Glycerol conductance and physical asymmetry of the Escherichia coli glycerol facilitator GlpF

The aquaglyceroporin GlpF is a transmembrane channel of Escherichia coli that facilitates the uptake of glycerol by the cell. Its high glycerol uptake rate is crucial for the cell to survive in very low glycerol concentrations. Although GlpF allows both influx and outflux of glycerol, its structure, similar to the structure of maltoporin, exhibits a significant degree of asymmetry. The potential of mean force characterizing glycerol in the channel shows a corresponding asymmetry with an attractive vestibule only at the periplasmic side. In this study, we analyze the potential of mean force, showing that a simplified six-step model captures the kinetics and yields a glycerol conduction rate that agrees well with observation. The vestibule improves the conduction rate by 40% and 75% at 10- micro M and 10-mM periplasmic glycerol concentrations, respectively. In addition, neither the conduction rate nor the conduction probability for a single glycerol (efficiency) depends on the orientation of GlpF. GlpF appears to conduct equally well in both directions under physiological conditions.     

Glycerol kinase of Escherichia coli is activated by interaction with the glycerol facilitator.

Glycerol transport is commonly cited as the only example of facilitated diffusion across the Escherichia coli cytoplasmic membrane. Two proteins, the glycerol facilitator and glycerol kinase, are involved in the entry of external glycerol into cellular metabolism. The glycerol facilitator is thought to act as a carrier or to form a selective pore in the cytoplasmic membrane, whereas the kinase traps the glycerol inside the cell as sn-glycerol-3-phosphate. We found that the kinetics of glycerol uptake in a facilitator-minus strain are significantly different from the kinetics of glycerol uptake in the wild type. Free glycerol was not observed inside wild-type cells transporting glycerol, and diffusion of glycerol across the cytoplasmic membrane was not the rate-limiting step for phosphorylation in facilitator-minus mutants. Therefore, the kinetics of glycerol phosphorylation are different, depending on the presence or absence of the facilitator protein. We conclude that there is an interaction between the glycerol facilitator protein and glycerol kinase that stimulates kinase activity, analogous to the hexokinase- and glycerol kinase-porin interactions in mitochondria.     

Disruption of glpF gene encoding the glycerol facilitator improves 1,3-propanediol production from glucose via glycerol in Escherichia coli

Engineered Escherichia coli has recently been applied to produce 1,3-propanediol (1,3-PDO) from glucose. A metabolic intermediate in the production pathway, glycerol, is partially secreted into the extracellular of E. coli through a glycerol facilitator encoded by glpF, and this secretion consequently decreases 1,3-PDO production. Therefore, we aimed to determine whether disrupting the glpF gene would improve 1,3-PDO production in E. coli. The intracellular glycerol concentration in a glpF-disruptant was 7·5 times higher than in a non-disruptant. The glpF-disrupted and non-disrupted E. coli strains produced 0·26 and 0·09 g l⁻¹ of 1,3-PDO, respectively, from 1% glucose after 72 h of cultivation. The specific growth rate (μ) and the 1,3-PDO yield from glucose (Y_P/S) in the disruptant were higher than those in the non-disruptant (ΔglpF, μ = 0·08 ± 0·00 h⁻¹, Y_P/S = 0·06 mol mol-glucose⁻¹; BW25113, μ = 0·06 ± 0·00 h⁻¹, Y_P/S = 0·02 mol mol-glucose⁻¹). Disruption of the glpF gene decreased the production of the by-product, acetic acid. These results indicated that disruption of glpF increased the intracellular concentration of glycerol and consequently increased 1,3-PDO production in E. coli.     

The recR locus of Escherichia coli K-12: molecular cloning, DNA sequencing and identification of the gene product.

The recR gene of Escherichia coli, which is associated with recBC-independent mechanisms of recombination and DNA repair, has been located between dnaZX and htpG on a 6.4 kb EcoRI fragment of DNA that has been cloned and analysed in lambda and plasmid vectors. Nucleotide sequencing of this interval revealed two open reading frames that constitute an operon lying immediately downstream of dnaZX. The second of these two reading frames was identified as recR. It encodes a polypeptide with a predicted molecular weight of 21,965 Daltons that migrates on SDS gels as a 26 kDa protein. The first gene of the operon encodes a polypeptide of 12,015 daltons. Its function is not known.     

Cloning the spoT gene of Escherichia coli: identification of the spoT gene product.

