Regulation of hydrogenase in Rhizobium japonicum: analysis of mutants altered in regulation by carbon substrates and oxygen

The synthesis of the H2 uptake system in free-living Rhizobium japonicum SR is repressed both by oxygen and by carbon substrates. Mutants selected for the ability to express hydrogenase in 10.0% partial pressure O2 were also less sensitive than the wild type to repression by carbon substrates such as arabinose, glycerol, gluconate, and succinate. The H2 uptake system in another class of mutants, previously shown to be hypersensitive to repression by O2, is also more sensitive to repression by carbon substrates. The oxygen- and carbon-insensitive mutants express the hydrogen uptake system during heterotrophic growth in the absence of hydrogen and thus can be considered constitutive (Hupc). The amount of cytochromes in the Hupc mutants is similar to that in the wild-type strain; however, the Hupc mutants contain greater methylene blue-dependent and O2-dependent hydrogenase activity, both as free-living cells and as bacteroids. Two-dimensional polyacrylamide gel electrophoresis revealed that during heterotrophic growth the Hupc mutant strain SR470 synthesized at least six peptides not found in the wild-type strain. The concentrations of cyclic AMP and guanosine tetraphosphate were similar in strain SR and the Hupc mutants during heterotrophic growth.     

Rhizobium japonicum mutant strains unable to grow chemoautotrophically with H2

Rhizobium japonicum strain SR grows chemoautotrophically on a mineral salts medium when incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow or that grow very poorly chemoautotrophically with H2 have been isolated from strain SR. The mutant isolation procedure involved mutagenesis with ethyl methane sulfonate, penicillin selection under chemoautotrophic growth conditions, and plating of the survivors onto medium containing carbon. The resulting colonies were replica plated onto medium that did not contain carbon, and the plates were incubated in an H2- and CO2-containing atmosphere. Mutant strains unable to grow under these conditions were chosen. Over 100 mutant strains with defects in chemoautotrophic metabolism were obtained. The phenotypes of the mutants fall into various classes. These include strains unable to oxidize H2 and strains deficient in CO2 uptake. Some of the mutant strains were capable of oxidizing H2 only when artificial electron acceptors were provided. Two mutant strains specifically lack activity of the key CO2-fixing enzyme ribulose 1,5-bisphosphate carboxylase. Other mutant strains lack both H2-oxidizing ability and ribulose 1,5-bisphosphate carboxylase activity.     

Carriers in electron transport from molecular hydrogen to oxygen in Rhizobium japonicum bacteroids.

An investigation has been conducted to identify electron transport carriers that participate in the oxidation of H2 by H2 uptake-positive strains of Rhizobium japonicum bacteroids. We have observed that the reduced form of dibromothymoquinone at a concentration of 0.2 mM strongly inhibited H2 uptake, endogenous respiration, and C2H2 reduction by bacteroid suspensions. Reduced dibromothymoquinone, however, failed to inhibit the transfer of electrons from H2 to methylene blue under anaerobic conditions, indicating that the hydrogenase per se is insensitive to this inhibitor. Metronidazole, at 1 mM, affected rates of H2 uptake and endogenous respiration only slightly, but strongly inhibited C2H2 reduction. Evidence for H2-dependent cytochrome reduction in an H2 uptake-positive strain of R. japonicum bacteroids is presented. In kinetic studies, the rates of reduction of the type b and c cytochromes in the presence of H2 were shown to be severalfold higher than the rates due to endogenous respiration alone. With hydrogenase-deficient mutants of R. japonicum, no measurable effect of H2 on cytochrome reduction was observed. Our results indicate that ubiquinone and cytochromes of types b and c are involved in the oxyhydrogen reaction in R. japonicum.     

