The Gene for 16S rRNA Methyltransferase (ksgA) Functions as a Multicopy Suppressor for a Cold-Sensitive Mutant of Era, an Essential RAS-Like GTP-Binding Protein in Escherichia coli

Abstract

Era, a Ras-like GTP-binding protein in Escherichia coli, has been shown to be essential for growth. However, its cellular functions still remain elusive. In this study, a genetic screening of an E. coli genomic library was performed to identify those genes which can restore the growth ability of a cold-sensitive mutant, Era(Cs) (E200K), at a restrictive temperature when expressed in a multicopy plasmid. Among eight suppressors isolated, six were located at 1 min of the E. coli genomic map, and the gene responsible for the suppression of Era(Cs) (E200K) was identified as the ksgA gene for 16S rRNA transmethylase, whose mutation causes a phenotype of resistance to kasugamycin, a translation initiation inhibitor. This is the first demonstration of suppression of impaired function of Era by overproduction of a functional enzyme. A possible mechanism of the suppression of the Era cold-sensitive phenotype by KsgA overproduction is discussed.

Era, an Escherichia coli Ras-like protein, has an intrinsic GTPase activity and sequence similarity with members of a family of GTP-binding proteins, such as the yeast RAS1 protein (1, 6). The era gene is highly conserved among prokaryotes (22, 31) and essential for cell growth (12, 19). Era has been shown to be associated with the cytoplasmic membrane (18). It has been suggested that Era may be involved in E. coli cell division (9) and in a checkpoint control in the E. coli cell cycle (4).

A temperature-sensitive allele, Era(Ts) (C8Y, 294::Tn10), was obtained by localized mutagenesis with a mini-Tn10 transposon (12), and its extragenic suppressors were isolated by ΔTn10-kan gene disruption mutation (24). Disruption of pstN, encoding a novel nitrogen-related enzyme, IIA (IIA^ntr), a member of the phosphoenolpyruvate-sugar phosphotransferase system, was found to be able to suppress the Era(Ts) phenotype. However, these disruptions did not affect classical nitrogen regulation or the expression of era, suggesting that the observed suppression was posttranslational. At present, it is not clear how the cellular function of PstN is related to the role of Era. Other extracellular suppressors for the Era(Ts) allele were suhB mutants (suhB2 and suhB10) (13). Mutant alleles of suhB have diverse effects on diverse cell activities, including protein export, stress response, DNA synthesis, and phospholipid biosynthesis. The mutant alleles of suhB were previously isolated as extragenic suppressors for the DNA synthesis mutant (dnaB121) (5), the protein secretion mutant (secY24) (24), and the heat shock response mutant (rpoH15) (30). The E. coli suhB gene product is homologous to mammalian inositol monophosphatase and has the inositol monophosphatase activity (20) for the phosphatidylinositol biosynthesis. Again, the functional link between suhB and era has not been established. A recent study has shown that a temperature-sensitive mutation in dnaG encoding a DNA primase required for DNA replication can be suppressed by an era mutation (P17R) or a reduced era expression, and again the exact mechanism for suppression of dnaG(Ts) by the era mutants is unknown (3).

Although many of the extragenic mutants were isolated to suppress era conditional mutants, none of the multicopy suppressors has been identified as directly suppressing the era mutant phenotype. The previous studies by localized error-prone random PCR led to isolation of several cold-sensitive Era mutants. Three recessive missense mutations in Era, N26S, A156D, and E200K, were reported to confer cold-sensitive phenotypes (16). Among these mutations, E200K was found to have a relatively tight cold-sensitive phenotype. In this study, we performed a genetic screening of an E. coli genomic library to search for genes which can suppress the cold-sensitive phenotype of the Era mutant when expressed in a multicopy plasmid.

Isolation of multicopy suppressors for Era(Cs) (E200K).

