Biomedical subjects

T J Larson

Publications and source records attributed to T J Larson.

51 records · Page 3Linked to original sources

PubMed1986

Physical and genetic structure of the glpD-malT interval of the Escherichia coli K-12 chromosome. Identification of two new structural genes of the glp-regulon.

A transducing lambda phage carrying glpD''lacZ, glpR, and malT was isolated from a strain harboring a glpD''lacZ fusion. Comparison of restriction endonuclease cleavage patterns of DNA isolated from this phage with that of the previously cloned malT region (Raibaud and Schwartz 1980) facilitated the construction of recombinant plasmids carrying different portions of the glpD-malT region. Results of minicell analysis and complementation studies showed that this region of the chromosome encodes at least five polypeptides. These included the previously identified glpD, glpR, and malT gene products. In addition, two new structural genes of the glp regulon (glpE and glpG) located between the glpD and glpR genes were identified. Hybrid plasmids carrying glpD''lacZ and glpR''lacZ fusions were constructed. Restriction endonuclease cleavage analysis of these two plasmids demonstrated that glpD and glpR are divergently transcribed.

Bacteriophage lambda↗

PubMed1985

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, Molecular↗

PubMed1984

Transposon Tn10-dependent expression of the lamB gene in Escherichia coli.

Among Tn10 insertions isolated in or near the malB region of Escherichia coli, one (zjb-729::Tn10) mapped between malK and lamB or late in malK and allowed MalT-independent expression of lamB. Tn10-dependent expression of a lamB-lacZ protein fusion was 25% of the expression of the fusion from the malK-lamB operon promoter in malTc constitutive strains. The maltoporin content of a strain carrying this Tn10 was about 20% that of a malTc malB+ strain. Transport of maltose at concentrations of below 10(-6) M was reduced about threefold. When maltoporin was present at about 50% of the level of malTc malB+ strains, maltose transport was largely restored. We conclude that maltoporin is not rate limiting for maltose transport in wild-type cells but becomes rate limiting when the ratio of maltoporin to other maltose transport components is reduced more than twofold.

Bacterial Outer Membrane Proteins↗

PubMed1984

sn-Glycerol-3-phosphate auxotrophy of plsB strains of Escherichia coli: evidence that a second mutation, plsX, is required.

sn-Glycerol-3-phosphate auxotrophs defective in phospholipid synthesis contain a Km-defective sn-glycerol-3-phosphate acyltransferase. Detailed genetic analysis revealed that two mutations were required for the auxotrophic phenotype. One mutation, in the previously described plsB locus (sn-glycerol-3-phosphate acyltransferase structural gene), mapped near min 92 on the Escherichia coli linkage map. Isolation of Tn10 insertions cotransducible with the auxotrophy in phage P1 crosses revealed that a second mutation was required with plsB26 to confer the sn-glycerol-3-phosphate auxotrophic phenotype. This second locus, plsX, mapped between pyrC and purB near min 24 on the E. coli linkage map. Tn10 insertions near plsX allowed detailed mapping of the genetic loci in this region. A clockwise gene order putA pyrC flbA flaL flaT plsX fabD ptsG thiK purB was inferred from results of two- and three-factor crosses. Strains harboring the four possible configurations of the mutant and wild-type plsB and plsX loci were constructed. Isogenic plsB+ plsX+, plsB+ plsX50, and plsB26 plsX+ strains grew equally well on glucose minimal medium without sn-glycerol-3-phosphate. In addition, plsX or plsX+ had no apparent effect on sn-glycerol-3-phosphate acyltransferase activity measured in membrane preparations. The molecular basis for the plsX requirement for conferral of sn-glycerol-3-phosphate auxotrophy in these strains remains to be established.

Acyltransferases↗

PubMed1983

Periplasmic glycerophosphodiester phosphodiesterase of Escherichia coli, a new enzyme of the glp regulon.

The promoter-proximal gene (glpT) of the glpT-glpQ operon of Escherichia coli encodes a membrane permease responsible for active transport of sn-glycerol 3-phosphate. Promoter-distal glpQ encodes a periplasmic protein which is not required for active transport of sn-glycerol 3-phosphate (Larson, T.J., Schumacher, G., and Boos, W. (1982) J. Bacteriol. 152, 1008-1021). This periplasmic protein has now been identified as a phosphodiesterase which hydrolyzes glycerophosphodiesters into sn-glycerol 3-phosphate plus alcohol. The enzyme exhibited broad substrate specificity with respect to the alcohol moiety; sn-glycerol 3-phosphate was released from glycerophosphoethanolamine, glycerophosphocholine, glycerophosphoglycerol, and bis(glycerophospho)glycerol. The enzyme was specific for glycerophosphodiesters; bis(p-nitrophenyl)phosphate, a substrate for other phosphodiesterases, was not hydrolyzed. In a coupled spectrophotometric assay utilizing sn-glycerol 3-phosphate dehydrogenase and NAD, apparent activity was optimal at pH 9 and was stimulated by Ca2+. The substrates of the phosphodiesterase had no affinity for the glpT-encoded active transport system. Thus, the glpQ gene product expands the catabolic capability of the glp regulon to include a variety of glycerophosphodiesters.

