# Discovering genetic determinants for cell-to-cell adhesion in two prevalent conjugative lactococcal plasmids

Guillermo Ortiz Charneco, Philip Kelleher, Andrius Buivydas, Paul de Waal, Irma M.H. van Rijswijck, Noël N.M.E. van Peij, Christian Cambillau, Jennifer Mahony, Douwe van Sinderen

► To cite this version:

Guillermo Ortiz Charneco, Philip Kelleher, Andrius Buivydas, Paul de Waal, Irma M.H. van Rijswijck, et al. Discovering genetic determinants for cell-to-cell adhesion in two prevalent conjugative lactococcal plasmids. Current Research in Microbial Sciences, 2024, 6, pp.100239. 10.1016/j.crmicr.2024.100239. hal-04877881

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Distributed under a Creative Commons Attribution 4.0 International License
Discovering genetic determinants for cell-to-cell adhesion in two
prevalent conjugative lactococcal plasmids

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Guillermo Ortiz Charneco¹, Philip Kelleher¹, Andrius Buivydas¹, Paul P. de Waal², Irma M. H. van Rijswijck², Noël N. M. E. van Peij², Christian Cambillau³, Jennifer Mahony¹, Douwe Van Sinderen¹*

¹School of Microbiology & APC Microbiome Ireland, University College Cork, Western Road, Cork, Ireland

²dsm-firmenich; Taste, Texture & Health, Center for Food Innovation, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands

³Architecture Fonction des Macromolécules Biologiques, Centre National de la Recherche Scientifique (CNRS), Campus de Luminy, Case 932, CEDEX 09, 13288 Marseille, France

* Correspondence:

Corresponding Author: d.vansinderen@ucc.ie

Keywords: lactococci, conjugation, adhesion, Gram-positive, fluorescence, cell clumping
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**Abstract**

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Plasmids pNP40 and pUC11B encode two prevalent yet divergent conjugation systems, which have been characterized in detail recently. Here, we report the elucidation of the putative adhesins of the pNP40 and pUC11B conjugation systems, encoded by *tra11*<sub>pNP40</sub> and *trs15*<sub>pUC11B</sub>, respectively. Despite their significant sequence divergence, *Tra11*<sub>pNP40</sub> and *Trs15*<sub>pUC11B</sub> represent the most conserved component between the pNP40- and the pUC11B-encoded conjugation systems and share similar peptidoglycan-hydrolase domains. Protein structure prediction using AlphaFold2 highlighted the structural similarities between their predicted domains, as well as the potential homo-dimeric state of both proteins. Expression of the putative surface adhesins resulted in a cell clumping phenotype not only among cells expressing these surface adhesins but also between adhesin-expressing and non-producing cells. Furthermore, mutant derivatives of plasmids pNP40 or pUC11B carrying a mutation in *tra11* or *trs15*, respectively, were shown to act as efficient donors provided the corresponding recipient expresses either *tra11*<sub>pNP40</sub> or *trs15*<sub>pUC11B</sub>, thus demonstrating *in trans* reciprocal complementarity of these proteins in conjugation systems.
40 **1 Introduction**

41 Lactococci, and in particular strains belonging to the *Lactococcus lactis* and *cremoris* species,
42 represent the most widely used starter cultures in the fermentation of dairy products. This is, in many
43 respects, due to their characteristically abundant plasmid content and their associated desirable and
44 technologically relevant traits (e.g. exopolysaccharide production, lactose metabolism, protease
45 activity and bacteriophage resistance) (Mills *et al.*, 2006; Fallico *et al.*, 2012; Ainsworth *et al.*, 2014).
46 Horizontal transfer of plasmids among *L. lactis/cremoris* strains is currently achieved predominantly
47 by conjugation and transduction (Ainsworth *et al.*, 2014). Conjugation among lactococcal strains
48 represents a very useful, non-recombinant tool for the development of novel strains with desirable traits
49 suited to the dairy environment (Ortiz Charneco *et al.*, 2023a).

50 Despite the economic significance of these Gram-positive lactococcal species, the molecular
51 process of conjugation has primarily been studied in Gram-negative organisms with very limited
52 studies in their Gram-positive counterparts. In Gram-negative bacteria, conjugation relies on pilus
53 formation to establish cell-to-cell contact between donor and recipient while Gram-positive bacteria
54 appear to rely on cell surface-associated adhesins (Kohler et al., 2019). For example, the conjugative
55 enterococcal plasmid pCF10 encodes several adhesin-related proteins, of which PrgA, PrgB and PrgC
56 were observed to promote a distinct cellular aggregation phenotype (Bhatty et al., 2015). Similarly, in
57 the model Gram-positive conjugative plasmid pIP501 from *Enterococcus faecalis*, TraO has been
58 reported to act as the surface adhesin (Kohler et al., 2018). No surface adhesin has yet been identified
59 for lactococcal conjugation systems; however, the sex factor of *L. cremoris* MG1363 has been
60 described as a cell-membrane-anchored protein, CluA, which is associated with a cell clumping
61 phenotype (Wels et al., 2019).

62 A recent study revealed the presence of 33 lactococcal plasmids among public databases with
conjugation-related genes similar to those of previously described lactococcal conjugative plasmids pMRC01, pAF22 and pNP40 (Coakley *et al.*, 1997; O'Driscoll *et al.*, 2004; Fallico *et al.*, 2012; Ortiz Charneco *et al.*, 2021). The conjugation-associated genes of plasmids pUC11B and pNP40 have recently been identified and characterized, although these efforts did not identify an obvious adhesin candidate responsible for cell-to-cell contact (Ortiz Charneco *et al.*, 2021; Ortiz Charneco *et al.*, 2023b).

