The PrfA protein of Listeria monocytogenes functions as a key regulatory factor for the coordinated expression of many virulence genes during bacterial infection of host cells . PrfA activityis controlled by multiple regulatory mechanisms, including anapparent requirement for either the presence of a cofactor orsome form of posttranslational modification that regulates theactivation of PrfA . In this study, we describe the identificationand characterization of a novel PrfA mutation that results inconstitutive activation of the PrfA protein . The PrfA L140Fmutation was found to confer high-level expression of PrfA-regulatedgenes and to be functionally dominant over the wild-type allele.The presence of the PrfA L140F mutation resulted in the aggregationof L . monocytogenes in broth culture and, unlike previouslydescribed prfA mutations, appeared to be slightly toxic to thebacteria . High-level PrfA-dependent gene expression showed noadditional increase in L . monocytogenes strains containing anadditional copy of prfA L140F despite a >4-fold increasein PrfA protein levels . In contrast, the introduction of multiplecopies of the wild-type prfA allele to L . monocytogenes resultedin a corresponding increase in PrfA-dependent gene expression,although overall expression levels remained far below thoseobserved for PrfA L140F strains . These results suggest a hierarchyof PrfA regulation, such that the relative levels of PrfA proteinpresent within the cell correlate with the levels of PrfA-dependentgene expression when the protein is not in its fully activatedstate; however, saturating levels of the protein are then quicklyreached when PrfA is converted to its active form . Regulationof the PrfA activation status must be an important facet ofL . monocytogenes survival, as mutations that result in constitutivePrfA activation may have deleterious consequences for bacterialphysiology.

 
Listeria monocytogenes is a ubiquitous gram-positive bacterial pathogen that can cause serious food-borne infections in immunocompromised individuals, pregnant women, and neonates [15, 18] . This facultativeintracellular pathogen is able to invade a wide variety of hostcells, gain entry to the cytosol, and subsequently replicateand spread to adjacent host cells [6, 7, 9, 10, 23, 24, 32, 34, 35, 46] . Several virulence factors important for the differentsteps of L . monocytogenes infection have been identified [reviewedin references 26, 41, and 48] . The majority of these virulencedeterminants are located within a 10-kb chromosomal region andare regulated by the positive transcriptional regulator PrfA.The prfA gene is also located within this cluster, which iscommonly referred to as the PrfA regulon [29, 30] . The prfA gene product is a key factor for L . monocytogenes pathogenesis, and bacterial strains lacking functional PrfA are essentially avirulent in mouse models of infection [13, 30, 31] . PrfA isa 27-kDa site-specific DNA-binding protein that recognizes a14-bp palindrome [PrfA box] within the –40 region of PrfA-dependentpromoters [1, 8, 13, 40].

Multiple mechanisms exist to regulate prfA expression and protein activity . Three promoters contribute to the transcriptional regulation of prfA . The promoters prfAp₁ and prfAp₂ are locatedimmediately upstream of the prfA coding region and are importantfor providing the initial levels of PrfA protein required forthe activation of gene products essential for bacterial escapefrom host cell vacuoles [12, 13] . These promoters are functionallyredundant in vivo and appear to contribute to both positiveand negative regulation of prfA expression [12, 13, 19] . Thethird promoter contributing to regulation of prfA is locatedupstream of the plcA gene and increases prfA expression viathe generation of a bicistronic plcA-prfA transcript; this promoteris PrfA dependent and represents a positive feedback loop forprfA expression [5, 13] . The 5' untranslated region of prfA mRNA has recently been shown to function as a thermosensor that regulates the translation of prfA mRNA in response to changes in temperature [22].

In addition to the regulation of prfA expression and mRNA translation, the activation state of PrfA appears to be regulated either through the binding of a cofactor or through some mode of posttranslational modification . The PrfA protein is a member of the Crp/Fnr family of transcriptional activators [25, 27, 41] and has significantstructural and functional homology to the Escherichia coli cyclicAMP [cAMP] receptor protein [Crp] [17, 39, 41] . Crp requiresthe binding of the cofactor cAMP for full activity, and cAMP-independent,constitutively active Crp* mutants have been described [14].A PrfA mutant analogous to Crp* has been identified in L . monocytogenes[37]; a glycine-to-serine substitution at position 145 in PrfAresulted in constitutively high expression of all PrfA-regulatedgenes [37] . The phenotype of the PrfA G145S mutant suggeststhat PrfA, like Crp, requires cofactor binding or modificationfor full activity [37] . Two additional mutations in PrfA leadingto activation of the protein have recently been identified [PrfAE77K and PrfA G155S]; both were found to result in increasedlevels of PrfA-dependent gene expression, but strains containingthe mutant alleles were phenotypically distinct, suggestingthat the mutations altered different aspects of PrfA function[43].

In this study, we describe a fourth PrfA mutation [PrfA L140F] that results in the constitutive activation of PrfA but is also functionally distinct from the three previously described constitutive PrfA mutations.

 
Bacterial strains and plasmids. The bacterial strains used in this study are listed in Table1 . E . coli DH5 was used as a host strain for recombinant plasmids.E . coli strain SM10 [44] was used as the donor strain for conjugation of pPL2 plasmid constructs . L . monocytogenes 10403S [serotype 1/2] is resistant to streptomycin and has a 50% lethal dosefor mice of 2 x 10⁴ CFU/ml [13]. L . monocytogenes wild-type[WT] strain 10403S containing a chromosomal actA-gus-plcB transcriptionalfusion [NF-L476] and NF-L758, a derivative of NF-L476 containingan erythromycin resistance gene [erm] inserted between orfZand orfB downstream of the PrfA regulon, have been described[43] . A 10403S L . monocytogenes strain containing a 339-bp in-frame deletion in prfA was generously provided by Hao Shen [University of Pennsylvania] and Jeff Miller [UCLA]; this strain was transduced with a phage U153 lysate [20] obtained from NF-L758, and theresulting erythromycin-resistant transductant was designated NF-L1003 . NF-L1003 therefore contains an in-frame deletion of prfA with a downstream copy of erm located between orfZ and orfB in the presence of a chromosomal actA-gus transcriptional fusion . L . monocytogenes strain NF-L879 was isolated as a mutant with high in vitro actA-gus expression following chemical mutagenesisof NF-L476 with ethylene methylsulfonate [EMS] [43] . The integrationvector pPL2 has been described [28] and was used for the constructionof the recombinant pPL2prfA constructs listed in Table 1 [describedbelow] . L . monocytogenes and E . coli were grown in brain heart infusion [BHI] broth [Difco Laboratories, Detroit, Mich.] andLuria broth [LB] [Invitrogen Corp., Grand Island, N.Y.], respectively.For selection of pPL2 or its recombinant plasmids, chloramphenicolat 25 µg/ml was used for E . coli; chloramphenicol at 7.5µg/ml was used for L . monocytogenes, and streptomycinat 200 µg/ml was used to select for L . monocytogenes followingconjugation . For solid media, 2.5% [wt/vol] agar was included.

