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Research Article
1 June 2007

Variable-Number Tandem Repeats as Molecular Markers for Biotypes of Pasteuria ramosa in Daphnia spp

ABSTRACT

Variable-number tandem repeats (VNTRs) have been identified in populations of Pasteuria ramosa, a castrating endobacterium of Daphnia species. The allelic polymorphisms at 14 loci in laboratory and geographically diverse soil samples showed that VNTRs may serve as biomarkers for the genetic characterization of P. ramosa isolates.
Pasteuria spp. are endospore-forming bacteria that are obligate parasites of cladoceran crustaceans and nematodes that develop through a water- or a soilborne stage (18). Their coevolution with their respective hosts has provided an opportunity to explore the genetic basis of host-parasite relationships in aquatic and soil environments. The type species for the genus is Pasteuria ramosa, which is found in Europe and North America and is related to Bacillus spp. by 16S rRNA gene homology (7). It is an endoparasite of Daphnia species, planktonic crustaceans that play an important role in the food chains of ponds. A single waterborne endospore may infect, geminate, and proliferate in the body cavity of its host to generate up to 80 million endospores. Transmission occurs horizontally with the infection of new hosts by mature spores released from dead infected hosts. The cost of infection is high, since hosts are completely castrated (8). Infective spores can survive for extended periods in soils, where they form long-lasting spore banks (9).
The infectivity of a spore, i.e., the ability of a spore to infect and propagate within a particular specimen of a Daphnia species, is dependent on the lineage of the parasite and the host (4, 6, 14). Until now, studies of the population genetics, evolution, and epidemiology of P. ramosa have been limited by the lack of genetic markers to distinguish among isolates. Sequence information from Pasteuria species is limited primarily to Pasteuria penetrans, a bacterium infecting phytopathogenic nematodes (5, 16, 19, 20). Identification of individual strains of P. ramosa is difficult because molecular methods used for genotype analyses, such as PCR of randomly amplified polymorphic DNA or restriction fragment length polymorphism analysis, are adversely affected by contamination with the DNA of their hosts. Here we have identified genetic markers based on short tandem repeats that may be used to distinguish isolates and to address the evolution of genetic variants in different environments.
Variable-number tandem repeats (VNTRs) comprised of short sequence repeats (SSRs) constitute a rich source of polymorphism and have been used extensively as markers for discrimination between strains within prokaryotic DNAs (12, 21). VNTR loci have even been found in genetically highly homogenous pathogens, such as Bacillus anthracis (1, 10, 13).
In this study, we describe nine VNTRs in noncoding and putative coding regions of the P. ramosa genome. Two laboratory isolates and bacteria from 11 soil samples collected in the United Kingdom, Belgium, and Russia (Table 1) were typed using these markers to assess the extent of polymorphism at these loci.
A cosmid library containing 25- to 40-kb inserts was generated using high-molecular-weight DNAs isolated from vegetative cells of the laboratory isolate P1 of Pasteuria ramosa. Screening for marker genes for P. ramosa and Daphnia by PCR indicated that approximately 90% of the DNA was P. ramosa DNA. This library was subjected to pyrosequencing (15) and provided contigs representing 3.6 Mb (the predicted genome size is 4 to 4.5 Mb). We searched for repetitive DNA in these contigs by using Tandem Repeats Finder software (2; http://tandem.bu.edu/trf/trf.html ). Short SSRs (repeat units of 3 to 6 nucleotides) were in a minority (6%) compared to repeats harboring 7 to 14 nucleotides (60%) or repeats of >15 nucleotides per unit (34%), which is rather uncommon for the relative abundance of prokaryotic SSRs (21). For DNA polymorphism analysis, we selected 14 SSRs harboring the largest number of repetitions in P1 (Table 2). Eight of these SSRs (indicated with asterisks in Table 2) were located within putative open reading frames (AMIGene Viewer [3; http://www.genoscope.cns.fr/agc/tools/amigene/Form/form.php ]), but no significant similarities were found compared to the corresponding amino acid sequences in the protein sequence databases at the National Center of Biotechnology Information database (Bethesda, MD).
