Protein and DNA Effectors Control the TraI Conjugative Helicase of Plasmid R1

The mechanisms controlling progression of conjugative DNA processing from a preinitiation stage of specific plasmid strand cleavage at the transfer origin to a stage competent for unwinding the DNA strand destined for transfer remain obscure. Linear heteroduplex substrates containing double-stranded DNA binding sites for plasmid R1 relaxosome proteins and various regions of open duplex for TraI helicase loading were constructed to model putative intermediate structures in the initiation pathway. The activity of TraI was compared in steady-state multiple turnover experiments that measured the net production of unwound DNA as well as transesterase-catalyzed cleavage at nic. Helicase efficiency was enhanced by the relaxosome components TraM and integration host factor. The magnitude of stimulation depended on the proximity of the specific protein binding sites to the position of open DNA. The cytoplasmic domain of the R1 coupling protein, TraDΔN130, stimulated helicase efficiency on all substrates in a manner consistent with cooperative interaction and sequence-independent DNA binding. Variation in the position of duplex opening also revealed an unsuspected autoinhibition of the unwinding reaction catalyzed by full-length TraI. The activity reduction was sequence dependent and was not observed with a truncated helicase, TraIΔN308, lacking the site-specific DNA binding transesterase domain. Given that transesterase and helicase domains are physically tethered in the wild-type protein, this observation suggests that an intramolecular switch controls helicase activation. The data support a model where protein-protein and DNA ligand interactions at the coupling protein interface coordinate the transition initiating production and uptake of the nucleoprotein secretion substrate.Controlled duplex DNA unwinding is a crucial prerequisite for the expression and maintenance of genomes. Genome-manipulating and -regulating proteins are central to that biological function in recognizing appropriate DNA targets at initiation sequences and unwinding the complementary strands to provide single-stranded DNA (ssDNA) templates for nucleic acid synthesis and other processing reactions. The protein machineries involved include nucleic acid helicases. DNA helicases are powerful enzymes that convert the energy of nucleoside triphosphate hydrolysis to directional DNA strand translocation and separation of the double helix into its constituent single strands (for reviews, see references 13, 14, 16, 38, 55, and 64). By necessity, these enzymes interact with DNA strands via mechanisms independent of sequence recognition. At replication initiation helicases gain controlled access to the double-stranded genome at positions determined by the DNA binding properties of initiator proteins that comprise an origin recognition complex (1, 9, 17, 31, 45, 66). The mechanisms supporting localized unwinding within the complex include initiator-induced DNA looping, wrapping, and bending and feature regions of low thermodynamic stability. The exposed ssDNA mediates helicase binding followed by directional translocation along that strand until the enzyme engages the duplex for unwinding.In the MOB_F family of conjugation systems, the plasmid DNA strand destined for transfer (T strand) is unwound from its complement by a dedicated conjugative helicase, TraI of F-like plasmids or TrwC of the IncW paradigm. These enzymes are remarkable in that the same polypeptides additionally harbor in a distinct domain a DNA transesterase activity. That function is required to recognize and cleave the precise phosphodiester bond, nic, in the T strand where unwinding of the secretion substrate begins. In current models the conjugative helicases are thus targeted to the transfer origin (oriT) of their cognate plasmid by the high-affinity DNA sequence interactions of their N-terminal DNA transesterase domains. In the bacterial cell, recruitment and activation of the conjugative helicase occur not on naked DNA but within an initiator complex called the relaxosome (67). For the F-like plasmid R1, sequence-specific DNA binding properties of the plasmid proteins TraI, TraY, TraM, and the host integration factor (IHF) direct assembly of the relaxosome at oriT (10, 12, 29, 33, 51, 52). Integration of protein TraM confers recognition features to the relaxosome, which permit its selective docking to TraD, the coupling protein associated with the conjugative type IV secretion system (T4CP) (2, 15, 49). In current models, the T4CP forms a hexameric translocation pore at the cytoplasmic membrane that not only governs substrate entry to the envelope spanning type IV secretion machinery but also provides energy for macromolecular transport via ATP hydrolysis (36, 50). These models propose that T4CPs provide not only a physical bridge between the plasmid and the type IV transporter but also a unique control function in distinguishing one plasmid (relaxosome) from another (7, 8). Before the current study (see accompanying report [41]), evidence indicating that regulation of the initiation of conjugative DNA processing also takes place at this interface had not been reported.F plasmid TraI protein, originally named Escherichia coli DNA helicase I, was initially characterized in the Hoffman-Berling laboratory (19). The purified enzyme exhibits properties in vitro consistent with its function in conjugative DNA strand transfer including a very high 1,100-bp/s rate of duplex unwinding, high processivity, and a 5′-to-3′ directional bias (relative to the strand to which it is bound) (34, 54). Together these features should readily support the observed rate of conjugative DNA translocation as well as concomitant replacement synthesis of the mobilized T strand from the 3′ OH product of nic cleavage.Comparatively little is known about the mechanisms of initiating TraI helicase activity. The enzyme requires ssDNA 5′ to the duplex junction (32), and a minimum length of 30 nucleotides (nt) is necessary to promote efficient duplex unwinding on substrates lacking oriT (11, 54). To our knowledge, oriT is the only sequence where the helicase activity is naturally initiated, however. Moreover, the unique fusion of a helicase to the site- and strand-specific DNA transesterase domains within MOB_F enzymes is expected to pose intriguing regulatory challenges during initiation. The combination within a single polypeptide of a site-specific DNA binding capacity with a helical motor activity would seem counterproductive. The extraordinary efficiency of these proteins in intercellular DNA strand transfer belies this prediction and instead hints strongly at a coordinated progression of the initiation pathway. Since relaxosome assembly is thus far insufficient to initiate helicase activity on supercoiled oriT substrates in vitro, we have developed a series of heteroduplex DNA substrates which support the unwinding reaction and model possible intermediate structures of R1 plasmid strand transfer initiation (10). In this system linear double-stranded DNA (dsDNA) substrates with a central region of sequence heterogeneity trap defined lengths of R1 oriT sequence in unwound conformation. Unexpectedly, efficient helicase activity initiated from a melted oriT duplex required ssDNA twice as long (60 nt) as that previously observed on substrates lacking this sequence (11).In the current report, we describe an application of these models where variation in the position of duplex opening in the vicinity of nic, as well as the additional presence of auxiliary relaxosome proteins, has revealed novel insights into control of a conjugative helicase involving both DNA and protein interactions. Moreover, we observe a sequence-independent stimulation of the unwinding reaction in the presence of T4CP TraD. These results support a model where docking of the preinitiation relaxosome assembly to the T4CP alters the composition and architecture of the complex in a manner essential to the subsequent initiation of T-strand unwinding.     

