enu

Bedf

DesiccationStress response

Saveismeq aericy 0.3

to conten wea et al.ecentut buttonymo

expression proles of bacteria in food matrices (Bergholz et al.,2009; Cretenet et al., 2011; Fratamico et al., 2011; Liu and Ream,2008; Makhzami et al., 2008; Raynaud et al., 2005; Sirsat et al.,

2. Materials and methods

2.1. Bacteria

We used S. Enteritidis str. ATCC BAA-1045 which was isolatedfrom recalled almonds implicated in an international outbreak ofsalmonellosis (Isaacs et al., 2005).

* Corresponding author. Tel.: 1 708 563 2980; fax: 1 708 563 1873.E-mail addresses: xiangyu.deng@gmail.com (X. Deng), zhangw@iit.edu

(W. Zhang).1 Current address: Kraft Foods Technology Center, 801 Waukegan Rd., Glenview,

Contents lists available at

Food Micr

w.

Food Microbiology 30 (2012) 311e315IL 60025, USA.States has demonstrated the need for basic understanding of thesurvival and persistence strategies of S. enterica in low aw foodcommodities. Despite extensive studies on the survival characteris-tics of S. enterica in various model systems with reduced aw (Archeret al., 1998; Gruzdev et al., 2011; Hiramatsu et al., 2005; Matticket al., 2000, 2001; Shachar and Yaron, 2006), little is known aboutthe physiology of this pathogen under desiccation and starvationstress and the underlyingmolecular mechanisms for its survival andpersistence in low aw foods.

A number of recent studies investigated the global gene

response to food and environmental stresses (e.g. detection of raretranscripts and non-coding regulatory RNAs).

In the present study, we demonstrate, for the rst time, the useof whole transcriptome sequencing (or RNA-seq) technology tostudy bacterial global gene expression in a food matrix. Specically,the transcriptomic response of S. Enteritidis to desiccation andstarvation stress in peanut oil (aw 0.3) was investigated as thisparticular Salmonella serotype has been implicated in a number offoodborne illness outbreaks associated with low aw foods (Gruzdevet al., 2011).1. Introduction

The ability of Salmonella entericalow water activity (aw) foods has beet al., 2000; Isaacs et al., 2005; KillaleRowe et al.,1987; Shohat et al.,1996). Rsalmonellosis associated with peanAnonymous, 2009) and pet food (An0740-0020/$ e see front matter 2011 Elsevier Ltd.doi:10.1016/j.fm.2011.11.001regulatory functions likely play roles in cross protecting Salmonella from desiccation and starvationstresses. In addition, non-coding RNAs may also play roles in Salmonella desiccation stress response. Thisis the rst report of using RNA-seq technology in characterizing bacterial transcriptomes in a food matrix.

2011 Elsevier Ltd. All rights reserved.

aminate and survive inll documented (Burnett, 1996; Kirk et al., 2004;multi-state outbreaks ofer (Anonymous, 2007;us, 2008) in the United

2011). Most of these studies relied on DNA array technology tocompare bacterial transcriptomes under various conditions ortreatments, based on which specic cellular responses to envi-ronmental cues were deduced. However, technical difcultiesassociated with isolating high quality bacterial RNA from complexfood matrices coupled with the relative low detection sensitivity ofDNAeDNA hybridization have restricted DNA array-based appli-cations in deciphering more subtle bacterial transcriptomicTranscriptome sequencingSalmonella entericadormant state with

2.2. Inoculation of peanut oil

We used peanut oil as the food matrix mainly because of itslow water activity (aw 0.3). Also, separation and recovery ofbacteria from peanut oil was effective. S. Enteritidis cells from10 ml of overnight Luria-Bertani (LB) culture (w109 CFU/ml) wereharvested by centrifugation at 4000 rpm for 5 min, washed threetimes with 1X phosphate buffered saline (PBS) at neutral pH, andair dried for 30 min. Dried cell pellets were thoroughly mixed

LB broth (10 ml overnight culture inoculated in 100 ml LB broth) at12 h (early stationary phase) and 312 h (long-term survival phase)after inoculation as described above.

2.4. RNA-seq analysis

After two consecutive rounds of DNase treatment (supplied byAMBION RiboPure kit), each DNA-free total RNA sample was sub-jected to whole transcriptome sequencing on an Illumina Genome

Table 1Summary of RNA-seq data.

