Biomedical subjects

G N Bennett

Publications and source records attributed to G N Bennett.

At least 73 records · Page 4Linked to original sources

PubMed1991

Cloning of an NADH-dependent butanol dehydrogenase gene from Clostridium acetobutylicum.

The acetone-butanol fermentation of C. acetobutylicum is characterized by the unique shift from acid to solvent production. The mechanism of the solventogenic switch involves the induction of several enzymes, including NADH-dependent butanol dehydrogenase (BDH) at the onset of solventogenesis. This enzyme is responsible for the final conversion of butyraldehyde to butanol, and is distinct from the NADPH-dependent alcohol dehydrogenase (ADH) also present in the organism. To characterize the genetic control of this gene, we have cloned and expressed it in E. coli. A lambda EMBL3 phage library of C. acetobutylicum DNA was screened via plaque hybridization using a [32P]-radiolabeled, 32-fold degenerate, 62-mer oligonucleotide probe. The probe was designed by reverse translation of the NH2-terminal amino acid sequence of purified BDH II. Southern blot experiments indicate that the phage insert was of clostridial origin and had no homology with the previously cloned NADPH-dependent ADH. Subcloning of DNA from purified positive plaques has localized the gene to a 3.5-kb EcoRI fragment from which the enzyme is well expressed. The sequence of the 25 NH2-terminal amino acids for the cloned enzyme purified from E. coli was determined and found to be identical to that for the clostridial NADH-dependent BDH II. Maxicell analysis of [35S]-radiolabeled plasmid-encoded proteins identified a species encoded by the clostridial insert with the expected Mr of 42 kD.

Alcohol Oxidoreductases↗

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↗

PubMed1991

Molecular cloning of an alcohol (butanol) dehydrogenase gene cluster from Clostridium acetobutylicum ATCC 824.

In Clostridium acetobutylicum, conversion of butyraldehyde to butanol is enzymatically achieved by butanol dehydrogenase (BDH). A C. acetobutylicum gene that encodes this protein was identified by using an oligonucleotide designed on the basis of the N-terminal amino acid sequence of purified C. acetobutylicum NADH-dependent BDH. Enzyme assays of cell extracts of Escherichia coli harboring the clostridial gene demonstrated 15-fold-higher NADH-dependent BDH activity than untransformed E. coli, as well as an additional NADPH-dependent BDH activity. Kinetic, sequence, and isoelectric focusing analyses suggest that the cloned clostridial DNA contains two or more distinct C. acetobutylicum enzymes with BDH activity.

Alcohol Dehydrogenase↗

PubMed1991

Partial characterization of a lysU mutant of Escherichia coli K-12.

The Escherichia coli K-12 strain GNB10181 shows no inducible lysyl-tRNA synthetase (LysRS) activity. Two-dimensional gel electrophoretic analysis of the polypeptides synthesized by this strain indicates that the normal lysU gene product, LysU, is absent. When both GNB10181 and its parent, MC4100, were grown at elevated temperatures (42 to 45 degrees C) no significant difference between their growth rates was observed. The lysU mutation was transferred to other E. coli K-12 backgrounds by using P1 transduction. The lysU transductants behaved comparably to their lysU+ parents at different growth temperatures. Therefore, the LysU proteins does not appear to be essential for growth at high temperatures, at least under the conditions examined here. In addition, lysU transductants were found to be defective for inducible lysine decarboxylase, (LDC), inducible arginine decarboxylase (ADI), and melibiose utilization (Mel), which are all missing in GNB10181. Complementation of the above missing functions was achieved by using the Clarke-Carbon plasmids pLC4-5 (LysU LDC) and pLC17-38 (LysU Mel ADI). From these experiments, it appears that GNB10181 has suffered a chromosomal deletion between 93.4 and 93.7 min, which includes the lysU gene. By using plasmid pLC17-38, the position of ADI on two-dimensional gels was identified. Finally, lysS delta lysU double mutants were constructed which can potentially be used as positive selection agents for the isolation of LysRS genes from other sources.

Cloning, Molecular↗

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↗

PubMed1990

Characterization and stereochemistry of cofactor oxidation by a type II dihydrofolate reductase.

Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are different both in sequence and in structure from known chromosomal DHFRs. These plasmid-derived DHFRs are responsible for conferring trimethoprim resistance to the host strain. A derivative of R388 DHFR, RBG200, has been cloned and overproduced [Vermersch, P. S., Klass, M. R., & Bennett, G. N. (1986) Gene 41, 289]. With this cloned and overproduced protein, a rapid purification procedure has been developed that yields milligram quantities of apparently homogeneous RBG200 DHFR with a specific activity 1.5-fold greater than that previously reported for the purified R388 protein [Amyes, S. G. B., & Smith, J. T. (1976) Eur. J. Biochem. 61, 597]. The pH versus activity profile and the native molecular weight of RBG200 DHFR were found to be similar to those previously reported for other type II DHFRs but different from those of the known chromosomal DHFRs. Stereospecifically labeled [4(S)-2H,4(R)-1H]NADPH was synthesized and used to determine the stereospecificity of NADPH oxidation by RBG200 DHFR. RBG200 DHFR was found to specifically transfer the pro-R hydrogen of NADPH to dihydrofolate, making it a member of the A-stereospecific class of dehydrogenases. Thus, although RBG200 DHFR is different both in sequence and in structure from known chromosomal enzymes, both enzymes catalyze identical hydrogen-transfer reactions. Two distinct binary RBG200 DHFR-NADP+ complexes were detected by monitoring the 1H NMR chemical shifts and line widths of the coenzyme in the presence of RBG200 DHFR.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1990

Methods for cloning key primary metabolic enzymes and ancillary proteins associated with the acetone-butanol fermentation of Clostridium acetobutylicum.

The unavailability of genetically defined mutants for complementation has intensified the problems inherent in cloning genes from C. acetobutylicum. The uniqueness of some of the pathways of this organism coupled with the relative inefficiency of transformation of clostridia and few characterized mutants in these pathways have made cloning these genes by traditional complementation methods impractical. Oligonucleotide hybridization techniques have been shown to circumvent many problems involved in detecting protein expression. The ease of hybridization screening of plaques allows phage libraries to be examined more readily than is generally the case with colony screening techniques. Recombinant lambda phages also contain more DNA per insert than most plasmid vectors can maintain, thus further decreasing the amount of screening necessary. Cosmid libraries, offering even greater length of individual inserts, can be screened in a similar manner, although such screening incorporates the limitations of colony screening techniques. It is true that the technique hinges on the ability to obtain an amino acid sequence from which an oligonucleotide can be designed. In the past, the ability to obtain sequences was limited because the quantity and number of purified proteins were limited or the proteins were amino-terminally blocked. However, recent technological advances in this area, such as high-resolution gel separation techniques coupled with microsequencing, have opened the door to proteins previously inaccessible. Deformylation methods have been developed to deblock amino-terminally formylated proteins, and successful internal amino acid sequence analysis by in situ protease digestion has also been reported using only picomolar quantities of proteins separated by one- or two-dimensional gel electrophoresis. Protein and DNA sequence data banks have been significantly upgraded in the past few years. A proposed oligonucleotide sequence can be evaluated to determine what other possible sequences have similar homology; moreover, protein similarity comparisons between related species might possibly supplant the need for protein isolation if regions of highly conserved amino acid sequences are found. To our knowledge, this represents the first reported use of oligonucleotide probe hybridization screening technology as a strategy for cloning solvent pathway genes of C. acetobutylicum. Despite the deleterious effects on hybridization inherent in the high A + T content of C. acetobutylicum gene specific-directed oligonucleotides, the technique has been shown to function with few modifications to previously recorded systems.

Acetone↗

PubMed1990

Purification of acetoacetate decarboxylase from Clostridium acetobutylicum ATCC 824 and cloning of the acetoacetate decarboxylase gene in Escherichia coli.

In Clostridium acetobutylicum ATCC 824, acetoacetate decarboxylase (EC 4.1.1.4) is essential for solvent production, catalyzing the decarboxylation of acetoacetate to acetone. We report here the purification of the enzyme from C. acetobutylicum ATCC 824 and the cloning and expression of the gene encoding the acetoacetate decarboxylase enzyme in Escherichia coli. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was screened by plaque hybridization, using oligodeoxynucleotide probes derived from the N-terminal amino acid sequence obtained from the purified protein. Phage DNA from positive plaques was analyzed by Southern hybridization. Restriction mapping and subsequent subcloning of DNA fragments hybridizing to the probes localized the gene within an approximately 2.1 kb EcoRI/Bg/II fragment. A polypeptide with a molecular weight of approximately 28,000 corresponding to that of the purified acetoacetate decarboxylase was observed in both Western blots (immunoblots) and maxicell analysis of whole-cell extracts of E. coli harboring the clostridial gene. Although the expression of the gene is tightly regulated in C. acetobutylicum, it was well expressed in E. coli, although from a promoter sequence of clostridial origin.

Amino Acid Sequence↗

PubMed1990

Cloning and expression of Clostridium acetobutylicum ATCC 824 acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase in Escherichia coli.

