REVIEW published: 18 March 2021 doi: 10.3389/fmolb.2021.648468 Multiple Layered Control of the Conjugation Process of the Bacillus subtilis Plasmid pLS20 Wilfried J. J. Meijer 1*, D. Roeland Boer 2, Saúl Ares 3, Carlos Alfonso 4, Fernando Rojo 5, Juan R. Luque-Ortega 6 and Ling Juan Wu 7* 1 Laboratory 402, Centro de Biología Molecular “Severo Ochoa” (CSIC-UAM), Universidad Autónoma, Canto Blanco, Madrid, Spain, 2ALBA Synchrotron Light Source, Barcelona, Spain, 3Laboratory 35, C. Grupo Interdisciplinar de Sistemas Complejos and Departamento de Biología de Sistemas, Centro Nacional de Biotecnología, CSIC, Madrid, Spain, 4Laboratory B08, Systems Biochemistry of Bacterial Division Lab, Centro de Investigaciones Biológicas Margarita Salas (CSIC), Madrid, Spain, 5Laboratory 216, Department of Microbial Biotechnology, Centro Nacional de Biotecnología, CSIC, Madrid, Spain, 6Laboratory S07, Molecular Interactions Facility, Centro de Investigaciones Biológicas Margarita Salas (CSIC), Madrid, Spain, 7Centre for Bacterial Cell Biology, Biosciences Institute, Newcastle University, Newcastle Upon Tyne, United Kingdom Edited by: Chew Chieng Yeo, Bacterial conjugation is the main horizontal gene transfer route responsible for the spread Sultan Zainal Abidin University, of antibiotic resistance, virulence and toxin genes. During conjugation, DNA is transferred Malaysia from a donor to a recipient cell via a sophisticated channel connecting the two cells. Reviewed by: Conjugation not only affects many different aspects of the plasmid and the host, ranging Günther Koraimann, University of Graz, Graz, Austria from the properties of the membrane and the cell surface of the donor, to other Imrich Barak, developmental processes such as competence, it probably also poses a burden on Institute of Molecular Biology (SAS), Slovakia the donor cell due to the expression of the large number of genes involved in the *Correspondence: conjugation process. Therefore, expression of the conjugation genes must be strictly Wilfried J. J. Meijer controlled. Over the past decade, the regulation of the conjugation genes present on the wmeijer@cbm.csic.es conjugative Bacillus subtilis plasmid pLS20 has been studied using a variety of methods Ling Juan Wu l.j.wu@newcastle.ac.uk including genetic, biochemical, biophysical and structural approaches. This review focuses on the interplay between RcopLS20, RappLS20 and Phr*pLS20, the proteins that control the Specialty section: activity of the main conjugation promoter Pc located upstream of the conjugation operon. This article was submitted to Molecular Recognition, Proper expression of the conjugation genes requires the following two fundamental a section of the journal elements. First, conjugation is repressed by default and an intercellular quorum- Frontiers in Molecular Biosciences signaling system is used to sense conditions favorable for conjugation. Second, Received: 31 December 2020 different layers of regulation act together to repress the Pc promoter in a strict manner Accepted: 08 February 2021 Published: 18 March 2021 but allowing rapid activation. During conjugation, ssDNA is exported from the cell by a Citation: membrane-embedded DNA translocation machine. Another membrane-embedded DNA Meijer WJ J, Boer DR, Ares S, translocation machine imports ssDNA in competent cells. Evidences are reviewed Alfonso C, Rojo F, Luque-Ortega JR and Wu LJ (2021) Multiple Layered indicating that conjugation and competence are probably mutually exclusive Control of the Conjugation Process of processes. Some of the questions that remain unanswered are discussed. the Bacillus subtilis Plasmid pLS20. Front. Mol. Biosci. 8:648468. Keywords: quorum sensing, Bacillus subtilis, Gram-positive bacteria, antibiotic resistance, horizontal gene transfer, doi: 10.3389/fmolb.2021.648468 DNA looping, transcriptional regulation, conjugation Frontiers in Molecular Biosciences | www.frontiersin.org 1 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation INTRODUCTION processing reaction. During stage 3) a sophisticated membrane-embedded translocation machinery, also called Horizontal Gene Transfer (HGT) refers to the process by which a transferosome, is synthesized through which the DNA is DNA element/region is transferred from one cell to another. transferred. The intercellular transferosome, which is a form of HGT occurs at large scale in bacteria, and a single HGT event can type IV secretion system, is composed of at least eight but often result in the acquisition of several to many genes by a cell and more different proteins (Christie et al., 2014; Christie, 2016; Li therefore plays a major role in bacterial evolution (Soucy et al., et al., 2019; Waksman, 2019). Moreover, most of these proteins 2015). Unfortunately, HGT also contributes importantly to the are present in multiple copies in the transferosome. The plasmid dissemination of antibiotic resistance, virulence and toxin genes. is recruited to the transferosome through interactions of an The spread of these genes has now become a major global ATPase, named coupling protein (CP) and which is located at problem, with serious consequences on both human lives and the cytoplasmic entry side of the transferosome, and the economy (Partridge et al., 2018; CDC, 2019; Lerminiaux and relaxosome proteins. Next, the relaxase pilots transfer of the Cameron, 2019). A better understanding of the HGT processes is ssDNA into the recipient cell in an energy consuming process needed to design drugs or strategies to impede the spread of that involves, besides the CP, one or two additional ATPases. antibiotic resistance and other pernicious genes. There are three Stage 4) is the establishment of the DNA once it has entered the main mechanisms responsible for HGT: transformation, recipient cell. Once transferred, the ssDNA has to be converted transduction and conjugation (Ochman et al., 2000; Frost into double-stranded DNA (dsDNA). Many conjugative elements et al., 2005; Thomas and Nielsen, 2005; Novick et al., 2010). encode a primase that is involved in the conversion of ss to Of these, conjugation is the mechanism that is majorly dsDNA. Finally, as most bacteria have evolved defense systems responsible for the spread of antibiotic resistance, toxin and that inactivate incoming foreign DNA, including conjugative virulence genes (Mazel and Davies, 1999; Davies and Davies, elements, conjugative elements encode proteins that transiently 2010). inactivate these defense mechanisms. So far, little is known about During conjugation, a DNA element is transferred from a this step and the proteins involved. donor cell to a recipient cell; hence, it requires direct contact Expression of the conjugation genes of most systems are between the two cells. Conjugative elements are often present on strictly controlled because of at least two reasons. First, plasmids, but they can also be integrated in a bacterial expression of the many proteins involved in the conjugation chromosome. These latter elements are named integrative and process imposes an energetic burden on the cell. Second, conjugative elements (ICE). Conjugation occurs in both Gram- activation of the conjugation process has major consequences positive (G+) and Gram-negative (G−) bacteria, and although for both the plasmid and the host. Conjugative plasmids replicate there are important differences (see below), the basics of the by the theta type of replication mechanism during vegetative conjugation process are conserved in G+ and G− bacteria. growth of the host. However, replication switches to the rolling- Conjugation can be divided into four discernable stages, which circle mode during conjugation in order to generate the ssDNA have been described in detail previously (Chen et al., 2005; molecule that is transferred to the recipient cell (Smillie et al., Arutyunov and Frost, 2013; Cabezon et al., 2014; Goessweiner- 2010; Cabezon et al., 2014). Moreover, probably all conjugative Mohr et al., 2014; Koraimann, 2018; Li et al., 2019; Waksman, plasmids possess a partitioning system to ensure that each 2019). Below we briefly summarize each stage, to illustrate the daughter cell receives a copy of the plasmid upon cell division large number of proteins involved in conjugation, and the (Baxter and Funnell, 2014; Brooks and Hwang, 2017; Bouet and complexity of the process in which the many proteins act Funnell, 2019). During conjugation, however, the plasmid is together in a temporally orchestrated manner. During stage 1) recruited to the entry site of the transferosome. The transfer competent donor cells select and attach to a suitable conjugation process also has consequences for the host cell, as recipient cell (also known as mating pair formation). This process it alters characteristics of the membrane and the cell surface. involves surface-located proteins, some of which are adhesion Studies on conjugative plasmids replicating in G- bacteria have proteins that establish contact with the recipient cell. The shown that the conjugation genes are strictly controlled by a adhesion proteins encoded by conjugative elements present in combination of plasmid and host-encoded factors. In particular, G− bacteria are generally located at the tip of a pilus structure. the mechanism controlling conjugation genes of the IncF Conjugative elements of G+ bacteria do not form so-called sex- incompatibility group has been studied in depth (Frost and pili but probably encode adhesions to fulfill this function. While Koraimann, 2010; Koraimann, 2018). Compared to G− some proteins are involved in establishing contact, other surface- bacteria, conjugation in G+ bacteria is less understood, and located proteins serve to inhibit conjugation between two donor little is known about the regulation of conjugation genes cells, in a process named exclusion. In stage 2) the conjugation (Singh and Meijer, 2014; Kohler et al., 2019). Exceptions are element is processed to generate the DNA that is transferred to the two related enterococcal plasmids pCF10 and pAD1. the recipient cell, which in most cases is single-stranded DNA Conjugation of these plasmids is induced by pheromones that (ssDNA). The DNA processing reaction requires a relaxase, are produced by