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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 Pcr[A.sub.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. Pcr[A.sub.Spn] displayed single-stranded DNA-dependent ATPase activity. Pcr[A.sub.Spn] showed 5'[right arrow]3' and 3'[right arrow]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. Pcr[A.sub.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, Pcr[A.sub.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 plasmid pLS1 encodes two transacting elements that regulate its copy number: protein RepA and antisense RNA II. In vitro transcription showed that RNAs for both repressors are synthesized from two promoters, PAB and PII. From PAB, genes encoding RepA (transcriptional repressor) and RepB (initiator of replication) are cotranscribed, the target of RepA being located within PAB. Mutants in repA or in PAB are still sensitive to RepA. However, cloning of the repA gene in a compatible replicon did not result in incompatibility towards pLS1. From PII, the 50-nucleotide RNA II is synthesized. The main incompatibility determinant towards pLS1 corresponds to the coding sequence for RNA II. The RNA II target could be reduced to 21 nucleotides, including the RepB initiation of translation signals. We propose that plasmids of the pLS1 family (pE194, pADB201, and pLB4) share functional and structural characteristics for the regulation of their copy numbers. [ABSTRACT FROM AUTHOR]

Fermentative processes by lactic acid bacteria can produce metabolites of interest to the health and food industries. Two examples are the production of B-group vitamins, and of prebiotic and immunomodulatory dextran-type exopolysaccharides. In this study, three riboflavin- and dextran-producing Weissella cibaria strains (BAL3C-5, BAL3C-7 and BAL3C-22) were used to develop a new method for selection and isolation of spontaneous riboflavin-overproducing W. cibaria mutants. This method was based on the selection of strains resistant to roseoflavin. The DNA sequencing of the FMN riboswitch of bacterial cell populations treated with various roseoflavin concentrations, revealed the existence of at least 10 spontaneous and random point mutations at this location. Folding and analysis of the mutated FMN riboswitches with the RNA fold program predicted that these mutations could result in a deregulation of the rib operon expression. When the roseoflavin-treated cultures were plated on medium supporting dextran synthesis, the most promising mutants were identified by the yellow color of their mucous colonies, exhibiting a ropy phenotype. After their isolation and recovery in liquid medium, the evaluation of their riboflavin production revealed that the mutant strains synthesized a wide range of riboflavin levels (from 0.80 to 6.50 mg/L) above the wild-type level (0.15 mg/L). Thus, this was a reliable method to select spontaneous riboflavin-overproducing and dextran-producing strains of W. cibaria. This species has not yet been used as a starter or adjunct culture, but this study reinforces the potential that it has for the food and health industry for the production of functional foods or as a probiotic. Furthermore, analysis of the influence of FMN present in the growth medium, on rib mRNA and riboflavin levels, revealed which mutant strains produce riboflavin without flavin regulation. Moreover, the BAL3C-5 C120T mutant was identified as the highest riboflavin-overproducer. Determination of its chromosomal DNA sequence and that of BAL3C-5, revealed a total identity between the 2 strains except for the C120T mutation at the FMN riboswitch. To our knowledge, this work is the first demonstration that only a single alteration in the genome of a lactic acid bacteria is required for a riboflavin-overproducing phenotype., 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 © 2023 Diez-Ozaeta, Martín-Loarte, Mohedano, Tamame, Ruiz-Masó, del Solar, Dueñas and López.)

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.)

