This part of the BaKillus-concept focuses on specific pathogen detection. Here we use quorum sensing to specifically recognize the presence of certain pathogens. The major aim of this subproject is transferring the quorum sensing two-component systems AgrC-II/AgrA of ''Staphylococcus aureus'' and ComDE of ''Streptococcus pneumoniae'' into ''Bacillus subtilis'', thus creating a pathogen detecting strain. With this strategy we will trigger our killing strategies only if a threshold concentration of autoinducers, produced by pathogens, is present in the environment Thereby we aim to drastically increase the specificity of our antimicrobial strategies compared to commonly used broad-spectrum antibiotic therapy.   

Quorum Sensing is a mechanism of bacterial cell-cell communication, granting bacteria the ability to measure the density of their surrounding cell population, and react to this by upregulation of specific genes.  

In ''S.  aureus'' this process is one of the major regulator of its virulence, making sure that energy-costly virulence factors are only produced when a large enough number of pathogens is present to overcome host-defence mechanisms.

In this mechanism a certain peptide termed autoinducing peptide (AIP) is produced from a pro-peptide called AgrD and further processed and exported by a membrane protein AgrB to create a cyclic AIP. When a certain threshold concentration of AIP in the environment is reached, AIPs activate the receptor-histidine kinase AgrC.  

Upon induction AgrC phosphorylates the intracellular response regulator AgrA with its cytosolic histidine kinase domain [http://www.annualreviews.org/doi/abs/10.1146/annurev.genet.42.110807.091640 (3)]. Phosphorylated AgrA then binds to distinct sites in the P2 and P3 promoter-region and activates these promoters. [http://jb.asm.org/content/193/21/6020 (2)]

The P2 promoter activates transcription of the ''agrBDCA''-Operon and thereby represents a positive feedback-loop. The P3 promoter activates transcription of the RNA-III transcript, involved in virulence gene expression [http://jb.asm.org/content/182/22/6517.full (4)]).

Interestingly, accumulated mutations in the hypervariable region of the ''agrBDCA''-operon have led to four different pherotypes of ''S. aureus'', using different AgrC-types, and producing different AIPs (AIPI, AIPII, AIPIII, AIPIV), which cross-inhibit each other and might correlate with certain diseases associated with ''S. aureus'' [http://www.sciencemag.org/content/287/5452/391.full.html (5)].

All over the world, ''Streptococcus pneumoniae'' causes the life threating invasive diseases pneumonia, sepsis and meningitis. In early childhood the diplococcus colonizes the epithelium of the upper respiratory tract as a commensal bacterium. Under certain circumstances,  it gets a threat by tissue invasion.  Still up to date, not much is known regarding the mechanism behind the shifting from a colonizer organism to an invader or how ''S. pneumoniae'' is able to cross the blood-brain barrier to cause meningitis [http://www.ncbi.nlm.nih.gov/pubmed/19880020 (6)].

To cross the line from an opportunist to a pathogen, ''S. pneumoniae'' needs to express specific virulence factors in a coordinated way.  Apart from virulence factors like the polysaccharide capsule, the pore forming toxin pneumolysin or surface proteins like PspA and PspC, also biofilm production plays an important role for persistence in the human nasopharynx and contributes to pneumonia and meningitis [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1618759 (7)].

One aspect of its infectiousness is the ability to perform natural transformation. Thus, it can take up extracellular DNA containing the information for resistance against diverse antibiotics and permanently incorporate the DNA into its genome via recombination.  The main [https://2014.igem.org/Team:LMU-Munich/Project/Problem problem] of treating ''S. pneumoniae'' infections is the increasing spread of ß-lactam resistance. Luckily, the molecular mechanism behind natural transformation in ''S. pneumoniae'' is the focus of current research.

In ''S. pneumoniae'', biofilm production and competence for natural genetic transformation are triggered by a two component regulatory system, responsive to environmental stimuli. There, a peptide pheromone called competence-stimulating-peptide (CSP) binds to and consequently activates the membrane-embedded sensor kinase ComD by changing the conformation of the polytopic kinase. ComD autophosphorylates and subsequently transphosphorylates the cognate response regulator ComE. ComE acts as transcription activator via binding to the -10 promoter regions of early and late competence genes. These ComE binding sites (CEbs) consist of a 9 bp direct repeat seperated by a stretch of 12 nucleotides.