Plasmid pAC19era(E200K) was transformed into strain CL213(Δera::Kan F′ lacI^q) cells (16), harboring a helper plasmid (pXC001 [19]) which contains a temperature-sensitive origin [ori(Ts)] and the wild-type era gene. pAC19era(E200K) is a derivative of a low-copy-number plasmid, pACYC184 (25), with an insertion of an NdeI- and TfiI-digested fragment of pUC19 at the EcoRV site of pACYC184. Relevant features of this plasmid include the p15A origin of replication, chloramphenicol resistance (Cm^r), and the lacZ promoter and the multiple cloning site from pUC19 (26). It further contains the era gene derived from EcoRI and XbaI digestion of pCLKS-ERA(E200K) (16) (blunt ended by Klenow enzyme in the presence of all four deoxynucleoside triphosphates) at the SmaI site in the multiple cloning site. The era gene in the plasmid is under the control of the lac promoter, which was designated pAC19era(E200K). Transformants were first isolated on Luria-Bertani (LB) agar plates containing chloramphenicol [for pAC19era(E200K)], kanamycin (for the chromosomal era deletion), and ampicillin (for pXC001) at 30°C. Single colonies were then picked and streaked on LB plates containing only chloramphenicol and kanamycin at 42°C in order to remove the Amp^r helper plasmid. A colony which was resistant to chloramphenicol (20 μg/ml) and kanamycin (50 μg/ml) but sensitive to ampicillin (50 μg/ml) at 42°C was selected and designated CS213. CS213 cells exhibited a cold-sensitive phenotype at 23°C or lower even in the presence of 0.5 mM isopropyl-β-thiogalactopyranoside (IPTG). This result demonstrates that CS213 cells do not carry the era⁺ helper plasmid and that the Era function of CS213 cells depends on Era(Cs) (E200K).

To examine whether there are genetic elements in the E. coli genome which are able to restore the growth ability of strain CS213 at low temperatures, CS213 cells were transformed with an E. coli genomic library in pUC19 (Amp^r). The library contained partially digested Sau3AI chromosomal DNA fragments from E. coli JM83, which were ligated to the BamHI-digested pUC19. Note that the pUC19-carried genomic library is compatible with pAC19era(E200K) because they contain different replication origins, ColE1 and p15A, respectively. Transformants were isolated for their ability to grow at 23°C on LB agar plates containing ampicillin. Plasmids from those colonies that gained the ability to grow at 23°C were purified and retransformed into CS213 cells to confirm their ability to suppress the cold-sensitive phenotype.

Identification of the gene responsible for the suppression of the Era(Cs) (E200K) cold-sensitive phenotype.

Of eight independent plasmids isolated, two suppressors (the EcoRI-HindIII fragments of the plasmids were labeled with [α³²P]dCTP with random primers) hybridized to a λ phage DNA containing the region encompassing the era gene, with the use of a screening filter consisting of an E. coli genomic λ phage array (Takara Shuzu Co., Kyoto, Japan), and all the remaining six plasmids were found to hybridize to another phage λ DNA containing the genomic region at 1 min on the E. coli chromosome. One of these plasmids was thus designated pES1. The inserted genomic element from plasmid pES1 was sequenced and found to contain six genes: an open reading frame of unknown function, pdxA, ksgA, apaG, apaH, and folA. To identify the exact gene that suppresses era(Cs), the pES1 DNA was digested with either XcmI or EcoRV, followed by self-ligation. The resulting plasmids (pES2 and pES3, respectively; see Fig. 1) were still capable of suppressing the era(Cs) phenotype. However, the plasmid treated with AccI followed by self-ligation after filling in the gaps with the Klenow enzyme (pES4) lost the suppression activity. These results indicate that the ksgA gene is responsible for the era(Cs) suppression. This was further confirmed by constructing a plasmid which contained only the ksgA gene (pKsgA [Fig. 1]). CS213 cells harboring this plasmid became capable of forming colonies on LB agar plates containing ampicillin at 23°C in contrast to CS213 cells harboring pUC19 (data not shown). It is important to note that the ksgA gene could not complement the null mutant alleles of era, indicating that the era(Cs) suppression by KsgA occurs at the level of the cold-sensitive EraE200K protein. The impaired function of EraE200K at low temperature is, therefore, somehow restored by the overexpression of 16S rRNA methyltransferase, the gene product of ksgA.

