Mapping of two ugp genes coding for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli.

Two genes, ugpA and ugpB, coding for a binding protein-dependent sn-glycerol-3-phosphate transport system, were mapped at 75.3 min on the Escherichia coli chromosome. A Tn10 insertion in ugpA resulted in loss of transport activity but still allowed the synthesis of the sn-glycerol-3-phosphate-binding protein. This Tn10 insertion was found to be linked by P1 transduction to pit, aroB, malA, asd, and livH with 2.5, 2.8, 25, 63.5, and 83% cotransduction frequency. An insertion of Mud (Ampr lac) in ugpB resulted in the loss of the binding protein. ugpB is closely linked to ugpA. It is either the structural gene for the binding protein or located proximal to it. The analysis of the crosses allowed the ordering of the markers in the clockwise direction as follows: aroB, malA, asd, ugpA, ugpB, livH, pit.     

Cloning of the ugp region containing the structural genes for the pho regulon-dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary Using a novel positive selection method for G3P transport activity, phages that carry either all or part of ugp, the genes of the pho regulon-dependent G3P transport system of Escherichia coli were isolated from a library of EcoRI fragments of Escherichia coli established in gt7. By subcloning EcoRI fragments carried by the different phages into the multicopy plasmids pACYC184 and pUR222, it was shown that two chromosomal fragments of 6.0 and 6.6 kb are required for the expression of ugp, whereas all the structural information is located on the 6.6 kb EcoRI fragment. A restriction map of the cloned DNA was established and the extent of ugp genes determined by Tn5 insertions. Using ugp-lacZ fusions, it could be shown that the ugp region consists of at least two different operons that are transcribed in the same direction (counterclockwise) on the E. coli chromosome.Abbreviations DHBP 3,4-dihydroxibutyl-1-phosphonate - G3P sn-glycerol-3-phosphate - G3PBP glycerol-3-phosphate binding protein - IPTG isopropyl--d-thiogalactopyranoside - XG 5-bromo-4-chloro-3-indolyl--d-galactopyranoside     

Carbon-starvation induction of the ugp operon, encoding the binding protein-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.

Summary The gene products of the ugp operon of Escherichia coli are responsible for the uptake of sn-glycerol-3-phosphate and certain glycerophosphodiesters. The regulation of ugp is mainly phoBR-dependent. Significant expression, however, can be observed even in the presence of high concentrations of phosphate, a condition which normally completely represses pho expression. Pho-independent ugp expression was found to be derepressed during the late logarithmic growth phase due to carbon starvation. Among different carbon sources tested, glucose caused the most complete repression. Addition of cAMP prevented glucose repression, indicating that a cAMP-CRP control mechanism may be directly or indirectly involved in the carbon-starvation response. This conclusion is supported by the fact that pho-independent ugp expression correlated with the presence of the cya and crp gene products.     

Characterization of the ugp region containing the genes for the phoB dependent sn-glycerol-3-phosphate transport system of Escherichia coli

Summary The ugp structural genes, coding for the pho regulon dependent sn-glycerol-3-phosphate transport system, were cloned in pBR322 and characterized. The expression of the cloned ugp system was phoB dependent. Cells containing the ugp plasmid overproduced the G3P binding protein upon phosphate starvation. Tn5 mutagenesis of the cloned DNA revealed that the ugp genes are organized in two separate operons which comprise at least four genes: ugpB and ugpD constitute one operon, ugpA and ugpC constitute the other. The structural gene for the G3P binding protein (G3PBP) is ugpB.The ugpC gene product was also synthesized in minicells as a polypeptide, with an apparent molecular weight of 40,000. No gene products could be assigned to the ugpA and ugpD genes. Hybridization experiments allowed the physical characterization of 20 kb of DNA adjacent to the ugp genes on the E. coli chromosome including the liv genes.     

A second transport system for sn-glycerol-3-phosphate in Escherichia coli.