Mutation of the genes encoding Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> causes a major reduction in the conjugation frequency of their respective conjugative plasmids (up to 1,000- and 4,500-fold, respectively) (Ortiz Charneco *et al.*, 2021; Ortiz Charneco *et al.*, 2023b) suggesting a near essential role in conjugation. In the current report, we performed an in-depth experimental and predictive analysis of the precise role played by Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> in conjugation. The generated results implicate Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> as adhesins involved in cell-to-cell contact during conjugation.
75 **2** Materials and Methods

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77 **2.1** Bacterial strains and growth conditions

78 Bacterial strains employed in this study are summarised in Supplementary Table S1. Overnight
79 cultures were incubated at 30 °C for 16 hours by inoculating bacterial cells from -80 °C glycerol stocks
80 into 10 mL of M17 supplemented with 0.5 % (v/v) glucose (GM17) containing either nisin (2.5 µg/mL,
81 for selection of pNP40), tetracycline (10 µg/mL, to select strains labouring pUC11B or pPTPi) or
82 erythromycin (5 µg/mL, which selected for strains containing pPEPi, pGFP8048E or pMC8048E).
83 Electrocompetent cells of L. cremoris were prepared as previously described (Holo and Nes, 1989).

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85 **2.2** Plasmids and constructs

86 All plasmids employed in this study are summarised in Supplementary Table S2. Purification of
87 plasmid DNA was performed using the GeneJET Plasmid MiniPrep Kit and following the
88 manufacturer's instructions (Thermo Scientific, USA). Newly constructed plasmids were generated by
89 conventional recombinant DNA techniques. PCR fragments used for cloning (Supplementary Table
90 S3) were digested with the same enzymes as the relevant cloning vector, and then ligated with T4 DNA
91 ligase. Ligation mixtures were subsequently introduced into competent L. cremoris NZ9000 (Kuipers
92 et al., 1998) by electroporation and transformants were screened by colony PCR to confirm the
93 presence of both plasmid and insert. Finally, the integrity of the construct sequence was verified by
94 Sanger sequencing (Genewiz, Azenta Life Sciences, Leipzig, Germany).
The fluorescent reporter plasmids pGFP8048E and pMC8048E were created using the backbone of plasmid pNZ8048E, which was in turn constructed using the backbone of pNZ8048 (De Ruyter *et al.*, 2009) and the erythromycin resistance gene from pNZ44E (Draper *et al.*, 2009; Ortiz Charneco *et al.*, 2024). The GFP-encoding gene from the fusion vector pHTP9 and the mCherry-encoding gene from the fusion vector pHTP1-mCherry (NZYTech, Portugal) were individually PCR-amplified allowing the incorporation of an artificial Shine-Dalgarno sequence at the 5'-end and a stop codon at the 3'-end of these genes, using oligonucleotides listed in Supplementary Table S3. These PCR-generated DNA fragments were subsequently digested with the same enzymes as vector pNZ8048E and ligated using T4 DNA ligase (Promega). Induced expression of these genes was achieved by the inclusion of nisin (10 ng/ml) in the growth medium. The proposed surface adhesin-encoding genes, *tra11*<sub>pNP40</sub> and *trs15*<sub>pUC11B</sub>, were PCR-amplified (incorporating an artificial Shine-Dalgarno sequence at the 5'-end of the gene) and individually cloned into the low-copy expression vectors pPTPi (O'Driscoll *et al.*, 2004) and pPEPi (Ortiz Charneco *et al.*, 2023b), generating constructs pPTPi::*tra11*<sub>pNP40</sub>, pPTPi::*trs15*<sub>pUC11B</sub>, pPEPi::*tra11*<sub>pNP40</sub> and pPEPi::*trs15*<sub>pUC11B</sub>. Expression of the relevant gene was induced by adding 10 ng/ml of nisin to the growth medium at the point of inoculation.

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## 2.3 Mating assays

Conjugation was performed using the spread solid mating approach, as previously described (Ortiz Charneco *et al.*, 2021). Briefly, overnight cultures of both donor and recipient were mixed in a 1:1 volume ratio (representing a 1:1.5 donor/recipient viable cell count ratio), centrifuged at 3,000 × g, resuspended in 200 µL of 5% reconstituted skim milk (RSM) supplemented with 2% glucose and evenly spread on 5% RSM, 2% glucose agar plates. Plates were subsequently incubated overnight at
117 30 °C, after which the cells were scraped from the plates and suspended in 4 mL of Ringer's solution,
118 serially diluted and plated onto GM17 agar plates supplemented with the relevant antibiotic.

