Overexpression of the cgtA (yhbZ,obgE) gene,coding for an essential GTP-binding protein,impairs the regulation of chromosomal functions in Escherichia coli

GTPases belonging to the Obg/Gtp1 subfamily are essential proteins in most bacterial species and are evolutionarily conservative from bacteria to humans. However, their specific functions in the regulation of cellular processes are largely unknown. Here we demonstrate that overproduction of a member of the Obg/Gtp1 subfamily, cgtA ( yhbZ, obgE) gene product, in Escherichia coli is deleterious for bacterial growth. However, syntheses of DNA, RNA, and proteins were not significantly affected under these conditions as measured by efficiency of incorporation of radioactive precursors. On the other hand, flow cytometry studies revealed that cgtA-overexpressing bacteria form enlarged cells with significantly changed distribution of chromosomal DNA. These results strongly suggest that overproduction of a GTP-binding protein from the Obg/Gtp1 subfamily impairs regulation of some chromosomal functions in E. coli, especially synchronization of DNA replication initiation and possibly also partitioning of daughter chromosomes after a replication round.     

A role for the common GTP-binding protein in coupling of chromosome replication to cell growth and cell division

Homologues of CgtA, the common GTP-binding protein of Vibrio harveyi, are present in diverse organisms ranging from bacteria to humans. In bacteria, proteins homologous to CgtA form a subfamily of small GTP-binding proteins, called Obg/Gtp1. Similarity between bacterial members of this subfamily and their eukaryotic homologues is as high as about 50%. Nevertheless, specific functions of these proteins remain largely unknown. Genes coding for CgtA-like proteins are essential in almost all species of bacteria. The only known exception is V. harveyi, whose cells survive disruption of the cgtA gene. Therefore, the V. harveyi cgtA insertional mutant is a very useful tool for studies on functions of CgtA. Here we demonstrate that under normal growth conditions, cells of the cgtA mutant are slightly larger than wild-type cells, whereas indirect inhibition of DNA replication initiation by addition of rifampicin results in significantly higher differences in average cell size between these two strains as measured by flow cytometry. These differences decreased when cell division was inhibited by cephalexin. DNA synthesis per cell mass was found to be increased in the cgtA mutant relative to wild-type V. harveyi strain, whereas the mutant cells grew slower than bacteria with functional cgtA gene. Kinetics of DNA replication after inhibition of cell division was also considerably different in wild-type and cgtA mutant strains. These results suggest that the cgtA gene product plays a role in coupling of DNA replication to cell growth and cell division.     

The Obg subfamily of bacterial GTP-binding proteins: essential proteins of largely unknown functions that are evolutionarily conserved from bacteria to humans

Members of the Obg subfamily of small GTP-binding proteins (called Obg, CgtA, ObgE or YhbZ in different bacterial species) have been found in various prokaryotic and eukaryotic organisms, ranging from bacteria to humans. Although serious changes in phenotypes are observed in mutant bacteria devoid of Obg or its homologues, specific roles of these GTP-binding proteins remain largely unknown. Recent genetic and biochemical studies, as well as determination of the structures of Obg proteins from Bacillus subtilis and Thermus thermophilus, shed new light on the possible functions of the members of the Obg subfamily and may constitute a starting point for the elucidation of their exact biological role.     

Biochemical and physiological characterization of the GTP-binding protein Obg of Mycobacterium tuberculosis

Background  

Obg is a highly conserved GTP-binding protein that has homologues in bacteria, archaea and eukaryotes. In bacteria, Obg proteins are essential for growth, and they participate in spore formation, stress adaptation, ribosome assembly and chromosomal partitioning. This study was undertaken to investigate the biochemical and physiological characteristics of Obg in Mycobacterium tuberculosis, which causes tuberculosis in humans.     

Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites

In bacteria, Ter sites bound to Tus/Rtp proteins halt replication forks moving only in one direction, providing a convenient mechanism to terminate them once the chromosome had been replicated. Considering the importance of replication termination and its position as a checkpoint in cell division, the accumulated knowledge on these systems has not dispelled fundamental questions regarding its role in cell biology: why are there so many copies of Ter, why are they distributed over such a large portion of the chromosome, why is the tus gene not conserved among bacteria, and why do tus mutants lack measurable phenotypes? Here we examine bacterial genomes using bioinformatics techniques to identify the region(s) where DNA polymerase III‐mediated replication has historically been terminated. We find that in both Escherichia coli and Bacillus subtilis, changes in mutational bias patterns indicate that replication termination most likely occurs at or near the dif site. More importantly, there is no evidence from mutational bias signatures that replication forks originating at oriC have terminated at Ter sites. We propose that Ter sites participate in halting replication forks originating from DNA repair events, and not those originating at the chromosomal origin of replication.     

Structural and biochemical analysis of the Obg GTP binding protein

The Obg nucleotide binding protein family has been implicated in stress response, chromosome partitioning, replication initiation, mycelium development, and sporulation. Obg proteins are among a large group of GTP binding proteins conserved from bacteria to man. Members of the family contain two equally and highly conserved domains, a C-terminal GTP binding domain and an N-terminal glycine-rich domain. Structural analysis of Bacillus subtilis Obg revealed respective domain architectures and how they are coupled through the putative switch elements of the C-terminal GTPase domain in apo and nucleotide-bound configurations. Biochemical analysis of bacterial and human Obg proteins combined with the structural observation of the ppGpp nucleotide within the Obg active sight suggest a potential role for ppGpp modulation of Obg function in B. subtilis.     

An essential GTP-binding protein functions as a regulator for differentiation in Streptomyces coelicolor

The Streptomyces coelicolor obg gene, which encodes a putative GTP-binding protein of the Obg/Gtp1 family, was characterized. The obg gene was essential for viability. Introduction of multiple copies of obg into wild-type S. coelicolor suppressed aerial mycelium formation. A single amino acid substitution at any of six positions was introduced into the GTP binding site of Obg, and the mutated proteins were expressed in wild-type cells. Obg^P168 → V exerted a more accentuated suppressive effect on aerial mycelium formation than did the wild-type Obg protein. In contrast, Obg^G171 → A accelerated the development of aerial mycelium. These results show that Obg protein functions as a pivotal regulator for the onset of cell differentiation through its ability to bind GTP. Western analysis revealed that expression of obg is regulated in a growth phase-dependent manner, indicating a sharp decrease just after onset of aerial mycelium development or at the end of vegetative growth. Obg was a membrane-bound protein as determined by immunoelectron microscopy.     

DNA elements modulating the <Emphasis Type="Italic">KARS12</Emphasis> chromosomal replicator in <Emphasis Type="Italic">Kluyveromyces lactis</Emphasis>

Eukaryotic chromosomal DNA replication is initiated by a highly conserved set of proteins that interact with cis-acting elements on chromosomes called replicators. Despite the conservation of replication initiation proteins, replicator sequences show little similarity from species to species in the small number of organisms that have been examined. Examination of replicators in other species is likely to reveal common features of replicators. We have examined a Kluyeromyces lactis replicator, KARS12, that functions as origin of DNA replication on plasmids and in the chromosome. It contains a 50-bp region with similarity to two other K. lactis replicators, KARS101 and the pKD1 replication origin. Replacement of the 50-bp sequence with an EcoRI site completely abrogated the ability of KARS12 to support plasmid and chromosomal DNA replication origin activity, demonstrating this sequence is a common feature of K. lactis replicators and is essential for function, possibly as the initiator protein binding site. Additional sequences up to 1 kb in length are required for efficient KARS12 function. Within these sequences are a binding site for a global regulator, Abf1p, and a region of bent DNA, both of which contribute to the activity of KARS12. These elements may facilitate protein binding, protein/protein interaction and/or nucleosome positioning as has been proposed for other eukaryotic origins of DNA replication.     

