Establishment genes present on pls20 family of conjugative plasmids are regulated in two different ways

microorganisms Article Establishment Genes Present on pLS20 Family of Conjugative Plasmids Are Regulated in Two Different Ways Jorge Val-Calvo 1, Andrés Miguel-Arribas 1, Fernando Freire 2, David Abia 2, Ling Juan Wu 3,* and Wilfried J.J. Meijer 1,*   Citation: Val-Calvo, J.; Miguel-Arribas, A.; Freire, F.; Abia, D.; Wu, L.J.; Meijer, W.J. Establishment Genes Present on pLS20 Family of Conjugative Plasmids Are Regulated in Two Different Ways. Microorganisms 2021, 9, 2465. https://doi.org/10.3390/ microorganisms9122465 Academic Editor: Imrich Barák Received: 26 October 2021 Accepted: 25 November 2021 Published: 29 November 2021 Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations. Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). 1Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Instituto de Biología Molecular “Eladio Viñuela” (CSIC), C. Nicolás Cabrera 1, Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain; [email protected] (J.V.-C.); [email protected] (A.M.-A.) 2Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Bioinformatics Facility, C. Nicolás Cabrera 1,Universidad Autónoma, Canto Blanco, 28049 Madrid, Spain; [email protected] (F.F.); [email protected] (D.A.) 3Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Richardson Road, Newcastle Upon Tyne NE4AX, UK *Correspondence: [email protected] (L.J.W.); [email protected] (W.J.J.M.); Tel.: +34-91-196-4539 (W.J.J.M.) Abstract: During conjugation, a conjugative DNA element is transferred from a donor to a recipient cell via a connecting channel. Conjugation has clinical relevance because it is the major route for spreading antibiotic resistance and virulence genes. The conjugation process can be divided into different steps. The initial steps carried out in the donor cell culminate in the transfer of a single DNA strand (ssDNA) of the conjugative element into the recipient cell. However, stable settlement of the conjugative element in the new host requires at least two additional events: conversion of the transferred ssDNA into double-stranded DNA and inhibition of the hosts’ defence mechanisms to prevent degradation of the transferred DNA. The genes involved in this late step are historically referred to as establishment genes. The defence mechanisms of the host must be inactivated rapidly and—importantly—transiently, because prolonged inactivation would make the cell vulnerable to the attack of other foreign DNA, such as those of phages. Therefore, expression of the establishment genes in the recipient cell has to be rapid but transient. Here, we studied regulation of the establishment genes present on the four clades of the pLS20 family of conjugative plasmids harboured by different Bacillus species. Evidence is presented that two fundamentally different mechanisms regulate the establishment genes present on these plasmids. Identification of the regulatory sequences were critical in revealing the establishment regulons. Remarkably, whereas the conjugation genes involved in the early steps of the conjugation process are conserved and are located in a single large operon, the establishment genes are highly variable and organised in multiple operons. We propose that the mosaical distribution of establishment genes in multiple operons is directly related to the variability of defence genes encoded by the host bacterial chromosomes. Keywords: conjugation; transcriptional regulation; Gram-positive bacteria; antibiotic resistance; establishment genes; Bacillus subtilis; plasmid; pLS20 1. Introduction Conjugation is a horizontal gene transfer (HGT) route by which a DNA element is transferred from a donor to a recipient cell via a connecting channel. Conjugation occurs at a large scale in both Gram-negative (G − ) and Gram-positive (G+) bacteria. Conjugative elements can be located in a bacterial chromosome or plasmid, and are named integrated conjugative elements (ICEs) or conjugative plasmid, respectively. The basic principles of the conjugation processes are conserved in G+ and G − bacteria. Conjugation can be divided into five discernible steps. The first step involves selection of and attachment to a recipient Microorganisms 2021,9, 2465. https://doi.org/10.3390/microorganisms9122465 https://www.mdpi.com/journal/microorganisms Microorganisms 2021,9, 2465 2 of 19 cell by the donor cell. The second step comprises the synthesis of a membrane-embedded DNA translocation machinery that is a T4-type secretion system (T4SS). In the third step, the conjugative DNA is processed in the donor cell in order to generate the single-stranded DNA (ssDNA) that is subsequently transferred through the translocation channel into the recipient cell. The final two steps take place in the recipient cell and are referred to as the establishment steps. Firstly, the transferred ssDNA must be circularised and converted into double-stranded plasmid DNA. Secondly, defence mechanisms that protect the cell against incoming foreign DNA must be inactivated. Most conjugation studies address aspects of the first three steps, but very little is known about the establishment step, particularly the way by which conjugative elements inhibit defence systems of the recipient cell. One well-known type of bacterial defence mechanism is the restriction–modification (RM) systems, which encode a restriction endonuclease (REase) and a methyltransferase (MTase). The MTase methylates specific short DNA sequences, and thereby prevents these sequences from being recognised and digested by the cognate REase of the RM system. The REase will digest any foreign DNA entering a cell that is not properly methylated (including conjugative DNA) [ 1 – 3 ]. This system can be considered