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• W2006366032 abstract "Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet. The E. coli IF-2, which participates exclusively in formylation-dependent protein initiation in E. coli, was unable to facilitate utilization of Met-tRNAfMet in initiation in P. aeruginosa. However, the E. coli IF-2 was made to function in formylation-independent protein initiation in P. aeruginosa by decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in the P. aeruginosa IF-2 prevented the protein from participating in formylation-independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMet in protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMet in protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination. Formylation of the initiator methionyl-tRNA (Met-tRNAfMet) was generally thought to be essential for initiation of protein synthesis in all eubacteria based on studies conducted primarily in Escherichia coli. However, this view of eubacterial protein initiation has changed because some bacteria have been demonstrated to have the capacity to initiate protein synthesis with the unformylated Met-tRNAfMet. Here we show that the Pseudomonas aeruginosa initiation factor IF-2 is required for formylation-independent protein initiation in P. aeruginosa, the first bacterium shown to have the ability to initiate protein synthesis with both the initiator formyl-methionyl-tRNA (fMet-tRNAfMet) and Met-tRNAfMet. The E. coli IF-2, which participates exclusively in formylation-dependent protein initiation in E. coli, was unable to facilitate utilization of Met-tRNAfMet in initiation in P. aeruginosa. However, the E. coli IF-2 was made to function in formylation-independent protein initiation in P. aeruginosa by decreasing the positive charge potential of the cleft that binds the amino end of the amino acid attached to the tRNA. Furthermore increasing the positive charge potential of this cleft in the P. aeruginosa IF-2 prevented the protein from participating in formylation-independent protein initiation. Thus, this is the first demonstration of a eubacterial IF-2 with an inherent capacity to facilitate utilization of Met-tRNAfMet in protein initiation, discounting the dogma that eubacterial IF-2 can only allow the use of fMet-tRNAfMet in protein initiation. Furthermore these findings give important clues to the basis for discriminating the initiator Met-tRNA by IF-2 and for the evolution of alternative mechanisms for discrimination. The initiator methionyl-tRNA is used to initiate protein synthesis in Archaebacteria and the cytoplasm of eukaryotes. In contrast, protein synthesis in eubacteria and in certain eukaryotic organelles, such as mitochondria and chloroplasts, can be initiated using the initiator formyl-methionyl-tRNA. Formylation is specific for the initiator methionyl-tRNA and is catalyzed by methionyl-tRNA formyltransferase (MTF) 1The abbreviations used are: MTF, methionyl-tRNA formyltransferase; Met-tRNAfMet, initiator methionyl-tRNA; fMet-tRNAfMet, initiator formyl-methionyl-tRNA; fMet, formyl-methionyl; IF, initiation factor; EF, elongation factor.1The abbreviations used are: MTF, methionyl-tRNA formyltransferase; Met-tRNAfMet, initiator methionyl-tRNA; fMet-tRNAfMet, initiator formyl-methionyl-tRNA; fMet, formyl-methionyl; IF, initiation factor; EF, elongation factor. (1Dickerman H.W. Smith B.C. J. Mol. Biol. 1971; 59: 425-445Crossref PubMed Scopus (7) Google Scholar, 2Dickerman H.W. Steers Jr., E. Redfield B.G. Weissbach H. J. Biol. Chem. 1967; 242: 1522-1525Abstract Full Text PDF PubMed Google Scholar, 3Guillon J.M. Mechulam Y. Blanquet S. Fayat G. J. Bacteriol. 