[[File:LMU14 ComDE.png|thumb|left|700px|'''Fig. 2''': Competence regulation in ''Streptococcus pneumoniae''. See text for more details. (Jonsborg et al. 2009)]]

A positive feedback loop ensures the availability of the two component system by activating the expression of the ''comCDE'' operon, where ''comDE'' encodes for the two component system ComDE and ''comC'' for the precursor peptide pre-CSP. Pre-CSP is cleaved to the mature peptide pheromone and transported to the extraplasmic room by the ABC transporter ComAB, whose expression is also activated by ComE. [http://www.ncbi.nlm.nih.gov/pubmed/?term=regulation+of+natural+genetic+transformation+and+acquisition+of+transforming+DNA (8)].  

In addition, ComE~P is able to trigger the expression of ''comX'' and ''comW '', encoding for the alternative σ-factor X, and its stability factor ComW. This alternative σ-factor is required for the transcription of the so called late competence genes involved in the process of DNA uptake and recombination.

We are working with the genomic DNA of the nonencapsulated apathogenic strain R6 of ''S. pneumoniae'', whose genome is completely sequenced [http://www.ncbi.nlm.nih.gov/pubmed/11544234 (9)]. CSP is a 17 amino acid long linear protein  with the following amino acid sequence: Glu-Met-Arg-Leu-Ser-Lys-Phe-Phe-Arg-Asp-Phe-Ile-Leu-Gln-Arg-Lys-Lys-OH [http://www.microbesonline.org/cgi-bin/fetchLocus.cgi?locus=134925&disp=4 (10)] with a charged N-terminal residue and a positively charged C-terminal tail [http://www.ncbi.nlm.nih.gov/pubmed/16484185 (11)].

The two component system [http://parts.igem.org/Part:BBa_K1351036 ''agrCII-A''] for ''S. aureus'' has been designed as a composite part derived from ''Staphylococcus aureus'' N315 gDNA.   

The two intermediated parts ''agrC-II'' and ''agrA'' were amplified by PCR using primers with modified RFC25 prefix and suffix. And then fused by using the restriction sites EcoRI and SpeI for ''agrC-II'', as well as XbaI and PstI for ''agrA'', thus creating the composite part [http://parts.igem.org/Part:BBa_K1351036 ''agrCII-A''].  

The operon encoding for the histidine sensor kinase ComD and the response regulator ComE ''comDE'' for ''S. pneumoniae'' sensing was designed in two different ways: On one hand, the native ''comDE'' operon with its native ribosome binding site (RBS) ([http://parts.igem.org/Part:BBa_K1351015 BBa_K1351015]), on the other hand the''comDE'' operon with the ''B. subtilis'' adapted RBS (TAAGGAGG) in the BioBrick standard RFC25 ([http://parts.igem.org/Part:BBa_K1351016 BBa_K1351016]). Unfortunately, the operon contains two EcoRI and one SpeI restriction site. The attempts to mutagenize these restriction sites were not completed successfully in time as one recognition site remained. Thus, the ''comDE'' operon could not been sent to the registry - but this will be accomplished as soon as possible.

In ''S. aureus'', the AgrA-sensitive promoters P2 [http://parts.igem.org/Part:BBa_K1351037 BBa_K1351037] and P3 [http://parts.igem.org/Part:BBa_K1351038 BBa_K1351038] with four AgrA binding sites downstream of the -35 signals were used for evaluation. The intergenic region with suspected regulator binding sites is shown in Fig. 3

[[File:LMU14 P2P3.jpg|center|thumb|700px|'''Fig. 3''': AgrA dependent promoters P2 and P3 with the intergenic region containing several regulator binding sites for AgrA (arrows) and SarA/R (dashed lines) (figure taken from Reyes et al. 2000)]]

For ''S. pneumoniae'' sensing, the two  ComE dependent promoters P_''comC'' [http://parts.igem.org/Part:BBa_K1351024 BBa_K1351024] and P_''comAB'' [http://parts.igem.org/Part:BBa_K1351025 BBa_K1351025] and additional promoters containing putative ComE binding sites (CEbs) P_''mreA'',  P_''natAB'') were taken for evaluation (see figure 4).

