HP1021 is a redox switch protein identified in Helicobacter pylori.

Helicobacter pylori is a gram-negative, microaerophilic, pathogenic bacterium and a widespread colonizer of humans. H. pylori has developed mechanisms that enable it to overcome the harsh environment of the human stomach, including reactive oxygen species (ROS). Interestingly, up to now no typical regulator dedicated to the oxidative-stress response has been discovered. In this work, we reveal that the inhibitor of replication initiation HP1021 functions as a redox switch protein in H. pylori and plays an important role in response to oxidative stress of the gastric pathogen. Each of the two predicted HP1021 domains contains three cysteine residues. We show that the cysteine residues of HP1021 are sensitive to oxidation both in vitro and in vivo, and we demonstrate that HP1021 DNA-binding activity to oriC depends on the redox state of the protein. Moreover, Zn2+ modulates HP1021 affinity towards oriC template DNA. Transcription analysis of selected H. pylori genes by RT-qPCR indicated that HP1021 is directly involved in the oxygen-dependent control of H. pylori fecA3 and gluP genes, which are implicated in response to oxidative stress. In conclusion, HP1021 is a redox switch protein and could be a target for H. pylori control strategies.

The role of Helicobacter pylori DnaA domain I in orisome assembly on a bipartite origin of chromosome replication.

The main roles of the DnaA protein are to bind the origin of chromosome replication (oriC), to unwind DNA and to provide a hub for the step-wise assembly of a replisome. DnaA is composed of four domains, with each playing a distinct functional role in the orisome assembly. Out of the four domains, the role of domain I is the least understood and appears to be the most species-specific. To better characterise Helicobacter pylori DnaA domain I, we have constructed a series of DnaA variants and studied their interactions with H. pylori bipartite oriC. We show that domain I is responsible for the stabilisation and organisation of DnaA-oriC complexes and provides cooperativity in DnaA-DNA interactions. Domain I mediates cross-interactions between oriC subcomplexes, which indicates that domain I is important for long-distance DnaA interactions and is essential for orisosme assembly on bipartite origins. HobA, which interacts with domain I, increases the DnaA binding to bipartite oriC; however, it does not stimulate but rather inhibits DNA unwinding. This suggests that HobA helps DnaA to bind oriC, but an unknown factor triggers DNA unwinding. Together, our results indicate that domain I self-interaction is important for the DnaA assembly on bipartite H. pylori oriC.

Putative Cooperative ATP-DnaA Binding to Double-Stranded DnaA Box and Single-Stranded DnaA-Trio Motif upon Helicobacter pylori Replication Initiation Complex Assembly.

oriC is a region of the bacterial chromosome at which the initiator protein DnaA interacts with specific sequences, leading to DNA unwinding and the initiation of chromosome replication. The general architecture of oriCs is universal; however, the structure of oriC and the mode of orisome assembly differ in distantly related bacteria. In this work, we characterized oriC of Helicobacter pylori, which consists of two DnaA box clusters and a DNA unwinding element (DUE); the latter can be subdivided into a GC-rich region, a DnaA-trio and an AT-rich region. We show that the DnaA-trio submodule is crucial for DNA unwinding, possibly because it enables proper DnaA oligomerization on ssDNA. However, we also observed the reverse effect: DNA unwinding, enabling subsequent DnaA-ssDNA oligomer formation-stabilized DnaA binding to box ts1. This suggests the interplay between DnaA binding to ssDNA and dsDNA upon DNA unwinding. Further investigation of the ts1 DnaA box revealed that this box, together with the newly identified c-ATP DnaA box in oriC1, constitute a new class of ATP-DnaA boxes. Indeed, in vitro ATP-DnaA unwinds H. pylori oriC more efficiently than ADP-DnaA. Our results expand the understanding of H. pylori orisome formation, indicating another regulatory pathway of H. pylori orisome assembly.

Structural Insights into New Bi(III) Coordination Polymers with Pyridine-2,3-Dicarboxylic Acid: Photoluminescence Properties and Anti-Helicobacter pylori Activity.

