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Development and application of a microtiter plate-based autoinduction bioassay for detection of the lantibiotic subtilin
Journal of Microbiological Methods 70 (2007) 179 – 185 www.elsevier.com/locate/jmicmeth
Development and application of a microtiter plate-based autoinduction bioassay for detection of the lantibiotic subtilin Michael Burkard, Karl-Dieter Entian, Torsten Stein ⁎ Center of Excellence: Macromolecular Complexes, Johann Wolfgang Goethe-University, Institute of Molecular Biosciences, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany Received 30 March 2007; received in revised form 23 April 2007; accepted 23 April 2007 Available online 1 May 2007
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 β-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. © 2007 Elsevier B.V. All rights reserved. Keywords: Bacillus subtilis; Lantibiotic; Pheromone; Subtilin; Two-component system
1. Introduction Because of their nanomolar effectiveness to kill a wide range of Gram-positive bacteria including various human pathogens, lantibiotics display a great potential for pharmaceutical application as resistance to classical antibiotics like vancomycin increases worldwide (Clardy et al., 2006; Hancock and Sahl, 2006; Levy and Marshall, 2004). The production of lantibiotics is a widespread property among Gram-positive bacteria (Chatterjee et al., 2005; Stein, 2005). Lantibiotics are a unique class of bacteriocins, which are ribosomally synthesized as precursor peptides and posttranslational modified by dehydration of serine and threonine residues and subsequent intramolecular addition to cysteine, resulting in formation of (β-methyl-) lanthionine thioether bridges, the characteristic structural elements for lantibiotics (Chatterjee et al., 2005; Schnell et al., 1988). The lantibiotics subtilin (Fig. 1) from Bacillus subtilis ATCC 6633 and the structural closely related nisin A from Lactococcus lactis exhibit bactericidal activity based upon ⁎ Corresponding author. Tel.: +49 69 798 29522; fax: +49 69 798 29527. E-mail address: [email protected] (T. Stein). 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.04.015
pore formation in the cytoplasm membrane using Lipid II, the hydrophobic carrier module for peptidoglycan monomers, as docking module as well as central constituent of the pore (Breukink et al., 2003; Hsu et al., 2004; Hyde et al., 2006). Biosynthesis of subtilin is regulated in a cell-density dependent manner according to a quorum sensing mechanism with the peptide itself as autoinducing agent resulting in signal transduction over twocomponent system SpaRK (Kleerebezem et al., 2004; Klein et al., 1993; Stein et al., 2002b). Binding of subtilin to the sensor domain of SpaK is followed by phosphorylation of the histidine kinase. Transfer of the phosphorylation to SpaR results in activation of the response regulator and furthermore transcriptional activation of three independent promoters preceding spaB, spaS and spaI (Kleerebezem et al., 2004; Stein et al., 2002b, 2003). Additionally, transcriptional regulation of spaRK expression is under control of transition state regulator sigma factor H and AbrB, the key regulator of late growth activities (Heinzmann et al., 2006; Stein et al., 2002b). A similar autoregulatory mechanism independent of regulation by SigH and AbrB is also described for nisin and mersacidin biosynthesis (Engelke et al., 1994; Kleerebezem, 2004; Kuipers et al., 1995; Schmitz et al., 2006).
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Fig. 1. Schematic representation of B. subtilis lantibiotics. Structure of the lantibiotics subtilin and ericin S produced by B. subtilis ATCC 6633 and A1/3, respectively. Amino acids (encircled) are given in one letter code. Posttranslationally modified residues are as follows: A–S–A, meso-lanthionine; Ab–S–A, (2S,3S,6R)-3methyllanthionine (the abbreviation Ab refers to aminobutyric acid); ΔA, 2,3-didehydroalanine; ΔB, (Z)-2,3-didehydobutyrine. Amino acid exchanges between subtilin and ericin S are indicated by arrows.
