Interaction between MazG and Era in the yeast two-hybrid system.
To identify E. coli
proteins that interact with Era, we constructed three E. coli
genomic libraries and screened the libraries using Era as bait in the yeast two-hybrid system. Potential interactions were selected in the yeast two-hybrid reporter strain PJ69-4A (16
). A library screen was performed as described in Materials and Methods. Library plasmids, which enabled PJ69-4A cells to grow on SD minimal medium lacking Trp, Leu, His, and Ade only in the presence of plasmid pGBD-Era, were sequenced and subjected to a BLAST search of the E. coli
genome database. Twelve positive library plasmids containing the various fragments from the mazG
gene were identified. The MazG protein consists of 263 amino acid residues. Eight positive plasmids contained a fragment from residues 58 to 263, three from residues 124 to 263, and one from residues 88 to 263. Sequence analysis revealed that all of the mazG
sequences in these plasmids were in the same reading frame as the GAL4
transcriptional activation domain.
Two-hybrid assays were further performed to unambiguously demonstrate that Era indeed interacts with MazG, excluding any effects from genes downstream of the mazG gene. For this purpose, the full-length mazG gene was cloned into the pGAD-C1 vector and cotransformed with pGBD-Era or pGBD-C1 vector into PJ69-4A yeast cells. In order to localize the interaction regions between Era and MazG proteins, a series of N- and C-terminal deletions of MazG were also constructed in pGAD-C1 and cotransformed with pGBD-Era or pGBD-C1 vector into PJ69-4A cells, as shown in Fig. . The cotransformants harboring pGAD-MazG, pGAD-MazGΔ(1-57), pGAD-MazGΔ(1-87), or pGAD-MazGΔ(1-123) with pGBD-Era were able to grow on SD minimal medium lacking Trp, Leu, His, and Ade, while the cotransformants with the pGBD-C1 vector were not. All of these cotransformants with pGBD-Era displayed a significant increase in β-galactosidase activity (Fig. ). Similar results were obtained in the reciprocal two-hybrid assays with the era gene inserted in the pGAD-C1 vector and mazG fragments inserted in the pGBD-C1 vector (data not shown). These data confirmed the initial two-hybrid library screen results, demonstrating that full-length MazG, MazGΔ(1-57), MazGΔ(1-87), and MazGΔ(1-123) can interact with Era. Further deletion from the N terminus of MazG [MazGΔ(1-149)] and deletions from the C terminus of MazG [MazGΔ(230-263) and MazGΔ(200-263)] (Fig. ) disrupted the two-hybrid interaction with Era. These results indicate that the C-terminal domain consisting of 140 amino acid residues of MazG is indispensable in the interaction with Era in the two-hybrid system.
FIG. 1. MazG and Era interaction in the yeast two-hybrid system. (A) The MazG C-terminal region is required for the interaction with Era. All mazG truncation mutations were constructed in pGAD-C1 as described in Materials and Methods. Numbers refer to the amino (more ...)
As shown in Fig. , a series of truncation mutations from the N and C termini of Era and a G2 region deletion mutation of Era were constructed in pGBD-C1 and cotransformed with pGAD-MazG into PJ69-4A cells. All of these cotransformed yeast cells were unable to grow on SD minimal medium in the absence of Trp, Leu, His, and Ade, indicating that these Era mutants were unable to interact with MazG. Therefore, both the N- and C-terminal regions of Era appear to be essential for the interaction with MazG. It is possible that both termini of Era contain residues involved in the interaction with MazG, or the whole structure of Era may be required for binding to MazG.
Interaction of MazG with Era in vitro.
In vitro experiments were performed to confirm the interaction between MazG and Era. MBP-MazG was mixed with Era, and then amylose resin was added to the mixture. Proteins binding to the amylose resin were eluted with maltose and were then subjected to SDS-PAGE. By Western blot analysis using the anti-Era antiserum as shown in Fig. , it was found that Era coeluted with MBP-MazG in the presence of GDP (Fig. , lane 2) or GTPγS (Fig. , lane 4), but not with MBP protein (Fig. , lanes 1 and 3). These results were reproducible, indicating that Era can form a complex with MBP-MazG by interacting with MazG. The complex formation seems to be stronger in the presence of GDP (Fig. , lane 2) than in the presence of GTPγS (Fig. , lane 4). In the absence of either nucleotide, the Era binding affinity to MazG was about at the same level as in the presence of GTPγS (data not shown).
