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Journal of Bacteriology, July 2008, p. 4870-4879, Vol. 190, No. 14
0021-9193/08/$08.00+0     doi:10.1128/JB.00358-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Xenorhabdus nematophila lrhA Is Necessary for Motility, Lipase Activity, Toxin Expression, and Virulence in Manduca sexta Insects{triangledown}

Gregory R. Richards, Erin E. Herbert, Youngjin Park,{dagger} and Heidi Goodrich-Blair*

Department of Bacteriology, University of Wisconsin-Madison, Madison Wisconsin

Received 11 March 2008/ Accepted 14 May 2008


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ABSTRACT
 
The gram-negative insect pathogen Xenorhabdus nematophila possesses potential virulence factors including an assortment of toxins, degradative enzymes, and regulators of these compounds. Here, we describe the lysR-like homolog A (lrhA) gene, a gene required by X. nematophila for full virulence in Manduca sexta insects. In several other gram-negative bacteria, LrhA homologs are transcriptional regulators involved in the expression (typically repression) of virulence factors. Based on phenotypic and genetic evidence, we report that X. nematophila LrhA has a positive effect on transcription and expression of certain potential virulence factors, including a toxin subunit-encoding gene, xptD1. Furthermore, an lrhA mutant lacks in vitro lipase activity and has reduced swimming motility compared to its wild-type parent. Quantitative PCR revealed that transcript levels of flagellar genes, a lipase gene, and xptD1 were significantly lower in the lrhA mutant than in the wild type. In addition, lrhA itself is positively regulated by the global regulator Lrp. This work establishes a role for LrhA as a vital component of a regulatory hierarchy necessary for X. nematophila pathogenesis and expression of surface-localized and secreted factors. Future research is aimed at identifying and characterizing virulence factors within the LrhA regulon.


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INTRODUCTION
 
Xenorhabdus nematophila is a gram-negative bacterial pathogen capable of infecting and killing several agriculturally relevant species of insects, including the tobacco hornworm, Manduca sexta (23, 41). In nature, X. nematophila forms a mutualistic alliance with the nematode Steinernema carpocapsae, which is the vector that carries the bacterium into insects (23, 72). When X. nematophila is experimentally injected into insects in the absence of the nematode, the bacterium evades and modulates insect immunity, and host death typically occurs within 48 h (19, 37, 54).

X. nematophila produces a plethora of putative virulence factors, including toxins, lipases, hemolysins, and proteases, which may contribute to disease and modulation of host immunity (7, 23). In many cases, the roles of individual factors in disease, as well as how they are coordinately regulated during the infection process, remain undetermined. However, some factors, such as the hemolysin XhlA, have been shown to be necessary for full virulence (16). In addition, several X. nematophila regulator mutants are defective in pathogenesis, including those with mutations in lrp (the gene encoding the leucine-responsive regulatory protein) (15), flhD (27), and the flhDC-dependent fliZ gene (44). Lrp is a global regulator in X. nematophila that affects at least 65% of the proteome and positively regulates the transcription of genes encoding lipase, a crystal protein, and two different hemolysins, as well as factors necessary for mutualistic colonization of the nematode host (13-16). FlhDC is the master regulator of flagellar synthesis and motility and is also necessary for the expression of lipase, protease, and hemolysin activities (16, 27, 53).

The loss of motility and lipase activity in both the lrp and the flhD mutants suggests that the loss of one or both of these activities could contribute to the attenuation of virulence of these mutants. However, the pleiotropic effects of FlhDC and Lrp complicate assigning specific roles in virulence to the genes they regulate. For example, the flhDC mutants are predicted to be defective in type III-like flagellar secretion and swimming, each of which can play distinct roles in pathogenesis (39). Although lipase activities have never been examined directly in X. nematophila, these activities have been implicated in the virulence of several bacterial pathogens (34, 36, 59, 67, 70, 71) and can contribute to pathogenesis through direct damage to host cells and tissues, by evasion or modulation of the immune response, and/or by acquisition of nutrients (1, 12, 24, 29, 42, 43, 51, 65, 66, 70).

To help reveal the identity and regulation of genetic factors potentially contributing to X. nematophila pathogenesis, we isolated mutants that were defective in the production of secreted lipase activity against Tween substrates in vitro. Here, we identify the lysR homolog A (lrhA) gene necessary for this activity. LrhA homologs are LysR-type transcriptional regulators involved in the expression of virulence factors in several gram-negative bacterial pathogens (5, 26, 31, 40, 45, 55). For example, in the plant pathogen Pectobacterium carotovorum (formerly Erwinia carotovora subsp. carotovora), the LrhA homolog HexA represses flagellar components and extracellular enzymes such as proteases and cellulases (31). In the insect pathogen Photorhabdus temperata, the hexA mutant is attenuated in virulence for Galleria mellonella insects, and HexA represses extracellular enzymes and bioluminescence but has no effect on motility (40). Escherichia coli LrhA represses the transcription of genes involved in motility, chemotaxis, and the formation of type I fimbriae (involved in adherence to host cells) and is necessary for biofilm formation (5, 45). Furthermore, E. coli LrhA has been shown to bind directly to the promoter of flhDC (45). Here, we present genetic evidence for the X. nematophila LrhA regulatory function and examine the role of lrhA in X. nematophila motility, secretion, and host interactions.