Transposon Tn5-Generated Bradyrhizobium japonicum Mutants Unable To Grow Chemoautotrophically with H(2)

Twelve Tn5-induced mutants of Bradyrhizobium japonicum unable to grow chemoautotrophically with CO(2) and H(2) (Aut) were isolated. Five Aut mutants lacked hydrogen uptake activity (Hup). The other seven Aut mutants possessed wild-type levels of hydrogen uptake activity (Hup), both in free-living culture and symbiotically. Three of the Hup mutants lacked hydrogenase activity both in free-living culture and as nodule bacteroids. The other two mutants were Hup only in free-living culture. The latter two mutants appeared to be hypersensitive to repression by oxygen, since Hup activity could be derepressed under 0.4% O(2). All five Hup mutants expressed both ex planta and symbiotic nitrogenase activities. Two of the seven Aut Hup mutants expressed no free-living nitrogenase activity, but they did express it symbiotically. These two strains, plus one other Aut Hup mutant, had CO(2) fixation activities 20 to 32% of the wild-type level. The cosmid pSH22, which was shown previously to contain hydrogenase-related genes of B. japonicum, was conjugated into each Aut mutant. The Aut Hup mutants that were Hup both in free-living culture and symbiotically were complemented by the cosmid. None of the other mutants was complemented by pSH22. Individual subcloned fragments of pSH22 were used to complement two of the Hup mutants.     

Expression of uptake hydrogenase and hydrogen oxidation during heterotrophic growth of Bradyrhizobium japonicum.

Strains I-110 ARS, SR, USDA 136, USDA 137, and AK13 1c of Bradyrhizobium japonicum induced Hup activity when growing heterotrophically in medium with carbon substrate and NH4Cl in the presence of 2% H2 and 2% O2. Hup activity was induced during heterotrophic growth in the presence of carbon substrates, which were assimilated during the time of H2 oxidation. Strains I-110 ARS and SR grown heterotrophically or chemoautotrophically for 3 days had similar rates of H2 oxidation. Similar rates of Hup activity were also observed when cell suspensions were induced for 24 h in heterotrophic or chemoautotrophic growth medium with 1% O2, 10% H2, and 5% CO2 in N2. These results are contrary to the reported repression of Hup activity by carbon substrates in B. japonicum. Bradyrhizobial Hup activity during heterotrophic growth was limited by H2 and O2 and repressed by aerobic conditions, and CO2 addition had no effect. Nitrogenase and ribulosebisphosphate carboxylase activities were not detected in H2-oxidizing cultures of B. japonicum during heterotrophic growth. Immunoblot analysis of cell extracts with antibodies prepared against the 65-kilodalton subunit of uptake hydrogenase indicated that Hup protein synthesis was induced by H2 and repressed under aerobic conditions.     

Nif- Hup- mutants of Rhizobium japonicum.

Two H2 uptake-negative (Hup-) Rhizobium japonicum mutants were obtained that also lacked symbiotic N2 fixation (acetylene reduction) activity. One of the mutants formed green nodules and was deficient in heme. Hydrogen oxidation activity in this mutant could be restored by the addition of heme plus ATP to crude extracts. Bacteroid extracts from the other mutant strain lacked hydrogenase activity and activity for both of the nitrogenase component proteins. Hup+ revertants of the mutant strains regained both H2 uptake ability and nitrogenase activity.     

Regulation of hydrogenase in Rhizobium japonicum.

Factors that regulate the expression of an H2 uptake system in free-living cultures of Rhizobium japonicum have been investigated. Rapid rates of H2 uptake by R. japonicum were obtained by incubation of cell suspensions in a Mg-phosphate buffer under a gas phase of 86.7% N2, 8.3% H2, 4.2% CO2, and 0.8% O2. Cultures incubated under conditions comparable with those above, with the exception that Ar replaced H2, showed no hydrogenase activity. When H2 was removed after initiation of hydrogenase derepression, further increase in hydrogenase activity ceased. Nitrogenase activity was not essential for expression of hydrogenase activity. All usable carbon substrates tested repressed hydrogenase formation, but none of them inhibited hydrogenase activity. No effect on hydrogenase formation was observed from the addition of KNO3 or NH4Cl at 10 mM. Oxygen repressed hydrogenase formation, but did not inhibit activity of the enzyme in whole cells. The addition of rifampin or chloramphenicol to derepressed cultures resulted in inhibition of enzyme formation similar to that observed by O2 repression. The removal of CO2 during derepression caused a decrease in the rate of hydrogenase formation. No direct effect of CO2 on hydrogenase activity was observed.     