Beyond DnaA: The Role of DNA Topology and DNA Methylation in Bacterial Replication Initiation

The replication of chromosomal DNA is a fundamental event in the life cycle of every cell. The first step of replication, initiation, is controlled by multiple factors to ensure only one round of replication per cell cycle. The process of initiation has been described most thoroughly for bacteria, especially Escherichia coli, and involves many regulatory proteins that vary considerably between different species. These proteins control the activity of the two key players of initiation in bacteria: the initiator protein DnaA and the origin of chromosome replication (oriC). Factors involved in the control of the availability, activity, or oligomerization of DnaA during initiation are generally regarded as the most important and thus have been thoroughly characterized. Other aspects of the initiation process, such as origin accessibility and susceptibility to unwinding, have been less explored. However, recent findings indicate that these factors have a significant role. This review focuses on DNA topology, conformation, and methylation as important factors that regulate the initiation process in bacteria. We present a comprehensive summary of the factors involved in the modulation of DNA topology, both locally at oriC and more globally at the level of the entire chromosome. We show clearly that the conformation of oriC dynamically changes, and control of this conformation constitutes another, important factor in the regulation of bacterial replication initiation. Furthermore, the process of initiation appears to be associated with the dynamics of the entire chromosome and this association is an important but largely unexplored phenomenon.     

Study of the DnaB:DciA interplay reveals insights into the primary mode of loading of the bacterial replicative helicase

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.     

Biochemical characterization of arabidopsis developmentally regulated G-proteins (DRGs)

Developmentally regulated G-proteins (DRGs) are a highly conserved family of GTP-binding proteins found in archaea, plants, fungi and animals, indicating important roles in fundamental pathways. Their function is poorly understood, but they have been implicated in cell division, proliferation, and growth, as well as several medical conditions. Individual subfamilies within the G-protein superfamily possess unique nucleotide binding and hydrolysis rates that are intrinsic to their cellular function, and so characterization of these rates for a particular G-protein may provide insight into its cellular activity. We have produced recombinant active DRG protein using a bacterial expression system and refolding, and performed biochemical characterization of their GTP binding and hydrolysis. We show that recombinant Arabidopsis thaliana atDRG1 and atDRG2a are able to bind GDP and GTP. We also show that DRGs can hydrolyze GTP in vitro without the assistance of GTPase-activating proteins and guanine exchange factors. The atDRG proteins hydrolyze GTP at a relatively slow rate (0.94 × 10⁻³ min⁻¹ for DRG1 and 1.36 × 10⁻³ min⁻¹ for DRG2) that is consistent with their nearest characterized relatives, the Obg subfamily. The ability of DRGs to bind nucleotide substrates without assistance, their slow rate of GTP hydrolysis, heat stress activation and domain conservation suggest a possible role as a chaperone in ribosome assembly in response to stress as it has been suggested for the Obg proteins, a different but related G-protein subfamily.     

Developmental stage influences chromosome segregation patterns and arrangement in the extremely polyploid,giant bacterium Epulopiscium sp. type B

Few studies have described chromosomal dynamics in bacterial cells with more than two complete chromosome copies or described changes with respect to development in polyploid cells. We examined the arrangement of chromosomal loci in the very large, highly polyploid, uncultivated intestinal symbiont Epulopiscium sp. type B using fluorescent in situ hybridization. We found that in new offspring, chromosome replication origins (oriCs) are arranged in a three‐dimensional array throughout the cytoplasm. As development progresses, most oriCs become peripherally located. Siblings within a mother cell have similar numbers of oriCs. When chromosome orientation was assessed in situ by labeling two chromosomal regions, no specific pattern was detected. The Epulopiscium genome codes for many of the conserved positional guide proteins used for chromosome segregation in bacteria. Based on this study, we present a model that conserved chromosomal maintenance proteins, combined with entropic demixing, provide the forces necessary for distributing oriCs. Without the positional regulation afforded by radial confinement, chromosomes are more randomly oriented in Epulopiscium than in most small rod‐shaped cells. Furthermore, we suggest that the random orientation of individual chromosomes in large polyploid cells would not hamper reproductive success as it would in smaller cells with more limited genomic resources.     