Sample Total number of reads Percentage of mapped readsa Number of reads mapped to CDS Percentage of genome represented

12 h LB (1) 27,151,557 92.98% 396,413 45.52%12 h LB (2) 33,274,075 92.45% 1,763,526 76.84%312 h LB (1) 29,017,764 92.24% 499,106 44.45%312 h LB (2) 34,958,141 90.48% 311,127 30.04%72 h peanut oil (1) 30,015,630 93.17% 228,119 1.52%72 h peanut oil (2) 30,131,874 94.30% 232,015 1.59%216 h peanut oil (1) 32,148,558 92.08% 228,255 4.42%216 h peanut oil (2) 30,918,263 91.55% 148,408 2.98%528 h peanut oil (1) 31,967,767 93.11% 210,987 1.45%528 h peanut oil (2) 32,179,987 90.45% 234,914 1.36%

a Based on default Bowtie setting at Galaxy which allowed a maximum of 2 mismatches in rst 28 bases (seed length 28 bp).

FPKM

12 h

30.24.12.37.

109.

X. Deng et al. / Food Microbiology 30 (2012) 311e315312with 10 ml peanut oil in centrifuge tubes by hand stirring andvortexing (nal cell density around 109 CFU/ml in peanut oil).Spiked peanut oil suspensions were kept horizontally in a shakingincubator at 300 rpm (to avoid the clumping of cells) at 25 C forup to 30 days.

2.3. RNA extraction

S. Enteritidis cells were harvested from peanut oil at specictime points (i.e. 72 h, 216 h and 528 h) by centrifugation at4000 rpm at room temperature for 5 min and immediately stabi-lized using QIAGEN RNAprotect bacterial reagent (QIAGEN Inc.,Valencia, CA). Total bacterial RNA was puried using the AMBIONRiboPure Bacterial Kit (Life Technologies Corporation, Carlsbad, CA).Total RNA from two biological replicates for each time point wasprepared from independent cultures on different days to evaluatethe reproducibility of RNA-seq data. For comparison purposes, totalRNA samples were also prepared from S. Enteritidis cells grown in

Table 2Genes transcribed in desiccated S. Enteritidis in LB broth and peanut oil.

Locus ID Description

SEN0011 Chaperone protein DnaKSEN0012 Chaperone protein DnaJSEN0135 N-acetylglucosamine deacetylaseSEN0598 Cold shock protein CspESEN0776 Starvation/stationary phase protection protein Dps

SEN0851 Cold shock-like protein CspD 19.SEN0984 Hypothetical protein 5.SEN1257 Hypothetical protein 20.SEN1835 Hypothetical protein 1.SEN2566 Sigma factor RpoE 13.SEN2602 Heat shock protein GrpE 6.SEN3194 Hypothetical protein 17.SEN3195 Hypothetical protein 17.SEN3391 Sigma factor 32 RpoD 13.SEN3472 Major cold shock protein 65.SEN3509 Mannitol repressor protein 4.SEN3510 Hypothetical protein 4.SEN3624 Heat shock chaperone IbpB 31.SEN3625 Heat shock protein IbpA 31.SEN4009 Putative stress-response protein 8.

e: Transcript was not detected in both replicates of the sample.a Abundance of transcripts were quantied as normalized number of fragments per kb Transcript was detected in only one replicate of the sample, of which the FPKM wasAnalyzer to generate 36-bp single-end reads. Library preparation,cluster generation and sequencing were performed according tomanufacturers recommendations (Illumina Inc., San Diego, CA).Prior to cluster generation, Duplex-Specic thermostable nuclease(DSN) (Evrogen, Moscow, Russia) normalization was performed toenrich rare transcripts (Zhulidov et al., 2004). Sequencing readswere mapped to a fully sequenced S. Enteritidis str. P125109genome (Thomson et al., 2008) using Bowtie (Langmead et al.,2009) and assembled into transcripts using Cufinks (Trapnellet al., 2010), both through the Galaxy server (Goecks et al., 2010).Annotation of the reference genome was then used to curate andanalyze assembled transcripts by self-written Python programsbased on Biopython modules (Cock et al., 2009). Sequencing dataand analysis history are publicly available at http://main.g2.bx.psu.edu/u/xdeng/h/salmonella-rna-seq. Quantitative reverse tran-scription PCR (qPCR or qRT-PCR) was used to validate the expres-sion of 12 genes identied by RNA-seq (Table 2) based on themethod described in (Wen et al., 2011).