119 To determine the possible role of Tra11 and Trs15 as surface adhesins of the pNP40 and pUC11B
120 conjugation systems, respectively, constructs pPEPi::tra11<sub>pNP40</sub> and pPEPi::trs15<sub>pUC11B</sub> were
121 individually introduced into *L. cremoris* NZ9000, and the resulting strains were subsequently used as
122 recipients in spread solid mating conjugation experiments using various donors. The donor strains
123 applied were *L. cremoris* NZ9000 strains carrying pNP40 or pUC11B, or *tra11<sup>-</sup>* or *trs15<sup>-</sup>* derivatives
124 of these respective plasmids (mutant nomenclature *L. cremoris* NZ9000 *tra11<sub>pNP40</sub>::Ter* or *L. cremoris*
125 NZ9000 *trs15<sub>pUC11B</sub>::Ter*, respectively). The mutant derivatives of pNP40 and pUC11B had been
126 constructed previously using an ssDNA recombineering approach (van Pijkeren and Britton, 2012;
127 Ortiz Charneco *et al.*, 2021; Ortiz Charneco *et al.*, 2023b). Prior to mixing donor and recipient cells,
128 expression of the proposed surface adhesins was induced in the recipient cultures by adding 10 ng/ml
129 nisin to the growth medium at the point of inoculation.

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## 2.4 Cell clumping assay

132 To ascertain if expression of the presumed surface adhesin-encoding genes, *tra11*<sub>pNP40</sub> and
133 *trs15*<sub>pUC11B</sub>, promoted a cell clumping phenotype in bacterial cells expressing these proteins, two
134 fluorescent reporter plasmids were constructed, pGFP8048E and pMC8048E, producing green and red
135 fluorescent proteins, respectively. These were introduced individually into *L. cremoris* NZ9000 pPTPi
136 (control), *L. cremoris* NZ9000 pPTPi::*tra11*<sub>pNP40</sub> and *L. cremoris* NZ9000 pPTPi::*trs15*<sub>pUC11B</sub>. The
137 resulting strains were used in different combinations in subsequent cell clumping assays. For cell
138 clumping assays, overnight cultures were diluted (on their own or in different strain combinations)
139 1:100 in 10 ml fresh GM17 supplemented with the relevant antibiotics and 10 ng/ml of nisin for
induction of the surface adhesins and/or the fluorescent proteins and grown to an OD<sub>600nm</sub> of 1.5. A
variety of induction conditions were evaluated including time of addition (from 0 to 120 minutes) and
the nisin concentration (1-20 ng/ml), and those stated above represent the optimal conditions identified
for the restoration of the *tra11<sub>pNP40</sub>* and *trs15<sub>pUC11B</sub>* mutants and the observed cell clumping phenotype.
Subsequently, 10 µL of each culture was spotted on the middle of a microscope slide, covered with a
cover slip following the wet mount method and visualized using a 60X objective lens of an upright
fluorescence microscope (BX53; Olympus) for imaging.

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148 **2.5 Comparative and functional analysis**

Sequence comparison at the protein level was performed using bi-directional BLAST alignment
(Altschul *et al.*, 1990). TMHMM v2.0 software was employed for the prediction of transmembrane
helices using the hidden Markov model (HMM) (Krogh *et al.*, 2001). HHpred software was used for
remote protein homology detection and structure prediction with pairwise comparison of profile HMM
(Söding *et al.*, 2005; Zimmermann *et al.*, 2018). Pfam (El-Gebali *et al.*, 2019) was used for the
identification of functional domains. Jalview V. 2.11 Desktop was employed to perform Clustal Omega
alignment of the *Tra11<sub>pNP40</sub>* and *Trs15<sub>pUC11B</sub>* proteins (Procter *et al.*, 2021), while SignalP 6.0 was
utilised for the prediction of secretion signals in protein sequences (Teufel *et al.*, 2022).

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158 **2.6 Protein structure predictions**

A Github AlphaFold2 notebook was used to perform the predictions and the modeling of homo-
multimers (Jumper *et al.*, 2021). Local Distance Difference Test (LDDT) evaluates local distance
differences of all atoms in a model with reference to an ensemble of equivalent structures. The pLDDT
162 (predicted LDDT-Cα) is a per-residue measure of local confidence on a scale from 0-100 (100 being the highest confidence level). The pLDDT values that are stored in the pdb file as B-factors, were plotted using Excel. Visual representations of the structures were prepared with ChimeraX (Pettersen et al., 2021).

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167 **2.7 Statistical data analysis**

168 Data in this study represent the means ± standard deviation (SD) of triplicate assays. Results were 169 analysed using the SigmaPlot 11.0 statistical package (SPSS), from Systat Software, Inc., San Jose 170 California USA. One-way analysis of variance ANOVA was performed to compare frequencies of 171 conjugation. A *P* value of ≤ 0.001 was considered very significant and is represented by two asterisks 172 "**" in the graphs.
173 **3** **Results**

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175 **3.1** **Comparative genomics**

176 The mechanism of plasmid conjugation in lactococcal species has long been a subject of study.
177 However, a critical component of this puzzle remains elusive: the surface adhesins responsible for
178 promoting the essential initial contact between donor and recipient cells during conjugation. Thus, the
179 objective of this study was to address this gap in our knowledge and enhance our understanding of the
180 adhesion process during plasmid-mediated conjugation in lactococcal species. Plasmids pNP40 and
181 pUC11B have recently been determined to represent two of the most prevalent lactococcal conjugative
182 systems among public databases (Ortiz Charneco et al., 2021). The pNP40 and pUC11B conjugation
183 systems have been selected for further characterization in this study based on their sequence
184 divergence, thus representing potentially distinct systems, and prevalence among public databases.