DNA sequence elements located immediately upstream of the -10 hexamer in Escherichia coli promoters: a systematic study

We have made a systematic study of how the activity of an Escherichia coli promoter is affected by the base sequence immediately upstream of the –10 hexamer. Starting with an activator-independent promoter, with a 17 bp spacing between the –10 and –35 hexamer elements, we constructed derivatives with all possible combinations of bases at positions –15 and –14. Promoter activity is greatest when the ‘non-template’ strand carries T and G at positions –15 and –14, respectively. Promoter activity can be further enhanced by a second T and G at positions –17 and –16, respectively, immediately upstream of the first ‘TG motif’. Our results show that the base sequence of the DNA segment upstream of the –10 hexamer can make a significant contribution to promoter strength. Using published collections of characterised E.coli promoters, we have studied the frequency of occurrence of ‘TG motifs’ upstream of the promoters’ –10 elements. We conclude that correctly placed ‘TG motifs’ are found at over 20% of E.coli promoters.     

High-throughput,cost-effective verification of structural DNA assembly

DNA ‘assembly’ from ‘building blocks’ remains a cornerstone in synthetic biology, whether it be for gene synthesis (∼1 kb), pathway engineering (∼10 kb) or synthetic genomes (>100 kb). Despite numerous advances in the techniques used for DNA assembly, verification of the assembly is still a necessity, which becomes cost-prohibitive and a logistical challenge with increasing scale. Here we describe for the first time a comprehensive, high-throughput solution for structural DNA assembly verification by restriction digest using exhaustive in silico enzyme screening, rolling circle amplification of plasmid DNA, capillary electrophoresis and automated digest pattern recognition. This low-cost and robust methodology has been successfully used to screen over 31 000 clones of DNA constructs at <$1 per sample.     

SYMBIOSIS: synthetic manipulable biobricks via orthogonal serine integrase systems

Serine integrases are emerging as one of the most powerful biological tools for synthetic biology. They have been widely used across genome engineering and genetic circuit design. However, developing serine integrase-based tools for directly/precisely manipulating synthetic biobricks is still missing. Here, we report SYMBIOSIS, a versatile method that can robustly manipulate DNA parts in vivo and in vitro. First, we propose a ‘keys match locks’ model to demonstrate that three orthogonal serine integrases are able to irreversibly and stably switch on seven synthetic biobricks with high accuracy in vivo. Then, we demonstrate that purified integrases can facilitate the assembly of ‘donor’ and ‘acceptor’ plasmids in vitro to construct composite plasmids. Finally, we use SYMBIOSIS to assemble different chromoprotein genes and create novel colored Escherichia coli. We anticipate that our SYMBIOSIS strategy will accelerate synthetic biobrick manipulation, genetic circuit design and multiple plasmid assembly for synthetic biology with broad potential applications.     