Two novel coordination polymers, [Bi2(2,3pydc)2(2,3pydcH)2(H2O)]n (1) and {(Et3NH)2[Bi(2,3pydc)(2,3pydcH)Cl2]}n (2) were prepared using as a prolinker pyridine-2,3-dicarboxylic acid (2,3pydcH2). The obtained complexes were fully characterized by elemental analysis, TG/DTG, FT-IR, solid-state photoluminescence, DFT calculations and single-crystal X-ray diffraction. The obtained complexes crystallized in the triclinic P-1 space group (1) and comprise dimeric units with two crystallographically different Bi(III) centers (polyhedra: distorted pentagonal bipyramid and bicapped trigonal prism) and monoclinic P21/c space group (2) with a distorted monocapped pentagonal bipyramid of Bi(III) center. The various coordination modes of bridging carboxylate ligands are responsible for the formation of 1D chains with 4,5C10 (1) and 2C1 (2) topology. The photoluminescence quantum yield for polymer 2 is 8.36%, which makes it a good candidate for more specific studies towards Bi-based fluorescent materials. Moreover, it was detected that polymer 1 is more than twice as active against H. pylori as polymer 2. It can be concluded that there is an existing relationship between the structure and the antibacterial activity because the presence of chloride and triethylammonium ions in the structure of complex 2 reduces the antibacterial activity.

Streptomycete origin of chromosomal replication with two putative unwinding elements.

DNA replication is controlled mostly at the initiation step. In bacteria, replication of the chromosome starts at a single origin of replication called oriC. The initiator protein, DnaA, binds to specific sequences (DnaA boxes) within oriC and assembles into a filament that promotes DNA double helix opening within the DNA unwinding element (DUE). This process has been thoroughly examined in model bacteria, including Escherichia coli and Bacillus subtilis, but we have a relatively limited understanding of chromosomal replication initiation in other species. Here, we reveal new details of DNA replication initiation in Streptomyces, a group of Gram-positive soil bacteria that possesses a long linear (8-10 Mbps) and GC-rich chromosome with a centrally positioned oriC. We used comprehensive in silico, in vitro and in vivo analyses to better characterize the structure of Streptomyces oriC. We identified 14 DnaA-binding motifs and determined the consensus sequence of the DnaA box. Unexpectedly, our in silico analysis using the WebSIDD algorithm revealed the presence of two putative Streptomyces DUEs (DUE1 and DUE2) located very near one another toward the 5' end of the oriC region. In vitro P1 nuclease assay revealed that DNA unwinding occurs at both of the proposed sites, but using an in vivo replication initiation point mapping, we were able to confirm only one of them (DUE2). The previously observed transcriptional activity of the Streptomyces oriC region may help explain the current results. We speculate that transcription itself could modulate oriC activity in Streptomyces by determining whether DNA unwinding occurs at DUE1 or DUE2.

Chaperone activity of serine protease HtrA of Helicobacter pylori as a crucial survival factor under stress conditions.

BACKGROUND: Serine protease HtrA exhibits both proteolytic and chaperone activities, which are involved in cellular protein quality control. Moreover, HtrA is an important virulence factor in many pathogens including Helicobacter pylori, for which the crucial stage of infection is the cleavage of E-cadherin and other cell-to-cell junction proteins. METHODS: The in vitro study of H. pylori HtrA (HtrAHp) chaperone activity was carried out using light scattering assays and investigation of lysozyme protein aggregates. We produced H. pylori ∆htrA deletion and HtrAHp point mutants without proteolytic activity in strain N6 and investigated the survival of the bacteria under thermal, osmotic, acidic and general stress conditions as well as the presence of puromycin or metronidazole using serial dilution tests and disk diffusion method. The levels of cellular and secreted proteins were examined using biochemical fraction and Western blotting. We also studied the proteolytic activity of secreted HtrAHp using zymography and the enzymatic digestion of ß-casein. Finally, the consequences of E-cadherin cleavage were determined by immunofluorescence microscopy. RESULTS: We demonstrate that HtrAHp displays chaperone activity that inhibits the aggregation of lysozyme and is stable under various pH and temperature conditions. Next, we could show that N6 expressing only HtrA chaperone activity grow well under thermal, pH and osmotic stress conditions, and in the presence of puromycin or metronidazole. In contrast, in the absence of the entire htrA gene the bacterium was more sensitive to a number of stresses. Analysing the level of cellular and secreted proteins, we noted that H. pylori lacking the proteolytic activity of HtrA display reduced levels of secreted HtrA. Moreover, we compared the amounts of secreted HtrA from several clinical H. pylori strains and digestion of ß-casein. We also demonstrated a significant effect of the HtrAHp variants during infection of human epithelial cells and for E-cadherin cleavage. CONCLUSION: Here we identified the chaperone activity of the HtrAHp protein and have proven that this activity is important and sufficient for the survival of H. pylori under multiple stress conditions. We also pinpointed the importance of HtrAHp chaperone activity for E- cadherin degradation and therefore for the virulence of this eminent pathogen.

