Environmental Microbiology (2009) 11(4), 733740

doi:10.1111/j.1462-2920.2008.01856.x

Minireview Applications of the rep-PCR DNA ngerprinting technique to study microbial diversity, ecology and evolution

Satoshi Ishii1 and Michael J. Sadowsky2,3,4* 1 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Tokyo, Japan. 2 Department of Soil, Water, and Climate, 3 BioTechnology Institute and 4Microbial and Plant Genomics Institute, University of Minnesota, St. Paul, MN, USA. Summary A large number of repetitive DNA sequences are found in multiple sites in the genomes of numerous bacteria, archaea and eukarya. While the functions of many of these repetitive sequence elements are unknown, they have proven to be useful as the basis of several powerful tools for use in molecular diagnostics, medical microbiology, epidemiological analyses and environmental microbiology. The repetitive sequence-based PCR or rep-PCR DNA ngerprint technique uses primers targeting several of these repetitive elements and PCR to generate unique DNA proles or ngerprints of individual microbial strains. Although this technique has been extensively used to examine diversity among variety of prokaryotic microorganisms, rep-PCR DNA ngerprinting can also be applied to microbial ecology and microbial evolution studies since it has the power to distinguish microbes at the strain or isolate level. Recent advancement in rep-PCR methodology has resulted in increased accuracy, reproducibility and throughput. In this minireview, we summarize recent improvements in rep-PCR DNA ngerprinting methodology, and discuss its applications to address fundamentally important questions in microbial ecology and evolution.

Introduction Microbial genomes contain a variety of repetitive DNA sequences, accounting for up to 5% of the genome (Ussery et al., 2004). Many of these repetitive DNA elements are of unknown function and have been localized to both intergenic and extragenic regions of the microbial genome. Recent data suggest that some of these repetitive elements may be involved in the synthesis or catabolism of RNA and DNA (Tobes and Ramos, 2005), or to be mediators of genome evolution (Schmidt and Anderson, 2006). These repetitive elements are typically comprised of duplicated genes, such as those encoding rRNA and tRNA, insertion sequence elements, transposons, short polynucleotide (37 mers) repeats, mosaic repetitive elements and other repetitive or palindromic sequences (Table 1) (Sadowsky and Hur, 1998; Versalovic and Lupski, 1998). The sizes of these interspersed repetitive elements vary from 15 bp (e.g. ve-time repeats of trinucleotides) to hundreds of base pairs in length (e.g. RLEP element) (Versalovic and Lupski, 1998). Analysis of the whole genome sequences of many microbial species has revelled the presence of numerous repetitive sequences (Ussery et al., 2004; Tobes and Ramos, 2005). While some repetitive elements (e.g. HRS1 and RLEP) are specically present in only a limited group of bacteria, others, such as the repetitive extragenic palindromic (REP) DNA sequences, have been found to be broadly distributed among many members of phylogenetically diverse bacteria (Sadowsky and Hur, 1998; Versalovic and Lupski, 1998; Tobes and Ramos, 2005). The use of repetitive elements for the analysis of bacterial genomes has proven to be a powerful tool for studies of microbial ecology, environmental microbiology, molecular diagnostics, medical microbiology and epidemiological analyses. Since each microbial strain or isolate has repetitive sequences located in distinct regions of their genome, the PCR technique, performed using sequences of these repetitive elements as primers and total genomic DNA as template, generates multiple amplicons that differ in size in direct proportion to the genomic

Received 14 October, 2008; accepted 27 November, 2008. *For correspondence. E-mail Sadowsky@umn.edu; Tel. (+1) 612 624 2706; Fax (+1) 612 625 2208.

2009 The Authors Journal compilation 2009 Society for Applied Microbiology and Blackwell Publishing Ltd

734 S. Ishii and M. J. Sadowsky

Table 1. Selected endogenous repetitive sequences found in microbial genomes Sequence name BIME BOXa ERICa HRS1 (GTG)5a IRU IS6110 MPTR REPa RLEP RSRja, RSRjb STRR Repetitive sequence type Mosaic repetitive element Mosaic repetitive element Large repetitive element Insertion sequence Polynucleotide repeat Large repetitive element Insertion sequence Polynucleotide repeat Palindromic sequence Large repetitive element Insertion sequences Polynucleotide repeat Reference Gilson et al. (1991) Martin et al. (1992) Hulton et al. (1991) Judd and Sadowsky (1993) Doll et al. (1993) Sharples and Lloyd (1990) Thierry et al. (1990) Hermans et al. (1992) Stern et al. (1984); Tobes and Ramos (2005) Woods and Cole (1990) Kaluza et al. (1985) Mazel et al. (1990)

a. Commonly used for the rep-PCR DNA ngerprinting. BIME, bacterial interspersed mosaic element; ERIC, enteric repetitive intergenic consensus; HRS1, hyper-reiterated sequences; IRU, intergenic repeat unit; MPTR, major polymorphic tandem repeat; STRR, short tandemly repeated repetitive sequences.

