Pathological phenotypes and in vivo DNA cleavage byunrestrained activity of a phosphorothioate-based restrictionsystem in Salmonella

Bo Cao,1,2 Qiuxiang Cheng,1 Chen Gu,2 Fen Yao,1

Michael S. DeMott,2 Xiaoqing Zheng,1 Zixin Deng,1

Peter C. Dedon2 and Delin You1*1State Key Laboratory of Microbial Metabolism andSchool of Life Sciences and Biotechnology, ShanghaiJiao Tong University, Shanghai, China.2Department of Biological Engineering and Center forEnvironmental Health Science, Massachusetts Instituteof Technology, Cambridge, MA, USA.

Summary

Prokaryotes protect their genomes from foreign DNAwith a diversity of defence mechanisms, including awidespread restriction–modification (R–M) systeminvolving phosphorothioate (PT) modification of theDNA backbone. Unlike classical R–M systems, highlypartial PT modification of consensus motifs in bacte-rial genomes suggests an unusual mechanism ofPT-dependent restriction. In Salmonella enterica, PTmodification is mediated by four genes dptB–E, whilerestriction involves additional three genes dptF–H.Here, we performed a series of studies to characterizethe PT-dependent restriction, and found that it pre-sented several features distinct with traditional R–Msystems. The presence of restriction genes in aPT-deficient mutant was not lethal, but insteadresulted in several pathological phenotypes. Subse-quent transcriptional profiling revealed the expressionof > 600 genes was affected by restriction enzymes incells lacking PT, including induction of bacteriophage,SOS response and DNA repair-related genes. Thesetranscriptional responses are consistent with theobservation that restriction enzymes caused exten-sive DNA cleavage in the absence of PT modificationsin vivo. However, overexpression of restriction geneswas lethal to the host in spite of the presence PTmodifications. These results point to an unusual

mechanism of PT-dependent DNA cleavage by restric-tion enzymes in the face of partial PT modification.

Introduction

Prokaryotes have evolved numerous systems to defendagainst foreign DNA, such as toxin–antitoxin (TA) pairs(Gerdes et al., 2005), abortive infection (Abi) (Chopinet al., 2005), clustered regularly interspaced short palin-dromic repeats-CRISPR-associated proteins (CRISPR-Cas) (Datsenko et al., 2012), and silencing by the histone-like nucleoid structuring (H-NS) proteins (Lucchini et al.,2006; Navarre et al., 2006). The best characterized ofthese defence systems are the methylation-basedrestriction–modification (R–M) systems, in which a DNAmethyltransferase modifies the host DNA in a specificpattern while a restriction enzyme recognizes and cleavesunmodified foreign DNA (Wilson and Murray, 1991).Among the tremendous diversity of Type I–III R–Msystems, all known DNA modifications were thought tooccur on the nucleobases until our recent discovery ofphosphorothioate (PT) modifications of the DNA back-bone, in which a non-bridging phosphate oxygen isreplaced with sulphur (Wang et al., 2007).

Modification of DNA with sequence- and stereo-specificPT involves a complex biosynthetic pathway requiring fourhorizontally transferred genes termed dndA, dndC, dndDand dndE in Streptomyces lividans 66, with gene nomen-clature based on the DNA degradation (dnd) phenotypeobserved during electrophoresis of genomic DNA frombacteria possessing the dnd gene cluster (Zhou et al.,2005). Both the PT modifications and the dnd genes aredistributed in a wide range of bacterial and archaealgenomes (Ou et al., 2009; Wang et al., 2011). In terms offunction, the protein product of dndB is predicted to be atranscriptional regulator (Zhou et al., 2005) and itsabsence leads to increased levels of PT modifications(Wang et al., 2007; 2011). DndA acts as a cysteine desul-phurase and assembles DndC a 4Fe-4S cluster protein(You et al., 2007), with some bacteria replacing DndA witha homologous cysteine desulphurase activity, such as IscSin Escherichia coli (An et al., 2012).DndC possesses ATP

Accepted 24 June, 2014. *For correspondence. E-mail dlyou@sjtu.edu.cn; Tel. (+86) 21 62932943; Fax (+86) 21 62932418.

