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Cytoplasmic membranes of Bacillus subtilis, grown in complex medium containing glucose, were fractionated into three membrane subfractions [light band (1.155–1.158 g/cm3); medium band (1.181–1.183 g/cm3); heavy band (1.21–1.25 g/cm3)] by sucrose density gradient centrifugation. Among these subfractions, the light and medium bands consisted mainly of membranes but the heavy band consisted of an irregular arrangement or aggregate of small globular protein components of 5–8 nm in diameter. We named this H‐protein. H‐protein formed trilamellar unit membrane structure when combined with lipid. In pulse‐labeling and pulse‐chase experiments with radioactive leucine, it was found that H‐protein consisted of the newest membrane protein synthesized in the cells and the label incorporated into H‐protein was shifted into light and medium band of the membranes during the chase. Cytochromes were not found in H‐protein. However, when H‐protein was incubated with haem α and protohaem, these compounds were incorporated into the apoproteins of the cytochromes present in H‐protein and form cytochromes a and b. Cytochromes were also formed in H‐protein which were isolated from the cells grown in the presence of haemin (haemin‐grown H protein). Succinate dehydrogenase activity was increased about 4‐fold by combining H‐protein or haemin‐grown H‐protein with lipid. H‐protein had no cytochrome oxidase activity; however, haemin‐grown H protein was found to have some of the activity and this was increased about 4‐fold by combining the protein with lipid. Haemin‐grown H protein was also found to form succinate: cytochrome c oxidoreductase when combined with lipid and vitamin K2. On the other hand, succinate oxidase was required for the addition of lipid, vitamin K2 and cytochrome c. NADH oxidase was also found in haemin‐grown H protein and was activated about 9‐fold in constituted reaction systems. Vesicles formed by haemin‐grown H protein and lipid, could accumulate alanine and proline by addition of NADH or reduced phenazine methosulfate. Alanine and proline was also accumulated into the vesicles when transport energy was supplied as a membrane potential introduced by K+‐diffusion via valinomycin. These results would indicate that H‐protein contains the apoprotein of cytochromes, and a carrier involved in the active transport of alanine and proline.

Crossbreeding is an effective approach to construct novel yeast strains with preferred characteristics; however, it is difficult to crossbreed strains of brewer's yeast, especially the bottom-fermenting yeast Saccharomyces pastorianus, because of the relative inefficiency of the available methods to obtain mating-competent cells (MCCs). Here, we describe a productive method for the isolation of MCCs without artificial genetic modification. We focused on the characteristics of two mating pheromone-supersensitive mutants, Δbar1 and Δsst2, that show a growth defect in the presence of the mating pheromone. When MCCs secreting α-factor and a-factor were spotted on to a lawn of MATa Δbar1 and MATα Δsst2, a halo was observed around the respective MCCs. This plate assay was successful in identifying MCCs from bottom-fermenting yeast strains. Furthermore, by selecting for cells that caused the growth defect in pheromone-supersensitive cells on cultures plates, 40 α/α-type and six a/a-type meiotic segregants of bottom-fermenting yeast strains were successfully isolated and crossed with tester strains to verify their mating type. This method of isolation is expected to be applicable to other industrial yeast strains, including wine, sake and distiller's yeasts, and will enable MCCs without genetic modifications to be obtained. As a result, it will be a useful tool for more convenient and efficient crossbreeding of industrial yeast strains that can be applied to practical brewing. Copyright © 2017 John Wiley & Sons, Ltd.

Alanine transport carrier was isolated and purified from H‐proteins of Bacillus subtilis. The purified carrier preparation was homogeneous in migration on polyacrylamide gels containing urea or sodium dodecyl sulfate. Electrophoresis on polyacrylamide gels containing dodecyl sulfate showed a single band of molecular weight of about 7500. 1 mol alanine was bound/mol carrier protein with a dissociation constant of 0.2 μM. The binding was inhibited by p‐chloromercuribenzoate and the inhibition was reversed by dithiothreitol.

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