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The bacterium Escherichia coli initiates replication once per cell cycle at a precise volume per origin and adds an on average constant volume between successive initiation events, independent of the initiation size. Yet, a molecular model that can explain these observations has been lacking. Experiments indicate that E. coli controls replication initiation via titration and activation of the initiator protein DnaA. In chapters 2, 3 and 4, we study by mathematical modelling how these two mechanisms interact to generate robust replication-initiation cycles. We first show that a mechanism solely based on titration generates stable replication cycles at low growth rates, but inevitably causes premature reinitiation events at higher growth rates. In this regime, the DnaA activation switch becomes essential for stable replication initiation. Conversely, while the activation switch alone yields robust rhythms at high growth rates, titration can strongly enhance the stability of the switch at low growth rates. Our analysis thus predicts that both mechanisms together drive robust replication cycles at all growth rates. In addition, it reveals how an origin-density sensor yields adder correlations. Initiating replication synchronously at multiple origins of replication allows the bacterium E. coli to divide even faster than the time it takes to replicate the entire chromosome in nutrient rich environments. What mechanisms give rise to synchronous replication initiation remains however poorly understood. In chapter 5, we identify four distinct synchronization regimes depending on two quantities: the duration of the so-called licensing period during which the initiation potential in the cell remains high after the first origin has fired and the duration of the blocking period during which already initiated origins remain blocked. For synchronous replication initiation, the licensing period must be long enough such that all origins can be initiated, but shorter than the blocking period to prevent reinitiation of origins that have already fired. We find an analytical expression for the degree of synchrony as a function of the duration of the licensing period, which we confirm by simulations. Our model reveals that the delay between the firing of the first and the last origin scales with the coefficient of variation (CV) of the initiation volume. Matching these to the values measured experimentally shows that the firing rate must rise with the cell volume with an effective Hill coefficient that is at least 20; the probability that all origins fire before the blocking period is over is then at least 94%. Our analysis thus reveals that the low CV of the initiation volume is a consequence of synchronous replication initiation. Finally, we show that the previously presented molecular model for the regulation of replication initiation in E. coli can give rise to synchronous replication initiation for biologically realistic parameters. Recent developments in synthetic biology may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. In chapter 6, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this chapter, we discuss potential paths towards an integrated synthetic cell cycle.

The bacterium Escherichia coli initiates replication once per cell cycle at a precise volume per origin and adds an on average constant volume between successive initiation events, independent of the initiation size. Yet, a molecular model that can explain these observations has been lacking. Experiments indicate that E. coli controls replication initiation via titration and activation of the initiator protein DnaA. In chapters 2, 3 and 4, we study by mathematical modelling how these two mechanisms interact to generate robust replication-initiation cycles. We first show that a mechanism solely based on titration generates stable replication cycles at low growth rates, but inevitably causes premature reinitiation events at higher growth rates. In this regime, the DnaA activation switch becomes essential for stable replication initiation. Conversely, while the activation switch alone yields robust rhythms at high growth rates, titration can strongly enhance the stability of the switch at low growth rates. Our analysis thus predicts that both mechanisms together drive robust replication cycles at all growth rates. In addition, it reveals how an origin-density sensor yields adder correlations. Initiating replication synchronously at multiple origins of replication allows the bacterium E. coli to divide even faster than the time it takes to replicate the entire chromosome in nutrient rich environments. What mechanisms give rise to synchronous replication initiation remains however poorly understood. In chapter 5, we identify four distinct synchronization regimes depending on two quantities: the duration of the so-called licensing period during which the initiation potential in the cell remains high after the first origin has fired and the duration of the blocking period during which already initiated origins remain blocked. For synchronous replication initiation, the licensing period must be long enough such that all origins can be initiated, but shorter than the blocking period to prevent reinitiation of origins that have already fired. We find an analytical expression for the degree of synchrony as a function of the duration of the licensing period, which we confirm by simulations. Our model reveals that the delay between the firing of the first and the last origin scales with the coefficient of variation (CV) of the initiation volume. Matching these to the values measured experimentally shows that the firing rate must rise with the cell volume with an effective Hill coefficient that is at least 20; the probability that all origins fire before the blocking period is over is then at least 94%. Our analysis thus reveals that the low CV of the initiation volume is a consequence of synchronous replication initiation. Finally, we show that the previously presented molecular model for the regulation of replication initiation in E. coli can give rise to synchronous replication initiation for biologically realistic parameters. Recent developments in synthetic biology may bring the bottom-up generation of a synthetic cell within reach. A key feature of a living synthetic cell is a functional cell cycle, in which DNA replication and segregation as well as cell growth and division are well integrated. In chapter 6, we describe different approaches to recreate these processes in a synthetic cell, based on natural systems and/or synthetic alternatives. Although some individual machineries have recently been established, their integration and control in a synthetic cell cycle remain to be addressed. In this chapter, we discuss potential paths towards an integrated synthetic cell cycle.

Shigella sonnei is one of the major causes of diarrhea and remained a critical microbe responsible for higher morbidity and mortality rates resulting from dysentery every year across the world. Antibiotic therapy of Shigella diseases plays a critical role in decreasing the prevalence as well as the fatality rate of this infection. However, the management of these diseases remains challenging, owing to the overall increase in resistance against many antimicrobials. The situation necessitates the rapid development of effective and feasible S. sonnei treatments. In the present study, the subtractive genomics approach was utilized to find the potential drug targets for S. sonnei strain Ss046. Various tools of bioinformatics were implemented to remove the human-specific homologous and pathogen-specific paralogous sequences from the bacterial proteome. Then, metabolic pathway and subcellular location analysis were performed of essential bacterial proteins to describe their role in various cellular processes. Only one essential protein i-e Chromosomal replication initiator protein DnaA was found in the proteome of the pathogen that could be used as a potent target for designing new drugs. 3D structure prediction of DnaA protein was carried out using Phyre 2. Molecular docking of 5000 phytochemicals was performed against DnaA to identify four top-ranked phytochemicals (Riccionidin A, Dothistromin, Fustin, and Morin) based on scoring functions and interaction with the active site. This study suggests that these phytochemicals could be used as antibacterial drugs to treat S. sonnei infections in the future. To confirm their efficacy and evaluate their drug potency, further in vitro analyses are required.

Bacterial genomes harbour cryptic prophages that are mostly transcriptionally silent with many unannotated genes. Still, cryptic prophages may contribute to their host fitness and phenotypes. In Bacillus subtilis, the yqaF-yqaN operon belongs to the prophage element skin, and is tightly repressed by the Xre-like repressor SknR. This operon contains several small ORFs (smORFs) potentially encoding small-sized proteins. The smORF-encoded peptide YqaH was previously reported to bind to the replication initiator DnaA. Here, using a yeast two-hybrid assay, we found that YqaH binds to the DNA binding domain IV of DnaA and interacts with Spo0A, a master regulator of sporulation. We isolated single amino acid substitutions in YqaH that abolished the interaction with DnaA but not with Spo0A. Then, using a plasmid-based inducible system to overexpress yqaH WT and mutant derivatives, we studied in B. subtilis the phenotypes associated with the specific loss-of-interaction with DnaA (DnaA_LOI). We found that expression of yqaH carrying DnaA_LOI mutations abolished the deleterious effects of yqaH WT expression on chromosome segregation, replication initiation and DnaA-regulated transcription. When YqaH was induced after vegetative growth, DnaA_LOI mutations abolished the drastic effects of YqaH WT on sporulation and biofilm formation. Thus, YqaH inhibits replication, sporulation and biofilm formation mainly by antagonizing DnaA in a manner that is independent of the cell cycle checkpoint Sda.