This work describes a method for deriving riboflavin overproducing strains of Weissella cibaria by exposing three strains (BAL3C-5, BAL3C-7, and BAL3C-22) isolated from dough to increasing concentrations of roseoflavin. By this procedure, we selected one mutant overproducing strain from each parental strain (BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2, respectively). Quantification of dextran and riboflavin produced by the parental and mutant strains in a defined medium lacking riboflavin and polysaccharides confirmed that riboflavin was only overproduced by the mutant strains, whereas dextran production was similar in both mutant and parental strains. The molecular basis of the riboflavin overproduction by the mutants was determined by nucleotide sequencing of their rib operons, which encode the enzymes of the riboflavin biosynthetic pathway. We detected a unique mutation in each of the overproducing strains. These mutations, which map in the sensor domain (aptamer) of a regulatory element (the so-called FMN riboswitch) present in the 5' untranslated region of the rib operon mRNA, appear to be responsible for the riboflavin-overproducing phenotype of the BAL3C-5 B2, BAL3C-7 B2, and BAL3C-22 B2 mutant strains. Furthermore, the molecular basis of dextran production by the six W. cibaria strains has been characterized by ( i ) the sequencing of their dsr genes encoding dextransucrases, which synthesize dextran using sucrose as substrate, and ( ii ) the detection of active Dsr proteins by zymograms. Finally, the parental and mutant strains were analyzed for in situ production of riboflavin and dextran during experimental bread making. The results indicate that the mutant strains were able to produce experimental wheat breads biofortified with both riboflavin and dextran and, therefore, may be useful for the manufacture of functional commercial breads., 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 © 2022 Hernández-Alcántara, Chiva, Mohedano, Russo, Ruiz-Masó, del Solar, Spano, Tamame and López.)

Manufacturing of probiotics and functional foods using lactic acid bacteria (LAB) that overproduce vitamin B 2 has gained growing interest due to ariboflavinosis problems affecting populations of both developing and affluent countries. Two isogenic Lactiplantibacillus plantarum strains, namely a riboflavin-producing parental strain (UFG9) and a roseoflavin-resistant strain (B2) that carries a mutation in the FMN-aptamer of the potential rib operon riboswitch, were analysed for production and intra- and extracellular accumulation of flavins, as well as for regulation of the rib operon expression. Strain B2 accumulated in the medium one of the highest levels of riboflavin+FMN ever reported for LAB, exceeding by ~ 25 times those accumulated by UFG9. Inside the cells, concentration of FAD was similar in both strains, while that of riboflavin+FMN was ~ 8-fold higher in B2. Mutation B2 could decrease the stability of the aptamer's regulatory P1 helix even in the presence of the effector, thus promoting the antiterminator structure of the riboswitch ON state. Although the B2-mutant riboswitch showed an impaired regulatory activity, it retained partial functionality being still sensitive to the effector. The extraordinary capacity of strain B2 to produce riboflavin, together with its metabolic versatility and probiotic properties, can be exploited for manufacturing multifunctional foods., (© 2021 The Authors. Microbial Biotechnology published by Society for Applied Microbiology and John Wiley & Sons Ltd.)

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.)

Riboflavin (vitamin B 2 ) is a vitamin of the B group involved in essential biological pathways, including redox reactions and the electron transport chain. Some lactic acid bacteria (LAB) can synthesize riboflavin and this capability is strain-dependent. In the last years, a growing interest has focused on the selection of riboflavin-overproducing food-grade LAB for the vitamin biofortification of fermented foods, as well as for the formulation of innovative functional products.In this chapter we report fast and inexpensive techniques in order to (1) screen LAB isolates able to produce riboflavin from different matrices, (2) select spontaneous roseoflavin-resistant riboflavin overproducing strains, and (3) quantify vitamin B 2 in culture media by fluorescence detection.These protocols could be useful to select new overproducing strains and/or species from different ecological niches, as well as to optimize the conditions for vitamin bioproduction.

Listeria monocytogenes can form long-lasting biofilms on food-contact surfaces. Lactic acid bacteria (LAB) have shown promise in antagonizing this microorganism in liquid media. However, the ecological relationships differ when cells are forming biofilms. In this work, we propose the use of Lactobacillus biofilms as surface "conditioners" to modulate the adhesion of L. monocytogenes. For this, the biofilm formation ability of Lactobacillus fermentum MP26 and Lactobacillus salivarius MP14 (human milk origin), fluorescently labeled by transfer of the mCherry-encoding pRCR12 plasmid, was first evaluated. Then, mature biofilms of these strains transformed with pRCR12 for expressing the fluorescent protein mCherry were used as adhesion substrate for GFP-tagged L. monocytogenes Scott A. The resulting biofilms were studied in terms of cellular population and attached biomass (cells plus matrix). Species distribution inside the biofilm structure was revealed by confocal laser scanning microscopy (CLSM). Although none of the Lactobacillus spp. strains reduced the adhesion of L. monocytogenes Scott A, species interactions seem to interfere with the synthesis of extracellular polymeric substances and species distribution inside the biofilms. In dual-species biofilms, CLSM images revealed that Lactobacillus cells were trapping those of L. monocytogenes Scott A. When surfaces were conditioned with Lactobacillus biofilms, the spatial distribution of L. monocytogenes Scott A cells was species-specific, suggesting these interactions are governing the ultimate biofilm structure. The results here obtained open new possibilities for controlling L. monocytogenes dispersal using these Lactobacillus spp. biofilms as a "natural" immobilization way. Whether species interactions could modify the virulence of L. monocytogenes still remains unclear., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.)

