Characterization of Streptococcus mutans strains deficient in EIIAB Man of the sugar phosphotransferase system

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is the major sugar uptake system in oral streptococci. The role of EIIAB(Man) (encoded by manL) in gene regulation and sugar transport was investigated in Streptococcus mutans UA159. The manL knockout strain, JAM1, grew more slowly than the wild-type strain in glucose but grew faster in mannose and did not display diauxic growth, indicating that EIIAB(Man) is involved in sugar uptake and in carbohydrate catabolite repression. PTS assays of JAM1, and of strains lacking the inducible (fruI) and constitutive (fruCD) EII fructose, revealed that S. mutans EIIAB(Man) transported mannose and glucose and provided evidence that there was also a mannose-inducible or glucose-repressible mannose PTS. Additionally, there appears to be a fructose PTS that is different than FruI and FruCD. To determine whether EIIAB(Man) controlled expression of the known virulence genes, glucosyltransferases (gtfBC) and fructosyltransferase (ftf) promoter fusions of these genes were established in the wild-type and EIIAB(Man)-deficient strains. In the manL mutant, the level of chloramphenicol acetyltransferase activity expressed from the gtfBC promoter was up to threefold lower than that seen with the wild-type strain at pH 6 and 7, indicating that EIIAB(Man) is required for optimal expression of gtfBC. No significant differences were observed between the mutant and the wild-type background in ftf regulation, with the exception that under glucose-limiting conditions at pH 7, the mutant exhibited a 2.1-fold increase in ftf expression. Two-dimensional gel analysis of batch-grown cells of the EIIAB(Man)-deficient strain indicated that the expression of at least 38 proteins was altered compared to that seen with the wild-type strain, revealing that EIIAB(Man) has a pleiotropic effect on gene expression.     

Fructose transport in Bacillus subtilis.

The transport of fructose in Bacillus subtilis was studied in various mutant strains lacking the following activities: ATP-dependent fructokinase (fruC), the fructose 1-phosphate kinase (fruB) the phosphofructokinase (pfk), the enzyme I of the phosphoenolpyruvate phosphotransferase system (the thermosensitive mutation ptsI1), and a transport activity (fruA). Combinations of these mutations indicated that the transport of fructose in Bacillus subtilis is tightly coupled to its phosphorylation either in fructose 1-phosphate, identified in vivo and in vitro or in fructose 6-phosphate identified by indirect lines of evidence. These steps of fructose metabolism were shown to depend on the activity of the enzyme I of the phosphoenolpyruvate phosphotransferase systems. The fruA mutations affect the transport of fructose when the bacteria are submitted to catabolite repression. The mutations were localized on the chromosome of Bacillus subtilis in a cluster including the fruB gene. When grown in a medium supplemented by a mixture of potassium glutamate and succinate the fruA mutants are able to carry on the two vectorial metabolisms generating fructose 6-phosphate as well as fructose 1-phosphate. A negative search of strictly negative transport mutants in fruA strains indicated that more than two structural genes are involved in the transport of fructose.     

Properties of mutants of bacteria belonging to the genus Erwinia devoid of common components of the phosphoenolpyruvate-dependent phosphotransferase system

Biochemical consequences of mutational damage to common components of the Erwinia phosphoenolpyruvate-dependent phosphotransferase system (the HPr protein and enzyme I) were studied. The transport of glucose, mannose, fructose, and mannitol in Erwinia was shown to require a preliminary induction of proteins of the phosphotransferase system. A drastic decrease in the rate of the transport of these carbohydrates was observed in ptsI and ptsH mutants. A disturbance in the common components suppresses the synthesis of inducible enzymes (beta-galactosidase, complexes of pectolyases and cellulases) and renders it resistant to catabolite repression by glucose, but mutants were shown to retain intracellular cAMP content. Erwinia mutants devoid of common components of the system lack phytopathogenic features. The appearance of an intact ptsI allele in the cell completely corrected pleiotropic disturbances in these mutants.     

