Structural insights of Mycobacterium GTPase-Obg and anti-sigma-F factor Usfx interaction.

An essential protein for bacterial growth, GTPase-Obg (Obg), is known to play an unknown but crucial role in stress response as its expression increases in Mycobacterium under stress conditions. It is well reported that Obg interacts with anti-sigma-F factor Usfx; however, a detailed analysis and structural characterization of their physical interaction remain undone. In view of above-mentioned points, this study was conceptualized for performing binding analysis and structural characterization of Obg-Usfx interaction. The binding studies were performed by surface plasmon resonance, while in silico docking analysis was done to identify crucial residues responsible for Obg-Usfx interaction. Surface plasmon resonance results clearly suggest that N-terminal and G domains of Obg mainly contribute to Usfx binding. Also, binding constants display strong affinity that was further evident by intermolecular hydrogen bonds and hydrophobic interactions in the predicted complex. Strong interaction between Obg and Usfx supports the view that Obg plays an important role in stress response, essentially required for Mycobacterium survival. As concluded by various studies that Obg is crucial for Mycobacterium survival under stress, this structural information may help us in designing novel and potential inhibitors against resistant Mycobacterium strains.

Sequence of the R1 plasmid and comparison to F and R100.

The R1 antibiotic resistance plasmid, originally discovered in a clinical Salmonella isolate in London, 1963, has served for decades as a key model for understanding conjugative plasmids. Despite its scientific importance, a complete sequence of this plasmid has never been reported. We present the complete genome sequence of R1 along with a brief review of the current knowledge concerning its various genetic systems and a comparison to the F and R100 plasmids. R1 is 97,566 nucleotides long and contains 120 genes. The plasmid consists of a backbone largely similar to that of F and R100, a Tn21-like transposon that is nearly identical to that of R100, and a unique 9-kb sequence that bears some resemblance to sequences found in certain Klebsiella oxytoca strains. These three regions of R1 are separated by copies of the insertion sequence IS1. Overall, the structure of R1 and comparison to F and R100 suggest a fairly stable shared conjugative plasmid backbone into which a variety of mobile elements have inserted to form an "accessory" genome, containing multiple antibiotic resistance genes, transposons, remnants of phage genes, and genes whose functions remain unknown.

Long-Term Stable and Tightly Controlled Expression of Recombinant Proteins in Antibiotics-Free Conditions.

Plasmid-based gene expression is a fundamental tool in the field of biotechnology. However, overexpression of genes of interest with multi-copy plasmids often causes detrimental effects on host cells. To overcome this problem, chromosomal integration of target genes has been used for decades; however, insufficient protein expression occurred with this method. In this study, we developed a novel cloning and expression system named the chromosomal vector (ChroV) system, that has features of stable and high expression of target genes on the F' plasmid in the Escherichia coli JM109(DE3) strain. We used an RMT cluster (KCTC 11994BP) containing a silent cat gene from a previous study to clone a gene into the F' plasmid. The ChroV system was applied to clone two model targets, GFPuv and carotenoids gene clusters (4 kb), and successfully used to prove the inducible tightly regulated protein expression in the F' plasmid. In addition, we verified that the expression of heterologous genes in ChroV system maintained stably in the absence of antibiotics for 1 week, indicating ChroV system is applicable to antibiotics-free production of valuable proteins. This protocol can be widely applied to recombinant protein expression for antibiotics-free, stable, and genome-based expression, providing a new platform for recombinant protein synthesis in E. coli. Overall, our approach can be widely used for the economical and industrial production of proteins in E. coli.

Structure of the Bacterial Sex F Pilus Reveals an Assembly of a Stoichiometric Protein-Phospholipid Complex.

Conjugative pili are widespread bacterial appendages that play important roles in horizontal gene transfer, in spread of antibiotic resistance genes, and as sites of phage attachment. Among conjugative pili, the F "sex" pilus encoded by the F plasmid is the best functionally characterized, and it is also historically the most important, as the discovery of F-plasmid-mediated conjugation ushered in the era of molecular biology and genetics. Yet, its structure is unknown. Here, we present atomic models of two F family pili, the F and pED208 pili, generated from cryoelectron microscopy reconstructions at 5.0 and 3.6 Å resolution, respectively. These structures reveal that conjugative pili are assemblies of stoichiometric protein-phospholipid units. We further demonstrate that each pilus type binds preferentially to particular phospholipids. These structures provide the molecular basis for F pilus assembly and also shed light on the remarkable properties of conjugative pili in bacterial secretion and phage infection.

