Formylation Is Not Essential for Initiation of Protein Synthesis in All Eubacteria*

Formylation of the initiator methionyl-tRNA, catalyzed by methionyl-tRNA formyltransferase, has long been regarded as essential for initiation of protein synthesis in eubacteria. Here, we show that this process is, in fact, dispensable in Pseudomonas aeruginosa. Disruption of the chromosomal methionyl-tRNA formyltransferase gene in P. aeruginosa resulted only in a moderate decrease in the rate of cell growth, whereas inEscherichia coli cell growth was severely impaired. The ability of the P. aeruginosa mutant strain to grow was not due to an additional copy of the methionyl-tRNA formyltransferase gene or to N-acylation of the methionyl moiety by a group other than formyl. These results indicate that P. aeruginosa can carry out formylation-independent initiation of protein synthesis, using the nonformylated methionyl-tRNA. Therefore, the dogma that eubacteria require formylation of the initiator methionyl-tRNA for initiation of protein synthesis may have been an invalid generalization of results obtained with E. coli.

Formylation of the initiator methionyl-tRNA, catalyzed by methionyl-tRNA formyltransferase, has long been regarded as essential for initiation of protein synthesis in eubacteria. Here, we show that this process is, in fact, dispensable in Pseudomonas aeruginosa. Disruption of the chromosomal methionyl-tRNA formyltransferase gene in P. aeruginosa resulted only in a moderate decrease in the rate of cell growth, whereas in Escherichia coli cell growth was severely impaired. The ability of the P. aeruginosa mutant strain to grow was not due to an additional copy of the methionyl-tRNA formyltransferase gene or to N-acylation of the methionyl moiety by a group other than formyl. These results indicate that P. aeruginosa can carry out formylation-independent initiation of protein synthesis, using the nonformylated methionyl-tRNA. Therefore, the dogma that eubacteria require formylation of the initiator methionyl-tRNA for initiation of protein synthesis may have been an invalid generalization of results obtained with E. coli.
Protein synthesis in eubacteria, and in the chloroplasts and mitochondria of eukaryotes, can be initiated using the initiator formyl-methionyl-tRNA (fMet-tRNA fMet ) 1 (1). Formylation is specific for the initiator methionyl-tRNA (Met-tRNA fMet ) and is catalyzed by methionyl-tRNA formyltransferase (MTF), which is encoded by the fmt gene (2)(3)(4)(5). The features in the Escherichia coli tRNA fMet required for formylation are the base-base mismatch between nucleotides 1 and 72 and the second and third base pairs of the acceptor stem (4 -6). The fMet moiety allows initiation factor IF-2 to recognize the initiator tRNA and reject other tRNAs. This conclusion is based on in vivo analysis of the effect of overproduction of IF-2 on the activity of E. coli tRNA fMet mutants defective in formylation (7,8) and on in vitro studies of IF-2 interaction with N-blocked aminoacyl-tRNA (9,10). The formyl group also prevents the tRNA fMet from partic-ipating in elongation by blocking binding of elongation factor EF-Tu (5,7).
Formylation of the Met-tRNA fMet is generally accepted as a key checkpoint required for initiation of protein synthesis in eubacteria. This dogma, based primarily on studies conducted in E. coli, was further substantiated by the finding that disruption of the E. coli chromosomal fmt gene severely curtailed cell growth (3). However, some earlier studies have obtained circumstantial evidence that questioned this generalization. A Streptococcus faecalis folate-deficient strain, which is unable to synthesize the formyl donor N 10 -formyltetrahydrofolate (fTHF), was shown to be unaffected in growth or viability (11). However, it was not clear that S. faecalis can initiate protein synthesis in the absence of formylation, since the strain also incurred additional chromosomal mutations that affect proper tRNA modification (11).
While the studies in S. faecalis were inconclusive, they hinted that formylation may not be a prerequisite for initiation of protein synthesis in all eubacteria. Therefore, we investigated the significance of the formylation step in Pseudomonas aeruginosa, a Gram-negative, opportunistic pathogen that is distantly related to E. coli. The results show that the process of initiation of protein synthesis in P. aeruginosa is not strictly dependent on formylation and can accommodate the use of non-N-blocked Met-tRNA fMet . The extent to which other eubacteria have this capacity to initiate translation thus becomes an open question.
Construct for Expression of the P. aeruginosa and E. coli MTFs in the E. coli fmt Mutant Strain-The open reading frame (ORF) of the fmt genes was cloned into the NcoI and BamHI sites in pACTN (15). In this construct, the fmt genes are under the control of the Lac promoter and the Shine-Dalgarno and transcription termination sequences of gene 10 of bacteriophage T7.