185 Previous functional analyses have elucidated several analogous functions between the two
186 conjugation systems, despite their overall low sequence similarity (Ortiz Charneco et al., 2023b). Even
187 though the overall divergence, both conjugation gene clusters share a set of three adjacent genes whose
188 products exhibit relatively high amino acid identity (Supplementary Figure S1). The first of this triplet,
189 i.e. *tra11*<sub>pNP40</sub> and *trs15*<sub>pUC11B</sub> for pNP40 and pUC11B, respectively, encode predicted cell wall-
190 degrading proteins, with 67.68 % amino acid identity. The second, represented by *tra10*<sub>pNP40</sub> and
191 *trs16*<sub>pUC11B</sub> (located pNP40 and pUC11B, respectively), encode predicted mating channel-related
192 proteins sharing 31.25 % amino acid identity, while the third, i.e. *tra09*<sub>pNP40</sub> in pNP40 and *trs17*<sub>pUC11B</sub>
193 in pUC11B, encode predicted thioredoxin-like proteins sharing 54.46 % amino acid identity. Apart
194 from these genes, the pNP40 and pUC11B conjugation gene clusters share little protein sequence
195 similarity.
## 3.2 Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> share similar functional domains

Despite recent functional studies, no cell surface adhesin-encoding genes have yet been proposed for either the pNP40- or the pUC11B-associated conjugation systems. Based on the presence of N-acetylmuramoyl-L-alanine amidase domains in Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> (described in the next paragraph), and their similarity to amidase domains present in surface adhesins of Gram-positive bacteria (Hirschhausen *et al.*, 2012; Kumar *et al.*, 2013), these two proteins were proposed as potential surface adhesins of their respective conjugation systems. Mutation of the corresponding genes had been shown to significantly diminish conjugation frequencies of pNP40 and pUC11B (Ortiz Charneco *et al.*, 2021; Ortiz Charneco *et al.*, 2023b).

A predictive analysis was performed using HHpred (Zimmermann *et al.*, 2018), TMHMM (Krogh *et al.*, 2001) and Pfam (El-Gebali *et al.*, 2019) in Tra11<sub>pNP40</sub> (434 aa) and Trs15<sub>pUC11B</sub> (423 aa) to search for protein similarity among public databases, the results of which are summarized in Figure 1A. Based on TMHMM analyses (Krogh *et al.*, 2001), both proteins contain a transmembrane domain in their N-terminal end, which may act as a membrane anchor, followed by an N-acetylmuramoyl-L-alanine amidase-like domain encompassing much of the remainder of the protein sequence. However, this latter domain is further divided in two sub-domains, i.e. a peptidoglycan-glycosyl hydrolase (PGH) domain (closer to the N-terminal end), followed by a linker of 44 amino acids, and a cysteine, histidine-dependent amidohydrolase/peptidase (CHAP) domain in their C-terminal end. Alignment of the Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> sequences using the Clustal Omega package (Fig. 1B) revealed that these two sub-domains (i.e., PGH and CHAP domain) are conserved between the two proteins.

A structure prediction of Tra11<sub>pNP40</sub> (434 aa) and Trs15<sub>pUC11B</sub> (423 aa) was implemented using AlphaFold2 (Jumper *et al.*, 2021) to detect functional domains in both Tra11 and Trs15 (Fig. 2A & B).
219 The Dali server reported an excellent match between the Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> peptidoglycan-
220 hydrolase (domain 1, Fig. 2C) and CHAP domains (domain 2, Fig. 2D) (root mean square deviation
221 (RMSD) of 0.533 Å between their domain 1 and 0.360 Å between their domain 2), denoting their quasi-
222 identical structures.

223 Domain 1 and domain 2 possess putative catalytic residues, a Glu/Asp (residues 96/151 and 89/144
224 for Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub>, respectively) catalytic dyad for domain 1 (Fig. 3A), and a Cys/His/Asp
225 (residues 317/382/377 and 310/375/370 for Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub>, respectively) catalytic triad
226 for the second domain (Fig. 3B). Domain 1 was superimposed onto a known structure, i.e. the autolysin
227 of *Listeria monocytogenes* (PDB: 3FI7), to identify the catalytic acidic amino acids. The *Listeria*
228 autolysin presents two catalytic residues Glu/Glu (residues 122/156) (Bublitz *et al.*, 2009). Domains 1
229 and 2 of Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> are both expected to participate in degradation of the cell-wall
230 peptidoglycan by hydrolyzing the polysaccharide bonds and the amido bond, respectively.

231 Tra11<sub>pNP40</sub> (Fig. 4A) and Trs15<sub>pUC11B</sub> (Fig. 4B) were predicted by AlphaFold2 to form a complex of
232 identical dimers (homodimers). Predicted aligned error (PAE) plots of Tra11<sub>pNP40</sub> (Fig. 4C) and
233 Trs15<sub>pUC11B</sub> (Fig. 4D) suggest that each monomer conformation is not only predicted with confidence,
234 but that homodimer packing is of relatively high confidence too. It is noteworthy that the performance
235 of AlphaFold2 is better for homo-oligomer prediction than for hetero-oligomer prediction, since for
236 the former the contact surface is usually larger compared to what would generally be the case for the
237 latter (Xiong *et al.*, 2022). The pLDDT values were stored as B-factors (Supplementary Figure S2).