distance between the binding sites of adjacent repetitive elements. Following electrophoresis, the distribution of the resolved amplicons generates a genomic DNA ngerprint pattern unique for each bacterial strain or isolate (Rademaker et al., 2008). This technique is most often referred to as repetitive sequence-based PCR (Versalovic et al., 1991) or repetitive extragenic palindromic PCR (rep-PCR), but other closely worded synonyms can also be frequently found in the literature. The rep-PCR DNA ngerprinting technique has proven to be a valuable tool to identify, track and examine diversity among medically and environmentally important microorganisms (Sadowsky and Hur, 1998; Versalovic et al., 1998; Rademaker et al., 2008). Most rep-PCR-based DNA ngerprinting studies have used short polytrinucleotides, such as (GTG)5, 3540 bp REP sequences, 124127 bp enterobacterial repetitive intergenic consensus (ERIC) sequences or the 154 bp BOX element as priming sites for PCR (Table 1) (Versalovic et al., 1991; 1994; Rademaker et al., 2008). Although other genomic DNA ngerprinting methods are also useful to group and identify microbes (e.g. pulsed-eld gel electrophoresis; PFGE), the rep-PCR DNA ngerprinting technique is a simple and rapid method that has the necessary resolving power for microbial identication at subspecies or strain level (Versalovic et al., 1998). In contrast, DNA:DNA hybridizations, 16S rRNA gene sequencing or ribotyping are more useful than rep-PCR in distinguishing bacteria at the generic or species level. While several comparative analyses have shown that rep-PCR and PFGE have nearly equal discriminating power, the former technique can be performed with standard equipment and with less labour, time and cost, and more throughput than PFGE (Olive and Bean, 1999). Hahm and colleagues (2003) compared rep-PCR, multiplex-PCR, PFGE, ribotyping and AFLP to characterize Escherichia coli strains, and concluded that rep-PCR

and PFGE were the most discriminative DNA ngerprinting methods available. Similarly, Foley and colleagues (2006) reported that rep-PCR, PFGE and multilocus sequence typing (MLST) had better discriminant power than did plasmid proling and antimicrobial susceptibility testing when subtyping Salmonella enterica serovar Typhimurium strains obtained from various animals. Given the usefulness of rep-PCR technique to genotypically characterize and track a large number of agriculturally, medically and environmentally important microorganisms, the objectives of this minireview are to: (i) summarize recent advances in rep-PCR DNA ngerprinting methodology, and (ii) describe recent applications of this tool that address fundamentally important questions in microbial ecology and microbial evolution. Other recent reviews concerning the rep-PCR technique are also available elsewhere (Sadowsky and Hur, 1998; Versalovic et al., 1998; Frye and Healy, 2006; Rademaker et al., 2008). Methodological considerations Several modications of the standard rep-PCR technique have been investigated to increase accuracy, reproducibility and throughput, and at the same time decrease time, labour and cost associated with this technology. The quality and ultimate usefulness of the DNA ngerprints generated by using rep-PCR are inuenced by both the PCR reaction itself and the method(s) used to resolve the generated amplicons into discrete bands. On the level of the PCR reaction itself, the improved quality of Taq DNA polymerase, which can generate larger sized DNA fragments (~10 kb) than previous formulations, was shown by Chokesajjawatee and colleagues (2008) to produce repPCR DNA ngerprints that clustered Vibrio strains similar to what was found by using DNA:DNA hybridizations, but with better resolution. This technique has been referred to