Strains containing phage Mucts inserted into glpT were isolated as fosfomycin-resistant clones. These mutants did not transport sn-glycerol-3-phosphate, and they lacked GLPT, a protein previously shown to be a product of the glpT operon. By plating these mutants on sn-glycerol-3-phosphate at 43 degrees C, we isolated revertants that regained the capacity to grow on G3P. Most of these revertants did not map in glpT and did not regain GLPT. These revertants exhibited a highly efficient uptake system for sn-glycerol-3-phosphate within an apparent Km of 5 micron. In addition, three new proteins (GP 1, 2, and 3) appeared in the periplasm of these revertants. None of these proteins were antigentically related to GLPT. However, like GLPT, GP1 exhibits abnormal behavior on sodium dodecyl sulfate-polyacrylamide gels. GP 2 is an efficient binding protein. The new uptake system showed different characteristics than the system that is coded for by the glpT operon. It was inhibited neither by phosphate nor fosfomycin. So far, none of the systems that transport organic acids in Escherichia coli could be implicated in the new sn-glycerol-3-phosphate uptake activity. The mutation ugp+, which was responsible for the appearance of the new transport system and the appearance of GP 1, 2, and 3 in the periplasm was cotransducible with araD by phage P1 transduction and was recessive in merodiploids.     

Regulation of Escherichia coli K-12 hexuronate system genes: exu regulon.

Two types of Escherichia coli K-12 regulatory mutants, partially or totally negative for the induction of the five catabolic enzymes (uronic isomerase, uxaC; altronate oxidized nicotinamide adenine dinucleotide: uxaB; mannonate hydrolyase, uxuA) and the transport system (exuT) of the hexuronate-inducible pathway, were isolated and analyzed enzymatically. Hexuronate-catabolizing revertants of the negative mutants showed a constitutive synthesis for some or all of these enzymes. Negative and constitutive mutations were localized in the same genetic locus, called exuR, and the following order for the markers situated between the min 65 and 68 was determined: argG--exuR--exuT--uxaC--uxaA--tolC. The enzymatic characterization of the pleiotropic negative and constitutive mutants of the exuR gene suggests that the exuR regulatory gene product exerts a specific and total control on the three exuT, uszB, and uxaC-uxaA operons of the galacturonate pathway and a partial control on the uxuA-uxuB operon of the glucuronate pathway. The analysis of diploid strains conatining both the wild type and a negative or constitutive allele of the exuR gene, as well as the analysis of thermosensitive mutants of the exuR gene, was in agreement with a negative regulatory mechanism for the control of the hexuronate system.     

Periplasmic protein related to the sn-glycerol-3-phosphate transport system of Escherichia coli.

Two-dimensional gel electrophoresis of shock fluids of Escherichia coli K-12 revealed the presence of a periplasmic protein related to sn-glycerol-3-phosphate transport (GLPT) that is under the regulation of glpR, the regulatory gene of the glp regulon. Mutants selected for their resistance to phosphonomycin and found to be defective in sn-glycerol-3-phosphate transport either did not produce GLPT or produced it in reduced amounts. Other mutations exhibited no apparent effect of GLPT. Transductions of glpT+ nalA phage P1 into these mutants and selection for growth on sn-glycerol-3-phosphate revealed a 50% cotransduction frequency to nalA. Reversion of mutants taht did not produce GLPT to growth on sn-glycerol-3-phosphate resulted in strains that produce GLPT. This suggests a close relationship of GLPT to the glpT gene and to sn-glycerol-3-phosphate transport. Attempts to demonstrate binding activity of GLPT in crude shock fluid towards sn-glycerol-3-phosphate have failed so far. However, all shock fluids, independent of their GLPT content, exhibited an enzymatic activity that hydrolyzes under the conditions of the binding assay, 30 to 60% of the sn-glycerol-3-phosphate to glycerol and inorganic orthophosphate.     

