Between feast and famine: endogenous inducer synthesis in the adaptation of Escherichia coli to growth with limiting carbohydrates.

Escherichia coli adapted to growth with low carbohydrate concentrations bypassed the requirement for exogenous inducer with at least three well-studied sugar regulons. Induction of mgl and gal genes became independent of added galactose in bacteria approaching stationary phase or during continuous culture with micromolar glucose in the medium. Bacteria became independent of exogenous induction because endogenous galactose and cyclic AMP (cAMP) pools were sufficient for high expression of mgl and gal genes under glucose limitation. Limitation-stimulated induction of mgl was dependent on a functional galETK operon for synthesis of the inducer galactose. Intracellular galactose levels were maximal not during starvation (or slow steady-state growth rates approaching starvation) but at fast growth rates with micromolar glucose. The extent of mgl/gal induction correlated better with inducer availability than with cAMP concentrations under all conditions tested. Endogenous inducer accumulation represents an adaptation to low-nutrient environments, leading to derepression of high-affinity transport systems like Mgl essential for bacterial competitiveness at low nutrient concentrations.     

Maltose and maltotriose can be formed endogenously in Escherichia coli from glucose and glucose-1-phosphate independently of enzymes of the maltose system.

The maltose system in Escherichia coli consists of cell envelope-associated proteins and enzymes that catalyze the uptake and utilization of maltose and alpha,1-4-linked maltodextrins. The presence of these sugars in the growth medium induces the maltose system (exogenous induction), even though only maltotriose has been identified in vitro as an inducer (O. Raibaud and E. Richet, J. Bacteriol., 169:3059-3061, 1987). Induction is dependent on MalT, the positive regulator protein of the system. In the presence of exogenous glucose, the maltose system is normally repressed because of catabolite repression and inducer exclusion brought about by the phosphotransferase-mediated vectorial phosphorylation of glucose. In contrast, the increase of free, unphosphorylated glucose in the cell induces the maltose system. A ptsG ptsM glk mutant which cannot grow on glucose can accumulate [14C]glucose via galactose permeases. In this strain, internal glucose is polymerized to maltose, maltotriose, and maltodextrins in which only the reducing glucose residue is labeled. This polymerization does not require maltose enzymes, since it still occurs in malT mutants. Formation of maltodextrins from external glucose as well as induction of the maltose system is absent in a mutant lacking phosphoglucomutase, and induction by external glucose could be regained by the addition of glucose-1-phosphate entering the cells via a constitutive glucose phosphate transport system. malQ mutants, which lack amylomaltase, are constitutive for the expression of the maltose genes. This constitutive nature is due to the formation of maltose and maltodextrins from the degradation of glycogen.     

Development of a LAC4 promoter-based gratuitous induction system in Kluyveromyces lactis

A gratuitous induction system based on the strong, indigenous LAC4 promoter was developed for Kluyveromyces lactis. To prevent consumption of the inducer galactose, a strain with a gal1-209 mutation was employed; this mutation disables the galactokinase function but retains the regulatory function for induction. The Escherichia coli lacZ gene (encoding beta-galactosidase) is functional in K. lactis and was used as the reporter gene downstream of the LAC4 promoter on a multicopy plasmid. The gal1-209 strain exhibited several unexpected phenomena, including partial consumption of the inducer galactose (although at a much slower rate relative to GAL1 strains) and growth inhibition at high concentrations of galactose. These unusual characteristics, however, did not prevent the successful construction of a strong gratuitous induction system. Due to the low rate of inducer consumption for the gratuitous strain, very low concentrations of galactose (1:20 galactose:glucose) resulted in high-level induction. Under these conditions, beta-galactosidase specific and volumetric activities were 4.2- and 5.5-fold higher, respectively, than those for the "GAL1" nongratuitous strain. This research demonstrated the improved productivity possible via LAC4 promoter-based gratuitous induction (and thus a more stable inducer concentration). The effects of various carbon source concentrations on growth and induction were also determined.     