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MATERIALS AND METHODS
 
Bacterial strains, plasmids, and growth conditions. All bacterial strains and plasmids utilized in these experiments are listed in Table 1. Luria-Bertani (LB) broth (48) was used to culture bacteria at 30°C. Media used to grow X. nematophila strains were either supplemented with 0.1% sodium pyruvate or stored in the dark (73). Unless stated otherwise, plasmids were introduced into X. nematophila strains through conjugation with E. coli S17-1({lambda}pir), as described previously (4, 72). Plasmids were maintained at the following antibiotic concentrations: ampicillin, 150 µg ml–1; chloramphenicol (Cm), 30 µg ml–1 (E. coli) or 15 µg ml–1 (X. nematophila); erythromycin (Erm), 200 µg ml–1; kanamycin (Km), 50 µg ml–1; rifampin (Rif), 100 µg ml–1.


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  		TABLE 1. Strains and plasmids used in this study

Molecular biological methods. This research was performed using standard molecular biological methods (58). DNA was PCR amplified using either ExTaq (Takara, Otsu, Shiga, Japan) or Platinum Pfx (Invitrogen, Carlsbad, CA) according to the manufacturers' instructions. To verify correct sequences, inserts of all constructs were sequenced at the University of Wisconsin Biotechnology Center, using Big Dye version 3.1 (Applied Biosystems, Foster City, CA). PCR purification, plasmid preparation, and gel extraction kits (Qiagen, Valencia, CA) were used according to the manufacturer's directions, as were the restriction enzymes (Promega, Madison, WI). The primers used in this work (Integrated DNA Technologies, Coralville, IA; University of Wisconsin Biotechnology Center, Madison, WI) are presented in Table 2.


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  		TABLE 2. Primers used in this study

Characterization of lrhA and flagellar gene mutations. Mutations in the lrhA, flhD, flgE, and fliC loci were obtained through mini-Tn10 random mutagenesis of wild-type X. nematophila. Mutations were identified based on the loss of in vitro lipase activity against Tween (lrhA) (63) or on the loss of in vitro motility (flagellar genes) (72). Locations of transposon-insertion mutations were ascertained through cloning and sequencing of the mutated regions. In the case of lrhA, arbitrary PCR (10, 52) was used to obtain the complete sequence, which was later verified in the X. nematophila genome sequence (http://www.xenorhabdus.org).

Construction of the xlpA and xptD1 mutants. The xlpA and xptD1 genes were found during a search for lipases and toxins, respectively, in the X. nematophila genome. The 4,176-nucleotide (nt) xptD1 (GenBank accession no. AJ308438) gene has been described previously (49), although only the 3' 3,840 nt were reported. For mutant construction, primers with engineered restriction sites (Table 2) were used to PCR amplify HGB800 chromosomal fragments located upstream (primers XlpAApaUpF and XlpABamUpR or XptD1SalUpF and XptD1BamUpR) and downstream (primers XlpABamDnF and XlpAXbaDnR or XptD1BamDnF and XptD1XbaDnR) of the region to be deleted (1,052 nt for xlpA and 4,019 nt for xptD1). These fragments were subsequently cloned, using the engineered restriction sites (Table 2), into pBluescript II SK+, and the Kmr cassette (BamHI digested from pEV2) was cloned into the unique BamHI site between them. The {Delta}xlpA::Km and {Delta}xptD1::Km constructs were then cloned, using the KpnI and XbaI restriction sites, into the suicide vector pKR100 to create pKRxlpKm and pKRxptD1Km. These constructs were conjugated from E. coli S17-1({lambda}-pir) into HGB800, and the allelic replacement in Kmr Cms exconjugants was verified by PCR amplification.

Construction of Tn7 complementation strains. lrhA and its native promoter region (estimated to be approximately 700 nt upstream of the lrhA translational start site) were PCR amplified using the primers HexAApaF and HexAKpnR with X. nematophila HGB081 genomic DNA. This PCR fragment was cloned into pCR2.1-TOPO and subsequently transferred to pEVS107, creating pEVSlrhA. pEVSlrhA and pEVS107 were transferred, using tri-parental conjugations, into HGB081 (Rifr wild-type X. nematophila strain AN6/1) and HGB760 (lrhA1::Tn10 AN6/1 Rifr) (4). The primers AttTn7EXT and ErmAnch1 were used to confirm proper transposition and insertion of the Tn7 constructs into the attTn7 site of the X. nematophila chromosome.

Construction and expression of the heterologous xlpA vector. The primers pBADxlpARBSF and pBADxlpAR were used to PCR amplify xlpA and its native ribosome binding site (but not its native promoter) from chromosomal DNA. This amplified region was cloned into pBluescript II SK+ through the use of the KpnI and XbaI restriction sites. Next, the xlpA fragment with its native ribosome binding site was gel extracted and cloned into pBAD18 (30) to create pBADxlpA. Either pBADxlpA or pBAD18 (containing no insert, as the negative control) was transformed into the chemically competent X. nematophila (74) HGB 081, lrhA1::Tn10, flhD4::Tn10, flgE1::Tn10, fliC3::Tn10, HGB 800, and xlpA1::Km strains. Expression was induced with 0.2% arabinose. Lipase activity was measured on Tween 20 plates as described previously (63), supplemented with either 15 µg ml–1 Cm or 15 µg ml–1 Cm plus 0.2% arabinose to induce xlpA expression.

