AJ Bjorkman

AN Lane

H Bauerschmitt

IM Keseler

JJE Bijlsma

KA Datsenko

KH Nierhaus

M Pech

S Shoji

T Kruse

T Ote

X Zhang

Y Qin

Fang Yang

Zhikai Li

Jia Hao

Yan Qin

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LETTEREF4 knockout E. coli cells exhibit lower levelsof cellular biosynthesis under acidic stressDear Editor,Recently, a novel elongation factor, termed EF4, was iden-tiﬁed in E. coli and shown to catalyze the back translocationof the E- and P-tRNAs (tRNA at E and P sites) toA- and P-sites (Qin et al., 2006). Despite a great number ofrecent biochemical studies, little remains known as to theprecise physiological role of EF4 in vivo. In E. coli, 80% ofEF4 molecules are associated with the cell membrane underoptimal conditions, but when cells grow at pH 6 in mediacontaining 100 mmol/L MgCl2, this percentage is reduced toless than 20% (Pech et al., 2011). Furthermore, it wasrevealed that overexpression of EF4 in E. coli severelyaffected cell growth (Qin et al., 2006). However, EF4knockout (ΔEF4, KO) cells exhibited no apparent phenotypein rich LB medium (Shoji et al., 2010). Under certain stres-ses, such as low temperature or high ionic stress, ΔEF4 cellswere out-competed by wildtype (WT) cells within a shortperiod of time (Shoji et al., 2010; Pech et al., 2011).The only organism for which EF4 was shown to beessential is the gastritis pathogen Helicobacter pylori. Asystematic knockout analysis in H. pylori revealed that EF4was essential for cell survival at hostile pH levels close to theacidic conditions in the stomach (Bijlsma, 2000). We there-fore wondered if low pH values represent a stress conditionunder which EF4 is required in E. coli. Because the ef4 KOstrain in Keio was not designed from the wildtype BW25113(Karim, 2010), we deleted ef4 in BW25113 strain by our-selves using the λ Red system (Datsenko and Wanner,2000) (Fig. 1A).In order to establish a relationship between EF4 depen-dency and pH conditions, we ﬁrst prepared LB medium with apH of 3, 5, or 7 and observed the growth of WTor ΔEF4 (KO)cells at each pH. As shown in Fig. 1B, KO cells grew as wellasWTcells at both pH 7 and pH 5, but neither could survive atpH 3. We then found a difference in cell growth at pH 4(Fig. 1D). WT cells grew vigorously after a relatively long lagphase, and ﬁnally reached saturation at an OD of 2 (A580). Incontrast, KO cells remained in lag phase for an extendedperiod of time, to ﬁnally reach saturation at about OD580 = 1.After a 10-fold dilution with pH 7 medium, WT cells regainedtheir normal growth, comparable to that observed for cellsgrown continuously at pH 7. In contrast, the KO strainunderwent a relatively long lag phase even after increase ofpH, and reached saturation at about OD580 = 2, as observedfor WT cells (Fig. 1D). We further found ΔEF4 cells with acomplementary plasmid survived under pH 4 and regainsimilar growth curves to WT cells under the same conditions(Fig. 1C and 1D). These results clearly demonstrate that EF4is required for the E. coli cell growth at pH 4.To investigate the overall impact of EF4 deletion on theprotein proﬁle of WT or ΔEF4 cells, two-dimensional elec-trophoresis (2-DE) was employed. No protein changes weredetected at pH 7 (Fig. S1). Intriguingly, despite the signiﬁcantslow-down in growth, only six proteins were downregulated inKO cells at pH 4 (Fig. 1E and 1F). Spots 1–7 were identiﬁedas tryptophan synthase subunit β (TrpB), D-ribose-bindingprotein mutant, glutamate-ammonia ligase, L-serine ammo-nia-lyase (TdcG), rod shape-determining protein (MreB),tRNA-modifying protein (YgfZ), and D-ribose-binding proteinmutant D67R, respectively. TrpB