International Journal of Food Microbiology 149 (2011) 159–170

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International Journal of Food Microbiology

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YkgM and ZinT proteins are required for maintaining intracellular zinc concentrationand producing curli in enterohemorrhagic Escherichia coli (EHEC) O157:H7 underzinc deficient conditions

Jeesun Lim a, Kang-Mu Lee a, So Hyun Kim a, Younghoon Kim b, Sae-Hun Kim b,Woojun Park c, Sungsu Park a,⁎a Department of Chemistry and Nano Sciences (BK21), Ewha Womans University, Seoul 120-750, South Koreab Division of Food Bioscience and Technology, Korea University, Seoul 136-713, South Koreac Division of Environmental Science and Ecological Engineering, Korea University, Seoul 136-713, South Korea

⁎ Corresponding author at: Department of ChemistrEwha Womans University, 11-1 Daehyun-Dong, SeodaemKorea. Tel.: +82 2 3277 4081; fax: +82 2 3277 3419.

E-mail address: nanopark@ewha.ac.kr (S. Park).

0168-1605/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.ijfoodmicro.2011.06.017

a b s t r a c t

a r t i c l e i n f o

Article history:Received 19 November 2010Received in revised form 18 April 2011Accepted 21 June 2011Available online 1 July 2011

Keywords:BiofilmEscherichia coli O157:H7ykgM genezinT geneZinc

Zn2+ uptake systems are required for many enteric pathogens to survive and form biofilm in zinc-deficientconditions. ykgM and zinT (formerly yodA), regulated by Zur (zinc uptake regulator), have been reported asbeing highly induced during zinc shortage. This work reports that ykgM and zinT in enterohemorrhagicEscherichia coli (EHEC) O157:H7 biofilms under fluidic conditions were highly expressed compared to those instationary-phase planktonic cells and a mutation of either ykgM or zinT genes led to the inhibition of curlibiosynthesis. Inductively coupled plasma mass spectroscopy showed that the ykgM and zinT mutantscontained lower concentrations of Zn2+ than the wild type. Both mutants were less attached to both the glasssurface of a microchannel and epithelial cells than the wild type. Quantitative reverse-transcription PCR dataindicated that the expression of csgA, which encodes the major curli subunit, was inhibited in both mutantswith a zinc deficiency. Scanning electron microscopy showed that the mutants grown under zinc-deficientcondition were covered with a lower amount of curli compared to the wild type and often becamefilamentous. Zn2+ supplementation restored curli production and prevented filamentation in the mutants.Overall, under zinc-deficient conditions, YkgM and ZinT proteins are required for maintaining optimal zincconcentration in EHEC and intracellular zinc deficiency inhibits curli production.

y and Nano Sciences (BK21),un-Gu, Seoul, 120-750, South

l rights reserved.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Zinc is an essential transition metal for all living cells. It serves as astructural or catalytic cofactor in a large number of proteins such as RNApolymerase (Scrutton et al., 1971), superoxide dismutase (Benov andFridovich, 1994), and zinc finger proteins (O'Connor et al., 1993). It wasshown that both growth and infection of enteropathogenic Escherichiacoli (EPEC) were inhibited at 0.4 mM or higher concentrations of zinc(Crane et al., 2007). Bacteria often experience zinc deficiencywhen theyinvade eukaryotic cells, as zinc ions (Zn2+) in the host cells areinaccessible to bacteria (Kimet al., 2004; Gabbianelli et al., 2011; Garridoet al., 2002; Outten and O'Halloran, 2001). They have to rely on Zn2+

import machinery to survive under such zinc-deficient conditions: Zur(zinc uptake regulator) responds to zinc shortages by up-regulatingznuABC, zinT (formerly yodA) and ykgM (Outten and O'Halloran, 2001;Petrarca et al., 2010). ZnuABC, a major zinc transporter, is important for

the uptake of Zn2+ fromoutside the cell and is thus required for bacterialsurvival and pathogenesis (Ammendola et al., 2007; Gunasekera et al.,2009; Sabri et al., 2009). For example, a uropathogenic E. coli (UPEC)ΔznuBmutant grew slowlywhen zinc deficient andwas not able to formbiofilms (Gunasekera et al., 2009). zinT encodes an auxiliary componentof ZnuABC. Its mutation led to the reduction of intracellular zincconcentration in Salmonella Typhimurium (reference deleted) but didnot affect the virulence of the microorganism in mice (Petrarca et al.,2010). ykgM is thought to encode a non-Zn2+

finger containingparalogue of the Zn2+

finger ribosome protein (Graham et al., 2009);its relation to Zn2+ uptake or pathogenesis is largely unknown.

Biofilms are bacterial communities covered by self-producedsubstances such as curli, cellulose, DNA, and proteins. The transitionfrom planktonic cells to biofilm begins with cells' interactions with asurface (O'Toole et al., 2000). Several extracellular structures,including lipopolysaccharide, flagella, pili, and curli are involved inthe initial adhesion of the cells to surfaces (Kikuchi et al., 2005). Curliare thin aggregative fimbriae, which contribute to bacterial adhesionto surfaces, cell aggregation, infection and biofilm formation (Gualdiet al., 2008; Kikuchi et al., 2005). Their biogenesis is subject to tightand complex regulation by several regulators such as HNS, OmpR, and

160 J. Lim et al. / International Journal of Food Microbiology 149 (2011) 159–170

RpoS proteins (Brown et al., 2001). The relationship of intracellularzinc concentration to curli biogenesis has not yet been reported.

Enterohemorrhagic E. coli (EHEC) O157:H7 is an important food-borne pathogen that causes hemorrhagic colitis (HC) and hemolyticuremic syndrome (HUS) (Park et al., 1999). Its sessile form on foodcontact surfaces is an important source of cross-contamination asbiofilms are more resistant to chlorine and antibiotics than planktoniccells. EHEC O157:H7 forms biofilm colonies in the guts of cattle, deer,and sheep, which can subsequently contaminate carcasses leading tofoodborne illness (humans) (Ryu and Beuchat, 2005). EHEC O157:H7within biofilms are difficult to remove because they aremore resistantto sanitizers and antibiotics than its planktonic cells (Ryu and Beuchat,2005). Bacteria form biofilms in response to specific environmentalcues, including the concentration of metal ions (O'Toole et al., 2000).Ferrous ion (Fe2+) is important for the infection and biofilmformation of pathogenic E. coli (O'Toole et al., 2000; Schaible andKaufmann, 2004). Unlike the well-studied Fe2+, Zn2+ has a lesser-known effect on biofilm formation and infection.

This study investigates how the proteins YkgM and ZinT affect theattachment and biofilm formation of EHEC O157:H7. For this purpose,we performed DNA microarray to compare the gene expressionpattern between stationary-phase planktonic cells biofilm. To inves-tigate the role of YkgM and ZinT proteins on biofilm formation andbacterial attachment, ykgM and zinT mutants were constructed andtheir biofilm formation and bacterial attachment capabilities werecompared to those of the wild type. The intracellular zinc concentra-tion in the mutants was compared to that of the wild type.

