characterization of the groesl operon inlisteria monocytogenes: … · encoding a homologue of the...

INFECTION AND IMMUNITY,0019-9567/01/$04.0010 DOI: 10.1128/IAI.69.6.3924–3932.2001

June 2001, p. 3924–3932 Vol. 69, No. 6

Copyright © 2001, American Society for Microbiology. All Rights Reserved.

Characterization of the groESL Operon in Listeria monocytogenes:Utilization of Two Reporter Systems (gfp and hly)

for Evaluating In Vivo ExpressionCORMAC G. M. GAHAN,* JAMES O’MAHONY, AND COLIN HILL

Department of Microbiology and National Food Biotechnology Centre,University College Cork, Cork, Ireland

Received 8 January 2001/Returned for modification 13 February 2001/Accepted 26 March 2001

The ability of intracellular pathogens to sense and adapt to the hostile environment of the host is animportant factor governing virulence. We have sequenced the operon encoding the major heat shock proteinsGroES and GroEL in the gram-positive food-borne pathogen Listeria monocytogenes. The operon has a con-served orientation in the order groES groEL. Upstream of groES and in the opposite orientation is a geneencoding a homologue of the Bacillus subtilis protein YdiL, while downstream of groEL is a gene encoding aputative bile hydrolase. We used both reverse transcriptase-PCR (RT-PCR) and transcriptional fusions to theUV-optimized Aequorea victoria green fluorescent protein (GFPUV) to analyze expression of groESL undervarious environmental stress conditions, including heat shock, ethanol stress, and acid shock, and duringinfection of J774 mouse macrophage cells. Strains harboring GFPUV transcriptional fusions to the promoterregion of groESL demonstrated a significant increase in fluorescence following heat shock that was detected byboth fluorimetry and fluorescence microscopy. Using both RT-PCR and GFP technology we detected expressionof groESL following internalization by J774 cells. Increased intracellular expression of dnaK was also deter-mined using RT-PCR. We have recently described a system which utilizes L. monocytogenes hemolysin as an invivo reporter of gene expression within the host cell phagosome (C. G. M. Gahan and C. Hill, Mol. Microbiol.36:498–507, 2000). In this study a strain was constructed in which hemolysin expression was placed under thecontrol of the groESL promoter. In this strain hemolysin expression during infection also confirms transcrip-tion from the groESL promoter during J774 and murine infection, albeit at lower levels than the knownvirulence factor plcA.

Listeria monocytogenes is a gram-positive food-borne patho-gen which poses a significant threat to the health of susceptibleindividuals (21). The ability of the pathogen to detect and reactto adverse environmental conditions is central to its capacity tocause disease. Listeria cells may encounter environmentalstresses such as heat, elevated osmolarity, and low pH whileresiding in foods, during survival of gastric passage and growthin the small intestine, and during intracellular pathogenesis(13). It has been demonstrated that an ability to adapt toenvironmental stress is essential for the realization of full vir-ulence potential in L. monocytogenes and in other intracellularpathogens, such as Salmonella enterica serovar Typhimurium(12, 23, 43, 48). In addition, for intracellular pathogens, per-turbations in host microenvironments, such as the low pH,oxidative stress, and low Mg21 of the host cell phagosome, arenecessary for triggering the synthesis of essential virulencefactors (16, 29). Environmental stimuli therefore function assignals allowing sequential induction and repression of appro-priate virulence factors during pathogenesis.

Following penetration of host cells, the production of list-eriolysin by L. monocytogenes causes lysis of the host cellphagosome, allowing escape of the bacterium into the host cellcytoplasm. It has been demonstrated that prior to lysis thephagosome becomes acidified and that this process is necessary

for optimal listeriolysin functionality (5). During this periodthe phagosome most likely represents a suboptimal environ-ment for the bacterium. Indeed, two-dimensional gel electro-phoresis of L. monocytogenes grown in cultured mammaliancells has revealed a pattern of protein synthesis that is distinctfrom that of cells grown under optimal conditions, suggestingbacterial adaptation to the host environment (25). Two-dimen-sional gel electrophoresis studies have also revealed a signifi-cant shift in protein synthesis in L. monocytogenes following anincrease in the acidity of the growth media to levels approxi-mating the pH of the host cell phagosome (39, 41, 42). Thedata indicate that Listeria cells are capable of reacting to theenvironment through upregulation in the synthesis of a num-ber of proteins. It is now evident that this process of bacterialadaptation is required for optimal virulence potential (14, 37,43).

Production of the heat shock proteins DnaK, DnaJ, GroES,and GroEL by bacteria is associated with exposure to environ-mental stress conditions. The molecular chaperonin proteinsGroES and GroEL are synthesized at elevated levels by bac-teria exposed to various environmental stressors. GroEL isamong the most highly conserved proteins in nature (46) andtogether with GroES functions to maintain protein integrityunder abusive environmental conditions (30). Studies using avariety of bacterial genera have also demonstrated elevatedsynthesis of GroEL following exposure to low pH, ethanol, salt,and bile salts, suggesting a role for this protein in the generalstress response (27, 32, 44). Indeed, evidence suggests a func-

* Corresponding author. Mailing address: Department of Microbi-ology, University College Cork, Cork, Ireland. Phone: 353-21-4902080.Fax: 353-21-4903101. E-mail: [email protected].

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tion for GroEL in bacterial growth at all temperatures, indi-cating a role in protein folding even under optimal growthconditions (17). Previous studies have utilized two-dimensionalgel electrophoresis to demonstrate the appearance of theGroEL protein following heat shock induction of L. monocy-togenes, and partial sequencing of the protein has been carriedout (25). However, despite studies with other organisms, thegroESL operon of L. monocytogenes has not previously beencharacterized.