catalyzes the last step of thetryptophan biosynthesis (Lane and Kirschner, 1991).D-ribose-binding protein serves as the primary chemore-ceptor for chemotaxis (Bjorkman et al., 1994). Glutamate-ammonia ligase catalyzes the single reaction in the glutaminebiosynthesis pathway (Keseler et al., 2013). Three genes(sdaA, sdaB, tdcG) code for three l-serine deaminases inE. coli, and participate in glycine, serine, and threoninemetabolism and cysteine metabolism, and null mutant cellsfor all three genes grow normally in glucose minimal medium.However, the mutant cells become very large, and many lyseupon subculturing in the same medium lacking tryptophan(Zhang and Newman, 2008). Deletion of mreB disrupts thetypical shape of E. coli, enlarging them before ﬁnally lysingthese cells (Kruse et al., 2005). YgfZ protein modiﬁes tRNAs,and its deletion mutant grows slowly relative toWT, especiallyat low temperatures (Ote et al., 2006). In summary, theseproteins are involved in protein biosynthesis, gluconeogen-esis, and cell metabolism processes. Intriguingly, however,none of the six proteins are essential for E. coli growth. Wespeculate that KO cells do not display signs of physicaldamage, and can be rescued from growth arrest upon sub-culturing in medium of pH 7. Hence, when grown to the sameA580, the cells showed similar protein expression proﬁles.Other conditions that lead to a slowing in growth upon ef4KO were shown to result in decreased translation (Pech© The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cn 563Protein Cell 2014, 5(7):563–567DOI 10.1007/s13238-014-0050-3 Protein&CellProtein&CellBA0.00.51.01.52.02.5A5800 50 100 150 200 250 300 350 400 450Time (min)pH 7 WTpH 7 KOpH 5 WTpH 5 KOpH 3 WTpH 3 KOA5800.00.51.01.52.02.5Time (min)0 100 200 300 400 500 600 700 800 900 1000 1100 120010× dilution in LB (pH 7)pH 4 RescuepH 7 WTpH 7 KOpH 4 WTpH 4 KOpH 7 RescueE56 12734pH 3 WT 1056 12734pH 3 KO 10DCGF1ef4H2H1TargetingconstructGenomeModifiedgenomeKanRKanRNheI siteef4    rplK ef4Modified pL11 plasmid    rplK pL11 plasmid12Time (min)0 3 6 100102030405060CPM  (×103 )/µg proteinpH 7 WTpH 7 KOpH 4 WTpH 4 KOTryptophan synthase subunit BetaD-ribose-binding protein mutantL-serine ammonia-lyase TdcGGlutamate-ammonia ligaseRod shape-determining protein EnvBtRNA-modifying protein YgfZD-ribose-binding protein mutant D67RSampleNo.NameMolecular    PIweight/DaQuantityratio(WT/KO)12345675.65.75.75.25.25.17.04.92.52.23.44.31.82.238489285154894752099381193605428498LETTER Fang Yang et al.564 © The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cnProtein&Cellet al., 2011). Similar phenotypes were observed in yeast,where EF4 was found to be located inside the mitochondria(Bauerschmitt et al., 2008). In yeast, loss of EF4 functioncaused growth defects under conditions of low temperatureand starvation, and importantly, decreased mitochondrialtranslation (Bauerschmitt et al., 2008). In order to investigatewhether translation was decreased in our EF4 KO strain atpH 4 we measured the S35-methionine incorporation rate toassay protein translation in vivo. Our analysis revealed thatprotein synthesis was inhibited at pH 4 even in WTcells, withlevels of protein synthesis at about 20% relative to that at pH 7(Fig. 1G). This result was consistent with the slower growthfound in WT cells at pH 4. Under normal pH 7 conditions,loss of EF4 made no signiﬁcant difference (Fig. 1G). Takentogether, these ﬁndings suggested a translation defectpresent in the ΔEF4 strain at pH 4. As a consequence, feweramid acids and tRNAs are needed, which in turn induces adecrease in expression of proteins participating in thesebiosynthesis