2. Materials and methods

2.1. Bacterial strains and growth conditions

Table 1 lists the bacterial strains and plasmids used in this study.EHEC O157:H7 ATCC 43894, which produces Shiga toxins 1 and 2, wasobtained from the American Type Culture Collection (Manassas, VA,USA). Competent E. coli SY327 (Miller and Mekalanos, 1988) wasobtained from the National BioResource Project (NIG, Shizuoka,Japan). Strains were routinely cultured at 37 °C in M9 medium(47.76 mM Na2HPO4, 22.06 mM KH2PO4, 8.56 mM NaCl, 18.7 mMNH4Cl, 0.4% glucose, 0.01% thiamine, 2 mM oMgSO4·7H2O and0.1 mM CaCl2) with shaking at 220 rpm. Kanamycin (50 μg/mL),ampicillin (100 μg/mL) and chloramphenicol (30 μg/mL) were addedas necessary. Isopropyl-beta-D-thiogalactoside (IPTG) was used at20 μM to induce expression of cloned genes in complemented strains.Unless otherwise stated, all other chemicals were obtained fromSigma-Aldrich. Bacterial stock cultures were stored at −70 °C in 20%glycerol.

Table 1E. coli strains and plasmids used in this study. AmpR, KmR and CmR are ampicillin, kanamyc

Strain or plasmid Genotype

StrainsE. coli DH5α F-λ-80 (lacZYA-argF) endA1 recA1E. coli SY327 Strain containing the pir factor, deE. coli O157:H7 ATCC43894 Shiga-toxin 1,2 producing E. coli (wE. coli O157:H7 ATCC43894 Δ ykgM ykgM gene knock-out mutantE. coli O157:H7 ATCC43894 Δ zinT zinT gene knock-out mutantE. coli O157:H7 ATCC43894 pYkgM pCA24N-ykgM in Δ ykgM mutantE. coli O157:H7 ATCC43894 pZinT pCA24N-zinT in Δ zinT mutant

PlasmidpUC19 Cloning vector, AmpR

pUC4K Cloning vector, KmR

pCVD442 R6K ori, mobRP4, bla, sacBpCA24N-ykgM CmR; lacIq, pCA24N PT5-lac::ykgM+,pCA24N-zinT CmR; lacIq, pCA24N PT5-lac::zinT+, epKEN-gfpmut2 Expression of enhanced GFP prote

2.2. Microfluidic device

For easy observation of biofilms, a microfluidic device comprising apolydimethylsiloxane (PDMS, Dow-Corning, Cortland, NY, USA) layerand a glass slidewas fabricated by soft lithography (Xia andWhitesides,1998). It consisted of a 3 cm×1 cm microchannel 400 μm deep and a1 cm square microchamber 400 μmdeep containing an array of 200 μmsquare pillars 400 μmhigh separated by gaps (100 μm) smaller than thediameters (150–212 μm)of the glass beads (Sigma-Aldrich) (Chen et al.,2009; Lim et al., 2008), which was contained in the pillars for easyextraction of RNA from biofilms formed in the microchannel. A syringepump(KD Scientific, Holliston,MA, USA)was connected through tubingto continuously supply the channel with medium.

To observe attached cells and biofilms in the microchannel by CSLM(Confocal Laser ScanningMicroscopy), the depth of themicrochannel inthe microfluidic device was decreased to 40 μm and glass beads werenot used in this case.

2.3. RNA isolation and microarray data analysis

Bacterial cells were induced to form biofilms in the microchannelpacked with glass beads as described by Lim et al. (2008). In brief, anovernight culture in M9 medium was diluted 100-fold with fresh M9medium and incubated at 37 °C with shaking at 220 rpm. When theOD600 nm of the culture reached 0.8, a 2 mL portion was introduced tothe microchannel containing fresh M9 medium. The microfluidicdevice was then incubated at 25 °C for 30 min to allow the bacteria toattach to the glass substrate. After this, fresh M9 medium flowed at5 μL/min into the microchannel for 4 days. The biofilm-covered glassbeads were taken from the device after the removal of the PDMS layer.Total RNA from the biofilms on the glass beads was isolated using aRNeasy Mini Kit (Qiagen, Valencia, CA, USA).

Total RNA was obtained from stationary phase planktonic cells bydiluting an overnight culture 1:100 in fresh M9 medium and incubatingfor 24 h with shaking at room temperature. Total RNAwas isolated withthe Mini Kit as described above. Each total RNA sample (4.25 μg) waslabeled with Cyanine-3 conjugated dCTP (Amersharm, Piscataway, NJ,USA) by a reverse transcription reaction using the reverse transcriptaseSuperScrip ll (Invitrogen, Carlsbad, CA, USA). The labeled cDNAwas thenconcentrated andwashed using a centrifugal filter device, Microcon YM-30, from Millipore (Billerica, MA, USA). Concentrated cDNA wasresuspended in 35 μL hybridization solution (Digital-Genomics, Seoul,Korea). The cDNA was then placed on TwinChip™ oligo-chip for EHECO157:H7 (DigitalGenomics) (Kimet al., 2009) and thechip in slideswerehybridized in MAUI® system (BioMicro Systems, Inc., Salt Lake, Utah,USA) at 42 °C for 12 h. The hybridized slides were washed and dried bycentrifuging at 3000 rpm for 20 s. Themicroarraywas then scannedwitha GenePix 4000B confocal laser scanner (Axon Instruments, Union City,

in, and chloramphenicol resistance, respectively.

Source/reference

hsdR17 deoR thi-1 supE44 gyrA96 relA1 Invitrogenl (lac pro), argE (am), rif, nalA, recA56 Miller and Mekalanos (1988)ild type) ATCC

This studyThis studyThis studyThis study

Yanisch-Perron et al. (1985)Oka et al. (1981)Philippe et al. (2004)

Expression of GFP protein Kitagawa et al. (2005)xpression of GFP protein Kitagawa et al. (2005)in, AmpR Cormack et al. (1996)

161J. Lim et al. / International Journal of Food Microbiology 149 (2011) 159–170

CA, USA) at 532 nm for Cy3. Image analysis was done by the use ofGenePixPro3.0 (Axon Instruments). Thepixel intensityof themicroarrayspots was first subtracted by the background pixel intensity and spotintensities were then normalized by using a per-spot and per-chipintensity-dependent (Lowess) normalization.

