JOURNAL OF BACTERIOLOGY, Feb. 2004, p. 611–622 Vol. 186, No. 30021-9193/04/$08.00�0 DOI: 10.1128/JB.186.3.611–622.2004Copyright © 2004, American Society for Microbiology. All Rights Reserved.

In Vivo Expression of the Mannose-Resistant Fimbriae ofPhotorhabdus temperata K122 during Insect Infection

L. M. Meslet-Cladiere,1 A. Pimenta,1,2 E. Duchaud,3 I. B. Holland,1 and M. A. Blight1*Institut de Genetique et Microbiologie, CNRS UMR 8621, Laboratoire de Pathogenese Comparee, Universite

Paris XI, 91405 Orsay Cedex,1 Laboratoire ERRMECe, Group d’Interactions Cellulaires, Universite deCergy-Pontoise, 95302 Cergy-Pontoise Cedex,2 and Atelier de BioInformatique (ABI),

75252 Paris Cedex 05,3 France

Received 12 August 2003/Accepted 24 October 2003

Photorhabdus temperata K122 is an entomopathogenic bacterium symbiotically associated with nematodes ofthe family Heterorhabditidae. Surface fimbriae are important for the colonization of many pathogenic bacteria,and here we report the nucleotide sequence and analysis of the expression of a 12-kbp fragment encoding themannose-resistant fimbriae of P. temperata (mrf). The mrf gene cluster contains 11 genes with an organizationsimilar to that of the mrp locus from Proteus mirabilis. mrfI (encoding a putative recombinase) and mrfA(encoding pilin), the first gene in an apparent operon of nine other genes, are expressed from divergentpromoters. The mrfI-mrfA intergenic region contains inverted repeats flanking the mrfA promoter. This regionwas shown to be capable of inversion, consistent with an ON/OFF regulation of the operon. In in vitro liquidcultures, both orientations were detected. Nevertheless, when we analyzed the expression of all of the genes inthe mrf locus by semiquantitative reverse transcription-PCR during infection of Galleria mellonella (greater waxmoth) larvae, expression of mrfA was not detected until 25 h postinfection, preceding the death of the larvaeat 32 h. In contrast, mrfJ (a putative inhibitor of flagellar synthesis) was expressed throughout infection.Expression of mrfI was also detected only late in infection (25 to 30 h), indicating a possible increase ininversion frequency at this stage. In both in vitro liquid cultures and in vivo larval infections, the distal genesof the operon were expressed at substantially lower levels than mrfA. These results indicate the complexregulation of the mrf cluster during infection.

Photorhabdus luminescens K122 is a gram-negative insectpathogen belonging to the gamma subdivision of the Enter-obacteriaceae (10, 16, 38, 39). In a recent reclassification, P.luminescens K122 was renamed Photorhabdus temperata (19).Despite the fact that it is a pathogen to insects, P. temperataalso forms a natural symbiotic association in the intestines ofnematodes of the family Heterorhabditidae (20). P. temperatapresents numerous advantages as a model system for the studyof both symbiosis and virulence in the same organism, since thebacteria and the nematode together constitute a highly efficientpathogenic interaction with a broad host range of insects.

For initiation of the pathogenic process, the nematode trans-ports the bacteria into the interior of insect larvae, where theyare released into the hemolymph. By releasing several toxins(6, 12, 18), the bacteria rapidly kill the larvae and multiply tolarge numbers (14). This virulence phase is followed, as thebacteria reach stationary phase, by the secretion of many hy-drolytic enzymes (proteases [13, 15, 40], lipase [42], phospho-lipase, and DNase) and other enzymes, which degrade themacromolecular structures of the larva. This phase, which canbe called the bioconversion phase, is essential for the devel-opment and reproduction of the hermaphrodite nematode (11,15). In addition, during the bioconversion phase, the bacteriasecrete a number of antibiotics which ensure that the insect

cadaver remains monoxenic (2, 24). With the completion ofnematode development and reproduction, the bacteria andinfective juvenile nematodes reestablish a symbiotic interac-tion. Finally, the infective juvenile nematodes leave the ca-daver and eventually enter a new host, thus repeating the lifecycle.

From the bacterial perspective, this system appears to rep-resent a true symbiosis, since the bacteria have never beenisolated in nature independently of the nematode (20). Nev-ertheless, the organism can be cultivated in the laboratory in avariety of media, and when P. temperata is grown in rich me-dium, it is frequently possible to identify two phenotypic forms,termed variants I and II. Phenotypic variant I cells are the onlyform found in nature to date. Variant II cells are distinguish-able by colony morphology, the qualitative and quantitativeprofile of proteins secreted to the medium (33, 42), a substan-tial reduction in the secretion of antibiotics, and the loss ofbioluminescence, which is normally displayed by variant I cellsas they enter stationary phase (22). In addition, variant II cellshave lost the capacity to enter into symbiosis with nematodehosts (7) but nevertheless remain virulent and kill larvae withgreat efficiency when injected directly into the hemocoel (41).

Pathogenesis in animals and humans is usually associatedwith several phases. These include the important early phase ofattachment and colonization of surfaces through the action ofa wide variety of fimbriae or pili and a variety of adhesins onthe surfaces of the bacteria (36). The paradigms for fimbrialexpression and regulation are pyelonephritis-associated pili(type P pili encoded by pap) and type 1 fimbriae (encoded byfim) (for a review, see reference 8). These structures and their

* Corresponding author. Mailing address: Institut de Genetique etMicrobiologie, CNRS UMR 8621, Laboratoire de Pathogenese Com-paree, Batiment 360 et 409, Universite Paris XI, 91405 Orsay Cedex,France. Phone: (33 1) 69158168. Fax: (33 1) 69156334. E-mail: mark.blight@igmors.u-psud.fr.