Mechanistic basis of plasmid-specific DNA binding of the F plasmid regulatory protein, TraM.

The conjugative transfer of bacterial F plasmids relies on TraM, a plasmid-encoded protein that recognizes multiple DNA sites to recruit the plasmid to the conjugative pore. In spite of the high degree of amino acid sequence conservation between TraM proteins, many of these proteins have markedly different DNA binding specificities that ensure the selective recruitment of a plasmid to its cognate pore. Here we present the structure of F TraM RHH (ribbon-helix-helix) domain bound to its sbmA site. The structure indicates that a pair of TraM tetramers cooperatively binds an underwound sbmA site containing 12 base pairs per turn. The sbmA is composed of 4 copies of a 5-base-pair motif, each of which is recognized by an RHH domain. The structure reveals that a single conservative amino acid difference in the RHH ß-ribbon between F and pED208 TraM changes its specificity for its cognate 5-base-pair sequence motif. Specificity is also dictated by the positioning of 2-base-pair spacer elements within sbmA; in F sbmA, the spacers are positioned between motifs 1 and 2 and between motifs 3 and 4, whereas in pED208 sbmA, there is a single spacer between motifs 2 and 3. We also demonstrate that a pair of F TraM tetramers can cooperatively bind its sbmC site with an affinity similar to that of sbmA in spite of a lack of sequence similarity between these DNA elements. These results provide a basis for the prediction of the DNA binding properties of the family of TraM proteins.

Tracking F plasmid TraI relaxase processing reactions provides insight into F plasmid transfer.

Early in F plasmid conjugative transfer, the F relaxase, TraI, cleaves one plasmid strand at a site within the origin of transfer called nic. The reaction covalently links TraI Tyr16 to the 5'-ssDNA phosphate. Ultimately, TraI reverses the cleavage reaction to circularize the plasmid strand. The joining reaction requires a ssDNA 3'-hydroxyl; a second cleavage reaction at nic, regenerated by extension from the plasmid cleavage site, may generate this hydroxyl. Here we confirm that TraI is transported to the recipient during transfer. We track the secondary cleavage reaction and provide evidence it occurs in the donor and F ssDNA is transferred to the recipient with a free 3'-hydroxyl. Phe substitutions for four Tyr within the TraI active site implicate only Tyr16 in the two cleavage reactions required for transfer. Therefore, two TraI molecules are required for F plasmid transfer. Analysis of TraI translocation on various linear and circular ssDNA substrates supports the assertion that TraI slowly dissociates from the 3'-end of cleaved F plasmid, likely a characteristic essential for plasmid re-circularization.

X-ray crystal structure of the bacterial conjugation factor PsiB, a negative regulator of RecA.

During bacterial conjugation, genetic material from one cell is transferred to another as single-stranded DNA. The introduction of single-stranded DNA into the recipient cell would ordinarily trigger a potentially deleterious transcriptional response called SOS, which is initiated by RecA protein filaments formed on the DNA. During F plasmid conjugation, however, the SOS response is suppressed by PsiB, an F-plasmid-encoded protein that binds and sequesters free RecA to prevent filament formation. Among the many characterized RecA modulator proteins, PsiB is unique in using sequestration as an inhibitory mechanism. We describe the crystal structure of PsiB from the Escherichia coli F plasmid. The stucture of PsiB is surprisingly similar to CapZ, a eukaryotic actin filament capping protein. Structure-directed neutralization of electronegative surfaces on PsiB abrogates RecA inhibition whereas neutralization of an electropositive surface element enhances PsiB inhibition of RecA. Together, these studies provide a first molecular view of PsiB and highlight its use as a reagent in studies of RecA activity.

Driving forces of gyrase recognition by the addiction toxin CcdB.