We have isolated five specialized transducing lambda bacteriophages (lambda dpyrE spoT) carrying the pyrE and spoT genes of Escherichia coli. A fragment from one of these phages was used as the source of DNA to clone the spoT and pyrE genes on a multicopy plasmid, pBR322. Insertions and deletions in this plasmid were obtained. These plasmids were used to transform a minicell-producing strain, and the gene products synthesized were determined. Our experiments demonstrate that the spoT and pyrE genes are separated by about 4 magadaltons and suggest that the spoT gene product is a protein whose molecular weight is 80,000. The strain in which the spoT+ allele is carried on a plasmid produced nine times more spoT gene activity than a normal spoT+ strain when assayed in crude extracts. This strain was used to prepare partially purified gene product, guanosine 5'-diphosphate, 3'-diphosphate pyrophosphatase. The enzyme has the following characteristics. (i) It hydrolyzes pyrophosphate from the 5'-pyrophosphate of guanosine 5'-diphosphate, 3'-diphosphate, yielding GDP and pyrophosphate. (ii) Its activity is strongly stimulated by Mn2+ and slightly stimulated by salt. (iii) Its activity is inhibited by uncharged tRNA. There are also two additional activities in the cell extract which degrade guanosine in 5'-diphosphate, 3'-diphosphate in vitro but which are not specified by the spoT gene.     

Galactofuranose biosynthesis in Escherichia coli K-12: identification and cloning of UDP-galactopyranose mutase.

We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[alpha-D-U-14C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic alpha-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.     

Glycerol metabolism in superoxide dismutase-deficient Escherichia coli.

Escherichia coli, which lacks cytoplasmic superoxide dismutases, exhibits various phenotypic deficits if grown aerobically. Here we report that sodAsodB E. coli cannot use glycerol under aerobic conditions. The reason is low activity of glycerol kinase (GK), the rate-limiting enzyme in glycerol metabolism. Superoxide does not inactivate GK, but makes it susceptible to inactivation by a heat-labile factor present in the cell-free extracts. This factor seems to be part of a proteolytic system, which recognizes and degrades oxidatively modified proteins.     

Restriction map of the Escherichia coli malA region and identification of the malT product.

A series of plaque-forming lambda h80 transducing phages carrying various portions of the malA region were isolated. A 5,800-base pair HindIII-EcoRI DNA fragment from one of these phages was cloned into pBR322 and shown to contain malT, which is the positive regulator gene of the maltose regulon, and most of malP, the structural gene for maltodextrin phosphorylase. A restriction map of the HindIII-EcoRI fragment was established, and it was correlated with the genetic map of the malA region (i) by mapping deletions which had been generated in vitro on the plasmid and (ii) by locating on the restriction map a DNA insertion of known genetic position. A 600-base pair HincII-HaeII segment was shown to contain all or part of the promoters for malT and malP, which are known to be transcribed in opposite directions. Strains carrying gene malT on a plasmid synthesized a 94,000-dalton polypeptide which was not produced by identical strains carrying similar plasmids in which malT was partially deleted. Estimates of the size of the malT gene support the conclusion that the 94,000-dalton polypeptide is the malT product.     

Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli.

The specificity of the glycerol facilitator (glpF) of Escherichia coli was studied with an osmotic method. This transport system allowed the entry of polyols (glycerol and erythritol), pentitols, and hexitols. The analogous sugars were not transported. However, urea, glycine, and DL-glyceraldehyde could use this pathway to enter the cell. The glpF protein allowed the rapid efflux of preequilibrated xylitol. Glycerol surprisingly did not inhibit the uptake of xylitol, and xylitol only slightly reduced the uptake of glycerol. The observation and the insensitivity of the xylitol transport to low temperature suggest that the facilitator behaves as a membrane channel.     

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.     

Cloning the polB gene of Escherichia coli and identification of its product

Using an in vivo mini-Mu cloning system, we have cloned the polB gene of Escherichia coli into the multicopy plasmid, pUC18. A chromosomal insert of 4.9 kilobases gave 30-40-fold overproduction of DNA polymerase II, and the cells containing the plasmid showed normal growth. The restriction pattern of the polB gene does not match that of either the polA gene or polC gene. Plasmid-directed protein synthesis demonstrates peptides of 99 and 82 kDa which are not expressed by derivative plasmids without DNA polymerase II activity. It appears from in situ gel assays and high performance liquid chromatography that 82- and 55-kDa proteins are derived from the 99-kDa protein by degradation, but all retain activity. DNA polymerase I or DNA polymerase III antibody does not inhibit the synthesis reaction of partially purified DNA polymerase II, but DNA polymerase II antibody does. By the criteria of restriction pattern of the polB gene, molecular weight of the protein, and antibody inhibition of reaction, DNA polymerase II can be demonstrated to be a distinct DNA polymerase.     

Antimonite is accumulated by the glycerol facilitator GlpF in Escherichia coli.

In a search for genes responsible for the accumulation of antimonite in Escherichia coli, TnphoA was used to create a pool of random insertional mutants, from which one antimonite-resistant mutant was isolated. Sequence analysis showed that the TnphoA insertion was located in the glpF gene, coding for the glycerol facilitator GlpF. The mutant was shown to be defective in polyol transport by GlpF. These results suggest that in solution Sb(III) is recognized as a polyol by the glycerol facilitator.     