plasmid-free recipient cells (for review see, which forms a nucleoprotein complex called the relaxosome Clewell, 2011; Dunny and Johnson, 2011; Chatterjee et al., that introduces a site- and strand-specific nick within the 2013; Dunny and Berntsson, 2016). origin of transfer (oriT). Host and/or plasmid-encoded The 65 kb plasmid pLS20 was originally identified in the proteins, named auxiliary proteins, often assist in the DNA Bacillus subtilis natto strain IFO3335 (Tanaka and Koshikawa, Frontiers in Molecular Biosciences | www.frontiersin.org 2 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation 1977) and is known to be conjugative in liquid as well as on solid growth phase of the recipient cells. Thus, pLS20 conjugation is media (Koehler and Thorne, 1987; Meijer et al., 1998; Itaya et al., strictly repressed except for a small window of time near the end 2006). Although very little was known about conjugation systems of the exponential growth phase. present on B. subtilis plasmids, as a host B. subtilis is one of the best studied G+ bacteria (Sonenshein et al., 1993; sonenshein et al., 2001). There are also other reasons why B. subtilis is an pLS20 CONTAINS A LARGE CONJUGATION interesting host to study conjugation. B. subtilis is closely related OPERON THAT IS PRECEDED BY A to fastidious and pathogenic bacilli, and more distantly related to the Gram+ pathogen Listeria. B. subtilis is a soil-dwelling DIVERGENTLY ORIENTED GENE, rcopLS20, bacterium that is found all over the world; therefore pLS20 ENCODING THE MASTER REGULATOR OF may be a representative of a group of globally distributed CONJUGATION conjugative plasmids. Finally, B. subtilis strains are also gut commensals in animals and humans (Cutting, 2011), so it is Our annotation of the plasmid revealed a large putative operon possible that B. subtilis plasmids play a role in HGT in the gut. spanning genes 28-74 in which some genes share similarity with Earlier studies on pLS20 have determined the replication conjugation genes (Singh et al., 2013; see Figure 1A for a schematic region of the plasmid (Meijer et al., 1995), and shown that view of plasmid pLS20). This indicated that genes 28-74 constitute a faithful segregation of the plasmid during cell division is large conjugation operon, which was later confirmed (see below). A ensured by the actin-like Alp7A protein (Derman et al., 2009). single divergently oriented gene, 27c, is located immediately However, little was known about the conjugation process and upstream of the conjugation operon. Its product, protein p27c, is how it was regulated. In this review we describe the advances that a putative Xre-type transcriptional regulator predicted to contain a have been made in recent years in understanding how expression typical DNA binding Helix-Turn-Helix (HTH) domain near its of the pLS20 conjugation genes is regulated, and identify the similarities and differences with two other conjugative systems of Gram+ origin. We also discuss future directions to unanswered questions. The most important gaps of knowledge concern structural insights in 1) how repressor molecules generate DNA looping, 2) how the antirepressor interacts with the repressor, and 3) if and how genes involved in different steps of the conjugation process–all located in a single large operon-are differentially expressed. More general reviews of regulation of conjugation in G+ and G− can be found elsewhere (Grohmann et al., 2003; Frost and Koraimann, 2010; Koraimann and Wagner, 2014; Singh and Meijer, 2014; Kohler et al., 2019). EVIDENCE THAT pLS20 CONJUGATION IS STRICTLY CONTROLLED AND NOT ACTIVATED BY PHEROMONES Determination of conjugation efficiencies as a function of growth revealed that maximum conjugation efficiencies, in the range of 1 × 10−3, could be obtained only during a short window of time FIGURE 1 | General characteristics of pLS20 and genetic organization of near the end of the exponential growth phase. Conjugation the pLS20 region involved in regulation of the conjugation genes. (A). Map of efficiency decreases sharply when donor cells enter the pLS20 (accession number AB615352.1). Different modules are indicated with stationary phase, and eventually declines below the detection colors. Light green box, region containing the three genes regulating the level (<1 × 10−8) (Singh et al., 2013). These results indicate that activity of the main conjugation promoter Pc (indicated with “R”); dark green box, conjugation operon (indicated with “C”). Purple box, origin of replication the conjugation process is strictly regulated. Conjugation region (indicated with “O”); orange box, partitioning genes (indicated with “P”); efficiencies, which are also low at early time points, increase and white box, module with unknown genes (indicated with “UF”). (B). Blow- during the exponential growth phase. However, this increase in up of the region encoding the genes rappLS20 (rap), phrpLS20 (phr) and rcopLS20 efficiency was not due to recipient-produced pheromones though (rco) involved in regulating the expression of the conjugation genes, together with the 5’ region of the conjugation operon. (C). Blow-up of the gene 28- as occurs for the enterococcal pheromone-responsive conjugative rcopLS20 intergenic region and its features involved in regulating expression of plasmids (Dunny, 2013), because similar results were obtained the conjugation genes. The position of the divergently oriented and when the growth medium was replaced by fresh medium before overlapping promoters Pc and Pr are indicated with green and red boxes, mating (Singh et al., 2013). This demonstrated that regulation of respectively. Transcription initiation sites are indicated with bent arrows and pLS20 conjugation is fundamentally different from the the RcopLS20 binding sites with blue triangles. The numbers at the top correspond to bp of the almost 600 bp intergenic region. The operators OI enterococcal pheromone-responsive plasmids. The narrow and OII, which are separated by a 75 bp spacer, are indicated at the bottom. time window of efficient conjugation does not depend on the Frontiers in Molecular Biosciences | www.frontiersin.org 3 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation N-terminus. Protein p27c has been shown to be the master regulator (Ramachandran et al., 2014). This approach revealed that of pLS20 conjugation and was named RcopLS20 (regulator of proper regulation required the presence of two DNA regions. conjugation of pLS20, see Figure 1B) (Singh et al., 2013). Ectopic These two regions contain 10 octamer boxes whose sequences are overexpression of rcopLS20 ( gene 27c) from a chromosomal location (nearly) identical to CAGTGAAA (see Figure 1C for a schematic blocked conjugation, and conjugation levels were constitutively high view). In the first region a cluster of six of these boxes, all in the in a derivative of pLS20 lacking the gene (pLS20rco). Plasmid same orientation, are located near, and overlapping with the Pr pLS20rco was very unstable in wild type B. subtilis cells and its and Pc promoters. The second region contains four clustered integrity required the ectopic expression of gene rcopLS20 in the host, boxes whose orientation is opposite to those present in the first suggesting that constitutive expression of the conjugation operon is region. The two clusters of boxes are separated by 75 bp. Binding generating high levels of stress to the host. Evidence that RcopLS20 is a of RcopLS20 to these boxes was demonstrated by DNase I transcriptional regulator was obtained by RNAseq analyses (Singh footprinting assays, and also by gel retardation experiments et al., 2013). Thus, genes within the conjugation operon were using fragments with/out mutation in the octamers expressed among the highest level of all plasmid-encoded genes (Ramachandran et al., 2014). The regions containing the four when samples were taken from donor cell cultures at late exponential and six octamers are named operator OI and OII, respectively (see growth phase, but their levels were >16 fold lower for samples of cells Figure 1C). in which rcopLS20 was ectopically overexpressed. The presence of two convergently oriented RcopLS20 operators raised the possibility that binding of RcopLS20 to these operators could result in DNA looping. However, the spacer region between PROMOTERS DRIVING EXPRESSION OF the two operators is only 75 bp. Due to intrinsic stiffness of DNA rcopLS20 AND THE CONJUGATION OPERON spacer regions in DNA-loops are generally larger than 90 bp. DNA loops containing smaller spacer lengths are only possible Several analyses, including RNAseq, deletion analyses and primer when the DNA region is intrinsically bent. Circular permutation extension experiments revealed that the promoters driving assays showed that the region containing the RcopLS20 operators is expression of the divergently oriented genes rcopLS20 and gene indeed intrinsically bent and that the center of the bent is located 28, the first gene in the conjugation operon, are located in the 595 in approximately the middle of the 75 bp spacer region between bps intergenic region (Ramachandran et al., 2014; see also operators OI and OII (Ramachandran et al., 2014). Several lines of Figure 1). The promoter upstream of rcopLS20 is named Pr, evidence supported that binding of RcopLS20 to operators OI and and the one upstream of the conjugation operon Pc. The Pc OII results in DNA looping. First, ultracentrifugation analysis promoter is located 462 bp upstream of gene 28 and, interestingly, showed that RcopLS20 forms tetramers in solution overlaps with the divergently oriented Pr promoter. The strength (Ramachandran et al., 2014), which is in line with the of the two promoters has been determined using transcriptional observation that proteins generating DNA looping commonly lacZ fusions (Ramachandran et al., 2014). These analyses showed form multimers in solution. Second, particularly for short spacer that the conjugation promoter Pc is a strong promoter, and regions, DNA looping requires a certain phasing between the two promoter Pr, driving expression of rcopLS20, is a very weak operators so that operator-bound proteins are in the required promoter whose activity was barely