Here we describe the sequence of groESL in L. monocyto-genes strain LO28. We also compare two reporter systemswhich utilize transcriptional fusions of genes to either gfpUV orhly in a single copy in situ in the listerial chromosome to allowin vivo monitoring of transcription from the groESL promoterregion. Both systems indicate increased expression of groESLduring infection of mice and within cultured mouse macro-phages.

MATERIALS AND METHODS

Bacterial strains and culture conditions. Strains and plasmids used in thisstudy are listed in Table 1. L. monocytogenes strain LO28 (serotype 1/2c) wasobtained from P. Cossart, Institut Pasteur, Paris, France. All Listeria strains werecultured in brain heart infusion (BHI) broth or tryptic soy broth-yeast extract(0.6%) (TSB-YE) with added antibiotics (Sigma Chemical Company, St. Louis,Mo.) when appropriate: erythromycin (5 mg/ml), kanamycin (50 mg/ml), or chlor-amphenicol (10 mg/ml). Escherichia coli strains were grown in Luria-Bertanibroth with 150 mg of erythromycin/ml added when appropriate.

Generation and screening of a Tn917 mutant bank. A Tn917 mutant bank wascreated using the temperature-sensitive plasmid pTV1-OK as described previ-ously (14). Isolated transformants were grown overnight in TSB-YE containingkanamycin at 30°C, subcultured into TSB-YE containing erythromycin (0.04mg/ml) at 42°C, and selected for kanamycin-sensitive Tn917 integrants on trypticsoy agar (TSA)-YE containing erythromycin. To isolate acid-sensitive Tn917mutants, 2,000 integrants were replica plated onto TSA-YE plates at pH 7 andTSA-YE plates adjusted to pH 5 with 3 M lactic acid. Two integrants demon-strated restricted growth on pH 5 plates, and one of them was selected for furtherstudy.

Sequence analysis of groESL. Inverse PCR and sequence analysis of theacid-sensitive Tn917 mutant of L. monocytogenes revealed insertion of the trans-poson downstream of a gene with homology to ydiL in B. subtilis (data notshown). Further sequencing of the inverse PCR product revealed the presence ofgroES and groEL genes. Another inverse PCR was carried out to generate a PCRproduct for further sequencing of groEL. Briefly, genomic DNA from wild-typeL. monocytogenes LO28 was digested with AvrII, and 5 ml was used in a ligation

mix with a total volume of 50 ml. PCR was carried out using the primers InEL-IN(59-GGAATCGCCTGCTCCTTCTAC-39) and InEL-OUT (59-CTCTACTCGCGCAGCTGTA-39) with Expand long-template Taq polymerase (Roche, Mann-heim, Germany) and appropriate conditions. The resulting 2.1-kb fragment wassequenced using a Beckman CEQ 2000 DNA analysis system. Confirmatorysequencing of the entire groESL operon was subsequently carried out. Homologysearches were performed against the GenBank database using the BLAST pro-gram.

Transcriptional analysis of groESL expression. L. monocytogenes LO28 wasgrown to an optical density at 600 nm (OD600) of 0.15 at 37°C. Cells were heatshocked at 45°C for various times. Total RNA was isolated using a hot acidphenol procedure (38). Briefly, 1-ml aliquots of culture were pelleted by centrif-ugation in an Eppendorf centrifuge model 5415C (12,000 rpm for 30 s) andimmediately frozen by immersion in a liquid N2 bath followed by storage at270°C. Pellets were thawed slowly on ice and resuspended in 500 ml of ice coldlysis buffer (20 mM sodium acetate [pH 5.2], 1 mM EDTA, 1% sodium dodecylsulfate). Cell suspensions were added to 500 ml of preheated (65°C) acid phenol-chloroform-isoamylalcohol (Sigma) and 200 mg of 425 to 600-mm glass beads(Sigma) and placed on a heating block (65°C) for 10 min with frequent vortexing.Suspensions were centrifuged for 10 min at 14,000 rpm, and the aqueous phasewas extracted with 500 ml of hot acid phenol-chloroform-isoamylalcohol andprecipitated in 2.5 volumes of ice cold ethanol for 1 h. Suspensions were pelletedby centrifugation in an Eppendorf centrifuge model 5415C (14,000 rpm for 15min), washed with 1 ml 70% ethanol, and resuspended in 100 ml of buffercontaining 10 mM MgCl2, 1 mM dithiothreitol, 10 mM Tris (pH 7), 1 mMEDTA, 5 U of DNAse I (Roche), and 5 U of RNasin (Roche). The RNA wasstored at 270°C until required for analysis.

For slot blot analysis, 1 mg of total RNA from each time point was denaturedin diethyl pyrocarbonate-treated water containing 1% dimethyl sulfoxide (Sigma)at 65°C for 15 min. The denatured RNA was then loaded onto a nylon membraneusing a vacuum slot blot manifold (Bio-Rad Laboratories, Hercules, Calif.) andfixed using a UV Stratalinker (Stratagene). The primers GSRA (59-GTAGAGCGGAACGTGTTAC-39) and GSF2 (59-GTAGTAGCCGTGAAAGC-39) wereused to generate a 400-bp fragment of groEL using standard PCR. This fragmentwas labeled using the Dig Hi Prime digoxigenin labeling kit (Roche). Ten nano-grams of labeled probe was used to hybridize with the immobilized RNA at 42°Covernight. The membrane was subsequently blocked, washed, and treated withalkaline phosphatase-conjugated antidigoxigenin antibody as instructed by themanufacturers (Roche). The membrane was subsequently exposed to KodakXR-Omat film, and signal intensities were compared using densitometry analysis(Phoretix, Newcastle upon Tyne, United Kingdom).