Analyses of enzyme II gene mutants for sugar transport and heterologous expression of fructokinase gene in Corynebacterium glutamicum ATCC 13032

Corynebacterium glutamicum ATCC 13032 has four enzyme II (EII) genes of the phosphotransferase system in its genome encoding transporters for sucrose, glucose, fructose, and an unidentified EII. To analyze the function of these EII genes, they were inactivated via homologous recombination and the resulting mutants characterized for sugar utilization. Whereas the sucrose EII was the only transport system for sucrose in C. glutamicum, fructose and glucose were each transported by a second transporter in addition to their corresponding EII. In addition, the ptsF ptsG double mutant carrying deletions in the EII genes for fructose and glucose accumulated fructose in the culture broth when growing on sucrose. As no fructokinase gene exists in the C. glutamicum genome, the fructokinase gene from Clostridium acetobutylicum was expressed in C. glutamicum and resulted in the direct phosphorylation of fructose without any fructose efflux. Accordingly, since fructokinase could direct fructose flux to the pentose phosphate pathway for the supply of NADPH, fructokinase expression may be a potential strategy for enhancing amino acid production.     

Properties of a Tn5 insertion mutant defective in the structural gene (fruA) of the fructose-specific phosphotransferase system of Rhodobacter capsulatus and cloning of the fru regulon.

In photosynthetic bacteria such as members of the genera Rhodospirillum, Rhodopseudomonas, and Rhodobacter a single sugar, fructose, is transported by the phosphotransferase system-catalyzed group translocation mechanism. Previous studies indicated that syntheses of the three fructose catabolic enzymes, the integral membrane enzyme II, the peripheral membrane enzyme I, and the soluble fructose-1-phosphate kinase, are coordinately induced. To characterize the genetic apparatus encoding these enzymes, a Tn5 insertion mutation specifically resulting in a fructose-negative, glucose-positive phenotype was isolated in Rhodobacter capsulatus. The mutant was totally lacking in fructose fermentation, fructose uptake in vivo, phosphoenolpyruvate-dependent fructose phosphorylation in vitro, and fructose 1-phosphate-dependent fructose transphosphorylation in vitro. Extraction of the membrane fraction of wild-type cells with butanol and urea resulted in the preparation of active enzyme II free of contaminating enzyme I activity. This preparation was used to show that the activity of enzyme I was entirely membrane associated in the parent but largely soluble in the mutant, suggesting the presence of an enzyme I-enzyme II complex in the membranes of wild-type cells. The uninduced mutant exhibited measurable activities of both enzyme I and fructose-1-phosphate kinase, which were increased threefold when it was grown in the presence of fructose. Both activities were about 100-fold inducible in the parental strain. Although the Tn5 insertion mutation was polar on enzyme I expression, fructose-1-phosphate kinase activity was enhanced, relative to the parental strain. ATP-dependent fructokinase activity was low, but twofold inducible and comparable in the two strains. A second fru::Tn5 mutant and a chemically induced mutant selected on the basis of xylitol resistance showed pleiotropic loss of enzyme I, enzyme II, and fructose-1-phosphate kinase. These mutants were used to clone the fru regulon by complementing the negative phenotype with a wild-type cosmid bank.     

Properties of Mutants of Bacteria Belonging the Genus Erwinia Devoid of Common Components of the Phosphoenolpyruvate-Dependent Phosphotransferase System

Biochemical consequences of mutational damage to common components of the Erwinia phosphoenolpyruvate-dependent phosphotransferase system (the HPr protein and enzyme I) were studied. The transport of glucose, mannose, fructose, and mannitol inErwinia was shown to require a preliminary induction of proteins of the phosphotransferase system. A drastic decrease in the rate of the transport of these carbohydrates was observed in ptsI and ptsH mutants. A disturbance in the common components suppresses the synthesis of inducible enzymes (-galactosidase, complexes of pectolate lyases and cellulases) and renders it resistant to catabolite repression by glucose, but mutants were shown to retain intracellular cAMP content. Erwinia mutants devoid of common components of the system lack phytopathogenic features. The appearance of an intact ptsI allele in the cell completely repaired pleiotropic disturbances in these mutants.     