Adaptation to life at micromolar nutrient levels: the regulation of Escherichia coli glucose transport by endoinduction and cAMP

Abstract: Free-living bacteria are expert in adapting to variations in nutrient availability, often using an array of transport systems of different affinities to scavenge for particular substrates (multiport). This review concentrates on the regulation of expression of different transporters contributing to multiport in response to varying nutrient levels. A novel mechanism of controlling bacterial transport affinity under sugar limitation is described. In particular, switching from glucose-rich to glucose-limited conditions results in Escherichia coli orchestrating outer membrane changes as well as the induction of a periplasmic binding protein-dependent (ABC-type) transport system. The changes leading to the high affinity transport pathway are directed towards uptake of rapidly utilisable concentrations and are optimal close to 10⁻⁶ M medium glucose. High affinity transport is absent under both glucose-rich 'feast' and glucose-starved 'famine' conditions hence high affinity transporters are not simply repressed by excess nutrient. Rather, the improvement in glucose scavenging involves induction of genes in 2 distinct regulons ( mgl/gal and mal/lamB ) through synthesis of 2 different endogenous inducer molecules (galactose, maltotriose). Endoinducer levels are tightly controlled by extracellular glucose concentration at different glucose-limited growth rates. Aside from endoinducers, the elevated intracellular level of cAMP plays a role in induction of the high-affinity pathway but CAMP-mediated relief from catabolite repression is not itself sufficient for high affinity transport. In contrast to the repressive role of glucose when present at millimolar concentrations, micromolar glucose also leads to the induction of transport systems for other sugars, further broadening the scavenging potential of nutrient-limited bacteria for other substrates.     

Constitutive expression in gal7 mutants of Kluyveromyces lactis is due to internal production of galactose as an inducer of the Gal/Lac regulon.

The induction process of the galactose regulon has been intensively studied, but until now the nature of the inducer has remained unknown. We have analyzed a delta gal7 mutant of the yeast Kluyveromyces lactis, which lacks the galactotransferase activity and is able to express the genes of the Gal/Lac regulon also in the absence of galactose. We found that this expression is semiconstitutive and undergoes a strong induction during the stationary phase. The gal1-209 mutant, which has a reduced kinase activity but retains its positive regulatory function, also shows a constitutive expression of beta-galactosidase, suggesting that galactose is the inducer. A gal10 deletion in delta gal7 or gal1-209 mutants reduces the expression to under wild-type levels. The presence of the inducer could be demonstrated in both delta gal7 crude extracts and culture medium by means of a bioassay using the induction in gal1-209 cells. A mutation in the transporter gene LAC12 decreases the level of induction in gal7 cells, indicating that galactose is partly released into the medium and then retransported into the cells. Nuclear magnetic resonance analysis of crude extracts from delta gal7 cells revealed the presence of 50 microM galactose. We conclude that galactose is the inducer of the Gal/Lac regulon and is produced via UDP-galactose through a yet-unknown pathway.     

Galactose repression of beta-galactosidase induction in Escherichia coli

Beggs, William H. (University of Minnesota, Minneapolis), and Palmer Rogers. Galactose repression of beta-galactosidase induction in Escherichia coli. J. Bacteriol. 91:1869-1874. 1966.-Galactose repression of beta-galactosidase induction in Escherichia coli was investigated to determine whether the galactose molecule itself is the catabolite repressor of this enzyme system. Without exception, beta-galactosidase induction by cells grown in a synthetic salts medium with lactate or glycerol as the carbon source was more strongly repressed by glucose than by galactose. This relationship existed even when the organism was previously grown in the synthetic medium containing galactose as the source of carbon. Two observations suggested that the ability of galactose to repress beta-galactosidase formation by Escherichia coli depends directly upon the cells' capacity to catabolize galactose. First, galactose repression of beta-galactosidase synthesis was markedly enhanced in bacteria tested subsequent to gratuitous induction of the galactose-degrading enzymes with d-fucose. Second, galactose failed to exert a repressive effect on beta-galactosidase in a galactose-negative mutant lacking the first two enzymes involved in galactose catabolism. Glucose completely repressed enzyme formation in this mutant. This same mutant, into which the genes for inducible galactose utilization had been introduced previously by transduction, again exhibited galactose repression. Pyruvate was found to be at least as effective as galactose in repressing beta-galactosidase induction by cells grown in synthetic salts medium plus glycerol. It is concluded that the galactose molecule itself is not the catabolite repressor of beta-galactosidase, but that repression is exerted through some intermediate in galactose catabolism.     

Effect of Galactose on β-Galactosidase Synthesis in Escherichia coli K-12

In wild-type strains of Escherichia coli K-12, the rate of thiomethylgalactoside (TMG)-induced beta-galactosidase synthesis is decreased in the presence of galactose or glucose. A spontaneous mutant of a K-12 strain, 58-161, which synthesizes beta-galactosidase at a low rate was isolated. In this mutant, galactose, after a lag of about one generation time, evoked the same final differential rate of enzyme synthesis as did the gratuitous inducer TMG. However, constitutive, TMG-induced and galactose-induced synthesis in the mutant were subject to inhibition by exogenous glucose. It is concluded that repression of beta-galactosidase synthesis derived from glucose is distinct from the inhibition derived from galactose.     