The property of subtilin and nisin as autoinducer of their own biosyntheses led to the development of protein expression systems that facilitate homologous and heterologous gene expression of membrane proteins or even toxic products by strict transcriptional control in dependence of autoinducer supplementation (Bongers et al., 2005; de Ruyter et al., 1996; Kleerebezem, 2004; Kuipers et al., 1995; Mierau and Kleerebezem, 2005). Lantibiotic-driven gene expression requires transcriptional fusion of the desired gene to a lantibiotic promoter region and the presence of the corresponding signal transduction system LanRK (Bongers et al., 2005; de Ruyter et al., 1996). Furthermore, versatile applications and bioassays for detection of nisin directly from foodstuff are described (Hakovirta et al., 2006; Reunanen and Saris, 2003). Here we describe the construction and application of a subtilin reporter strain for qualitative and quantitative determination of subtilin in biological samples. The subtilin bioassay was optimized for microtiter plate analyses and applied for the detection of subtilin and subtilin-isoform producing B. subtilis strains originating from both strain collections and natural isolates. 2. Materials and methods 2.1. Bacterial strains, growth conditions and plasmids B. subtilis cells were grown either in a modified medium according to Landy complemented with 0.5% yeast extract (Landy et al., 1948; Stein et al., 2004) or in TY (0.8% tryptone, 0.5% yeast extract, 0,5% NaCl). Liquid cultures were grown under intensive shaking at 37 °C. L. lactis was grown in M17 medium at 30 °C. The strains used in this study are listed in Table 1. For the chromogenic plate assay TY medium containing 0.3 M NaCl and 1.5% agar was complemented with X-Gal to a final concentration of 50 μg/mL. In the case of antibiotic resistance markers, 5 μg/mL chloramphenicol and 100 μg/mL spectinomycin were used for selection of B. subtilis. Selection of E. coli was performed by adding 100 μg/mL ampicillin to the media. The B. subtilis spaS gene including the native promoter sequence was PCR-amplified with primers CISub_1 (5′-GGTTACAGCGGTATCGGTC-
GACTTCTGCTTGC-3′) and CISub_i (5′-GGTGTGAATATAAAAGCTTTCACACCCGTCG-3′). The plasmid pMB32 was constructed after digestion with SalI and HindIII and ligation into plasmid pSD27 digested with the same enzymes. pMB32 was used to complement the Sub− phenotype after integration into the amyE locus of B. subtilis by double homologous recombination. Recombinant plasmids amplified in E. coli DH5α and isolated due to an alkaline extraction procedure (Birnboim and Doly, 1979) were transformed into B. subtilis ΔspaS using standard protocols (Spizizen et al., 1966). Purification of plasmids was performed by the use of Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, USA). Established protocols were used for molecular biology techniques and PCR was performed according to standard procedures using a Tpersonal 48 thermocycler (Biometra). 2.2. Chromogenic plate assay for subtilin monitoring For the detection of subtilin induction the reporter strain B. subtilis ΔspaS amyE::PspaS–lacZ was generated by transformation of strain ΔspaS with plasmid pSB5, which enables chromosomal integration of lacZ under control of spaS promoter region into the amyE locus of B. subtilis. The B. subtilis strains to be tested were streaked out on TY 0.3 M NaCl plates supplemented with X-Gal against the indicator strain. The plates were incubated for 36–48 h at room temperature until clear discoloring of the reporter strain according to induction of lacZ expression was observed. 2.3. Subtilin induced lacZ gene expression in the subtilin reporter strain For detection of subtilin-induced expression of β-galactosidase in a concentration dependent manner reporter, overnight grown cells (TY, 37 °C) of the subtilin reporter strain B. subtilis ΔspaS amyE::PspaS–lacZ with an OD600 of 7–8 were diluted into fresh TY medium (5 mL test tubes) to an OD600 of 0.1. To induce lacZ gene expression the reporter strain was incubated with different amounts of RP-HPLC-purified subtilin for 6 h under intensive shaking at 37 °C.
M. Burkard et al. / Journal of Microbiological Methods 70 (2007) 179–185 Table 1 Strains and plasmids used in this study Bacillus subtilis strains Description ATCC 6633 ΔspaS
Wild type, subtilin producer (Sub+) spaS::spec (Specr), Sub−
ΔspaS amyE:: PspaS–spaS ΔspaS amyE:: PspaS–lacZ 168 A1/3
DspaS complemented with pMB32, (Specr, Neor), Sub+ spaS transformed with pSB5, (Specr, Cmr), Sub− Used for genome sequencing, Sub− Wild type, producer of ericin A and ericin S Wild type, Sub− Wild type, Sub− Wild type, Sub− Wild type, Sub− Wild type, Sub− Wild type, Sub+ Wild type, Sub+ wild type, Sub+ Wild type, Sub+ Wild type, Sub+
DSM 10T DSM 1088 DSM 2109 DSM 3258 DSM 60015 DSM 618 a DSM 1087 a DSM 6395 a DSM 6405 a DSM 8439 a
Isolated Bacillus subtilis strains: HI Wild type, Sub− IP
Wild type, Sub−
HS a
Wild type, Sub+
N5 a
Wild type, Sub+
Additional strains: Micrococcus luteus ATCC 9341 Lactococcus lactis 6F3
Indicator organism for antimicrobial activity of Bacillus subtilis strains Nisin A producer
Reference ATCC Stein et al. (2004) This work This work DSM 402 (Stein et al., 2002a,b) DSM DSM DSM DSM DSM DSM DSM DSM DSM DSM
Helfrich, submitted Helfrich, submitted Helfrich, submitted Helfrich, submitted
ATCC Fuchs et al. (1975)
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producing B. subtilis strains 5 μL culture supernatants of the respective strain after overnight growth (OD600 ≈ 12–14) in Landy medium were added to 200 μL of the reporter strain culture in a microtiter plate. 2.4. β-Galactosidase activity assays 10 μL aliquots of the samples, either from microtiter plates or test tubes, were added to 990 μL Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM βmercaptoethanol, pH 7.0) and cells were permeabilized by the addition of 20 μL toluene. After removal of toluene by incubation at 37 °C for 45 min the β-galactosidase reaction was started by adding 200 μL of o-nitrophenyl-β-D-galactosid (ONPG, 4 mg/mL in Z-buffer) at 28 °C to the samples. Addition of 500 μL 1 M Na2CO3 solution stopped the reaction. Determination of enzyme activities was performed according to standard methods described previously (Stein et al., 2002b; Zuber and Losick, 1983). Standard deviations are calculated by at least two independent approaches. 2.5. Isolation of B. subtilis strains from soil Soil samples were resuspended in sterile H2O under vigorous stirring. To enrich spore-forming bacteria 10 mL of the sample was sedimented and pasteurized (20 min, 80 °C). Aliquots were spread out on TY plates and incubated at 37 °C. Single colonies exhibiting antimicrobial activity against Micrococcus luteus (Gutowski-Eckel et al., 1994) were isolated and 16 S rDNA-typed B. subtilis strains were chosen for further investigations (M. Helfrich, K.-D. Entian, and T. Stein, submitted). 3. Results and discussion
Plasmids pSD27
pMB32 pSB5
pMLK83 derivative (gusA gene was cut out with EcoRI), for marker exchange recombination into B. subtilis amyE (Apr, Neor) spaS gene including native promoter in pSD27 (Apr, Neor) spaS promoter region (202 bp) in front of lacZ, for marker exchange recombination into B. subtilis amyE (Apr, Cmr)
Düsterhus, This work
This work (Stein et al., 2002a,b)
a The Sub+ phenotype (this work) was confirmed by mass spectrometry and sequencing of spaS structural gene (Helfrich, M., Entian, K.-D., Stein, T., submitted for publication).
