A two-component ribonucleotidyl transferase from E. coli

Some properties of an enzyme designated as a two-component ribonucleotidyl transferase from E. coli are presented. The enzyme in the presence of magnesium ions catalyzes the synthesis of polyribonucleotide chains using all four nucleoside triphosphates as substrates. The enzyme consists of two components; component A in the presence of Mg²⁺ catalyzes the synthesis of homo- and heteropolymers using ATP, CTP and UTP but not GTP as substrates. Component B itself does not catalyze any synthesis at all, but its addition to component A affects this component in two ways: quantitatively—the activity of component A considerably increases, and qualitatively—both components together are capable of catalyzing the synthesis of polyribonucleotides consisting of all four ribonucleotides.     

The two-component system ScnRK of Streptococcus mutans affects hydrogen peroxide resistance and murine macrophage killing

To survive macrophage killing is critical in the pathogenesis of viridians streptococci-induced infective endocarditis (IE). Streptococcus mutans, an opportunistic IE pathogen, generally does not survive well phagocytic killing in murine macrophage RAW 264.7 cells. A putative two-component system (TCS), ScnR/ScnK from S. mutans, was investigated to elucidate the mechanisms underlying bacteria-cellular interaction in this study. Both the wild-type and mutant strains were phagocytosed by RAW 264.7 cells at a comparable rate and an increased intracellular susceptibility during a 5 h incubation period was observed with the scnRK-null mutants. The amount of reactive oxygen species (ROS) in activated macrophages was reduced significantly after ingesting wild-type, but not scnRK-null mutant strains, suggesting that increased macrophage killing of these mutants is due to the impaired ability of S. mutans to counteract ROS. Additionally, both scnR- or scnRK-null mutants were more susceptible to hydrogen peroxide. Interestingly, scnRK expression was unaffected by hydrogen peroxide. These experimental results indicate that scnRK is important in counteracting oxidative stress in S. mutans, and decreased susceptibility to phagocytic killing is at least partly attributable to inhibition of intracellular ROS formation.     

Investigating the importance of charged residues in lantibiotics

Lantibiotics are antimicrobial peptides which can have a broad spectrum activity against many Gram positive pathogens. Many of these peptides contain charged amino acids which may be of critical importance with respect to antimicrobial activity. We have recently carried out an in-depth bioengineering based investigation of the importance of charged residues in a representative two peptide lantibiotic, lacticin 3147, and here we discuss the significance of these findings in the context of other lantibiotics and cationic antimicrobial peptides.     

A two-component theory of encephalization in mammals

The evolutionary increase in relative brain size in mammals is shown to be correlated with an addition of neuronal elements to the cerebral cortex. From a theory of encephalization, based on these findings, it appears that the number of modules (or neurons) in the cerebral cortex associated with higher order brain functions at the same time depends upon the size of the animal. This supports the thesis that the two major components, which determine the volume of the brain, viz. a body size related component and a non-somatic evolutionary component, are inter-related.     

Centriole duplication: A lesson in self-control

In interphase and mitosis, centrosomes play a major role in the spatial organization of the microtubule network. Alterations in centrosome number and structure are associated with genomic instability and occur in many cancers. Centrosome duplication is controlled by centriole replication. In most dividing animal cells, centrioles duplicate only once per cell cycle at a site adjacent to existing centrioles. The conserved protein kinase Polo-like kinase 4 (Plk4) has a key role in controlling centriole biogenesis. Overexpression of Plk4 drives centrosome amplification and is associated with tumorigenesis in flies. By contrast, haploinsufficiency of Plk4 promotes cytokinesis failure, leading to an increased incidence of tumors in mice. Recent studies have shown that Plk4 is a low abundance protein whose stability is linked to the activity of the enzyme. We discuss how this autoregulatory feedback loop acts to limit the damaging effects caused by too much or too little Plk4.Key words: centrosome, centriole, polo-like kinase 4, Plk4, SAK, SCF, phosphodegron, β-TrCP, aneuploidyCentrosomes are the major microtubule organizing centers of animal cells and play a particularly important role during mitosis where they organize the two opposite poles of the bipolar microtubule spindle apparatus upon which chromosomes are segregated. Although centrosomes are not strictly essential for the formation of the mitotic/meiotic spindle, whenever they are present they play a dominant role in guiding spindle formation.^1,2 Extra copies of centrosomes frequently result in errors in spindle assembly that give rise to chromosome missegregation and the production of aneuploid daughter cells.^3,4 Almost one hundred years ago, Theodor Boveri proposed that centrosome amplification can contribute to tumorigenesis.⁵ Since then supernumerary centrosomes have been reported in a variety of different tumor cells in vitro and in vivo and are a consistent feature of aneuploid tumors.^6–13 However, despite the large body of circumstantial evidence linking extra centrosomes to the development of cancer, it remains unclear whether supernumerary centrosomes actively contribute to tumorigenesis or arise as a byproduct of cellular transformation.     

Cross-reactivity of bacteriocins from lactic acid bacteria and lantibiotics in a nisin bioassay and ELISA

A number of bacteriocins from lactic acid bacteria and lantibiotics were tested for cross-reactivity in a nisin ELISA and bioassay. The bacteriocins showed no cross-reactivity, reflecting their structural dissimilarity from nisin. The lantibiotic subtilin which shares many common structural features with nisin, showed a high cross-reactivity in both the ELISA and the bioassay suggesting possible modifications to nisin to enhance its activity. Gallidermin did not cross react in the ELISA but did produce a zone of inhibition in the less specific bioassay. Other lantibiotics tested did not react in either assay.     

Lichenicidin rational site-directed mutagenesis library: A tool to generate bioengineered lantibiotics

Lantibiotics are ribosomally synthesized and posttranslationally modified antimicrobial peptides that arise as an alternative to the traditional antibiotics. Lichenicidin is active against clinically relevant bacteria and it was the first lantibiotic to be fully produced in vivo in the Gram-negative host Escherichia coli. Here, we present the results of a library of lichenicidin mutants, in which the mutations were generated based on the extensive bibliographical search available for other lantibiotics. The antibacterial activity of two-peptide lantibiotics, as is lichenicidin, requires the synergistic activity of two peptides. We established a method that allows screening for bioactivity which does not require the purification of the complementary peptide. It is an inexpensive, fast and user-friendly method that can be scaled up to screen large libraries of bioengineered two-peptide lantibiotics. The applied system is reliable and robust because, in general, the results obtained corroborate structure–activity relationship studies carried out for other lantibiotics.