2009 The Authors Journal compilation 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 733740

Rep-PCR DNA ngerprinting 735 as long-range rep-PCR (Brumlik et al., 2001; Chokesajjawatee et al., 2008). Deciencies in the resolution of gel-based electrophoresis systems have also been shown to limit the ability of rep-PCR DNA ngerprinting to differentiate among closely related or genetically near-identical strains (Johnson et al., 2004). The uorophore-enhanced repPCR (FERP) technique allows precise size calling of uorescently labelled DNA ngerprint bands relative to uorescently labelled internal DNA size markers applied in the same lane as the samples to be analysed (Versalovic et al., 1995; Rademaker et al., 2008). While the original FERP method was relatively expensive and required an automated system for fragment separation and detection, such as a capillary gel sequencing unit (Rademaker et al., 2008), Johnson and colleagues (2004) reported the development of a horizontal, uorophoreenhanced rep-PCR (HFERP) technique that used conventional horizontal agarose gel electrophoresis coupled to the use of uorescently labelled amplicons and uorescently labelled internal size standards. Since the internal size standard was applied in every lane of the gel, each band in each DNA ngerprint pattern could be normalized to the internal standard. This reduced error due to inherent problems associated with gel-based electrophoresis, and genetically identical strains were found to have DNA ngerprints that were > 92100% similar. This technology has been successfully used to characterize and determine the potential sources of many thousands E. coli strains (Johnson et al., 2004; Ishii et al., 2007). Recently, a semi-automated rep-PCR DNA ngerprinting system has become commercially available. The DiversiLab system (bioMrieux, Marcy eEtoile, France) uses a microuidics device to separate rep-PCR amplicon rapidly and reliably (Healy et al., 2005), which can be subsequently analysed using web-based DiversiLab software and libraries of ngerprints from several bacterial (Cangelosi et al., 2004; Freeman et al., 2005; Ross et al., 2005; Harrington et al., 2007; Vogel et al., 2007), archaeal (Cleland et al., 2008) and eukaryote species (Wise et al., 2007). This semi-automated rep-PCR system was reported to have discriminant power equal to PFGE when typing Streptococcus pneumoniae, and was capable of distinguishing among Mycobacterium tuberculosis and Mycobacterium avium complex strains with a resolution equivalent to IS6110-RFLP analysis (Cangelosi et al., 2004; Freeman et al., 2005). Computer-assisted DNA ngerprint acquisition and data analysis has also increased the accuracy of the rep-PCR DNA ngerprinting technique. While it is possible to analyse rep-PCR DNA ngerprinting data by eye when a sample set is relatively small and banding patterns are relatively simple, this analysis is still subjective and labour-intensive (Rademaker et al., 2008). This problem is amplied when large data sets are analysed and DNA ngerprints contain many closely spaced bands. Several free and commercially available computer programs have proven especially useful in the rapid, bias-free, analysis of DNA ngerprint patterns, and the subsequent statistical analysis of the data. Computer-assisted DNA ngerprint analysis involves three distinct steps: (i) ngerprint acquisition, (ii) ngerprint normalization and (iii) statistical analysis of ngerprint data. The R program, a free resource at http://www.r-project.org, and clustering algorisms have been shown to increase the accuracy and reproducibility of analyses of multiple rep-PCR DNA ngerprinting data sets (S. Ishii and K. Kadota, unpubl. data). For more comprehensive analyses, commercially available software is frequently used. Most studies have used the GelCompar and BioNumerics (Applied Math, Kortrijk, Belgium) software packages which facilitate ngerprint acquisition and normalization, and allow the user to determine correlations between banding patterns, draw dendrograms and perform a variety of statistical analyses, including correspondence, clustering and discriminant analyses. Computer-assisted rep-PCR DNA ngerprinting data analysis is described in more detail elsewhere (Rademaker and de Bruijn, 2008). Applications of rep-PCR DNA ngerprinting for environmental microbiology The rep-PCR technique has been extensively used over the last decade for molecular epidemiological applications and to classify human and plant pathogens, plant symbionts, soil bacteria, and a large number of other environmentally and medically relevant microorganisms (see reviews by Sadowsky and Hur, 1998; Versalovic et al., 1998; Frye and Healy, 2006; Rademaker et al., 2008). While several studies have used rep-PCR DNA ngerprinting to establish associations between particular microbial strains or pathovars with specic human, animal or plant diseases (Versalovic et al., 1998), the technique has also proven useful to determine sources of microorganism and their transmission in disease outbreaks. For example, the source of microbes responsible for a large tuberculosis outbreak among homeless persons in Seattle, Washington, was identied by using rep-PCR DNA ngerprinting system and the Bacterial Barcodes (Athens, GA) mycobacterial primer kit (Freeman et al., 2005). Sources of non-disease-causing microorganisms have also been investigated by using rep-PCR DNA ngerprinting. For example, while E. coli is currently used as an indicator of faecal contamination, the potential source(s) of this bacterium in the environment is usually not known (Ishii and Sadowsky, 2008). Rep-PCR and HFERP DNA ngerprinting have both been used in an attempt to differ-