Evolving origin-of-transfer sequences on staphylococcal conjugative and mobilizable plasmids—who’s mimicking whom?

In Staphylococcus aureus, most multiresistance plasmids lack conjugation or mobilization genes for horizontal transfer. However, most are mobilizable due to carriage of origin-of-transfer (oriT) sequences mimicking those of conjugative plasmids related to pWBG749. pWBG749-family plasmids have diverged to carry five distinct oriT subtypes and non-conjugative plasmids have been identified that contain mimics of each. The relaxasome accessory factor SmpO, encoded by each conjugative plasmid, determines specificity for its cognate oriT. Here we characterized the binding of SmpO proteins to each oriT. SmpO proteins predominantly formed tetramers in solution and bound 5′-GNNNNC-3′ sites within each oriT. Four of the five SmpO proteins specifically bound their cognate oriT. An F7K substitution in pWBG749 SmpO switched oriT-binding specificity in vitro. In vivo, the F7K substitution reduced but did not abolish self-transfer of pWBG749. Notably, the substitution broadened the oriT subtypes that were mobilized. Thus, this substitution represents a potential evolutionary intermediate with promiscuous DNA-binding specificity that could facilitate a switch between oriT specificities. Phylogenetic analysis suggests pWBG749-family plasmids have switched oriT specificity more than once during evolution. We hypothesize the convergent evolution of oriT specificity in distinct branches of the pWBG749-family phylogeny reflects indirect selection pressure to mobilize plasmids carrying non-cognate oriT-mimics.     

Single-Stranded DNA Binding by F TraI Relaxase and Helicase Domains Is Coordinately Regulated

Transfer of conjugative plasmids requires relaxases, proteins that cleave one plasmid strand sequence specifically. The F plasmid relaxase TraI (1,756 amino acids) is also a highly processive DNA helicase. The TraI relaxase activity is located within the N-terminal ∼300 amino acids, while helicase motifs are located in the region comprising positions 990 to 1450. For efficient F transfer, the two activities must be physically linked. The two TraI activities are likely used in different stages of transfer; how the protein regulates the transition between activities is unknown. We examined TraI helicase single-stranded DNA (ssDNA) recognition to complement previous explorations of relaxase ssDNA binding. Here, we show that TraI helicase-associated ssDNA binding is independent of and located N-terminal to all helicase motifs. The helicase-associated site binds ssDNA oligonucleotides with nM-range equilibrium dissociation constants and some sequence specificity. Significantly, we observe an apparent strong negative cooperativity in ssDNA binding between relaxase and helicase-associated sites. We examined three TraI variants having 31-amino-acid insertions in or near the helicase-associated ssDNA binding site. B. A. Traxler and colleagues (J. Bacteriol. 188:6346-6353) showed that under certain conditions, these variants are released from a form of negative regulation, allowing them to facilitate transfer more efficiently than wild-type TraI. We find that these variants display both moderately reduced affinity for ssDNA by their helicase-associated binding sites and a significant reduction in the apparent negative cooperativity of binding, relative to wild-type TraI. These results suggest that the apparent negative cooperativity of binding to the two ssDNA binding sites of TraI serves a major regulatory function in F transfer.Transfer of conjugative plasmids between bacteria contributes to genome diversification and acquisition of new traits. Conjugative plasmids encode most proteins required for transfer of one plasmid strand from the donor to the recipient cell (reviewed in references 11, 24, and 43). In preparation for transfer, a complex of proteins assembles at the plasmid origin of transfer (oriT). Within this complex, called the relaxosome, a plasmid-encoded relaxase or nickase binds and cleaves one plasmid strand at a specific oriT site (nic). As part of the cleavage reaction, the relaxase forms a covalent linkage between an active-site tyrosyl hydroxyl oxygen and a single-stranded DNA (ssDNA) phosphate, yielding a 3′ ssDNA hydroxyl (19, 30). Upon initiation of transfer, the plasmid strands are separated, and the cut strand is transported into the recipient. The relaxase is likely transferred into the recipient (12, 31) while still physically attached to plasmid DNA. The transferred relaxase may then join the ends of the ssDNA plasmid copy in the final step of plasmid transfer. Complementary strand synthesis in the donor and the recipient generates a double-stranded plasmid that is competent for further transfer. Successful conjugation requires effective temporal regulation, yet the mechanisms governing this regulation are poorly understood.The F plasmid oriT is ∼500 bp long and includes multiple binding sites for integration host factor (IHF), TraY, and TraM and a single site for TraI, the F relaxase (11). IHF, TraY, and TraM, participants in the relaxosome, bind double-stranded DNA to facilitate the action of TraI, perhaps by creating or stabilizing the ssDNA conformation around nic required for TraI recognition. The F TraI minimal high-affinity binding site includes ∼15 nucleotides around nic (39), and throughout the text, we refer to oligonucleotides that contain the TraI wild-type (wt) or variant binding site as oriT oligonucleotides. F TraI is 192 kDa (42), and in addition to its relaxase activity, TraI has a 5′-to-3′ helicase activity (4). These activities must be physically joined to allow efficient plasmid transfer (29), yet how the two activities are coordinated is a mystery. The relaxase region of F TraI has been defined as the N-terminal ∼300 amino acids (aa) (6, 40). Conserved helicase motifs, including those associated with an ATPase, lie between amino acids 990 and 1450. The C-terminal region (positions 1450 to 1756) plays an important role in bacterial conjugation, possibly involving protein-protein interactions with TraM (32) and/or inner membrane protein TraD (28).The 70-kDa central region of TraI that lies between the relaxase and helicase domains has been implicated in two functions. Haft and colleagues described TraI variants with 31-amino-acid insertions in this TraI region that facilitated plasmid transfer with greater efficiency than that afforded by the wild-type protein when these proteins are expressed at high levels (16). On the basis of this observation, the authors proposed that the region participated in a negative regulation of transfer. Matson and Ragonese demonstrated that this central region is required for TraI helicase function, likely due to participation in ssDNA recognition essential for the helicase activity (28). We wondered whether the proposed regulatory and ssDNA binding roles of the central region are linked and whether this region might help modulate TraI helicase and relaxase activities. Our objectives in this study were to confirm the role of the central region in ssDNA recognition, to assess the affinity and specificity of the ssDNA recognition by the central region, and to determine whether the relaxase and central domain ssDNA binding sites demonstrate cooperativity in binding. Our work yielded two significant and surprising results. First, the binding site within the TraI central region binds ssDNA with high affinity and significant sequence specificity, both unusual characteristics for a helicase. Second, the central region and relaxase ssDNA binding sites show an apparent strong negative cooperativity of binding, possibly explaining the role of the central region as a negative regulator and providing clues about how the timing of conjugative transfer might be regulated.     