Genetic Diversity as Consequence of a Microaerobic and Neutrophilic Lifestyle.

As a neutrophilic bacterium, Helicobacter pylori is growth deficient under extreme acidic conditions. The gastric pathogen is equipped with an acid survival kit, regulating urease activity by a pH-gated urea channel, opening below pH 6.5. After overcoming acid stress, the bacterium's multiplication site is situated at the gastric mucosa with near neutral pH. The pathogen exhibits exceptional genetic variability, mainly due to its capability of natural transformation, termed competence. Using single cell analysis, we show here that competence is highly regulated in H. pylori. DNA uptake complex activity was reversibly shut down below pH 6.5. pH values above 6.5 opened a competence window, in which competence development was triggered by the combination of pH increase and oxidative stress. In contrast, addition of sublethal concentrations of the DNA-damaging agents ciprofloxacin or mitomycin C did not trigger competence development under our conditions. An oxygen-sensitive mutant lacking superoxide dismutase (sodB) displayed a higher competent fraction of cells than the wild type under comparable conditions. In addition, the sodB mutant was dependent on adenine for growth in broth and turned into non-cultivable coccoid forms in its absence, indicating that adenine had radical quenching capacity. Quantification of periplasmically located DNA in competent wild type cells revealed outstanding median imported DNA amounts of around 350 kb per cell within 10 min of import, with maximally a chromosomal equivalent (1.6 Mb) in individual cells, far exceeding previous amounts detected in other Gram-negative bacteria. We conclude that the pathogen's high genetic diversity is a consequence of its enormous DNA uptake capacity, triggered by intrinsic and extrinsic oxidative stress once a neutral pH at the site of chronic host colonization allows competence development.

Recent Advances in Helicobacter pylori Replication: Possible Implications in Adaptation to a Pathogenic Lifestyle and Perspectives for Drug Design.

DNA replication is an important step in the life cycle of every cell that ensures the continuous flow of genetic information from one generation to the next. In all organisms, chromosome replication must be coordinated with overall cell growth. Helicobacter pylori growth strongly depends on its interaction with the host, particularly with the gastric epithelium. Moreover, H. pylori actively searches for an optimal microniche within a stomach, and it has been shown that not every microniche equally supports growth of this bacterium. We postulate that besides nutrients, H. pylori senses different, unknown signals, which presumably also affect chromosome replication to maintain H. pylori propagation at optimal ratio allowing H. pylori to establish a chronic, lifelong infection. Thus, H. pylori chromosome replication and particularly the regulation of this process might be considered important for bacterial pathogenesis. Here, we summarize our current knowledge of chromosome and plasmid replication in H. pylori and discuss the mechanisms responsible for regulating this key cellular process. The results of extensive studies conducted thus far allow us to propose common and unique traits in H. pylori chromosome replication. Interestingly, the repertoire of proteins involved in replication in H. pylori is significantly different to that in E. coli, strongly suggesting that novel factors are engaged in H. pylori chromosome replication and could represent attractive drug targets.

The atypical response regulator HP1021 controls formation of the Helicobacter pylori replication initiation complex.

The replication of a bacterial chromosome is initiated by the DnaA protein, which binds to the specific chromosomal region oriC and unwinds duplex DNA within the DNA-unwinding element (DUE). The initiation is tightly regulated by many factors, which control either DnaA or oriC activity and ensure that the chromosome is duplicated only when the conditions favor the survival of daughter cells. The factors controlling oriC activity often belong to the protein families of two-component systems. Here, we found that Helicobacter pylori oriC activity is controlled by HP1021, a member of the atypical response regulator family. HP1021 protein specifically interacts with H. pylori oriC at HP1021 boxes (5'-TGTT[TA]C[TA]-3'), which overlap with three modules important for oriC function: DnaA boxes, the hypersensitivity (hs) region and the DUE. Consequently, HP1021 binding to oriC precludes DnaA-oriC interactions and inhibits DNA unwinding at the DUE. Thus, HP1021 constitutes a negative regulator of the H. pylori orisome assembly in vitro. Furthermore, HP1021 boxes were found upstream of at least 70 genes, including those encoding CagA and Fur proteins. We postulate that HP1021 might coordinate chromosome replication, and thus bacterial growth, with other cellular processes and conditions in the human stomach.

Helicobacter pylori oriC--the first bipartite origin of chromosome replication in Gram-negative bacteria.