Regulation of the pho regulon in Escherichia coli K-12. Genetic and physiological regulation of the positive regulatory gene phoB

phoB is a positive regulatory gene for phoA, which codes for alkaline phosphatase, as well as for other genes belonging to the phosphate (pho) regulon whose expression is inducible by phosphate limitation in Escherichia coli. A hybrid plasmid that contains a phoB-lacZ fused gene was constructed in vitro. This plasmid enabled us to study phoB gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoB gene expression in these strains was studied under limited and excess phosphate conditions. It was found that the regulation of phoB expression was very similar to that of phoA expression. Expression of both genes was induced by phosphate starvation. Both genes were constitutively expressed in phoR, phoS, phoT and phoU mutants and were not expressed in a phoR-phoM double mutant. The implications of these findings for the regulatory mechanism of the pho regulon are discussed.     

Repressor for the sn-glycerol-3-phosphate regulon of Escherichia coli K-12: cloning of the glpR gene and identification of its product

The glpR gene encoding the repressor for the glp regulon of Escherichia coli was cloned from a library of HindIII DNA fragments established in bacteriophage lambda. Phages harboring glpR were isolated by selection for sn-glycerol-3-phosphate dehydrogenase function encoded by glpD, which is adjacent to glpR on the E. coli linkage map. Restriction endonuclease analysis and recloning of DNA fragments localized glpR to a 3-kilobase-pair EcoRI-SalI segment of DNA. Strains exhibiting constitutive expression of the glp operons were strongly repressed after introduction of multicopy plasmids containing the glpR gene. Analysis of proteins labeled in minicells harboring either glpR+ recombinant plasmids or a glpR::Tn5 derivative showed that the glpR gene product is a protein with an apparent molecular weight of 33,000.     

Pi exchange mediated by the GlpT-dependent sn-glycerol-3-phosphate transport system in Escherichia coli.

The GlpT system for sn-glycerol-3-phosphate transport in Escherichia coli is shown to catalyze a rapid efflux of Pi from the internal phosphate pools in response to externally added Pi or glycerol-3-phosphate. A glpR mutation, which results in constitutive expression of the GlpT system, is responsible for this rapid Pi efflux and the arsenate sensitivity of several laboratory strains, including the popular strain C600. Glucose and other phosphotransferase system sugars inhibit Pi efflux by repressing glpT expression.     

Escherichia coli K-12 mutants that allow transport of maltose via the beta-galactoside transport system.

We have isolated mutants of Escherichia coli that have an altered beta-galactoside transport system. This altered transport system is able to transport a sugar, maltose, that the wild-type beta-galactoside transport system is unable to transport. The mutation that alters the specificity of the transport system is in the lacY gene, and we refer to the allele as lacYmal. The lacYmal allele was detected originally in strains in which the lac genes were fused to the malF gene. Thus, as a result of gene fusion and isolation of the lacYmal mutation, a new transport system was evolved with regulatory properties and specificity similar to those of the original maltose transport system. Maltose transport via the lacYmal gene product is independent of all of the normal maltose transport system components. The altered transport system shows a higher affinity than the wild-type transport system for two normal substrates of the beta-galactoside transport system, thiomethyl-beta-D-galactoside and o-nitrophenyl-beta-D-galactoside.     

Regulation of the pho regulon of Escherichia coli K-12. Cloning of the regulatory genes phoB and phoR and identification of their gene products

