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Applied and Environmental Microbiology, January 2008, p. 259-264, Vol. 74, No. 1
0099-2240/08/$08.00+0     doi:10.1128/AEM.01778-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genetic and Immunological Comparison of the Cladoceran Parasite Pasteuria ramosa with the Nematode Parasite Pasteuria penetrans{triangledown}

Liesbeth M. Schmidt,1 Laurence Mouton,2 Guang Nong,1 Dieter Ebert,2 and James F. Preston1*

University of Florida Department of Microbiology and Cell Science, Gainesville, Florida,1 Zoologisches Institut, Universitat Basel, Basel, Switzerland2

Received 31 July 2007/ Accepted 5 October 2007


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pasteuria penetrans, an obligate endospore-forming parasite of Meloidogyne spp. (root knot nematodes), has been identified as a promising agent for biocontrol of these destructive agricultural crop pests. Pasteuria ramosa, an obligate parasite of water fleas (Daphnia spp.), has been shown to modulate cladoceran populations in natural ecosystems. Selected sporulation genes and an epitope associated with the spore envelope of these related species were compared. The sigE and spoIIAA/spoIIAB genes differentiate the two species to a greater extent than 16S rRNA and may serve as probes to differentiate the species. Single-nucleotide variations were observed in several conserved genes of five distinct populations of P. ramosa, and while most of these variations are silent single-nucleotide polymorphisms, a few result in conservative amino acid substitutions. A monoclonal antibody directed against an adhesin epitope present on P. penetrans P20 endospores, previously determined to be specific for Pasteuria spp. associated with several phytopathogenic nematodes, also detects an epitope associated with P. ramosa endospores. Immunoblotting provided patterns that differentiate P. ramosa from other Pasteuria spp. This monoclonal antibody thus provides a probe with which to detect and discriminate endospores of different Pasteuria spp. The presence of a shared adhesin epitope in two species with such ecologically distant hosts suggests that there is an ancient and ecologically significant recognition process in these endospore-forming bacilli that contributes to the virulence of both species in their respective hosts.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Pasteuria spp. are gram-positive endospore-forming obligate parasitic bacteria that have the unique distinction of being hosted by organisms in two distinct phyla, the Nematoda and the Arthropoda. These bacteria include parasites of phytopathogenic nematodes (4, 5, 13, 16, 19, 24, 26) and aquatic cladocerans (Moinidae and Daphinidae) (15) that suppress fecundity in populations occurring in natural environments. The ability of Pasteuria penetrans to suppress the growth of root knot nematodes supports its use as a benign alternative to chemical nematicides (6, 8, 11, 12, 18, 20). Pasteuria ramosa, first described by Metchnikoff in 1888 (22), is as the type species of the genus Pasteuria. The planktonic crustacean Daphnia magna is a vital component of the food chain in freshwater ecosystems, and fluctuations in populations have a profound effect on pond ecology. As one of several naturally occurring parasites of the Daphnidae (16), P. ramosa is thought to play a significant role in the temporal distribution of Daphnia spp. in natural ecosystems (29).

Species assignments for several phytopathogenic Pasteuria spp. and P. ramosa are based on 16S rRNA sequences, morphological properties of mature endospores, and host preferences (2, 4, 14, 16, 19, 24). The phylogenetic relationships based on highly conserved sporulation transcription factors (24, 28, 31) and multiple genetic loci (9) further define the position of P. penetrans in relation to genomically defined Bacillus spp. All of these characteristics indicate that P. ramosa is the most phylogenetically distinct species for which comparisons in this genus have been made. To determine evolutionary relationships, contiguous sequences of the spoIIAA/spoIIAB genes encoding highly conserved sporulation factors have been compared (3, 24). Significant sequence differences clearly distinguished P. ramosa and P. penetrans and also distinguished isolates of P. ramosa obtained from different locations based on the presence of single-nucleotide polymorphisms (SNPs). Isolates of P. penetrans have been shown to harbor silent SNPs in the spoIIAA/spoIIAB genes, and in some cases these SNPs may serve as markers that correlate with virulence for a specific host (23).