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associated adhesin proteins permit the colonization of epithe-lial cells through attachment of bacteria to specific surfaceglycoproteins (3, 17, 28, 30). Another class of fimbriae, themannose-resistant fimbriae, have also been shown to be essen-tial for the virulence of several bacterial species. Mannose-resistant pili are structures 6 to 7 nm in diameter. They arecomposed of large numbers of a single pilin molecule of about17 kDa, and they carry specific adhesins and a few copies ofother proteins localized at their tips (36). The genes encodingthe different polypeptides of the mannose-resistant pilus areorganized in an operon; the best-studied example is present inProteus mirabilis, which promotes urogenital infections in hu-mans (4, 29, 44).

In this study, we have identified and characterized the com-position and expression of the mrf operon of P. temperataK122, encoding a mannose-resistant pilus, in both variant I andvariant II cells in in vitro liquid culture and during in vivoinfection of an insect host, Galleria mellonella (greater waxmoth).

MATERIALS AND METHODS

Bacterial strains. P. temperata K122 was obtained from D. Clarke (21). Esch-erichia coli DH5� [supE44 �lacU169 (�80lacZ�M15) hsdR17 recA1 endA1gyrA96 thi-1 relA1] was used as a host strain for all transformations (except for thelibrary construction) and as a negative control for all experiments.

The P. temperata K122 plasmid library was transformed into XL1-Blue MRF�Supercompetent cells (Stratagene).

Plasmids. Plasmid pPTF 100 contains the entire mannose-resistant fimbrialmrf operon. It was isolated from a genomic library of P. temperata K122 byhybridization against mrfA and mrfG genes.

Media and chemicals. All bacteria were grown in Luria broth (LB) at 29°C.When DH5�/pPTF 100 was grown, ampicillin was added to a final concentrationof 100 �g/ml. All enzymes were supplied by Promega, unless otherwise stated.

DNA probes were labeled with [�-32P]dCTP (Amersham) by using the Re-diprime II kit (Amersham). After labeling, they were purified on Microspincolumns of Sephacryl S-200 HR (Amersham).

Insect infection. An overnight culture of P. temperata K122 was diluted in LBto 5 � 105 cells � ml�1. Ten microliters of this suspension was injected into thehemolymph of G. mellonella 5th-instar larvae. Injected larvae were incubated at28°C, and at various times after infection, larvae were crushed, following freezingin liquid nitrogen, with a mortar and pestle. Larvae were obtained from G.Dumond, Earl La Teigne Doree, St. Germain les Vergnes, France.

Chromosomal DNA extraction from P. temperata. A 30-ml overnight culture ofP. temperata was centrifuged at 10,000 � g for 15 min. The cell pellet wasresuspended in 3 ml of lysis buffer (10 mM NaCl, 20 mM Tris-HCl [pH 8], 1 mMEDTA, 0.5% sodium dodecyl sulfate [SDS], 100 mg of proteinase K � ml�1), andthe mixture was incubated at 50°C overnight. The lysate was treated with 3 ml ofphenol-chloroform (21:1, vol/vol) and centrifuged as described above, and theDNA was precipitated from the aqueous phase in the presence of 0.3 M sodiumacetate and 2 volumes of ethanol.

Recombinant DNA techniques. Plasmid DNA was isolated by using minicol-umns (Macherey-Nagel) as specified by the manufacturer. Electroporation,transformation, and restriction endonuclease digestion were performed accord-ing to the methods of Sambrook et al. (35).

Genomic library of P. temperata K122. Genomic DNA was partially digestedwith Sau3AI and separated on a sucrose density gradient. Fragments isolated inthe range of 5 to 15 kbp were cloned into plasmid pUC18, which had previouslybeen linearized with BamHI, and dephosphorylated with bacterial alkaline phos-phatase (Pharmacia Amersham Biotech).

RNA preparation. (i) Bacterial RNA (E. coli and P. temperata). Total RNA wasprepared from bacterial cultures by using the SV Total RNA Isolation kit asspecified by the manufacturer (Promega). Each RNA sample was used as atemplate in a 35-cycle PCR as a control to ensure the absence of DNA contam-ination.

(ii) RNA from infected larvae. Total RNA was obtained from whole infectedlarvae (1 larva per time point) by using the TRI reagent (Sigma) and was treatedwith 5 U of DNase RQ1 (Promega). Then the RNA was precipitated in thepresence of 0.3 M sodium acetate and 2 volumes of ethanol. Fifty nanograms of

each total-RNA sample was used as a template in a 35-cycle PCR, as a controlto ensure the absence of DNA contamination.

Hemagglutination assays:. P. temperata variant I and variant II bacteria weregrown to either exponential or stationary phase in LB liquid culture with orwithout agitation or were resuspended in LB from colonies on LB agar plates.Cells were washed in 1� phosphate-buffered saline (PBS) and resuspended to afinal concentration of 106 bacteria per ml. Serial dilutions of resuspended cellswere made by factors of 2, and 50 �l was added to 50 �l of horse erythrocytes thathad been washed and resuspended in 144 mM NaCl in the wells of a microtiterplate. Mannose resistance was determined by using erythrocytes resuspended in144 mM NaCl–50 mM mannose. Plates were incubated for 30 min at 25°C, andhemagglutination was determined by the accumulation of precipitated erythro-cyte pellets at the bottoms of the wells.

DNA sequencing and analysis. DNA templates were sequenced by using theBig Dye Terminator RR Mix (Applied Biosystems). Reactions were run on anABI 373 DNA sequencer (Perkin-Elmer Applied Biosystems). Data banksearches and analysis were performed with BLASTX and BLAST software(http://www.ncbi.nlm.nih.gov/BLAST/).

