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B S Hartley

Publications and source records attributed to B S Hartley.

At least 37 records · Page 2Linked to original sources
PubMed1985

Structure of wild-type and mutant repressors and of the control region of the rbt operon of Klebsiella aerogenes.

Pentitol metabolism in Klebsiella aerogenes is encoded by continuous ribitol (rbt) and D-arabitol (dal) operons transcribed in bipolar fashion and sandwiched between long stretches of homologous DNA. The operons are separated by a central control region (2.2 kb) which encodes both the repressors and all the control sequences. The rbt repressor (270 amino acids) shows homology to the Escherichia coli lac repressor and other DNA-binding proteins. It is transcribed from the strand opposite the rbt operon and the intervening control region (254-bp) contains features which reflect the complex regulation. A rbt-constitutive mutant strain used in previous studies of experimental enzyme evolution encodes a truncated rbt-peptide of 133 residues due to a frameshift mutation.

Amino Acid Sequence↗
PubMed1985

Nucleotide sequence of the thymidylate synthetase gene (thyP3) from the Bacillus subtilis phage phi 3T.

The thyP3 gene, encoding thymidylate synthetase, from the Bacillus subtilis phage phi 3T has been cloned and the nucleotide sequence determined. The derived amino acid sequence indicates a subunit Mr of 32 748. The primary amino acid sequence is compared with the sequences of the analogous proteins specified by Escherichia coli (thyA), Lactobacillus casei, (thyA) and phage T4 (td). Extensive conservation exists in all four sequences implying a shared tertiary structure.

Amino Acid Sequence↗
PubMed1985

Transcription of the Bacillus subtilis bacteriophage phi 3T in vitro.

The in vitro transcription pattern of Bg/II-digested phi 3T DNA is described. Eight Bg/II fragments that hybridized to in vitro transcription products were unequivocally identified. A further hybridizing region corresponding to a Bg/II triplet was also revealed, giving a total of nine to 11 Bg/II fragments. These represent 47 to 53% of the phi 3T genome. Transcription was shown to initiate within Bg/II fragments B, G, C, H, I, F and J, implying that all of these contain at least one promoter. The relevance of these data to the construction of a cloning vector based on phi 3T is discussed.

Bacillus subtilis↗
PubMed1984

Molecular cloning, DNA structure and expression of the Escherichia coli D-xylose isomerase.

The D-xylose isomerase (EC 5.3.1.5) gene from Escherichia coli was cloned and isolated by complementation of an isomerase-deficient E. coli strain. The insert containing the gene was restriction mapped and further subcloning located the gene in a 1.6-kb Bg/II fragment. This fragment was sequenced by the chain termination method, and showed the gene to be 1002 bp in size. The Bg/II fragment was cloned into a yeast expression vector utilising the CYCl yeast promoter. This construct allowed expression in E. coli grown on xylose but not glucose suggesting that the yeast promoter is responding to the E. coli catabolite repression system. No expression was detected in yeast from this construct and this is discussed in terms of the upstream region in the E. coli insert with suggestions of how improved constructs may permit achievement of the goal of a xylose-fermenting yeast.

Aldose-Ketose Isomerases↗
PubMed1983

The amino acid sequence of the tyrosyl-tRNA synthetase from Bacillus stearothermophilus.

The primary structure of the tyrosyl-tRNA synthetase (TyrTS) of Bacillus stearothermophilus has been deduced from the nucleotide sequence of the cloned gene and from the amino acid sequence of peptides isolated from the purified enzyme. TyrTS (B. stearothermophilus) has a molecular weight of 47316 and the sequence is 56% homologous with that of TyrTS (Escherichia coli). The binding domain for the substrate intermediate tyrosyl adenylate is located in the N-terminal portion of the polypeptide and is highly conserved in both enzymes. Several lysine residues, which are shielded from acetylation in the TyrTS-tRNATyr complex, are also located in a stretch of highly conserved sequence.

Amino Acid Sequence↗
PubMed1983

Industrial prospects for thermophiles and thermophilic enzymes.

Reasons for enzyme instability are discussed. Thermophiles are a promising source of more stable intracellular enzymes. This aids their purification as well as providing desirable industrial properties. The organisms themselves have advantages for high temperature fermentations, e.g. for ethanol production. Systems for cloning genes into them are under development. An example is given of genetic and physiological manipulation of a fast-fermenting Bacillus stearothermophilus to increase ethanol yields at 70 degrees C similar to those obtained with yeasts.

Anaerobiosis↗
PubMed1982

Ribitol dehydrogenase messenger RNA from an enzyme superproducer strain of Klebsiella aerogenes. Purification, cell-free translation and studies in vitro and in vivo.

