CpxRA Influences Xenorhabdus nematophila Colonization Initiation and Outgrowth in Steinernema carpocapsae Nematodes through Regulation of the nil Locus

The gammaproteobacterium Xenorhabdus nematophila mutualisticallycolonizes an intestinal region of a soil-dwelling nematode andis a blood pathogen of insects. The X. nematophila CpxRA two-componentregulatory system is necessary for both of these host interactions(E. Herbert et al., Appl. Environ. Microbiol. 73:7826-7836,2007). Mutualistic association of X. nematophila with its nematodehost consists of two stages: initiation, where a small numberof bacterial cells establish themselves in the colonizationsite, and outgrowth, where these cells grow to fill the space.In this study, we show that the Cpx system is necessary forboth of these stages. X. nematophila ${Delta}$ cpxR1 colonized fewer nematodesthan its wild-type parent and did not achieve as high a densityas did the wild type within a portion of the colonized nematodes.To test whether the ${Delta}$ cpxR1 host interaction phenotypes are dueto its overexpression of mrxA, encoding the type I pilin subunitprotein, we assessed the colonization phenotype of a ${Delta}$ cpxR1 ${Delta}$ mrxA1double mutant. This mutant displayed the same colonization defectas ${Delta}$ cpxR1, indicating that CpxR negative regulation of mrxA doesnot play a detectable role in X. nematophila-host interactions.CpxR positively regulates expression of nilA, nilB, and nilCgenes necessary for nematode colonization. Here we show thatthe nematode colonization defect of the ${Delta}$ cpxR1 mutant is rescuedby elevating nil gene expression through mutation of nilR, anegative regulator of nilA, nilB, and nilC. These data suggestthat the nematode colonization defect previously observed in ${Delta}$ cpxR1 is caused, at least in part, by altered regulation ofnilA, nilB, and nilC.

INTRODUCTION

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INTRODUCTION

As part of its natural life cycle the gammaproteobacterium Xenorhabdusnematophila lives within two animal hosts: it is a mutualisticsymbiont of the soil-dwelling nematode, Steinernema carpocapsae,and a pathogen toward a number of insect orders (14, 15, 18).X. nematophila colonizes a modified intestinal region of a free-living,nonfeeding stage of S. carpocapsae, which serves as its vectorinto the blood system (hemocoel) of susceptible insect hosts(15). X. nematophila is released from its nematode vector intothe hemocoel and within 1 to 2 days kills the insect (15). Theinsect cadaver serves as a nutrient source for nematode andbacterium reproduction. Once nutrients inside the insect hostare depleted, X. nematophila reassociates with the vector stageof the nematode, which migrates into the soil to seek a newinsect host and repeat the cycle (16).

The S. carpocapsae vector-stage association with X. nematophilais species specific: of all Xenorhabdus species tested to date,only X. nematophila can colonize S. carpocapsae nematodes (1,6). In a fully mature vector-stage nematode, the intestinalcolonization site, known as a receptacle (48), or vesicle (2)can contain >100 CFU of X. nematophila (33). This bacterialpopulation is founded by one or a few X. nematophila cells,and the process of colonization includes distinguishable initiationand outgrowth stages (33). Within the vesicle, X. nematophilacells adhere to a nematode-derived structure termed the intravesicularstructure that is surrounded by a mucuslike material (32). Thesedata have led to a model in which colonization initiation byX. nematophila cells includes binding to a specific sugar associatedwith the intravesicular structure (32). Newly colonized S. carpocapsaenematodes contain very few X. nematophila cells that increasein number as the nematodes age, reaching a fully colonized levelafter $~$ 6 days (33). X. nematophila mutants defective in bothinitiation and outgrowth have been isolated, lending insightinto gene products and physiological processes necessary fornematode colonization by this bacterium (6, 23, 32, 49). Forexample, metabolic mutants defective in the biosynthesis ofmethionine or threonine can initiate colonization but do notgrow within the vesicle (13, 34), while mutants lacking thesigma factor homolog RpoS cannot initiate colonization (23,49).