1993; 175: 4507-4514Crossref PubMed Google Scholar, 4Guillon J.M. Meinnel T. Mechulam Y. Lazennec C. Blanquet S. Fayat G. J. Mol. Biol. 1992; 224: 359-367Crossref PubMed Scopus (84) Google Scholar, 5Lee C.P. Seong B.L. RajBhandary U.L. J. Biol. Chem. 1991; 266: 18012-18017Abstract Full Text PDF PubMed Google Scholar, 6Varshney U. Lee C.P. Seong B.L. RajBhandary U.L. J. Biol. Chem. 1991; 266: 18018-18024Abstract Full Text PDF PubMed Google Scholar). In vitro studies of protein synthesis using extracts from Escherichia coli showed that translation was stimulated by fMet-tRNAfMet (7Ebbole D.J. Zalkin H. J. Biol. Chem. 1987; 262: 8274-8287Abstract Full Text PDF PubMed Google Scholar). Furthermore inhibition of formylation in E. coli by using trimethoprim to impair folate metabolism caused the cells to grow very slowly. These early studies led to the general belief that formylation of the Met-tRNAfMet is a key step in initiation of protein synthesis in all eubacteria. This view was reinforced by the findings that disruption of the MTF gene in E. coli (8Guillon J.M. Mechulam Y. Schmitter J.M. Blanquet S. Fayat G. J. Bacteriol. 1992; 174: 4294-4301Crossref PubMed Scopus (130) Google Scholar), Streptococcus pneumoniae (9Margolis P. Hackbarth C. Lopez S. Maniar M. Wang W. Yuan Z. White R. Trias J. Antimicrob. Agents Chemother. 2001; 45: 2432-2435Crossref PubMed Scopus (69) Google Scholar), and Bacillus subtilis (10Kobayashi K. Ehrlich S.D. Albertini A. Amati G. Andersen K.K. Arnaud M. Asai K. Ashikaga S. Aymerich S. Bessieres P. Boland F. Brignell S.C. Bron S. Bunai K. Chapuis J. Christiansen L.C. Danchin A. Debarbouille M. Dervyn E. Deuerling E. Devine K. Devine S.K. Dreesen O. Errington J. Fillinger S. Foster S.J. Fujita Y. Galizzi A. Gardan R. Eschevins C. Fukushima T. Haga K. Harwood C.R. Hecker M. Hosoya D. Hullo M.F. Kakeshita H. Karamata D. Kasahara Y. Kawamura F. Koga K. Koski P. Kuwana R. Imamura D. Ishimaru M. Ishikawa S. Ishio I. Le Coq D. Masson A. Mauel C. Meima R. Mellado R.P. Moir A. Moriya S. Nagakawa E. Nanamiya H. Nakai S. Nygaard P. Ogura M. Ohanan T. O'Reilly M. O'Rourke M. Pragai Z. Pooley H.M. Rapoport G. Rawlins J.P. Rivas L.A. Rivolta C. Sadaie A. Sadaie Y. Sarvas M. Sato T. Saxild H.H. Scanlan E. Schumann W. Seegers J.F. Sekiguchi J. Sekowska A. Seror S.J. Simon M. Stragier P. Studer R. Takamatsu H. Tanaka T. Takeuchi M. Thomaides H.B. Vagner V. van Dijl J.M. Watabe K. Wipat A. Yamamoto H. Yamamoto M. Yamamoto Y. Yamane K. Yata K. Yoshida K. Yoshikawa H. Zuber U. Ogasawara N. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4678-4683Crossref PubMed Scopus (1132) Google Scholar) severely curtailed cell growth, and the MTF gene is present in all eubacterial genomes sequenced to date. However, this dogma of eubacterial protein initiation is no longer valid because some bacteria have been shown to initiate protein synthesis independently of formylation (11Margolis P.S. Hackbarth C.J. Young D.C. Wang W. Chen D. Yuan Z. White R. Trias J. Antimicrob. Agents Chemother. 2000; 44: 1825-1831Crossref PubMed Scopus (117) Google Scholar, 12Newton D.T. Creuzenet C. Mangroo D. J. Biol. Chem. 1999; 274: 22143-22146Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 13Samuel C.E. Rabinowitz J.C. J. Biol. Chem. 1974; 249: 1198-1206Abstract Full Text PDF PubMed Google Scholar, 14Delk A.S. Rabinowitz J.C. Nature. 