[[File:LMU14 ComEdep promoters.png|center|thumb|700px|'''Fig. 4''': ComE dependent promoters containing bases matching (blue) and differing (red) from the CEbs consensus sequence. (figure modified from Martin et al. 2013)]]

The BIG result we obtained in the night of the wiki freeze performing a [https://2014.igem.org/Team:LMU-Munich/Notebook/Protocols luminescence assay] with different constructs in ''B. subtilis'' P_''xyl'' - ''comDE'' P_''QS'' - ''lux'' based on [http://parts.igem.org/Part:BBa_K1351016 BBa_K1351016]. Three different P_''QS'' were tested: P_''comC'', P_''comAB'', P_''mreA''. The ''comDE'' operon expression was induced with 0 %, 0,02 % and 0,2 % xylose. Adding 0 mg/ml CSP, 100 mg/ml CSP and 1000 mg/ml CSP ''luxABCDE'' expression is only activated if ''B. subtilis'' was able to sense CSP due to the ''comDE'' operon.

For P_''mreA'', no significant luminescence output could be measured concerning differing CSP concentrations. Nevertheless, at a xylose concentration of 0,02 % and induction of 1000 mg/ml CSP P_''comC'' and P_''comAB'' showed a 3 fold increased luminescence output compared with an induction of 0 mg/ml and 100 mg/ml CSP.

Surface display of the pathogen-binding peptides is achieved by translational fusions to naturally occurring surface proteins of ''B. subtilis''. Cell wall-binding modules of autolysins are used for non-covalent interaction with the ''B. subtilis'' cell wall, whereas a sortase-based system functions via covalent attachment to the cell wall. In both cases, a linker between cell wall anchor and peptide allows flexibility of the fusion protein and increases its range. Peptides binding to different receptors on the surface of pathogens mediate specific binding.

As depicted in figure 1A, cell-wall binding domains (CWB) of LytC or LytE ([http://parts.igem.org/wiki/index.php?title=Part:BBa_K1351006 BBa_K1351006] or [http://parts.igem.org/wiki/index.php?title=Part:BBa_K1351007 BBa_K1351007]) should be translationally fused to a protein of interest for non-covalent anchoring of the latter to the cell wall. These CWB domains also included the native signal peptide (SP) of the respective proteins to direct export via the Sec pathway. For covalent anchoring, signal peptide ([http://parts.igem.org/Part:BBa_K1351008 BBa_K1351008]) and cell wall anchoring domain (CWAD, [http://parts.igem.org/Part:BBa_K1351010 BBa_K1351010]) of the sortase substrate YhcR and a 57 amino acid linker ([http://parts.igem.org/Part:BBa_K1351009 BBa_K1351009]) were assembled with the protein of interest as depicted in figure 1B to generate a YhcR-like fusion.

To evaluate the functionality of the constructs, the latter should be cloned into the replicative ''B. subtilis'' vector pBS0K under the control of the inducible promoter P_''spac''. Two different proteins should be for successful display: First, a superfolder GFP variant (sfGFP) was used for fluorescence microscopy to check if the constructs locate to the cell wall as shown in figure 2. This variant of GFP had been shown previously to fold in the periplasm of gram-negative bacteria [10] and could thus be expected to be fuctional also in the extracellular environment, in contrast to normal GFP that does not fold under reducing conditions. As controls for the sfGFP constructs, only the signal peptides without the respective cell wall binding or anchoring domains were fused to sfGFP, which should lead to secretion of the fusion into the growth medium.

Secondly, fusions to alkaline phosphatase (PhoA) should be used to detect surface accessibility of the presented protein, as PhoA is only active when exported out of the cytoplasm [11]. Agar plates containing the PhoA substrate XP (5-bromo-4-chloro-3-indolyl-phosphate) indicate enzyme activity by turning blue where the substrate is hydrolyzed and further oxidized to an indigo dye [12]. PhoA was also fused to intracellular GFP as a negative control and to an extracellular domain of LiaI as a positive control. The PhoA-deficient ''B. subtilis'' strain MH3402 will be used for expression of all PhoA constructs.