a

LB 312 h LB 72 h oil 216 h oil 528 h oil

99 17.84 1.16 e e21 17.84 e 1.91 e03 16.45 4.23 5.63 1.0533 21.77 13.29 9.81 1.98b

46 12.48 1.41b 3.72 2.04

71 8.75 1.56 e e65 3.64 5.16 17.77 16.2933 17.82 3.70 8.19 1.8550 10.31 9.99 13.03 1.9297 24.68 1.43 1.17 0.9701 5.91 0.58 4.73 1.7926 6.17 28.29 17.27 5.0026 6.17 28.29 17.27 5.0078 26.09 1.81 9.05 1.5777 33.84 1.35 3.82 1.5340 10.44 2.34 5.78 1.8040 10.44 2.34 5.78 1.5594 31.88 1.82 11.19 1.26b

94 31.88 1.46 11.19 e74 6.37 2.60 0.92b e

ilo bases per million reads (FPKM).shown.

3. Results and discussions

Deep transcriptome sequencing generated 27e35 million readsper sample (Table 1). An average of 98.4% of these reads weremapped to rRNA and tRNA sequences, whereas 0.15e1.7 millionreads per sample were mapped to protein-coding sequences (CDS)and other rare RNA species (e.g. non-coding RNA) in the S. Enter-itidis str. P125109 genome. The output of CDS-targeting RNA in thisstudy was comparable with those of other bacterial RNA-seqstudies (Oliver et al., 2009; Perkins et al., 2009; Yoder-Himeset al., 2009). We observed high level of ribosomal RNA (rRNA)degradation in S. Enteritidis after 6 h inoculation in LB broth and atvarious time points during the incubation in peanut oil. Thisobservation was consistent with a previous report that extensiverRNA fragmentation and degradation was found upon the transi-tion from exponential to stationary phase in S. enterica (Hsu et al.,1994). Degraded rRNA was thought to be a source of nutrientsthat could be utilized by bacteria during starvation or upon otherenvironmental stresses (Deutscher, 2006).

A drastic difference in transcriptomic activities was detectedbetween S. Enteritidis cells inoculated in LB broth and those inpeanut oil (Fig. 1). Nearly half of the S. Enteritidis genome wastranscribed under starvation stress in LB broth at 312 h; however,only about 1.36e4.42% of the genome was actively transcribedunder both starvation and desiccation stresses in peanut oil at 216 hand 518 h. This observation suggests that starved S. Enteritidis cellsin low aw peanut oil are likely in a physiological state distinct fromthose starved in LB broth. Two possible scenarios may partiallyexplain the fact that much fewer transcripts were detected indesiccated S. Enteritidis cells. First, desiccated S. Enteritidis cellsmay be in a metabolically dormant state marked by low tran-scriptional activity as found in other nonspore-forming bacterialspecies under starvation stress (Jones and Lennon, 2010; Lewis,2007). Second, as a survival mechanism, desiccated S. Enteritidiscells may increase the degradation of RNA molecules in order toobtain more nutrients. It was reported that prolonged starvation inbatch cultures can induce a growth advantage in stationary phase(GASP) phenotype in enterobacteria such as Escherichia coli and

cle shreepons

X. Deng et al. / Food Microbiology 30 (2012) 311e315 313Fig. 1. A circular map of S. Enteritidis transcripts detected by RNA-seq. The innermost cirthe two blue circles show genes expressed in 12 h and 312 h LB broth, respectively; the tgenes detected in both biological replicates were shown. Desiccation-induced stress-res

from A through L: dnaK, dnaJ, cspE, dps, cspD, sigE, grpE, sigma-32, major cold shock protein, ibRNA, rprA RNA, ryfA RNA, tke1 RNA, csrB RNA, RNase P RNA, sraJ RNA and csrC RNA.hows nucleotide coordinates on the S. Enteritidis str. P125109 genome. From inside out:red circles show genes expressed in 72 h, 216 h, and 528 h peanut oil, respectively. Onlye genes and non-coding RNAs were labeled at corresponding genomic locations. Genes,

pA, ibpB, and putative stress-response protein. Non-coding RNAs, from 1 to 9: sraC/ryeA

nome bi

obioS. enterica (Finkel and Kolter, 1999; Lewis, 2007; Martinez-Garciaet al., 2003). During GASP, genetic mutants tter for the adverseenvironment keep emerging and replacing less competitive cells,causing dynamic uctuations in the cell population. We observedthat S. Enteritidis cells in peanut oil at 216 h appeared to haveincreased population (between 120 h and 360 h) and transcrip-tional activity (compared with 72 h and 528 h samples). Furtherstudies are necessary to determine whether GASP phenotype ispresent in S. Enteritidis under desiccation stress.