Current evidence suggests that, although X. nematophila growthoccurs in the nematode vesicle, it is a nutrient-limited andstressful environment (7, 19). When released from this environmentinto insect hemolymph, X. nematophila is expected to encountera relatively nutrient rich environment (7, 17, 20, 38, 41) butalso faces insect immune responses. We do not yet understandfully which characteristics of these environments are sensedby X. nematophila or how this information is transduced to theexpression of symbiotic activities appropriate for each host.However, one regulator involved in this process is the CpxRAtwo-component signaling system, which is necessary for interactionswith both hosts and is hypothesized to help regulate transitionsbetween the nematode and insect environments (21). In otherbacteria, the Cpx system senses cell surface-related parameters,such as proximity to a surface (39), changes in external conditions(27, 31, 36, 37, 50), or misfolded proteins in the periplasm(11). In response to sensor kinase (CpxA) activation by thesesignals, the response regulator, CpxR, regulates expressionof genes that function in cell envelope integrity (43), motilityand chemotaxis (10), biofilm formation (12), adherence and invasionof host cells (24, 31), pilus formation (25), type III secretion(35), and protein folding and degradation (8, 9, 42, 44).

When the cpxR gene is deleted in X. nematophila, the bacteriumis less effective at killing a model insect host, Manduca sexta,and colonizes S. carpocapsae nematodes at lower levels thanits wild-type parent (21). Phenotypic and gene expression studiesof a cpxR deletion mutant ( ${Delta}$ cpxR1) led to the identificationof multiple genes in the CpxR regulon, including genes knownor thought to be involved in one or both host interactions.One of these genes is mrxA, encoding the X. nematophila typeI pilin subunit (21) reported to be involved in X. nematophila-nematodemutualism (3) and virulence toward insects (3, 28-30). Previousstudies have shown that the pilin subunit is toxic toward Helicoverpaarmigera insects in a purified form (28-30) and that these pilinsubunits are found in outer membrane vesicle blebs, which, perhapsnot coincidentally, are also toxic toward H. armigera (28).In the ${Delta}$ cpxR1 mutant, mrxA transcript is increased >3-foldover that of wild-type cells, indicating that the ${Delta}$ cpxR1 mutantcell surface may contain an increased number of pili comparedto wild-type cells. This has the potential to severely affecthost interactions of a cell due to the potential of pili tostimulate immune systems (either of the nematode or insect)or attach to host ligands or other structures (26, 46).

Three other X. nematophila CpxR regulon members—nilA,nilB, and nilC (21)—are each necessary for nematode colonizationand are thought to be involved in initiation of colonization,perhaps through signaling or attaching to a nematode surface(4-6). nilA, nilB, and nilC are encoded at the same locus, termedthe symbiosis region 1 (23). nilA and nilB are cotranscribed,while nilC is divergently transcribed (23). NilC is a periplasmicallyoriented outer membrane lipoprotein (4), NilB is an outer membraneprotein of unknown function (A. Bhasin and H. Goodrich-Blair,unpublished data), while NilA is predicted to be an inner membraneprotein (23).

Both nil promoters are repressed by the synergistic activitiesof Lrp, the leucine-responsive regulatory protein that alsoplays a role in nematode mutualism and insect virulence, andNilR, a small helix-turn-helix containing protein (4, 5, 7).Maximal expression of the nil genes in liquid culture occursin strains in which both nilR and lrp are disrupted (5). Derepressionof the nil genes is thought to be necessary for successful colonizationof nematodes, since a X. nematophila strain carrying an ectopiccopy of NilR showed a 60-fold lower colonization level comparedto the control strain (5).

The goal of the present study was to elucidate the mechanism(s)by which the Cpx signal transduction system contributes to X.nematophila mutualistic nematode colonization. Specifically,we tested whether CpxR regulation of mrxA and nil genes influencescolonization and whether CpxR contributes to colonization initiation,outgrowth, or both.

