Evidence for Regulation of Gluconeogenesis by the Fructose Phosphotransferase System in Salmonella typhimurium A genetic locus designated fruR , previously mapped to min 3 on the Salmonella typhimurium chromosome , gave rise to constitutive expression of the fructose ( fru ) regulon and pleiotropically prevented growth on all Krebs cycle intermediates .
Regulatory effects offruR were independent of cyclic AMP and its receptor protein and did not prevent uptake of Krebs cycle intermediates .
Instead , the phosphotransferase system appeared to regulate gluconeogenesis by controlling the activities of phosphoenolpyruvate carboxykinase and phosphoenolpyruvate synthase .
The phosphotransferase system ( PTS ) , found in bacteria capable of anaerobic-growth , initiates the metabolism of numerous sugars via glycolysis .
This enzyme system has evolved to an almost unexcelled degree of complexity ( 11 ) , functioning in several catalytic and regulatory capacities ( 9 , 13 ) .
Results of a recent study in our laboratory suggest that this system may have evolved from a primordial PTS which was specific for a single sugar , fructose ( 13 ) .
This proposal places the fructose system in a central position in the evolutionary scheme , suggesting that the fructose-specific proteins of the PTS might possess some unique and important features .
The discovery of a fructose-inducible HPr-like protein ( 14 ) supports this contention .
In the present study , the effects of the constitutive expression of the fructose ( fru ) regulon are shown to exert pleiotropic effects on the utilization of Krebs cycle intermediates and other metabolites .
The locus ( fruR ) , which when defective causes constitutive expression of the fru genes , was mapped precisely , and its effects on the synthesis of PTS enzymes and gluconeogenic enzymes were measured .
The loss offruR function reduced the activities of two key gluconeogenic enzymes , phosphoenolpyruvate ( PEP ) carboxykinase and PEP synthase .
We eliminated the possibilities that the regulatory mechanism involves cyclic AMP , its receptor protein , and the permeases responsible for inducer uptake .
Our results suggest that the fructose PTS negatively controls gluconeogenesis .
( Preliminary results were presented at a meeting of the Royal Netherlands Academy of Arts and Science in September 1985 well the annual meeting of the American as as at Society for Microbiology in March 1986 .
) Many of the bacterial strains used in this study and the procedures used for their isolation were described in the accompanying study ( 3 ) .
The fruRSJ : : TnJO mutation was crossed into other strains by bacteriophage P22 transduction ( 5 ) and selection for tetracycline resistance .
In vitro assays for the proteins of the PTS ( 15 ) , fructose-i-phosphate kinase ( 6 ) , PEP synthase ( 4 ) , and PEP carboxykinase ( 7 ) have been previously described .
The procedure used for the measurement of the uptake of radioactive substrates by intact bacteria has also been reported previously ( 15 ) .
Materials used in this study were from commercial sources and were of the highest purity available .
The effects of a single fruR mutation on the utilization of a variety of compounds in several different genetic backgrounds are summarized in Table 1 .
The fruR5J : : TnJO mutation did not prevent the rapid utilization of sugars or PEP for growth .
By contrast , growth on minimal-agar plates containing as the sole carbon source acetate , pyruvate , alanine , or any of a variety of Krebs cycle intermediates was completely prevented by the loss offruR function regardless of the genetic background ( Table 1 ) .
The negative regulatory effect of the fruR mutation was not altered by point or deletion mutations in the pts operon or by a crp mutation which renders catabolite-sensitive operons independent of cyclic AMP ( 8 ) .
Similarly , exogenous cyclic AMP did not allow-growth offruR mutants on Krebs cycle intermediates .
An additional allele , dadRJ8 ( 16 ) , a promoter mutation which renders D-amino-acid dehydrogenase and the utilization of alanine independent of cyclic AMP , was also without effect .
Uptake studies revealed that the fruRSi : : TnJO mutants still accumulated [ ' 4C ] citrate and [ ' 4C ] succinate after growth in a nutrient medium supplemented with either of Fructose + + + + -- + + Mannitol + + - + -- + + Glycerol + + - + -- + + + + + + + + Galactose + + PEP + + + + + + + + Acetate + - + - + - + - Pyruvate + - + - + - + - Alanine + - + - + - + - Citrate + - + - + - + - Isocitrate + - + -- + - Fumarate + - + -- + - Malate + - + -- + - Oxaloacetate + - + - + - + - aEach pair of strains is isogenic except for the presence or absence of the fruR51 : TnJO mutation .