PCR. DNA fragments spanning the invertible mrfA promoter element wereamplified by 30 cycles of PCR. The PCR was performed on a GeneAmp PCRsystem 2700 (Applied Biosystems) using Taq DNA polymerase. The oligonucle-otides used for amplification of mrfA were A5-5 (5� TCACCCCTGAAACAGTTG 3�) and A5-3 (5� CCTGAAGATAAGCAGCGA 3�), and those used foramplification of mrfG were mrfG-5 (5� CGTGTTGCGCGTTGGTC 3�) andmrfG-3 (5� CTACCCGCACTTAATGC 3�).

Primer extension. Primer extension experiments were performed using twooligonucleotides: Con2Bis (5� GAAGAAGAACGACCG 3�), to identify thetranscriptional start site of the operon upstream of mrfA, and Seq 2700 (5�CAGCATGACGACCTTGACG 3�), for the analysis of mrfI. Two micrograms ofRNA extracted from P. temperata variant I cells in the exponential-growth phasewas denatured at 100°C for 5 min in the presence of the corresponding oligo-nucleotide. The sample was cooled at 50°C for 5 min. A mixture containing theMoloney murine leukemia virus (M-MLV) buffer (5�), 1 U of RNasin (Pro-mega), 0.5 �l of 100 mM dGTP, 0.5 �l of 100 mM dTTP, 0.5 �l of 100 mM dATP,0.5 �l of [�-32P]dCTP, and 1 �l of M-MLV reverse transcriptase (Promega) wasprepared. The mixture was incubated for 10 min at 37°C. Following the additionof 0.5 �l of 100 mM dCTP, the sample was incubated at 37°C for 50 min andpurified on Microspin columns of Sephacryl S-200 HR (Amersham). The sam-ples were denatured at 100°C in the presence of 6 �l of stop buffer (Promega)and loaded onto a 6% polyacrylamide DNA sequencing gel (Ultrapure Sequa-gel-6; National Diagnostics). A DNA sequencing ladder provided the markersfor accurate determination of the transcript initiation site.

Western blotting. Cells (0.25 OD450 [optical density at 450 nm] unit) werecentrifuged and resuspended in PBS. Proteins were precipitated with 10% tri-chloroacetic acid for 30 min on ice, washed with 80% ice-cold acetone, denaturedin loading buffer containing -mercaptoethanol as described by Sambrook et al.(35), and then incubated at 100°C for 10 min. Samples were subjected to SDS-polyacrylamide gel electrophoresis on an 11% acrylamide gel (BDH LaboratorySupplies), and proteins were transferred to a nitrocellulose membrane (Trans-Blot; Bio-Rad Laboratories) in 25 mM Tris–192 mM glycine buffer. Westernblots were incubated for 1 h with a rabbit polyclonal antiserum against MrpA ofP. mirabilis (25) at a dilution of 1:20,000. Following three 15-min washes in anexcess of PBS, blots were incubated with a 1:10,000 dilution of the secondaryantibody, an alkaline phosphatase-conjugated goat anti-rabbit antibody (Pro-mega). Blots were developed with the ECL detection system (Amersham) ac-cording to the manufacturer’s instructions.

Northern blotting. Northern blotting was performed according to the methodof Sambrook et al. (35). Four to 30 �g of total RNA was denatured at 50°C for1 h in 0.5 M dimethyl sulfoxide–0.1 M phosphate buffer–144 mM glyoxal. De-natured RNA was separated on a 1% agarose gel in 10 mM phosphate buffer.Following migration, RNA was transferred in 20� SSC (1� SSC is 0.15 M NaClplus 0.015 M sodium citrate) to a nylon membrane (Hybond N�; Amersham).Probes were labeled with [�-32P]dCTP by using the Rediprime II kit and werepurified on Sephacryl S-200 HR Microspin columns (Amersham). Purifiedprobes were added to the prehybridization solution, and the mixture was incu-bated at 65°C overnight. Membranes were washed twice in 2� SSC–0.5% SDS at65°C, air dried, and exposed to X-ray film (Kodak).

Semiquantitative RT-PCR (Q-RT-PCR). Single-step reverse transcription-PCRs (RT-PCRs) were performed using the Lightcycler RNA amplification kitwith SYBR Green I (Roche Molecular Biochemicals). Reverse transcription wasperformed at 55°C for 10 min, followed by PCR (55 cycles of 0 s at 94°C, 5 s at55°C, and 20 s at 72°C). Single-point fluorescence acquisition from the incorpo-ration of SYBR Green into the double-stranded PCR product was measured at

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the end of each cycle. Melting analysis was performed at the end of each PCR todetermine the homogeneity of PCR products, and results were confirmed by themigration of PCR bands on 1.5% agarose gels. The efficiency (E) of each pair ofPCR primers for each of the mrf genes, and that for the control P. temperataK122 16S gene, was determined by PCR of serial dilutions of P. temperata K122genomic DNA (100 ng to 10 pg); E was consistently 1.93 0.05. The pair ofoligonucleotide primers for the 16S RNA was specific for P. temperata and wasobtained from R. ffrench-Constant (15). All semiquantitative RT-PCRs wereperformed with four replicate reactions.

Immunogold electron microscopy. P. temperata K122 variants I and II weregrown either on LB agar or in aerated or static liquid LB. For labeling experi-ments, bacterial suspensions were prepared in PBS. Immunogold labeling wasperformed as described by Zhao et al. (44). Diluted cultures were placed on anN Formvar grid, from which the excess liquid was removed after a 5-min incu-bation. The grid was air dried and blocked for 30 min in the presence of PBScontaining 1% bovine serum albumin (BSA). The grid was incubated with aprimary antiserum solution (1/100) against MrpA for 1 h (25) and then washedtwice for 5 min in PBS–1% BSA. Gold-labeled goat anti-rabbit immunoglobulinG (AuroProbe; Amersham Pharmacia Biotech) was used as a secondary antibodyfor 2 h. Grids were washed twice with PBS-BSA, and bacteria coupled withantisera were fixed with 3% glutaraldehyde for 5 min. Following a wash in sterilewater, grids were stained with 5% uranyl acetate for 10 s, and observations wereperformed by transmission electron microscopy using a Philips EM 208 at 80 kV.