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pyrophosphatase activity (Zhou et al., 2005), while a DndDhomologue in Pseudomonas fluorescens Pf0-1, SpfD, hasATPase activity that is possibly related to DNA structurealteration or nicking during PT incorporation (Yao et al.,2009). DNA nicking activity may also be associated with E.coli DndE (Hu et al., 2012). Many bacterial strains havebeen found to possess the dnd homologues, includingwide-ranging genera from diverse habitats, such as soil-dwelling and marine species, aerobic and anaerobicmicrobes, and human saprophytes and pathogens (Ouet al., 2009).

Recent evidence points to host defence as a majorfunction for PT modifications. We recently demonstratedthat PT modifications protect Salmonella enterica SerovarCerro 87 against unmodified plasmid DNA during trans-formation, albeit with a lower efficiency compared to thetraditional R–M system (Xu et al., 2010). In S. enterica, agene cluster homologous to dndB–E, namely dptB–E, isrequired for PT modification, with an endogenous cysteinedesulphurase homologous to E. coli iscS replacing dndA(An et al., 2012). The PT-dependent restriction phenotypein S. enterica is conferred by an additional three-genecluster, named as dptF–H (Xu et al., 2010). The geneclusters of dptB–E and dptF–H are present in a widevariety of bacteria with a similar genomic organization (Xuet al., 2010), which suggests widespread use of PTmodifications-dependent R–M system in prokaryotes.

A highly unusual feature of the PT-based R–M system isthe fact that the only apparent consensus sequence for thebistranded modification sites in Escherichia coli B7A,which shares dnd/dpt genes that are highly homologous toS. enterica, involves GPSAAC/GPSTTC and only 12% ofthese sites contain a PT modification (Cao et al., 2014).This partial modification phenomenon led us to pursue aseries of experiments to characterize the dptF–HPT-dependent restriction system, with the discovery ofseveral unusual features that differ from classical R–Msystems.

Results

Phenotypic changes caused by loss of dptB–E in theface of active dptF–H

To understand the PT-dependent restriction mechanism,we undertook studies to define the effects of expression ofthe dptF–H restriction genes in S. enterica mutants lackingPT modifications (ΔdptB–E). Interestingly, unlike manyrestriction systems, the presence of dptF–H in thePT-deficient mutant was not lethal, but resulted in a rangeof abnormal phenotypes. All of the pathological pheno-types were resolved either by additional deletion ofdptF–H or by complementation of the mutant strain with adptB–E PT modification gene cluster-containing plasmid.A

summary of the colony and cell morphology and growthphenotypes altered by dptF–H in the absence of dptB–E isshown in Fig. 1.

One feature of the growth of the ΔdptB–E mutant wasthe accumulation of a white mucoid material during late-exponential phase in liquid culture (Fig. 1A). In terms ofmacroscopic phenotypes, the ΔdptB–E mutant displayedaltered colony morphology on solid medium, with a faintyellow colour and a rougher surface, suggesting defec-tive growth (Fig. 1B). When grown in liquid culture,microscopic analysis revealed an elongated morphologyfor the mutant cells, which is consistent with failed cytoki-nesis (Fig. 1C). Meanwhile, ΔdptB–E grew at a slowerrate than the wild-type strain and ΔdptB–H mutant(Fig. 1D). Based on these changes and on a proteomicanalysis of mucoid material apparent late-exponentialphase in liquid culture (Fig. 1A and B), which showedenrichment in elongation factors, RecA, ribosomal pro-teins, and outer membrane proteins (Table S1), weassessed the membrane integrity of ΔdptB–E mutant bypropidium iodide (PI) fluorescence flow cytometry(Gregori et al., 2001; Su et al., 2009). This approach isbased on the fact that DNA-binding PI is excluded fromcells with intact membranes, while cells with damagedmembranes allow entry and DNA binding by PI, resultingin an increased cellular fluorescence that can be quan-tified by flow cytometry. Both the wild-type strain and theΔdptB–H mutant showed identical levels PI fluorescenceand no changes as a function of cell state, as shown inFig. 2 (representative flow cytometry histograms areshown in Fig. S1). In contrast, ΔdptB–E showed a shift tohigher levels of PI fluorescence (∼ 16%) during late-exponential and stationary phase (Fig. 2).