Initiation of chromosomal replication requires dynamic nucleoprotein complexes. In most eubacteria, the origin oriC contains multiple DnaA box sequences to which the ubiquitous DnaA initiators bind. In Escherichia coli oriC, DnaA boxes sustain construction of higher-order complexes via DnaA–DnaA interactions, promoting the unwinding of the DNA unwinding element (DUE) within oriC and concomitantly binding the single-stranded (ss) DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, oriC sequences are highly diverse. The present study investigated the design of oriC (tma-oriC) from Thermotoga maritima, an evolutionarily ancient eubacterium. The minimal tma-oriC sequence includes a DUE and a flanking region containing five DnaA boxes recognized by the cognate DnaA (tmaDnaA). This DUE was comprised of two distinct functional modules, an unwinding module and a tmaDnaA-binding module. Three direct repeats of the trinucleotide TAG within DUE were essential for both unwinding and ssDUE binding by tmaDnaA complexes constructed on the DnaA boxes. Its surrounding AT-rich sequences stimulated only duplex unwinding. Moreover, head-to-tail oligomers of ATP-bound tmaDnaA were constructed within tma-oriC, irrespective of the directions of the DnaA boxes. This binding mode was considered to be induced by flexible swiveling of DnaA domains III and IV, which were responsible for DnaA–DnaA interactions and DnaA box binding, respectively. Phasing of specific tmaDnaA boxes in tma-oriC was also responsible for unwinding. These findings indicate that a ssDUE recruitment mechanism was responsible for unwinding and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria.

Most organisms possess several cell cycle checkpoints to preserve genome stability in periods of stress. Upon starvation, the absence of chromosomal duplication in the bacterium Escherichia coli is ensured by holding off commencement of replication. During normal growth, accumulation of the initiator protein DnaA along with cell cycle changes in its activity, ensure that DNA replication starts only once per cell cycle. Upon nutrient starvation, the prevailing model is that an arrest in DnaA protein synthesis is responsible for the absence of initiation. Recent indications now suggest that DnaA degradation may also play a role. Here we comment on the implications of this potential new layer of regulation.

Most bacteria replicate and segregate their DNA concomitantly while growing, before cell division takes place. How bacteria synchronize these different cell cycle events to ensure faithful chromosome inheritance by daughter cells is poorly understood. Here, we identify Cell Cycle Regulator protein interacting with FtsZ (CcrZ) as a conserved and essential protein in pneumococci and related Firmicutes such as Bacillus subtilis and Staphylococcus aureus. CcrZ couples cell division with DNA replication by controlling the activity of the master initiator of DNA replication, DnaA. The absence of CcrZ causes mis-timed and reduced initiation of DNA replication, which subsequently results in aberrant cell division. We show that CcrZ from Streptococcus pneumoniae interacts directly with the cytoskeleton protein FtsZ, which places CcrZ in the middle of the newborn cell where the DnaA-bound origin is positioned. This work uncovers a mechanism for control of the bacterial cell cycle in which CcrZ controls DnaA activity to ensure that the chromosome is replicated at the right time during the cell cycle., In this Article, the authors identify CcrZ as a protein that coordinates DNA replication and cell division in Streptococcus pneumoniae and other Firmicutes. CcrZ is localized to the division site by binding directly to the divisome protein FtsZ, and there it activates DnaA, the master initiator of DNA replication, through a still unknown mechanism.

Genome replication is a fundamental requirement for the proliferation of all cells. Throughout the domains of life, conserved DNA replication initiation proteins assemble at specific chromosomal loci termed replication origins and direct loading of replicative helicases (1). Despite decades of study on bacterial replication, the diversity of bacterial chromosome origin architecture has confounded the search for molecular mechanisms directing the initiation process. Recently a basal system for opening a bacterial chromosome origin (oriC) was proposed (2). In the model organism Bacillus subtilis, a pair of double-stranded DNA (dsDNA) binding sites (DnaA-boxes) guide the replication initiator DnaA onto adjacent single-stranded DNA (ssDNA) binding motifs (DnaA-trios) where the protein assembles into an oligomer that stretches DNA to promote origin unwinding. We report here that these core elements are predicted to be present in the majority of bacterial chromosome origins. Moreover, we find that the principle activities of the origin unwinding system are conserved in vitro and in vivo. The results suggest that this basal mechanism for oriC unwinding is broadly functionally conserved and therefore may represent an ancestral system to open bacterial chromosome origins.

Sixty years ago, bacterial cell size was found to be an exponential function of growth rate. Fifty years ago, a more general relationship was proposed, in which cell mass was equal to the initiation mass multiplied by 2 to the power of the ratio of the total time of C and D periods to the doubling time. This relationship has recently been experimentally confirmed by perturbing doubling time, C period, D period, or initiation mass. However, the underlying molecular mechanism remains unclear. Here, we developed a theoretical model for initiator protein DnaA mediating DNA replication initiation in Escherichia coli. We introduced an initiation probability function for competitive binding of DnaA-ATP and DnaA-ADP at oriC. We established a kinetic description of regulatory processes (e.g., expression regulation, titration, inactivation, and reactivation) of DnaA. Cell size as a spatial constraint also participates in the regulation of DnaA. By simulating DnaA kinetics, we obtained a regular DnaA oscillation coordinated with cell cycle and a converged cell size that matches replication initiation frequency to the growth rate. The relationship between the simulated cell size and growth rate, C period, D period, or initiation mass reproduces experimental results. The model also predicts how DnaA number and initiation mass vary with perturbation parameters, comparable with experimental data. The results suggest that 1) when growth rate, C period, or D period changes, the regulation of DnaA determines the invariance of initiation mass; 2) ppGpp inhibition of replication initiation may be important for the growth rate independence of initiation mass because three possible mechanisms therein produce different DnaA dynamics, which is experimentally verifiable; and 3) perturbation of some DnaA regulatory process causes a changing initiation mass or even an abnormal cell cycle. This study may provide clues for concerted control of cell size and cell cycle in synthetic biology.