Nucleotide sequence accession number. The nucleotide sequence of the P.temperata K122 mrf operon has been deposited in GenBank under accession no.AF396083.

RESULTS

Identification of a gene, mrfA, encoding a mannose-resistantpilus in P. temperata K122. In the course of an investigation ofdifferential gene expression in P. temperata K122 variant I andII cells, using RNA fingerprinting by arbitrarily primed PCR(43) and BLASTX and BLASTN analysis, we identified, forthe first time in this organism, a gene fragment with 90%similarity to mrpA, which encodes a mannose-resistant pilin inP. mirabilis. Although the P. temperata gene, which we desig-nated mrfA, appeared to be expressed in both variant I andvariant II cells in liquid culture, we considered it worthy offurther investigation, in anticipation that it may play an impor-

tant role in pathogenicity toward insects. In addition, in theseexperiments we also identified a fragment of a second genewith strong homology to another gene in the P. mirabilis mrpoperon, mrpG, encoding a minor component of the pilus.

Screening of a P. temperata library for the intact mrf operon.A plasmid bank of P. temperata grown on LB agar medium plusampicillin was screened by hybridization with a probe for themrfA gene as described in Materials and Methods. Severalpositive clones were obtained, and one was selected for furtherstudy. The plasmid was extracted, and the DNA was amplifiedby PCR using primers derived from mrfA and mrfG. The plas-mid was analyzed by various restriction enzymes, and the re-sults indicated an insert of approximately 12 kbp possessingboth mrfA and mrfG, as shown by Southern blotting. More-over, the extremities of the insert did not correspond to genesin the mrp operon from P. mirabilis, indicating that the entiremrf operon was present on this fragment.

DNA sequence of the P. temperata K122 mrf operon. DNAsequencing of the insert was achieved by walking along thefragment, and the sequence was aligned and compared withthe P. mirabilis sequence as described in Materials and Meth-ods, by using FredFrap and Autoassembler.

As shown in Fig. 1, the resulting sequence (GenBank acces-sion no. AF396083) contains 11 open reading frames (ORFs),with a predicted stem-loop structure between mrfA and mrfB,together with a Rho independent terminator following the lastgene, mrfJ. The mrf genes are organized in an apparent operon(Fig. 1a) like the mrp operon from P. mirabilis (Fig. 1b). Sim-ilarly, the mrf locus has an additional gene, mrfI, on the op-posing strand and upstream of mrfA, as in P. mirabilis. How-ever, we also noted some differences between the mrf and mrpoperons. Most clearly, the mrf operon contains an additionalgene, mrfX, upstream of mrfE. Although this gene displays44.8% identity with mrfE at the DNA level (Table 1), there isonly 20.7% identity at the protein level. Similarly, mrfX and

FIG. 1. Genetic organization of the P. temperata K122 mrf (GenBank accession no. AF396083) (a) and P. mirabilis mrp (GenBank accessionno. Z32686) (b) operons. “�1” indicates transcription initiation sites. The stem-loop following mrfA is predicted on the basis of sequence data.The predicted function of each gene, based on the data for P. mirabilis, is given. Arrows indicate direction of transcription.

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mrpE exhibit 43.1% DNA identity and 20.5 and 32.7% aminoacid identity and similarity, respectively. The origin of mrfXand the function of MrfX remain unknown, although a data-base BLASTP search indicates the presence of a conservedfimbrial motif (motif pfam00419.8), and the closest databasehomologue is the long polar fimbrial protein A precursor fromSalmonella enterica serovar Typhimurium LT2 (GenBank ac-cession no. AAL22500).

Table 1 further details the DNA and amino acid compari-sons between the conserved P. temperata mrf and P. mirabilismrp ORFs. Overall, genes encoding the major fimbrial subunit(mrfA and mrpA), minor subunit (mrfF and mrpF), regulatorprotein (mrfI and mrpI), outer membrane usher (mrfC andmrpC), and periplasmic chaperone (mrfD and mrpD) are allhighly conserved at both the DNA and amino acid sequencelevels. The exceptions are mrfD and mrpD, where DNA con-servation is relatively low (58.9%) while amino acid sequencesimilarity is high (82.6%). Genes encoding the putative fim-brial anchor (mrfB), adaptor/initiator proteins (mrfE andmrfG), adhesin (mrfH), and repressor of flagellar synthesis(mrfJ) exhibit lower levels of DNA and amino acid sequenceconservation.

The intergenic distance between the divergent genes mrfAand mrfI in P. temperata is slightly longer (684 bp) than thatbetween mrpI and mrpA in P. mirabilis (678 bp), and as de-scribed below, the most distal gene, mrfJ, unlike mrpJ, canapparently be transcribed independently of the rest of theoperon, at least under some conditions.

P. temperata-mediated horse erythrocyte hemagglutination.In view of the differences between the mrf and mrp operons, wetested if mrf encodes fimbriae functionally similar to thoseencoded by mrp by examining mannose-resistant erythrocytehemagglutination. P. temperata variant I and II cells from ex-ponential- and stationary-phase liquid LB cultures grown withor without agitation and from resuspended colonies from LBplates were tested for their abilities to hemagglutinate horseerythrocytes (see Materials and Methods). In all cases, addi-tion of bacteria to erythrocyte suspensions in PBS resulted inbacterial-dose-dependent hemagglutination. Addition of 50mM mannose to hemagglutination tests had no significant ef-fect. Therefore, we concluded that the mrf fimbriae, like themrp fimbriae in P. mirabilis, are indeed mannose resistant.

Primer extension analysis to identify transcription startsites. Primer extension analysis was performed as described inMaterials and Methods following total-RNA extraction fromvariant I cells growing exponentially in LB medium. As shownin Fig. 2a, the results of the first extension analysis demon-strated that a transcriptional start site was detected at nucle-otide C3260, upstream of, and on the coding strand for, mrfA.