1974; 252: 106-109Crossref PubMed Scopus (26) Google Scholar).Pseudomonas aeruginosa was the first bacterium shown to have the capacity to use Met-tRNAfMet to initiate protein synthesis (12Newton D.T. Creuzenet C. Mangroo D. J. Biol. Chem. 1999; 274: 22143-22146Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Subsequently it was demonstrated that formylation of Met-tRNAfMet was not necessary either for protein initiation in Staphylococcus aureus (11Margolis P.S. Hackbarth C.J. Young D.C. Wang W. Chen D. Yuan Z. White R. Trias J. Antimicrob. Agents Chemother. 2000; 44: 1825-1831Crossref PubMed Scopus (117) Google Scholar), Haemophilus influenzae (15Akerley B.J. Rubin E.J. Novick V.L. Amaya K. Judson N. Mekalanos J.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 966-971Crossref PubMed Scopus (324) Google Scholar), and Saccharomyces cerevisiae mitochondria under certain growth conditions (16Vial L. Gomez P. Panvert M. Schmitt E. Blanquet S. Mechulam Y. Biochemistry. 2003; 42: 932-939Crossref PubMed Scopus (13) Google Scholar, 17Li Y. Holmes W.B. Appling D.R. RajBhandary U.L. J. Bacteriol. 2000; 182: 2886-2892Crossref PubMed Scopus (59) Google Scholar, 18Tibbetts A.S. Oesterlin L. Chan S.Y. Kramer G. Hardesty B. Appling D.R. J. Biol. Chem. 2003; 278: 31774-31780Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Streptococcus faecalis is another bacterium that may also be capable of formylation-independent protein initiation based on evidence obtained from early studies of a S. faecalis mutant strain unable to synthesize formyltetrahydrofolate, the formyl donor in the Met-tRNAfMet formylation reaction (13Samuel C.E. Rabinowitz J.C. J. Biol. Chem. 1974; 249: 1198-1206Abstract Full Text PDF PubMed Google Scholar, 19Samuel C.E. D'Ari L. Rabinowitz J.C. J. Biol. Chem. 1970; 245: 5115-5121Abstract Full Text PDF PubMed Google Scholar). Thus, unlike E. coli, S. pneumoniae and B. subtilis, which are dependent on fMet-tRNAfMet for protein initiation, P. aeruginosa, S. aureus, H. influenzae, and possibly S. faecalis can perform initiation with either fMet-tRNAfMet or Met-tRNAfMet. The mechanism, however, that allows utilization of Met-tRNAfMet in protein initiation in these bacteria is not understood. Furthermore why some bacteria have the ability to initiate protein synthesis with both fMet-tRNAfMet and Met-tRNAfMet and others with only fMet-tRNAfMet is not understood.The mechanism that facilitates the use of fMet-tRNAfMet in bacteria that require formylation for protein initiation is better understood and is absolutely dependent on the initiation factor IF-2 (20Sacerdot C. Vachon G. Laalami S. Morel-Deville F. Cenatiempo Y. Grunberg-Manago M. J. Mol. Biol. 1992; 225: 67-80Crossref PubMed Scopus (42) Google Scholar, 21Laalami S. Putzer H. Plumbridge J.A. Grunberg-Manago M. J. Mol. Biol. 1991; 220: 335-349Crossref PubMed Scopus (54) Google Scholar). IF-2 selects the fMet-tRNAfMet over all other tRNAs for utilization in protein initiation by recognizing the fMet moiety as well as the C × A mismatch at the end of the acceptor stem of the initiator tRNA (22Mangroo D. RajBhandary U.L. J. Biol. Chem. 1995; 270: 12203-12209Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 23Guillon J.M. Heiss S. Soutourina J. Mechulam Y. Laalami S. Grunberg-Manago M. Blanquet S. J. Biol. Chem. 1996; 271: 22321-22325Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 24Hartz D. McPheeters D.S. Gold L. Genes Dev. 1989; 3: 1899-1912Crossref PubMed Scopus (190) Google Scholar, 25Sundari R.M. Stringer E.A. Schulman L.H. Maitra U. J. Biol. Chem. 1976; 251: 3338-3345Abstract Full Text PDF PubMed Google Scholar, 26Mayer C. Kohrer C. Kenny E. Prusko C. RajBhandary U.L. Biochemistry. 