 

Unfortunately, the status of these constructs is still "cloning".

Evaluation of peptides binding to ''S. pneumoniae'' was done by a biochemical pull-down assay with their binding partner NADH oxidase (NOX). As shown in figure 3, NOX was bound to streptavidin-coupled beads via an N-terminal fused Strep-Tag II, for which the pASK-IBA expression vector system was used. ''S. pneumoniae''-specific peptides C4P, CSP and L5P ([http://parts.igem.org/Part:BBa_K1351001 BBa_K1351001], [http://parts.igem.org/Part:BBa_K1351002 BBa_K1351002] and [http://parts.igem.org/Part:BBa_K1351003 BBa_K1351003], respectively) were fused C-terminally to a maltose-binding protein (MBP) and a 6xHis-tag into the pKLD66 vector. This was achieved by digestion of the BioBricks and pKLD66 with NotI and HindIII and ligation of the products. The MBP prevents degradation of the short peptides by proteases and was used for detection of the peptide fusions on an SDS gel. To increase specificity of the detection, the 6xHis-tag was used for Western Blot analysis of the pulled down proteins. Additionally, ''S. pneumoniae'' itself was used as cellular beads for a pull down of the same peptide-MBP-His constructs, again followed by analysis with SDS gel and Western Blot.

[[File:LMU14_Adhesion_Results_Figure3.PNG|thumb|center|700px|'''Figure 3:‎SDS-PAGE of the lysates and the fractions of the incubated S. pneumoniae cells.''' E. coli cells carrying the plasmids coding for C4P, CSP and A5P were lysed. ''S. pneumoniae'' cells were resuspended in the lysate and incubated for 30 minutes, followed by pelleting to separate the supernatant (SP) from the cell pellet (P).  3 μl of sample were applied per lane.]]

The gel of the SDS-PAGE (Fig. 3) shows strong bands for the lysates at 40kDa, indicating a high amount of the His-MBP tagged peptides. The empty vector shows a relatively low expression of His-MBP.  The pellet fractions of the ''S. pneumoniae'' cells incubated with the tagged C4P and CSP peptides show light bands at the same height indicating adhesion of the peptides to the cells (Fig. 4, white arrows). Although the cells incubated in buffer alone also show a band at the same height indicating a native protein of that size (Fig. 3, black arrows) the amount of protein is clearly less. To determine whether the band represents the His-MBP tagged peptides, a western blot was performed (Figure 4).

[[File:LMU14_Adhesion_Results_Figure5.PNG|thumb|center|800px|'''Figure 4: Western-Blots of the lysates and the fractions of the incubated ''S. pneumoniae'' cells.''' Duplicates of the gel from Figure 4 (A) and another gel loaded with 10 μl/ lane (B) were processed by western blot with Anti-His antibodies.]]

The blot shows a clear signal for the applied lysates and all the pellet fractions of the cells incubated with the peptides, indicating their adhesion to ''S.pneumoniae''. The band for the His-MBP of empty vector is missing (or very weak), which might be due to the lower expression, but one can clearly detect the bands corresponding to the His-MBP in the SDS-PAGE (Fig. 4). Our results strongly indicate that the previously described adhesion of the peptides to ''S. pneumoniae'' works. Thus, our BioBricks [http://parts.igem.org/Part:BBa_K1351001 BBa_K1351001], [http://parts.igem.org/Part:BBa_K1351002 BBa_K1351002] and [http://parts.igem.org/Part:BBa_K1351003 BBa_K1351003] should serve as a tool for adhesion to ''S. pneumoniae''.