Effects of unrestrained PT-dependent restriction activityon gene expression

To further explore the observations of abnormal pheno-types of the PT-deficient mutant, we performed compre-hensive transcriptional profiling to identify changes in geneexpression caused by loss of dptB–E in S. enterica. In thisstudy, we used 8797 probes that covered all genes ofpublished S. enterica genomes in GenBank, including theseven dpt genes, and three independent biologicalreplicates with cell cultures in early-exponential, late-exponential and stationary phases. The resulting microar-ray data reflected 5413 genes, which is consistent withother Salmonella strains possessing ∼ 4000–6000 genes(McClelland et al., 2001; Parkhill et al., 2001; Deng et al.,2003; Chiu et al., 2005). With the criterion of at leasta ± 2-fold change with a P value of ≤ 0.05 to define signifi-cantly up- or downregulated genes, the filtered data fromthe ΔdptB–E and ΔdptB–H mutants at each growth phasecan be found at the NCBI Gene Expression Omnibus

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website (Geo Accession GSE59225; http://www.ncbi.nml.nih.gov/geo/).

Based on these statistical parameters, the ΔdptB–Hmutant displayed a transcriptional profile similar to thewild-type strain, with 23 and 17 differentially expressedgenes at the early- and late-exponential phase respec-tively. However, relative to wild-type cells, the ΔdptB–Estrain showed 116 genes altered at their early-exponentialphase, and 235 and 683 at late-exponential and station-ary phase respectively (Fig. S2). Analysis of thesechanges in gene expression by hierarchical clustering isshown in Fig. 3, from which it is apparent that most of theupregulated genes in the early-exponential phase werecontinuously expressed at higher levels throughout thecourse of growth. Meanwhile, upregulation of other genesbegan at the late-exponential phase. Notably, downregu-lated genes were significantly outnumbered by upregu-lated genes. The microarray data were corroborated byreal-time reverse transcription-polymerase chain reactionanalysis of six representative genes, including the PTrestriction gene dptF, flagellar biosynthesis gene fliH and

Fig. 1. Phenotypic alterations in the ΔdptB–E mutant.A. White mucoid material was observed in ΔdptB–E mutant grown in liquid culture, but not ΔdptB–H.B. Altered colony morphology on the solid media, with recovery provided by the dptB–E-containing plasmid (pJTU1238).C. Microscopic analysis of cell mobility and division.D. Growth rate curves in liquid culture.

Fig. 2. Analysis of membrane defects in the ΔdptB–E mutant atdifferent growth phases. Membrane permeability was quantified byPI staining and flow cytometry, as described in Experimentalprocedures. Data represent mean ± SD for three biologicalreplicates.

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the four DNA repair genes recA, recN, ruvA and nerE(Fig. S3).