2009 The Authors Journal compilation 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 733740

736 S. Ishii and M. J. Sadowsky entiate among genetically near-identical, but ecotypically distinct E. coli originating from various animal host sources (Dombek et al., 2000; Johnson et al., 2004). The overall basis for this approach is that the rep-PCR DNA ngerprint patterns of environmental E. coli samples should be similar to the ngerprints of E. coli in knownsource libraries, thus allowing identication of unknown isolates (Johnson et al., 2004; Ishii et al., 2007). While large databases are necessary to adequately represent genotypic diversity in E. coli populations obtained from multiple host animals, rep-PCR DNA ngerprinting appears to be a useful tool to analyse faecal contamination in environments containing a limited number of potential input sources (Ishii and Sadowsky, 2008). For example, Ishii and colleagues (2007) successfully applied HFERP DNA ngerprinting to determine the potential sources of E. coli contaminating a beach near Duluth, Minnesota. The technique was found to be useful in differentiating E. coli originating from treated wastewater, in spring samples, from those coming from waterfowl, which were present on the beach in the summer to fall months. Similarly, Vogel and colleagues (2007) used automated rep-PCR (the DiversiLab system) and reported that cattle and wildlife were the main sources of E. coli in river water and sediment samples in south-central Nebraska. The HFERP DNA ngerprinting method has also been used to examine the presence, genotypic distribution, survival and persistence of specic E. coli clones in the environment (Byappanahalli et al., 2006; Ishii et al., 2006). Ishii and colleagues (2006) reported that indigenous soilborne E. coli clones can be found to overwinter in soils form northern Minnesota over multiple seasons. Similarly, the clonality of Bacteroides in faecal samples from humans who received a 7-day antibiotic treatment, and the shift in population structure of acetic acid bacteria during wine fermentation have also been examined by using rep-PCR DNA ngerprinting (Gonzlez et al., 2005; Jernberg et al., 2007). The rep-PCR DNA ngerprinting technique has been used to examine genetic diversity among nitrogen-xing bacteria, including species members within the genera Rhizobium and Bradyrhizobium (Sadowsky and Hur, 1998), and has proven to be useful to examine the phylogeny of Nitrobacter strains isolated from geographically and ecophysiologically different origins (Vanparys et al., 2007). Moreover, due to its high resolving power, the technique was also found to be useful in distinguishing among closely related plant pathogens, including disease-causing and -suppressive Streptomyces strains (Sadowsky et al., 1996), and to type Xanthomonas sp. strains to the subspecies, strain or pathovar level (Rademaker et al., 2005). Although the rep-PCR DNA ngerprinting technique has mostly been used as genotyping and strain-tracking tools, it also has other uses. For example, rep-PCR has been used to generate speciesand strain-specic DNA probes by targeting rep-PCR amplicons in Campylobacter (Giesendorf et al., 1993), Helicobacter (Kwon et al., 1998) and Burkholderia (Matheson et al., 1997). In this case, DNA bands specic to species or strains of interests were isolated directly from gels (Giesendorf et al., 1993; Kwon et al., 1998), or by cloning of rep-PCR products (Matheson et al., 1997). After specicity testing, these fragments can be useful as hybridization probes for detection of specic bacterial species or strains in environmental samples. Microbial genome evolution While rep-PCR DNA ngerprint patterns are thought to be stable over many generations of microbial growth (Kang and Dunne, 2003), they are susceptible to changes over time caused by polymorphisms, prophage insertions, loss or rearrangement of indigenous plasmids, recombination between repeated sequences, and introgression of genomic DNA via horizontal gene transfer (Sadowsky and Hur, 1998). Since all of these factors alter the genome structure of microorganisms, rep-PCR ngerprinting can be used to study microbial genome evolution and molecular phylogeny, and the plasticity of microbial genomes. Escherichia coli reference strains, such as those in the ECOR collection (Ochman and Selander, 1984), are often used for studies of microbial evolution and phylogeny. While initial studies performed using multilocus enzyme electrophoresis (MLEE) classied the ECOR strains into six phylogenetic groups (A, B1, B2, C, D and E) (Goullet and Picard, 1989), strains within a given phylogenetic group could not be clustered based on rep-PCR DNA ngerprinting patterns (Johnson and OBryan, 2000). Similar trends were also reported for analyses performed using microsatellites (short-motif repeats) to characterize ECOR strains (Metzgar et al., 2001). Ishii and co-workers also noted that there was not a direct correlation between HFERP DNA ngerprint patterns and phylogenetic groups among a larger set of E. coli isolates (n = 1531) obtained from humans and 12 animal sources, an example of which is shown in Fig. 1 (S. Ishii, K.P. Meyer and M.J. Sadowsky, unpublished). This disparity extends beyond phylogenetic group classication; the PFGE (Ohnishi et al., 2002) and HFERP (L.K. Johnson, J.A. Ferguson and M.J. Sadowsky, unpubl. data) techniques have also revealed great genetic diversity among DNA ngerprinting of E. coli O157:H7 serotype strains. This lack of correlation may be due, in part, to the hypermutable microsatellites, effects of prophages (Metzgar et al., 2001; Ohnishi et al., 2002) or lack of sensitivity. In addition, while MLEE detects polymorphic variation in enzymes revealed following electrophoresis, serotyping is based on variation in somatic (O), agella (H) and capsular (K) antigens as