Lantibiotics are promising candidates for future antimicrobials, as they inhibit the growth of many clinically relevant pathogens comprising even multidrug-resistant bacteria. Moreover, lantibiotics have only low tendency to generate resistance. Both features make them highly attractive for medical applications. [http://www.unboundmedicine.com/medline/citation/24210177/Lantibiotics:_promising_candidates_for_future_applications_in_health_care_[1]] [http://www.ncbi.nlm.nih.gov/pubmed/16205711[2]]

[http://www.ncbi.nlm.nih.gov/pubmed/?term=Quorum+sensing+control+of+lantibiotic+production%3B+nisin+and+subtilin+autoregulate+their+own+biosynthesis[3] ]]]

[[File:LMU14 killing subtilin genecluster.png|thumb|800px|center|Fig. 2. Organisation of the subtilin biosynthetic gene cluster.  [http://www.ncbi.nlm.nih.gov/pubmed/?term=Quorum+sensing+control+of+lantibiotic+production%3B+nisin+and+subtilin+autoregulate+their+own+biosynthesis [3] ]]]

<i>Table 1: The role of the different proteins of the ''spa''-locus</i>

Of course, the producer needs to be immune against the antimicrobial agent it produces. ''B. subtilis'' ATCC6633 protects itself against subtilin by two mechanisms that both act independently and confer some level of resistance, however full resistance is only achieved when both mechanisms are active. SpaI is a membrane-anchored lipoprotein. It is suggested that it binds the antimicrobial peptide and by this keeps it away from the membrane. SpaFEG is a LanFEG-like ABC-transporter that works by exporting subtilin from the cytoplasmic membrane to the culture supernatant. [http://www.ncbi.nlm.nih.gov/pubmed/15659659 [4]] [http://www.ncbi.nlm.nih.gov/pubmed/?term=Alkhatib%2C+Z.%3B+Abts%2C+A.%3B+Mavaro%2C+A.%3B+Schmitt%2C+L.%3B+Smits%2C+S.+H.%3A+Lantibiotics%3A+how+do+producers+become+self-protected%3F+Journal+of+biotechnology+2012%2C+159%2C+145-54. [5]]

[[File:LMU14 killing subtilin regulation.png|thumb|400px|right|Fig. 3. Dual control of subtilin biosynthesis and immunity. [http://www.ncbi.nlm.nih.gov/pubmed/11972779 [6] ]]]

Lantibiotics are active against a wide range of Gram-positive bacteria including multidrug-resistant pathogens. Some lantibiotics have a dual mode of action, subtilin is proposed to be among them. Subtilin forms a complex with the cell wall precursor lipid II, whereby the pyrophosphate moiety may play a key role for target recognition. Thereby, the cell wall biosynthesis is inhibited. Furthermore, the complexes aggregate and form a pore in the bacterial membrane by using lipid II as a docking molecule. This leads to a depolarisation of the membrane potential, the efflux of cytoplasma and in turn to rapid cell death. Just like nisin, which is highly similar, subtilin is still active in the nanomolar range and thus serves excellently as alternative killing strategy against multidrug-resistant pathogens. [http://www.ncbi.nlm.nih.gov/pubmed/?term=Molecular+Mechanism+of+Target+parisot [7]] [http://www.researchgate.net/publication/7875075_Mode_of_action_of_lipid_II-targeting_lantibiotics [8]]

 

[[File:LMU14 killing subtilin modeofaction.png|frame|150px|center|Fig. 4. Proposed mechanism of pyrophosphate-mediated target engagement by the lanthionine antibiotic subtilin. Membrane breaches occur in a lipid II-mediated fashion and cell wall synthesis is impeded through engagement of pyrophosphate-containing intermediates into complexes.  [http://www.ncbi.nlm.nih.gov/pubmed/?term=Molecular+Mechanism+of+Target+parisot [7] ]]]

[[File:LMU14 killing subtilin growthcurve pneumo.png|thumb|600px|center|Fig. 1. Growth curve of ''S. pneumoniae'' measured in the plate reader in 100 µl THB broth.]]

[[File:LMU14 killing subtilin Pneumo-halo.png|thumb|400px|center|Fig. 2. The spot-on-lawn assay shows a clear halo around the ''B. subtilis'' ATCC6633 spot. No clearing zone is visible around ''B. subtilis'' W168.]]