 
Preparation of bacteriophage U153 lysates. High-titer U153 lysates were prepared as described previouslywith minor modifications [20] . Dilutions of a starting lysate were prepared in TM buffer [10 mM Tris-HCl, pH 7.5, 10 mM MgSO₄], and 100 µl of diluted lysate was added to sterile 13-by 100-mm glass tubes containing 100 µl of mid-log-phaseL . monocytogenes grown in LB with shaking at 30°C . Bacterialcultures were incubated with U153 lysate dilutions at room temperature[RT] for 40 min, and then 3 ml of molten LB plus 0.75% [wt/vol]agar-10 mM MgSO₄-10 mM CaCl₂ was added to each tube, followedby gentle mixing and immediate pouring of the tube contentsonto LB-10 mM MgSO₄-10 mM CaCl₂ plates, which were then incubatedat RT overnight . Plates containing U153 plaques just touchingone another [giving the lawn of L . monocytogenes a lacy appearance]were selected for the preparation of fresh bacteriophage lysates.TM buffer [2 ml] was added to the selected plates, and the plateswere incubated for 20 min at RT . The soft top agar layer wasthen broken up with a sterile glass spreader, and the agar andliquid were transferred into a sterile centrifuge tube and vortexed.The supernatant was recovered after centrifugation at 7,000 x g for 10 min, and chloroform was added to 10% [vol/vol] finalvolume . After the chloroform was mixed in and allowed to settle,bacteriophage lysates in the supernatant were transferred tosterile tubes and mixed with equal volumes of 100% glycerol.The lysates obtained were generally on the order of 10⁸ to 10⁹ PFU/ml.

U153 bacteriophage-mediated transduction. High-titer U153 lysates [50 to 200 µl] prepared from strainNF-L758 [NF-L476 with an erm insertion downstream of the PrfAregulon] were mixed with 200 µl of mid-exponential-phaseL . monocytogenes mutant strain NF-L879 [grown at 30°C withshaking] in the presence of 10 mM MgSO₄ and 10 mM CaCl₂, andthe mixture was incubated at RT for 1 h with occasional gentlemixing . An aliquot of 3 ml of warm BHISA [BHI plus 0.75% [wt/vol]agar-10 mM sodium citrate, pH 7.5] containing 50 µg of5-bromo-4-chloro-3-indolyl-ß-D-glucuronide salt [XG;Inalco]/ml was added with gentle mixing to the bacteriophage-L.monocytogenes suspension, erythromycin at 1.3 µg/ml wasincluded to induce erm expression, and the mixture was immediatelypoured onto BHI plates containing 10 mM sodium citrate [pH 7.5]and 50 µg of XG/ml . After incubation at 37°C for 2 h, another layer of BHISA containing 50 µg of XG/ml and13 µg of erythromycin/ml was poured onto the plates, whichwere again incubated at 37°C . Bacterial transductants wereusually visible within 1 to 2 days, and Erm^r transductants werescored for blue or white colony color . Mutants containing mutationsclosely linked to the erm gene produced white transductantson indicator plates at a frequency that correlated with thedistance of the mutation from the erm marker [distance = [1– the cube root of the transduction frequency] x phage size [40.8 kb for U153]] [20] . Transductants isolated afterphage infection of mutant NF-L879 were either dark blue or white.The dark-blue ones retained the mutation that conferred a high level of in vitro actA-gus expression, whereas the white coloniescontained the WT copy of prfA from NF-L758.

Construction of pPL2 recombinant plasmids. Genomic DNA was isolated from the L . monocytogenes WT strainNF-L476 and the EMS mutant strain NF-L879 for splicing by overlapextension PCR [21] to clone the WT or mutant prfA genes withthe prfA and plcA promoters and their PrfA-binding palindromesinto the integrative vector pPL2 . The primers 5'SacI-pPlcA [5'-GGGAATTCGGTATCAAATAAAACG-3'; the added SacI site is underlined] and 3'SalI prfA tx term [5'-AATCGCTTTCTTTACTGCAGGA-3';the added SalI site is underlined] [sequences of both primerswere provided by D . Higgins, Harvard Medical School, Boston,Mass.] were used as the external primer pair for splicing byoverlap extension PCR with the internal primer pairs delplcAR[5'-TACTTTGTTGTTTAATGCTGCATTAAAATAAATTGG-3'] and delplcAF [5'-CCAATTTATTTTAATGCAGCATTAAACAACAAAGTA-3'].The internal primers generated a 453-bp in-frame deletion inplcA . An 500-bp product was obtained from PCR with 5'SacI-pPlcAand delplcAR, and a 1.2-kb fragment was amplified with delplcAFand 3'SalI prfA tx term . The two overlapping products formedwere purified with a QIAquick gel extraction kit [Qiagen Inc.,Valencia, Calif.] and subjected to a second PCR containing onlythe two external primers . The final product [1.7 kb] was gelpurified, digested with SacI and SalI, and ligated to pPL2 cutwith the same enzymes for transformation into E . coli DH5 . Afterthe inserted fragments were checked by sequencing, each of the recombinant plasmids was electroporated into E . coli SM10 for conjugation into recipient L . monocytogenes strains.