Binding of the DnaA protein to oriC leads to DNA melting within the DNA unwinding element (DUE) and initiates replication of the bacterial chromosome. Helicobacter pylori oriC was previously identified as a region localized upstream of dnaA and containing a cluster of DnaA boxes bound by DnaA protein with a high affinity. However, no unwinding within the oriC sequence has been detected. Comprehensive in silico analysis presented in this work allowed us to identify an additional region (oriC2), separated from the original one (oriC1) by the dnaA gene. DnaA specifically binds both regions, but DnaA-dependent DNA unwinding occurs only within oriC2. Surprisingly, oriC2 is bound exclusively as supercoiled DNA, which directly shows the importance of the DNA topology in DnaA-oriC interactions, similarly as previously presented only for initiator-origin interactions in Archaea and some Eukaryota. We conclude that H. pylori oriC exhibits bipartite structure, being the first such origin discovered in a Gram-negative bacterium. The H. pylori mode of initiator-oriC interactions, with the loop formation between the subcomplexes of the discontinuous origin, resembles those discovered in Bacillus subtilis chromosome and in many plasmids, which might suggest a similar way of controlling initiation of replication.

Profiling of the Helicobacter pylori redox switch HP1021 regulon using a multi-omics approach.

The gastric human pathogen Helicobacter pylori has developed mechanisms to combat stress factors, including reactive oxygen species (ROS). Here, we present a comprehensive study on the redox switch protein HP1021 regulon combining transcriptomic, proteomic and DNA-protein interactions analyses. Our results indicate that HP1021 modulates H. pylori's response to oxidative stress. HP1021 controls the transcription of 497 genes, including 407 genes related to response to oxidative stress. 79 proteins are differently expressed in the HP1021 deletion mutant. HP1021 controls typical ROS response pathways (katA, rocF) and less canonical ones, particularly DNA uptake and central carbohydrate metabolism. HP1021 is a molecular regulator of competence in H. pylori, as HP1021-dependent repression of the comB DNA uptake genes is relieved under oxidative conditions, increasing natural competence. Furthermore, HP1021 controls glucose consumption by directly regulating the gluP transporter and has an important impact on maintaining the energetic balance in the cell.

The structure of a DnaA/HobA complex from Helicobacter pylori provides insight into regulation of DNA replication in bacteria.

Bacterial DNA replication requires DnaA, an AAA+ ATPase that initiates replication at a specific chromosome region, oriC, and is regulated by species-specific regulators that directly bind DnaA. HobA is a DnaA binding protein, recently identified as an essential regulator of DNA replication in Helicobacter pylori. We report the crystal structure of HobA in complex with domains I and II of DnaA (DnaA(I-II)) from H. pylori, the first structure of DnaA bound to one of its regulators. Biochemical characterization of the complex formed shows that a tetramer of HobA binds four DnaA(I-II) molecules, and that DnaA(I-II) is unable to oligomerize by itself. Mutagenesis and protein-protein interaction studies demonstrate that some of the residues located at the HobA-DnaA(I-II) interface in the structure are necessary for complex formation. Introduction of selected mutations into H. pylori shows that the disruption of the interaction between HobA and DnaA is lethal for the bacteria. Remarkably, the DnaA binding site of HobA is conserved in DiaA from Escherichia coli, suggesting that the structure of the HobA/DnaA complex represents a model for DnaA regulation in other Gram-negative bacteria. Our data, together with those from other studies, indicate that HobA could play a crucial scaffolding role during the initiation of replication in H. pylori by organizing the first step of DnaA oligomerization and attachment to oriC.

Challenging the "gold standard" of colony-forming units - Validation of a multiplex real-time PCR for quantification of viable Campylobacter spp. in meat rinses.

