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Some transcription factors bind DNA motifs containing direct or inverted sequence repeats. Preference for each of these DNA topologies is dictated by structural constraints. Most prokaryotic regulators form symmetric oligomers, which require operators with a dyad structure. Binding to direct repeats requires breaking the internal symmetry, a property restricted to a few regulators, most of them from the AraC family. The KorA family of transcriptional repressors, involved in plasmid propagation and stability, includes members that form symmetric dimers and recognize inverted repeats. Our structural analyses show that ArdK, a member of this family, can form a symmetric dimer similar to that observed for KorA, yet it binds direct sequence repeats as a non-symmetric dimer. This is possible by the 180° rotation of one of the helix–turn–helix domains. We then probed and confirmed that ArdK shows affinity for an inverted repeat, which, surprisingly, is also recognized by a non-symmetrical dimer. Our results indicate that structural flexibility at different positions in the dimerization interface constrains transcription factors to bind DNA sequences with one of these two alternative DNA topologies., This work was supported by the Spanish Ministry of Economy, Industry and Competitiveness [BIO2016-77883-C2-2-P and FIS2015-72574-EXP (AEI/FEDER, EU), to D.R.B., BFU2017-86378-P to F.dlC.] and by the Spanish Ministryof Science (MCI/AEI/FEDER,UE) [PGC2018-093885-BI00 and PID2021-122164NB-I00 to G.M., PID2020- 117028GB-I00 to D.R.B. and PID2019-110216GB-I00 to R. F-L.].

15 p.-6 fig.1 graph. abst., 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., Ministry of Science and Innovation of Spain [BFU2017-83720-P, PID2020-120141GB-100 to M.C., 2022AEP028 (RT12018-097114-B-I00), BFU2010-19597 to G.dS.]; RYC fellowship [RYC-2011-09071 to C.M.]; CSIC [2020AEP116 to M.C.]. Funding for open access charge: CSIC.

15 p.-4 fig.-3 tab., Quorum sensing allows bacterial cells to communicate through the release of soluble signaling molecules into the surrounding medium. It plays a pivotal role in controlling bacterial conjugation in Gram-positive cells, a process that has tremendous impact on health. Intracellular regulatory proteins of the RRNPP family are common targets of these signaling molecules. The RRNPP family of gene regulators bind signaling molecules at their C-terminal domain (CTD), but have highly divergent functionalities at their N-terminal effector domains (NTD). This divergence is also reflected in the functional states of the proteins, and is highly interesting from an evolutionary perspective. RappLS20 is an RRNPP encoded on the Bacillus subtilis plasmid pLS20. It relieves the gene repression effectuated by RcopLS20 in the absence of the mature pLS20 signaling peptide Phr*pLS20. We report here an in-depth structural study of apo and Phr*pLS20-bound states of RappLS20 at various levels of atomic detail. We show that apo-RappLS20 is dimeric and that Phr*pLS20-bound Rap forms NTD-mediated tetramers. In addition, we show that RappLS20 binds RcopLS20 directly in the absence of Phr*pLS20 and that addition of Phr*pLS20 releases RcopLS20 from RappLS20. This allows RcopLS20 to bind the promotor region of crucial conjugation genes blocking their expression., Ministry of Economy and Competitiveness of the Spanish Government [BFU2016-75471-C2-1-P (AEI/FEDER,EU) to C.A., BIO2013-41489-P (AEI/FEDER, EU) to W.M. which also funded AM-A, BIO2016-77883-C2-2-P (AEI/FEDER, EU) to R.B., BIO2016-77883-C2-2-P(AEI/FEDER, EU) also supported N.B.]; ‘Fundación Ramón Areces’ and ‘Banco de Santander’ to the Centro de Biología Molecular ‘Severo Ochoa’.Funding for open access charge: Ministry of Economy and Competitiveness of the Spanish Government [BIO2016-77883-C2-1-P and BIO2016-77883-C2-2-P].