Insect virulence assays. Tobacco hornworm (M. sexta) eggs (W. Goodman, University of Wisconsin-Madison; North Carolina State University) were reared to the fourth-instar larvae stage on an artificial gypsy moth wheat germ diet (MP Biomedicals, Aurora, OH) as described previously (72). Stationary-phase cultures of X. nematophila strains were assessed for virulence, as explained previously (16, 50). Briefly, overnight cultures grown at 30°C in LB broth were subcultured at 1:100 dilutions into fresh LB broth and grown for 18 to 24 h. The strains were then washed in phosphate-buffered saline, diluted, and plated on LB agar for the calculation of CFU. Each treatment was injected into 10 larvae per experiment, using a 30-gauge syringe (Hamilton, Reno, NV), and survival was monitored for at least 72 h postinjection. Logarithmic-phase X. nematophila cultures were assayed with the following modifications: subcultures at 1:100 dilution in fresh LB broth were incubated at 30°C until they reached an optical density at 600 nm (OD600) of 0.8. Cultures were then injected at a level of approximately 102 CFU.

Phenotypic and nematode colonization assays. Nematode cocultivations were executed as described previously (33). Briefly, each X. nematophila strain was grown on lipid agar plates to which sterile S. carpocapsae nematode eggs were added. Nematodes reared on these plates were collected, surface sterilized, and sonicated. The sonicated nematode solution was serially diluted, and this information was used to calculate the average CFU per nematode. Bacterial growth rate was based on the OD600 of cultures grown for 24 h in LB broth and was calculated from the exponential phase of growth. Phenotypic plate analyses were performed as described previously to assay motility (72), lipase activity (63), protease activity (6), hemolytic activity (33, 57) on agar containing 5% defibrinated sheep blood (Colorado Serum Company, Denver, CO), and antibiotic activity (47, 72) against Bacillus subtilis. Quantification was achieved by measuring the diameter of the zone of clearing (on antibiotic plates) or swimming (on motility plates). Phenotypic experiments were conducted a minimum of twice, with a minimum of two replicates per experiment.

qPCR detection of transcript levels. Measurement of transcript levels, using quantitative PCR (qPCR), was performed as described previously (16). Using the TRIzol extraction procedure (Invitrogen, Carlsbad, CA), complete cellular RNA was isolated from X. nematophila cultures at an OD600 of 0.9 (log phase) or after 22 h of growth (stationary phase). Residual DNA was removed from the cellular RNA samples with DNase I (Boehringer, Mannheim, Germany). Then, cDNA was synthesized by using random hexamer primers (Integrated DNA Technologies, Coralville, IA) and avian myeloblastosis virus (AMV) reverse transcriptase (Promega, Madison, WI). Next, the cDNA samples were subjected to qPCR in duplicate 25-µl reaction mixtures containing iQ SYBR Green Supermix (Bio-Rad, Hercules, CA) and the relevant primers (listed in Table 2). Water was added in lieu of the cDNA template as a negative control. qPCRs were performed on a Bio-Rad iCycler machine, and the resulting data were analyzed using Bio-Rad iCycler iQ software. Transcript levels of recA, detected with the primers RecAminFor and RecAminRev, were used to normalize cycle threshold results between strain cDNA samples. Statistical analysis was performed with normalized cycle numbers, and data are presented in a form that accounts for the twofold change in the amount of product per cycle.

Statistics. Transcript level cycle numbers and data for motility, antibiotic production, nematode colonization, and virulence LT50 (the time period by which 50% of the insects injected with that treatment died) values were analyzed with either an unpaired t test or one-way analysis of variance (ANOVA) with Tukey's posttest at a 95% confidence interval, using GraphPad Prism software, version 3.0a for Macintosh (GraphPad Software, San Diego, CA). The percent survival virulence data were analyzed using either one-way ANOVA or the stratified log rank test to compare survival curves in SAS software (version 9.1.3; SAS Institute, Cary, NC).

Nucleotide sequence accession number. The lrhA locus with the promoter region was submitted to GenBank under the accession number EF219056.


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RESULTS
 
Identification of the X. nematophila lrhA mutant defective in lipase activity. A library of approximately 6,000 kanamycin-resistant mini-Tn10 transposon insertion mutants was screened for the loss of in vitro lipase activity on Tween agar plates (6, 63), and 7 lipase-deficient mutants were obtained. The disrupted gene in one X. nematophila lipase-deficient mutant was named lrhA, based on its similarity to the E. coli gene encoding the LysR-type transcriptional regulator LrhA. The X. nematophila LrhA protein is predicted to be 330 amino acids in length and is 57% identical and 76% similar to the E. coli homolog. In addition, X. nematophila LrhA is similar to the LrhA homologs (HexA) of the insect pathogen Photorhabdus temperata (85% identity, 91% similarity) and the plant pathogen Pectobacterium carotovorum (57% identity, 76% similarity) (2). The arrangement of the X. nematophila lrhA locus is similar to that of these three bacteria, with lrhA flanked upstream by a divergently oriented gene predicted to encode an aminotransferase and downstream by a tandemly oriented gene predicted to encode a NADH dehydrogenase subunit (Fig. 1). LrhA has a helix-turn-helix domain from amino acids 13 to 72 and a LysR substrate-binding domain from amino acids 94 to 293 (22).


