IHF-binding Site Research Articles

Bending is required for activation of dsz operon by the TetR family protein (DszGR).

The dsz operon responsible for the biodesulfurization of organosulfurs is under the control of a 385bp long promoter. Recently, a TetR family protein was identified which served as an activator of operon. Here we report that the TetR family protein (WP_058249973.1), named DszGR can specifically activate the dsz operon. Direct binding of the DszGR to DNA was observed at single molecule level by AFM. It was found that the binding of DszGR to the promoter DNA induces a bend by about∼40-50° degrees which may not be enough for the activation of the promoter. Thus, bendability in the promoter sequence was analyzed. The results show that the promoter has a curvature at around -235 and -200bp with respect to dszA start codon. On mutating this region, a decrease in activity of the promoter was observed. Our results suggest that the DszGR protein binds to the upstream sequences and induces a bend, which is facilitated by further bending of the DNA which is required for dsz promoter activity. IHF binding site present in the promoter, and a significant reduction in desulphurization activity in the absence of either IHF subunits, suggested role of IHF in regulation of the dsz operon.

The DnaA Cycle in Escherichia coli: Activation, Function and Inactivation of the Initiator Protein.

This review summarizes the mechanisms of the initiator protein DnaA in replication initiation and its regulation in Escherichia coli. The chromosomal origin (oriC) DNA is unwound by the replication initiation complex to allow loading of DnaB helicases and replisome formation. The initiation complex consists of the DnaA protein, DnaA-initiator-associating protein DiaA, integration host factor (IHF), and oriC, which contains a duplex-unwinding element (DUE) and a DnaA-oligomerization region (DOR) containing DnaA-binding sites (DnaA boxes) and a single IHF-binding site that induces sharp DNA bending. DiaA binds to DnaA and stimulates DnaA assembly at the DOR. DnaA binds tightly to ATP and ADP. ATP-DnaA constructs functionally different sub-complexes at DOR, and the DUE-proximal DnaA sub-complex contains IHF and promotes DUE unwinding. The first part of this review presents the structures and mechanisms of oriC-DnaA complexes involved in the regulation of replication initiation. During the cell cycle, the level of ATP-DnaA level, the active form for initiation, is strictly regulated by multiple systems, resulting in timely replication initiation. After initiation, regulatory inactivation of DnaA (RIDA) intervenes to reduce ATP-DnaA level by hydrolyzing the DnaA-bound ATP to ADP to yield ADP-DnaA, the inactive form. RIDA involves the binding of the DNA polymerase clamp on newly synthesized DNA to the DnaA-inactivator Hda protein. In datA-dependent DnaA-ATP hydrolysis (DDAH), binding of IHF at the chromosomal locus datA, which contains a cluster of DnaA boxes, results in further hydrolysis of DnaA-bound ATP. SeqA protein inhibits untimely initiation at oriC by binding to newly synthesized oriC DNA and represses dnaA transcription in a cell cycle dependent manner. To reinitiate DNA replication, ADP-DnaA forms oligomers at DnaA-reactivating sequences (DARS1 and DARS2), resulting in the dissociation of ADP and the release of nucleotide-free apo-DnaA, which then binds ATP to regenerate ATP-DnaA. In vivo, DARS2 plays an important role in this process and its activation is regulated by timely binding of IHF to DARS2 in the cell cycle. Chromosomal locations of DARS sites are optimized for the strict regulation for timely replication initiation. The last part of this review describes how DDAH and DARS regulate DnaA activity.

Emergent Properties in Complex Synthetic Bacterial Promoters.

Regulation of gene expression in bacteria results from the interplay between hundreds of transcriptional factors (TFs) at target promoters. However, how the arrangement of binding sites for TFs generates the regulatory logic of promoters is not well-known. Here, we generated and fully characterized a library of synthetic complex promoters for the global regulators, CRP and IHF, in Escherichia coli, which are formed by a weak -35/-10 consensus sequence preceded by four combinatorial binding sites for these two TFs. Using this approach, we found that while cis-elements for CRP preferentially activate promoters when located immediately upstream of the promoter consensus, binding sites for IHF mainly function as "UP" elements and stimulate transcription in several different architectures in the absence of this protein. However, the combination of CRP- and IHF-binding sites resulted in emergent properties in these complex promoters, where the activity of combinatorial promoters cannot be predicted from the individual behavior of its components. Taken together, the results presented here add to the information on architecture-logic of complex promoters in bacteria.