For reverse transcriptase PCR (RT-PCR) analysis cDNA synthesis was carriedout by adding 1 mg of total RNA to 4 ml of 53 RT buffer (Roche), 2 ml of 100mM dithiothreitol, 0.5 ml of a deoxynucleoside triphosphate mix (dATP, dCTP,dGTP, and dTTP; each 10 mM), 1 ml (40 U) of RNasin, 100 ng of the randomprimer p(dN)6, and 1 ml of Expand reverse transcriptase (Roche). The reactionmixture was incubated at 37°C for 2 h. PCR was carried out using the followingprimers: for groEL, GSRA and GSF2; for dnaK, DNAK-F (59-GCTGGTCTTG

TABLE 1. Bacterial strains and plasmids

Strain or plasmid Characteristics Reference or source

StrainsE. coli EC1000 Kmr, MC1000 derivative carrying a single copy of pWV01 repA in glgB 45L. monocytogenes LO28 Wild-type strain, serovar 1/2c P. CossartL. monocytogenes LO28Dhly Dhly, L. monocytogenes LO28 24L. monocytogenes gro-gfp Emr, gro-gfp transcriptional fusion in LO28 This studyL. monocytogenes plc-gfp Emr, plcA-gfp transcriptional fusion in LO28 This studyL. monocytogenes gro-hly Emr, gro-hly transcriptional fusion in LO28Dhly This studyL. monocytogenes plc-hly Emr, plcA-hly transcriptional fusion in LO28Dhly (previously designated

L. monocytogenes PLC1)24

L. monocytogenes pGFP-Int Emr Cmr, LO28 containing pGFP-Int and pVE6007 This studyL. monocytogenes CGNeg Emr, hly fusion strain, negative hemolysis under all conditions tested 24

Plasmids 45pORIX Emr, lacZ derivative of pORI13 (REF) with single BlnI site downstream of

multiple cloning site; Ori1 Rep224

pGFP-Int Emr, derivative of pORIX containing promoterless gfpuv downstream of MCS This studypCOR2 Emr, derivative of pORIX containing promoterless hly downstream of MCS 24pVE6007 Cmr, temperature sensitive derivative of pWV01 36pNF579 pKSV7 plasmid containing gfpuv N. E. Freitag

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AAGTAGAAC-39) and DNAK-R (59-GTTCATCAAATTTAGCACGAGT-39);for plcA, PLCA-X (59-TTCGGGGAAGTCCATGATTAG-39) and PLCA-Y (59-CACTACTCCCAGAACAGACACG-39); and for 16S RNA, 16sRNA-E (59-TTAGCTAGTTGGTAGGGT-39) and 16sRNA-B (59-AATCCGGACAACGCTTGC-39). PCRs were carried out for 16, 22, or 30 cycles to allow optimalquantitation of PCR products. cDNA was added to PCRs for groEL, plcA, ordnaK at levels which gave similar band intensities for 16S RNA (control) reac-tions.

Construction of gfpuv transcriptional gene fusion strains. The RepA2 pORIsystem for generating single plasmid insertions into the chromosome has beendescribed previously (34, 36). We have created a pORIX derivative which con-tains a single BlnI cut site downstream of a multiple cloning site (24). In order tocreate a plasmid with UV-optimized green fluorescent protein (GFPUV) as areporter, we amplified gfpuv from plasmid pNF579 (a gift from N. E. Freitag,Detroit, Mich.) using primers CGFP-1 (59-CCCCTAGGAGGAGGAAAAATATGAGTAAAGGAGAAGAAC-39) and CGFP-2 (59-GCCCTAGGTTATTTGTAGAGCTCATCCATG-39) designed to contain BlnI cut sites (underlined).High-fidelity Vent DNA polymerase (New England Biolabs, Beverly, Mass.) wasused for PCRs. The resulting fragment was digested with BlnI and cloned intosimilarly digested pORIX in E. coli EC1000 (45) to create the plasmid pGFP-Int.The orientation of the gfpuv ligation was checked using PCR. Plasmids containingpromoter regions upstream of gfpuv in pGFP-Int were created by amplifyingappropriate regions from L. monocytogenes LO28 genomic DNA and cloninginto the multiple cloning site using E. coli EC1000 as the host. Primers GRO1(59-CTTCTTATAGATCTCGTTATGAAGCTT-39), containing a BglII cut site(underlined), and GRO2 (59-CTTTGGCAGAGTCTAGAAATCCAATCC-39),containing an XbaI cut site, were used to amplify a region containing the putativepromoter region of groESL including the CIRCE (for “controlling invertedrepeat of chaperone expression”) element. Primers PLC1A (59-GGTTGGATCCGATAATCTAGACTATCG-39) (XbaI cut site) and PLC3 (59-TTCGCTTCTGCAGATGAAACGC-39) (PstI cut site) were used to amplify a region contain-ing the plcA promoter (24).

Plasmids were electroporated into L. monocytogenes LO28 containing theRepA1 helper plasmid pVE6007 (Cmr) using a standard protocol (40) andincubated at 30°C. Plasmid integration resulted following growth at 42°C inantibiotic-free BHI broth and plating onto prewarmed BHI plates containingerythromycin at 42°C. Individual colonies were replica plated onto BHI-chlor-amphenicol and BHI-erythromycin plates followed by incubation at 30°C todetermine loss of pVE6007 (Cms) and integration of pGFP-Int (Emr).