2009 The Authors Journal compilation 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 733740

Rep-PCR DNA ngerprinting 737

Fig. 1. Dendrogram showing the relatedness of E. coli strains isolated from humans, cows and sheep as determined by HFERP DNA ngerprint analysis performed using the BOXA1R primer. Phylogenetic groups were determined by using multiplex PCR as previously described (Clermont et al., 2000). Ten strains each were randomly chosen from a collection (n = 1531) of E. coli strains obtained from humans and 12 animal sources. Relationships between DNA ngerprints were determined by using Pearsons curve-based correlation coefficient and the dendrogram was constructed by using the UPGMA clustering method.

detected by several immunological-based assays. Thus, these methods use indirect gross changes in enzyme mobility or serological reaction to categorize strains. In contrast, since genome ngerprinting methods, such as rep-PCR and PFGE, directly detect changes in nucleotide base composition, they provide a simple, rapid and direct method to identify possible rearrangements, or compositional changes in microbial genome structure. Rep-PCR DNA ngerprinting has been recently used to examine the loss of virulence genes from E. coli in the environment. Duriez and colleagues (2008) reconstituted E. coli populations by mixing 48 different strains with autoclaved manure slurry and used rep-PCR DNA ngerprinting to examine clonality of populations. They reported that virulence genes were lost from specic clonal populations during manure storage (within 3 weeks), suggesting that gene content within the populations were changing in

response to the survival and/or adaptation to this new environment. These authors also observed the appearance of new rep-PCR DNA ngerprint patterns that were different from those of the initial 48 strains, indicating the emergence of new genotypes during manure storage, potentially due to prophage-mediated genome rearrangements. Concluding remarks Although the majority of the rep-PCR DNA ngerprinting studies have been performed for epidemiological and strain tracking purposes, this technique also has great versatility and can be used to study microbial ecology and microbial evolution. This is chiey due to the fact that the rep-PCR technique has the sensitivity to accurately genotype microbes at the strain or isolate level. As such, it has

2009 The Authors Journal compilation 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 733740

738 S. Ishii and M. J. Sadowsky great versatility for investigations of microbial community structure, population dynamics, strain survival and host microbe associations. Currently, the isolation of the target microorganisms is a prerequisite for performing rep-PCR DNA ngerprinting, due to the complexity of banding patterns in microbial community DNA. However, whole genome amplication (e.g. multiple displacement amplication; Binga et al., 2008) can be coupled to the rep-PCR technique to allow for the determination of the genotypes of single cells without cultivation. If the resulting ngerprints are reproducible, it will open a new door for the study of the uncultured microbial world at the strain rather than population or community levels.
Clermont, O., Bonacorsi, S., and Bingen, E. (2000) Rapid and simple determination of the Escherichia coli phylogenetic group. Appl Environ Microbiol 66: 45554558. Doll, L., Moshitch, S., and Frankel, G. (1993) Poly(GTG)5associated proles of Salmonella and Shigella genomic DNA. Res Microbiol 144: 1724. Dombek, P.E., Johnson, L.K., Zimmerley, S.T., and Sadowsky, M.J. (2000) Use of repetitive DNA sequences and the PCR to differentiate Escherichia coli isolates from human and animal sources. Appl Environ Microbiol 66: 25722577. Duriez, P., Zhang, Y., Lu, Z., Scott, A., and Topp, E. (2008) Loss of virulence genes in Escherichia coli populations during manure storage on a commercial swine farm. Appl Environ Microbiol 74: 39353942. Foley, S.L., White, D.G., McDermott, P.F., Walker, R.D., Rhodes, B., Fedorka-Cray, P.J., et al. (2006) Comparison of subtyping methods for differentiating Salmonella enterica serovar Typhimurium isolates obtained from food animal sources. J Clin Microbiol 44: 35693577. Freeman, R., Kato-Maeda, M., Hauge, K.A., Horan, K.L., Oren, E., Narita, M., et al. (2005) Use of rapid genomic deletion typing to monitor a tuberculosis outbreak within an urban homeless population. J Clin Microbiol 43: 5550 5554. Frye, S.R., and Healy, M. (2006) Molecular strain typing using repetitive sequence based PCR. In Advanced Techniques in Diagnostic Microbiology. Tang, Y.W., and Stratton, C. (eds). New York, NY, USA: Springer, pp. 444471. Giesendorf, B.A., van Belkum, A., Koeken, A., Stegeman, H., Henkens, M.H., van der Plas, J., et al. (1993) Development of species-specic DNA probes for Campylobacter jejuni, Campylobacter coli, and Campylobacter lari by polymerase chain reaction ngerprinting. J Clin Microbiol 31: 1541 1546. Gilson, E., Saurin, W., Perrin, D., Bachellier, S., and Hofnung, M. (1991) Palindromic units are part of a new bacterial interspersed mosaic element (BIME). Nucleic Acids Res 10: 13751383. Gonzlez, ., Hierro, N., Poblet, M., Mas, A., and Guillamn, J.M. (2005) Application of molecular methods to demonstrate species and strain evolution of acetic acid bacteria population during wine production. Int J Food Microbiol 102: 295304. Goullet, P., and Picard, B. (1989) Comparative electrophoretic polymorphism of esterases and other enzymes in Escherichia coli. J Gen Microbiol 135: 135143. Hahm, B.K., Maldonado, Y., Schreiber, E., Bhunia, A.K., and Nakatsu, C.H. (2003) Subtyping of foodborne and environmental isolates of Escherichia coli by multiplex-PCR, repPCR, PFGE, ribotyping and AFLP. J Microbiol Methods 53: 387399. Harrington, S.M., Stock, F., Kominski, A.L., Campbell, J.D., Hormazabal, J.C., Livio, S., et al. (2007) Genotypic analysis of invasive Streptococcus pneumoniae from Mali, Africa, by semiautomated repetitive-element PCR and pulsed-eld gel electrophoresis. J Clin Microbiol 45: 707714. Healy, M., Huong, J., Bittner, T., Lising, M., Frye, S., Raza, S., et al. (2005) Microbial DNA typing by automated repetitive-sequence-based PCR. J Clin Microbiol 43: 199 207.