As ''S. pneumoniae'' is a target of the BaKillus killing strategies, a spot-on-lawn-assay should reveal the inhibitory impact of subtilin. Therefore, spots of the ''B. subtilis'' W168 wild type and the subtilin-producer ''B. subtilis'' ATCC6633, respectively, were spotted on a  ''S. pneumoniae'' lawn. As depicted in figure 2 and 3, the subtilin producer significantly inhibits the growth of ''S. pneumoniae''. In contrast,  ''B. subtilis'' W168 wild type does not show inhibitory impact. The experiment was performed three times and the mean value and the standard deviation are illustrated in the graph below.

[[File:LMU14 killing subtilin Pneumo-inhibition diagramm.png|thumb|700px|center|Fig. 3. Analysis of subtilin impact on ''S. pneumoniae''.]]

Moreover, the impact of subtilin on ''Staphylococcus aureus'' was tested in collaboration with iGEM Team Groningen, as they have access to S2 laboratories, in which ''S. aureus'' and even multidrug-resitant species are handled. A member of Team Groningen kindly performed spot-on-lawn assays with ''B. subtilis'' W168 wild type and the subtilin-producer ''B. subtilis'' ATCC6633 on different ''S. aureus'' species. The tested strains were CAL, NWZ (both MRSA, obtained at the UMCG hospital) and CECT240 (''Staphylococcus aureus'' subsp. ''aureus'', Rosenbach 1884). Strains were plated out in soft agar and spots from overnight cultures of ''Bacillus subtilis'' grown in either complex medium (LB) or defined medium (SMM) were added on top. [[File:LMU14 killing subtilin MRSA.png|thumb|700px|center|Fig. 4. Spot-on-lawn assay with the subtilin producer and on three different ''S. aureus'' lawns]]

[[File:LMU14 killing MRSA diagramm.png|thumb|500px|center|Fig. 5 Bar chart of the ''S. aureus'' spot-on-lawn assay.]]

The aim of this approach was to transfer the complete subtilin gene cluster into our working horse ''Bacillus subtilis'' W168 by maintaining the native regulation mainly mediated by the ''spaRK'' two-component system. So far, the 12 kb-locus is assumed to be transferred into our ''Bacillus'' wild type but only half of the locus is sequenced. Unfortunately, no subtilin production could be ascertained. This could be due to an incorrect sequence or the fact that the native regulation of ''B. subtilis'' ATCC6633 does not work in our wild type. Our next steps are the full sequencing of the putative ''spa''-gene cluster and an attempt to transfer it into ''B.subtilis'' W168 Δ''abrB'', a mutant that lacks the repressing transition state regulator.

This approach aims to "BioBrick" and by this standardize the subtilin gene cluster according to its transcriptional entities. In contrast to the heterologous expression, the BioBricks will be under quorum sensing dependent regulation.

<b>Modification & Transport</b>

The BioBrick [http://parts.igem.org/Part:BBa_K1351041 BBa K1351041], encoding SpaBTC, is mainly responsible for the modification and the export of the subtilin propeptide. After the removal of 6 BioBrick restriction sites within the three genes, it is now in the RFC10 BioBrick standard.

[http://parts.igem.org/Part:BBa_K1351012 BBa K1351012] and [http://parts.igem.org/Part:BBa_K1351013 BBa K1351013] are coding for SpaS, the subtilin pro-peptide. Their main difference are their overhangs, one being in RFC25, which allows the tagging or translational fusion of [http://parts.igem.org/Part:BBa_K1351012 BBa K1351012], whereas the other can be used according to the RFC10 BioBrick standard. Unfortunately non of them could be evaluated so far, as the development of the immunity BioBrick as well as of the modification and export BioBrick were required beforehand.

 

[[File:LMU14 killing subtilin Immunity assay.png|thumb|700px|center|Fig. 6. Supernatant-in-lawn assay shows smaller halo diameters for ''B. subtilis'' with BioBRick-acquired immunity. ]]

