The post-translationally modified, antimicrobial peptide nisin is secreted by strains of Lactococcus lactis that contain the chromosomally located nisin biosynthetic gene cluster nisABTCIPRKFEG. When a 4-base pair deletion is introduced into the structural nisA gene (⌬nisA), transcription of ⌬nisA is abolished. Transcription of the ⌬nisA gene is restored by adding subinhibitory amounts of nisin, nisin mutants, or nisin analogs to the culture medium, but not by the unmodified precursor peptide or by several other antimicrobial peptides. Upon disruption of the nisK gene, which encodes a putative sensor protein that belongs to the class of twocomponent regulators, transcription of ⌬nisA was no longer inducible by nisin. Fusion of a nisA promoter fragment to the promoterless reporter gene gusA resulted in expression of gusA in L. lactis NZ9800 (⌬nisA) only upon induction with nisin species. The expression level of gusA was directly related to the amount of inducer that was added extracellularly. These results provide insight into a new mechanism of autoregulation through signal transduction in prokaryotes and demonstrate that antimicrobial peptides can exert a second function as signaling molecules.
Characterization of the nisin gene cluster nisABTCIPR of Lactococcus lactis. Requirement of expression of the nisA and nisI genes for development of immunity Kuipers, Oscar P.; Beerthuyzen, Marke M.; Siezen, Roland J.; Vos, Willem M. de
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 promoters in the nisin gene cluster nisABTCIPRKFEG of Lactococcus lactis were characterized by primer extension and transcriptional fusions to the Escherichia coli promoterless -glucuronidase gene (gusA). Three promoters preceding the nisA, nisR, and nisF genes, which all give rise to gusA expression in the nisin-producing strain L. lactis NZ9700, were identified. The transcriptional autoregulation of nisA by signal transduction involving the sensor histidine kinase NisK and the response regulator NisR has been demonstrated previously (O. P. Kuipers, M. M. Beerthuyzen, P. G. G. A. de Ruyter, E. J. Luesink, and W. M. de Vos, J. Biol. Chem. 270: 27299-27304, 1995), and therefore the possible nisin-dependent expression of gusA under control of the nisR and nisF promoters was also investigated. The nisR promoter was shown to direct nisin-independent gusA expression in L. lactis MG1363, which is a nisin-transposon-and plasmid-free strain. L. lactis NZ9800, which does not produce nisin because of a deletion in the nisA gene, containing the nisF-gusA fusion plasmid, gave rise to -glucuronidase production only after induction by nisin. A similar regulation was found in L. lactis NZ3900, which contains a single copy of the nisR and nisK genes but no other genes of the nisin gene cluster. In contrast, when the nisK gene was disrupted, no -glucuronidase activity directed by the nisF promoter could be detected even after induction with nisin. These results show that, like the nisA promoter, the nisF promoter is nisin inducible. The nisF and nisA promoter sequences have significant similarities and contain a conserved region that could be important for transcriptional control.
Many food fermentations are performed using mixed cultures of lactic acid bacteria. Interactions between strains are of key importance for the performance of these fermentations. Yogurt fermentation by Streptococcus thermophilus and Lactobacillus bulgaricus (basonym, Lactobacillus delbrueckii subsp. bulgaricus) is one of the best-described mixed-culture fermentations. These species are believed to stimulate each other's growth by the exchange of metabolites such as folic acid and carbon dioxide. Recently, postgenomic studies revealed that an upregulation of biosynthesis pathways for nucleotides and sulfur-containing amino acids is part of the global physiological response to mixed-culture growth in S. thermophilus, but an in-depth molecular analysis of mixed-culture growth of both strains remains to be established. We report here the application of mixedculture transcriptome profiling and a systematic analysis of the effect of interaction-related compounds on growth, which allowed us to unravel the molecular responses associated with batch mixed-culture growth in milk of S. thermophilus CNRZ1066 and L. bulgaricus ATCC BAA-365. The results indicate that interactions between these bacteria are primarily related to purine, amino acid, and long-chain fatty acid metabolism. The results support a model in which formic acid, folic acid, and fatty acids are provided by S. thermophilus. Proteolysis by L. bulgaricus supplies both strains with amino acids but is insufficient to meet the biosynthetic demands for sulfur and branched-chain amino acids, as becomes clear from the upregulation of genes associated with these amino acids in mixed culture. Moreover, genes involved in iron uptake in S. thermophilus are affected by mixed-culture growth, and genes coding for exopolysaccharide production were upregulated in both organisms in mixed culture compared to monocultures. The confirmation of previously identified responses in S. thermophilus using a different strain combination demonstrates their generic value. In addition, the postgenomic analysis of the responses of L. bulgaricus to mixed-culture growth allows a deeper understanding of the ecology and interactions of this important industrial food fermentation process.Fermented dairy products are typically produced using mixed cultures of lactic acid bacteria, a prominent group of Gram-positive bacteria. Yogurt is milk fermented by the lactic acid bacteria Streptococcus thermophilus and Lactobacillus bulgaricus (basonym, Lactobacillus delbrueckii subsp. bulgaricus). These bacteria stimulate each other's growth and acid production. This mutual stimulation is based on the exchange of growth enhancing metabolites (for a recent review, see reference 30). S. thermophilus is suggested to provide L. bulgaricus with formic acid (12), folic acid (10, 36), and carbon dioxide (14), compounds that are all associated with purine biosynthesis either as precursors or as cofactors. Other metabolic interactions exist at the level of nitrogen metabolism. Typically, the nonproteolytic S. therm...
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