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.

molecular mass and occurring as monomers in their active form) are involved in a number of essential processes, for example: signal transduction, protein synthesis and translocation, and cell cycle regulation (for reviews see, for example, Sprang, 1997;Caldon & March, 2003).Perhaps surprisingly, relatively little information is currently available about the roles of GTP-binding proteins in prokaryotes.
The best known prokaryotic small GTP-binding protein is Era (named for 'Escherichia coli Ras-like protein').This protein is essential for bacterial growth, and mutants in the era gene reveal pleiotropic phenotypes, including alterations in the regulation of carbon metabolism, the stringent response, and cell division (Lerner & Inouye, 1991;Britton et al., 1997;1998).Era bears an RNA-binding motif (Chen et al., 1999) and binds to the 30S ribosomal subunit (Sayed et al., 1999).
Apart from Era, a group of other small GTP-binding proteins (revealing GTPase activities) was discovered in prokaryotic cells.Interestingly, members of this group have homologues in diverse organisms ranging from bacteria to humans (Okamoto & Ochi, 1998;Czyż et al., 2001;Sikora-Borgula et al., 2002;Ishikawa et al., 2003).Recently, Leipe et al. (2002) presented an excellent work in which all available sequences and structures of the most abundant group of GTPases (so called P-loop GTPases) were analyzed, and their classification was proposed.According to this classification (shown in a graphic form in Fig. 1), a subfamily of proteins named Obg, after its best investigated member -the Obg protein of Bacillus subtilis, has been distinguished.The Obg subfamily, together with four other subfamilies (DRG, YyaF/YchF, Ygr210, and NOG1) forms the OBG family, which includes both bacterial and eukaryotic GTP-binding proteins.Together with the HflX family, OBG forms the OBG-HflX superfamily (Leipe et al., 2002).
Examples of bacterial members of the Obg subfamily are Obg proteins from B. subtilis, Streptomyces griseus and S. coelicolor, CgtA proteins from Caulobacter crescentus, Escherichia coli (E. coli CgtA is also called ObgE) and Vibrio harveyi, and YhbZ from Haemophilus influenzae (Trach & Hoch, 1989;Maddock et al., 1997;Okamoto & Ochi, 1998;Czyż et al., 2001;Kobayashi et al., 2001;Dutkiewicz et al., 2002).Below, some of these proteins are described in more detail in the light of results of recent studies that provided basic information about their structure and possible functions.

