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Karldieter Entian - One of the best experts on this subject based on the ideXlab platform.

  • specificity of Subtilin mediated activation of histidine kinase spak
    Applied and Environmental Microbiology, 2017
    Co-Authors: Christoph Geiger, Tobias Spies, Peter Kotter, Sophie Marianne Korn, Karldieter Entian
    Abstract:

    Autoinduction via two-component systems is a widespread regulatory mechanism that senses environmental and metabolic changes. Although the lantibiotics nisin and Subtilin are closely related and share the same lanthionine ring structure, they autoinduce their biosynthesis in a highly specific manner. Subtilin activates only the two-component system SpaRK of Bacillus subtilis, whereas nisin activates solely the two-component system NisRK of Lactococcus lactis To identify components that determine the specificity of Subtilin autoinduction, several variants of the respective lantibiotics were analyzed for their autoinductive capacities. Here, we show that amino acid position 20 is crucial for SpaK activation, as an engineered nisin molecule with phenylalanine at position 20 (nisin N20F) was able to activate SpaK in a specific manner. In combination with the N-terminal tryptophan of Subtilin (nisin I1W/N20F), SpaK autoinduction reached almost the level of Subtilin-mediated autoinduction. Furthermore, the overall structure of Subtilin is also important for its association with the histidine kinase. The destruction of the second lanthionine ring (Subtilin C11A, ring B), as well as mutations that interfere with the flexibility of the hinge region located between lanthionine rings C and D (Subtilin L21P/Q22P), abolished SpaK autoinduction. Although the C-terminal part of Subtilin is needed for efficient SpaK autoinduction, the destruction of lanthionine rings D and E had no measurable impact. Based on these findings, a model for the interaction of Subtilin with histidine kinase SpaK was established.IMPORTANCE Although two-component systems are important regulatory systems that sense environmental changes, very little information on the molecular mechanism of sensing or the interaction of the sensor with its respective kinase is available. The strong specificity of linear lantibiotics such as Subtilin and nisin for their respective kinases provides an excellent model system to unravel the structural needs of these lantibiotics for activating histidine kinases in a specific manner. More than that, the biosyntheses of lantibiotics are autoinduced via two-component systems. Therefore, an understanding of their interactions with histidine kinases is needed for the biosynthesis of newly engineered peptide antibiotics. Using a Bacillus subtilis-based reporter system, we were able to identify the molecular constraints that are necessary for specific SpaK activation and to provide SpaK specificity to nisin with just two point mutations.

  • autoinduction specificities of the lantibiotics Subtilin and nisin
    Applied and Environmental Microbiology, 2015
    Co-Authors: Tobias Spies, Peter Kotter, Sophie Marianne Korn, Karldieter Entian
    Abstract:

    The biosynthesis of the lantibiotics Subtilin and nisin is regulated by autoinduction via two-component systems. Although Subtilin is structurally closely related to nisin and contains the same lanthionine ring structure, both lantibiotics specifically autoinduce their biosynthesis. Subtilin and also the Subtilin-like lantibiotics entianin and ericin autoinduce the two-component system SpaRK of Bacillus subtilis, whereas the biosynthesis of nisin is autoinduced via the two-component system NisRK of Lactococcus lactis. Autoinduction is highly specific for the respective lantibiotic and therefore of major importance for the functional expression of genetically engineered Subtilin-like lantibiotics. To identify the structural features required for Subtilin autoinduction, Subtilin-nisin hybrids and specific point mutations of amino acid position 1 were generated. For Subtilin autoinduction, the N-terminal tryptophan is the most important for full SpaK activation. The failure of Subtilin to autoinduce the histidine kinase NisK mainly depends on the N-terminal tryptophan, as its single exchange to the aliphatic amino acid residues isoleucine, leucine, and valine provided NisK autoinduction. In addition, the production of Subtilin variants which did not autoinduce their own biosynthesis could be rescued upon heterologous coexpression in B. subtilis DSM15029 by the autoinducing Subtilin-like lantibiotic entianin.