The transcription factor RpoS (σS) of Escherichia coli controls a large number of genes important for tolerance of a variety of stress conditions. IraD promotes the post-translation stability of RpoS by inhibition of RssB, an adaptor protein for ClpXP degradation. We have previously documented DNA damage induction of iraD expression, independent of the SOS response. Both iraD and rpoS are required for tolerance to DNA damaging treatments such as H2O2 and the replication inhibitor azidothymidine in the log phase of growth. Using luciferase gene fusions to the 672 bp iraD upstream region, we show here that both promoters of iraD are induced by AZT. Genetic analysis suggests that both promoters are repressed by DnaA-ATP, partially dependent on a putative DnaA box at −81 bp, and regulated by RIDA (regulatory inactivation of DnaA), dependent on the DnaN processivity clamp. By electrophoretic mobility shift assays we show that purified DnaA protein binds to the iraD upstream region, so DnaA regulation of IraD is likely to be direct. DNA damage induction of iraD during log phase growth is abolished in the dnaA-T174P mutant, suggesting that DNA damage, in some way, relieves DnaA repression, possibly through the accumulation of replication clamps and enhanced RIDA. We demonstrate that the RNA-polymerase associated factor, SspA (stringent starvation protein A), induced by the accumulation of ppGpp, also affects IraD expression, with a positive effect on constitutive expression and a negative effect on AZT-induced expression, in a fashion independent of DnaA.SIGNIFICANCEDNA damage can lead to cell death or genomic instability. Cells have evolved transcriptional responses that sense DNA damage and up-regulate tolerance and repair factors; the LexA/RecA-regulated SOS response in E. coli was the first example of such a system. This work describes an alternative DNA damage response, controlled by DnaA and the IraD post-translational regulator of RpoS. The cellular signals for this response, we propose, are empty replication processivity (β) clamps that accumulate at replication blocks. IraD expression is also regulated by stringent starvation protein, SspA, induced by nutrient deprivation. This SOS-independent DNA damage response integrates a signal of incomplete replication with starvation to modulate expression of genes that promote the completion of replication.

DnaA is the initiator protein of chromosome replication, but the regulation of its homoeostasis in enterobacteria is not well understood. The DnaA level remains stable at different growth rates, suggesting a link between metabolism and dnaA expression. In a bioinformatic prediction, which we made to unravel targets of the sRNA rnTrpL in Enterobacteriaceae, the dnaA mRNA was the most conserved target candidate. The sRNA rnTrpL is derived from the transcription attenuator of the tryptophan biosynthesis operon. In Escherichia coli, its level is higher in minimal than in rich medium due to derepressed transcription without external tryptophan supply. Overexpression and deletion of the rnTrpL gene decreased and increased, respectively, the levels of dnaA mRNA. The decrease of the dnaA mRNA level upon rnTrpL overproduction was dependent on hfq and rne. Base pairing between rnTrpL and dnaA mRNA in vivo was validated. In minimal medium, the oriC level was increased in the ΔtrpL mutant, in line with the expected DnaA overproduction and increased initiation of chromosome replication. In line with this, chromosomal rnTrpL mutation abolishing the interaction with dnaA increased both the dnaA mRNA and the oriC level. Moreover, upon addition of tryptophan to minimal medium cultures, the oriC level in the wild type was increased. Thus, rnTrpL is a base-pairing sRNA that posttranscriptionally regulates dnaA in E. coli. Furthermore, our data suggest that rnTrpL contributes to the DnaA homoeostasis in dependence on the nutrient availability, which is represented by the tryptophan level in the cell.

Aim: The rapid emergence of β-lactam resistance in gram-negative bacteria is a major problem in the treatment of infections caused by pathogenic bacterial strains, in particular Klebsiella pneumoniae. In our study, we are presenting a systems biology approach to understand the role of SHV-11 gene in drug resistance mechanism patterns in K. pneumoniae strain. Results: From the results, we have observed that the SHV-11 gene has a role in drug resistance mechanism along with its functional partner genes gyrA, parC, glsA, osmE, yjhA, yhdT, rimL, pepB, KPN_00437, and KPN_01875. We have also observed that of 51 genes, 27 genes were enriched in various Gene Ontology terms such as DNA metabolic process, DNA repair, and response to stress. The genes gyrA, parC, gyrB, parE, recA, dnaA, polB, dnaK, mutS, and dnaN constitute >41% of the total interactions; thus, these genes can be considered as hub nodes in the network, and they can be used as the potential drug targets. Conclusions: Drug exposure leads to the DNA damage in bacterial spp. We observed that the SHV11 gene along with the functional partners help in maintaining the genomic integrity by withstanding the environmental stress by inducing DNA damage repair mechanism. Our results provide a detailed understanding on the role of SHV-11 gene in drug resistance mechanisms in K. pneumoniae, and we are of the opinion that our results will be useful for researchers exploring the antibiotic resistance mechanisms in pathogenic bacteria.

Initiation of Escherichia coli chromosome replication is controlled by the DnaA initiator protein. Both rifampicin‐mediated inhibition of transcription and ppGpp‐induced changes in global transcription stops replication at the level of initiation. Here, we show that continued DnaA protein synthesis allows for replication initiation both during the rifampicin treatment and during the stringent response when the ppGpp level is high. A reduction in or cessation of de novo DnaA synthesis, therefore, causes the initiation arrest in both cases. In accordance with this, inhibition of translation with chloramphenicol also stops initiations. The initiation arrest caused by rifampicin was faster than that caused by chloramphenicol, despite of the latter inhibiting DnaA accumulation immediately. During chloramphenicol treatment transcription is still ongoing and we suggest that transcriptional events in or near the origin, that is, transcriptional activation, can allow for a few extra initiations when DnaA becomes limiting. We suggest, for both rifampicin treated cells and for cells accumulating ppGpp, that a turn‐off of initiation from oriC requires a stop in de novo DnaA synthesis and that an additional lack of transcriptional activation enhances this process, that is, leads to a faster initiation stop., Both rifampicin and high levels of ppGpp affect the E. coli RNA polymerase (RNAP), and lead to reduced dnaA gene transcription and cessation of initiation of chromosome replication (a). Replication initiation does however continue when DnaA is de novo synthesized from a rifampicin‐resistant and ppGpp‐insensitive T7 RNAP‐dependent promoter (b). Therefore, both rifampicin and ppGpp inhibit initiation of replication primarily by limiting DnaA synthesis.

An aerobic, oligotrophic, Gram-positive, non-sporulating, motile, rod-shaped, palladium-leaching bacterial strain, Deinococcus sp. KR-1, was previously isolated from pond water collected in Japan. This strain grew at 10 °C to 40 °C (optimum 30 °C), at pH 4.5 to 11.5 (optimum pH 8.0), and in the presence of 2.0% NaCl. Its major cellular fatty acids were C15: 1ω6 and C16 : 1ω7c. The quinone system was menaquinone 8. Multilocus sequence analysis based on partial sequences of four housekeeping genes (atpD, dnaA, gyrB and rpoB) showed that branching of Deinococcus sp. KR-1 was distant to those of Deinococcus type strains. The genome average nucleotide identity value between strain KR-1 and its closest related Deinococcus type strain was less than 95.69%. Based on its phenotypic, chemotaxonomic and phylogenetic data, strain KR-1 (= HUT-8138T = KCTC 33977T) can be considered a novel species within the genus Deinococcus with the proposed name Deinococcus kurensis sp. nov.