2003; 42: 4787-4799Crossref PubMed Scopus (30) Google Scholar). Therefore, IF-2 also plays an important role in preventing the participation of unformylated Met-tRNAfMet in protein initiation in bacteria such as E. coli. In this study, we showed that the P. aeruginosa IF-2 is responsible for formylation-independent protein initiation in P. aeruginosa by using a formylation-deficient P. aeruginosa strain. In contrast, the E. coli IF-2 was unable to facilitate the use of Met-tRNAfMet in protein initiation in P. aeruginosa. It is, therefore, likely that IF-2 allows formylation-independent protein initiation in other eubacteria that have the capacity to initiate protein synthesis with Met-tRNAfMet. A structural model of the tRNA-binding domains of the P. aeruginosa and E. coli IF-2s was established using the solution structure of the Bacillus stearothermophilus IF-2 tRNA-binding domain (27Meunier S. Spurio R. Czisch M. Wechselberger R. Guenneugues M. Gualerzi C.O. Boelens R. EMBO J. 2000; 19: 1918-1926Crossref PubMed Scopus (63) Google Scholar). This structure is similar to the crystal structure of the Thermus aquaticus elongation factor EF-Tu. The co-crystal structure of the T. aquaticus EF-Tu complexed with the E. coli cysteinyl-tRNACys has also been determined (28Nissen P. Thirup S. Kjeldgaard M. Nyborg J. Struct. Fold. Des. 1999; 7: 143-156Abstract Full Text Full Text PDF Scopus (228) Google Scholar), allowing superimposition of the tRNA onto the tRNA-binding sites of the P. aeruginosa and E. coli IF-2s. Analysis of the IF-2 structures indicated that the cleft containing the amino end of the amino acid attached to the tRNA is positively charged. However, this charge is larger for the E. coli IF-2 compared with that of the P. aeruginosa IF-2. Increasing the positive charge potential of this cleft by site-directed mutagenesis in the P. aeruginosa IF-2 impaired the ability of the protein to facilitate formylation-independent protein initiation, whereas reduction of the positive charge potential of the E. coli IF-2 allowed the protein to facilitate utilization of the Met-tRNAfMet in protein initiation. This suggests that the reduced positive charge potential of the cleft is a key determinant allowing the P. aeruginosa IF-2 to bind both the formylated and unformylated Met-tRNAfMet and facilitate their use in protein initiation in P. aeruginosa. Interestingly the positive charge potential of this cleft of the bovine and S. cerevisiae mitochondria IF-2s, which have been shown to allow formylation-independent mitochondrial protein initiation in a S. cerevisiae strain with an inactivated mitochondrial MTF gene, is lower than that of the P. aeruginosa IF-2. Thus, it may be possible to use this structural characteristic and the P. aeruginosa formylation-defective strain as a tool to predict whether a bacterium is capable of both formylation-dependent and -independent protein initiation or relies primarily on formylation-dependent initiation.MATERIALS AND METHODSThe plasmids pEX18AP, pUCGM, pUCP26, pACTN, and pUCP26-PAMTF; the P. aeruginosa fmt mutant strain; and P. aeruginosa PAO1 were described previously (12Newton D.T. Creuzenet C. Mangroo D. J. Biol. Chem. 1999; 274: 22143-22146Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). Rabbit anti-IF-2 was provided by Dr. U. L. RajBhandary, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA.Constructs for Expression of the P. aeruginosa and E. coli IF-2s in the P. aeruginosa fmt Mutant Strain—A 3.5-kilobase pair fragment containing the P. aeruginosa infB gene encoding IF-2 was prepared by PCR using P. aeruginosa