DnaA structure, function, and dynamics in the initiation at the chromosomal origin

Escherichia coli DnaA is the initiator of chromosomal replication. Multiple ATP-DnaA molecules assemble at the oriC replication origin in a highly regulated manner, and the resultant initiation complexes promote local duplex unwinding within oriC, resulting in open complexes. DnaB helicase is loaded onto the unwound single-stranded region within oriC via interaction with the DnaA multimers. The tertiary structure of the functional domains of DnaA has been determined and several crucial residues in the initiation process, as well as their unique functions, have been identified. These include specific DNA binding, inter-DnaA interaction, specific and regulatory interactions with ATP and with the unwound single-stranded oriC DNA, and functional interaction with DnaB helicase. An overall structure of the initiation complex is also proposed. These are important for deepening our understanding of the molecular mechanisms that underlie DnaA assembly, oriC duplex unwinding, regulation of the initiation reaction, and DnaB helicase loading. In this review, we summarize recent progress on the molecular mechanisms of the functions of DnaA on oriC. In addition, some members of the AAA+ protein family related to the initiation of replication and its regulation (e.g., DnaA) are briefly discussed.     

The twisted 'life' of DNA in the cell: bacterial topoisomerases

DNA topoisomerases are essential to the cell for the regulation of DNA supercoiling levels and for chromosome decatenation. The proposed mechanisms for these reactions are essentially the same, except that a change in supercoiling is due to an intramolecular event, while decatenation requires an intermolecular event. The characterized bacterial topoisomerases appear capable of both types of reaction in vitro. Four DNA topoisomerases have been identified in Escherichia coli. Topoisomerase I, gyrase, and topoisomerase IV normally appear to have distinct essential functions within the cell, Gyrase and topoisomerase I are responsible for the regulation of DNA supercoiling. Both gyrase and topoisomerase IV are necessary for chromosomal decatenation. Multiple topoisomerases with distinct functions may give the cell more precise control over DNA topology by allowing tighter regulation of the principal enzymatic activities of these different proteins.     

Autonomous plasmid‐like replication of a conjugative transposon

Integrative and conjugative elements (ICEs), a.k.a. conjugative transposons, are mobile genetic elements involved in many biological processes, including pathogenesis, symbiosis and the spread of antibiotic resistance. Unlike conjugative plasmids that are extra‐chromosomal and replicate autonomously, ICEs are integrated in the chromosome and replicate passively during chromosomal replication. It is generally thought that ICEs do not replicate autonomously. We found that when induced, Bacillus subtilis ICEBs1 undergoes autonomous plasmid‐like replication. Replication was unidirectional, initiated from the ICEBs1 origin of transfer, oriT, and required the ICEBs1‐encoded relaxase NicK. Replication also required several host proteins needed for chromosomal replication, but did not require the replicative helicase DnaC or the helicase loader protein DnaB. Rather, replication of ICEBs1 required the helicase PcrA that is required for rolling circle replication of many plasmids. Transfer of ICEBs1 from the donor required PcrA, but did not require replication, indicating that PcrA, and not DNA replication, facilitates unwinding of ICEBs1 DNA for horizontal transfer. Although not needed for horizontal transfer, replication of ICEBs1 was needed for stability of the element. We propose that autonomous plasmid‐like replication is a common property of ICEs and contributes to the stability and maintenance of these mobile genetic elements in bacterial populations.     

Genome Sizes of Desulfovibrio desulfuricans, Desulfovibrio vulgaris, and Desulfobulbus propionicus Estimated by Pulsed-Field Gel Electrophoresis of Linearized Chromosomal DNA

Pulsed-field gel electrophoresis (PFGE) of linearized, full-length chromosomal DNA was used to estimate the genome sizes of three species of sulfate-reducing bacteria. Genome sizes of Desulfovibrio desulfuricans, Desulfovibrio vulgaris, and Desulfobulbus propionicus were estimated to be 3.1, 3.6, and 3.7 Mb, respectively. These values are double the genome sizes previously determined for two Desulfovibrio species by two-dimensional agarose gel electrophoresis of DNA cut with restriction enzymes. PFGE of full-length chromosomal DNA could provide a generally applicable method to rapidly determine bacterial genome size and organization. Received: 1 October 1996 / Accepted: 5 November 1996     