Conjugation and plasmid integration. Conjugation of the pPL2 recombinant plasmids from E . coli SM10into recipient strains was performed as previously described[28] with slight modifications . Briefly, the bacterial strainswere grown at 30°C with shaking to mid-log phase [opticaldensity at 600 nm [OD₆₀₀], 0.6]. E . coli donor strains weregrown in LB containing 25 µg of chloramphenicol/ml, andL . monocytogenes recipient strains were grown in BHI . The donorculture [250 µl] was mixed with 150 µl of recipientculture, put onto a 0.45-µm-pore-size HA-type filter [Millipore,Billerica, Mass.] on a BHI plate without antibiotics, and incubatedat 30°C for 2 h . The bacterial cells were then resuspended in 2 ml of BHI, and aliquots [50 to 200 µl] were mixedwith 3 ml of LB top agar and overlaid onto BHI plates with 7.5µg of chloramphenicol/ml and 200 µg of streptomycin/ml.The plates were then incubated at 30°C overnight and shiftedto 37°C for 2 to 3 days . Individual colonies were screenedby PCR . The colonies were picked with sterile toothpicks, streakedonto a fresh selection plate, and then boiled at 100°C for10 min in 75 µl of sterile water . PCR was performed with5 µl of the boiled bacterial suspension . The primer pairNC16 [5'-GTCAAAACATACGCTCTTATC-3'] and PL95 [5'-ACATAATCAGTCCAAAGTAGATGC-3']amplified a 499-bp product in strains that are lysogenic orcontain the integration vector at tRNA^Arg-attBB' [28] . The primerpairs 5'SacI-pPlcA and 3'SalI prfA tx term amplified a single2.1-kb product from NF-L476 or when the vector alone [pPL2]was integrated into the chromosome; a single 1.8-kb fragmentwas amplified from NF-L1003 [prfA] and its pPL2 integrant . Anadditional band at 1.7-kb was obtained if a pPL2 recombinantplasmid carrying a prfA allele was integrated into the chromosome.Potential mutant insertion strains were confirmed by PCR usingisolated genomic DNA.

Determination of hemolytic activity. Overnight cultures of L . monocytogenes grown without shakingin BHI at 37°C were diluted 1:10 in fresh BHI broth andgrown with shaking at 37°C . The culture supernatants wereassayed for hemolytic activity with sheep erythrocytes as previouslydescribed [4] . Hemolytic activity was determined as the reciprocalof the supernatant dilution at which 50% lysis of erythrocyteswas observed and then normalized to the OD₅₉₅ of the bacterialcultures.

Measurement of GUS activity. Overnight cultures of L . monocytogenes grown without shakingin BHI at 37°C were diluted 1:20 into fresh BHI and grownwith shaking at 37°C . Chloramphenicol at 7.5 µg/mlwas included in the medium for L . monocytogenes strains integratedwith the pPL2 vector or a pPL2 recombinant plasmid . At varioustime points, the OD₅₉₅ was determined for each culture by usinga spectrophotometer [UV-1201 UV-VIS spectrophotometer; ShimadzuScientific Instruments, Inc., Columbia, Md.] . Bacterial pelletswere harvested from 1-ml culture suspensions by centrifugation,washed once with ABT buffer [1 M potassium phosphate [pH 7.0],0.1 M NaCl, 1% Triton], and resuspended in 100 µl or 1ml of the same buffer . ß-Glucuronidase [GUS] activity was measured as described by Youngman [51] with the substitutionof 4-methylumbelliferyl-ß-D-glucuronide [Sigma, St.Louis, Mo.] in place of 4-methylumbelliferyl-ß-D-galactoside.

Motility assays. Swimming motility was evaluated on semisolid [0.3% [wt/vol]agar] BHI medium treated with 0.2% [wt/vol] activated charcoalas previously described [43] . The plates were inoculated with2 µl of mid-log-phase [OD₆₀₀, 0.7] bacterial culturesgrown in BHI at 37°C; chloramphenicol at 7.5 µg/ml was included in the media for strains with the pPL2 vector ora pPL2 recombinant plasmid integrated into the chromosome . Theplates were incubated overnight at 37°C . Motility was quantifiedas the diameter of the swimming colony minus the diameter ofa nonmotile flaA L . monocytogenes deletion mutant [43] . Results were obtained from duplicate samples of two independent experiments for each strain.

Plaque formation in murine L2 fibroblasts. Plaque assays were performed with murine L2 fibroblasts as describedpreviously [45], with a multiplicity of infection of 1:3 . Plaquesize was measured using a comparator [Finescale, Orange, Calif.].Chloramphenicol at 5 µg/ml was included in the tissue culture media for L2 cells infected by L . monocytogenes strains integrated with the pPL2 vector or a pPL2 recombinant plasmid. The average diameter of 10 plaques from at least three independent experiments was determined for each strain.

PlcB activity assay. PlcB activities were assayed by streaking bacterial coloniesonto BHI medium overlaid with molten agar containing activated-charcoal-treated[0.2% [wt/vol]] BHI and a 5% [[vol/vol]] concentration of a1:1 egg yolk-phosphate-buffered saline [PBS] solution . The plateswere then incubated at 37°C or room temperature for 1 to2 days.

PrfA monoclonal antibodies. Monoclonal antibody MAB-prfA55 derived against recombinant PrfAprotein containing an N-terminal six-residue histidine tag wasgenerated by the Hope Heart Institute [Seattle, Wash.].