processes, such as TrpB, glutamate-ammonialigase, TdcG, and YgfZ. This decrease in translation furtherimpairs the response of E. coli cells to the acidic growthconditions, and the expression of proteins required directlyor indirectly for adaption to this environment also changed(Fig. 1E and 1F). Collectively, our results suggested that thelack of EF4 affects translation and growth only in unfavorableconditions. Importantly, its high degree of sequence con-servation throughout evolution indicates that EF4 may playessential roles for the survival in unfavorable growth condi-tions other than extreme temperature, high metal concen-trations, or low pH, and be in part an evolutionary responseto temporary/transient unfavorable conditions.Because of the association of EF4 with ribosomalmovement, and the roles of EF4 in translational processes,we examined the polysome patterns of ΔEF4 cellsthroughout the complete growth process, including lagphase (A580 = 0.1), log phase (A580 = 0.4–0.6), semi-logphase (A580 = 0.8), and stationary phase (A580 = 1.0). Whencells grew at pH 7, the polysome proﬁle of ΔEF4 cells wassimilar to that of WT cells (Qin et al., 2006). Cells in the logphase (A580 = 0.4–0.6) had an active translation system andprotein biosynthesis was efﬁcient. All the classical peaks ofribosomes or ribosomal subunits were present under normalconditions pH 7 (Fig. 2A and 2C). When the pH was loweredto 4, WT cells showed the similar polysome pattern to that atpH 7 (Fig. 2A and 2B). However, the quantity of ribosomes inΔEF4 cells was reduced to 40% of that at A580 = 0.1, andsome non-classical ribosomal subunits appeared as indi-cated with arrows in the top panel of Fig. 2D. Compared tothe position of the 30S peak in Fig. 2C, the non-classicalpeak had a smaller sedimentation coefﬁcient, indicating thatthe biogenesis of the 30S subunit was hindered. This situ-ation became more profound when A580 reached 0.4. Inaddition to premature 30S subunits, there were premature50S peaks, as shown in panel 2 of Fig. 2D. As A580 reached0.6, there were no further non-classical peaks, but the totalquantity of ribosomes did not increase relative to that atA580 = 0.1. In addition, the ratio between polysome and 70Sribosomes suggests that the translation efﬁciency was low.At A580 ≥ 0.8, ΔEF4 cells had the same polysome pattern,but exhibited lower levels of polysome and 70S ribosomesthan that at pH 7, and the total quantity of ribosomes wasmuch lower than the 4S fraction (Fig. 2D). We also noted thatthe total quantity of ribosomes in ΔEF4 cells from differentgrowth phases was always much lower than that of 4S,which represents the background absorbance of cell lysatesat 260 nm (Fig. 2). Our results suggested that ΔEF4 cellstook 10 h to overcome subunit maturation defects, reﬂectingthe inhibition of ribosomal biogenesis in ΔEF4 cells at pH 4.In contrast, the WT strain succeeded in overcoming low pH-induced problems, even though the lag phase was pro-longed to 6 h (Figs. 1 and 2). This data also provides apossible explanation for the null-phenotype observed byShoji et al. (Shoji et al., 2010), who analyzed pH conditionsranging from 3.5 to 12. The difference observed herebetween the two strains only became apparent after 7 h ofculture. In addition, the 70S ribosomes and the 30S/50Ssubunits had reached an equilibrium indicating that thetranslation machinery will not work efﬁciently (Nierhaus andWilson, 2004). In addition, rescued ΔEF4 cells showedclassical polysome patterns (Fig. 2E and 2F).In summary, our results showed slower growth of ΔEF4cells and a lower level of intracellular biosynthesis underFigure 1. Effects of