2.4. qRT-PCR analysis

To validate the increased expression of ykgM and zinT genes inbiofilm, two-step real-timequantitative reverse transcription PCR (qRT-PCR) was performed by GenoCheck Co. (Ansan-City, Korea). Total RNAwas harvested from independent stationary phase planktonic cells andbiofilm cells in the microchannel and prepared exactly as describeabove. The specific primers used in the qRT-PCR assays were designedaccording to the EHEC O157:H7 EDL933 genome sequence (http://www.ncbi.nlm.nih.gov/) (Table 2). The amplification and detection ofDNA were performed by a Prism 7900 Sequence Detection System(Applied Biosystems, Foster City, CA, USA). Quantification of geneexpression was performed using Sequence Detection System softwareversion 2.1 (Applied Biosystems). A gel-purified standardDNA templaterepresenting each target and reference gene was diluted in series from1010 to 103 and employed in qRT-PCR to plot standard curves. Theamplification efficiency of each target (ykgM and zinT) and reference(rrsD) genewas derived from the standard curve of cycle threshold (CT)versus copies.

To evaluate the effects of the ykgM and zinT mutations on theexpression of the genes (adrA, csgA, fliC, and fimH) related to bacterialattachment, qRT-PCR was performed in both zinT and ykgM mutants.In brief, precultures in M9 medium were diluted 100-fold with freshM9 medium and incubated with shaking at 37 °C. When OD600 nm ofthe cultures reached 1.0, total RNA was isolated. The amplificationefficiency of each target (adrA, csgA, fimH and fliC) and reference(rrsD) gene was derived from a standard curve of cycle threshold (CT)versus copies. Results were expressed as ratios of ykgM, zinT, csgA,fimH, and fliC mRNA to rrsD mRNA (Lee et al., 2009).

2.5. Construction of knock-out mutants and complemented strains

ΔykgM and ΔzinT mutants were constructed by the followingprocedure (Philippe et al., 2004). PCR primers homologous to the 5′

Table 2Oligonucleotide primers used in this study.

Primers Nucleotide sequence (5

For construction of deletion mutantΔykgM-Forward-1 AGCGCGCATGCTTATTGAΔykgM-Reverse-1 TGCGCGGATCCTTTGTGAΔykgM-Forward-2 AGCGCGGATCCAGCGAAΔykgM-Reverse-2 TGCGCGAGCTCAAGCCAΔzinT-Forward-1 AGCGCGCATGCAACCAGΔzinT-Reverse-1 TGCGCGGATCCCAGGATΔzinT-Forward-2 AGCGCGGATCCGGGGCGΔzinT-Reverse-2 TGCGCGAGCTCTGCCGCA

For real-time PCRrrsD-Forward ATACCGCATAACGTCGCArrsD-Reverse ATATTCCCCACTGCTGCCzinT-Forward GTCTTTCAGAAGAAAGCzinT-Reverse CTATGGAATTCAACAATGykgM-Forward TACTGTGGTGTTCCACGAykgM-Reverse CCTGTATAGAACGGGTGcsgA-Forward TACCCAGCATGGTGGTGcsgA-Reverse GGTGGAATTAGATGCAGfimH-Forward CGATGATTTCCAGTTTGTfimH-Reverse CGGAGAGGTAATACCCCadrA-Forward TGTCTGAATTTGATGGGGadrA-Reverse GACAATAATGGGAAGGGfliC-Forward CACAGTTGGCGGCGTAGfliC-Reverse AAGGGTGGCAGCTAACG

and 3′ ends of the ykgM and zinT genes were designed according to theEHEC O157:H7 EDL933 genome sequence (http://www.ncbi.nlm.nih.gov/) (Table 2). Sequences of ca. 500 bp flanking the 5′ terminus of theykgM and zinT genes were PCR amplified from the EHEC O157:H7chromosome using theΔykgM-1 andΔzinT-1 primer pairs, respectively,in order to construct the disruption vectors. The 3′ flanking sequenceswere amplified using the ΔykgM-2 and ΔzinT-2 primer pairs, respec-tively. The 5′ fragments were digested with SphI and BamHI, andinserted into SphI and BamHI site of pUC19 (Yanisch-Perron et al.,1985). The 3′ fragmentswere digestedwith BamHI and SacI, and clonedinto BamHI and SacI sites of pUC19 containing 5′ fragments, generatingpSSP1. pSSP1 digested with BamHI was ligated with BamHI fragmentcontaining kanamycin resistance gene from pUC4K as a selectionmarker, to construct pSSP2 (Oka et al., 1981). The inserted fragments inpS22P2 were confirmed by DNA sequencing (SolGent Co., Daejeon,Korea). The SphI and SacI treated-fragmentswere recovered frompSSP2andwere cloned into SphI and SacI sites of the suicide vector, pCVD442,containing the ampicillin-resistance gene (Philippe et al., 2004),yielding pSSP3. pSSP3 was then transformed into EHEC O157:H7. Thegene disruption of ykgM and zinT in EHEC O157:H7 after the first andsecond crossovers was detected by their ampicillin sensitivity andkanamycin resistance, respectively. Inactivation of ykgM and zinT geneswas verified by qRT-PCR.

For construction of complemented strains, pCA24N-ykgM andpCA24N-zinT plasmids containing intact ykgM and zinT ORFs (openreading frames) obtained from National BioResource Project (Tsukuba,Japan) (Kitagawa et al., 2005) were then transformed into ΔykgM andΔzinT mutants.

2.6. Biofilm formation in the microchannel

Fluorescent observation of biofilms under fluidic conditions wasachieved through transformation of strains with pKEN-gfpmut2(Cormack et al., 1996) by electroporation at 1.8 kV. The strains weregrown in M9 medium containing ampicillin and introduced into themicrochannel as described above. The three-dimensional structures ofbiofilms were observed by CLSM (LSM510, Carl Zeiss, Jena, Germany)equipped with LSM510 Version 3.2 operation software at excitationand emission wavelengths (488 nm and 507 nm) for visualizing theGFP (green fluorescent protein). CLSM images were quantified using

′–3′) Source

TACGCACACGCCG This studyAGACGAATCGCCA This studyGAAGTGGTCGAGGA This studyTCAAAGCAATCCCC This studyCCAAATTTTGGTCC This studyGGATATTGGGCTTCA This studyTGATGAAAGTCCTTA This studyCAAAATGGTTATG This study

AG Lee et al. (2009)TC Lee et al. (2009)GGATG This studyCCGT This studyCACC This studyCGATT This studyGTAAC This studyTCTGGTC This studyGTGGA This studyAGGTTT This studyGC This studyAGAGC This studyATTAT This studyCAT This study

Table 3Genes affected in expression: biofilm cells versus planktonic cells.