After optimization the subtilin induction step was performed in microtiter plates: 180 μL aliquots of TY medium containing 0.3 M NaCl were inoculated with maximal 20 μL of the preculture resulting of an OD600 of 0.1. These cultures were incubated with different amounts of RP-HPLC-purified subtilin (20 μL aliquots in sterile water) which led to an increase of the volume of less than 10%. The microtiter plate was incubated for 6 h at 37 °C and the OD600 of the reporter strain was in the range between 0.3 and 0.4. For identification of several subtilin
3.1. Development of the subtilin autoinduction bioassay A highly interesting phenomenon among lantibiotics is their peptide pheromone action that autoregulates their biosyntheses. To establish a subtilin reporter system on the basis of the interaction between the histidine kinase SpaK and subtilin in the genetic background of the subtilin producer B. subtilis ATCC 6633, a spaS gene deletion mutant was used (Fig. 2A), which was unable to produce subtilin (Sub − phenotype) (Klein et al., 1992). Importantly, this strain still contains an intact two-component regulatory system spaRK (Fig. 2B). The subtilin promoter spaS was translationally fused to the reporter gene lacZ on plasmid pSB5 carrying a chloramphenicol resistance gene (Stein et al., 2002b) and finally the subtilin reporter strain (spaS::spec; amyE::PspaS– lacZ) was obtained after chromosomal integration of the PspaS–lacZ fusion into ΔspaS cells (Fig. 2C). The subtilin reporter strain (Fig. 2E, vertical streak) enables monitoring of subtilin production (Fig. 2D) as expression of the lacZ gene is governed by the native two-component regulatory system SpaRK. The native subtilin producer B. subtilis ATCC 6633 as well as ΔspaS cells complemented with spaS (both Sub+
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Fig. 2. Construction of the subtilin reporter strain. A) The subtilin gene cluster and the amylase locus of the B. subtilis ATCC 6633 wild type strain (Subtilin+). B) Cells with deleted spaS gene abolished subtilin production (ΔspaS, Subtilin−). C) The lacZ gene under control of the native promotor PspaS was chromosomally integrated into the amylase locus of the ΔspaS strain after transformation of plasmid pSB5 resulting in the subtilin reporter strain (spaS::spec amyE::PspaS–lacZ). D) Schematic signal transduction pathway in the subtilin reporter strain includes sensing of subtilin by the histidine kinase SpaK and phosphotransfer to SpaR followed by the transcriptional activation of the PspaS–lacZ-fusion. E) Chromogenic plate assay using X-Gluc as substrate for the visualization of subtilin induced lacZ expression. The strains B. subtilis ATCC 6633 (Subtilin+), the deletion mutant ΔspaS (Subtilin−), and ΔspaS complemented with PspaS–spaS (Subtilin+) chromosomally integrated into the amyE-site were tested for subtilin production after streaking them out against the subtilin indicator strain. The Subtilin+ phenotype is indicated by discoloration of the reporter strain according to an induction of lacZ expression (see part D).
