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R L Fuchs

Publications and source records attributed to R L Fuchs.

30 records · Page 2Linked to original sources
PubMed1991

Broad host-range vector for efficient expression of foreign genes in gram-negative bacteria.

A broad host-range expression plasmid was constructed comprising the incQ replicon, the recA promoter from Escherichia coli and the g10-L ribosome binding site (RBS) derived from bacteriophage T7. The structural genes for porcine somatotropin (pst) and E. coli beta-galactosidase (lacZ) were used to monitor gene expression in a diverse collection of Gram-negative bacterial hosts: Escherichia coli, Pseudomonas aeruginosa, Pseudomonas syringae, Pseudomonas putida, Pseudomonas fluorescens, Pseudomonas testosteroni, Serratia marcescens and Erwinia herbicola. The E. coli recA promoter was functional in this wide range of hosts and was inducible by the addition of nalidixic acid. Moreover, the level of lacZ expression was often at least as high as that observed in E. coli. Previous studies had shown that the g10-L RBS was superior to a simple "consensus" RBS sequence for expression of foreign genes in E. coli. Here we demonstrate a 38 to 70 fold increase in expression in two Pseudomonas hosts using the g10-L RBS, indicating that the translational enhancer present in the g10-L RBS is also functional in other bacteria. The juxtaposition of these transcriptional and translational elements in a broad host-range vector provides a simple way to evaluate alternate hosts for recombinant protein production.

Cloning, Molecular↗
PubMed1990

Purification and characterization of Bacillus thuringiensis var. tenebrionis insecticidal proteins produced in E. coli.

Native and single amino acid variants of the Bacillus thuringiensis var. tenebrionis insecticidal proteins were expressed in Escherichia coli, purified and examined for biological and biochemical properties. A novel, pH dependent, preferential precipitation method was implemented to purify Escherichia coli produced Bacillus thuringiensis var. tenebrionis proteins, which are active against Colorado potato beetle (Leptinotarsa decemlineata) larvae. Cysteine residues of the native Bacillus thuringiensis var. tenebrionis protein were replaced by serine residues by site-directed mutagenesis to investigate the biological and structural importance of the individual cysteine residues. Sulfhydryl determination of the native and amino acid variant Bacillus thuringiensis var. tenebrionis proteins revealed that the native protein contains no disulfide bonds. Modification of the carboxyl terminal cysteine residue (amino acid 540) caused complete inactivation of the protein. Native, truncated and single amino acid variants (other than at amino acid 540) exhibited insecticidal activities comparable to each other and to solubilized crystals from the original strain.

Animals↗
PubMed1990

Specificity and efficacy of purified Bacillus thuringiensis proteins against agronomically important insects.

The host range and relative efficacy of three purified Bacillus thuringiensis insect control proteins were determined against 17 different agronomically important insects representing five orders and one species of mite. The three B. thuringiensis proteins were single gene products from B. thuringiensis ssp. kurstaki HD-1 (CryIA(b)) and HD-73 (CryIA(c)), both lepidopteran-specific proteins, and B. thuringiensis ssp. tenebrionis (CryIIIA), a coleopteran-specific protein. Seven insects showed sensitivity to both B. thuringiensis ssp. kurstaki proteins, whereas only 1 of the 18 insects was sensitive to B. thuringiensis ssp. tenebrionis protein. The level of B. thuringiensis ssp. kurstaki protein required for 50% mortality (LC50) varied by 2000-fold for these 7 insects. A larval growth inhibition assay was developed to determine the amount of B. thuringiensis ssp. kurstaki protein required to inhibit larval growth by 50% (EC50). This extremely sensitive assay enabled detection of B. thuringiensis ssp. kurstaki HD-73 levels as low as 1 ng/ml.

Animals↗
PubMed1990

Insect resistant cotton plants.