Expression of cytochrome o in hydrogen uptake constitutive mutants of Rhizobium japonicum.

Mutant strains of Rhizobium japonicum constitutive for H2 uptake activity (Hupc) contained significantly more membrane-bound b-type cytochrome than did the wild type when grown heterotrophically. The Hupc strains contained approximately three times more dithionite- and NADH-reducible CO-reactive b-type cytochrome than did the wild type; the absorption features of the CO spectra were characteristic of cytochrome o. This component, designated cytochrome b', was not reduced by NADH in the presence of cyanide. Cytochrome o from the wild type (SR) and cytochrome b' from mutants SR476 and SR481 bound to CO with similar dissociation constants of 5.4, 7.4, and 5.6 microM, respectively. NADH-dependent reduction of cytochrome b' from SR476 and SR481 and the cytochrome o from SR followed pseudo-first-order kinetics with similar rate constants. Based on these spectral, ligand-binding, and kinetic measurements, it was concluded that cytochrome b' expressed by the Hupc mutants is equivalent to cytochrome o found in the wild type. H2, NADH, and succinate each reduced the same amount of total b-type cytochrome in membranes from SR481, and the rate of H2-dependent cytochrome o reduction was significantly less than with succinate or NADH as the reductants. It was concluded that neither cytochrome o nor any b-type cytochrome expressed by the Hupc mutants was unique to the H2 oxidation system. At low O2 concentrations, the inhibition of H2 and NADH oxidase activities by CO closely paralleled the binding of CO to cytochrome o rather than cytochromes a3 or c'. This suggested that NADH and H2 oxidation involved primarily cytochrome o as the terminal oxidase at low O2 tensions.     

Revertible hydrogen uptake-deficient mutants of Rhizobium japonicum.

We have developed mutants of Rhizobium japonicum which are deficient in H2 uptake capacity (Hup-) and which spontaneously revert to the parent type at a frequency consistent with that of a single-point mutation (ca. 1.0 x 10(-09)). The mutagenesis by nitrous acid and the selection of the Hup- phenotype by using penicillin and chemolithotrophy as enrichment for chemolithotrophy-deficient strains are described. Two mutants retain low but reproducible levels of ribulose bisphosphate-dependent CO2 fixation when grown on a low-carbon medium under an atmosphere of 1% O2, 4% H2, 5% CO2, and 90% N2. Neither O2 nor the artificial electron acceptors phenazine methosulfate or methylene blue supported detectable H2 uptake by the free-living Hup- mutants or by their bacteroids. Plant growth experiments under bacteriologically controlled conditions were conducted to assess the mutants' performance as inocula for soybean plants. Plants inoculated with Hup- strains had lower dry weights and contained less total N than did plants inoculated with the parent Hup+ strain. Use of either the Hup- mutants or the Hup+ parent strain as inocula, however, did not significantly affect the acetylene-reducing activity or the fresh weight of nodules. These results, obtained with apparently isogenic lines of H2 uptake-deficient R. japonicum, provide strong support for a beneficial role of the H2 uptake phenotype in legume symbiosis.     

Competitive inhibition of an energy-dependent nickel transport system by divalent cations in Bradyrhizobium japonicum JH.

Both nickel-specific transport and nickel transport by a magnesium transporter have been described previously for a variety of nickel-utilizing bacteria. The derepression of hydrogenase activity in Bradyzhizobium japonicum JH and in a gene-directed mutant of strain JH (in an intracellular Ni metabolism locus), strain JHK7, was inhibited by MgSO4. For both strains, Ni2+ uptake was also markedly inhibited by Mg2+, and the Mg(2+)-mediated inhibition could be overcome by high levels of Ni2+ provided in the assay buffer. The results indicate that both B. japonicum strains transport Ni2+ via a high-affinity magnesium transport system. Dixon plots (1/V versus inhibitor) showed that the divalent cations Co2+, Mn2+, and Zn2+, like Mg2+, were competitive inhibitors of Ni2+ uptake. The KiS for nickel uptake inhibition by Mg2+, Co2+, Mn2+, and Zn2+ were 48, 22, 12, and 8 microM, respectively. Cu2+ strongly inhibited Ni2+ uptake, and molybdate inhibited it slightly. Respiratory inhibitors cyanide and azide, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, and ionophores nigericin and valinomycin significantly inhibited short-term (5 min) Ni2+ uptake, showing that Ni2+ uptake in strain JH is energy dependent. Most of these conclusions are quite different from those reported previously for a different B. japonicum strain belonging to a different serogroup.     