1. Ribitol dehydrogenase messenger RNA, from a strain of Klebsiella aerogenes that had been evolved to superproduce this enzyme, has been purified in a single step by labelling extracted polysomes with rabbit anti(ribitol dehydrogenase) and immunoprecipitating with sheep anti-(rabbit IgG). 2. The extracted mRNA is stable in a protein synthesis system in vitro and directs synthesis 35-40-times more efficiently than RNA from coliphages MS2 or Q beta, to give ribitol dehydrogenase as sole major product. 3. Its size distribution shows a major band of 1500 nucleotides plus fragments 400-1400 nucleotides, with only traces of size 2400-3000 nucleotides. Only the latter could encode both proteins of the operon: ribitol dehydrogenase and D-ribulokinase. 4. Ribitol dehydrogenase mRNA represents 24% of total mRNA in cells harvested just after a 'switch' point' in mid-exponential phase. About half of the polysomes containing this mRNA are unattached to DNA, whereas only 3% of other mRNAs are unattached to DNA. 5. This mRNA is not outstandingly stable in vivo, though there are indications that it may be more stable than average. Hence the high level of synthesis of ribitol dehydrogenase (up to 30% of total protein in an extract) seems to be due to very efficient transcription and translation from multiple copies of a constitutive rbtD gene.

DNA-Directed RNA Polymerases↗
PubMed1981

Purification and properties of D-ribulokinase and D-xylulokinase from Klebsiella aerogenes.

The D-ribulokinase and D-xylulokinase of Klebsiella aerogenes were purified to homogeneity from Escherichia coli K12 construct strains that synthesized these enzymes constitutively. The D-ribulokinase, which is encoded in the ribitol operon, is active as a dimer of 60 000 subunit mol.wt., whereas the D-xylulokinase, which is encoded in the D-arabitol operon, is active as a dimer of 54 000 subunit mol.wt. The amino acid compositions and N-terminal sequences of both pentulokinases are reported. The Kapp. values of the enzymes for their D-pentulose substrates were determined, and the D-ribulokinase was shown to have a low-affinity side-specificity for ribitol and D-arabitol. These results are discussed in the context of the evolution of the Klebsiella aerogenes pentitol operons.

Amino Acids↗
PubMed1981

Sequence of a secondary phage lambda attachment site located between the pentitol operons of Klebsiella aerogenes.

We have determined the nucleotide sequence of a secondary phage lambda attachment site (att) located between the structural genes of the ribitol and D-arabitol catabolic operons of Klebsiella aerogenes. The core region of this secondary attachment site (sequence: GGTTTTTTCGATTAT) shows considerable homology with the 15-base-pair core region common to both the phage att and the primary bacterial att of Escherichia coli K12 (sequence: GCTTTTTTACTAA); however, there is no such clear homology between the sequences flanking the cores of the primary att and this secondary att. Integration of phage lambda into the K. aerogenes secondary att occurred by recombination between the core region of the phage att and an oligo(T.A) stretch located within the K. aerogenes secondary att.

Attachment Sites, Microbiological↗
PubMed1981

Petite-negative mutants of Saccharomyces cerevisiae.

A series of yeast mutants has been isolated with the inability to grow on fermentable carbon sources whilst growing normally on ethanol media. One of the mutants, namely MC16/206 lacks pyruvate decarboxylase activity and does not grow on glucose at 37 degrees C but grows on both ethanol and glucose at 27 degrees C. In this strain rho- petites are non-viable.

Alcohol Oxidoreductases↗
PubMed1981

Structure of an experimentally evolved gene duplication encoding ribitol dehydrogenase in a mutant of Klebsiella aerogenes.

We have previously described a system of experimental evolution in which many of the mutants of Klebsiella aerogenes selected for faster growth on xylitol ('evolvants') synthesized elevated levels of ribitol dehydrogenase and have presented genetic evidence implicating gene duplication in the enzyme superproduction in some of the evolvants. Here we describe a physical approach to the screening for gene duplications and subsequent structure determination. Nick-translated, cloned ribitol operon (rbt) DNA was used as a hybridization probe to identify fragments containing rbt operon sequences in restriction digests of total bacterial DNA. Whilst several of the evolvants probably harbour duplications spanning the entire rbt operon, one of the spontaneously arising evolvants (strain A3) was shown to harbour a small (5.8 kilobase pairs) direct DNA repeat which encodes the dehydrogenase (but not the kinase) of the closely linked D-arabitol operon as well as the dehydrogenase (but not the kinase) of the rbt operon. The hybridization data suggest that there are 4 to 5 copies of the repeat arranged contiguously on the chromosome. The genetic instability of strain A3, the rbt fragment hybridization pattern of an A3 segregant and the activities of the pentitol catabolic enzymes in A3 are all consistent with the proposed gene duplication structure.

Base Sequence↗
PubMed1980

Minicircular ColE1-related DNA in strains of Klebsiella aerogenes selected for fast growth on xylitol.

We have previously described a large family of mutants of Klebsiella aerogene which were selected by continuous on xylitol and which superproduce ribitol dehydrogenase. One of these strains was found to harbour a high copy number 2.1 x 10(6) dalton plasmid. This plasmid is a deletion derivative of a low copy number 3.5 x 10(6) dalton plasmid present in the ancestral strain of K. aerogenes. However, since these plasmids do not contain the genes required for pentitol catabolism and some enzyme-superproducing strains have lost all DNA homologous to the plasmids, they are not implicated in the fast growth on xylitol. The plasmids contain regions of homology with the Escherichia coli plasmid ColE1.

