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Archives of Microbiology ISSN 0302-8933 Arch MicrobiolDOI 10.1007/s00203-012-0798-x

The inner membrane protein, YhiM, isnecessary for Escherichia coli (E. coli)survival in acidic conditions

Tuan M. Nguyen & Rebecca L. Sparks-Thissen

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DOI 10.1007/s00203-012-0798-x

SHORT COMMUNICATION

The inner membrane protein, YhiM, is necessary for Escherichia coli (E. coli) survival in acidic conditions

Tuan M. Nguyen · Rebecca L. Sparks-Thissen

Received: 8 September 2011 / Revised: 18 January 2012 / Accepted: 27 January 2012© Springer-Verlag 2012

Abstract Escherichia coli must be able to survive extremeacidic conditions. We were interested in determining the roleof the inner membrane protein YhiM in survival in acidicconditions. Previous data demonstrated that the yhiM genewas upregulated in acidic conditions (Tucker et al. in J Bac-teriol. 184:6551–6558, 2002). We therefore tested tn10 inser-tions into the yhiM gene for their ability to survive at low pH(pH 2.5). We show that YhiM was required for survival atpH 2.5. We also tested the YhiM dependence of the diVerentacid resistance pathways. YhiM was required for the RpoS,glutamine and lysine–dependent acid resistance pathways. Incontrast, YhiM was not required for the arginine-dependentacid resistance pathway.

Keywords E. coli · Acid resistance · Inner membrane protein · Acid stress

Introduction

Many bacteria possess systems to allow the bacterium tosurvive in conditions of stress, even if they do not normallyreside in those conditions. Enteric bacteria such as Esche-richia coli (E. coli) normally colonize the intestines ofwarm-blooded animals. They must transit through thestomach and survive the presence of gastric acids in orderto reach the intestine. Acid resistance, therefore, is a neces-sary adaptation to allow the bacterium to transit through the

stomach, which has a pH of 2.5, on its way to the intestine(Slonczewski et al. 2009).

There are Wve documented acid resistance pathways inE. coli. The Wrst pathway requires the alternative sigma factorRpoS (�38, katF) and cAMP receptor protein (Foster 2004).RpoS is induced under many conditions of cellular stressincluding growth in acidic conditions (McCann et al. 1991).This system is repressed by the addition of glucose andinduced in Luria Burtani (LB) broth (Castanie-Cornet et al.1999), but the mechanism of action of this pathway is poorlyunderstood. The second pathway is dependent on the pres-ence of glutamic acid and uses the decarboxylases GadA andB and the inner membrane antiporter GadC to convert gluta-mate to gamma-amino butyric acid (GABA) and CO2 whileconsuming a cytoplasmic proton (Bearson et al. 1997; Foster2004). The third pathway consists of the arginine decarboxyl-ase AdiA, which converts arginine to agmatine and CO2. Theagmatine is then pumped out of the cell by AdiC (Iyer et al.2003). The fourth pathway utilizes the lysine decarboxylaseLcdI (CadA), which converts lysine into cadaverine and CO2.The cadaverine is then pumped out of the cell using a lysine-cadaverine antiporter (CadB) (Soksawatmaekhin et al. 2004).The Wfth system is not well understood but uses the ornithinedecarboxylase SpeF and the ornithine-putrescine antiporterPotE (Kashiwagi et al. 1992).

YhiM is an inner membrane protein of unknown function(Rudd 2000). Expression of YhiM has been shown to beinduced under acid stress (Tucker et al. 2002). Expression ofyhiM has also been shown to be regulated by RpoS (�38), asigma factor commonly induced under conditions of cellularstress (Mitchell et al. 2007; Zhang et al. 1998). Since the yhiMtranscript is regulated by genes involved in protection of cellsin acidic conditions and is upregulated under conditions of acidstress (Tucker et al. 2002), we hypothesized that YhiM mightplay a role in protection of cells from acid stress. Our data

Communicated by Erko Stackebrandt.

T. M. Nguyen · R. L. Sparks-Thissen (&)Department of Biology, Wabash College, 301 W. Wabash Avenue, Crawfordsville, IN 47933, USAe-mail: thissenr@wabash.edu

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indicate that YhiM deWcient cells do not survive at pH 2.5 butgrow normally at pH 7 and pH 8. YhiM also is necessary forthe RpoS, glutamine, and lysine–dependent systems. YhiMdoes not play a role in arginine-dependent acid resistance.

Is yhiM necessary for E. coli growth or survival in acidic conditions?

Previous studies have shown that YhiM is upregulatedunder conditions of acid stress (Tucker et al. 2002). Thecontribution of YhiM to survival and growth of E. coliunder acidic challenge was therefore analyzed by using twoindependent tn10 insertions into yhiM (Table 1). Cells weregrown overnight in LB and then diluted 1:1,000 inLB + 100 mM MES pH 7, LB + 100 mM MOPS pH 8 orLB pH 2.5. Growth of both NVU 31 (yhiM167::tn10) andNVU 44 (yhiM90::tn10) was normal in LB + 100 mMESpH 7 and LB + 100 mM MOPS pH 8, suggesting that YhiMis not required for cell replication (Fig. 1a, c). Both NVU31 and NVU44 could not survive in LB pH 2.5 (Fig. 1ep = 0.004). This data suggest that the yhiM gene is neces-sary and survival of cells in acidic conditions.