Construct for Expression of the P. aeruginosa MTF in the P. aeruginosa fmt Mutant Strain-A 1.4-kilobase pair KpnI fragment containing the wild type P. aeruginosa fmt gene was inserted into the same site in the P. aeruginosa shuttle vector pUCP26. The cloned fragment is under the control of the Lac promoter.
Disruption of the Chromosomal P. aeruginosa fmt Gene-The P. aeruginosa fmt gene on a 6-kilobase pair fragment was isolated previously from a genomic library (GenBank TM accession number AF073952) (16). A 4.5-kilobase pair SmaI segment containing the fmt gene and flanking sequences was obtained from the 6-kilobase pair fragment and inserted into the same site of pEX100T. An 840-base pair fragment containing the gentamycin resistance gene (Gm R ) was obtained by PCR amplification using pUCGM as the template. The fragment was digested with MfeI and inserted into the same site in the fmt gene on pEX100T. The pEX100Tfmt::Gm R construct was used to replace the chromosomal MTF gene of the P. aeruginosa PAO1 strain (12). Gene replacement was verified by PCR analysis using primers complementary to the 5Ј and 3Ј ends of the fmt ORF. The sequence of the primers are 5Ј-TTTTCCATGGCGATGAGCCAAGCATTGCGC-3Ј and 5Ј-TTTG-GATCCTCATTGGCCGAGCACCTG-3Ј.
Preparation of Cell Extracts-The wild type and mutant fmt P. aeruginosa strains harboring the pUCP26 vector without the fmt gene were grown overnight at 37°C in Luria-Bertani medium supplemented with 60 g/ml tetracycline. An aliquot of the cultures were diluted * This work was supported by a research grant from the Natural Sciences and Engineering Research Council of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Measurement of MTF Activity-The initiator tRNA substrate was acylated with methionine at 37°C for 30 min. The incubation mixture (20 l) consisted of 20 mM imidazole HCl, pH 7.5, 150 mM NH 4 Cl, 10 mM MgCl 2 , 0.1 mM EDTA, 10 g/ml bovine serum albumin, 100 M methionine (specific activity: 13,000 -15,000 cpm/pmol), 2 mM ATP, 5 M tRNA fMet , and an excess of MetRS (1 g). To measure the formylation activity in cell extracts, 5 l of 1.8 mM of fTHF in 20 mM imidazole HCl, pH 7.5, and 5 l of appropriately diluted MTF were added, and the incubation mixture was incubated for 10 min at 37°C. The reaction was terminated by the addition of 30 l of 360 mM copper sulfate in 1.1 M Tris, pH 7.3. After 5 min an aliquot was spotted on Whatman No. 3MM filter paper and washed sequentially with 10% trichloroacetic acid containing 0.2% casamino acids, 5% trichloroacetic acid containing 0.2% casamino acids, and cold 95% ethanol. The retained radioactivity was determined by liquid scintillation counting.
Determination of the Amount of N-Blocked Met-tRNA fMet in the Wild Type and Mutant fmt P. aeruginosa Strains-The wild type and mutant strains carrying the pUCP26 vector were grown at 37°C in 100 ml of Luria-Bertani medium containing 60 g/ml tetracycline to an A 600 of 1. The cells were pelleted and resuspended in 1 ml of 300 mM sodium acetate, pH 4.8, buffer containing 10 mM NaEDTA (3). Total tRNA was extracted from the cells with phenol and purified under acidic conditions at 4°C (3). A portion of the tRNA samples was completely deacylated by a 90-min incubation at 37°C in 0.5 ml of 2 M Tris-HCl, pH 8.0. Cleavage of the ester linkage of aminoacyl-tRNAs in another portion of the samples was carried out for 30 min at 37°C in 0.5 ml of 200 mM sodium acetate, pH 5.0, buffer containing 10 mM CuSO 4 . 0.5 A 260 units of tRNA from the fully deacylated and CuSO 4 -treated samples were acylated with methionine for 5 min using a large excess of MetRS. Formylation was carried out for 5 min using a large excess of affinity purified P. aeruginosa MTF. The amount of N-blocked Met-tRNA fMet in each strain was determined by the difference between formyl acceptance of the two tRNA samples and expressed as a percent of the total tRNA fMet in the completely deacylated sample.

RESULTS AND DISCUSSION
In vitro studies of protein synthesis using extracts from E. coli showed that translation was stimulated by fMet-tRNA fMet (17). Furthermore, inhibition of formylation in E. coli by using trimethoprim to impair folate metabolism caused the cells to grow very slowly (18 -20). These early studies led to the belief that formylation is an important prerequisite for initiation of protein synthesis in eubacteria. Recently, this view was supported by the finding that inactivation of the E. coli chromosomal fmt gene, which codes for the formylating enzyme, severely curtailed cell growth (3). Both in vitro and in vivo studies showed that the formyl group is important for selection of the initiator tRNA by IF-2 at the initiation site of the ribosome and for preventing misappropriation of the tRNA in elongation (5,(7)(8)(9)(10).
