Food Microbiology 30 (2012) 311e315

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Food Microbiology
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Short communication

Transcriptome sequencing of Salmonella enterica serovar Enteritidis under desiccation and starvation stress in peanut oil
Xiangyu Deng 1, Zengxin Li, Wei Zhang*
Institute for Food Safety and Health, Illinois Institute of Technology, 6502 S. Archer Rd, Bedford Park, IL 60501, USA

a r t i c l e i n f o
Article history: Received 24 August 2011 Received in revised form 7 October 2011 Accepted 2 November 2011 Available online 13 November 2011 Keywords: Transcriptome sequencing Salmonella enterica Desiccation Stress response

a b s t r a c t
It is well recognized that Salmonella can survive long-term starvation and desiccation stresses and contaminate foods that have intermediate to low water activities; however, little is known about the specic molecular mechanisms underlying its survival and persistence in low water activity foods. In this study, we used the RNA-seq approach to compare the transcriptomes (27e33 million 36-bp reads per sample) of a Salmonella enterica subsp. enteric serovar Enteritidis strain ATCC BAA-1045 after inoculation in peanut oil (water activity 0.30) for 72 h, 216 h and 528 h to those grown in Luria-Bertani (LB) broth for 12 h and 312 h. Our results showed that desiccated Salmonella cells in peanut oil were in a physiologically dormant state with <5% of its genome being transcribed compared to 78% in LB broth. Among the few detected transcripts in peanut oil, genes involved in heat and cold shock response, DNA protection and regulatory functions likely play roles in cross protecting Salmonella from desiccation and starvation stresses. In addition, non-coding RNAs may also play roles in Salmonella desiccation stress response. This is the rst report of using RNA-seq technology in characterizing bacterial transcriptomes in a food matrix. 2011 Elsevier Ltd. All rights reserved.

1. Introduction The ability of Salmonella enterica to contaminate and survive in low water activity (aw) foods has been well documented (Burnett et al., 2000; Isaacs et al., 2005; Killalea et al., 1996; Kirk et al., 2004; Rowe et al., 1987; Shohat et al., 1996). Recent multi-state outbreaks of salmonellosis associated with peanut butter (Anonymous, 2007; Anonymous, 2009) and pet food (Anonymous, 2008) in the United States has demonstrated the need for basic understanding of the survival and persistence strategies of S. enterica in low aw food commodities. Despite extensive studies on the survival characteristics of S. enterica in various model systems with reduced aw (Archer et al., 1998; Gruzdev et al., 2011; Hiramatsu et al., 2005; Mattick et al., 2000, 2001; Shachar and Yaron, 2006), little is known about the physiology of this pathogen under desiccation and starvation stress and the underlying molecular mechanisms for its survival and persistence in low aw foods. A number of recent studies investigated the global gene 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.,
* 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, IL 60025, USA. 0740-0020/$ e see front matter 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fm.2011.11.001

2011). Most of these studies relied on DNA array technology to compare bacterial transcriptomes under various conditions or treatments, based on which specic cellular responses to environmental cues were deduced. However, technical difculties associated with isolating high quality bacterial RNA from complex food matrices coupled with the relative low detection sensitivity of DNAeDNA hybridization have restricted DNA array-based applications in deciphering more subtle bacterial transcriptomic response to food and environmental stresses (e.g. detection of rare transcripts and non-coding regulatory RNAs). In the present study, we demonstrate, for the rst time, the use of whole transcriptome sequencing (or RNA-seq) technology to study bacterial global gene expression in a food matrix. Specically, the transcriptomic response of S. Enteritidis to desiccation and starvation stress in peanut oil (aw 0.3) was investigated as this particular Salmonella serotype has been implicated in a number of foodborne illness outbreaks associated with low aw foods (Gruzdev et al., 2011). 2. Materials and methods 2.1. Bacteria We used S. Enteritidis str. ATCC BAA-1045 which was isolated from recalled almonds implicated in an international outbreak of salmonellosis (Isaacs et al., 2005).