MATERIALS AND METHODS

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MATERIALS AND METHODS

Bacterial strains and culture conditions.
Bacterial strains and plasmids used in the present study arelisted in Table 1. All strains were grown in the dark in LBmedium or LB agar plus 0.1% pyruvate at 30°C unless otherwisespecified. Antibiotics were used at the following concentrations:ampicillin (150 µg ml^–1), chloramphenicol (15 µgml^–1 for X. nematophila and 30 µg ml^–1 forE. coli), gentamicin (25 µg ml^–1), kanamycin (50µg ml^–1), and streptomycin (12.5 µg ml^–1for X. nematophila and 25 µg ml^–1 for E. coli).Sucrose was used at a 5% final concentration.

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TABLE 1. Bacterial strains and plasmids used in this study

Molecular biological methods.
Standard biological methods were used in the present study (45).Plasmid and PCR purification kits (Zymo Research, Orange, CA)were used according to the manufacturer's recommendations. DNAamplification was performed by using ExTaq (Takara Otsu, Shiga,Japan) according to the manufacturer's directions.

β-Galactosidase assays.
β-Galactosidase assays were performed as previously described(4) on overnight cultures inoculated from LB medium plus ampicillinagar X. nematophila colonies. X. nematophila lacZ fusion strainswere created by triparental mating of pTn7-nilA, pTn7-nilB,or pTn7-nilC (5) as previously described (21).

Bacterial mutant construction.
The cpxR gene was deleted in a ${Delta}$ nilR17::Kan background (5) byallelic exchange using pKRcpxRStr (21). Candidate colonies resistantto ampicillin, kanamycin, and streptomycin, but sensitive tochloramphenicol were chosen. A ${Delta}$ cpxR1/ ${Delta}$ mrxA1:: ${Omega}$ Kan mutant was createdby conjugating pPK13 (3) into the ${Delta}$ cpxR1 mutant (21) background(HGB1230). Candidate colonies resistant to ampicillin, kanamycin,streptomycin, and sucrose but sensitive to chloramphenicol andgentamicin were chosen. The presence of an insertion withinmrxA was confirmed by Southern analysis as described previously(3). Both mutants were tested for the presence of the appropriateinsertions by PCR amplification of the loci. Primers EH72 (5'-GAATAGCGGAGTTTGTTATCG-3')and EH15 (5'-CACTGGTAACAAGGAGTAAGC-3') amplified the cpxR locus.EH193 (5'-GGATCCATGAAACTTAACACAATTGGC-3') and EH194 (5'-CTCGAGGAGGAAATATGTCACCATCGG-3')amplified the mrxA locus.

Nematode colonization.
Colonization assays were performed as previously described (4)using five replicates per bacterial strain per experiment. Day1 of the assay corresponds to the day infective juvenile (IJ)nematodes were taken from water traps and surface sterilized.IJs were then kept in closed tissue culture flasks at room temperaturein the dark for the remainder of the experiment. For all timepoints, 1,000 IJs were sonicated, dilution plated onto LB plates,and grown overnight at 30°C to determine the average bacterialCFU/IJ for the nematode population. For microscopic analysis,nematodes were paralyzed with levamisole (Sigma, St. Louis,MO) and viewed as previously described (33). Between 100 and1,000 IJs were viewed per time point per bacterial strain.

Nematode survival and development.
Bacterial strains were grown in liquid culture overnight, platedon lipid agar plates, and grown overnight in the dark at 30°C.Nematode eggs were plated in equal volumes onto the bacteriallawns and, at various time points after hatching, the nematodeswere washed off the plates with water, and a portion was countedand viewed microscopically to determine the total number ofnematodes present, as well as the approximate stage of development.