  • synthesis and succinylation of Subtilin like lantibiotics are strongly influenced by glucose and transition state regulator abrb
    Applied and Environmental Microbiology, 2015
    Co-Authors: Sophie Bochmann, Peter Kotter, Tobias Spies, Karldieter Entian
    Abstract:

    Subtilin and the closely related entianin are class I lantibiotics produced by different subspecies of Bacillus subtilis. Both molecules are ribosomally synthesized peptide antibiotics with unusual ring structures. Subtilin-like lantibiotics develop strong antibiotic activities against various Gram-positive organisms with an efficiency similar to that of nisin from Lactococcus lactis. In contrast to nisin, Subtilin-like lantibiotics partially undergo an additional posttranslational modification, where the N-terminal tryptophan residue becomes succinylated, resulting in drastically reduced antibiotic activities. A highly sensitive high-performance liquid chromatography (HPLC)-based quantification method enabled us to determine entianin and succinylated entianin (S-entianin) concentrations in the supernatant during growth. We show that entianin synthesis and the degree of succinylation drastically change with culture conditions. In particular, increasing glucose concentrations resulted in higher entianin amounts and lower proportions of S-entianin in Landy-based media. In contrast, no succinylation was observed in medium A with 10% glucose. Interestingly, glucose retarded the expression of entianin biosynthesis genes. Furthermore, deletion of the transition state regulator AbrB resulted in a 6-fold increased entianin production in medium A with 10% glucose. This shows that entianin biosynthesis in B. subtilis is strongly influenced by glucose, in addition to its regulation by the transition state regulator AbrB. Our results suggest that the mechanism underlying the succinylation of Subtilin-like lantibiotics is enzymatically catalyzed and occurs in the extracellular space or at the cellular membrane.

  • entianin a novel Subtilin like lantibiotic from bacillus subtilis subsp spizizenii dsm 15029t with high antimicrobial activity
    Applied and Environmental Microbiology, 2011
    Co-Authors: Sebastian W Fuchs, Torsten Stein, Michael Karas, Thorsten W Jaskolla, Sophie Bochmann, Peter Kotter, Thomas A Wichelhaus, Karldieter Entian
    Abstract:

    Lantibiotics, such as nisin and Subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and Subtilin autoinduction are available. In this report, we used the previously reported Subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel Subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029T. Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from Subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated Subtilin, B. subtilis DSM 15029T secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated Subtilin (S-Subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-Subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.

  • maturation and processing of spai the lipoprotein involved in Subtilin immunity in bacillus subtilis atcc 6633
    Microbiological Research, 2010
    Co-Authors: Torsten Stein, Prakash M Halami, A Chandrashekar, Karldieter Entian
    Abstract:

    SpaI is a small lipoprotein that provides Bacillus subtilis with autoimmunity against the lantibiotic Subtilin. We have investigated the maturation of SpaI through the lipoprotein biosynthesis pathway, and analyzed the consequences of maturations in the acylation of the target lipobox in Subtilin immunity. Further specificity of lipid acylation of the cysteine within the conserved sequence of the candidate lipobox (LSAC) was studied by site-directed mutagenesis. The mutants LSAA and LSCA blocked lipid attachment to the SpaI protein. Cell-wall stress-sensing B. subtilis BSF 2470 was exploited to study the function of each mutant upon heterologous expression. This system allowed the monitoring of β-galactosidase activity to the added Subtilin at a sublethal dose. Mutants exhibited 2-fold reduction in β-Gal activity, suggesting their contribution in Subtilin autoimmunity.

Torsten Stein - One of the best experts on this subject based on the ideXlab platform.

  • Oxygen-Limiting Growth Conditions and Deletion of the Transition State Regulator Protein Abrb in Bacillus subtilis 6633 Result in an Increase in Subtilosin Production and a Decrease in Subtilin Production
    Probiotics and Antimicrobial Proteins, 2019
    Co-Authors: Torsten Stein
    Abstract:

    It has been recently shown, that certain strains/isolates of Bacillus subtilis can be used as a probiotic for humans. The production of the macrocyclic sactibiotic subtilosin in B. subtilis ATCC 6633 is highly regulated. To improve the subtilosin productivity of B. subtilis , different growth conditions were compared for maximal expression of the sbo promoter that regulates the expression of the subtilosin biosynthetic gene cluster. Oxygen-limiting conditions led to a strong increase of sbo promoter activities compared to aerobic conditions, and accordingly, the subtilosin amount determined by reversed phase HPLC (7.8 mg/L) was 15-fold superior to the amount of aerobic grown cultures (0.5 mg/L). A further promising enhancement of the subtilosin yield was achieved using a deletion mutant that is avoiding the general transition state regulator protein AbrB. The subtilosin titer of 42 mg/L produced by Δ abrB cells grown under oxygen-limiting conditions corresponds to an 84-fold increase compared to the subtilosin titer obtained from B. subtilis wild type cells propagated in aerobic conditions. Furthermore, evidence is provided that oxygen-limiting conditions led to a strong decrease in the productivity of the lantipeptide Subtilin suggesting contrary regulatory mechanisms for the B. subtilis antimicrobials Subtilin and subtilosin.