Labeling of bacterial cells with fluorescent proteins allows tracking the bacteria in competition and interactomic in vivo and in vitro studies. During the last years, a few plasmid vectors have been developed aimed at the fluorescent labeling of specific members of the lactic acid bacteria (LAB), a heterogeneous group that includes microorganisms used in the food industry, as probiotics, or as live vectors for mucosal vaccines. Successful and versatile labeling of a broad range of LAB not only requires a vector containing a promiscuous replicon and a widely recognized expression system for the constitutive or regulated expression of the fluorescence determinant, but also the knowledge of the main features of the entire plasmid/host/fluorescent protein ensemble. By using the LAB model species Lactococcus lactis , we have compared the utility properties of a set of labeling vectors constructed by combining a promiscuous replicon (pMV158 or pSH71) of the pMV158 plasmid family with the gene encoding either the EGFP or the mCherry fluorescent protein placed under control of promoter P X or P M from the pneumococcal mal gene cluster for maltosaccharide uptake and utilization, respectively. Some vectors carrying P M also harbor the malR gene, whose product represses transcription from this promoter, thus enabling maltose-inducible synthesis of the fluorescent proteins. We have determined the plasmid copy number (PCN) and segregational stability of the different constructs, as well as the effect of these features on the fitness and fluorescence intensity of the lactococcal host. Constructs based on the pSH71 replicon had a high copy number (∼115) and were segregationally stable. The copy number of vectors based on the pMV158 replicon was lower (∼8-45) and varied substantially depending on the genetic context of the plasmid and on the bacterial growth conditions; as a consequence, inheritance of these vectors was less stable. Synthesis of the fluorescent proteins encoded by these plasmids did not significantly decrease the host fitness. By employing inducible expression vectors, the fluorescent proteins were shown to be very stable in this bacterium. Importantly, conditions for accurate quantification of the emitted fluorescence were established based on the maturation times of the fluorescent proteins.

Although differing in size, encoded traits, host range, and replication mechanism, both narrow-host-range theta-type conjugative enterobacterial plasmid R1 and promiscuous rolling-circle-type mobilizable streptococcal plasmid pMV158 encode a transcriptional repressor protein, namely CopB in R1 and CopG in pMV158, involved in replication control. The gene encoding CopB or CopG is cotranscribed with a downstream gene that encodes the replication initiator Rep protein of the corresponding plasmid. However, whereas CopG is an auto-repressor that inhibits transcription of the entire copG-repB operon, CopB is expressed constitutively and represses a second, downstream promoter that directs transcription of repA . As a consequence of the distinct regulatory pathways implied by CopB and CopG, these repressor proteins play a different role in control of plasmid replication during the steady state: while CopB has an auxiliary role by keeping repressed the regulated promoter whenever the plasmid copy number is above a low threshold, CopG plays a primary role by acting coordinately with RNAII. Here, we have studied the role of the regulatory circuit mediated by these transcriptional repressors during the establishment of these two plasmids in a new host cell, and found that excess Cop repressor molecules in the recipient cell result in a severe decrease in the frequency and/or the velocity of appearance of transformant colonies for the cognate plasmid but not for unrelated plasmids. Using the pMV158 replicon as a model system, together with highly sensitive real-time qPCR and inverse PCR methods, we have also analyzed the effect of CopG on the kinetics of repopulation of the plasmid in Streptococcus pneumoniae . We show that, whereas in the absence of CopG pMV158 repopulation occurs mainly during the first 45 min following plasmid transfer, the presence of the transcriptional repressor in the recipient cell severely impairs the replicon repopulation and makes the plasmid replicate at approximately the same rate as the chromosome at any time after transformation, which results in maximal plasmid loss rate in the absence of selection. Overall, these findings indicate that unrepressed activity of the Cop-regulated promoter is crucial for the successful colonization of the recipient bacterial cells by the plasmid.