We have expressed truncated forms of the insect control protein genes of Bacillus thuringiensis var. kurstaki HD-1(cryIA(b) and HD-73 (cryIA(c) in cotton plants at levels that provided effective control of agronomically important lepidopteran insect pests. Total protection from insect damage of leaf tissue from these plants was observed in laboratory assays when tested with two lepidopteran insects, an insect relatively sensitive to the B.t.k. insect control protein, Trichoplusia ni (cabbage looper) and an insect that is 100 fold less sensitive, Spodoptera exigua (beet armyworm). Whole plants, assayed under conditions of high insect pressure with Heliothis zea (cotton bollworm) showed effective square and boll protection. Immunological analysis of the cotton plants indicated that the insect control protein represented 0.05% to 0.1% of the total soluble protein. We view these results as a major step towards the agricultural use of genetically modified plants with insect resistance in this valuable, high acreage crop.

Amino Acid Sequence↗
PubMed1990

Cloning of two genes from Bacillus circulans WL-12 which encode 1,3-beta-glucanase activity.

Two genes encoding distinct 1,3-beta-glucanases have been cloned from Bacillus circulans and expressed in Escherichia coli. A cosmid library of B. circulans WL-12 DNA was constructed in the broad-host-range cosmid pLAFR1 and screened in E. coli for clones which exhibited 1,3-beta-glucanase activity. Two 1,3-beta-glucanase-positive clones were identified which contained genes encoding two independent 1,3-beta-glucanases as shown by biochemical, physical and molecular analyses. The cosmids, designated pMON5401 (27.1 kb insert) and pMON5402 (24.7 kb insert), encoded 68 kDa and 40 kDa 1,3-beta-glucanases, respectively. Both 1,3-beta-glucanases were purified from their respective E. coli strains, characterized biochemically, and were shown to exhibit lytic activity against purified yeast cell wall preparations.

Amino Acid Sequence↗
PubMed1986

Cloning of a Serratia marcescens Gene Encoding Chitinase.

Serratia marcescens, a chitinase-producing microorganism, was shown to produce five unique chitinolytic proteins with subunit molecular masses of 21, 36, 48, 52, and 57 kilodaltons. A cosmid library of S. marcescens DNA was constructed in the broad-host-range cosmid pLAFR1 and screened in Escherichia coli for clones capable of degrading chitin. A total of four independent clones (22- to 27-kilobase inserts) were isolated, characterized by restriction endonuclease digestion, and shown to share a common 9.5-kilobase EcoR1 fragment apparently encoding the same 57-kilodalton chitinase, the most abundant chitinase produced by S. marcescens. Chitinase expression from these constructs in both E. coli and Pseudomonas fluorescens 701E1 is apparently driven by an S. marcescens promoter. The significantly higher chitinase levels produced in E. coli relative to those in P. fluorescens 701E1 suggest that E. coli may recognize this promoter sequence more efficiently than P. fluorescens.

Journal Article↗
PubMed1985

Fine structure analysis of Salmonella typhimurium glutamate synthase genes.

Glutamate synthase activity is required for the growth of Salmonella typhimurium on media containing a growth-rate-limiting nitrogen source. Mutations that alter glutamate synthase activity had been identified in the gltB gene, but it was not known which of the two nonidentical subunits of the enzyme was altered. To examine the gene-protein relationship of the glt region, two nonsense mutations were identified and used to demonstrate that gltB encodes the large subunit of the enzyme. Six strains with independent Mu cts d1 (lac bla) insertions were isolated, from which a collection of deletion mutations was obtained. The deletions were transduced with the nonsense mutations and 38 other glt point mutations to construct a fine-structure genetic map. Chromosome mobilization studies, mediated by Hfr derivatives of Mu cts d1 lysogens, showed that gltB is transcribed in a clockwise direction, as shown in the S. typhimurium linkage map. Studies of the polar effects of three Mu cts d1 insertions indicated that the gene for the small subunit maps clockwise to gltB and that the two genes are cotranscribed to form a glt operon.

Bacteriophages↗
PubMed1985

In vivo synthesis of histidine by a cloned histidine ammonia-lyase in Escherichia coli.