phenotypes) provoked discoloring of the reporter strain (Fig. 2E, horizontal streaks). 3.2. Concentration dependency of the subtilin autoinduction bioassay Assays of subtilin mediated induction of lacZ expression was initially carried out by addition of purified subtilin to the growing reporter strain in 5 mL test tubes. Incubation of exponentially grown cells of the subtilin indicator strain for 6 h with subtilin lead to optimal concentration dependent β-galactosidase activities (Fig. 3A). Linear dose–response relationship was detectable in the subtilin concentration range between 0.1 and 1 μg/mL. Higher subtilin concentrations (up to 10 μg/mL) did not further increase β-galactosidase activities due to a saturation of the signaling capacity. To increase the throughput of our subtilin detection system the assay has been performed in 96-well microtiter plates. Remarkably, utilizing optimized conditions, even 10 ng/mL
subtilin could be detected (Fig. 3B, insert). Moreover, a linear dose–response relationship for subtilin induced β-galactosidase expression was observed between 0.01 and 0.1 μg/mL subtilin. Maximal β-galactosidase activities (about 15 × 10 3 Miller Units) were already achieved with 2 μg/mL subtilin (Fig. 3B) corresponding to 50–100-fold higher activities as compared to non-induced conditions. We could significantly (four-fold) increase the sensitivity of our subtilin detection system after optimizing the concentration of NaCl (Fig. 3C), maximal signal transduction was obtained with 0.3 M NaCl. A possible explanation for this observation is the prohibition of unspecific electrostatic interactions of the cationic subtilin with under the growth conditions negatively charged B. subtilis cell wall and/ or cytoplasmic membrane. The signal transduction system SpaRK is highly specific for the cognate autoinducer subtilin: Addition of the structurally closely related lantibiotic nisin A, sharing a similar arrangement of intramolecular lanthionine rings, in different concentrations did not induce the expression of lacZ (data not shown).
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amount to obtain comparable reporter gene activities. However, the throughput of the subtilin monitoring system is restricted by the subsequent β-galactosidase determination which has to be carried out in large tubes. Here, about 50 determinations can be performed in parallel. 3.3. Application of the subtilin autoinduction bioassay: Identification of subtilin producing B. subtilis wild type strains Although many different B. subtilis strains are allocated in culture collections, the production of the lantibiotic subtilin so far is merely described for a single B. subtilis strain (ATCC 6633). In order to apply our developed subtilin monitoring assay for the identification of novel subtilin producing strains, culture supernatants of different B. subtilis wild-type strains were analyzed. Interestingly, 5 μL culture supernatants from five culture collection strains (DSM 618, 1087, 6395, 6405, and 8439) as well as two natural isolated B. subtilis strains HS and N5 (Helfrich, M., Entian, K.-D., and Stein, T., submitted for publication) were able to induce the subtilin reporter strain (Fig. 4; 7.5–13 × 103 Miller Units). In contrast, the strains DSM 10T, 1088, 2109, 3258 and 60015, as well as 168 (used for genome sequencing and known as subtilin non-producer) as well as two further natural isolated B. subtilis strains (HI and IP) were identified as subtilin non-producer (background values were between 100 and 200 Miller Units). Remarkably, the culture supernatant of B. subtilis A1/3 (Stein et al., 2002a), producer of the natural subtilin variant ericin S (Fig. 1) also induces the subtilin induction system. This suggests, that the subtilin-specific sensor kinase SpaK recognizes a certain structural spectrum, for example ericin S with four amino acid exchanges as compared to subtilin. This demonstrates the use of the described microtiter plate assay for elucidation of structural prerequisites for lantibiotic recognition. Furthermore, the nisin containing culture supernatant of strain L. lactis 6F3 did not effect lacZ gene expression of the subtilin reporter strain. 3.4. Conclusions
Fig. 3. Subtilin autoinduction bioassay using the subtilin reporter strain. A) Standard curve for subtilin induced β-galactosidase activity of the subtilin reporter strain using test tubes (see also Table 1 and Fig. 2, part C). B) Standard curve for subtilin induced β-galactosidase activity of the subtilin reporter strain using 96-well microtiter plates. The insert shows details of the graph in the lower concentration range (0–0.1 μg/mL). C) The subtilin reporter strain was inoculated with 0.1 μg/mL subtilin and different NaCl-concentrations (assay volume: 200 μL in microtiter plates). The means of two independent induction experiments are shown, for each two β-galactosidase determinations have been performed.
The developed microtiter plate assay allows high-throughput quantitative subtilin monitoring. The main advantage of the developed methodology is an increased subtilin sensitivity. Both, scale-down of the assay volume from 5 mL to 200 μL in microtiter plates as well as optimization of the NaCl concentration during the induction step lead to a 160-fold reduction of the required subtilin
Lantibiotic biosyntheses for subtilin, nisin, and mersacidin is regulated in a cell-density dependent manner according to a quorum sensing mechanism (Kuipers et al., 1995; Schmitz et al., 2006; Stein et al., 2002b). As the lantibiotics subtilin, nisin, or mersacidin act as pheromones (third component) to induce their cognate, typical two-component system histidine kinase LanK and response regulator protein LanR, the term three-component regulation system has been established (Eijsink et al., 2002; Fabret et al., 1999; Quadri, 2002). In this work we constructed and functionally characterized a β-galactosidase expression system under the control of external subtilin application which can be used both in qualitative and quantitative determination of subtilin in biosamples. Application of this system lead to the identification of novel subtilin producing Bacillus strains, five from culture collections and two isolated from soil samples. These results indicate a more common microbial and ecological distribution of subtilin producing B. subtilis strains as previously assumed and could lead to future identifications of
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Fig. 4. Identification of subtilin producing B. subtilis wild-type strains. Subtilin induced β-galactosidase activity of the subtilin reporter strain by addition of 5 μL culture supernatants from different B. subtilis strains using microtiter plates. To illustrate the specificity of the assay, the nisin-containing culture supernatant of L. lactis 6F3 was added as a control. The means of two independent induction experiments are shown, for each two β-galactosidase determinations have been performed.
