(glpA-lac) glpR arcBI zgi::Tn1O P1(ECL594) x ECL526 This study a I(glpA-lac) refers to the fusion glpAlOI::Xpl(209) (8); D(glpA-lacZ+Y) refers to the stabilized glpAlOl::Mu dl (9). VOL. 172, 1990 MULTIPLE CONTROLS OF gipA AND glpD 181 TABLE 2. Expression of gipT in F(gIpA-lac) and I[9glpAJ(Oxr)-lac] strains G3P uptake (nmollmin per mg of protein) under P-Galactosidase activity (U) under the following the following growth conditionsa: growth conditionsa: Strain Genotype +02 02 +02 -02 -GF +GF -GF +GF -GF +GF -GF +GF ECL389 F)(gIpA-lac) 0 22 0 31 16 54 14 420 ECL503 D[glpA1(Oxr)-1ac] 0 18 0 33 44 590 68 2,200 a Cells were grown aerobically (+02) or anaerobically (-02) on oxylose medium. -GF, glycerol and fumarate omitted; +GF, glycerol and fumarate added. inheritance of the desired mutation was confirmed by genetic transport at 30°C as described previously (5), except that 5 linkages, cell growth, or enzyme assay. ml of buffer was used to wash the cells at the end of the Growth conditions. For routine cultures, LB medium assay. Specific activity was expressed in nanomoles per (1.0% tryptone, 0.5% yeast extract, 0.5% NaCl) was used. minute per milligram of protein. For enzyme and transport assays, cells were grown at 37°C in a mineral medium (pH 7.0) buffered by 0.1 M phosphate RESULTS (29) and supplemented with 0.03% casein acid hydrolysate (to prime growth). As the principal carbon and energy Mutant with a 41(glpA-lac) operon resistant to aerobic source, additional appropriate compounds were added. Aer- repression. Strain ECL503 was selected from strain ECL389 obic cultures were grown with vigorous rotatory agitation ['F(glpA-lac)] as a spontaneous mutant which grew aerobi- and harvested in mid-exponential phase (approximately 100 cally as a large colony on agar containing 5 mM lactose as Klett units; no. 42 filter). A control experiment showed that the principal carbon and energy source and 0.2 mM glycerol the specific activity of ,-galactosidase in a 'D(glpA-lac) glpR as the inducer.
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