low pH on the growth and proteintranslation of ef4-deleted cells. (A) Construction of a single-gene deletion mutant using the λ Red system. H1 and H2:homologous to chromosomal sequences of the deleted gene;Step ① indicates that the λ-mediated recombination. Thetargeting construct containing selection marker KarR wastransferred to strain BW25113, which contained plasmidsexpressing the components of λ Red system. Then homologousrecombination was performed, and the genomic ef4 gene wasreplaced by KarR. Finally, the resistance gene was eliminatedusing a helper plasmid. (B) E. coliWTcells and ΔEF4 cells (KO)were grown in LB medium at pH 3, 5 or 7. (C) Construction ofthe EF4 rescues plasmid. Step① indicates the redundant NheIrestriction site of pL11 plasmid containing tac promoter wasmutated by PCR ampliﬁcation. Step ② indicates the ef4 geneampliﬁed from E. coli genomic DNA and mutant pL11 digestedby NheI enzyme before ligation to form the EF4 rescue plasmid.(D) E. coli WT cells, ΔEF4 cells, and rescued cells were grownin LB medium at either pH 4 or pH 7. After cells reachedstationary phase, each of the samples was diluted 10 times inLB medium (pH 7). (E) 2-DE images of total proteins in E. coli atpH 4. The number in red circle represents the number ofproteins downregulated in ΔEF4 cells. (F) Quantitative analysisof differentially expressed protein spots from (E). (G) Analysis oftranslation efﬁciency in vivo by 35S-methionine incorporation.At times indicated, samples were taken from each culture, andthe incorporation of radioactive material was determined.sEF4 is essential for optimal growth under low pH stress LETTER© The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cn 565Protein&Cellacidic stress. In agreement with growth defects, the EF4-depleted strain exhibited a very slower protein translation atpH 4 in vivo. However, there was no difference between WTand ΔEF4 cells at normal condition pH 7 (Figs. 1 and 2). Thisphenotype suggested that EF4 plays an essential and spe-ciﬁc role in increasing the ﬁtness of E. coli under unfavorableto extreme environmental conditions, where it ensuresappropriate/adequate regulation of protein synthesis. Our45 15Sucrosegradient (%)0.10.40.60.81.0DC FEBA45 15154545 15)7Hp(eucseR)7Hp(eucseR)7Hp(OK)4Hp(TW KO (pH 4)45 15A580 WT (pH 7)    154570SPolysome50S30S4S0.00.20.40.00.20.40.00.20.40.00.10.20.00.20.40.00.20.40.00.20.40.00.20.40.00.10.20.00.20.40.00.20.40.00.20.40.00.20.40.00.20.40.00.10.20.00.20.40.00.20.40.00.20.40.00.20.40.00.20.40.00.10.20.00.20.40.00.20.40.00.20.40.00.20.40.00.20.40.00.10.20.00.20.40.00.20.40.00.20.4A260 A260 A260 A260 A260 A260A260 A260 A260 A260 A260 A260A260 A260 A260 A260 A260 A260A260 A260 A260 A260 A260 A260A260 A260 A260 A260 A260 A260Figure 2. Polysome proﬁle of ΔEF4 E. coli cells during different growth phases at pH 7 and pH 4. Cell extracts were preparedfrom ΔEF4 strains during different growth phases (OD580 = 0.1, 0.4, 0.6, 0.8 or 1.0). The gradient was fractionated while monitoringthe absorbance at 260 nm (A260). The dotted vertical line indicates the monomer peak. (A and B) Polysome proﬁles of WTcells grownat either pH 7 or pH 4. The ribosome peak-subunits (30S, 50S), monomer (70S) and polysome, and 4S peak are indicated. Thepolysome pattern of cells growing at pH 4 showed no obvious difference to those from cells growing under normal conditions.(C) Polysome proﬁles of ΔEF4 E. coli cells grown at pH 7. (D) Polysome proﬁle of ΔEF4 E. coli cells grown at pH 4. Black arrowsindicate non-classical ribosomal subunits. Note that units on the Y-axis for pH 4 samples are half of those for the pH 7 sample. Thequantity of ribosomes in pH 4 samples was signiﬁcantly reduced compared to that in pH 7 with only few polysomes detected.