Coenzyme A (CoA)-transferase (acetoacetyl-CoA:acetate/butyrate:CoA-transferase [butyrate-acetoacetate CoA-transferase] [EC 2.8.3.9]) of Clostridium acetobutylicum ATCC 824 is an important enzyme in the metabolic shift between the acid-producing and solvent-forming states of this organism. The purification and properties of the enzyme have recently been described (D. P. Weisenborn, F. B. Rudolph, and E. T. Papoutsakis, Appl. Environ. Microbiol. 55:323-329, 1989). The genes encoding the two subunits of this enzyme have been cloned by using synthetic oligodeoxynucleotide probes designed from amino-terminal sequencing data from each subunit of the CoA-transferase. A bacteriophage lambda EMBL3 library of C. acetobutylicum DNA was prepared and screened by using these probes. Subsequent subcloning experiments established the position of the structural genes for CoA-transferase. Complementation of Escherichia coli ato mutants with the recombinant plasmid pCoAT4 (pUC19 carrying a 1.8-kilobase insert of C. acetobutylicum DNA encoding CoA-transferase activity) enabled the transformants to grow on butyrate as a sole carbon source. Despite the ability of CoA-transferase to complement the ato defect in E. coli mutants, Southern blot and Western blot (immunoblot) analyses showed that neither the C. acetobutylicum genes encoding CoA-transferase nor the enzyme itself shared any apparent homology with its E. coli counterpart. Polypeptides of Mr of the purified CoA-transferase subunits were observed by Western blot and maxicell analysis of whole-cell extracts of E. coli harboring pCoAT4. The proximity and orientation of the genes suggest that the genes encoding the two subunits of CoA-transferase may form an operon similar to that found in E. coli.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetates↗

PubMed1989

Regulation of lysine decarboxylase activity in Escherichia coli K-12.

The biodegradative lysine decarboxylase of E. coli has been reported to attain a higher specific activity when grown to saturation in the presence of excess lysine under conditions of low pH and absence of aeration. In order to examine possible sources of the pH and anaerobic regulation, a series of isogenic strains of E. coli K-12 were constructed. The effects of cadR-, fnr-, cya-, crp- and pgi- mutations on lysine decarboxylase expression were examined. Cultures were grown in a lysine supplemented rich medium at pH 5.5, pH 6.8, and pH 8.0 with and without aeration and the enzyme was assayed from log phase cultures. The results suggested that the pH and air responses were independent and that these known regulatory processes are not responsible for this regulation of the biodegradative lysine decarboxylase.

Anaerobiosis↗

PubMed1989

Construction of lac fusions to the inducible arginine- and lysine decarboxylase genes of Escherichia coli K12.

The induction of several amino acid decarboxylases under anaerobic conditions at low pH has been known for many years, but the mechanism associated with this type of regulation has not been elucidated. To study the regulation of the biodegradative arginine and lysine decarboxylases of Escherichia coli K12, Mudlac fusions to these genes were isolated. Mudlac fusion strains deficient for lysine decarboxylase or arginine decarboxylase were identified using decarboxylase indicator media and analysed for their regulation of beta-galactosidase expression. The position of the Mudlac fusion in lysine decarboxylase-deficient strains has been mapped to the cadA gene at 93.7 minutes, while the Mudlac fusions exhibiting a deficiency in the inducible arginine decarboxylase have been mapped to 93.4 minutes.

Amino Acid Sequence↗

PubMed1989

Isolation and Characterization of Mutants of Clostridium acetobutylicum ATCC 824 Deficient in Acetoacetyl-Coenzyme A:Acetate/Butyrate:Coenzyme A-Transferase (EC 2.8.3.9) and in Other Solvent Pathway Enzymes.

Mutants of Clostridium acetobutylicum ATCC 824 exhibiting resistance to 2-bromobutyrate or rifampin were isolated after nitrosoguanidine treatment. Mutants were screened for solvent production by using an automated alcohol test system. Isolates were analyzed for levels of butanol, ethanol, acetone, butyrate, acetate, and acetoin in stationary-phase batch cultures. The specific activities of NADH- and NADPH-dependent butanol dehydrogenase and butyraldehyde dehydrogenase as well as those of acetoacetyl-coenzyme A:acetate/butyrate:coenzyme A-transferase (butyrate-acetoacetate coenzyme A-transferase [EC 2.8.3.9]) (CoA-transferase), butyrate kinase, and phosphotransbutyrylase were measured at the onset of stationary phase. Rifampin-resistant strain D10 and 2-bromobutyrate mutant R were found to be deficient in only CoA-transferase, while several other mutants exhibited reduced butyraldehyde dehydrogenase and butanol dehydrogenase activities as well. The colony morphology of 2-bromobutyrate mutant R was similar to that of the parent on RCM medium; however, it had about 1/10 the level of CoA-transferase and increased levels of butanol dehydrogenase and butyraldehyde dehydrogenase. A nonsporulating, spontaneously derived degenerated strain exhibited reduced levels of butyraldehyde dehydrogenase, butanol, dehydrogenase, and CoA-transferase compared with those of the original strain. When C. acetobutylicum ATCC 824 was grown on medium containing low levels of 2-bromobutyrate, an altered colony morphology was observed. Not all strains resistant to 2-bromobutyrate (12 mM) were non-solvent-producing strains.