RESULTS

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RESULTS

cpxR contributes to initiation and outgrowth during nematode mutualism.
We previously reported that ${Delta}$ cpxR1 mutant cells are recoveredfrom a population of colonized nematodes at an average of $~$ 38%that of wild-type cells (21). To determine whether this nematodecolonization defect is due to an inability of nematodes to surviveor reproduce on lawns of ${Delta}$ cpxR1 mutant cells, populations ofnematodes growing on either wild-type X. nematophila, a ${Delta}$ cpxR1mutant, or the complemented ${Delta}$ cpxR1 mutant ( ${Delta}$ cpxR1 Tn7-cpxRA) weremonitored for 2 weeks to assess differences in nematode viabilityor development. At various time points after plating nematodeeggs on bacterial lawns, the nematodes were rinsed off the lipidagar plates with water, counted microscopically to quantifytotal numbers, and classified by approximate stage of growth.No obvious differences were observed, either in number or growthstage of the nematodes present (data not shown).

One possible explanation for the ${Delta}$ cpxR1 mutant colonization defectis that, compared to the number of wild-type X. nematophilacells in colonized nematodes, ${Delta}$ cpxR1 mutant-colonized nematodeseach contain only 38% as many mutant cells, which would indicatean inability of the ${Delta}$ cpxR1 mutant to maintain a fully colonizedstate or to grow past a certain limiting threshold within thenematode. A second possibility is that 38% of the nematodesexposed to the ${Delta}$ cpxR1 mutant are fully colonized and that theremaining nematodes are uncolonized, which would indicate adefect in the ${Delta}$ cpxR1 mutant's ability to initiate nematode colonization.To distinguish between these two possibilities, we used greenfluorescent protein-labeled wild-type X. nematophila and ${Delta}$ cpxR1mutant cells to quantify by fluorescence microscopy the numberof fully colonized (Fig. 1A), partially colonized (containingfewer than 10 bacterial cells within the vesicle) (Fig. 1B)and uncolonized (Fig. 1C) S. carpocapsae nematodes for eachbacterial strain. In this experiment, wild-type X. nematophilacells fully colonized 89% of the nematodes counted, while theremaining 11% of nematodes counted were either uncolonized orpartially colonized (Fig. 2A, day 1). However, of nematodesgrown on the ${Delta}$ cpxR1 mutant, only 49% were fully colonized, while40% of nematodes were uncolonized and 11% were partially colonized(Fig. 2B, day 1).

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FIG. 1. A ${Delta}$ cpxR1 mutant displays variable distribution within nematodes. Fluorescence microscope images of S. carpocapsae bacterial receptacles displaying various states of colonization by the ${Delta}$ cpxR1 mutant are shown. The bottom row are fluorescence micrographs of nematode receptacles either fully colonized (A), partially colonized (containing 10 bacterial cells or fewer) (B), or uncolonized (C). The top-row images are phase-contrast micrographs of nematodes with false-colored fluorescence micrographs overlaid. All images were taken at a magnification of x1,000. Bars, 10 µm.

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FIG. 2. A ${Delta}$ cpxR1 mutant is defective in nematode colonization initiation and outgrowth. The proportion of fully colonized, partially colonized, and uncolonized nematode vesicles in nematodes colonized by wild-type X. nematophila (A) or the ${Delta}$ cpxR1 mutant (B) over 39 days is shown. (C) The relative colonization level for the nematode population (as determined by dilution plating nematode sonicates) between the ${Delta}$ cpxR1 mutant and the wild type was consistent over 39 days. Average values from three experiments are shown. Different letters indicate values that are statistically different from each other. Error bars indicate the standard error. WT, wild type.

