CHARACTERIZATION AND CLASSIFICATION OF MICROBES
BY REP-PCR GENOMIC FINGERPRINTING AND COMPUTER-ASSISTED PATTERN
J.L.W. RADEMAKER1 and F.J. DE BRUIJN1,2,3.
1MSU-DOE Plant Research Laboratory, 2Department
of Microbiology, 3NSF Center for Microbial Ecology, Michigan State
University, East Lansing, MI 48824, USA.
The identification and classification of bacteria
are of crucial importance in environmental, industrial, medical
and agricultural microbiology and microbial ecology. A number
of different phenotypic and genotypic methods are presently being
employed for microbial identification and classification (see
Fig. 1 and Louws et al. 1996). Each of these methods permits
a certain level of phylogenetic classification, from the genus,
species, subspecies, biovar to the strain specific level (Fig.
1). Moreover, each method has its advantages and disadvantages,
with regard to ease of application, reproducibility, requirement
for equipment and level of resolution (Akkermans et al.
Generally, DNA-based methods are emerging as the more reliable, simple and inexpensive ways to identify and classify microbes. In fact, the assignment of genera/species has traditionally been based on DNA-DNA hybridization methods (Wayne et al. 1987) and modern phylogeny is increasingly based on 16S rRNA sequence analysis (Woese 1987; Stackebrandt and Goebel 1994). Here, we describe a method referred to as rep-PCR genomic fingerprinting, a DNA amplification based technique, which has been found to be extremely reliable, reproducible, rapid and highly discriminatory (Versalovic et al. 1994; Louws et al. 1996).
Rep-PCR genomic fingerprinting makes use of DNA primers
complementary to naturally occurring, highly conserved, repetitive
DNA sequences, present in multiple copies in the genomes of most
Gram-negative and several Gram-positive bacteria (Lupski and Weinstock
1992). Three families of repetitive sequences have been identified,
including the 35-40 bp repetitive extragenic palindromic (REP)
sequence, the 124-127 bp enterobacterial repetitive intergenic
consensus (ERIC) sequence, and the 154 bp BOX element (Versalovic
et al. 1994). These sequences appear to be located in distinct,
intergenic positions around the genome. The repetitive elements
may be present in both orientations, and oligonucleotide primers
have been designed to prime DNA synthesis outward from the inverted
repeats in REP and ERIC, and from the boxA subunit of BOX, in
the polymerase chain reaction (PCR) (Versalovic et al. 1994).
The use of these primer(s) and PCR leads to the selective amplification
of distinct genomic regions located between REP, ERIC or BOX elements.
The corresponding protocols are referred to as REP-PCR, ERIC-PCR
and BOX-PCR genomic fingerprinting respectively, and rep-PCR genomic
fingerprinting collectively (Versalovic et al. 1991; 1994).
The amplified fragments can be resolved in a gel matrix, yielding
a profile referred to as a rep-PCR genomic fingerprint (Versalovic
et al. 1994; see Fig. 2). These fingerprints resemble "bar
code" patterns analogous to UPC codes used in grocery
stores (Lupski 1993).
The rep-PCR genomic fingerprints generated from bacterial isolates permit differentiation to the species, subspecies and strain level.
Rep-PCR genomic fingerprinting protocols have been developed in collaboration with the group led by Dr. J.R. Lupski at Baylor College of Medicine (Houston, Texas) and have been applied successfully in many medical, agricultural, industrial and environmental studies of microbial diversity (Versalovic et al. 1994). In addition to studying diversity, rep-PCR genomic fingerprinting has become a valuable tool for the identification and classification of bacteria, and for molecular epidemiological studies of human and plant pathogens (van Belkum et al. 1994; Louws et al. 1996 and references therein; Versalovic et al. 1997).