Quantitation of fluorescence. gfpUV fusion strains or controls were grownovernight in BHI broth containing 5 mg of erythromycin/ml at 30°C, and 500 mlwas used to inoculate 10 ml of antibiotic-free BHI broth at 30°C. For growth withfluorescence analysis, cells were immediately incubated at 43°C. For analysis ofvarious stresses, fresh inocula were grown at 30°C for 1 h before the addition ofethanol (4% [vol/vol] final concentration), hydrogen peroxide (0.1% [vol/vol]final concentration), or bile salts no. 3 (Oxoid) (0.08% [wt/vol] final concentra-tion, pH 6.8), reduction in pH to pH 5.0 (with HCl), or shift in temperature to43°C. Cells were subjected to various stresses (except heat stress) at 30°C. Atappropriate time points, the OD600 of cultures was measured, cells were resus-pended to an OD600 of 0.2, and 1 ml of cells was washed once in phosphate-buffered saline (PBS) and resuspended in 200 ml of PBS. Cultures were diluted1:2 in PBS, and 100 ml was used to measure fluorescence. Fluorescence of strainswas measured using a Wallac 1420 multilabel counter (Perkin Elmer Life Sci-ences, Wellesley, Mass.) fitted with a 395-nm excitation filter and a 535-nmemission filter. Specific fluorescence intensity is the raw fluorescence intensitydivided by the OD600. Relative fluorescence intensity is the specific fluorescenceintensity test value minus the intensity for the negative control (L. monocytogenesLO28, no plasmid) for each time point.

J774 infection with gfpUV fusion strains. J774 mouse macrophage cells weregrown on coverslips in antibiotic-free Dulbecco’s modified Eagle medium(DMEM) (Gibco Laboratories, Grand Island, N.Y.) in tissue culture petri dishes(Gibco). Monolayers were infected with appropriate gfpUV fusion strains or anegative control (L. monocytogenes containing pGFP-Int) at a multiplicity ofinfection of 100 CFU/cell. After 1 h, monolayers were washed once with DMEMand subsequently incubated with DMEM containing 15 mg of gentamicin/ml fora further 6 h. Cells were then washed three times with PBS and mounted onslides for microscopic analysis. Tandem samples were also assayed for bacterialnumbers by the addition of an infected coverslip to 10 ml of ice-cold steriledistilled water, serial dilution, and plating onto BHI agar plates. For microscopicanalysis, samples were fixed by the addition of a drop of 3.7% formaldehyde inPBS for 5 min at room temperature, washed with PBS, covered with Micromountmounting medium (Surgipath Medical Industries, Richmond, Ill.), and allowed

to set overnight. Slides were examined by confocal microscopy (Bio-Rad). Theparameters used for each confocal examination were identical.

RT-PCR from L. monocytogenes infecting J774 cells. J774 cells were grown inantibiotic-free DMEM in tissue culture petri dishes and were infected at amultiplicity of infection of approximately 100 bacteria per cell with L. monocy-togenes LO28. Infected cells were incubated for 30 min, and following this periodgentamicin was added to a final concentration of 15 mg/ml. After a furtherdefined incubation time, monolayers were washed twice in PBS, and internalizedbacteria were released by lysis of J774 cells with ice-cold sterile distilled water.Bacteria were pelleted by centrifugation, and RNA extraction was carried out asdetailed above. Control bacterial suspensions were grown overnight in BHIbroth, washed twice in PBS, and resuspended in antibiotic-free DMEM for 90min under the same conditions as J774 cells.

Creation of a hly-groESL promoter fusion strain. The pCOR2-based systemfor creating transcriptional fusions to hly has been described previously (24). Inthis study we cloned the putative promoter region from groESL, generated usingGRO1 and GRO2 primers, into pCOR2 and forced integration of the plasmidinto the chromosome of the hly mutant strain L. monocytogenes LO28Dhly. Theresulting strain is referred to as the L. monocytogenes gro-hly strain.

Both L. monocytogenes gro-hly and L. monocytogenes plc-hly (24) were analyzedfor virulence in a murine model of infection and compared to a hemolysin-negative fusion strain, L. monocytogenes CGNeg (24). Eight- to twelve-week-oldfemale BALB/c mice were inoculated intraperitoneally with 2 3 108 CFU of theappropriate inoculum in 200 ml of PBS. Numbers of bacteria surviving in mouselivers and spleens were determined 2 days postinfection (24).

Survival and growth of fusion strains was determined in J774 cells using amodification of a previously described procedure (22). J774 cells were grown in24-well tissue culture plates (Gibco) in antibiotic-free DMEM. Washed bacterialsuspensions were added to individual wells at a multiplicity of infection of 5bacteria per cultured cell, and plates were centrifuged at 150 3 g for 10 min toincrease contact between bacteria and macrophages. Plates were incubated for1 h to allow uptake of Listeria cells, and growth medium was removed andreplaced with DMEM containing gentamicin (15 mg/ml). Plates were incubatedfor 30 min, and at this stage (T0) and at various intervals, wells were washed twicewith PBS and monolayers were lysed with ice-cold sterile distilled water. Bacte-rial counts were determined by serial dilution and plating onto BHI agar plates.

Nucleotide sequence accession number. The nucleotide sequence data re-ported in this study have been submitted to GenBank and assigned accessionnumber AF335323.