SDS-PAGE and Western immunoblotting. To isolate cell lysates, overnight cultures of L . monocytogenesstrains were diluted 1:20 in 30 ml of fresh BHI broth and grownto an OD₅₉₅ of 0.7 [chloramphenicol at 7.5 µg/ml was includedin the media for strains with the pPL2 vector or a pPL2 recombinantplasmid integrated into the chromosome] . Each culture was centrifugedat 5,000 x g for 10 min, washed in 1x PBS [Invitrogen, GrandIsland, N.Y.], and centrifuged again . Each pellet was resuspendedin 400 µl of lysis buffer [50 mM Tris-HCl, pH 7.5, 1 mMdithiothreitol, 0.1% [vol/vol] Triton X-100] . Bacterial suspensionswere mixed with 200 mg of glass beads [Sigma] and disruptedusing a Mini-Beadbeater [Biospec Products, Barttesville, Okla.]at maximum speed for 20 s . Samples were kept on ice and then centrifuged at top speed in a microcentrifuge for 1 min to recover the supernatants . Protein concentrations were determined using the DC Protein Assay kit [Bio-Rad Laboratories, Hercules, Calif.] following instructions provided by the manufacturer . Equivalent amounts of total protein from each culture lysate were mixedwith 4x sample buffer [0.25 mM Tris-HCl, pH 6.8, 8% [wt/vol]sodium dodecyl sulfate [SDS], 40% [vol/vol] glycerol, 4% [vol/vol] 2-mercaptoethanol, 0.04% bromophenol blue] . Samples were heatedto 100°C for 5 min prior to SDS- polyacrylamide gel electrophoresis [SDS-PAGE] on 15% polyacrylamide gels . The proteins were transferred to nitrocellulose membranes at 100 V for 1.5 h . Immunoblotingfor PrfA or PrfA derivatives was performed at RT . The membraneswere briefly washed with 1x PBS, blocked with 5% [wt/vol] nonfatdry milk in 1x PBS for 1 h, and then incubated overnight witha 1:100 dilution of the monoclonal antibody MAB-prfA55 in 1x PBS with 5% [wt/vol] nonfat dry milk and 0.05% [vol/vol] Tween 20 . After three 10-min washes in 1x PBS, the membranes wereincubated with a 1:2,000 dilution of an alkaline phosphatase-conjugatedgoat anti-mouse antibody [Sigma, St . Louis, Mo.] for 30 min.Following three 10-min PBS washes and equilibration of the membranesfor 5 min in detection buffer [100 mM Tris-HCl, 100 mM NaCl,50 mM MgCl₂, pH 9.5], PrfA proteins were detected by using SigmaFast 5-bromo-4-chloro-3-indolyl phosphate-nitroblue tetrazoliumtablets, a colorimetric alkaline phosphatase substrate . Forquantitative comparison of PrfA protein levels visualized by Western analysis, serial dilutions of protein extracts were compared to determine the relative amounts of PrfA producedby different strains.

Intracellular growth in macrophage-like J774 cells. To examine intracellular growth, cell monolayers were grownon circular coverslips [12-mm diameter] in tissue culture dishes.Bacterial cultures were grown overnight without shaking in BHIat 37°C; chloramphenicol at 7.5 µg/ml was includedin the media for strains with the pPL2 vector or a pPL2 recombinantplasmid integrated into the chromosome . The cultures were washedwith PBS and then used to infect J774 cells at a multiplicityof infection of 1:20 . After infection for 30 min, the monolayerswere washed three times with 37°C PBS prior to the additionof 5 ml of 37°C medium, and gentamicin was added to a finalconcentration of 10 µg/ml at 1 h postinfection to killany remaining extracellular bacteria . At various time points,the coverslips were removed to determine the number of intracellularbacteria . The cell monolayers on each coverslip were lysed byvortexing them for 10 s in 5 ml of sterile water, and dilutionsof the cell lysate were plated on LB plates . Bacterial CFU weredetermined for each strain in triplicate after overnight incubationat 37°C on LB plates.

 
Identification of a novel prfA mutation that results in constitutive activation of PrfA. Previous experiments using EMS chemical mutagenesis resultedin the isolation of 43 L . monocytogenes mutants that exhibitedhigh-level in vitro expression of gene products normally inducedwithin the cytosol of infected host cells [43] . These mutantswere identified by screening for isolates exhibiting increasedexpression of actA, a gene normally expressed at low levelsin bacteria grown outside of host cells, through the use ofa transcriptional actA-gus reporter gene fusion located withinthe bacterial chromosome . Mutants with increased GUS activitywere identified by the dark-blue color of the colonies on solidmedia containing the indicator substrate XG . One mutant, NF-L879,exhibited stable high-level in vitro actA expression based onthe dark-blue color of the colonies and was selected for furtherstudy.

To determine whether the mutation in NF-L879 was associatedwith a gene located within or near the prfA locus, the L . monocytogenes-transducing bacteriophage U153 [20] was used to transduce an erythromycinresistance [erm] gene marker linked to the PrfA regulon fromstrain NF-L758 to the NF-L879 mutant strain . NF-L758 is a derivativeof the WT strain NF-L476 [actA gus], which contains a copy oferm inserted downstream of orfXYZ at the 3' end of the PrfAregulon . If the mutation in NF-L879 was closely linked to thePrfA regulon, then transduction of NF-L879 with a phage lysatederived from NF-L758 would result in a population of white transductantswith WT levels of actA-gus expression on XG medium, as wellas a population of dark-blue transductants that retained theNF-L879 mutation . The frequency of white versus blue transductantswould reflect how closely linked the NF-L879 mutation was tothe erm marker . If the mutation in NF-L879 was not linked tothe PrfA regulon, then transduction of NF-L879 with the NF-L758-derivedphage lysate would result in only dark-blue transductants . Transductionof NF-L879 resulted in the isolation of white colonies on XGmedium at a frequency of 47% [31 white colonies identified froma total of 66 transductants], indicating that the mutation inNF-L879 conferring high-level in vitro actA expression was closelylinked [within 9 kb] to the PrfA regulon . DNA sequence analysisof NF-L879 prfA revealed the presence of a CTT-to-TTT mutationwithin the prfA coding region, which would result in the replacementof a leucine with a phenylalanine at amino acid 140 of the proteinsequence [PrfA L140F] [Fig . 1] . This mutation maps near thesite of another prfA mutation that has been described [37], PrfA G145S [or PrfA*], which is believed to lock the protein into a constitutively activated state [37, 49] . No other mutationswere found within the 9-kb region encompassing the prfA regulonof NF-L879.

 
Several attempts were made to confirm that the PrfA mutationwas responsible for the high actA expression phenotype via the introduction of the PrfA L140F mutation into the parent strain, NF-L476, by allelic replacement . These attempts proved unsuccessful, despite the fact that other PrfA mutations resulting in increased virulence gene expression [PrfA G145S, PrfA E77K, and PrfA G155S] have been successfully introduced into NF-L476 using this approach[43 and data not shown] . The NF-L879 mutant had no obvious growthdefect in comparison to the WT parent strain, NF-L476 [datanot shown], but interestingly, cultures of NF-L879 grown overnightin broth culture without shaking were repeatedly found to settleto the bottoms of the culture tubes rather than to remain insuspension [Fig . 2], suggesting that the mutant cells possessedaltered surface structure or cell density . A comparative Gramstain of WT and mutant bacteria taken from settled broth culturesrevealed no obvious changes in cell septation or cell shape[data not shown]; thus, the bacteria were apparently not settlingas a result of cell filamentation.