A functional interaction between the putative primosomal protein DnaI and the main replicative DNA helicase DnaB in Bacillus

In Gram negative Escherichia coli there are two well-characterised primosomal assembly processes, the PriA- and DnaA-mediated cascades. The presence of PriA and DnaA proteins in Gram positive Bacillus spp. supports the assumption that both the PriA- and DnaA-mediated primosomal assembly cascades also operate in these organisms. However, the lack of sequence homology between the rest of the primosomal proteins indicates significant differences between these two bacterial species. Central to the process of primosomal assembly is the loading of the main hexameric replicative helicase (DnaB in E.coli and DnaC in Bacillus subtilis) on the DNA. This loading is achieved by specialised proteins known as ‘helicase loaders’. In E.coli DnaT and DnaC are responsible for loading DnaB onto the DNA during primosome assembly, in the PriA- and DnaA-mediated cascades, respectively. In Bacillus the identity of the helicase loader is still not established unequivocally. In this paper we provide evidence for a functional interaction between the primosomal protein DnaI from B.subtilis and the main hexameric replicative helicase DnaB from Bacillus stearothermophilus. Our results are consistent with the putative role of DnaI as the ‘helicase loader’ in the Gram positive Bacillus spp.     

Conjugative plasmids: vessels of the communal gene pool

Comparative whole-genome analyses have demonstrated that horizontal gene transfer (HGT) provides a significant contribution to prokaryotic genome innovation. The evolution of specific prokaryotes is therefore tightly linked to the environment in which they live and the communal pool of genes available within that environment. Here we use the term supergenome to describe the set of all genes that a prokaryotic ‘individual’ can draw on within a particular environmental setting. Conjugative plasmids can be considered particularly successful entities within the communal pool, which have enabled HGT over large taxonomic distances. These plasmids are collections of discrete regions of genes that function as ‘backbone modules’ to undertake different aspects of overall plasmid maintenance and propagation. Conjugative plasmids often carry suites of ‘accessory elements’ that contribute adaptive traits to the hosts and, potentially, other resident prokaryotes within specific environmental niches. Insight into the evolution of plasmid modules therefore contributes to our knowledge of gene dissemination and evolution within prokaryotic communities. This communal pool provides the prokaryotes with an important mechanistic framework for obtaining adaptability and functional diversity that alleviates the need for large genomes of specialized ‘private genes’.     