- , No growth after 48 h on solid minimal-medium for noncarbohydrates or no fermenation after 24 h on eosin-methylene blue fermentation plates containing the indicated sugar at a concentration of 0.5 % .
Furthermore , transfer of galactose-grown LJ712 cells to minimal-citrate medium led to immediate growth stasis .
These results suggest that the fruR5 : : TnJO mutation may give rise to the negative growth phenotype by preventing gluconeogenesis .
A natural isolate of Escherichia coli which expressed the fructose ( fru ) regulon constitutively was shown previously to contain a mutant gene which maps at about 1 min on the E. coli chromosome between ara and leu ( 10 ) .
Phage P22 were therefore grown on strain LJ702 and used to transduce SB1340 ( leuBJ29 ara-9 ; provided by P. E. Hartman ) to tetracycline resistance .
Leucine prototrophy and arabinose fermentation were scored in 262 transductants .
Tetr and ara + exhibited 13 % contransduction , whereas Tetr and leuB + showed 42 % cotransduction .
These results suggest that the clockwise gene order is fruR leu ara and thatfruR is approximately 0.2 min from leu and 0.4 min from ara .
fruR mutants of E. coli can not grow in the presence of the toxic fructose analog xylitol ( 10 ) .
The same phenomenon was observed in Salmonella typhimurium .
Xylitol-resistant mutants usually gave rise to a fructose-negative phenotype .
Of those which were mannitol positive , many had gained the ability to grow on some or all of the Krebs cycle intermediates listed in Table 1 .
When fruR strains were mutagenized and the ability to grow on minimal-citrate plates was selected , a significant proportion of the resulting mutants lost the ability to ferment fructose .
Mutants selected for either xylitol resistance or growth on minimal citrate retained tetracycline resistance .
Enzyme assays revealed that the fruRSi : : TnJO mutation resulted in the constitutive expression of all known fructose ( fru ) regulon enzymes ( Table 2 ) .
fru mutants which regained the ability to utilize Krebs cycle intermediates exhibited low activities for some or all of these enzymes ( Table 2 ) .
The results of enzyme analyses reported in Table 2 do not provide unequivocal evidence for or against the involvement of any one enzyme of the fructose PTS in the regulation of the utilization of Krebs cycle intermediates .
However , the isolation of fructose-specific mutants by selection for growth on citrate suggests that one or more of the fructose enzymes may play a causal role in the regulation of growth on gluconeogenic substances .
Calvo et al. ( 2 ) identified a presumed regulatory locus , likely to befruR , which prevented both the utilization of any Krebs cycle intermediate and the expression of PEP synthase .
However , a deficiency in PEP synthase alone can not account for the growth phenotype ( 1 ) .
Both PEP synthase and PEP carboxykinase exhibited lower activities in strain LJ716 than in the isogenic parental strain LJ715 .
Data for PEP carboxykinase are included in Table 2 .
Significantly , this activity was partially restored by mutations which specifically reduced enzyme IIIf '' activity .
This result suggests a possible involvement of enzyme IIfrU or FPr or both in the regulation of the gluconeogenic enzymes .
A complex of enzyme Il/ru and FPr has been purified to near homogeneity ( S. Sutrina and M. H. Saier , Jr. , unpublished results ) .
However , inclusion of this purified protein preparation only weakly inhibited the activity of PEP carboxy-kinase in high-speed supernatants ( 14 ) derived from various Salmonella strains .
We recently observed that E. coli strains which constitutively express the glucitol ( gut ) operon , encoded on a multicopy plasmid , exhibited the same growth properties as the fruR mutants described in Table 1 .
Genetic loss of plasmid-encoded enzyme IIIguf ( but not the enzyme lIgUt ) resulted in restoration of the normal growth phenotype .
The negative growth phenotype was found to correlate with low PEP carboxykinase activity ( M. Yamada and M. H. Saier , Jr. , unpublished results ) .
The involvement of enzyme lllgu ' is therefore suggested .
This work was supported by Public Health Service grants 5 RO1 Al 21702 and 2 RO1 Al 14176-09A1 from the National Institute of Allergy and Infectious Diseases .
We gratefully acknowledge the expert assistance of Brad Schnierow , who performed the Krebs cycle intermediate uptake experiments .
Bacterial strains were generously provided by P. E. Hartman , J. R. Roth , D. Botstein , and P. Postma .