Bacterial Proteins↗

PubMed1983

sn-Glycerol-3-phosphate transport in Salmonella typhimurium.

Salmonella typhimurium contains a transport system for sn-glycerol-3-phosphate that is inducible by growth on glycerol and sn-glycerol-3-phosphate. In fully induced cells, the system exhibited an apparent Km of 50 microM and a Vmax of 2.2 nmol/min . 10(8) cells. The corresponding system in Escherichia coli exhibits, under comparable conditions, a Km of 14 microM and a Vmax of 2.2 nmol/min . 10(8) cells. Transport-defective mutants were isolated by selecting for resistance against the antibiotic fosfomycin. They mapped in glpT at 47 min in the S. typhimurium linkage map, 37% cotransducible with gyrA. In addition to the glpT-dependent system, S. typhimurium LT2 contains, like E. coli, a second, ugp-dependent transport system for sn-glycerol-3-phosphate that was derepressed by phosphate starvation. A S. typhimurium DNA bank containing EcoRI restriction fragments in phage lambda gt7 was used to clone the glpT gene in E. coli. Lysogens that were fully active in the transport of sn-glycerol-3-phosphate with a Km of 33 microM and a Vmax of 2.0 nmol/min . 10(8) cells were isolated in a delta glpT mutant of E. coli. The EcoRI fragment harboring glpT was 3.5 kilobases long and carried only part of glpQ, a gene distal to glpT but on the same operon. The fragment was subcloned in multicopy plasmid pACYC184. Strains carrying this hybrid plasmid produced large amounts of cytoplasmic membrane protein with an apparent molecular weight of 33,000, which was identified as the sn-glycerol-3-phosphate permease. Its properties were similar to the corresponding E. coli permease. The presence of the multicopy glpT hybrid plasmid had a strong influence on the synthesis or assembly of other cell envelope proteins of E. coli. For instance, the periplasmic ribose-binding protein was nearly absent. On the other hand, the quantity of an unidentified E. coli outer membrane protein usually present only in small amounts increased.

Biological Transport↗

PubMed1982

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

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 lambda 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.

Biological Transport↗

PubMed1982

Identification of the glpT-encoded sn-glycerol-3-phosphate permease of Escherichia coli, an oligomeric integral membrane protein.

A collection of hybrid plasmids carrying either the wild-type or mutated glpT gene was generated in vitro and used to characterize the glpT-dependent active transport system for sn-glycerol-3-phosphate in Escherichia coli K-12. Restriction endonuclease analysis and recloning of DNA fragments localized glpT to a 3-kilobase pair PstI-HpaI segment of DNA. Comparison of DNA carrying glpT-lacZ fusions with DNA carrying intact glpT allowed determination of the direction of transcription. Through characterization of the proteins synthesized by strains harboring hybrid plasmids carrying amber, missense, or deletion mutations in glpT, it was shown that glpT is a promoter-proximal gene in an operon consisting of at least two genes. The gene product of glpT, the sn-glycerol-3-phosphate permease, was found associated with the inner membrane. It could be solubilized by treatment with sodium dodecyl sulfate at 50 degrees C. Its molecular weight, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was dependent upon sample treatment before electrophoresis. The apparent molecular weight was 44,000 when membrane fractions were heated to 50 degrees C; subsequent treatment at 95 degrees C modified the protein such that it migrated faster (apparent molecular weight = 33,000). Several missense mutations in glpT were negatively dominant over wild-type glpT, indicating that the active form of the permease is multimeric. A gene (named glpQ) promoter distal to glpT codes for a periplasmic protein. This protein had previously been named GLPT protein to indicate its relationship to the glpT gene. The present report demonstrates that it is not the gene product of glpT and is not required for active transport of sn-glycerol-3-phosphate.

Cell Membrane↗

PubMed1980

Membrane phospholipid synthesis in Escherichia coli. Cloning of a structural gene (plsB) of the sn-glycerol-3-phosphate acyl/transferase.