Figure 1
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  		FIG. 1. (A) The lrhA locus of X. nematophila. Black arrows denote the genes and their transcriptional orientation. The inverted triangle represents the position of the mini-Tn10 Kmr transposon insertion. The areas of lrhA predicted to encode a helix-turn-helix motif and a LysR substrate domain are designated by gray and hatched boxes, respectively. Genes were named based on their similarities to E. coli homologs. (B) Upstream region of lrhA containing potential Lrp binding sites. Sites are underlined and were identified based on a match among at least 10 of 15 of the E. coli Lrp binding consensus sequence elements, YAGHAWATTWTDCTR, in which Y = C or T; H = A, T, or C; W = A or T; D = G, A, or T; R = A or G (18). The bent arrow denotes the start of the lrhA open reading frame.

X. nematophila lrhA is necessary for virulence in M. sexta larvae. To determine if LrhA contributes to X. nematophila virulence, we tested the abilities of logarithmic-phase (Fig. 2A) and stationary-phase (Fig. 2B) cultures of the lrhA1::Tn10 mutant to kill M. sexta larvae. (Logarithmic cultures of X. nematophila tend to kill more readily and at lower doses than stationary cultures.) In both cases, the mutant was significantly reduced in its ability to cause mortality in M. sexta, killing fewer than 10% of the insects, compared to a rate of 95% for the wild type (P < 0.001). A wild-type copy of lrhA, introduced by using a Tn7 that transposes to the attTn7 site on the X. nematophila chromosome (4, 46), restored virulence to the mutant, demonstrating that the virulence defect is due to the lrhA mutation and is unlikely to be caused by polar effects on expression of the downstream nuoA gene. The wild-type strain carrying an extra copy of lrhA exhibited a slight but significant decrease in killing relative to that of the wild type, suggesting that LrhA activity may be influenced by its titer.


Figure 2
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  		FIG. 2. The X. nematophila lrhA1::Tn10 mutant exhibits reduced virulence in M. sexta. Approximately 102 logarithmic-phase (A) or 104 stationary-phase (B) X. nematophila cultures were injected into M. sexta, and survival was monitored over the course of 72 h. In each case, one representative experiment is shown. The wild type (squares) or the lrhA1::Tn10 mutant (circles) carrying Tn7 (closed symbol and line) or a wild-type copy of lrhA in Tn7 (open symbol and dashed line) was injected. One-way ANOVA was performed with composite data for all replicates (n = 4 for stationary-phase injections and n = 3 for log-phase injections), and different letters indicate significant differences (P < 0.001).

The lrhA1::Tn10 mutant has reduced motility and flagellar gene expression. lrhA is predicted to encode a transcriptional regulator and is necessary for lipase activity. To identify other possible LrhA-dependent activities, the lrhA1::Tn10 mutant was tested for a variety of qualitative (in vitro dye binding and hemolysin and protease activity) and quantitative (in vitro motility, antibiotic production, and growth rate) phenotypes (Table 3). The lrhA strain exhibited a reproducible defect in motility compared to that of the wild type. However, no other detectable defects were observed. The introduction of a wild-type copy of lrhA into the mutant restored both lipase activity and motility, demonstrating that the lrhA mutation is responsible for these defects.


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  		TABLE 3. Selected phenotypes of the X. nematophila lrhA mutant

In E. coli and P. temperata, LrhA regulates protein levels of the stationary phase sigma factor RpoS (26, 40). Because RpoS is necessary for X. nematophila colonization of the nematode host (72), we also tested the colonization ability of the lrhA1::Tn10 mutant and found it to be no different from that of the wild type (Table 3). In addition, the lrhA1::Tn10 mutant exhibited RpoS levels that were similar to those of the wild type (detected during Western blotting with anti-X. nematophila RpoS antibodies [E. I. Vivas and H. Goodrich-Blair, unpublished] in stationary-phase cultures; data not shown).

To further examine the potential regulation of motility by X. nematophila LrhA, we examined the expression of flagellar genes, using quantitative PCR. flhD transcript levels in the lrhA mutant were approximately 25% of those of the wild type, providing evidence that LrhA is a positive regulator of flhD (Fig. 3). Conversely, lrhA transcript levels in the flhD4::Tn10 mutant were no different from those in the wild type (data not shown). Similarly, putative flagellar regulon members fliA (predicted to encode the class II flagellar sigma factor {sigma}28), flgE (predicted to encode the class II hook protein required for flagellar secretion), and fliC (predicted to encode the class III flagellin subunit) each showed significantly lower RNA levels in the lrhA1::Tn10 mutant relative to those of the wild type (Fig. 3). Introduction of a wild-type copy of lrhA into the mutant restored the flhD transcript level to that of the wild type (106.1% of that of the wild type; P > 0.05; n = 12). Therefore, LrhA positively influences X. nematophila flagellar gene expression and motility.