The part was evaluated with a variation of an spot-on-lawn assay. In this case, holes were cut out of agar plates on which either ''B. subtilis'' W168 wild type or ''B. subtilis'' W168 ''amyE''::P_''xyl''_''spaIFEG'' were plated. The holes were then filled with different amounts of sterile supernatant from overnight cultures of W168 or ATCC6633, respectively. The xylose-containing plates were incubated at 37 °C for 24 h and resulting halo diameters were measured. The supernatant derived from the negative control (W168) did not cause any growth inhibition to neither the wild type, nor to the strain who carries the immunity BioBrick (Figure 6). The subtilin-containing supernatant from ''B. subtilis'' ATCC6633 however, was able to inhibit the growth of both bacterial lawns. For the lowest amounts (32 µl), no significant difference between the halo diameters could be determined. The larger the amount of supernatant, and presumably, the higher the amount of subtilin in there, the bigger the zones of inhibition. For the largest volume (128 µl), a two-sided T-test could determine the p-value below 0.1 (p=0,007762603) and by this prove the functionality of this BioBrick. This is a major step for this project, as the immunity is the base for any strain which produces antimicrobial peptides. Now further steps can be taken and the remaining BioBricks can be evaluated.

 

 

 

[[File:Supernatant in-lawn.png|thumb|700px|center|Fig. 7 Evaluation of the ''spaIFEG'' BioBrick ]]

During stationary phase of a ''Bacillus subtilis'' culture it has been observed that some cells start producing a toxin which causes cell lysis of sister cells whereas the toxin-producer cells stay unaffected. [http://www.sciencemag.org/content/301/5632/510.full [1]] This social and heterogenous behavior is called cannibalism and seems to be an even more promising basis for constructing the BaKillus “suicide switch“ since it has been shown that the toxin also kills a variety of Gram-positive bacteria, including MRSA-strains. [http://www.pnas.org/content/107/37/16286.full [2]]

As soon as BaKillus detects QS-molecules of ''S. aureus'' or ''S. pneumoniae'' the cannibalism toxin SDP is produced and secreted. This leads to the co-expression and co-regulation of a transmembrane protein responsible for the immunity to SDP by binding the extracellular toxin. Simultaneously to the SDP production, the expression of an alternative σ-factor initiates a delayed suicide process by activating either one or both of two types of RNA: an antisense RNA, complementary to the mRNA of the immunity and a small RNA fsrA which has a binding site in the added 5'-UTR of the immunity coding mRNA. As a result the translation of the immunity protein is inhibited and the immunity is lost with the decay of the existing immunity protein. Finally BaKillus is now sensitve to the SDP making cell lysis inevitable.

[http://www.sciencemag.org/content/301/5632/510.full [1]] Gonzalez-Pastor, J. E., et al. (2003). "Cannibalism by sporulating bacteria." Science 301(5632): 510-513.

[http://www.pnas.org/content/107/37/16286.full [2]] Liu, W. T., et al. (2010). "Imaging mass spectrometry of intraspecies metabolic exchange revealed the cannibalistic factors of Bacillus subtilis." Proc Natl Acad Sci U S A  

'''The ''sdp''-System of ''B. subtilis''''' consists of two operons: The ''sdpABC'' operon, coding for the production and secretion of the cannibalism toxin SDP and the ''sdpRI'' operon responsible for the regulation and production of the immunity protein SdpI (Fig. 1).  

[[File:LMU14 suicide background Fig.1.png|thumb|800px|center|Fig. 1.  Gene organization for the ''sdpABC'' ''sdpRI'' operons. The hairpin symbolizes thetranscriptional terminators. [http://www.sciencemag.org/content/301/5632/510.full [1] ]]]  

In vegetative cells, both operons are repressed by the unstable AbrB regulator. However, during early stages of sporulation AbrB itself is repressed by the master regulator of sporulation Spo0A, making ''sdpABC'' and ''sdpRI'' accessible for RNA polymerase. [http://www.sciencemag.org/content/301/5632/510.full [1]]

=== The ''sdpABC'' Operon – Production and Secretion of the Cannibalism Toxin SDP ===

The production of the Cannibalism Toxin SDP is a multi-step process.  The ''sdpC'' sequence encodes the Pro-SdpC1-203,,which is translated by the ribosome.It is a precursor peptide which needs to be processed by a signal peptidases and the two membrane proteins SdpA and SdpB to become functional. This active form of SDP is a 42-amino-acid antimicrobial peptide (AMP) containing a disulfide bond between two cysteine residues located at the N-terminus.(Fig. 2). [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697648/ [2]]