Campylobacter jejuni is the leading bacterial food-borne pathogen in Europe. Despite the accepted limits of cultural detection of the fastidious bacterium, the "gold standard" in food microbiology is still the determination of colony-forming units (CFU). As an alternative, a live/dead differentiating qPCR has been established, using propidium monoazide (PMA) as DNA-intercalating crosslink agent for inactivating DNA from dead, membrane-compromised cells. The PMA treatment was combined with the addition of an internal sample process control (ISPC), i.e. a known number of dead C. sputorum cells to the samples. The ISPC enables i), monitoring the effective reduction of dead cell signal by the light-activated DNA-intercalating dye PMA, and ii), compensation for potential DNA losses during processing. Here, we optimized the method for routine application and performed a full validation of the method according to ISO 16140-2:2016(E) for the quantification of live thermophilic Campylobacter spp. in meat rinses against the classical enumeration method ISO 10272-2:2017. In order to render the method applicable and cost-effective for practical application, the ISPC was lyophilized to be distributable to routine laboratories. In addition, a triplex qPCR was established to simultaneously quantify thermophilic Campylobacter, the ISPC and an internal amplification control (IAC). Its performance was statistically similar to the two duplex qPCRs up to a contamination level of 4.7 log10Campylobacter per ml of meat rinse. The limit of quantification (LOQ) of the alternative method was around 20 genomic equivalents per PCR reaction, i.e. 2.3 log10 live Campylobacter per ml of sample. The alternative method passed a relative trueness study, confirming the robustness against different meat rinses, and displayed sufficient accuracy within the limits set in ISO 16140-2:2016(E). Finally, the method was validated in an interlaboratory ring trial, confirming that the alternative method was fit for purpose with a tendency of improved repeatability and reproducibility compared to the reference method for CFU determination. Campylobacter served as a model organism, challenging CFU as "gold standard" and could help in guidance to the general acceptance of live/dead differentiating qPCR methods for the detection of food-borne pathogens.

Regulation of the initiation of chromosomal replication in bacteria.

The initiation of chromosomal replication occurs only once during the cell cycle in both prokaryotes and eukaryotes. Initiation of chromosome replication is the first and tightly controlled step of a DNA synthesis. Bacterial chromosome replication is initiated at a single origin, oriC, by the initiator protein DnaA, which specifically interacts with 9-bp non-palindromic sequences (DnaA boxes) at oriC. In Escherichia coli, a model organism used to study the mechanism of DNA replication and its regulation, the control of initiation relies on a reduction of the availability and/or activity of the two key elements, DnaA and the oriC region. This review summarizes recent research into the regulatory mechanisms of the initiation of chromosomal replication in bacteria, with emphasis on organisms other than E. coli.

Establishment of serine protease htrA mutants in Helicobacter pylori is associated with secA mutations.

Helicobacter pylori plays an essential role in the pathogenesis of gastritis, peptic ulcer disease, and gastric cancer. The serine protease HtrA, an important secreted virulence factor, disrupts the gastric epithelium, which enables H. pylori to transmigrate across the epithelium and inject the oncogenic CagA protein into host cells. The function of periplasmic HtrA for the H. pylori cell is unknown, mainly due to unavailability of the htrA mutants. In fact, htrA has been described as an essential gene in this bacterium. We have screened 100 worldwide H. pylori isolates and show that only in the N6 strain it was possible to delete htrA or mutate the htrA gene to produce proteolytically inactive HtrA. We have sequenced the wild-type and mutant chromosomes and we found that inactivation of htrA is associated with mutations in SecA - a component of the Sec translocon apparatus used to translocate proteins from the cytoplasm into the periplasm. The cooperation of SecA and HtrA has been already suggested in Streptococcus pneumonia, in which these two proteins co-localize. Hence, our results pinpointing a potential functional relationship between HtrA and the Sec translocon in H. pylori possibly indicate for the more general mechanism responsible to maintain bacterial periplasmic homeostasis.

Properties of the HtrA Protease From Bacterium Helicobacter pylori Whose Activity Is Indispensable for Growth Under Stress Conditions.

The protease high temperature requirement A from the gastric pathogen Helicobacter pylori (HtrA Hp ) belongs to the well conserved family of serine proteases. HtrA Hp is an important secreted virulence factor involved in the disruption of tight and adherens junctions during infection. Very little is known about the function of HtrA Hp in the H. pylori cell physiology due to the lack of htrA knockout strains. Here, using a newly constructed ΔhtrA mutant strain, we found that bacteria deprived of HtrA Hp showed increased sensitivity to certain types of stress, including elevated temperature, pH and osmotic shock, as well as treatment with puromycin. These data indicate that HtrA Hp plays a protective role in the H. pylori cell, presumably associated with maintenance of important periplasmic and outer membrane proteins. Purified HtrA Hp was shown to be very tolerant to a wide range of temperature and pH values. Remarkably, the protein exhibited a very high thermal stability with the melting point (Tm) values of above 85°C. Moreover, HtrA Hp showed the capability to regain its active structure following treatment under denaturing conditions. Taken together, our work demonstrates that HtrA Hp is well adapted to operate under harsh conditions as an exported virulence factor, but also inside the bacterial cell as an important component of the protein quality control system in the stressed cellular envelope.

Structure and Function of the Campylobacter jejuni Chromosome Replication Origin.