RESULTS

Sequence analysis of groESL in L. monocytogenes. We ini-tially located the groESL operon through genetic analysis of anacid-sensitive transposon mutant of L. monocytogenes LO28.This mutant was one of two mutants, isolated from a bank of2,000 tested, which demonstrated slow growth at pH 5 but notat a neutral pH (data not shown). Initial sequencing revealedthe presence of the transposon in a region downstream of agene which is a homologue of the Bacillus subtilis ydiL gene.The transposon does not disrupt ydiL in L. monocytogenes,and further genetic analysis of this mutant is ongoing to de-termine the exact effect of the mutation. However, sincegroESL is upstream of ydiL in B. subtilis, we continued se-quencing this region in L. monocytogenes. Further analysisdetermined that, as in B. subtilis, the L. monocytogenes groESLoperon lies upstream of ydiL.

Analysis of this region revealed two open reading framesencoding proteins with significant homologies to GroES andGroEL in gram-positive bacteria. Protein homology searchesshowed that the first open reading frame encoded a proteinthat is 77% identical to the GroES protein of B. subtilis and71% identical to the GroES protein of Bacillus stearother-mophilus. The downstream region encodes a protein with highidentity (86%) with GroEL in B. subtilis and significant iden-tities with GroEL (Hsp60) in a number of bacterial species.Further downstream in L. monocytogenes is an open reading

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frame which differs from the corresponding region in B. subtilisand encodes a putative protein with significant identity (52%over 105 amino acids) with bile salt hydrolase in Lactobacillusplantarum (Fig. 1).

Downstream of the putative promoter region of groESL inL. monocytogenes is a conserved CIRCE element. These reg-ulatory elements are highly conserved in heat shock genes(dnaK and groESL) of gram-positive bacteria (28, 46). InL. monocytogenes, as in all other gram-positive bacteria, thetwo inverted repeats are separated by a 9-bp spacer region(28). As in B. subtilis, Lactobacillus helveticus, and Lactobacil-lus zeae, the putative initiation codon is UUG rather thanAUG, and this may serve to limit expression of the gene at thelevel of translation (47). Between the end of the groEL geneand the start of the putative bile hydrolase is a likely rho-independent transcriptional terminator (DG 5 213.4 kcal/mol).

Heat shock and transcriptional analysis of groESL inL. monocytogenes. Initial experiments showed that L. monocy-togenes cells subjected to heat shock at 45°C for 30 min developincreased heat resistance (to 55°C) relative to nonadaptedcontrols (data not shown). This demonstrates the presence inL. monocytogenes of an adaptive heat shock response which inother organisms involves the induction of GroES and GroEL.To examine transcriptional induction of groEL, we isolatedtotal RNA from bacterial cultures subjected to a shift in tem-perature from 37 to 45°C. Cells in this study were demonstrablymore resistant to lethal heat treatment than nonshocked con-trols (data not shown). Total RNA concentrations examinedfor each sample were identical based upon gel visualizationand UV spectrophotometric (Genequant) analysis. RNA wasanalyzed for specific groEL mRNA using either RNA slotblotting or RT-PCR (Fig. 2). The results indicate the presenceof transcript at low levels at 37°C prior to heat shock. However,5 min following heat shock there is a clear increase in tran-scription of groEL, reaching high levels after 15 min. Densi-

tometry analysis of slot blots suggested a 4.6-fold increase intranscription 10 min following heat shock, with a 4.9-fold in-crease after 15 min relative to control (nonshocked) cells.Finally, RT-PCR using a forward primer on groES and a re-verse primer on groEL produced a product of the predictedsize, indicating that both groES and groEL can be transcribedas a single mRNA (data not shown).

Design of the gfpUV-based integrating plasmid pGFP-Int.We have previously described a system to create single plasmidinsertions into the chromosome of L. monocytogenes to allowmonitoring of gene expression using hemolysin (24). The sys-tem was originally described for Lactococcus lactis and utilizesa RepA2 integrating plasmid in which the desired promoterelement is cloned upstream of a promoterless reporter gene.The RepA2 plasmid can then be introduced into a host strainharboring a temperature-sensitive RepA1 helper plasmid(pVE6007) (34). Growth of the strain at the permissive tem-perature (30°C) allows stable replication of both plasmids dueto in trans complementation from helper to pORI. However,an increase in growth temperature results in curing of thehelper plasmid, leading to the loss of the RepA1 phenotypeand forced integration of the RepA2 plasmid at a site ofhomology provided by cloned host DNA (Fig. 3A).

We have created a RepA2 plasmid (pGFP-Int) based on thelactococcal plasmid pORI13, which contains a promoterlesscopy of gfpuv (15) downstream of the multiple cloning site (seeMaterials and Methods). Cloning the promoter regions of ei-ther groESL (Pgro) or plcA (Pplc) into pGFP-Int permits thecreation of single-copy chromosomal fusions to gfpuv in theL. monocytogenes chromosome (Fig. 3A).

In order to determine the efficacy of the system we measuredthe specific fluorescence intensity of L. monocytogenes gro-gfpduring growth at an elevated temperature (43°C). Specificfluorescence intensity represents raw fluorescence data nor-malised for cell density (OD600). Growth at this temperatureresulted in a fourfold increase in specific fluorescence intensityrelative to the wild type, indicative of expression of GFP fromPgro (Fig. 3B). The fluorescence intensity reached a peak be-tween 6 and 8 h of growth. No significant increase in fluores-cence was seen in L. monocytogenes gro-gfp grown at 30 or 37°Cfor 8 h (data not shown).