Relationships Between the Regulation of the Lactose and Galactose Operons of Escherichia coli

A group of structurally related compounds, including galactose, fucose, and a number of galactosides, are regulatory effectors for both the lac and gal operons of Escherichia coli. Although a common set of effectors exists, each operon appears to be regulated independently of the other. Experiments with various regulatory mutants have shown, first, that the presence of the proteins of one operon is without effect on the regulation of the other and, second, that the influence an effector has on one operon is independent of the presence or the functional state of the regulatory genes of the other operon. It is unlikely, therefore, that the two operons share a common regulatory macromolecule. Both gal R(-) and gal o(c) regulatory mutants are equally resistant to repression by glucose and galactosides. It has been possible to show, in the gal operon, that induction and repression are competitive processes. For this operon, the differential rate of enzyme synthesis is set by the relative intracellular concentrations of inducer (fucose) and repressor (isopropylthiogalactoside).     

Sugar transport by the bacterial phosphotransferase system. Regulation of other transport systems (lactose and melibiose)

The role of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) in the phenomenon of inducer exclusion was examined in whole cells of Salmonella typhimurium which carried the genes of the Escherichia coli lactose operon on an episome. In the presence of the PTS substrate methyl alpha-D-glucopyranoside, the extent of accumulation of the lactose analog methyl beta-D-thiogalactopyranoside was reduced. A strain carrying a mutation in the gene for Enzyme I was hypersensitive to the PTS effect, while a crr mutant strain was completely resistant. Influx, efflux, and exchange of galactosides via the lactose "permease" were inhibited by methyl alpha-glucoside. This inhibition occurred in the presence of metabolic energy poisons, and therefore does not involve either the generation of metabolic energy or energy-coupling to the lactose transport system. When the cellular content of the lactose permease was increased by induction with isopropyl beta-D-thiogalactopyranoside, cells gradually became less sensitive to inducer exclusion. The extent of inhibition of methyl beta-thiogalactoside accumulation by methyl alpha-glucoside was shown to be dependent on the relative cellular content of the PTS and lactose system. The data were consistent with an hypothesis involving partial inactivation of galactoside transport due to interaction between a component of the PTS and the lactose permease. By examination of the effects of the PTS and lactose uptake and melibiose permease-mediated uptake of methyl beta-thiogalactoside, it was further shown that the manner in which inducer exclusion is expressed is independent on the routes available to the non-PTS sugar for exit from the cell.     

Transient repression of catabolite-sensitive enzyme synthesis elicited by 2,4-dinitrophenol.

Transient inhibition of catabolic enzyme synthesis in Escherichia coli occurred when a low concentration of 2,4-dinitrophenol (DNP) was simultaneously added with inducer. Using mutant strains defective for gamma-gene product or constitutive for lac enzymes, it was found that the inhibition is not due to the exclusion of inducer by uncoupling. The addition of cyclic adenosine 3',5'-monophosphate overcame repression. The components of the lac operon coordinately responded to DNP inhibition. From deoxyribonucleic acid-ribonucleic acid hybridization experiments, it was found that the inhibition of beta-galactosidase induction occurred at the level of messenger ribonucleic acid synthesis specific for the lac operon. It seems probable that DNP represses induction in a similar manner to that of transient repression observed upon the addition of glucose. Furthermore, it was found that transient repression disappeared if cells were preincubated with DNP before induction. This indicates that new contact of cells with DNP is obligatory for transient repression. From these results, it is suggested that the cell membrane may be responsible for regulation of catabolite-sensitive enzyme synthesis.     

Permease-specific mutations in Salmonella typhimurium and Escherichia coli that release the glycerol, maltose, melibiose, and lactose transport systems from regulation by the phosphoenolpyruvate:sugar phosphotransferase system.