an innate defence mechanism. Another well-known system, which can be considered as an adaptive defence mechanism, is the CRISPR–Cas system [ 4 ]. Additional defence systems have been discovered in recent years [ 5 ]. For a conjugation event to be successful, the defence systems have to be evaded or inactivated, implying that conjugative elements encode inhibitors of bacterial defence systems. Although this topic has been studied little, it is known for more than two decades that many conjugative plasmids contain an anti-restriction gene whose encoded product is able to inhibit a restriction enzyme [ 6 – 9 ], i.e., anti-restriction genes are typical establishment genes. Importantly, inactivation of the bacterial defence systems must occur temporarily, because prolonged inactivation would make the cell vulnerable to entry of other foreign DNA, e.g., phage DNA. This implies that anti-restriction genes and other establishment genes must be regulated in a special way, such that they are expressed rapidly and transiently upon arrival of the plasmid in the recipient cell. In previous work, we showed that the conjugative plasmid p576 from Bacillus pumilis NRS576 contains an anti-restriction gene, and also encodes a transcriptional regulator, named Reg p576 , that efficiently represses the promoter of the anti-restriction gene [ 10 ]. While the DNA, but not the repressor protein, is transferred during conjugation, the anti-restriction gene will be expressed in the recipient cell soon after it enters the cell until sufficient repressor protein is produced to repress the promoter again. Reg p576 also regulates its own expression and it represses two other promoters controlling the expression of four genes, which presumably also play a role in establishment. In other words, plasmid p576 contains an establishment regulon whose genes are regulated by Regp576. Plasmid p576 shares similarity with the conjugative plasmid pLS20 from Bacillus subtilis. Although pLS20 is among the best-studied conjugative plasmids in G+ bacteria, its establishment genes have not been studied so far. Very recently, we have found that pLS20 is the prototype of a new family of conjugative plasmids that includes p576 [ 11 ]. The family has over 30 members and the plasmids are hosted by Bacillus species that are distributed worldwide. In this work, we studied the establishment regulons present on the pLS20 family of plasmids. We found that all plasmids contain an establishment regulon and that these regulons contain at least one gene that interferes with RM systems. Interestingly, these establishment genes are regulated by two fundamentally different systems, prototyped by the systems present on p576 and pLS20, respectively. Furthermore, whereas the conjugation genes are highly conserved between members of the pLS20 family of plasmids, there is a high level of variation in the establishment genes. We discuss the possibility that the diversity of the establishment genes is directly related to the defence genes encoded by the bacterial genome. Microorganisms 2021,9, 2465 3 of 19 2. Materials and Methods 2.1. Bacterial Strains, Plasmids, and Oligonucleotides Bacterial strains were grown in lysogeny broth (LB) or LB agar plates (LB with 1.5% agar [ 12 ]). Where appropriate, the following antibiotics were added: ampicillin (100 µ g/mL) for E. coli; or spectinomycin (100 µ g/mL) for B. subtilis. The strains, plasmids, and oligonucleotides used are listed in supplementary Tables S1–S3, respectively. All oligonucleotides were purchased from Integrated DNA Technologies (Belgium). 2.2. Construction of Plasmids and Strains Standard molecular methods were used to manipulate DNA [ 13 ]. The correctness of all constructs was verified by sequence analysis. All enzymes were purchased from New England Biolabs, USA. E. coli transformation was carried out using standard methods [ 13 ]. Competent B. subtilis 168 cells were prepared as described before [ 14 ]. Transformants were selected on LB agar plates supplemented with appropriate antibiotics. The following strategy was used to construct derivatives of B. subtilis 168 that contain at their amyE locus a cassette in which the wild-type or derivative of the EGeRS1-B region of pLS20 is placed between the inducible IPTG P spank promoter and the superfolder gfp gene (for simplicity, named here gfp). First, the intergenic region between pLS20cat genes 82c and 85, encompassing EGeRS1-B, or a subregion, was amplified by PCR using pairs of primers listed in supplemental Table S3. The amplified product was purified, digested with SalI, and cloned into the unique SalI site that is present on the amyE integration vector pAND101 in between the P spank promoter and the gfp gene [ 15 ]. The ligation mixtures were used to transform competent E. coli XL1-Blue cells. Recombinant plasmids were identified by colony PCR. The orientation of the insert was determined by PCR using primer pDR111_U in combination with one of the two oligonucleotides used to amplify the corresponding fragment. The names of the pAND101 derivatives are listed in supplemental Table S2. Constructed plasmids were then used to transform competent B. subtilis 168 cells, and spectinomycin-resistant transformants were tested for the loss of amylase activity to identify clones resulting from double crossover events at amyE. The resulting strains, which are listed in supplemental Table S1, contain the configuration Pspank-[fragment X]-gfp. 2.3. Flow Cytometry Promoter strength quantification using fluorescence and flow cytometry was carried out as described before [ 16 ]. In short, overnight grown samples in LB medium at 37 ◦ C were diluted to an OD 600 ≈ 0.025 in fresh 37 ◦ C LB supplemented with or without IPTG (1 mM), and grown again until OD 600 ≈ 0.8–1. For each culture, a 2 mL sample was pelleted, washed twice with 2 mL of filtered 1xPBS, and resuspended in 1 mL of filtered 1xPBS. Fluorescence value is expressed as the mean value of the Geomean value of 100,000 cells measured in three independent experiments. 