FIG. 1.

Identification of the gene responsible for the suppression of the era cold-sensitive phenotype. pES1-derived subclones were constructed as follows. pES1 was digested with EcoRV to remove the apaH gene and self-ligated to construct pES2. pES3 was constructed by self-ligation of the fragment after XcmI digestion. pES4 was constructed by religation of the fragment after AccI digestion followed by Klenow fill-in reaction. pKsgA was constructed by cloning the fragment between AccI and EcoRV on the pUC19 vector. X, XcmI; A, AccI; E, EcoRV. ORF, open reading frame.

Multicopy suppression of the Era (E200K) cold-sensitive phenotype by ksgA.

CS213 cells grew at 37°C at almost the same rate as their parental strain, CL83 (reference 15 and data not shown). When the culture was shifted to 17°C, CS213 cells grew slower than the wild-type cells and almost stopped growing after 24 h as the cell density increased approximately sixfold (data not shown). Next, cells grown for 24 h at 17°C were examined by 4,6-diamino-2-phenylindole (DAPI) staining. Wild-type cells contained either one or two nucleoids (Fig. 2A), while CS213 cells became elongated and filamentous (Fig. 2B, upper panel); importantly, approximately 50% of them contained four well-segregated nucleoids, and the remaining cells also contained at least two nucleoids (Fig. 2B, lower panel). These results indicate that CS213 cells are defective in cell division at low temperature, while DNA replication and nucleoid segregation stay normal. When CS213 cells transformed with pKsgA were incubated at 17°C for 72 h, they divided normally with one or two nucleoids per cell (Fig. 2D, lower panel) in normal-sized cells (Fig. 2D, upper panel; compare with Fig. 2A, upper panel). In contrast, in CS213 cells transformed with pUC19 segregation of nucleoids became abnormal after 72 h of incubation at 17°C (Fig. 2C). These results clearly demonstrate that ksgA is capable of suppressing the impaired function of Era(Cs) (E200K).

FIG. 2.

Cellular morphology of wild-type and era mutant cells. Cells were visualized under a phase-contrast microscope or a Fluo-phase microscope after DAPI staining as described previously by Hiraga et al. (11). (A and B) Wild-type CL83 (A) and mutant CS213 (B), both carrying control plasmid pUC19. They were cultured at 17°C for 24 h. (C and D) CS213 cells carrying pUC19 (C) and pKsgA (D) were cultured for 72 h at 17°C. (Upper panels) Phase-contrast microscopy. (Lower panels) Fluo-phase micrography (A and B) and fluorescent micrographs after DAPI staining (C and D). For microscopy images, Kodak 400 Elite slide films were used to capture phase and fluorescence images obtained with a Zeiss Axioskop Zeiss plan-NEOFLUAR 100× microscope (NA = 1.3; oil-immersion objective; 100-W HBO lamp). A DAPI filter set (a 360- to 370-nm excitation filter and a 420-nm barrier filter) was used for DNA staining. Images were scanned from slides with a Kodak 3025 slide scanner and imported into Adobe Photoshop software.

Effects of overexpression of KsgA on era expression.

It has been speculated that 16S RNA methyltransferase, the ksgA product, may play a role in translation initiation and translational fidelity (23, 29). Therefore, it is possible that overproduction of KsgA may cause overproduction of Era (E200K), which may overcome the defective cold-sensitive phenotype. To test this possibility, Western blot analysis was carried out to compare the levels of Era production between cells carrying the vector and those with the suppressor pKsgA plasmid. As shown in Fig. 3, Era expression in CS213 cells with pKsgA (lanes 3) was identical with the Era production in the wild-type cells (lanes 2), indicating that KsgA overproduction did not affect the Era synthesis. In addition, it has been shown that overexpression of mutant EraE200K still exhibited the cold-sensitive phenotype in era null mutation cells (16).

FIG. 3.