detectable. Surprisingly, configuration to generate the DNA loop. Enlarging the spacer analyses of additional strains containing, besides a region between the two operators with a half helical turn transcriptional lacZ fusion, either pLS20 or a copy of rcopLS20 (i.e., 5 bp) disrupted RcopLS20 mediated regulation of the Pc under the control of an inducible promoter, revealed that promoter in vivo (Ramachandran et al., 2014). Finally, RcopLS20 does not just repress the strong promoter Pc, but also RcopLS20-mediated DNA looping was further supported by gel regulates its own promoter, Pr. At low expression levels RcopLS20 retardation and ultracentrifugation experiments, which also stimulates the activity of Pr but at high expression level it provided important insights into the mode of DNA binding represses its own promoter (Ramachandran et al., 2014). It is (Singh et al., 2020). DNA looping may be generated as a important to mention that the maximum strength of the Pr consequence of one of the two fundamentally different modes promoter is about 50–75-fold lower than that of the Pc by which tetrameric RcopLS20 units bind DNA, as schematically promoter (Singh et al., 2020). In summary, RcopLS20 represses shown in Figure 2A. The difference between these two modes of the strong promoter Pc and simultaneously regulates the activity DNA binding can be distinguished by gel retardation experiments of its own promoter Pr (Ramachandran et al., 2014). using DNA fragments containing only one operator, as the second binding mode generates a maximum of two retarded species, while the first binding mode would allow the generation REGULATION OF Pc AND Pr BY THE of more than two retarded species. Retardations using DNA TETRAMERIC RcopLS20 INVOLVES DNA fragments containing either operator OI or OII both generated a maximum of two retarded species (Ramachandran et al., 2014), LOOPING indicating that DNA looping occurs through interaction between The binding regions of RcopLS20 were initially determined by two RcopLS20 tetramers each bound to an operator. This analyzing the response of Pr and Pc promoters to ectopically conclusion was supported by additional gel retardation expressed rcopLS20, using strains containing lacZ fused to experiments using two DNA fragments, each containing one promoter-containing fragments of different lengths operator but having different lengths and distinct flanking Frontiers in Molecular Biosciences | www.frontiersin.org 4 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation FIGURE 2 | Probable mode of DNA binding by RcopLS20 to the intrinsically bent DNA region encompassing RcopLS20 operators OI and OII. (A). Schematic representation of two different binding modes by which tetrameric DNA binding proteins can bind a DNA fragment containing one operator. Binding modes 1 and 2 can generate up to four and two different protein-bound DNA configurations, respectively. (B–D). Schematic view of conjugation unrepressed state and repressed state involving DNA looping due to binding of RcopLS20 to operators OI and OII according to binding mode 2 explained in panel A. (B). Unrepressed state. The conjugation operon starting with gene 28 is derepressed when the operators OI and OII are not bound by RcopLS20 tetramers. (C). RcopLS20 tetramers can bind to operators OI and OII, resulting in a local high concentration and probably a specific configuration stimulating interaction between the two tetramers. (D). Repressed state. Interaction between the two RcopLS20 tetramers bound to operators OI and OII results in repression of the conjugation genes and simultaneous activation of rcopLS20. Blue triangles: RcopLS20 binding sites. Blue U shapes: RcopLS20 tetramers. sequences, allowing the fragments to be distinguished by forms dimers. In addition, the region separating the operators Southern blotting. Gel retardation using a mixture of the small is about 2.3 kb in phage λ, and 75 bp in pLS20. and large DNA fragment resulted in the appearance of an additional retarded species composed of a small and a large DNA fragment (Singh et al., 2020). Furthermore, SAXS RcopLS20-MEDIATED REPRESSION IS analysis showed that RcopLS20 forms complexes of increased size at concentrations above 200 μM, indicating that RcopLS20 RELIEVED BY THE QUORUM tetramers can interact with each other (Crespo et al., 2020), which SENSING-RESPONSIVE ANTI-REPRESSOR would be expected if DNA binding occurs via the second mode. RappLS20 BELONGING TO THE RRNPP The combined evidences strongly indicate that efficient FAMILY OF PROTEINS repression of the Pc promoter and simultaneous activation of the Pr promoter by RcopLS20 is achieved through DNA looping as Expression of the conjugation genes requires suspension of a consequence of interaction between the RcopLS20 tetramers RcopLS20-mediated repression of the conjugation promoter Pc. bound to operators OI and OII. We cannot fully excluded the In case of pLS20, the repressor RcopLS20 is inactivated by an possibility that, in addition to DNA looping, binding of RcopLS20 antirepressor, RappLS20. Annotation of pLS20cat revealed that a also introduces other effects on the DNA configuration or two-gene rap-phr cassette is located downstream of rcopLS20, with orientation. direction of transcription convergent to that of rcopLS20 (see rcopLS20 is under the control of a very weak promoter (see Figure 1B). Rap proteins belong to a large family of signal- above). Nonetheless, despite the low levels of RcopLS20 present in peptide receptor proteins of the so-called RRNPP family, which is the cell, the conjugation process is strictly controlled as the named after its prototypical members Rap, Rgg, NprR, PlcR and number of transconjugants is below the detection level under PrgX (for review see, Declerck et al., 2007; Rocha-Estrada et al., conjugation-unfavorable conditions. Probably, this is achieved 2010; Dunny and Berntsson, 2016; Neiditch et al., 2017). RRNPP through DNA looping because it causes a high local proteins, which are encoded by many bacteria belonging to the concentration of the transcriptional regulator at the right phylum of Firmicutes, are generally co-transcribed with a phr location, which has been shown to be able to increase its gene encoding the cognate signaling peptide. The Phr signaling specificity and affinity, and concomitantly controls the peptides are synthesized as a pre-proprotein. After being secreted stochasticity of cellular processes (Vilar and Saiz, 2005; Oehler they are cleaved again to generate the mature peptide, which in and Muller-Hill, 2010). Thus, DNA looping allows strict most cases correspond to the C-terminal region of the pre- regulation at low protein concentration, while remaining proprotein, and which can be imported into the cell by the sensitive for accurate activation of conjugation when oligopeptide permease system (Pottathil and Lazazzera, 2003; appropriate conditions –as detailed in the following section- Monnet et al., 2016). The chromosome of B. subtilis contains occur. Examples of DNA looping playing a crucial role in eight rap-phr cassettes, and some Bacillus plasmids also contain transcriptional regulation have been described before. One of such cassettes. Many of these rap-phr cassettes, including those the best-studied examples is DNA looping exerted by the CI on plasmids, function to interfere with developmental processes protein and which is crucial for the genetic switch between the such as sporulation, competence and degradative enzyme lysogenic and lytic state of the Escherichia coli phage λ (for review production (Perego, 2001; Bongiorni et al., 2006; Smits et al., see, Dodd et al., 2005; Oppenheim et al., 2005). There are clear 2007; Parashar et al., 2013). Ectopically overexpressing the rap- differences between the DNA looping mechanisms of λ and phr cassette of pLS20 in B. subtilis did not seem to affect pLS20. Whereas RcopLS20 forms tetramers in solution, CI developmental processes of the host. However, it strongly Frontiers in Molecular Biosciences | www.frontiersin.org 5 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation affected the conjugation process, resulting in 1) increased of an additional strain containing gfp fused to the upstream maximum conjugation levels, 2) overexpression of genes region of phrpLS20 revealed that the expression of phrpLS20 located in the conjugation operon, and 3) allowing indeed is controlled by a second promoter, named Pphr, whose conjugation to occur during a much broader time window. activity is about two-fold higher than that of Prap. Unlike the Pc Moreover, conjugation levels were severely affected for a and Pr promoters, the activities of Prap and Pphr were not affected pLS20 derivative lacking rap-phr (Singh et al., 2013). Addition by the presence of pLS20 in the cell, indicating that RcopLS20 does of a synthetic peptide corresponding to the five C-terminal not regulate these promoters. The absence of RcopLS20 binding residues of the unprocessed Phr peptide also inhibited motifs in the vicinity of the Prap and Pphr promoters supports this conjugation, showing that the activity of Rap is regulated by conclusion (Singh et al., 2020). In summary, 1) the conjugation the mature signaling peptide (Singh et al., 2013). Based on these genes are under the control of the strong Pc promoter, 2) the results the genes were named rappLS20 and phrpLS20 and the rcopLS20 and rappLS20 genes, driving expression of the proteins mature Phr peptide was referred to as Phr*pLS20. regulating the activity of the Pc promoter, are preceded by very weak promoters, 3) expression of phr is controlled by Prap and additionally by the two-fold stronger promoter Pphr, presumably MINIMAL IN VIVO REGULATORY CIRCUIT to accommodate for the dilution effect upon secretion, and 4) OF pLS20 CONJUGATION RcopLS20 represses Pc and regulates the Pr promoter, but not promoters Prap and Pphr. The results outlined above suggest that RcopLS20, RappLS20 and Phr*pLS20 are the only pLS20-encoded proteins involved in regulation of the conjugation genes. This was confirmed using PROMOTER Pc IS HOMOGENEOUSLY an in vivo system in which the regulatory components were EXPRESSED uncoupled from their native setting in a B. subtilis strain where the rcopLS20 and