Inducer Exclusion Research Articles

Pentose utilization by the ruminal bacterium Ruminococcus albus.

Ruminococcus albus is an important fibrolytic ruminal bacteria which degrades hemicellulose and ferments the resulting pentose sugars. However, little information is available on the utilization of pentoses by this organism or the effect of hexose sugars on pentose metabolism. Enzymatic studies indicated that R. albus metabolized pentoses via the pentose phosphate pathway and possessed constitutive transketolase activity. Cellobiose was preferred over xylose and arabinose, and it appeared that the disaccharide decreased pentose metabolism by repression of transport activity and catabolic enzymes (isomerases and kinases). Glucose and xylose were co-utilized, and transport studies suggested that there was a common transport system for both sugars. In contrast, glucose was preferred over arabinose and the hexose noncompetitively inhibited the transport of arabinose. Since R. albus lacks a glucose phosphotransferase system, the inhibition of arabinose uptake could not be explained by previously described models of inducer exclusion involving such a system. Because accumulation of radiolabeled xylose, arabinose, and glucose proceeded in the absence of a proton motive force and since transport was correlated with the intracellular ATP concentration, it appeared that monosaccharide uptake was driven by ATP hydrolysis.

Genetic characterization of a Rhizobium meliloti lactose utilization locus.

We identified several linked genes of a lactose regulon in Rhizobium meliloti. These were lacZ, the structural gene for beta-galactosidase; lacR, the lactose repressor gene; and two genes encoding proteins of unknown function, lacW and lacX. Insertion mutants in lacW and lacZ belonged to a single genetic complementation group, and lacW appeared to lie upstream of lacZ in an operon. Expression of lacZ, lacW and lacX was repressed by lacR, and expression of lacZ and lacW was derepressed by lactose. lacZ was not required for induction of lacW by lactose, suggesting that lactose itself, rather than a processed form of lactose, may be the actual inducer molecule. Expression of all three genes was repressed by succinate, and the lacR independence of this repression showed that inducer exclusion could not be the sole mechanism. This pattern of lac gene organization and regulation differs in several ways from that observed in enteric bacteria.

Catabolite repression of the Bacillus subtilis gnt operon mediated by the CcpA protein.

Inducer exclusion was not important in catabolite repression of the Bacillus subtilis gnt operon. The CcpA protein (also known as AlsA) was found to be necessary for catabolite repression of the gnt operon, and a mutation (crsA47, which is an allele of the sigA gene) partially affected this catabolite repression.

A Regulatory Element in the CHA1 Promoter Which Confers Inducibility by Serine and Threonine on Saccharomyces cerevisiae Genes

CHA1 of Saccharomyces cerevisiae is the gene for the catabolic L-serine (L-threonine) dehydratase, which is responsible for biodegradation of serine and threonine. We have previously shown that expression of the CHA1 gene is transcriptionally induced by serine and threonine. Northern (RNA) analysis showed that the additional presence of good nitrogen sources affects induction. This may well be due to inducer exclusion. To identify interactions of cis-acting elements with trans activators of the CHA1 promoter, we performed band shift assays of nuclear protein extracts with CHA1 promoter fragments. By this approach, we identified a protein-binding site of the CHA1 promoter. The footprint of this protein contains the ABF1-binding site consensus sequence. This in vitro binding activity is present irrespectively of CHA1 induction. By deletion analysis, two other elements of the CHA1 promoter, UAS1CHA and UAS2CHA, which are needed for induction of the CHA1 gene were identified. Each of the two sequence elements is sufficient to confer serine and threonine induction upon the CYC1 promoter when substituting its upstream activating sequence. Further, in a cha4 mutant strain which is unable to grow with serine or threonine as the sole nitrogen source, the function of UAS1CHA, as well as that of UAS2CHA, is obstructed.

A regulatory element in the CHA1 promoter which confers inducibility by serine and threonine on Saccharomyces cerevisiae genes.