Role of the phosphoenolpyruvate-dependent fructose phosphotransferase system in the utilization of mannose by Escherichia coli.

Mutants of Escherichia coli devoid of the membrane-spanning proteins PtsG and PtsMP, which are components of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) and which normally effect the transport into the cells of glucose and mannose, do not grow upon or take up either sugar. Pseudorevertants are described that take up, and grow upon, mannose at rates strongly dependent on the mannose concentration in the medium (apparent Km > 5 mM); such mutants do not grow upon glucose but are derepressed for the components of the fructose operon. Evidence is presented that mannose is now taken up via the fructose-PTS to form mannose 6-phosphate, which is further utilized for growth via fructose 6-phosphate and fructose 1,6-bisphosphate.     

Enzymes II of the phosphotransferase system do not catalyze sugar transport in the absence of phosphorylation.

In Salmonella typhimurium, glucose, mannose, and fructose are normally transported and phosphorylated by the phosphoenolpyruvate:sugar phosphotransferase system. We have investigated the transport of these sugars and their non-metabolizable analogs in mutant strains lacking the phospho-carrier proteins of the phosphoenolpyruvate:sugar phosphotransferase system, the enzymes I and HPr, to determine whether the sugar-specific, membrane-bound components of the phosphonenolpyruvate: sugar phosphotransferase system, the enzymes II, can catalyze the uptake of these sugars in the absence of phosphorylation. This process does not occur. We have also isolated mutant strains which lack enzyme I and HPr, but have regained the ability to grow on mannose or fructose. These mutants contained elevated levels of mannokinase (fructokinase). In addition, growth on mannose required constitutive synthesis of the galactose permease. When strains were constructed which lacked the galactose permease, they were unable to grow even on high concentrations of mannose, although elevated levels of mannokinase (fructokinase) were present. These results substantiate the conclusion that the enzymes II of the phosphoenolpyruvate:sugar phosphotransferase system are unable to carry out facilitated diffusion.     

Xylitol and D-arabitol toxicities due to derepressed fructose, galactitol, and sorbitol phosphotransferases of Escherichia coli.

d-Arabitol was observed to be toxic to many laboratory strains of Escherichia coli K-12, and xylitol was found to be toxic to an existing E. coli C mutant strain. Fructose-specific components of the phosphoenolpyruvate:sugar phosphotransferase system are required for xylitol toxicity. Selection for xylitol resistance results in Fru(-) strains blocked in fructose phosphotransferase. Introduction of the ptsF or ptsI mutation into a xylitol-sensitive strain eliminates sensitivity. [(14)C]fructose uptake experiments imply that the mutation to xylitol sensitivity, which is co-transducible with ara and leu, results in derepression of normally inducible fructose phosphotransferase. Wild-type strains also become xylitol sensitive if induced by (and then removed from) fructose. Xylitol toxicity is prevented by fructose in both wild-type and mutant strains. Circumstances causing xylitol, a new food additive, to become toxic to an otherwise insensitive wild-type organism have not been reported previously. The d-arabitol-sensitive laboratory strains are galactitol (dulcitol) utilizers, although most other strains are not. Selection for d-arabitol resistance results in Gat(-) strains blocked in a constitutive galactitol-specific component of the phosphotransferase system. A mutation causing d-arabitol sensitivity occurred many years ago in AB284, the parent of AB311, AB312, AB313, and many other strains. d-Arabitol sensitivity also occurs in sorbitol-constitutive strains and is shown, like the previous two instances of pentitol toxicities, to result from a constitutive phosphotransferase, which is blocked in mutants selected for resistance.     

