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Biomedical subjects

G N Bennett

Publications and source records attributed to G N Bennett.

At least 19 recordsLinked to original sources
PubMed1992

Vector construction, transformation, and gene amplification in Clostridium acetobutylicum ATCC 824.

In order to alter the primary metabolism of C. acetobutylicum, we have constructed E. coli- or B. subtilis-C. acetobutylicum shuttle vectors that could be used to deliver homologous fermentative genes into C. acetobutylicum ATCC 824. The plasmid copy number and plasmid stability in C. acetobutylicum for several of these plasmids were determined. We have also developed a protocol for the electrotransformation of C. acetobutylicum ATCC 824. Difficulty in the transformation of C. acetobutylicum ATCC 824 with vectors containing DNA from E. coli plasmids was found to be due to the existence of a restriction system in this strain. This type II restriction endonuclease, named Cac824I, recognizes the sequence 5'-GCNGC-3' and cuts ColE1 plasmids frequently. One of the vectors, pFNK1, possessing a variety of unique cloning sites was used in the amplification of one acid (PTB) and one solvent (AADC) formation gene. The corresponding enzyme activities were amplified in C. acetobutylicum as shown by enzyme assays and SDS-PAGE gels of cell extracts.

Bacillus subtilis↗
PubMed1992

Expression of cloned homologous fermentative genes in Clostridium acetobutylicum ATCC 824.

We have previously cloned the acetone-formation pathway gene, encoding acetoacetate decarboxylase (adc), and butyrate-formation pathway gene, encoding phosphotransbutyrylase (ptb), of Clostridium acetobutylicum ATCC 824 in Escherichia coli. Here we report their subcloning in Bacillus subtilis and transfer to strain ATCC 824 via electrotransformation, where the corresponding enzyme activities were expressed at elevated levels, using pFNK1, a new B. subtilis/C. acetobutylicum shuttle vector. Plasmid pFNK1 was used because shuttle vectors that function in E. coli were unable to electrotransform ATCC 824 unless they became deleted in the E. coli-plasmid regions. The difficulties with shuttle vectors that function in E. coli are probably due to the presence of a restriction endonuclease in ATCC 824. This endonuclease recognizes the sequence 5'-GCNGC-3', which is prevalent in E. coli plasmids but occurs infrequently in pFNK1 and C. acetobutylicum genes. Cloning of genes in C. acetobutylicum is critical for redirecting the cellular metabolism (metabolic engineering) as well as for genetic studies of this industrial organism.

Bacillus subtilis↗
PubMed1992

Molecular characterization of two Clostridium acetobutylicum ATCC 824 butanol dehydrogenase isozyme genes.

A 4-kb segment of DNA containing two previously cloned butanol dehydrogenase (BDH) isozyme genes (D. Petersen, R. Welch, F. Rudolph, and G. Bennett, J. Bacteriol. 173:1831-1834, 1991) was sequenced. Two complete open reading frames (ORFs) were identified (bdhA and bdhB), along with a third truncated ORF (ORF1). The translation products of bdhA and bdhB corresponded to the N-terminal sequences of the purified BDH I and BDH II proteins, respectively. The two isozymes had a high amino acid identity (73%) and showed homology to a newly described class of alcohol dehydrogenases. Northern blots revealed that bdhA and bdhB did not form an operon. Primer extension experiments located single transcriptional start sites 37 and 58 bp upstream of the start codons of bdhA and bdhB, respectively. The -10 and -35 promoter regions for these genes were almost identical. bdhA and bdhB were found to be induced or derepressed immediately prior to significant butanol production in controlled pH 5.0 batch fermentations.

Alcohol Oxidoreductases↗
PubMed1992

Nucleotide sequence of the Escherichia coli cad operon: a system for neutralization of low extracellular pH.

Lysine decarboxylase of Escherichia coli has been the subject of enzymological studies, and the gene encoding lysine decarboxylase (cadA) and a regulatory gene (cadR) have been mapped. This enzyme is induced at low pH in the presence of lysine and achieves maximal level under anaerobic conditions. The induction of lysine decarboxylase increases the pH of the extracellular medium and provides a distinctive marker in tests of clinical strains. We report the sequence of the cad operon encoding lysine decarboxylase, a protein of 715 amino acids, and another protein, CadB, of 444 amino acids. The amino acid sequence of lysine decarboxylase showed high homology to that of the lysine decarboxylase of Hafnia alvei with less homology to the sequence of speC, which encodes the biosynthetic ornithine decarboxylase of E. coli. The cadA and cadB genes were separately cloned and placed under the control of lac and tac promoters, respectively, to facilitate independent study of their physiological effects. The cadB gene product had a mobility characteristic of a smaller protein on protein gels, analogous to that found for some other membrane proteins. The CadB sequence showed homology to that of ArcD of Pseudomonas aeruginosa, encoding an arginine/ornithine antiporter. Excretion studies of various strains, the coinduction of cadB and cadA, and the attractive physiological role for an antiport system led to a model for the coupled action of cadA and cadB in uptake of lysine, the reduction of H+ concentration, and excretion of cadaverine.