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Development and application of a microtiter plate-based autoinduction bioassay for detection of the lantibiotic subtilin Michael Burkard, Karl-Dieter Entian, Torsten Stein ⁎ Center of Excellence: Macromolecular Complexes, Johann Wolfgang Goethe-University, Institute of Molecular Biosciences, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany Received 30 March 2007; received in revised form 23 April 2007; accepted 23 April 2007 Available online 1 May 2007
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 β-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. © 2007 Elsevier B.V. All rights reserved. Keywords: Bacillus subtilis; Lantibiotic; Pheromone; Subtilin; Two-component system
1. Introduction Because of their nanomolar effectiveness to kill a wide range of Gram-positive bacteria including various human pathogens, lantibiotics display a great potential for pharmaceutical application as resistance to classical antibiotics like vancomycin increases worldwide (Clardy et al., 2006; Hancock and Sahl, 2006; Levy and Marshall, 2004). The production of lantibiotics is a widespread property among Gram-positive bacteria (Chatterjee et al., 2005; Stein, 2005). Lantibiotics are a unique class of bacteriocins, which are ribosomally synthesized as precursor peptides and posttranslational modified by dehydration of serine and threonine residues and subsequent intramolecular addition to cysteine, resulting in formation of (β-methyl-) lanthionine thioether bridges, the characteristic structural elements for lantibiotics (Chatterjee et al., 2005; Schnell et al., 1988). The lantibiotics subtilin (Fig. 1) from Bacillus subtilis ATCC 6633 and the structural closely related nisin A from Lactococcus lactis exhibit bactericidal activity based upon ⁎ Corresponding author. Tel.: +49 69 798 29522; fax: +49 69 798 29527. E-mail address: [email protected] (T. Stein). 0167-7012/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.mimet.2007.04.015
pore formation in the cytoplasm membrane using Lipid II, the hydrophobic carrier module for peptidoglycan monomers, as docking module as well as central constituent of the pore (Breukink et al., 2003; Hsu et al., 2004; Hyde et al., 2006). Biosynthesis of subtilin is regulated in a cell-density dependent manner according to a quorum sensing mechanism with the peptide itself as autoinducing agent resulting in signal transduction over twocomponent system SpaRK (Kleerebezem et al., 2004; Klein et al., 1993; Stein et al., 2002b). Binding of subtilin to the sensor domain of SpaK is followed by phosphorylation of the histidine kinase. Transfer of the phosphorylation to SpaR results in activation of the response regulator and furthermore transcriptional activation of three independent promoters preceding spaB, spaS and spaI (Kleerebezem et al., 2004; Stein et al., 2002b, 2003). Additionally, transcriptional regulation of spaRK expression is under control of transition state regulator sigma factor H and AbrB, the key regulator of late growth activities (Heinzmann et al., 2006; Stein et al., 2002b). A similar autoregulatory mechanism independent of regulation by SigH and AbrB is also described for nisin and mersacidin biosynthesis (Engelke et al., 1994; Kleerebezem, 2004; Kuipers et al., 1995; Schmitz et al., 2006).
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Fig. 1. Schematic representation of B. subtilis lantibiotics. Structure of the lantibiotics subtilin and ericin S produced by B. subtilis ATCC 6633 and A1/3, respectively. Amino acids (encircled) are given in one letter code. Posttranslationally modified residues are as follows: A–S–A, meso-lanthionine; Ab–S–A, (2S,3S,6R)-3methyllanthionine (the abbreviation Ab refers to aminobutyric acid); ΔA, 2,3-didehydroalanine; ΔB, (Z)-2,3-didehydobutyrine. Amino acid exchanges between subtilin and ericin S are indicated by arrows.