Figure 3
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  		FIG. 3. Transcript levels of X. nematophila flagellar genes. Total cellular RNA was extracted from logarithmic-phase cultures of the wild type (filled bars) or the lrhA1::Tn10 mutant (open bars), and cDNA was analyzed by qPCR. Levels of flhD (the master flagellar regulator), fliA ({sigma}28), flgE (the hook), and fliC (the flagellin subunit) transcripts are shown. Levels of transcript are reported as percentages of the wild-type strain derived from arbitrary units of transcript. Bars with asterisks are significantly different from the respective wild-type control (P < 0.05, n = 6).

To determine if the loss of motility of lrhA could explain its virulence defects, we examined the virulence of flagellar mutants, including flhD4::Tn10, flgE1::Tn10, and fliC3::Tn10, in M. sexta insects (Table 4). Significantly more M. sexta insects survived when they were injected with the flhD4::Tn10 and flgE1::Tn10 mutants than with wild-type X. nematophila or the fliC3::Tn10 mutant (P < 0.0001; Table 4). Also, flhD4::Tn10 and flgE1::Tn10 exhibited higher LT50 values than the wild type or the fliC3::Tn10 mutant. However, due to variability between experiments, only the flgE1::Tn10 mutant LT50 value was significantly higher than that of the wild type (P < 0.05; Table 4). These data are consistent with those of a previous report that found flhD contributes to X. nematophila F1 virulence in a different insect host; a flhD mutant required a longer time to kill Spodoptera littoralis than wild-type X. nematophila did (27). Thus, FlhDC regulation and flagellar secretion appear to contribute to virulence, but motility itself does not. Since none of the motility mutants displayed a defect in eliciting insect death as severe as that of the lrhA1::Tn10 mutant, these data further demonstrate that the virulence phenotype of the lrhA1::Tn10 mutant cannot be explained solely by its defects in flagellar motility and/or secretion.


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  		TABLE 4. Selected phenotypes of X. nematophila flagellar mutants

xlpA encodes a potential lipase that is likely secreted via the flagellar apparatus. To examine the role of LrhA in regulating lipase activity, we first identified a putative lipase gene within the X. nematophila genome. This confirmed the findings of another group who conducted a similar survey (53). Both studies revealed an open reading frame (http://www.xenorhabdus.org), designated xlpA and predicted to encode a 374-amino- acid protein with characteristic lipase sequence motifs (3, 35, 36) and sequence similarity to the Yersinia enterocolitica lipase YplA (38% identical, 59% similar). YplA is a virulence factor that can be secreted through both the type III secretion system and the flagellar apparatus, which is itself a type III-like secretion system (59, 60, 75, 76). We deleted X. nematophila xlpA and replaced it with a kanamycin-resistant cassette to create xlpA2::Km. This mutant had no detectable in vitro lipase activity against Tween substrates 20, 40, and 60 (data not shown), consistent with the conclusions of Park and Forst (53) that XlpA is the lipase responsible for the Tween activity of X. nematophila. The xlpA2::Km mutant exhibited wild-type levels of virulence in M. sexta insects (survival ± standard error of 0% ± 0%, compared to 0% ± 0% for the wild-type, log-phase cells injected; P > 0.05; n = 3). Thus, the loss of XlpA lipase activity does not explain the virulence defect of the lrhA1::Tn10 mutant.

The xlpA locus has no associated transporter and no predicted type I or type II secretion signal (20, 56). In addition, the X. nematophila genome lacks homologs encoding a nonflagellar type III secretion apparatus (9). These facts suggest that XlpA is secreted through the flagellar type III-like secretion apparatus. The lipase activity phenotypes of flagellar mutants support this notion: the flhD4::Tn10 and flgE1::Tn10 mutants are predicted to lack a complete secretion apparatus, and neither mutant produces in vitro activity against Tween. In contrast, fliC3::Tn10, which lacks the flagellin subunit but is predicted to have a functional flagellar secretion apparatus, does produce lipase activity (Table 4). Taken together, these data suggest that the XlpA lipase is secreted through the flagellar apparatus, consistent with the conclusions of Park and Forst (53).

LrhA positively regulates expression and secretion of X. nematophila xlpA. Our data indicate two possible mechanisms by which LrhA could affect X. nematophila lipase activity: xlpA transcription and/or export of XlpA through the flagellar apparatus. To determine if LrhA affects lipase activity at the transcriptional level, we quantified xlpA transcript in the lrhA1::Tn10 and flhD4::Tn10 regulator mutants. Levels of xlpA transcript were approximately 49% of those of the wild type in the lrhA1::Tn10 mutant and approximately 36% of the wild-type levels in the flhD4::Tn10 mutant (Fig. 4). Introduction of a wild-type copy of lrhA into the lrhA1::Tn10 mutant restored xlpA transcript levels to approximately wild-type levels (74.1% of that of the wild type; P > 0.05; n = 6). Thus, LrhA and FlhDC positively affect xlpA transcription. (The regulation by FlhDC is consistent with the findings of Park and Forst, who reported that xlpA transcript is reduced relative to that of the wild type in both the X. nematophila flhC and fliA mutants (53). However, xlpA transcript levels in lrhA1::Tn10 are roughly half that of the wild type, while in vitro lipase activity is virtually abolished. It is possible that a decrease in XlpA secretion due to the reduced production of the flagellar apparatus in the lrhA1::Tn10 mutant also contributes to the in vitro lipase phenotype.


