Die Verteilung der loeslichen lytischen Transglycosylase in Escherichia coli und ihre Rolle bei der Bakterienlyse durch den Phagen MS2 Elektronenmikroskopische und physiologische Untersuchungen

Available from TIB Hannover: DW 3502 / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEDEGerman

Specific localization of the lysis protein of bacteriophage MS2 in membrane adhesion sites of Escherichia coli.

Specific localization of the lysis (L) protein of bacteriophage MS2 in the cell wall of Escherichia coli was determined by immunoelectron microscopy. After induction of the cloned lysis gene, the cells were plasmolyzed, fixed, and embedded in either Epon or Lowicryl K4M. Polyclonal L-protein-specific antiserum was purified by preabsorption to membranes from cells harboring a control plasmid. Protein A-gold was used to label the protein-antibody complexes. Between 42.8% (Lowicryl) and 33.8% (Epon) of the label was found in inner and outer membranes, but 30.3% (Lowicryl) and 32.8% (Epon) was present mostly in clusters in the adhesion sites visible after plasmolysis. The remaining label (26.9 and 33.4%, respectively) appeared to be present in the periplasmic space but may also have been part of membrane junctions not visible because of poor contrast of the specimen. In contrast, a quite different distribution of the L protein was found in cells grown under conditions of penicillin tolerance, i.e., at pH 5, a condition that had previously been shown to protect cells from L-protein-induced lysis. At tolerant conditions, only 21.0% of the L protein was in the adhesion sites; most of the protein (68.2%) was found in inner and outer membranes. It is concluded that lysis of the host, E. coli, was a result of the formation of specific L-protein-mediated membrane adhesion sites

Subcellular distribution of the soluble lytic transglycosylase in Escherichia coli.

The localization of the major autolytic enzyme, the soluble lytic transglycosylase, in the different cell compartments of Escherichia coli was investigated by immunoelectron microscopy. Ultrathin sections were labeled with a specific antiserum against purified soluble lytic transglycosylase, and the antibody-enzyme complexes were visualized with colloidal protein A-gold. A preferential localization of the lytic transglycosylase in the envelope was observed, with only 20 to 30% of the enzyme left in the cytoplasm. Most of the enzyme associated with the cell wall was tightly bound to the murein sacculus. Sacculi prepared by boiling of cells in 4% sodium dodecyl sulfate could be immunolabeled with the specific antiserum, indicating a surprisingly strong interaction of the lytic transglycosylase with murein. The enzyme-substrate complex could be reconstituted in vitro by incubating pronase-treated, protein-free murein sacculi with purified lytic transglycosylase at 0 degrees C. Titration of sacculi with increasing amounts of enzyme indicated a limiting number of binding sites for about 1,000 molecules of enzyme per sacculus. Ruptured murein sacculi obtained after penicillin treatment revealed that the enzyme is exclusively bound to the outer surface of the sacculus. This finding is discussed in the light of recent evidence suggesting that the murein of E. coli might be a structure of more than one layer expanding by inside-to-outside growth of patches of murein

On the embryonic origin of adult melanophores: the role of ErbB and Kit signalling in establishing melanophore stem cells in zebrafish

Pigment cells in vertebrates are derived from the neural crest (NC), a pluripotent and migratory embryonic cell population. In fishes, larval melanophores develop during embryogenesis directly from NC cells migrating along dorsolateral and ventromedial paths. The embryonic origin of the melanophores that emerge during juvenile development in the skin to contribute to the striking colour patterns of adult fishes remains elusive. We have identified a small set of melanophore progenitor cells (MPs) in the zebrafish (Danio rerio, Cyprinidae) that is established within the first 2 days of embryonic development in close association with the segmentally reiterated dorsal root ganglia (DRGs). Lineage analysis and 4D in vivo imaging indicate that progeny of these embryonic MPs spread segmentally, giving rise to the melanophores that create the adult melanophore stripes. Upon depletion of larval melanophores by morpholino knockdown of Mitfa, the embryonic MPs are prematurely activated; their progeny migrate along the spinal nerves restoring the larval pattern and giving rise to postembryonic MPs associated with the spinal nerves. Mutational or chemical inhibition of ErbB receptors blocks all early NC migration along the ventromedial path, causing a loss of DRGs and embryonic MPs. We show that the sparse like (slk) mutant lacks larval and metamorphic melanophores and identify kit ligand a (kitlga) as the underlying gene. Our data suggest that kitlga is required for the establishment or survival of embryonic MPs. We propose a model in which DRGs provide a niche for the stem cells of adult melanophores