Hda, a novel DnaA-related protein, regulates the replication cycle in Escherichia coli

The bacterial DnaA protein binds to the chromosomal origin of replication to trigger a series of initiation reactions, which leads to the loading of DNA polymerase III. In Escherichia coli, once this polymerase initiates DNA synthesis, ATP bound to DnaA is efficiently hydrolyzed to yield the ADP-bound inactivated form. This negative regulation of DnaA, which occurs through interaction with the beta-subunit sliding clamp configuration of the polymerase, functions in the temporal blocking of re-initiation. Here we show that the novel DnaA-related protein, Hda, from E.coli is essential for this regulatory inactivation of DnaA in vitro and in vivo. Our results indicate that the hda gene is required to prevent over-initiation of chromosomal replication and for cell viability. Hda belongs to the chaperone-like ATPase family, AAA(+), as do DnaA and certain eukaryotic proteins essential for the initiation of DNA replication. We propose that the once-per-cell-cycle rule of replication depends on the timely interaction of AAA(+) proteins that comprise the apparatus regulating the activity of the initiator of replication.     

Structure and interactions of the Bacillus subtilis sporulation inhibitor of DNA replication,SirA, with domain I of DnaA

Chromosome copy number in cells is controlled so that the frequency of initiation of DNA replication matches that of cell division. In bacteria, this is achieved through regulation of the interaction between the initiator protein DnaA and specific DNA elements arrayed at the origin of replication. DnaA assembles at the origin and promotes DNA unwinding and the assembly of a replication initiation complex. SirA is a DnaA‐interacting protein that inhibits initiation of replication in diploid Bacillus subtilis cells committed to the developmental pathway leading to formation of a dormant spore. Here we present the crystal structure of SirA in complex with the N‐terminal domain of DnaA revealing a heterodimeric complex. The interacting surfaces of both proteins are α‐helical with predominantly apolar side‐chains packing in a hydrophobic interface. Site‐directed mutagenesis experiments confirm the importance of this interface for the interaction of the two proteins in vitro and in vivo. Localization of GFP–SirA indicates that the protein accumulates at the replisome in sporulating cells, likely through a direct interaction with DnaA. The SirA interacting surface of DnaA corresponds closely to the HobA‐interacting surface of DnaA from Helicobacter pylori even though HobA is an activator of DnaA and SirA is an inhibitor.     

The Escherichia coli GTPase ObgE modulates hydroxyl radical levels in response to DNA replication fork arrest

Obg proteins are universally conserved GTP-binding proteins that are essential for viability in bacteria. Homologs in different organisms are involved in various cellular processes, including DNA replication. The goal of this study was to analyse the structure-function relationship of Escherichia?coli ObgE with regard to DNA replication in general and sensitivity to stalled replication forks in particular. Defined C-terminal chromosomal deletion mutants of obgE were constructed and tested for sensitivity to the replication inhibitor hydroxyurea. The ObgE C-terminal domain was shown to be dispensable for normal growth of E.?coli. However, a region within this domain is involved in the cellular response to replication fork stress. In addition, a mutant obgE over-expression library was constructed by error-prone PCR and screened for increased hydroxyurea sensitivity. ObgE proteins with substitutions L159Q, G163V, P168V, G216A or R237C, located within distinct domains of ObgE, display dominant-negative effects leading to hydroxyurea hypersensitivity when over-expressed. These effects are abolished in strains with a single deletion of the iron transporter TonB or combined deletions the toxin/antitoxin modules RelBE/MazEF, strains both of which have been shown to be involved in a pathway that stimulates hydroxyl radical formation following hydroxyurea treatment. Moreover, the observed dominant-negative effects are lost in the presence of the hydroxyl radical scavenger thiourea. Together, these results indicate involvement of hydroxyl radical toxicity in ObgE-mediated protection against replication fork stress.