yciM is an essential gene required for regulation of lipopolysaccharide synthesis in Escherichia coli

yciM is an essential gene required for regulation oflipopolysaccharide synthesis in Escherichia coli

S. Mahalakshmi,†,‡ M. R. Sunayana,‡ L. SaiSree andManjula Reddy*CSIR-Centre for Cellular and Molecular Biology,Hyderabad, India 500007.

Summary

The outer membrane of Gram-negative bacteria is anasymmetric lipid bilayer consisting of an essentialglycolipid lipopolysaccharide (LPS) in its outer leafletand phospholipids in the inner leaflet. Here, we showthat yciM, a gene encoding a tetratricopeptide repeatprotein of unknown function, modulates LPS levelsby negatively regulating the biosynthesis of lipid A,an essential constituent of LPS. Inactivation of yciMresulted in high LPS levels and cell death in Escheri-chia coli; recessive mutations in lpxA, lpxC or lpxDthat lower the synthesis of lipid A, or a gain of functionmutation in fabZ that increases the formation of mem-brane phospholipids, alleviated the yciM mutant phe-notypes. A modest increase in YciM led to significantreduction of LPS and increased sensitivity to hydro-phobic antibiotics. YciM was shown to regulate LPS byaltering LpxC, an enzyme that catalyses the first com-mitted step of lipid A biosynthesis. Regulation of LpxCby YciM was contingent on the presence of FtsH, anessential membrane-anchored protease known todegrade LpxC, suggesting that FtsH and YciM act inconcert to regulate synthesis of lipid A. In summary,this study demonstrates an essential role for YciM inregulation of LPS biosynthesis in E. coli.

Introduction

The cell envelope of Gram-negative bacteria is made up ofthree distinct layers: the outer membrane (OM), the pepti-doglycan wall (PG), and the inner membrane (IM). The OMserves as an effective permeability barrier to prevent theentry of hydrophobic as well as large hydrophilic moleculesand is responsible for the intrinsic resistance of Escheri-

chia coli to antibiotics, detergents and dyes. Structurally,OM is an asymmetric lipid bilayer with the outer leafletmade up of a unique and essential glycolipid calledlipopolysaccharide (LPS) and the inner leaflet composedof phospholipids. LPS is the major immunogenic surfacemolecule consisting of three covalently linked moieties:lipid A, a proximally located hydrophobic anchor thatserves as an endotoxin; a distal O-antigen chain; and acore oligosaccharide that connects these two domains(Nikaido, 1996; Raetz, 1996; Bos et al., 2007).

LPS is essential for growth of most Gram-negative bac-teria. Any perturbations in synthesis, assembly or functionof LPS affect normal barrier properties of OM leading toaltered permeability to various hydrophobic antibiotics,detergents, and dyes (Vaara, 1992; Nikaido, 2003). Thecomponents of LPS are synthesized on the cytoplasmicsurface of IM as discrete units and are transported into theperiplasmic side of IM before being assembled to form afunctional LPS molecule (Schnaitman and Klena, 1993;Raetz, 1996; Bos et al., 2007; Raetz et al., 2007). Thefirst step in the synthesis of lipid A is fatty acylation ofa sugar-nucleotide, UDP-N-acetylglucosamine by LpxA(Fig. 1). This monoacylated molecule is then irreversiblydeacetylated by LpxC for further acylation by LpxD, thethird enzyme in the biosynthetic machinery of lipid A. Inboth the acylation steps catalysed by LpxA and LpxD, thefatty acyl donor is R-3-hydroxymyristoyl-ACP generated bythe activity of fabZ encoding an ACP dehydrase. R-3-hydroxymyristoyl-ACP also serves as an acyl donor for thesynthesis of membrane phospholipids, and hence is situ-ated at a crucial metabolic branch point in the biosynthesisof membrane components (Raetz and Dowhan, 1990;Mohan et al., 1994).

Subsequent steps catalysed by LpxH, LpxB, and LpxK,respectively, result in the synthesis of an intermediate, lipidIVA. Two molecules of an eight carbon sugar, keto-deoxy-D-manno-octulosonic acid (Kdo) are then added to lipid IVA

by WaaA (or KdtA), a glycosyl transferase forming Kdo2-IVA, the minimal essential lipid A required for growth ofE. coli. Kdo2-IVA is further acylated sequentially by WaaM(LpxL) and WaaN (LpxM) to finally yield Kdo2-lipid A(Fig. 1). Subsequently, the core oligosaccharide is addedto Kdo2-lipid A molecule and the nascent core-lipid A isflipped across IM to the periplasmic surface of IM (Trent,2004; Raetz et al., 2007). Here, the Kdo2-lipidA is ligated to

Accepted 1 November, 2013. *For correspondence. E-mail [email protected]; Tel. (+91) 40 27192523; Fax (+91) 40 27160310.†Present address: Department of Microbiology, Osmania University,Hyderabad, India 500007. ‡These authors contributed equally to thework.

Molecular Microbiology (2014) 91(1), 145–157 ■ doi:10.1111/mmi.12452First published online 21 November 2013

© 2013 John Wiley & Sons Ltd

mailto:[email protected]

mailto:[email protected]

the O-antigen polymer and transported to the outer leafletof OM by Lpt system (Bos et al., 2007; Ruiz et al., 2009).However, most laboratory strains of E. coli such as K12and B are deficient in the synthesis of O-antigen andcontain LPS molecules that lack O-antigen (Nikaido,1996).

Lipid A synthesis is regulated at the deacetylationstep catalysed by lpxC gene product, UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase asthis is the first committed step of lipid A biosyntheticpathway (Young et al., 1995; Sorensen et al., 1996). LpxCis modulated at the level of proteolysis by an essentialmembrane-bound AAA-type metalloprotease, FtsH inE. coli (Ogura et al., 1999; Ito and Akiyama, 2005; Führeret al., 2006). Absence of FtsH stabilizes LpxC leading toenhanced levels of LPS and cell death (Ogura et al., 1999;Führer et al., 2006). In addition to regulating LpxC, FtsH isalso involved in the turnover of glycosyl transferase(WaaA) that ligates Kdo residues to lipid IVA moleculeensuring a balanced synthesis of mature LPS (Katz andRon, 2008). Furthermore, LpxC enzyme levels and activityincrease 5- to 10-fold if the early steps in lipid A biosynthe-sis are blocked either by mutations in lpxA, lpxD or bytreatment of cells with sublethal concentrations of an inhibi-tor of LpxC; these findings point to the existence of an asyet unidentified pathway that involves feedback regulationof LpxC synthesis and activity (Onishi et al., 1996;Sorensen et al., 1996; Raetz et al., 2007).