Regulatory dynamics in the ternary DnaA complex for initiation of chromosomal replication in Escherichia coli.

In Escherichia coli, the level of the ATP–DnaA initiator is increased temporarily at the time of replication initiation. The replication origin, oriC, contains a duplex-unwinding element (DUE) flanking a DnaA-oligomerization region (DOR), which includes twelve DnaA-binding sites (DnaA boxes) and the DNA-bending protein IHF-binding site (IBS). Although complexes of IHF and ATP–DnaA assembly on the DOR unwind the DUE, the configuration of the crucial nucleoprotein complexes remains elusive. To resolve this, we analyzed individual DnaA protomers in the complex and here demonstrate that the DUE–DnaA-box-R1–IBS–DnaA-box-R5M region is essential for DUE unwinding. R5M-bound ATP–DnaA predominantly promotes ATP–DnaA assembly on the DUE-proximal DOR, and R1-bound DnaA has a supporting role. This mechanism might support timely assembly of ATP–DnaA on oriC. DnaA protomers bound to R1 and R5M directly bind to the unwound DUE strand, which is crucial in replication initiation. Data from in vivo experiments support these results. We propose that the DnaA assembly on the IHF-bent DOR directly binds to the unwound DUE strand, and timely formation of this ternary complex regulates replication initiation. Structural features of oriC support the idea that these mechanisms for DUE unwinding are fundamentally conserved in various bacterial species including pathogens.

Initiation of DNA Replication at the Chromosomal Origin of E. coli, oriC.

The Escherichia coli chromosomal origin consists of a duplex-unwinding region and a region bearing a DNA-bending protein, IHF-binding site, and clusters of binding sites for the initiator protein DnaA. ATP-DnaA molecules form highly organized oligomers in a process stimulated by DiaA, a DnaA-binding protein. The resultant ATP-DnaA complexes promote local unwinding of oriC with the aid of IHF, for which specific interaction of DnaA with the single-stranded DNA is crucial. DnaA complexes also interact with DnaB helicases bound to DnaC loaders, promoting loading of DnaB onto the unwound DNA strands for bidirectional replication. Initiation of replication is strictly regulated during the cell cycle by multiple regulatory systems for oriC and DnaA. The activity of oriC is regulated by its methylation state, whereas that of DnaA depends on the form of the bound nucleotide. ATP-DnaA can be yielded from initiation-inactive ADP-DnaA in a timely manner depending on specific chromosomal DNA elements termed DARS (DnaA-reactivating sequences). After initiation, DnaA-bound ATP is hydrolyzed by two systems, yielding ADP-DnaA. In this review, these and other mechanisms of initiation and its regulation in E. coli are described.

Novel DNA packaging recognition in the unusual bacteriophage N15

Phage lambda׳s cosB packaging recognition site is tripartite, consisting of 3 TerS binding sites, called R sequences. TerS binding to the critical R3 site positions the TerL endonuclease for nicking cosN to generate cohesive ends. The N15 cos (cosN15) is closely related to cosλ, but whereas the cosBN15 subsite has R3, it lacks the R2 and R1 sites and the IHF binding site of cosBλ. A bioinformatic study of N15-like phages indicates that cosBN15 also has an accessory, remote rR2 site, which is proposed to increase packaging efficiency, like R2 and R1 of lambda. N15 plus five prophages all have the rR2 sequence, which is located in the TerS-encoding 1 gene, approximately 200bp distal to R3.An additional set of four highly related prophages, exemplified by Monarch, has R3 sequence, but also has R2 and R1 sequences characteristic of cosB–λ. The DNA binding domain of TerS-N15 is a dimer.

Transposon-mediated directed mutation controlled by DNA binding proteins in Escherichia coli.

Transposon-mediated directed mutation controlled by DNA binding proteins in Escherichia coli.