Endospore envelope peptides of Pasteuria spp. and biotypes associated with several species of phytopathogenic nematodes have been compared based on immunodetection with a monoclonal antibody (MAb) raised to P. penetrans biotype P20. This antibody is specific for an epitope shared by different polypeptides, resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), and detected on immunoblots, and the "antigenic ladder" distinguishes Pasteuria spp. and biotypes exhibiting host preferences (24). This antibody was developed for environmental detection of P. penetrans and showed no cross-reactivity with endospore-forming bacteria outside the genus Pasteuria (27). The epitope recognized contains a putative β-1,4-linked N-acetyl-D-glucosamine carbohydrate residue (10) that is uniformly distributed on the surface of P. penetrans endospores (27) and is formed in the late stages of spore maturation (7). In the studies described here genetic and immunological approaches were employed to compare and define these obligate parasites of phylogenetically diverse hosts.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial isolates.
P. penetrans isolate P20 originating from Meloidogyne arenaria (Neal) Chitwood race 1 from Levy County, FL, was grown on tomato (Lycopersicon esculentum Mill. cv. Rutgers) in greenhouses. P. ramosa isolates P1 through P5 originated from the following locations: P1 originated from a single infected D. magna female in Gaarzerfeld, Germany; P2 was obtained from pond sediments in Kains in northern England; P3 was obtained from 10 hosts originating from a rock pool in southern Finland; P4 is a mixture of eight lines from eight D. magna females originating from Belgium; and P5 was obtained from sediments originating from a pond at the Moscow Zoo in Moscow, Russia. P. penetrans endospores were cultured and harvested as described previously (27).

Genomic DNA extraction.
All chemicals and reagents used were reagent grade, enzyme grade, or molecular grade. P. penetrans vegetative cells were harvested from 14- to 21-day-old M. arenaria race 1-infected plants as described previously (28). Vegetative cells obtained from live 2-week-old P. ramosa P1-infected D. magna were processed as described previously (27) and placed in 50 µl of 0.01 M Tris-HCl (pH 7.0)-0.01 M EDTA-0.15 M NaCl (TNE buffer). To this preparation 10 µl of a 100-mg/ml lysozyme solution was added, and the resulting solution was incubated at 37°C for 1 h. DNA was extracted using a Qiagen stool DNA kit (Qiagen, Valencia, CA). The DNA was stored at –20°C until it was used. P. penetrans vegetative cells were washed in 0.01 M Tris-HCl (pH 8.0)-0.15 M NaCl (T-NaCl) and resuspended in 1 ml of T-NaCl with 5.0 mM MgCl2. To this preparation 50 µl DNase I and 50 µl RNase were added. The solution was incubated at 37°C for 1 h and centrifuged at 10,000 x g for 5 min, and the pellet was resuspended in T-NaCl containing 10 mM EDTA. The cells were washed and resuspended in 50 µl TNE buffer, and to this preparation 20 µl of a 100-mg/ml lysozyme solution was added. The sample was incubated for 1 h at 37°C. DNA was extracted using a Qiagen DNeasy kit (Qiagen, Valencia, CA). Cell breakage was augmented by repeatedly freezing the sample (three times) in liquid N2 and thawing it in a 65°C water bath. The genomic DNA was stored at –20°C prior to use.

Extraction of bacterial DNA for comparisons of P. ramosa isolate P1 to P5 nucleotide sequences was performed using an EZNA tissue DNA kit (Peqlab, Erlangen, Germany).

PCR amplification, cloning, and restriction digestion.
Primers were designed and used to amplify partial coding sequences in P. ramosa (Table 1). PCR amplicons were purified using a GenElute gel extraction kit (Sigma, Buchs, Switzerland) and either sequenced directed or cloned into the TOPO TA pCR2.1 vector, transformed into chemically competent Escherichia coli TOP10 (Invitrogen, Carlsbad, CA), and prepared for DNA sequencing.


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  		TABLE 1. DNA primers and fluorescent probe used in this study to amplify and detect selected genes

 
Double restriction digestion of the spoIIAA/spoIIAB amplicon sequence of P. penetrans and P. ramosa was performed using Sau3AI and RsaI with bovine serum albumin in NEbuffer I (New England Biolabs, Ipswitch, MA) at 37°C. The digest was resolved on a 1% agarose gel and visualized by staining in 0.1% ethidium bromide.