The property of subtilin and nisin as autoinducer of their own biosyntheses led to the development of protein expression systems that facilitate homologous and heterologous gene expression of membrane proteins or even toxic products by strict transcriptional control in dependence of autoinducer supplementation (Bongers et al., 2005; de Ruyter et al., 1996; Kleerebezem, 2004; Kuipers et al., 1995; Mierau and Kleerebezem, 2005). Lantibiotic-driven gene expression requires transcriptional fusion of the desired gene to a lantibiotic promoter region and the presence of the corresponding signal transduction system LanRK (Bongers et al., 2005; de Ruyter et al., 1996). Furthermore, versatile applications and bioassays for detection of nisin directly from foodstuff are described (Hakovirta et al., 2006; Reunanen and Saris, 2003). Here we describe the construction and application of a subtilin reporter strain for qualitative and quantitative determination of subtilin in biological samples. The subtilin bioassay was optimized for microtiter plate analyses and applied for the detection of subtilin and subtilin-isoform producing B. subtilis strains originating from both strain collections and natural isolates. 2. Materials and methods 2.1. Bacterial strains, growth conditions and plasmids B. subtilis cells were grown either in a modified medium according to Landy complemented with 0.5% yeast extract (Landy et al., 1948; Stein et al., 2004) or in TY (0.8% tryptone, 0.5% yeast extract, 0,5% NaCl). Liquid cultures were grown under intensive shaking at 37 °C. L. lactis was grown in M17 medium at 30 °C. The strains used in this study are listed in Table 1. For the chromogenic plate assay TY medium containing 0.3 M NaCl and 1.5% agar was complemented with X-Gal to a final concentration of 50 μg/mL. In the case of antibiotic resistance markers, 5 μg/mL chloramphenicol and 100 μg/mL spectinomycin were used for selection of B. subtilis. Selection of E. coli was performed by adding 100 μg/mL ampicillin to the media. The B. subtilis spaS gene including the native promoter sequence was PCR-amplified with primers CISub_1 (5′-GGTTACAGCGGTATCGGTC-
GACTTCTGCTTGC-3′) and CISub_i (5′-GGTGTGAATATAAAAGCTTTCACACCCGTCG-3′). The plasmid pMB32 was constructed after digestion with SalI and HindIII and ligation into plasmid pSD27 digested with the same enzymes. pMB32 was used to complement the Sub− phenotype after integration into the amyE locus of B. subtilis by double homologous recombination. Recombinant plasmids amplified in E. coli DH5α and isolated due to an alkaline extraction procedure (Birnboim and Doly, 1979) were transformed into B. subtilis ΔspaS using standard protocols (Spizizen et al., 1966). Purification of plasmids was performed by the use of Wizard® SV Gel and PCR Clean-Up System (Promega Corporation, USA). Established protocols were used for molecular biology techniques and PCR was performed according to standard procedures using a Tpersonal 48 thermocycler (Biometra). 2.2. Chromogenic plate assay for subtilin monitoring For the detection of subtilin induction the reporter strain B. subtilis ΔspaS amyE::PspaS–lacZ was generated by transformation of strain ΔspaS with plasmid pSB5, which enables chromosomal integration of lacZ under control of spaS promoter region into the amyE locus of B. subtilis. The B. subtilis strains to be tested were streaked out on TY 0.3 M NaCl plates supplemented with X-Gal against the indicator strain. The plates were incubated for 36–48 h at room temperature until clear discoloring of the reporter strain according to induction of lacZ expression was observed. 2.3. Subtilin induced lacZ gene expression in the subtilin reporter strain For detection of subtilin-induced expression of β-galactosidase in a concentration dependent manner reporter, overnight grown cells (TY, 37 °C) of the subtilin reporter strain B. subtilis ΔspaS amyE::PspaS–lacZ with an OD600 of 7–8 were diluted into fresh TY medium (5 mL test tubes) to an OD600 of 0.1. To induce lacZ gene expression the reporter strain was incubated with different amounts of RP-HPLC-purified subtilin for 6 h under intensive shaking at 37 °C.
M. Burkard et al. / Journal of Microbiological Methods 70 (2007) 179–185 Table 1 Strains and plasmids used in this study Bacillus subtilis strains Description ATCC 6633 ΔspaS
Wild type, subtilin producer (Sub+) spaS::spec (Specr), Sub−
ΔspaS amyE:: PspaS–spaS ΔspaS amyE:: PspaS–lacZ 168 A1/3
DspaS complemented with pMB32, (Specr, Neor), Sub+ spaS transformed with pSB5, (Specr, Cmr), Sub− Used for genome sequencing, Sub− Wild type, producer of ericin A and ericin S Wild type, Sub− Wild type, Sub− Wild type, Sub− Wild type, Sub− Wild type, Sub− Wild type, Sub+ Wild type, Sub+ wild type, Sub+ Wild type, Sub+ Wild type, Sub+
DSM 10T DSM 1088 DSM 2109 DSM 3258 DSM 60015 DSM 618 a DSM 1087 a DSM 6395 a DSM 6405 a DSM 8439 a
Isolated Bacillus subtilis strains: HI Wild type, Sub− IP
Wild type, Sub−
HS a
Wild type, Sub+
N5 a
Wild type, Sub+
Additional strains: Micrococcus luteus ATCC 9341 Lactococcus lactis 6F3
Indicator organism for antimicrobial activity of Bacillus subtilis strains Nisin A producer
Reference ATCC Stein et al. (2004) This work This work DSM 402 (Stein et al., 2002a,b) DSM DSM DSM DSM DSM DSM DSM DSM DSM DSM
Helfrich, submitted Helfrich, submitted Helfrich, submitted Helfrich, submitted
ATCC Fuchs et al. (1975)
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producing B. subtilis strains 5 μL culture supernatants of the respective strain after overnight growth (OD600 ≈ 12–14) in Landy medium were added to 200 μL of the reporter strain culture in a microtiter plate. 2.4. β-Galactosidase activity assays 10 μL aliquots of the samples, either from microtiter plates or test tubes, were added to 990 μL Z-buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, 50 mM βmercaptoethanol, pH 7.0) and cells were permeabilized by the addition of 20 μL toluene. After removal of toluene by incubation at 37 °C for 45 min the β-galactosidase reaction was started by adding 200 μL of o-nitrophenyl-β-D-galactosid (ONPG, 4 mg/mL in Z-buffer) at 28 °C to the samples. Addition of 500 μL 1 M Na2CO3 solution stopped the reaction. Determination of enzyme activities was performed according to standard methods described previously (Stein et al., 2002b; Zuber and Losick, 1983). Standard deviations are calculated by at least two independent approaches. 2.5. Isolation of B. subtilis strains from soil Soil samples were resuspended in sterile H2O under vigorous stirring. To enrich spore-forming bacteria 10 mL of the sample was sedimented and pasteurized (20 min, 80 °C). Aliquots were spread out on TY plates and incubated at 37 °C. Single colonies exhibiting antimicrobial activity against Micrococcus luteus (Gutowski-Eckel et al., 1994) were isolated and 16 S rDNA-typed B. subtilis strains were chosen for further investigations (M. Helfrich, K.-D. Entian, and T. Stein, submitted). 3. Results and discussion
Plasmids pSD27
pMB32 pSB5
pMLK83 derivative (gusA gene was cut out with EcoRI), for marker exchange recombination into B. subtilis amyE (Apr, Neor) spaS gene including native promoter in pSD27 (Apr, Neor) spaS promoter region (202 bp) in front of lacZ, for marker exchange recombination into B. subtilis amyE (Apr, Cmr)
Düsterhus, This work
This work (Stein et al., 2002a,b)
a The Sub+ phenotype (this work) was confirmed by mass spectrometry and sequencing of spaS structural gene (Helfrich, M., Entian, K.-D., Stein, T., submitted for publication).