Some circumstantial evidence obtained from studies conducted in S. faecalis and Bacillus subtilis questioned the importance of formylation in initiation of protein synthesis in eubacteria. In contrast to E. coli, B. subtilis was capable of growth when formylation was blocked by trimethoprim inhibition of folate metabolism (21). Similarly, a S. faecalis mutant deficient in folate synthesis was not affected in growth (11). In the case of S. faecalis, the selection conditions used to isolate the mutant strain resulted in additional chromosomal mutations, which affected proper tRNA modification. This observation suggests that growth of B. subtilis could be due to compensatory chromosomal mutations. Therefore, it is not clear whether formylation is required for initiation of protein synthesis in these organisms.
Disruption of the P. aeruginosa Chromosomal MTF Gene-To investigate the importance of Met-tRNA fMet formylation in P. aeruginosa, the chromosomal fmt gene was replaced with a mutant copy by homologous recombination (12). The fmt gene used in this study was isolated previously from a genomic library by functional complementation of a MTF-deficient E. coli strain (16). A 4.5-kilobase pair SmaI segment containing the fmt gene as well as flanking sequences was obtained from the genomic clone and inserted into the same site in the suicide vector pEX100T. The fmt gene was inactivated by insertion of the Gm R cassette into a MfeI site. P. aeruginosa transformants containing the pEX100Tfmt::Gm R vector in the chromosome were identified by selecting for carbenicillin and gentamycin resistance. Resolution of the plasmid was achieved by subjecting the merodiploid strain to sucrose counter-selection in the presence of gentamycin.
Several sucrose-and gentamycin-resistant colonies were observed within 24 h of incubation at 37°C. Replacement of the wild type fmt gene with the mutant copy was verified by PCR analysis using primers complementary to the 5Ј and 3Ј ends of the fmt ORF (Fig. 1). Two products of about 1.7-and 1-kilobase pair, corresponding to the fmt::Gm R fragment (compare lanes 1 and 4) and the wild type fmt ORF (compare lanes 1 and 3), respectively, were obtained when the chromosomal DNA from the merodiploid strain was used (lane 1). The presence of the fmt::Gm R fragment and absence of the wild type fmt gene confirmed replacement of the chromosomal copy in one of the isolates (lane 2).
Formylation Is Less Important in P. aeruginosa than in E. coli-The P. aeruginosa MTF mutant strain grew with a doubling time of 152 min ( Fig. 2A). Complementation with the wild type fmt gene, provided in trans on the plasmid pUCP26, decreased the doubling time of the mutant strain to 56 min. This is comparable with that of the wild type strain harboring the pUCP26 vector with or without the fmt gene ( Fig. 2A). This finding established that the reduced growth rate of P. aeruginosa was due specifically to inactivation of the chromosomal fmt gene. The E. coli MTF mutant, constructed as described previously (3,16,22), doubled every 474 min, whereas the mutant strain expressing the wild type E. coli or P. aeruginosa fmt ORFs grew with a doubling time of 42-46 min (Fig. 2B). The residual growth of the E. coli mutant strain is probably due to some low level utilization of the unformylated Met-tRNA fMet in initiation. The growth rate of the MTF-deficient E. coli strain was reduced by 12-fold and that of the P. aeruginosa mutant strain was reduced by 3-fold. The distinct growth phenotype of the two mutant strains suggests that formylation is less important in P. aeruginosa than in E. coli.
Growth of the P. aeruginosa fmt mutant strain was not due to the presence of a homologue of MTF or to the mutant MTF enzyme being active. This conclusion is based on measurement of formylation activity in cell extracts prepared from the wild type and mutant strains (Fig. 3). The total activity in cell extracts prepared from the wild type parental strain increased with the amount of protein assayed. In contrast, no formylation activity was detected in cell extracts prepared from the mutant strain even at high protein levels. These results show that the P. aeruginosa fmt mutant strain was devoid of formylating activity.
