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DNA replication is essential to all living organisms as it ensures the fidelity of genetic material for the next generation of dividing cells. One of the simplest replication initiation mechanisms is the rolling circle replication. In the streptococcal plasmid pMV158, which confers antibiotic resistance to tetracycline, replication initiation is catalysed by RepB protein. The RepB N-terminal domain or origin binding domain binds to the recognition sequence (bind locus) of the double-strand origin of replication and cleaves one DNA strand at a specific site within the nic locus. Using biochemical and crystallographic analyses, here we show how the origin binding domain recognises and binds to the bind locus using structural elements removed from the active site, namely the recognition α helix, and a β-strand that organises upon binding. A new hexameric structure of full-length RepB that highlights the great flexibility of this protein is presented, which could account for its ability to perform different tasks, namely bind to two distinct loci and cleave one strand of DNA at the plasmid origin., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Plasmid vectors constitute a valuable tool for homologous and heterologous gene expression, for characterization of promoter and regulatory regions, and for genetic manipulation and labeling of bacteria. During the last years, a series of vectors based on promiscuous replicons of the pMV158 family have been developed for their employment in a variety of Gram-positive bacteria and proved to be useful for all above applications in lactic acid bacteria. A proper use of the plasmid vectors requires detailed knowledge of their main replicative features under the changing growth conditions of the studied bacteria, such as the acidification of the culture medium by lactic acid production. Initiation of pMV158 rolling-circle replication is catalyzed by the plasmid-encoded RepB protein, which performs a sequence-specific cleavage on one of the parental DNA strands and, as demonstrated in this work, establishes a covalent bond with the 5'-P end generated in the DNA. This covalent adduct must last until the leading-strand termination stage, where a new cleavage on the regenerated nick site and a subsequent strand-transfer reaction result in rejoining of the ends of the cleaved parental strand, whereas hydrolysis of the newly-generated adduct would release the protein from a nicked double-stranded DNA plasmid form. We have analyzed here the effect of pH on the different in vitro reactions catalyzed by RepB and on the in vivo replication ability of plasmid pMV158. We show that acidic pH greatly impairs the catalytic activity of the protein and reduces hydrolysis of the covalent RepB-DNA adduct, as expected for the nucleophilic nature of these reactions. Conversely, the ability of pMV158 to replicate in vivo , as monitored by the copy number and segregational stability of the plasmid in Lactococcus lactis , remains almost intact at extracellular pHs ranging from 7.0 to 5.0, and a significant reduction (by ∼50%) in the plasmid copy number per chromosome equivalent is only observed at pH 4.5. Moreover, the RepB to pMV158 molar ratio is increased at pH 4.5, suggesting the existence of compensatory mechanisms that operate in vivo to allow pMV158 replication at pH values that severely disturb the catalytic activity of the initiator protein., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Valdelvira, Bordanaba-Ruiseco, Martín-Huestamendía, Ruiz-Masó and del Solar.)

Bacillus subtilis PcrA abrogates replication-transcription conflicts in vivo and disrupts RecA nucleoprotein filaments in vitro . Inactivation of pcrA is lethal. We show that PcrA depletion lethality is suppressed by recJ (involved in end resection), recA (the recombinase), or mfd (transcription-coupled repair) inactivation, but not by inactivating end resection ( addAB or recQ ), positive and negative RecA modulators ( rarA or recX and recU ), or genes involved in the reactivation of a stalled RNA polymerase ( recD 2, helD, hepA , and ywqA ). We also report that B. subtilis mutations previously designated as recL16 actually map to the recO locus, and confirm that PcrA depletion lethality is suppressed by recO inactivation. The pcrA gene is epistatic to recA or mfd , but it is not epistatic to addAB, recJ, recQ, recO16, rarA, recX, recU, recD 2, helD, hepA , or ywqA in response to DNA damage. PcrA depletion led to the accumulation of unsegregated chromosomes, and this defect is increased by recQ, rarA , or recU inactivation. We propose that PcrA, which is crucial to maintain cell viability, is involved in different DNA transactions., (Copyright © 2020 Moreno-del Alamo, Torres, Manfredi, Ruiz-Masó, del Solar and Alonso.)