 
As an alternative approach to introduction of the PrfA L140Fmutation by allelic exchange, an L . monocytogenes site-specificphage integration vector, pPL2 [28], was used to introduce a single copy of a WT or mutant prfA allele into strain NF-L1003, which contains an in-frame deletion within prfA in addition to the actA-gus reporter gene fusion . Recombinant pPL2 plasmids integrated at the phage attachment site of the PSA prophage within the tRNA^Arg gene that is present only once in the genome of L . monocytogenes [16, 28] . To provide all promoters knownto contribute to prfA expression, coding sequences for WT PrfAor PrfA L140F were introduced in the presence of both of theprfAp₁ and prfAp₂ promoters, as well as the upstream plcA promoter.During the cloning process, another PrfA mutation [PrfA L147P][Fig . 1] was fortuitously generated, and this mutation was alsoindependently introduced for comparison into pPL2 for integrationinto the chromosome of NF-L1003 . The three integrants generatedwere NF-L1041 [containing an integrated copy of WT prfA [WTi]],NF-L1011 [containing an integrated copy of prfA L140F [L140Fi]],and NF-L1042 [containing an integrated copy of prfA L147P [L147Pi]][Table 1] . The L140Fi integrants were isolated at the same frequencyand with the same colony growth characteristics as prfA, L147Pi,and pPL2 integrants on agar plates [data not shown]; however,unshaken overnight cultures of NF-L1011 [prfA + L140Fi] settledto the bottoms of tubes in a manner identical to that of theoriginal EMS mutant strain, NF-L879 [Fig. 2] . In addition, incontrast to NF-L1041 [prfA + WTi] or NF-L1042 [prfA + L147Pi],the NF-L1011 [prfA + L140Fi] integrant was found to be unstablein the absence of drug selection when grown in BHI broth culture[data not shown], suggesting that the presence of the L140Fiallele may adversely affect some aspect of L . monocytogenesgrowth.

Analysis of PrfA protein levels in prfA strains containing integrated copies of WT or mutant prfA. To compare the levels of PrfA present in prfA integrant strains, total cell extracts derived from broth cultures grown to similar mid-log-phase densities were examined by Western analysis usinga mouse monoclonal antibody directed against PrfA . No PrfA wasdetected in the prfA parent strain NF-L1003 or in the vectorcontrol integrant NF-L1009 [prfA + pPL2i] [Fig . 3A] . The EMSmutant NF-L879 produced increased levels of PrfA protein incomparison to the WT strain NF-L476 [Fig . 3A] . Similarly, largeramounts of PrfA protein were detected in the integrant strainNF-L1011 [prfA + L140Fi] than in the control strain NF-L1041[prfA + WTi] [Fig . 3A] . Interestingly, no PrfA was detected from the PrfA L147P integrant NF-L1042 [Fig . 3A], even whena rabbit polyclonal antibody against PrfA was used for detection [data not shown] . The PrfA L147P mutation therefore appearedto result in an unstable, rapidly degraded form of the protein.For quantitative comparison of PrfA protein levels, serial dilutionsof protein extracts were compared [Fig . 3B and C] . The EMS mutant NF-L879 produced about two to four times more PrfA protein than the WT strain NF-L476 [Fig . 3B], and the integrant strain NF-L1011[prfA + L140Fi] produced at least four times more PrfA proteinthan the control strain NF-L1041 [prfA + WTi] [Fig . 3C] . Theseresults demonstrated that the introduction of the integratedprfA mutant and WT alleles into L . monocytogenes prfA strainsresulted in levels of PrfA protein that were comparable to WT and mutant protein levels produced by NF-L476 and NF-L879 strains.

 
In vitro expression of virulence genes by prfA integrant strains. PrfA is the major regulatory element controlling the transcriptionof many L . monocytogenes virulence genes [30, 31]; thus, theeffects of the PrfA mutations on the expression of several PrfA-dependentgenes were examined . Listeriolysin O [LLO], encoded by hly,is important for L . monocytogenes to escape from primary hostcell vacuoles and is normally expressed during bacterial growthin standard broth culture [3, 4, 11, 33] . LLO activity can bereadily detected in culture supernatants . In contrast to hly,actA is expressed at low-to-undetectable levels in broth-growncultures but is highly induced following entry of L . monocytogenesinto the host cell cytosol [2, 3, 11, 24, 33] . Following bacterial spread into adjacent host cells, the activity of the phospholipase PlcB is important for bacterial escape from double-membranevacuoles, and similar to actA, plcB is normally expressed at low-to-undetectable levels in bacteria grown in standard broth culture [38].

Relative amounts of secreted LLO were determined by measuring bacterial supernatants derived from mid-log-phase cultures for hemolytic activity against sheep red blood cells . Supernatants derived from the prfA strain NF-L1003, the vector control integrantNF-L1009 [prfA + pPL2i], and the PrfA L147P integrant strainNF-L1042 [prfA + L147Pi] exhibited very little hemolytic activity[Table 2] . The EMS mutant strain NF-L879 produced significantly larger amounts of LLO activity than the WT strain NF-L476 [Table 2], indicating that the PrfA L140F mutation is capable of elevatingthe expression of PrfA-dependent gene products distinct fromactA . The integrated WTi allele in NF-L1041 [prfA + WTi] complementedthe prfA deletion in the NF-L1003 background, and similarly,the integrated L140Fi allele conferred levels of hemolytic activityon NF-L1011 [prfA + L140Fi] that were comparable to those observedfor NF-L879 [Table 2].