Grouping b number Gene Fold Function of description

Zur regulated B0296 ykgM 26.03 Putative ribosomal proteinB1973 zinT 77.18 Cd(II) induced; hypothetical protein

Fur ragulated B0596 entA −2.89 2,3-dihydro-2,3-dihydroxybenzoate dehydrogenase, enterochelin biosynthesisB0585 fes −2.62 Enterochelin esteraseB1681 ynhC −4.81 orf, hypothetical proteinB1680 sufS −6.85 orf, hypothetical protein, selenocysteine lyase, PLP-dependentB2676 nrdF −5.88 Ribonucleoside-diphosphate reductase 2, beta chain, fragB2155 cirA −3.38 Outer membrane receptor for iron-regulated colicin I receptor; porin; requires tonB gene product

Information storage and processing B0953 rmf 3.46 Ribosome modulation factoB2616 recN −5.08 Protein used in recombination and DNA repairB2892 recJ −2.75 ssDNA exonuclease, 5′ –N 3′ specificB2699 recA −7.30 DNA strand exchange and renaturation, DNA-dependent ATPase, DNA- and ATP-dependent coproteaseB3645 dinD −2.53 DNA-damage-inducible proteinB0231 dinP −6.99 Damage-inducible protein P; putative tRNA synthetaseB1601 dinI −5.15 Damage-inducible protein IB4044 dinF −4.69 DNA-damage-inducible protein FB3984 rplA −6.49 50S ribosomal subunit protein L1, regulates synthesis of L1 and L11B3318 rplW −5.85 50S ribosomal subunit protein L23B3319 rplD −5.35 50S ribosomal subunit protein L4, regulates expression of S10 operonB3320 rplC −4.74 50S ribosomal subunit protein L3B3317 rplB −3.89 50S ribosomal subunit protein L2B3315 rplV −3.69 50S ribosomal subunit protein L22B3301 rplO −2.80 50S ribosomal subunit protein L15B3983 rplK −2.62 50S ribosomal subunit protein L11

Metabolism B3784 rbsD 7.01 D-ribose high-affinity transport system; membrane-associated proteinB3751 rbsB 2.64 D-ribose periplasmic binding proteinB3749 rbsA 2.66 ATP-binding component of D-ribose high-affinity transport systemB3845 fadA 3.11 Thiolase I; 3-ketoacyl-CoA thiolase; acetyl-CoA transferaseB2344 fadL 3.37 Transport of long-chain fatty acids; sensitivity to phage T2B1805 fadD 3.78 acyl-CoA synthetase, long-chain-fatty-acid–CoA ligaseB3846 fadB 6.64 4-enzyme protein: 3-hydroxyacyl-CoA dehydrogenase; 3-hydroxybutyryl-CoA epimerase; delta(3)-cis-deltB0726 sucA 4.86 2-oxoglutarate dehydrogenase (decarboxylase component)B4079 fdhF 6.80 Selenopolypeptide subunit of formate dehydrogenase HB3926 glpK 6.78 Glycerol kinaseB3927 glpF 7.05 Facilitated diffusion of glycerolB2240 glpT 3.74 sn-glycerol-3-phosphate permease

Metabolism B3828 metR 7.70 Regulator for metE and metHB3829 metE 8.58 Tetrahydropteroyltriglutamate methyltransferaseB2942 metK 5.37 Methionine adenosyltransferase 1 (AdoMet synthetase); methyl and propylamine donor,

corepressor of met genesB4013 metA 9.55 Homoserine transsuccinylase/homoserine O-succinyltransferaseB4014 aceB 7.90 Malate synthase AB3938 metJ 5.29 Repressor of all met genes but metFB3939 metB 12.91 Cystathionine gamma-synthaseB3940 metL 3.87 Aspartokinase II and homoserine dehydrogenase IIB3941 metF 22.46 5,10-methylenetetrahydrofolate reductaseB3008 metC 3.27 Cystathionine beta-lyase (beta-cystathionase)B0972 hyaA 13.87 Hydrogenase-1 small subunit, respiratory genes/energy metabolism, carbon:aerobic respirationB0973 hyaB 3.68 Hydrogenase-1 large subunit, respiratory genesB0974 hyaC 2.73 Probable Ni/Fe-hydrogenase 1 b-type cytochrome subunit, respiratory genesB0975 hyaD 2.61 Processing of HyaA and HyaB proteins, respiratory genesB0977 hyaF 6.74 Nickel incorporation into hydrogenase-1 proteins, respiratory genesB0978 appC 3.23 Probable third cytochrome oxidase, subunit IB1468 narZ 2.72 Cryptic nitrate reductase 2, alpha subunitB2469 narQ 2.77 Sensor for nitrate reductase system, protein histidine kinase (acts on NarP and narL)B4107 phnB 3.51 orf, hypothetical proteinB1020 phoH 3.12 PhoB-dependent, ATP-binding pho regulon component; may be helicase; induced by P starvationB0523 purE 2.74 Phosphoribosylaminoimidazole carboxylase = AIR carboxylase, catalytic subunitB1131 purB 3.40 Adenylosuccinate lyaseB3895 fdhD −2.65 Affects formate dehydrogenase-NB1227 narI −3.58 Nitrate reductase 1, cytochrome b(NR), gamma subunit

Fimbriae B0535 fimZ 2.566 Fimbrial Z protein; probable signal transducerB4312 fimB 3.206 Recombinase involved in phase variation; regulator for fimAB1038 csgF 2.634 Curli production assembly/transport component, 2nd curli operonB1039 csgE 2.753 Curli production assembly/transport component, 2nd curli operonB1042 csgA 3.641 Curlin major subunit, coiled surface structures; crypticB1041 csgB 4.5 Minor curlin subunit precursor, similar ro CsgAB1043 csgC 5.879 Putative curli production protein

Motility B1938 fliF −4.59 Flagellar biosynthesis; basal-body MS(membrane and supramembrane)-ring and collar proteinB1945 fliM −3.79 Flagellar biosynthesis, component of motor switch and energizing, enabling rotation and determiningB1948 fliP −3.79 Flagellar biosynthesisB1941 fliI −3.45 Flagellum-specific ATP synthaseB1942 fliJ −2.95 Flagellar fliJ proteinB1922 fliA −2.86 Flagellar biosynthesis; alternative sigma factor 28; regulation of flagellar operonsB1939 fliG −2.58 Flagellar biosynthesis, component of motor switching and energizing, enabling rotation and determini

162 J. Lim et al. / International Journal of Food Microbiology 149 (2011) 159–170

Table 3 (continued)

Grouping b number Gene Fold Function of description

B1944 fliL −2.56 Flagellar biosynthesisB1947 fliO −2.53 Flagellar biosynthesis

Cell division B0084 ftsI 4.27 Septum formation; penicillin-binding protein 3; peptidoglycan synthetaseB3463 ftsE −3.46 ATP-binding component of a membrane-associated complex involved in cell divisionB3462 ftsX −2.79 Cell division membrane proteinB0095 ftsZ −2.85 Cell division; forms circumferential ring; tubulin-like GTP-binding protein and GTPaseB0958 sulA −4.55 Suppressor of lon; inhibits cell division and ftsZ ring formationB4317 fimD −6.33 Outer membrane protein; export and assembly of type 1 fimbriae, interruptedB1303 pspF −2.85 psp operon transcriptional activatorB1307 pspD −2.51 Phage shock protein