THE Obg PROTEIN OF BACILLUS SUBTILIS
The obg gene was discovered in B. subtilis by Trach and Hoch (1989).Its name is for 'Spo0B-associated GTP-binding protein' since the obg gene product was found to be involved in the control of sporulation.Namely, genetic studies led to proposals that Obg may regulate initiation of sporulation (Trach & Hoch, 1989;Vidwans et al., 1995).Results of other experiments suggested that Obg may be involved in the control of DNA replication (Kok et al., 1994), but the proposed hypothesis was highly speculative.Subsequent studies demonstrated that the obg gene function is necessary for stress-dependent activation of transcription factor s B (Scott & Haldenwang, 1999).
It was proposed that Obg can function by sensing intracellular GTP level (Kok et al., 1994) and may be required to stimulate the activity of the phosphorelay system (Vidwans et al., 1995).More recent evidence points to a role of Obg in ribosome function.In B. subtilis cells, the Obg protein exists as a large cytoplasmic complex, coelutes with ribosomal subunits and specifically interacts with the ribosomal protein L13 (Scott et al., 2000).
As can be deduced from the description presented above, Obg, one of six essential GTP-binding proteins discovered in B. subtilis (Morimoto et al., 2002), is involved in many crucial physiological processes, but its exact cellular function is unknown.Recent studies on the structure of this protein shed new light on a possible role for this protein.Buglino et al. (2002) have determined the 2.6 C resolution X-ray structure for Obg.This structure reveals common features of the G domain.Such a domain typically contains six b strands and five a helices (see Wittinghofer, 2002).In fact, the whole Obg protein consists of three domains.The N-terminal domain (residues 1-158) contains a sequence highly conserved among other members of the OBG family, but it does not reveal structural similarity to any other known protein.The second domain (residues 161-342) is a conserved GTP-binding domain similar to that found in small Ras-like GTPases.The C-terminal domain (residues 342-428) of Obg is structurally similar to a domain found in bacterial stress response proteins, among others the SpoT protein, and is called TGS.
The global structure of Obg from Thermus thermophilus (Fig. 2), described recently by Kukimoto-Niino et al. (2004), is similar to that of B. subtilis.However, detailed analysis of the structural data led to a suggestion about a possible domain rearrangement of Obg, and a potential role of its C-terminal domain in the regulation of nucleotide binding (Kukimoto-Niino et al., 2004).
The discovery of the TGS domain in the Obg protein was intriguing.SpoT is a protein bearing two opposite enzymatic activities: that of guanosine-5¢-diphosphate-3¢-diphosphate (ppGpp) synthetase and of ppGpp-ase (see Mechold et al., 2002 and references therein).ppGpp is a specific nucleotide, synthesized extensively in amino acid-starved cells, which plays a role as a starvation alarmone and a global regulator of gene expression due to its direct interaction with RNA polymerase (for a review see Cashel et al., 1996).More detailed structural studies revealed that Obg contains a bound nucleotide.This nucleotide was identified as ppGpp by Buglino et al. (2002).These authors speculated that the Obg protein may have evolved to recognize ppGpp in response to changes in the cellular environment.
It is of special interest that the biochemical properties of the Obg protein of B. subtilis and its homologue from human cells have been found to be very similar (Buglino et al., 2002).These results were found intriguing because it was generally believed that ppGpp is not involved in eukaryotic stress response (see Wittinghofer, 2002).However, recent studies demonstrated that ppGpp is produced in eukaryotic cells, namely in a unicellular photosynthetic organism, Chlamydomonas reinhardtii (Kasai et al., 2002), and also possibly in Arabidopsis (van der Biezen et al., 2000).In C. reinhardtii, ppGpp localizes to chloroplasts (Kasai et al., 2002).These results imply that, contrary to previous assumptions, ppGpp may be involved in the intracellular signaling in plants.Thus, a lack of ppGpp function in animal and human cells seems not to be so obvious how as it was assumed a few years ago.

CAULOBACTER CRESCENTUS
A homologue of the B. subtilis Obg protein was found in C. crescentus, a bacterium that has a special developmental program, in which two types of cells are produced.This protein was named CgtA, for 'Caulobacter GTP-binding protein' (Maddock et al., 1997).
CgtA is essential for cell viability and is present at low levels throughout the cell cycle (Maddock et al., 1997).Furthermore, it was shown that the CgtA protein displays unique guanine nucleotide binding and exchange parameters (Lin et al., 1999;Lin & Maddock, 2001;Lin et al., 2001).Whereas the well-studied eukaryotic Ras-like proteins bind guanine nucleotides with high affinity, CgtA binds them with only moderate affinity.In addition, CgtA exchanges GDP and GTP rapidly.These biochemical features are consistent with a model in which the intracellular guanine nucleotide pools govern the guanine nucleotide occupancy of CgtA.
The biochemical data described above suggest a model in which in the absence of a guanine nucleotide dissociation inhibitor, the guanine nucleotide bound state of CgtA would be controlled by the relative ratio of cellular concentrations of GTP and GDP.Similarly to the Obg protein of B. subtilis, CgtA of C. crescentus cofractionates with ribosomes (Lin et al., 2004).Therefore, one could also speculate that CgtA might either be involved in ribosome assembly or in monitoring the assembly-state of the ribosomes.However, perhaps unexpectedly, recent studies on conditional C. crescentus cgtA mutants revealed that the lethal phenotype of cgtA dysfunction is not due to impaired ribosome function (Datta et al., 2004).Moreover, the same studies revealed that CgtA is necessary for DNA replication and progression through the cell cycle (Datta et al., 2004).Definitely, further studies are necessary to understand the precise role of CgtA in C. crescentus cells.