In addition, by using primer Seq2700 (bp 2655 to 2673;located within the 3� end of mrfI), we identified a transcrip-tional start site upstream of mrfI (data not shown). However,we cannot indicate the nucleotide with precision in this case,since the sequencing ladder was illegible. We attributed this toa possible mixed population of DNA molecules being in theON and OFF states (see below) with respect to the mrfI-mrfAintergenic region, thus resulting in a double DNA sequencingladder.

Subsequent studies of the expression of mrf genes duringinfection of larvae (see below) also indicated that mrfJ, whoseequivalent in P. mirabilis encodes a repressor of flagellar syn-thesis (27), might in fact be transcribed independently of theother mrf genes. However, our attempts at locating a transcrip-tion start site upstream of mrfJ were not conclusive.

Sequence analysis of the phase inversion region in the mrfoperon. Regulation of phase variation expression of the fimtype 1 fimbrial operon, similarly to P. mirabilis mrp regulation,involves inversion of a short DNA element, fimS, betweenfimE and fimA (1). Regulation of fim expression involves otherrecombinases, FimB and FimE, together with the regulatoryproteins Lrp (leucine-responsive regulatory protein) and IHF(integration host factor), which stimulate recombinase activity(8). In contrast, recombinase activity is repressed by H-NS(37). Expression of the pap operon does not involve an invert-ible element but is regulated by Lrp, together with PapB andPapI, H-NS, the cyclic AMP receptor protein (CRP), and Dammethylation of two GATC sites within the papI-papB inter-genic region (see reference 8). In both the fim and pap oper-ons, Lrp, together with regulators specific to each system, isinvolved in the formation of a nucleoprotein complex in theregulatory region, binding to a consensus motif, 5�-GN2-3

TTT(T)-3� (32). In the case of the pap system, this also impli-cates the Dam methylation sites.

Inspection of the DNA sequence of the mrfA locus identifieda 319-bp sequence containing the two 25-bp inverted repeatsflanking a 269-bp core. In contrast to those in P. mirabilis, the25-bp inverted repeats are not identical but have two mis-matches (indicated by the asterisks): 2966-AAACT�AAACAG�AATGCACCAAACTGT-2991 and 3259-ACAGTTTGGTGCATTT�TGTTTG�GTTT-3284. The positions of therelevant control elements of the mrfI-mrfA intergenic region inthe ON position are shown in Fig. 2b. Sequence analysis of thisregion not only reveals the inverted-repeat motifs but alsoidentifies 14 consensus Lrp binding sites (seven 5� to 3� andseven 3� to 5�), a GATC Dam methylation site consensus(positions 3342 to 3345), putative �35 (TTACTC; bp 3214 to3220) and �10 (TAGTA; bp 3238 to 3242; 18 bp upstream ofthe transcription initiation site, C3260) sequences, and a ribo-some binding site (GGAAT; bp 3402 to 3406) 8 bp upstream ofthe mrfA initiation codon.

In vitro inversion of the mrfI-mrfA inversion element. Figure3a indicates the two alternative positions of the inverted ele-

TABLE 1. Global alignmenta comparison between individual mrpand mrf genes and encoded protein sequences

Mrf vs Mrpcomparison

% DNAsimilarity

% Amino acididentity

% Amino acidsimilarity

I 67.1 73.3 84.8A 63.7 66.7 77.2B 51.9 41.9 55.5C 61.3 60.1 76.8D 58.9 61.7 82.6E 53.4 44.3 63.5F 62.3 57.1 71.8G 56.0 48.2 64.9H 56.6 52.5 71.2J 55.1 52.3 65.8MrfX vs MrpE 43.1 20.5 32.7MrfX vs MrfE 44.8 20.7 32.3

a Alignments were performed with the algorithm from EMBOSS (34) avail-able at http://www.ebi.ac.uk/emboss/align/index.html.

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ment for transcription of mrfA, the ON and the OFF positions.The two alternative positions should be detectable by restric-tion enzyme digestion of the intergenic and flanking regions,yielding two AflII fragments of different lengths.

Accordingly, a 1,327-bp fragment from a site in mrfI or mrfA(Fig. 3a) was first amplified by PCR from genomic DNA iso-lated at different times throughout the growth of a culture of P.temperata variant I and was then digested with AflII. The re-sults (Fig. 3b) identified two intense bands corresponding tothe intergenic region in the ON position. However, two addi-tional bands of lower intensity, with sizes corresponding to theOFF position, were also detected. This indicated that a sub-stantial proportion (approximately 40%) of variant I cells un-

der these conditions have an inactive mrfA promoter. Further-more, the relative proportions of ON and OFF bands appearto remain constant throughout growth.

When an identical experiment was performed with variant IIcells, the results (Fig. 3c) were quite clear-cut, with the wholepopulation displaying the ON position.

Expression of mrfA in liquid cultures. Cultures of both vari-ant I and variant II bacteria were grown in LB medium, andsamples were removed for mRNA and protein analysisthroughout growth (Fig. 4a). Total RNA was extracted, dena-tured, and analyzed by Northern blotting followed by probingwith a denatured [�-32P]dCTP-labeled mrfA probe. Figure 4bshows that the mrfA transcript could be detected throughout

FIG. 2. (a) �1 position of mrfA. Asterisk indicates the �1 nucleotide in the sequence, corresponding to the mrfA transcriptional initiation site.(b) Organization of the mrfA promoter and invertible element. Inverted repeats are boxed, and the mrfA transcriptional start site, C3260, is enlargedin the second repeat. mrfI and mrfA initiation codons are boldfaced and underlined. The mrfA ribosome binding site (RBS) and �10 and �35sequences are italicized and underlined. Putative Lrp binding sites are indicated by arrows, and the putative dam methylation site (positions 3343to 3346) is indicated by a dumbbell. Numbers correspond to sequence positions in GenBank accession no. AF396083.