In this study, we demonstrate the existence of an addi-tional player, YciM, a conserved tetratricopeptide repeat(TPR) protein of unknown function in the regulation of lipidA biosynthesis. Absence of yciM increases LpxC enzymelevels resulting in a higher amount of LPS ultimatelyleading to cell death. Conversely, a modest increase ofyciM significantly reduces the amount of LpxC leading to

lowered LPS levels and increased OM permeability tovarious hydrophobic antibiotics, indicating that YciM nega-tively regulates lipid A synthesis. In addition, we show thatthe regulation of LPS by YciM is mediated by FtsH, anessential membrane-bound AAA-protease known todegrade LpxC suggesting that YciM may modulate theproteolytic activity of FtsH towards LpxC. In summary,these results demonstrate a novel regulation of lipid Abiosynthesis which is a potential target for development ofantibacterial therapeutic agents.

Results

yciM is essential for growth and viability of E. coli K12

During the course of our studies, we observed that strainJW1272 from the Keio mutant collection (Baba et al.,2006), carrying a deletion of yciM gene (BW25113ΔyciM::Kan; Table 1), grew very poorly on nutrient agar(NA) at 42°C, although it was able to grow very well on LBor minimal A plates. However, when we attempted totransfer the yciM::Kan deletion from strain JW1272 byP1-phage mediated transduction into either MG1655 (wild-type K12 strain) or BW25113 (parental strain of the Keiocollection), no transductants were obtained on LB orminimalAplates. On the other hand, the yciM::Kan deletioncould be introduced into a Kan-sensitive yciM+ derivative ofJW1272 (constructed by crossing out the region encom-passing the yciM::Kan deletion of JW1272 with linkedtrpB::Tet marker; Table 1); these observations suggestedthat the yciM gene is essential for the growth of E. coli, andthat the original JW1272 strain carries suppressor muta-tion (s) elsewhere on the chromosome enabling its growth.We further confirmed the requirement of yciM for thegrowth of E. coli by successful introduction of yciM::Kan

Fig. 1. Schematic diagram depicting the Kdo2-lipid A biosynthesis. The precursor of lipid A synthesis is UDP-N-acetylglucosamine(UDP-GlcNAc) and the fatty acyl donor for both lpxA and lpxD is 3-hydroxy-myristoyl acyl carrier protein (3-OH-C14-ACP). lpxC encodes thedeacetylase which is the rate-limiting enzyme of this pathway. Of the nine genes required for the synthesis of Kdo2-lipid A, the first sevengenes are absolutely essential for synthesis of lipid A; waaM is required for high-temperature survival and waaN is not essential for growth ofE. coli (Trent, 2004; Raetz et al., 2007).

146 S. Mahalakshmi, M. R. Sunayana, L. SaiSree and M. Reddy ■

© 2013 John Wiley & Sons Ltd, Molecular Microbiology, 91, 145–157

deletion into MG1655 strain carrying a plasmid-borne copyof yciM (pMN101) but not into MG1655 carrying theplasmid vector alone.

To further examine these observations, we constructedstrain MR703 (MG1655 ΔyciM::Kan carrying a conditionalIPTG-dependent replication vector, pAM34 with a clonedcopy of yciM, pMN103). The plasmid pAM34 is unique inthat the replication of the plasmid itself is dependent on thepresence of IPTG (Gil and Bouche, 1991); hence strainMR703 is yciM+ on IPTG-supplementation and yciM− inabsence of IPTG. As shown in Fig. 2A, this strain wasextremely sick on LB and did not grow at all on minimal Aplates at restrictive conditions (without IPTG) whereas itgrew very well at the permissive conditions (with IPTG).

Further, the terminal phenotype in cells of strain MR703grown under restrictive conditions was significant cell lysisas evidenced by decrease in optical density and colonyforming units (Fig. 2B). The lysing cells exhibited morpho-logical aberrations and cell filamentation as visualized byDIC and live-dead cell staining (Fig. 2C).

The structural organization of the yciM locus indicatedthat it is the second gene in a bicistronic operon, yciSM,located at 28.85 min on the E. coli chromosome. Toexamine the role of the upstream yciS gene, we con-structed MG1655 ΔyciS::Kan deletion strain (MR704) anda minor effect on cell viability was noticed from the size ofthe colonies growing on plates at restrictive conditions(Fig. 2A). However, this effect disappeared when the kana-

Table 1. Bacterial strains and plasmids used in this study.

Strain Relevant characteristics Source or reference

BW25113 lacIq rrnB3 ΔlacZ4787 Δ(araBAD)567 Δ(rhaBAD)568 hsdR514 Baba et al. (2006)CAG18455 MG1655 trpB83::Tn10 Lab collectionD22 lpxC101 proA23 lac-28 tsx-81 trp-30 his-51 rpsL173 tufA1 ampCp-1 CGSCa; Normark et al. (1969)JW1272 BW25113 ΔyciM::Kan Baba et al. (2006)MC4100 F− araD139 Δ(argF-lac)U169 rpsL150 relA1 flb5301 deoC1 thiA1 ptsF25 rbsR Lab collectionMG1655 rph1 ilvG rfb-50 (wild-type) Lab collectionRL436 Hfr (PO3) lpxD36(ts) relA1 spoT1 metB1 CGSCa; Lathe et al. (1980)SM101 F− thr-1 araC14 lpxA2(ts) tsx-78 Δ(galK-attLAM)99 hisG4(Oc) rfbC1 rpsL136

xylA5 mtl-1 thiE1CGSCa; Galloway and Raetz (1990)

