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B E Davidson

Publications and source records attributed to B E Davidson.

At least 37 records · Page 2Linked to original sources
PubMed1995

Lactococcus lactis glyceraldehyde-3-phosphate dehydrogenase gene, gap: further evidence for strongly biased codon usage in glycolytic pathway genes.

The gene gap, encoding glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12), was isolated from a genomic library of Lactococcus lactis LM0230 DNA. Plasmids containing the L. lactis gene were able to complement a gap mutant of Escherichia coli. The nucleotide sequence of gap predicted a polypeptide chain of 337 amino acids for the enzyme and a subunit molecular mass of 36,043. The codon usage in gap and four other glycolytic genes from L. lactis showed a high degree of bias, when compared with 84 other chromosomal genes. Northern blot analysis of total L. lactis RNA showed that gap hybridized strongly with a 1.3 kb transcript. The 5' end of the transcript was determined by primer extension analysis to be a C located 35 bp upstream from the gap start codon. These transcript analyses, and the orientation of the open reading frames in the DNA flanking gap, indicated that in L. lactis gap is expressed on a monocistronic transcript. Nucleotide sequencing indicated that the DNA adjacent to gap did not encode other glycolytic pathway enzymes. The DNA sequence flanking gap contained two open reading frames (ORF156 and ORF211) of unknown function. The 3' end of a clpA homologue was identified in the sequence upstream of ORF156. The location of gap on the L. lactis DL11 chromosome map was determined to be between map coordinates 0.530 and 0.660.

Amino Acid Sequence↗
PubMed1995

Analysis of an Escherichia coli mutant TyrR protein with impaired capacity for tyrosine-mediated repression, but still able to activate at sigma 70 promoters.

In Escherichia coli, TyrR represses and activates transcription of operons required for tyrosine, phenylalanine and tryptophan biosynthesis and uptake. The TyrR central domain is homologous with NtrC and some other bacterial regulatory proteins, although TyrR regulates sigma 70, not sigma 54, promoters. We isolated a central domain TyrR mutant (TyrR E274Q) by substitution of a normally conserved amino acid. The mutant was unable to bring about tyrosine-mediated repression of aroF, aroL, tyrB, and tyrP and had diminished capability for tyrosine- and phenylalanine-mediated repression of aroP. In contrast, it was able to effect wild-type levels of phenylalanine-mediated repression of aroG, tryptophan-mediated repression of aroP and transcriptional activation of mtr and tyrP. The binding of purified TyrR E274Q to ATP (a requirement for tyrosine binding) and to the strong TyrR box of tyrP operator DNA were normal, but tyrosine binding and tyrosine-dependent hexamerization were significantly impaired. These properties are consistent with the proposal that self association is essential for tyrosine-mediated repression by TyrR but not for tyrosine- or phenylalanine-mediated activation. E274 of TyrR must participate in either the binding of tyrosine, or the coupling of ATP binding with a conformational change that alters the affinity of the ATP-dependent aromatic amino acid-binding site.

Adenosine Triphosphatases↗
PubMed1995

Evidence for two aromatic amino acid-binding sites, one ATP-dependent and the other ATP-independent, in the Escherichia coli regulatory protein TyrR.

In Escherichia coli, genetic regulation of aromatic amino acid biosynthesis and uptake is effected by the protein TyrR, which acts via ligand-mediated repression and activation. Characterization of the interactions of tyrosine, phenylalanine and tryptophan with TyrR revealed the presence of two separate aromatic amino acid-binding sites, one ATP-dependent, the other ATP-independent. Binding to the ATP-dependent site induces the self-association of TyrR. Using sedimentation equilibrium analyses, dissociation constants for this site in the dimeric and hexameric forms of TyrR were determined to be 330 microM and 24 microM, respectively, for tyrosine, and 55 mM and 3.7 mM, respectively, for phenylalanine. Tryptophan bound with a strength similar to that of phenylalanine, and both phenylalanine and tryptophan competed with the binding of tyrosine. The ATP-independent site, which has not been observed previously, was characterized by ultraviolet (u.v.) difference spectroscopy and a sedimentation-velocity meniscus-depletion method. Phenylalanine bound co-operatively to this site, exhibiting half-saturation at 260 microM. Tryptophan competed weakly with phenylalanine, half-saturation occurring at 1.2 mM. No binding of tyrosine to this site could be detected. We propose that the binding of phenylalanine or tryptophan to this ATP-independent site is responsible for phenylalanine- and tryptophan-mediated regulation by TyrR.

Adenosine Triphosphate↗
PubMed1995

Sequence analysis of the Lactococcus lactis temperate bacteriophage BK5-T and demonstration that the phage DNA has cohesive ends.