Si+ hybrid ColE1 plasmids of the Clarke-Carbon collection (Clarke, C., and Carbon, J. (1976) Cell 9, 91-99) which eliminate the sn-glycerol 3-phosphate growth requirement of a mutant of Escherichia coli with a Km defect in sn-glycerol-3-phosphate acyltransferase (plsB) were identified. Marked overproduction of a plasmid-encoded sn-glycerol-3-phosphate acyltransferase with a wild type Km in a host plsB- background indicates that the hybrid plasmids carry a structural gene for this enzyme. In addition, all of these plasmids suppress the phenotype of a mutation in a second locus involved in phospholipid biosynthesis, dgk (diglyceride kinase), and one of them also bears the dnaB structural gene. Diglyceride kinase activity is also overproduced in these strains. The linkage of plsB, dgk and dnaB loci was confirmed by transduction analysis which demonstrated the clockwise gene order malB, dnaB, dgk, plsB, and uvrA near Minute 91 on the E. coli linkage map. This is in contrast to the previously reported co-transduction of plsB with dctA near Minute 78 (Cronan, J. E., Jr., and Bell, R. M. (1974) J. Bacteriol., 120, 227-233). Recloning of restriction endonuclease fragments and in vitro mutagenesis have localized the dgk, and plsB loci to a 2.2-megadalton DNA segment, and have demonstrated that diglyceride kinase and sn-glycerol-3-phosphate acyltransferase activities reside in separate polypeptides. Availability of these clones and mutationally altered derivatives has allowed the identification of a single polypeptide (Mr = 83,000) corresponding to the sn-glycerol-3-phosphate acyltransferase and purification of this membrane-bound enzyme to near homogeneity (Larson, T. J., Lightner, V. A., Green, P. R., Modrich, P., and Bell, R. M. (1980) J. Biol. Chem. 255, 9421-9426). The size of the plsB polypeptide indicates that a major fraction of the DNA segment to which this gene has been localized is involved in coding for the sn-glycerol-3-phosphate acyltransferase.

Acyltransferases↗

PubMed1980

Membrane phospholipid synthesis in Escherichia coli. Identification of the sn-glycerol-3-phosphate acyltransferase polypeptide as the plsB gene product.

A collection of hybrid plasmids bearing a structural gene, plsB, for the sn-glycerol-3-phosphate acyltransferase of Escherichia cole (Lightner, V. A., Larson, T. J., Tailleur, P., Kantor, G. D., Raetz, C. R. H., Bell, R. M., and Modrich, P. (1980) J. Biol. Chem. 255, 9413-9420) was employed to identify the membrane protein which is the sn-glycerol-3-phosphate acyltransferase. Strains containing these hybrid plasmids exhibited a marked increase in sn-glycerol-3-phosphate acyltransferase activity which was quantitatively extracted from membrane preparations with Triton X-100. Analysis of polypeptides present in detergent extracts of membranes from strains harboring the hybrid plasmids revealed a marked overproduction of a protein with an apparent molecular weight of 83,000, which was also the major protein labeled in minicells containing these hybrid plasmids. The labeled 83,000-dalton protein cochromatographed with sn-glycerol-3-phosphate acyltransferase activity on DEAE-cellulose. Utilization of three hybrid plasmids bearing amber mutations within the plsB gene demonstrated that the 83,000-dalton protein is the sn-glycerol-3-phosphate acyltransferase. Analysis of Bam HI deletion plasmids demonstrated that a 2.3-megadalton DNA fragment is necessary and sufficient for expression of the plsB gene. The sn-glycerol-3-phosphate acyltransferase was purified to near homogeneity from Triton X-100 extracts of membranes from overproducing strains. The preparations had reconstitutable specific activity of 2.5 micromol/min/mg and contained a single polypeptide with an apparent molecular weight of 83,000.

Acyltransferases↗

PubMed1977

Gene cloning for the isolation of enzymes of membrane lipid synthesis: phosphatidylserine synthase overproduction in Escherichia coli.

We have screened a bank of 2000 E. coli strains carrying hybrid ColE1 plasmids [Clarke, L. & Carbon, J. (1976) Cell 9, 91-99] for those that correct the temperature sensitivity of a mutant in CDP-1,2-diacyl sn-glycerol:L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase). Two hybrid plasmids of this kind (pLC34-44 and pLC34-46) were identified and characterized. Strains carrying these plasmids overproduce the synthase by 6- to 15-fold, as demonstrated by assays of extracts and purification to homogeneity of the overproduced enzyme. The overproduced synthase, like the wild-type enzyme, is found associated predominately with the ribosomal fraction of crude cell extracts. Because the membrane phospholipid composition of these overproducers is not greatly altered, we suggest that the synthase is normally present in excess.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗

PubMed1976

Membrane-associated phosphatidylglycerophosphate synthetase from Escherichia coli: purification by substrate affinity chromatography on cytidine 5'-diphospho-1,2-diacyl-sn-glycerol sepharose.