chromosomal DNA as the template and the oligonucleotides 5′-GGT GGT GGA TCC GCC ACG CTC AGT CCT CGG TCT-3′ and 5′-ACC ACC GGA TCC GCC AGT TGA CCG GCT GGA CCC-3′; the fragment was digested with BamHI and cloned into the same site in pUCP26 to produce pUCPLIF2. A 2.5-kilobase pair fragment of the coding sequence of the P. aeruginosa infB gene was isolated by PCR using the pUCPLIF2 plasmid as a template and 5′-TTT TGA ATT CTC ATG ACG CAA GTC ACG GTG-3′ and 5′-CGG TTC TAG ATC AAA GGC TGC GTG CGA CTT-3′; the DNA fragment was inserted into the EcoRI and XbaI sites in pUCP26. The infB gene in the pUCPIF2 plasmid is under the control of the lacZ promoter in pUCP26. A 2.7-kilobase pair fragment of the open reading frame of the E. coli infB gene was isolated by PCR using E. coli chromosomal DNA and the oligonucleotides 5′-ACC ACC CCA TGG GCA TGA CAG ATG TAA CGA TTA A-3′ and 5′-GGT GGT GGA TCC TTA AGC AAT GGT ACG TTG GAT-3′; the fragment was digested with NcoI and BamHI and ligated into the same sites in pACTN. The pACTN-ECIF2 construct was used as a template to prepare an EcoRI-BamHI fragment of the E. coli infB gene by PCR. The primers used were 5′-ACC ACC GAA TTC ATG ACA GAT GTA ACG ATT AA-3′ and 5′-GGT GGT GGA TCC TTA AGC AAT GGT ACG TTG GAT-3′; the fragment was cloned into the same sites in pUCP26.Disruption of the Chromosomal P. aeruginosa infB Gene—An EcoRI-XbaI fragment containing the coding sequence of the P. aeruginosa infB gene was obtained from pUCP26IF2 and ligated into the same sites in pEX18APΔK, which lacks the KpnI site in the multiple cloning site. The infB gene is under the control of the lacZ promoter in the plasmid. An 840-base pair fragment containing the gentamycin resistance gene (GmR) was cloned into the KpnI site of the infB gene in pEX18APΔK, generating pEX18APΔKinfB::GmR. The chromosomal infB gene in the P. aeruginosa PAO1 strain harboring pUCPLIF2 was replaced using the pEX18APΔKinfB::GmR construct. Gene replacement was verified by PCR analysis using the following primers: P1, 5′-GCG ACG GAG AGA CTC CAG CTC-3′; P2, 5′-AGG AAG CCG TGC AGC ACG CGA-3′; P3, 5′-GTT ACG CAG CAG GGC AGT CGC-3′; P4, 5′-GGC GGT ATC CGG CGA GAT CG-3′.Preparation of Cell Extracts for Western Blot Analyses—The wild type and mutant fmt P. aeruginosa strains harboring pUC26 without or with the P. aeruginosa fmt gene were grown overnight at 37 °C in Luria-Bertani medium supplemented with 60 μg/ml tetracycline (12Newton D.T. Creuzenet C. Mangroo D. J. Biol. Chem. 1999; 274: 22143-22146Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). An aliquot of the cultures was diluted into 3 ml of fresh medium containing tetracycline and grown for 3–5 h at 37 °C. Cells from 1.2 ml of culture were pelleted by centrifugation and lysed (12Newton D.T. Creuzenet C. Mangroo D. J. Biol. Chem. 1999; 274: 22143-22146Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). The cell lysate was centrifuged, and an aliquot of the supernatant (6 μg of total protein) was subjected to SDS-PAGE on a 10% polyacrylamide gel. The separated proteins were transferred electrophoretically onto polyvinylidene difluoride membrane. Western blot analysis was performed using the enhanced chemiluminescence protocol as described by the supplier.Analysis of the in Vivo Formylation Status of the P. aeruginosa Initiator tRNA—The various P. aeruginosa strains were grown at 37 °C in Luria-Bertani medium supplemented with 60 μg/ml tetracycline. The cells were pelleted by centrifugation and resuspended in 1 ml of 300 mm sodium acetate, pH 4.8 buffer containing 10 mm NaEDTA. Total RNA was