Given the prolonged lag phase of ${Delta}$ cpxR1 mutant growth in LB comparedto wild-type cells (21), these results raised a third possiblescenario, in which the ${Delta}$ cpxR1 mutant grows more slowly in thevesicle but, given time, would eventually achieve full colonizationlevels in all nematodes in which colonization was initiated.Therefore, we continued our observation of nematode vesiclesfor 39 days past the initial surface sterilization to determinewhether the ${Delta}$ cpxR1 mutant showed delayed outgrowth. At days 4,6, 11, and 39 after the initial surface sterilization, the nematodeswere sonicated and plated to determine the average bacterialCFU per nematode, and the proportion of colonized versus uncolonizednematodes was assessed microscopically. Over 39 days, the averagecolonization of the ${Delta}$ cpxR1 mutant (as determined by dilutionplating the sonicated nematodes) consistently remained at ca.26 to 54% that of nematodes colonized by wild-type X. nematophilacells (Fig. 2C). When fluorescent nematode vesicles were countedmicroscopically, wild-type X. nematophila cells fully colonized89 to 95% of nematodes counted over the course of the experiment,as expected (Fig. 2A). The ${Delta}$ cpxR1 mutant, however, fully colonizedonly 44 to 50% of the nematodes counted at all time points,and the number of fully colonized nematodes did not increasesignificantly over time. The percentage of partially colonizedvesicles in both wild-type and ${Delta}$ cpxR1-colonized nematodes decreasedslightly during the course of the experiment (Fig. 2B).

Eliminating aberrant mrxA expression does not rescue ${Delta}$ cpxR1 host interaction defects.
CpxR negatively influences the expression of mrxA (21), whichencodes the X. nematophila type I pilin subunit protein. MrxAhas been implicated in both mutualism (3) and the pathogenesis(3, 28-30) of X. nematophila, and we therefore hypothesizedthat abnormally high levels of the mrxA transcript in the ${Delta}$ cpxR1mutant (21) may contribute to ${Delta}$ cpxR1 mutant-host interactiondefects. For example, increased pili on the cell surface couldmask lectins or receptors necessary for physical interactionswithin the nematode colonization site. Alternatively, withinthe insect or nematode host, an increase in surface pili onthe ${Delta}$ cpxR1 mutant may trigger an immune response (21). To testthese ideas, we deleted mrxA in a ${Delta}$ cpxR1 mutant background. IfmrxA overexpression is responsible for the nematode colonizationdefect seen in the ${Delta}$ cpxR1 mutant, then colonization may be restoredto wild-type levels in the double mutant. One caveat of thisapproach is that mrxA itself was recently implicated as beingnecessary for nematode colonization (3). However, in contrastto that earlier study (3), we saw no decrease in nematode colonizationof our ${Delta}$ mrxA1 mutant, which colonized at 121% ± 6% thatof the wild type (P < 0.05) (Fig. 3A). The absence of mrxA-encodedpilin subunit did not rescue the colonization defect of the ${Delta}$ cpxR1 mutant (Fig. 3A). In this experiment, the ${Delta}$ cpxR1 mutantcolonized nematodes at 67% ± 4% that of the wild type(P < 0.001), while the ${Delta}$ cpxR1 ${Delta}$ mrxA1 mutant colonized at 73%± 6%, a difference statistically different from the wildtype (P < 0.01) but not different from the ${Delta}$ cpxR1 mutant (Fig.3A).

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FIG. 3. Eliminating aberrant mrxA expression in a ${Delta}$ cpxR1 mutant does not rescue nematode colonization or virulence toward insects. For all graphs, error bars represent the standard error, and treatments labeled with different letters are significantly different from each other (P < 0.05). (A) The relative nematode colonization of ${Delta}$ mrxA1, ${Delta}$ cpxR1, and ${Delta}$ cpxR1 ${Delta}$ mrxA1 mutants is expressed as a percentage of the wild-type colonization. The average of two or more experiments is shown. (B) The percent mortalities of insects injected with wild type, ${Delta}$ cpxR1, ${Delta}$ mrxA1, and ${Delta}$ cpxR1 ${Delta}$ mrxA1 mutants are shown. The data represent the average of two or more experiments. WT, wild type.