  • entianin a novel Subtilin like lantibiotic from bacillus subtilis subsp spizizenii dsm 15029t with high antimicrobial activity
    Applied and Environmental Microbiology, 2011
    Co-Authors: Sebastian W Fuchs, Torsten Stein, Michael Karas, Thorsten W Jaskolla, Sophie Bochmann, Peter Kotter, Thomas A Wichelhaus, Karldieter Entian
    Abstract:

    Lantibiotics, such as nisin and Subtilin, are lanthionine-containing peptides that exhibit antimicrobial as well as pheromone-like autoinducing activity. Autoinduction is specific for each lantibiotic, and reporter systems for nisin and Subtilin autoinduction are available. In this report, we used the previously reported Subtilin autoinduction bioassay in combination with mass spectrometric analyses to identify the novel Subtilin-like lantibiotic entianin from Bacillus subtilis subsp. spizizenii DSM 15029T. Linearization of entianin using Raney nickel-catalyzed reductive cleavage enabled, for the first time, the use of tandem mass spectrometry for the fast and efficient determination of an entire lantibiotic primary structure, including posttranslational modifications. The amino acid sequence determined was verified by DNA sequencing of the etnS structural gene, which confirmed that entianin differs from Subtilin at 3 amino acid positions. In contrast to B. subtilis ATCC 6633, which produces only small amounts of unsuccinylated Subtilin, B. subtilis DSM 15029T secretes considerable amounts of unsuccinylated entianin. Entianin was very active against several Gram-positive pathogens, such as Staphylococcus aureus and Enterococcus faecalis. The growth-inhibiting activity of succinylated entianin (S-entianin) was much lower than that of unsuccinylated entianin: a 40-fold higher concentration was required for inhibition. For succinylated Subtilin (S-Subtilin), a concentration 100-fold higher than that of unsuccinylated entianin was required to inhibit the growth of a B. subtilis test strain. This finding was in accordance with a strongly reduced sensing of cellular envelope stress provided by S-entianin relative to that of entianin. Remarkably, S-entianin and S-Subtilin showed considerable autoinduction activity, clearly demonstrating that autoinduction and antibiotic activity underlie different molecular mechanisms.

  • maturation and processing of spai the lipoprotein involved in Subtilin immunity in bacillus subtilis atcc 6633
    Microbiological Research, 2010
    Co-Authors: Torsten Stein, Prakash M Halami, A Chandrashekar, Karldieter Entian
    Abstract:

    SpaI is a small lipoprotein that provides Bacillus subtilis with autoimmunity against the lantibiotic Subtilin. We have investigated the maturation of SpaI through the lipoprotein biosynthesis pathway, and analyzed the consequences of maturations in the acylation of the target lipobox in Subtilin immunity. Further specificity of lipid acylation of the cysteine within the conserved sequence of the candidate lipobox (LSAC) was studied by site-directed mutagenesis. The mutants LSAA and LSCA blocked lipid attachment to the SpaI protein. Cell-wall stress-sensing B. subtilis BSF 2470 was exploited to study the function of each mutant upon heterologous expression. This system allowed the monitoring of β-galactosidase activity to the added Subtilin at a sublethal dose. Mutants exhibited 2-fold reduction in β-Gal activity, suggesting their contribution in Subtilin autoimmunity.