Cloning and Identification of Conjugative Transfer Origins in the Rhizobium meliloti Genome

A simple approach was used to identify Rhizobium meliloti DNA regions with the ability to convert a nontransmissible vector into a mobilizable plasmid, i.e., to contain origins of conjugative transfer (oriT, mob). RecA-defective R. meliloti merodiploid populations, where each individual contained a hybrid cosmid from an R. meliloti GR4 gene library, were used as donors en masse in conjugation with another R. meliloti recipient strain, selecting transconjugants for vector-encoded antibiotic resistance. Restriction analysis of cosmids isolated from individual transconjugants resulted in the identification of 11 nonoverlapping DNA regions containing potential oriTs. Individual hybrid cosmids were confirmed to be mobilized from the original recA donors at frequencies ranging from 10⁻² to 10⁻⁵ per recipient cell. DNA hybridization experiments showed that seven mob DNA regions correspond to plasmid replicons: four on symbiotic megaplasmid 1 (pSym1), one on pSym2, and another two on each of the two cryptic plasmids harbored by R. meliloti GR4. Another three mob clones could not be located to any plasmid and were therefore preliminarily assigned to the chromosome. With this strategy, we were able to characterize the oriT of the conjugative plasmid pRmeGR4a, which confirmed the reliability of the approach to select for oriTs. Moreover, transfer of the 11 mob cosmids from R. meliloti into Escherichia coli occurred at frequencies as high as 10⁻¹, demonstrating the R. meliloti gene transfer capacity is not limited to the family Rhizobiaceae. Our results show that the R. meliloti genome contains multiple oriTs that allow efficient DNA mobilization to rhizobia as well as to phylogenetically distant gram-negative bacteria.     

Plastid Proteomic Analysis in Tomato Fruit Development

To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of ‘Micro-Tom’ fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared ‘Micro-Tom’ results with those from two other varieties, ‘Black’ and ‘White Beauty’. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of ‘Micro-Tom’, and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared ‘Micro-Tom’ fruits with ‘Black’ and ‘White Beauty’ using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein) and HrBP1 (harpin binding protein-1) in the ‘Black’ and ‘White Beauty’ varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin) in 2D-PAGE results, however the number of spots and their isoelectric points differed between ‘Micro-Tom’ and ‘Black’/‘White Beauty’. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast.     

The R1162 Mob Proteins Can Promote Conjugative Transfer from Cryptic Origins in the Bacterial Chromosome

The mobilization proteins of the broad-host-range plasmid R1162 can initiate conjugative transfer of a plasmid from a 19-bp locus that is partially degenerate in sequence. Such loci are likely to appear by chance in the bacterial chromosome and could act as cryptic sites for transfer of chromosomal DNA when R1162 is present. The R1162-dependent transfer of chromosomal DNA, initiated from one such potential site in Pectobacterium atrosepticum, is shown here. A second active site was identified in Escherichia coli, where it is also shown that large amounts of DNA are transferred. This transfer probably reflects the combined activity of the multiple cryptic origins in the chromosome. Transfer of chromosomal DNA due to the presence of a plasmid in the cytoplasm describes a previously unrecognized potential for the exchange of bacterial DNA.The discovery over 50 years ago of bacterial mating by Lederberg and Cavalli-Sforza (summarized by Cavalli-Sforza [10]) was a major step in the development of modern microbial genetics. It was later realized that chromosomal DNA transfer was due to the integration of a plasmid, the F factor. This plasmid is able to effect its own transfer and, in the integrated state, chromosomal DNA as well. A unique site on the plasmid, the origin of transfer (oriT), is essential for this process. A complex of plasmid- and host-encoded proteins assemble at oriT (15); the F-encoded relaxase then cleaves one of the DNA strands, forming a covalent, protein-DNA intermediate (27) that is delivered to the type IV secretion apparatus, also encoded by F (24). The possibility of low-level transfer initiating from chromosomal sites was raised early in the history of conjugation (12). However, it is now clear that the exacting architecture of the oriT DNA-protein complex, both for the F factor and related oriTs, results in a very high degree of sequence and structural specificity, and no secondary origins in the chromosome have been identified.The broad-host-range plasmid R1162 (RSF1010), like the F factor, is also transferred from cell to cell. Although the mechanism of transfer is similar for the two plasmids, the oriTs are structurally very different. In prior studies we have determined that the R1162 oriT is small, structurally simple, and able to accommodate base changes at different positions without a complete loss of function (5, 21). As a result of this relaxed specificity, the R1162 mobilization (Mob) proteins can activate the related but different oriT of another plasmid, pSC101 (28).The R1162 oriT is shown in Fig. ​Fig.1.1. The R1162 relaxase MobA interacts with the core region, highly conserved in the R1162 Mob family (5), and the adjacent, inner arm of the inverted repeat (23). The protein forms at nic a tyrosyl phosphodiester linkage with the 5′ end of the DNA strand (30), which is then unwound from its complement as the protein-DNA complex is passed into the recipient cell. Circular plasmid DNA is reformed by a reverse of the initial protein-DNA transesterification, with the relaxase now binding to the 3′ end of the transferred DNA. This binding requires the complete inverted repeat, which probably forms a hairpin loop to recreate the double-stranded character of the relaxase binding site on duplex DNA.Open in a separate windowFIG. 1.Base sequence of R1162 oriT on the nicked strand. The relaxase cleaves at nic; subsequent transfer is 5′ to 3′ so that most of oriT is transferred last. Below is the consensus sequence of DNA active for initiation of transfer.An important feature of the steps in DNA processing at the R1162 oriT is that an inverted repeat is not required for the initiation of transfer or passage of DNA into a new cell. As a result of this and the permissiveness of the relaxase to base changes within oriT, different sites in the chromosome that are not part of a plasmid oriT might nevertheless be capable of initiating transfer when cloned into a plasmid. We previously identified one such site in the chromosome of Erwinia carotovora subsp. atroseptica (now Pectobacterium atrosepticum) (21). In addition, by testing libraries of oriTs with one or more mutations for relaxase-induced nicking, we found that a large population of DNAs with different sequences was potentially active (21). The consensus sequence for activity, derived from these studies, is shown in Fig. ​Fig.11.I show here that ectopic sites on the bacterial chromosome, active for the initiation of transfer of plasmid DNA, can also serve for the transfer of chromosomal DNA when the Mob proteins are provided in trans. Thus, the presence of R1162 in the cell can result in a cryptic sexuality in bacteria, resulting in the transfer of chromosomal genes. Although such events are likely to be infrequent, the broad-host-range of R1162 and its close relatives, with their ability to reside stably in the cytoplasms of many different bacteria, could be another source of gene exchange for a variety of different species.     

