Difference between revisions of "Part:BBa K1351043"

Latest revision as of 18:36, 30 October 2014

Canibalism toxin SDP of B. subtilis

This part was generated with the RFC10 standard and has the following prefix and suffix:

Sites of restriction enzymes generating compatible overhangs have the same color: EcoRI and PstI in blue, NotI in green, XbaI and SpeI in red.

This part is used in the 2014 LMU-Munich iGEM project [http://2014.igem.org/Team:LMU-Munich BaKillus].

Background

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).

Fig. 1. Gene organization for the sdpABC sdpRI operons. The hairpin symbolizes thetranscriptional terminators. [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. [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). [2]]

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. [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. [3]

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. [4]

Sources

[1] Gonzalez-Pastor, J. E. (2011). "Cannibalism: a social behavior in sporulating Bacillus subtilis." FEMS Microbiol Rev 35(3): 415-424.

[2] Perez Morales, T. G., et al. (2013). "Production of the cannibalism toxin SDP is a multistep process that requires SdpA and SdpB." J Bacteriol 195(14): 3244-3251.

[3] Lamsa, A., et al. (2012). "The Bacillus subtilis cannibalism toxin SDP collapses the proton motive force and induces autolysis." Mol Microbiol 84(3): 486-500.

Design

Production of the toxin SDP and 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. The construct was then tested by spot-on-lawn assays on B. subtilis W168 and B. subtilis W168 ∆sdpI mutant lawns.

Results

Fig. 1. Spot-on-lawn assays on Bacillus subtilis W168 and Bacillus subtilis W168 ΔsdpI lawns

The functionality of our BioBrick was tested by performing spot-on-lawn assays. For the lawns we chose B. subtilis W168 wildtype and W168 sdpI mutant strains. Three different strains were spotted from overnight cultures: B. subtilis W168 wildtype as a positive control, W168 ΔsdpAB and W168 ΔsdpC both carrying our inducible BioBrick (Fig. 1).

As a result you can see a xylose dependent SDP overexpression in the sdpAB and sdpC mutant strains causing clearly recognizable zones of clearance on both lawns, whereas the wildtype W168 shows no clearance. The effect is higher in the sdpAB mutant strain than in the sdpC mutant.This is probably due to the fact, that in the sdpAB mutant the native sdpC is still intact and therefore even more sdpC is produced than in the sdpC mutant.

Furthermore we proved once more that in the presence of the toxin SDP, loss of the immunity protein SdpI leads to significant cell death.

Sequence and Features