[[File:Background Fig.2.png|thumb|800px|center|Fig. 2. SDP production requires multiple steps. In the cytosol, the full length SdpC (pro-SdpC1-203) is secreted via the Sec pathway. Following secretion, the signal peptidases SipS and SipT cleave the N-terminal signal peptide sequence of SdpC. Disulfide bond formation occurs independently of SdpAB. Finally, posttranslational cleavage of SdpC occurs via SdpAB to produce a 42-amino-acid SDP that will be secreted extracellulary as the active SDP peptide. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3697648/ [2] ]]]  

SDP has been shown to be a very effective AMP against a variety of Gram-positive bacteria in the Phylum of the Firmicutes (Fig. 3). It rapidly collapses the proton motive force (PMF), thus inducing autolysis. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3839633/ [3]]

[[File:Background Fig.3.png|thumb|600px|center|Fig. 3. SDP inhibition curves for pathogenic microbes. Relative growth of the strains named abovewith the presence of increasing concentrations of SDP is shown in the curve. As a negative control the gram-negative bacteria ''K. pneumoniae'' and ''P. aeruginosa'' are depicted, which are unaffected by the toxin SDP, as it specifically targets gram-positive bacteria. ''B. subtilis'' is a gram-positive bacteria, but expresses the immunity protein SdpI and is therefore relatively resistent to the toxin SDP. the ''Stapylococcus'' species (also MRSA) though are quite drastically reduced in the presence of the SDP. [http://www.pnas.org/content/107/37/16286.full [4] ]]]

=== The ''sdpIR'' Operon – Production and Regulation of the Immunity Protein SdpI ===

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

By the activation of the QS-dependent promoter P_''QS'', BaKillus produces the Cannibalism Toxin SDP (Fig. 1A). For evaluation purposes we cloned the ''sdpABC'' operon under the control of inducable xylose promoter P_''xyl'' (Fig. 1B) into a ''∆sdpAB'' and a ''∆sdpC'' mutant strain of ''B. subtilis'' W168.

 

 

 

 

 

A time delayed loss of the immunity protein SdpI in BaKillus can be achieved by interposing the alternative σ-factor ECF41_{''bli aa 1-204''} [http://parts.igem.org/Part:BBa_K823043 BBa_K823043] which is also under the control of the quorum sensing promoter P_''QS'' (Fig.3A). When expressed, ECF41_{''bli aa 1-204''} binds to the target promoter P_''ydfG'' [http://parts.igem.org/Part:BBa_K823041 BBa_K823041] leading either to the expression of fsrA, a small RNA, or transcription of sdpI-antisense, a RNA, a complementary RNA to the sdpI mRNA (Fig. 3B).  

 

 

For the loss of immunity we implemented two strategies, both causing translational inhibition of the ''sdpI'' mRNA.  

The first strategy is based on an artificial synthesized RNA called sdpI-antisense RNA, which is complementary to the sdpI mRNA sequence. The sdpI-antisense sequence is regulated by P_''ydfG'' (Fig. 4A) which is activated by the binding of Ecf41_{''bli aa 1-204''}. As a consequence the sdpI-antisense RNA and the sdpI mRNA form a double-stranded duplex which is inaccessible for the ribosome making the cell SDP-sensitive.

 

 

The second strategy involves fsrA, a small RNA regulated by P_ydfG which binds to its binding site sdhC' (the 5'-UTR of sdhC mRNA) which is cloned between P_''sdpRI'' and ''sdpI'' (Fig. 5A). Binding of fsrA to the sdhC' site on the transcript inhibits the ribosomal translation process of the sdpI-mRNA.

To evaluate this small RNA and its binding site in ''B. subtilis'' we designed two strains. One ''∆abrB ∆sdpI'' mutant strain where we replaced the P_''ydfG'' promoter with the P_''xyl'' promoter (Fig. 5B). The second construct containing fsrA under control of P_''xyl'' and replaced P_''sdpRI''-''sdhC'sdpI'' with the P_''lepA''-''sdhC'gfp'', where GFP is consecutively expressed, (Fig. 5C) was transformed into ''B. subtilis'' W168.

 

[[File:Design Fig.5.png|600px|center|Fig. 5.]]

 

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