Alcohol Oxidoreductases↗
PubMed1980

A switch from translational control to transcriptional control of protein synthesis in mid-exponential growth phase of bacterial cultures. Specific radioimmune labelling of ribitol-dehydrogenase-synthesising polysomes from Klebsiella aerogenes in the presence of heparin.

1. We present evidence suggesting a sudden switch from translational control to transcriptional control of protein synthesis in mid-exponential growth of bacterial batch cultures. At a critical cell density a switch from large to small polysomes occurs during a short period of exponential growth. The profile of specific polysomes engaged in synthesis of a constitutive enzyme, ribitol dehydrogenase, changes at the same point but in an opposite way: a linear profile peaking at monosomes changes to a dome-shaped profile peaking at about 15 ribosomes/mRNA, which persists into late exponential phase despite a gradual reduction in the total polysome population. The switch in the pattern of protein synthesis is exhibited dramatically by changes in the specific activity or ribitol dehydrogenase in cell extracts at different stages of batch culture. In early exponential phase the specific activity of the enzyme is constant, but it begins to rise suddenly, at the same point at which the polysome profiles change, and continues to increase up to the end of exponential phase. This effect is exhibited by the strains of Klebsiella aerogenes that are inducible for (in the presence of the inducer), consitutive for, or superproducers of ribitol dehydrogenase, and it appears to be unrelated to catabolite repression. 2. The above results depend on improved techniques for production of large amounts of bacterial polysomes and the ability to label nascent peptides attached to polysomes very specifically with radioactive antibody to ribitol dehydrogenase. Our success was due to the observation that sodium heparin completely abolishes non-specific interactions of the antibody with the polysomes.

Bacterial Proteins↗
PubMed1979

Purification and properties of Klebsiella aerogenes D-arabitol dehydrogenase.

An Escherichia coli K12 strain was constructed that synthesized elevated quantities of Klebsiella aerogenes D-arabitol dehydrogenase; the enzyme accounted for about 5% of the soluble protein in this strain. Some 280 mg of enzyme was purified from 180 g of cell paste. The purified enzyme was active as a monomer of 46,000 mol.wt. The amino acid composition and kinetic constants of the enzyme for D-arabitol and D-mannitol are reported. The apparent Km for D-mannitol was more than 3-fold that for D-arabitol, whereas the maximum velocities with both substrates were indistinguishable. The enzyme purified from the E. coli K12 construct was indistinguishable by the criteria of molecular weight, electrophoretic mobility in native polyacrylamide gel and D-mannitol/D-arabitol activity ratio from D-arabitol dehydrogenase synthesized in wild-type K. aerogenes. Purified D-arabitol dehydrogenase showed no immunological cross-reaction with K. aerogenes ribitol dehydrogenase. During electrophoresis in native polyacrylamide gels, oxidation by persulphate catalysed the formation of inactive polymeric forms of the enzyme. Dithiothreitol and pre-electrophoresis protected against this polymerization.

Amino Acids↗
PubMed1979

Evolution of enzyme structure.

Three-dimensional structures of enzymes offer evidence about their evolution. There are clear examples of divergent families (e.g. mammalian serine proteases) and convergence (e.g. chymotrypsin and subtilisin). Topological similarities in dehydrogenases may reflect an ancient divergence or merely chemical constraints on protein architectures. Further experimental evidence is desirable to back up arguments based on molecular morphology. By growing microorganisms on novel foodstuffs in a chemostat, one can focus selective pressure on a specific enzyme activity. Experiments will be described in which such pressure is focused on pentitol metabolism. Examination of the fine structure of the genes responsible for this pentitol metabolism has given clues about the volution of metabolic pathways.

Amino Acid Sequence↗
PubMed1979

Purification, properties and specificity of the restriction endonuclease from Bacillus stearothermophilus.

The restriction endonuclease BstI was purified from 70kg of Bacillus stearothermophilus. The final product is at least 97% pure as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; this major protein species co-migrates with the enzyme activity on native polyacrylamide-gel electrophoresis and isoelectric focusing. Pure restriction endonuclease BstI has a subunit mol.wt. of 26,000 and is probably a loosely associated dimer. The enzyme shows maximum activity at pH values between 7 and 9.5, and in the presence of 0.5-2mM-Mg2+. NaCl inhibits the restriction enzyme activity. Restriction endonuclease BstI cleaves DNA in a position identical with that cleaved by endonuclease BamHI (for Bacillus amyloliquefaciens), i.e.: (formula: see text). In the presence of high concentrations of enzyme, DNA cleavage occurs at secondary sites. This side-specificity is enhanced by the addition of glycerol. Preliminary studies indicate that these sites are of the type: (formula: see text).

Chemical Phenomena↗