The complete phosphotransferase system in Escherichia coli

We here tabulate and describe all currently recognized proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) and their homologues encoded within the genomes of sequenced E. coli strains. There are five recognized Enzyme I homologues and six recognized HPr homologues. A nitrogen-metabolic PTS phosphoryl transfer chain encoded within the rpoN and ptsP operons and a tri-domain regulatory PTS protein encoded within the dha (dihydroxyacetone catabolic) operon, probably serve regulatory roles exclusively. In addition to several additional putative regulatory proteins, there are 21 (and possibly 22) recognized Enzyme II complexes. Of the 21 Enzyme II complexes, 7 belong to the fructose (Fru) family, 7 belong to the glucose (Glc) family, and 7 belong to the other PTS permease families. All of these proteins are briefly described, and phylogenetic data for the major families are presented.     

Vinylglycolate resistance in Escherichia coli.

Escherichia coli K-12 vinylglycolate-resistant mutants have been isolated and characterized. Two of the mutants, JSH 150 and JSH 151, have been determined to be double mutants, lacking both membrane-bound L-and D-lactate dehydrogenases. The lactate transport system is intact in all strains; both radioactive lactate and vinylglycolate are actively taken up. Likewise, the phosphoenolypyruvate-dependent phosphotransferase system for hexose uptake is active. Vinylglycolate, previously shown to inhibit the phosphoenolpyruvate-dependent phosphotransferase system, has very little effect in the double mutants. The extent of vinylglycolate inhibition in other mutants seems directly related to the activity of the lactate dehydrogenases. This indicates that vinylglycolate is oxidized to 2-keto-3-butenoate before inactivating the phosphoenolpyruvate-dependent phosphotransferase system. These results were found in whole cells and confirmed in isolated membrane vesicles.     

Phosphoenolpyruvate:hexose phosphotransferase systems in Lactobacillus species]

The substrate range of phosphoenolpyruvate:hexose phosphotransferase systems (hexose-PTSs) in Lactobacillus casei subsp. casei LAC3 and L. acidophilus LAC5 was examined. Strain LAC3 demonstrated PTS activities for glucose (Glc), mannose (Man), glucosamine (GcN), 2-deoxyglucose (2DG) and fructose (Fru), while strain LAC5 showed the activities only for Man and Fru. These activities were all constitutive. Growth of both strains was strongly inhibited by 2DG. 2DG-resistant mutants DG329 and DG504 were isolated, respectively, from strains LAC3 and LAC5. Mutant DG329 grown on Glc was defective in all the above-described activities observed with strain LAC3, whereas no defect in PTS activities was found in mutant DG504. Mutant DG329, however, showed some inducible activities for Man and Fru when grown on Man, Fru or Scr. These results strongly suggest that strain LAC3 has inducible PTS(s) specific for Man and/or Fru besides the well-known, broadly specific, constitutive Man-PTS, and also that strain LAC5 lacks the Man-PTS, but has other constitutive PTS(s) specific for Man and/or Fru. L. fermentum LAC12 had the Man-PTS as reported previously (Nagasaki et al., 1992), but had no inducible activities like those found in strain LAC3.     

Plasmid-mediated uptake and metabolism of sucrose by Escherichia coli K-12.

The conjugative plasmid pUR400 determines tetracycline resistance and enables cells of Escherichia coli K-12 to utilize sucrose as the sole carbon source. Three types of mutants affecting sucrose metabolism were derived from pUR400. One type lacked a specific transport system (srcA); another lacked sucrose-6-phosphate hydrolase (scrB); and the third, a regulatory mutant, expressed both of these functions constitutively (scrR). In a strain harboring pUR400, both transport and sucrose-6-phosphate hydrolase were inducible by fructose, sucrose, and raffinose; if a scrB mutant was used, fructose was the only inducer. These data suggested that fructose or a derivative acted as an endogenous inducer. Sucrose transport and sucrose-6-phosphate hydrolase were subject to catabolite repression; these two functions were not expressed in an E. coli host (of pUR400) deficient in the adenosine 3-,5'-phosphate receptor protein. Sucrose uptake (apparent Km = 10 microM) was dependent on the scrA gene product and on the phosphoenolpyruvate-dependent sugar:phosphotransferase system (PTS) of the host. The product of sucrose uptake (via group translocation) was identified as sucrose-6-phosphate, phosphorylated at C6 of the glucose moiety. Intracellular sucrose-6-phosphate hydrolase catalyzed the hydrolysis of sucrose-6-phosphate (Km = 0.17 mM), sucrose (Km = 60 mM), and raffinose (Km = 150 mM). The active enzyme was shown to be a dimer of Mr 110,000.     