To gain insight into the functional significance of thedptF–H-dependent changes in gene expression, we thenannotated and categorized the significantly changedgenes according to the NCBI Clusters of OrthologousGroups (COG) database. Strikingly, one of the mostprominent groups of upregulated genes representedDNA repair pathways, including base-excision repair,homologous recombination, and the SOS responsegenes (Fig. 3). These genes were induced at the early-exponential phase and increased to a significantly highlever at stationary phase, which is consistent with DNAdamage arising from DptF–H in the ΔdptB–E mutant.Furthermore, 31 prophage genes were also induced atearly-exponential phase and accumulated to an expres-sion level 100-fold higher than that in the wild-type strain(Fig. 3). The ΔdptB–E mutant also showed upregulationof genes involved in cell mobility (23 genes), includingflagellar biosynthesis; DNA replication, transcription andtranslation (41 genes); energy production and conversion(39 genes), especially anaerobic metabolism; membraneproteins and colanic acid capsullar biosynthesis (8genes); as well as 101 genes with unknown function. Thedownregulated genes corresponded to basal metabolism(166 genes), cell division (2 genes), biofilm formation (3genes), and 97 genes of unknown function. These tran-scriptional changes are consistent with the proteincontent of the mucoid debri (Table S1), with upregulationof 30 ribosomal protein genes, an elongation factor,

RecA, and 17 outer membrane proteins. For the slowedcell division phenotype, we found that the sulA gene foran SOS-dependent cell division inhibitor was upregu-lated by 21-fold, while two cell division genes (ftsA, ftsQ)were downregulated. Reduction of biofilm formationregulatory gene bssR (yliH) and activation of the lyticbacteriophage are consistent with the rough, less shinycolony morphology (Fig. 1) (Levine, 1957; Park et al.,2007), while genes responsible for cell division suppres-sion, lytic bacteriophage activation and SOS responsecould cause the slower growth phenotype (Fig. 1).

dptF–H expression in the absence of PT is associatedwith DNA cleavage in vivo

Given the phenotypic and gene expression evidence fora DNA damage response in the ΔdptB–E mutant strain,we used a derivative of the TUNEL (terminal deoxynu-cleotidyl transferase-mediated dUTP nick and labelling)assay to quantify DNA strand breaks in wild-type andmutant S. enterica strains. As a routinely used approachto monitor DNA strand breaks in vivo in both eukaryoticand prokaryotic cells, the TUNEL assay is based onTdT-mediated labelling of the 3′-OH of DNA breaks withfluorescently labelled dUTP (Rohwer and Azam, 2000;Loo, 2011), here using FITC-dUTP. Single-cell fluores-cence analysis was then accomplished by flow cytom-etry, using PI staining to gate out cell-sized particulatelacking DNA. Three independent samples of the wild-type strain and the ΔdptB–E and ΔdptB–H mutants were

Fig. 3. Hierarchical clustering analysis ofmicroarray data of ΔdptB–E and ΔdptB–Hmutants in different growth phases. Clusteringwas performed on fold-change data for eachgene (rows), with green indicatingdownregulation and red indicatingupregulation relative to the wild-type strain;the inset shows the colour code for thefold-change values. Columns A, B, Crepresent the ΔdptB–E strain grown atearly-exponential, late-exponential andstationary phase respectively. Columns D andE represent the ΔdptB–H strain at early- andlate-exponential phases respectively.

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collected at three different growth phases for quantifica-tion of DNA breaks using the TUNEL assay, with flowcytometric analysis of a total of 30 000 events for eachsample.

As shown in Fig. 4, ΔdptB–E cells exhibited a signifi-cantly higher level of TUNEL signal than ΔdptB–H andwild-type strain in all three growth phases, and the level ofTUNEL signal kept increasing along the course of itsgrowth. In order to quantitatively analyse the cells contain-ing DNA strand breaks, we defined them as TUNEL-positive cells that showed FITC fluorescent signalexceeding that of 99% of the wide-type cells. With thisdefinition, we found that ∼ 50% of ΔdptB–E cells wereobserved as TUNEL-positive at early-exponential phase,while the percentage increased to more than 80% whengrown to late-exponential and stationary phases. Theseresults indicate that DptF–H induces DNA breaks in theabsence of PT modifications, suggesting a PT-dependent,DNA cleavage-associated R–M system.