Gyrase, an essential bacterial topoisomerase, is the target of several antibiotics (e.g. quinolones) as well as of bacterial toxin CcdB. This toxin, encoded by Escherichia coli toxin-antitoxin module ccd, poisons gyrase by causing inhibition of both transcription and replication. Because the molecular driving forces of gyrase unfolding and CcdB-gyrase binding were unknown, the nature of the CcdB-gyrase recognition remained elusive. Therefore, we performed a detailed thermodynamic analysis of CcdB binding to several fragments of gyrase A subunit (GyrA) that contain the CcdB-binding site. Binding of CcdB to the shorter fragments was studied directly by isothermal titration calorimetry. Its binding to the longer GyrA59 fragment in solution is kinetically limited and was therefore investigated via urea induced unfolding of the GyrA59-CcdB complex and unbound GyrA59 and CcdB, monitored by circular dichroism spectroscopy. Model analysis of experimental data, in combination with the relevant structural information, indicates that CcdB binding to gyrase is an enthalpic process driven mainly by specific interactions between CcdB and the highly stable dimerization domain of the GyrA. The dissection of binding energetics indicates that CcdB-gyrase recognition is accompanied by opening of the tower and catalytic domain of GyrA. Such extensive structural rearrangements appear to be crucial driving forces for the functioning of the ccd toxin-antitoxin module.

The structure of F-pili.

Exchange of DNA between bacteria involves conjugative pili. While the prevailing view has been that F-pili are completely retracted before single-stranded DNA is passed from one cell to another, it has recently been reported that the F-pilus, in addition to establishing the contact between mating cells, serves as a channel for passing DNA between spatially separated cells during conjugation. The structure and function of F-pili are poorly understood. They are built from a single subunit having only 70 residues, and the small size of the subunit has made these filaments difficult to study. Here, we have applied electron cryo-microscopy and single-particle methods to solve the long-existing ambiguity in the packing geometry of F-pilin subunits. We show that the F-pilus has an entirely different symmetry from any of the known bacterial pili as well as any of the filamentous bacteriophages, which have been suggested to be structural homologs. Two subunit packing schemes were identified: one has stacked rings of four subunits axially spaced by approximately 12.8 A, while the other has a one-start helical symmetry with an axial rise of approximately 3.5 A per subunit and a pitch of approximately 12.2 A. Both structures have a central lumen of approximately 30 A diameter that is more than large enough to allow for the passage of single-stranded DNA. Remarkably, both schemes appear to coexist within the same filaments, in contrast to filamentous phages that have been described as belonging to one of two possible symmetry classes. For the segments composed of rings, the twist between adjacent rings is quite variable, while the segments having a one-start helix are in multiple states of both twist and extension. This coexistence of two very different symmetries is similar to what has recently been reported for an archaeal Methanococcus maripaludis pili filament and an archaeal Sulfolobus shibatae flagellar filament.

Examination of an inverted repeat within the F factor origin of transfer: context dependence of F TraI relaxase DNA specificity.

Prior to conjugative transfer of plasmids, one plasmid strand is cleaved in a site- and strand-specific manner by an enzyme called a relaxase or nickase. In F and related plasmids, an inverted repeat is located near the plasmid strand cleavage site, and others have proposed that the ability of this sequence to form a hairpin when in single-stranded form is important for transfer. Substitutions were introduced into a cloned F oriT region and their effects on plasmid transfer were assessed. For those substitutions that substantially reduced transfer, the results generally correlated with effects on in vitro binding of oligonucleotides to the F TraI relaxase domain rather than with predicted effects on hairpin formation. One substitution shown previously to dramatically reduce both plasmid transfer and in vitro binding to a 17-base oligonucleotide had little apparent effect on binding to a 30-base oligonucleotide that contained the hairpin region. Results from subsequent experiments strongly suggest that the relaxase domain can bind to hairpin oligonucleotides in two distinct manners with different sequence specificities, and that the protein binds the oligonucleotides at the same or overlapping sites.

Inter- and intramolecular determinants of the specificity of single-stranded DNA binding and cleavage by the F factor relaxase.