P. aeruginosa Has the Capacity to Initiate Protein Synthesis with Non-N-blocked Met-tRNA fMet -We investigated whether N-acylation of the Met-tRNA fMet by a functional group other than the formyl group could account for the growth of the P. aeruginosa fmt mutant strain. Total tRNAs were isolated from the wild type and mutant strains, under conditions that stabilize the aminoacyl ester linkage (3). One portion of the sample was completely deacylated, by incubating the tRNA under alkaline conditions. A second portion was treated with CuSO 4 , which cleaves the ester bond of aminoacyl-tRNA but not that of N-acyl-Met-tRNA fMet . Both tRNA samples were re-acylated with methionine, and formyl acceptance was measured using P. aeruginosa MTF overproduced in E. coli and purified by affinity chromatography (15). The amount of N-blocked Met-tRNA fMet in the wild type and mutant MTF strains was determined by the difference between formyl acceptance of the completely deacylated and CuS0 4 -treated tRNA samples. Based on FIG. 2. Growth phenotype of the P. aeruginosa and E. coli MTF mutant strains. A, the P. aeruginosa PAO1 strains harboring the pUCP26 vector without (q, ) and with the P. aeruginosa fmt gene (f, OE) were grown at 37°C in Luria-Bertani medium containing 60 g/ml tetracycline. B, the E. coli MTF-deficient strain harboring the pACTN vector without (OE) and with the E. coli (q) or P. aeruginosa (f) fmt gene was grown at 37°C in 100 ml of Luria-Bertani medium supplemented with 100 g/ml ampicillin, 10 g/ml tetracycline, 25 g/ml kanamycin, and 1 mM isopropyl-␤-D-thiogalactopyranoside. At the times indicated the optical density of the cultures was measured at 600 nm. The doubling time for each strain is shown in the inset. The P. aeruginosa initiator tRNA sequence was obtained by a BLAST search of the National Center for Biotechnology Information P. aeruginosa data base for sequences homologous to that of the E. coli initiator tRNA. The boxes indicate the determinants that are important for MTF recognition of the initiator tRNA. The arrows denote the most crucial of the three formylation determinants. The asterisks signify the nucleotides that are different between the P. aeruginosa and E. coli tRNAs. this analysis, 80% of the Met-tRNA fMet in the wild type strain was N-blocked (Fig. 4). However, only the unblocked Met-tRNA fMet species was found in the mutant strain. This result showed that the Met-tRNA fMet in the MTF-deficient strain was not N-acylated by another group. Taken altogether, the results indicate that protein synthesis in the MTF-deficient P. aeruginosa strain is initiated with the initiator methionyl-tRNA and not with N-blocked Met-tRNA fMet .
In archaebacteria and the cytoplasm of eukaryotes, protein synthesis is initiated with the initiator Met-tRNA (23). These initiator tRNAs contain an A:U base pair which is, in part, required for initiation factor (eIF) recognition of the tRNA (24). In contrast, eubacterial initiator tRNAs contain a C ϫ A mismatch between nucleotides 1 and 72. The mismatch in the E. coli initiator tRNA is a key formylation determinant (4 -6). Therefore, for initiation in the absence of formylation, it is possible that P. aeruginosa is using an initiator tRNA with functional properties comparable with those of eukaryotes. A search of the P. aeruginosa genome sequence data base identified two initiator tRNA genes encoding tRNAs that are almost identical. The sequences of the initiator tRNAs are homologous to the E. coli tRNA fMet (Fig. 5). Moreover, the P. aeruginosa tRNAs contain a C ϫ A mismatch between nucleotides 1 and 72. This feature was used by the P. aeruginosa MTF to recognize the initiator tRNA, since overproduction of the enzyme increased the initiator activity of a formylation-defective tRNA fMet mutant with a base pair between nucleotides 1 and 72 (data not shown). These results suggest that P. aeruginosa is using the tRNA fMet and not a unique initiator tRNA to initiate protein synthesis in the absence of formylation.
IF-2 is essential for initiation of protein synthesis in E. coli (25). The protein has a higher affinity for the fMet-tRNA fMet than for the Met-tRNA fMet , indicating that the formyl group is an IF-2 recognition element (9,10). In vitro studies showed that IF-2 selects the fMet-tRNA fMet at the initiation site of the preinitiation complex (9). Therefore, in E. coli, IF-2 is partly responsible for excluding the participation of the nonformylated Met-tRNA fMet in initiation. This is consistent with the observation that in the MTF-deficient E. coli strain, initiation of protein synthesis is severely impaired. The eIF-2s of archaebacteria and eukaryotic cytoplasm are involved in selecting the initiator Met-tRNA for utilization in initiation of translation. A BLAST search of the P. aeruginosa genome data base identified sequences that were homologous to the E. coli IF-2 gene but none that were homologous for the eIF-2 gene. Therefore, it is conceivable that the P. aeruginosa IF-2 has dual substrate specificity and facilitates utilization of both Met-tRNA fMet and fMet-tRNA fMet in initiation or that an unidentified initiation factor, which only recognizes the Met-tRNA fMet species, is involved.
We have shown that P. aeruginosa can carry out formylationindependent initiation of protein synthesis, using the Met-tRNA fMet . This finding represents the first direct evidence that eubacteria have the ability to initiate translation with fMet-tRNA fMet and Met-tRNA fMet . While it is not known whether this phenomenon is unique to P. aeruginosa, it opens the possibility that other eubacteria may have the capacity to use both forms of the tRNA fMet for initiation. The availability of various fmt sequences will facilitate investigation of this question in both Gram-positive and Gram-negative bacteria.