Bacterial plasmids provide useful model systems to study, at the biochemical, biophysical and structural levels, the protein-protein, protein-DNA and RNA-RNA interactions which constitute the molecular basis of essential processes such as DNA replication, and gene expression and the regulation thereof. In our research group we have spent many years studying the replication of the promiscuous streptococcal plasmid pMV158 and its control, which is effected via transcriptional repression and transductional inhibition of the gene that encodes the protein that initiates the replicative process. These studies have highlighted, in many aspects, the singularity of the replicon pMV158 among prototype plasmids replicating by ‘rolling circle’. Many of these aspects could underlie the great promiscuity of this plasmid. The following elements of the basic replicon of pMV158 have been analyzed.

- RepB still remains the only Rep plasmid initiator of the HUH endonuclease super-family with a resolved molecular structure. Its hexameric conformation is unique among the Reps encoded by plasmids that replicate by ‘rolling circle’. Each RepB protomer consists of an N-terminal domain (the so-called Origin Binding Domain, OBD), which contains both the catalytic activities and the capacity to specifically recognize the plasmid origin, that is linked by a short hinge region to a C-terminal Oligomerization Domain (OD), which is involved in the hexamerization of the protein. The high flexibility of the hinge allows the OBDs to adopt a large variety of orientations relative to the rigid cylindrical scaffold formed by the ODs. By means of the construction and characterization of protein mutants we are studying the underlying molecular bases in i) the specific recognition of the origin DNA by RepB, ii) the catalytic activity of RepB in the initiation  and termination stages of replication, and  iii) the possible participation of RepB in the unwinding of the DNA. This latter study will also involve the use of a variety of mutants that we have constructed in the host PcrA helicase. The model derived from these analyses could help to explain pMV158’s great promiscuity.

- CopG is one of the elements that controls the replication of pMV158 and acts by repressing the transcription of the copG-repB operon. CopG is the smallest transcriptional repressor so far characterized. The high affinity and specificity of CopG for its DNA target is based on the sequential and cooperative binding of dimers of CopG to the 4 sub-sites that constitute its operator. In the absence of cooperative interactions, CopG only binds to the primary site of the operator. By means of high resolution ‘footprinting’ we have determined the order of binding of CopG to the operator. The mode of binding of CopG to DNA (unique among the proteins in the RHH super-family) throws light on the mechanism used by this transcriptional repressor to remove the RNA polymerase firmly bound to the promoter. In addition to its role in the control of the steady-state replication of pMV158, CopG plays a key role during the establishment of the plasmid in a new host since the unrepressed activity of the CopG-regulated promoter is crucial for the successful colonization of the recipient bacterial cells by the plasmid. Thus, CopG could be used as an immunity factor to prevent "infection" of a potential host bacterium with the target donor plasmid. These findings can be extrapolated to other proteins that repress transcription of rep genes of rolling-circle- or theta-replicating plasmids.

- RNAII is the other element involved in the control of replication of pMV158. This is a small ‘antisense’ RNA coded in cis, complimentary to the copG-repB region of the mRNA which is situated immediately at 5’ from the signals of the initiation of the transduction of repB. Contrary to what happens in the majority of systems that control plasmid replication by using antisense RNA, the interaction between RNAII and its target in the mRNA copG-repB does not require formation of a ‘kissing complex’, but it needs the 3’ region of the 5’ single-stranded tail of RNAII instead. For its part, the inhibitory capacity of RNAII lies at the 5’ region of the above mentioned 5’-terminal tail. The characteristics of RNAII make it potentially useful for the construction of vectors for gene silencing in Gram-positive bacteria. Furthermore, the characterization of the replicon of pMV158 has allowed us to define an ever increasing family of plasmids isolated from a wide variety of bacteria (the majority belonging to the phylum Firmicutes; many of them being LAB). Our group has used some of these replicons to construct vectors for gene cloning and expression, for characterization of promoters and regulatory regions, as well as for fluorescent labeling of bacteria.

Drawing on the experience of our group in the analysis of plasmid replication and regulation of gene expression, we have characterized the replicon and the expression of the gene that encodes the dextransucrase of plasmid pMN1, which was isolated from Lactobacillus sakei and which is responsible for dextran production by the bacterial host. Likewise, in the frame of a collaboration with the group of Prof. Jesús Murillo (Universidad Pública de Navarra, Pamplona, Spain), we have contributed to the preliminary characterization of a second replicon from  Pseudomonas syringae virulence plasmid pPsv48C, which appears to be a natural chimera between the gene encoding a newly described replication protein and a putative replication control region present in the widespread family of PFP virulence plasmids.