The TraI protein of conjugative plasmid F factor binds and cleaves a single-stranded region of the plasmid prior to transfer to a recipient. TraI36, an N-terminal TraI fragment, binds ssDNA with a subnanomolar K(D) and remarkable sequence specificity. The structure of the TraI36 Y16F variant bound to ssDNA reveals specificity determinants, including a ssDNA intramolecular 3 base interaction and two pockets within the protein's binding cleft that accommodate bases in a knob-into-hole fashion. Mutagenesis results underscore the intricate design of the binding site, with the greatest effects resulting from substitutions for residues that both contact ssDNA and stabilize protein structure. The active site architecture suggests that the bound divalent cation, which is essential for catalysis, both positions the DNA by liganding two oxygens of the scissile phosphate and increases the partial positive charge on the phosphorus to enhance nucleophilic attack.

The 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different antibiotic-resistance determinants, two iron-acquisition systems and other putative virulence-associated functions.

The antibiotic-multiresistance IncF plasmid pRSB107 was isolated by a transformation-based approach from activated-sludge bacteria of a wastewater-treatment plant. It confers resistance to ampicillin, penicillin G, chloramphenicol, erythromycin, kanamycin, neomycin, streptomycin, sulfonamides, tetracycline and trimethoprim and against mercuric ions. Complete sequencing of this plasmid revealed that it is 120 592 bp in size and has a G+C content of 53.1 mol%. The plasmid backbone is composed of three replicons, RepFIA, RepFIB and RepFII, which are almost identical to corresponding regions located on the F-plasmid and on R100. The three replicons encode replication initiation (rep) and replication control, multimer resolution (mrs), post-segregational killing of plasmid-free cells (psk) and active plasmid partitioning (sopABC locus). Part of the F-leading region and remnants of the F-homologous DNA-transfer (tra) module complete the pRSB107 backbone. Plasmid pRSB107 contains a complex, highly mosaic 35 991 bp antibiotic-resistance region consisting of a Tn21- and a Tn10-derivative and a chloramphenicol-resistance module. The Tn21 derivative is composed of a mercury-resistance region (mer), a Tn4352B-like kanamycin/neomycin-resistance transposon, a streptomycin/sulfonamide-resistance module, remnants of the beta-lactam-resistance transposon Tn1, a macrolide-resistance module flanked by copies of IS26 and IS6100, remnants of Tn402 integrating a class 1 integron and the Tn21-specific transposition module. A truncated version of the tetracycline-resistance transposon Tn10 and the chloramphenicol acetyltransferase gene catA complete the pRSB107 resistance region. In addition to antibiotic resistance, pRSB107 encodes the following putative virulence-associated functions: (i) an aerobactin iron-acquisition siderophore system (iuc/iut); (ii) a putative high-affinity Fe(2+) uptake system which was previously identified on a pathogenicity island of Yersinia pestis and in the genome of the phytopathogen Erwinia carotovora subsp. atroseptica SCRI1043; (iii) an sn-glycerol-3-phosphate transport system (ugp); and (iv) the virulence-associated genes vagCD having a possible function in stable plasmid inheritance. All the accessory modules are framed by insertion sequences, indicating that pRSB107 was gradually assembled by integration of different horizontally acquired DNA segments via transposition or homologous recombination.

Thirty-eight C-terminal amino acids of the coupling protein TraD of the F-like conjugative resistance plasmid R1 are required and sufficient to confer binding to the substrate selector protein TraM.

Coupling proteins (CPs) are present in type IV secretion systems of plant, animal, and human pathogens and are essential for DNA transfer in bacterial conjugation systems. CPs connect the DNA-processing machinery to the mating pair-forming transfer apparatus. In this report we present in vitro and in vivo data that demonstrate specific binding of CP TraD of the IncFII R1 plasmid transfer system to relaxosomal protein TraM. With overlay assays and enzyme-linked immunosorbent assays we showed that a truncated version of TraD, termed TraD11 (DeltaN155), interacted strongly with TraM. The apparent TraD11-TraM association constant was determined to be 2.6 x 10(7) liters/mol. Electrophoretic mobility shift assays showed that this variant of TraD also strongly bound to TraM when it was in complex with its target DNA. When 38 amino acids were additionally removed from the C terminus of TraD, no binding to TraM was observed. TraD15, comprising the 38 amino-acid-long C terminus of TraD, bound to TraM, indicating that the main TraM interaction domain resides in these 38 amino acids of TraD. TraD15 exerted a dominant negative effect on DNA transfer but not on phage infection by pilus-specific phage R17, indicating that TraM-TraD interaction is important for conjugative DNA transfer but not for phage infection. We also observed that TraD encoded by the closely related F factor bound to TraM encoded by the R1 plasmid. Our results thus provide evidence that substrate selection within the IncF plasmid group is based on TraM's capability to select the correct DNA molecule for transport and not on substrate selection by the CP.