Purification of the mannitol-specific enzyme II of the Escherichia coli phosphoenolpyruvate:sugar phosphotransferase system.

The inducible, mannitol-specific Enzyme II of the phosphoenolpyruvate:sugar phosphotransferase system has been purified approximately 230-fold from Escherichia coli membranes. The enzyme, initially solubilized with deoxycholate, was first subjected to hydrophobic chromatography on hexyl agarose and then purified by several ion exchange steps in the presence of the nonionic detergent, Lubrol PX. The purified protein appears homogeneous by several criteria and probably consists of a single kind of polypeptide chain with a molecular weight of 60,000 (+/- 5%). In addition to catalyzing phosphoenolpyruvate-dependent phosphorylation of mannitol in the presence of the soluble enzymes of the phosphotransferase system, the purified Enzyme II also catalyzes mannitol 1-phosphate:mannitol transphosphorylation in the absence of these components. A number of other physical and catalytic properties of the enzyme are described. The availability of a stable, homogeneous Enzyme II should be invaluable for studying the mechanism of sugar translocation and phosphorylation catalyzed by the bacterial phosphotransferase system.     

Pyruvate formation during the catabolism of simple hexose sugars by Escherichia coli: studies with pyruvate kinase-negative mutants.

Escherichia coli K-12 mutants lacking the adenosine 5'-monophosphate-activated pyruvate kinase have been isolated accidentally and used to prepare further mutants additionally devoid of the fructose bisphosphate-activated pyruvate kinase. Such double mutants totally devoid of pyruvate kinase activity still grow well under aerobic conditions on sugars that are catabolized by the phosphoenolpyruvate (PEP):sugar phosphotransferase system, but they grow poorly on non-phosphotransferase system sugars. This suggests that although pyruvate kinase plays a major role in the formation of pyruvate from PEP during growth on non-phosphotransferase system sugars, the operation of the PEP:sugar phosphotransferase system can contribute significantly to pyruvate production from PEP. In the absence of pyruvate kinase and an active PEP:sugar phosphotransferase system the methylglyoxal glycolytic bypass may also function to some extent for the formation of pyruvate during the catabolism of simple hexose sugars. No unique physiological role can yet be ascribed to the adenosine 5'-monophosphate-activated pyruvate kinase as a result of these studies.     

Fructose catabolism in Xanthomonas campestris pv. campestris. Sequence of the PTS operon, characterization of the fructose-specific enzymes.

In Xanthomonas campestris pv. campestris, fructose is transported and phosphorylated into fructose 1-phosphate through a phosphoenolpyruvate-dependent phosphotransferase system. The nucleotide sequence of the fruA gene encoding the phosphotransferase system permease specific of fructose (EIIFru) was determined. The fructose 1-phosphate produced by the phosphotransferase system is phosphorylated into fructose 1,6-bisphosphate by a 1-phosphofructokinase. This enzyme was characterized and the corresponding gene (fruK) was sequenced. Sequence comparisons revealed that FruK is a member of a new family of ATP-binding proteins composed of sugar (or sugar-phosphate) kinases. In phosphotransferase system-deficient strains, fructose can still be transported by an unidentified permease. The intracellular fructose is then phosphorylated by a multimeric fructokinase of 135 kDa specific for fructose and inhibited by fructose, fructose 1,6-bisphosphate, and mannose. Several other enzymes of fructose metabolism were assayed and a potential pathway for fructose catabolism is presented.