Overexpression of dptF–H is lethal to the host in thepresence of PT modifications

Given that dptF, G, and H reside within an operon inSalmonella, these three enzymes are likely to assembleand perform their function as a complex rather than asindividual proteins. We then attempted to heterologouslyexpress the dptF, G and H operon in E. coli BL21 (DE3), astrain that does not possess dnd/dpt genes. Not surpris-ingly, the dptFGH-containing plasmid could not be trans-formed into the host E. coli lacking PT modifications,suggesting a lethal effect to the host by the restrictionenzymes in the absence of PT modification. However,overexpression of the restriction gene operon also resultedin a lethal effect (i.e. failure of transformation) in hoststrains possessing PT modifications, such as wild-typeSalmonella or an E. coli strain harbouring a dptB–E-containing plasmid. Considering the partial or incompletestate of PT modifications in bacterial genomes (Cao et al.,

Fig. 4. A–C. TUNEL assay analysis of DptF–H-induced DNA stand-breaks in the dptB–E mutant at early-exponential (A), late-exponential (B)and stationary phase (C).D. Comparison of TUNEL-positive cells of dptB–E mutant in different growth phases.E. Percentage of TUNEL-positive cells in dptB–E mutant at different growth phases.

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2014) and the fact that, in most R–M systems, the pres-ence of the DNA modification prevented DNA cleavage byrestriction enzymes, the lethal effects of overexpression ofDndF–H restriction enzymes in the presence of a normalcomplement of PT modifications suggests that the PTmodification-based R–M system involves controlled andbalanced restriction activity.

Discussion

Of the diverse defence mechanisms in bacteria, R–Msystems are the only ones that discriminate foreign DNAdirectly by modification of self DNA. In most R–Msystems, restriction and modification enzymes occur inparallel since the restriction enzyme usually causes lethaleffects in the modification-deficient strains (Boyer, 1964;Suri and Bickle, 1985). All known R–M systems are basedon DNA nucleobase modifications until the recent discov-ery of the PT-dependent restriction system in bacteria (Xuet al., 2010). However, unlike traditional R–M systems,each member of the set of all possible PT modificationsites in a bacterial cell is not always modified, such thatany cell will contain unmodified consensus sites that

should be susceptible to restriction enzyme cleavage(Cao et al., 2014). Considering the fact that, in most R–Msystems, all possible sites are modified to prevent DNAcleavage by restriction enzymes, the existence of partialmodification with PT points to a highly unusual restrictionmechanism in the PT R–M system and some kind ofregulation of the restriction enzymes in the wild-typestrain. This is entirely consistent with our observationsthat PT-dependent restriction enzymes presented severalunusual features in the PT-deficient mutant, as summa-rized in Fig. 5.

Both the transcriptional profiling data and evidence forDNA cleavage establish that the PT-based restrictionsystem involves DNA cleavage. It is thus not surprisingthat expression of restriction enzymes in a PT-deficient S.enterica mutant led to a pathological phenotype, which isbest explained as unrestrained DNA cleavage by restric-tion enzymes in the absence of DNA modifications. This isconsistent with similar observations in other R–M systems(Makovets et al., 1999).

The presence of DNA-cleaving restriction activity incells containing unmodified consensus sites points tosome kind of mechanism for controlling the activity of the

Fig. 5. Summary of the effects of PT-dependent restriction enzymes on the host strain.

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PT-based restriction system. This is illustrated by theobservation that overexpression of the restriction genes islethal even in a host strain possessing normal levels of PTmodifications, which is consistent with lethal DNA cleav-age when restriction enzymes are expressed at levels thatexceed the capacity of the cell to control their activity. Inmost R–M systems, all possible consensus sites aremodified to prevent cleavage by restriction enzymes. Sothe toxic consequences of DptF–H overexpression in thepresence of PT modifications and the phenomenon ofpartial modification with PT in wild-type cells suggestsome kind of mechanism for regulating restriction activityin the PT R–M system. For example, Clp protease hasbeen shown to control restriction endonuclease activity ina Type I R–M system in E. coli and to prevent lethal DNAcleavage in the absence of DNA modifications (Makovetset al., 1999).