  • development and application of a microtiter plate based autoinduction bioassay for detection of the lantibiotic Subtilin
    Journal of Microbiological Methods, 2007
    Co-Authors: Michael Burkard, Karldieter Entian, Torsten Stein
    Abstract:

    Production of the lantibiotic Subtilin in Bacillus subtilis ATCC 6633 is regulated in a quorum sensing-like mechanism with Subtilin acting as autoinducer and signal transduction via the Subtilin-specific two-component regulation system SpaRK. Here, we report the construction and application of a Subtilin reporter strain in which Subtilin induced lacZ gene expression in a B. subtilis ATCC 6633 spaS gene deletion mutant is monitored and visualized by the beta-galactosidase in a chromogenic plate assay. A quantitative microtiter plate Subtilin bioassay was developed and optimized. Maximal sensitivity of the system was achieved after 6 h of incubation of the reporter strain together with Subtilin in a medium containing 300 mM NaCl. This sensitive and unsusceptible method was applied to identify Subtilin producing B. subtilis wild type strains from both, culture collections and soil samples. The B. subtilis lantibiotic ericin S with four amino acid exchanges compared to Subtilin induces the Subtilin reporter strain, in contrast to the structurally closely related Lactococcus lactis lantibiotic nisin. These observations suggest a certain substrate specificity of the histidine kinase SpaK, which however, also would allow the identification of Subtilin-isoform producing microorganisms.

  • structure function relationships of the lanthionine cyclase spac involved in biosynthesis of the bacillus subtilis peptide antibiotic Subtilin
    Biochemistry, 2007
    Co-Authors: Markus Helfrich, Karldieter Entian, Torsten Stein
    Abstract:

    Biosynthesis of the lantibiotic Subtilin in Bacillus subtilis is accomplished by a synthetase complex consisting of the dehydratase SpaB, cyclase SpaC, and transporter SpaT. Genetically engineered Subtilin cyclases SpaC and related NisC and EriC proteins involved in biosynthesis of the lantibiotics nisin and ericin A/S, respectively, were analyzed to functionally substitute native SpaC in vivo. We could show for the first time posttranslational modification of a lantibiotic precursor peptide (Subtilin) by a hybrid lantibiotic synthetase (SpaBT/EriC). Genetically engineered SpaC alanine replacement mutants revealed the essentiality of residues His231, Trp302, Cys303, Tyr304, Gly305, Cys349, and His350, as well as the conserved C-terminal motif Lys437-Ala438-Leu439-Leu440-Ile441 for Subtilin biosynthesis. Assignment of these strictly conserved lantibiotic cyclase residues to the NisC structure [Li, B., Yu, J. B., Brunzelle, J. S., Moll, G. N., van der Donk, W. A., and Nair, S. K. (2006) Science, 311, 1464−1...

J N Hansen - One of the best experts on this subject based on the ideXlab platform.

  • identification and characterization of the structural and transporter genes for and the chemical and biological properties of sublancin 168 a novel lantibiotic produced by bacillus subtilis 168
    Journal of Biological Chemistry, 1998
    Co-Authors: Sun H Paik, A Chakicherla, J N Hansen
    Abstract:

    An antimicrobial peptide produced by Bacillus subtilis 168 was isolated and characterized. It was named sublancin 168, and its behavior during Edman sequence analysis and its NMR spectrum suggested that sublancin is a dehydroalanine-containing lantibiotic. A hybridization probe based on the peptide sequence was used to clone the presublancin gene, which encoded a 56-residue polypeptide consisting of a 19-residue leader segment and a 37-residue mature segment. The mature segment contained one serine, one threonine, and five cysteine residues. Alkylation of mature sublancin showed no free sulfhydryl groups, suggesting that one sulfydryl had formed a beta-methyllanthionine bridge with a dehydrobutyrine derived by posttranslational modification of threonine; with the other four cysteines forming two disulfide bridges. It is unprecedented for a lantibiotic to contain a disulfide bridge. The sublancin leader was similar to known type AII lantibiotics, containing a double-glycine motif that is typically recognized by dual-function transporters. A protein encoded immediately downstream from the sublancin gene possessed features of a dual-function ABC transporter with a proteolytic domain and an ATP-binding domain. The antimicrobial activity spectrum of sublancin was like other lantibiotics, inhibiting Gram-positive bacteria but not Gram-negative bacteria; and like the lantibiotics nisin and Subtilin in its ability to inhibit both bacterial spore outgrowth and vegetative growth. Sublancin is an extraordinarily stable lantibiotic, showing no degradation or inactivation after being stored in aqueous solution at room temperature for 2 years. The fact that sublancin is a natural product of B. subtilis 168, for which a great deal of genetic information is available, including the entire sequence of its genome, suggests that sublancin will be an especially good model for studying the potential of lantibiotics as sources of novel biomaterials.