Previous studies of various bacteria have shown increasedexpression of GroEL following exposure to low pH, ethanol,and bile salts (27, 44). We used the gro-gfp strain to determinetranscription from Pgro under various stress conditions. Cells

FIG. 1. Molecular organization of groES and groEL in L. monocy-togenes LO28. CIRCE tandem repeat elements are boxed. The puta-tive start codon, based upon BLAST homology searches, is underlined.Possible promoter regions (210 and 235 sites) are indicated. ydiL wasidentified on the basis of homology with B. subtilis ydiL, a gene en-coding a putative transmembrane protein. The arrow indicates thedirection of the putative bile acid hydrolase gene based upon homol-ogy with a gene encoding a conjugated bile salt hydrolase (M96175) inL. plantarum.

FIG. 2. Analysis of the transcription of L. monocytogenes groELfollowing heat shock using slot blot analysis or RT-PCR. L. monocy-togenes RNA was isolated prior to heat shock (0 min) or at 5, 10, or 15min (p) following a shift from 37 to 45°C. Total bacterial RNA con-centrations were determined by Genequant analysis, and identicalconcentrations of RNA were analyzed for specific groEL RNA in eachcase. RT-PCR represents 16 cycles of PCR.



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were exposed to various stressors for 8 h, and fluorescenceintensity was determined using a fluorimeter (see Materialsand Methods). Data is presented as fluorescence intensity rel-ative to that of unlabeled wild-type cells subjected to eachstress. While ethanol, acid, and bile salts were capable of stim-ulating significant (P , 0.05) increases in fluorescence, thelevels of expression never reached those of heat-shocked cellsunder the conditions examined (Fig. 3C). Peroxide stress didnot result in significant induction of GFP under the conditionsused (Fig. 3C). The overall results are similar to those of arecent study utilizing gfpuv fusions to analyze dnaK expressionin E. coli (11).

groEL expression within J774 mouse macrophage cells. Wehave used a number of approaches to analyze transcriptionof groEL within the cultured mouse macrophage cell lineJ774. J774 cells were grown on coverslips and infected witheither L. monocytogenes gro-gfp, L. monocytogenes plc-gfp, orL. monocytogenes(pGFP-Int) (negative control). Cells weregrown for 6 h and then analyzed using confocal laser micros-copy for fluorescent bacterial cells. In random microscopicfields, fluorescent bacteria were clearly visualized within J774cells infected with gro-gfp and plc-gfp strains but not with thenegative control (Fig. 4). This is consistent with expression ofGFPUV from both Pgro and Pplc during infection. Similar num-bers of fluorescent bacteria were seen in macrophages infectedwith L. monocytogenes gro-gfp and L. monocytogenes plc-gfp.Fluorescent bacteria could be visualized in directly adjacentfields of J774 cells infected with gro-gfp and plc-gfp strains butnot with the negative control. J774 cells infected with unstimu-lated gfpUV fusion strains for 1 h were negative for fluorescentbacteria, suggesting that longer time periods are required forexpression of GFPUV (data not shown). Bacterial plate countsfrom duplicate coverslips demonstrated that all coverslips, in-cluding those with L. monocytogenes(pGFP-Int)-infected cells,contained similarly high numbers of bacteria (;106 CFU/cov-erslip).

A previous study used RT-PCR to determine virulence geneexpression by L. monocytogenes infecting cultured mammaliancells (8). Here we used RT-PCR to detect groEL mRNA pro-duced by wild-type L. monocytogenes during infection of J774cells. Total RNA was isolated from L. monocytogenes-infectedJ774 cells at 1 and 3 h postinfection. Total cDNA was subse-quently analyzed for groEL, dnaK, plcA, and 16S RNA (con-trol). cDNA was added such that PCR products for 16S RNAwere of similar intensity for all samples after 16 cycles of PCR,indicating similar amounts of total bacterial RNA in each sam-ple (Fig. 5). This allowed the analysis of the same cDNA withdifferent primer pairs. The data indicate a clear and substantialincrease in expression of groEL during J774 infection. Simi-larly, there is a clear increase in expression of dnaK under

FIG. 3. (A) Site-directed integration of pGFP-Int into the chromo-some of L. monocytogenes LO28. The promoter region of groESL wascloned into the multiple cloning site of the RepA2 plasmid pGFP-Int(Emr). This construct was used to transform L. monocytogenes con-taining the temperature-sensitive, RepA1 plasmid pVE6007 (Cmr)and incubated at 30°C. Upon temperature upshift, the pVE6007 plas-mid no longer supports replication of pGFP-Int, which integrates intothe chromosome at the point of homology provided by the cloned

L. monocytogenes DNA. Integrants are Cms and Emr. (B) Specificfluorescence intensity of L. monocytogenes gro-gfp and L. monocyto-genes LO28 (wild type) during growth at 43°C. Data are representativeof duplicate experiments. (C) Fluorescence intensity of L. monocyto-genes gro-gfp exposed to acid (HCl, pH 5.0), bile salts (0.08% [wt/vol]),ethanol (4% [vol/vol]), heat (43°C), and hydrogen peroxide (0.1% [vol/vol]). Fluorescence data are relative to the negative control (L. mono-cytogenes LO28) and are the means plus standard deviations for trip-licate experiments.



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these conditions. There is also an increase in transcription ofthe virulence factor plcA during infection relative to the con-trol (T0). A repeat experiment demonstrated similar results.

Evidence for in vivo expression of groESL using a hemolysinfusion strain. We have previously described a system for iso-lation of in vivo-expressed promoters in L. monocytogeneswhich relies upon expression of the hemolysin gene (hly) fromrandom promoter elements (24). Here we describe the use ofthis system for the analysis of in vivo expression from a specificpromoter, Pgro.