In summary, we have presented evidence that, whiletraditional and PT-dependent R–M systems share acommon function of restriction of unmodified foreign DNA,the PT-dependent restriction enzymes present severalunusual features. Deletion of PT modification genes wasnot lethal to the host strain and caused phenotypic altera-tions, transcriptional profiling change and DNA damage,with overexpression of restriction enzymes causing lethal-ity even in the presence of the PT modifications. Theseresults provide new insights into an unusual R–M systemin bacteria.

Experimental procedures

Bacterial strains and culture conditions

Salmonella enterica serovar Cerro 87 wild-type strain har-boured both the dptB–E gene cluster (modification genes) andthe dptF–H (restriction genes); the ΔdptB–E mutant strain wasnamed XTG102 in our previous study (Xu et al., 2010);ΔdptB–E mutants harbouring a complementary dptB–E inpUC18 (pJTU1238; Xu et al., 2010), namely ΔdptB–E (dptB–E), and the empty pUC18, namely ΔdptB–E (pUC18), wereused for phenotypic description. The ΔdptB–H mutant wasconstructed with the same strategy used for generation ofXTG102, which was based on double-crossover homologousrecombination (Xu et al., 2010). Briefly, the left and right armsfor homologous recombination to generate ΔdptB–H mutantwere amplified by PCR using genomic DNA as the template.PCR primers dptBLL/dptBLR (dptLL: 5′-GACCTCGAGTTGGTTTTCAATA-3′, XhoI underlined; dptLR: 5′-AAGCAACCGTGTCAAGGTAATTGGCGTTGCTGCGTGGTTA-3′) wereused for the 752 bp left arm and dptHRR/dptHRL (dptHRR5′-CCGAGGCTCCAGCATTCACAGTGGTCGCACTAACACTGGA-3′; dptHRL: 5′-CAAGGATCCCAAAGAAATGTTCGCAAC-3′, BamHI) were for right arm. The two 40 bp overlappedarms were ligated by PCR and then cloned into the thermo-sensitive plasmid pKOV-Kan through the designed endonu-clease sites. The plasmid construct was then transformed intowild-type S. enterica to generate the ΔdptB–H mutant, which

grew on LA plates with15% sucrose at 43°C (Xu et al., 2010).Cells were grown at 37°C on solid or liquid Luria–Bertani (LB)medium supplemented with appropriate antibiotics unless oth-erwise stated. To obtain cells from different growth phases, theovernight bacterial cultures were diluted into 5 ml of LBmedium at a concentration of 5000 cells ml−1 and the cellgrowth was monitored by the optical density 600 nm (OD600).The cells reached OD600 of 0.8, 2.0 and 3.5 were considered tobe at their early-, late-exponential and stationary phaserespectively.

Phenotypic characterization of bacterial strains

For microscopic analysis of the colony morphology on solidmedia, strains were diluted to an appropriate concentrationand grown overnight on LA plates. The colonies forming onthe plates during overnight growth were then monitoreddirectly by microscopy. To study cell mobility and division,the strains were grown in liquid LB medium to exponentialphase and the morphology was then observed by micros-copy following methylene blue staining (400× magnifica-tion). To study growth rate, 1000 cells (based on OD600) ofeach strain were introduced into 10 ml fresh LB mediumand growth was monitored by OD600 every 1–2 h. After 16 h,growth curves were plotted based on three independentreplicates for each strain.

Proteomic analysis of mucoid material accumulated byΔdptB–E mutant

To gain insight into the mucoid material produced by ΔdptB–Emutant during late-exponential phase in liquid culture, weperformed a proteomic analysis of trypsin-derived peptidesusing chromatography (HPLC)-coupled tandem mass spec-trometry. In this assay, the digestion mixture containingtrypsin-digested mucoid material was resolved on an AgilentZorbax 300SB-C8 column (2.1 × 150 mm, 3.5 μm) with a gra-dient 5%–95% acetonitrile in 0.1% formic acid over 60 min ata flow of 0.2 ml min−1, and then monitored the peptides byAgilent 6410 QTOF MS (4 GHz high resolution, 50–1700 m/z mass range mode, positive ion mode) and MS-MS analysis(30 eV for fragmentation). Agilent MassHunter software wasused to analyse the peptide mass values. All the data fromMS were submitted to the online peptide database (http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=PMF) to identify the peptides from the digestedmucoid material.