The Lactococcus lactis temperate bacteriophage BK5-T is a type phage in the lactococcal phage classification (A. W. Jarvis, G. F. Fitzgerald, M. Mata, A. Mercenier, H. Neve, I. B. Powell, C. Ronda, M. Saxelin, and M. Teuber, Intervirology 32:2-9, 1991). The nucleotide sequence of 18,935 bp of the genome of BK5-T was determined and analyzed for the presence of open reading frames and other structural features. Thirty-two open reading frames longer than 60 codons were identified, and these appeared to be grouped into at least seven transcriptional units. A search of the nucleotide sequence for restriction sites identified a small number of discrepancies with the previously published physical map of the BK5-T genome (G. Lakshmidevi, B. E. Davidson, and A. J. Hillier, Appl. Environ. Microbiol. 54:1039-1045, 1988). Subsequent analysis of restriction digests of BK5-T DNA which were heated prior to electrophoresis indicated that BK5-T DNA was not terminally redundant as previously reported but contained cohesive ends.

Amino Acid Sequence↗
PubMed1995

Identification of prophage genes expressed in lysogens of the Lactococcus lactis bacteriophage BK5-T.

Bacteriophage BK5-T is a small isometric-headed temperate phage that infects Lactococcus lactis subsp. cremoris. Northern (RNA) analysis of mRNA produced by lysogenic strains containing BK5-T prophage revealed four major BK5-T transcripts that are 0.8, 1.3, 1.8, and 1.8 kb in size and enabled a transcription map of the prophage genome to be prepared. The position and size of each transcript corresponded closely to the position and size of open reading frames predicted from the nucleotide sequence of BK5-T. Analysis of the transcripts suggested that one of them was derived from the gene encoding the BK5-T integrase and another was from the gene encoding the BK5-T homolog of the lambda cI repressor. Computer analysis of the nucleotide sequence upstream of the BK5-T cI homolog predicted the presence of a pair of divergent promoters and three inverted repeat sequences, features characteristic of temperature-phage immunity regions. By analogy with lambda, the three inverted repeat sequences could be binding sites for cI or Cro homologs and the two divergent promoters could initiate transcription through the BK5-T equivalents of cI and cro.

Amino Acid Sequence↗
PubMed1995

Spontaneous deletion mutants of the Lactococcus lactis temperate bacteriophage BK5-T and localization of the BK5-T attP site.

Spontaneous deletion mutants of the temperate lactococcal bacteriophage BK5-T were obtained when the phage was grown vegetatively on the indicator strain Lactococcus lactis subsp. cremoris H2. One deletion mutant was unable to form stable lysogens, and analysis of this mutant led to the identification of the BK5-T attP site and the integrase gene (int). The core sequences of the BK5-T attP and host attB regions are conserved in a number of lactococcal phages and L. lactis strains.

Amino Acid Sequence↗
PubMed1995

Genomic organization of Lactococci.

The genus Lactococcus contains four species of which L. lactis is the most thoroughly studied. Its genome is A+T-rich and consists of a circular chromosome of 2.0 to 2.7 Mbp, a wide range of plasmids, and frequently one or more prophages. Insertion sequence elements are commonly present in both the chromosome and the plasmids, while one conjugative transposon of 68 kbp has been described. Genetic maps of the chromosomes of a number of different L. lactis strains have been determined and found to differ quite significantly. For example, the maps of two L. lactis subsp. cremoris strains, MG1363 and FG2, have an inversion of approximately 40% of the chromosome when compared with the maps of two L. lactis subsp. lactis strains, DL11 and IL1403. Other differences indicative of smaller scale translocations and inversions are also found. Chromosomal rearrangements have also been induced in the laboratory by incubation of L. lactis cultures with infecting lytic phage and by treatment with mutagens. These results are indicative of a great deal of plasticity in the lactococcal genome.

Bacteriophages↗
PubMed1994

Ligand-induced self-association of the Escherichia coli regulatory protein TyrR.

