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PubMed1995

Genes involved in immunity to the lantibiotic nisin produced by Lactococcus lactis 6F3.

The lantibiotic nisin is produced by several strains of Lactococcus lactis. The complete gene cluster for nisin biosynthesis in L. lactis 6F3 comprises 15 kb of DNA. As described previously, the structural gene nisA is followed by the genes nisB, nisT, nisC, nisI, nisP, nisR, and nisK. Further analysis revealed three additional open reading frames, nisF, nisE, and nisG, adjacent to nisK. Approximately 1 kb downstream of the nisG gene, three open reading frames in the opposite orientation have been identified. One of the reading frames, sacR, belongs to the sucrose operon, indicating that all genes belonging to the nisin gene cluster of L. lactis 6F3 have now been identified. Proteins NisF and NisE show strong homology to members of the family of ATP-binding cassette (ABC) transporters, and nisG encodes a hydrophobic protein which might act similarly to the immunity proteins described for several colicins. Gene disruption mutants carrying mutations in the genes nisF, nisE, and nisG were still able to produce nisin. However, in comparison with the wild-type strain, these mutants were more sensitive to nisin. This indicates that besides nisI the newly identified genes are also involved in immunity to nisin. The NisF-NisE ABC transporter is homologous to an ABC transporter of Bacillus subtilis and the MbcF-MbcE transporter of Escherichia coli, which are involved in immunity to subtilin and microcin B17, respectively.

ATP-Binding Cassette Transporters↗

PubMed1996

Structure-activity relationships in the peptide antibiotic nisin: role of dehydroalanine 5.

A mutant of the peptide antibiotic nisin in which the dehydroalanine residue at position 5 has been replaced by an alanine has been produced and structurally characterized. It is shown to have activity very similar to that of wild-type nisin in inhibiting growth of Lactococcus lactis and Micrococcus luteus but is very much less active than nisin as an inhibitor of the outgrowth of spores of Bacillus subtilis. These observations, which parallel those of W. Liu and J. N. Hansen (Appl. Environ. Microbiol. 59:648-651, 1993) on the corresponding mutant of the related antibiotic subtilin, are discussed in terms of the mechanism(s) of action of these antibiotics.

Alanine↗

PubMed1993

Antibiotics synthesized by posttranslational modification.

Peptides that have antimicrobial activity are synthesized by many prokaryotic and eukaryotic organisms. Antimicrobial peptides commonly contain unusual amino acids that contribute to their properties and functions. Although bacteria synthesize most of these peptides by nonribosomal mechanisms, this review focuses on those that are synthesized by pathways that involve posttranslational modification of ribosomally synthesized precursor peptides. A particularly interesting class of these antimicrobial peptides is the lantibiotics, of which nisin and subtilin are the longest-known examples, although nearly a dozen new lantibiotics have been discovered in recent years. The fact that the lantibiotic structures are derived from gene-encoded peptides means that structural analogs of natural lantibiotics can be constructed by mutagenesis of their structural genes. Recent advances in our understanding of the molecular genetics of lantibiotics has made the construction of novel lantibiotics with enhanced chemical and antimicrobial properties possible. This review describes these advances and proposes future trends of research, as well as potential application of engineered lantibiotics, in the context of the general field of antimicrobial peptides.

Amino Acid Sequence↗

PubMed1995

Cyclic AMP modulates the rate of 'constitutive' exocytosis of apical membrane proteins in Madin-Darby canine kidney cells.

Madin-Darby canine kidney and other epithelial cell lines (e.g. Caco-2, MCF-10A and MCF-7) develop intracellular vacuoles composed of apical membrane displaying microvilli (VACs) when impaired from forming normal cell-to-cell contacts. In a previous publication, we showed that VACs are rapidly exocytosed upon treatment with 8-Br-3',5'-cyclic adenosine monophosphate (8-Br-cAMP), a membrane-permeable analog of cAMP, and that this exocytosis correlates with variations in the cellular cAMP concentration in response to the cell-cell contacts. In the present work, we tested the hypothesis that cAMP may be a positive modulator of the 'constitutive' exocytic pathway. To mimic conditions in cells with incomplete intercellular contacts, the intracellular levels of cAMP were decreased by means of two independent approaches: (i) pores were induced in the plasma membrane with the polypeptidic antibiotic subtilin, thus allowing small molecules (including cAMP) to permeate and move out of the cytoplasm; and (ii) adenylate cyclase and protein kinase A were blocked with specific inhibitors. In all cases, the intracellular levels of cAMP were measured and, in porated cells, equilibrated to simulate the corresponding physiological intracellular concentrations. The decrease in cAMP within the physiological range resulted in a decreased rate of transport of an apical marker of the constitutive pathway (influenza virus hemagglutinin) from the trans-Golgi network to the apical plasma membrane. Likewise, the delivery of a number of cellular apical proteins to the plasma membrane was retarded at low cAMP concentrations. The inhibitors of adenylate cyclase failed to block basolateral delivery of vesicular stomatitis virus G protein. This differential modulatory effect may represent a differentiation-dependent control of the insertion of apical membrane in epithelial cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

Molecular biology of antibiotic production in Bacillus.

Several species of the genus Bacillus produce peptide antibiotics which are synthesized either through a ribosomal or non-ribosomal mechanism. The antibiotics gramicidin, tyrocidine, and bacitracin are synthesized nonribosomally by the multienzyme thiotemplate mechanism. Surfactin and mycobacillin are also synthesized nonribosomally but by a mechanism that, apparently, is distinct from that of the multienzyme thiotemplate. Other antibiotics such as subtilin are gene encoded and are ribosomally synthesized. Molecular genetic and DNA sequence analysis have shown that biosynthesis genes for some antibiotics are clustered into polycistronic transcription units and are under the control of global regulatory systems that govern the expression of genes that are induced when Bacillus cells enter stationary phase of growth. Future experiments involving the molecular dissection of peptide antibiotic biosynthesis genes in Bacillus will be attempted in hopes of further examining the mechanism and regulation of antibiotic production.