Ten primer sets were designed to amplify these 14 SSRs (Table 2) by using Primer3 software (17; http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi ). Bacterial DNA extraction from infected Daphnia cells was carried out with an EZNA tissue DNA kit (Peqlab) according to the manufacturer's instructions. For pond sediment samples, successful detection of microbial DNA requires adequate purification from the coextracted contaminants that inhibit PCR, such as humic and fulvic acids (22); therefore, we used a SoilMaster DNA extraction kit (Epicentre). Endospores of P. ramosa in pond sediments were subjected to mechanical disruption before extracting the DNA. Bead mill homogenization was carried out with a high-speed (5,000 rpm) bead beater (BioSpec Products, Inc.) after suspending 200 mg of soil samples in 250 μl of soil DNA extraction buffer and 2 μl of proteinase K (50 μg/μl) in tubes containing glass beads (0.5-, 0.1-, and 1-mm diameter). Tubes were subjected to bead beating at 5,000 rpm for one cycle of 10 s, one cycle of 20 s, and three cycles of 30 s successively and then centrifuged at 4,500 rpm for 15 min at 10°C. DNAs were extracted from the supernatant following the kit procedure. PCR amplifications were performed in 25-μl volumes containing 1× PCR buffer [Tris-HCl, pH 8.7, KCl, (NH4)2SO4, 15 mM MgCl2], a 200 μM concentration of each deoxynucleoside triphosphate, a 200 nM concentration of each primer, 0.5 U of HotStarTaq DNA polymerase (QIAGEN GmbH), and 2 μl of template DNA. The PCR cycling conditions were as follows: 15 min at 94°C; 42 cycles of 30 s at 94°C, 30 s at 50°C (primer-specific annealing temperature), and 1 min at 72°C; and a final elongation step for 10 min at 72°C.
Polymorphism was checked for each of the 14 SSRs in the two laboratory isolates by sequencing the PCR products (Fasteris SA, Inc.). Five SSRs, all situated within putative coding regions, did not show variation in the number of repeats, an observation which has been confirmed with four other laboratory isolates (originating from the United States, United Kingdom, Russia, and Belgium). The nine other SSRs (shown in bold in Table 2) showed polymorphisms and were chosen to study diversity in field samples. For genotyping, forward primers were fluorescently labeled. Allele sizes were determined by separation of the PCR products in an ABI PRISM 310 DNA sequencer (Applied Biosystems). Fragment lengths were assigned by Genemapper, using a GeneScan-500 (6-carboxytetramethylrhodamine) size standard. The results are presented in Table 3. In some samples, more than one allele was found for a given primer set. The allele numbers ranged from three for Pr SSR3 to eight for Pr SSR4. We did not find any correlation between the repeat copy number and the allelic variability (Spearman rank test; rho = −0.42; P = 0.26). For some loci, e.g., Pr SSR3, distinct alleles were found for each of the three geographical locations studied. Others showed polymorphism within a studied location (Pr SSR4) or between two samples collected from the same pond during successive years (Pr SSR6, Oxford, pond 8).
For the sequence amplified by the primer set Pr2, which was located within an open reading frame, length variation did not change the reading frame for the putative encoded protein. However, it is known that VNTRs have the potential to affect metabolic regulation, antigenic variation, or environmental adaptation (11). Moreover, extragenic VNTRs can have pronounced effects on adjacent gene expression (21). The biological significance of P. ramosa VNTRs is unknown, but the identification of VNTRs can be a starting point for such research.
These VNTRs are the first molecular markers reported that have allowed the differentiation of populations of Pasteuria spp. as a function of environmental distribution. Moreover, the use of VNTRs for analyzing P. ramosa spore diversity in sediment samples raises the possibility of in situ analysis without isolating bacteria. This approach will facilitate epidemiological, genetic, and ecological studies of this nonculturable bacterium and will be valuable in determining the basis for host preference and virulence of Pasteuria spp. as parasites of phytopathogenic nematodes.
TABLE 1.
TABLE 1. P. ramosa isolates used in this study
IsolateOriginGeographic region, site of isolationaYear of isolation
P1Lab strainGaarzerfeld, Germany1997
P3Lab strainTvärminne, Finland2002
Moscow Zoo 97SedimentMoscow, Russia, zoological garden1997
Moscow NJK 97SedimentMoscow, Russia, Novodevichiy Monastery1997
Moscow V 97SedimentMoscow, Russia, Vorantzovsky Park1997
Oxford Pond 8 96SedimentOxford, United Kingdom, pond 81996
Oxford Pond 8 97SedimentOxford, United Kingdom, pond 81997
Oxford Pond 11 97SedimentOxford, United Kingdom, pond 111997
Oxford Pond 12 97SedimentOxford, United Kingdom, pond 121997
Oxford Pond 17 97SedimentOxford, United Kingdom, pond 171997