Hemolysin is expressed optimally from its own promoterwithin the phagosome of infected cells and functions to allowphagosomal lysis and escape of the bacterium into the host cellcytoplasm where bacterial division can take place. In this study,we uncoupled hemolysin from its normal regulation by placinga promoterless copy of hly under the influence of Pgro in an hlymutant host strain (Fig. 6A). Hemolysin expression is low fromthis fusion strain as measured on blood agar plates at 37°C andis similar to fusions to previously described in vivo-inducedgenes (24). The L. monocytogenes gro-hly strain is capable ofreaching high numbers in the spleens of infected mice relativeto the L. monocytogenes CGNeg control strain (Fig. 6B). Thisis indicative of in vivo expression of hly from Pgro. However, inthe spleens of infected mice, the fusion strain does not reachthe same high levels as the L. monocytogenes plc-hly strain orthe wild-type L. monocytogenes LO28, which causes lethalitywhen administered at similar levels (24). This indicates thatwhile some expression from Pgro does take place in vivo, the lev-els of expression and/or the time course of expression is not suf-ficient for a full restoration of virulence potential. Data frominfected livers demonstrated identical trends (data not shown).

We have further analyzed growth of these fusion strainsduring infection of J774 cells (Fig. 6C). Analysis of cell num-bers at various time points during infection demonstrates thathemolysin-negative cells are incapable of growth in this cellline under the conditions used. The L. monocytogenes plc-hlyfusion strain grows rapidly in J774 cells at rates similar to thoseof wild-type Listeria cells (data for the wild type not shown).This is most likely due to rapid production of hemolysin fromthe PrfA-regulated plcA promoter, resulting in escape from thephagosome and growth of the strain in the cytoplasm. In con-trast, growth of the L. monocytogenes gro-hly strain occurs at a

later stage, suggesting a lag in expression of hemolysin fromthe groESL promoter in strains confined within the phagosome.

DISCUSSION

We have sequenced the groESL operon in L. monocytogenesLO28 and have analyzed expression of groEL under variousstress conditions, including infection of mouse macrophages.The groESL operon of L. monocytogenes demonstrates a con-served organization, with groES followed by groEL. The regionupstream of the operon contains a homologue of the B. subtilisgene ydiL. However, in contrast to the gene organization inB. subtilis, downstream of groEL in L. monocytogenes is a geneencoding a putative bile hydrolase (33). In addition, the reg-ulatory region of the listerial groESL contains a distinctiveCIRCE element, an inverted repeat which in other organismsacts as a negative cis element preventing excessive expressionunder normal growth conditions and facilitating heat shockinduction of the operon (46). Analysis of putative open readingframes suggests that the GroES and GroEL proteins demon-strate highest identity to B. subtilis proteins.

Previous studies have demonstrated that L. monocytogenesexhibits a typical heat shock response to mildly elevated growthtemperatures, which serves to protect cells against normally

FIG. 5. Analysis of the transcription of L. monocytogenes genesfollowing growth in J774 macrophage cells. J774 cells were infectedwith wild-type L. monocytogenes LO28 for 1 or 3 h (p), cells were lysedwith sterile distilled water, and total RNA was isolated from intracel-lular bacterial cells. Controls (T0) were exposed to DMEM for 90 minand washed in sterile distilled water prior to RNA isolation. TotalcDNA was used for PCRs (30 cycles) at levels which gave similarintensities for 16S RNA reactions (16 cycles).

FIG. 4. Fluorescence of L. monocytogenes cells incubated with J774 mouse macrophages. Monolayers were grown on glass coverslips andinfected with L. monocytogenes pGFP-Int for 6 h (A), with heat-shocked (43°C) L. monocytogenes gro-gfp for 1 h (B), with L. monocytogenes gro-gfpfor 6 h (C), or with L. monocytogenes plc-gfp for 6 h (D). Coverslips were prepared as described in Materials and Methods and examined by confocalmicroscopy.



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lethal temperatures (10, 18). This adaptive response involvesthe induction of both GroEL and DnaK heat shock proteins,which can be detected at 30 min after heat shock (45°C) (25).Here we show that mild heat shock (45°C) results in increasedtranscription of groEL as rapidly as 5 min following tempera-ture upshift and reaches high levels at 15 min following heatshock. This finding reflects a rapid response to the environ-mental insult and is similar to data obtained with other gram-positive organisms (4, 7).

In order to facilitate analysis of expression from the groESLpromoter and other promoter regions, we have developed asystem which utilizes the UV-optimized GFPUV from Aequoriavictoria as a marker of gene expression. The system employsthe stable integration of a promoter probe vector in single copyin the chromosome and thereby overcomes possible artifactsassociated with plasmid copy number, which may be encoun-tered especially under environmental stress conditions. A sig-nificant increase in fluorescence intensity was evident followinggrowth of the L. monocytogenes gro-gfp fusion strain at a hightemperature (43°C). Levels of fluorescence were similar tothose in a recent study of heat shock gene expression in E. coliusing a gfpuv fusion (11). The delay in fluorescent signal notedin the present study is due to the time necessary for chro-mophore formation and has been documented previously (3,11). However, the data indicate that the transcriptional fusionof the groESL promoter to gfpuv is a useful marker of geneexpression.

Previous studies have demonstrated increased expression ofgroEL under a range of environmental stress conditions. Stressconditions encountered during infection, such as low pH, bilesalts, and oxidative stress, have been shown to elicit groESLexpression in a variety of organisms (19, 27, 32). In addition,ethanol has been shown to efficiently induce groESL expres-sion (44). We have used the L. monocytogenes gro-gfp strain todemonstrate expression following exposure to low pH, bilesalts, and ethanol, suggesting that these environmental insultshave the potential to induce gene expression. However, asnoted in similar studies, heat shock remains the most potentinducer of groESL expression under the conditions analyzed(6, 19, 27). Given the acidity of the macrophage phagosomefollowing infection (5), it is notable that exposure of L. mono-cytogenes to pH 5.0 is capable of eliciting groESL expression.