2.4. In Silico Analyses The homologous genes of the studied plasmids were initially identified using the Get_Homologues or Blast tools. The InterProScan program was used to detect conserved domains or classify proteins into families already described. The homology relationships detected were represented on the sequences of the plasmids, giving rise to a comparative genetic map. Genetic maps were made using the GenomeDiagram module (Biopython project), and subsequently edited with Inkscape software to manually annotate and represent the homology relationships on the plasmid maps. The EGeRS1 regions were identified as duplications in the plasmids on dot plot graphs (plasmid sequence against itself) or by direct observation of the sequences. The ViennaRNA Web Services toolkit was used to analyse the secondary structures of the EGeRS1 regions, either in RNA or ssDNA [ 17 ]. The RNAfold program was used to predict secondary structures of each EGeRS region, while the RNAalifold program [ 18 ] was used to predict conserved secondary structures in MSA of the EGeRS regions. The parameters used in Microorganisms 2021,9, 2465 4 of 19 both programs were the default values, except selecting the Turner model (2004) for RNA and Matthews model (2004) for ssDNA. The alignments used as input to the RNAalifold program were performed with the Pro-Coffee algorithm with standard parameters [ 19 ]. Presentation of the alignments was prepared using the Esprit3.0 program [ 20 ], while the arc diagrams were made using the R-CHIE web server [ 21 ]. The software used to generate the phylogenetic tree was IQ-TREE [ 22 ]. The IQ-TREE software was allowed to determine the substitution model to be used [ 23 ], and the ultrafast bootstrap statistical method was applied (1000 replicates). The phylogenetic trees were drawn using the FigTree v1.4.4 software (http://tree.bio.ed.ac.uk/, accessed on 7 September 2021, Andrew Rambout research group, Edinburgh, UK). The phylogenetic tree was unrooted, although a root was subsequently added, located in the midpoint. The sequences of the EGeRS regions of plasmids pLS20, pBatNRS213, pBamB1895, pBglSRCM103574, pBliYNP2 † , and pBspNMCC4 † were aligned by the Pro-Coffee algorithm. The likelihood tree was made from 30 sequences with 1240 base pairs. The model selected by ModelFinder was K2P + R2. The tree with the highest log likelihood (−6291.1512) is shown. 3. Results 3.1. pLS20 Contains an Anti-Restriction Gene Similar to the One on Plasmid p576, but Both Genes Are Preceded by Very Different Sequences In previous work, we found that the B. pumilus plasmid p576 contains an antirestriction gene, ardC p576 , which is a typical establishment gene [ 10 ]. The upstream sequences hold the key for the transient expression of ardC p576 after the plasmid has entered a recipient cell. Sequences sharing between 50 and 68% identity to those upstream of ardC p576 are also present upstream of some other p576 genes/operons, and these genes were found to be regulated in an identical or similar way as ardC p576 , strongly indicating that these genes also play a role in establishment of the plasmid in the recipient cell. In other words, the conserved upstream sequences of the typical establishment gene ardC p576 formed the crux in identifying the establishment regulon of p576 [ 10 ]. A schematic view of the regulatory mechanism of the establishment operons on plasmid p576 is shown in supplemental Figure S1. We wondered if we could use the same strategy to identify the establishment regulon of plasmid pLS20 from B. subtilis that is related to p576 [24,25]. Like p576, pLS20 contains an anti-restriction gene (gene 82c, according to our annotation, and renamed here ardC pLS20 ) whose encoded product shares 51% identity with ArdC p576 (supplemental Figure S2). Like ardC p576 of p576, ardC pLS20 would be expressed only transiently after conjugative transfer of pLS20 into the recipient cell. However, the sequences upstream of ardC pLS20 are very different from those upstream of the p576 establishment genes: the region is about fivefold larger, and it contains multiple inverted repeated sequences that are absent in p576 (Figure 1A,C) . The presence of completely different sequences upstream of ardC pLS20 and upstream of ardC p576 and other p576 establishment genes strongly suggests that they are regulated in different ways. Microorganisms 2021,9, 2465 5 of 19 Microorganisms 2021, 9, x FOR PEER REVIEW 5 of 19 Figure 1. The region upstream of ardC pLS20 , which is different from the upstream region of ardC p576 , contains multiple inverted repeated sequences predicted to generate stable secondary structures in ssDNA or RNA. (A) The 600 bp region immediately upstream of the ribosome binding site (RBS) of ardC pLS20 contains multiple inverted repeated sequences. Inverted repeated sequences are indicated with coloured arrows shown above the sequence. The inverted repeated sequences that are separated by sequences of more than 100 bp, which themselves can contain other inverted repeated sequences, are boxed. (B) Centroid secondary structure predicted to be formed according to the RNAfold web server (rna.tbi.univie.ac.at//cgibin/RNAWebSuite/RNAfold.cgi) when the upstream region is in its single-stranded form. A very similar secondary structure is predicted when the region is transcribed into RNA. The sequence immediately upstream of the RBS is predicted to form a branch of ⁓300 nt with four ramifications (positioned at the right side of the image). Upstream of this