Bacterial conjugation is an important route for horizontal gene transfer. The initial step in this process involves a macromolecular protein-DNA complex called the relaxosome, which in plasmids consists of the origin of transfer (oriT) and several proteins that prepare the transfer. The relaxosome protein named relaxase introduces a nick in one of the strands of the oriT to initiate the process. Additional relaxosome proteins can exist. Recently, several relaxosome proteins encoded on the Bacillus subtilis plasmid pLS20 were identified, including the relaxase, named RelpLS20, and two auxiliary DNA-binding factors, named Aux1pLS20 and Aux2pLS20. Here, we extend this characterization in order to define their function. We present the low-resolution SAXS envelope of the Aux1pLS20 and the atomic X-ray structure of the C-terminal domain of Aux2pLS20. We also study the interactions between the auxiliary proteins and the full-length RelpLS20, as well as its separate domains. The results show that the quaternary structure of the auxiliary protein Aux1pLS20 involves a tetramer, as previously determined. The crystal structure of the C-terminal domain of Aux2pLS20 shows that it forms a tetramer and suggests that it is an analog of TraMpF of plasmid F. This is the first evidence of the existence of a TraMpF analog in gram positive conjugative systems, although, unlike other TraMpF analogs, Aux2pLS20 does not interact with the relaxase. Aux1pLS20 interacts with the C-terminal domain, but not the N-terminal domain, of the relaxase RelpLS20. Thus, the pLS20 relaxosome exhibits some unique features despite the apparent similarity to some well-studied G- conjugation systems.

The principal route for dissemination of antibiotic resistance genes is conjugation by which a conjugative DNA element is transferred from a donor to a recipient cell. Conjugative elements contain genes that are important for their establishment in the new host, for instance by counteracting the host defense mechanisms acting against incoming foreign DNA. Little is known about these establishment genes and how they are regulated. Here, we deciphered the regulation mechanism of possible establishment genes of plasmid p576 from the Gram-positive bacterium Bacillus pumilus. Unlike the ssDNA promoters described for some conjugative plasmids, the four promoters of these p576 genes are repressed by a repressor protein, which we named Reg576. Reg576 also regulates its own expression. After transfer of the DNA, these genes are de-repressed for a period of time until sufficient Reg576 is synthesized to repress the promoters again. Complementary in vivo and in vitro analyses showed that different operator configurations in the promoter regions of these genes lead to different responses to Reg576. Each operator is bound with extreme cooperativity by two Reg576-dimers. The X-ray structure revealed that Reg576 has a Ribbon-Helix-Helix core and provided important insights into the high cooperativity of DNA recognition., Economy and Competitiveness of the Spanish Government [BFU2016-75471-C2-1-P (AEI/FEDER, EU) to C.A., BIO2013-41489-P (AEI/FEDER, EU) and BIO2016-77883-C2-1-P (AEI/FEDER, EU) to W.M., BIO2016-77883-C2-2-P (AEI/FEDER, EU) to R.B., BIO-2015-66203-P (AEI/FEDER, EU) to F.R., FIS2016-78313-P (AEI/FEDER, EU) to S.A.]; BIO2013-41489-P (AEI/FEDER, EU) and BIO2016-77883-C2-1-P (AEI/FEDER, EU) also supported J.V., A.M., and C.G.; Wellcome Investigator Award [209500 to Jeff Errington] supported L.W; ‘Ramón y Cajal’ Contract Supported S.A.; ‘Agencia Estatal de Investigaci ´on’ (AEI); ‘Fondo Europeo de Desarrollo Regional (FEDER); European Union (EU)