  • use of alkaline phosphatase as a reporter polypeptide to study the role of the Subtilin leader segment and the spat transporter in the posttranslational modifications and secretion of Subtilin in bacillus subtilis 168
    Applied and Environmental Microbiology, 1997
    Co-Authors: G Izaguirre, J N Hansen
    Abstract:

    The Subtilin leader segment of preSubtilin was fused to alkaline phosphatase (AP), which was used as a reporter polypeptide to study the role of the Subtilin leader segment in posttranslational modifications during the conversion of preSubtilin to Subtilin and in the translocation of preSubtilin from the cytoplasm of Bacillus subtilis 168 to the extracellular medium. It was observed that the Subtilin leader segment could be utilized by a wild-type transporter, but secretion was enhanced if the Subtilin transporter was available. The Subtilin leader was not cleaved away from the AP component of the precursor until the precursor had been transported to the cell wall, and none of the AP was released into the medium until after cleavage had occurred. The role of SpaT, which is an ABC transporter that has been implicated in Subtilin secretion, was explored by making a large in-frame deletion from the central region of SpaT and observing the effect on translocation of the AP reporter. Instead of having an effect on translocation, the deletion disrupted proteolytic cleavage of the Subtilin leader segment and release of the AP reporter into the medium. The AP that was secreted by means of the Subtilin leader segment had not undergone any posttranslational modifications, as assessed by amino acid composition analysis and enzymatic activity analysis.

  • characterization of a chimeric prou operon in a Subtilin producing mutant of bacillus subtilis 168
    Journal of Bacteriology, 1995
    Co-Authors: Yuhmei Lin, J N Hansen
    Abstract:

    The ability to respond to osmotic stress by osmoregulation is common to virtually all living cells. Gram-negative bacteria such as Escherichia coli and Salmonella typhimurium can achieve osmotolerance by import of osmoprotectants such as proline and glycine betaine by an import system encoded in an operon called proU with genes for proteins ProV, ProW, and ProX. In this report, we describe the discovery of a proU-type locus in the gram-positive bacterium Bacillus subtilis. It contains four open reading frames (ProV, ProW, ProX, and ProZ) with homology to the gram-negative ProU proteins, with the B. subtilis ProV, ProW, and ProX proteins having sequence homologies of 35, 29, and 17%, respectively, to the E. coli proteins. The B. subtilis ProZ protein is similar to the ProW protein but is smaller and, accordingly, may fulfill a novel role in osmoprotection. The B. subtilis proU locus was discovered while exploring the chromosomal sequence upstream from the spa operon in B. subtilis LH45, which is a Subtilin-producing mutant of B. subtilis 168. B. subtilis LH45 had been previously constructed by transformation of strain 168 with linear DNA from B. subtilis ATCC 6633 (W. Liu and J. N. Hansen, J. Bacteriol. 173:7387-7390, 1991). Hybridization experiments showed that LH45 resulted from recombination in a region of homology in the proV gene, so that the proU locus in LH45 is a chimera between strains 168 and 6633. Despite being a chimera, this proU locus was fully functional in its ability to confer osmotolerance when glycine betaine was available in the medium. Conversely, a mutant (LH45 deltaproU) in which most of the proU locus had been deleted grew poorly at high osmolarity in the presence of glycine betaine. We conclude that the proU-like locus in B. subtilis LH45 is a gram-positive counterpart of the proU locus in gram-negative bacteria and probably evolved prior to the evolutionary split of prokaryotes into gram-positive and gram-negative forms.

  • the antimicrobial effect of a structural variant of Subtilin against outgrowing bacillus cereus t spores and vegetative cells occurs by different mechanisms
    Applied and Environmental Microbiology, 1993
    Co-Authors: J N Hansen
    Abstract:

    Subtilin is a ribosomally synthesized antimicrobial peptide that contains several unusual amino acids as a result of posttranslational modifications. Site-directed mutagenesis was employed to construct a structural variant of Subtilin in which the unusual dehydroalanine (Dha) residue at position 5 was changed to alanine. Proton nuclear magnetic resonance spectroscopy, amino acid composition, and N-terminal sequence analysis established that the mutation did not disrupt posttranslational processing of the precursor peptide. This mutant Subtilin was devoid of antimicrobial activity as assessed by its lack of inhibitory effects on outgrowth of Bacillus cereus T spores. However, this same mutant Subtilin was fully active with respect to its ability to induce lysis of vegetative B. cereus T cells. Because an intact Dha-5 residue is required in the one instance but not in the other, it was concluded that the molecular mechanism by which Subtilin inhibits (without lysis) spore outgrowth is not the same as the mechanism by which it inhibits (with lysis) vegetative cells.

