Structure, organization and characterization of the gene cluster involved in the production of microcin E492, a channel-forming bacteriocin.

Microcin E492 is a low-molecular-weight, channel-forming bacteriocin produced and excreted by Klebsiella pneumoniae RYC492. A 13 kb chromosomal DNA fragment from K. pneumoniae RYC492 was sequenced, and it was demonstrated by random Tn5 mutagenesis that most of this segment, which has at least 10 cistrons, is needed for the production of active microcin and its immunity protein. Genes mceG and mceH correspond to an ABC exporter and its accessory protein, respectively, and they are closely related to the colicin V ABC export system. The microcin E492 system also requires the product of gene mceF as an additional factor for export. Despite the fact that this bacteriocin lacks post-translational modifications, genes mceC, mceI and mceJ are needed for the production of active microcin. Genes mceC and mceI are homologous to a glycosyl transferase and acyltransferase, respectively, whereas mceJ has no known homologue. Mutants in these three genes secrete an inactive form of microcin, able to form ion channels in a phospholipidic bilayer, indicating that the mutation of these microcin genes does not alter the process of membrane insertion. On the other hand, microcin isolated from mutants in genes mceC and mceJ has a lethal effect when incubated with spheroplasts of sensitive cells, indicating that the microcin defects in these mutants are likely to alter receptor recognition at the outer membrane. A model for synthesis and export is proposed as well as a novel maturation pathway that would involve conformational changes to explain the production of active microcin E492.

Gap junctions in the chicken pineal gland.

The chicken pineal gland, which contains a heterogeneous cell population, sustains a circadian rhythm of activity. Synchronization of cellular activity of heterogeneous cells might be facilitated by gap junctional intercellular channels which are permeable to ions and second messengers. To test this possibility, we looked for morphologically identifiable gap junctions between the different pineal cells, used antibodies and cDNA probes to screen for the presence of connexins, and tested for functional intercellular coupling. By transmission electron microscopy and immunocytochemistry, gap junctions and connexins were observed between pinealocyte cell bodies, stromal cells, astrocytes, and astrocyte and pinealocyte processes. Two gap junctional proteins, connexin43 and connexin45, were detected by immunocytochemistry, immunoblotting and RNA blot analysis. Functional intercellular coupling was observed in the gland by transfer of low molecular weight dyes. Dye transferred between homologous and heterologous cells. These data suggest that homologous and heterologous gap junctions may provide a mechanism for coordination of the cellular responses of the elements of the biological clock which are induced by lighting cues to produce the circadian rhythm of pineal activity.