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most of the exponential-growth phase in variant I cells but thendisappeared in stationary phase. Similar results were obtainedfor variant II cells (data not shown). Furthermore, the esti-mated size of the mrfA transcript (700 bp) is consistent with thesize of the mrfA gene (540 bp) plus the 155-bp untranslatedleader, indicating that transcription termination occurs at thestem-loop structure downstream of mrfA. We were unable todetect a larger operon transcript by Northern blotting. Thismay be attributed to the limit of detection in total-RNA sam-ples by this technique.

In contrast to the expression of mrfA, Northern blot analysisof mrfG expression failed to detect any transcript, suggestingthat expression of the distal genes is regulated in some way,presumably involving the mrfA distal stem-loop structure. Ex-pression of mrfA in both variant I and variant II cells was alsoexamined by semiquantitative RT-PCR. As shown in Fig. 5, theresults confirmed expression in exponentially growing cells(taken at 5 h of growth from the culture for which results areshown in Fig. 4a) at significantly higher levels, as might beexpected from the results shown in Fig. 3c for the variant IIpopulation. Importantly, semiquantitative RT-PCR now also

detected transcripts from mrfG (data not shown), although atlevels 50- to 70-fold lower than those of mrfA in variant I andII populations, respectively. This supports the idea that mostmrfA transcripts may terminate at the predicted stem-loopbetween mrfA and mrfB (see Fig. 1a).

Samples were also analyzed by SDS-polyacrylamide gel elec-trophoresis and Western blotting with an antibody to the P.mirabilis MrpA protein (see Materials and Methods). The re-sults for variant I cells showed that the MrfA protein waspresent throughout the growth phase and remained quite sta-ble during stationary phase (Fig. 4c). Essentially identicalresults were obtained for variant II cells (data not shown).Subsequent studies with immunogold electron microscopyconfirmed that Mrf fimbriae are indeed present on the P.temperata K122 cell surface.

Immunogold detection of Mrf fimbriae. The experimentsdescribed above clearly demonstrated the expression of mrfA,encoding the pilin, in both variant I and variant II cells growingexponentially. The results also indicated a lower-level expres-sion of the downstream genes necessary for production ofcomplete pili. As shown in Fig. 6, by using an antibody to the

FIG. 4. Production of mrfA transcripts and the MrfA protein during the growth of variant I cells in LB culture. (a) An overnight culture of P.temperata K122 variant I was diluted in LB medium to an OD450 of 0.1. Growth at 29°C was monitored by measurement of OD450 at each hour.(b) Northern blot of total RNA during growth. mrfA was used as a probe. (c) Western blot analysis of MrfA during growth, using an antiserumagainst MrpA.

FIG. 3. (a) (i) Organization of the mrfA-mrfI intergenic region, showing the invertible element bearing the mrfA promoter (shaded oval) flankedby two inverted repeats (solid boxes). (ii and iii) Schematic representations of the PCR products obtained after amplification of the intergenicregion. The relative positions of the AflII site and the sizes of the fragments obtained after restriction digestion are indicated, allowing fordiscrimination between the ON (ii) and OFF (iii) states of the invertible element. (b and c) Analysis of the inversion states of the intergenic elementduring growth of P. temperata variants I and II. Growth times (in hours) are given at the top of the upper gel. P, overnight culture. (b) PCR productsof the variant I intergenic region digested with AflII and analyzed on a 2% agarose gel. (c) A similar experiment with variant II cells.

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homologous MrpA pilin from P. mirabilis (a gift from H. Mo-bley), we were able to confirm by immunogold electron mi-croscopy the production of a small number of approximately300-nm-long Mrf fimbriae in both variant I and variant II cells.Analysis of cells from colonies grown on solid LB agar mediumor from liquid LB culture throughout the growth phase dem-onstrated that Mrf fimbriae are always present on the surfaceof P. temperata K122 and in approximately similar numbers.Notably, however, in Fig. 6, very few intact fimbriae can bedetected, indicating their apparent fragility under these condi-tions.

RT-PCR analysis of the expression of the mrf operon duringinfection. Previous studies have indicated that following injec-tion of G. mellonella larvae with approximately 5,000 cells of P.temperata, death of larvae occurs after approximately 30 h. Thebacteria accumulate to 1010 cells after 48 h, and the specificsecreted metalloprotease PrtA, associated with the bioconver-sion phase (13), is first detected 5 to 10 h after the death of thelarvae (15). In this study, we analyzed the expression of the mrfoperon during the infection of G. mellonella larvae by usingsemiquantitative RT-PCR. Since it is generally accepted thatprokaryotic 16S rRNA genes, like eukaryotic actin, are consti-

FIG. 5. Relative abundance and expression of mrfA transcripts (with 16S RNA used as a standard) at different growth points in liquid culturesof P. temperata variants I and II and E. coli (used as a negative control). Bar 1, P. temperata K122 variant I cells growing exponentially; bar 2, variantI cells in stationary phase; bar 3, variant II cells in exponential phase; bar 4, variant II cells in stationary phase; bar 5, E. coli cells in exponentialphase.

FIG. 6. Immunogold detection of P. temperata K122 variant I and variant II Mrf fimbriae. Cells were grown on LB agar. Arrows indicate thelocalization of mannose-resistant fimbriae on cells.

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tutively expressed at a constant level under most growth con-ditions, the bacterial 16S rRNA gene was used as an internalstandard for expression studies.

G. mellonella larvae were infected with 5,000 variant I bac-teria as described in Materials and Methods. Total RNA wasextracted from individual larvae at intervals up to 38 h. First, asshown in Fig. 7a, the accumulation of 16S RNA itself duringinfection was measured by semiquantitative RT-PCR. Thisreflects the growth of the bacteria, with a short, approximately5-h lag, an exponential phase, and entry into stationary phaseat approximately 30 h, with an exponential generation time ofapproximately 2 h 26 min. By use of primers specific for eachgene in the mrf locus, the relative levels of transcription ofthese genes were determined by comparison with the level of16S RNA (see Materials and Methods). Total-RNA sampleswere tested by PCR with all oligonucleotide pairs, and all

samples used in Q-RT-PCR were negative for DNA contam-ination.