Figure 4
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  		FIG. 4. Transcript levels of xlpA in the lrhA1::Tn10 and flhD4::Tn10 mutants. Total cellular RNA was extracted from logarithmic-phase cultures of the wild type (filled bar), the lrhA1::Tn10 mutant (open bar), or the flhD4::Tn10 mutant (hatched bar), and cDNA was analyzed by qPCR. Levels of transcript are reported as the percentages of the wild-type strain derived from arbitrary units of transcript. Bars with different letters are significantly different from each other. (P < 0.01, n ≥ 6).

To determine if LrhA also has posttranscriptional influence on XlpA secretion through the flagellar export apparatus, xlpA was ectopically expressed in pBAD18 (pBAD18/xlpA), which has an arabinose-inducible promoter (30), to remove it from its normal regulation by LrhA and FlhDC. This xlpA construct was introduced into the wild type and the lrhA1::Tn10 mutant, as well as into flgE1::Tn10 (which lacks a component of the flagellar secretion apparatus; serves as the negative control), fliC3::Tn10 (which lacks motility but possesses secretion; serves as the positive control), and xlpA2::Km (which lacks lipase activity but is able to secrete; serves as a control for ectopic xlpA expression) (Table 5).


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  		TABLE 5. Influence of ectopic xlpA expression on lipase activitya

As expected, both the wild type and the fliC3::Tn10 positive control expressed lipase activity that increased upon arabinose induction, while the lipase-minus phenotype of the xlpA2::Km mutant was rescued by arabinose induction of pBAD18/xlpA. When pBAD18/xlpA was induced, lrhA1::Tn10 expressed some in vitro lipase activity but not as much as the uninduced wild type. Since this defect in lipase activity is independent of xlpA transcriptional regulation by LrhA, it is likely due to a decrease in XlpA secretion through the flagellar apparatus in the lrhA1::Tn10 mutant relative to that of the wild type. However, flgE1::Tn10, which presumably lacks a functional secretion apparatus containing pBAD18/xlpA, expressed some lipase activity when induced. This could be because the overexpression of xlpA allowed some XlpA to escape through the partially constructed flagellar apparatus, resulting in a "leaky" phenotype. In fact, when pBAD18/xlpA was introduced into flhD4::Tn10 (which is predicted to completely lack the flagellar secretion machinery), no lipase activity was observed upon induction (Table 5). (Negative controls of all of the strains containing only an "empty" pBAD18 vector had the same lipase phenotypes as their parent strains.) Taken together, these data suggest that low expression of the flagellar export apparatus components in lrhA1::Tn10 prevents wild-type secretion of lipase and that LrhA directly or indirectly controls lipase activity at the levels of transcription and secretion.

LrhA positively regulates expression of the X. nematophila toxin gene xptD1. Our data indicate that the positive influence of LrhA on motility and lipase activity cannot by itself account for its crucial role in virulence. Therefore, LrhA likely regulates other virulence determinants. To further explore the LrhA regulon, we examined the expression of a gene predicted to encode a component of a high-molecular-weight protein toxin complex (TC). Xenorhabdus and Photorhabdus spp. produce high-molecular-weight protein TCs with three subunits, A, B, and C. The A subunit appears to be the active component, with the other two serving as potentiators (21). We assessed the expression of the A subunit gene, xptD1, in the wild type and the lrhA1::Tn10 mutant, since expression of this gene is reduced in strains that have undergone virulence modulation (Y. Park and H. Goodrich-Blair, unpublished data) (54). qPCR revealed that levels of the xptD1 transcript are lower in lrhA1::Tn10 than in the wild type (20.5% that of the wild type; P < 0.05; n = 3). Introduction of a wild-type copy of lrhA into the mutant restored the xptD1 transcript level to that of the wild type (128.9% of that of the wild type; P > 0.05; n = 3). To date, there are no reports of regulators controlling the expression of Xenorhabdus TC genes or of the mechanisms of toxin secretion. Although the oral activity of TCs in feeding assays against insects has been well documented (17, 21, 38, 49, 61, 62), a direct role for TCs in Xenorhabdus virulence has not been described previously. We created an insertion deletion mutant, {Delta}xptD1-13::Km, and found it was significantly reduced in virulence compared to that of the wild type (53.3% ± 3.3%, percent survival ± standard error, compared to 3.3% ± 3.3% of the wild type; n = 3; P < 0.05; approximately 106 stationary-phase cells injected). Therefore, the loss of xptD1 expression likely contributes to the virulence defect of the lrhA mutant.