FISH.
A protocol for performing fluorescence in situ hybridization (FISH) was developed from methods described by Amann et al. (1); this protocol has been described previously (28). A 5'-fluorescein-conjugated DNA probe, 5'-fluorescein-CTACGAACAGCCCTATCTATG corresponding to a segment of the P. ramosa sigE gene, was added to 20 µl of hybridization buffer (20 mm Tris-HCl [pH 7.2], 0.90 M NaCl, 0.01% SDS, 20% formamide) and applied to coverslips. Hybridization to fixed cells was performed at 48°C for 1.5 h in a moist chamber. Each coverslip was washed in 1.5 ml of distilled H2O and air dried prior to mounting in Gel/Mount (Biomedia, Foster City, CA). Preparations were stored in the dark and visualized with a Nikon epifluorescent microscope fitted with an Episcopic fluorescence attachment that housed a 495-nm excitation filter under a x40 fluorescent or x100 differential interference contrast objective with oil.

Immunoblotting of endospore peptides.
P. ramosa P1 endospores were recovered from D. magna cadavers provided by one of us (Dieter Ebert). Pasteuria endospore surface proteins used for SDS-PAGE immunoblotting were extracted as previously described (27). Host cadavers containing mature endospores were gently crushed (to limit maceration of host tissue) with a Teflon mortar in a sterile 1.5-ml microcentrifuge tube containing sterile deionized water. The homogenate was passed through a woven polyester 21-µm mesh filter (Spectra mesh) housed in a 13-mm Swinnex disk holder (Millipore) to separate the debris. The endospore cell pellet was then washed several times in sterile deionized water and stored at –20°C prior to use. Pasteuria endospores remain viable after freezing.

Endospore surface antigens were solubilized by incubation for 2 h at 37°C in a solution containing 6.0 M urea, 3.0 mM dithiothreitol, and 5.0 mM 2-[N-cyclohexylamino]-ethanesulfonic acid buffer (pH 10). Samples were prepared for gel loading in nonreducing treatment buffer (50 mM Tris-HCl [pH 6.8], 2.0% SDS, 10% glycerol, 0.10% bromophenol blue). Peptides were resolved on a 4 to 20% precast gradient polyacrylamide gel (Bio-Rad, Hercules, CA) and processed as previously described (27).


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Morphological comparison and FISH.
Figure 1 shows various life stage morphologies, visualized by differential interference contrast microscopy, for P. ramosa (Fig. 1A) and P. penetrans (Fig. 1B) which were recovered from infected cadavers. Early vegetative stage growth of P. ramosa is shown in Fig. 1A. Intermediate stages in the process of development leading to sporulation are represented by the formation of tetrads and dyads, which are developmental stages that define Pasteuria spp., including P. ramosa and P. penetrans (22, 25, 26). Premature endospores of P. ramosa are "grape-like" cells, and mature spores of P. ramosa and P. penetrans are disk-shaped cells.


Figure 1
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  		FIG. 1. (A and B) Phase-contrast micrographs comparing various life stage morphologies of (A) P. ramosa P1 and (B) P. penetrans P20. An early vegetative stage of P. ramosa P1 is shown in the inset in panel A. Intermediate stages of sporulation are represented by the formation of tetrads and dyads. Endospores are "grape-like" and disk-shaped cells in P. ramosa P1 and P. penetrans P20, respectively. The spore cortex is easily recognized as a dense spherical body at the apex of the cell. Micrographs were taken under oil (magnification, x1,000). The line in panel B indicates 3 nm. (C) FISH of a sigE-specific gene probe hybridizing to P. ramosa P1 vegetative cells. The micrograph was taken with a Nikon epifluorescent microscope equipped with a 495-nm excitation filter under oil (magnification, x1,000).

 
A probe designed using the unique sequence of the P. ramosa sigE gene allowed detection of this gene by FISH. The results show that this probe hybridized to chromosomal DNA present in the early stages of vegetative development, thus validating the origin of the gene sequence (Fig. 1C).

Genomic comparisons.
Phylogenetic analysis of the partial coding sequence of sigE, spoIIAA/spoIIAB, and the 16S rRNA gene placed P. ramosa and P. penetrans together in a distinct clade within the gram-positive endospore-forming bacilli. Nucleic acid sequence alignment of sigE revealed 72.8% pairwise identity between P. ramosa and P. penetrans. The regions of the sigE gene corresponding to specific probe sequences are shown in a sequence alignment in Fig. 2. The partial coding sequences for P. penetrans and P. ramosa spoIIAA/spoIIAB exhibited 69.7% pairwise identity. Double restriction digestion of the partial coding sequences of the spoIIAA/spoIIAB genes using Sau3AI and RsaI enabled differentiation of the species (Fig. 3). Digestion of the P. ramosa P1 and P. penetrans P20 spoIIAA/spoIIAB PCR amplicons using Sau3AI and RsaI resulted in cleavage of the 577-bp parent amplicon that produced 367- and 210-bp fragments for P. penetrans P20 and 327- and 250-bp fragments for P. ramosa P1.