Split-Cre Complementation Restores Combination Activity on Transgene Excision in Hair Roots of Transgenic Tobacco

The Cre/loxP system is increasingly exploited for genetic manipulation of DNA in vitro and in vivo. It was previously reported that inactive ‘‘split-Cre’’ fragments could restore Cre activity in transgenic mice when overlapping co-expression was controlled by two different promoters. In this study, we analyzed recombination activities of split-Cre proteins, and found that no recombinase activity was detected in the in vitro recombination reaction in which only the N-terminal domain (NCre) of split-Cre protein was expressed, whereas recombination activity was obtained when the C-terminal (CCre) or both NCre and CCre fragments were supplied. We have also determined the recombination efficiency of split-Cre proteins which were co-expressed in hair roots of transgenic tobacco. No Cre recombination event was observed in hair roots of transgenic tobacco when the NCre or CCre genes were expressed alone. In contrast, an efficient recombination event was found in transgenic hairy roots co-expressing both inactive split-Cre genes. Moreover, the restored recombination efficiency of split-Cre proteins fused with the nuclear localization sequence (NLS) was higher than that of intact Cre in transgenic lines. Thus, DNA recombination mediated by split-Cre proteins provides an alternative method for spatial and temporal regulation of gene expression in transgenic plants.     

NAR Breakthrough Article: Telomere-associated proteins add deoxynucleotides to terminal proteins during replication of the telomeres of linear chromosomes and plasmids in Streptomyces

Typical telomeres of linear chromosomes and plasmids of soil bacteria Streptomyces consist of tightly packed palindromic sequences with a terminal protein (‘TP’) covalently attached to the 5′ end of the DNA. Replication of these linear replicons is initiated internally and proceeds bidirectionally toward the telomeres, which leaves single-strand overhangs at the 3′ ends. These overhangs are filled by DNA synthesis using the TPs as the primers (‘end patching’). The gene encoding for typical TP, tpg, forms an operon with tap, encoding an essential telomere-associated protein, which binds TP and the secondary structures formed by the 3′ overhangs. Previously one of the two translesion synthesis DNA polymerases, DinB1 or DinB2, was proposed to catalyze the protein-primed synthesis. However, using an in vitro end-patching system, we discovered that Tpg and Tap alone could carry out the protein-primed synthesis to a length of 13 nt. Similarly, an ‘atypical’ terminal protein, Tpc, and its cognate telomere-associated protein, Tac, of SCP1 plasmid, were sufficient to achieve protein-primed synthesis in the absence of additional polymerase. These results indicate that these two telomere-associated proteins possess polymerase activities alone or in complex with the cognate TPs.     