Histidine ammonia-lyase catalyzes the first step in histidine catabolism, the deamination of histidine to urocanate and ammonia. In vitro experiments have shown that histidine ammonia-lyase also can catalyze the reverse (amination) reaction, histidine synthesis, relatively efficiently under extreme reaction conditions (4 M NH4OH, pH 10). An Escherichia coli hisB deletion strain was transformed with a pBR322 derivative plasmid (pCB101) containing the entire Klebsiella aerogenes histidine utilization (hut) operon to determine whether the catabolic histidine ammonia-lyase could function biosynthetically in vivo to satisfy the histidine auxotrophy. Although the initial construct did not grow on media containing urocanate and ammonia as a source of histidine, spontaneous mutants possessing this ability were isolated. Four mutants characterized grew at doubling times of 4 h compared with 1 h when histidine was present, suggesting that histidine synthesis, although unequivocally present, remained growth limiting. Each mutant contained a plasmid-encoded mutation which eliminated urocanase activity, the second enzyme in the Hut catabolic pathway. This genetic block led to the accumulation of high intracellular levels of urocanate, which was subsequently converted to histidine via histidine ammonia-lyase, thus satisfying the histidine auxotrophic requirement.

Ammonia-Lyases↗
PubMed1982

Identification of the structural genes for glutamate synthase and genetic characterization of this region of the Salmonella typhimurium chromosome.

Salmonella typhimurium cells require glutamate synthase activity for growth in media containing a growth rate-limiting nitrogen source. Although this enzyme plays a critical role in ammonia assimilation, little is known about the organization and regulation of the structural genes for its two subunits. To identify the location of the structural genes, mutants having heat-labile glutamate synthase activities were isolated and characterized. Mutations that altered glutamate synthase activity were mapped at 69 U on the S. typhimurium chromosome. Four strains with independent Tn10 insertions in this region were constructed and used for mutant selection and for positioning mutations affecting glutamate synthase activity relative to other genetic markers. In contrast to results obtained with Escherichia coli mutants, there was no linkage between mutations affecting glutamate synthase activity and the argG gene. The results of a combination of transduction experiments demonstrated the gene order argG-glnF-gltB-cod-argR-envB-aroE for S-typhimurium.

Chromosome Mapping↗
PubMed1980

Comparative properties of glutamine synthetases I and II in Rhizobium and Agrobacterium spp.

Some properties of glutamine synthetase I (GSI) and GSII are described for a fast-growing Rhizobium sp. (Rhizobium trifolii T1), a slow-growing Rhizobium sp. (Rhizobium japonicum USDA 83), and Agrobacterium tumefaciens C58. GSII of the fast-growing Rhizobium sp. and GSII of the Agrobacterium sp. were considerably more heat labile than GSII of the slow-growing Rhizobium sp. As previously shown in R. japonicum 61A76, GSI became adenylylated rapidly in all species tested in response to ammonium. GSII activity disappeared within one generation of growth in two of the strains, but the disappearance of GSII activity required two generations in another. Isoactivity points for transferase assay, which were derived from the pH curves of adenylylated GSI and deadenylylated GSI, were approximately pH 7.8 for both R. trifolii and A. tumefaciens. No isoactivity point was found for R. japonicum under the standard assay conditions used. When the feedback inhibitor glycine was used to inhibit differentially the adenylylated GSI and deadenylylated GSI of R. japonicum, an isoactivity point was observed at pH 7.3. Thus, the transferase activity of GSI could be determined independent of the state of adenylation. A survey of 23 strains of bacteria representing 11 genera indicated that only Rhizobium spp. and Agrobacterium spp. contained GSII. Thus, this enzyme appears to be unique for the Rhizobiaceae.

Ammonium Chloride↗
PubMed1979

The purification and characterization of ADP-glucose pyrophosphorylase A from developing maize seeds.

ADPglucose pyrophosphorylase A (ATP:alpha-D-glucose-1-phosphate adenylyltransferase, EC 2.7.7.27) from developing maize (Zea mays) endosperm was purified 129 fold to apparent homogeneity. The molecular weight estimated by gel filtration and by polyacrylamide gel electrophoresis was 375 000 and 400 000, respectively. The preparation gave a single protein band after SDS-polyacrylamide gel electrophoresis suggesting a monomer mol. wt. of 96 000. It was concluded that ADPglucose pyrophosphorylase A in maize endosperm is a tetramer of four similar molecular weight subunits. Values for the Km for glucose 1-phosphate and ATP were 3.8 . 10(-5) and 1.8 . 10(-4) M, respectively (using the homogeneous preparation).

Adenosine Diphosphate Glucose↗