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240 **3.3** *Two potential surface adhesins restore a conjugation deficiency phenotype of*
241 *corresponding mutants through reciprocal, in trans complementation*

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Following the *in silico* analysis, *tra11*<sub>pNP40</sub> and *trs15*<sub>pUC11B</sub> were selected as candidate surface adhesin-encoding genes. To experimentally evaluate the proposed surface adhesin functionality, *tra11*<sub>pNP40</sub> and *trs15*<sub>pUC11B</sub> were individually cloned into a nisin-inducible expression vector and introduced into *L. cremoris* NZ9000 and employed as recipients in conjugation experiments using a donor strain harbouring either the *tra11*<sup>-</sup> and *trs15*<sup>-</sup> mutated derivatives of pNP40 or pUC11B. It was hypothesized that the adhesin could be expressed in either the donor or recipient cell since either would promote cell-to-cell contact. Therefore, a recipient cell expressing one of the proposed adhesins would be expected to restore (reciprocally and *in trans*) conjugation frequencies in an otherwise deficient conjugation process caused by a mutation of the proposed surface adhesin-encoding gene in the conjugative plasmid of the donor. Expression of *tra11*<sub>pNP40</sub> or *trs15*<sub>pUC11B</sub> (Materials and Methods; by adding 10 ng/mL of nisin to the growth medium) in a recipient *L. cremoris* NZ9000 resulted in very significant (*P* ≤ 0.001) increases in the conjugation frequencies between the negative control harbouring pPEPi and those harbouring the pPEPi::*tra11*<sub>pNP40</sub> and pPEPi::*trs15*<sub>pUC11B</sub> constructs (Fig. 5).

Conjugation frequencies of *tra11*<sub>pNP40</sub>::Ter increased from 5.97 × 10<sup>-4</sup> % (*L. cremoris* NZ9000 pPEPi recipient) to 4.1 × 10<sup>-1</sup> % with *L. cremoris* NZ9000 pPEPi::*tra11*<sub>pNP40</sub> as recipient, and to 5.2 × 10<sup>-1</sup> % with *L. cremoris* NZ9000 pPEPi::*trs15*<sub>pUC11B</sub> as recipient, representing a 680-fold and 870-fold increase in conjugation frequency, respectively. Similarly, employing *trs15*<sub>pUC11B</sub>::Ter as donor, very significant (*P* ≤ 0.001) differences were observed in the conjugation frequencies between the recipient harbouring the empty vector (3.38 × 10<sup>-4</sup> %) and those expressing *trs15*<sub>pUC11B</sub> (6.47 × 10<sup>-1</sup> %) and *tra11*<sub>pNP40</sub> (8.3 × 10<sup>-1</sup> %), in which conjugation of plasmid pUC11B increased more than 1,900-fold and 2,400-fold, respectively.
Overall, conjugation frequencies of strains carrying *tra11*<sub>pNP40</sub> and *trs15*<sub>pUC11B</sub> mutations in the pNP40 and pUC11B conjugation gene cluster, respectively, increased very significantly (*P* ≤ 0.001) when intact copies of either gene were expressed *in trans* in a recipient strain. Furthermore, both genes displayed a similar reciprocal restorative effect in either mutant derivative of the two conjugation systems, indicative of an analogous function. Additionally, expression of either gene in a recipient cell during conjugation with a donor harbouring intact copies of either conjugative plasmid showed no significant (*P* ≤ 0.001) differences with the control.

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272 **3.4 The proposed surface adhesins promote a clumping phenotype**

To further substantiate the hypothesis that *Tra11*<sub>pNP40</sub> and *Trs15*<sub>pUC11B</sub> represent surface adhesins, the genes encoding these proteins were individually cloned into the low copy vector pPTPi and introduced into *L. cremoris* NZ9000 strains harbouring one of the reporter plasmids, pGFP8048E or pMC8048E. These strains were subsequently co-cultured in different combinations to determine if any cell-to-cell contact could be observed after *Tra11*<sub>pNP40</sub> and/or *Trs15*<sub>pUC11B</sub> induction, which may or may not be visible in cultures but which would be visible in microscopic images.

When two strains were co-cultured, in which one strain harboured one of the reporter plasmids and the other strain harboured the empty vector pPTPi (Fig. 6A), no cell clumping was observed irrespective of nisin addition. However, *L. cremoris* NZ9000 cells expressing either *tra11*<sub>pNP40</sub> (Fig. 6B) or *trs15*<sub>pUC11B</sub> (Fig. 6C) showed a distinct clumping phenotype when cultured separately and after nisin induction. This was observed on imaging analysis, but it is worth noticing that this cell clumping phenotype could also be partially observed in the tubes, in which some cell clumps could be seen after vortexing of the tubes (data not shown). Additionally, *L. cremoris* NZ9000 cells expressing *tra11*<sub>pNP40</sub> displayed cell clumping when co-cultured with *L. cremoris* NZ9000 cells not expressing either of the
287 proposed surface adhesins (Fig. 6D), and similarly *L. cremoris* NZ9000 cells expressing Trs15<sub>pUC11B</sub>

288 clumped together with *L. cremoris* NZ9000 cells (Fig. 6E). Finally, this clumping phenotype was also

289 observed when *L. cremoris* NZ9000 cells producing Tra11<sub>pNP40</sub> were co-cultured together with *L. cremoris* NZ9000 pPTPi::trs15<sub>pUC11B</sub> (Fig. 6F).

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4 Discussion

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Despite the economic significance of lactococci for the dairy industry, lactococcal plasmid-encoded
conjugation has been less extensively characterized compared to certain other Gram-positive
conjugative plasmids, such as the enterococcal model plasmids pCF10 and pIP501 (Goessweiner-Mohr
et al., 2014; Kohler et al., 2019). The lactococcal conjugative plasmids pNP40 and pUC11B have been
subject to recent characterization studies (Ortiz Charneco et al., 2021; Ortiz Charneco et al., 2023b),
which showed that both plasmids contain two prevalent yet distinct gene clusters responsible for
conjugation. Unraveling their mechanistic details will provide insights into other similar systems
present among conjugative plasmids present in lactococci and other Gram-positive bacteria.