Analyses of the sedimentation properties of the Escherichia coli regulatory protein TyrR indicated that it undergoes a ligand-induced hexamerization. This phenomenon was observed at protein concentrations approximating to those found in vivo. In the absence of added ligands, TyrR sedimented as a single molecular species with a sedimentation coefficient of 5.3 S and a relative molecular mass of 113,000. Given a subunit relative molecular mass of 57,640 for TyrR, it was concluded that this species is a dimer. Similar sedimentation properties were observed when TyrR was sedimented in the presence of either tyrosine, phenylalanine, ATP or ATP gamma S, a non-hydrolysable analogue of ATP. However, in the presence of saturating ATP gamma S and 500 microM tyrosine or 25 mM phenylalanine the sedimentation behaviour of TyrR yielded relative molecular masses of 340,000 and 310,000, respectively, indicative of hexamer formation. The sedimentation data obtained across a range of TyrR concentrations fitted equally well to dimer-hexamer and dimer-tetramer-hexamer models. For the latter model, the predicted overall association constant was 3.2 x 10(13) M-2 at saturating tyrosine, while the relative values of the association constants for the two individual steps indicated a concerted mechanism with the tetramer a minor component. There was no indication of dimer dissociation when dilute TyrR solutions (100 nM) were sedimented. A model to explain the role of hexamerization in tyrosine-mediated repression of transcription by TyrR is proposed. It is suggested that the hexameric form of TyrR is the active repressing species, interacting with two or three specific sequences (TyrR boxes) in the targeted regulatory DNA. The hexamerization reaction that takes place when the tyrosine concentration rises is envisaged as occurring in situ on the DNA, with a TyrR dimer that permanently occupies one of the TyrR boxes acting as a nucleation site for the development of the hexamer-DNA complex.

Adenosine Triphosphate↗
PubMed1994

Purification of the Escherichia coli regulatory protein TyrR and analysis of its interactions with ATP, tyrosine, phenylalanine, and tryptophan.

A plasmid that directs the overexpression of the Escherichia coli regulatory protein TyrR was constructed. Cell extracts of an E. coli strain harboring the plasmid were used to develop a two-step procedure for purifying homogeneous TyrR. The weight-average molecular weight of the pure protein was determined by sedimentation equilibrium analyses to be 110,000 +/- 5,000, indicating that native TyrR is a homodimer. The binding of ligands to TyrR was investigated by the techniques of sedimentation velocity meniscus depletion and steady state dialysis. One mol of ATP bound per mol of TyrR subunit with half-maximal saturation at 5-7 microM ATP. ATP binding curves exhibited positive cooperativity, with a value of 1.3 for the Hill constant, nH. The binding was not significantly affected by the presence of either 500 microM tyrosine or 2 mM phenylalanine. Binding of tyrosine to TyrR (40 microM subunit) could not be detected in the absence of ATP, indicating that the TyrR-tyrosine complex has a dissociation constant (Kd) in excess of 180 microM. However, binding was observed in the presence of saturating ATP (200 microM), where 1 mol of tyrosine bound per mol of TyrR subunit with half-maximal saturation at 50 microM tyrosine. The binding exhibited positive cooperativity (nH of 1.2). There was no detectable binding of either phenylalanine or tryptophan to TyrR (40 microM) in the absence or presence of 200 microM ATP, indicating that any binding of these amino acids to TyrR or TyrR.ATP also has a Kd in excess of 180 microM. Each of these amino acids was found to inhibit the binding of tyrosine by TyrR.ATP when present in large molar excess (20 microM tyrosine and 2 or 10 mM phenylalanine or tryptophan), indicating that TyrR binds each of these amino acids, albeit more weakly than it binds tyrosine.

Adenosine Triphosphate↗
PubMed1994

Analysis of the cos region of the Lactococcus lactis bacteriophage sk1.

The location, structure and nature of the cos site of the Lactococcus lactis bacteriophage sk1 was determined using a Taq DNA polymerase runoff sequencing technique. The cos site contains a single-stranded 3' overhang of 11 nucleotides. The region surrounding cos contains several features which may be involved in the binding and catalytic action of a phage terminase. These include four putative terminase-binding sites which show some homology to lambda R-sites, an 11-bp direct repeat, a 10-bp inverted repeat, a string of eight consecutive C residues and six copies of the pentanucleotide, AATCT. The spacing between adjacent copies of the pentanucleotides would place them on the same side of the DNA helix.

Bacteriophages↗
PubMed1994

Conservation of gene arrangement and an unusual organization of rRNA genes in the linear chromosomes of the Lyme disease spirochaetes Borrelia burgdorferi, B. garinii and B. afzelii.