Acknowledgements
We thank John Ferguson for helping with HFERP analyses and gures, and Keishi Senoo for valuable discussions. This project was supported, in part, by grants from the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN), the Bio-oriented Technology Research Advancement Institution, Japan (to S.I. and K.S.), and by grants, from the Minnesota Sea Grant College Program, supported by the NOAA Office of Sea Grant, United States Department of Commerce, under Grant NA03OAR4170048, and the University of Minnesota Agricultural Experiment Station (to M.J.S.).

References
Binga, E.K., Lasken, R.S., and Neufeld, J.D. (2008) Something from (almost) nothing: the impact of multiple displacement amplication on microbial ecology. ISME J 2: 233241. Brumlik, M.J., Szymajda, U., Zakowska, D., Liang, X., Redkar, R.J., Patra, G., and Del Vecchio, V.G. (2001) Use of long-range repetitive element polymorphism-PCR to differentiate Bacillus anthraces strains. Appl Environ Microbiol 67: 30213028. Byappanahalli, M.N., Whitman, R.L., Shively, D.A., Sadowsky, M.J., and Ishii, S. (2006) Population structure, persistence, and seasonality of autochthonous Escherichia coli in temperate, coastal forest soil from a Great Lake watershed. Environ Microbiol 8: 504513. Cangelosi, G.A., Freeman, R.J., Lewis, K.N., LivingstonRosanoff, D., Shah, K.S., Milan, S.J., and Goldberg, S.V. (2004) Evaluation of a high-throughput repetitivesequence-based PCR system for DNA ngerprinting of Mycobacterium tuberculosis and Mycobacterium avium complex strains. J Clin Microbiol 42: 26852693. Chokesajjawatee, N., Zo, Y.-G., and Colwell, R.R. (2008) Determination of clonality and relatedness of Vibrio cholerae isolates by genomic ngerprinting, using long-range rep-PCR. Appl Environ Microbiol 74: 53925401. Cleland, D., Krader, P., and Emerson, D. (2008) Use of the DiversiLab repetitive sequence-based PCR system for genotyping and identication of Archaea. J Microbiol Methods 73: 172178.

