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.     

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.     

Regulation of ugp, the sn-glycerol-3-phosphate transport system of Escherichia coli K-12 that is part of the pho regulon.

The expression of the ugp-dependent sn-glycerol-3-phosphate transport system that is part of the pho regulon was studied in mutants of Escherichia coli K-12 containing regulatory mutations of the pho regulon. The phoR and phoST gene products exerted a negative control on the expression of ugp. Induction of the system was positively controlled by the phoB, phoM, and phoR gene products. Using a ugp-lacZ operon fusion, we showed that the ugp and phoA genes were coordinately derepressed and repressed.     

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.     

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     

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.     

Identification and DNA sequence of tdcR, a positive regulatory gene of the tdc operon of Escherichia coli

Summary Efficient in vivo expression of the biodegradative threonine dehydratase (tdc) operon of Escherichia coli is dependent on a regulatory gene, tdcR. The tdcR gene is located 198 base pairs upstream of the tdc operon and is transcribed divergently from this operon. The nucleotide sequence of tdcR and two unrelated reading frames has been determined. The deduced amino acid sequence of TdcR indicates that is is a polypeptide of M_r 12000 with 99 amino acid residues and contains a potential helix-turnhelix DNA binding motif. Deletion analysis and minicell expression of the tdcR gene suggest that TdcR may serve as a trans-acting positive activator for the tdc operon.     

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.     

Molecular analysis of the Escherichia coli hns gene encoding a DNA-binding protein, which preferentially recognizes curved DNA sequences.

Summary We previously demonstrated that the E. coli protein, H-NS (or Hla), encoded by the gene hns (or osmZ or bglY preferentially recognizes curved DNA sequences in vitro. In order to gain further insight into the complex function of H-NS and the significance of DNA curvature, we constructed a structurally defined hns deletion mutant on the E. coli chromosome. The hns deletion mutant thus obtained showed a variety of phenotypes previously for other lesions in hns. It was further demonstrated that, in this hns deletion background, numerous E. coli cellular proteins were either strongly expressed or remarkably repressed, as compared to their expression levels in wild-type cells.     

Characterization of three genes in the dam-containing operon of Escherichia coli

The dam-containing operon in Escherichia coli is located at 74 min on the chromosomal map and contains the genes aroK, aroB, a gene called urf74.3, dam and trpS. We have determined the nucleotide sequence between the dam and trpS genes and show that it encodes two proteins with molecular weights of 24 and 27 kDa. Furthermore, we characterize the three genes urf74.3, 24kDa, 27kDa and the proteins they encode. The predicted amino acid sequences of the 24 and 27 kDa proteins are similar to those of the CbbE and CbbZ proteins, respectively, of the Alcaligenes eutrophus cbb operon, which encodes enzymes involved in the Calvin cycle. In separate experiments, we have shown that the 24 kDa protein has d-ribulose-5-phosphate epimerase activity (similar to CbbE), and we call the gene rpe. Similarly, the 27 kDa protein has 2-phosphoglycolate phosphatase activity (similar to CbbZ), and we name the gene gph. The Urf74.3 protein, with a predicted molecular weight of 46 kDa, migrated as a 70 kDa product under denaturing conditions. Overexpression of Urf74.3 induced cell filamentation, indicating that Urf74.3 directly or indirectly interferes with cell division. We present evidence for translational coupling between aroB and urf74.3 and also between rpe and gph. Proteins encoded in the dam superoperon appear to be largely unrelated: Dam, and perhaps Urf74.3, are involved in cell cycle regulation, AroK, AroB, and TrpS function in aromatic amino acid biosynthesis, whereas Rpe and Gph are involved in carbohydrate metabolism.     

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.     

Nucleotide sequence and gene-polypeptide relationships of the glpABC operon encoding the anaerobic sn-glycerol-3-phosphate dehydrogenase of Escherichia coli K-12.

The nucleotide sequence of a 4.8-kilobase SacII-PstI fragment encoding the anaerobic glycerol-3-phosphate dehydrogenase operon of Escherichia coli has been determined. The operon consists of three open reading frames, glpABC, encoding polypeptides of molecular weight 62,000, 43,000, and 44,000, respectively. The 62,000- and 43,000-dalton subunits corresponded to the catalytic GlpAB dimer. The larger GlpA subunit contained a putative flavin adenine dinucleotide-binding site, and the smaller GlpB subunit contained a possible flavin mononucleotide-binding domain. The GlpC subunit contained two cysteine clusters typical of iron-sulfur-binding domains. This subunit was tightly associated with the envelope fraction and may function as the membrane anchor for the GlpAB dimer. Analysis of the GlpC primary structure indicated that the protein lacked extended hydrophobic sequences with the potential to form alpha-helices but did contain several long segments capable of forming transmembrane amphipathic helices.     

Increased expression of the Escherichia coli umuDC operon restores SOS mutagenesis in lexA41 cells

Summary The lexA41 allele of Escherichia coli encodes a semidefective mutant repressor that is also resistant to RecA facilitated cleavage. Cells harboring the lexA41 allele were found previously to repress only a subset of operons in the SOS regulon. lexA41 cells cannot promote SOS mutagenesis, presumably because one or more operons required for mutagenesis are repressed by this mutant repressor. Using the lac regulatory system to increase the expression of the umuDC operon, we were able to restore mutagenesis in the lexA41 mutant. We conclude that the products of the umuDC operon appear to be uniquely limiting in this mutant.     

GroEL/GroES chaperone and Lon protease regulate expression of the Vibrio fischeri lux operon in Escherichia coli

Chaperone GroEL/GroES and Lon protease were shown to play a role in regulating the expression of the Vibrio fischeri lux operon cloned in Escherichia coli cells. The E. coli groE mutant carrying a plasmid with the full-length V. fischeri lux regulon showed a decreased bioluminescence. The bioluminescence intensity was high in E. coli cells with mutant lonA and the same plasmid. Bioluminescence induction curves lacked the lag period characteristic of lon ⁺ strains. Regulatory luxR of V. fischeri was cloned in pGEX-KG to produce the hybrid gene GST-luxR. The product of its expression, GST-LuxR, was isolated together with GroEL and Lon upon affinity chromatography on a column with glutathione-agarose, suggesting complexation of LuxR with these proteins. It was assumed that GroEL/GroES is involved in LuxR folding, while Lon protease degrades LuxR before its folding into an active globule or after denaturation.