Stress response B0623 cspE 5.05 Cold shock proteinB0989 cspH 5.16 Cold shock-like proteinB1823 cspC 12.72 Cold shock proteinB4062 soxS 3.44 Regulation of superoxide response regulonB4249 yjgI 3.76 Putative oxidoreductaseB0606 ahpF 3.87 Alkyl hydroperoxide reductase, F52a subunit; detoxification of hydroperoxidesB1521 uxaB 5.71 Altronate oxidoreductaseB0599 ybdH 9.80 Putative oxidoreductaseB1287 yciW 2.98 Putative oxidoreductaseB2468 yffG 3.19 Putative oxidoreductase, Fe–S subunitB1166 ymgB −22.68 orf, hypothetical protein/we showed that YmgB represses biofilm formation in rich medium. (Journal)B4063 soxR −4.78 Redox-sensing activator of soxS

RpoS-regulated genes B4069 acs 56.38 Acetyl-CoA synthetaseB3049 glgS 5.66 Glycogen biosynthesis, rpoS dependentB3426 glpD 3.96 sn-glycerol-3-phosphate dehydrogenase (aerobic)B3509 hdeB 2.61 orf, hypothetical protein/acid resistance hdeABD (biofilm increase-hdeA mutant)B0972 hyaA 13.87 Hydrogenase-1 small subunit, respiratory genesB0973 hyaB 3.68 Hydrogenase-1 large subunit, respiratory genesB0974 hyaC 2.73 Probable Ni/Fe-hydrogenase 1 b-type cytochrome subunit, respiratory genesB0975 hyaD 2.61 Processing of HyaA and HyaB proteins, respiratory genesB0977 hyaF 6.74 Nickel incorporation into hydrogenase-1 proteins, respiratory genesB3432 glgB −2.52 1,4-alpha-glucan branching enzymeB4187 aidB −2.99 Putative acyl coenzyme A dehydrogenase

AI-2 signaling B1513 lsrA 2.773 Putative ATP-binding component of a transport systemB1516 lsrB 4.912 Putative LACI-type transcriptional regulatorB1518 lsrG 2.827 orf, hypothetical protein, autoinducer-2 (AI-2) modifying protein LsrG

Other B3528 dctA 15.12 Uptake of C4-dicarboxylic acidsB0346 mhpR 14.38 Transcriptional regulator for mhp operonB0034 caiF 12.08 Transcriptional regulator of cai operonB0365 tauA 11.75 Taurine transport system periplasmic proteinB2552 hmpA −7.75 Dihydropteridine reductase, ferrisiderophore reductase activityB2675 nrdE −5.52 Ribonucleoside-diphosphate reductase 2, alpha subunit

Unknown gene B3512 yhiE 35.45 orf, hypothetical proteinB0936 ycbO 27.81 orf, hypothetical proteinB1426 ydcH 26.92 orf, hypothetical proteinB0935 ycbN 14.71 orf, hypothetical proteinB1824 yobF 12.62 orf, hypothetical proteinB4047 yjbL 9.91 orf, hypothetical proteinB2238 yfaH 8.552 orf, hypothetical proteinB2462 ypfE 8.52 orf, hypothetical proteinB1970 yedX 8.35 orf, hypothetical proteinB1689 ydiL 8.03 orf, hypothetical proteinB1541 ydfZ 6.18 orf, hypothetical proteinB1837 yebW 6.01 orf, hypothetical proteinB0572 ylcB −20.66 Putative resistance proteinB0258 ykfC −12.11 orf, hypothetical proteinB2432 yfeY −5.81 orf, hypothetical proteinB0795 ybhG −3.51 Putative membrane proteinB3512 yhiE 35.45 orf, hypothetical proteinB0936 ycbO 27.81 orf, hypothetical proteinB1426 ydcH 26.92 orf, hypothetical proteinB0935 ycbN 14.71 orf, hypothetical proteinB1824 yobF 12.62 orf, hypothetical proteinB4047 yjbL 9.91 orf, hypothetical proteinB2238 yfaH 8.552 orf, hypothetical proteinB2462 ypfE 8.52 orf, hypothetical proteinB1970 yedX 8.35 orf, hypothetical proteinB1689 ydiL 8.03 orf, hypothetical proteinB1541 ydfZ 6.18 orf, hypothetical proteinB1837 yebW 6.01 orf, hypothetical proteinB0572 ylcB −20.66 Putative resistance proteinB0258 ykfC −12.11 orf, hypothetical proteinB2432 yfeY −5.81 orf, hypothetical proteinB0795 ybhG −3.51 Putative membrane protein

163J. Lim et al. / International Journal of Food Microbiology 149 (2011) 159–170

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ISA3D software (Hwang et al., 2008) to evaluate surface coverage,mean thickness and biovolume of the biofilms. Three independentexperiments were performed for each strain and statistical signifi-cance between the biofilms formed by the wild type and each mutantwere determined using Student's t-test.

2.7. Estimation of intracellular Zn2+ concentration in planktonic cells

To measure intracellular Zn2+ concentration in planktonic cells, abacterial preculture in M9mediumwas diluted 100-fold with freshM9medium and incubated at 37 °C with shaking at 220 rpm for 12 h.Bacterial cells in the culture were harvested by centrifuging (Micro 17R,Hanil Science Industrial, Incheon, Korea) at 7500×g for 15 min. Thepellets were washed three times with modified defined medium A(mDMA) (11.3 g K2PO4, 5.4 g NaH2PO4, 200 mg MgSO4, 10 mg CaCl2,0.1 mg Na2B4O7, 0.1 mgNa2MoO4, 0.26 g EDTA, and 2 g NH4Cl per liter)to reduce the Zn2+ contamination (Kershaw et al., 2007). Zn2+

concentration in 10 mg of the cells was measured by inductivelycoupled plasma mass spectroscopy (ICP-MS, SCIEX - ELAN 6100,PerkinElmer Inc. Salem, MA, USA) at the National Center for Inter-University Research Facilities (Seoul National University, Seoul, Korea).

2.8. Bacterial adhesion to abiotic and biotic surfaces

Bacterial attachment to an abiotic surface was studied with apreculture of each strain constitutively expressing GFP in M9 mediumdiluted 100-fold with fresh M9 medium and incubated at 37 °C withshaking at 220 rpm. When the OD600 of the culture reached 1.0, it wasallowed to flow at 10 μL/min into themicrochannel for 1 h at 37 °C. Themicrochannel lacked glass beads, therefore, bacterial cells attached tothe glass side of themicrochannelweremeasured. Bacterial cells unableto adhere to the glass were flushed away for 10 min with fresh M9medium at the same flow rate. Fluorescence images of the bacteriaattached to the glass in the microchannel were taken by CLSM and thepercentage surface coverage of bacteria was quantified using Image Jsoftware (NIH, Bethesda, MD, USA). The level of bacterial attachmentwas calculated with respect to that of the wild type.