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.

Rolling-circle replication of streptococcal plasmid pMV158 is controlled by the concerted action of two trans-acting elements, namely transcriptional repressor CopG and antisense RNAII, which inhibit expression of the repB gene encoding the replication initiator protein. The pMV158-encoded antisense RNAII exerts its activity of replication control by inhibiting translation of the essential repB gene. RNAII is the smallest and simplest among the characterized antisense RNAs involved in control of plasmid replication. Structure analysis of RNAII revealed that it folds into an 8-bp-long stem containing a 1-nt bulge and closed by a 6-nt apical loop. This hairpin is flanked by a 17-nt-long single-stranded 5'-tail and an 8-nt-long 3'-terminal U-rich stretch. Here, the 3' and 5' regions of the 5'-tail of RNAII are shown to play a critical role in the binding to the target mRNA and in the inhibition of repB translation, respectively. In contrast, the apical loop of the single hairpin of RNAII plays a rather secondary role and the upper stem region hardly contributes to the binding or inhibition processes. The entire 5'-tail is required for efficient inhibition of repB translation, though only the 8-nt-long region adjacent to the hairpin seems to be essential for rapid binding to the mRNA. These results show that a "kissing" interaction involving base-pairing between complementary hairpin loops in RNAII and mRNA is not critical for efficient RNA/RNA binding or repB translation inhibition. A singular binding mechanism is envisaged whereby initial pairing between complementary single-stranded regions in the antisense and sense RNAs progresses upwards into the corresponding hairpin stems to form the intermolecular duplex.

Antisense RNAII is a replication control element encoded by promiscuous plasmid pMV158. RNAII binds to its complementary sequence in the copG-repB mRNA, thus inhibiting translation of the replication initiator repB gene. In order to initiate the biochemical characterization of the pMV158 antisense RNA-mediated control system, conditions for in vitro transcription by T7RNA polymerase were set up that yielded large amounts of antisense and target run-off products able to bind to each other. The run-off antisense transcript was expected, and confirmed, to span the entire RNAII as synthesized by the bacterial RNA polymerase, including the intrinsic transcription terminator at its 3'-terminus. On the other hand, two different target transcripts, mRNA₆₀ and mRNA₈₀, were produced, characterized and tested for efficient binding to the antisense product. The mRNA₆₀ and mRNA₈₀ run-off transcripts supposedly spanned 60 and 80 nucleotides, respectively, on the copG-repB mRNA and lacked terminator-like structures at their 3'-termini. Probing of the sequence and conformation of the main products, along with modeling of their secondary structures, showed that both target transcripts were actually longer-than-expected, and contained a 3'-terminal hairpin wherein the extra nucleotides base-paired to the expected 3'-terminus of the corresponding run-off transcript. These longer products were proposed to arise from the RNA-dependent polymerizing activity of T7RNA polymerase on correct run-off transcripts primed by extremely short 3'-selfcomplementarity. Seizing of the target mRNA sequence complementary to the 5'-terminus of RNAII in a stable 3'-terminal hairpin generated by this activity seemed to cause a 3-fold decrease in the efficiency of binding to the antisense RNA., (Copyright © 2013 Elsevier Inc. All rights reserved.)

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.)

Promiscuous, rolling-circle replication plasmid pMV158 determines tetracycline resistance to its host and can be mobilized by conjugation. Plasmid pLS1 is a deletion derivative of pMV158 that has lost its conjugative mobilization ability. Both plasmids replicate efficiently and are stably inherited in Streptococcus pneumoniae. We have analyzed the effect of pMV158 and pLS1 carriage on the bacterial growth rate. Whereas the parental plasmid does not significantly modify the cell doubling time, pLS1 slows down the growth of the bacterial host by 8-9%. The bases of the differential burden caused by pMV158 and pLS1 carriage are not yet understood. The negligible cost of the pMV158 parental natural plasmid on the host might explain the prevalence of small, multicopy, rolling-circle replication plasmids, even though they lack any selectable trait., (Copyright © 2012 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.)