branch, another branch of ⁓300 nt would be formed, with two and three ramifications predicted to be formed with low and high probability, respectively. Colours indicate the degree of probability that the predicted secondary structure is formed. Red nucleotides reflect very high probabilities. Levels of decreasing probabilities are indicated by nucleotides in orange, yellow, green, light blue, Figure 1. The region upstream of ardC pLS20 , which is different from the upstream region of ardC p576 , contains multiple inverted repeated sequences predicted to generate stable secondary structures in ssDNA or RNA. ( A ) The 600 bp region immediately upstream of the ribosome binding site (RBS) of ardC pLS20 contains multiple inverted repeated sequences. Inverted repeated sequences are indicated with coloured arrows shown above the sequence. The inverted repeated sequences that are separated by sequences of more than 100 bp, which themselves can contain other inverted repeated sequences, are boxed. ( B ) Centroid secondary structure predicted to be formed according to the RNAfold web server (rna.tbi.univie.ac.at//cgibin/RNAWebSuite/RNAfold.cgi) when the upstream region is in its single-stranded form. A very similar secondary structure is predicted when the region is transcribed into RNA. The sequence immediately upstream of the RBS is predicted to form a branch of ~300 nt with four ramifications (positioned at the right side of the image). Upstream of this branch, another branch of ~300 nt would be formed, with two and three ramifications predicted to be formed with low and high probability, respectively. Colours indicate the degree of probability that the predicted Microorganisms 2021,9, 2465 6 of 19 secondary structure is formed. Red nucleotides reflect very high probabilities. Levels of decreasing probabilities are indicated by nucleotides in orange, yellow, green, light blue, and dark blue colours. ( C ) The 140 bp region of plasmid p576 located upstream of ardC p576 . The promoter sequences of P ardCp576 are shown on a blue background and the − 35 and − 10 boxes are indicated. The Reg p576 operator is shown on a red background. The two short inverted repeats, indicated with blue and green convergent arrows, most likely function to enhance the half-life time of the RNA molecule [ 10 ]. 3.2. Sequences Highly Similar to Those Upstream of ardC pLS20 Are also Present Upstream of Four Operons on pLS20: Identification of the Establishment Regulon of pLS20 Further analysis of the pLS20 revealed that sequences sharing an identity ranging between 62 and 95% to the region upstream of ardC pLS20 were present at four other positions on pLS20, all confined within a region spanning about 20% of the pLS20 genome (see Figure 2A ). An alignment of these five conserved sequences, ranging from 420 to 750 bp, is shown in Figure 2B. Interestingly, all five sequences are located immediately upstream of a likely ribosomal binding site (RBS), and each is followed by a putative operon (see Figure 2A ). To distinguish between them, we refer to these five operons as operon A to E, which are composed of the following pLS20 genes: 79c-78c-77c-76c (operon A); genes 82c-81c (operon B, gene 82c =ardC pLS20 ); genes 90c-89c-88c-87c-86c (operon C); genes 6c-5c4c-3c-2c (operon D); and genes 9c-8c-7c (operon E) (see Figure 2A). By analogy with the situation on plasmid p576, it is likely that the upstream sequences are crucial for regulating the expression of the genes present in the downstream operons of pLS20. In other words, the 19 genes from these five operons probably form the establishment regulon of pLS20. We refer to the conserved sequences upstream of the putative establishment operons present on pLS20 and p576 as establishment gene regulatory sequence (EGeRS) type 1 (or EGeRS1) and EGeRS type 2 (or EGeRS2), respectively. To discriminate between the five EGeRS1 regions present on pLS20, their EGeRS1 names are extended with the letter corresponding to the downstream operon; hence, the region upstream of ardC pLS20 is named EGeRS1-B. Although the EGeRS1 sequences are 62 to 95% identical to each other, there are some noteworthy differences. For instance, the 420 bp EGeRS1-A is smaller than the approximate 610 bp long regions of EGeRS1-B, D, and E, because it lacks the 180 bp sequence located at the 5 0 side of the EGeRS1 regions in B, D, and E. Finally, EGeRS1-C has a size of 751 bp due to an internal duplication of 131 bp plus 9 extra bp (Figure 2B). 1 Figure 2. A conserved region of about 600 bp is present upstream of five putative operons on pLS20. ( A ) Circular map of pLS20. The different operons/modules are indicated with thick coloured lines on the inner circle: green, conjugation operon; Microorganisms 2021,9, 2465 7 of 19 yellow, origin of replication; brown, partitioning genes; and blue, establishment genes. Genes are indicated with wide arrows on the outer ring. Greenand blue-coloured arrows represent conjugation and establishment genes, respectively. Other genes are coloured grey, except the gene encoding the repressor of the conjugation genes, which is shown in red. The blow-up shown in the lower panel shows the organisation of the five establishment operons encompassing genes 76c to 9c, corresponding to plasmid positions 41,801–54,600 of pLS20cat (accession number AB615352). Positions of repeated sequences are indicated with yellow boxes and numbered A to E. Genes are indicated with wide arrows. The green arrow indicates the position and orientation of the conjugation genes. ( B ) Alignment of the five conserved regions located upstream of the putative operons on pLS20. Note that region “A” is shorter due to the absence of about 195 bp at the 5 0 end that is conserved in the other four regions. Region “C” is longer due to an internal repeat of 131 bp (underlined with a grey arrow; the black arrow indicates the duplicated segment). Each sequence is located just upstream of the RBS of the first gene of the operon. Residues conserved in all five sequences are shown in a black background, while residues conserved in at least four sequences are boxed. 