IHF is required for the transcriptional regulation of the Desulfovibrio vulgaris Hildenborough orp operons.

Transcriptional activation of σ54-dependent promoters is usually tightly regulated in response to environmental cues. The high abundance of potential σ54-dependent promoters in the anaerobe bacteria, Desulfovibrio vulgaris Hildenborough, reflects the high versatility of this bacteria suggesting that σ54 factor is the nexus of a large regulatory network. Understanding the key players of σ54-regulation in this organism is therefore essential to gain insights into the adaptation to anaerobiosis. Recently, the D. vulgaris orp genes, specifically found in anaerobe bacteria, have been shown to be transcribed by the RNA polymerase coupled to the σ54 alternative sigma factor. In this study, using in vitro binding experiments and in vivo reporter fusion assays in the Escherichia coli heterologous host, we showed that the expression of the divergent orp promoters is strongly dependent on the integration host factor IHF. Bioinformatic and mutational analysis coupled to reporter fusion activities and mobility shift assays identified two functional IHF binding site sequences located between the orp1 and orp2 promoters. We further determined that the D. vulgaris DVU0396 (IHFα) and DVU1864 (IHFβ) subunits are required to control the expression of the orp operons suggesting that they form a functionally active IHF heterodimer. Interestingly results obtained from the in vivo inactivation of DVU0396, which is required for orp operons transcription, suggest that several functionally IHF active homodimer or heterodimer are present in D. vulgaris.

Transcriptional Organization and Regulatory Elements of a Pseudomonas sp. Strain ADP Operon Encoding a LysR-Type Regulator and a Putative Solute Transport System

The atzS-atzT-atzU-atzV-atzW gene cluster of the Pseudomonas sp. strain ADP atrazine-degradative plasmid pADP-1, which carries genes for an outer membrane protein and the components of a putative ABC-type solute transporter, is located downstream from atzR, which encodes the LysR-type transcriptional regulator of the cyanuric acid-degradative operon atzDEF. Here we describe the transcriptional organization of these genes. Our results show that all six genes are cotranscribed from the PatzR promoter to form the atzRSTUVW operon. A second, stronger promoter, PatzT, is found within atzS and directs transcription of the four distal genes. PatzT is σ(N) dependent, activated by NtrC in response to nitrogen limitation with the aid of IHF, and repressed by AtzR. A combination of in vivo mutational analysis and primer extension allowed us to locate the PatzT promoter and map the transcriptional start site. Similarly, we used deletion and point mutation analyses, along with in vivo expression studies and in vitro binding assays, to locate the NtrC, IHF, and AtzR binding sites and address their functionality. Our results suggest a regulatory model in which NtrC activates PatzT transcription via DNA looping, while AtzR acts as an antiactivator that diminishes expression by interfering with the activation process.

Neisserial Correia repeat-enclosed elements do not influence the transcription of pil genes in Neisseria gonorrhoeae and Neisseria meningitidis.

Two human-specific neisserial pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, require the expression of type IV pili (tfp) for initial attachment to the host during infection. However, the mechanisms controlling the assembly and functionality of tfp are poorly understood. It is known that the gonococcal pilE gene, encoding the major subunit, is positively regulated by IHF, a multifunctional DNA binding protein. A neisserial specific repetitive DNA sequence, termed the Correia repeat-enclosed element (CREE) is situated upstream of three pil loci: pilHIJKX (pilH-X), pilGD, and pilF. CREEs have been shown to contain strong promoters, and some CREE variants contain a functional IHF binding site. CREEs might therefore be involved in the regulation of tfp biogenesis in pathogenic Neisseria. Site-directed and deletion mutagenesis on promoter::cat reporter constructs demonstrated that transcription of pilH-X and pilGD is from a σ(70) promoter and is independent of the CREE. The insertion of a CREE in the pilF promoter region in N. meningitidis generated a functional σ(70) promoter. However, there is also a functional promoter at this position in N. gonorrhoeae, where there is no CREE. These results suggest CREE insertion in these three pil loci does not influence transcription and that IHF does not coordinately regulate tfp biogenesis.

