THE PEPTIDE

In our system, the hexapeptide SAIRGA is the signalling molecule used for cell-to-cell communication [1]. To perform its quorum sensing function in the natural phi3T phage infection, SAIRGA needs to be secreted out of the cell. It is produced as an immature pre-pro-peptide, AimP, that upon secretion is cleaved extracellularly to remove the secretion signal and release the mature hexapeptide. The mature peptide then enters cells and binds to its receptor protein AimR. Binding of SAIRGA to AimR blocks the activator function of AimR that, in turn, facilitates a switch from lysogenic-to-lytic viral cycle. By acting as a negative regulator of AimR, the SAIRGA signal makes the lysogeny-to-lysis switch of phi3T phage dependent on the “quorum” of Bacillus cells. In our system, the hexapeptide SAIRGA is a key component which is responsible for cell-to-cell communication (ref. Erez, Zohar, et al. "Communication between viruses guides lysis–lysogeny decisions." Nature 541.7638 (2017): 488). To perform its function, SAIRGA needs to be secreted. It is produced as an immature peptide, AimP, that, after secretion is cleaved and its secretion signal removed to release the mature hexapeptide. Only the mature peptide, SAIRGA, is able to bind to AimR, its receptor.

DESIGN

In Bacillus subtilis, SAIRGA is produced with a specific secretion tag (ref. Erez, Zohar, et al. "Communication between viruses guides lysis–lysogeny decisions." Nature 541.7638 (2017): 488). This secretion tag is cleaved after the secretion of SAIRGA by a Bacillus extracellular protease. Since we had no further information about this protease, we could not investigate the ability of Escherichia coli to produce a similar enzyme. Consequently, to produce SAIRGA in E. coli, we have decided to replace this tag by an E. coli secretion signal. We have investigated three different secretion signals: OmpA (BBa_K208003), PelB (BBa_J32015) and TAT of csp2 gene of Corynebacterium glutamicum (Uniprot Q04985).

Part Name	CDS Sequence	Part Number
AimP	MKKVFFGLVILTALAISFVAGQQSVSTASASDEVTVASAIRGA	BBa_
OmpA_SAIRGA	MKKTAIAIAVALAGFATVAQASAIRGA	BBa_
PelB_SAIRGA	MKYLLPTAAAGLLLLAAQPAMASAIRGA	BBa_
Tat_SAIRGA	MFNNRIRTAALAGAIAISTAASGVAIPAFASAIRGA	BBa_

To express them in E. coli, we have codon optimized all sequences for E. coli DH5α, have designed specific RBSs with the Salis RBS Calculator (ref. A. Espah Borujeni, A.S. Channarasappa, and H.M. Salis, "Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites", Nucleic Acid Research, 2013 ; H.M. Salis, E.A. Mirsky, C.A. Voigt, Nat. Biotech., 2009) and have placed these peptides under the control of the constitutive strong promoter J23100 (BBa_J23100). Thus, we generated four SAIRGA expression plasmids (BBa_xxx to BBa_xxy).

EXPERIMENTS

E. coli DH5α harboring the SAIRGA expression plasmids were grown at 30°C and 37°C in mineral salts medium (ref. Hall Mol Biol Evol, 1998, 15, 1−5.) containing 2 mg/mL glucose and 35 μg/mL chloramphénicol. During the culturing, samples were prelevated, centrifuged and the supernatant was first sterilised by filtering through a 0.22 µm membrane, then loaded on a Pall Nanosep centrifugal device with Omega membrane (MWCO 3 kDa). The thus recovered low molecular weight fraction of the culturing media was analysed by LC-MS: ultra high pressure liquid column chromatography (UPLC) on a HSST3-C18 column (Waters) coupled with a Xevo G2-S Qtof mass spectrometer (Waters). The chromatography was performed using a gradient of using a (A) H2O-1% formic acid / (B) ACN-1%formic acid with the following.... The column was heates at 45°C and the ions were detected in resolution negative mode. The pure synthetic SAIRGA peptide was readily detected by LC-MS. As shown in figures 1 and 2, a peak of 572 Da, which correspond to SAIRGA’s monoisotopic mass [M-H]- was observed at a retention time of 3.9 min (figure 3). The area under the curve is proportional to the concentration of SAIRGA (figure 4).

**Figure 2**: Mass spectrometry analysis of SAIRGA’s fraction obtained with 5 µM of pure synthetic SAIRGA peptide (zoom on SAIRGA’s peak).

**Figure 4**: Standard curve of the area under the curve obtained by UPLC as a function of the concentration of SAIRGA.

RESULTS

LC-MS analysis of samples isolated from the fermentation broth of the E. coli strains harbouring a SAIRGA expression plasmid was performed. As shown in Figures 5 and 6, the part OmpA_SAIRGA (BBa_xx) is able to produce a compound with a monoisotopic mass a peak of 572 Da, which correspond to SAIRGA’s monoisotopic mass [M-H]- at a retention time of 3.9 min. The size or the area under the peak is increasing with time (figure 7). This suggests that OmpA is an efficient E. coli secretion tag which allows the release of the mature peptide SAIRGA in the culturing media.

**Figure 5**: Mass spectrometry of culturing media taken after 2, 4, 6 and 8 hours of incubation of E. coli cells harboring pSB1C3_OmpA_SAIRGA. The negative control has been performed with en empty pSB1C3 (bottom red chromatogram). The positive control (pure synthetic SAIRGA at 5 µM) is represented on the top red chromatogram.

**Figure 7**: SAIRGA production at 37°C by E. coli cells harboring the various SAIRGA expression plasmids (BBa_xxx to BBa_yyy). The negative control has been performed with en empty pSB1C3.

**Figure 8**: Growth curves of the differents clones at 37°C

The other parts have not shown any peak at 572 Da, which suggests that the original Bacillus secretion tag, PelB and Tat are not able to release the mature peptide. Our hypothesis is that, in each case, the pro-peptide is produced and released but its secretion tag is not cleaved by the cell.

As shown in Figure 9, the cells which have been grown at 30°C have not shown any significant production of SAIRGA.

**Figure 9**: SAIRGA production at 30°C by E. coli cells harboring the various SAIRGA expression plasmids (BBa_xxx to BBa_yyy). The negative control has been performed with en empty pSB1C3.

Moreover, after 24h of incubation, we were unable to detect the SAIRGA peptide in the culturing media (Figures 10 and 11). This is probably due to the exhaustion of the medium: the cells might have used SAIRGA as a source of amino acids.

CONCLUSION

We have successfully built a part, OmpA_SAIRGA (BBa_xxx), which is able to produce and release the mature hexapeptide SAIRGA in the culturing media of E. coli cells harboring this part equipped with a RBS and under the control of a constitutive promoter (BBa_xxx).

REFERENCES

[1] Erez Z, Steinberger-Levy I, Shamir M, Doron S, Stokar-Avihail A, Peleg Y, Melamed S, Leavitt A, Savidor A, Albeck S, Amitai G, Sorek R. Communication between viruses guides lysis-lysogeny decisions. Nature (2017) 541, 488-493.