 
The PrfA L140F mutation was originally identified based on its apparent ability to confer high levels of actA expression in bacteria grown outside of host cells . Quantitative analysisof levels of actA expression was performed for prfA WT and mutant strains by measuring levels of GUS activity in strains containing actA-gus transcriptional fusions . As mentioned previously, the PrfA L140F integration appeared to be unstable under nonselective conditions, and all integrant strains were therefore grown inthe presence of chloramphenicol . The inclusion of selective concentrations of chloramphenicol did not affect the growthrates of integrant strains compared to the rate observed forNF-L476, NF-L879, or NF-L1003 [prfA] grown in BHI alone [Fig.4A] . The prfA control strains NF-L1003 and NF-L1009 [prfA +pPL2i] had almost undetectable levels of GUS activity, and thesame was observed for the integrant NF-L1042 [prfA + L147Pi][Fig . 4B]; all of these strains did not produce detectable PrfAprotein as observed by Western analysis [Fig . 3A] . The integrationof a WTi copy into the prfA deletion strain NF-L1003 completely restored actA expression levels in NF-L1041 [prfA + WTi] tothose observed for the WT strain NF-L476 [Fig. 4B] . actA expressionof the original EMS mutant NF-L879 was observed to peak duringexponential growth, and levels of expression were up to 37-foldhigher than the expression levels observed for strains containinga single copy of the WT prfA allele [NF-L476 and NF-L1041] [Fig.4C] . The actA expression profile of integrant NF-L1011 [prfA + L140Fi] was very similar to that of NF-L879 [Fig . 4C], indicatingthat the L140F mutation in PrfA was responsible for the highin vitro actA induction in the original EMS mutant . NF-L1011[prfA + L140Fi] produced increased levels of PrfA protein in comparison to the original NF-L879 mutant strain [Fig . 3A]; the similar levels of actA expression observed for NF-L879 and NF-L1011, therefore, suggest that the mutant PrfA L140F reached a level of PrfA saturation beyond which there was no furtherincrease in the induction of actA expression despite increased production of PrfA.

 
The expression of plcB is coregulated with that of actA [42]. PlcB activity was not observed for the prfA control strainsNF-L1003 and NF-L1009 [prfA + pPL2i] or for the integrant NF-L1042[prfA + L147Pi] [Fig . 5] . No PlcB activity was observed for WT strain NF-L476 and the integrant NF-L1041 [prfA + WTi] [Fig.5] . In contrast, NF-L1011 [prfA + L140Fi] exhibited increasedlevels of PlcB activity similar to that observed for the originalEMS mutant NF-L879 [Fig. 5], even in media not treated withactivated charcoal [treatment of BHI broth with activated charcoalhas been reported to induce the expression of plcB [38]] [data not shown].

 
In summary, the introduction of a WT copy of prfA via vector integration at the tRNA^Arg-attBB' site was sufficient to fully complement PrfA-dependent gene expression in the prfA deletion strain NF-L1003 . The introduction of the PrfA L140F mutation demonstrated that this mutation was sufficient for generatingthe high-level in vitro actA-gus expression observed for theEMS mutant NF-L879; production of LLO and PlcB was also increasedin both strains . In contrast, the introduction of the PrfA L147Pmutation did not lead to the induction of PrfA-dependent geneexpression, indicating that this mutation results in an inactiveor unstable form of the protein.

The PrfA L140F mutation is phenotypically dominant to WT PrfA. The PrfA protein is thought to be active as a multimer basedon its similarity to E . coli Crp and on the recently solvedPrfA crystal structure [27, 49; http://www.rcsb.org/pdb/] . It seemed possible, therefore, that strains producing both thePrfA WT and PrfA L140F proteins might exhibit altered patternsof PrfA-dependent gene expression as a result of PrfA heterodimer formation . The PrfA L140F and PrfA L147P mutations, with thetwo prfA promoters and the plcA promoter, were therefore introduced into the chromosome of WT strain NF-L476 to examine the potential effects on PrfA-dependent gene expression . prfA merodiploid integrants NF-L1008 [WT + L140Fi] and NF-L1040 [WT + L147Pi] were generated, along with strain NF-L1006 with an integrated vector alone [WT + pPL2i] and a WTi merodiploid strain, NF-L1039 [WT + WTi], as controls . All strains exhibited apparently normal growth characteristics in broth cultures [data not shown] . Similar to the EMS mutant NF-L879 and the NF-L1011 [prfA + L140Fi] integrant,unshaken overnight cultures of NF-L1008 [WT + L140Fi] were observedto settle to the bottoms of culture tubes [Fig . 1].

Western analysis of PrfA protein levels in the vector control integrant NF-L1006 [WT + pPL2i] and the merodiploid NF-L1040 [WT + L147Pi] demonstrated levels of PrfA protein comparable to those of the WT strain NF-L476 [Fig . 6A] . This result suggestedthat mutant PrfA L147P protein was not produced in NF-L1040 [WT + L147Pi] and that the presence of the mutant allele did not affect the expression or stability of WT PrfA . Increased amounts of PrfA protein were detected in extracts derived from NF-L1008 [WT + L140Fi] [Fig . 6A], with protein levels at leastfourfold higher than in the vector control integrant NF-L1006[WT + pPL2i] [Fig . 6B] . Two copies of the WTi allele in NF-L1039[WT + WTi] also led to a >4-fold increase in PrfA synthesis[Fig . 6A and C].

 
The various prfA merodiploid strains were then examined for PrfA-dependent gene expression . The vector control integrantNF-L1006 [WT + pPL2i] had actA expression levels similar to those observed for WT strain NF-L476, as did the NF-L1040 [WT+ L147Pi] integrant [Fig . 7B] . Despite the high level of PrfA protein produced by NF-L1039 [WT + WTi] [Fig . 6A and C], thisstrain had only a twofold increase in actA expression in comparisonto WT strains at both 3 and 5 h of growth [Fig. 7B] . actA expressionlevels from the NF-L1008 [WT + L140Fi] integrant were the sameas those from the original EMS mutant NF-L879, indicating theapparent dominance of PrfA L140F over WT PrfA [Fig . 7C].

 
Similar results were obtained when the strains were examinedfor LLO activity . NF-L1006 [WT + pPL2i] and NF-L1040 [WT + L147Pi] produced levels of LLO that were the same as those producedby WT NF-L476, and NF-L1039 [WT + WTi] LLO activity was approximately twofold higher than that of strains with only one copy of WTi [Table 2] . Integrant NF-L1008 [WT + L140Fi] retained high hemolyticactivity, confirming the dominance of the PrfA L140F mutationover the WT [Table 2] . An increase in PlcB activity was observedonly for the NF-L1008 strain [WT + L140Fi] [Fig . 5] . If therewas increased PlcB activity in NF-L1039 [WT + WTi], it was toolow to be detected using this assay.