Construction of an Efficient In Vitro System for Analysis of Transcription from Sigma 54-Dependent pspA Promoter

IbsC is a small toxin protein in Escherichia coli, whose expression is repressed by a cis-acting small noncoding RNA, SibC (renamed from RygC or QUAD1c). Overexpression of IbsC or the absence of SibC transcription induces the expression of both psp operon (pspABCDE) and pspG gene encoding phage shock proteins, whose expression were known to be induced by multiple environment stresses or agents such as filamentous phage infection, osmotic shock, continued incubation in stationary phase, heat shock, and ethanol treatment. Although exogenous overexpression of IbsC leads to cell death, physiological roles of IbsC remain unknown especially because the strain without ibsC gene in the chromosome shows no growth defects. Considering that only Psp proteins are induced by expression of IbsC, not by other similar small toxin proteins such as LdrD, ShoB, and TisB, the role of IbsC might specifically be related to phage shock proteins. The psp operon is transcribed from pspA promoter by RNA polymerase holoenzyme containing the alternative stress-responsive sigma factor 54 (σ). Transcription from pspA promoter requires PspF as an activator protein that binds to the upstream region of the core promoter elements. Furthermore, PspA inhibits the σ-dependent pspA transcription by interacting with PspF and exerting its negative effects on transcription activation by PspF. Since the IbsC expression causes the induction of pspA transcription, it is attempting to see how the signal of IbsC gets transduced into the transcription activation. The mechanism involved in linking IbsC to pspA transcription is essential for understanding not only the physiological functions of IbsC, but also the regulation mechanism of transcription from σdependent promoters in response to environmental stresses. To understand the molecular mechanism of the pspA induction by IbsC, it is necessary to know which factors are involved in the activation of pspA transcription under conditions of IbsC expression. Although an in vitro transcription system for pspA promoter analysis was previously set up, it is difficult to identify in vitro transcripts because their transcription termination sites are unclear. To overcome the drawback of the previous in vitro transcription system, in this study, we constructed plasmid pPR56 containing a transcription fusion of pspA promoter and rnpB terminator by replacing the rnpB promoter-containing DNA fragment of pLMd23-wt with the pspA promoter-containing fragment. The pspA promoter-containing fragment spanning from −320 through +56 of pspA, which also includes two UAS sequences (UAS I and UAS II) as PspF-binding sites and the IHF binding site, was subcloned into the BamHI/ EcoRI linearlized pLMd23-wt not having the rnpB promotercontaining DNA fragment, to generate the fusion plasmid pPR56 (Fig. 1). The rnpB terminator region in the fusion construct contained the rnpB sequence from +331 to +1286, which includes the three rnpB terminators T1, T2, and T3 leading to transcription termination at +413, +526, and +638, respectively. Therefore, this pPR56 construct was designed to generate pspA-rnpB fusion transcripts of 146 nt terminating at T1 (pspAT1), 259 nt at T2 (pspAT2), and 371 nt at T3 (pspAT3) if transcription starts at the transcription initiation site of pspA. Using supercoiled plasmid pPR56 DNA as a template, in vitro transcription was carried out by adding Eσ and PspF. The pspAT1 transcript of 145 nt was produced as a major band although minor pspAT2 and pspAT3 products were also observed (Fig. 2). The increased abundance of three transcripts was observed with the incremental amount of PspF protein, while RNA I of 108 nt (a transcript transcribed from its own σ specific promoter of the plasmid DNA) was not produced. In contrast, the same transcription reaction performed with Eσ did not produce the pspAT transcripts. Instead, this reaction generated RNA I. All our data confirmed that the pspA transcripts were produced from the σ-specific pspA promoter of pPR56. To

Direct Interaction between Quorum-sensing Regulator SmcR and RNA Polymerase Is Mediated by Integration Host Factor to Activate vvpE Encoding Elastase in Vibrio vulnificus