23 p.-5 fig.-2 tab., Antibiotic resistance is a serious global problem. Antibiotic resistance genes (ARG), which are widespread in environmental bacteria, can be transferred to pathogenic bacteria via horizontal gene transfer (HGT). Gut microbiomes are especially apt for the emergence and dissemination of ARG. Conjugation is the HGT route that is predominantly responsible for the spread of ARG. Little is known about conjugative elements of Gram-positive bacteria, including those of the phylum Firmicutes, which are abundantly present in gut microbiomes. A critical step in the conjugation process is the relaxase-mediated site-and strand-specific nick in the oriT region of the conjugative element. This generates a single-stranded DNA molecule that is transferred from the donor to the recipient cell via a connecting channel. Here we identified and characterized the relaxosome components oriT and the relaxase of the conjugative plasmid pLS20 of the Firmicute Bacillus subtilis. We show that the relaxase gene, named rel(LS20), is essential for conjugation, that it can function in trans and provide evidence that Tyr26 constitutes the active site residue. In vivo and in vitro analyses revealed that the oriT is located far upstream of the relaxase gene and that the nick site within oriT is located on the template strand of the conjugation genes. Surprisingly, the Rel(LS20) shows very limited similarity to known relaxases. However, more than 800 genes to which no function had been attributed so far are predicted to encode proteins showing significant similarity to RelLS20. Interestingly, these putative relaxases are encoded almost exclusively in Firmicutes bacteria. Thus, RelLS20 constitutes the prototype of a new family of relaxases. The identification of this novel relaxase family will have an important impact in different aspects of future research in the field of HGT in Gram-positive bacteria in general, and specifically in the phylum of Firmicutes, and in gut microbiome research., Work in the Meijer lab was funded by the Spanish government through grant Bio2013- 41489-P of the Ministry of Economy and Competitiveness, and through grant Bio2016- 77883-C2-1-P of the Ministry of Economy, Industry and Competitiveness; the former grant also funded AMA and CGC. The Spanish government also supported DRB, JRLO, and CA.DRB was funded by grant Bio2016-77883-C2-2-P of the Ministry of Economy, Industry and Competitiveness, and JRLO and CA were supported by grant BFU2014-52070-C2-2-P of the Ministry of Economy and Competitiveness to CA.LJW's work was supported by Wellcome Trust grant WT098374AIA to Jeff Errington.

Relaxases are metal-dependent nucleases that break and join DNA for the initiation and completion of conjugative bacterial gene transfer. Conjugation is the main process through which antibiotic resistance spreads among bacteria, with multidrug-resistant staphylococci and streptococci infections posing major threats to human health. The MOBV family of relaxases accounts for approximately 85% of all relaxases found in Staphylococcus aureus isolates. Here, we present six structures of the MOBV relaxase MobM from the promiscuous plasmid pMV158 in complex with several origin of transfer DNA fragments. A combined structural, biochemical, and computational approach reveals that MobM follows a previously uncharacterized histidine/metal-dependent DNA processing mechanism, which involves the formation of a covalent phosphoramidate histidine-DNA adduct for cell-to-cell transfer. We discuss how the chemical features of the high-energy phosphorus-nitrogen bond shape the dominant position of MOBV histidine relaxases among small promiscuous plasmids and their preference toward Gram-positive bacteria., The Institute for Research in Biomedicine Barcelona is the recipient of a Severo Ochoa Award of Excellence from the Spanish Ministry of Economy and Competitiveness (MINECO). The Molecular Biology Institute, Barcelona Spanish Research Council’s Structural Biology Unit is the recipient of a Maria de Maeztu Award of Excellence from MINECO. This work was supported by grants from MINECO (BFU2008-02372/ BMC, CSD-2006-23, BFU2011-22588, CSD-2008/00013, BIO2013-49148-C2-2-R, BFU2014-53550-P, BIO2015-69085-REDC, and BIO2015-64802), the Generalitat de Catalunya (2014-SGR134 and 2014-SGR1530), and the European Commission [project no. 260644, ERC_SimDNA, and the H2020 program (MuG and BioExcel Projects)]. R.P. and H.G. received a La Caixa/Institute for Research in Biomedicine Barcelona International PhD and Juan de La Cierva fellowship, respectively. M.O. is a Catalan Institution for Research and Advanced Studies Academia researcher.

Isoquinoline 1-oxidoreductase (IOR) from Brevundimonas diminuta is a mononuclear molybdoenzyme of the xanthine-dehydrogenase family of proteins and catalyzes the conversion of isoquinoline to isoquinoline-1-one. Its primary sequence and behaviour, specifically in its substrate specificity and lipophilicity, differ from other members of the family. A crystal structure of the enzyme is expected to provide an explanation for these differences. This paper describes the crystallization and preliminary X-ray diffraction experiments as well as an optimized purification protocol for IOR. Crystallization of IOR was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement. However, phases need to be improved in order to obtain a more interpretable electron-density map. publishersversion published