Amino Acid Sequence↗
PubMed1992

Regulation of the Escherichia coli cad operon: location of a site required for acid induction.

The cad operon encodes lysine decarboxylase and a protein homologous to amino acid antiporters. These two genes are induced under conditions of low pH, anaerobiosis, and excess lysine. The upstream regulatory region of the cad operon has been cloned into lacZ expression vectors for analysis of the sequences involved in these responses. Deletion analysis of the upstream region and cloning of various fragments to make cadA::lacZ or cadB::lacZ protein fusions or operon fusions showed that cadA was translated more efficiently than cadB and localized the pH-responsive site to a region near an upstream EcoRV site. Construction of defined end points by polymerase chain reaction further localized the left end of the regulatory site. The presence of short fragments bearing the regulatory region on high-copy-number plasmids greatly reduced expression from the chromosomal cad operon, suggesting that titration of an essential activator protein was occurring. With nonoptimal polymerase chain reaction conditions, a set of single point mutants were made in the upstream regulatory region. Certain of these altered regulatory regions were unable to compete for the regulatory factor in vivo. The locations of these essential bases indicate that a sequence near the EcoRV site is very important for the activator-DNA interaction. In vivo methylation experiments were conducted with cells grown at pH 5.5 or at pH 8, and a difference in protection was observed at specific G residues in and around the region defined as important in pH regulation by the mutation studies. This work defines essential sequences for acid induction of this system involved in neutralization of extracellular acid.

Base Sequence↗
PubMed1991

Stability of mutant type II dihydrofolate reductase proteins in suppressor strains.

In the course of study of a (Glu-58 to Gln-58) mutant type II dihydrofolate reductase (DHFR), it was found that the altered DHFR was poorly produced in vivo. Investigations with several common laboratory Escherichia coli strains including htpR and lon strains bearing plasmids expressing the Gln-58 DHFR indicated a correlation of rapid degradation with the presence of a sup+ phenotype. The supo strain MC1061(p3) was transformed with a series of plasmids containing the Gln-58 DHFR gene with and without an additional supF gene, and expression levels were compared. The supF+ constructs exhibited little accumulation of the Gln-58 DHFR, while reasonable levels were found in the supo cases. Experiments with extracts of plasmid-free sup+ and supo strains showed rapid degradation by certain strains compared to MC1061(p3) and this degradation was not dependent upon ATP. In another route to increasing the stability of labile DHFR derivatives, mutagenesis of a strain bearing a N-terminally shortened Gln-58 DHFR was performed. Selection and analysis of a trimethoprim-resistant stable mutant showed that this DHFR gene contained a triple repeat of leu-pro-ser in the enzymatically non-essential N-terminal portion of the protein.

Amino Acid Sequence↗
PubMed1991

Cloning of the Clostridium acetobutylicum ATCC 824 acetyl coenzyme A acetyltransferase (thiolase; EC 2.3.1.9) gene.

Thiolase (acetyl coenzyme A acetyltransferase; EC 2.3.1.9) from Clostridium acetobutylicum is a key enzyme in the production of acids and solvents in this organism. The purification and properties of the enzyme have already been described (D. P. Wiesenborn, F. B. Rudolph, and E.T. Papoutsakis, Appl. Environ. Microbiol. 54:2717-2722, 1988). The thl gene encoding the thiolase has been cloned by using primary antibodies raised to the purified enzyme. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by immunoblots with the antithiolase antibodies. Phage DNA was purified from positive plaques, and restriction enzyme digests identified an approximately 4.8-kb AccI fragment common to all positive plaques. A corresponding fragment was also found in AccI digests of C. acetobutylicum chromosomal DNA. The fragment was purified and EcoRI linkers were attached before being subcloned into pUC19. Maxicell analysis showed the production of an approximately 42-kDa protein, whose size corresponded to the molecular size of the purified thiolase, from the clostridial insert. Enzyme activity assays and Western blot (immunoblot) analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell extracts of Escherichia coli harboring the cloned thl confirmed the presence of the thiolase encoded within the cloned DNA.