Construction of an L . monocytogenes strain containing two copies of L140Fi. To examine the potential effects on PrfA-dependent gene expressionthat would result from the presence of multiple copies of thePrfA L140F mutation, the L140Fi allele was introduced via integrationinto the EMS mutant NF-L879 to generate NF-L1067 [L140F + L140Fi].Unshaken overnight cultures of NF-L1067 were observed to settleto the bottoms of culture tubes [Fig . 1], although NF-L1067had no obvious growth defects in broth culture [data not shown]. Approximately two- to fourfold more PrfA protein was producedin NF-L1067 than in its EMS mutant parent strain, NF-L879 [Fig. 6D], but hly, actA, and plcB expression levels were not foundto differ significantly between the two strains [Table 2 andFig . 5 and 7D] . These data indicate that the induction of PrfA-dependent gene expression was already saturated with the amount of activated PrfA protein synthesized from one L140Fi allele, and therefore, the introduction of a second copy resulted in no additional increase.

Intracellular growth and cell-to-cell spread of prfA mutant strains in tissue culture cells. The capacity of L . monocytogenes to replicate within the cytosoland spread to adjacent cells can be assessed by visualizingzones of cell clearing [plaques] in monolayers of infected tissueculture cells [45] . Strains that are deficient in intracellulargrowth and/or cell-to-cell spread fail to form plaques or formplaques of reduced size . It has been demonstrated that strainslacking functional PrfA do not escape from host cell vacuolesand are unable to replicate within cells or spread to adjacentcells; these strains do not form plaques in monolayers of mouseL2 fibroblasts [13, 30] . As anticipated, strains NF-L1003 [prfA], NF-L1009 [prfA + pPL2i], and NF-L1042 [prfA + L147Pi] failedto form plaques in monolayers of L2 fibroblasts [Table 2] . TheEMS mutant NF-L879 formed plaques that were 79% the size ofthose formed by WT NF-L476 [Table 2] . In the presence of chloramphenicol,integrant strain NF-L1041 [prfA + WTi] formed plaques smallerthan those formed by NF-L476 [63.5% ± 6.4% [average ±standard deviation with NF-L476 set at 100%]]; however, in theabsence of the antibiotic, plaque formation was comparable tothat of the WT [data not shown] . Chloramphenicol therefore appearedto reduce the efficiency of plaque formation, despite the presenceof the cat gene in the pPL2 integrant strains . The inclusionof chloramphenicol was necessary to ensure the stability ofthe integrated L140Fi allele; therefore, the drug was includedfor all pPL2 integrants, and the plaque size of NF-L1041 [prfA + WTi] was set at 100% for comparison with mutant prfA integrantstrains . The integrant strain NF-L1011 [prfA + L140Fi] was stillfound to form reproducibly smaller plaques than strain NF-L1041[prfA + WTi]; however, the plaques were only slightly [10%] smaller than those formed in the presence of the WTi allele [Table 2] . L . monocytogenes strains containing the L140Fi allelemay therefore be slightly deficient in invasion, intracellularreplication, and/or cell-to-cell spread . No significant differenceswere observed in plaque size or frequency for any of the variousprfA integrant strains in the WT background or, interestingly,for NF-L1067 containing two copies of L140Fi [Table 2] . Experimentsexamining bacterial infection of the macrophage-like cell lineJ774 indicated that all integrant strains had similar intracellulargrowth for at least 7 h postinfection [data not shown].

L . monocytogenes strains containing L140Fi exhibit decreased swimming motility. Previous work has demonstrated that two strains of L . monocytogenesthat contained constitutively activated forms of PrfA protein[PrfA E77K and PrfA G155S] exhibited decreased swimming motilityin soft agar [43] . We therefore examined the swimming motilityof the PrfA L140F strains . The EMS mutant NF-L879 and integrantsNF-L1008 [WT + L140Fi], NF-L1011 [prfA + L140Fi], and NF-L1067[L140F + L140Fi] were all strikingly deficient in swimming motility,while the other integrants were similar in motility to WT strainNF-L476 [Table 2] . Strains containing constitutively activatedforms of PrfA [such as PrfA L140F] may thus be deficient innutrient acquisition and exhibit decreased fitness for survivalin environments outside of host cells.

 
The transcriptional regulator PrfA of L . monocytogenes tightly coordinates the expression of many virulence determinants to facilitate bacterial survival both inside and outside of hostcells . Regulation of prfA expression and PrfA activity occurson multiple levels and includes transcriptional, posttranscriptional, and posttranslational regulatory mechanisms, all of which are necessary for pathogenesis [5, 12, 13, 19, 22] . In this study,we have identified a novel mutation within PrfA [PrfA L140F] that results in a constitutively activated form of the protein and high-level virulence gene expression in bacteria grown outsideof host cells . The discovery of the PrfA L140F mutation bringsthe total number of PrfA-activating mutations defined to four,and each mutation appears to have its own distinguishable effecton PrfA function . Deciphering how specific structural alterationsin the PrfA protein alter distinct functional aspects of thiskey virulence regulator will further illuminate how L . monocytogenes controls virulence gene expression in response to environmentalcues.

In contrast to the three previously described mutations within PrfA that lead to the protein's constitutive activation [PrfAG145S, PrfA G155S, and PrfA E77K], the PrfA L140F mutation conferredsome degree of toxicity upon bacterial cells . Multiple attemptsto reconstruct the PrfA L140F mutation in WT L . monocytogenesby allelic replacement proved unsuccessful . In addition, whenthe mutant allele was integrated into an ectopic location onthe L . monocytogenes chromosome [as in strain NF-L1011 [prfA + L140Fi]], the L140Fi allele could not be stably maintained in the absence of selective pressure [unlike the WT allele, which was stable without selection] . The observation that unshaken overnight cultures of L . monocytogenes strains harboring the L140Fi allele settled to the bottoms of culture tubes suggests that the presence of PrfA L140F results in an altered bacterial surface structure or cell density [Fig . 1] . Altered bacterial cell physiology or morphology probably reflects changes in the spectrum of gene products normally produced by the bacteriaduring growth in broth culture; these products are likely tobe either directly or indirectly regulated by PrfA . Preliminaryanalysis of L . monocytogenes membranes and secreted proteinsof PrfA L140F strains using denaturing PAGE indicates readilydetectable changes in the abundance of several polypeptide species[K . K . Y . Wong, unpublished data] . The PrfA-directed alterationsin L . monocytogenes bacterial surfaces or cell physiology mayfunction to promote bacterial survival in the cytosol of infectedhost cells, an environment demonstrated to induce PrfA activation[42] . It is notable that, in contrast to the pPL2-prfA L140F reconstructed L . monocytogenes strains, the PrfA L140F mutation is stably maintained in the original NF-L879 mutant isolate;it is possible that this strain contains additional mutation[s]that alleviate the toxicity associated with this PrfA mutantallele . NF-L879, however, formed plaques in fibroblast monolayersthat were significantly smaller than those formed by WT strainsand smaller than those formed by the isogenic NF-L1011 [prfA + L140Fi] mutant [79% ± 3% for NF-L879 versus 90% ±2% for NF-L1011] . It is possible, therefore, that the mutationin NF-L879 that compensates for the presence of the L140Fi allele adversely affects the ability of L . monocytogenes to spreadto adjacent cells . Alternatively, NF-L879 may contain an independent mutation, unrelated to the presence of L140Fi, that reduces cell-to-cell spread . We do not believe that the L140Fi suppressor mutation is present in either NF-L1011 or NF-L1008, as these strains do not stably maintain the L140Fi allele in the absence of drug selection, and multiple L . monocytogenes isolates obtained from independent pPL2-prfA L140F vector transformations were indistinguishable from NF-L1011 and NF-L1008 in independent assays [K . K . Y . Wong, unpublished data] . Additionally, L . monocytogenes integrants containing the pPL2-prfA L140F plasmid were isolated at the same frequency and with the same colony growth characteristics on agar plates as integrants containing the pPL2-prfA or pPL2 vector, a result that suggests that the toxicity associated with the presence of L140Fi is not severe.