Effects of overexpression of KsgA on the expression level of the era gene. CS213 cells transformed with pUC19 or pKsgA were cultured at 37°C. Cells were harvested during experimental growth, and the same amounts of cell lysates were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to the polyvinylidene difluoride membrane (Millipore) for Western blot analysis with the purified Era antibody (B). The antibody was purified as described previously (18). Western blotting was performed with the ECL Western blotting detection system (Amersham Life Science) according to the manufacturer’s instructions. The same filter was washed with 50 mM NaHPO₄ (pH 7.6) overnight and then stained with Coomassie blue to visualize the protein pattern (A). Lanes 1, 0.25 μg of purified Era protein; lanes 2, cell lysate of CS213 transformed with pUC19; lanes 3, cell lysate of CS213 transformed with pKsgA.

Possible roles of KsgA in the suppression of the Era (E200K) mutation.

The ksgA gene product is 16S rRNA methyltransferase, which methylates two highly conserved adjacent adenosine residues in a loop region at the 3′ end of 16S rRNA, which is located close to the sequence complementary to the Shine-Dalgarno sequence (8). The Shine-Dalgarno sequence exists upstream of the initiation codon in mRNAs playing an essential role in binding of ribosomes to mRNAs. It has been proposed that 16S rRNA methyltransferase is a ribosome-associated protein involved in functions of 30S ribosomes for the initiation of protein translation (23). Among widely divergent species of prokaryotes, 16S rRNA methyltransferase is highly conserved, implying its important role in cellular function in the prokaryotes, although the ksgA gene has been shown to be dispensable in E. coli (17). Interestingly, Saccharomyces cerevisiae has a gene equivalent to the ksgA gene encoding 18S rRNA methyltransferase, which is essential for normal cell growth (14).

Downstream of ksgA, there is the apaH gene in the same operon, which encodes a hydrolase for the degradation of AppppA dinucleotides (known as alarmone) in E. coli (7). Notably, the kasugamycin-resistant phenotype caused by a mutation in the ksgA gene can be reverted to kasugamycin sensitive by an additional mutation in the apaH gene (17). This effect may be due to an elevated level of Ap4A, which might cause a conformational change in the unmethylated 16S rRNA to a conformation similar to that of the methylated 16S rRNA so that kasugamycin regains its inhibitory activity. It has been proposed that elevated levels of alarmone Ap4A due to the mutation of apaH might cause an effect similar to that caused by the methylation of the highly conserved adenosine doublet at the 3′ end of 16S rRNA (17).

Recently, the apaH gene was found to be involved in cell division and identified as one of the cfc genes (control of the frequency of cell division) (21). A high level of Ap4A due to the mutations in apaH can lead to increasing frequency of cell division, producing unusually small cells. These authors propose that Ap4A functions as a signal for induction of cell division. If the methylation of the conserved adenosine doublet at 16S rRNA may cause an effect similar to that of Ap4A on 16S rRNA function (17), it is reasonable to speculate that overexpression of KsgA may cause an effect on cellular physiology similar to that caused by an elevated level of alarmone Ap4A. This effect would then enhance cell division frequency. Since the era mutation in CS213 led to a block in cell division, causing formation of elongated cells at low temperature, overexpression of KsgA enhancing cell division frequency may complement the defective cell division caused by the EraE200K mutant.

Alternately, the mechanism of suppression of the Era cold-sensitive phenotype by overexpression of KsgA may be due to the protein-protein interaction between cell division protein Era and the ksgA gene product, provided that there may be a concerted obligatory interaction of cell division and translation initiation. It is important to note that 16S rRNA methyltransferases are highly conserved in the genomes of a number of prokaryotes whose sequences have been recently determined (2) and deposited in GenBank. At present, it is unknown why and how era mutations can be suppressed by mutations in other genes with seemingly quite different functions, such as pstN and suhB, and how an Era mutation can suppress a dnaG mutant. Recently, it has been reported that a partially defective Era GTPase mutation suppresses several temperature-sensitive lethal alleles involved in chromosomal replication and segregation but not in cell division (4). These authors suggest that Era may function in cell cycle progression and the initiation of cell division. The present result, however, is the first demonstration of suppression of defective Era by overproduction of a functional enzyme, and further characterization of the suppression mechanism of the cold-sensitive Era mutant will provide an insight into the exact role of Era.