LrhA regulation of protease and hemolysin expression. The lrhA1::Tn10 mutation leads to a decrease in flhDC expression, and FlhDC is known to positively influence protease and hemolysin activities (16, 27, 53). Consistent with these facts, transcript levels of the genes encoding the protease (xptA) and the hemolysin (xaxA) showed a slight but reproducible decrease in the lrhA1::Tn10 mutant compared to that of the wild type, although only the xaxA decrease was statistically significant (xaxA, 24.2% of that of the wild type; P < 0.05; xptA, 68.8% of that of the wild type; P > 0.05; n = 9). Despite the potential influence of LrhA on xptA and xaxA transcription, the lrhA mutant does not display the predicted defects in the corresponding qualitatively measured in vitro C1 hemolysin (8) and the XptA protease (11, 53) activities encoded by these genes (Table 3). However, unlike lipase activity, hemolysins and protease are secreted independently of the LrhA-influenced flagellar export machinery (53; C. Lipke and H. Goodrich-Blair, unpublished). Thus, a likely explanation for this apparent discrepancy is that the reduction in FlhDC-dependent transcription of xptA and xaxA in the lrhA mutant is not severe enough to be detectable in the corresponding in vitro assays for hemolysin and protease activity that were utilized here, due to the absence of additional impacts on secretion.

Lrp positively regulates lrhA transcript levels in X. nematophila. Analysis of the X. nematophila lrhA promoter region revealed several potential binding sites for the global regulator Lrp (Fig. 1B). Like the lrhA1::Tn10 mutant, an X. nematophila lrp-2::Km insertion-deletion mutant is deficient in virulence in M. sexta, as well as deficient in in vitro lipase activity, and this mutant is nonmotile in vitro. However, the lrp-2::Km mutant has pleiotropic phenotypes that indicate the Lrp regulon is broader than that of LrhA (15). These facts suggest that Lrp may regulate lrhA. To address this, we examined transcript levels in both mutants, using qPCR. In the lrp-2::Km mutant, the lrhA transcript levels were approximately 6.8% of that of the wild type (n = 6; P < 0.0001). Introduction of a wild-type copy of lrp into the mutant restored the lrhA transcript to wild-type levels (84.1% of that of the wild type; P > 0.05; n = 6). Thus, Lrp appears to positively regulate the lrhA transcription. Conversely, lrp RNA levels were unaffected in the lrhA mutant (data not shown).


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DISCUSSION
 
X. nematophila lrhA is necessary for virulence and is part of a regulatory hierarchy controlling motility, lipase activity, and toxin expression. X. nematophila possesses numerous predicted virulence factors that may contribute to its ability to kill diverse insect hosts, but little is known about the regulation of these factors or their precise involvement in the infection process. In this study, we demonstrate that X. nematophila lrhA is necessary for virulence in M. sexta insects and positively affects X. nematophila in vitro motility and lipase activity. LrhA affects these phenotypes by positively influencing the transcription of genes in the FlhDC flagellar regulon, including the lipase-encoding gene xlpA (Fig. 4). In addition, LrhA positively regulates expression of the TC toxin-encoding gene xptD1 and is itself positively regulated by the global regulator Lrp. To our knowledge, this is the first report linking lrhA to the Lrp regulon in any system. This laboratory has previously shown that CpxRA, a global regulator in X. nematophila that affects both mutualism and pathogenesis, positively regulates lrhA (32). These results help place LrhA in the context of an emerging regulatory hierarchy controlling aspects of X. nematophila pathogenesis, motility, and exoenzyme production (Fig. 5).


Figure 5
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  		FIG. 5. Model of LrhA regulation of motility and lipase activity in X. nematophila. Transcriptional regulation is indicated by solid arrows, while open arrows indicate influence on flagellar export. Genes whose transcription is positively regulated (whether directly or indirectly) by LrhA are illustrated, as is the regulation of lrhA by Lrp and pxRA. Effects on downstream transcription of motility, lipase, and toxin genes are also shown, as is secretion of the lipase XlpA. The dashed arrow indicates potential interactions between the CpxR and Lrp regulators.

The virulence defect of the lrhA1::Tn10 mutant is more severe than those of individual mutants defective in activities it regulates, including the production of flagella and toxin or lipase activities (Fig. 2 and Table 4) (27, 53). This suggests that infection by X. nematophila is multifactorial, with redundancy and overlap in the actions of individual virulence factors. For example, X. nematophila possesses three distinct hemolysins and lipase and lecithinase activities and encodes at least six TC A subunits, which may each have distinct target specificities (8, 16, 23, 69) (http://www.xenorhabdus.org). Therefore, a regulator mutant that is defective in the expression of multiple factors would logically have a more severe reduction in virulence than a mutant that is defective in the production of only one factor. The lrhA mutant virulence defect is also more severe than that of the flhD regulatory mutants that lack expression of flagella, hemolysin, and lipase activities, suggesting that key LrhA-dependent virulence factors fall outside the flagellar regulon. Examination of the LrhA and FlhDC regulons will likely reveal numerous factors utilized by X. nematophila during pathogenesis.

LrhA regulon: control of flagellar and lipase transcription and lipase secretion through the flagellar type III-like secretion apparatus. Our results demonstrate that LrhA affects the expression of lipase activity by controlling both the transcription of xlpA (Fig. 4) and the secretion of the XlpA lipase through the flagellar secretion apparatus (Table 5). LrhA regulation of xlpA at both the level of transcription and the level of secretion likely explains the severe lipase-deficiency phenotype of the lrhA1::Tn10 mutant. LrhA likely influences both xlpA transcription and flagellar secretion by activating transcription of the genes encoding the master flagellar regulator FlhDC (Fig. 3), although it may also act directly at the xlpA promoter.