Figure 2
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  		FIG. 2. Nucleic acid alignment of the sigE partial coding sequence using ClustalW, version 1.83. The top line shows the homologous region associated with the P. penetrans P20-specific fluorescein-conjugated probe; the middle line shows the homologous region for the P. ramosa P1-specific fluorescein-conjugated probe; and the bottom line shows the conserved region which is the target for the general Pasteuria probe.

 

Figure 3
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  		FIG. 3. Double restriction digestion of a PCR-amplified spoIIAA/spoIIAB partial coding sequence using Sau3AI and RsaI. Lane 1, undigested spoIIAA/spoIIAB amplicon from P. penetrans P20; lane 2, double digest of P. penetrans P20; lane 3, undigested spoIIAA/spoIIAB amplicon from P. ramosa P1; lane 4, double digest of P. ramosa P1. The numbers at left represent base pairs.

 
Nucleotide sequence variability between populations of P. ramosa.
Partial coding sequences of nine housekeeping genes from five distinct populations of P. ramosa that infect D. magna were sequenced and compared to determine the level of conservation between populations (Table 2). The results indicated that SNPs were present in the partial coding sequences of five of the nine genes analyzed. The genes expressing SNPs included spoIIAA/spoIIAB, atpG, rspA, pheT, and pabA. The genes not displaying SNPs included sigE, spo0A, polC, and the 16S rRNA gene. Silent SNPs were observed in the spoIIAA/spoIIAB amplicon for all five P. ramosa populations in various locations. SNPs were observed to result in conservative amino acid substitutions in three instances or in 21.4% of the sequences analyzed, and these substitutions are not likely to contribute to significant biochemical changes in protein function. Percent identity for a given gene sequence in the five populations was shown to range from >99.1 to 100%.


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  		TABLE 2. Comparison of selected partial coding sequences of conserved housekeeping genes for distinct populations of P. ramosa P1 through P5

 
Immunogenic comparison of endospore envelope proteins.
Labile endospore coat polypeptides of P. penetrans and P. ramosa in endospore coat extracts were resolved by SDS-PAGE and detected on immunoblots with MAb 2A41D10 raised to whole spores of P. penetrans P20, which provided an immunological comparison of the two species of Pasteuria (Fig. 4). The blot demonstrates that there is a universally conserved epitope that is shared by P. penetrans and P. ramosa endospores. The antigenic ladder revealed distribution patterns that were significantly different in the two species. The phytopathogenic nematode parasite contained a more diverse molecular weight array of antigenically reactive peptides than the cladoceran parasite, with some peptides that overlapped and some peptides that were distinct in the two species.


Figure 4
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  		FIG. 4. SDS-PAGE Western blot showing the results of an immunological comparison of P. penetrans P20 and P. ramosa P1 using MAb 2A41D10. Lane 1, P. penetrans P20 endospore coat extract; lane 2, P. ramosa P1 endospore coat extract.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This study revealed that P. penetrans and P. ramosa, which are obligate parasites that are associated with hosts belonging to two distant phyla, the Nematoda and the Arthropoda, are indeed closely related both genetically and immunogenically.

The process of sporulation is tightly regulated in the endospore-forming bacilli (30), and the sigma factor genes principally regulating this pathway (17, 21) are highly conserved between Clostridium spp. and Bacillus spp. (3). Phylogenetic analysis of P. penetrans and P. ramosa based on the sporulation factors sigE and spoIIAB showed that these organisms are both members of a distinct clade within the gram-positive endospore-forming bacilli, supporting previous findings for spo0A (31). Sporulation genes are desirable targets for detection and biotyping or ecotyping of Pasteuria spp. based on their level of conservation and the fact that there is a greater degree of nucleotide sequence variability between and within Pasteuria populations in these genes than in the 16S rRNA gene. To investigate these genes as targets for detection, a specific probe for sigE was designed and used to detect P. ramosa by in situ hybridization. The fluorescein-conjugated gene probe was shown to specifically recognize the sigE sequence in P. ramosa in vegetative cells, thus providing qualitative evidence that the sequence obtained indeed originated from the target organism and was not derived from other host- or culture-associated microparasites or epibionts (29).