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Although several conjugation-related functions have been identified or predicted in both systems, one key component of Gram-positive-related conjugation remained unknown i.e. the functionality that is responsible for the establishment of cell-to-cell contact. The *tra11*<sub>pNP40</sub> and *trs15*<sub>pUC11B</sub> genes were identified as potential surface adhesin-encoding genes of the pNP40- and pUC11B-encoded conjugation machinery, respectively, based on conserved N-acetylmuramoyl-L-alanine amidase domains. These domains have been reported to be a characteristic of proteins with functional roles as both cell wall hydrolases and adhesins, such as Rv3717 from *Mycobacterium tuberculosis* (Kumar *et al.*, 2013) and other Gram-positive pathogenic bacteria, such as Aas from *Staphylococcus saprophyticus* (Henderson and Martin, 2011) and Aaa from *Staphylococcus aureus* (Hirschhaysen *et al.*, 2012). Both *Tra11*<sub>pNP40</sub> and *TRS15*<sub>pUC11B</sub> share a transmembrane domain in their N-terminal end, which suggests an anchoring role to the membrane. The protruding section of both proteins contains one conserved cell wall hydrolase domain at their N-terminal end, followed by a non-conserved region and a conserved CHAP domain within their C-terminal portion.
However, no conserved domains were observed between Tra11<sub>pNP40</sub> or Trs15<sub>pUC11B</sub> and the characterized chromosomally encoded clumping factor in *L. cremoris* MG1363, CluA (Stentz *et al.*, 2006), or to other surface adhesins such as PrgC in the *E. faecalis* pCF10 plasmid (Bhatty *et al.*, 2015) and TraO, the surface adhesin of the model Gram-positive conjugative plasmid, pIP501 (Kohler *et al.*, 2018). For instance, both TraO and PrgC were shown to possess transmembrane domains in their C-terminal ends, highly repetitive sequence motifs of three-residue periodicity and an LPxTG cell wall anchor motif (Navarre and Schneewind, 1999; Goessweiner-Mohr *et al.*, 2014). Neither Tra11<sub>pNP40</sub> nor Trs15<sub>pUC11B</sub> presented any significant similarities to either protein. Beyond their transmembrane domain located in their amino-terminus, no mechanism of action that would promote cell-to-cell binding has been observed. It is noteworthy that the cell wall in lactic acid bacteria has only been studied in recent years, and that characterization efforts regarding surface adhesins in lactococcal species have been scarce (Chapot-Chartier, 2014).

Our results provide evidence to the possible role of proteins Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> as surface adhesins, and their function appears to be interchangeable between the two conjugation systems which are otherwise divergent on a protein similarity level from each other beyond the previously discussed gene triplet (Supplementary Figure S1). Furthermore, expression of either potential surface adhesin in a recipient cell during conjugation with donors harbouring intact copies of either conjugative plasmid showed no significant differences in conjugation frequencies when compared to the control. This phenomenon was further corroborated during the cell clumping assays, in which it was observed that cells expressing either Tra11<sub>pNP40</sub> or Trs15<sub>pUC11B</sub> displayed a significant cell clumping phenotype against cells not expressing either of these proteins. Additionally, cells expressing Tra11<sub>pNP40</sub> were observed to clump together with cells expressing Trs15<sub>pUC11B</sub>, and *vice versa*. These results suggest that either there is a different exclusion mechanism preventing mating of two donor cells during conjugation that is not related to the presence of these proposed surface adhesins in two different cells,
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or that there is no exclusion mechanism in lactococcal conjugation, and that donor cells may exchange
plasmids via conjugation with each other as well as with other recipients.

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Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> do not present any recognizable cell wall-binding domains with conserved sequences to other adhesins, which introduce a challenge when trying to determine their mechanism of action. Based on their predicted three-dimensional structures, their catalytic domains and the observed conjugation and clumping assays, they could weaken the cell wall on the donor strain to facilitate anchoring of the conjugation machinery encoded by the conjugative plasmid. This presumption may be explained by our conjugation results, in which (reciprocal) *in trans* complementation of the proposed surface adhesins from pNP40 and pUC11B significantly increase conjugation of the adhesin-deficient versions of these conjugative plasmids when compared to the mutants, although conjugation frequencies are not completely restored, which could be due to the inherent obstacles of *in trans* complementation, imperfect timing or suboptimal level of expression. After weakening the donor's peptidoglycan layer, they could protrude from the cell wall, exposing their peptidoglycan hydrolase and CHAP domains to the extracellular environment. Upon contact with other possible recipient cells, these domains may be responsible for the degradation of the peptidoglycan layer of these recipients to promote the extension of the conjugation machinery from the donor to the recipient cell and facilitate membrane fusion. The CHAP domains presented some conserved sequences from the core of other Gram-positive CHAP amidase domains, such as PcsB, an essential putative peptidoglycan hydrolase from *Streptococcus pneumoniae* (Rigden *et al.*, 2003; Sham *et al.*, 2011), suggesting these are functional and act as the catalytic domains of both Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub>. Based on our AlphaFold2 structure predictions, the catalytic triad of domain 2 from both Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> (the CHAP domains) is formed by Cys/His/Asp residues. Other previously characterized CHAP domains present similar catalytic triads but with Glu as the last residue, like the proteolytic triad formed by Cys/His/Glu in the CHAP domain of *Staphylococcus saprophyticus* (Rossi

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361 et al., 2009) or the Cys/His/Glu catalytic triad present in the CHAP domain of the endolysin LysK
362 from *Staphylococcus aureus* bacteriophage K (Sanz-Gaitero et al., 2014).

