The complete DNA sequence of the yeast Saccharomyces cerevisiae chromosome XI has been determined. In addition to a compact arrangement of potential protein coding sequences, the 666,448-base-pair sequence has revealed general chromosome patterns; in particular, alternating regional variations in average base composition correlate with variations in local gene density along the chromosome. Significant discrepancies with the previously published genetic map demonstrate the need for using independent physical mapping criteria.

Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a non-fermentable carbon source leads to selective and rapid degradation of fructose-1,6-bisphosphatase. This so called catabolite inactivation of the enzyme is brought about by the ubiquitin-proteasome system. To identify additional components of the catabolite inactivation machinery, we isolated three mutant strains, gid1, gid2, and gid3, defective in glucose-induced degradation of fructose-1,6-bisphosphatase. All mutant strains show in addition a defect in catabolite inactivation of three other gluconeogenic enzymes: cytosolic malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase. These findings indicate a common mechanism for the inactivation of all four enzymes. The mutants were also impaired in degradation of short-lived N-end rule substrates, which are degraded via the ubiquitin-proteasome system. Sitedirected mutagenesis of the amino-terminal proline residue yielded fructose-1,6-bisphosphatase forms that were no longer degraded via the ubiquitin-proteasome pathway. All amino termini other than proline made fructose-1,6-bisphosphatase inaccessible to degradation. However, the exchange of the amino-terminal proline had no effect on the phosphorylation of the mutated enzyme. Our findings suggest an essential function of the amino-terminal proline residue for the degradation process of fructose-1,6-bisphosphatase. Phosphorylation of the enzyme was not necessary for degradation to occur.

Bacillus subtilis ATCC 6633 produces the cationic pore-forming lantibiotic subtilin, which preferentially acts on gram-positive microorganisms; self protection of the producer cells is mediated by the four genes spaIFEG. To elucidate the mechanism of subtilin autoimmunity, we transferred different combinations of subtilin immunity genes under the control of an inducible promoter into the genome of subtilin-sensitive host strain B. subtilis MO1099. Recipient cells acquired subtilin tolerance through expression of either spaI or spaFEG, which shows that subtilin immunity is based on two independently acting systems. Cells coordinately expressing all four immunity genes acquired the strongest subtilin protection level. Quantitative in vivo peptide release assays demonstrated that SpaFEG diminished the quantity of cell-associated subtilin, suggesting that SpaFEG transports subtilin molecules from the membrane into the extracellular space. Homology and secondary structure analyses define SpaFEG as a prototype of lantibiotic immunity transporters that fall into the ABC-2 subfamily of multidrug resistance proteins. Membrane localization of the lipoprotein SpaI and specific interaction of SpaI with the cognate lantibiotic subtilin suggest a function of SpaI as a subtilin-intercepting protein. This interpretation was supported by hexahistidine-mediated 0-Å cross-linking between hexahistidinetagged SpaI and subtilin.Bacillus subtilis strain ATCC 6633 produces the cationic peptide antibiotic (lantibiotic) subtilin. Lantibiotics contain unusual thioether amino acids, such as meso-lanthionine and 3-methyl-lanthionine (17), which are incorporated into prepeptides through extensive posttranslational modifications (25,32,41). The subtilin and the closely related ericin gene clusters (35) encompass genes for posttranslational modification (18), transport (18), immunity (20), and regulation (19). Extracellular B. subtilis serine proteases are involved in the final processing step (7, 37). Subtilin biosynthesis and immunity are under the control of the two-component regulatory system SpaK/ SpaR (histidine kinase and response regulator, respectively) and the alternative sigma factor H (36, 38).Lantibiotics act against a wide range of gram-positive bacteria. The antimicrobial action of nisin produced by Lactococcus lactis, a structurally close relative of subtilin, is based on voltage-dependent pore formation that affects the efflux of small molecules and finally the collapse of the proton motive force (for a review see reference 4). The Bacto prenol-bound peptidoglycan precursor lipid II appears to be both a docking molecule assisting membrane targeting (5) and an integral constituent of the lethal pore itself (14). Gram-positive lantibiotic-producing strains need efficient countermeasures to obviate the lethal action of their own products (31). The nisin self protection (immunity) system is composed of ABC transporter homologue NisFEG and lipoprotein NisI (39).In the present study we report on the establishment of subtilin immunit...

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Eight different Bacillus subtilis strains and Bacillus atrophaeus were found to produce the bacteriocin subtilosin A. On the basis of the subtilosin gene (sbo) sequences two distinct classes of B. subtilis strains were distinguished, and they fell into the two B. subtilis subspecies (B. subtilis subsp. subtilis and B. subtilis subsp. spizizenii). The entire sequence of the subtilosin gene cluster of a B. subtilis subsp. spizizenii strain, B. subtilis ATCC 6633, was determined. This sequence exhibited a high level of homology to the sequence of the sbo-alb gene locus of B. subtilis 168. By using primer extension analysis the transcriptional start sites of sbo in B. subtilis strains ATCC 6633 and 168 were found to be 47 and 45 bp upstream of the sbo start codon, respectively. Our results provide insight into the incipient evolutionary divergence of the two B. subtilis subspecies.Almost 4% of the 4.2-Mbp Bacillus subtilis 168 genome codes for proteins similar to the proteins involved in the biosynthesis of antimicrobial metabolites (17). However, B. subtilis 168 produces only a few antibiotics because several of the biosynthetic pathways are not functional, most likely because of the X-ray mutation of the original Marburg strain (6). In contrast, various other B. subtilis wild-type strains produce characteristic cocktails of numerous peptide antibiotics (1, 18). For example, a well-established bioindicator strain for sterilization control, ATCC 6633 (11), was investigated with respect to biosynthesis of the lantibiotic subtilin (4,8,16,27) and its regulation (26,28). In a series of B. subtilis strains production of the nonribosomally synthesized cyclic lipopeptides surfactin, fengycin, and the iturins, including mycosubtilin, with different compositions has been observed (9,18,20,31).Subtilosin is a macrocyclic bacteriocin with three intramolecular bridges (14,19). An acidic isoelectric point differentiates subtilosin from the basic lantibiotics (13, 24). Subtilosin transcription is increased under oxygen-limited and anaerobic conditions (22; T. Stein, S. Düsterhus, A. Stroh, and K.-D. Entian, 10th Int. Conf. Bacilli, abstr. P103, p. 65, 1999). The production of mature subtilosin is based on the expression of the sbo-alb gene cluster encompassing the subtilosin structural gene sbo and genes involved in posttranslational modification and processing of presubtilosin and in immunity (34, 35).Here we describe subtilosin production by eight different B. subtilis wild-type strains and Bacillus atrophaeus. The sbo genes of these organisms, as well as the entire subtilosin gene cluster of B. subtilis ATCC 6633, were sequenced in order to analyze the genetic variation between B. subtilis wild-type strains. MATERIALS AND METHODSStrains and plasmids. Strains used in this work are listed in Table 1. Recombinant plasmids were amplified in Escherichia coli DH5␣ or TG1 grown in Luria-Bertani medium (GIBCO, Neu-Isenburg, Germany). B. subtilis was grown either on TY (0.8% tryptone, 0.5% yeast extract [Difco, Detroit, Mich.], 0.5% N...