The ability to assign a phenotype based on host preference with a particular genotype is a critical factor in further understanding the influence of Pasteuria spp. on soil and water ecology. Nucleic acid sequence alignment analysis of the spoIIAA/spoIIAB gene cluster with only 69.7% identity, along with the presence of SNPs, suggested that this genomic region is a good candidate for genetic isotyping of Pasteuria isolates. Different restriction digestion fragments of the partial coding sequences of the P. penetrans and P. ramosa spoIIAA/spoIIAB amplicons illustrated this capability. A comparison of partial coding sequences of nine housekeeping genes from five distinct populations of P. ramosa infecting D. magna showed the presence of SNPs in five of the nine housekeeping genes investigated, indicating the existence of genetic ecotypes within a species infecting a common host. These data support previous findings showing that there are SNPs in spoIIAB in two biotypes of P. penetrans infecting different Meloidogyne sp. hosts (23). Variations in the nucleotide sequence resulting in silent SNPs and conservative amino acid substitutions observed in isolated populations of P. ramosa may reflect a correlation with host preference predicated by more substantive variations in genes associated with virulence. Comparisons of sequences of sporulation genes from additional isolates of Pasteuria spp. obtained from members of the Nematoda and Arthropoda could provide an opportunity to further evaluate these genes as candidates for genetic markers to distinguish Pasteuria ecotypes and biotypes.

Of considerable interest was the finding that a conserved epitope was present on the endospore envelope of two ecologically divergent Pasteuria spp. The antigenic ladders shown in an SDS-PAGE immunoblot, while distinctly different for the two species, provided evidence that a universally conserved chemistry is associated with endospore surface or coat proteins. The MAb used to probe the immunogenic profile of the endospore envelope was previously shown to be specific for Pasteuria based on challenges with a number of soilborne gram-positive endospore-forming bacteria (27) and was also shown to detect epitopes on a variety of Pasteuria isolates obtained from other phytopathogenic nematodes (24). The antigenic ladders have been shown to differ even at the biotype level for all isolates examined so far when comparisons between isolates from different hosts and host strains have been made. These results demonstrate that Pasteuria spp. harbor a unique spore envelope epitope, presented late in sporulation (7), that is universal and distinct from the epitopes of other closely related organisms. Because Pasteuria, like most pathogenic bacteria, must first recognize and bind to a host ligand as a prelude to infection, the common endospore epitope found here may well represent a virulence determinant critical to the infection process for both species that warrants further evaluation.

The evolution of the obligate host-parasite relationships of P. ramosa with Daphnia spp. and of P. penetrans with Meloidogyne spp. suggests that there are common developmental requirements in both the cladoceran and nematode hosts. All Pasteuria spp. suppress or eliminate the fecundity of their hosts (16). Both the cladoceran and nematode hosts reproduce by cyclic parthenogenesis (i.e., alteration of asexual and sexual reproduction) and produce eggs having a chitinous shell. Based on lectin blotting with wheat germ agglutinin, the epitope recognized by MAb 2A41D10 contains β-1,4-linked N-acetyl-D-glucosamine residues (10, 24). It may be that a common glycochemistry and the clonal population dynamics of the host population contribute to the interaction between the host and the parasite and thereby confer virulence to a Pasteuria sp. or biotype for a particular host.


   ACKNOWLEDGMENTS
 
This work was supported by USDA/CSREES project 50554, USDA/CSREES multistate project NE1019, and University of Florida IFAS Agricultural Experiment Station CRIS projects FLA-MCS-04353 and FLA-MCS-04080. D.E. and L.M. were supported by the Swiss National Funds.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology and Cell Science, University of Florida, Gainesville, FL 32611. Phone: (352) 392-5923. Fax: (352) 392-5922. E-mail: jpreston{at}ufl.edu Back

{triangledown} Published ahead of print on 12 October 2007. Back


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Applied and Environmental Microbiology, January 2008, p. 259-264, Vol. 74, No. 1
0099-2240/08/$08.00+0     doi:10.1128/AEM.01778-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.




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