The exopolysaccharide synthesized by Lactobacillus sakei MN1 is a dextran with antiviral and immunomodulatory properties of potential utility in aquaculture. In this work we have investigated the genetic basis of dextran production by this bacterium. Southern blot hybridization experiments demonstrated the plasmidic location of the dsrLS gene, which encodes the dextransucrase involved in dextran synthesis. DNA sequencing of the 11,126 kbp plasmid (pMN1) revealed that it belongs to a family which replicates by the theta mechanism, whose prototype is pUCL287. The plasmid comprises the origin of replication, repA, repB , and dsrLS genes, as well as seven open reading frames of uncharacterized function. Lb. sakei MN1 produces dextran when sucrose, but not glucose, is present in the growth medium. Therefore, plasmid copy number and stability, as well as dsrLS expression, were investigated in cultures grown in the presence of either sucrose or glucose. The results revealed that pMN1 is a stable low-copy-number plasmid in both conditions. Gene expression studies showed that dsrLS is constitutively expressed, irrespective of the carbon source present in the medium. Moreover, dsrLS is expressed from a monocistronic transcript as well as from a polycistronic repA-repB-orf1-dsrLS mRNA. To our knowledge, this is the first report of a plasmid-borne dextransucrase-encoding gene, as well as the first time that co-transcription of genes involved in plasmid maintenance and replication with a gene encoding an enzyme has been established.

Plasmids are a main factor for the evolution of bacteria through horizontal gene exchange, including the dissemination of pathogenicity genes, resistance to antibiotics and degradation of pollutants. Their capacity to duplicate is dependent on their replication determinants (replicon), which also define their bacterial host range and the inability to coexist with related replicons. We characterize a second replicon from the virulence plasmid pPsv48C, from Pseudomonas syringae pv. savastanoi, 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. We present extensive evidence of this type of chimerism in structurally similar replicons from species of Pseudomonas , including environmental bacteria as well as plant, animal and human pathogens. We establish that these replicons consist of two functional modules corresponding to putative control (REx-C module) and replication (REx-R module) regions. These modules are functionally separable, do not show specificity for each other, and are dynamically exchanged among replicons of four distinct plasmid families. Only the REx-C module displays strong incompatibility, which is overcome by a few nucleotide changes clustered in a stem-and-loop structure of a putative antisense RNA. Additionally, a REx-C module from pPsv48C conferred replication ability to a non-replicative chromosomal DNA region containing features associated to replicons. Thus, the organization of plasmid replicons as independent and exchangeable functional modules is likely facilitating rapid replicon evolution, fostering their diversification and survival, besides allowing the potential co-option of appropriate genes into novel replicons and the artificial construction of new replicon specificities.

Initiation of plasmid rolling circle replication (RCR) is catalyzed by a plasmid-encoded Rep protein that performs a Tyr- and metal-dependent site-specific cleavage of one DNA strand within the double-strand origin ( dso ) of replication. The crystal structure of RepB, the initiator protein of the streptococcal plasmid pMV158, constitutes the first example of a Rep protein structure from RCR plasmids. It forms a toroidal homohexameric ring where each RepB protomer consists of two domains: the C-terminal domain involved in oligomerization and the N-terminal domain containing the DNA-binding and endonuclease activities. Binding of Mn 2+ to the active site is essential for the catalytic activity of RepB. In this work, we have studied the effects of metal binding on the structure and thermostability of full-length hexameric RepB and each of its separate domains by using different biophysical approaches. The analysis of the temperature-induced changes in RepB shows that the first thermal transition, which occurs at a range of temperatures physiologically relevant for the pMV158 pneumococcal host, represents an irreversible conformational change that affects the secondary and tertiary structure of the protein, which becomes prone to self-associate. This transition, which is also shown to result in loss of DNA binding capacity and catalytic activity of RepB, is confined to its N-terminal domain. Mn 2+ protects the protein from undergoing this detrimental conformational change and the observed protection correlates well with the high-affinity binding of the cation to the active site, as substituting one of the metal-ligands at this site impairs both the protein affinity for Mn 2+ and the Mn 2+ -driven thermostabilization effect. The level of catalytic activity of the protein, especially in the case of full-length RepB, cannot be explained based only on the high-affinity binding of Mn 2+ at the active site and suggests the existence of additional, lower-affinity metal binding site(s), missing in the separate catalytic domain, that must also be saturated for maximal activity. The molecular bases of the thermostabilizing effect of Mn 2+ on the N-terminal domain of the protein as well as the potential location of additional metal binding sites in the entire RepB are discussed.

DNA replication initiation is a vital and tightly regulated step in all replicons and requires an initiator factor that specifically recognizes the DNA replication origin and starts replication. RepB from the promiscuous streptococcal plasmid pMV158 is a hexameric ring protein evolutionary related to viral initiators. Here we explore the conformational plasticity of the RepB hexamer by i) SAXS, ii) sedimentation experiments, iii) molecular simulations and iv) X-ray crystallography. Combining these techniques, we derive an estimate of the conformational ensemble in solution showing that the C-terminal oligomerisation domains of the protein form a rigid cylindrical scaffold to which the N-terminal DNA-binding/catalytic domains are attached as highly flexible appendages, featuring multiple orientations. In addition, we show that the hinge region connecting both domains plays a pivotal role in the observed plasticity. Sequence comparisons and a literature survey show that this hinge region could exists in other initiators, suggesting that it is a common, crucial structural element for DNA binding and manipulation.