Peanut butter production typically includes multiple steps ofdrying and roasting, therefore, bacterial resistance to desiccationand heat is considered critical for S. enterica survival and persis-tence in this food (Archer et al., 1998; Gruzdev et al., 2011; Matticket al., 2000, 2001; Shachar and Yaron, 2006). Among the consid-erably few genes expressed in peanut oil-treated cells, transcriptionof 12 genes were detected in both biological replicates at the threesampling time points (Table 2). Four of these genes encode proteinsinvolved in bacterial stress response to temperature shift, includingheat shock sigma factor RpoH (Landick et al., 1984), extreme heatand cell envelope stress sigma factor RpoE (Hiratsu et al., 1995),heat shock protein GrpE (Ang et al., 1986), and a major cold shockprotein CspA (Goldstein et al., 1990). Several additional stress-response genes were also detected in the majority of the vesamples. In spite of the overall metabolic dormancy and elevateddegradation of transcriptome in desiccated S. Enteritidis cells inpeanut oil, a few critical genes maintained active transcription,which may in turn assist the persister cells to survive subsequentheat and other environmental challenges. In addition to protein-coding genes, a number of non-coding regulatory RNAs (ncRNA)were also detected in starved and desiccated S. Enteritidis cells inpeanut oil (Table 3). Notably, transcription of the enterobacter-specic csrB and csrC ncRNAs was detected only in the starved S.Enteritidis cells in peanut oil but not in the starved S. Enteritidiscells in LB broth. Both ncRNAs bind to mRNA of the global carbon

Table 3Non-coding RNA transcribed in desiccated S. Enteritidis in peanut oil.

Genome location Annotation

503792.503915 Signal recognition particle RNA1226143.1226322 sraC/ryeA RNA (Wassarman et al., 2001)1784211.1784419 rprA RNA (Majdalani et al., 2001)2675687.2676039 ryfA RNA (Wassarman et al., 2001)2708464.2708604 tke1 RNA (Rivas et al., 2001)2796024.2796426 Transfer messenger RNA (Williams and Bartel, 1998)3005411.3005789 csrB RNA (Liu et al., 1997)3302193.3302585 RNase P RNA (Brown, 1999)4012003.4012218 craJ RNA (Argaman et al., 2001)4078959.4079170 csrC RNA (Argaman et al., 2001)

Only ncRNAs detected in both replicates under at least one condition are listed; ge(Thomson et al., 2008); , detected in one biological replicate; e, not detected in on

X. Deng et al. / Food Micr314storage regulator gene CsrA and regulate carbon ux and metabo-lism (Argaman et al., 2001; Liu et al., 1997). The functions of thesencRNAs and their potential roles in mediating energy metabolismin persister cells are yet to be identied.

In summary, low water activity in foods can reduce bacterialgrowth and metabolism. Desiccation in turn can trigger a numberof stress response mechanisms in the bacteria that enhancebacterial resistance to other stresses and prolong bacterial survivalin the product. S. enterica has been implicated in numerous humandisease outbreaks associated with a wide variety of low moisturefoods. Yet, it remains a mystery how this pathogen manages tosurvive in these foods where free water and nutrients are limitedfor bacterial growth. This study provides an initial assessment of S.Enteritidis transcriptomic stress response to desiccation in peanutoil as it relates to the survival and persistence of this pathogen inother low aw foods. A better understanding of the specicmolecular determinants employed by this pathogen to resistdesiccation stress in low aw foods may enlight new strategies formore effective intervention and control.

Acknowledgments

This study was supported by the Food Research Initiative Grantno. 2010-65201-20593 from the USDA National Institute of FoodAgriculture, Food Safety and Epidemiology: Biological Approachesfor Food Safety program (program code 93231).

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Transcriptome sequencing of Salmonella enterica serovar Enteritidis under desiccation and starvation stress in peanut oil1. Introduction2. Materials and methods2.1. Bacteria2.2. Inoculation of peanut oil2.3. RNA extraction2.4. RNA-seq analysis

3. Results and discussionsAcknowledgmentsReferences