rappLS20 genes are placed under different The multi-layered regulation of the Pc promoter in inducible promoters, and a lacZ reporter gene fused to the combination with the Rap/Phr-based quorum sensing main conjugation promoter Pc. The main conjugation mechanism might be expected to cause heterogeneous promoter Pc was active in the absence of both rcopLS20 and activation of the Pc promoter in a population of donor cells. rappLS20 expression. Pc was repressed when rcopLS20 alone was This seemed not to be the case though, because flow cytometry induced, strongly supporting the view that RcopLS20 is directly analyses of samples of B. subtilis cells having a chromosomal responsible for repressing the Pc promoter, which is in line with cassette containing the Pc-gfp fusion, or those harboring in in vitro results (see above). The Pc promoter became active when addition pLS20, both showed homogeneous activity of the Pc both rcopLS20 and rappLS20 were induced, showing that RappLS20 promoter (Singh et al., 2020). In this set up, the Pc promoter alone is sufficient to relieve RcopLS20-mediated repression of the was present on the bacterial chromosome whereas proteins Pc promoter. Finally, when both rcopLS20 and rappLS20 were regulating its activity were encoded by the resident plasmid induced in the presence of the mature peptide Phr*pLS20 containing its own Pc promoter. A homogeneous pattern of Pc (added to the growth medium), the Pc promoter remained promoter activity was also found for cells containing a inactive, demonstrating that Phr*pLS20 is required and derivative of pLS20cat in which a copy of the gfp gene was sufficient to inactivate RappLS20 (Singh et al., 2020). placed on pLS20 behind gene 28, the first gene of the conjugation operon. Together, these results strongly indicate that the multi-levels of regulation results in a sensitive genetic RELATIVE PROMOTER STRENGTHS switch that activates the Pc promoter in a coordinated manner in most or all pLS20-containing cells (Singh et al., 2020). This The interplay between RcopLS20, RappLS20 and Phr*pLS20 requires does not automatically imply that activation of the Pc promoter an understanding of the relative strengths of their promoters, as guarantees successful plasmid transfer or even the initiation of well as the activity of promoter Pc, which these proteins regulate. the conjugation process in all cells. In fact, in most if not all The strengths of Pc, Pr and the promoter driving expression of conjugation systems studied, the full conjugation process rappLS20 and phrpLS20, named Prap, were determined using strains appears to be activated only in a subpopulation of the containing gfp reporter cassettes in which either of these donor cells (reviewed in, Koraimann and Wagner, 2014; promoters was fused to the gfp gene (Singh et al., 2020). The Stingl and Koraimann, 2017). In the case of pLS20, results obtained with the gfp-fusion strains confirmed that Pc and checkpoints may be present downstream the Pc promoter. Pr are a strong and a very weak promoter, respectively. Prap was Moreover, successful transfer may not occur even when all found to be a weak promoter, whose strength was similar to the Pr conjugation genes are expressed due to, for example, promoter in cells containing pLS20, and about 50 to 75-fold lower unsuccessful mating pair formation or failure of the plasmid than that of Pc (see Figure 3). to establish itself in the new host. Moreover, it should be noted Prap drives the expression of rappLS20 and phrpLS20. However, that the laboratory conditions under which conjugation is the concentration of PhrpLS20 will drop once it is exported from studied are very different from natural conditions in which the cell and it is plausible that proper signaling of Phr*pLS20 environmental fluctuations at macro and microscale are likely requires higher expression levels than that of RappLS20. Analysis to affect individual cells or subpopulations. Frontiers in Molecular Biosciences | www.frontiersin.org 6 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation FIGURE 3 | Relative strength of promoters Pc, Pr, Prap and Pphr determined by FACS analysis of strains containing transcriptional gfp fusions. Relative promoter strengths determined by cytometry using strains containing promoter Pc, Pr, Prap or Pphr transcriptionally fused to gfp. A negative control strain (-) and two positive control strains that contain gfp fused to the IPTG-inducible promoter Pspank or Physpank were included. Values correspond to late exponentially growing cultures (OD600 between 0.8-1). Light and dark colored bars reflect strains lacking or containing pLS20spec, respectively. The figure is adapted from (Singh et al., 2020). RappLS20 ACTIVATES CONJUGATION BY 2011). Several Rap proteins, including RapA, RapB, RapE, DETACHING RcopLS20 FROM ITS RapH and RapJ, can interrupt the phosphorylation of Spo0A by dephosphorylating Spo0F∼P. RapA was the first Rap member OPERATORS AND FORMING discovered to have this activity and was named Rap, which stands HETEROCOMPLEXES WITH RcopLS20 for Response regulator Aspartate Phosphatase (Perego, 2001). Finally, in the third subset of RRNPP proteins, the effector All members of the RRNPP family of proteins analyzed so far, are domain binds to its cognate effector protein, which impedes all-helical proteins composed of a large C-terminal domain the function of its cognate effector protein (in)directly by (CTD) and a much smaller N-terminal domain (NTD). modulating the expression of the differentiation pathway Whereas the large CTD interacts with its cognate signaling involved. Examples of these are RapC, RapF, RapG, RapH and peptide resulting in conformational changes that affects the RapK (note that not all Rap proteins have phosphatase activity). It activity of the protein, the small NTD constitutes the effector is also worth mentioning that the effector domain of some domain that interacts with a target molecule ( D’Andrea and RRNPP proteins, for instance RapH and NprR, exhibit both Regan, 2003; Rocha-Estrada et al., 2010; Neiditch et al., 2017). activities. Although the effector domains of all RRNPP proteins are Based on the features of the known RRNPP proteins, one composed of three α-helices, they exert their regulatory possibility of how RappLS20 might activate the pLS20 conjugation functions in three fundamentally different ways. In the first genes is by competing with RcopLS20 for binding to DNA near the subset of RRNPP proteins these three α-helices have evolved Pc promoter. However, both gel retardation and analytical into Helix-Turn-Helix (HTH) DNA binding motifs, which allow ultracentrifugation (AUC) analyses showed that RappLS20 has them to negatively regulate protein expression by binding to no DNA binding activity, and the NTD of RappLS20 does not DNA. Examples of such RRNPP proteins are PlcR from Bacillus adopt a HTH configuration (Crespo et al., 2020; Singh et al., thuringiensis (Declerck et al., 2007) and PrgX from Enterococcus 2020). Instead, RappLS20 seems to exert its antirepressor activity faecalis (Shi et al., 2005; Dunny and Berntsson, 2016). Particularly by binding directly to RcopLS20, as bacterial two-hybrid analysis the PrgX protein is interesting in the context of this review showed that RappLS20 and RcopLS20 interact in vivo (Singh et al., because it regulates the conjugation genes present on the 2020). In vitro evidence that RappLS20 interacts directly with plasmid pCF10 (see below). In the second subset of RRNPP RcopLS20 has also been obtained by SAXS and analytical size proteins, the effector domain has phosphatase activity. In B. exclusion chromatography (Crespo et al., 2020). Furthermore, subtilis, the developmental process of sporulation is induced AUC analyses provided conclusive evidence that RappLS20 forms by the phosphorylated form of the master regulator Spo0A, predominantly dimers in solution. Sedimentation velocity Spo0A∼P. Spo0A phosphorylation is controlled by a experiments of mixtures of RappLS20 and RcopLS20 resulted in phosphorelay consisting of several kinases and two the appearance of a novel species whose deduced molecular phosphorelay proteins, Spo0F and Spo0B (Hoch, 1993; weight was compatible with a complex of one RappLS20 dimer Grossman, 1995; Molle et al., 2003; Chastanet and Losick, and one RcopLS20 tetramer (Singh et al., 2020). This conclusion Frontiers in Molecular Biosciences | www.frontiersin.org 7 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation was reinforced by multi-signal sedimentation velocity (MSSV) THE ACTIVATION STATE OF THE analysis from which it was determined that the heterocomplex CONJUGATION GENES IS ULTIMATELY was composed of a stoichiometry of 2.1 mol of RcopLS20 per mol of RappLS20. More importantly, besides demonstrating that DETERMINED BY THE SIGNALING RappLS20 interacts with free RcopLS20, AUC analyses also PEPTIDE Phr*pLS20 showed that RappLS20 is able to interact with DNA-bound RcopLS20, and that this results in the release of RcopLS20 from The presence of the mature signaling peptide Phr*pLS20 the DNA and the concomitant formation of RappLS20/RcopLS20 suppresses the RappLS20–mediated activation of the complexes. Complementary gel retardation experiments conjugation genes (see above). For other RRNPP members it provided evidence that RappLS20 preferentially interacts with has been shown that signaling peptides interact directly with the RcopLS20 tetramers involved in DNA looping (Singh et al., CTDs of their cognate RRNPP proteins, causing inactivation 2020). In summary, RappLS20 appears to activate the through peptide-induced conformational changes of the RRNPP conjugation genes by preferentially interacting with RcopLS20 protein. As outlined below, this is also the case for RappLS20 and involved in DNA-looping, resulting in the detachment of Phr*pLS20. Phr*pLS20 binds directly to RappLS20 (see below) and the RcopLS20 from its operators, and forming a heterocomplex in dissociation constant, Kd, for Phr*pLS20 was determined to be 2.1 which one RappLS20 dimer binds one RcopLS20 tetramer. A and 7.4 μM by ultracentrifugation and fluorescence anisotropy schematic view of how RappLS20 activates the conjugation approaches, respectively (Crespo et al., 2020; Singh