Several carbohydrate permease systems in Salmonella typhimurium and Escherichia coli are sensitive to regulation by the phosphoenolpyruvate:sugar phosphotransferase system. Mutant Salmonella strains were isolated in which individual transport systems had been rendered insensitive to regulation by sugar substrates of the phosphotransferase system. In one such strain, glycerol uptake was insensitive to regulation; in another, the maltose transport system was resistant to inhibition; and in a third, the regulatory mutation specifically rendered the melibiose permease insensitive to regulation. An analogous mutation in E. coli abolished inhibition of the transport of beta-galactosides via the lactose permease system. The mutations were mapped near the genes which code for the affected transport proteins. The regulatory mutations rendered utilization of the particular carbohydrates resistant to inhibition and synthesis of the corresponding catabolic enzymes partially insensitive to repressive control by sugar substrates of the phosphotransferase system. Studies of repression of beta-galactosidase synthesis in E. coli were conducted with both lactose and isopropyl beta-thiogalactoside as exogenous sources of inducer. Employing high concentrations of isopropyl beta-thiogalactoside, repression of beta-galactosidase synthesis was not altered by the lactose-specific transport regulation-resistant mutation. By contrast, the more severe repression observed with lactose as the exogenous source of inducer was partially abolished by this regulatory mutation. The results support the conclusions that several transport systems, including the lactose permease system, are subject to allosteric regulation and that inhibition of inducer uptake is a primary cause of the repression of catabolic enzyme synthesis.     

Allelism of IMP1 and GAL2 genes of Saccharomyces cerevisiae.

Cloning and characterization of the previously described Saccharomyces cerevisiae IMP1 gene, which was assumed to be a nuclear determinant involved in the nucleomitochondrial control of the utilization of galactose, demonstrate allelism to the GAL2 gene. Galactose metabolism does not necessarily involve the induction of the specific transport system coded by GAL2/IMP1, because a null mutant takes up galactose and grows on it. Data on galactose uptake are presented, and the dependence on ATP for constitutive and inducible galactose transport is discussed. These results can account for the inability of imp1/gal2 mutants to grow on galactose in a respiration-deficient background. Under these conditions, uptake was affected at the functional level but not at the biosynthetic level.     

Primary structure and properties of the Na+/glucose symporter (Sg1S) of Vibrio parahaemolyticus.

Previously, we cloned and sequenced a DNA fragment from Vibrio parahaemolyticus and found four open reading frames (ORFs). Here, we clearly demonstrate that one of the ORFs, ORF1, is the gene (sglS) encoding a Na+/glucose symporter (SglS). We characterize the Na+/glucose symporter produced in Escherichia coli mutant (JM1100) cells which lack original glucose transport activity and galactose transport activity. We also show that phlorizin, a potent inhibitor of the SGLT1 Na+/glucose symporter of animal cells, inhibited glucose transport, but not galactose transport, via the SglS system.     

Functional characterization of roles of GalR and GalS as regulators of the gal regulon.

An isorepressor of the gal regulon in Escherichia coli, GalS, has been purified to homogeneity. In vitro DNase I protection experiments indicated that among operators of the gal regulon, GalS binds most strongly to the external operator of the mgl operon, which encodes the high-affinity beta-methylgalactoside galactose transport system, and with less affinity to the operators controlling expression of the gal operon, which codes for enzymes of galactose metabolism. GalS has even less affinity for the external operator of galP, which codes for galactose permease, the major low-affinity galactose transporter in the cell. This order of affinities is the reverse of that of GalR, which binds most strongly to the operator of galP and most weakly to that of mgl. Our results also show that GalS, like its homolog, GalR, is a dimeric protein which in binding to the bipartite operators of the gal operon selectively represses its P1 promoter. Consistent with the fact that GalR is the exclusive regulator of the low-affinity galactose transporter, galactose permease, and that the major role of GalS is in regulating expression of the high-affinity galactose transporter encoded by the mgl operon, we found that the DNA binding of GalS is 15-fold more sensitive than that of GalR to galactose.     

The biochemical and genetic basis for high frequency thiomethyl galactoside resistance in lambda,lambdadg lysogens of Escherichia coli.

In a culture of Escherichia coli K12 gal (lambdadg), cells which form large colonies on agar plates containing galactose and thiomethyl beta-D-galactoside (TMG) appear at high frequency. These clones are resistant to growth inhibition by TMG on galactose minimal medium. Biochemical studies of the steady-state levels of galactokinase and UDPgalactose 4-epimerase suggest that the resistant clones have extra copies of the genes for the galactose-metabolizing enzymes. The mutation for TMG resistance is not located in either the bacterial or the bacteriophage genome, but is probably due to an aberrant association between cell and prophage DNA. Mapping the TMG-resistant characteristic by phage P1 indicates that TMG-resistant bacteria posses at least two GAL+ OPERONS, ONE OF WHICH IS COTRANSDUCIBLe with bio+. In addition, TMG-resistant bacteria behave like lambdadg polylysogens when challenged with the phage lambdaI90c17. From these genetic experiments we conclude that TMG-resistant bacteria arise by duplication of the lambdadg prophage. Finally, gal+ bacteria which carry a single, additional, lambdadg prophage are TMG-resistant. TMG resistance is probably a gal+ gene dosage effect.     