AtzR is a LysR-type regulator responsible for activation of the cyanuric acid utilization operon atzDEF. AtzR binds the PatzDEF promoter region at a strong recognition element, designated the repressor binding site, and a weaker binding determinant, the activator binding site (ABS). AtzR activates transcription in response to two dissimilar signals, nitrogen limitation and cyanuric acid. In the present work we analyse the structure and function of the cis-acting elements involved in AtzR activation of atzDEF. Hydroxyl radical footprinting assays revealed that the ABS is composed of three functional subsites spaced at one helix-turn intervals. Two modes of interaction with the ABS are detected in vitro: AtzR binds at the ABS-2 and ABS-3 subsites in the absence of inducer, and relocates to interact with the ABS-1 and ABS-2 subsites in the presence of cyanuric acid. In vivo mutational analysis indicates that ABS-1 and ABS-2 are required for full PatzDEF activation in all conditions. In contrast, ABS-3 acts as a 'subunit trap' that hinders productive AtzR interactions with ABS-1 and ABS-2. Our results strongly suggest an activation model in which cyanuric acid and nitrogen limitation cooperate to reposition AtzR from an inactive, ABS-3 bound configuration to an active, ABS-1- and ABS-2-bound configuration.

Replication of the promiscuous plasmid pMV158 requires expression of the initiator repB gene, which is controlled by the repressor CopG. Genes repB and copG are co-transcribed from promoter P(cr). We have studied the interactions between RNA polymerase, CopG and the promoter to elucidate the mechanism of repression by CopG. Complexes formed at 0 degrees C and at 37 degrees C between RNA polymerase and P(cr) differed from each other in stability and in the extent of the DNA contacted. The 37 degrees C complex was very stable (half-life of about 3 h), and shared features with typical open complexes generated at a variety of promoters. CopG protein repressed transcription from P(cr) at two different stages in the process leading to the initiation complex. First, CopG hindered binding of RNA polymerase to the promoter. Second, CopG was able to displace RNA polymerase once the enzyme has formed a stable complex with P(cr). A model for the CopG-mediated disassembly of the stable RNA polymerase-P(cr) promoter complex is presented.

RepB initiates plasmid rolling-circle replication by binding to a triple 11-bp direct repeat (bind locus) and cleaving the DNA at a specific distant site located in a hairpin loop within the nic locus of the origin. The structure of native full-length RepB reveals a hexameric ring molecule, where each protomer has two domains. The origin-binding and catalytic domains show a three-layer alpha-beta-alpha sandwich fold. The active site is positioned at one of the faces of the beta-sheet and coordinates a Mn2+ ion at short distance from the essential nucleophilic Y99. The oligomerization domains (ODs), each consisting of four alpha-helices, together define a compact ring with a central channel, a feature found in ring helicases. The toroidal arrangement of RepB suggests that, similar to ring helicases, it encircles one of the DNA strands during replication to confer processivity to the replisome complex. The catalytic domains appear to be highly mobile with respect to ODs. This mobility may account for the adaptation of the protein to two distinct DNA recognition sites.

This study reports the development of several cloning vectors for bifidobacteria based on the replicon of pBC1, a cryptic plasmid from Bifidobacterium catenulatum L48 thought to replicate via the theta mode. These vectors, in which antibiotic resistance genes encoding either erythromycin or tetracycline resistance acted as selection markers, were able to replicate in a series of eight Bifidobacterium species at frequencies ranging from 4.0 x 10(1) to 1.0 x 10(5) transformants microg(-1) but not in Lactococcus lactis or Lactobacillus casei. They showed a relative copy number of around 30 molecules per chromosome equivalent and a good segregational stability, with more than 95% of the cells retaining the vectors after 80 to 100 generations in the absence of selection. Vectors contain multiple cloning sites of different lengths, and the lacZalpha peptide gene was introduced into one of the molecules, thus allowing the easy selection of colonies harboring recombinant plasmids in Escherichia coli. The functionality of the vectors for engineering Bifidobacterium strains was assessed by cloning and examining the expression of an alpha-l-arabinofuranosidase gene belonging to Bifidobacterium longum. E. coli and Bifidobacterium pseudocatenulatum recombinant clones were stable and showed an increase in alpha-arabinofuranosidase activity of over 100-fold compared to that of the untransformed hosts.