The decision whether to replicate DNA is crucial for cell survival, not only to proliferate in favorable conditions, but also to adopt to environmental changes. When a bacteria encounters stress, e.g. starvation, it launches the stringent response, to arrest cell proliferation and to promote survival. During the stringent response a vast amount of polymer composed of phosphate residues, i.e. inorganic polyphosphate (PolyP) is synthesized from ATP. Despite extensive research on PolyP, we still lack the full understanding of the PolyP role during stress. It is also elusive what is the mechanism of DNA replication initiation arrest in starved Escherichia coli cells. Here, we show that during stringent response PolyP activates Lon protease to degrade selectively the replication initiaton protein DnaA bound to ADP, but not ATP. In contrast to DnaA-ADP, the DnaA-ATP does not interact with PolyP, but binds to dnaA promoter to block dnaA transcription. The systems controlling the ratio of nucleotide states of DnaA continue to convert DnaA-ATP to DnaA-ADP, which is proteolysed by Lon, thereby resulting in the DNA replication initiation arrest. The uncovered regulatory mechanism interlocks the PolyP-dependent protease activation with the ATP/ADP cycle of dual-functioning protein essential for bacterial cell proliferation.

In bacterial DNA replication, the initiator protein DnaA binds to the multiple DnaA box sequences located at oriC to facilitate the unwinding of duplex DNA strands. The cyanobacterium Synechococcus elongatus PCC 7942, which contains multiple chromosomal copies per cell, has DnaA box (like sequences around the oriC region, which is located upstream of dnaN. We previously observed the binding of DnaA around the oriC region; however, the DNA-binding specificity of DnaA to DnaA box sequences has not been examined. Here, we analyzed the binding specificity of DnaA protein to the DnaA box in S. elongatus by using bio-layer interferometry (BLI), a method for monitoring intermolecular interactions. We observed that recombinant DnaA protein recognized specifically the DnaA box sequence TTTTCCACA in vitro. In addition, DNA binding activity was significantly increased by R328H mutation of DnaA. This is the first report to characterize DnaA binding to the DnaA box sequence in cyanobacteria.

Flow cytometry is a high-throughput technique that analyzes individual particles as they pass through a laser beam. These particles can be individual cells and by detecting cell-scattered light their number and relative size can be measured as they pass through the beam. Labeling of molecules, usually via a fluorescent reporter, allows the amount of these molecules per cell to be quantified. DNA content can be estimated using this approach and here we describe how flow cytometry can be used to assess the DNA content of Escherichia coli cells.

Streptomyces DNA replication starts with the DnaA binding to the origin of replication. Differently to most bacteria, cytokinesis only occurs during sporulation. Cytokinesis is modulated by the divisome, an orderly succession of proteins initiated by FtsZ. Here, we characterised SCO2102, a protein harbouring a DnaA II protein–protein interaction domain highly conserved in Streptomyces. The ΔSCO2102 knockout shows highly delayed sporulation. SCO2102-mCherry frequently co-localises with FtsZ-eGFP during sporulation and greatly reduces FtsZ-eGFP Z-ladder formation, suggesting a role of SCO2102 in sporulation. SCO2102 localises up-stream of SCO2103, a methylenetetrahydrofolate reductase involved in methionine and dTMP synthesis. SCO2102/SCO2103 expression is highly regulated, involving two promoters and a conditional transcription terminator. The ΔSCO2103 knockout shows reduced DNA synthesis and a non-sporulating phenotype. SCO2102-mCherry co-localises with SCO2103-eGFP during sporulation, and SCO2102 is essential for the SCO2103 positioning at sporulating hyphae, since SCO2103-eGFP fluorescent spots are absent in the ΔSCO2102 knockout. We propose a model in which SCO2102 positions SCO2103 in sporulating hyphae, facilitating nucleotide biosynthesis for chromosomal replication. To the best of our knowledge, SCO2102 is the first protein harbouring a DnaA II domain specifically found during sporulation, whereas SCO2103 is the first methylenetetrahydrofolate reductase found to be essential for Streptomyces sporulation.

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.

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.

Expression of the Escherichia coli dnaN-encoded β clamp at ≥10-fold higher than chromosomally expressed levels impedes growth by interfering with DNA replication. A mutant clamp (β(E202K) bearing a glutamic acid-to-lysine substitution at residue 202) binds to DNA polymerase III (Pol III) with higher affinity than the wild-type clamp, suggesting that its failure to impede growth is independent of its ability to sequester Pol III away from the replication fork. Our results demonstrate that the dnaN(E202K) strain underinitiates DNA replication due to insufficient levels of DnaA-ATP and expresses several DnaA-regulated genes at altered levels, including nrdAB, that encode the class 1a ribonucleotide reductase (RNR). Elevated expression of nrdAB was dependent on hda function. As the β clamp-Hda complex regulates the activity of DnaA by stimulating its intrinsic ATPase activity, this finding suggests that the dnaN(E202K) allele supports an elevated level of Hda activity in vivo compared with the wild-type strain. In contrast, using an in vitro assay reconstituted with purified components the β(E202K) and wild-type clamp proteins supported comparable levels of Hda activity. Nevertheless, co-overexpression of the nrdAB-encoded RNR relieved the growth defect caused by elevated levels of the β clamp. These results support a model in which increased cellular levels of DNA precursors relieve the ability of elevated β clamp levels to impede growth and suggest either that multiple effects stemming from the dnaN(E202K) mutation contribute to elevated nrdAB levels or that Hda plays a noncatalytic role in regulating DnaA-ATP by sequestering it to reduce its availability. IMPORTANCE DnaA bound to ATP acts in initiation of DNA replication and regulates the expression of several genes whose products act in DNA metabolism. The state of the ATP bound to DnaA is regulated in part by the β clamp-Hda complex. The dnaN(E202K) allele was identified by virtue of its inability to impede growth when expressed ≥10-fold higher than chromosomally expressed levels. While the dnaN(E202K) strain exhibits several phenotypes consistent with heightened Hda activity, the wild-type and β(E202K) clamp proteins support equivalent levels of Hda activity in vitro. Taken together, these results suggest that β(E202K)-Hda plays a noncatalytic role in regulating DnaA-ATP. This, as well as alternative models, is discussed.

Escherichia coli coordinates replication and division cycles by initiating replication at approximately the same size per chromosome at all growth rates. By tracking replisomes in individual cells through thousands of division cycles, we have dissected the mechanism behind this precise process. We have characterized wild-type cells grown under different conditions and also many mutants related to the expression and binding states of the initiator protein DnaA. This rich data set allowed us to compare the relative importance of all previously described control systems. We found that the replication initiation size regulation is not strongly dependent on the absolute concentration of DnaA, nor does it depend on active dnaA expression. Replication initiation is also not consistently triggered by cell division or replication termination. In contrast, some of the factors that convert DnaA between its ATP- and ADP-bound states have a strong effect on initiation size. We suggest a plausible model for DnaA-ATP mediated triggering of initiation at fast growth, where regulatory inactivation of DnaA (RIDA) is the main system for monitoring the number of chromosomes during active replication.