RNA isolation and purification

Total RNA from bacteria in different growth phases wasextracted using TRIZOL Reagent (Life technologies, Carls-bad, CA, USA) following the manufacturer’s instructions andRNA integrity was determined using an Agilent Bioanalyser2100 and the resulting RIN number (Agilent technologies,Santa Clara, CA, USA). Quantified total RNA was furtherpurified using an RNeasy micro kit (QIAGEN, GmBH,Germany) and RNase-Free DNase Set (QIAGEN, GmBH,Germany).

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cDNA synthesis, labelling, and microarray hybridization

Purified total RNA was amplified and labelled using the LowRNA Input Linear Amplification kit (Agilent technologies,Santa Clara, CA, USA), 5-(3-aminoallyl)-UTP (Ambion,Austin, TX, USA), Cy3 NHS ester (GE healthcare Bio-sciences, Pittsburgh, PA, USA), following the manufacturer’sinstructions. Labelled cDNA were purified by RNeasy mini kit(QIAGEN, GmBH, Germany). A designed 8∗15K DNA Micro-array (Design ID 030218, Agilent), with 8797 probes thatcovered all the genes of 13 published Salmonella genome,was applied to microarray hybridization. During the hybridi-zation, each slide was hybridized with 1.65 μg of Cy3-labelledcRNA using Gene Expression Hybridization Kit (Agilenttechnologies, Santa Clara, CA, USA) in Hybridization Oven(Agilent technologies, Santa Clara, CA, USA), according tothe manufacturer’s instructions. After 17 h hybridization,slides were washed in staining dishes (Thermo Shandon,Waltham, MA, USA) with Gene Expression Wash Buffer Kit(Agilent technologies, Santa Clara, CA, USA), Stabilizationand Drying Solution (Agilent technologies, Santa Clara, CA,USA), following the manufacturer’s instructions.

Data acquisition and analysis of the microarray

After hybridization, microarray slides were scanned using anAgilent Microarray Scanner (Agilent technologies, SantaClara, CA, USA) and Feature Extraction software 10.7 (Agilenttechnologies, Santa Clara, CA, USA) with default settings:scan resolution, 5 μm; PMT, 100%/10%. For data analysis,signal intensities from each array were normalized using theQuantile algorithm in Gene Spring Software 11.0 (Agilenttechnologies, Santa Clara, CA, USA). Changes in geneexpression were expressed as signal log ratios (base 2), andanalysis of variance (ANOVA) was used to compare geneexpression values in each mutant strain, using wild-type cellsas the baseline for comparison. Experiments for array analysiswere performed based on biological replicates with threeindependent experiments for each strain. Transcripts werefiltered based on a P value, obtained from t-test by R-software(http://www.r-project.org/), threshold of 0.05 as well as a fold-change threshold of 2. Differentially regulated genes wereannotated and categorized according to general function bythe NCBI Clusters of Orthologous Groups (COG) database. Inaddition, Gene Cluster 3.0 (http://bonsai.hgc.jp/∼mdehoon/software/cluster/) and Java TreeView (http://jtreeview.sourceforge.net/) tools were used to generate the heat map.