After optimization the subtilin induction step was performed in microtiter plates: 180 μL aliquots of TY medium containing 0.3 M NaCl were inoculated with maximal 20 μL of the preculture resulting of an OD600 of 0.1. These cultures were incubated with different amounts of RP-HPLC-purified subtilin (20 μL aliquots in sterile water) which led to an increase of the volume of less than 10%. The microtiter plate was incubated for 6 h at 37 °C and the OD600 of the reporter strain was in the range between 0.3 and 0.4. For identification of several subtilin
3.1. Development of the subtilin autoinduction bioassay A highly interesting phenomenon among lantibiotics is their peptide pheromone action that autoregulates their biosyntheses. To establish a subtilin reporter system on the basis of the interaction between the histidine kinase SpaK and subtilin in the genetic background of the subtilin producer B. subtilis ATCC 6633, a spaS gene deletion mutant was used (Fig. 2A), which was unable to produce subtilin (Sub − phenotype) (Klein et al., 1992). Importantly, this strain still contains an intact two-component regulatory system spaRK (Fig. 2B). The subtilin promoter spaS was translationally fused to the reporter gene lacZ on plasmid pSB5 carrying a chloramphenicol resistance gene (Stein et al., 2002b) and finally the subtilin reporter strain (spaS::spec; amyE::PspaS– lacZ) was obtained after chromosomal integration of the PspaS–lacZ fusion into ΔspaS cells (Fig. 2C). The subtilin reporter strain (Fig. 2E, vertical streak) enables monitoring of subtilin production (Fig. 2D) as expression of the lacZ gene is governed by the native two-component regulatory system SpaRK. The native subtilin producer B. subtilis ATCC 6633 as well as ΔspaS cells complemented with spaS (both Sub+
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Fig. 2. Construction of the subtilin reporter strain. A) The subtilin gene cluster and the amylase locus of the B. subtilis ATCC 6633 wild type strain (Subtilin+). B) Cells with deleted spaS gene abolished subtilin production (ΔspaS, Subtilin−). C) The lacZ gene under control of the native promotor PspaS was chromosomally integrated into the amylase locus of the ΔspaS strain after transformation of plasmid pSB5 resulting in the subtilin reporter strain (spaS::spec amyE::PspaS–lacZ). D) Schematic signal transduction pathway in the subtilin reporter strain includes sensing of subtilin by the histidine kinase SpaK and phosphotransfer to SpaR followed by the transcriptional activation of the PspaS–lacZ-fusion. E) Chromogenic plate assay using X-Gluc as substrate for the visualization of subtilin induced lacZ expression. The strains B. subtilis ATCC 6633 (Subtilin+), the deletion mutant ΔspaS (Subtilin−), and ΔspaS complemented with PspaS–spaS (Subtilin+) chromosomally integrated into the amyE-site were tested for subtilin production after streaking them out against the subtilin indicator strain. The Subtilin+ phenotype is indicated by discoloration of the reporter strain according to an induction of lacZ expression (see part D).