MR701 JW1272 yciM+ KanS trpB83::Tn10 This studyMR702 MG1655/pMN103 This studyMR703 MG1655 ΔyciM::Kan/pMN103 This studyMR704 MG1655 ΔyciS::Kan/pMN103 This studyMR705 MG1655 ΔyciSM::Cm/pMN103 This studyMR706 MG1655 lpxC1272 leuB::Tn10 This studyMR707 MR706 ΔyciSM::Cm This studyMR708 MG1655 lpxD14 (skp::Tn10dTet) This studyMR709 MR708 ΔyciSM::Cm This studyMR710 MG1655 lpxD36 zae-502::Tn10 This studyMR711 MR710 ΔyciSM::Cm This studyMR712 MG1655/pfabZ This studyMR713 MR712 ΔyciM::Kan This studyMR714 MG1655/pMN104 This studyMR716 MG1655/pMN104/pMN105 This studyMR718 MC4100 ΔyciS::lacZ-Kan/pMN102 This studyMR719 MC4100 ΔyciM::lacZ-Kan/pMN102 This studyMR720 MR705 sfhC21 zad-22::Tn10 This studyMR721 MG1655 ftsH1 zgj-3198::Tn10Kan This studyMR722 MR705 ftsH1 zgj-3198::Tn10Kan This study

PlasmidspAM34 pMB1-based, IPTG-dependent replicon, AmpR, SpcR Gil and Bouche (1991)pCL1920 pSC101-based, SpcR Lerner and Inouye (1990)pDSW210 ColE1, AmpR, P206 (trc) promoter Weiss et al. (1999)pMA2 pSC101 (Ts), AmpR Reddy (2007); Japanese cloning

vector collectionpKGE137 KanR, FRT, lacZY+, Ori-R6K Ellermeier et al. (2002)ASKA-fabZ CmR, pCA24N-fabZ Kitagawa et al. (2006)pMN101 pCL1920-yciM This studypMN102 pMA2-yciM This studypMN103 pAM34-yciM This studypMN104 pDSW210-yciM This studypMN105 pCL1920-lpxC This studypMN106 pMU2385-plpxC (plpxC::lacZ) This study

a. Coli Genetic Stock Center.

Regulation of lipid A synthesis by yciM 147


mycin determinant from yciS::Kan deletion was flipped outindicating that this defect is likely due to polarity of Kaninsertion on the expression of downstream yciM gene. Astrain with a complete deletion of yciSM locus carrying a

cloned copy of only yciM gene (MG1655/ΔyciSM::Cm/pMN103, designated MR705) also behaved identical tothat of the single yciM deletion strain, MR703, showing thatabsence of yciM alone is responsible for the loss of viability

Fig. 2. yciM is essential for growth and survival of E. coli.A. Growth of mutants lacking yciSM, yciM or yciS. Cultures of WT (MG1655), ΔyciSM, ΔyciM or ΔyciS strains carrying the plasmidpAM34-yciM (pMN103) were grown overnight in LB-IPTG (500 μM) broth, serially diluted and 5 μl from each dilution were spotted on indicatedplates. Growth was scored after 18–24 h at 37°C. MM (Minimal A; Miller, 1992) plates were supplemented with 0.2% glucose as sole carbonsource. Although faint growth was observed on LB plates in case of ΔyciSM or ΔyciM strains, these could not be further purified. As bothΔyciSM and ΔyciM mutant strains exhibited similar growth defects and phenotypes, these two strains were used interchangeably depending onthe availability of the antibiotic resistance determinant.B. Growth of mutants lacking yciSM or yciM. Strains MR703 (ΔyciM/pMN103; blue) or MR705 (ΔyciSM/pMN103; red) were grown overnight inLB-IPTG (500 μM) and next morning, they were diluted 1:500 into either LB (closed squares) or LB supplemented with 500 μM IPTG (opensquares) and grown at 37°C. Growth was monitored by measuring absorbance at 600 nm (i) and also by determining the colony-forming units(cfu ml−1) (ii) at regular intervals.C. Cells lacking yciM undergo lysis and cell death. Strains MG1655/pAM34 (I); MG1655/pAM34-yciM (II) and MG1655 ΔyciM/pAM34-yciM(MR703; III) were grown overnight in LB-IPTG broth and next morning diluted 1:500 into fresh LB broth (without IPTG; restrictive conditions inwhich pAM34 based vectors will not be able to replicate) and grown at 37°C. At each hour, 1 ml aliquots were withdrawn, cells were stainedusing LIVE/DEAD BacLight bacterial viability kit (Molecular Probes) and observed by DIC (differential interference contrast) microscopy andfluorescence microscopy using GFP and DsRed filters on a Zeiss apotome microscope. Representative pictures at 3 h of incubation areshown. In this assay, live cells stain green and dead cells stain red. Scale bar represents 5 μm.



in these mutants (Fig. 2A and B). In summary, the resultsabove demonstrate that yciM is fundamental for growth ofE. coli under standard laboratory conditions and that strainJW1272 is viable owing to the presence of compensatoryor suppressor mutation (s) unlinked to yciM locus.

Mutations that lower lipid A synthesis suppress theessentiality of yciM

To understand the basis of yciM essentiality, we identifiedthe mutation responsible for the viability of JW1272 withthe aid of a transposon-tagging approach as described inExperimental procedures. The suppressor was found to bea recessive mutation in lpxC that is due to T-to-A conver-sion in codon 186 resulting in a change in the amino acidsequence from isoleucine to asparagine (lpxC-I186N,hereafter referred to as lpxC1272). Fig. 3A shows theextent of suppression conferred by lpxC1272 allele instrain MR705. A known allele of lpxC (lpxC101 or envA1;Normark et al., 1969; Beall and Lutkenhaus, 1987) alsosuppressed the growth defects of both yciM and yciSMdeletion mutants to a moderate extent (data not shown).

The lpxC1272 allele alone (reconstructed in the wild-type strain background MG1655, designated MR706;Table 1) conferred sensitivity to growth on NA at 42°C,

indicating that the initial phenotype observed in strainJW1272 is essentially due to lpxC mutation. It is interest-ing to note that in the chemical genomics screen ofNichols et al. (2011), JW1272 exhibited a strong correla-tion with asmB mutations that are allelic to lpxC (Kloseret al., 1996), suggesting that most likely the lpxC suppres-sor mutation would have arisen during the construction ofyciM deletion mutant. In an earlier study, strain JW1272was identified as a mutant deficient in biofilm formationand was also shown to have synthetic lethal phenotypeswith surA and yfgA (rodZ) which code for a periplasmicpeptidyl-prolyl isomerase and a cytoskeletal membraneprotein respectively (Niba et al., 2007); however, the sig-nificance of these observations remains to be examined.