Boer, D. R., Ruiz-Masó, J. A., López-Blanco, J. R., Blanco, A. G., Vives-Llàcer, M., Chacón, P., Usón, I., Gomis-Rüth, F. X., Espinosa, M., Llorca, O., del Solar, G.*, and Miquel Coll* (* co-corresponding authors) (2009). Plasmid replication initiator RepB forms a hexamer reminiscent of ring helicases and has mobile nuclease domains. EMBO J. 28: 1666-1678.

Hernández-Arriaga, A. M., Rubio-Lepe, T. S., Espinosa, M. and del Solar, G. (2009). Repressor CopG prevents access of RNA polymerase to promoter and actively dissociates open complexes. Nucl. Acids Res. 37: 4799-4811.

Ruiz-Masó, J. A., López-Aguilar, C., Nieto, C., Sanz, M., Burón, P., Espinosa, M. and del Solar, G. (2012). Construction of a plasmid vector based on the pMV158 replicon for cloning and inducible gene expression in Streptococcus pneumoniae. Plasmid 67: 53–59.

López-Aguilar, C., Ruiz-Masó, J. A., Rubio-Lepe, T. S., Sanz, M., del Solar, G. (2013). Translation initiation of the replication initiator repB gene of promiscuous plasmid pMV158 is led by an extended non-SD sequence. Plasmid 70: 69-77.

López-Aguilar, C. and del Solar, G. (2013). Probing the sequence and structure of in vitro synthesized antisense and target RNAs from the replication control system of plasmid pMV158. Plasmid 70: 94-103.

Ruiz-Masó, J. A., Machón, C., Bordanaba-Ruiseco, L., Espinosa, M., Coll, M. and del Solar, G. (2015). Plasmid Rolling-Circle Replication. Microbiol Spectrum 3(1): PLAS-0035-2014. DOI: 10.1128 /microbiolspec.PLAS-0035-2014.

López-Aguilar, C., Romero-López, C., Espinosa, M., Berzal-Herranz, A. and del Solar, G. (2015). The 5′-tail of antisense RNAII of pMV158 plays a critical role in binding to the target mRNA and in translation inhibition of repB. Front. Genet. 6:225. doi: 10.3389/fgene.2015.00225.

Boer, D. R., Ruiz-Masó, J. A., Rueda, M., Petoukhov, M. V., Machón, C., Svergun, D. I., Orozco, M., del Solar, G.* and Coll, M.* (* co-corresponding authors) (2016).  Conformational plasticity of RepB, the replication initiator protein of promiscuous streptococcal plasmid pMV158. Sci. Rep. 6, 20915; doi: 10.1038/srep20915.

Ruiz-Masó, J.A., Luengo, L.M., Moreno-Córdoba, I., Díaz-Orejas, R. and del Solar, G. (2017). Successful establishment of plasmids R1 and pMV158 in a new host requires the relief of the transcriptional repression of their essential rep genes. Front. Microbiol. 8:2367. doi: 10.3389/fmicb.2017.02367.

Bardaji, L., Añorga, M., Ruiz-Masó, J.A., del Solar, G. and Murillo, J. (2017). Plasmid replicons from Pseudomonas are natural chimeras of functional, exchangeable modules. Front. Microbiol. 8:190. doi: 10.3389/fmicb.201700190.

Nácher-Vázquez, M., Ruiz-Masó, J. A., Mohedano, M. L., del Solar, G., Aznar, R. and López, P. (2017). Dextransucrase expression is concomitant with that of replication and maintenance functions of the pMN1 plasmid in Lactobacillus sakei MN1. Front. Microbiol. 8:2281.

Garay-Novillo, J.N., García-Morena, D., Ruiz-Masó, J.A., Barra, J.L. and del Solar, G. (2019). Combining modules for versatile and optimal labeling of lactic acid bacteria: two pMV158-family promiscuous replicons, a pneumococcal system for constitutive or inducible gene expression, and two fluorescent proteins. Front. Microbiol. 10:1431. doi: 10.3389/fmicb.2019.01431. 

Moreno-del Alamo,  M., Torres, R., Manfredi, C., Ruiz-Masó,  J.A., del Solar, G. and Alonso, J.C. (2020). Bacillus subtilis PcrA couples DNA replication, transcription, recombination and segregation. Front. Mol. Biosci. 7:140. doi: 10.3389/fmolb.2020.00140.

Jara, J., Pérez-Ramos, A., del Solar, G., Rodríguez, J.M., Fernández, L. and Orgaz, B. (2020). Role of Lactobacillus biofilms in Listeria monocytogenes adhesion to glass surfaces. Int. J. Food Microbiol. 334. doi: 10.1016/j.ijfoodmicro.2020.108804.