Streptococcus pyogenes is a significant pathogen that causes skin and upper respiratory tract infections and non-suppurative complications, such as acute rheumatic fever and post-strep glomerulonephritis. Multidrug resistance has emerged in S. pyogenes strains, making them more dangerous and pathogenic. Hence, it is necessary to identify and develop therapeutic methods that would present novel approaches to S. pyogenes infections. In the current study, a subtractive proteomics approach was employed to core proteomes of four strains of S. pyogenes using several bioinformatic software tools and servers. The core proteome consists of 1324 proteins, and 302 essential proteins were predicted from them. These essential proteins were analyzed using BLASTp against human proteome, and the number of potential targets was reduced to 145. Based on subcellular localization prediction, 46 proteins with cytoplasmic localization were chosen for metabolic pathway analysis. Only two cytoplasmic proteins, i.e., chromosomal replication initiator protein DnaA and two-component response regulator (TCR), were discovered to have the potential to be novel drug target candidates. Three-dimensional (3D) structure prediction of target proteins was carried out via the Swiss Model server. Molecular docking approach was employed to screen the library of 1000 phytochemicals against the interacting residues of the target proteins through the MOE software. Further, the docking studies were validated by running molecular dynamics simulation and highly popular binding free energy approaches of MM-GBSA and MM-PBSA. The findings revealed a promising candidate as a novel target against S. pyogenes infections.

The ability to regulate DNA replication initiation in response to changing nutrient conditions is an important feature of most cell types. In bacteria, DNA replication is triggered by the initiator protein DnaA, which has long been suggested to respond to nutritional changes, nevertheless the underlying mechanisms remain poorly understood. Here, we report a novel mechanism that adjusts DnaA synthesis in response to nutrient availability in Caulobacter crescentus. By performing a detailed biochemical and genetic analysis of the dnaA mRNA we identified a sequence downstream of the dnaA start codon that inhibits DnaA translation elongation upon carbon exhaustion. Our data show that the corresponding peptide sequence, but not the mRNA secondary structure or the codon choice, are critical for this response, suggesting that specific amino acids in the growing DnaA nascent chain tune translational efficiency. Our study provides new insights into DnaA regulation and highlights the importance of translation elongation as a regulatory target. We propose that nascent chain sequences, like the one described, might constitute a general strategy for modulating the synthesis rate of specific proteins under changing conditions.

Genome duplication is a critical event in the reproduction cycle of every cell. Because all daughter cells must inherit a complete genome, chromosome replication is tightly regulated, with multiple mechanisms focused on controlling when chromosome replication begins during the cell cycle. In bacteria, chromosome duplication starts when nucleoprotein complexes, termed orisomes, unwind replication origin (oriC) DNA and recruit proteins needed to build new replication forks. Functional orisomes comprise the conserved initiator protein, DnaA, bound to a set of high and low affinity recognition sites in oriC. Orisomes must be assembled each cell cycle. In Escherichia coli, the organism in which orisome assembly has been most thoroughly examined, the process starts with DnaA binding to high affinity sites after chromosome duplication is initiated, and orisome assembly is completed immediately before the next initiation event, when DnaA interacts with oriC’s lower affinity sites, coincident with origin unwinding. A host of regulators, including several transcriptional modulators, targets low affinity DnaA-oriC interactions, exerting their effects by DNA bending, blocking access to recognition sites, and/or facilitating binding of DnaA to both DNA and itself. In this review, we focus on orisome assembly in E. coli. We identify three known transcriptional modulators, SeqA, Fis (factor for inversion stimulation), and IHF (integration host factor), that are not essential for initiation, but which interact directly with E. coli oriC to regulate orisome assembly and replication initiation timing. These regulators function by blocking sites (SeqA) and bending oriC DNA (Fis and IHF) to inhibit or facilitate cooperative low affinity DnaA binding. We also examine how the growth rate regulation of Fis levels might modulate IHF and DnaA binding to oriC under a variety of nutritional conditions. Combined, the regulatory mechanisms mediated by transcriptional modulators help ensure that at all growth rates, bacterial chromosome replication begins once, and only once, per cell cycle.

The Escherichia coli chromosome is a circular double helix. DNA polymerase, therefore, cannot use it directly as a template for polymerization until it has first been unwound. The DnaA protein opens the chromosome at a unique and specific site (oriC), which allows the polymerase to begin DNA replication. François Jacob and Sydney Brenner predicted the existence of the initiator protein, DnaA, back in the early 1960s. In order to demonstrate the existence of the hypothetical initiator, identification and isolation of dnaA mutants were undertaken. In the following, I recount, in a historical setting, the original research done on the identification and isolation of dnaA mutants.

In bacteria, AAA+ proteases such as Lon and ClpXP specifically degrade substrates to promote growth and stress responses. Here, we find that an ATP-binding mutant of ClpX suppresses physiological defects of a Lon-deficient strain by shifting ClpXP protease specificity toward normally Lon-restricted substrates and away from normal ClpXP targets. Reconstitution with purified proteins assigns these effects to changes in direct recognition and processing of substrates. We show that wildtype ClpXP specificity can be similarly altered when ATP hydrolysis is reduced, which unexpectedly accelerates degradation of some substrates. This activation corresponds with changes in ClpX conformation, leading to a model where ClpX cycles between capture and processive states depending on ATP loading. Limiting ATP binding alters dynamics between states affording better recognition of unorthodox substrates, but worse degradation of proteins specifically bound by the processive state. Thus, AAA+ protease specificities can be directly tuned by differences in ATP hydrolysis rates. HighlightsO_LIA mutation in the Walker B region of ClpX induces recognition of new substrates. C_LIO_LIProteases are optimized for specific functions but barrier to recognize new substrates is easily overcome. C_LIO_LIExpanding substrate recognition by a protease comes at the cost of reducing native substrate degradation. C_LIO_LIDecreasing ATP enhances ClpXP mediated degradation of certain classes of substrates. C_LIO_LIClpX adopts distinct conformational states to favor better recognition of some substrates over others. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=183 HEIGHT=200 SRC="FIGDIR/small/456811v1_ufig1.gif" ALT="Figure 1"> View larger version (40K): org.highwire.dtl.DTLVardef@fe7c1corg.highwire.dtl.DTLVardef@1862957org.highwire.dtl.DTLVardef@1044c0forg.highwire.dtl.DTLVardef@11d7168_HPS_FORMAT_FIGEXP M_FIG C_FIG In a wildtype cell, AAA+ proteases Lon and ClpXP promote normal growth by degrading distinct substrates. ClpX*P can compensate for the absence of the Lon protease by tuning ClpXP substrate specificity to better degrade Lon-privileged substrates (such as DnaA, SciP, and misfolded proteins) but this comes at the cost of native ClpXP substrates (such as ssrA-tagged proteins and CtrA). We propose that ClpX alternates between a closed and open conformation and promoting one state over the other leads to alterations in substrate specificity. In the presence of ClpX* or in ATP-limited conditions, the open state is favored, allowing capture and recognition of substrates such as casein. The balance shifts to the closed state under high ATP conditions, allowing degradation of substrates such as GFP-ssrA, which preferentially bind the closed state.