et al., 2020). genes is shown in Figure 4. Although AUC analysis showed that RappLS20 forms predominantly dimers in solution (Crespo et al., 2020, see also above), it seems to have an intrinsic ability to form tetramers at higher concentrations, as 1.1% of RappLS20 was detected as tetramers in solution at 4.5 μM, and this percentage increased to 3.6% at 25 μM. Interestingly, AUC analysis showed that in the presence of Phr*pLS20, RappLS20 forms almost exclusively tetramers in solution (Crespo et al., 2020). The structures of the apo and peptide-bound forms of RappLS20 have been determined recently, which show that RappLS20 forms the typical structure of RRNPP family proteins (Crespo et al., 2020). RappLS20 is an all α-helical protein (17 antiparallel helices) that is composed of a large 14-helix C-terminal domain (CTD) that can bind the signal peptide, and a much smaller 3-helix N-terminal domain (NTD). The three helices of the NTD do not form a HTH topology, but have an approximate parallel configuration. The N- and CTDs are separated by a 13- residue long flexible loop that connects α-helices three and four. RappLS20 dimerizes through interactions between the CTDs, and these interactions involve helices H5-H7, H16-H17 and the C-terminal residues. The structure of the peptide-bound form of RappLS20 showed that Phr*pLS20 binds in a cavity of the CTD. Binding of the Phr*pLS20 peptide causes the N-terminal domains to swing outwards thereby favoring RappLS20 to form tetramers (Crespo et al., 2020). Interestingly, the dimer-dimer interface is principally formed between the NTDs of each dimer, resulting in a so-called FIGURE 4 | Overview of the current understanding of how the pLS20 conjugation genes are regulated by RcopLS20, RappLS20 and Phr*pLS20. Stage foot-2-foot interaction (see stage III in Figure 4 for a schematic I, as default, the two RcopLS20 tetramers bound to operators OI and OII interact view). The small NTDs of RRNPP proteins form the effector with each other resulting in DNA-looping. This configuration causes a domain and this is most likely also the case for RappLS20, implying steady state in which the strong conjugation promoter Pc is repressed and the that the NTD of RappLS20 interacts with RcopLS20. Due to the weak Pr promoter driving expression of rcopLS20 is simultaneously stimulated. Stage II, the conjugation genes are activated when two apo-form of RappLS20 particular foot-2-foot configuration induced upon peptide dimers detach two RcopLS20 tetramers from the DNA through direct binding, the N-terminal effector domain is no longer capable interaction and generating RappLS20:RcopLS20 heterocomplexes in a of interacting with RcopLS20 to relieve repression, providing the stoichiometry of 1:2. Stage III, binding of the Phr*pLS20 signaling peptide molecular mechanism by which Phr*pLS20 inactivates the provokes a conformational change of RappLS20 that stimulates tetramerization antirepressor activity of RappLS20. Phr*pLS20 can also bind to of RappLS20 via the so-called foot-2-foot configuration in which the N-terminal effector domains interact with each other and are, presumably, no longer RappLS20 complexed with RcopLS20. This conclusion is based available to interact with RcopLS20. The liberated RcopLS20 tetramers are then on the results of size exclusion chromatography and SAXS free to bind its operators thereby returning the system to the default experiments, which showed that the addition of Phr*pLS20 conjugation repressed “OFF” state (stage I). peptide to preformed RappLS20/RcopLS20 complexes resulted in Frontiers in Molecular Biosciences | www.frontiersin.org 8 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation disruption of these complexes and the appearance of elution and identical; position 2 concerns a conserved substitution of SAXS patterns that were similar to those of RcopLS20 alone and Lysine to Arginine, and position 5 a change from Tyrosine to RappLS20-Phr*pLS20 complexes (i.e., RappLS20 tetramers) (Crespo Isoleucine. PhrF* has been shown to cross talk with RappLS20 et al., 2020). (Singh et al., 2020). Thus, PhrF* can interact with RappLS20 with a Kd that is only 2.5-fold higher than that of Phr*pLS20 as determined by AUC (5.3 and 2.1 μM, respectively), and RECIPROCAL INHIBITION OF interaction with PhrF* results in tetramerization of RappLS20. CONJUGATION AND COMPETENCE More importantly, PhrF* is able to inactivate RappLS20 in vivo when tested using the minimal regulatory system of pLS20 PATHWAYS conjugation (see above). Under certain growth conditions, B. subtilis can develop the state of natural competence in which cells can bind and adsorb extracellular DNA into the cytosol after which it can be CURRENT VIEW OF THE SIGNAL-PEPTIDE integrated into the bacterial genome via homologous MEDIATED REGULATION OF pLS20 recombination (Dubnau, 1991; Hamoen et al., 2003). The processes of conjugation and competence have in common CONJUGATION that they transport DNA via a sophisticated membrane- All the evidences obtained so far show that the conjugation genes embedded DNA translocation machinery, but in opposite of pLS20 are regulated in a highly sophisticated manner ensuring directions: DNA is exported in the case of conjugation, but that conjugation is only activated under conditions that favor a imported during competence development. In both cases, only successful conjugation event, i.e. when potential recipient cells are one of the two DNA strands (i.e., an ssDNA strand) is present. A schematic view of the current knowledge of how the transported, and various other similarities exist between conjugation genes are regulated is presented in Figure 4. The competence and conjugation related ssDNA transfer machines main and strong conjugation promoter Pc located upstream of the (Chen et al., 2005). However, simultaneous import and export of conjugation operon is repressed by RcopLS20, which also controls large ssDNA regions by the same cell may be incompatible for the activity of its own weak promoter Pr to ensure that only low several reasons. For instance, simultaneous production and levels of RcopLS20 are produced. Despite these low levels, the assembly of the ssDNA translocation machineries for tetrameric RcopLS20 efficiently represses the Pc promoter. This is competence and conjugation may interfere with each other most likely achieved through DNA looping that ensures high and/or they may compete for the same cellular positions. In local concentration of RcopLS20 and simultaneous binding to addition, the recombination enzymes synthesized during multiple binding sites in the two operators. The low levels of competence may act on ssDNA of the conjugative element. RcopLS20 probably are also crucial to allow for a sensitive and The following evidence indeed supports that conjugation and rapid switch to activate the conjugation genes when favorable competence are incompatible processes and that at least two conditions occur. Activation of the conjugation genes occurs by reciprocal mechanisms have evolved to avoid simultaneous the action of the dimeric form of RappLS20 that relieves RcopLS20- activation of the competence and conjugation pathways in the mediated repression of the Pc promoter. Binding of RappLS20 to same cell. Competence development is strongly suppressed in RcopLS20 results in detachment of RcopLS20 from the DNA, and cells containing pLS20 by a plasmid-encoded protein called hence it is the apo form of RappLS20 that activates conjugation. RokpLS20. RokpLS20 shares similarity with B. subtilis Rok, which Accumulating levels of Phr*pLS20 cause inactivation of RappLS20 is a repressor of comK that encodes the transcriptional activator by inducing a conformational change of RappLS20 that favors the of the recombination and the structural competence genes. As for formation of tetramers. Intriguingly, tetramerization occurs Rok, RokpLS20 suppresses competence by repressing comK (Singh through interactions between the N-terminal effector domains et al., 2012). with the consequence that these are presumably no longer Like conjugation, competence is also induced by quorum available to interact with RcopLS20. Consequently, RappLS20 sensing signals. The two-component ComP-ComA system tetramerization results in the liberation of RcopLS20 from the plays a crucial role in the induction of competence: upon RappLS20/RcopLS20 complexes, which are then available to bind its sensing the proper signals, the membrane-embedded kinase operators and thereby returning the system to the default “OFF” ComP activates ComA through phosphorylation which then state. Therefore, as schematically shown in Figure 5, the activates competence genes, leading ultimately to the activation state of the pLS20 conjugation genes is ultimately activation of the master regulator gene comK (Hamoen et al., regulated by the concentration of the Phr*pLS20 signaling 2003). RapF, encoded by the B. subtilis chromosome, inhibits peptide. When all or a majority of the cells within a stimulation of the competence pathway by interacting with population contain the plasmid, Phr*pLS20 concentrations will ComA thereby preventing ComA from activating gene be high and conjugation will be inhibited. The conjugation genes expression. RapF is cotranscribed with phrF that encodes the will become activated when Phr*pLS20 concentrations are low, pre-proprotein which, after being processed, generates the which occurs when donor cells form a minority of the population signaling peptide PhrF*. High levels of PhrF* stimulate and are surrounded by many potential recipient cells. In fact, the competence by inactivating RapF. Intriguingly, Phr*pLS20 and presence of recipient cells has a dual effect on lowering the PhrF* are very similar: residues at positions 1, 3 and 4 are Phr*pLS20 concentration: 1) the relative number of donor cells, Frontiers in Molecular Biosciences | www.frontiersin.org 9 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation FIGURE 5 | The concentration of the signaling peptide Phr*pLS20 determines the activation state of the conjugation genes. Schematic view of how a high and low donor/recipient ratio (left and right panel, respectively) affects the Phr*pLS20 concentration, and thereby the activation