In a number of other facultative intracellular pathogens,GroEL is clearly expressed following penetration of host cells(2, 9, 35, 49). However, examinations of heat shock gene ex-pression in S. enterica serovar Typhimurium suggest thatGroEL expression is increased during growth in J774 cells (9)but not in U937 cells (1). Two previous studies have failed todetect an increase in synthesis of GroEL by L. monocytogenesinfecting J774 cells (26) or peritoneal macrophages (31). Bothstudies utilized pulse-labeling of bacterial proteins followingmacrophage uptake and two-dimensional gel electrophoresisfor protein analysis. In contrast, we have adopted a geneticapproach to study the possible role of groESL in pathogenesis.Since GroEL may play a role in cellular physiology even underoptimal growth conditions (17), a deletion of groEL wouldmost likely have pleiotropic effects. Instead, we examined tran-scription of groEL during growth of L. monocytogenes in J774mouse macrophage cells using two approaches, RT-PCR andGFP technology, that have been used previously to study ex-pression of virulence genes during infection (8, 20). Both ap-proaches show a clear and significant increase in expression ofgroESL following uptake by macrophages. Infection of J774cells with the gro-gfp fusion strain results in induction of fluo-rescence and is comparable to the fluorescence of a plc-gfpcontrol strain as visualized using confocal laser microscopy.Using RT-PCR, a significant increase in bacterial groELmRNA is evident following infection of J774 cells. Increasedexpression of the bacterial virulence gene plcA is also apparent.

FIG. 6. Analysis of promoter-hly fusion strains for growth in J774cells and during murine infection. (A) Representation of the hly fusionto the groESL promoter region in L. monocytogenes gro-hly. (B) Anal-ysis of levels of L. monocytogenes strains in the spleens of infectedmice. Mice were infected intraperitoneally with L. monocytogenes gro-hly, L. monocytogenes plc-hly, or L. monocytogenes CGNeg, and bacte-rial numbers in the spleens were determined at 2 days postinfection.Data are means 6 standard deviations for four mice per group. (C)Growth of fusion strains in J774 cells. J774 cells were infected withL. monocytogenes gro-hly, L. monocytogenes plc-hly, or L. monocyto-genes CGNeg, and numbers of intracellular bacteria were determinedat specific time points postinfection. Data are means 6 standard de-viations for three wells per time point.



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Interestingly, the heat shock gene dnaK is also expressed atelevated levels postinfection. This may reflect a recent findingthat DnaK is required for efficient entry into macrophages(26).

While we provide clear evidence for increased transcriptionof groESL during macrophage infection, as mentioned previ-ously another study failed to detect an increase in GroELprotein in L. monocytogenes infecting the same mouse cell line(25). This may simply be a result of the differences in ap-proaches used. RT-PCR is a sensitive technique capable of de-tecting slight changes in levels of transcript, whereas largerincreases may be necessary for visualization on protein gels.Alternatively, the increased transcription of groESL seen inour study may not result in increased GroES or GroEL trans-lation. In L. monocytogenes, as in L. helveticus, L. zeae, andB. subtilis, the putative start codon for groES is UUG ratherthan AUG. In other bacterial genera it is likely that non-AUGinitiation codons function to limit translation (47). In addition,other mechanisms exist to limit protein expression at the trans-lational level and may play a role during infection. The signif-icant increase in transcription of groESL during infection maynot therefore result in a detectable increase in protein levels.This hypothesis will require further investigation.

Utilization of a recently described promoter probe vectorsystem (24) has provided further evidence for increased tran-scription of groESL during infection. We have constructed aL. monocytogenes strain in which hemolysin is expressed fromthe promoter region of groESL but not from its native pro-moter. During infection, hemolysin can therefore function as areporter of groESL expression. Indeed, the L. monocytogenesgro-hly fusion strain demonstrated increased survival potentialin mice relative to a negative control, indicative of expressionof hemolysin from the groESL promoter during infection.However, the strain did not reach the same high levels inmouse tissues as a positive control in which hemolysin is ex-pressed from the PrfA-dependent plcA promoter. Similarly,the gro-hly strain demonstrated a significantly increased growthpotential in J774 cells relative to the negative control but didnot reach the same levels as the plc-hly strain. However, thegro-hly fusion strain exhibited a lag period during which growthdid not take place, most likely due to delayed expression ofhemolysin from the groESL promoter in bacteria residing with-in the phagosome. It is envisaged that the application ofhemolysin as an in vivo reporter of expression from knownpromoter regions will provide a useful tool for analysis ofin vivo-induced loci.

Our results describe the gene sequence and organization ofthe groESL operon in L. monocytogenes. The data demonstrateexpression from the groESL promoter during infection butpossibly at lower levels than specialized virulence factors, suchas plcA, which are part of the PrfA regulon. The data suggestthat infection represents a significant stress for the bacteriumand results in expression of general stress proteins.

ACKNOWLEDGMENTS

We thank Nancy Freitag for providing plasmid pNF579 and BerniceRea for assistance with the confocal microscopy study.

C.G.M.G. is supported by a Health Research Board PostdoctoralResearch Fellowship and by BioResearch Ireland. This work has alsobeen funded by the Food Sub-Programme administered by the De-

partment of Agriculture, Food and Forestry, and is supported by na-tional and EU funds.

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