The mechanisms responsible for helicase loading during the initiation of chromosome replication in bacteria are unclear. Here we report both a positive and a negative mechanism for directing helicase recruitment in the model organism Bacillus subtilis. Systematic mutagenesis of the essential replication initiation gene dnaD and characterization of DnaD variants revealed protein interfaces required for interacting with the master initiator DnaA and with a specific single-stranded DNA (ssDNA) sequence located in the chromosome origin (DnaD Recognition Element, “DRE”). We propose that the location of the DRE within the replication origin orchestrates recruitment of helicase to achieve bidirectional DNA replication. We also report that the developmentally expressed repressor of DNA replication initiation, SirA, acts by blocking the interaction of DnaD with DnaA, thereby inhibiting helicase recruitment to the origin. These findings significantly advance our mechanistic understanding of helicase recruitment and regulation during bacterial DNA replication initiation. Because DnaD is essential for the viability of clinically relevant Gram-positive pathogens, DnaD is an attractive target for drug development.

Topoisomerase I (Topo I) of Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including ΔtopA, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn lead to transcription-replication conflicts and to cSDR.ImportanceIn all life forms, double helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli. That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability, and suggests that in its absence aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.

The Escherichia coli YfiF protein is functionally unknown, being predicted as a transfer RNA/ribosomal RNA (tRNA/rRNA) methyltransferase. We find that absence of the yfiF gene delays initiation of chromosome replication and the delay is reversed by ectopic expression of YfiF, whereas excess YfiF causes an early initiation. A slight decrease in both cell size and number of origin per mass is observed in ΔyfiF cells. YfiF does not genetically interact with replication proteins such as DnaA, DnaB, and DnaC. Interestingly, YfiF is associated with ribosome modulation factor (RMF), hibernation promotion factor (HPF), and the tRNA methyltransferase TrmL. Defects in replication initiation of Δrmf, Δhpf, and ΔtrmL can be rescued by overexpression of YfiF, indicating that YfiF is functionally identical to RMF, HPF, and TrmL in terms of replication initiation. Also, YfiF interacts with the rRNA methyltransferase RsmC. Moreover, the total amount of proteins and DnaA content per cell decreases or increases in the absence of YfiF or the presence of excess YfiF. These facts suggest that YfiF is a ribosomal dormancy-like factor, affecting ribosome function. Thus, we propose that YfiF is involved in the correct timing of chromosome replication by changing the DnaA content per cell as a result of affecting ribosome function.

Cells respond to genome damage by inducing restorative programs, typified by the SOS response of Escherichia coli. Streptococcus pneumoniae (the pneumococcus), with no equivalent to the SOS system, induces the genetic program of competence in response to many types of stress, including genotoxic drugs. The pneumococcal competence regulon is controlled by the origin-proximal, auto-inducible comCDE operon. It was previously proposed that replication stress induces competence through continued initiation of replication in cells with arrested forks, thereby increasing the relative comCDE gene dosage and expression and accelerating the onset of competence. We have further investigated competence induction by genome stress. We find that absence of RecA recombinase stimulates competence induction, in contrast to SOS response, and that double-strand break repair (RexB) and gap repair (RecO, RecR) initiation effectors confer a similar effect, implying that recombinational repair removes competence induction signals. Failure of replication forks provoked by titrating PolC polymerase with the base analogue HPUra, over-supplying DnaA initiator, or under-supplying DnaE polymerase or DnaC helicase stimulated competence induction. This induction was not correlated with concurrent changes in origin-proximal gene dosage. Our results point to arrested and unrepaired replication forks, rather than increased comCDE dosage, as a basic trigger of pneumococcal competence.

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.

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria., Graphical Abstract Graphical AbstractDciA is the antecedent but unrelated to the helicase loaders DnaC/I. Structurally, the N-terminal domain of DciA is related to the NTD of DnaA, whereas its CTD is unfolded. DciA associates with DnaB according to a 3/6 stoichiometry which loads the helicase more efficiently on DNA. We identified in DnaB proteins a determinant residue that discriminates DciA- and DnaC/I-helicases and which happens to be critical in vivo. The determinant residue is located at the center of the DciA binding site.

In Escherichia coli, repression of phosphatidylglycerol synthase A gene (pgsA) lowers the levels of membrane acidic phospholipids, particularly phosphatidylglycerol (PG), causing growth-arrested phenotype. The interrupted synthesis of PG is known to be associated with concomitant reduction of chromosomal content and cell mass, in addition to accumulation of unprocessed outer membrane lipoprotein intermediate, pro-Lpp, at the inner membrane. However, whether a linkage exists between the two altered-membrane outcomes remains unknown. Previously, it has been shown that pgsA+ cells overexpressing mutant Lpp(C21G) protein have growth defects similar to those caused by the unprocessed pro-Lpp intermediate in cells lacking PG. Here, we found that the ectopic expression of DnaA(L366K) or deletion of fis (encoding Factor for Inversion Stimulation) permits growth of cells that otherwise would be arrested for growth due to accumulated Lpp(C21G). The DnaA(L366K)-mediated restoration of growth occurs by reduced expression of Lpp(C21G) via a σE-dependent small-regulatory RNA (sRNA), MicL-S. In contrast, restoration of growth via fis deletion is only partially dependent on the MicL-S pathway; deletion of fis also rescues Lpp(C21G) growth arrest in cells lacking physiological levels of PG and cardiolipin (CL), independently of MicL-S. Our results suggest a close link between the physiological state of the bacterial cell membrane and DnaA- and Fis-dependent growth.

The eubacterial β sliding clamp (DnaN) plays a crucial role in DNA metabolism through direct interactions with DNA, polymerases, and a variety of protein factors. A canonical protein-DnaN interaction has been identified in Escherichia coli and some other species, during which protein partners are tethered into the conserved canonical hydrophobic crevice of DnaN via the consensus β-binding motif. Caulobacter crescentus is an excellent research model for use in the investigation of DNA replication and cell-cycle regulation due to its unique asymmetric cell division pattern with restricted replication initiation; however, little is known about the specific features of C. crescentus DnaN (CcDnaN). Here, we report a significant divergence in the association of CcDnaN with proteins based on docking analysis and crystal structures that show that the β-binding motifs of its protein partners bind a novel pocket instead of the canonical site. Pull-down and isothermal titration calorimetry results revealed that mutations within the novel pocket disrupt protein-CcDnaN interactions. It was also shown by replication and regulatory inactivation of DnaA assays that mediation of protein interaction by the novel pocket is closely related to the performance of CcDnaN during replication and the DnaN-mediated regulation process. Moreover, assessments of clamp competition showed that DNA does not compete with protein partners when binding to the novel pocket. Overall, our structural and biochemical analyses provide strong evidence that CcDnaN employs a noncanonical protein association pattern.

