Biosynthesis of the lantibiotic peptide nisin by Lactococcus lactis NIZO R5 relies on the presence of the conjugative transposon TnS276 in the chromosome. A 12-kb DNA fragment of T M276 including the nisA gene and about 10 kb of downstream DNA was cloned in L. lactis, resulting in the production of an extracellular nisin precursor peptide. This peptide reacted with antibodies against either nisin A or the synthetic leader peptide, suggesting that it consisted of a fully modified nisin with the nisin leader sequence still attached to it. This structure was confirmed by N-terminal sequencing and 'H-nuclear magnetic resoqnnce analysis of the purified peptide. Deletion studies showed that the nisR gene is essential for the production of this intermediate. The deduced amino acid sequence of the nisR gene product indicated that the protein belongs to the family of two-component regulators. The deduced amino acid sequence of NisP, the putative product of the gene upstream of nisR, showed an N-terminal signal sequence, a catalytic domain with a high degree of similarity to those of subtilisin-like serine proteases, and a putative C-terminal membrane anchor. Cell extracts of Escherichia coli overexpressing nisP were able to cleave the nisin precursor peptide, producing active, mature nisin. A similar activation was obtained with whole cells but not with membrane-free extracts ofL. lactis strains carrying TnS276 in which the nisA gene had been inactivated. The results indicate that the penultimate step in nisin biosynthesis is secretion of precursor nisin without cleavage of the leader peptide, whereas the last step is the cleavage of the leader peptide sequence from the fully maturated nisin peptide.Nisin is a 34-residue antibacterial peptide that is produced by several strains of Lactococcus lactis and strongly inhibits the growth of a broad range of gram-positive bacteria (24). Insertion of the peptide into the cytoplasmic membrane of the target cell renders the membrane permeable to monovalent cations (18) and dissipates the proton motive force of the cell (18, 37). The mature peptide displays several unusual features, such as the dehydrated residues dehydroalanine and dehydrobutyrine, which are derived from Ser and Thr residues, respectively (22, 24), and lanthionine and P-methyllanthionine residues, which form five intramolecular thioether bridges. Molecular structures similar to those in mature nisin are found in several other antibacterial peptides secreted, e.g., by Bacillus subtilis (subtilin) (21) or Staphylococcus epidermidis (epidermin and Pep5) (2, 42), together forming the group of the lantibiotics (47; for a review, see reference 25).Recently, it was reported that two widely distributed natural variants of nisin exist, named nisin A and nisin Z, which differ in a single amino acid residue (11,36). The structural genes for both nisin A, nisA, and nisin Z, nisZ, have been cloned and sequenced elsewhere (5,14,26,36).These studies showed that both nisin variants are ribosomally synthesized as 57-amino-acid pre...

The mitochondrial matrix enzyme manganese superoxide dismutase (SOD) of Saccharomyces cerevisiae is encoded in the nucleus. It is synthesized as a precursor with an NH2‐terminal extension of 26 amino acids which is cleaved off during import into the mitochondrion. Fusions between the NH2‐terminal 34 amino acids of SOD and the cytosolic proteins invertase of yeast and mouse dihydrofolate reductase (DHFR) were tested for in vitro binding and import into mitochondria. Efficient translocation over the mitochondrial membranes takes place in the case of the SOD‐DHFR fusion. The SOD‐invertase fusion protein does not get translocated and binds to the organelle with only low efficiency. Yeast transformants harbouring the SOD‐invertase fusion gene accumulate approximately 95% of the hybrid protein in the cytosol. The remaining material is found in the interior of the mitochondrion, loosely attached to the inner membrane. We conclude that the pre‐sequence of SOD is able to deliver a passenger protein to the mitochondrion. The efficiency of protein delivery and translocation across the membrane is, however, influenced by the passenger protein.

Genes coding for the 40 kilodaltons (kDa), 17-kDa, 14-kDa and 11-kDa subunits of the ubiquinol-cytochrome c reductase in yeast are present in single copies in the haploid genome. We have mapped each gene to a unique genomic environment and demonstrate that integration of cloned segments into nuclear DNA by homologous crossing-over with the endogenous gene results in the replacement of the corresponding chromosomal restriction fragment by fragments of predicted sizes. Chromosomal mapping, carried out by the procedure of Falco and Botstein 1983, indicates that the gene for the 17-kDa subunit lies on chromosome VI and that for the 11-kDa subunit on chromosome XII.

Southern blot analysis of genomic DNA and gene-cloning experiments have recently shown that the human ZP3 gene is not a single-copy gene. The human genome harbors sequences encoding a protein of 424 amino acids and, in addition, a polymorphic locus with the potential to give rise to a probably nonfunctional polypeptide of 372 residues. In this report it is shown, by screening of a panel of human × hamster hybrid cell lines, that both the ZP3 and ZP3P loci are located on human chromosome 7.