We also obtained additional suppressors in strainMR703 after random transposon insertional mutagenesisas described in Experimental procedures. Genetic andmolecular characterization of an insertion mutation thatconferred very significant suppression to yciM deletionmutant showed an insertion after codon 79 in a genecalled skp located at 4 min. However, subsequent studiesshowed that the resultant suppression phenotype of yciMdeletion is a consequence of a polar effect of the insertionon the expression of downstream lpxD gene, as a plasmidencoding lpxD alone was able to abrogate the suppressionconferred by this mutation whereas a plasmid carrying skpwas unable to do so (for convenience sake, this allele ishereafter referred to as lpxD14). In addition, a Δskp::Kaninsertion-deletion from Keio collection (Baba et al., 2006)also suppressed the yciM growth defects, presumably dueto the polar effect of the Kan insertion on lpxD. The growthdefect of yciM mutant was also abolished by introductionof lpxD36, a known temperature-sensitive allele of lpxD(Lathe et al., 1980) as shown in Fig. 3A. A temperature-sensitive mutation in lpxA (lpxA2; Galloway and Raetz,1990) also weakly suppressed the absence of yciM at30°C, although at 37°C, the lpxA2 mutant itself was notviable. On the other hand, deletion mutations in waaM,waaN or lpxT involved in the maturation of lipid A did notrescue the growth phenotypes of yciM deletion mutant(data not shown).

Absence of yciM results in increased LPS

As mutations in lpxA, lpxC or lpxD that are known to lowerlipid A biosynthesis (Raetz, 1996) suppressed the yciMgrowth defects, we speculated that yciM may have a rolein lipid A (or LPS) metabolism or regulation. Hence, wemeasured the levels of LPS in strains MR703 (ΔyciM::Kan/pMN103) and MR705 (ΔyciSM::Cm/pMN103) grown atrestrictive conditions (without IPTG), by estimating theamount of Kdo and also by quantifying LPS using SDS-PAGE. Data from Fig. 4B and Table 2 indicate that theamount of LPS is around twofold higher in yciM or yciSM

Fig. 3. Growth of yciSM deletion mutant and its suppressors.A. Suppressors of yciSM growth defect. Strain MR705 and itsderivatives carrying lpxC1272, lpxD14, lpxD36, or sfhC21 alleleswere grown in LB with IPTG, serially diluted and 5 μl from eachdilution were spotted on MM plates with (500 μM) or without IPTG.As controls, WT (MG1655) carrying either empty vector (pAM34) orpMN103 (pAM34-yciM) are also grown under similar conditions.B. Multiple copies of fabZ suppress the growth defect of yciMdeletion mutant. Strain MG1655 (WT) carrying a plasmid encodingfabZ and its yciM deletion derivative were grown with 20 μM IPTG,serially diluted and 5 μl spotted on LB plates with (20 μM IPTG forinduction of fabZ expression) or without IPTG.



mutant cells compared with that of the parental strainimplicating a role for YciM in regulation of LPS.

To examine the basis of suppression of YciM− phenotypeby lpxC or lpxD mutations, LPS levels in these single anddouble mutants were also measured. Mutations in lpxC(lpxC1272) or lpxD (lpxD14) lowered the LPS levels both inthe wild-type as well as in the yciSM deletion strain, sup-porting the notion that the suppression of yciSM growthdefects is most likely due to decreased LPS (or lipid A)synthesis (Fig. 4C and Table 2). However, although bothlpxC1272 and lpxD14 single mutants showed approxi-mately 50% decrease in LPS (Fig. 4C, lanes 2 and 4), theLPS level of lpxC1272 yciSM double mutant was compa-rable to that of single lpxC1272 mutant (Fig. 4C, lanes 4and 5) whereas in lpxD14 yciSM double mutant the LPSwas elevated approximately twofold (similar to that of thelevel seen in the wild-type) compared with the singlelpxD14 mutant (Fig. 4C, lanes 2 and 3). In addition,lpxC101 and lpxD36 alleles also behaved similarly in that

the LPS levels of lpxC101 yciSM strain remained like thatof lpxC101 single mutant whereas in lpxD36 yciSM strain,the LPS levels were higher compared with lpxD36 alone(Fig. S1).

Mutations that lower lipid A synthesis alter the barrierproperties of OM leading to hyperpermeability (andhence, increased sensitivity) to hydrophobic antibiotics(Vaara, 1992; Nikaido, 2003). As expected, strains carry-ing either lpxC1272 (MR706), lpxD14 (MR708) or lpxD36(MR710) alleles were hypersensitive to antibiotics such asvancomycin and nalidixic acid (Fig. 4A). Interestingly,absence of yciSM reversed the antibiotic hypersensitivityphenotypes of both lpxD14 and lpxD36 mutants but not oflpxC1272 (Fig. 4A). This observation correlates well withthe results above wherein the LPS levels of lpxD14 yciSMstrain but not of lpxC1272 yciSM are restored back to thewild-type level (Fig. 4C; Table 2) and these results arediscussed below.

Increased FabZ also suppresses the absence of yciM

It is known from earlier studies that balanced synthesis ofOM components is crucial for growth. The toxic effectsof high LPS are alleviated by increasing the formationof membrane phospholipids by multiple copies of FabZthat increases the pool of R-3-hydroxymyristoyl-ACP, acommon precursor of both lipid A and phospholipid synthe-sis (Ogura et al., 1999). Conversely, recessive mutations infabZ suppress the effects of lpxA or lpxC by balancing theratio of LPS to phospholipid (Mohan et al., 1994; Kloseret al., 1998; Zeng et al., 2013). Therefore, we examinedthe effect of multiple copies of fabZ in yciM mutant back-ground and observed that increased expression ofplasmid-borne Plac-fabZ (achieved by addition of 20 μM