3.3. Features of the EGeRS1 Sequences Two conspicuous features characterise the EGeRS1 regions. First, their GC content is significantly higher than the mean GC content of pLS20 (51.4 versus 37.7%, respectively). Second, they contain multiple inverted repeated sequences (Figure 1A). When present in ssDNA or RNA, these sequences are predicted to form complex secondary structures. Since the DNA is transferred into the recipient cell as ssDNA, it is very possible that secondary structures form temporally soon after transfer into the recipient cell. According to the RNAfold web server, EGeRS1 sequences are predicted to form very stable secondary structures, with calculated free energies of − 152.1/ − 71 kcal/mol (EGeRS1-A; RNA/DNA) or around − 220/ − 110 kcal/mol (other EGeRS1s; RNA/DNA). Figure 1B shows an example of the predicted secondary structures formed in the ssDNA of EGeRS1-B. The RNAalifold web server of the RNAWebServer was used to predict a consensus structure of the five EGeRS1 sequences (see supplementary Figure S3). The predicted structure shows that the region of about 325 nt located immediately upstream of the RBSs, which is conserved in all five EGeRS1 sequences, would form a branch containing four ramifications. Except for EGeRS1-A, which lacks the 180 bp at the 5 0 end, the other EGeRS1 sequences were predicted to generate an additional branch. In the case of the EGeRS1 sequences B, D, and E, this branch would form two stem-loops. Due to the 131 bp internal duplication in EGeRS1-C, these upstream secondary structures are more complex. The predicted secondary structures may be important for function, although, at present, we do not have evidence that the regulatory mechanism involving the EGeRS1 regions depends on ssDNA or RNA. 3.4. Functional Analysis of pLS20 EGeRS1-B •EGeRS1-B does not contain a constitutive promoter The EGeRS2 sequences of p576, i.e., the sequences preceding the p576 establishment genes, contain a strong promoter [ 10 ]. We tested whether EGeRS1 sequences also contain a promoter by constructing strains containing a transcriptional fusion of (parts of) the EGeRS1-B region with a gfp reporter gene (see Materials and Methods) and measuring fluorescence levels by flow cytometry. Figure 3A shows a schematic presentation of the different EGeRS1-B regions fused to gfp. In short, fragments were cloned in between the IPTG-inducible promoter P spank and the gfp reporter gene present on the amyE integration vector pAND101 [ 15 ], and the resulting cassette “P spank -[fragment X]-gfp” was subsequently placed at the amyE locus of the B. subtilis chromosome. Since the P spank promoter is tightly repressed in the absence of the inductor [ 16 ], any fluorescence detected in the absence of the inducer IPTG is due to promoter activity originating from within the cloned fragment. These strains can also be exploited to study possible effects of the cloned insert on upstream initiated transcription by comparing fluorescence levels in the absence and presence of IPTG (see below). The results of the cytometry analysis are presented in Figure 3B. As expected, very low fluorescence levels (~2 arbitrary units (AU)) were obtained for the negative control strain AND101 when cells were grown in the absence of IPTG (see Figure 3B, Microorganisms 2021,9, 2465 8 of 19 AND101, green bar), demonstrating that the P spank promoter is tightly repressed under these conditions. Importantly, very low fluorescence levels, similar to those obtained with the negative control strain AND101, were also obtained for strain JV62A containing the entire EGeRS1-B region fused to gfp in its native orientation. This result suggested that either the cloned EGeRS1-B region did not contain a constitutive promoter, or the activity of the promoter was inhibited or masked by some DNA sequences that were also present in the cloned fragment. To test the latter possibility, strains containing progressive deletions at either the 5 0 or the 3 0 end were constructed (Figure 3A, strains JV63A to JV65A, and JV66 to JV69). However, low fluorescence levels were also obtained for these strains ( Figure 3B ). Next, we constructed strains JV62B–JV65B that contained the EGeRS1-B region in the reverse orientation compared to strains JV62A–JV65A (see Figure 3A) to eliminate the possibility that the promoter activity might have been obscured by a convergently oriented promoter also present in the cloned fragment. Again, no promoter activity was detected in these strains. We then considered the possibility that a promoter was located further upstream of EGeRS1-B. Therefore, the regions cloned in strains JV66 to JV69 were extended at their 5 0 ends to include the 338 bp sequence upstream of EGeRS1-B, which ended in the divergently oriented gene 85. However, promoter activity was still not observed for strains JV66–JV69, indicating that the EGeRS1-B upstream region did not contain a constitutive promoter. Moreover, contrary to the situation on p576, manual inspection of these sequences did not result in the identification of sequences sharing clear similarities to the consensus sequences of σA -dependent promoters (not shown). Together, these results suggest that EGeRS1-B and sequences upstream do not contain a constitutive σA -dependent promoter that would transcribe the downstream-located ardC pLS20 establishment gene, unlike the situation in p576, where the upstream region of ardC p576 contains a strong promoter [ 10 ]. However, we could not exclude the possibility that the EGeRS1-B region contains a promoter that can be induced under certain conditions. Microorganisms 2021, 9, x FOR PEER REVIEW 8 of 19 under these conditions. Importantly, very low fluorescence levels, similar to those obtained with the negative control strain AND101, were also obtained for strain JV62A containing the entire EGeRS1-B region fused to gfp in its native orientation. This result suggested that either the cloned EGeRS1-B region did not contain a constitutive promoter, or the activity of the promoter was inhibited or masked by some DNA sequences that were also present in the cloned fragment. To test the latter possibility, strains containing progressive deletions at either the 5′ or the 3′ end were constructed (Figure 3A, strains JV63A to JV65A, and JV66 to JV69). However, low fluorescence levels were also obtained for these strains (Figure 3B). Next, we constructed strains JV62B–JV65B that contained the EGeRS1-B region in the reverse orientation compared to strains JV62A–JV65A (see Figure 3A) to eliminate the possibility that the promoter activity might have been obscured by a convergently oriented promoter also present in the cloned fragment. Again, no promoter activity was detected in these strains. We then considered the possibility that a promoter was located further upstream of EGeRS1-B. Therefore, the regions cloned in strains JV66 to JV69 were extended at their 5′ ends to include the 338 bp sequence upstream of EGeRS1B, which ended in the divergently oriented gene 85. However, promoter activity was still not observed for strains JV66–JV69, indicating that the EGeRS1-B upstream region did not contain a constitutive promoter. Moreover, contrary to the situation on p576, manual inspection of these sequences did not result in the identification of sequences sharing clear similarities to the consensus sequences of σ A -dependent promoters (not shown). Together, these results suggest that EGeRS1-B and sequences upstream do not contain a constitutive σ A -dependent promoter that would transcribe the downstream-located ardC pLS20 establishment gene, unlike the situation in p576, where the upstream region of ardC p576 contains a strong promoter [10]. However, we could not exclude the possibility that the EGeRS1-B region contains a promoter that can be induced under certain conditions. . Figure 3. EGeRS1-B located upstream of ardC pLS20 does not contain a regular promoter, but its presence prevents readthrough from upstream promoters. (A) Schematic overview of the gfp fusions generated. 5′ deletion fragments were cloned in both orientations between the IPTG-inducible promoter P spank and the gfp reporter gene in vector pAND101. 3′ deletion fragments were cloned in the native orientation. Names of the B. subtilis strains containing these gfp fusions at the chromosomal amyE locus are shown at the left; names ending with letter “A” and “B” correspond to strains containing the cloned fragment in the native and reverse orientations, respectively. The secondary structures predicted to form in ssDNA or RNA are indicated with arcs above the lines representing the cloned regions. (B) Fluorescence levels of cells taken from late exponentially growing cultures (OD 600 = 1) of strains grown in the absence (black bars) or presence of 1 mM IPTG (green bars). Strain AND101 (P spank -gfp) served as a control. Error bars indicate standard deviations. Each experiment was performed at least three times. Figure 3. EGeRS1-B located upstream of ardC pLS20 does not contain a regular promoter, but its presence prevents readthrough from upstream promoters. ( A ) Schematic overview of the gfp fusions generated. 5 0 deletion fragments were cloned in both orientations between the IPTG-inducible promoter P spank and the gfp reporter gene in vector pAND101. 3 0 deletion fragments were cloned in the native orientation. Names of the B. subtilis strains containing these gfp fusions at the chromosomal amyE locus are shown at the left; names ending with letter “A” and “B” correspond to strains containing the cloned fragment in the native and reverse orientations, respectively. The secondary structures predicted to form in ssDNA or RNA are indicated with arcs above the lines representing the cloned regions. ( B ) Fluorescence levels of cells taken from late exponentially growing cultures (OD 600 = 1) of strains grown in the absence (black bars) or presence of 1 mM IPTG (green bars). Strain AND101 (P spank -gfp) served as a control. Error bars indicate standard deviations. Each experiment was performed at least three times. Microorganisms 2021,9, 2465 9 of 19 •EGeRS1-B sequence prevents readthrough of upstream promoters We next considered the possibility that the presence of EGeRS1-B might interfere with progression of transcripts initiated at upstream promoters by determining the levels of fluorescence produced by the upstream-located P spank promoter. As expected, under the conditions where P spank was activated (i.e., strains grown in the presence of 1 mM IPTG), high fluorescence levels were obtained for the control strain AND101 (Figure 3B, green bar). Interestingly, very low fluorescence levels were obtained for strains JV62A–JV65A containing the EGeRS1-B region in its native orientation. In the case of JV62A containing the entire EGeRS1-B region, only background levels of fluorescence were obtained. Strain JV65A, which contained only the 215 bp 3 0 region of EGeRS1-B, also produced very low levels of fluorescence. Thus, the presence of the ~3 0 half of the EGeRS1-B region, which is conserved in all five EGeRS1 regions, was sufficient for interfering with transcripts starting at the upstream P spank promoter and preventing the expression of the downstream gfp gene. This