312 Table 1 Summary of RNA-seq data. Sample 12 h LB (1) 12 h LB (2) 312 h LB (1) 312 h LB (2) 72 h peanut oil (1) 72 h peanut oil (2) 216 h peanut oil (1) 216 h peanut oil (2) 528 h peanut oil (1) 528 h peanut oil (2)
a

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Total number of reads 27,151,557 33,274,075 29,017,764 34,958,141 30,015,630 30,131,874 32,148,558 30,918,263 31,967,767 32,179,987

Percentage of mapped readsa 92.98% 92.45% 92.24% 90.48% 93.17% 94.30% 92.08% 91.55% 93.11% 90.45%

Number of reads mapped to CDS 396,413 1,763,526 499,106 311,127 228,119 232,015 228,255 148,408 210,987 234,914

Percentage of genome represented 45.52% 76.84% 44.45% 30.04% 1.52% 1.59% 4.42% 2.98% 1.45% 1.36%

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

2.2. Inoculation of peanut oil We used peanut oil as the food matrix mainly because of its low water activity (aw 0.3). Also, separation and recovery of bacteria from peanut oil was effective. S. Enteritidis cells from 10 ml of overnight Luria-Bertani (LB) culture (w109 CFU/ml) were harvested by centrifugation at 4000 rpm for 5 min, washed three times with 1X phosphate buffered saline (PBS) at neutral pH, and air dried for 30 min. Dried cell pellets were thoroughly mixed with 10 ml peanut oil in centrifuge tubes by hand stirring and vortexing (nal cell density around 109 CFU/ml in peanut oil). Spiked peanut oil suspensions were kept horizontally in a shaking incubator at 300 rpm (to avoid the clumping of cells) at 25  C for up to 30 days. 2.3. RNA extraction S. Enteritidis cells were harvested from peanut oil at specic time points (i.e. 72 h, 216 h and 528 h) by centrifugation at 4000 rpm at room temperature for 5 min and immediately stabilized using QIAGEN RNAprotect bacterial reagent (QIAGEN Inc., Valencia, CA). Total bacterial RNA was puried using the AMBION RiboPure Bacterial Kit (Life Technologies Corporation, Carlsbad, CA). Total RNA from two biological replicates for each time point was prepared from independent cultures on different days to evaluate the reproducibility of RNA-seq data. For comparison purposes, total RNA samples were also prepared from S. Enteritidis cells grown in
Table 2 Genes transcribed in desiccated S. Enteritidis in LB broth and peanut oil. Locus ID Description

LB broth (10 ml overnight culture inoculated in 100 ml LB broth) at 12 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 by AMBION RiboPure kit), each DNA-free total RNA sample was subjected to whole transcriptome sequencing on an Illumina Genome Analyzer to generate 36-bp single-end reads. Library preparation, cluster generation and sequencing were performed according to manufacturers recommendations (Illumina Inc., San Diego, CA). Prior to cluster generation, Duplex-Specic thermostable nuclease (DSN) (Evrogen, Moscow, Russia) normalization was performed to enrich rare transcripts (Zhulidov et al., 2004). Sequencing reads were mapped to a fully sequenced S. Enteritidis str. P125109 genome (Thomson et al., 2008) using Bowtie (Langmead et al., 2009) and assembled into transcripts using Cufinks (Trapnell et al., 2010), both through the Galaxy server (Goecks et al., 2010). Annotation of the reference genome was then used to curate and analyze assembled transcripts by self-written Python programs based on Biopython modules (Cock et al., 2009). Sequencing data and analysis history are publicly available at http://main.g2.bx.psu. edu/u/xdeng/h/salmonella-rna-seq. Quantitative reverse transcription PCR (qPCR or qRT-PCR) was used to validate the expression of 12 genes identied by RNA-seq (Table 2) based on the method described in (Wen et al., 2011).