Plasmids are DNA entities that undergo controlled replication independent of the chromosomal DNA, a crucial step that guarantees the prevalence of the plasmid in its host. DNA replication has to cope with the incapacity of the DNA polymerases to start de novo DNA synthesis, and different replication mechanisms offer diverse solutions to this problem. Rolling-circle replication (RCR) is a mechanism adopted by certain plasmids, among other genetic elements, that represents one of the simplest initiation strategies, that is, the nicking by a replication initiator protein on one parental strand to generate the primer for leading-strand initiation and a single priming site for lagging-strand synthesis. All RCR plasmid genomes consist of a number of basic elements: leading strand initiation and control, lagging strand origin, phenotypic determinants, and mobilization, generally in that order of frequency. RCR has been mainly characterized in Gram-positive bacterial plasmids, although it has also been described in Gram-negative bacterial or archaeal plasmids. Here we aim to provide an overview of the RCR plasmids' lifestyle, with emphasis on their characteristic traits, promiscuity, stability, utility as vectors, etc. While RCR is one of the best-characterized plasmid replication mechanisms, there are still many questions left unanswered, which will be pointed out along the way in this review.

RepB is the pMV158-encoded protein that initiates rolling-circle replication of this promiscuous plasmid. Availability of RepB is rate-limiting for the plasmid replication process, and therefore the repB gene encoding the protein is subjected to strict control. Two trans-acting plasmid elements, CopG and the antisense RNAII, are involved in controlling the synthesis of the initiator at the transcriptional and translational level, respectively. In addition to this dual control of repB expression that senses and corrects fluctuations in plasmid copy number, proper availability of RepB also relies on the adequate functionality of the transcription and translation initiation regulatory signals. Translation of repB has been postulated to depend on an atypical ribosome binding site that precedes its start codon, although such a hypothesis has never been proved. To define sequences involved in translation of repB, several mutations in the translation initiation region of the repB mRNA have been characterized by using an Escherichia coli in vitro expression system wherein the synthesis of RepB was detected and quantified. We showed that translation of repB is not coupled to that of copG and depends only on its own initiation signals. The atypical ribosome binding site, as it was defined, is not involved in translation initiation. However, the sequence just upstream of the repB start codon, encompassing the proximal box of the atypical ribosome binding site and the four bases immediately downstream of it, is indeed important for efficient translation of repB. The high degree of conservation of this sequence among the rep genes of plasmids of the same pMV158 family supports its relevancy as a translation initiation signal in mRNAs without a recognizable Shine-Dalgarno sequence., (Copyright © 2013 Elsevier Inc. All rights reserved.)

We report the construction of a plasmid vector designed for regulated gene expression in Streptococcus pneumoniae. The new vector, pLS1ROM, is based on the replicon of the streptococcal promiscuous rolling circle replication (RCR) plasmid pMV158. We inserted the controllable promoter P(M) of the S. pneumoniaemalMP operon, followed by a multi-cloning site sequence aimed to facilitate the insertion of target genes. The expression from P(M) is negatively regulated by the transcriptional repressor MalR, which is released from the DNA operator sequence by growing the cells in maltose-containing media. To get a highly regulated expression of the target gene, MalR was provided in cis by inserting the malR gene under control of the constitutive P(tet) promoter, which in pMV158 directs expression of the tetL gene. To test the functionality of the system, we cloned the reporter gene gfp from Aequorea victoria, encoding the green fluorescent protein (GFP). Pneumococcal cells harboring the recombinant plasmid rendered GFP fluorescence in a maltose-dependent mode with undetectable background levels in the absence of the inducer. The new vector, pLS1ROM, exhibits full structural and segregational stability and constitutes a valuable tool for genetic manipulation and regulated gene expression in S. pneumoniae., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Plasmids replicating by the rolling circle mode usually possess a single site for binding of the initiator protein at the origin of replication. The origin of pMV158 is different in that it possesses two distant binding regions for the initiator RepB. One region was located close to the site where RepB introduces the replication-initiating nick, within the nic locus; the other, the bind locus, is 84 bp downstream from the nick site. Binding of RepB to the bind locus was of higher affinity and stability than to the nic locus. Contacts of RepB with the bind and nic loci were determined through high-resolution footprinting. Upon binding of RepB, the DNA of the bind locus follows a winding path in its contact with the protein, resulting in local distortion and bending of the double-helix. On supercoiled DNA, simultaneous interaction of RepB with both loci favoured extrusion of the hairpin structure harbouring the nick site while causing a strong DNA distortion around the bind locus. This suggests interplay between the two RepB binding sites, which could facilitate loading of the initiator protein to the nic locus and the acquisition of the appropriate configuration of the supercoiled DNA substrate.