The results of this analysis are presented in Fig. 7b. The firstgene whose expression was detected in infected larvae was mrfJat 5 h, and its levels remained relatively high to the end of theinfection. This result clearly supports the independent expres-sion of mrfJ, encoding a putative inhibitor of flagellar biosyn-thesis, from its own promoter. In contrast, mrfA expression wasdetected only at 25 h, about 5 to 6 h before the death of thelarvae in nonsacrificed controls. Expression of several down-stream genes of the mrf operon was detected between 30 and38 h. However, the levels of the latter, for example, mrfB, mrfC,mrfF, and mrfG, were on average 2 orders of magnitude lowerthan the level of mrfA, again suggesting a role for the predictedstem-loop following the mrfA gene, in vivo as in vitro (see Fig.1a), in downregulating expression of the distal genes. Barely

FIG. 7. (a) Growth curve of P. temperata K122 variant I bacteria in infected G. mellonella larvae as determined from the Q-RT-PCRcrossing-point data obtained by amplification of 16S mRNA. (b) Expression patterns of different genes of the mannose-resistant fimbrial locusduring infection of G. mellonella by P. temperata K122 variant I.

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detectable levels of expression were obtained for the mrfD andmrfX genes, and no expression was observed for mrfH. Weshould stress that all oligonucleotide primer pairs, includingthose for mrfH, were verified for PCR efficiency (see Materialsand Methods) and that E values were 1.93 0.05 in all cases(theoretical maximum � 2). Moreover, expression of mrfHcould be demonstrated by Q-RT-PCR from total RNA isolatedfrom exponentially growing P. temperata K122 cells grown invitro in LB liquid culture. Therefore, the failure to detect mrfHexpression or the low-level expression of mrfD and mrfX in invivo infection samples was not due to a physical detection limitbut rather to some factor related to conditions of extractionfrom larvae.

The analysis shown in Fig. 7b indicates that mrfA transcrip-tion is blocked in the early stages of growth of the bacteria,despite the fact that the liquid-grown cells used for the inoc-ulum normally contain a majority (60%) with the invertibleregion in the ON position. The regulation of transcriptionunder these infection conditions may therefore be complex. Inthis context, it is interesting that, as also shown in Fig. 7b, theexpression of mrfI is detected at approximately 30 h, consistentwith a possibly increased frequency of switching of the invert-ible mrfA promoter region at this time.

DISCUSSION

Surface appendages such as pili or fimbriae are now wellestablished as important pathogenicity factors in several bac-teria (30). In P. mirabilis the mannose-resistant MR/P fimbriaeare essential for uropathogenic infections leading to bladdercolonization in mice (26). Such MR/P fimbriae are also impli-cated in pellicle formation (26) and swarming in this organism(23). As in other cases, MR/P fimbriae are therefore likely tobe involved both in interbacterial interactions and in adhesionto host cell surface receptors. Previous studies with Xenorhab-dus nematophilus, a related insect pathogen also capable of ahighly specific symbiotic relationship with nematodes (31),have indicated that adhesion is important for colonization ofthe nematode gut, although the molecular basis of this phe-nomenon was not investigated (5). In this study, we have iden-tified and characterized the genetic locus, mrf, in P. temperata.The mrf operon is highly similar to the mrp operon encodingthe mannose-resistant pili in P. mirabilis. These pili are relatedto the type P and type 1 pili in E. coli and are secreted by the“chaperone-usher” pathway (36).

The mrf operon, like its mrp counterpart, contains the highlyconserved structural genes for the outer membrane usher, thePapD-like chaperone, and different components of the pilusitself. The latter include the major pilin, encoded by the firstgene, mrfA, and the putative adhesin, encoded by mrfH. Whilethe structural components of pili are well conserved, the globalregulation of expression of different fimbrial operons in differ-ent bacteria varies considerably, although the different regula-tors are usually encoded at either end of the correspondingoperon. These regulatory mechanisms include modulation ofthe methylation of GATC sites within the promoter region inE. coli (9), trans-acting regulatory factors (Lrp, H-NS, IHF),and site-specific recombination switches (inversion) of the pro-moter. In fact, as in the mrp locus from P. mirabilis (44), wealso identified a putative recombinase gene, mrfI, transcribed

in the opposite direction from mrfA, in the mrf locus. Thepresence of inverted repeats upstream of mrfA, and directevidence from PCR analysis for inversions in this region,clearly shows that the mrf operon is also regulated by an in-version mechanism, controlling an ON/OFF switch. Similarly,both the mrp and mrf operons contain a predicted stem-loopstructure between the first and the second genes in the operonwhich could participate in the modulation of transcription toensure that the pilin subunit, MrfA, is produced in excess ofthe other Mrf proteins. Indeed, both in in vitro liquid culturesand in vivo, in the insect, mrfA messenger was detected inexcess (up to 2 orders of magnitude) of the transcripts for thedownstream genes. By using primer extension analysis, tran-scriptional start sites for the divergent mrfI and mrfA geneswere confirmed, as found in P. mirabilis for the mrp operon.

Several studies have previously indicated that the productionof pili or flagella, required for relatively sedentary versus mo-tile behavior, respectively, might be mutually exclusive (23).Indeed, Li et al. (27) have shown that the MrpJ protein in P.mirabilis is a repressor of flagellar synthesis, although the mo-lecular basis of this effect was not elucidated. The MrfJ proteinis 52.3% identical to MrpJ and is highly likely to fulfill a similarfunction in regulating motility in P. temperata.