Background:One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited.Materials and Methods:Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria.Results:The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen.Conclusion:We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

Escherichia coli replication initiator protein DnaA binds ATP with high affinity but the amount of ATP required to initiate replication greatly exceeds the amount required for binding. Previously, we showed that ATP-DnaA, not ADP-DnaA, undergoes a conformational change at the higher nucleotide concentration, which allows DnaA oligomerization at the replication origin but the association state remains unclear. Here, we used Small Angle X-ray Scattering (SAXS) to investigate oligomerization of DnaA in solution. Whereas ADP-DnaA was predominantly monomeric, AMP–PNP–DnaA (a non-hydrolysable ATP-analog bound-DnaA) was oligomeric, primarily dimeric. Functional studies using DnaA mutants revealed that DnaA(H136Q) is defective in initiating replication in vivo. The mutant retains high-affinity ATP binding, but was defective in producing replication-competent initiation complexes. Docking of ATP on a structure of E. coli DnaA, modeled upon the crystallographic structure of Aquifex aeolicus DnaA, predicts a hydrogen bond between ATP and imidazole ring of His136, which is disrupted when Gln is present at position 136. SAXS performed on AMP–PNP–DnaA (H136Q) indicates that the protein has lost its ability to form oligomers. These results show the importance of high ATP in DnaA oligomerization and its dependence on the His136 residue.

Chromosome replication is a fundamental process in all domains of life. To accurately transmit genetic material to offspring, the initiation of chromosome replication is tightly regulated to ensure that it occurs only once in each cell division cycle. In the model bacterium Caulobacter crescentus, the CtrA response regulator inhibits the origin of replication at the pre-replication stage. Inactivation of CtrA permits the universal DnaA initiator to form an initiation complex at the origin, leading to replication initiation. Subsequently, the initiation complex is inactivated to prevent extra initiation. Whereas DNA replication occurs periodically in exponentially growing cells, replication initiation is blocked under various stress conditions to halt cell cycle progression until the normal condition is restored or the cells adapt to the stress. Thus, regulating the initiation complex plays an important role in not only driving cell cycle progression, but also maintaining cell integrity under stress. Multiple regulatory signaling pathways controlling CtrA and DnaA have been identified and recent studies have advanced our knowledge of the underlying mechanistic and molecular processes. This review focuses on how bacterial cells control replication initiation, highlighting the latest findings that have emerged from studies in C. crescentus.

Pseudoalteromonas marina is one of the potential pathogens that cause green spot disease (GSD) in Pyropia yezoensis. To prevent GSD from development and spread, an effective method to detect the pathogen at early GSD infection stages need to be established. In this research, PCR methods were established targeting the dnaA gene (encoding chromosome replication initiator protein) and the dnaN gene (encoding β sliding clamp of DNA polymerase III protein) to detect P. marina with three primer pairs pws-dnaA2 (Forward, 5′-ACCGCATTAACGAACTACTCGTG-3′; Reverse, 5′-TGCCATTACCTACAGCATGG-3′), pcs-dnaN2 (Forward, 5′-CTTACAACGTTATCAGCGGC-3′; Reverse, 5′-GTTGAGTATTAAGTGATTGAGTAAGC-3′) or pws-dnaN3 (Forward, 5′-ACTTACAACGTTATCAGCGGC-3′; Reverse, 5′-ACTGCTGTTTGAGTCTGCTAAC-3′). Three PCR methods corresponding to the three primer pairs sufficiently distinguished P. marina from 22 bacterial species, thus resulting in detection limits of 4 to 4×102 CFU cells or 2.37×101 to 2.37×103 fg of P. marina DNA per PCR reaction. In an artificial infection experiment of P. yezoensis infected with P. marina, all established PCRs successfully detected P. marina at early GSD infection stages. The results show that the established PCRs are specific and sensitive, and are potential for applications in early diagnosis of GSD in Pyropia.

A previous study reported on the isolation of 430 polysaccharide (gum)-producing bacteria from a South African sugarcane processing factory and the identification of isolates by comparative 16S rRNA gene sequencing. A large number of isolates (202) belonged to the genus Weissella and clustered with reference strains of Weissella cibaria and Weissella confusa. In this study, we identified 147 strains as W. cibaria and 55 as W. confusa based on phylogenetic analyses of pheS and dnaA gene sequences of representative isolates. We also included atpA gene sequence analysis of Weissella isolates as potential future phylogenetic marker to differentiate amongst strains of W. cibaria and W. confusa.

The decision to initiate DNA replication is a critical step in the cell cycle of all organisms. In nearly all bacteria, replication initiation requires the activity of the conserved replication initiation protein DnaA. Due to its central role in cell cycle progression, DnaA activity must be precisely regulated. This review summarizes the current state of DnaA regulation in the asymmetrically dividing α-proteobacterium Caulobacter crescentus, an important model for bacterial cell cycle studies. Mechanisms will be discussed that regulate DnaA activity and abundance under optimal conditions and in coordination with the asymmetric Caulobacter cell cycle. Furthermore, we highlight recent findings of how regulated DnaA synthesis and degradation collaborate to adjust DnaA abundance under stress conditions. The mechanisms described provide important examples of how DNA replication is regulated in an α-proteobacterium and thus represent an important starting point for the study of DNA replication in many other bacteria. This article is part of a Special Issue entitled: Dynamic gene expression, edited by Prof. Patrick Viollier.

The genome of Sinorhizobium meliloti, a model for studying plant-bacteria symbiosis, contains eight genes coding for LuxR-like proteins. Two of these, SinR and ExpR, are essential for quorum sensing (QS). Roles and regulation surrounding the others are mostly unknown. Here, we reveal the DNA recognition sequence and regulon of the LuxR-like protein SMc00877. Unlike ExpR, which uses the long-chain acyl homoserine lactones (AHLs) as inducers, SMc00877 functioned independently of AHLs and was even functional in Escherichia coli. A target of SMc00877 is SinR, the major regulator of AHL production in S. meliloti. Disruption of SMc00877 decreased AHL production. A weaker production of AHLs resulted in smaller microcolonies, starting from single cells, and delayed AHL-dependent regulation. SMc00877 was expressed only in growing cells in the presence of replete nutrients. Therefore, we renamed it NurR (nutrient sensitive LuxR-like regulator). We traced this nutrient-sensitive expression to transcription control by the DNA replication initiation factor, DnaA, which is essential for growth. These results indicate that NurR has a role in modulating the threshold of QS activation according to growth. We propose growth behavior as an additional prerequisite to population density for the activation of QS in S. meliloti.

Summary Bacterial antibiotic resistance, a global health threat, is caused by plasmid transfer or genetic mutations. Quinolones are important antibiotics, partially because they are fully synthetic and resistance genes are unlikely to exist in nature; nonetheless, quinolone resistance proteins have been identified. The mechanism by which plasmid‐borne quinolone resistance proteins promotes the selection of quinolone‐resistant mutants is unclear. Here, we show that QnrB increases the bacterial mutation rate. Transcriptomic and genome sequencing analyses showed that QnrB promoted gene abundance near the origin of replication (oriC). In addition, the QnrB expression level correlated with the replication origin to terminus (oriC/ter) ratio, indicating QnrB‐induced DNA replication stress. Our results also show that QnrB is a DnaA‐binding protein that may act as an activator of DNA replication initiation. Interaction of QnrB with DnaA promoted the formation of the DnaA‐oriC open complex, which leads to DNA replication over‐initiation. Our data indicate that plasmid‐borne QnrB increases bacterial mutation rates and that genetic changes can alleviate the fitness cost imposed by transmitted plasmids. Derivative mutations may impair antibiotic efficacy and threaten the value of antibiotic treatments. Enhanced understanding of how bacteria adapt to the antibiotic environment will lead to new therapeutic strategies for antibiotic‐resistant infections.