Amino Acid Sequence↗

PubMed1992

Engineering dehydrated amino acid residues in the antimicrobial peptide nisin.

The small antimicrobial peptide nisin, produced by Lactococcus lactis, contains the uncommon amino acid residues dehydroalanine and dehydrobutyrine and five thio ether bridges. Since these structures are posttranslationally formed from Ser, Thr, and Cys residues, it is feasible to study their role in nisin function and biosynthesis by protein engineering. Here we report the development of an expression system for mutated nisin Z (nisZ) genes, using nisin A producing L. lactis as a host. Replacement by site-directed mutagenesis of the Ser-5 codon in nisZ by a Thr codon, led to a mutant with a dehydrobutyrine instead of a dehydroalanine residue at position 5, as shown by NMR. Its antimicrobial activity was 2-10-fold lower relative to wild-type nisin Z, depending on the indicator strain used. In another mutagenesis study a double mutation was introduced in the nisZ gene by replacing the codons for Met-17 and Gly-18 by codons for Gln and Thr, respectively, as in the third lanthionine ring of the related antimicrobial peptide subtilin from Bacillus subtilis. This resulted in the simultaneous production of two mutant species, one containing a Thr residue and the other containing a dehydrobutyrine residue at position 18, both having different bacteriocidal properties.

Amino Acid Sequence↗

PubMed1988

Structure, expression, and evolution of a gene encoding the precursor of nisin, a small protein antibiotic.

We have cloned and sequenced a gene (spaN) from Streptococcus lactis ATCC 11454 which encodes the peptide precursor of the small protein antibiotic nisin. The encoded precursor is 57 amino acids long, with a 23-residue leader region and a 34-residue structural region. The structural region contains serines, threonines, and cysteines at exactly the positions required to give mature nisin by a series of post-translational modifications involving dehydration of serines and threonines to dehydro forms, and cross-linking with cysteine residues. S1 mapping revealed a 267-nucleotide transcript of the nisin gene that is expressed during vegetative growth and stationary phase. It has a half-life of 7-10 min. The absence of an identifiable promoter or rho-independent terminator and the detection of two different 5'-ends of the transcript suggested it is a processing product from a larger RNA. This may represent a polycistronic mRNA which may also encode proteins involved in processing the nisin precursor peptide. Open reading frames were found in regions flanking the nisin gene. The one downstream had a ribosome binding site and appeared to be transcribed by read-through from the nisin gene. The one upstream had significant homology to a putative transposase from the Escherichia coli IS2 insertion element. Comparison of gene sequence homologies between nisin and the other lanthionine antibiotics, subtilin and epidermin, indicated that they all evolved from a common ancestor. Corresponding leader peptide sequences showed mediocre amino acid homology, but nearly perfect hydropathic homologies, suggesting a common function. It is proposed that this function includes recognition signals or other information required for post-translational processing.

Amino Acid Sequence↗

PubMed1994

Site-directed mutagenesis of conserved serines in rat histidase. Identification of serine 254 as an essential active site residue.

We have identified serine 254 as an essential residue in rat histidase (histidine ammonia-lyase, EC 4.3.1.3). Histidase and phenylalanine ammonia-lyase are the only two enzymes that have been postulated to require the modified amino acid, dehydroalanine, for enzyme activity. In the bacterial peptides nisin and subtilin, and in the pyruvoyl enzymes, the precursor for dehydroalanine is a serine. To determine whether serine may be the dehydroalanine precursor in rat histidase, we substituted four highly conserved serines with alanines, and expressed the mutated histidase cDNAs in COS cells, which have no endogenous histidase activity. Substitution of serines 223, 254, 508, and 533 resulted in the production of approximately equal amounts of histidase protein with histidase activities of 2.4, 0.0, 75, and 16%, respectively. The abrogation of histidase activity by the substitution of alanine for serine 254, together with the modification by L-cysteine of the corresponding residue in Pseudomonas putida histidase (Hernandez, C., Stroh, J. G., and Phillips, A. T. (1993) Arch. Biochem. Biophys. 307, 126-132), is strong evidence that this residue is the precursor of the essential electrophilic moiety of histidase.

Amino Acid Sequence↗

PubMed1996

Genetic determinants for the biosynthesis of nisin, a bacteriocin produced by Lactococcus lactis.

In the past, the genetic determinants for nisin biosynthesis were thought to be plasmid-located. However, it has been shown that production of nisin, immunity to nisin, and other properties such as the fermentation of sucrose, are encoded on 70 kb conjugative transposons that are chromosomally located. The extrachromosomal location of the nisin genes has not been substantiated by experiments that unequivocally show plasmid transfer. Two natural variants of nisin have been identified, nisin A and nisin Z, encoded by the genes nisA and nisZ, respectively. Both genes have been cloned and sequenced and differ only in a single base pair. Approximately 12 kb downstream from the structural gene has been cloned and sequenced, and a further 10 genes involved in the biosynthesis of nisin have been identified. The nisB and nisC gene products are involved in nisin maduration, the nisT in its secretion and the nisP in its processing. The nisR and nisK gene products have a regulatory role and the nisI, nisF, nisE and nisG are involved in immunity to nisin. All these genes display significant homology to the corresponding genes of the related lantibiotics subtilin and epidermin.

Amino Acid Sequence↗