In order to further confirm Era-MazG complex formation, MBP-Era and 35S-labeled MazG produced in a cell-free system were mixed and protein complexes were trapped by amylose resin as mentioned above. The proteins eluted from the resin with maltose were subjected to SDS-PAGE, and then 35S-labeled MazG was detected by autoradiography. It was found that 35S-labeled MazG could form a complex with MBP-Era in the presence of GDP (Fig. , lane 2) or GTPγS (Fig. , lane 4), but not with MBP (Fig. , lanes 1 and 3). It appeared that more 35S-labeled MazG was trapped on the resin with MBP-Era in the presence of GDP (Fig. , lane 2) than in the presence of GTPγS (Fig. , lane 4). The results of the in vitro experiments were consistent with the results from the yeast two-hybrid system that Era is able to interact with MazG and furthermore suggested that GDP-bound Era may bind more strongly to MazG than to GTP-bound Era.
Expression and purification of MazG.
After E. coli BL21 harboring pET11a-MazG was induced with isopropyl-β-d-thiogalactopyranoside (IPTG), MazG was produced to about 30% of the total protein and formed a major band with an approximate molecular mass of 30 kDa on the SDS-PAGE gel (not shown). MazG was purified by gel filtration followed by Q-Sepharose column chromatography to near homogeneity. When analyzed by gel filtration, the purified MazG was eluted at around 60 kDa, indicating that MazG exists as a dimer (data not shown).
MazG has no effect on the GTPase activity of Era.
In order to examine whether the interaction between Era and MazG has any effect on the GTPase activity of Era, Era was incubated with MazG in the presence of [α-32P]GTP at 37°C. Nucleotide analysis was performed by polyethyleneimine thin-layer chromatography. It was found that MazG had no significant effect on the GTPase activity of Era, while, surprisingly, MazG could efficiently convert GTP to GMP, suggesting that MazG may have a nucleoside triphosphate pyrophosphohydrolase activity against GTP. Era also had no effect on the GTP hydrolysis activity of MazG (Fig. ). As the GDP-bound Era has a stronger binding affinity to MazG than GTP-bound Era, GDP was added into the reaction mixture in 5 to 10 times molar excess of GTP to examine the effect of GDP on GTP hydrolysis. It was found that the GTPase activity of Era was significantly inhibited with higher GDP concentrations but was not affected by adding MazG into the reaction mixture (data not shown), indicating that the interaction between Era and MazG does not modulate their individual GTP hydrolysis activities.
FIG. 3. Effect of MazG on the GTPase activity of Era. MazG was incubated with increasing concentrations of Era and 100 μM GTP with 5 μCi of [α-32P]GTP in 20 μl of reaction buffer at 37°C for 30 min as described in Materials (more ...) Properties of MazG.
We further attempted to characterize the nucleoside triphosphate pyrophosphohydrolase activity by incubating the purified MazG with [α-32P]ATP and [α-32P]GTP at 37°C, respectively. Nucleotide analysis was performed by polyethyleneimine-cellulose thin-layer chromatography. It was revealed that MazG not only converted GTP to GMP (Fig. , lane 4) but also converted ATP to AMP (Fig. , lane 2). When [γ-32P]GTP was used as the substrate for MazG and the reaction products were separated by paper chromatography and visualized by autoradiography, a strong radioactive signal was observed at the position of the pyrophosphate marker (Fig. , lane 2). When yeast inorganic pyrophosphatase was added into the reaction mixture, the radioactive pyrophosphate spot disappeared with the concomitant appearance of a new spot corresponding to the position of the inorganic phosphate marker (Fig. , lane 3). These results indicate that MazG has nucleoside triphosphate pyrophosphohydrolase activity hydrolyzing GTP to GMP and PPi. When the GTP hydrolysis activity of MazG was tested in the presence of the unlabeled nucleotides as competitors (200-fold excess), GTP, ATP, CTP, and UTP effectively competed with GTP to block the reaction. On the other hand, GDP, GMP, and GTPγS had no significant influence on the hydrolysis of GTP (Fig. ). It was also found that dGTP, dATP, dCTP, and dTTP effectively competed with GTP to block the reaction (data not shown).
FIG. 4. Nucleoside triphosphate pyrophosphohydrolase activity of MazG. (A) Conversion of ATP to AMP and of GTP to GMP by MazG. Reactions were performed in 20 μl of reaction buffer containing 10 μM ATP with 1 μCi of [α-32P]ATP (lanes (more ...)