It has been suggested that quorum sensing is an important signal transduction system regulating the expression of numerous virulence genes in bacterial pathogens. We previously revealed that SmcR, a LuxR homologue of Vibrio vulnificus, activates promoter S, an RpoS-dependent promoter of vvpE encoding a potential virulence factor elastase and binds in vitro to a binding site centered at -196.5. In this study, chromatin immunoprecipitation assays and promoter deletion analyses demonstrated that SmcR binds to the vvpE regulatory region in vivo and directly interacts with RNAP for activation of the vvpE expression. A search for regulatory genes involved in the regulation of elastase production singled out ihfA, which encodes for a subunit of integration host factor (IHF). Levels of both elastase activity and vvpE transcript decreased significantly as a result of inactivation of ihfA, and primer extension analyses demonstrated that IHF regulates the vvpE transcription by activating PS. Direct binding of IHF to the two distinct binding sites centered at -174 and -131, respectively, was determined using an electrophoretic mobility shift assay and a DNase I protection assay. Chromatin immunoprecipitation assays revealed that the interaction of SmcR with RNAP in vivo was mediated by IHF. Collectively, the results proposed a model whereby IHF positions SmcR to contact RNAP by looping the vvpE regulatory DNA, thus allowing precise control of the expression level of VvpE during the pathogenesis of V. vulnificus.

IHF-binding sites inhibit DNA loop formation and transcription initiation

Transcriptional activation of enhancer and σ54-dependent promoters requires efficient interactions between enhancer-binding proteins (EBP) and promoter bound σ54-RNA polymerase (Eσ54) achieved by DNA looping, which is usually facilitated by the integration host factor (IHF). Since the lengths of the intervening region supporting DNA-loop formation are similar among IHF-dependent and IHF-independent promoters, the precise reason(s) why IHF is selectively important for the frequency of transcription initiation remain unclear. Here, using kinetic cyclization and in vitro transcription assays we show that, in the absence of IHF protein, the DNA fragments containing an IHF-binding site have much less looping-formation ability than those that lack an IHF-binding site. Furthermore, when an IHF consensus-binding site was introduced into the intervening region between promoter and enhancer of the target DNA fragments, loop formation and DNA-loop-dependent transcriptional activation are significantly reduced in a position-independent manner. DNA-looping-independent transcriptional activation was unaffected. The binding of IHF to its consensus site in the target promoters clearly restored efficient DNA looping formation and looping-dependent transcriptional activation. Our data provide evidence that one function for the IHF protein is to release a communication block set by intrinsic properties of the IHF DNA-binding site.

Initiator titration complex formed at datA with the aid of IHF regulates replication timing in Escherichia coli

The initiation of replication in Escherichia coli is negatively controlled by a mechanism referred to as 'initiator titration', a process by which the initiator protein, DnaA, is titrated to newly replicated binding sequences on the chromosome to reduce the initiation potential for replication. Initiator titration occurs predominantly at the datA locus that binds exceptionally large amounts of DnaA molecules to prevent aberrant initiations. We found that this was enabled by integration host factor (IHF). Within datA, there is a consensus IHF recognition sequence between the two DnaA recognition sequences (DnaA boxes) essential for its function. Binding of IHF to this site was demonstrated both in vitro and in vivo. Disruption of the core sequence in the consensus of the IHF-binding resulted in increased origin concentration as observed in Delta datA cells. Furthermore, the number of DnaA molecules bound to datA was reduced in cells carrying a disruption in the IHF-binding core sequence. The IHF-binding site and the essential DnaA boxes had to be located at a proper distance and orientation to maintain the accurate initiation timing. Therefore, IHF is a unique element in the control of replication initiation that acts negatively at datA, while known to act as a positive regulator at oriC.