probably indicates that, in its native setting, the EGeRS1-B region prevents expression of the ardC pLS20 gene resulting from readthrough of upstream-located promoter(s), thereby contributing to the strict control of genes ardC pLS20 and gene 81c, which is likely to be important for proper functioning and fitness of the host cell. We also tested the effect of the EGeRS1-B sequences when present in the reverse orientation. As shown in Figure 3B , moderate to high fluorescence levels were obtained for strains JV62B–JV65B; therefore, when present in the reverse orientation, the EGeRS1-B sequences did not block or majorly interfere with transcription progression. In summary, the above results suggest that, contrary to EGeRS2 regions present on plasmid p576, the EGeRS1 regions on pLS20 do not contain a constitutive σA -dependent promoter responsible for the expression of the downstream-located establishment genes. However, it seems that the presence of the EGeRS1-B region prevents read through of upstream promoter(s), thereby contributing to proper expression of the ardCpLS20 establishment gene. 3.5. Establishment Genes Present on pLS20 Family Plasmids Are Regulated by One of the Two Different Mechanisms Recently, we showed that pLS20 is the prototype of a family of related plasmids present in different Bacillus species. To gain insights into their phylogenetic relationships, two maximum likelihood trees were constructed. One of these was based on the replication region and the other on a concatenated sequence of nine conserved orthologous genes. This analysis resulted in two very similar trees in which these plasmids were divided into four clades [ 11 ]. pLS20 belongs to clade I, together with 23 other plasmids, and p576 belongs to clade II, together with six other plasmids. Clade III comprises three plasmids, and clade IV is constituted by a single member. Since the establishment genes/operons of pLS20 and p576 are preceded by completely different sequences, we wondered whether the upstream (regulatory) sequences of the establishment genes/operons in the other pLS20 family plasmids were similar to those present on pLS20 or p576, or perhaps would be preceded by different sequences. This analysis revealed that all pLS20 family plasmids contain multiple copies of sequences that were >60% identical to either EGeRS1 of pLS20 or EGeRS2 of p576. Moreover, as in p576 and pLS20, the conserved sequences were located upstream of the putative establishment genes/operons (see below). These results strongly indicate that the establishment genes present on the pLS20 family of plasmids are regulated by one of the two different mechanisms that are exemplified by those present on pLS20 and p576. Besides p576, EGeRS2 sequences are present on six other plasmids that all belong to clade II. All the other plasmids of the pLS20 family belonging to clades I, III, and IV contain EGeRS1-like sequences. 3.6. Analysis of the EGeRS1 Sequences Clade I, III, and IV plasmids each contain multiple EGeRS1 sequences. To see how similar these sequences were, and whether their similarities correlated with the different Microorganisms 2021,9, 2465 16 of 19 restriction genes [ 34 ] that are also present on many pLS20 family plasmids (this work). We have considered the possibility that the EGeRS1 sequences might constitute ssDNA promoters. However, this seems highly unlikely for the following reasons. First, the EGeRS sequences are much longer than the ssDNA promoters, and no putative promoter could be identified in the predicted secondary structure generated upon hybridisation of the EGeRS1 sequences. Second, and more importantly, the DNA strand that is nicked by the relaxase is the strand that is transferred into the recipient cell. The DNA strand that is nicked by the relaxase of pLS20 has been determined [ 35 ], and this strand corresponds to the non-template strand, i.e., the transferred ssDNA strand cannot function as a template for generation of the establishment gene mRNA. 4.2. Establishment Regulons Present on pLS20 Family of Plasmids and Implications of Establishment Regulons in General Several conclusions can be drawn from the identification of the establishment operons on the pLS20 family of plasmids described here, and also from the published results. For instance, based on their role in the conversion of ss to dsDNA and/or inhibition of host-encoded defence mechanism, it is clear that the establishment genes play a crucial role in the conjugation process, and hence should be considered as part of the conjugation module, together with the genes in the large conjugation operon. This conclusion is supported by the demonstrated importance of ard genes in the establishment of conjugative plasmids in the new host [ 36 – 39 ]. Therefore, most genes present on the pLS20 family plasmids are related to the conjugation process, e.g., 69 of the 92 genes (75%) of pLS20 form part of the conjugation module. The work presented here also revealed two interesting differences between the genes of the pLS20 family plasmids that are involved in the first three steps of conjugation (i.e., recipient cell selection and attachment, synthesis of the translocation machinery, and DNA processing), and those corresponding to the fourth step of the conjugation process (i.e., establishment genes). First, there is an overall high level of similarity between the genes involved in the first three steps [ 11 ], but the establishment genes involved in step four tend to be more variable. Second, while the genes involved in the first three steps are all located in one single large operon, the establishment genes are distributed in multiple operons, which cluster together on the plasmid. Based on the arguments described below, we propose that the organisation of establishment