Plasmid R1 Conjugative DNA Processing Is Regulated at the Coupling Protein Interface

Selective substrate uptake controls initiation of macromolecular secretion by type IV secretion systems in gram-negative bacteria. Type IV coupling proteins (T4CPs) are essential, but the molecular mechanisms governing substrate entry to the translocation pathway remain obscure. We report a biochemical approach to reconstitute a regulatory interface between the plasmid R1 T4CP and the nucleoprotein relaxosome dedicated to the initiation stage of plasmid DNA processing and substrate presentation. The predicted cytosolic domain of T4CP TraD was purified in a predominantly monomeric form, and potential regulatory effects of this protein on catalytic activities exhibited by the relaxosome during transfer initiation were analyzed in vitro. TraDΔN130 stimulated the TraI DNA transesterase activity apparently via interactions on both the protein and the DNA levels. TraM, a protein interaction partner of TraD, also increased DNA transesterase activity in vitro. The mechanism may involve altered DNA conformation as TraM induced underwinding of oriT plasmid DNA in vivo (ΔL_k = −4). Permanganate mapping of the positions of duplex melting due to relaxosome assembly with TraDΔN130 on supercoiled DNA in vitro confirmed localized unwinding at nic but ruled out formation of an open complex compatible with initiation of the TraI helicase activity. These data link relaxosome regulation to the T4CP and support the model that a committed step in the initiation of DNA export requires activation of TraI helicase loading or catalysis.Type IV secretion systems (T4SS) in gram-negative bacteria mediate translocation of macromolecules out of the bacterial cell (14). The transmission of effector proteins and DNA into plant cells or other bacteria via cell-cell contact is one example of their function, and conjugation systems as well as the transferred DNA (T-DNA) delivery system of the phytopathogen Agrobacterium tumefaciens are prototypical of the T4SS family. Macromolecular translocation is achieved by a membrane-spanning protein machinery comprised of 12 gene products, VirB1 to VirB11 and an associated factor known as the coupling protein (VirD4) (66). The T4SS-associated coupling protein (T4CP) performs a crucial function in recognition of appropriate secretion substrates and governing entry of those molecules to the translocation pathway (7, 8, 10, 30, 41). In conjugation systems substrate recognition is applied to the relaxosome, a nucleoprotein complex of DNA transfer initiator proteins assembled specifically at the plasmid origin of transfer (oriT). In current models, initiation of the reactions that provide the single strand of plasmid (T-strand) DNA for secretion to recipient bacteria is expected to resemble the initiation of chromosomal replication (for reviews, see references 18, 54, and 81). Controlled opening of the DNA duplex is required to permit entry of the DNA processing machinery. The task of remodeling the conjugative oriT is generally ascribed to two or three relaxosome auxiliary factors, of host and plasmid origin, which occupy specific DNA binding sites at this locus. Intrinsic to the relaxosome is also a site- and strand-specific DNA transesterase activity that breaks the phosphodiester backbone at nic (5). Upon cleavage, the transesterase enzyme (also called relaxase) forms a reversible phosphotyrosyl linkage to the 5′ end of the DNA. Duplex unwinding initiating from this site produces the single-stranded T strand to be exported. A wealth of information is available supporting the importance of DNA sequence recognition and binding by relaxosome components at oriT to the transesterase reaction in vitro and for effective conjugative transfer (for reviews, see references 18, 54, and 81). On the other hand, the mechanisms controlling release of the 3′-OH generated at nic and the subsequent DNA unwinding stage remain obscure.Equally little is known about the process of nucleoprotein uptake by the transport channel. DNA-independent translocation of the relaxases TrwC (R388), MobA (RSF1010), and VirD2 (Ti plasmid) has been demonstrated; thus, current models propose that the relaxase component of the protein-DNA adduct is the substrate actively secreted by the transport system after interaction with the T4CP (42, 66). Cotransport of the covalently linked single-stranded T strand occurs concurrently (42). The mechanisms underlying relaxosome recognition by T4CPs are not understood. Direct interactions have been observed biochemically between the RP4 TraG protein and relaxase proteins of the cognate plasmid (65) and heterologous relaxosomes that it mobilizes (73, 76). TrwB of R388 interacts in vitro with relaxase TrwC and an auxiliary component, TrwA (44). TraD proteins of plasmid R1 and F are known to interact with the auxiliary relaxosome protein TraM (20) via a cluster of C-terminal amino acids (3, 62). Extensive mutagenic analyses (45) plus recent three-dimensional structural data for a complex of the TraM tetramerization domain and the C-terminal tail of TraD (46) have provided more detailed models for the intermolecular contacts involved in recognition.Application of the Cre recombinase assay for translocation of conjugative relaxases as well as effector proteins to eukaryotic cells is currently the most promising approach to elucidate protein motifs recognized by T4CPs (56, 68, 78, 79). Despite that progress, the nature of the interactions between a T4CP and its target protein that initiate secretion and the mechanisms controlling this step remain obscure. In contrast to systems dedicated specifically to effector protein translocation, conjugation systems mobilize nucleoprotein complexes that additionally exhibit catalytic activities, which can be readily monitored. These models are therefore particularly well suited to investigate aspects of regulation occurring at the physical interface of a T4CP and its secretion substrate. For this purpose the MOB_F family of DNA-mobilizing systems is additionally advantageous, since DNA processing within this family features the fusion of a dedicated conjugative helicase to the DNA transesterase enzyme within a single bifunctional protein. The TraI protein of F-like plasmids, originally described as Escherichia coli DNA helicase I (1, 2, 23), and the related TrwC protein of plasmid R388 (25) are well characterized (reviewed in reference 18). Early work by Llosa et al. revealed a complex domain arrangement for TrwC (43). Similar analyses with TraI identified nonoverlapping transesterase and helicase domains (6, 77), while the remaining intermediate and C-terminal regions of the protein additionally provide functions essential to effective conjugative transfer (49, 71). The ability to physically separate the catalytic domains of TraI and TrwC has facilitated a detailed biochemical characterization of their DNA transesterase, ATPase, and DNA-unwinding reactions. Nonetheless, failure of the physically disjointed polypeptides to complement efficient conjugative transfer when coexpressed indicates a role(s) for these proteins in the strand transfer process that goes beyond the need for their dual catalytic activities (43, 50). The assignment of additional functional properties to regions within TraI is a focus of current investigation (16, 29, 49).In all systems studied thus far, conditions used to reconstitute relaxosomes on a supercoiled oriT plasmid have not supported the initiation steps necessary to enable duplex unwinding by a conjugative helicase. The question remains open whether additional protein components are required and/or whether the pathway of initiation is subject to specific repression. In the present study, we applied the IncFII plasmid R1 paradigm to investigate the potential for interaction between purified components of the relaxosome and its cognate T4CP, TraD, to exert regulatory effects on relaxosome activities in vitro. In this and in the accompanying report (72), we present evidence for wide-ranging stimulatory effects of the cytoplasmic domain of TraD protein and its interaction partner TraM on multiple aspects of relaxosome function.     