CHA1 of Saccharomyces cerevisiae is the gene for the catabolic L-serine (L-threonine) dehydratase, which is responsible for biodegradation of serine and threonine. We have previously shown that expression of the CHA1 gene is transcriptionally induced by serine and threonine. Northern (RNA) analysis showed that the additional presence of good nitrogen sources affects induction. This may well be due to inducer exclusion. To identify interactions of cis-acting elements with trans activators of the CHA1 promoter, we performed band shift assays of nuclear protein extracts with CHA1 promoter fragments. By this approach, we identified a protein-binding site of the CHA1 promoter. The footprint of this protein contains the ABF1-binding site consensus sequence. This in vitro binding activity is present irrespectively of CHA1 induction. By deletion analysis, two other elements of the CHA1 promoter, UAS1CHA and UAS2CHA, which are needed for induction of the CHA1 gene were identified. Each of the two sequence elements is sufficient to confer serine and threonine induction upon the CYC1 promoter when substituting its upstream activating sequence. Further, in a cha4 mutant strain which is unable to grow with serine or threonine as the sole nitrogen source, the function of UAS1CHA, as well as that of UAS2CHA, is obstructed.

Evidence that the glutamine-stimulated loss of nitrate reductase protein from the yeast Candida nitratophila is not the result of inducer exclusion.

Synthesis of nitrate reductase protein and increases in nitrate reductase activity occurred in cultures of the yeast Candida nitratophila when they were incubated in medium containing ammonium nitrate. Similar treatment with glutamine plus nitrate resulted in little increase in nitrate reductase activity, in cultures grown previously with reduced nitrogen compounds, and decreases in enzyme activity, in cultures adapted to nitrate. Labelling studies conducted in vivo revealed a rapid cessation of de novo nitrate reductase synthesis when glutamine was supplied to nitrate-adapted cultures in the presence of nitrate. Intracellular glutamine concentrations increased rapidly under these conditions and these cultures exhibited high glutamine: glutamate ratios. As nitrate was taken up in the presence of glutamine in these experiments, it is concluded that the glutamine-stimulated inhibition of nitrate reductase synthesis is a consequence of repression and rapid turnover of nitrate reductase mRNA and not inducer (nitrate) exclusion.

Functional exchangeability of the ABC proteins of the periplasmic binding protein-dependent transport systems Ugp and Mal of Escherichia coli.

The periplasmic binding protein-dependent transport systems Ugp and Mal of Escherichia coli transport sn-glycerol-3-phosphate and maltose, respectively. The UgpC and MalK proteins of these transport systems, which couple energy to the transport process by ATP-hydrolysis, are highly homologous, suggesting that they might be functionally exchangeable. Complementation experiments showed that UgpC expression could restore growth of a malK mutant on maltose as a carbon source, provided that it was expressed at a sufficiently high level in the absence of the integral inner membrane components UgpA and/or UgpE of the Ugp system. Conversely, MalK expression could complement ugpC mutants and restore the utilization of sn-glycerol-3-phosphate as a phosphate source. The hybrid transporters appeared to be less efficient than the wild-type systems. The complementation of ugpC mutations by MalK was strongly inhibited by the presence of glucose or alpha-methylglucoside, which are substrates of the phosphotransferase system. This inhibition is probably due to hypersensitivity of the hybrid UgpBAE-MalK transporter to inducer exclusion. UgpC expression did not complement the regulatory function of MalK in mal gene expression. The exchangeability of UgpC and MalK indicates that these proteins do not contribute to a substrate-binding site conferring substrate specificity to the transporter. These are the first examples of functional, hybrid periplasmic permeases in which the energy-coupling components could be functionally exchanged.

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.

Simultaneous uptake and utilisation of glucose and xylose byClostridium thermohydrosulfuricum

Growth studies of Clostridium thermohydrosulfuricum Rt8.B1 demonstrated that glucose and xylose were used simultaneously when supplied together at nonlimiting concentrations in pH-controlled batch culture. Under conditions of hyperbolic growth, both catabolite repression and inducer exclusion were absent. Glucose did not repress xylose metabolism (i.e. xylose permease and xylose isomerase genes were expressed in the presence of glucose and were not subject to catabolite inhibition when glucose was added to cultures growing on high concentrations of xylose). The kinetics of glucose and xylose utilisation indicated that separate systems were present for the uptake of these substrates when supplied together. Glucose utilisation was biphasic, indicating high- and low-affinity systems for glucose uptake. Xylose utilisation was directly proportional to the xylose concentration, suggesting a facilitated diffusion mechanism was operative for uptake.