Journal Article↗

PubMed1988

Synthesis and expression of a gene for a mini type II dihydrofolate reductase.

The Type II dihydrofolate reductases (DHFR) are resistant to the folate analogs, trimethoprim and methotrexate. The monomer is very small (MW 9,000) and has no structural homology with other known DHFR types. A dhfr structural gene was synthesized which incorporates many unique restriction sites (Nco I, Nhe I, Pvu I, Hind III, Sma I, Bgl II, Xho I, and Ban I) within the coding sequence. This gene encodes a small DHFR (68 amino acids) which is 10 amino acids shorter at the amino-terminus than natural Type II DHFRs. The last 60 residues of the synthetically encoded protein are identical in sequence to R388 DHFR. The enzyme is functional and relatively stable, as evidenced by trimethoprim resistance conferred to cells expressing the synthetic gene. The gene was cloned onto a high-copy-number plasmid, pPV7SYN5, in which a trp-lac promoter drives transcription of both the dhfr gene and the primer for plasmid replication (RNA II). High levels of the small DHFR are accumulated in stationary phase cultures of MC1061(p3) containing pPV7SYN5 without the addition of IPTG.

Amino Acid Sequence↗

PubMed1988

Cloning and expression of Clostridium acetobutylicum phosphotransbutyrylase and butyrate kinase genes in Escherichia coli.

A 13.6-kilobase (kb) Sau3AI restriction endonuclease fragment of Clostridium acetobutylicum DNA cloned into pBR322 enabled Escherichia coli ato mutants to grow on butyrate as a sole carbon source (But+). Complementation of the ato defect by the recombinant plasmid pJC6 was due to expression of the genes for phosphotransbutyrylase (PTB) and butyrate kinase (BK). Both genes were efficiently expressed in E. coli, as their products were readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts. PTB was found to have a polypeptide subunit molecular weight of approximately 31,000, while that of BK was approximately 39,000. Deletion analysis and Tn5 mutagenesis of plasmid pJC7 (a But+ subclone containing a 4.4-kb BamHI fragment from the insert of pJC6) localized the PTB and BK genes within a region spanning approximately 2.9 kb. Preliminary evidence suggests that the two genes may form an operon that is transcribed as a single unit from a promoter of clostridial origin within the 4.4-kb insert of pJC7.

Acetyltransferases↗

PubMed1987

Formation of MboII vectors and cassettes using asymmetric MboII linkers.

Class-IIS restriction endonucleases such as MboII cleave DNA at a specified distance away from their recognition sequences. This feature was exploited to cleave DNA at previously inaccessible locations by preparing special asymmetric linker/adapters containing the MboII recognition sequence. These could be joined to DNA fragments and subsequently cleaved by MboII. Attachment of a 3' phosphate to one of the two different oligodeoxynucleotides comprising the asymmetric duplex prevented ligation at the improper end of the linker. Plasmids were constructed containing a unique BamHI or BclI site between the recognition and cleavage site of MboII. These sites were used to introduce a foreign fragment into the plasmid at a position permitting MboII to cleave within the newly inserted fragment. Once cleaved at the unique MboII site, another DNA fragment was inserted. DNA was thus inserted at a sequence not previously accessible to specific cleavage by a restriction enzyme. A cassette containing an identifiable marker, the lac operator, between two oppositely oriented MboII/BamHI linkers was made and tested in a random insertion linker mutagenesis experiment.

Base Sequence↗

PubMed1987

The use of a selectable FokI cassette in DNA replacement mutagenesis of the R388 dihydrofolate reductase gene.

FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5' end consisting of four nucleotides, 10-13 residues 3' from the nonpalindromic recognition sequence, GGATG. Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments. The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites. FokI excises the cassette and several base pairs of the neighboring vector sequence. The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA. A cassette which contains a tyrosine tRNA suppressor gene (supF) is selectable by the suppression of amber mutations in the recipient host. A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes. An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described.

Base Sequence↗

PubMed1986

The effect of nalidixic acid on expression from related E. coli promoters.

The effect of the DNA gyrase inhibitor, nalidixic acid, on expression from E. coli promoters was studied using the pKO-1, galactokinase expression vector system. Expression from a series of related hybrid promoters, tet promoter variants and the trp promoter flanked by oligonucleotide blocks was measured after incubation with nalidixic acid. Expression from the pBR322 tet promoter and tet promoter mutants within the -10 region was reduced after the drug treatment. The lacUV5, trp, and tettrp promoters were essentially unaffected while the trplac and the trptet promoters were stimulated. Studies of the trp promoter flanked by upstream or downstream oligonucleotide blocks revealed similar responses to the trp promoter parent control plasmids.

Base Sequence↗