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Are specialised ABC transporters responsible for the circularisation of type I circular bacteriocins?

The authors have withdrawn their manuscript whilst they perform further analysis to verify some interpretations and conclusions. Therefore, the authors do not wish this work to be cited as a reference. If you have any questions, please contact the corresponding author.


Introduction
Circular bacteriocins are secreted antimicrobial proteins [2] found thus far in Gram-positive 43 bacteria only [1,3,4], which are ligated at the N and C termini by a peptide bond [5,6]. 44 Circularisation gives them a number of advantageous traits such as thermostability, pH 45 tolerance, proteolytic resistance and temporal stability [7][8][9]. This also renders them 46 resistant to exopeptidases. The circularity does not have to directly contribute to 47 HMMER [43] (https://www.ebi.ac.uk/Tools/hmmer/, date accessed: 2/9/19) was used to 87 identify the N-terminal ABC transporter domain from the gene clusters of the circular 88 bacteriocins (Data S1). This region (starting at residue 1) was aligned against known 89 peptidase-domains from ABC transporters to determine any putative peptidase domains 90 using BLAST [44] (https://blast.ncbi.nlm.nih.gov/Blast.cgi, date accessed: 2/9/19). Once 91 confirmed, sequences of the N-terminal domains of these circular bacteriocin associated-92 ABC transporters were aligned using Clustal Omega and a phylogenetic tree was built using 93

FigTree. 94
Hydrophobicity profiles 95 Hydrophobicity profiles were generated using the protscale website 96 https://web.expasy.org/protscale/ with a sliding window of 9 [45]. Data (Table S1), which  110   allow signal peptide cleavage and translocation of post-translationally modified bacteriocins  111 epidermin and subtilin, respectively [47][48][49]. We hypothesize that circular bacteriocin-112 associated ABC transporters are not only performing the cleavage of the signal peptide, but 113 also the subsequent ligation and translocation across the cell membrane. Recently, it has 114 been shown that ABC transporter EnkT from Enterococcus faecium NKR-5-3 could cleave 115 signal sequences and export several non-circular bacteriocins [50]. and Glu 145 (numbered based off plantacyclin B21AG circABC transporter sequence) across 119 every sequence. The ubiquitously conserved Gly-X-X-Gly-X-Gly-Lys-Thr/Ser motif forms a 120 mononucleotide or ATP/GTP binding site [51,52]. The conserved Ser 121 , Asp 144 and Glu 145 121 indicate these proteolytic domains are likely serine-carboxyl peptidases or sedolisins, relying 122 on a SED catalytic triad [53]. 123 Sedolisins are peptidases with activity at low pH [54], which matches the general activity 124 and low pH local environment of L. plantarum species growing fermentatively [55]. Ser 121 is 125 the nucleophile involved in amide bond breakage, while Glu 145 acts as a general base for 126 proton transfer from serine during nucleophilic attack, then acts as an acid to protonate the 127 leaving group. Asp 144 acts as a general acid forming the oxyanion hole to protonate the 128 glutamate as well as forming the acyl intermediate [53,56]. 129 To further corroborate this hypothesis, the 3D structure of the plantacyclin B21AG circABC transporter was predicted to see if these residues would be predicted to be within potential catalytic proximity (Figure 2). The ABC transporter ATP-binding protein from Thermotoga maritima (PDB accession 4YER) had the closest structural homology (TM score of 0.891, RMSD of 1.42) and was used for this analysis. This protein was also included in the multiple sequence alignment ( Figure S1) and is also likely a sedolisin due to the conserved SED triad.
This also showed ADP molecules bound to the Gly-X-X-Gly-X-Gly-Lys-Thr/Ser ATP/GTP binding motif. These ABC transporters exist functionally as dimers [57,58] as seen in Figure   2. Existing as a dimer may allow the SED triad to be in close enough proximity, given they are all found within the main groove of the structure. It is possible the catalytic Asp and Glu from chain A may form the triad with Ser from chain B, and vice versa. We hypothesise that this peptidase also performs circularisation after cleavage (Error! Reference source not found.). As the circABC transporters contains a SED motif allowing amide bond cleavage, the opposite reaction (amide bond formation) should also be occurring. Similarly to a ribosome, the peptidase likely shields the catalytic active site from water [59], encouraging the hydrophobic bacteriocin pre-peptide (containing the leader signal sequence) to fit and bind for subsequent cleavage of the signal sequence. Energy from the hydrolysis of 2 ATP at the ATP binding domain of the ABC transporter produces ~30.5 kJ/mol -ΔG [60], allowing this reaction to take place. Cleavage of the amide bond between the C terminus of the signal sequence and the N terminus of the mature peptide results in the addition of a single water molecule and 8 -16 kJ/mol -ΔG are produced via hydrolysis of the amide bond [61,62].
The equilibrium constant for peptide bond formation in water is extremely small [63], which may explain why both termini of circular bacteriocins are hydrophobic [1], as they will favour low-water environments located in the SED active site within the circABC transporter. Circular bacteriocins are unique in this aspect, as hydrophobicity profile comparisons to other non-circular bacteriocins do not have dual hydrophobic termini ( Figure 3, Data S2).

Conclusions
We propose that the bacteriocin circularisation mechanism involves the cleavage of a signal peptide by the peptidase domain of the bacteriocin-associated ABC transporter followed by subsequent amide bond formation with the nascent N terminus, driven by an SED catalytic triad in a uniquely hydrophobic environment. This is coupled to rapid ATP/GTP dependent translocation for extracellular export to ensure overall thermodynamic feasibility.