Physical maps of the chromosomes of the Lyme disease spirochaetes Borrelia garinii and Borrelia afzelii have been elucidated for the enzymes CspI, SgrAI, I-CeuI, SmaI, EagI, BssHII, MluI and ApaI by two-dimensional pulsed-field gel electrophoresis techniques. The maps contain 42 sites for B. garinii and 32 for B. afzelii. The mapping studies showed that the two chromosomes are linear DNA molecules of 953 and 948 kbp, respectively. A comparison of the physical maps of B. garinii and B. afzelii and the published map of the other Lyme disease spirochaete, Borrelia burgdorferi [Davidson, B.E., MacDougall, J. & Saint Girons, I. (1992) J Bacteriol 174, 3766-3774] revealed that the three chromosomes have few endonuclease sites in common, apart from a cluster in rrl (encoding 23S rRNA) and rrs (encoding 16S rRNA). Cloned borrelial genes were used as specific hybridization probes to construct genetic maps, using the physical maps as a basis. The resulting maps contain 41 genetic loci for B. burgdorferi, 39 for B. garinii, and 33 for B. afzelii. In contrast to the physical maps, the three genetic maps are closely related, with no detectable differences in gene order along the entire length of the chromosome. It is concluded that the chromosomes of these three borrelial species have undergone no major rearrangements, deletions or insertions during their evolution from a common ancestor. Detailed mapping of the region of the B. garinii and B. afzelii chromosomes that encodes rRNA revealed that each chromosome contains one copy of rrs separated by 5 kbp from two copies each of rrl and rrf (encoding 5S rRNA). (ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed1994

Temporal transcription map of the Lactococcus lactis bacteriophage sk1.

Bacteriophage sk1 is a small isometric-headed lytic phage that infects Lactococcus lactis. The phage has a linear double-stranded DNA genome of 28 kbp, with cohesive ends. RNA was prepared from phage-infected L. lactis cells harvested at various intervals after infection, and the RNA molecules were resolved by electrophoresis. Northern blots of these gels were hybridized with sk1 DNA probes and the results obtained from these experiments, together with the results of primer extension analyses, enabled a transcription map of the phage genome to be prepared. Three classes of phage transcripts, designated as early, middle or late based on their time of appearance, were detected. Seven partially overlapping early transcripts were detected; these were transcribed from a 10 kbp region of the phage. The nine middle transcripts were derived from a 2 kbp region, limited by cos at one end and the start of the early transcripts at the other. The early and middle transcripts were transcribed divergently from a region mapping at 26 kbp on the sk1 physical map. The four late transcripts were derived from a 16 kbp region of the phage limited at one end by cos. The late transcripts were transcribed in the opposite direction to the early transcripts and three of the late transcripts terminated in the same region of the phage genome as three of the early transcripts.

Amino Acid Sequence↗
PubMed1993

Stability analysis of the Lactococcus lactis DRC1 lactose plasmid using pulsed-field gel electrophoresis.

Pulsed-field agarose gel electrophoresis of SmaI digests of genomic DNA was used to examine lactose plasmid copy number and stability in Lactococcus lactis. In L. lactis strain DRC1, the plasmid was found to exist as a single-copy plasmid. Transconjugants of strain HID113 carrying this plasmid were unstable. Variants were isolated with improved phenotypic stability resulting from improved maintenance of the lactose plasmid or from integration of part of the plasmid into the lactococcal chromosome.

Conjugation, Genetic↗
PubMed1993

Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon.

Amino Acid Sequence↗
PubMed1992

Mapping of genes on the linear chromosome of the bacterium Borrelia burgdorferi: possible locations for its origin of replication.

Molecular clones of Borrelia burgdorferi, aetiologic agent of Lyme borreliosis, were isolated and analysed by DNA sequence determination. This procedure yielded B. burgdorferi homologues of gidA, gyrB, gyrA, ftsA and ftsZ. The genes were located on the physical map of the B. burgdorferi linear chromosome. Also mapped were the genes fla and p60 while dnaA was mapped using a heterologous probe. gyrA and gyrB were found to be in tandem and were mapped, along with dnaA at the centre of the chromosome. gidA was located close to the left hand extremity of the chromosome. Because gyrB, dnaA and gidA are normally located within 50 kb of the origin of replication (oriC), we propose two possible sites for oriC in the B. burgdorferi linear chromosome.

Amino Acid Sequence↗
PubMed1992

Phage DNA synthesis and host DNA degradation in the life cycle of Lactococcus lactis bacteriophage c6A.

Bacteriophage c6A is a lytic phage that infects strains of Lactococcus lactis. Infection of L. lactis strain C6 resulted in inhibition of culture growth within 10 min, mature intracellular phage particles appeared after 17.5 min, and cell lysis occurred after 25 min. A culture of strain C6 carrying 3H-labelled DNA was infected with c6A, and the fate of the radiolabel was monitored. The results showed that degradation of host cell DNA began within 6 min of infection and that the breakdown products were incorporated into progeny c6A DNA. Quantitative DNA hybridizations indicated that synthesis of phage DNA began within 6 min of infection and continued at an approximately constant rate throughout the latent period.

Bacteriophages↗