2009 The Authors Journal compilation 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 733740

Rep-PCR DNA ngerprinting 739

Hermans, P.W.M., van Soolingen, D.V., and van Embden, J.D.A. (1992) Characterization of a major polymorphic tandem repeat in Mycobacterium tuberculosis and its potential use in the epidemiology of Mycobacterium kansaii and Mycobacterium gordonae. J Bacteriol 174: 4157 4165. Hulton, C.S.J., Higgins, C.F., and Sharp, P.M. (1991) ERIC sequences, a novel family of repetitive elements in the genome of Escherichia coli, Salmonella typhimurium and other enterobacterial. Mol Microbiol 5: 825834. Ishii, S., and Sadowsky, M.J. (2008) Escherichia coli in the environment: implications for water quality and human health. Microbes Environ 23: 101108. Ishii, S., Ksoll, W.B., Hicks, R.E., and Sadowsky, M.J. (2006) Presence and growth of naturalized Escherichia coli in temperate soils from Lake Superior watersheds. Appl Environ Microbiol 72: 612621. Ishii, S., Hansen, D.L., Hicks, R.E., and Sadowsky, M.J. (2007) Beach sand and sediments are temporal sinks and sources of Escherichia coli in Lake Superior. Environ Sci Technol 41: 22032209. Jernberg, C., Lofmark, S., Edlund, C., and Jansson, J.K. (2007) Long-term ecological impacts of antibiotic administration on the human intestinal microbiota. ISME J 1: 5666. Johnson, J.R., and OBryan, T.T. (2000) Improved repetitiveelement PCR ngerprinting for resolving pathogenic and nonpathogenic phylogenetic groups within Escherichia coli. Clin Vaccine Immunol 7: 265273. Johnson, L.K., Brown, M.B., Carruthers, E.A., Ferguson, J.A., Dombek, P.E., and Sadowsky, M.J. (2004) Sample size, library composition, and genotypic diversity among natural populations of Escherichia coli from different animals inuence accuracy of determining sources of fecal pollution. Appl Environ Microbiol 70: 44784485. Judd, A.K., and Sadowsky, M.J. (1993) The Bradyrhizobium japonicum serocluster 123 hyperreiterated DNA region, HRS1, has DNA and amino acid sequence homology to IS1380, an insertion sequence from Acetobacter pasteurianus. Appl Environ Microbiol 59: 16561661. Kaluza, K., Hahn, M., and Hennecke, H. (1985) Repeated sequences similar to insertion elements clustered around the nif region of the Rhizobium japonicum genome. J Bacteriol 162: 535542. Kang, H.P., and Dunne, W.M. (2003) Stability of repetitivesequence PCR patterns with respect to culture age and subculture frequency. J Clin Microbiol 41: 26942696. Kwon, D.-H., El-Zaatari, A.K., Woo, J.-S., Perng, C.L., Graham, D.Y., and Go, M.F. (1998) REP-PCR fragments as biomarkers for differentiating gastroduodenal diseasespecic Helicobacter pylori strains. Digest Dis Sci 43: 980 987. Martin, B., Humbert, O., Camara, M., Guenzi, E., Walker, J., Mitchell, T., et al. (1992) A highly conserved repeated DNA element located in the chromosome of Streptococcus pneumoniae. Nucleic Acids Res 20: 34793483. Matheson, V.G., Munakata-Marr, J., Hopkins, G.D., McCarty, P.L., Tiedje, J.M., and Forney, L.J. (1997) A novel means to develop strain-specic DNA probes for detecting bacteria in the environment. Appl Environ Microbiol 63: 28632869. Mazel, D., Houmard, J., Castets, A.M., and de Marsac, N.T. (1990) Highly repetitive DNA sequences in cyanobacterial genomes. J Bacteriol 172: 27552761. Metzgar, D., Thomas, E., Davis, C., Field, D., and Wills, C. (2001) The microsatellites of Escherichia coli: rapidly evolving repetitive DNAs in a non-pathogenic prokaryote. Mol Microbiol 39: 183190. Ochman, H., and Selander, R.K. (1984) Standard reference strains of Escherichia coli from natural populations. J Bacteriol 157: 690693. Ohnishi, M., Terajima, J., Kurokawa, K., Nakayama, K., Murata, T., Tamura, K., et al. (2002) Genomic diversity of enterohemorrhagic Escherichia coli O157 revealed by whole genome PCR scanning. Proc Natl Acad Sci USA 99: 1704317048. Olive, D.M., and Bean, P. (1999) Principles and applications of methods for DNA-based typing of microbial organisms. J Clin Microbiol 37: 16611669. Rademaker, J.L.W., and de Bruijn, F.J. (2008) Computerassisted analysis of molecular ngerprint proles and database construction. In Molecular Microbial Ecology Manual. Kowalchuck, G.A., de Bruijn, F.J., Head, I.M., Akkermans, A.D.L., and van Elsas, J.D. (eds). Dordrecht, the Netherlands: Springer, pp. 13971446. Rademaker, J.L.W., Louws, F.J., Schultz, M.H., Rossbach, U., Vauterin, L., Swings, J., and de Bruijn, F.J. (2005) A comprehensive species to strain taxonomic framework for Xanthomonas. Phytopathology 95: 10981111. Rademaker, J.L.W., Louws, F.J., Versalovic, J.V., and de Bruijn, F.J. (2008) Characterization of the diversity of ecologically important microbes by rep-PCR genomic ngerprinting. In Molecular Microbial Ecology Manual. Kowalchuck, G.A., de Bruijn, F.J., Head, I.M., Akkermans, A.D.L., and van Elsas, J.D. (eds). Dordrecht, the Netherlands: Springer, pp. 611644. Ross, T.L., Merz, W.G., Farkosh, M., and Carroll, K.C. (2005) Comparison of an automated repetitive sequencebased PCR microbial typing system to pulsed-eld gel electrophoresis for analysis of outbreaks of methicillinresistant Staphylococcus aureus. J Clin Microbiol 43: 56425647. Sadowsky, M.J., and Hur, H.-G. (1998) Use of endogenous repeated sequences to ngerprint bacterial genomic DNA. In Bacterial Genomics: Physical Structure and Analysis. de Bruijn, F.J., Lupski, J.R., and Weinstock, G.M. (eds). New York, NY, USA: Chapman & Hall, pp. 399413. Sadowsky, M.J., Kinkel, L.L., Bowers, J.H., and Schottel, J.L. (1996) Use of repetitive intergenic DNA sequences to classify pathogenic and disease-suppressive Streptomyces strains. Appl Environ Microbiol 62: 34893493. Schmidt, A.L., and Anderson, L.M. (2006) Repetitive DNA elements as mediators of genomic change in response to environmental cues. Biol Rev 81: 531543. Sharples, G.J., and Lloyd. R.G. (1990) A novel repeated DNA sequence located in the intergenic regions of bacterial chromosomes. Nucleic Acids Res 18: 65036508. Stern, M.J., Ames, G.F.-L., Smith, N.H., Robinson, E.C., and Higgins, C.F. (1984) Repetitive extragenic palindromic sequences, a major component of the bacterial genome. Cell 37: 10151026. Thierry, D., Cave, M.D., Eisenach, K.D., Crawford, J.T., Bates, J.H., Gicquel, B., and Guesdon, J.L. (1990) IS6110,