Since mrxA has also been reported to play a role in insect virulence,we assessed whether the absence of mrxA rescues the virulencedefect of the ${Delta}$ cpxR1 mutant. However, virulence of the doublemutant was not significantly different from that of the ${Delta}$ cpxR1mutant (Fig. 3B). Wild-type cells and the ${Delta}$ mrxA1 mutant killed100% ± 0% of M. sexta insects by 72 h after injection;however, the ${Delta}$ cpxR1 and ${Delta}$ cpxR1 ${Delta}$ mrxA1 mutants each killed significantlyfewer insects than did the wild type (P < 0.01) at 35% ±5% and 40% ± 10% of the insects injected, respectively(Fig. 3B). This finding is also in contrast to previously reporteddata indicating that an mrxA mutant has reduced mortality towardH. armigera insects (3).

Elevated expression of nilA, nilB, and nilC, rescues the colonization defect of the ${Delta}$ cpxR1 mutant.
CpxR positively regulates expression of three genes—nilA,nilB, and nilC (21)—that encode colonization factors (4,23). We hypothesized that the colonization defect of the ${Delta}$ cpxR1mutant is caused by reduced expression of these genes. nilRencodes a repressor of nilA, nilB, and nilC expression (5),and an insertion mutation within nilR elevates nilABC expressionabove that of wild-type X. nematophila levels (5). To test whetherelevation of nilABC expression in a ${Delta}$ cpxR1 mutant would rescuethe mutant's nematode colonization defect, we created a ${Delta}$ nilR17 ${Delta}$ cpxR1 double mutant that expresses greater than wild-type levelsof nilA, nilB, and nilC (Fig. 4) and assessed its ability tocolonize nematodes (Fig. 5). While a ${Delta}$ cpxR1 mutant colonizednematodes at ca. 47% ± 5% that of the wild-type X. nematophilain this experiment (P < 0.001), the ${Delta}$ nilR17 ${Delta}$ cpxR1 double mutantcolonized at 79% ± 7% that of the wild type (P > 0.05)(Fig. 5). As expected (5), the ${Delta}$ nilR17 single mutant colonizednematodes at 117% ± 10%, a level not significantly differentfrom that of the wild type (P > 0.05) (Fig. 5).

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FIG. 4. A mutation in nilR elevates nilABC expression in a ${Delta}$ cpxR1 mutant. The relative expression of nilA, nilB, and nilC (expressed as a percentage of the wild-type activity) was measured by assaying the β-galactosidase activities of reporter fusions in wild-type, ${Delta}$ cpxR1, ${Delta}$ nilR17, and ${Delta}$ nilR17 ${Delta}$ cpxR1 strain backgrounds. The data points represent individual colonies tested, and bars represent strain averages. Different letters indicate values that are significantly different from the wild type (P < 0.01). Outlier ${Delta}$ cpxR1 mutant colonies (circled) were left out of strain averages and statistical analysis. WT, wild type.

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FIG. 5. A mutation in nilR rescues the nematode colonization defect of a ${Delta}$ cpxR1 mutant. The relative nematode colonization of ${Delta}$ cpxR1, ${Delta}$ nilR17, and ${Delta}$ nilR17 ${Delta}$ cpxR1 mutants is expressed as a percentage of the wild-type colonization. The average of two or more experiments is shown. Error bars represent the standard error. Different letters indicate values that are significantly different from wild-type colonization levels (P < 0.001). WT, wild type.

Interestingly, while a majority of the ${Delta}$ cpxR1 single colony isolatestested demonstrated the previously reported reduction in nilgene expression, several ${Delta}$ cpxR1 clones expressing lacZ fusionsto one of the nil promoters exhibited abnormally high expressionof nilA, nilB, or nilC (139, 111, and 458%, respectively, comparedto that of the wild type) (Fig. 4). These clones, whose expressionvaried from the mean by more than twofold the standard deviationvalue, were not counted toward the average gene expression presentedfor a given strain. Potential causes of this phenomenon arediscussed below.

DISCUSSION

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DISCUSSION

In X. nematophila, the Cpx system is necessary for colonizationof S. carpocapsae nematodes and virulence in M. sexta insects(21). In the present study we have shown that cpxR impacts twodistinct stages of nematode colonization: initiation and outgrowth.Furthermore, we show that the likely cause of the colonizationdefect is that the ${Delta}$ cpxR1 mutant does not express wild-type levelsof nil genes encoding membrane-localized proteins necessaryfor nematode colonization.