THE CgtA (ObgE) PROTEINS OF BACTERIA THAT DO NOT SPORULATE AND DO NOT HAVE A SPECIAL DEVELOPMENTAL PROGRAM
The first obg gene was discovered in B. subtilis, and was considered to be involved in the regulation of sporulation (Trach & Koch, 1989).Extensive biochemical studies were performed with the CgtA protein from C. crescentus, a bacterium that have a special developmental program, in which two types of cells are produced (see preceding paragraph).However, homologues of Obg/CgtA proteins are present in all bacteria investigated thus far (Czy¿ et al., 2001 and references therein).Therefore, it is perhaps surprising that relatively little information is available about the member of the Obg subfamily from E. coli.Arigoni et al. (1998) demonstrated that the gene coding for this protein, formerly catalogued as yhbZ, is essential for E. coli growth, but no specific function of this gene was determined at that time.Currently, even the nomenclature of this gene is controversial.It was proposed to call this gene obgE, for 'obg of E. coli' (Kobayashi et al., 2001).However, the name obg was established for 'Spo0B-associated GTP-binding protein' of B. subtilis, whereas E. coli does not sporulate and there is no homologue or analogue of the Spo0B protein in this bacterium.Therefore, the name obgE was considered as confusing by Dutkiewicz et al. (2002) who proposed the name cgtA, which is identical to that of the C. crescentus gene coding for a member of the Obg subfamily.However, different etymology was proposed, namely 'common GTP-binding protein' (Dutkiewicz et al., 2002), which would better symbolize the features of this gene than the names yhbZ or obgE.Kobayashi et al. (2001) constructed a temperature-sensitive cgtA (obgE) allele, which was expressed from a plasmid in E. coli cells devoid of a functional chromosomal copy of this gene.Studies on such a strain indicated that partitioning of daughter chromosomes after a replication round is impaired in the absence of CgtA (ObgE) function.Similar conclusions were presented on the basis of studies on another Gram-negative, non-sporulating and non-differentiating bacterium, Vibrio harveyi (S³omiñska et al., 2002).Moreover, a weak DNA binding activity of the CgtA (ObgE) protein was found in vitro (Kobayashi et al., 2001).Experiments with the use of bacteria overproducing E. coli CgtA (ObgE) suggested that this protein may have a role in synchronization of DNA replication, although the kinetics of DNA, RNA and protein syntheses were not significantly affected under these conditions (Dutkiewicz et al., 2002).
Contrary to the suggestions of Kobayashi et al. (2001), it has recently been demonstrated that E. coli CgtA (ObgE) protein is involved in DNA replication regulation.Namely, although replication of ColE1-like plasmids was not affected in the cgtA mutant, dysfunction of this gene caused a strong inhibition of l plasmid DNA replication (Ulanowska et al., 2003).Bacteriophage l development was also severely impaired in the cgtA mutant.Replication of other plasmid replicons (derivatives of F, R1, R6K and RK2) was moderately influenced by the cgtA mutation (Ulanowska et al., 2003).
It is worth mentioning that two other essential GTP-binding proteins of B. subtilis, Bex and YqeH, appear to participate in the regulation of chromosome replication (Morimoto et al., 2002).It seems that DNA synthesis per se is not affected by CgtA in E. coli, and that this protein may control replication initiation indirectly, by regulation of the function(s) or production of one or more replication factors.In fact, it was found that level of the host-encoded replication protein DnaA is significantly decreased in the cgtA mutant (Ulanowska et al., 2003).This indicates that CgtA is involved in the regulation of gene expression, which had not been noticed previously.
CgtA-mediated gene expression seems also to play a crucial role in the mechanism of enhancement of survival of cells after UV irradiation.Expression of the cgtA gene was found to be enhanced after UV-irradiation of cells (Zielke et al., 2003).Moderate overexpression of cgtA resulted in increased UV-resistance of E. coli wild-type and dnaQ strains but not of uvrA, uvrB, umuC and recA mutants (Zielke et al., 2003), suggesting that the RecA-dependent excinuclease and error-prone DNA repair systems may be stimulated by CgtA.It was also demonstrated that the basal level of the RecA protein was lower in a temperature-sensitive cgtA mutant of E. coli than in the cgtA + strain, and unlike in wild-type bacteria, no significant increase in the recA gene expression was observed after UV irradiation of this cgtA mutant (Zielke et al., 2003).Therefore, it appears that the cgtA gene product is involved in DNA repair processes, most probably by stimulation of recA gene expression and resultant activation of RecA-dependent DNA repair pathways.Interestingly, the impairment of DNA repair efficiency in cgtA mutants was used in a new microbiological test to detect mutagenic pollution of the environment (Czyż et al., 2000;2002;2003;Węgrzyn & Czyż, 2003).Overexpression of the cgtA (obgE) gene suppressed the phenotype normally associated with defects in the rrmJ (ftsJ) gene, coding for an rRNA methyltransferase, by an unknown mechanism (Tan et al., 2002).
Very recent studies by Foti et al. (2005) provided another support for the proposal that CgtA is involved in DNA replication regulation.They have isolated an E. coli mutant in the cgtA gene (called again obgE by them) which was very sensitive to various DNA replication inhibitors.Genetic analysis suggested that chromosome brakes and regressed replication forks may accumulate in this mutant.Overproduction of CgtA caused spreading of the SeqA protein within the cell (SeqA is normally localized to replication forks, and can be observed as specific foci).Therefore, Foti et al. (2005) proposed that CgtA is involved in promotion of bacterial cell survival when replication forks are arrested.Moreover, report by Foti et al. (2005) opens many more questions and provide further suggestions about involvement of CgtA in the regulation of chromosomal DNA replication at various stages, including the initiation step.
Similarly to the Obg protein from B. subtilis and CgtA from C. crescentus, the CgtA protein from E. coli cofractionates with the large ribosomal subunit (Wout et al., 2004).In the light of the discovery that ppGpp may be bound to CgtA, it was intriguing that CgtA may directly interact with SpoT (a ppGpp synthetase and hydrolase), as revealed by coprecipitation experiments and by two-hybrid assays (Wout et al., 2004).Undoubtedly, studies on the putative interplay between GTP-binding proteins and those involved in ppGpp metabolism may lead to very interesting discoveries which might tell us more about the physiological functions of CgtA and related proteins.