extracted from the cells with phenol and precipitated at 4 °C. The RNA pellet was dissolved in 10 mm sodium acetate, pH 4.8 buffer containing 1 mm NaEDTA, and an aliquot was subjected to electrophoresis on a 6.5% polyacrylamide gel containing 8 m urea at 4 °C using 100 mm sodium acetate buffer, pH 5.0 (22Mangroo D. RajBhandary U.L. J. Biol. Chem. 1995; 270: 12203-12209Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 29Varshney U. Lee C.P. RajBhandary U.L. J. Biol. Chem. 1991; 266: 24712-24718Abstract Full Text PDF PubMed Google Scholar). The separated RNAs were transferred electrophoretically onto Nytran membrane. The membranes were incubated at 37 °C for 4 h in prehybridization solution consisting of 4× SSPE (1× SSPE = 0.18 m NaCl, 10 mm NaH2PO4, 1 mm Na2EDTA), 250 μg/ml sheared and denatured salmon sperm DNA, 0.1% sodium dodecyl sulfate, and 10× Denhardt solution (1× Denhardt = 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone 40, and 0.02% Ficoll). Hybridization was performed at 37 °C overnight in prehybridization solution containing 5′-end [32P]-labeled oligonucleotide (1–2 × 106 cpm/ml). The membranes were washed twice for 30 min at room temperature and once for 30 min at 38 °C with 1× SSPE and 0.1% sodium dodecyl sulfate and subjected to autoradiography.Site-directed Mutagenesis—Site-specific mutation was introduced into the P. aeruginosa and E. coli infB open reading frames on pUCP26 using Pfu DNA polymerase and the Stratagene QuikChange mutagenesis protocol.Comparative Modeling—The program ClustalW (30Thompson J.D. Higgins D.G. Gibson T.J. Nucleic Acids Res. 1994; 22: 4673-4680Crossref PubMed Scopus (55157) Google Scholar) was used to derive multiple sequence alignments of prokaryotic IF-2 and of EF-Tu and IF-2 tRNA-binding domains. The 3D-PSSM web server (31Kelley L.A. MacCallum R.M. Sternberg M.J. J. Mol. Biol. 2000; 299: 499-520Crossref PubMed Scopus (1120) Google Scholar) was used to construct sequence-to-structure alignments and to derive templates for model construction. Models were then constructed in the program SPDBV (www.expasy.org/spdbv/; Ref. 32Guex N. Peitsch M.C. Electrophoresis. 1997; 18: 2714-2723Crossref PubMed Scopus (9462) Google Scholar). SPDBV was also used for energy minimization, calculating electrostatic potential surfaces, and superimposition of structures.RESULTS AND DISCUSSIONOverexpression of the P. aeruginosa IF-2 Compensates for the Loss of Formylation in P. aeruginosa—Bacteria such as E. coli, which are primarily dependent on formylation-dependent protein initiation, are unable to initiate protein synthesis without the activity of IF-2. This has been exemplified by the finding that overexpression of the E. coli IF-2 did not alleviate the severe growth defect of an E. coli mutant strain lacking a functional MTF (23Guillon J.M. Heiss S. Soutourina J. Mechulam Y. Laalami S. Grunberg-Manago M. Blanquet S. J. Biol. Chem. 1996; 271: 22321-22325Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, overexpression of IF-2 has been shown to increase the initiator activity of a formylation-defective E. coli initiator tRNA mutant in vivo, suggesting that IF-2 may be able to facilitate utilization of Met-tRNAfMet in protein initiation in E. coli (23Guillon J.M. Heiss S. Soutourina J. Mechulam Y. Laalami S. Grunberg-Manago M. Blanquet S. J. Biol. Chem. 1996; 271: 22321-22325Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Therefore, it is possible that protein initiation using the unformylated Met-tRNAfMet in the P. aeruginosa MTF mutant strain is due to increased expression of the infB gene encoding