Belgium OM1SedimentHeverlee, Belgium, pond OM12006
Belgium OM3SedimentHeverlee, Belgium, pond OM32006
Belgium NeeryseSedimentNeerijse, Belgium2006
a
In Russia, samples were collected from three places in Moscow, namely, the zoological garden, the Vorantzovsky Park of the German Embassy (7 km south of the zoo), and a pond close to the Novodevichiy Monastery (4 km south of the zoo). UK samples were collected from four ponds located within 1 to 2 km of each other, 25 km south of Oxford. Belgium samples came from two adjacent ponds in Heverlee and one pond in Neerijse, a village close to Heverlee.
TABLE 2.
TABLE 2. Primer sequences and repeat motif attributes of 14 P. ramosa SSRs
Primer namePrimer sequenceSSR namecSSR motifRepeat size (nt)No. of repetitions in isolate P1Smallest-largest no. of repetitionsSize range of replicons (nt)No. of alleles
Pr1 fwdACCTAAAGAACAGGAATATCTGGAPr SSR1AAACTAACA973-9195-2495
Pr1 revGCATGGAATGATTTTTGCTG       
Pr2 fwdaCTGCTGGATGGATGGACTACGTGAPr SSR2.1*CCTGGTAAA943-4259-3043
Pr2 revACCGGTCCCGTAGGTATAGGPr SSR2.2*CATCCTGGTGGTCCTTGG1832-4  
Pr3 fwdGGACCAATCGAACCAGGTATPr SSR3*GG(A/G)CCGATGb975-8356-3833
Pr3 revAACGGTTTCTTCGCTTGTTG       
Pr4 fwdGGTAACCCTGGATGTCCTGAPr SSR4TT(A/G)CTTTAb81610-19329-3938
Pr4 revATCCCGTTACAAATGGGACA       
Pr5 fwdaCCCTAAAGGAGACCCAGGAGPr SSR5.1*TGGAGCACC93  1
Pr5 revTGAATCGCACTATTACTTGGAAAPr SSR5.2*AAAGGCGAT917   
Pr6 fwdAACATAAGGGATTAAGGAATGTCPr SSR6TTTTTCTTTTCT1261-6235-2954
Pr6 revTGGAAAAGAAAAGGCATTAGC       
Pr7 fwdAACGTACTGACAAACCAAACCAPr SSR7AACAACC(T/C)Cb9114-11109-1727
Pr7 revAATTTTTCTTAGATTGCTAGGTTGA       
Pr8 fwdGCATCAAATACAAAAACAAATGAAGPr SSR8AGAATATGAAGAAGATGC1844-8386-4585
Pr8 revTGTTTCTCTCGCGTTTCCTT       
Pr9 fwdATACGACGAACGGAAcAAGAPr SSR9AGCAACAAC952-8151-2056
Pr9 revAACCAAAGAATTAACGCCATT       
Pr10 fwdaCATTACTGATTAAGCCGGAATCTAPr SSR10.1*GTTGCTCCG94  1
Pr10 revTCGCAAGCTAATATACCAGGAAPr SSR10.2*ACAGGACCATTTATACCC182   
  Pr SSR10.3*GTAGGACCTGTACGTCCA182   
a
Primers that amplify more than one closely located repeat motif.
b
Imperfect repeats. The nucleotide at the left in parentheses can be replaced by the one at the right.
c
*, located within putative coding region. SSRs in bold were found to be polymorphic.
TABLE 3.
TABLE 3. Amplicon sizes of fragments containing SSRs found in P. ramosa isolates
StrainAmplicon size (nt) with primer       
 Pr1Pr2Pr3Pr4Pr6Pr7Pr8Pr9
Lab P1213286374369295172386178
 231       
Lab P3222304383393283145422169
 249       
Moscow Zoo 97195259374329271109404169
 249286383369 118  
    377 163  
      172  
Moscow NJK 97195259374361271109404169
    369 145422 
      163  
      172  
Moscow V 97249286374369271109458169
  304   145  
      154  
      163  
      172  
Oxford Pond 8 96195259374369271109440169
  286  283145  
      154  
      163  
Oxford Pond 8 97195286374369235109440169
     271145  
      154  
      163  
Oxford Pond 11 97195286383369235109440169
     271145458 
      154  
      163  
Oxford Pond 12 97195286374369271109440151
  304383  145 169
      163  
      172  
Oxford Pond 17 97195259374369271109440169
   383377 145  
      154  
      163  
Belgium OM1231286356321271109404187
      136 196
      145 205
      154  
      163  
Belgium OM3231259356321271109404187
  286 353 145 196
      154  
      163  
Belgium Neeryse231304356321271109404178
    345 145  
      154  
      163  

Acknowledgments

We thank Isabelle Colson and Louis Du Pasquier for helpful advice and Tom Little, Ellen Decaestecker, and Lev Yampolsky for providing samples.
This work was supported by the Swiss Nationalfonds and the Freiwilige Akademische Geselschaft, Basel, Switzerland, and by USDA/CSREES project 50554, USDA/CSREES multistate project NE1019, and the University of Florida Agricultural Experiment Station under CRIS projects FLA-MCS-04353 and FLA-MCS-04080.

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Published In

cover image Applied and Environmental Microbiology
Applied and Environmental Microbiology
Volume 73Number 111 June 2007
Pages: 3715 - 3718
PubMed: 17400766

History

Received: 11 October 2006
Accepted: 24 March 2007
Published online: 1 June 2007

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Authors

Laurence Mouton [email protected]
Zoologisches Institut der Universität Basel, Evolutionsbiologie, Vesalgasse 1, 4051 Basel, Switzerland
Guang Nong
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611-0700
James F. Preston
Department of Microbiology and Cell Science, University of Florida, Gainesville, Florida 32611-0700
Dieter Ebert
Zoologisches Institut der Universität Basel, Evolutionsbiologie, Vesalgasse 1, 4051 Basel, Switzerland

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