Acetyl-CoA C-Acetyltransferase↗
PubMed1990

Targeted neuronal lesion induced by photosensitizing dyes.

Free radical-induced phototoxicity mediated by laser irradiation was investigated in the rabbit facial nerve. Azure-C, mesoporphyrin, or the dye conjugated to the protein carrier horseradish peroxidase were injected into the levator alae nasi muscle. Two to 7 days after uptake and laser exposure, nerve sections showed varying degrees of cellular modifications including: severe membrane degradation and associated lipid peroxide granules, distended mitochondria, and mitochondrial loss. Immunoblots of homogenates from treated nerves revealed specific changes in neurofilament and myelin basic protein. The site specific damage produced in vivo by photosensitizing dye resembles abnormalities in aging neurons and in Batten's disease, both hypothesized to be cases of free radical-peroxidation reactions. These reactions differ from those found in transection and crush lesions.

Animals↗
PubMed1979

The N protein of bacteriophage lambda, defined by its DNA sequence, is highly basic.

Nucleotide sequence has been determined for the restriction fragments and cloned DNA from the pL-N-tL1 region of bacteriophage lambda. A unique reading frame for the N gene is defined by the absence of natural nonsense codons and by the presence of seven nonsense codons generated by mutations in N. This reading frame is initiated at two alternative ATG codons, the second of which is probably the in vivo translation start. Reading is stopped at a single TAG codon. The protein coded is therefore 133 or, more probably, 107 amino acids long, rich in lysine, arginine and proline.

Bacteriophage lambda↗
PubMed1976

Guanosine tetraphyosphate and its analogues. Chemical synthesis of guanosine 3',5'-dipyrophosphate, deoxyguanosine 3',5'-dipyrophosphate, guanosine 2',5'-bis(methylenediphosphonate), and guanosine 3',5'-bis(methylenediphosphonate).

A new procedure for the synthesis of the pyrophosphate bond has been employed in the preparation of nucleoside dipyrophosphates from nucleoside 3',5'-diphosphates. The method makes use of a powerful phosphorylating agent generated in a mixture of cyanoethyl phosphate, dicyclohexylcarbodiimide, and mesitylenesulfonyl chloride in order to avoid possible intramolecular reactions between the two phosphate groups on the sugar ring. That such reactions can readily occur was shown by the facile cyclization of deoxyguanosine 3',5'-diphosphate to P1,P2-deoxyguanosine 3',5'-cyclic pyrophosphate in the presence of dicyclohexylcarbodiimide alone. The phosphorylation reagent was initially tested in the conversion of deoxyguanosine 3',5'-diphosphate to the corresponding 3',5'-dipyrophosphate and was then used to phosphorylate 2'-O-(alpha-methoxyethyl)guanosine 3',5'-diphosphate, which had been prepared from 2'-O-(alpha-methoxyethyl)guanosine. In the latter case, the addition of the two beta phosphate groups was accomplished in 40% yield. Removal of the methoxyethyl group from the phosphorylated product gave guanosine 3',5'-dipyrophosphate, which was shown to be identical with guanosine tetraphosphate prepared enzymatically from a mixture of GDP and ATP. A modification of published procedures was also necessary to effect the synthesis of guanosine bis(methylenediphosphonate). Guanosine was treated with methylenediphosphonic acid and dicyclohexylcarbodiimide in the absence of added base. The product consisted of a mixture of guanosine 2',5' - and 3',5'-bis(methylenediphosphonate), which was resolved by anion-exchange chromatography. The 2',5' and 3',5' isomers are interconvertible at low pH, with the ultimate formation of an equilibrium mixture having a composition ratio of 2:3. The predominant constituent of this mixture has been unequivocally identified as the 3',5' isomer by synthesis from 2'-O-tetrahydropyranylguanosine.

Alkaline Phosphatase↗
PubMed1976

Nucleotide sequence of region preceding trp mRNA initiation site and its role in promoter and operator function.

The nucleotide sequence of the region preceding the transcription initiation site of the tryptophan operon of Escherichia coli was determined. Essentially all of the trp operator precedes the transcribed portion of the operon. The deduced sequence contains the recognition site of endonuclease Hpa I. This site is protected from Hpa I cleavage by RNA polymerase and by trp repressor. Regions of 2-fold symmetry are present in the DNA sequence.

Base Sequence↗