For the study of bacterial attachment to a biotic surface, apreculture of each strain was incubated with HT-29 colonic epithelialcells (Korean Cell Line Bank, Seoul, Korea) by a previously describedmethod with some modification (Kim et al., 2006). In detail, HT-29cells were maintained in RPMI 1640 (Gibco, Grand Island, NY, USA)supplemented with 10% fetal bovine serum (FBS), penicillin 100 unitand streptomycin 100 μg/mL at 37 °C in the presence of 5% CO2. Forthe attachment assay, 105 epithelial cells were placed into a 6-welldish and incubated in RPMI 1640 with the above supplementation for24 h in the presence of 5% CO2. After incubation, cells were washedthree times with phosphate-buffered saline (PBS, pH 7.4) and 1.5 mLRPMI 1640 containing ampicillin (100 μg/mL) was added to each well.Bacterial preculture in M9 medium was diluted 100-fold with freshM9 medium and incubated at 37 °C and 220 rpm until OD600 nm

reached 0.5. A 1 mL portion was then centrifuged and washed threetimes with PBS. The bacterial pellets were resuspended in 500 μL ofRPMI 1640. The bacterial suspension was added to the HT-29 cells inthe 6-well dish and incubated at 37 °C for 3 h in the presence of 5%CO2. After the incubation, the wells werewashed three timeswith PBSand the HT-29 cells infected with EHEC O157:H7 were lysed using0.1% Triton X-100 in PBS. The lysates were diluted in a series from 101

to 103 with PBS and spread on LB plates. The plates were incubated at37 °C and colony forming units (CFU) were counted. The relativebinding efficiency of each strain was calculated by dividing thepercentage of HT-29 cells with attached bacteria of interest with thepercentage of HT-29 cells with attached wild type bacteria asdescribed previously (Ho et al., 2008).

2.9. Scanning electron microscopy (SEM)

To compare curli production in the wild type and its mutants, apreculture of each strain in M9 medium was diluted 2-fold with freshM9 medium, and 12 mL of the bacterial suspension was evenlydistributed into a 6-well dish. A small fragment (ca. 4 mm2) of a siliconwafer (4science Inc., Seoul, Korea) was added to each well, and the dishwas incubated for 5 h at 25 °Cwithout shaking. Samples were preparedfor SEM as previously described with the following modification (Mayand Okabe, 2008). Bound cells on the silicon fragments were washedwith PBS before cells were fixed with 2.5% glutaraldehyde solution(Fluka, Milwaukee, WI, USA) in PBS for 2 h. Samples were washed withtriple distilled water and post-fixed in 1% osmium tetroxide (Fluka) inPBS for 1 h. Samples were dehydrated by sequentially immersing insolutions of increasing ethanol concentrations (75, 80, 85, 90, 95, and100%), and then coated with a 10 nm platinum layer by sputtering(E1010, Hitachi, Tokyo, Japan). SEM images of the samples were takenwith JSM-6700F (JEOL, Tokyo, Japan).

3. Results

3.1. Comparison of gene expression between planktonic and biofilm cells

Gene expression in planktonic and biofilm cells was compared byDNA microarray analysis performed by isolating RNA from stationaryphase planktonic cells and biofilm cells on glass beads. Fig. 1A and Bshows differently induced and repressed classified by functionalcategories which were based on the previous reports from Beloin etal. (2004) and Sigdel et al. (2006). Genes related to metabolism(carbohydrate metabolism, lipid metabolism, amino acid metabolism,and tryptophanmetabolism) and stress response were up-regulated inbiofilms, which are similar to gene expression patterns from planktoniccells in stationary phase (Beloin et al., 2004). Table 3 lists 137differentially expressed genes with changes of at least 2.5 fold (p-valuesb0.05) between planktonic and biofilm cells. Of the 137 genes, 88were up-regulated and 49 were down-regulated in the biofilms, withgenes related to metabolism (of carbohydrates, lipids, amino acids, andtryptophan) and stress response being up-regulated. The genes (soxS,ahpF, norV, and fucO) involved in resistance to oxidative stresswere alsoup-regulated 3.4, 3.9, 2.2 and 2.2 fold. Genes related to cell division,motility, information storage and information processing were down-regulated. Genes involved in flagella biosynthesis (fliA, fliF, fliG, fliI, fliJ,fliL, fliM, fliO, and flip) were down-regulated (2.9–3.8 fold, respectively),while genes related to fimbriae biosynthesis (fimB, fimZ, csgA, csgB, csgC,csgE, and csgF)were up-regulated (3.2, 2.6, 3.6, 4.5, 5.9, 2.8, and 2.6 fold).lsrA and lsrB encoding the transporter of autoinducer-2were inducedupby 2.8, and 4.9 fold, respectively. RpoS-regulated genes, related to thecellular response to osmotic stress, oxidative DNA damage, andstarvation, were up-regulated. Interestingly, expression of genes(entA, fes, ynhS, sufS, nrdF and cirA) regulated by ferric uptake regulator(Fur) was repressed, while the expression of ykgM and zinT genes,regulated by Zn2+ uptake regulator (Zur), exhibited significant in-creases of 26.0 and 77.2 fold, respectively. To validate the relatively highincreases in ykgM and zinT genes, their expression levels in EHEC O157:H7 biofilms were quantified by qRT-PCR (Fig. 1C), which showed thattheir expression increased by 36.0 and 54.7 fold, respectively, confirm-ing the microarray analysis data.

3.2. Effects of ykgM and zinT mutations on biofilm formation underfluidic and static conditions

In order to investigate the role of ykgM and zinT in biofilm formation,thebiofilm capabilities ofΔykgM andΔzinTmutants and their respectivecomplemented strains were compared with that of the wild type in themicrochannel. There was no growth inhibition in any of the strainswhen they were grown in M9 medium, but the growth of the

Fig. 1. Classification of induced and repressed genes in biofilm by microarray analysis (A–B) and expression levels of ykgM and zinT genes confirmed by qRT-CR (C). The induced andrepressed genes (A and B) were classified as functional groups according to the classification of Beloin et al. (2004) and Sigdel et al. (2006). Selected genes showed significantchanges of≥2.5 fold and p-values≤0.05. qRT-PCR data (C) show that the expression of ykgM and zinT genes in biofilms was significantly increased, compared with expression in thestationary phase. ThemRNA levels of each genewere adjusted to that of rrsDmRNA, which was used as the internal standard. Error bars indicate standard deviations from themeans.

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complemented strainswas slightly inhibitedafter theaddition of IPTGathigher than 20 μM (data not shown). Therefore, 20 μM IPTGwas addedto the complemented strains. All strains were incubated in themicrochannel for 4 days. The CLSM images (Fig. 2A–E) show that bothwild type and complemented strains formed more biofilms than theΔykgM and ΔzinT mutants. This result was supported by the ISAD data(Fig. 2F), which show significantly thicker (N15 μmvs. b5 μm) (pb0.05,n=3) biofilms with higher surface coverage (80% vs. 10%) and greaterbiovolume (400 mm3 vs. 15 mm3) formed by both the wild type andcomplemented strains compared to either of themutants. These resultsshow that biofilm formation in EHEC O157:H7 was greatly affected bymutation of either the ykgM or zinT genes under fluidic conditions.