Based on our work and that of others (53), it is now well established that the X. nematophila flagellar system functions both in motility and in secretion of at least one nonflagellar substrate and that the loss of either of these functions could explain the attenuated virulence of the flhDC mutants (Table 4) (27). We show here that while the flgE2::Tn10 flagellar secretion mutant has a virulence defect, the nonmotile fliC3::Tn10 mutant does not, indicating that flagellar secretion but not swimming motility plays a role in virulence in X. nematophila. To date, XlpA lipase is the only nonflagellar factor known to be secreted through the flagellar type III-like secretion apparatus (Table 4 and 5) (53). Since the xlpA mutant has no observable defect in virulence in our assays, the delayed killing phenotype of the flgE mutant likely results from the loss of secretion of additional compounds. One such possible factor is xptD1. Our data show that LrhA controls the transcription of this gene and that an xptD1 mutant displays attenuated virulence for M. sexta insects. Recent findings demonstrated that Yersinia pestis TC genes are regulated by YitR, a LysR-type regulator (but not a LrhA homolog) (25) and that the TC can be secreted through the type III secretion pathway. Therefore, it is plausible that, like the secretion of lipase, the X. nematophila TC, including XptD1, is secreted through the flagellar export apparatus. If so, it will be of interest to determine if xptD1, like xlpA, is regulated as a member of the flagellar transcriptional regulon.

LrhA positively influences the expression of multiple genes. The lrhA mutant displays reduced lipase activity, motility, and transcript levels (of the flagellar genes xlpA and xptD1) relative to that of the wild type, highlighting the positive effect of X. nematophila LrhA on regulation. This differs from other LrhA homologs, which typically act as repressors of motility, virulence factors, and secretion activities. For example, in the plant pathogen P. carotovorum, the LrhA homolog HexA represses virulence factors including flagellar components and extracellular enzymes such as proteases and cellulases (31). In addition, E. coli lrhA mutants exhibit increased motility (45). Therefore, our data suggest that X. nematophila LrhA operates differently than its homologs in other bacteria. However, we have not ruled out the possibility that LrhA acts indirectly by repressing the expression of a repressor of motility, toxin expression, and lipase activity.

It is particularly relevant to compare the regulatory role of the LrhA homolog of X. nematophila with that of P. temperata, as these two bacteria share similar life styles as nematode mutualists and insect pathogens (28). In P. temperata, the LrhA homolog HexA represses expression of multiple secretion activities, including lipase, protease, and antibiotic, and undefined factors necessary for growth and development of the nematode host (40). Furthermore, the P. temperata hexA mutant has a competitive virulence defect for Galleria mellonella insects (40). Thus, HexA/LrhA appears to have broader effects in P. temperata than in X. nematophila. These broad effects are more reminiscent of X. nematophila Lrp, since the lrp mutants exhibit defects in multiple secreted enzyme activities, nematode interactions, and virulence (15). In P. temperata, but not X. nematophila, hexA/lrhA is necessary for the expression of RpoS. Therefore, in both organisms, hexA/lrhA is necessary for virulence and is involved in regulating aspects of gene expression, but the mode of regulation (positive in X. nematophila versus negative in P. temperata) and the regulatory hierarchy in which the LrhA homolog functions are distinct.

In sum, this work establishes a role for LrhA in the virulence of X. nematophila for M. sexta insects and as a vital member of an intricate regulatory hierarchy downstream of Lrp and upstream of the flagellar regulon. Our work shows furthermore that the LrhA regulon extends to the toxin gene xptD1 and, likely, to additional as-yet-unidentified virulence genes. Thus, we are beginning to understand the regulatory hierarchy that X. nematophila utilizes to coordinately control its multiple virulence factors during the infection process, and further examination of LrhA regulation and the LrhA-dependent regulon will lend important insights into this area.


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ACKNOWLEDGMENTS
 
We thank W. Goodman for supplying M. sexta eggs for some experiments, M. Clayton and T.-L. Lin for invaluable statistical assistance, G. B. Templeton for initial work on motility, and E. I. Vivas and S. Gilmore for work on initial screens for lipase and motility mutants.

This work was supported by an Investigators in Pathogenesis of Infectious Disease award from the Burroughs Wellcome Foundation and National Institutes of Health (NIH) grant GM59776, both awarded to H. Goodrich-Blair. Additionally, G. R. Richards and E. E. Herbert received support from National Institutes of Health National Research Service Award T32 G07215 from the NIGMS and T32 AI007414 from the NIAID, respectively.


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FOOTNOTES
 
* Corresponding author. Mailing address: 1550 Linden Drive, Madison, WI 53706. Phone: (608) 265-4537. Fax: (608) 262-9865. E-mail: hgblair{at}bact.wisc.edu Back

{triangledown} Published ahead of print on 23 May 2008. Back

{dagger} Present address: Department of Entomology, 413 Biological Science Bldg., University of Georgia, Athens, GA 30602. Back


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Journal of Bacteriology, July 2008, p. 4870-4879, Vol. 190, No. 14
0021-9193/08/$08.00+0     doi:10.1128/JB.00358-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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