Insights into the architecture and stoichiometry of Escherichia coli PepA*DNA complexes involved in transcriptional control and site-specific DNA recombination by atomic force microscopy

Multifunctional Aminopeptidase A (PepA) from Escherichia coli is involved in the control of two distinct DNA transaction processes: transcriptional repression of the carAB operon, encoding carbamoyl phosphate synthase and site-specific resolution of ColE1-type plasmid multimers. Both processes require communication at a distance along a DNA molecule and PepA is the major structural component of the nucleoprotein complexes that underlie this communication. Atomic Force Microscopy was used to analyze the architecture of PepA·carAB and PepA·cer site complexes. Contour length measurements, bending angle analyses and volume determinations demonstrate that the carP1 operator is foreshortened by ∼235 bp through wrapping around one PepA hexamer. The highly deformed part of the operator extends from slightly upstream of the –35 hexamer of the carP1 promoter to just downstream of the IHF-binding site, and comprises the binding sites for the PurR and RutR transcriptional regulators. This extreme remodeling of the carP1 control region provides a straightforward explanation for the strict requirement of PepA in the establishment of pyrimidine and purine-specific repression of carAB transcription. We further provide a direct physical proof that PepA is able to synapse two cer sites in direct repeat in a large interwrapped nucleoprotein complex, likely comprising two PepA hexamers.

Loss of Hda activity stimulates replication initiation from I‐box, but not R4 mutant origins in Escherichia coli

Initiation of chromosome replication in Escherichia coli is limited by the initiator protein DnaA associated with ATP. Within the replication origin, binding sites for DnaA associated with ATP or ADP (R boxes) and the DnaA(ATP) specific sites (I-boxes, tau-boxes and 6-mer sites) are found. We analysed chromosome replication of cells carrying mutations in conserved regions of oriC. Cells carrying mutations in DnaA-boxes I2, I3, R2, R3 and R5 as well as FIS and IHF binding sites resembled wild-type cells with respect to origin concentration. Initiation of replication in these mutants occurred in synchrony or with slight asynchrony only. Furthermore, lack of Hda stimulated initiation in all these mutants. The DnaA(ATP) containing complex that leads to initiation can therefore be formed in the absence of several of the origin DnaA binding sites including both DnaA(ATP) specific I-boxes. However, competition between I-box mutant and wild-type origins, revealed a positive role of I-boxes on initiation. On the other hand, mutations affecting DnaA-box R4 were found to be compromised for initiation and could not be augmented by an increase in cellular DnaA(ATP)/DnaA(ADP) ratio. Compared with the sites tested here, R4 therefore seems to contribute to initiation most critically.

IHF- and SeqA-binding sites, present in plasmid cloning vectors, may significantly influence activities of promoters

Escherichia coli Integration Host Factor (IHF) regulates transcription from some bacterial and phage promoters by affecting DNA topology. Here we demonstrate that IHF affects transcription from bacteriophage λ pR promoter and the ptac promoter located on plasmids that contain IHF-binding sites. The IHF consensus sites are abundant and they can bind the IHF protein as shown in in vitro studies. The SeqA protein has a role in the complex regulation of pR activity, together with other factors altering DNA topology. Down-regulation of the transcription from ptac in the absence of IHF, together with IHF- and SeqA-mediated effects on pR, suggest that DNA topology cannot be underestimated when assessing in vivo promoters’ activity. This may have a significant impact on experiments employing recombinant genes cloned in plasmids and on choosing appropriate plasmid vectors.

The interplay of StyR and IHF regulates substrate-dependent induction and carbon catabolite repression of styrene catabolism genes in Pseudomonas fluorescens ST

BackgroundIn Pseudomonas fluorescens ST, the promoter of the styrene catabolic operon, PstyA, is induced by styrene and is subject to catabolite repression. PstyA regulation relies on the StyS/StyR two-component system and on the IHF global regulator. The phosphorylated response regulator StyR (StyR-P) activates PstyA in inducing conditions when it binds to the high-affinity site STY2, located about -40 bp from the transcription start point. A cis-acting element upstream of STY2, named URE, contains a low-affinity StyR-P binding site (STY1), overlapping the IHF binding site. Deletion of the URE led to a decrease of promoter activity in inducing conditions and to a partial release of catabolite repression. This study was undertaken to assess the relative role played by IHF and StyR-P on the URE, and to clarify if PstyA catabolite repression could rely on the interplay of these regulators.ResultsStyR-P and IHF compete for binding to the URE region. PstyA full activity in inducing conditions is achieved when StyR-P and IHF bind to site STY2 and to the URE, respectively. Under catabolite repression conditions, StyR-P binds the STY1 site, replacing IHF at the URE region. StyR-P bound to both STY1 and STY2 sites oligomerizes, likely promoting the formation of a DNA loop that closes the promoter in a repressed conformation. We found that StyR and IHF protein levels did not change in catabolite repression conditions, implying that PstyA repression is achieved through an increase in the StyR-P/StyR ratio.ConclusionWe propose a model according to which the activity of the PstyA promoter is determined by conformational changes. An open conformation is operative in inducing conditions when StyR-P is bound to STY2 site and IHF to the URE. Under catabolite repression conditions StyR-P cellular levels would increase, displacing IHF from the URE and closing the promoter in a repressed conformation. The balance between the open and the closed promoter conformation would determine a fine modulation of the promoter activity. Since StyR and IHF protein levels do not vary in the different conditions, the key-factor regulating PstyA catabolite repression is likely the kinase activity of the StyR-cognate sensor protein StyS.