Photorhabdus species display a characteristic phenotypicvariation, in some cases in response to stress conditions,whereby stationary-phase cultures of variant II cells lack thecharacteristic bioluminescence, strong yellow pigmentation,and many proteins normally secreted to the medium (22). Ourresults clearly showed that while laboratory cultures of variantI populations displayed both ON and OFF variations for themrfI-mrfA intergenic region, variant II cells were all apparentlylocked in the ON position. Unfortunately, neither the physio-logical significance of this phase change nor its molecular basisis known; therefore, it is not clear how the conversion to theON state is effected in variant II cells. Furthermore, the inter-genic region between mrfI and mrfA, containing the inversionelement, also contains 14 consensus Lrp binding sites and onepotential methylation GATC sequence (positions 3342 to3345) between the mrfA ribosome binding site and the �10promoter sequence on the untranslated 5� leader. We cannot,therefore, exclude the formal possibility that these sequencesare also involved in regulation of mrf expression.

From previous studies with P. luminescens W14 (15) andfrom the present study with the related species P. temperataK122, the time course and program of events during infectionof G. mellonella larvae may be summarized as follows. Theinjected bacteria grow exponentially during most of the 10- to30-h postinfection period; the larvae then appear to die be-tween 30 and 40 h (particularly in the case of K122), while thebioconversion phase, signaled by the production of a secretedprotease (13) and the production of the orange pigment se-creted by the K122 strain, commences around 40 to 48 h (datanot shown). In liquid cultures of Photorhabdus, protease andpigment production occurs at the onset of stationary phase. Inthe case of P. luminescens W14 (15), the presence of at leastone secreted toxin can be detected in infected larvae afterabout 18 h, consistent with a role in the killing of host cells. Inthe present study, which used Q-RT-PCR, the major pilingene, mrfA, apparently was transcribed only between 20 and25 h postinfection. This time point apparently corresponds to

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the mid-exponential phase of growth of strain K122 in thelarvae, according to the accumulation of 16S mRNA (Fig. 7a),which is still many hours before the death of the larvae. Inter-estingly, in liquid culture (Fig. 4b), early-exponential-phasecells also appear to express very low levels of mrfA messengercompared to those expressed by late-exponential-phase cells.This temporal control of mrfA expression indicates that the mrfoperon may be regulated in other ways in addition to theON/OFF promoter inversion. Moreover, since the inoculumused in these infection experiments usually contains a majorityof cells (60%) with the mrf operon in the ON position (Fig. 3b),these results imply that early in infection the ON switch isinverted or, alternatively, that transcription from cells withmrfA in the ON position is repressed by some other mecha-nism. In either case, the results suggest that Mrf pili may bemost important for some late stages of infection. Expression ofother downstream genes in the operon was also detected be-tween 25 and 30 h postinfection, but, as found in liquid cul-tures, their levels were substantially lower than that of mrfA.Every effort was made to ensure the efficacy of the oligonucle-otide probes, including that for mrfH, which, while detectingthe product in liquid cultures, failed to detect anything inlarvae. It is also unlikely that the difficulties with some distalgenes or the absence of mrfA transcripts early in infection isdue to an inherent limit in transcript detection, since mrfJmessenger was detectable from the earliest times.

As a further illustration of the possible complexity of mrfexpression in vivo, transcription of the presumed recombinasegene, mrfI, was also detected in these experiments only afterabout 25 to 30 h. As in the case of P. mirabilis, we haveidentified a transcriptional start for this gene, which is quitedistinct from that of the inverting pA promoter and outside theinversion element. However, these results indicate that in thedifferent cells of the bacterial population in the infected larvae,both mrfI and mrfA can be activated in the later stages ofinfection, with the implication that some cells may have the mrfoperon in the ON state and others may have it in the OFFstate. This remains to be tested. The presence of two opposedpromoters, pI and pA, in the mrfI-mrfA intergenic region, to-gether with a putative GATC Dam methylation site and Lrpbinding motifs, is similar to the situation in the papI-papBintergenic region. The presence of an inversion element andLrp binding sites is similar to that in the fimE-fimA intergenicregion. Regulation of expression of the P. temperata mrf and P.mirabilis mrp fimbrial operons may, therefore, possess ele-ments from both of the well-characterized paradigms of fim-brial expression, fim and pap. The in vivo Q-RT-PCR experi-ments (Fig. 7b) analyzing mrfJ expression demonstrate aprofile clearly different from that of the other mrf genes. Weattempted to identify an independent mrfJ transcriptional startsite and promoter element upstream of mrfJ by primer exten-sion, but results were inconclusive. This intriguing profile formrfJ expression will be studied further.

Overall, the in vivo experiments and analysis of mrfJ expres-sion suggest that P. temperata K122 flagellar synthesis is regu-lated even early during infection. In addition, however, pilinsynthesis is also blocked and is renewed only after 25 h ormore. This perhaps may coincide with a new bacterial coloni-zation phase, not yet identified, preceding larval death, whichrequires these surface structures. However, it is not possible to

determine from these experiments whether the production ofMrf pili is specifically required for the eventual killing of thehost or for some other function.

Although much remains to be determined concerning thenature of the molecular switches which control the differentstages of Mrf pilus production in vivo, the results presentedhere further illuminate the differential regulation of gene ex-pression in the pathogenicity of P. temperata and in particularstrongly suggest that the mannose-resistant pili play an impor-tant role in this process.

ACKNOWLEDGMENTS

We thank D. Jaillard for technical assistance with electron micros-copy and Y. Zivanovic for helping with the DNA sequence assemblyand alignments. We are very grateful to H. L. Mobley for antibodiesagainst MrpA and to G. Dumond for kindly providing G. mellonellalarvae.

This work was supported by the CNRS and Universite Paris-Sud.L.M.M-C. acknowledges the receipt of a Fondation pour la RechercheMedicale (FRM) fellowship.

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