In contrast with the results in the microchannel, biofilm formationof EHEC O157:H7 in microtiter plates was not affected by mutation of

Fig. 2. CLSM micrographs of biofilms formed by wild type (A), ΔykgM mutant (B), ΔzinT mBiofilms were formed at 5 μL/min for 4 days. Size makers indicate 20 μm. (F) The surface coError bars indicate standard deviations of three independent experiments. Representative i

either the ykgM or zinT genes (Fig. 3). Taken together with the resultsfrom the microchannel tests, these results suggest that initialattachment more critically affect biofilm formation in fluidic condi-tions than when static.

3.3. Effects of ykgMand zinTmutations on intracellular Zn2+ concentration

To determine whether YkgM and ZinT proteins are important tomaintaining zinc homeostasis in planktonic cells, intracellular Zn2+

concentrations in wild type and mutant planktonic cells grown in zinc-deficient conditions were measured by ICP-MS. They were 3.33±0.06 mM, 0.63±0.14 mM, and 0.69±0.22 mM in the wild type, ΔykgMand ΔzinT mutants, respectively, indicating that zinc concentration inEHEC O157:H7 was greatly decreased by the mutations (Fig. 4).

utant (C), pYkgM complementation strain (D), and pZinT complementation strain (E).verage, mean thickness, and biovolume of biofilm were quantified by ISA3D software.mages are shown.

Fig. 5. Attachment of EHEC O157:H7 wild type and ΔykgM and ΔzinT mutants to glasssubstrate (A) and HT-29 cells (B). (A) Attachment to the glass substrate was quantifiedby calculation of surface coverage. (B) Attachment to HT-29 cells was quantified bydetermining CFUs. Error bars indicate standard deviations of three independentexperiments. Representative images are shown.

Fig. 3. Biofilms formedbyEHECO157:H7 strains (thewild type,ΔykgMandΔzinTmutants)grown inpolystyrene 96-wellmicrotiter plates under static condition at 25 °C for 24 h. Theamount of EPS was quantified by the crystal violet assay. Error bars indicate the standarddeviation of three independent experiments. Representative images are shown.

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3.4. Effects of ykgM and zinT mutations on bacterial attachment tomicrochannel and epithelial cells

To confirm whether YkgM and ZinT proteins are important toinitial attachment, bacterial attachment assays were performed onabiotic (glass) and biotic (HT-29 cell) surfaces. Attachment of themutants to glass was less than that of the wild type. The relativebinding efficiencies of the ΔykgM and ΔzinT mutants to themicrochannel were respectively only 45.9±12.2% and 26.4±14.9%of the wild type and are significantly lower than the binding efficiencyof the wild type (pb0.05, Student's t-test) (Fig. 5A).

Compared with the wild type, there was less attachment of themutants to HT-29 cells. Fig. 5B shows the relative binding efficienciesof ΔykgM and ΔzinTmutants: 44.8±3.1% and 9.8±4.5%, respectively.This indicates that this gene is required for EHEC O157:H7 attachmentto the host cells.

3.5. Effects of ykgM and zinT mutations on expression of genes related tobacterial attachment

To evaluate the effects of the ykgM and zinT mutations on genesrequired for bacterial attachment, the expression of the genes adrA,csgA, fimH, and fliC in ΔykgM and ΔzinT mutants under zinc-deficientconditions was quantified by qRT-PCR. Fig. 6 shows that theexpression of csgA in both mutants was significantly reducedcompared with the wild type. Expression of the other genes (adrA,fimH, and fliC) was not significantly changed. This indicates that bothykgM and zinT are required for the production of curli, encoded bycsgA, under zinc-deficient conditions.

Fig. 4. Intracellular Zn2+ concentration in planktonic cells measured by ICP-MS. Errorbars indicate standard deviations of three independent experiments.

3.6. Effects of ykgM and zinT mutations on curli production andfilamentation

To validate the qRT-PCR result that both ykgM and zinT are requiredfor the production of curli under zinc-deficient conditions, SEM wasused as elsewhere described (May and Okabe, 2008) to compare curliproduction in themutants under zinc-deficient conditions to that in the

Fig. 6. qRT-PCR of csgA, fimH, adrA, and fliC genes related to bacterial attachment. ThemRNA levels of each gene were adjusted to that of rrsD mRNA, used as internalstandard. The error bars indicate standard deviations from means.

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wild type. Even under the zinc-deficiency, the wild type maintained itsrod shape and formed curli-like structures on its surface (Fig. 7A–B),while both ΔykgM and ΔzinT mutants (Fig. 7C–F) became elongated,over 5 μm, assuming filamentous form. Many of the ΔzinTmutant cellsappeared damaged.

To determine if zinc deficiency inhibits curli production and inducesfilamentation in themutants, theywere incubatedwith siliconwafers inM9medium supplemented with 50 μMZnSO4. In this case, bothΔykgM(Fig. 7G and H) and ΔzinT (Fig. 7I and J) mutants were covered withcurli-like structures and their shapes were similar to those of the wildtype. Taken tighter with the SEM images (Fig. 7A–F) under zinc-deficient condition, these results indicate that ykgM and zinT arerequired for curli production andmaintaining the normal shape of EHECO157:H7 cells under zinc-deficient conditions.

Fig. 7. SEM images of the wild type and ΔykgM and ΔzinT mutants grown under zinc-deficieare covered with curli-like structures with neither damaged nor filamentous cells, while Δykdamaged and filamentous cells. With zinc (G–J), both ΔykgM (G and H) and ΔzinT (I andstructures and lack damaged and filamentous cells. Each strain was incubated with fragmentat room temperature without shaking for 5 h before imaging. c, d, and f labels stand curli, c

4. Discussion

There have been relatively few reports regarding the geneexpression in EHEC O157:H7 biofilms. This work compares geneexpressions of stationary phase cells and those in biofilms using DNAmicroarrays. The stationary phase was chosen as the previousmicroarray data (Beloin et al., 2004) had suggested that E. coli K-12biofilms are more closely related to the stationary than theexponential phase. The microarray data (Table 3) indicate that 137genes were differentially expressed in EHEC O157:H7 biofilms, mostof them related to metabolism and stress response regulated by RpoS.It has also been reported that cells in biofilms were metabolicallyactive (Beloin et al., 2004) and the rpoS gene is important for biofilmformation of nonpathogenic E. coli (Domka et al., 2007). Different

nt and zinc-replete conditions. Under zinc shortage (A–F), the wild type cells (A and B)gM (C and D) and ΔzinT (E and F) mutants lack the curli-like structures and have manyJ) mutants appear similar to the wild type (A and B); they are covered with curli-likes of silicon wafer in either M9medium or M9medium supplemented with 50 μM ZnSO4

ells with cell wall damage and filamentation, respectively.

Fig. 7 (continued).