Fig. 4. Effect of yciM/SM deletions in mutants defective in lipid Abiosynthesis.A. Antibiotic hypersensitivity phenotypes of MG1655 (WT) and itsderivatives carrying lpxC1272 or lpxD14 single mutations orlpxC1272 yciSM or lpxD14 yciSM double mutations on LB platessupplemented with either vancomycin (Van; 80 μg ml−1) or nalidixicacid (Nal; 1 μg ml−1).B. Silver-stained tricine-SDS-PAGE gel showing LPS levels ofyciM/SM deletion mutants. Strains MG1655 (WT), ΔyciM or ΔyciSMcarrying pAM34-yciM (pMN103) were grown in LB-500 μM IPTGovernight and next morning, subcultured 1:500 into LB brothwithout IPTG. The cultures were grown for 120 min and cells wereharvested before lysis followed by preparation of cell lysates asdescribed in Experimental procedures. Total protein was estimatedand protein equivalent to 15 μg was electrophoresed and stainedfor LPS.C. Silver-stained tricine-SDS-PAGE gel showing LPS levels of WT,lpxD14, lpxD14 yciSM, lpxC1272 and lpxC1272 yciSM strains.Strains were grown in LB to an OD600 of 1.0 and cell lysates wereprepared. Total protein was estimated and an equivalent of 25 μgprotein was loaded in each lane.

Table 2. Kdo values of various E. coli strains.

Relevant genotypeμg of Kdo per mgof proteina

Wild-type (MG1655) 9.0 ± 0.5ΔyciSMb 18.0 ± 2.0ΔyciMb 20.0 ± 1.8lpxC1272 7.3 ± 0.5lpxC1272 ΔyciSM 7.5 ± 0.6lpxD14 7.9 ± 0.4lpxD14 ΔyciSM 10.5 ± 0.3cMG1655/ptrc::yciM 8.3 ± 0.2cMG1655/ptrc::yciM (100 μM IPTG) 6.4 ± 0.2dΔyciM/pfabZ 18.0 ± 0.9dΔyciM/pfabZ (20 μM IPTG) 6.2 ± 0.5

a. Kdo values were determined from three independent experiments.b. Strains MR703 and MR705 were grown at restrictive conditions(LB) for 120 min and processed before lysis.c. Strain MR714 grown with (100 μM) and without IPTG.d. Strain MR713 grown with (20 μM) and without IPTG.



IPTG to the growth medium) suppressed the yciM growthdefects extremely well (Fig. 3B) and also lowered the levelof LPS (Table 2). In addition, a gain of function allele of fabZ(sfhC21, which is known to suppress ftsH mutations byincreasing the formation of phospholipids; Ogura et al.,1999) restored growth equally well to the yciSM deletionmutant (Fig. 3A).

yciM overexpression leads to a decrease in LPS levelswhich is reversed upon LpxC overexpression

As the experiments above suggested involvement ofyciM in modulation of LPS levels, we examined theeffect of additional copies of yciM on cell growth andpermeability. For this purpose, yciM was cloned inpDSW210 (Weiss et al., 1999), a medium-copy-number,ColE1-based vector under the control of an attenuatedtrc promoter (Ptrc::yciM; designated pMN104) and intro-duced into MG1655. Increasing the expression of yciM(by addition of 50 μM IPTG) resulted in severe hyper-sensitivity to various antibiotics such as vancomycin,rifampin and nalidixic acid indicating a defect in OMbarrier functions (Fig. 5A). Likewise, there was a signifi-cant reduction in LPS levels when yciM is present inmultiple copies (Fig. 5B and Table 2). All these pheno-types could be reversed by a moderate increase in theexpression of lpxC which is known to increase the fluxinto lipid A biosynthesis (Fig. 5A).

YciM regulates LpxC, the rate-limiting enzyme of lipid Abiosynthetic pathway

Since LpxC is the rate-limiting enzyme in lipid A biosyn-thetic pathway and that absence of yciM was not able toelevate the LPS level in lpxC mutants in our studies(Fig. 4A and C), we reasoned that YciM may regulate lipidA synthesis at the step of LpxC. Accordingly, we examined

the level of LpxC protein in various mutants by westernanalysis using anti-LpxC antisera. Fig. 6A shows approxi-mately fivefold upregulation of LpxC enzyme levels inabsence of yciM or yciSM, clearly suggesting that LPSoverproduction in these mutants is due to increasedamount of LpxC. Likewise, there was approximatelytwofold decrease in LpxC levels in presence of morecopies of yciM (Fig. 6B, lanes 1 and 2).

To examine whether these alterations are due to tran-scriptional regulation of lpxC, a β-galactosidase reportergene was fused to the promoter of lpxC on a low-copy-number plasmid (as described in Experimental proce-dures) and introduced into appropriate mutant strains.But, there was no significant difference in the expressionof lpxC-lacZ fusion during the absence or overexpres-sion of yciM (Table S1). An lpxC-GFP promoter fusionon a low-copy-number plasmid (Zaslaver et al., 2006)also behaved similarly in that there was no alteration inthe expression of GFP both in absence of yciM and inpresence of additional copies of yciM (data not shown)suggesting a post-transcriptional regulation of LpxC byYciM.

YciM-mediated regulation of LpxC is dependent onfunctional FtsH protease

As mentioned above, LpxC is regulated by FtsH, amembrane-anchored essential metalloprotease at thelevel of proteolysis, and as a consequence, ftsH mutantsaccumulate LpxC leading to lethal overproduction of LPSand cell death (Ogura et al., 1999; Führer et al., 2006). Asabsence of either FtsH or YciM results in similar growthdefects and that both the mutants are suppressed byfactors that lower lipid A synthesis or increase the mem-brane phospholipid formation (Fig. 3; Ogura et al., 1999),we speculated that FtsH and YciM may participate in acommon pathway to regulate the levels of LpxC. Toexamine whether YciM regulates LpxC via FtsH, LpxC

Fig. 5. Effect of multiple copies of yciM onOM permeability and LPS levels.A. Antibiotic hypersensitivity of strainsoverexpressing yciM. MG1655/pDSW210(empty vector control), MG1655/pMN104(Ptrc::yciM) or MG1655/pMN104(Ptrc::yciM)/pMN105 (pCL-lpxC) were grownand serial dilutions were spotted on LB plates(supplemented with 50 μM IPTG to elevatethe expression of yciM) containing eithervancomycin (Van; 125 μg ml−1) or nalidixicacid (Nal; 2 μg ml−1).B. Silver-stained tricine-SDS-PAGE gelshowing LPS levels in MG1655 carryingpDSW210 (vector) or pMN104 (Ptrc::yciM).Strains were grown in presence of either 100or 200 μM IPTG to induce the expression ofyciM and 25 μg protein was loaded in eachlane.



levels were measured in an ftsH mutant strain (carrying atemperature-sensitive allele, ftsH1) with additional copiesof yciM (MR721/pMN104). Figure 6B shows higher levelsof LpxC in ftsH1 mutant as expected; however, overex-pression of yciM did not decrease the LpxC levels in ftsH1strain background (Fig. 6B, lanes 3 and 4) unlike that of inthe wild-type strain (Fig. 6B, lanes 1 and 2). Moreover,LpxC levels remained unaltered in an ftsH1 mutant straincarrying a deletion of yciSM compared with either of thesingle mutants (Fig. 6C). Both these experiments showedthat regulation of LpxC by YciM is mediated through func-tional FtsH protease.