FPKMa 12 h LB 312 h LB 17.84 17.84 16.45 21.77 12.48 8.75 3.64 17.82 10.31 24.68 5.91 6.17 6.17 26.09 33.84 10.44 10.44 31.88 31.88 6.37 72 h oil 1.16 e 4.23 13.29 1.41b 1.56 5.16 3.70 9.99 1.43 0.58 28.29 28.29 1.81 1.35 2.34 2.34 1.82 1.46 2.60 216 h oil e 1.91 5.63 9.81 3.72 e 17.77 8.19 13.03 1.17 4.73 17.27 17.27 9.05 3.82 5.78 5.78 11.19 11.19 0.92b 528 h oil e e 1.05 1.98b 2.04 e 16.29 1.85 1.92 0.97 1.79 5.00 5.00 1.57 1.53 1.80 1.55 1.26b e e

SEN0011 SEN0012 SEN0135 SEN0598 SEN0776 SEN0851 SEN0984 SEN1257 SEN1835 SEN2566 SEN2602 SEN3194 SEN3195 SEN3391 SEN3472 SEN3509 SEN3510 SEN3624 SEN3625 SEN4009

Chaperone protein DnaK Chaperone protein DnaJ N-acetylglucosamine deacetylase Cold shock protein CspE Starvation/stationary phase protection protein Dps Cold shock-like protein CspD Hypothetical protein Hypothetical protein Hypothetical protein Sigma factor RpoE Heat shock protein GrpE Hypothetical protein Hypothetical protein Sigma factor 32 RpoD Major cold shock protein Mannitol repressor protein Hypothetical protein Heat shock chaperone IbpB Heat shock protein IbpA Putative stress-response protein

30.99 24.21 12.03 37.33 109.46 19.71 5.65 20.33 1.50 13.97 6.01 17.26 17.26 13.78 65.77 4.40 4.40 31.94 31.94 8.74

e: Transcript was not detected in both replicates of the sample. a Abundance of transcripts were quantied as normalized number of fragments per kilo bases per million reads (FPKM). b Transcript was detected in only one replicate of the sample, of which the FPKM was shown.

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3. Results and discussions Deep transcriptome sequencing generated 27e35 million reads per sample (Table 1). An average of 98.4% of these reads were mapped to rRNA and tRNA sequences, whereas 0.15e1.7 million reads per sample were mapped to protein-coding sequences (CDS) and other rare RNA species (e.g. non-coding RNA) in the S. Enteritidis str. P125109 genome. The output of CDS-targeting RNA in this study was comparable with those of other bacterial RNA-seq studies (Oliver et al., 2009; Perkins et al., 2009; Yoder-Himes et al., 2009). We observed high level of ribosomal RNA (rRNA) degradation in S. Enteritidis after 6 h inoculation in LB broth and at various time points during the incubation in peanut oil. This observation was consistent with a previous report that extensive rRNA fragmentation and degradation was found upon the transition from exponential to stationary phase in S. enterica (Hsu et al., 1994). Degraded rRNA was thought to be a source of nutrients that could be utilized by bacteria during starvation or upon other environmental stresses (Deutscher, 2006).

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

Fig. 1. A circular map of S. Enteritidis transcripts detected by RNA-seq. The innermost circle shows nucleotide coordinates on the S. Enteritidis str. P125109 genome. From inside out: the two blue circles show genes expressed in 12 h and 312 h LB broth, respectively; the three red circles show genes expressed in 72 h, 216 h, and 528 h peanut oil, respectively. Only genes detected in both biological replicates were shown. Desiccation-induced stress-response genes and non-coding RNAs were labeled at corresponding genomic locations. Genes, from A through L: dnaK, dnaJ, cspE, dps, cspD, sigE, grpE, sigma-32, major cold shock protein, ibpA, ibpB, and putative stress-response protein. Non-coding RNAs, from 1 to 9: sraC/ryeA RNA, rprA RNA, ryfA RNA, tke1 RNA, csrB RNA, RNase P RNA, sraJ RNA and csrC RNA.