Nitrogen catabolite repression of arginase (CAR1) expression in Saccharomyces cerevisiae is derived from regulated inducer exclusion

Expression of the Saccharomyces cerevisiae arginase (CAR1) gene is regulated by induction and nitrogen catabolite repression (NCR). Arginine was demonstrated to be the native inducer. CAR1 sensitivity to NCR has long been accepted to be accomplished through a negative control mechanism, and cis-acting sites for it have been hypothesized. In search of this negatively acting site, we discovered that CAR1 sensitivity to NCR derives from regulated inducer (arginine) exclusion. The route of catabolic entry of arginine into the cell, the general amino acid permease (GAP1), is sensitive to NCR. However, CAR1 expression in the presence of sufficient intracellular arginine is NCR insensitive.

Mutational analysis of the enzyme III Glc of the phosphoenolpyruvate phosphotransferase system in Escherichia coli

The phosphoenolpyruvate phosphotransferase system (PTS) component EIII Glc is responsible for transport and phosphorylation of glucose via EII Glc. It also regulates the catabolism of other carbon sources, such as lactose and maltose, by modulating both the intracellular concentrations of the corresponding inducers and of cAMP. Mutational analysis of EIII Glc was performed in order to identify crucial residues mediating the interactions between EIII Glc and its target proteins. Such mutations were isolated by in vitro hydroxylamine mutagenesis of the cloned EIII Glc gene, crr. Five mutated EIII Glc impaired in the function of inducer exclusion were obtained. However, these mutations did not abolish the function of EIII Glc in the transport and phosphorylation of glucose, nor in activation of adenylate cyclase. A single amino acid change was found for each mutation, which is located in a restricted area of the polypeptide chain: Gly 47 → Ser 47 for the HA2 and HA5 mutations, Ala 76 → Thr 76 for HA4 mutation and Ser 78 → Phe 78 for HA3 mutation, indicative of quaternary interactions between the corresponding region of EIII Glc and its target protein(s).

The activities of the Escherichia coli MalK protein in maltose transport, regulation, and inducer exclusion can be separated by mutations

The maltose regulon consists of several genes encoding proteins involved in the uptake and utilization of maltose and maltodextrins. Five proteins make up a periplasmic binding-protein-dependent active transport system. One of these proteins, MalK, contains an ATP-binding site and is thought to couple the hydrolysis of ATP to the accumulation of substrate. Beside its function in transport, MalK has two additional roles: (i) it negatively regulates mal regulon expression and (ii) it serves as the target for regulation of transport activity by enzyme IIIGlc of the phosphotransferase system. To determine whether the three functions of MalK are separable, we have isolated and characterized three classes of malK mutations. The first type (class I) exhibited constitutive mal gene expression but still allowed normal transport of maltose; the second type (class II) lacked the ability to transport maltose but retained the ability to repress the mal genes. Class I mutations were localized in the last third of the gene, at amino acids 267 (Trp to Gly) and 346 (Gly to Ser). Mutations of class II were found at the positions 137 (Gly to Ala), 140 (delta Gln Arg), and 158 (Asp to Asn). These mutations are near or within the region of MalK that exhibits extensive homology to the B site of an ATP-binding fold. In addition, site-directed mutagenesis was used to add or remove one amino acid in the A site of the ATP-binding fold. Plasmids carrying these mutations also behaved as class II mutants. The third class of malK mutations resulted in resistance to the enzyme IIIGlc-mediated inhibitory effects of alpha-methylglucoside. These mutations did not interfere with the regulatory function of MalK. One of these mutations (exchanging a serine at position 282 for leucine) is located in a short stretch of amino acids that exhibits homology to a sequence in the Escherichia coli Lac permease in which alpha-methylglucoside-resistant mutations have been found.

Regulation of the maltose transport system of Escherichia coli by the glucose-specific enzyme III of the phosphoenolpyruvate-sugar phosphotransferase system. Characterization of inducer exclusion-resistant mutants and reconstitution of inducer exclusion in proteoliposomes.

Maltose transport in Escherichia coli is regulated at the protein level by the glucose-specific enzyme III (IIIglc) of the phosphoenolpyruvate-sugar phosphotransferase system, by a mechanism known as inducer exclusion. We have isolated and characterized four mutants in the maltose transport system, all of which are in malK, which are resistant to inducer exclusion. The mutations in three of these mutants fall within the COOH-terminal domain of MalK and suggest the first reported function for this domain. Two of these are in a region which shows sequence similarity to lacY and melB, both of which are also regulated by IIIglc, and thus may define a IIIglc-binding domain. We have also reconstituted inducer exclusion in proteoliposomes made from membranes overexpressing the maltose permease. Maltose transport is inhibited by 50-60% when IIIglc is included in the intravesicular space. The inhibition is due to a decrease in the Vmax of transport by a factor of 2. IIIglc does not affect the coupling of ATP hydrolysis to maltose transport, since the ratio of ATP hydrolyzed/maltose transported remained constant in the presence and absence of IIIglc. Finally, the Ki for IIIglc was 40 microM, roughly the same as the in vivo concentration of IIIglc.