Protein p56 (56 amino acids) from the Bacillus subtilis phage 29 inactivates the host uracil-DNA glycosylase (UDG), an enzyme involved in the base excision repair pathway. At present, p56 is the only known example of a UDG inhibitor encoded by a non-uracil containing viral DNA. Using analytical ultracentrifugation methods, we found that protein p56 formed dimers at physiological concentrations. In addition, circular dichroism spectroscopic analyses revealed that protein p56 had a high content of beta-strands (around 40%). To understand the mechanism underlying UDG inhibition by p56, we carried out in vitro experiments using the Escherichia coli UDG enzyme. The highly acidic protein p56 was able to compete with DNA for binding to UDG. Moreover, the interaction between p56 and UDG blocked DNA binding by UDG. We also demonstrated that Ugi, a protein that interacts with the DNA-binding domain of UDG, was able to replace protein p56 previously bound to the UDG enzyme. These results suggest that protein p56 could be a novel naturally occurring DNA mimicry.

PcrA is a chromosomally encoded DNA helicase of gram-positive bacteria involved in replication of rolling circle replicating plasmids. Efficient interaction between PcrA and the plasmid-encoded replication initiator (Rep) protein is considered a requirement for the plasmid to replicate in a given host, and thus, the ability of a Rep protein to interact with heterologous PcrA helicases has been invoked as a determinant of plasmid promiscuity. We characterized transcription of the Streptococcus pneumoniae pcrA gene in its genetic context and studied the biochemical properties of its product, the PcrA(Spn) helicase. Transcription of the pneumococcal pcrA gene was directed by promoter Pa, consisting of an extended -10 box. Promoter Pa also accounted for expression of a second essential gene, radC, which was transcribed with much lower efficiency than pcrA, probably due to the presence of a terminator/attenuator sequence located between the two genes. PcrA(Spn) displayed single-stranded DNA-dependent ATPase activity. PcrA(Spn) showed 5'-->3' and 3'-->5' helicase activities and bound efficiently to partially duplex DNA containing a hairpin structure adjacent to a 6-nucleotide 5' or 3' single-stranded tail and one unpaired (flap) nucleotide in the complementary strand. PcrA(Spn) interacted specifically with RepC, the initiator of staphylococcal plasmid pT181. Although the pneumococcal helicase was able to initiate unwinding of the RepC-nicked pT181 DNA, it was much less processive in this activity than the cognate staphylococcal PcrA protein. Accordingly, PcrA(Spn) was inefficient in in vitro replication of pT181, and perhaps as a consequence, this plasmid could not be established in S. pneumoniae.

The promiscuous rolling circle (RC)-replicating plasmid pMV158 encodes the 210-amino-acid initiator of replication protein, RepB. The protein relaxes supercoiled cognate DNA in a topoisomeraseI-like manner. A new vector and procedure for overproduction, scaling-up, and purification of the protein has been developed. RepB purified as a hexamer in solution, as shown by analytical ultracentrifugation assays. Circular dichroism (CD) of RepB indicated that the protein has an estimated content of around 33% alpha-helices and 20% beta-strands. Characterisation of temperature-induced transitions of the protein showed an irreversible change in the spectra when the temperature was raised above 35 degrees C, indicating that the protein undergoes a conformational change that could account for the relatively high optimal temperature of the RepB-mediated cleavage.

The cryptic plasmid pt38 (2911 bp) of Streptococcus thermophilus ST2783, a strain isolated from Bulgarian yogurt, was subcloned and sequenced. Five ORFs (ORF1 to ORF5) were identified, although putative transcription initiation and termination signals, and Shine-Dalgarno sequence could only be localized for three of them (ORF1, ORF2, and ORF5). ORF2 would specify a 142-amino acid protein sharing a high degree of homology with plasmid-born low-molecular-weight heat stress proteins described in a variety of S. thermophilus strains. On the other hand, ORF1 would encode a 311-residue protein, which was found to be almost identical to the putative Rep proteins of previously sequenced S. thermophilus rolling circle-replicating plasmids. Intracellular single-stranded pt38 DNA was detected, showing that, in fact, the plasmid replicates via a rolling circle mechanism. A putative double-strand origin with significant homology to that of pC194, and a ssoA-type single-strand origin were also identified on the nucleotide sequence of pt38. A DNA region that can be transcribed into a small RNA (ctRNA) complementary to the leader segment of the rep (ORF1) mRNA is proposed to be involved in the control of plasmid replication. In vitro synthesis of this ctRNA was observed, and this constitutes the first report on the existence of such antisense RNAs, likely acting as regulatory elements, in S. thermophilus plasmids.