To test whether YciM is a general modulator of theproteolytic activity of FtsH, we examined the levels ofRpoH (σ32), another known target of FtsH (Herman et al.,1995; Tomoyasu et al., 1995) in various strain back-grounds (wild-type, ΔyciM, ΔyciSM and ftsH1) by westernanalysis using anti-RpoH antisera (Fig. S2). RpoH wasnot stabilized in absence of YciM indicating that YciM maynot have an effect on all the targets of FtsH protease.However, as expected, RpoH levels were high in the ftsH1mutant background (Fig. S2).

LpxC increase in mutants defective in early steps oflipid A biosynthesis is dependent on YciM

We also observed that single mutations in lpxA, lpxC orlpxD that lower the lipid A synthesis increased the LpxCenzyme levels approximately two- to sixfold (Fig. 6A; Fig.S3) confirming the previous findings of Sorensen et al.(1996). However, the LpxC level in each of these mutantscarrying a deletion of yciSM (i.e. lpxC1272 yciSM; lpxC101yciSM; lpxD14 yciSM and lpxD36 yciSM) was not additive,but similar to that of the single yciSM deletion mutantraising an interesting possibility that the feedback regula-tion of LpxC in mutants defective in early steps of lipid Abiosynthesis is dependent on the presence of YciM(Fig. 6A; Fig. S3).

In addition, the elevated LpxC levels observed in thelpxD yciSM mutants (Fig. 6A, lanes 5 and 6; Fig. S3)allowed us to explain our earlier findings in which the lpxDyciSM double mutants showed increased amounts of LPSleading to restoration of antibiotic hypersensitivity pheno-types of lpxD single mutants (Fig. 4A and C). In otherwords, higher amounts of functional LpxC in absence of

Fig. 6. Western analysis for levels of LpxC in various strains.A. LpxC levels in yciM/SM deletion mutants and their suppressors. WT, ΔyciM or ΔyciSM strains carrying the plasmid pMN103 (pAM34-yciM)were grown overnight in LB-IPTG and next morning diluted 1:500 in LB and grown for 120 min (approximately to an OD600 of 0.4) and celllysates were prepared. Strains carrying lpxC1272, lpxD14 or their yciSM derivatives are also grown in LB till an OD600 of 0.4 followed by celllysate preparation. Total protein was estimated and equal amount of protein was loaded in each lane of an SDS-PAGE gel followed byWestern blotting with anti-LpxC antiserum. The upper box depicts the levels of LpxC whereas the lower box represents an approximately65 kDa protein band from the same blot that has been stained by Ponceau which served as loading and transfer control. The band intensitieswere quantified using Image-J software and the fold-change is indicated below the blots.B. LpxC levels in WT or ftsH1 mutant carrying either pDSW210 (vector control) or pMN104 (ptrc::yciM). The strains are grown overnight in LBand subcultured 1:250 into LB with 100 μM IPTG and grown initially at 30°C till an OD600 of 0.15 and then shifted to 42°C and further grownfor 150 min. Cells are harvested and processed as described above.C. LpxC levels of WT, ftsH1, yciSM or ftsH1 yciSM mutants. All these four strains carrying pMN103 (pAM34-yciM) were grown overnight inLB-IPTG and next morning, subcultured 1:250 and grown at 30°C till an OD600 of 0.15 after which they were shifted to 42°C and further grownfor 105 min. Cells are harvested and processed as described above.



yciM results in elevated LPS in lpxD14 and lpxD36mutants. However, in lpxC mutants, the accumulation ofmutant (non-functional) LpxC enzyme may not contributeto an increase in LPS synthesis and thus do not alter theantibiotic-hypersensitivity phenotypes of lpxC mutants(Fig. 4A and C).

yciS and yciM show transcriptional upregulation byincreased RpoH

To examine the physiological regulation of yciSM operon,transcriptional lacZ fusions of yciS and yciM were con-structed on the chromosome as described in Experimen-tal procedures. As these reporter gene fusion strains aredeleted for yciS or yciM structural genes, they are con-structed in strains carrying a plasmid-borne copy of yciM,pMN102 (Table 1). The β-galactosidase values of theselacZ fusions indicated that yciSM operon has a moderate-to-strong promoter (Table S2). However, the lacZ expres-sion levels in these strains were not altered either bydecrease or increase of yciM or by presence of lpxC orlpxD mutant alleles, indicating that yciSM is not feedbackregulated by lipid A at the transcriptional level. In a globalgene expression study, it was earlier reported that theexpression of yciS and yciM are upregulated by increasedcopies of RpoH (Zhao et al., 2005; Nonaka et al., 2006).We experimentally validated these observations byshowing that the lacZ expression of both yciS and yciMfusion strains is enhanced approximately threefold byincreased expression of RpoH but not of RpoE (Table S2).However, the significance of this observation remains tobe understood as expression of yciS/M was unaltered byvariations in temperature, osmolarity or pH (data notshown), although in expression profiling experiments,yciM mRNA was reported to be induced at high tempera-ture (47°C) and also at acidic pH conditions in presence ofcadmium (Worden et al., 2009; Murata et al., 2011).