state of the conjugation process. Donor and recipient cells are indicated with orange and green rectangles, respectively. Red oval lines, pLS20; purple four-pointed star, signaling peptide. The amplified regions in the lower part illustrate secretion of the signaling peptide by donor cells, and its adsorption by donor and recipient cells as indicated by straight and bent arrows, respectively. Note that for simplicity only the mature form of the signaling peptide is shown. See text for details. which produce Phr*pLS20, will be lower in populations containing pLS20 from B. subtilis, pCF10 from Enterococcus faecalis, and recipient cells, and 2) recipient cells can actively adsorb the those of the ICEBs1 element that is present in many B. subtilis signaling peptide. Finally, it is not known if the RappLS20- strains. The N-terminal effector domain of the pCF10-encoded Phr*pLS20 complexes are destined for rapid degradation, or RRNPP protein PrgX has evolved into a HTH DNA-binding whether there exists a mechanism to recycle RappLS20 by domain. Remarkably, PrgX can interact with two signal peptides removing Phr*pLS20 from the complex, thereby stimulating the that cause opposing effects on its DNA binding. One of these activation of the conjugation pathway. It should be noted, signal peptides, iCF10, is encoded by the plasmid, and the other, however, that the promoter Prap driving expression of rappLS20 cCF10 (the pheromone) by recipient cells. PrgX bound to the and phrpLS20 has been shown to be constitutive, thus resulting in plasmid-encoded iCF10 adopts a conformation in which PrgX de novo synthesis of cytosolic RappLS20, and PhrpLS20 that is binds DNA resulting in repression of the conjugation genes. destined for secretion (Singh et al., 2020). However, when PrgX binds to cCF10, DNA is released resulting in activation of the conjugation genes (for recent review see, Dunny and Berntsson, 2016). The regulatory system of pCF10 COMPARISON OF REGULATION OF differs therefore in two important aspects from that of pLS20. First, in the case of pCF10 recipient cells are directly responsible CONJUGATION GENES OF pLS20 AND for activation of the conjugation genes, and second, regulation of OTHER CONJUGATIVE ELEMENTS the conjugation genes is controlled by one instead of two REGULATED BY RRNPP PROTEINS regulatory proteins (PrgX vs. RappLS20 and RcopLS20). The conjugation genes of ICEBs1 are repressed by a regulator Signaling peptide receptor proteins belonging to the RRNPP named ImmR; an analogue of RcopLS20. Relieve of ImmR- family all have a similar structure that is composed of a small mediated repression can occur in two ways. First, as a 3-helix N-terminal effector domain and a much larger CTD that, consequence of RecA-dependent SOS response to DNA upon interaction with its cognate signal peptide, undergoes a damage, or second, by a mechanism that involves the ICE- conformational change affecting the functionality of the protein. encoded RRNPP protein RapI (Auchtung et al., 2007; Bose Despite these conserved features, evolutionary adaptations have et al., 2008). However, activation of the conjugation genes by led to major effects on the mechanistic action of these proteins. RapI occurs in a different way to RappLS20. RapI is required but This is exemplified here by comparing the three RRNPP members not sufficient to activate the conjugation genes; a protease that regulate the conjugation genes of the conjugative plasmids encoded by gene immA that degrades ImmR in a RapI- Frontiers in Molecular Biosciences | www.frontiersin.org 10 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation dependent manner is also needed. The exact mechanism of how domains. Various experimental approaches have shown that RapI stimulates degradation of ImmR by ImmA is not yet known RcopLS20 does not bind DNA when it forms a complex with (Bose et al., 2008). RappLS20 (Crespo et al., 2020; Singh et al., 2020). This may be The presence and absence of a protease dedicated to degrade because RappLS20 binds the DNA binding domain of RcopLS20. the repressor is likely to have important effects on reversibility of Alternatively, the formation of an RcopLS20/RappLS20 complex switching on and off of the conjugation pathway. Similar to may induce a conformational change in RcopLS20 affecting its RcopLS20, ImmR represses the conjugation promoter and DNA binding ability. Answers to several or all of these questions simultaneously activates its own promoter (Auchtung et al., may be obtained by unraveling the apo structure of RcopLS20 and 2007). Degradation of ImmR will therefore result in activation the structures of RcopLS20 complexed with DNA and with of the conjugation genes and concomitantly inhibits de novo RappLS20. ImmR synthesis, causing conjugation of the ICEBs1 to be an The activation of several developmental processes in B. irreversible process. In the case of pLS20, conjugation may be a subtilis such as sporulation, competence and motility reversible process, or at least it may be more flexible than the depends on stochastic variability in expression of a master ICEBs1 system, because, rather than being degraded, RcopLS20 regulator that results in heterogeneic behavior of genetically becomes temporally sequestered by RappLS20. Inactivation of identical cells within a population (Korobkova et al., 2004; RappLS20 by PhrpLS20 results in the release of RcopLS20, which Dubnau and Losick, 2006; Veening et al., 2008). A benefit of can then bind to DNA and again repress the conjugation genes. heterogeneity can be division of labor, where a subpopulation Interaction of RappLS20 with RcopLS20 might inactivate RcopLS20 of cells produces products for the benefit of the entire by altering its oligomerization state, in a similar way that RapF community. Heterogeneity may also lead to the so-called causes dissociation of its dimeric interacting partner ComA upon bet-hedging strategies in which a subpopulation of binding (Griffith and Grossman, 2008; Baker and Neiditch, 2011; differentiated cells is generated even in the absence of Wolf et al., 2016). However, results of different analyses conditions favoring the differentiation process. Like division compellingly show that RcopLS20 is released as functional of labor, this strategy is beneficial for the community at the tetramers from the RappLS20-RcopLS20 complex upon addition population level because the differentiated cells are adapted to of Phr*pLS20, lending further support for the view that activation adverse conditions that may arise suddenly. However, the of the pLS20 conjugation genes can be a reversible process. process of conjugation is an energy consuming process and has major impacts on cell surface and membrane components, requiring tight repression at times when conditions for UNANSWERED QUESTIONS AND FUTURE successful DNA transfer are not apt. Instead of a PERSPECTIVES heterogeneic behavior resulting in a bet-hedging strategy, the conjugation process must be strictly controlled, allowing Studies of the past years have resulted in major advances in its activation to occur only under conditions that are understanding how the conjugation genes of pLS20 are regulated. favourable for successful transfer events, and this requires Yet, multiple questions remain unanswered. Several of these an efficient and sensitive genetic switch from an “OFF” to concern multimerization of RcopLS20 and how RcopLS20 “ON” state only under conditions that favor successful plasmid interacts with DNA and with RappLS20. In silico analysis transfer. Several features described above contribute to this indicates that RcopLS20 contains a HTH motif in its efficient switch, such as overlapping divergent promoters, low N-terminal region, which probably will be responsible for levels of RcopLS20 and RappLS20, and DNA looping. We DNA binding. Therefore, the CTD of RcopLS20 is presumably consider it possible that the multimerization state of involved in tetramerization, but how the RcopLS20 monomers RcopLS20, and particularly the apo and peptide bound forms interact to form a tetramer is unknown. Currently, it is also not of RappLS20 may also contribute to the sensitivity and efficiency exactly known how two tetramers interact when they are bound of the switch. to DNA and induce DNA looping. Binding to DNA may generate Conjugation and competence appear to be incompatible a conformational change in the tetrameric structure of RcopLS20 processes. Although no evidence is currently available, one or that favors octamerization. Alternatively, octamerization may be two-way interference may also exist between conjugative favored by a local high concentration as the consequence of plasmids and phage infection. Some Bacillus phages use a RcopLS20 binding to the intrinsically bent region containing quorum sensing mechanism that plays a crucial role in operators OI and OII. The interacting surface between entering the lytic or the lysogenic cycle (Erez et al., 2017). RcopLS20 and RappLS20 is also not known. RappLS20 may bind Perhaps, cross-talk exists between this quorum sensing system the DNA binding surface of RcopLS20 in an analogous way as and the one regulating conjugation. RapF interacts with the DNA binding domain of ComA (Baker The work summarized in this review shows that conjugation is and Neiditch, 2011). However, the observation that RappLS20 exquisitely controlled at the level of the Pc promoter. However, appears to bind preferentially to RcopLS20 tetramers that are the conjugation operon is very large and contains many genes involved in DNA looping (see above) suggests that RappLS20 that are involved in different stages of the conjugation process, binds preferentially, but not exclusively, to the RcopLS20 surface and it is very well possible that subsets of conjugation genes need generated upon dimerization of two RcopLS20 tetramers; this to be expressed differentially or temporally. This may be achieved, interface most likely does not involve the DNA binding at least in part, by different translation initiation signals, and Frontiers in Molecular Biosciences | www.frontiersin.org 11 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation protein/RNA stability, but we have recently obtained evidence AUTHOR