Regulation of assimilatory nitrate reductase formation in Klebsiella aerogenes W70

Klebsiella aerogenes W70 could grow aerobically with nitrate or nitrite as the sole nitrogen source. The assimilatory nitrate reductase and nitrite reductase responsible for this ability required the presence of either nitrate or nitrite as an inducer, and both enzymes were repressed by ammonia. The repression by ammonia, which required the NTR (nitrogen regulatory) system (A. Macaluso, E. A. Best, and R. A. Bender, J. Bacteriol. 172:7249-7255, 1990), did not act solely at the level of inducer exclusion, since strains in which the expression of assimilatory nitrate reductase and nitrite reductase was was independent of the inducer were also susceptible to repression by ammonia. Insertion mutations in two distinct genes, neither of which affected the NTR system, resulted in the loss of both assimilatory nitrate reductase and nitrite reductase. One of these mutants reverted to the wild type, but the other yielded pseudorevertants at high frequency that were independent of inducer but still responded to ammonia repression.

Carbohydrate uptake in the oral pathogen Streptococcus mutans: mechanisms and regulation by protein phosphorylation

Streptococcus mutans is the primary etiological agent of dental caries in man and other animals. This organism and other related oral streptococci use carbohydrates almost exclusively as carbon and energy sources, fermenting them primarily to lactic acid which initiates erosion of tooth surfaces. Investigations over the past decade have shown that the major uptake mechanism for most carbohydrates in S. mutans is the phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS), although non-PTS systems have also been identified for glucose and sucrose. Regulation of sugar uptake occurs by induction/repression and inducer exclusion mechanisms in S. mutans, but apparently not by inducer expulsion as is found in some other streptococci. In addition, ATP-dependent protein kinases have also been indentified in S. mutans and other oral streptococci, and a regulatory function for at least one of these has been postulated. Among a number of proteins that are phosphorylated by these enzymes, the predominant soluble protein substrate is the general phospho-carrier protein of the PTS, HPr, as had previously been observed in a variety of Gram-positive bacteria. Recent results have provided evidence for a role for ATP-dependent phosphorylation of HPr in the coordination of sugar uptake and its catabolism in S. mutans. In this review, these results are summarized, and directions for future research in this area are discussed.

Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

The bacterial phosphotransferase system (PTS) functions in a variety of regulatory capacities. One of the best characterized of these is the process by which the PTS regulates inducer uptake and catabolite repression. Early genetic and physiological evidence supported a mechanism whereby the phosphorylation state of an enzyme of the PTS, the enzyme III specific for glucose (IIIGlc), allosterically inhibits the activities of a number of permeases and catabolic enzymes, the lactose, galactose, melibiose, and maltose permeases, as well as glycerol kinase. Extensive biochemical evidence now supports this model. Evidence is also available showing that substrate binding to those target proteins enhances their affinities for IIIGlc. In the case of the lactose permease, this positively cooperative interaction represents a well documented example of transmembrane signaling, demonstrated both in vivo and in vitro. Although the PTS-mediated regulation of cyclic AMP synthesis (catabolite repression) is not as well defined from a mechanistic standpoint, a model involving allosteric activation of adenylate cyclase by phospho-IIIGlc, together with the evidence supporting it, is presented. These regulatory mechanisms may prove to be operative in gram-positive as well as gram-negative bacteria, but the former organisms may have introduced variations on the theme by covalently attaching IIIGlc-like moieties to some of the target permeases and catabolic enzymes. It appears likely that the general process of PTS-catalyzed protein phosphorylation-dephosphorylation will prove to be important to the regulation of numerous bacterial physiological processes, including chemotaxis, intermediary metabolism, gene transcription, and virulence.

Protein phosphorylation and allosteric control of inducer exclusion and catabolite repression by the bacterial phosphoenolpyruvate: sugar phosphotransferase system.