Competitive inhibition of an energy-dependent nickel transport system by divalent cations in Bradyrhizobium japonicum JH.

Both nickel-specific transport and nickel transport by a magnesium transporter have been described previously for a variety of nickel-utilizing bacteria. The derepression of hydrogenase activity in Bradyzhizobium japonicum JH and in a gene-directed mutant of strain JH (in an intracellular Ni metabolism locus), strain JHK7, was inhibited by MgSO4. For both strains, Ni2+ uptake was also markedly inhibited by Mg2+, and the Mg(2+)-mediated inhibition could be overcome by high levels of Ni2+ provided in the assay buffer. The results indicate that both B. japonicum strains transport Ni2+ via a high-affinity magnesium transport system. Dixon plots (1/V versus inhibitor) showed that the divalent cations Co2+, Mn2+, and Zn2+, like Mg2+, were competitive inhibitors of Ni2+ uptake. The KiS for nickel uptake inhibition by Mg2+, Co2+, Mn2+, and Zn2+ were 48, 22, 12, and 8 microM, respectively. Cu2+ strongly inhibited Ni2+ uptake, and molybdate inhibited it slightly. Respiratory inhibitors cyanide and azide, the uncoupler carbonyl cyanide m-chlorophenylhydrazone, the ATPase inhibitor N,N'-dicyclohexylcarbodiimide, and ionophores nigericin and valinomycin significantly inhibited short-term (5 min) Ni2+ uptake, showing that Ni2+ uptake in strain JH is energy dependent. Most of these conclusions are quite different from those reported previously for a different B. japonicum strain belonging to a different serogroup.     

Characterization of sugar transport in 2-deoxy-d-glucose resistant mutants of yeast

Summary A number of 2-deoxy-d-glucose (2-DOG) resistant mutants exhibiting resistance to glucose repression were isolated from variousSaccharomyces yeast strains. Most of the mutants isolated were observed to have improved maltose uptake ability in the presence of glucose. Fermentation studies indicated that maltose was taken up at a faster rate and glucose taken up at a slower rate in the mutant strains compared to the parental strains, when these sugars were fermented together. When these sugars were fermented separately, only the 2-DOG resistant mutant obtained fromSaccharomyces cerevisiae strain 1190 exhibited alterations in glucose and maltose uptake compared to the parental strain. Kinetic analysis of sugar transport employing radiolabelled glucose and maltose indicated that both glucose and maltose were transported with higher rates in the mutant strain. These results suggested that the high affinity glucose transport system was regulated by glucose repression in the parental strain but was derepressed in the mutant.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N(2) Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO(3)-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a "conventional" Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Nickel uptake in Bradyrhizobium japonicum.