CONTRIBUTIONS that at least one other layer of expression is involved. pLS20cat gene 29 encodes a surface-located protein that exerts exclusion; WM and LW wrote the paper. DB, SA, FR, CA, and JRL-O edited i.e., it inhibits futile transfer of the plasmid between donor cells, the paper, WM and SA made Figures. thereby contributing to efficient transfer to plasmid-free recipient cells (Gago-Cordoba et al., 2019). Proper functioning of exclusion systems require that the gene(s) involved are expressed in all FUNDING donor cells, but gene 29 is located inside the conjugation operon. This apparent paradox was solved by the identification of a weak Ministry of Science and Innovation of the Spanish Government promoter located upstream of gene 29, and a similar strategy is (PID2019 108778GB C21 (AEI/FEDER, EU)) to WM, (BIO2016- probably used in other conjugative systems as well (Gago- 77883-C2-2-P and FIS2015-72574-EXP (AEI/FEDER, EU)), to Cordoba et al., 2019). It is possible that the conjugation DB, (PID2019-104544GBI00 (AEI/FEDER/EU)) to CA, operon contains additional constitutive or regulated promoters (PID2019-109320GB-100/AEI/10.13039/501100011033) to SA, that may modulate the expression of subsets of genes within the (PGC2018-094143-B-I00, (AEI/FEDER, EU)) to FR, and operon. (FIS2016-78313-P) to SA. The Ministry of Science and Contrary to most other conjugative systems, pLS20 conjugates Innovation of the Spanish Government also funded the Centro efficiently in liquid medium and therefore it will have an Nacional de Biotecnología (CNB) by means of the “Severo important role in gene transfer under natural conditions. Ochoa” Center of Excellence grant SEV 2017-0712. A However, the predominant lifestyle of bacteria, including B. Wellcome Investigator grant (209500) to Jeff Errington subtilis, is forming colonies or biofilms (Donlan, 2002). pLS20 supported LW. Institutional grants from the ‘Fundación also conjugates efficiently on solid medium (Koehler and Thorne, Ramón Areces’ and ‘Banco de Santander’ supported the 1987; Meijer et al., 1998). As described above, the activation state Centro de Biología Molecular ‘Severo Ochoa’. of the conjugation process is ultimately determined by the concentration of the Phr*pLS20 signaling peptide. The results summarized in this review are based on conjugation ACKNOWLEDGMENTS experiments carried out in liquid medium, and particularly using shaking cultures. Under these conditions, the We thank members of the different labs for useful discussions, concentration of the exported signal peptide will be instantly and Daniel Zeigler of the Bacillus Genetic Stock Center for homogenized, which is very different from the situation in which providing plasmids and strains essential for many experiments donor cells form part of a colony or biofilm where secretion of the described in this review. We acknowledge the publication fee signal peptide will lead to a concentration gradient. It will be support of the CSIC Open Access Publication Support Initiative interesting to see how the conjugation genes are regulated under through its ‘Unit of Information Resources for Research’ such conditions. (URICI). Cabezón, E., Ripoll-Rozada, J., Peña, A., De la Cruz, F., and Arechaga, I. (2014). REFERENCES Towards an integrated model of bacterial conjugation. FEMS Microbiol. Rev. 39, 81–95. doi:10.1111/1574-6976.12085 Arutyunov, D., and Frost, L. S. (2013). F conjugation: back to the beginning. CDC (2019). Antibiotic resistance threats in the United States. Atlanta, GA: U.S. Plasmid 70 (1), 18–32. doi:10.1016/j.plasmid.2013.03.010 Department of health and human services. Auchtung, J. M., Lee, C. A., Garrison, K. L., and Grossman, A. D. (2007). Chastanet, A., and Losick, R. (2011). Just-in-time control of Spo0A synthesis in Identification and characterization of the immunity repressor (ImmR) that Bacillus subtilis by multiple regulatory mechanisms. J. Bacteriol. 193 (22), controls the mobile genetic element ICEBs1 of Bacillus subtilis. Mol. Microbiol. 6366–6374. doi:10.1128/JB.06057-11 64 (6), 1515–1528. doi:10.1111/j.1365-2958.2007.05748.x Chatterjee, A., Cook, L. C., Shu, C. C., Chen, Y., Manias, D. A., Ramkrishna, D., Baker, M. D., and Neiditch, M. B. (2011). Structural basis of response regulator et al. (2013). Antagonistic self-sensing and mate-sensing signaling controls inhibition by a bacterial anti-activator protein. PLoS Biol. 9 (12), e1001226. antibiotic-resistance transfer. Proc. Natl. Acad. Sci. U.S.A. 110 (17), 7086–7090. doi:10.1371/journal.pbio.1001226 doi:10.1073/pnas.1212256110 Baxter, J. C., and Funnell, B. E. (2014). Plasmid partition mechanisms. Microbiol. Chen, I., Christie, P. J., and Dubnau, D. (2005). The ins and outs of DNA transfer in Spectr. 2 (6) 1–20. doi:10.1128/microbiolspec.PLAS-0023-2014 bacteria. Science 310 (5753), 1456–1460. doi:10.1126/science.114021 Bongiorni, C., Stoessel, R., Shoemaker, D., and Perego, M. (2006). Rap phosphatase Christie, P. J. (2016). The mosaic type IV secretion systems. EcoSal Plus 7 (1), 10. of virulence plasmid pXO1 inhibits Bacillus anthracis sporulation. J. Bacteriol. doi:10.1128/ecosalplus.ESP-0020-2015 188 (2), 487–498. doi:10.1128/JB.188.2.487-498.2006 Christie, P. J., Whitaker, N., and González-Rivera, C. (2014). Mechanism and Bose, B., Auchtung, J. M., Lee, C. A., and Grossman, A. D. (2008). A conserved anti- structure of the bacterial type IV secretion systems. Biochim. Biophys. Acta 1843 repressor controls horizontal gene transfer by proteolysis. Mol. Microbiol. 70 (8), 1578–1591. doi:10.1016/j.bbamcr.2013.12.019 (3), 570–582. doi:10.1111/j.1365-2958.2008.06414.x Clewell, D. B. (2011). Tales of conjugation and sex pheromones: a plasmid and Bouet, J. Y., and Funnell, B. E. (2019). Plasmid localization and partition in enterococcal odyssey. Mob. Genet. Elem. 1 (1), 38–54. doi:10.4161/mge.1.1.15409 Enterobacteriaceae. EcoSal Plus 8 (2) 1–23. doi:10.1128/ecosalplus.ESP-0003-2019 Crespo, I., Bernardo, N., Miguel-Arribas, A., Singh, P. K., Luque-Ortega, J. R., Brooks, A. C., and Hwang, L. C. (2017). Reconstitutions of plasmid partition Alfonso, C., et al. (2020). Inactivation of the dimeric RappLS20 anti-repressor of systems and their mechanisms. Plasmid 91, 37–41. doi:10.1016/j.plasmid.2017. the conjugation operon is mediated by peptide-induced tetramerization. 03.004 Nucleic Acids Res. 48, 8113. doi:10.1093/nar/gkaa540 Frontiers in Molecular Biosciences | www.frontiersin.org 12 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation Cutting, S. M. (2011). Bacillus probiotics. Food Microbiol. 28 (2), 214–220. doi:10. Itaya, M., Sakaya, N., Matsunaga, S., Fujita, K., and Kaneko, S. (2006). 1016/j.fm.2010.03.007 Conjugational transfer kinetics of pLS20 between Bacillus subtilis in liquid D’Andrea, L. D., and Regan, L. (2003). TPR proteins: the versatile helix. Trends medium. Biosci. Biotechnol. Biochem. 70, 740–742. doi:10.1271/bbb.70.740 Biochem. Sci. 28 (12), 655–662. doi:10.1016/j.tibs.2003.10.007 Koehler, T. M., and Thorne, C. B. (1987). Bacillus subtilis (natto) plasmid pLS20 Davies, J., and Davies, D. (2010). Origins and evolution of antibiotic resistance. mediates interspecies plasmid transfer. J. Bacteriol. 169 (11), 5271–5278. doi:10. Microbiol. Mol. Biol. Rev. 74 (3), 417–433. doi:10.1128/MMBR.00016-10 1128/jb.169.11.5271-5278.1987 Declerck, N., Bouillaut, L., Chaix, D., Rugani, N., Slamti, L., Hoh, F., et al. (2007). Kohler, V., Keller, W., and Grohmann, E. (2019). Regulation of gram-positive Structure of PlcR: insights into virulence regulation and evolution of quorum conjugation. Front. Microbiol. 10, 1134. doi:10.3389/fmicb.2019.01134 sensing in gram-positive bacteria. Proc. Natl. Acad. Sci. U.S.A. 104 (47), Koraimann, G. (2018). Spread and persistence of virulence and antibiotic resistance 18490–18495. doi:10.1073/pnas.0704501104 genes: a ride on the F plasmid conjugation module. EcoSal Plus 8 (1) 1–23. Derman, A. I., Becker, E. C., Truong, B. D., Fujioka, A., Tucey, T. M., Erb, M. L., doi:10.1128/ecosalplus.ESP-0003-2018 et al. (2009). Phylogenetic analysis identifies many uncharacterized actin-like Koraimann, G., and Wagner, M. A. (2014). Social behavior and decision making in proteins (Alps) in bacteria: regulated polymerization, dynamic instability and bacterial conjugation. Front. Cell Infect. Microbiol. 4, 54. doi:10.3389/fcimb. treadmilling in Alp7A. Mol. Microbiol. 73 (4), 534–552. doi:10.1111/j.1365- 2014.00054 2958.2009.06771.x Korobkova, E., Emonet, T., Vilar, J. M., Shimizu, T. S., and Cluzel, P. (2004). From Dodd, I. B., Shearwin, K. E., and Egan, J. B. (2005). Revisited gene regulation in molecular noise to behavioural variability in a single bacterium. Nature 428 bacteriophage lambda. Curr. Opin. Genet. Dev. 15 (2), 145–152. doi:10.1016/j. (6982), 574–578. doi:10.1038/nature02404 gde.2005.02.001 Lerminiaux, N. A., and Cameron, A. D. S. (2019). Horizontal transfer of antibiotic Donlan, R. M. (2002). Biofilms: microbial life on surfaces. Emer. Infect. Dis. 8 (9), resistance genes in clinical environments. Can. J. Microbiol. 65 (1), 34–44. 881–890. doi:10.3201/eid0809.020063 doi:10.1139/cjm-2018-0275 Dubnau, D. (1991). Genetic competence in Bacillus subtilis. Microbiol. Rev. 55, Li, Y. G., Hu, B., and Christie, P. J. (2019). Biological and structural diversity of type 395–424. doi:10.1128/mmbr.55.3.395-424.1991 IV secretion systems. Microbiol. Spectr. 7 (2) 1–10. doi:10.1128/microbiolspec. Dubnau, D., and Losick, R. (2006). Bistability in bacteria. Mol. Microbiol. 61 (3), PSIB-0012-2018 564–572. doi:10.1111/j.1365-2958.2006.05249.x Mazel, D., and Davies, J. (1999). Antibiotic resistance in microbes. Cell Mol. Life Dunny, G. M. (2013). Enterococcal sex pheromones: signaling, social behavior, and Sci. 56 (9–10), 742–754. doi:10.1007/s000180050021 evolution. Annu. Rev. Genet. 47, 457–482. doi:10.1146/annurev-genet-111212- Meijer, W. J., de Boer, A., van Tongeren, S., Venema, G., and Bron, S. (1995). 133449 Characterization of the replication region of the Bacillus subtilis plasmid pLS20: a Dunny, G. M., and Berntsson, R. P. (2016). Enterococcal sex pheromones: novel type of replicon. Nucleic Acids Res. 23, 3214–3223. doi:10.1093/nar/23.16.3214 evolutionary pathways to complex, two-signal systems. J. Bacteriol. 