Mutations that affect outer membrane assembly ororganization also suppress the yciM essentiality

We obtained and characterized several transposon inser-tion mutations that were weak suppressors of the growthdefect in the yciM deletion mutant MR703. The insertionswere identified to be in genes encoding, (1) a lipopolysac-charide kinase (rfaP), (2) UDP-glucose pyrophosphory-lase (galU), (3) a major OM lipoprotein (lpp) and (4) aprotein of unknown function (ybcN). The correspondingdeletion mutations from the Keio collection also showedsimilar weak suppression phenotypes (Fig. S4). Further-more, deletion mutations in genes encoding other outermembrane proteins such as ompA, tolA, pal also weaklysuppressed the yciM growth defects. Though the basis ofthis suppression is not clear, it is possible that these

mutations decrease the lethality associated with high LPSlevels by affecting the formation and/or assembly of func-tional LPS molecules or by altering the effective concen-tration of LPS in the OM. Alternatively, some of thesemutations may alter the ratio of LPS to phospholipid andthus partially contribute to viability. As described earlier forthe lpxD alleles (Fig. 4A), the antibiotic sensitivity pheno-types of some of the above mutants were suppressed byabsence of yciM indicating that increased LPS in thesedouble mutants is able to reverse the OM permeabilitydefects (data not shown).

Discussion

In this study, we demonstrate that YciM, encoded by agene of a hitherto unknown function, modulates cellularLPS levels by regulating LpxC, the rate-limiting enzyme oflipid A biosynthesis. Previous studies have shown thatexpression of LpxC is regulated by FtsH-mediated prote-olysis as also by conditions that inhibit early steps of lipidA synthesis (Sorensen et al., 1996; Ogura et al., 1999).Our results show that YciM and FtsH participate in acommon pathway to regulate the LpxC levels implicatinga role for YciM in modulation of proteolytic activity of FtsHtowards LpxC.

Deficiency of YciM, FtsH or overexpression of LpxCresult in lethal overproduction of LPS

Inactivation of yciM leads to high LPS levels and celldeath (Figs 2 and 4B). As mutations that lower the syn-thesis of lipid A suppress the lethality of yciM by decreas-ing the levels of LPS (Fig. 4C; Table 2), the most likelycause of cell death in yciM deletion mutants is high LPS.It is known that in the absence of FtsH protease, LpxCenzyme is stabilized leading to high LPS levels eventuallycausing cell death (Ogura et al., 1999). Likewise, over-expression of LpxC is toxic to cells as it leads to over-production of LPS (Sullivan and Donachie, 1984; Bealland Lutkenhaus, 1987; Young et al., 1995; Führer et al.,2006). However, it is not clear why excess LPS leads tolethality. During FtsH inactivation, abnormal membranousstructures form in periplasm, which are thought to bedeleterious to the cell (Ogura et al., 1999). Alternatively,excess LPS production may lead to depletion of fatty acyldonor molecules needed for the synthesis of membranephospholipids.

Physiological role of yciM

Regulation of lipid A synthesis is crucial as very high orvery low amounts of lipid A (or LPS) is detrimental to thegrowth of a cell. As mentioned above, lipid A is regulatedat the step of deacetylation as it is the first committed step



in its biosynthesis. Our results show that the deacetylaseenzyme, LpxC is regulated by YciM and this regulation isdependent on functional FtsH protease (Fig. 6A and B).However, another well-studied target of FtsH protease,RpoH, is not stabilized in absence of YciM indicating thatYciM is a specific modulating factor for FtsH-dependentproteolysis of LpxC.

FtsH protease is well conserved in all eubacteria,mitochondria and chloroplasts (Ito and Akiyama, 2005;Langklotz et al., 2012). On the other hand, yciM gene iscompletely absent in Gram-positive organisms and is con-served only in a few members of β- and γ-proteobacteriaof Gram-negative class. However, it is found to be verywell conserved in the enterobacteriaceae family. In thiscontext, it is noteworthy to mention that in Pseudomonasaeruginosa (that belongs to γ-proteobacteria) and also inorganisms belonging to α-proteobacteria such as Agro-bacterium tumefaciens and Caulobacter crescentus inwhich degradation of LpxC is independent of FtsH pro-tease (Langklotz et al., 2011), yciM gene is also absent,lending support to the presumed function of YciM.

In addition to LpxC, FtsH protease degrades a variety ofcellular targets including phage λ cII, SecY, and RpoH(σ32). It is known that degradation of these targets by FtsHis facilitated by specific adapter proteins; for, e.g. degra-dation of λ cII is modulated by HflKC complex whereasdegradation of RpoH requires the presence of DnaK/Jchaperone system (Ito and Akiyama, 2005; Langklotzet al., 2012). These facts also point to the possibility ofYciM being a specific adapter protein for modulation ofLpxC proteolysis.

Our results also indicate that the feedback regulation ofLpxC enzyme observed in mutants that are defective inearly steps of lipid A biosynthesis is possibly mediated byYciM (Fig. 6A). Although, it is not clear how YciM facilitatesthis regulation, one attractive possibility is that YciM com-municates the feedback signal to FtsH protease to specifi-cally alter LpxC to maintain optimal levels of lipid A. Insupport of this, recently, Schakermann et al. (2013) haveshown that the FtsH-mediated stabilization of LpxC islinked to cellular growth rate leading them to speculate theexistence of additional factor (s) that modulate the LpxCproteolysis.

Structurally, YciM contains six tetratricopeptide (34-amino acid) repeats and a C-terminal Zinc-finger domain.In general, TPR domain proteins mediate protein–proteininteractions and facilitate the assembly of multiproteincomplexes and are involved in a variety of biological pro-cesses including cell cycle regulation, transcriptionalcontrol, mitochondrial and peroxisomal protein transport,and bacterial virulence functions (Blatch and Lassle, 1999;D’Andrea and Regan, 2003). Interestingly, an instance of aTPR domain of a eukaryotic protein phosphatase being

activated by polyunsaturated fatty acids and anionic phos-pholipids is earlier known (Sinclair et al., 1999).

To sum up, this study demonstrates an essential role forYciM in lipid A regulation in E. coli. Our results suggestthat YciM could be an adapter protein of FtsH protease tofacilitate regulated proteolysis of LpxC to maintain optimalcellular lipid A levels. It would be worthwhile to test thismodel and also examine the mechanistic aspects of LpxCregulation by YciM.