To determine whether the survival defect of yhiMmutants under acidic conditions was speciWc to yhiMmutants in the UCB background, we repeated the experi-ment using a yhiM mutant, JW5944, from the Keio collec-tion (Baba et al. 2006). Similar to the previous data, bothJW5944 and its parental strain BW25113 grew at similarrates in both LB + 100 mM MES pH 7 and LB + 100mMMOPS pH 8 (Fig. 1b, d). JW5944 had signiWcantly less sur-vival at pH 2.5 than BW25113 (p = 0.02518). Takentogether, these results suggest that YhiM plays a role in sur-vival of E. coli in extreme acidic conditions.

Does yhiM play a role in RpoS-dependent acid resistance system?

Previous data showed that the sigma factor RpoS has a positiveeVect on yhiM expression (Zhang et al. 1998). We thereforehypothesized that the RpoS-dependent acid resistance systemwas YhiM-dependent. To test the RpoS-dependent system,

we grew cells overnight in LB-MES (pH 5.5) and diluted inLB-MES (pH 2.5) or EG (pH 2.5) (Castanie-Cornet et al.1999). When RpoS was repressed, the wild type and bothyhiM mutant strains could not survive in pH 2.5 media(Fig. 2a). When the RpoS-dependent AR system was acti-vated, only the wild type cells were able to survive(Fig. 2a). Our data indicate that the RpoS-dependent acidresistance system requires yhiM.

Does yhiM play a role in glutamate-dependent acid resistance system?

It has been shown that transcription of yhiM is directly regu-lated by RcsB-P/GadE complex, which is a central regulatorof the glutamate-dependent acid resistance system (Krin et al.2010). We therefore tested whether the glutamate-dependentacid resistance system was dependent on YhiM. The gluta-mate-dependent AR system was tested by growing cells over-night in LB + 0.4% glucose (LBG), which represses theRpoS-dependent oxidative system, and diluting the cultureinto EG (pH 2.5) or EG (pH 2.5) supplemented with gluta-mate (Castanie-Cornet et al. 1999). Disruption of the yhiMgene attenuated the function of this system. When none of theacid resistance pathways were turned on, both the wild typeand yhiM mutant cells could not survive in pH 2.5 media(Fig. 2b). When the glutamate-dependent acid resistance sys-tem was activated, the yhiM mutant survival increasedslightly but was not rescued to the same extent as UCB(p < 0.0001) (Fig. 2b). The absence of yhiM diminished thefunction of the glutamate-dependent acid resistance system.

Does yhiM play a role in arginine-dependent AR system?

Since our data indicated that YhiM is required for survival inacidic conditions and is required for RpoS and glutamate-dependent acid resistance system, we wanted to knowwhether YhiM was also required in other acid resistance sys-tems. We Wrst tested whether YhiM was required for arginine-dependent acid resistance by growing UCB or yhiM mutantcells in BHI supplemented with 0.4% glucose (BHIG). Cells

Table 1 Strains used in this study

Strain Genotype References

UCB HfrH-797 thi (McIver et al. 1995)

NVU31 HfrH-797 thi yhiM167::tn10 This study

NVU44 HfrH-797 thi yhiM90::tn10 This study

BW25113 �(araD-araB) 567�lacZ4787(::rrnB-3) �–rph-1�(rhaD-rhaB568 hsdR514

(Baba et al. 2006)

JW5944 �(araD-araB) 567�lacZ4787(::rrnB-3) �–rph-1�(rhaD-rhaB568 hsdR514 yhiM747(del)::kan

(Baba et al. 2006)

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were then diluted in either EG (pH 2.5) or EG (pH 2.5) sup-plemented with arginine (Castanie-Cornet et al. 1999). Dis-ruption of the yhiM gene had no eVect upon the function ofthis system. When none of the acid resistance pathways wasturned on, both the wild type and yhiM mutant cells could notsurvive in pH 2.5 media (Fig. 2c). When the arginine-depen-dent acid resistance system was activated, both wild type andyhiM mutant cells were rescued to the same extent(p = 0.9912) (Fig. 2c). The absence of YhiM does not aVectthe function of the arginine-dependent AR system.

Does yhiM play a role in the lysine-dependent acid resistance system?

We next tested whether YhiM was necessary for lysine-dependent survival in acidic conditions. We grew either

UCB or yhiM mutant cells in LBG overnight. Cells werethen diluted in either EG (pH 2.5) or EG (pH 2.5) supple-mented with lysine. Neither UCB nor yhiM mutant cellscould survive in the absence of lysine. In contrast, UCB butnot yhiM mutant cells survived in presence of lysine.