Free-living Bradyrhizobium japonicum grown heterotrophically with 1 microM 63Ni2+ accumulated label. Strain SR470, a Hupc mutant, accumulated almost 10-fold more 63Ni2+ on a per-cell basis than did strain SR, the wild type. Nongrowing cells were also able to accumulate nickel over a 2-h period, with the Hupc mutant strain SR470 again accumulating significantly more 63Ni2+ than strain SR. These results suggest that this mutant is constitutive for nickel uptake as well as for hydrogenase expression. The apparent Kms for nickel uptake in strain SR and strain SR470 were found to be similar, approximately 26 and 50 microM, respectively. The Vmax values, however, were significantly different, 0.29 nmol of Ni/min per 10(8) cells for SR and 1.40 nmol of Ni/min per 10(8) cells for SR470. The uptake process was relatively specific for nickel; only Cu2+ and Zn2+ (10 microM) were found to appreciably inhibit the uptake of 1 microM Ni, while a 10-fold excess of Mg2+, Co2+, Fe3+, or Mn2+ did not affect Ni2+ uptake. The lack of inhibition by Mg2+ indicates that nickel is not transported by a magnesium uptake system. Nickel uptake was also inhibited by cold (53% inhibition at 4 degrees C) and slightly by the ionophores nigericin and carbonyl cyanide m-chlorophenylhydrazone. Other ionophores did not appreciably affect nickel uptake, even though they significantly stimulated O2 uptake. The cytochrome c oxidase inhibitors azide, cyanide, and hydroxylamine did not inhibit Ni2+ uptake, even at concentrations (of cyanide and hydroxylamine) that inhibited O2 uptake. The addition of oxidizable substrates such as succinate or gluconate did not increase nickel uptake, even though they increased respiratory activity. Nickel update showed a pH dependence with an optimum at 6.0. Most (approximately 85%) of the 63Ni2+ taken up in 1 min by strain SR470 was not exchangeable with cold nickel.     

Nickel is a component of hydrogenase in Rhizobium japonicum

The derepression of H2-oxidizing activity in free-living Rhizobium japonicum does not require the addition of exogenous metal to the derepression media. However, the addition of EDTA (6 microM) inhibited derepression of H2 uptake activity by 80%. The addition of 5 microM nickel to the derepression medium overcame the EDTA inhibition. The addition of 5 microM Cu or Zn also relieved EDTA inhibition, but to a much lesser extent; 5 microM Fe, Co, Mg, or Mn did not. The kinetics of induction and magnitude of H2 uptake activity in the presence of EDTA plus Ni were similar to those of normally derepressed cells. Nickel also relieved EDTA inhibition of methylene blue-dependent Hup activity, suggesting that nickel is involved directly with the H2-activating hydrogenase enzyme. Adding nickel or EDTA to either whole cells or crude extracts after derepression did not affect the hydrogenase activity. Cells were grown in 63Ni and the hydrogenase was subsequently purified by gel electrophoresis. 63Ni comigrated with the H2-dependent methylene blue reducing activity on native polyacrylamide gels and native isoelectric focusing gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the nickel-containing hydrogenase band revealed a single polypeptide with a molecular weight of ca. 67,000. We conclude that the hydrogenase enzyme in R. japonicum is a nickel-containing metalloprotein.     

Effect of pH on tritium exchange and hydrogen production and uptake in free-living cells and in bacteroids of Bradyrhizobium japonicum

Soybean nodule bacteroids and Bradyrhizobium japonicum free-living cells induced for H2-uptake hydrogenase, actively catalyze the evolution of H2 in a reaction highly dependent on the pH. The optimal pHs for the evolution and uptake reactions were 4.0 and 7.5-8.0, respectively. No differences were found between free-living cells and bacteroids with respect to hydrogen acceptor specificity, although absolute rates of H2 uptake were higher for free-living cells. Both types of cells were able to evolve hydrogen from reduced methyl viologen at low pH. These intact cells also catalyzed the exchange reaction between tritium and water in the absence of oxygen. The pH profile of the exchange activity showed two peaks at values near the optimal pHs for the evolution and uptake reactions.     

Isolation of genes (nif/hup cosmids) involved in hydrogenase and nitrogenase activities in Rhizobium japonicum.

