ABSTRACT
To improve the production of the lantibiotic subtilin in Bacillus subtilis ATCC 6633, two genetic engineering strategies were followed. Firstly, additional copies of subtilin self-protection (immunity) genes spaIFEG have been integrated into the genome of the producer strain. Their expression significantly enhanced the subtilin tolerance level, and concomitantly, the subtilin yield 1.7-fold. Secondly, a repressor of subtilin gene expression, the B. subtilis general transition state regulator protein AbrB, was deleted. A sixfold enhancement of the subtilin yield could be achieved with the abrB deletion mutant; however, the produced subtilin fraction predominantly consists of succinylated subtilin species with less antimicrobial activity compared to unmodified subtilin.
Subject(s)
Bacillus subtilis/genetics , Bacteriocins/biosynthesis , ATP-Binding Cassette Transporters/genetics , Anti-Bacterial Agents/biosynthesis , Anti-Bacterial Agents/chemistry , Anti-Bacterial Agents/pharmacology , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Bacteriocins/chemistry , Bacteriocins/genetics , Bacteriocins/pharmacology , DNA-Binding Proteins/genetics , Drug Tolerance , Gene Deletion , Gene Dosage , Genetic Engineering , Genome, Bacterial , Lipoproteins/genetics , Membrane Proteins/genetics , Peptides/chemistry , Peptides/genetics , Peptides/pharmacology , Succinic Acid/analysis , Transcription Factors/geneticsABSTRACT
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-A cross-linking between hexahistidine-tagged SpaI and subtilin.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/genetics , Peptides/genetics , Amino Acid Sequence , Bacterial Proteins/immunology , Bacteriocins , Base Sequence , DNA Primers , Escherichia coli/genetics , Genes, Bacterial , Molecular Sequence Data , Peptides/immunology , Plasmids , Polymerase Chain Reaction , Sequence Alignment , Sequence Homology, Amino AcidABSTRACT
The subtilin gene cluster (spa) of Bacillus subtilis ATCC 6633 is organized in transcriptional units spaBTC, spaS, spaIFEG and spaRK. Specific binding of the response regulator protein SpaR to spaB, spaS and spaI DNA promoter fragments was shown by means of electromobility shift assays. A repeated pentanucleotide sequence spaced by six nucleotides was identified as SpaR binding motif (spa-box). Saturating mutational analysis of the spa-box by single- and multiple-base-pair substitutions revealed the consensus motif (A/T)TGAT for optimal SpaR binding with the second, third and fifth position being absolutely conservative. Variations in the spacer size between the two pentanucleotide repeats revealed a strong conservation of their relative location. Only DNA with a proximal arrangement of two pentanucleotide repeats showed affinity to SpaR. A 2:1 stoichiometry between SpaR and DNA was obtained by optical biosensor analyses, which corresponds to the binding of two SpaR proteins per spa-box.
Subject(s)
Anti-Bacterial Agents/biosynthesis , Bacillus subtilis/metabolism , Peptides , Transcriptional Activation , Amino Acid Sequence , Anti-Bacterial Agents/metabolism , Bacillus subtilis/immunology , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Bacteriocins , Binding Sites , Biosensing Techniques , DNA Mutational Analysis/methods , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Lipoproteins/genetics , Lipoproteins/metabolism , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Optics and Photonics , Regulatory Sequences, Nucleic Acid , Repetitive Sequences, Nucleic Acid , Sequence Homology, Amino Acid , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
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 NisFEG, 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 hexahistidine-tagged NisI from Escherichia coli interacted specifically with nisin and not with subtilin. This suggests a function of NisI as a nisin-intercepting protein.
Subject(s)
Bacillus subtilis/genetics , Bacterial Proteins/metabolism , Lactococcus lactis/genetics , Lipoproteins/metabolism , Membrane Proteins , ATP-Binding Cassette Transporters/genetics , ATP-Binding Cassette Transporters/immunology , ATP-Binding Cassette Transporters/metabolism , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/pharmacology , Bacillus subtilis/drug effects , Bacillus subtilis/immunology , Bacillus subtilis/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/immunology , Biological Transport/physiology , Cell Fractionation , Drug Resistance, Bacterial , Gene Expression Regulation, Bacterial , Genes, Bacterial , Lactococcus lactis/drug effects , Lactococcus lactis/immunology , Lipoproteins/genetics , Lipoproteins/immunology , Multigene Family , Nisin/genetics , Nisin/metabolism , Nisin/pharmacology , Operon , Promoter Regions, Genetic , Subtilisin/genetics , Subtilisin/metabolism , Subtilisin/pharmacology , Transcription, GeneticABSTRACT
The production of the peptide antibiotic (lantibiotic) subtilin in Bacillus subtilis ATCC 6633 is highly regulated. Transcriptional organization and regulation of the subtilin gene cluster encompassing 11 genes was characterized. Two polycistronic mRNAs encoding transcript spaBTC (6.8 kb) and encoding transcript spaIFEG (3.5 kb) as well as the monocistronic spaS (0.3 kb) mRNA were shown by Northern hybridization. Primer extension experiments and beta-galactosidase fusions confirmed three independent promoter sites preceding genes spaB, spaS and spaI. beta-Galactosidase expression of spaB, spaS and spaI promoter lacZ fusions initiated in mid-exponential growth. Maximal activities were reached at the transition to stationary growth and were collinear with subtilin production. The lacZ activity was dependent on co-expression with the two-component regulatory system spaRK. The presence of subtilin was needed for efficient expression of all three promoter lacZ fusions. This suggests a transcriptional autoregulation according to a quorum-sensing mechanism with subtilin as autoinducer and signal transduction via SpaRK. Additionally, spaR expression was found to be under positive control of the alternative sigma factor H. Deletion of sigma H strongly decreased subtilin production. Full subtilin production could be restored after in-trans complementation of spaR. Deletion of the major B. subtilis transition state regulator AbrB strongly increased subtilin production. These results show that the spaRK two-component regulatory system, and hence subtilin biosynthesis and immunity, is under dual control of two independent regulatory systems: autoinduction via subtilin and transcriptional regulation via sigma factor H.