PERSPECTIVES
The evolutionary conservation of the members of the Obg subfamily of GTP-binding proteins, from bacteria to humans, is comparable to that found in a group of heat shock proteins (Leipe et al., 2002).This implies crucial functions of Obg-like proteins in cells of most, if not all, organisms.Recent studies have revealed that these proteins are involved in many basic cellular processes, including chromosomal functions, reactions of the protein synthesis machinery, control of gene expression, and regulation of stress responses and developmental processes.Determination of the Obg protein structure of B. subtilis, construction of mutants in the homologous genes of other bacteria, and purification and basic biochemical characterization of Obg and CgtA (from C. crescentus) allowed us to learn about basic functions of these very important proteins (Caldon & March, 2003).Nevertheless, it is still unknown what is the primary role of the members of the Obg subfamily, and how so many different cellular processes can be regulated by these relatively small proteins.
We are grateful to Janine Maddock for many fruitful discussions, and to members of our team who have participated in projects on Obg-like proteins.

Figure 1 .
Figure 1.GTPases and related ATPases which bear the P-loop structure, classified according to the proposal of Leipe et al. (2002).The schematic classification is shown at the levels of classes, superfamilies and families.

Figure 2 .
Figure 2. A model for Thermus thermophilus Obg protein.Particular colors represent: cyan, the N-terminal domain; orange, the C-terminal domain; yellow, the switch-I region of the G domain; green, the switch-II region of the G domain; purple and red, other parts of the G domain.The atomic coordinates of the Obg protein from T. thermophilus are available from the Protein Data Bank, with the accession code 1UDX.This figure is reprinted from: Kukimoto-Niino M, et al., (2004) J Mol Biol.; 337: 761-770, with permission from Elsevier.