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Specific DNA recognition mediated by a type IV pilin Ana Cehovina, Peter J. Simpsonb, Melanie A. McDowellc, Daniel R. Browna, Rossella Noscheseb,...
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1. Give the name of the journalyear2. What are the last names3. What type of organismgenusspecies

 and the  in which this article was published.

 of the authors? What are their universities?

 is being studied? Give  and  names.

4. What is the hypothesis of the experiment? 

5.  Describe each graph in detail, making sure to discuss how each graph relates to the hypothesis of the paper. 

6. a. What is the major conclusion of the study? 

6. b. What will the researchers do next, based on the current results of this study?

Specifc DNA recogniton mediaTed by a Type IV pilin Ana Cehovin a , PeTer J. Simpson b , Melanie A. McDowell c , Daniel R. Brown a , Rossella Noschese b , MiTchell Palle± a , Jacob Brady b , GeoFrey S. Baldwin b , Susan M. Lea c , STephen J. Ma±hews b , and Vladimir Pelicic a,1 a Medical Research Council CenTre ²or Molecular BacTeriology and In²ecton, Secton o² Microbiology and b Division o² Molecular Biosciences, Imperial College London, London SW7 2AZ, UniTed Kingdom; and c Sir William Dunn School o² PaThology, UniversiTy o² Ox²ord, Ox²ord OX1 3RE, UniTed Kingdom EdiTed by Emil C. GoTschlich, ³he Rocke²eller UniversiTy, New York, NY, and approved January 10, 2013 (received ²or review OcTober 31, 2012) NaTural Trans²ormaton is a dominanT ²orce in bacTerial evoluton by promotng horizonTal gene Trans²er. ³his process may have devasTatng consequences, such as The spread o² antbiotc resisTance or The emergence o² highly virulenT clones. However, upTake and recombinaton o² ²oreign DNA are mosT o´en deleTerious To compeTenT species. ³here²ore, model naTurally Trans²ormable Gramnegatve bacTeria, including The human paThogen Neisseria meningitdis, have evolved means To pre²erentally Take up homoTypic DNA conTaining shorT and genus-specifc sequence mot²s. DespiTe decades o² inTense investgatons, The DNA upTake sequence recepTor in Neisseria species has remained elusive. We show here, using a multdisciplinary approach combining biochemisTry, molecular genetcs, and sTrucTural biology, ThaT meningococcal Type IV pili bind DNA Through The minor pilin ComP via an elecTropositve sTripe ThaT is predicTed To be exposed on The flamenTs sur²ace and ThaT ComP displays an exquisiTe binding pre²erence ²or DNA upTake sequence. Our fndings illuminaTe The earliesT sTep in naTural Trans²ormaton, reveal an unconventonal mechanism ²or DNA binding, and suggesT ThaT selectve DNA upTake is more widespread Than previously ThoughT. DNA recepTor | genetc compeTence atural transformation is the process by which competent bacteria take up free DNA that is abundant in many environments and incorporate it into their genomes through homologous recombination. This widespread property is shared by dozens of species (1), and was key to one of the most important discoveries in biology (that DNA carries genetic information) (2). Critically, transformation is a powerful mechanism for generating genetic diversity through horizontal transfer of genes. For example, the naturally competent Neisseria meningitidis, which is a human pathogen of global importance, displays an extensive genetic diversity, with as much as 10% of its gene content differing between isolates (3). This diversity is key to the success of the meningococcus in competing with our immune system and has dire practical consequences, such as the dif f cult design of vaccines providing universal protection against meningococcal disease (4). In other species, horizontal gene transfer may also have dramatic consequences, such as the unabated spread of antibiotic resistance or the emergence of highly virulent clones. N Our understanding offreeDNAtransport fromtheextracellular milieu into the bacterial cytoplasm during transformation remains fragmentary, but the following model is widely accepted (5). Competent bacteria f rst bind DNA at their surface before it is taken up by type IV pili (Tfp) that are long, hair-like f laments found in a myriad of species (6) or shorter, evolutionarily related competence pseudopili. In Gram-negative bacteria, DNA uptake is de f ned as the crossing of the outer membrane that is thought to occur through