A ${Delta}$ cpxR1 mutant shows a defect in the colonization of S. carpocapsaenematodes, where, compared to wild-type cells, between 38 and67% as many mutant cells are released from sonicated nematodes(21). Because these numbers represent an average number of bacterialcells recovered from a nematode population, we assessed whetherthe ${Delta}$ cpxR1 mutant colonized only a portion of the nematode populationor whether the entire population of nematodes was colonizedto a lower level. We have shown, by fluorescence microscopyof nematodes cultivated with green fluorescent protein-labeledX. nematophila strains, that the ${Delta}$ cpxR1 mutant failed to colonize40% of the nematode population (Fig. 2). This indicates thatthe defect in this mutant is due in large part to an inabilityto initiate colonization of the nematode host.

The colonization and virulence defects of the ${Delta}$ cpxR1 mutant werenot rescued by deletion of mrxA (Fig. 3), a gene negativelyregulated by CpxR (21). This indicates that elevated mrxA expressiondoes not cause the host interaction defects of the ${Delta}$ cpxR1 mutant.We found that an mrxA mutant does not have host interactionphenotypes, in contrast to a recent report stating that an mrxAmutant has decreased virulence toward H. armigera insects andreduced levels of nematode colonization (3). The authors ofthat study supplied the mrxA mutant construct that was usedto create the strain used in the present study. Furthermore,quantitative PCR analysis confirmed that mrxA transcript isnot expressed in the mrxA mutant made for the present study.Therefore, the discrepancies between the two reports cannotbe explained by differences in the type of mutation. Both studiesused X. nematophila ATCC 19061, although the strains were acquiredat different times and may have diverged. The strain backgroundused in the present study was the source of genomic DNA forsequencing (https://www.genoscope.cns.fr/agc/mage). The genomesequence contains another type I fimbrial subunit homologue,annotated as fimA (XN_978_8275/XN_1735). fimA is 48% identicalin nucleotide sequence to mrxA and shares 36% amino acid identity.Therefore, our ${Delta}$ mrxA1 mutant strain may not display defects inhost interactions due to functional redundancy with fimA. Furtherexperimentation will be required to determine whether this isthe case and whether the loss of both type I fimbrial genescauses a decrease in host interactions in X. nematophila.

Our findings indicate that the overall colonization defectsof the ${Delta}$ cpxR1 mutant are due to insufficient levels of Nil colonizationfactors necessary for initiation. However, our data do not clarifywhether CpxR transcriptional regulation of nil gene expressionis necessary for colonization or whether overexpression of thenil genes simply is sufficient to overcome other defects ofthe ${Delta}$ cpxR1 mutant. For example, the ${Delta}$ cpxR1 mutant may have generalenvelope defects that destabilize Nil proteins in the membrane.Elevated expression of nil genes by removal of NilR-mediatedrepression might allow the expression of sufficient levels ofNil proteins to counteract such a general membrane defect.