Campylobacter jejuni is the leading bacterial cause of foodborne infections worldwide. However, our understanding of its cell cycle is poor. We identified the probable C. jejuni origin of replication (oriC) - a key element for initiation of chromosome replication, which is also important for chromosome structure, maintenance and dynamics. The herein characterized C. jejuni oriC is monopartite and contains (i) the DnaA box cluster, (ii) the DnaA-dependent DNA unwinding element (DUE) and (iii) binding sites for regulatory proteins. The cluster of five DnaA boxes and the DUE were found in the dnaA-dnaN intergenic region. Binding of DnaA to this cluster of DnaA-boxes enabled unwinding of the DUE in vitro. However, it was not sufficient to sustain replication of minichromosomes, unless the cluster was extended by additional DnaA boxes located in the 3' end of dnaA. This suggests, that C. jejuni oriC requires these boxes to initiate or to regulate replication of its chromosome. However, further detailed mutagenesis is required to confirm the role of these two boxes in initiation of C. jejuni chromosome replication and thus to confirm partial localization of C. jejuni oriC within a coding region, which has not been reported thus far for any bacterial oriC. In vitro DUE unwinding by DnaA was inhibited by Cj1509, an orphan response regulator and a homolog of HP1021, that has been previously shown to inhibit replication in Helicobacter pylori. Thus, Cj1509 might play a similar role as a regulator of C. jejuni chromosome replication. This is the first systematic analysis of chromosome replication initiation in C. jejuni, and we expect that these studies will provide a basis for future research examining the structure and dynamics of the C. jejuni chromosome, which will be crucial for understanding the pathogens' life cycle and virulence.

The Role of the N-Terminal Domains of Bacterial Initiator DnaA in the Assembly and Regulation of the Bacterial Replication Initiation Complex.

The primary role of the bacterial protein DnaA is to initiate chromosomal replication. The DnaA protein binds to DNA at the origin of chromosomal replication (oriC) and assembles into a filament that unwinds double-stranded DNA. Through interaction with various other proteins, DnaA also controls the frequency and/or timing of chromosomal replication at the initiation step. Escherichia coli DnaA also recruits DnaB helicase, which is present in unwound single-stranded DNA and in turn recruits other protein machinery for replication. Additionally, DnaA regulates the expression of certain genes in E. coli and a few other species. Acting as a multifunctional factor, DnaA is composed of four domains that have distinct, mutually dependent roles. For example, C-terminal domain IV interacts with double-stranded DnaA boxes. Domain III drives ATP-dependent oligomerization, allowing the protein to form a filament that unwinds DNA and subsequently binds to and stabilizes single-stranded DNA in the initial replication bubble; this domain also interacts with multiple proteins that control oligomerization. Domain II constitutes a flexible linker between C-terminal domains III-IV and N-terminal domain I, which mediates intermolecular interactions between DnaA and binds to other proteins that affect DnaA activity and/or formation of the initiation complex. Of these four domains, the role of the N-terminus (domains I-II) in the assembly of the initiation complex is the least understood and appears to be the most species-dependent region of the protein. Thus, in this review, we focus on the function of the N-terminus of DnaA in orisome formation and the regulation of its activity in the initiation complex in different bacteria.

Architecture of bacterial replication initiation complexes: orisomes from four unrelated bacteria.

Bacterial chromosome replication is mediated by single initiator protein, DnaA, that interacts specifically with multiple DnaA boxes located within the origin (oriC). We compared the architecture of the DnaA-origin complexes of evolutionarily distantly related eubacteria: two Gram-negative organisms, Escherichia coli and Helicobacter pylori, and two Gram-positive organisms, Mycobacterium tuberculosis and Streptomyces coelicolor. Their origins vary in size (from approx. 200 to 1000 bp) and number of DnaA boxes (from 5 to 19). The results indicate that: (i) different DnaA proteins exhibit various affinities toward single DnaA boxes, (ii) spatial arrangement of two DnaA boxes is crucial for the H. pylori and S. coelicolor DnaA proteins, but not for E. coli and M. tuberculosis proteins, and (iii) the oriC regions are optimally adjusted to their cognate DnaA proteins. The primary functions of multiple DnaA boxes are to determine the positioning and order of assembly of the DnaA molecules. Gradual transition from the sequence-specific binding of the DnaA protein to binding through co-operative protein-protein interactions seems to be a common conserved strategy to generate oligomeric initiator complexes bound to multiple sites within the chromosomal, plasmid and virial origins.