John D. Brooker

Effect of hydrolysable and condensed tannins on growth, morphology and metabolism of Streptococcus gallolyticus (S. caprinus) and Streptococcus bovis

Streptococcus gallolyticus (S. caprinus) was resistant in vitro to at least 7% (w/v) tannic acid and 4% (w/v) acacia condensed tannin, levels 10-fold greater than those tolerated by S. bovis. Growth of S. gallolyticus in liquid medium was characterized by a lag period which increased, and a growth rate which decreased, with increasing tannin concentration. S. gallolyticus was also more tolerant to the presence of simple phenolic acid monomers than was S. bovis, but the lag period was still concentration dependent. Gallate decarboxylase activity in S. gallolyticus was elevated in the presence of tannic acid or gallic acid but not with other phenolic acids. Scanning electron microscopic analysis showed that both the size and shape of S. gallolyticus and S. bovis changed in response to tannin but only S. gallolyticus was surrounded by an extracellular polysaccharide matrix which accumulated in a tannin-concentration-dependent fashion. Washing of the cells to remove extracellular polysaccharide increased the lag period of S. gallolyticus in the presence of 1% (w/v) tannic acid from 4 h to 6 h. In contrast, increasing extracellular polysaccharide synthesis in S. bovis did not increase its tolerance to tannic acid. These data demonstrate that S. gallolyticus has developed a number of mechanisms to reduce the potential effect of tannins on cell growth, and that these mechanisms provide the organism with a selective advantage over S. bovis when grown in the presence of tannins.

Hemin inhibits transfer of pre-δ-aminolevulinate synthase into chick embryo liver mitochondria

Abstract Pulse labelling studies in chick embryo livers show that hemin prevents the transfer of drug induced pre-δ-aminolevulinate synthase from the cytosol into the mitochondria, leading to an accumulation of precursor in the cytosol. No effect of hemin was observed on the transfer of pre-pyruvate carboxylase into mitochondria. These results eliminated a general toxic effect of hemin on mitochondrial import of proteins and are consistent with the view that hemin specifically inhibits the transfer of ALA synthase.

Genetic diversity in ruminal isolates ofSelenomonas ruminantium

Diversity in the ruminal bacterial speciesSelenomonas ruminantium has been investigated by DNA fingerprinting, DNA-DNA hybridization, plasmid analysis, bacteriophage sensitivity, and monoclonal antibody-based immunoassay. Twenty different isolates from the sheep rumen were initially classified morphologically and by carbon source utilization. DNA fingerprint analyses and quantitative genomic DNA hybridizations showed that limited grouping of these isolates was possible, with the largest group comprising four isolates, and two other groups comprising two isolates each. The remaining isolates were unique. Plasmids in four different size classes, 2.5, 3.7, 6.5 and 12.0 kbp, were identified, but these did not appear in all isolates. There was no apparent relationship between DNA fingerprint pattern and plasmid content. Only three isolates were sensitive to theS. ruminantium-specific temperate bacteriophage S-1. These data indicate that substantial genetic diversity exists within the ruminal speciesS. ruminantium, but that at least one strain may represent up to 20% of isolates.

Evidence for a cytosolic precursor of chick embryo liver mitochondrial δ-aminolevulinate synthase

Following the recent demonstration [Borthwick, I.A., Srivastava, G., Brooker, J.D., May, B.K. and Elliott, W.H. (1982) Eur. J. Biochem. in press] that chick embryo liver mitochondrial delta-aminolevulinate synthase has a minimum molecular weight of 68,000 (rather than the hitherto accepted value of 49,000), we have shown that the primary translation product of delta-aminolevulinate synthase mRNA is a protein of molecular weight 74,000. This protein has for the first time been shown to occur in the cytosol fraction of drug-treated chick embryo livers. This form does not occur in mitochondria nor does the smaller mitochondrial form occur in the cytosol. It is concluded that the 74,000 molecular weight protein is a precursor which is processed during transport into the mitochondria. In vivo labelling experiments are consistent with this conclusion.

Gene knockout of the intracellular amylase gene by homologous recombination in Streptococcus bovis.

Abstract.Streptococcus bovis expresses two different amylases, one intracellular and the other secreted. A suicide vector containing part of the intracellular α-amylase gene from Streptococcus bovis WI-1 was recombined into the S. bovis WI-1 chromosome to disrupt the endogenous gene. Recombination was demonstrated by Southern blot, and zymogram analysis confirmed the loss of the intracellular amylase. Amylase activity in cell-free extracts of the recombinant grown in the presence of 1% starch was only 7% of wild type. The rate of logarithmic growth of the recombinant was 15–20% of the wild type in medium containing either 1% glucose, starch, or cellobiose. Revertants and non-amylase control recombinants had logarithmic growth rates that were the same as wild type. Plasmid transformants containing multiple copies of the cloned gene expressed up to threefold higher levels of intracellular amylase activity than wild type but did not demonstrate elevated growth rates. These results suggest that a critical level of expression of the intracellular amylase gene may be important for rapid growth of the bacterium.

Purification of rat liver mitochondrial δ-aminolaevulinate synthase

Abstract Mitochondrial δ-aminolaevulinate synthase was purified from drug-induced adult rat liver and identified for the first time as a protein of minimum molecular weight 70,000. The enzyme was also identified in mitochondria by pulse-labelling and immunoprecipitation and shown to have a molecular weight of 70,000. The purified enzyme was degraded by papain to smaller forms of molecular weight about 56,000 with no loss of enzyme activity. In vitro translation experiments suggest that the enzyme is synthesized initially as a larger precursor of molecular weight 76,000.

Effect of heme on the activity of chick embryo liver mitochondrial δ-aminolevulinate synthase

We have examined the effect of heme on the activity of native δ‐aminolevulinate synthase isolated from drug‐induced chick embryo liver mitochondria. The enzyme was not inhibited by concentrations of heme up to 1nM and this finding makes it improbable that heme acts physiologically to control mitochondrial δ‐aminolevulinate synthase activity.

Lysogenic bacteriophage M1 from Selenomonas ruminantium: isolation, characterization and DNA sequence analysis of the integration site

Bacteriophage M1 from the ruminal bacterium Selenomonas ruminantium strain ML12 comprises a 30 nm icosahedral capsid, a 25 nm tail and 48 kb of linear dsDNA with cohesive ends. A restriction map of the phage genome has been constructed. The presence of bacteriophage M1 in the rumen has been demonstrated by PCR amplification and Southern blot analysis of DNA from rumen bacterial samples obtained from ten different sheep. Lysogeny was demonstrated by hybridization of M1 DNA to host chromosomal DNA and by identification and cloning of a 2.3 kb region of the phage containing the predicted attP domain which promotes chromosomal integration. DNA sequencing of the attP region demonstrated two major ORFs surrounding the predicted attP site and structural analysis of this region revealed a motif comprising three different inverted repeats surrounding a 12 bp palindrome. Analysis of the translated amino acid sequence upstream of the attP site demonstrated the presence of conserved residues found within integrase proteins of several temperate phages of different bacterial species.