Experimental procedures

Growth media and conditions

The defined and nutrient media were, respectively, minimalA medium (supplemented with 0.2% D-glucose as theC-source) or Luria–Bertani medium (Miller, 1992). Nutrientagar contained 0.5% bacto-peptone and 0.3% beef extract(Miller, 1992). Unless otherwise indicated, growth tempera-ture was 37°C. The following antibiotics were used at theindicated concentrations (μg ml−1): ampicillin (Amp), 50;kanamycin (Kan), 50; chloramphenicol (Cm), 20; spectinomy-cin (Spc), 50; and tetracycline (Tet), 15.

Bacterial strains, phages and plasmids

The bacterial strains and plasmids used in this study arelisted in Table 1. Unless otherwise indicated, all strains arederivatives of E. coli K12 MG1655. The authenticity of thedeletion alleles of the Keio mutant collection (Baba et al.,2006) used in this study was confirmed by linkage analysis,PCR, sequencing and phenotype if known. Complete dele-tion of yciSM locus was made on the chromosome by recom-bineering as described earlier (Yu et al., 2000). Marker-lessstrains were made by flipping out the antibiotic resistancemarker (KanR) using pCP20 plasmid that encodes a Flprecombinase as described earlier (Datsenko and Wanner,2000). Phage P1kc was from our laboratory stock.

Molecular and genetic techniques

Standard protocols were followed for experiments involvingrecombinant DNA and plasmid manipulations (Sambrook andRussel, 2001). Transpositions, P1-phage mediated transduc-tions and β-galactosidase assays were performed usingstandard methods as described (Miller, 1992). Transposontagging and identification of the suppressor mutation inJW1272 were done using conventional conjugational andtransductional mapping techniques (Miller, 1992) and finallyby sequencing and complementation studies.

Identification of transposon insertion mutations thatsuppressed the yciM mutant phenotypes

Strain MR703 (MG1655 ΔyciM::Kan/pMN103) was subjectedto random insertion mutagenesis with transposon Tn10dTetfollowing infection with phage λ1098, as described previously



(Miller, 1992). The TetR transposon-mutagenized library wasscreened for colonies that grew well on plates without IPTG(restrictive condition in which the plasmid pMN103 is unableto replicate leading to the manifestation of YciM− phenotype).The precise location of the insertions that were responsiblefor suppression of YciM− phenotype was identified by cloningthe TetR insertion along with flanking sequences onto theplasmid pCL1920, followed by sequencing the gene junctionsusing the outwardly directed Tet primers.

Growth and OM permeability assays

The viability of each strain was measured by applying 5–10 μlaliquots of various dilutions (10−2, 10−4, 10−5, 10−6 and 10−7) ofovernight cultures onto indicated plates and incubating themat 37°C for 20 to 36 h. Outer membrane barrier function wasexamined using hypersensitivity to various antibiotics such asvancomycin, nalidixic acid or rifampin at the indicated con-centrations. Strains of interest were grown, serially dilutedand appropriate dilutions were placed on indicated plates andincubated generally for 16–24 h.

Microscopy and viability measurements

To monitor growth, indicated cells were grown overnight inpermissive conditions and next day they were washed anddiluted 1:500 into fresh medium and grown at both permissiveand restrictive conditions. At indicated time points, absorb-ance at 600 nm and colony forming units were measured andadditional 0.5 ml culture was drawn for microscopy. Cell viabil-ity was also measured after staining cells with LIVE/DEADBaclight bacterial viability kit (Molecular Probes, Invitrogen).Cells were immobilized on a thin agarose pad and visualizedon a Zeiss apotome microscope by DIC (Nomarski optics) andfluorescence microscopy using GFP and DsRed filters.

Determination of Kdo (keto-deoxy-D-manno-8-octanoic acid)

Kdo was measured essentially as described earlier (Karkhaniset al., 1978). Strains of interest were grown in LB broth,washed, resuspended in 10 mM HEPES buffer (pH 7.4) andcell lysates were prepared by sonication. Kdo was measuredfollowing acid hydrolysis of these lysates as described earlier(Ogura et al., 1999) and the values are expressed per mgof protein. Protein concentrations were determined usingQuickstart-Bradford reagent from Bio-Rad with bovine serumalbumin (BSA) as the standard.

Quantification of LPS

LPS levels were measured using the cell lysates prepared asdescribed above. The cell lysates (normalized to the proteinconcentrations) were mixed with equal volume of tricinesample buffer (100 mM Tris-HCl, pH 6.8, 24% w/v glycerol,8% w/v SDS, 5% v/v 2-β-mercaptoethanol, 0.02% w/v bromo-phenol blue), and boiled for 10 min. To 50 μl of boiled sample,10 μl of proteinase K solution (2.5 mg ml−1 in sample buffer)was added and incubated further at 60°C for 60 min followed

by centrifugation at 16 000 g for 30 min. The supernatantswere loaded on 18% tricine-SDS polyacrylamide gels andLPS was visualized by silver staining (Austin et al., 1990).LPS was quantified using Image-J densitometric software.

Western analysis

Strains of interest were grown in LB broth, washed, resus-pended in 10 mM HEPES buffer (pH 7.4) and cell lysateswere prepared by sonication. Total protein was estimated andequal amount of protein was electrophoresed by SDS-PAGE.Western analysis was done as described earlier (Sambrookand Russel, 2001). LpxC antiserum (a kind gift from FNarberhaus) and RpoH antibodies (ab26890; purchasedfrom AbCam) were used at 1:20000 and 1:2000 dilutionrespectively. Appropriate secondary anti-rabbit-HRP wasused at a dilution of 1:10000. Blots were developed usingECL chemiluminiscent detection reagents (Roche) and quan-tified using Image-J software.

The details of plasmid constructions, oligonucleotides usedand additional experimental procedures are described in theSupplemental information. It also includes additional refer-ences, four figures and two tables.

Acknowledgements

We thank NBRP: E. coli for the Keio mutant collection andASKA plasmids; Coli Genetic Stock Centre for lpx mutants;Carol Gross for ftsH1 and sfhC21 mutants; Thomas Silhavyand Dante Ricci for support and valuable discussions; and JGowrishankar for advice on the manuscript. We would like tothank Jan Tommassen for helpful suggestions and FranzNarberhaus for generous sharing of LpxC antisera. This workwas supported in part by funds from Council of Scientific andIndustrial Research (CSIR), and Department of Biotechnol-ogy, Government of India.

Conflict of interest

The authors declare that they have no conflict of interest.

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