The bacterial phosphotransferase system (PTS) functions in a variety of regulatory capacities. One of the best characterized of these is the process by which the PTS regulates inducer uptake and catabolite repression. Early genetic and physiological evidence supported a mechanism whereby the phosphorylation state of an enzyme of the PTS, the enzyme III specific for glucose (IIIGlc), allosterically inhibits the activities of a number of permeases and catabolic enzymes, the lactose, galactose, melibiose, and maltose permeases, as well as glycerol kinase. Extensive biochemical evidence now supports this model. Evidence is also available showing that substrate binding to those target proteins enhances their affinities for IIIGlc. In the case of the lactose permease, this positively cooperative interaction represents a well documented example of transmembrane signaling, demonstrated both in vivo and in vitro. Although the PTS-mediated regulation of cyclic AMP synthesis (catabolite repression) is not as well defined from a mechanistic standpoint, a model involving allosteric activation of adenylate cyclase by phospho-IIIGlc, together with the evidence supporting it, is presented. These regulatory mechanisms may prove to be operative in gram-positive as well as gram-negative bacteria, but the former organisms may have introduced variations on the theme by covalently attaching IIIGlc-like moieties to some of the target permeases and catabolic enzymes. It appears likely that the general process of PTS-catalyzed protein phosphorylation-dephosphorylation will prove to be important to the regulation of numerous bacterial physiological processes, including chemotaxis, intermediary metabolism, gene transcription, and virulence.

Regulation of the galactose-inducible lac operon and the histidine utilization operons in pts mutants of Klebsiella aerogenes.

Galactose appears to be the physiological inducer of the chromosomal lac operon in Klebsiella aerogenes. Both lactose and galactose are poor inducers in strains having a functional galactose catabolism (gal) operon, but both are excellent inducers in gal mutants. Thus the slow growth of K. aerogenes on lactose reflects the rapid degradation of the inducer. Several pts mutations were characterized and shown to affect both inducer exclusion and permanent catabolite repression. The beta-galactosidase of pts mutants cannot be induced at all by lactose, and pts mutants appear to have a permanent and constitutive inducer exclusion phenotype. In addition, pts mutants show a reduced rate of glucose metabolism, leading to slower growth on glucose and a reduced degree of glucose-mediated permanent catabolite repression. The crr-type pseudorevertants of pts mutations relieve the constitutive inducer exclusion for lac but do not restore the full level of glucose-mediated permanent catabolite repression and only slightly weaken the glucose-mediated inducer exclusion. Except for weakening the glucose-mediated permanent catabolite repression, pts and crr mutations have no effect on expression of the histidine utilization (hut) operons.

MAL63 codes for a positive regulator of maltose fermentation in Saccharomyces cerevisiae.

Genetic analysis of the MAL6 locus has previously yielded mal6 mutants which fall into a single complementation group and which are noninducible for maltase and maltose permease. However, the strains used in these studies contained additional partially functional copies of MAL1 (referred to as MAL1g) and MAL3 (referred to as MAL3g). Using a strain lacking MALg genes, we have isolated two classes of mutants and these classes correspond to mutations in MAL63 and MAL61, two genes of the MAL6 complex. Disruptions of MAL63 are noninducible for maltase and maltose permease and for their corresponding mRNAs. The mal6 mutants are shown to map to MAL63. Inducer exclusion as a cause of the noninducible phenotype of the mal63 mutations has been eliminated by constructing a mal63 mutant in a strain constitutive for maltose permease; the strain remains noninducible. These results rigorously demonstrate that MAL63 is a regulatory gene which plays a positive role in the regulation of maltose fermentation.

Two functionally different glucose phosphotransferase transport systems in Streptococcus mutans and Streptococcus sobrinus.

Abstract Spontaneous phosphotransferase negative mutants of Streptococcus mutans GS5‐2 (mutant GM500) and of Streptococcus sobrinus 27352 (mutant MM202) were isolated by screening for 2‐deoxyglucose resistance using lactose as the growth substrate. Measurements of phosphoenolpyruvate: phosphotransferase activities with isolated membranes and purified Enzyme I and HPr indicated that the mutant strains were defective at the level of membrane components of the phosphoenolpyruvate: mannose phosphotranferase system but still possessed phosphoenolpyruvate: glucose phosphotransferase activity. These results were corroborated by uptake studies performed with resting cells. Our results suggest that glucose is transported by 2 distinct phosphoenolpyruvate: phosphotransferase systems in S. mutans and S. sobrinus, one of which is also capable of transporting mannose. This glucose‐mannose transport system is involved in catabolite repression and inducer exclusion, and controls, by way of these mechanisms, the metabolism of lactose.