The membrane-associated cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDP-diglyceride):sn-glycerol-3-phosphate phosphatidyltransferase (EC 2.7.8.5) from Escherichia coli has been solubilized wiTriton X-100 and purified 6000-fold to 85% of homogeneity. The major purification was attained using several modifications of the the CDP-diglyceride Sepharose affinity chromatography system described by Larson et al. (Larson, T.J., Hirabayashi, T., and Dowhan, W. (1976), Biochemistry 15, 974). The native enzyme in Triton X-100 had an apparent molecular weight of over 200 000, as judged by Sepharose 6B gel filtration. The apparent size of the native enzyme appeared to be due to its association with Triton X-100, as judged by sucrose gradient centrifugation, polyacrylamide gel electrophoresis, and the lack of affinity for ion-exchange resins. The minimum subunit molecular weight of the enzyme, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, was 24 000. This low molecular weight is consistent with the stability of enzyme to heat, urea, or sodium dodecyl sulfate denaturation. The purified enzyme had an absolute requirement for magnesium ion (KM = 50 mM) and Triton X-100 (0.5-6%) for activity when either CDP-diglyceride or dCDP-diglyceride was used as substrate. Kinetic analysis of the enzymatic reaction indicated an ordered sequential Bi-Bi reaction with the liponucleotide forming a dead-end complex at high concentration, which inhibited both the forward and reverse reactions. The enzyme would not hydrolyze the pyrophosphate bond of its lipid substrate or the phosphate esters of its lipid product but would catalyze a cytidine 5'-monophosphate dependent exchange reaction between glycero-3-phosphate and phosphatidylglycerophosphate.

Chromatography, Affinity↗

PubMed1976

Ribosomal-associated phosphatidylserine synthetase from Escherichia coli: purification by substrate-specific elution from phosphocellulose using cytidine 5'-diphospho-1,2-diacyl-sn-glycerol.

Cytidine 5'-diphospho-1,2-diacyl-sn-glycerol (CDPdiglyceride):L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthetase) is bound tightly to the ribosomes in crude extracts of Escherichia coli. After separation of the enzyme from the ribosomes by the method of Raetz and Kennedy (Raetz, C.R.H., and Kennedy, E.P. (1974), J. Biol. Chem. 249, 5038), we have purified the enzyme to 97% of homogenekty. The major portion of the overall 5500-fold purification was attained by substrate-specific elution from phosphocellulose using CDP-diglyceride in the presence of detergent. The purified enzyme migrated as a single band with an apparent minimum molecular weight of 54 000 when subjected to electrophoresis on polyacrylamide disc gels containing sodium dodecyl sulfate. The purified enzyme catalyzed exchange reactions between cytidine 5'- monophosphate (CMP) and CDP-diglyceride and between serine and phosphatidylserine. The enzyme also catalyzed the hydrolysis of CDP-diglyceride to form CMP and phosphatidic acid. dCDP-diglyceride was equivalent to CDP-diglyceride in all reactions catalyzed by the enzyme. In addition, the purified enzyme catalyzed the formation of phosphatidylglycerol or phosphatidylglycerophosphate at a very slow rate when serine was replaced as substrate by glycerol or sn-glycero-3-phosphate, respectively. These results suggest catalysis occurs via a ping-pong mechanism through the formation of a phosphatidyl-enzyme intermediate.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗

PubMed1976

Phosphatidylglycerol biosynthesis in Bacillus licheniformis Resolution of membrane-bound enzymes by affinity chromatography on cytidinediphospho-sn-1,2-diacylglycerol Sepharose.

Cytidinediphospho-sn-1,2-diaclglycerol (CDP-diglyceride) has been covalently linked to Sephrose 4B via adipic acid dihydrazide spacer arm forming an effective affinity chromatography column. This liponucleo-tide ligand and sn-glycero-3-phosphate are subtracts for the formation of 3-sn-phoshatidyl-1'-sn-glycero-3'-phosphate (PGP) catalyzed in both eukaryotic and prokaryotic organisms by sn-glycero-3-phosphate: CMP phosphatidlytranferase (PGP synthetase). Using this CDP-diglyceride Sephrose affinity column we were able to resolve the membrane associated 3-sn-phosphatidyl'1-sn-glycerol (PG) synthesizing system present in Bacillus licheniformis into two activities. A PGP synthetase activity was adsorbed to the affinity column and was eluted using buffer containg CDP-diglyceride; a PGP phosphatease acactivity had no affinity for the column. Both PGP synthase and PGP phosphatase of B. licheniformis were associated with a membrane component of the cell as evidenced by sucrose gradient centrifugation, differential centrifugation, and solubilization by buffers containing detergent...

Bacillus↗

PubMedDate not supplied

Resuscitating and transforming hospice volunteer services.

This article is designed for hospice leaders and volunteers to explore creative ways to deal with the stagnancy that can occur in volunteer programs. Specific strategies, such as nurturing caregivers, transitioning into a gift-based program, and integrating spirituality and creativity into volunteer efforts, provide the focus of the article.

Hospices↗