phenotypes) provoked discoloring of the reporter strain (Fig. 2E, horizontal streaks). 3.2. Concentration dependency of the subtilin autoinduction bioassay Assays of subtilin mediated induction of lacZ expression was initially carried out by addition of purified subtilin to the growing reporter strain in 5 mL test tubes. Incubation of exponentially grown cells of the subtilin indicator strain for 6 h with subtilin lead to optimal concentration dependent β-galactosidase activities (Fig. 3A). Linear dose–response relationship was detectable in the subtilin concentration range between 0.1 and 1 μg/mL. Higher subtilin concentrations (up to 10 μg/mL) did not further increase β-galactosidase activities due to a saturation of the signaling capacity. To increase the throughput of our subtilin detection system the assay has been performed in 96-well microtiter plates. Remarkably, utilizing optimized conditions, even 10 ng/mL
subtilin could be detected (Fig. 3B, insert). Moreover, a linear dose–response relationship for subtilin induced β-galactosidase expression was observed between 0.01 and 0.1 μg/mL subtilin. Maximal β-galactosidase activities (about 15 × 10 3 Miller Units) were already achieved with 2 μg/mL subtilin (Fig. 3B) corresponding to 50–100-fold higher activities as compared to non-induced conditions. We could significantly (four-fold) increase the sensitivity of our subtilin detection system after optimizing the concentration of NaCl (Fig. 3C), maximal signal transduction was obtained with 0.3 M NaCl. A possible explanation for this observation is the prohibition of unspecific electrostatic interactions of the cationic subtilin with under the growth conditions negatively charged B. subtilis cell wall and/ or cytoplasmic membrane. The signal transduction system SpaRK is highly specific for the cognate autoinducer subtilin: Addition of the structurally closely related lantibiotic nisin A, sharing a similar arrangement of intramolecular lanthionine rings, in different concentrations did not induce the expression of lacZ (data not shown).
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amount to obtain comparable reporter gene activities. However, the throughput of the subtilin monitoring system is restricted by the subsequent β-galactosidase determination which has to be carried out in large tubes. Here, about 50 determinations can be performed in parallel. 3.3. Application of the subtilin autoinduction bioassay: Identification of subtilin producing B. subtilis wild type strains Although many different B. subtilis strains are allocated in culture collections, the production of the lantibiotic subtilin so far is merely described for a single B. subtilis strain (ATCC 6633). In order to apply our developed subtilin monitoring assay for the identification of novel subtilin producing strains, culture supernatants of different B. subtilis wild-type strains were analyzed. Interestingly, 5 μL culture supernatants from five culture collection strains (DSM 618, 1087, 6395, 6405, and 8439) as well as two natural isolated B. subtilis strains HS and N5 (Helfrich, M., Entian, K.-D., and Stein, T., submitted for publication) were able to induce the subtilin reporter strain (Fig. 4; 7.5–13 × 103 Miller Units). In contrast, the strains DSM 10T, 1088, 2109, 3258 and 60015, as well as 168 (used for genome sequencing and known as subtilin non-producer) as well as two further natural isolated B. subtilis strains (HI and IP) were identified as subtilin non-producer (background values were between 100 and 200 Miller Units). Remarkably, the culture supernatant of B. subtilis A1/3 (Stein et al., 2002a), producer of the natural subtilin variant ericin S (Fig. 1) also induces the subtilin induction system. This suggests, that the subtilin-specific sensor kinase SpaK recognizes a certain structural spectrum, for example ericin S with four amino acid exchanges as compared to subtilin. This demonstrates the use of the described microtiter plate assay for elucidation of structural prerequisites for lantibiotic recognition. Furthermore, the nisin containing culture supernatant of strain L. lactis 6F3 did not effect lacZ gene expression of the subtilin reporter strain. 3.4. Conclusions
Fig. 3. Subtilin autoinduction bioassay using the subtilin reporter strain. A) Standard curve for subtilin induced β-galactosidase activity of the subtilin reporter strain using test tubes (see also Table 1 and Fig. 2, part C). B) Standard curve for subtilin induced β-galactosidase activity of the subtilin reporter strain using 96-well microtiter plates. The insert shows details of the graph in the lower concentration range (0–0.1 μg/mL). C) The subtilin reporter strain was inoculated with 0.1 μg/mL subtilin and different NaCl-concentrations (assay volume: 200 μL in microtiter plates). The means of two independent induction experiments are shown, for each two β-galactosidase determinations have been performed.
The developed microtiter plate assay allows high-throughput quantitative subtilin monitoring. The main advantage of the developed methodology is an increased subtilin sensitivity. Both, scale-down of the assay volume from 5 mL to 200 μL in microtiter plates as well as optimization of the NaCl concentration during the induction step lead to a 160-fold reduction of the required subtilin
Lantibiotic biosyntheses for subtilin, nisin, and mersacidin is regulated in a cell-density dependent manner according to a quorum sensing mechanism (Kuipers et al., 1995; Schmitz et al., 2006; Stein et al., 2002b). As the lantibiotics subtilin, nisin, or mersacidin act as pheromones (third component) to induce their cognate, typical two-component system histidine kinase LanK and response regulator protein LanR, the term three-component regulation system has been established (Eijsink et al., 2002; Fabret et al., 1999; Quadri, 2002). In this work we constructed and functionally characterized a β-galactosidase expression system under the control of external subtilin application which can be used both in qualitative and quantitative determination of subtilin in biosamples. Application of this system lead to the identification of novel subtilin producing Bacillus strains, five from culture collections and two isolated from soil samples. These results indicate a more common microbial and ecological distribution of subtilin producing B. subtilis strains as previously assumed and could lead to future identifications of
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Fig. 4. Identification of subtilin producing B. subtilis wild-type strains. Subtilin induced β-galactosidase activity of the subtilin reporter strain by addition of 5 μL culture supernatants from different B. subtilis strains using microtiter plates. To illustrate the specificity of the assay, the nisin-containing culture supernatant of L. lactis 6F3 was added as a control. The means of two independent induction experiments are shown, for each two β-galactosidase determinations have been performed.
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