IF-2. To investigate this possibility, Western blot analysis was used to assess the IF-2 level in total extract from the wild type and mutant P. aeruginosa strains (Fig. 1). The amount of IF-2 is about the same in the MTF mutant strain harboring the pUCP26 vector without (lane 3) or with the P. aeruginosa fmt gene encoding MTF (lane 4). This is comparable to the IF-2 level in the wild type strain carrying pUCP26 without (lane 1) or with the fmt gene (lane 2). Thus, increased expression of the IF-2 gene is not the reason why the P. aeruginosa MTF mutant strain can initiate protein synthesis without formylation. However, findings presented below suggest that the P. aeruginosa IF-2 is responsible for formylation-independent protein initiation in the MTF mutant strain.We have established previously that growth of the P. aeruginosa MTF mutant strain on rich medium at 37 °C is only slightly slower compared with the wild type strain (12Newton D.T. Creuzenet C. Mangroo D. J. Biol. Chem. 1999; 274: 22143-22146Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). However, on minimal medium, the mutant strain (Fig. 2, left panel, sector 2) was found to grow significantly slower than the wild type strain within 15 h of incubation (Fig. 2, left panel, sector 3). Growth of the MTF mutant strain on minimal medium was observed when the incubation was continued for another 33 h (Fig. 2, right panel, sector 2), indicating that the P. aeruginosa MTF mutant strain is viable. We have proposed previously that the P. aeruginosa IF-2 has dual substrate specificity and is responsible for formylation-independent protein initiation in P. aeruginosa (12Newton D.T. Creuzenet C. Mangroo D. J. Biol. Chem. 1999; 274: 22143-22146Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). To test this notion, growth of the P. aeruginosa MTF mutant strain on minimal medium was used to assess whether overproduction of the P. aeruginosa IF-2 facilitates the use of unformylated Met-tRNAfMet in protein initiation (Fig. 2). The wild type P. aeruginosa strains carrying the pUCP26 plasmid without (sector 3) and with the P. aeruginosa fmt (left panel, sector 1) or infB (left panel, sector 4) gene grows on minimal medium within 15 h of incubation at 37 °C. However, no significant growth of the MTF mutant strain harboring pUCP26 alone (left panel, sector 2) was observed within 15 h. In contrast, the mutant strain grows when the P. aeruginosa IF-2 (left panel, sector 5) or MTF (left panel, sector 6) was overproduced. These data suggest that IF-2 is allowing utilization of the initiator Met-tRNA in protein initiation in the P. aeruginosa MTF mutant strain.Fig. 2Complementation of the P. aeruginosa MTF mutant strain with the P. aeruginosa IF-2 or MTF gene. The wild type and mutant MTF P. aeruginosa strains harboring the pUCP26 vector without and with the infB or fmt gene encoding for IF-2 and MTF, respectively, were grown as described under “Materials and Methods.” The cultures were diluted into M9 glucose medium, streaked on M9 glucose medium containing 60 μg/ml tetracycline, and incubated at 37 °C.View Large Image Figure ViewerDownload (PPT)To exclude the possibility that overexpression of the P. aeruginosa IF-2 is causing N-acylation of the methionyl moiety of Met-tRNAfMet by another group, the N-acylation status of Met-tRNAfMet in the P. aeruginosa MTF mutant strain was assessed (Fig. 3). Total RNA, isolated under acidic conditions to prevent hydrolysis of the ester bond between the tRNA and the amino acid, was separated by polyacrylamide gel electrophoresis, and Northern blot