Energetics of the sequence-specific binding of single-stranded DNA by the F factor relaxase domain.

Transfer of conjugative plasmids between bacteria requires the activity of relaxases or mobilization proteins. These proteins nick the plasmid in a site- and strand-specific manner prior to transfer of the cut strand from donor to recipient. TraI36, the relaxase domain of TraI from plasmid F factor, binds a single-stranded DNA (ssDNA) oligonucleotide containing an F factor sequence with high affinity and sequence specificity. To better understand the energetics of this interaction, we examined the temperature, salt, and pH dependence of TraI36 recognition. Binding is entropically driven below 25 degrees C and enthalpically driven at higher temperatures. van't Hoff analysis yields an estimated deltaC(P)(0) of binding (-3300 cal x mol(-1) x K(-1)) that is larger and more negative than that observed for most double-stranded DNA (dsDNA)-binding proteins. Based on analyses of circular dichroism data and the crystal structure of the unliganded protein, we attribute the deltaC(P)(0) to both burial of hydrophobic surface area and coupled folding and binding of the protein. The salt dependence of the binding indicates that several ssDNA phosphates are buried in the complex, and the pH dependence of the binding suggests that some of these ssDNA phosphates form ionic interactions with basic residues of the protein. Although data are available for relatively few sequence-specific ssDNA-binding proteins, sufficient differences exist between TraI36 and other proteins to indicate that, like dsDNA-binding proteins, ssDNA-binding proteins use different motifs and combinations of forces to achieve specific recognition.

Reconstitution of F factor DNA replication in vitro with purified proteins.

Jacob, Brenner, and Cuzin pioneered the development of the F plasmid as a model system to study replication control, and these investigations led to the development of the "replicon model" (Jacob, F., Brenner, S., and Cuzin, F. (1964) Cold Spring Harbor Symp. Quant. Biol. 28, 329-348). To elucidate further the mechanism of initiation of replication of this plasmid and its control, we have reconstituted its replication in vitro with 21 purified host-encoded proteins and the plasmid-encoded initiator RepE. The replication in vitro was specifically initiated at the F ori (oriV) and required both the bacterial initiator protein DnaA and the plasmid-encoded initiator RepE. The wild type dimeric RepE was inactive in catalyzing replication, whereas a monomeric mutant form called RepE(*) (R118P) was capable of catalyzing vigorous replication. The replication topology was mostly of the Cairns form, and the fork movement was unidirectional and mostly from right to left. The replication was dependent on the HU protein, and the structurally and functionally related DNA bending protein IHF could not efficiently substitute for HU. The priming was dependent on DnaG primase. Many of the characteristics of the in vitro replication closely mimicked those of in vivo replication. We believe that the in vitro system should be very useful in unraveling the mechanism of replication initiation and its control.

A mob of reps.

Emerging structural results confirm that the large Rolling Circle Replication initiator superfamily is composed of two classes of proteins that are circularly permutated with respect to each other, as initially suggested by sequence analysis. The two classes are united by the same endonucleolytic mechanism and a conserved Mg(2+) binding site containing multiple histidine ligands unique to this superfamily.

Structural insights into single-stranded DNA binding and cleavage by F factor TraI.

Conjugative plasmid transfer between bacteria disseminates antibiotic resistance and diversifies prokaryotic genomes. Relaxases, proteins essential for conjugation, cleave one plasmid strand sequence specifically prior to transfer. Cleavage occurs through a Mg(2+)-dependent transesterification involving a tyrosyl hydroxyl and a DNA phosphate. The structure of the F plasmid TraI relaxase domain, described here, is a five-strand beta sheet flanked by alpha helices. The protein resembles replication initiator protein AAV-5 Rep but is circularly permuted, yielding a different topology. The beta sheet forms a binding cleft lined with neutral, nonaromatic residues, unlike most single-stranded DNA binding proteins which use aromatic and charged residues. The cleft contains depressions, suggesting base recognition occurs in a knob-into-hole fashion. Unlike most nucleases, three histidines but no acidic residues coordinate a Mg(2+) located near the catalytic tyrosine. The full positive charge on the Mg(2+) and the architecture of the active site suggest multiple roles for Mg(2+) in DNA cleavage.