Of the 60% of nematodes successfully colonized by the ${Delta}$ cpxR1mutant, 18% were only partially colonized (Fig. 2), suggestingthat, in some nematodes, the mutant fails to grow. A similarcombination of initiation and outgrowth defects has been positedfor a nilA mutant that, unlike nilB and nilC mutants, is ableto partially colonize a fraction (35%) of nematodes (6, 34).Therefore, the defects of the ${Delta}$ cpxR1 mutant in both initiationand outgrowth potentially are due to insufficient expressionof nil genes. Alternatively, CpxR may be necessary for acquisitionof nutrients within the vesicle. Although the vesicle has sufficientlevels of many amino acids and vitamins to support bacterialgrowth (34), it likely is nutrient limited. This idea is basedon findings that X. nematophila grows slowly in the vesicle( $~$ 10 h per doubling compared to 1 h per doubling in LB medium)(33) and that X. nematophila lrp mutants show delayed colonyformation on LB plates when recovered from nematodes or minimalmedium but not when recovered from insect hemolymph or LB medium(7). Two pieces of evidence argue against the idea that theoutgrowth defects of the ${Delta}$ cpxR1 mutant are due to nutrient limitation.First, the ${Delta}$ cpxR1 mutant does not have a growth defect in definedmedium containing leucine, glutamate, pyruvate, aspartic acid,and glucose (G. R. Richards, unpublished data), although growthof the ${Delta}$ cpxR1 mutant in medium lacking sugars has not been tested.Second, metabolic mutants defective in the synthesis of methionineor threonine do not grow in the nematode vesicle. Such mutantsexhibit spheroplast morphology and are eventually cleared fromthe population (34). Such spheroplast morphology has not beenobserved in colonizing ${Delta}$ cpxR1 mutant cells (Fig. 1), and thesemutant cells remain at stable levels over time (Fig. 2).

Unexpectedly, within the ${Delta}$ cpxR1 population, several coloniesdisplayed abnormally high levels of nil gene expression comparedto the majority of other colonies in the population (21) (Fig.4, circled data points). This phenomenon was seen in both previouslystocked (21) and newly constructed nil fusion strains (datafrom both are shown in Fig. 4). There are several possible explanationsto explain individual colonies with CpxR-independent expressionof nil genes, including the presence of suppressor mutations,such as in nilR. Alternatively, nil gene expression may be affectedby an as-yet-undescribed mechanism of phenotypic variation,in which a subpopulation of cells has altered regulation. Forexample, Lrp is necessary for the repression of nil gene expressionand is known to control pleiotropic phenotypes in X. nematophila(7). Therefore, variation in Lrp activity or levels in subpopulationsof the ${Delta}$ cpxR1 mutant could cause variations in nil expression.In E. coli, Lrp and CpxR are known to compete for binding sitesupstream of the operon encoding Pap pili (22). Binding of phosphorylatedCpxR prevents the Lrp-mediated transition between phase OFFand phase ON states of pap gene expression (22). Although noCpxR consensus binding sites were identified upstream of thenil genes in X. nematophila (21), a similar regulatory mechanismacting directly or indirectly to influence nil gene expressionmay occur in X. nematophila. Consistent with this hypothesis,variant colonies with elevated levels of nil gene expressionwere not detected in the wild-type background, raising the possibilitythat the absence of cpxR increases the appearance of such variants.

X. nematophila was recently reported to undergo virulence modulation,in which a subpopulation of wild-type cells exhibited attenuatedvirulence and immune suppression in M. sexta insects but wild-typecolonization levels in nematodes (40). Although the mechanismscontrolling virulence modulation are not known, it is possiblethat nil gene expression is affected by this phenomenon. Toaddress this possibility, it will be of interest to assess thecolonization and virulence phenotypes of individual ${Delta}$ cpxR1 clonesexhibiting elevated nil gene expression and, conversely, tomonitor nil gene expression in virulence-modulated strains.

ACKNOWLEDGMENTS

This research was supported by an Investigators in Pathogenesisof Infectious Disease Award from the Burroughs-Wellcome Foundationand by National Institutes of Health (NIH) grant GM59776. E.E.H.T.was supported by an NIH National Research Service Award (T32AI007414).

We thank Nirupama Banerjee for the gift of plasmid pPK13 andElizabeth Hussa for experimental support.

FOOTNOTES

* Corresponding author. Mailing address: Department of Bacteriology, University of Wisconsin—Madison, 1550 Linden Dr., Madison, WI 53706. Phone: (608) 265-4537. Fax: (608) 262-9865. E-mail: hgblair{at}bact.wisc.edu

Published ahead of print on 17 April 2009.

Present address: Laboratory of Cell Biology, Center for CancerResearch, National Cancer Institute, National Institutes ofHealth, Bethesda, MD 20892.

REFERENCES

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