Whole genome sequencing (WGS) is able to differentiate closely related Mycobacterium tuberculosis variants within the same transmission cluster. Our aim was to evaluate if this higher discriminatory power may help identify and characterize more actively transmitted variants and understand the factors behind their success. We selected a robust MIRU-VNTR-defined cluster from Almería, Spain (22 cases throughout 2003–2019). WGS allowed discriminating, within the same epidemiological setting, between a successfully transmitted variant and seven closely related variants that did not lead to secondary cases, or were involved in self-limiting transmission (one single secondary case). Intramacrophagic growth of representative variants was evaluated in an in vitro infection model using U937 cells. Intramacrophage multiplication ratios (CFUs at Day 4/CFUs at Day 0) were higher for the actively transmitted variant (range 5.3–10.7) than for the unsuccessfully transmitted closely related variants (1.5–3.95). Two SNPs, mapping at the DNA binding domain of DnaA and at kdpD, were found to be specific of the successful variant.

Replication fork stalling generates a variety of responses, most of which cause an increase in single-stranded DNA. ssDNA is a primary signal of replication distress that activates cellular checkpoints. It is also a potential source of genome instability and a substrate for mutation and recombination. Therefore, managing ssDNA levels is crucial to chromosome integrity. Limited ssDNA accumulation occurs in wild-type cells under stress. In contrast, cells lacking the replication checkpoint cannot arrest forks properly and accumulate large amounts of ssDNA. This likely occurs when the replication fork polymerase and helicase units are uncoupled. Some cells with mutations in the replication helicase (mcm-ts) mimic checkpoint-deficient cells, and accumulate extensive areas of ssDNA to trigger the G2-checkpoint. Another category of helicase mutant (mcm4-degron) causes fork stalling in early S-phase due to immediate loss of helicase function. Intriguingly, cells realize that ssDNA is present, but fail to detect that they accumulate ssDNA, and continue to divide. Thus, the cellular response to replication stalling depends on checkpoint activity and the time that replication stress occurs in S-phase. In this review we describe the signs, signals, and symptoms of replication arrest from an ssDNA perspective. We explore the possible mechanisms for these effects. We also advise the need for caution when detecting and interpreting data related to the accumulation of ssDNA.

DNA replication is a highly regulated process that is primarily controlled at the step of initiation. In the gram-positive bacterium Bacillus subtilis the replication initiator DnaA, is regulated by YabA, which inhibits cooperative binding at the origin. Mutants lacking YabA have increased and asynchronous initiation. We found that under conditions of rapid growth, the dnaA1 mutation that causes replication over-initiation, was synthetic lethal with a deletion of yabA. We isolated several classes of suppressors of the lethal phenotype of the ΔyabA dnaA1 double mutant. Some suppressors (dnaC, cshA) caused a decrease in replication initiation. Others (relA, nrdR) stimulate replication elongation. One class of suppressors decreased levels of the replicative helicase, DnaC, thereby limiting replication initiation. We found that decreased levels of helicase were sufficient to decrease replication initiation under fast growth conditions. Our results highlight the multiple mechanisms cells use to regulate DNA replication.

The structure and function of bacterial chromosomes are dynamically regulated by a wide variety of nucleoid-associated proteins (NAPs) and DNA superstructures, such as DNA supercoiling. In Escherichia coli, integration host factor (IHF), a NAP, binds to specific transcription promoters and regulatory DNA elements of DNA replication such as the replication origin oriC: binding to these elements depends on the cell cycle but underlying mechanisms are unknown. In this study, we combined GeF-seq (genome footprinting with high-throughput sequencing) with synchronization of the E. coli cell cycle to determine the genome-wide, cell cycle-dependent binding of IHF with base-pair resolution. The GeF-seq results in this study were qualified enough to analyze genomic IHF binding sites (e.g., oriC and the transcriptional promoters of ilvG and osmY) except some of the known sites. Unexpectedly, we found that before replication initiation, oriC was a predominant site for stable IHF binding, whereas all other loci exhibited reduced IHF binding. To reveal the specific mechanism of stable oriC–IHF binding, we inserted a truncated oriC sequence in the terC (replication terminus) locus of the genome. Before replication initiation, stable IHF binding was detected even at this additional oriC site, dependent on the specific DnaA-binding sequence DnaA box R1 within the site. DnaA oligomers formed on oriC might protect the oriC–IHF complex from IHF dissociation. After replication initiation, IHF rapidly dissociated from oriC, and IHF binding to other sites was sustained or stimulated. In addition, we identified a novel locus associated with cell cycle-dependent IHF binding. These findings provide mechanistic insight into IHF binding and dissociation in the genome.

The studies presented in this thesis aimed to better understand specific C. difficile physiological processes: genome maintenance, DNA replication, and toxin regulation. As the available tools to investigate C difficile were insufficient, a secondary goal was to develop novel methods for C. difficile studies. We developed a luciferase toolkit for use in C. difficile, not only for gene expression analysis, but also for protein-protein interaction studies in vivo, secretion of proteins or the analysis of complexes present at the cell surface. We investigated fluorescence microscopy for C. difficile, by comparing different fluorescent systems previously described, and introduced the use of the fluorophore HaloTag. Additionally, we analyzed C. difficile intrinsic fluorescence and report the potential limitations for live-cell microscopy. The development of the tools enabled the assessment of the localization of the TcdC C-terminus and the characterization of the HupA protein. Finally, we provide new insights into the chromosome remodelling and DNA replication by studying DnaA-dependent unwinding and characterizing the histone-like protein HupA. In the discussion, we present the implications of our findings and provide perspectives for further research on TcdC-mediated toxin regulation and chromosome dynamics, specifically during DNA replication.

Background: The availability of nutrients impacts cell size and growth rate in many organisms. Research in E. coli has traditionally focused on the influence of exogenous nutrient sources on cell size through their effect on growth and cell cycle progression. Utilising a set of mutants where three genes involved in glycogen degradation - glycogen phosphorylase (glgP), glycogen debranching enzyme (glgX) and maltodextrin phosphorylase (malP) - were disrupted, we examined if degradation of this energy storage compound affects cell size. Results: It was found that mutations to malP increased cell lengths and resulted in substantial heterogeneity of cell size. This was most apparent during exponential growth and the phenotype was unaccompanied by alterations in Z-ring occurrence, cellular FtsZ levels and generation times. ∆malP mutant cells did, however, show abnormal nuclear staining patterns and accumulate increased DnaA amounts at late growth stages, indicating a potential effect on DNA replication. Conclusion: This study reveals a link between glycogen metabolism and cell size in E. coli. A disrupted malP allele is associated with cell size heterogeneity and elongation at all stages of growth, without detectable changes to mass doubling time, FtsZ protein levels or division ring frequency.