363 Our results indicate that the N-terminal peptidoglycan hydrolase domains act as the substrate-
364 binding domains of both proteins, although no significant similarities to other peptidoglycan-binding
365 proteins were found. However, additional experimental data will be required to propose a more detailed
366 mechanism of action, and future studies will focus on the elucidation of the function and mode of action
367 of Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub>. Introducing mutations in the proposed catalytic sites of each
368 peptidoglycan-active domain will facilitate an evaluation of the effect of such mutations on the *in trans*
369 complementation and cell clumping. Additionally, it will provide insights into the domains that are
370 essential to establish cell binding, which are currently unknown. Further assays should also determine
371 if Tra11<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> possess the hereby proposed cell wall-degrading activity, while
372 transmission electron microscopy analysis may provide key insights into the exact effect that these
373 proteins have on the cell walls and membranes of donor and recipient cells.

374 To our knowledge, no surface adhesins have yet been identified in any lactococcal conjugation
375 system to date, and in this study genes *tra11*<sub>pNP40</sub> and *trs15*<sub>pUC11B</sub> are hereby proposed to encode surface
376 adhesins of the conjugation machineries of plasmids pNP40 and pUC11B, respectively. These two
377 genes are part of a gene triplet that encodes proteins that are reasonably well conserved between the
378 two systems, and future investigation may focus on these co-localised genes as well to determine if
379 and how they functionally participate in this part of the conjugation too. The work described herein
380 provides a promising starting point for future studies on lactococcal plasmid-encoded adhesion
381 proteins. In turn, this could enhance efforts to boost conjugation frequencies and facilitate the
382 generation of more robust starter cultures in the dairy industry using means of natural transformation.
383 **5** Funding

384 This publication has emanated from research conducted with the financial support of Science
385 Foundation Ireland under Grant Numbers 12/RC/2273 and 17/SP/4678, which is co-funded by dsm-
386 firmenich, Taste, Texture & Health.

387

388 **6** Acknowledgements

389 The authors would like to acknowledge the Department of Anatomy & Neuroscience Imaging
390 Centre, BioSciences Institute, University College Cork, for assistance in preparing and imaging
391 specimens for this research.

392

393 **7** References

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**Declaration of Interest Statement:** PPdW, IMHvR and NNMEvP are employed by the company dsm-firmenich. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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## 8 Figure captions

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**Figure 1.** (A) Functional domains present in Trall<sub>pNP40</sub> and Trs15<sub>pUC11B</sub>, based on sequence and structural similarities using HHpred, TMHMM and Pfam databases; TMD: transmembrane domain.

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**PGH:** peptidoglycan hydrolase domain. **CHAP:** cysteine, histidine-dependent amidohydrolase peptidase domain. **AMD:** N-acetylmuramoyl-L-alanine amidase domain. (B) Jalview of a Clustal Omega alignment for the Trall<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> proteins. Conserved amino acids are highlighted with two shades of blue based on conservation, (light blue aminoacid with strongly similar properties; dark blue: aminoacid fully conserved) while uncoloured amino acids correspond to non-conserved regions.

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**Figure 2.** Trall<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> predicted structures and domain comparison. (A) Ribbon representation of the predicted structure of Trall<sub>pNP40</sub>, rainbow-coloured from N-terminus to C-terminus. The two predicted domains are outlined in dotted rectangles. (B) Ribbon representation of the predicted structure of Trs15<sub>pUC11B</sub>, rainbow-coloured from N-terminus to C-terminus. The two predicted domains are indicated by dotted rectangles. (C) Superposition of the Domain 1 from Trs15<sub>pUC11B</sub> (coloured) and Trall<sub>pNP40</sub> (grey). (D) Superposition of the Domain 2 from Trs15<sub>pUC11B</sub> (coloured) and Trall<sub>pNP40</sub> (grey). The TMHMM predicted and disordered trans-membrane domain in

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the N-terminus of Trall<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> were removed. Abbreviations: **Ct**: C-terminus; **RMSD**: Root Mean Square Deviation.

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**Figure 3.** Trall<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> predicted catalytic domains. (A) Superposition of the Domain 1 from Trs15<sub>pUC11B</sub> (pink) and Trall<sub>pNP40</sub> (beige) with the autolysin domain of L. monocytogenes (PDB: 3FI7). The catalytic residues Glu/Asp are marked by dotted rectangles. (B) Superposition of the Domain 2 from Trs15<sub>pUC11B</sub> (green) and Trall<sub>pNP40</sub> (orange). The catalytic residues Cys/His/Asp are marked by dotted rectangles.

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**Figure 4.** Trall<sub>pNP40</sub> and Trs15<sub>pUC11B</sub> predicted dimerisation. (A) Spatial structure of the Trall<sub>pNP40</sub> homodimer. (B) Spatial structure of the Trs15<sub>pUC11B</sub> homodimer. (C, D) Heatmap of the predicted aligned error (PAE) of Trall<sub>pNP40</sub> and Trs15<sub>pUC11B</sub>, respectively. Colour at the “x” and “y” axis indicates AlphaFold2’s expected position error at residue “x” if the predicted structures were aligned on residue “y”.