The relative levels of PrfA protein have been shown to be important for optimal induction of virulence gene expression [5, 12, 13,36] . PrfA synthesis increases upon entry of L . monocytogenesinto host cells [36], and this increase is necessary to direct the expression of gene products required for bacterial spread to adjacent cells [5, 12, 13] . The data presented here indicatethat saturating levels of virulence gene expression are morerapidly achieved by activated forms of PrfA . For example, theintroduction of a second WT copy of prfA into L . monocytogenesdoubled the production of LLO and ActA [Table 2 and Fig . 7].However, no significant increase in either actA or hly expression was observed for L . monocytogenes strains containing two L140Fi alleles [NF-L1067 [L140F + L140Fi]] versus those with one copy [NF-L879 and NF-L1011 [prfA + L140Fi]] [Table 2 and Fig . 4 and 7], even though the presence of a second copy increased thelevels of PrfA protein produced in these strains [Fig. 6] . Theseresults suggest that the expression of PrfA-dependent genesis most sensitive to variations in PrfA protein levels whenPrfA is present in its nonactivated state.

The apparent phenotypic dominance of the L140Fi allele over the WT allele is interesting in light of predictions that PrfAis active as a multimer . These predictions are based upon thehomology PrfA shows with Crp and upon the recently solved crystalstructure of PrfA [37, 49; http://www.rcsb.org/pdb/] . The merodiploidintegrant NF-L1008 [WT + L140Fi] presumably produces both WTPrfA and PrfA L140F, but this has not been definitely demonstrated.The PrfA L140F protein would be predicted to activate expressionnot only from its own upstream plcA promoter but also from thatof the WT copy, thus leading to high-level expression of bothproteins . It is possible that PrfA L140F has sufficiently highpromoter binding affinity to outcompete the WT protein at allpromoters examined; it has been reported that WT PrfA couldcompete and neutralize the activation of virulence genes byan activated PrfA form [PrfA G145S] if the WT PrfA was producedin trans from a multicopy plasmid [37] . We favor the possibilitythat PrfA L140F may induce conformational changes in the WTprotein upon multimerization that lead to activation of bothPrfA molecules . Alternatively, the PrfA L140F mutation may serveto stabilize the formation of active PrfA dimers.

As mentioned above, the crystal structure of PrfA has recently been solved and released [Protein Data Bank accession code 1OMI[http://www.rcsb.org/pdb/]] . The structure obtained indicatesthat PrfA is structurally similar to the cAMP receptor protein[Crp] from E . coli [50] . Figure 8 depicts the locations of thePrfA L140F and the PrfA L147P mutations within the WT structure.Also depicted is the original PrfA G145S mutation, the firstmutation identified that resulted in a constitutively activeform of PrfA [PrfA*] [37] . The PrfA L140F and L147P mutationsare located very close to the DNA-binding helix-turn-helix [HTH]domain of PrfA . The phenylalanine side chain of PrfA L140F,based on its location within the WT structure, might possiblyalter the positioning of the HTH domain to facilitate the interactionof this region with DNA; confirmation of this hypothesis awaitsfurther structural and functional analyses of PrfA L140F . Incontrast, proline substitutions often introduce dramatic turnsinto protein chains, and thus, the PrfA L147P substitution mightresult in improper folding and protein degradation, consistentwith the failure of this strain to produce detectable PrfA protein.

 
Finally, it has been proposed that L . monocytogenes is a bacterium that must mediate a balance between virulence for mammalian hosts and survival in environments outside of host cells [43]. L . monocytogenes strains containing the prfA E77K or prfA G155Sallele are fully virulent [or, in the case of PrfA G155S, hypervirulent]in murine models of infection; however, these mutants are severelycompromised in swimming motility, a behavior that is likelyto be important for nutrient acquisition outside of host cells [43] . Similarly, strains containing the PrfA L140F mutationwere found to be defective for swimming motility [Table 2].Elucidating how the activation status of PrfA contributes tothe survival of L . monocytogenes in its varied environmentswill aid our understanding of how L . monocytogenes is able toadapt and flourish in such a wide diversity of habitats.

 
We thank Darren Higgins for the pPL2 plasmid, the SM10/pPL2strain, and the sequences for primers 5'SacI-pPlcA and 3'SalIprfA tx term . We thank Hao Shen and Jeff Miller for the giftof the L . monocytogenes prfA deletion strain and Daniel Portnoyand Richard Calendar for the gift of the bacteriophage U153.We also thank members of the Freitag laboratory for helpfuldiscussions and the reviewers of the manuscript for insightfulcomments.

This work was supported by Public Health Service grant AI41816 [N.E.F.] from the National Institutes of Health and by the M.J . Murdock Charitable Trust.

 
* Corresponding author . Mailing address: Seattle Biomedical Research Institute, 307 Westlake Ave . N., Suite 500, Seattle, WA 98109-5219 . Phone: [206] 256-7345 . Fax: [206] 256-7229 . E-mail: nancy.freitag@sbri.org.

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