Quantitative real-time reverse-transcription PCR(qRT-PCR)

For qRT-PCR, RNA isolation and purification were performedas described above. To synthesize cDNA, 3 μg of purifiedtotal RNA was amplified in a 20 μl reaction volume usingRevertAid™ H Minus Reverse Transcriptase and RandomHexamer Primer (Fermentas). cDNA (50 ng) was used astemplate for each qPCR experiment using the primers listedin Table S2. The 23S rRNA gene and the 16S rRNA genewere used as multiple internal references. The qPCR reac-tions were performed according to the protocol of the

Maxima® SYBR Green/ROX qPCR Master Mix, and theprogram was carried out in the Applied Biosystems 7500 FastReal-Time PCR System. The relative transcript copy numberfor each target gene was shown as the ratio between thesamples in different growth phases and the wild-type con-trols. It was calculated using the comparative Ct method(Pfaffl et al., 2002). For each gene, the transcript copynumber of wild-type strain in 8 h growth phase was assigneda value of 1.0. Accordingly, in the other sample and growthphases, values higher than 2 or lower than 0.5 suggested thatthe transcription of the target gene was significantly changed.All the quantitative real-time PCR assays were carried out intriplicate for each culture and then repeated three times withRNA isolated from independent cultures.

Membrane integrity test assay

The fluorescent reporter dye propidium iodide (PI) was used tomonitor the membrane permeability of the bacterial strains atdifferent growth phase. Three independent biological repli-cates of each strain were grown as described above and ∼ 107

cells were harvested at each different growth phases. Afterwashing twice with PBS, the cells were incubated in the dark atambient temperature for 20 min and then analysed by flowcytometry. The data were collected with the FSC threshold setat 40 000 on a Becton Dickinson Accuri C6 flow cytometer.Thirty thousand qualified events were collected for eachsample with their FL-2 (488→550 nm) values recorded.FlowJo software was then used to analyse fluorescencevalues. Data analysis of the fluorescence values of PI-stainedcells was averaged from three independent biologicalreplicates.

Detection of DNA strand breaks

Three independent biological replicates for each strain weregrown as described above, with ∼ 107 cells at different growthphases collected and washed twice with PBS. The cells werethen resuspended in 1 ml of 4% paraformaldehyde and incu-bated for 30 min on ice. The cells was subsequently collectedby centrifugation, washed twice with PBS, and resuspendedin 200 ml of ice-cold PBS. Then 1 ml ice-cold 70% ethanol(v/v, in H2O) was added to each sample and the sampleswere stored at −20°C overnight for permeabilization. The nextday, the cells were collected by centrifuge at 8000 g for 2 minand washed once with PBS. For the TUNEL assay, the Apo-Direct Kit (BD Bioscience) was used to detect the DNAstrand-breaks in each strain, according to the manufacturer’sinstructions. After staining, the samples were analysed usinga Becton Dickinson Accuri C6 flow cytometer. The data werecollected using a dual threshold of FSC 40 000 and FL-2 500,with both FL-1 channel (488 nm→510 nm) and FL-2 channel(488 nm→550 nm) recorded, and then analysed with theFlowJo software. A TUNEL-positive cell was defined as onewhose FITC fluorescence intensity exceeded that detected in99% of the wild-type cells at each growth phase.

Overexpression of the dptF–H operon

The dptF–H operon was derived from the plasmid pJTU3818,which contained both dptB–E and dptF–H gene clusters (Xu

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et al., 2010). Two strategies were used to clone the dptF–Hoperon: NsiI-digested dptF–H-containing fragment frompJTU3818 was cloned to the PstI (NsiI isocandamer) treatedpBluScript SK+ (Life Technologies, NY); or the dptB–E genecluster in pJTU3818 was removed by ScaI and PvuII partialdigestion, with dptF–H left alone in the plasmid. Both plasmidconstructs were then transformed into E. coli DH10b, E. coliBL21 or wild-type S. enterica by electroporation (Micropulser,Bio-Rad).

Acknowledgements

This work was supported by grants from the National ScienceFoundation of China (31170085 and 31070058); the Ministryof Science and Technology (973 and 863 Programs); Shang-hai Pujiang Program from the Shanghai Municipal Council ofScience and Technology (12PJD021); China ScholarshipCouncil; and the National Science Foundation of the UnitedStates (CHE-1019990).

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