secretin channels, which are involved in the emergence of Tfp onto the bacterial surface. In Gram-positive species, DNA uptake consists in the crossing of the thick layer of peptidoglycan. The f nding that PilT, which is a cytoplasmic ATPase powering the remarkable capacity of Tfp to retract and generate mechanical force (7), is necessary for DNA uptake (8) suggests that Tfp/pseudopili pull DNA through the outer membrane/peptidoglycan on retraction (5). DNA then interacts with ComE/ComEA (9, 10) and is delivered to a conserved translocase machinery in the cytoplasmicmembranethat further transports itinto thecytoplasm. Finally, DNA is integrated in the bacterial chromosome in a RecAdependent manner, but it can also be used as a template for the repair of DNA damage or a source of food (5). How Tfp/pseudopili interact with DNA remains enigmatic for several reasons. (i) High nonspeci f c binding of DNA to whole bacteria (11, 12) has been a hinder to genetic studies. (ii) ELISA using puri f ed f laments has revealed only weak DNA binding with biological signi f cance that is unclear (13, 14). (iii) Various biochemical approaches have only identi f ed DNA binding proteins that are involved at late stages during transformation (15, 16). Nevertheless, the fact that Neisseria species and Pasteurellaceae favor uptake of homotypic DNA by selectively recognizing genus-speci f c DNA uptake sequence (DUS) motifs [or uptake signal sequence (USS) as they are known in Pasteurellaceae] is clear evidence of the existence of speci f c surfaceexposed DNA receptors (5, 17, 18). None of the known DNA binding proteins involved in transformation show preference for DNA containing DUS/USS, and the nature of the DUS/USS receptors has, thus, remained mysterious. Although no type IV pilin has ever been reported to bind DNA, the following observations prompted us to address the question of whether ComP might be the elusive DUS receptor in Neisseria species. (i) ComP is one of three minor (low abundance) pilins in pathogenic Neisseria species (with PilV and PilX) that modulate Tfp-mediated properties without affecting Tfp biogenesis (19 22). ComP has a prominent role in competence (20, 21) at the level of DNA uptake (23). (ii) ComP displays unparalleled sequence conservation for a surface-exposed protein in N. meningitidis, because it was found to be virtually identical in a large collection of disease isolates (24). This f nding is consistent with the hypothesis that sequence conservation of DUS should be matched by sequence conservation of its cognate receptor. To test the ability of ComP to bind DNA, we have, therefore, carried out and report here a multidisciplinary analysis combining biochemistry, molecular genetics, and structural biology. ResulTs ComP Is Highly Conserved ³hroughouT The Genus Neisseria. The 5 atGCCGTCTGAA-3 DUS motif (less conserved nucleotides are in lowercase) (17, 25) is highly overrepresented within the genomes of all sequenced Neisseria species (on average, every kilobase), with single base variationsin some ofthem(26). Therefore,wereasoned MICROBIOLOGY www.pnas.org/cgi/doi/10.1073/pnas.1218832110 PNAS | µebruary 19, 2013 | vol. 110 | no. 8 | 3065–3070
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Author contribuTons: S.J.M. and V.P. designed research; A.C., P.J.S., M.A.M., D.R.B., R.N., M.P., J.B., and V.P. performed research; G.S.B. and S.M.L. contributed new reagents/ analyTc tools; A.C., P.J.S., G.S.B., S.M.L., S.J.M., and V.P. analyzed data; and S.J.M. and V.P. wrote the paper. ±he authors declare no conFict of interest. ±his arTcle is a PNAS Direct Submission. ²reely available online through the PNAS open access opTon. Data deposiTon: ±he NMR, atomic coordinates, chemical shi³s, and restraints data have been deposited in the Protein Data Bank, www.pdb.org (PDB ID code 2M3K ) . 1 ±o whom correspondence should be addressed. E-mail: [email protected] ±his arTcle contains supporTng informaTon online at www.pnas.org/lookup/suppl/doi:10. 