RNA as the stone guest of protein aggregation

The study of prions as infectious aggregates dates several decades. From its original formulation, the definition of a prion has progressively changed to the point that many aggregation-prone proteins are now considered bona fide prions. RNA molecules, not included in the original ‘protein-only hypothesis’, are also being recognized as important factors contributing to the ‘prion behaviour’, that implies the transmissibility of an aberrant fold. In particular, an association has recently emerged between aggregation and the assembly of prion-like proteins in RNA-rich complexes, associated with both physiological and pathological events. Here, we discuss the historical rising of the concept of prion-like domains, their relation to RNA and their role in protein aggregation. As a paradigmatic example, we present the case study of TDP-43, an RNA-binding prion-like protein associated with amyotrophic lateral sclerosis. Through this example, we demonstrate how the current definition of prions has incorporated quite different concepts making the meaning of the term richer and more stimulating. An important message that emerges from our analysis is the dual role of RNA in protein aggregation, making RNA, that has been considered for many years a ‘silent presence’ or the ‘stone guest’ of protein aggregation, an important component of the process.     

Exploitation of Plasmid pMRC01 To Direct Transfer of Mobilizable Plasmids into Commercial Lactococcal Starter Strains

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 × 10⁻⁴ transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01. All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the oriT-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that oriT of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.     

Mobilisation of the streptococcal plasmid pMV158: interactions of MobM protein with its cognate oriT DNA region

The streptococcal plasmid pMV158 encodes the relaxase protein, MobM, involved in its mobilisation. Purified MobM protein specifically cleaved supercoiled or single-stranded DNA containing the plasmid origin of transfer, oriT. Gel retardation and DNase I footprinting assays performed with DNA fragments containing the plasmid oriT provided evidence for specific binding of MobM by oriT DNA. Dissection of the MobM-binding sequence revealed that the oriT region protected by MobM spanned 28 nucleotides, and includes an inversely repeated sequence, termed IR2. MobM exhibits a high degree of similarity with the mob gene product of the Streptococcus ferus plasmid pVA380-1. Although the origins of transfer of pMV158 and pVA380-1 show 20% sequence divergence in a 24-bp sequence included in their oriT regions, the pMV158 MobM was able to cleave a supercoiled derivative of pVA380-1 in vitro.