198 (11), Meijer, W. J., Wisman, G. B., Terpstra, P., Thorsted, P. B., Thomas, C. M., 1556–1562. doi:10.1128/JB.00128-16 Holsappel, S., et al. (1998). Rolling-circle plasmids from Bacillus subtilis: Dunny, G. M., and Johnson, C. M. (2011). Regulatory circuits controlling complete nucleotide sequences and analyses of genes of pTA1015, pTA1040, enterococcal conjugation: lessons for functional genomics. Curr. Opin. pTA1050 and pTA1060, and comparisons with related plasmids from gram- Microbiol. 14 (2), 174–180. doi:10.1016/j.mib.2011.01.008 positive bacteria. FEMS Microbiol. Rev. 21 (4), 337–368. doi:10.1111/j.1574- Erez, Z., Steinberger-Levy, I., Shamir, M., Doron, S., Stokar-Avihail, A., Peleg, Y., 6976.1998.tb00357.x et al. (2017). Communication between viruses guides lysis-lysogeny decisions. Molle, V., Fujita, M., Jensen, S. T., Eichenberger, P., González-Pastor, J. E., Liu, J. S., Nature 541 (7638), 488–493. doi:10.1038/nature21049 et al. (2003). The Spo0A regulon of Bacillus subtilis. Mol. Microbiol. 50 (5), Frost, L. S., and Koraimann, G. (2010). Regulation of bacterial conjugation: 1683–1701. doi:10.1046/j.1365-2958.2003.03818.x balancing opportunity with adversity. Future Microbiol. 5 (7), 1057–1071. Monnet, V., Juillard, V., and Gardan, R. (2016). Peptide conversations in gram- doi:10.2217/fmb.10.70 positive bacteria. Crit. Rev. Microbiol. 42 (3), 339–351. doi:10.3109/1040841X. Frost, L. S., Leplae, R., Summers, A. O., and Toussaint, A. (2005). Mobile genetic 2014.948804 elements: the agents of open source evolution. Nat. Rev. Microbiol. 3, 722–732. Neiditch, M. B., Capodagli, G. C., Prehna, G., and Federle, M. J. (2017). Genetic and doi:10.1038/nrmicro1235 structural analyses of RRNPP intercellular peptide signaling of gram-positive Gago-Córdoba, C., Val-Calvo, J., Miguel-Arribas, A., Serrano, E., Singh, P. K., Abia, bacteria. Annu. Rev. Genet. 51, 311–333. doi:10.1146/annurev-genet-120116- D., et al. (2019). Surface exclusion revisited: function related to differential 023507 expression of the surface exclusion system of Bacillus subtilis plasmid pLS20. Novick, R. P., Christie, G. E., and Penadés, J. R. (2010). The phage-related Front. Microbiol. 10, 1502. doi:10.3389/fmicb.2019.01502 chromosomal islands of gram-positive bacteria. Nat. Rev. Microbiol. 8 (8), Goessweiner-Mohr, N., Arends, K., Keller, W., and Grohmann, E. (2014). 541–551. doi:10.1038/nrmicro2393 Conjugation in gram-positive bacteria. Microbiol. Spectr. 2 (4) 1–19. doi:10. Ochman, H., Lawrence, J. G., and Groisman, E. A. (2000). Lateral gene transfer and 1128/microbiolspec.PLAS-0004-2013 the nature of bacterial innovation. Nature 405 (6784), 299–304. doi:10.1038/ Griffith, K. L., and Grossman, A. D. (2008). A degenerate tripartite DNA-binding 35012500 site required for activation of ComA-dependent quorum response gene Oehler, S., and Müller-Hill, B. (2010). High local concentration: a fundamental expression in Bacillus subtilis. J. Mol. Biol. 381 (2), 261–275. doi:10.1016/j. strategy of life. J. Mol. Biol. 395 (2), 242–253. doi:10.1016/j.jmb.2009.10.056 jmb.2008.06.035 Oppenheim, A. B., Kobiler, O., Stavans, J., Court, D. L., and Adhya, S. (2005). Grohmann, E., Muth, G., and Espinosa, M. (2003). Conjugative plasmid transfer in Switches in bacteriophage lambda development. Annu. Rev. Genet. 39, 409–429. gram-positive bacteria. Microbiol. Mol. Biol. Rev. 67 (2), 277–contents. doi:10. doi:10.1146/annurev.genet.39.073003.113656 1128/mmbr.67.2.277-301.2003 Parashar, V., Konkol, M. A., Kearns, D. B., and Neiditch, M. B. (2013). A plasmid- Grossman, A. D. (1995). Genetic networks controlling the initiation of sporulation encoded phosphatase regulates Bacillus subtilis biofilm architecture, and the development of genetic competence in Bacillus subtilis. Annu. Rev. sporulation, and genetic competence. J. Bacteriol. 195 (10), 2437–2448. Genet. 29, 477–508. doi:10.1146/annurev.ge.29.120195.002401 doi:10.1128/JB.02030-12 Hamoen, L. W., Venema, G., and Kuipers, O. P. (2003). Controlling competence in Partridge, S. R., Kwong, S. M., Firth, N., and Jensen, S. O. (2018). Mobile genetic Bacillus subtilis: shared use of regulators. Microbiology (Reading, Engl) 149, elements associated with antimicrobial resistance. Clin. Microbiol. Rev. 31 (4), 9–17. doi:10.1099/mic.0.26003-0 e00088-17. doi:10.1128/CMR.00088-17 Hoch, J. A. (1993). “spo0 genes, the phosphorelay, and the initiation of Perego, M. (2001). A new family of aspartyl phosphate phosphatases targeting the sporulation,” in Bacillus subtilis and other gram-positive bacteria: sporulation transcription factor Spo0A of Bacillus subtilis. Mol. Microbiol. 42 biochemistry, physiology, and molecular genetics. Editors A. L. Sonenshein, (1), 133–143. doi:10.1046/j.1365-2958.2001.02611.x J. A. Hoch, and R. Losick (Washington, DC: American Society for Microbiology Pottathil, M., and Lazazzera, B. A. (2003). The extracellular Phr peptide-Rap phosphatase (ASM)), 747–755. signaling circuit of Bacillus subtilis. Front. Biosci. 8, 32–45. doi:10.2741/913 Frontiers in Molecular Biosciences | www.frontiersin.org 13 March 2021 | Volume 8 | Article 648468 Meijer et al. Regulation of pLS20 Conjugation Ramachandran, G., Singh, P. K., Luque-Ortega, J. R., Yuste, L., Alfonso, C., Rojo, F., Sonenshein, A. L., Hoch, J. A., and Losick, R. (2001). Bacillus subtilis and its closest et al. (2014). A complex genetic switch involving overlapping divergent relatives: from genes to cells. Washington, DC: ASM Press. promoters and DNA looping regulates expression of conjugation genes of a Soucy, S. M., Huang, J., and Gogarten, J. P. (2015). Horizontal gene transfer: gram-positive plasmid. PLoS Genet. 10 (10), e1004733. doi:10.1371/journal. building the web of life. Nat. Rev. Genet. 16 (8), 472–482. doi:10.1038/nrg3962 pgen.1004733 Stingl, K., and Koraimann, G. (2017). Prokaryotic information games: how and Rocha-Estrada, J., Aceves-Diez, A. E., Guarneros, G., and de la Torre, M. (2010). when to take up and secrete DNA. Curr. Top. Microbiol. Immunol. 413, 61–92. The RNPP family of quorum-sensing proteins in gram-positive bacteria. Appl. doi:10.1007/978-3-319-75241-9_3 Microbiol. Biotechnol. 87 (3), 913–923. doi:10.1007/s00253-010-2651-y Tanaka, T., and Koshikawa, T. (1977). Isolation and characterization of four types Shi, K., Brown, C. K., Gu, Z. Y., Kozlowicz, B. K., Dunny, G. M., Ohlendorf, D. H., of plasmids from Bacillus subtilis (natto). J. Bacteriol. 131, 699–701. doi:10. et al. (2005). Structure of peptide sex pheromone receptor PrgX and PrgX/ 1128/JB.131.2.699-701.1977 pheromone complexes and regulation of conjugation in Enterococcus faecalis. Thomas, C. M., and Nielsen, K. M. (2005). Mechanisms of, and barriers to, Proc. Natl. Acad. Sci. U.S.A. 102 (51), 18596–18601. doi:10.1073/pnas10.1073/ horizontal gene transfer between bacteria. Nat. Rev. Microbiol. 3 (9), 711–721. pnas.0506163102 doi:10.1038/nrmicro1234 Singh, P. K., and Meijer, W. J. (2014). Diverse regulatory circuits for transfer of Veening, J. W., Smits, W. K., and Kuipers, O. P. (2008). Bistability, epigenetics, and conjugative elements. FEMS Microbiol. Lett. 358 (2), 119–128. doi:10.1111/ bet-hedging in bacteria. Annu. Rev. Microbiol. 62, 193–210. doi:10.1146/ 1574-6968.12526 annurev.micro.62.081307.163002 Singh, P. K., Ramachandran, G., Durán-Alcalde, L., Alonso, C., Wu, L. J., and Vilar, J. M., and Saiz, L. (2005). DNA looping in gene regulation: from the assembly Meijer, W. J. (2012). Inhibition of Bacillus subtilis natural competence by a of macromolecular complexes to the control of transcriptional noise. Curr. native, conjugative plasmid-encoded comK repressor protein. Environ. Opin. Genet. Dev. 15 (2), 136–144. doi:10.1016/j.gde.2005.02.005 Microbiol. 14 (10), 2812–2825. doi:10.1111/j.1462-2920.2012.02819.x Waksman, G. (2019). From conjugation to T4S systems in gram-negative bacteria: Singh, P. K., Ramachandran, G., Ramos-Ruiz, R., Peiró-Pastor, R., Abia, D., Wu, L. a mechanistic biology perspective. EMBO Rep. 20 (2), e47012. doi:10.15252/ J., et al. (2013). Mobility of the native Bacillus subtilis conjugative plasmid embr.201847012 pLS20 is regulated by intercellular signaling. PLoS Genet. 9 (10), e1003892. Wolf, D., Rippa, V., Mobarec, J. C., Sauer, P., Adlung, L., Kolb, P., et al. (2016). The doi:10.1371/journal.pgen.1003892 quorum-sensing regulator ComA from Bacillus subtilis activates transcription Singh, P. K., Serrano, E., Ramachandran, G., Miguel-Arribas, A., Gago-Cordoba, using topologically distinct DNA motifs. Nucleic Acids Res. 44 (5), 2160–2172. C., Val-Calvo, J., et al. (2020). Reversible regulation of conjugation of Bacillus doi:10.1093/nar/gkv1242 subtilis plasmid pLS20 by the quorum sensing peptide responsive anti-repressor RappLS20. Nucleic Acids Res. 48 (19), 10785–10801. doi:10.1093/nar/gkaa797 Conflict of Interest: The authors declare that the research was conducted in the Smillie, C., Garcillán-Barcia, M. P., Francia, M. V., Rocha, E. P., and De la Cruz, F. absence of any commercial or financial relationships that could be construed as a (2010). Mobility of plasmids. Microbiol. Mol. Biol. Rev. 74 (3), 434–452. doi:10. potential conflict of interest. 1128/MMBR.00020-10 Smits, W. K., Bongiorni, C., Veening, J. W., Hamoen, L. W., Kuipers, O. P., and Copyright © 2021 Meijer, Boer, Ares, Alfonso, Luque-Ortega and Wu. This is an Perego, M. (2007). Temporal separation of distinct differentiation pathways by open-access article distributed under the terms of the Creative Commons Attribution a dual specificity Rap-Phr system in Bacillus subtilis. Mol. Microbiol. 65 (1), License (CC BY). The use, distribution or reproduction in other forums is permitted, 103–120. doi:10.1111/j.1365-2958.2007.05776.x provided the original author(s) and the copyright owner(s) are credited and that the Sonenshein, A. L., Hoch, J. A., and Losick, R. (1993). Bacillus subtilis and other original publication in this journal is cited, in accordance with accepted academic Gram-positive bacteria; Biochemistry, physiology, and molecular genetics. practice. No use, distribution or reproduction is permitted which does not comply Washington, DC: American Society for Microbiology (ASM). with these terms. 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