The chemical composition of the cell wall of Sz. pombe is known as b-1,3-glucan, b-1,6-glucan, a-1,3-glucan and a-galactomannan; however, the three-dimensional interactions of those macromolecules have not yet been clarified. Transmission electron microscopy reveals a three-layered structure: the outer layer is electron-dense, the adjacent layer is less dense, and the third layer bordering the cell membrane is dense. In intact cells of Sz. pombe, the high-resolution scanning electron microscope reveals a surface completely filled with a-galactomannan particles. To better understand the organization of the cell wall and to complement our previous studies, we set out to locate the three different types of b-glucan by immuno-electron microscopy. Our results suggest that the less dense layer of the cell wall contains mainly b-1,6-branched b-1,3-glucan. Occasionally a line of gold particles can be seen, labelling fine filaments radiating from the cell membrane to the a-galactomannan layer, suggesting that some of the radial filaments contain b-1,6-branched b-1,3-glucan. b-1,6-glucan is preferentially located underneath the a-galactomannan layer. Linear b-1,3-glucan is exclusively located in the primary septum of dividing cells. b-1,6-glucan only labels the secondary septum and does not co-localize with linear b-1,3-glucan, while b-1,6-branched b-1,3-glucan is present in both septa. Linear b-1,3-glucan is present from early stages of septum formation and persists until the septum is completely formed; then just before cell division the label disappears. From these results we suggest that linear b-1,3-glucan is involved in septum formation and perhaps the separation of the two daughter cells. In addition, we frequently found b-1,6-glucan label on the Golgi apparatus, on small vesicles and underneath the cell membrane. These results give fresh evidence for the hypothesis that b-1,6-glucan is synthesized in the endoplasmic reticulum-Golgi system and exported to the cell membrane.

The classification feast͞famine regulatory proteins (FFRPs) encompasses archaeal DNA-binding proteins with Escherichia coli transcription factors, the leucine-responsive regulatory protein and the asparagine synthase C gene product. In this paper, we describe two forms of the archaeal FFRP FL11 (pot0434017), both assembled from dimers. When crystallized, a helical cylinder is formed with six dimers per turn. In contrast, in solution, disks are formed, most likely consisting of four dimers each; an observation by cryoelectron microscopy. Whereas each dimer binds a 13-bp sequence, different forms will discriminate between promoters, based on the numbers of repeating 13-bp sequences, and types of linkers inserted between them, which are either of 7-8 or Ϸ18 bp. The amino acid sequences of these FFRPs are designed to form the same type of 3D structures, and the transition between their assembly forms is regulated by interaction with small molecules. These considerations lead us to propose a possible mechanism for regulating a number of genes by varying assembly forms and by combining different FFRPs into these assemblies, responding to environmental changes.

Transcriptional repressor FL11 from the hyperthermophilic archaeon, Pyrococcus OT3, was crystallized in its dimer form in complex with a DNA duplex, TGAAAWWWTTTCA. Chemical contacting of FL11 to the terminal 5 bps, and DNA bending by propeller twisting at WWW confirmed specificity of the interaction. Dimer-binding sites were identified in promoters of approximately 200 transcription units coding, for example, H+-ATPase and NAD(P)H dehydrogenase. In the presence of lysine, four FL11 dimers were shown to assemble into an octamer, thereby covering the fl11 promoter. In the "feast" mode, when P. OT3 grows on amino acids, the FL11 octamer will terminate transcription of fl11, as was shown in vitro, thereby derepressing transcription of many metabolic genes. In the "famine" mode in the absence of lysine, approximately 6000 FL11 dimers present per cell will arrest growth. This regulation resembles global regulation by Escherichia coli leucine-responsive regulatory protein, and hints at a prototype of transcription regulations now highly diverged.