Cloning and identification of the product of the dnaE gene of Escherichia coli

We successively subcloned the dnaE gene of Escherichia coli into pBR322, resulting in a plasmid that contains 4.6 kilobases of E. coli DNA. This plasmid can complement a dnaE temperature-sensitive mutation. A restriction map of the dnaE gene and the surrounding 10.7-kilobase region of the E. coli chromosome was determined. A unique HindIII restriction endonuclease site within the cloned segment of DNA was identified as a site required for expression of the dnaE gene. By using the maxicell plasmid-directed protein synthesizing system, we demonstrated that dnaE codes for the alpha subunit of DNA polymerase III.     

The identification of the Escherichia coli fts Y gene product: an unusual protein

The ftsYEX operon in Escherichia coli encodes three proteins, two of which (FtsE and FtsX) are known to be required for cell division. Although FtsE and FtsX have been identified using SDS-PAGE, the FtsY protein has not. We have used in vitro insertion mutagenesis to identify FtsY as a 92 kD polypeptide in maxicell experiments, although predictions from the DNA sequence estimated FtsY to be 54 kD. Results suggest that this disparity could be due to the unusually high percentage of acidic residues within the protein. Complementation tests indicated the presence of a promoter within ftsY required for expression of ftsE and ftsX. The FtsY protein exhibits sequence homology with the SR alpha protein of eukaryotes which is involved in protein secretion. The essential nature of the ftsY gene was also demonstrated.     

Cloning of the aroP gene and identification of its product in Escherichia coli K-12.

The aroP gene of Escherichia coli K-12 was located in a ca. 1.2-kilobase region of DNA. The aroP gene product was identified as a membrane-bound protein with an apparent molecular weight of approximately 37,000.     

Glycerol elicits energy taxis of Escherichia coli and Salmonella typhimurium.

Escherichia coli and Salmonella typhimurium show positive chemotaxis to glycerol, a chemical previously reported to be a repellent for E. coli. The threshold of the attractant response in both species was 10(-6) M glycerol. Glycerol chemotaxis was energy dependent and coincident with an increase in membrane potential. Metabolism of glycerol was required for chemotaxis, and when lactate was present to maintain energy production in the absence of glycerol, the increases in membrane potential and chemotactic response upon addition of glycerol were abolished. Methylation of a chemotaxis receptor was not required for positive glycerol chemotaxis in E. coli or S. typhimurium but is involved in the negative chemotaxis of E. coli to high concentrations of glycerol. We propose that positive chemotaxis to glycerol in E. coli and S. typhimurium is an example of energy taxis mediated via a signal transduction pathway that responds to changes in the cellular energy level.     

Analysis of the ruv locus of Escherichia coli K-12 and identification of the gene product.

The ruv gene of Escherichia coli, which is associated with inducible mechanisms of DNA repair and recombination, has been cloned into the low-copy plasmid vector pHSG415. The recombinant plasmid pPVA101 fully complements the DNA repair-deficient phenotype of ruv mutants. Restriction endonuclease analysis of this plasmid revealed a 10.6-kilobase (kb) HindIII DNA insert which contained a 7.7-kb PstI fragment identified as being from the chromosomal ruv region. Deletion analysis and Tn1000 insertional inactivation of ruv function located the ruv coding region to a 2.2-kb section of the cloned DNA fragment. A comparison of the proteins encoded by ruv wild-type and mutant plasmids identified the gene product as a protein of molecular weight 41,000.     

Glycerol metabolism in superoxide dismutase-deficient Escherichia coli

Escherichia coli, which lacks cytoplasmic superoxide dismutases, exhibits various phenotypic deficits if grown aerobically. Here we report that sodAsodB E. coli cannot use glycerol under aerobic conditions. The reason is low activity of glycerol kinase (GK), the rate-limiting enzyme in glycerol metabolism. Superoxide does not inactivate GK, but makes it susceptible to inactivation by a heat-labile factor present in the cell-free extracts. This factor seems to be part of a proteolytic system, which recognizes and degrades oxidatively modified proteins.     

Cloning of the Escherichia coli endo-1,4-D-glucanase gene and identification of its product

A plasmid (pYP17) containing a genomic DNA insert from Escherichia coli K-12 that confers the ability to hydrolyze carboxymethylcellulose (CMC) was isolated from a genomic library constructed in the cosmid vector pLAFR3 in E. coli DH5α. A small 1.65-kb fragment, designated bcsC (pYP300), was sequenced and found to contain an ORF of 1,104 bp encoding a protein of 368 amino acid residues, with a calculated molecular weight of 41,700 Da. BcsC carries a typical prokaryotic signal peptide of 21 amino acid residues. The predicted amino acid sequence of the BcsC protein is similar to that of CelY of Erwinia chrysanthemi, CMCase of Cellulomonas uda, EngX of Acetobacter xylinum, and CelC of Agrobacterium tumefaciens. Based on these sequence similarities, we propose that the bcsC gene is a member of glycosyl hydrolase family 8. The apparent molecular mass of the protein, when expressed in E. coli, is approximately 40 kDa, and the CMCase activity is found mainly in the extracellular space. The enzyme is optimally active at pH 7 and a temperature of 40° C. Received: 6 February 1998 / Accepted: 6 November 1998