We report here results of crystallographic studies at 3.0 Å resolution of complexes of phosphate ligands with aspartate carbamoyltransferase from Escherichia coli. Specifically, we interpret the binding of CTP, ATP, 5-bromo-CTP, 8-bromo-GTP. formycin A 5′-triphosphate. 3,N⁶-etheno-ATP. phosphate/carbamoyl-d.l-aspartate and pyrophosphate to the catalytic and regulatory chains of the enzyme.We observed two modes of binding of ligands to the phosphate crevice of the catalytic chain. Pyrophosphate and phosphate penetrate deeply into the cleft between the two domains of a catalytic monomer. In contrast. ATP, CTP. formycin A 5′-triphosphate and 3,N⁶-etheno-ATP bind to an exposed region of this cleft through their β and γ phosphates. Although the β and γ phosphates of 8-bromo-GTP bind to the same region as do the non-brominated nucleotides. 8-bromo-GTP interacts with the protein through all three of its phosphates and its ribose.Ser52, Arg54. Thr55, Arg105, His134. Gln137 and Arg167 are residues of the catalytic chain near density corresponding to phosphate ligands. The interactions of phosphate ligands are consistent with results of nuclear magnetic resonance, kinetics and equilibrium binding studies.Nucleoside triphosphates also bind to the regulatory chain in two modes. ATP and CTP bind in similar conformations to nearly the same site of the allosteric domain. The effector 8-bromo-GTP interacts at a location that does not overlap with the ATP-CTP site. The phosphates are in an extended conformation for all effectors. Furthermore, ATP. 5-bromo-CTP and 8-bromo-GTP bind to the protein in the anti conformation.Interactions of ATP and CTP with the protein are essentially consistent with the proposals put forward by London &; Schmidt (1972). We suggest, however, a modification of the London &; Schmidt model on the basis of our results with 8-bromo-GTP. In addition, we propose that the allosteric binding sites of nucleoside triphosphates are coupled to each other through the N-terminal segments of monomers of a regulatory dimer.     

Only one gene is required for the glpT-dependent transport of sn-glycerol-3-phosphate in Escherichia coli

Summary Deletion and point mutants defective in the glpT-dependent sn-glycerol-3-phosphate transport system were isolated and located on the Escherichia coli chromosome. They mapped in glpT in the clockwise order gyrA, glpA, glpT at around 48 min on the Escherichia coli linkage map. The mutations within glpT were ordered by deletion mapping, three factor crosses, and by crosses involving transducing bacteriophages carrying glpT-lac operon fusions. Results obtained using these fusion phages indicated that glpT is transcribed in the counterclockwise direction on the E. coli linkage map.Complementation analysis using these mutants revealed only one complementation group. Thus, one gene is necessary and sufficient for the proton motive force-dependent sn-glycerol-3-phosphate transport system.     

Cell-free synthesis of proteins related to sn-glycerol-3-phosphate transport in Escherichia coli.

An Escherichia coli periplasmic protein (GlpT) related to sn-glycerol-3-phosphate transport was synthesized in a cell-free system directed by hybrid plasmic ColE1-glpT DNA. The in vitro product cross-reacted with antisera against the purified protein. The ColE1-glpT DNA-directed cell-free system was induced by sn-glycerol-3-phosphate and phosphonomycin and was dependent on cyclic AMP. The in vitro-synthesized protein showed the characteristics of a multimeric protein, as did the purified periplasmic protein. The main proportion of the newly synthesized product had a higher molecular weight than the mature protein found in the periplasm of cells and showed a more positive charge in two-dimensional gel electrophoresis. Thus, a proportion of this protein is presumed to be synthesized in vitro as a precursor. The cell-free system yielded a second protein that is likely to be also coded for by the glpT operon. This protein had a molecular weight of approximately 33,000 in sodium dodecyl sulfate-acrylamide gel electrophoresis and behaved like an intrinsic membrane protein.     

D-serine transport system in Escherichia coli K-12

The d-serine transport system in Escherichia coli K-12 was studied by use of a mutant unable to form d-serine deaminase, yet resistant to d-serine. The mutant is greatly impaired in its ability to accumulate d-serine, d-alanine, and glycine. Transport of l-alanine is partially affected but transport of l-serine is unaffected. The mutant is also resistant to d-cycloserine, indicating that d-serine is transported by the system responsible for uptake of d-cycloserine. The d-serine transport system is not inducible, but appears to be formed constitutively, as are the transport systems of most amino acids. The transport mutation appears to be multistep and maps to the right of malB on the E. coli linkage map.