Nisin-producing Lactococcus lactis strains show a high degree of resistance to the action of nisin, which is based upon expression of the self-protection (immunity) genes nisI, nisF, nisE, and nisG. Different combinations of nisin immunity genes were integrated into the chromosome of a nisin-sensitive Bacillus subtilis host strain under the control of an inducible promoter. For the recipient strain, the highest level of acquired nisin tolerance was achieved after coordinated expression of all four nisin immunity genes. But either the lipoprotein NisI or the ABC transporter-homologous system Nis-FEG, respectively, were also able to protect the Bacillus host cells. The acquired immunity was specific to nisin and provided no tolerance to subtilin, a closely related lantibiotic. Quantitative in vivo peptide release assays demonstrated that NisFEG diminished the quantity of cell-associated nisin, providing evidence that one role of NisFEG is to transport nisin from the membrane into the extracellular space. NisI solubilized from B. subtilis membrane vesicles and recombinant hexahistidinetagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.In recent years peptide antibiotics have gained increasing attention as therapeutics (1, 2) and food preservatives (3). Nisin represents the most prominent member of lantibiotics, peptide antibiotics with intramolecular lanthionine bridges (4 -9). The nisin producer Lactococcus lactis 6F3 contains a gene cluster encoding proteins for the biosynthesis and transport (10 -14), immunity (15), and regulation (16 -18) of nisin. Subtilin (19,20) and ericin S (21) produced by Bacillus subtilis ATCC 6633 and A1/3, respectively, are closely related lantibiotics. Lantibiotics form voltage-dependent pores in the bacterial cytoplasmic membrane that are lethal for the target cells but also for the producer. For nisin, the mode of action was investigated in several model systems such as black lipid bilayers and membrane vesicles (22)(23)(24)(25). Recent findings demonstrated that specific binding of nisin to the cell wall precursor lipid II coincides with pore formation (26, 27). Specific selfprotection (immunity) mechanisms are necessary to protect the lantibiotic-producing organisms from the action of their own lantibiotics. For nisin and subtilin (28) Although numerous genes involved in lantibiotic immunity are known, the mechanism by which the encoded proteins confer immunity remain unclear. For full nisin or subtilin immunity, both are required, i.e. the lipoprotein as well as the immunity transporter. The lack of each component diminished the tolerance to nisin (35) or subtilin (28) significantly. Here we report for the first time on the establishment of nisin immunity in the heterologous host B. subtilis. Functional analyses of its different components provided evidence that NisI acts as a nisin-sequestering protein and that NisFEG acts as a nisin exporter that expels nisin molecules fro...

A 3-5 kb EcoRl-BamHI fragment of Baci//us subtilis chromosomal DNA carrying the ribR gene, involved in regulation of the B. subtilis riboflavin operon, was cloned in the B. subtilis-€scherichia coli shuttle vector pCB2O. DNA sequence analysis of this fragment revealed several ORFs, one of which encodes a polypeptide of 230 amino acids with up to 45% sequence identity with FAD synthetases from a number of micro-organisms, such as Corynebacterium ammoniagenes, E. coli and Pseudomonas fluorescens, and also to the ribC gene product of B. subtilis. The ribR gene was amplified by PCR, cloned and expressed in E. coli. Measurement of f lavokinase activity in cell extracts demonstrated that ribR encodes a monof unctional f lavokinase which converts riboflavin into FMN but not to FAD, and is specific for the reduced form of riboflavin.Keywords : Bacillus subtilis, flavinogenesis, riboflavin, operon regulation, flavokinase INTRODUCTIONThe Bacillus subtilis riboflavin operon comprises a cluster of five non-overlapping genes that encode the enzymes which catalyse the reactions for de novo riboflavin biosynthesis starting from GTP. The operon is located at 209" on the B. subtilis genetic map. The functional organization of the riboflavin operon has been described in detail in several articles (Perkins & Pero, 1993;Mironov et al., 1994). The expression of the operon is negatively regulated by the product of the ribC gene, which is situated at 147" on the B. subtilis chromosome and appears to encode a flavin-activated aporepressor. A possible repressor binding site is situated in a region of 294 bp between the promoter and the first structural gene of the operon (Kil et al., 1992). Mutations in this region designated rib0 mutations are cis-dominant and, like ribC mutations, have a phenotype of riboflavin overproduction, resulting in accumulation of the vitamin in the growth medium. While the exact mechanism of regulation is still un- The EMBL accession number for the sequence referred t o in this paper is Y09721.known (Azevedo et al., 1993) it is evident that the regulatory system of the operon is not limited to the ri6C gene and the rib0 regulatory region. We have examined B. subtilis mutants which carry the constitutive ri6Cl mutation and are partially resistant to the riboflavin analogue 7,8-dimethyl-l0-( O-methylacetoxime)-isoalloxazine (MO), in which the ribityl moiety is replaced with -CH,-CH = N-O-CH, (Fig. 1). A number I. M. S O L O V I E V A a n d OTHERSof these mutants had completely lost the ability to oversynthesize riboflavin but retained the original ri6Cl mutation in the chromosome (Kreneva & Perumov, 1994). This was interpreted as the restoration of regulatory activity due to a mutation at a previously unknown locus, resulting in expression of a product that could also regulate expression of the riboflavin operon of B. subtilis. This effect is only seen in ri6C constitutive mutants and not in the background of rib0 regulatory mutations.Two mutations which decrease the constitutive expression of the B. subtil...

The riboflavin kinase encoding gene ribR is situated within a 12 genes locus ytmI-ytnM of the Bacillus subtilis chromosome. Here we demonstrate that ribR is transcribed as part of a 10 kb ytmI-ytnM operon. The riboflavin overproduction phenotype of B. subtilis ribC mutant strains, which is a result of the strongly reduced flavokinase activity of the riboflavin kinase/FAD synthetase RibC, was suppressed by ribR expression. Analysis of mutations with an upregulated ribR gene revealed 2 different groups of mutants. One class of mutants contained base substitutions in an 8 nucleotide sequence of the promoter region of the ytmI-ytnM operon. A second class of mutants had single point mutations within the yrzC gene or in the RBS of this gene. Dot-blot analysis of ytmI-ytnM transcription and the results of in trans complementation experiments for the yrzC mutants confirmed a role of the yrzC gene product as a negative regulator for the ytmI-ytnM operon.