(E) Polysome proﬁle of KO cells overexpressing EF4 grown at pH 7. The ribosome peaks showed nearly the same classical patternsas WTat pH 7. (F) Polysome proﬁle of EF4-rescued KO cells grown at pH 4. The polysome quantity and patterns under this conditiondid not change signiﬁcantly compared to those of cells grown at pH 7. However, the polysome quantity was higher than that in ΔEF4E. coli cells at pH 4, with polysome patterns more classical.LETTER Fang Yang et al.566 © The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cnProtein&Celldata presented here provide the ﬁrst time evidence that thetranslation factor EF4, contribute to protein synthesis andcell growth at unfavorable conditions, explaining why noapparent role was described at optimal growth conditionsbefore.FOOTNOTESThis work was supported by grants to Y.Q. from the National BasicResearch Program (973 Program) (Nos. 2012CB911001 and2013CB531200) and the National Natural Science Foundation ofChina (Grant Nos. 31170756, 31322015, 31270847, and 31000596),CAS grant (KSZD-EW-Z-003), National Laboratory of Biomacro-molecules in Institute of Biophysics. We thank Dejiu Zhang for helpwith the manuscript preparation, and Dr. Joy Fleming for criticalreading of the manuscript. We thank Dr. Yong Tao of the Institute ofMicrobiology, CAS for his kind gifts of plasmids for E. coli λ Red-mediated homologous recombination.Fang Yang, Zhikai Li, Jia Hao, and Yan Qin declare that theyhave no conﬂict of interest. All institutional and national guidelinesfor the care and use of laboratory animals were followed.Fang Yang1,2, Zhikai Li1, Jia Hao1, Yan Qin1&1 Laboratory of Noncoding RNA, Institute of Biophysics, ChineseAcademy of Sciences, Beijing 100101, China2 University of Chinese Academy of Sciences, Beijing 100049,China& Correspondence: qiny@ibp.ac.cn (Y. Qin)OPEN ACCESSThis article is distributed under the terms of the Creative CommonsAttribution License which permits any use, distribution, andreproduction in any medium, provided the original author(s) andthe source are credited.REFERENCESBauerschmitt H, Funes S, Herrmann JM (2008) The membrane-bound GTPase Guf1 promotes mitochondrial protein synthesisunder suboptimal conditions. 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Proc Natl Acad SciUSA 108:3199–3203Qin Y, Polacek N, Vesper O, Staub E, Einfeldt E, Wilson DN,Nierhaus KH (2006) The highly conserved LepA is a ribosomalelongation factor that back-translocates the ribosome. Cell127:721–733Shoji S, Janssen BD, Hayes CS, Fredrick K (2010) Translation factorLepA contributes to tellurite resistance in Escherichia coli butplays no apparent role in the ﬁdelity of protein synthesis.Biochimie 92:157–163Zhang X, Newman E (2008) Deﬁciency in l-serine deaminase resultsin abnormal growth and cell division of Escherichia coli K-12. MolMicrobiol 69:870–881Electronic supplementary material The online version of thisarticle (doi:10.1007/s13238-014-0050-3) contains supplementarymaterial, which is available to authorized users.EF4 is essential for optimal growth under low pH stress LETTER© The Author(s) 2014. This article is published with open access at Springerlink.com and journal.hep.com.cn 567Protein&Cell

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EF4 knockout E. coli cells exhibit lower levels of cellular biosynthesis under acidic stress

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EF4 knockout E. coli cells exhibit lower levels of cellular biosynthesis under acidic stress

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