FIG. 5. GTP hydrolysis activity of MazG in the presence of nucleotide competitors. The GTP hydrolysis activity assays were carried out in 20 μl of reaction buffer containing 10 μM GTP with 1 μCi of [α-32P]GTP and 0.5 μg (more ...)
Next, the activity of MazG toward each of the eight canonical ribo- and deoxynucleoside triphosphates was determined by the colorimetric assay as described in Materials and Methods. MazG hydrolyzed all eight of the canonical ribo- and deoxynucleoside triphosphates to their respective monophosphates and PPi. Pi was not detected unless inorganic pyrophosphatase was added to the reaction mixtures. At a fixed concentration of 4 mM, the most preferred substrate for MazG was found to be dTTP, followed by dATP and dCTP (Fig. ). It seems that MazG has higher activity towards deoxynucleoside triphosphates than ribonucleoside triphosphates, indicating that MazG recognizes not only the nucleotide base but also the sugar group.
FIG. 6. Substrate specificity of MazG. The hydrolysis of various substrates was detected with a standard colorimetric assay as described in Materials and Methods. The hydrolysis activity with each substrate was depicted relative to the activity with dTTP, which (more ...)
Similar to other nucleoside triphosphate pyrophosphohydrolases, the nucleotide hydrolysis by MazG requires divalent cation Mg2+ or Mn2+ (5 mM). The addition of EDTA effectively blocked its activity. The effect of pH on the nucleotide hydrolysis activity of MazG was analyzed under various pH conditions. The nucleotide hydrolysis activity of MazG has an optimum at pH 9.5 (not shown).
What is the role of MazG?
There are 52 protein sequences homologous to E. coli MazG, two of them from Archaea, and the rest from bacteria. Sequence alignments of MazG proteins from E. coli, Yersinia pestis, Vibrio cholerae, Pasteurella multocida, Haemophilus influenzae, Caulobacter crescentus, Agrobacterium tumefaciens, and Thermotoga maritima are shown in Fig. . MazG is highly conserved and the sequence similarities to E. coli MazG in these bacteria are present throughout the sequence.
FIG. 7. Sequence alignments of MazG proteins. Sequence alignments of eight bacterial MazG proteins are shown. The ClustalW program was used for alignment analysis. Identical residues among the eight different proteins are shown in black boxes, and similar residues (more ...)
In E. coli
, the mazG
gene is located downstream of mazEF
, which is a chromosomal “addiction module” proposed to be responsible for programmed cell death (2
). The relationship between mazG
is still unknown. In order to discover if mazG
is an essential gene for E. coli
, we have constructed a mazG
deletion strain by replacing the mazG
gene with a kanamycin-resistant gene as described in Materials and Methods. The mazG
deletion strain was able to form colonies under several growth conditions such as low (15°C) and high (37 and 42°C) temperatures on LB medium or M9 medium, indicating that the mazG
function is not required under normal growth conditions.
Some other E. coli
proteins, such as MutT and Orf17, also catalyze the hydrolysis of ribo- and deoxynucleoside triphosphates, yielding inorganic pyrophosphate and nucleoside monophosphates (6
). However, they have different and more substrate specificities than MazG. dATP is the preferred substrate for Orf17, and dGTP is the preferred substrate for MutT, while MazG does not have a strong substrate specificity among the eight canonical nucleoside triphosphates. Moreover, there is no significant amino acid sequence similarity between MazG and MutT or Orf17. It has been shown that MutT hydrolyzes 8-oxo-dGTP, the mutagenic form of dGTP, thus preventing AT-to-CG mutations (21
). Orf17 is of unknown function and does not have antimutator properties (25
). In order to find if MazG is also involved in preventing mutations, the mutation frequencies of the mazG
deletion strain and its parent strain were determined on LB plates containing different antibiotics. As shown in Table , frequencies of spontaneous mutation to rifampin, nalidixic acid, and streptomycin resistance did not significantly increase in the mazG
deletion strain, indicating that MazG does not play a role as an antimutator.
Spontaneous mutation frequencies of the mazG deletion strain
Although our two-hybrid system and in vitro experiments demonstrated their physical interaction, the significance of the interaction between MazG and Era remains elusive at present. It is possible that another protein factor(s) may be needed for their functional interaction. It is interesting that GDP-bound Era binds more tightly to MazG than GTP-bound Era, suggesting that Era may play a role as a molecular switch in regulating the function associated with MazG. Further studies on the function of MazG may provide insights into our understanding of the physiological role of MazG and the functional significance of its interaction with Era.