1073/pnas.1218832110/-/DCSupplemental . that, if ComP was the DUS receptor in Neisseria species, it must be conserved beyond N. meningitidis. Accordingly, ComP orthologs were found in all sequenced Neisseria species ( Table S1 ). Strikingly, inthecladecomprisingN.meningitidis,N.gonorrhoeae,N.lactamica, and N. polysaccharea, we found that ComP was virtually identical with a maximum of two nonconservative substitutions over the 149 residues of the preprotein ( Fig. S1 ). Such 99% sequence identity is in line with that of key essential proteins, such as the components of the RNA polymerase (encoded by rpo genes) or the ribosome (encoded by rps, rpl, and rpm genes), and is consistent with a key cellular function for ComP. ComP Is Required for E´cient DNA Binding by Puriµed ±fp. As the identi µ cation of the DUS receptor has been impossible by genetic means because of the high nonspeci µ c binding of DNA to whole bacteria (11, 12), we µ rst tested the ability of µ laments puri µ ed from a WT strain and an isogenic comP mutant to bind DNA by ELISA. To use highly puri µ ed pili in this assay, we engineered the 8013-PilE His6 variant of N. meningitidis 8013, in which the last µ ve residues in the major pilus subunit PilE were replaced by a polyHis tag, and designed a metal af µ nity chromatography methodology to purify Tfp ( Fig. S2 ). We phenotypically characterized 8013-PilE His6 and found that it is piliated (slightly less than the parent 8013 strain) and displays all of the classical Tfp-linked phenotypes ( Fig. S2 ) . Critically, transformation frequencies in 8013-PilE His6 and its parent strain 8013 were comparable, showing that the poly-His tag does not interfere with Tfp-mediated DNA uptake. We, therefore, puri µ ed pili from 8013- PilE His6 (WT) and its isogenic comP mutant and loaded serial dilutions of equal amounts of µ bers (as assessed by PilE immunobloting) in the wells of streptavidin-coated microtiter plates, in which a biotinylated double-stranded (ds) 22-mer primer centered on DUS was previously immobilized. Quanti µ cation by ELISA showed that, although WT Tfp bound DNA in a concentration-dependent and saturable manner, µ laments of a comP mutant were dramatically impaired for DNA binding (Fig. 1). This µ nding provided evidence linking ComP with DNA binding. ComP Is the only Pilus Component Capable of Binding DNA. To determine whether ComP has intrinsic DNA binding activity, we puri µ ed this protein and the other three known pilin components of N. meningitidis pili (PilE, PilV, and PilX). Using a strategy previously validated during the structural characterization of PilX (19), the soluble portion of these proteins in strain 8013, without their highly conserved and hydrophobic N-terminal α -helices (27), was fused to maltose binding protein (MBP). The fusion proteins are directed to the periplasm, which is important for disul µ de bond formation, a characteristic feature of type IV pilins (27). After a two-step puri µ cation that yielded pure proteins, we tested the ability of these four pilins to interact with DNA using agarose EMSAs (10). As a target, we used the pSY6 plasmid that contains one DUS, which has been widely used to measure transformation ef µ ciencies in Neisseria species (13). These experiments showed that ComP is the only pilus component capable of binding DNA, because no shift was seen with the other pilins with as much as 10 μ M pure protein (Fig. 2A). The ladder pattern observed with increasing concentrations of ComP most likely indicates that this protein was able to bind to multiple sites in the target plasmid. We further characterized ComP s DNA binding activity by using a more sensitive acrylamide EMSA and a ds 22-mer DUS primer labeled with biotin as a target ( Table S2 ) . This assay con µ rmed that ComP is capable of binding DNA (Fig. 2B). A shift was observed in a concentration-dependent manner with as little as 0.2 μ M protein, indicating a signi µ cant af µ nity of ComP for DNA. ComP Interacts Be¶erwith DUS. We next used a range of approaches to determine whether ComP has a higher af µ nity for DUS. Using acrylamide EMSA, we performed competition assays by assessing the effect of an excess of unlabeled ds primer on the formation of ComP DUS complex obtained with 0.4 μ M protein. Although unlabeled DUS ef µ ciently competed with biotinylated DUS in a dose-dependent manner (Fig. 3A), which was demonstrated by the gradual disappearance of the ComP DUS complex, a scrambled primer (SUD; in which every second base of DUS was changed) was incapable of competing, even at very high concentration. Similar results were obtained with another 3066 | www.pnas.org/cgi/doi/10.1073/pnas.1218832110 Cehovin et al.
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