New criteria for selecting the origin of DNA replication in Wolbachia and closely related bacteria

BackgroundThe annotated genomes of two closely related strains of the intracellular bacterium Wolbachia pipientis have been reported without the identifications of the putative origin of replication (ori). Identifying the ori of these bacteria and related alpha-Proteobacteria as well as their patterns of sequence evolution will aid studies of cell replication and cell density, as well as the potential genetic manipulation of these widespread intracellular bacteria.ResultsUsing features that have been previously experimentally verified in the alpha-Proteobacterium Caulobacter crescentus, the origin of DNA replication (ori) regions were identified in silico for Wolbachia strains and eleven other related bacteria belonging to Ehrlichia, Anaplasma, and Rickettsia genera. These features include DnaA-, CtrA- and IHF-binding sites as well as the flanking genes in C. crescentus. The Wolbachia ori boundary genes were found to be hemE and COG1253 protein (CBS domain protein). Comparisons of the putative ori region among related Wolbachia strains showed higher conservation of bases within binding sites.ConclusionThe sequences of the ori regions described here are only similar among closely related bacteria while fundamental characteristics like presence of DnaA and IHF binding sites as well as the boundary genes are more widely conserved. The relative paucity of CtrA binding sites in the ori regions, as well as the absence of key enzymes associated with DNA replication in the respective genomes, suggest that several of these obligate intracellular bacteria may have altered replication mechanisms. Based on these analyses, criteria are set forth for identifying the ori region in genome sequencing projects.

The F plasmid‐encoded TraM protein stimulates relaxosome‐mediated cleavage at oriT through an interaction with TraI

Conjugative DNA transfer is a highly conserved process for the direct transfer of DNA from a donor to a recipient. The conjugative initiator proteins are key players in the DNA processing reactions that initiate DNA transfer - they introduce a site- and strand-specific break in the DNA backbone via a transesterification that leaves the initiator protein covalently bound on the 5'-end of the cleaved DNA strand. The action of the initiator protein at the origin of transfer (oriT) is governed by auxiliary proteins that alter the architecture of the DNA molecule, allowing binding of the initiator protein. In the F plasmid system, two auxiliary proteins have roles in establishing the relaxosome: the host-encoded IHF and the plasmid-encoded TraY. Together, these proteins direct the loading of TraI which contains the catalytic centre for the transesterification. The F-oriT sequence includes a binding site for another plasmid-encoded protein, TraM, which is required for DNA transfer. Here the impact of TraM protein on the formation and activity of the F plasmid relaxosome has been examined. Purified TraM stimulates the formation of relaxed DNA in a reaction that requires the minimal components of the relaxosome, TraI, TraY and IHF. Unlike TraY and IHF, TraM is not essential for the formation of the relaxosome in vitro and TraM cannot substitute for either TraY or IHF in this process. The TraM binding site sbmC, along with both IHF binding sites, is essential for stimulation of the relaxase reaction. In addition, stimulation of transesterification appears to require the C-terminal domain of TraI suggesting that TraM and TraI may interact through this domain on TraI. Taken together, these results provide additional evidence of a role for TraM as a component of the relaxosome, suggest a previously unknown interaction between TraI and TraM, and allow us to propose a molecular role for the C-terminal domain of TraI.