genes on a plasmid in a cluster of variable operons has coevolved with the variable clusters of defence mechanisms encoded by bacterial chromosomes. In the last decade or so, it has become clear that bacteria encode (many) more defence mechanisms than the RM and CRISPR–cas systems to protect them against incoming foreign DNA, such as that of phages and conjugative elements [ 5 ]. There are striking similarities in the organisation and distribution between the defence genes encoded by bacterial chromosomes and the establishment genes and anti-defence genes present on the pLS20 family of plasmids. Like chromosomal defence genes, establishment genes of the pLS20 family are organised in cluster(s), and there is a big variety of establishment genes harboured by the individual plasmids. Thus, according to the so-called “pan-immune model”, a group of related bacterial species together possess a large arsenal of defence mechanisms but individual cells contain only a subset of these defence genes, probably because they pose a high fitness cost to a bacterial cell [ 5 ]. The clustered defence mechanisms on bacterial genomes are referred to as defence islands [ 40 ]. In analogy, we propose to name the clustered establishment operons as establishment or anti-defence islands. Establishment and anti-defence operons are also clustered on other conjugative plasmids, such as the F-like plasmid that are controlled by ssDNA promoters, and on at least several other conjugative plasmids and mobile elements [ 41 – 43 ], suggesting that island-like organisation of establishment/anti-defence genes/operons is a general feature. Although the origin and mechanism(s) responsible for the formation and plasticity of defence islands are unknown, these regions often also contain mobility genes (e.g., transposases and recombinases, and even conjugation genes), suggesting that horizontal gene transfer events play a role in this [ 40 , 44 , 45 ]. In addition, or alternatively, Microorganisms 2021,9, 2465 17 of 19 regions containing defence clusters may be integration hotspot sites for acquired genes, or they may reflect functional links, including possible coregulation [5,46,47]. At this moment, we do not know why the establishment/anti-defence genes on conjugative elements and other mobile elements are in clusters, but (several of) the arguments proposed to explain the clustered organisation of defence genes on chromosomes may also be accountable for this. Particularly, coregulation may play an important role in clustering of these genes, as we have shown here that the establishment genes present on at least the pLS20 family and the F-like plasmids are regulated “en bloc” by one of the three different mechanisms. In addition, the variability of the establishment/anti-defence genes and their organisation in multiple, rather than one, operons may be directly related to the way the defence genes are organised on bacterial chromosomes. Thus, a given cluster of establishment/anti-defence genes on conjugative elements would suffice to transiently inhibit a subset of defence systems harboured by a recipient cell. Interactions between mobile genetic elements, including conjugative elements, and the host cells would be the driving force for rapid evolutionary alterations in defence and anti-defence genes. One reason why the establishment/anti-defence genes are organised in multiple operons may be that it favours shuffling of the individual operons between plasmids. In summary, based on the results obtained here, we propose that, besides the antirestriction genes, several or most of the other establishment genes present on pLS20 family plasmids, and conjugative plasmids in general, function to transiently inhibit known and unknown defence mechanisms encoded by bacterial chromosomes. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10.339 0/microorganisms9122465/s1, Figure S1: Schematic presentation of the regulatory mechanism of the establishment genes on the pLS20-related plasmid p576. Figure S2: Alignment between the Shigella flexneri encoded antirestriction protein ArdC (ArdC/pSa) and the putative antirestriction proteins encoded by p576 (ArdC/p576) and pLS20 (ArdC/pLS20). Figure S3: Predicted conserved secondary structure of the EGeRS1 sequences when present in ssDNA or RNA. Figure S4: All seven clade II pLS20 family plasmids encode a homolog of Regp 576 that in p576 is responsible for regulating the establishment genes. Figure S5: Conserved promoters flanked by the Reg operators in the seven clade II plasmids of the pLS20 family. Table S1: Strains used. Table S2: Plasmids. Table S3: oligonucleotides used. Table S4: Putative establishment genes identified on clade II pLS20 family plasmids. Table S5: Putative establishment genes identified on plasmids pLS20, pBatNRS213, pBamB1895, pBglSRCM103574, pBliYNP2†, pBspNMCC4† and pBS72. Author Contributions: Conceptualization, J.V.-C., L.J.W. and W.J.J.M.; Data curation, J.V.-C., F.F., D.A. and W.J.J.M.; Formal analysis, J.V.-C., A.M.-A., F.F. and D.A.; Funding acquisition W.J.J.M.; Investigation, J.V.-C., F.F., A.M.-A., L.J.W. and W.J.J.M.; Methodology, J.V.-C., A.M.-A., F.F., D.A., L.J.W. and W.J.J.M.; Project administration, W.J.J.M.; Resources, W.J.J.M.; Software, D.A.; Supervision, W.J.J.M.; Validation, D.A.; Visualization, J.V.-C. and W.J.J.M.; Writing—original draft, W.J.J.M.; Writing—review and editing, L.J.W. and W.J.J.M. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the MINISTRY OF SCIENCE AND INNOVATION OF THE SPANISH GOVERNMENT grants (bio2016-77883-C2-1-P) and (PID2019-108778GB-C21 (AEI/FEDER, EU)) to W.J.J.M., which also funded J.V-C. and A.M-A. A Wellcome Investigator grant (209500) to Jeff Errington supported L.J.W. 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