Crystallization and preliminary X-ray characterization of the relaxase domain of F factor TraI.

Conjugative plasmids are capable of transferring a copy of themselves in single-stranded form from donor to recipient bacteria. Prior to transfer, one plasmid strand must be cleaved in a sequence-specific manner by a relaxase or mobilization protein. TraI is the relaxase for the conjugative plasmid F factor. A 36 kDa N-terminal fragment of TraI possesses the single-stranded DNA-binding and cleavage activity of the protein. Crystals of the 36 kDa TraI fragment in native and selenomethionine-labeled forms were grown by sitting-drop vapor-diffusion methods using PEG 1000 as the precipitant. Crystallization in the presence of chloride salts of magnesium and strontium was required to obtain crystals yielding high-resolution diffraction. To maintain high-resolution diffraction upon freezing, crystals had to be soaked in crystallization buffer with stepwise increases of ethylene glycol. The resulting crystals were trigonal and diffracted to a resolution of 3.1 A or better using synchrotron radiation.

Subdomain organization and catalytic residues of the F factor TraI relaxase domain.

TraI from conjugative plasmid F factor is both a "relaxase" that sequence-specifically binds and cleaves single-stranded DNA (ssDNA) and a helicase that unwinds the plasmid during transfer. Using limited proteolysis of a TraI fragment, we generated a 36-kDa fragment (TraI36) retaining TraI ssDNA binding specificity and relaxase activity but lacking the ssDNA-dependent ATPase activity of the helicase. Further proteolytic digestion of TraI36 generates stable N-terminal 26-kDa (TraI26) and C-terminal 7-kDa fragments. Both TraI36 and TraI26 are stably folded and unfold in a highly cooperative manner, but TraI26 lacks affinity for ssDNA. Mutational analysis of TraI36 indicates that N-terminal residues Tyr(16) and Tyr(17) are required for efficient ssDNA cleavage but not for high-affinity ssDNA binding. Although the TraI36 N-terminus provides the relaxase catalytic residues, both N- and C-terminal structural domains participate in binding, suggesting that both domains combine to form the TraI relaxase active site.

TraY DNA recognition of its two F factor binding sites.

F factor TraY, a ribbon-helix-helix DNA-binding protein, performs two roles in bacterial conjugation. TraY binds the F origin of transfer (oriT) to promote nicking of plasmid DNA prior to conjugative transfer. TraY also binds the P(Y) promoter to up-regulate tra gene expression. The two plasmid regions bound by TraY share limited sequence identity, yet TraY binds them with similar affinities. TraY recognition of the two sites was first probed using in vitro footprinting methods. Hydroxyl radical footprinting at both oriT and P(Y) sites indicated that bound TraY protected the DNA backbone bordering three adjacent DNA subsites. Analytical ultracentrifugation results for TraY:oligonucleotide complexes were consistent with two of these subsites being bound cooperatively, and the third being occupied at higher TraY concentrations. Methylation protection and interference footprinting identified several guanine bases contacted by or proximal to bound TraY, most located within these subsites. TraY affinity for variant oriT sequences with base substitutions at or near these guanine bases suggested that two of the three subsites correspond to high-affinity, cooperatively bound imperfect inverted GA(G/T)A repeats. Altering the spacing or orientation of these sites reduced binding. TraY mutant R73A failed to protect two symmetry-related oriT guanine bases in these repeats from methylation, identifying possible direct TraY-DNA contacts. The third subsite appears to be oriented as an imperfect direct repeat with its adjacent subsite, although base substitutions at this subsite did not reduce binding. Although unusual for ribbon-helix-helix proteins, this binding site arrangement occurs at both F TraY sites, consistent with it being functionally relevant.