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Table 3 Non-coding RNA transcribed in desiccated S. Enteritidis in peanut oil. Genome location 503792.503915 1226143.1226322 1784211.1784419 2675687.2676039 2708464.2708604 2796024.2796426 3005411.3005789 3302193.3302585 4012003.4012218 4078959.4079170 Annotation Signal recognition particle RNA sraC/ryeA RNA (Wassarman et al., 2001) rprA RNA (Majdalani et al., 2001) ryfA RNA (Wassarman et al., 2001) tke1 RNA (Rivas et al., 2001) Transfer messenger RNA (Williams and Bartel, 1998) csrB RNA (Liu et al., 1997) RNase P RNA (Brown, 1999) craJ RNA (Argaman et al., 2001) csrC RNA (Argaman et al., 2001) 12 h LB e e e e e e e e e e 312 h LB e e e e e e 72 h oil e e 216 h oil e 528 h oil e e e

Only ncRNAs detected in both replicates under at least one condition are listed; genomic locations and annotation are based on reference genome S. Enteritidis P125109 (Thomson et al., 2008); , detected in one biological replicate; e, not detected in one biological replicate.

S. enterica (Finkel and Kolter, 1999; Lewis, 2007; Martinez-Garcia et al., 2003). During GASP, genetic mutants tter for the adverse environment keep emerging and replacing less competitive cells, causing dynamic uctuations in the cell population. We observed that S. Enteritidis cells in peanut oil at 216 h appeared to have increased population (between 120 h and 360 h) and transcriptional activity (compared with 72 h and 528 h samples). Further studies are necessary to determine whether GASP phenotype is present in S. Enteritidis under desiccation stress. Peanut butter production typically includes multiple steps of drying and roasting, therefore, bacterial resistance to desiccation and heat is considered critical for S. enterica survival and persistence in this food (Archer et al., 1998; Gruzdev et al., 2011; Mattick et al., 2000, 2001; Shachar and Yaron, 2006). Among the considerably few genes expressed in peanut oil-treated cells, transcription of 12 genes were detected in both biological replicates at the three sampling time points (Table 2). Four of these genes encode proteins involved in bacterial stress response to temperature shift, including heat shock sigma factor RpoH (Landick et al., 1984), extreme heat and cell envelope stress sigma factor RpoE (Hiratsu et al., 1995), heat shock protein GrpE (Ang et al., 1986), and a major cold shock protein CspA (Goldstein et al., 1990). Several additional stressresponse genes were also detected in the majority of the ve samples. In spite of the overall metabolic dormancy and elevated degradation of transcriptome in desiccated S. Enteritidis cells in peanut oil, a few critical genes maintained active transcription, which may in turn assist the persister cells to survive subsequent heat and other environmental challenges. In addition to proteincoding genes, a number of non-coding regulatory RNAs (ncRNA) were also detected in starved and desiccated S. Enteritidis cells in peanut oil (Table 3). Notably, transcription of the enterobacterspecic csrB and csrC ncRNAs was detected only in the starved S. Enteritidis cells in peanut oil but not in the starved S. Enteritidis cells in LB broth. Both ncRNAs bind to mRNA of the global carbon storage regulator gene CsrA and regulate carbon ux and metabolism (Argaman et al., 2001; Liu et al., 1997). The functions of these ncRNAs and their potential roles in mediating energy metabolism in persister cells are yet to be identied. In summary, low water activity in foods can reduce bacterial growth and metabolism. Desiccation in turn can trigger a number of stress response mechanisms in the bacteria that enhance bacterial resistance to other stresses and prolong bacterial survival in the product. S. enterica has been implicated in numerous human disease outbreaks associated with a wide variety of low moisture foods. Yet, it remains a mystery how this pathogen manages to survive in these foods where free water and nutrients are limited for bacterial growth. This study provides an initial assessment of S. Enteritidis transcriptomic stress response to desiccation in peanut oil as it relates to the survival and persistence of this pathogen in other low aw foods. A better understanding of the specic

molecular determinants employed by this pathogen to resist desiccation stress in low aw foods may enlight new strategies for more effective intervention and control. Acknowledgments This study was supported by the Food Research Initiative Grant no. 2010-65201-20593 from the USDA National Institute of Food Agriculture, Food Safety and Epidemiology: Biological Approaches for Food Safety program (program code 93231). References
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