Coregulation of beta-galactoside uptake and hydrolysis by the hyperthermophilic bacterium Thermotoga neapolitana

Regulation of the beta-galactoside transport system in response to growth substrates in the extremely thermophilic anaerobic bacterium Thermotoga neapolitana was studied with the nonmetabolizable analog methyl-beta-D-thiogalactopyranoside (TMG) as the transport substrate. T. neapolitana cells grown on galactose or lactose accumulated TMG against a concentration gradient in an intracellular free sugar pool that was exchangeable with external galactose or lactose and showed induced levels of beta-galactosidase. Cells grown on glucose, maltose, or galactose plus glucose showed no capacity to accumulate TMG, though these cells carried out active transport of the nonmetabolizable glucose analog 2-deoxy-D-glucose. Glucose neither inhibited TMG uptake nor caused efflux of preaccumulated TMG; rather, glucose promoted TMG uptake by supplying metabolic energy. These data show that beta-D-galactosides are taken up by T. neapolitana via an active transport system that can be induced by galactose or lactose and repressed by glucose but which is not inhibited by glucose. Thus, the phenomenon of catabolite repression is present in T. neapolitana with respect to systems catalyzing both the transport and hydrolysis of beta-D-galactosides, but inducer exclusion and inducer expulsion, mechanisms that regulate permease activity, are not present. Regulation is manifest at the level of synthesis of the beta-galactoside transport system but not in the activity of the system.     

Biosynthesis and structure of lipopolysaccharide in an outer membrane-defective mutant of Escherichia coli J5.

Membrane-defective mutants of Escherichia coli J5 were isolated on the basis of supersensitivity to the antibiotic novobiocin. These mutants display an increased sensitivity to a wide range of antibiotics and to several dyes and detergents. In addition, several mutants leak the periplasmic enzymes, alkyline phosphatase and ribonuclease. This evidence indicates an outer membrane defect in these mutants. The inner and outer membranes of one mutant were separated and subjected to compositional analysis. A deficiency in galactose containing lipopolysaccharide in the outer membrane of the mutant was observed. Two possible causes of this deficiency were examined and discounted: defective galactose uptake into the cell, and defective translocation of lipopolysaccharide from the inner membrane. Extraction and chemical analysis of mutant and wild type lipopolysaccharides suggests that the mutant is defective in the enzyme which transfers glucose to the growing lipopolysaccharide core, UDPglucose transferase. Thus, the mutant's deficiency in galactose-containing lipopolysaccharide can be ascribed to the fact that addition of glucose to the lipopolysaccharide core is a prerequisite for galactose addition. The physiological implications of this alteration are discussed.     

Characteristics of Saccharomyces cerevisiae gal1 Delta and gal1 Delta hxk2 Delta mutants expressing recombinant proteins from the GAL promoter

Galactose can be used not only as an inducer of the GAL promoters, but also as a carbon source by Saccharomyces cerevisiae, which makes recombinant fermentation processes that use GAL promoters complicated and expensive. To overcome this problem during the cultivation of the recombinant strain expressing human serum albumin (HSA) from the GAL10 promoter, a gal1 Delta mutant strain was constructed and its induction kinetics investigated. As expected, the gal1 Delta strain did not use galactose, and showed high levels of HSA expression, even at extremely low galactose concentrations (0.05-0.1 g/L). However, the gal1 Delta strain produced much more ethanol, in a complex medium containing glucose, than the GAL1 strain. To improve the physiological properties of the gal1 Delta mutant strain as a host for heterologous protein production, a null mutation of either MIG1 or HXK2 was introduced into the gal1 Delta mutant strain, generating gal1 Delta mig1 Delta and gal1 Delta hxk2 Delta double strains. The gal1 Delta hxk2 Delta strain showed a decreased rate of ethanol synthesis, with an accelerated rate of ethanol consumption, compared to the gal1 Delta strain, whereas the gal1 Delta mig1 Delta strain showed similar patterns to the gal1 Delta strain. Furthermore, the gal1 Delta hxk2 Delta strain secreted much more recombinant proteins (HSA and HSA fusion proteins) than the other strains. The results suggest that the gal1 Delta hxk2 Delta strain would be useful for the large-scale production of heterologous proteins from the GAL10 promoter in S. cerevisiae.