Our data indicate that YhiM plays a role in the sur-vival of E. coli in acidic conditions. It is necessary forsurvival in RpoS, glutamate and lysine-dependent but notarginine-dependent mechanism. The mechanism thatYhiM mediates this acid resistance is unclear. One possi-bility is that yhiM deWcient cells do not properly regulateexpression of the outer membrane proteins OmpC andOmpF, both of which have been shown to be regulatedunder acidic conditions (Heyde and Portalier 1987). Inaddition, we have previously shown that the bacterio-phage T4, which uses the OmpC as its receptor, fails toattach to the cell surface (unpublished observations).

Fig. 1 Growth of yhiM mutants at acidic pH. Overnight cultures of UCB, NVU 44, NVU31 (a, c, e) or BW25113 and JW5944 (b, d, f) were diluted 1:1,000 into LB pH 8 (a, b) LB pH 7 (c, d) or LB pH 2.5 (e, f)

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This suggests that expression of OmpC may be altered inthe absence of YhiM.

The signiWcance of a protein being involved in RpoS, glu-tamate, and lysine but not arginine-dependent acid resistanceis unclear. There is known overlap between the RpoS- andglutamate-dependent pathway. This involves the H-NS pro-tein, which has also been shown to be involved in regulatingyhiM expression (Zhang et al. 1998). In addition, the T7 5.5protein binds to the H-NS protein and can inhibit its oligo-merization with DNA (Ali et al. 2011). This suggests that thevirus needs to regulate H-NS and that improper regulation ofH-NS in the absence of YhiM may eVect both viral replica-tion and survival in acidic conditions.

We would like to thank Andrew Gilman for his experi-mental input in designing these experiments. This workwas supported by the Byron K. Trippet and Norman Tree-ves funds at Wabash College.

Materials and methods

Strains used

All strains are described in Table 1. The E. coli strain UCBused for all studies (McIver et al. 1995) was a gift fromBruce Voyles (Grinnell College). BW25113 and JW 5933

were obtained from the E. coli Genetic Stock Center (NewHaven, CT).

Mutagenesis of E. coli

Overnight cultures of UCB were diluted 1:1,000 in 20 mlLB and then grown for 3 h at 30°C. �NK561 (Silhavy et al.1984) was then added to the culture at an MOI = 10. Cul-tures were then incubated for 20 min at 30°C. A volume of800 �l of 1 M sodium citrate and 20 ml of LB were thenadded. Cultures were then incubated for an additional hourat 30°C. Cells were then spun at for 10 min at6,000£g. Cell pellets were resuspended in 2.5 mlLB + 20 mM sodium citrate. Then, 100 �l aliquots wereplated on LB agar plates containing 5 �g/ml tetracycline.IdentiWcation of the location of the tn10 insertion wasobtained by inverse PCR (Hersh et al. 1996).

Growth in acidic and basic conditions

To investigate the role of yhiM in growth and survival ofE. coli in acidic conditions, wild type and �yhiM mutant cellswere grown overnight at 37°C in LB [(10 g/l tryptone, 5 g/lyeast extract, 10 g/l NaCl), pH 7] and diluted 1:1,000 intoLB (pH 5) or LB (pH 2.5). LB pH 7 was buVered in100 mM 2-N-morpholinoethanesulfonic acid (MES). LB

Fig. 2 YhiM dependence of acid resistance systems. Over-night cultures were diluted 1:1,000 in EG pH 2.5 (no acid resistance) and a LB–MES pH 2.5 (to induce expression of RpoS) or b EG pH 2.5 + glutamine or c EG pH 2.5 + arginine or d EG pH 2.5 + lysine

RpoS dependentacid resistance

UCB

NVU 44

NVU 31UCB

NVU 44

NVU 310

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100

150

-RpoS + RpoS%

su

rviv

al

Glutamine dependentacid resistance

UCB

NVU 44

NVU 31UCB

NVU 44

NVU 310

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- glutamine + glutamine

% s

urv

ival

acid resistance

UCB

NVU 44

NVU 31UCB

NVU 44

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-arginine + arginine

% s

urv

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Lysine dependentacid resistance

UCB

NVU 44

NVU 31UCB

NVU 44

NVU 310

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- lysine + lysine

% s

urv

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A B

C DArginine dependent

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pH 8 was buVered in 100 mM 4-Morpholinepropanesulf-onic acid (MOPS).

Acid resistance system assays

Cells were grown overnight at 37°C in either LB broth con-taining 0.4% glucose (LBG), BHIG and LB broth buVeredwith 100 mM MES; pH 5.5. The overnight stationary-phasecultures were diluted 1:1,000 into LB-MES (pH 2.5), mini-mal E medium (Vogel and Bonner 1956) containing 0.4%glucose (EG; pH 2.5) or EG (pH 2.5) supplemented whereindicated with 1.5 mM glutamate, arginine or lysine. Forthe glucose-repressed oxidative system, cells were grownovernight in LB-MES (pH 5.5) and diluted in LB-MES (pH2.5). The glutamate and lysine dependent acid resistancesystems was tested by growing cells overnight in LBG,which represses the RpoS-dependent oxidative system, anddiluting the culture into EG (pH 2.5) supplemented withglutamate or lysine. The arginine-dependent acid resistancesystem was tested by growing cells in BHIG overnight anddiluting the culture into EG supplemented with arginine(Castanie-Cornet et al. 1999).

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