analysis was used to detect the tRNAfMet (lane 1), fMet-tRNAfMet, and Met-tRNAfMet species. In the wild type P. aeruginosa strain tRNAfMet was entirely present in the fMet form (lanes 2 and 5), whereas only the Met-tRNAfMet species was detected in the P. aeruginosa MTF mutant strain (lanes 3 and 8). The fMet form of the RNA was found in the MTF mutant strain overproducing MTF (lanes 4 and 6), but only the Met-tRNAfMet species was observed when IF-2 was overproduced (lane 7). These results verify that the P. aeruginosa MTF mutant strain is devoid of MTF and are consistent with IF-2 facilitating formylation-independent protein initiation.Fig. 3Analysis of the formylation status of the initiator tRNA in wild type and mutant MTF P. aeruginosa strains overproducing IF-2 or MTF. Total RNA was separated on a 6.5% polyacrylamide gel containing 8 m urea at 4 °C using 100 mm sodium acetate buffer, pH 5.0, and transferred onto Nytran membrane. The various forms of the initiator tRNA were detected with 5′-CGGGTTATGAGCCCG-3′, which is complementary to nucleotides 28–42 of the anticodon stem loop.View Large Image Figure ViewerDownload (PPT)Disruption of the infB Gene in P. aeruginosa Is Lethal—The P. aeruginosa IF-2 appears to be responsible for utilization of both fMet-tRNAfMet and Met-tRNAfMet in initiation. If this is the case, then disruption of the P. aeruginosa chromosomal infB gene is expected to be lethal. To this test this possibility, we attempted to replace the chromosomal infB gene with a disrupted copy by homologous recombination in P. aeruginosa with or without the pUCPLIF2 plasmid. The two strains were transformed with the suicide vector pEX18APΔKinfB::GmR, and transformants containing the suicide 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 counterselection in the presence of gentamycin.Gentamycin- and sucrose-resistant transformants were obtained within 24 h of incubation at 37 °C. Replacement of the wild type infB gene with the mutant copy in several gentamycin- and sucrose-resistant transformants was assessed by PCR analysis as shown in Fig. 4. Two products of ∼2 and 1 kilobase pair, corresponding to the expected size of the wild type infB gene and the infB::GmR fragment, respectively, were observed in the gentamycin- and sucrose-resistant transformants with (left panel, lanes 6 and 7) and without (lanes 3–5) the pUCPLIF2 plasmid when primers P1 and P2, which are complementary to a segment of the 3′- and 5′-ends of the infB gene, respectively, were used. The 1-kilobase pair fragment but not the 2-kilobase pair fragment was observed in the wild type P. aeruginosa strain without (lane 1) and with the pUCPLIF2 plasmid (lane 2). A 1.6-kilobase pair fragment, corresponding to the expected size of the mutant infB::GmR gene, was detected in the transformants with (middle panel, lanes 6 and 7) and without (lanes 4 and 5) pUCPLIF2 using P3, which is complementary to the 5′-end of the GmR gene and P1. As expected this fragment was not observed in the wild type P. aeruginosa strain without (lane 2) and with (lane 3) pUCPLIF2 but was detected when the pEX18APΔKinfB::GmR plasmid was used as the template (lane 1). These results indicated that the mutant copy of the infB gene is present in the chromosome of transformants with and without the pUCPLIF2 plasmid. To as" @default.
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• W2006366032 title "The Pseudomonas aeruginosa Initiation Factor IF-2 Is Responsible for Formylation-independent Protein Initiation in P. aeruginosa" @default.
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