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**Figure 5.** Conjugation frequencies between *L. cremoris* NZ9000 pNP40, pUC11B and the adhesin-deficient derivatives as donors strains against the recipients *L. cremoris* NZ9000 pPEPi, pPEPi::trall<sub>pNP40</sub> or pPEPi::trs15<sub>pUC11B</sub>. Frequency of conjugation of the mutated version of either pNP40 or pUC11B is compared against the frequency of conjugation of the respective wild-type plasmid. A *P*-value ≤ 0.001 was considered very significant and is represented by two asterisks “**”. Presented data are the mean of three replicates ± standard deviation.

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**Figure 6.** Fluorescent microscopy images of *L. cremoris* NZ9000 derivatives. (A) NZ9000 pPTPi (green) and NZ9000 pPTPi (red); (B) NZ9000 pPTPi::trall<sub>pNP40</sub> (green); (C) NZ9000 pPTPi::trs15<sub>pUC11B</sub> (red); (D) NZ9000 pPTPi::trall<sub>pNP40</sub> (green) and NZ9000 pPTPi (red); (E) NZ9000 pPTPi::trall<sub>pNP40</sub> (green) and NZ9000 pPTPi::trs15<sub>pUC11B</sub> (red); (F) NZ9000 pPTPi (green) and NZ9000 pPTPi::trs15<sub>pUC11B</sub> (red).

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**Supplementary Figure S1.** Comparative gene map of the conjugation gene clusters of pNP40 and pUC11B. The figure details the genetic organization of both conjugation gene clusters and their shared amino acid identity (indicated by shaded areas). The predicted conjugation-related functions (Ortiz Charneco *et al.*, 2023b) are indicated by coloured arrows.

**Supplementary Figure S2.** Excel plot of the predicted Local Distance Difference Test (pLDDT) displa

<figure><img src="image_2.png" /></figure>

B.

Tra11 1 MASKAVVGAVATTVSGIISFPFELIVGLVIVIVMVVCVFTEQNDNCVKPVDPQ---HEEVGKPAKSIDDFIKSHKEA 74
Trs15 1 --- -MFVT---KAKISIAKKIIWLVAGL--- -FILIFMVSGHEEDECIPSNSDSNTTEDLKKVPKSVEDFVKSHKES 67

Tra11 75 YIQSWKAGGF LPSAIAQS MVENG F NFT N P S GTSFWKA HNMGGVKTS KLSDFPITIATYGKDSIDITGT KAGTSVGD 151
Trs15 68 YILSWKAGGF LPSAIAQTMVENG F NFT N P S GTSFWKA HNMGGVKTS KKE DFPVTLASYGNEAVDITGT KAGTSVGD 144

Tra11 152 NTGGAYTYFKDYNAGIVGKAEFMAHQTLYGAINNTNPQATLTAIYMGGWATDPKYLS SLLSAYNTLGQNYKFIDDE 228
Trs15 145 NTGGAYTYFKDYNAGIVGKS EFMAHQS LYTGAINNTDGISSLTAIFKGGWATDPNYLVSLIGAYQS LGO KFKWIDKE 221

Tra11 229 AIKYGKTPYQANHIMPNVIPNNDPHKNDSKNTNEVNA DNDGCVVDPNT---SNGKNGAPVLDLPKEYIGKLTLPM 302
Trs15 222 AIEKYGQAPYKAGNALKNQIDKTT---NKKEASQSDTDEDGCVVEQTPSKEATGTNSAPTLAPPKEYEGKLMKP 295

Tra11 303 DKNYSGNSYPFGQCTWGVYNRLAQIGMPIEWFSGD S GNGGNWGA SA KAKGYKVEKGKPKIWGWGASVPAQVAGSDPV 379
Trs15 296 DNKNYAGNSYPFGQCTWGVYNRMAQLGTPIEWFSGD S GNGGSWGA SARAKGYKVEKGTPHVGWGASFYGGLAGSDPN 372

Tra11 380 AGHIAVVEYINPDGSI LVSETNVVKAGTGTRSWRVLSKE TVSQILF I QGKAQHGK 434
Trs15 373 AGHIAVVEYVNEDGSI LVSETNVVSPNSGTRSWRVINKATVAAQIDF I QGRK--- 423
<figure><img src="image_3.png" /><figcaption>B.</figcaption></figure>

<figure><img src="image_4.png" /><figcaption>C.</figcaption></figure>

<figure><img src="image_5.png" /><figcaption>D.</figcaption></figure>

<figure><img src="image_6.png" /></figure>
<figure><img src="image_7.png" /><figcaption>B. Trs15<sub>pUC11B</sub>/Tra11<sub>pNP40</sub> – Domain 2</figcaption></figure>

<figure><img src="image_8.png" /></figure>
<caption>A.</caption>

Tra11<sub>pNP40</sub>

<figure><img src="image_9.png" /><figcaption>B.</figcaption></figure>

Trs15<sub>pUC11B</sub>

<figure><img src="image_10.png" /><figcaption>C.</figcaption></figure>

Predicted Aligned Error
Dimer interactions

<figure><img src="image_11.png" /><figcaption>D.</figcaption></figure>

Predicted Aligned Error
Dimer interactions

<figure><img src="image_12.png" /></figure>
<figure><img src="image_13.png" /></figure>

567 ying domains 1 and 2 from Tra11 (A) and Trs15 (B).