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from previous reports (Beloin et al., 2004; Domka et al., 2007), thedata here show that ykgM and zinTwere highly expressed in biofilms.These genes along with znuA, that encodes the metallochaperonecomponent of the ZnuABC Zn2+ importer, have been reported asbeing highly induced under severe zinc-deficiencies (Graham et al.,2009). znuA was not induced in this work. Although UPEC was notable to form biofilm under zinc-deficient conditions, there has beenno report about the induction of ykgM and zinT genes in biofilms.Makarova et al. (2001) predicted that YkgM is a non-Zn2+

finger parolog of Zn2+-binding ribosomal proteins and has lost theZn2+-binding motif. Hemm et al. (2010) showed that the expressionof ykgM gene was repressed by Zur, a transcription regulating geneexpression in response to zinc levels, and the expression of ykgM wasderepressed during zinc shortage. It was known that the expression ofzinT increased during zinc shortage as well (Graham et al., 2009).Since zinT and ykgM genes are only induced during zinc shortage, itwas implied that EHEC O157:H7 experienced severe zinc deficiency inthe fluidic condition tested here. Thus, ykgM and zinT genes arehypothesized as being required for biofilm formation under fluidicand nutrient limited conditions. This hypothesis was supported inpart by the results that both ykgM and zinT mutants were less able toform biofilm in the microchannel (Fig. 2), while they were able toform biofilm in microtiter plates similar to the wild type (Fig. 3). TheICP-MS data (Fig. 4) also indicate that intracellular zinc concentrationsin the mutants were several times lower than in the wild type,suggesting that M9 medium is not a zinc-replete medium and EHECneeds YkgM as well as ZinT proteins to maintain zinc homeostasis inM9 medium. There was a similar report (Petrarca et al., 2010) thatmutation of zinT gene led to the reduction of intracellular zincconcentration in Salmonella Typhimurium (Petrarca et al., 2010),whereas it is the first report that intracellular zinc concentration wasreduced by the mutation in ykgM. Graham et al. (2009) hypothesizedthat during zinc shortage YkgM derepressed by Zur becomespreferentially bound to the ribosome, allowing the metal ion to be

recycled within the ribosome. Based on this hypothesis, we canexplain that the reduced zinc concentration in ygkM mutant could becaused by the disturbance of zinc recycling process in the ribosomerepresenting an enormous zinc reservoir (Gabriel and Helmann,2009).

With limited nutrients, microorganisms tend to attach to thesurface and form biofilm. In the microtiter plate, bacteria may havebeen more able to attach to the surface in comparison with themicrochannel where they may have required appendages such asflagella and curli to adhere to the glass surface because of the flow.Thus, a study of the effect of ykgM and zinT mutations on bacterialattachment and the production of the appendages was required. Theinitial attachment of bacteria is crucial to biofilm formation andinfection. Both ykgM and zinT mutants displayed less attachment toboth abiotic and biotic surfaces (Fig. 5). Since the attachmentexperiment to the abiotic surface was conducted in the microchannelfilled with zinc-deficient M9 medium, it is not surprising to observereduced attachment of the mutants. ZinT has been shown to berequired for EHEC adherence to cultured HeLa cell monolayers (Ho etal., 2008). However, the relation of YkgM to adherence to a bioticsurface has not been reported. The results herein suggest that duringzinc shortage, YkgM and ZinT are essential for initial attachment toboth abiotic and biotic surfaces. Since the data (Figs. 2 and 4) indicatethat zinc deficiency in EHEC due to ykgM or zinT mutations led to theinhibition of bacterial attachment, which is affected by various cellsurface appendages such as curli, cellulose and flagella (Van Houdtand Michiels, 2005), clarification is required of which bacterialappendage is affected by the ykgM and zinT mutations during zincshortage. The qRT-PCR results (Fig. 6) show that the expression ofcsgA is inhibited in the ΔykgM and ΔzinT mutants, while theexpression of the genes encoding the other cell surface appendagessuch as pili and flagella, as well as genes related to cellulose synthesiswere not inhibited. CsgA protein is a major subunit of curli, which arethin aggregative fimbriae and very important for initial attachment

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during biofilm development and infection (Kikuchi et al., 2005; VanHoudt and Michiels, 2005). It has been reported that curli are moreimport than flagella and type I pili to infection and biofilm formationin E. coli (Ma and Wood, 2009). The DNA microarray data herein alsoindicate the induction of curli synthesis genes (csgA, csgB, csgC, csgEand csgF) in EHEC O157:H7 biofilm (Table 3). The inhibition of curlisynthesis in bothmutants is further supported by the SEM images thatshow the surface of the wild type cell covered with curli under zinc-deficient conditions (Fig. 7A–B), while the surfaces of the mutantswere not covered with curli under the same conditions (Fig. 7C–F).Curli were restored in the mutants by addition of ZnSO4 (Fig. 7G–J).Taken together with the qRT-PCR data (Fig. 6), the SEM imagessuggest that deletion of either ykgM or zinT negatively affects curlibiogenesis under zinc-deficient conditions. Although curli productionresponds to environmental cues such as temperature, salt andnutrient limitation (nitrogen, phosphate, and iron), it has not beenreported that a zinc shortage can inhibit curli production.

Interestingly, both ykgM and zinT mutants were elongated to over5 μm (Fig. 7C and E). Such filamentation was not observed in ΔykgMand ΔzinT mutants when supplemented with Zn2+ (Fig. 7G–E). It hasbeen suggested that filamentation is part of bacteria's survivalstrategy under environmental stresses such as ROS (reactive oxygenspecies) or ultraviolet radiation (Justice et al., 2008). It has also beenreported that resistance to oxidative stress is reduced in zinc-deficientconditions and that Zn2+ is required to protect cell membranes fromlipid oxidation caused by ROS (Kim et al., 2006; Sabri et al., 2009). TheICP-MS results indicate that intracellular Zn2+ concentrations in themutants were several times lower than in the wild type, implying thatthe mutants experienced extreme zinc shortages. Taken together, it issuggested that ΔykgM and ΔzinT mutants become elongated with theextreme zinc shortage leading to DNA damage.

5. Conclusion

ykgM and zinT genes were highly induced in EHEC biofilm underfluidic conditions. Intracellular Zn2+ shortage in EHEC due tomutation in either of these genes led to the inhibition of bacterialattachment and biofilm formation through reduced curli biogenesis.These results suggest that both ykgM and zinT genes play importantroles in bacterial surface attachment under zinc-deficient conditions.

Acknowledgments

This research was supported equally by the programs (R11-2005-008-02001-01 and R01-2008-000-20593-0) of the National ResearchFoundation (NRF) in Korea and the grant (10816) from the SeoulResearch and Business Development Program. E. coli SY327 andpCA24N-ykgM and pCA24N-zinT plasmids were obtained fromNational BioResource Project (NBRP, Japan). We also thank ProfessorChung-Hak Lee and Byung-KooK Whang at Seoul National Universityfor allowing the use of ISA3D software.

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