2009 The Authors Journal compilation 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 733740

740 S. Ishii and M. J. Sadowsky

an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res 18: 188. Tobes, R., and Ramos, J.-L. (2005) REP code: dening bacterial identity in extragenic space. Environ Microbiol 7: 225228. Ussery, D.W., Binnewies, T.T., Gouveia-Oliveira, R., Jarmer, H., and Hallin, P.F. (2004) Genome update: DNA repeats in bacterial genomes. Microbiology 150: 35193521. Vanparys, B., Spieck, E., Heylen, K., Wittebolle, L., Geets, J., Boon, N., and De Vos, P. (2007) The phylogeny of the genus Nitrobacter based on comparative rep-PCR, 16S rRNA and nitrite oxidoreductase gene sequence analysis. Syst Appl Microbiol 30: 297308. Versalovic, J., and Lupski, J.R. (1998) Interspersed repetitive sequences in bacterial genomes. In Bacterial Genomics: Physical Structure and Analysis. de Bruijn, F.J., Lupski, J.R., and Weinstock, G.M. (eds). New York, NY, USA: Chapman & Hall, pp. 3848. Versalovic, J., Koeuth, T., and Lupski, J.R. (1991) Distribution of repetitive DNA sequences in eubacteria and application to ngerprinting of bacterial genomes. Nucleic Acids Res 24: 68236831. Versalovic, J., Schneider, M., de Bruijn, F.J., and Lupski, J.R. (1994) Genomic ngerprinting of bacteria using repetitive sequence based PCR (rep-PCR). Methods Cell Mol Biol 5: 2540. Versalovic, J., Kapur, V., Koeuth, T., Mazurek, G.H., Whittam, T.S., Musser, J.M., and Lupski, J.R. (1995) DNA ngerprinting of pathogenic bacteria by uorophoreenhanced repetitive sequence-based polymerase chain reaction. Arch Pathol Lab Med 119: 2329. Versalovic, J.V., de Bruijn, F.J., and Lupski, J.R. (1998) Repetitive sequence-based PCR (rep-PCR) DNA ngerprinting of bacterial genomes. In Bacterial Genomes: Physical Structure and Analysis. de Bruijn, F.J., Lupski, J.R., and Weinstock, G.M. (eds). New York, NY, USA: Chapman & Hall, pp. 437454. Vogel, J.R., Stoeckel, D.M., Lamendella, R., Zelt, R.B., Santo Domingo, J.W., Walker, S.R., and Oerther, D.B. (2007) Identifying fecal sources in a selected catchment reach using multiple source-tracking tools. J Environ Qual 36: 718729. Wise, M.G., Healy, M., Reece, K., Smith, R., Walton, D., Dutch, W., et al. (2007) Species identication and strain differentiation of clinical Candida isolates using the DiversiLab system of automated repetitive sequencebased PCR. J Med Microbiol 56: 778787. Woods, S.A., and Cole, S.T. (1990) A family of dispersed repeats in Mycobacterium leprae. Mol Microbiol 4: 1745 1751.

2009 The Authors Journal compilation 2009 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology, 11, 733740