Recombinant cosmids containing a Rhizobium japonicum gene involved in both hydrogenase (Hup) and nitrogenase (Nif) activities were isolated. An R. japonicum gene bank utilizing broad-host-range cosmid pLAFR1 was conjugated into Hup- Nif- R. japonicum strain SR139. Transconjugants containing the nif/hup cosmid were identified by their resistance to tetracycline (Tcr) and ability to grow chemoautotrophically (Aut+) with hydrogen. All Tcr Aut+ transconjugants possessed high levels of H2 uptake activity, as determined amperometrically. Moreover, all Hup+ transconjugants tested possessed the ability to reduce acetylene (Nif+) in soybean nodules. Cosmid DNAs from 19 Hup+ transconjugants were transferred to Escherichia coli by transformation. When the cosmids were restricted with EcoRI, 15 of the 19 cosmids had a restriction pattern with 13.2-, 4.0-, 3.0-, and 2.5-kilobase DNA fragments. Six E. coli transformants containing the nif/hup cosmids were conjugated with strain SR139. All strain SR139 transconjugants were Hup+ Nif+. Moreover, one nif/hup cosmid was transferred to 15 other R. japonicum Hup- mutants. Hup+ transconjugants of six of the Hup- mutants appeared at a frequency of 1.0, whereas the transconjugants of the other nine mutants remained Hup-. These results indicate that the nif/hup gene cosmids contain a gene involved in both nitrogenase and hydrogenase activities and at least one and perhaps other hup genes which are exclusively involved in H2 uptake activity.     

Nitrogen fixation in transposon mutants from Bradyrhizobium japonicum USDA 110 impaired in nitrate reductase

Tn5 transposon mutagenesis was carried out in Bradyrhizobium japonicum strain USDA 110 to produce defective mutants. From over one thousand clones expressing low levels of nitrate reductase activity as free-living bacteria, approximately five percent had significantly different ratios of nodulation, N2 fixation or nitrate reductase activity compared to the wild strain when determined in bacteroids from soybean nodules. Tn5 insertions were checked previously and mutants were arranged into four different groups. Only one of these groups, designated AN, was less effective at N2 fixation than the wild strain, suggesting a mutation in a domain shared by nitrogenase and NR. The remaining groups of insertions successfully nodulated and were as effective at N2 fixation as the wild strain, but showed diminished ability to reduce nitrate both in nodules and in the isolated bacteroids when assayed in vitro with NADH or methyl viologen as electron donors. PCR amplification demonstrated that Tn5 insertions took place in different genes on each mutant group and the type of mutant (CC) expressing almost no nitrate reductase activity under all treatments seemed to possess transposable elements in two genes. Induction of nitrate reductase activity by nitrate was observed only in those clones expressing a low constitutive activity (AN and AE). Nitrate reductase activity in bacteroids along nodule growth decreased in all groups including the ineffective AN group, whose nodulation was highly inhibited by nitrate at 5 mmol/L N. Host-cultivar interaction seemed to influence the regulation of nitrate reductase activity in bacteroids. Total or partial repression of nitrate reductase activity in bacteroids unaffected by N2 fixation (CC, AJ and AE groups) improved nodule resistance to nitrate and N yields of shoots over those of the wild strain. These observations may suggest that some of the energy supplied to bacteroids was wasted by its constitutive NRA.     

High-affinity glucose transport in Saccharomyces cerevisiae is under general glucose repression control.

Saccharomyces cerevisiae mutants defective in growth on low glucose concentration (lgn mutants) were isolated and screened for abnormal glucose transport. Nine complementation groups were identified, falling into two broad groups: those unable to significantly derepress high-affinity (low-Km) glucose uptake (lgn1, lgn4, lgn5, lgn7, and lgn8), and those with elevated repressed levels of high-affinity uptake that either derepress to normal or near normal levels of high-affinity uptake with loss of low-affinity transport (lgn2 and lgn3) or derepress only slightly, appearing to have an intermediate yet constitutive level of high-affinity transport (lgn6 and lgn9). Further analysis of the lgn mutations revealed pleiotropic phenotypes most consistent with the true defect being in regulation or expression of glucose repression and derepression. The kinetics of glucose uptake in strains carrying known mutations preventing derepression of glucose-repressible functions (snf1, snf2, snf4, and snf6) demonstrated that three of these mutations (snf1, snf4, and snf6) were similarly defective in derepression of high-affinity glucose uptake. The snf2 and snf5 mutations had no apparent effect on glucose uptake. Two mutations resulting in constitutive expression of glucose-repressible functions, cid1 and reg1, resulted in constitutive expression of high-affinity glucose uptake. These data support the conclusion that high-affinity glucose uptake in Saccharomyces cerevisiae is under general glucose repression control. The implications of other properties of these mutants are discussed.