  • enhancement of the chemical and antimicrobial properties of Subtilin by site directed mutagenesis
    Journal of Biological Chemistry, 1992
    Co-Authors: Wei Liu, J N Hansen
    Abstract:

    Subtilin and nisin are gene-encoded antibiotic peptides that are ribosomally synthesized by Bacillus subtilis and Lactococcus lactis, respectively. Gene-encoded antibiotics are unique in that their structures can be manipulated by mutagenesis of their structural genes. Although Subtilin and nisin share considerable structural homology, Subtilin has a greater tendency than nisin to undergo spontaneous inactivation. This inactivation is a accompanied by chemical modification of the dehydroalanine at position 5 (DHA5) with a kinetic first-order t1/2 of 0.8 days. It was hypothesized that the R group carboxyl of Glu4 in Subtilin participates in the chemical modification of the adjacent DHA5. Noting that nisin has Ile at position 4, site-directed mutagenesis was used to change Glu4 of Subtilin to Ile, in order to eliminate this carboxyl-group participation. The DHA5 of this mutant Subtilin (E4I-Subtilin) underwent modification with a t1/2 of 48 days, which is 57-fold slower than natural Subtilin, and the rate of loss of biological activity dropped by a like amount. These results suggest that an intact DHA5 is critical for Subtilin activity against bacterial spore outgrowth. A double mutant of Subtilin, in which the DHA5 residue of E4I-Subtilin was mutated to Ala was devoid of detectable inhibition against spore outgrowth. The specific activity of E4I-Subtilin was 3-4-fold higher than natural Subtilin, suggesting that an increase in the hydrophobicity of the N-terminal end of the molecule enhances activity. These are the first mutants of Subtilin that have been reported, and E4I-Subtilin is the first example of any lantibiotic whose properties have been improved by mutagenesis. In order to carry out the mutagenesis, a host-vector pair was constructed that permits a deletion replacement in which the natural Subtilin gene is replaced by the mutant gene at the normal location in the chromosome. This maintains normal gene dosage and regulatory responses, as well as eliminates ambiguities caused by expression of the normal and mutant genes in the same cell.

Stefan Heinzmann - One of the best experts on this subject based on the ideXlab platform.

  • engineering bacillus subtilis atcc 6633 for improved production of the lantibiotic Subtilin
    Applied Microbiology and Biotechnology, 2006
    Co-Authors: Stefan Heinzmann, Karldieter Entian, Torsten Stein
    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.

  • expression and functional analysis of the Subtilin immunity genes spaifeg in the Subtilin sensitive host bacillus subtilis mo1099
    Journal of Bacteriology, 2005
    Co-Authors: Torsten Stein, Stefanie Dusterhus, Stefan Heinzmann, Stefan Borchert, Karldieter Entian
    Abstract:

    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.

  • the spa box for transcriptional activation of Subtilin biosynthesis and immunity in bacillus subtilis
    Molecular Microbiology, 2003
    Co-Authors: Torsten Stein, Stefan Heinzmann, Peter Kiesau, Bettina Himmel, Karldieter Entian
    Abstract:

    Summary 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.

  • function of lactococcus lactis nisin immunity genes nisi and nisfeg after coordinated expression in the surrogate host bacillus subtilis
    Journal of Biological Chemistry, 2003
    Co-Authors: Torsten Stein, Stefan Heinzmann, Irina M Solovieva, Karldieter Entian
    Abstract:

    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.

  • dual control of Subtilin biosynthesis and immunity in bacillus subtilis
    Molecular Microbiology, 2002
    Co-Authors: Torsten Stein, Stefan Heinzmann, Stefan Borchert, Peter Kiesau, Stephan Kloss, Klein Cora, Markus Helfrich, Karldieter Entian
    Abstract:

    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.

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