This chapter also describes the application of computer
assisted analysis of rep-PCR generated genomic fingerprints for
the identification and classification of microbes using cluster
analysis algorithms. Cluster analysis is the art of finding groups
in data, and bacterial classification and taxonomy are principal
applications of this methodology (see Fig. 2B). We will describe
the generation of raw data, the comparison of fingerprints, and
the different algorithms used to find groupings in the data, and
to identify specific strains in a database using their genomic
In this second section we will provide an overview of the different methodologies and protocols used to implement rep-PCR genomic fingerprinting. One distinct advantage of the rep-PCR genomic fingerprinting method is that the primers used work in a variety of Gram-negative and Gram-positive bacteria (see Versalovic et al. 1991; 1994; Louws et al. 1996). This means that no previous knowledge of the genomic structure or nature of indigenous repeated sequences is necessary. It also bypasses the need to identify suitable arbitrary primers by trial and error, that is inherent in the RAPD protocol (see Welsh and McClelland 1990).
Sample preparation is simple and rapid and genomic fingerprints can be obtained from a variety of different templates (see next section). Many samples can be prepared in a short time. PCR amplification requires 5-7 hours. Electrophoresis on agarose gels can be performed in eight hours, but 18 hours is preferred for better resolution of the complex fingerprints on long (24 cm) gels. Therefore rep-PCR fingerprinting, including pattern analysis by eye or using a computer, can be performed in two days. In this section, we will primarily focus on examples involving the analysis of plant-associated and other soil bacteria. For a discussion of medical applications, consult reviews by Versalovic et al. (1994, 1997).
Correct pattern imaging, visual interpretation or
conversion to computer processable data, will be described in
the third section. Important parameters that will be discussed
include choice of size marker standards for multiple gel comparison
and database construction, determination of proximity coefficients,
and use of appropriate clustering methods for phylogenetic analysis.
Template preparation for rep-PCR genomic fingerprinting.
Several methods of template preparation can be used
for rep-PCR mediated genomic fingerprinting. The method used depends
on the nature of the microbes to be analyzed, their receptiveness
to lysing (releasing DNA), size of pools to be analyzed, level
of resolution desired and time available. Rep-PCR genomic fingerprints
have been obtained from purified DNA, whole cells from pure liquid
cultures or cultures from plates, as well as directly from extracts
of plant lesions or nodules (Versalovic et al. 1994; 1997;
Louws et al. 1994, 1995, 1996; Nick and Lindstrom 1994;
Schneider and de Bruijn 1996; Vera Cruz et al. 1996). Here
we will focus on the whole cell and purified DNA based methods,
and not discuss the plant tissue related approaches. For a detailed
description of the latter, see Schneider and de Bruijn (1996)
and Louws et al. (1996).
- Whole cells from pure liquid cultures.
Whole cells obtained from a liquid culture can be
directly used in rep-PCR amplification reactions. Generally, washing
the cells improves the quality of the rep-PCR reactions. Using
this method rep-PCR genomic fingerprints have been generated from
for example A. caulinodans and R. meliloti (Schneider
and de Bruijn 1996).
1. Take 3 ml of a liquid culture (OD600 0.65 - 0.95).
2. Spin down the cells.
3. Wash the cell pellet with 1 M NaCl. Repeat the washing several times for cultures producing a lot of polysaccharides.
4. Resuspend the cell pellet in 100 µl double distilled water. Store aliquots at -20_C (optional).
5. Use 1-2 µl of template per PCR reaction (see
- Whole cells from single colonies on plates.
Whole cells obtained from single colonies on plates
can also be used directly in the rep-PCR reaction. Using this
method rep-PCR genomic fingerprints have been obtained from Rhizobium
sp., Clavibacter michiganensis subsp., E.coli, various
xanthomonads and pseudomonads, as well as from a large collection
of unidentified 3 CBA degraders and subsurface microbes (de Bruijn
1992; Louws et al. 1994, 1995; Judd et al. 1993;
Zlatkin et al. 1996; Schneider and de Bruijn 1996; M.H.
Schultz, J.L.W. Rademaker and F.J. de Bruijn unpublished results).
1. Remove a small portion of a well-defined single colony directly off a fresh plate using a 1 ml disposable inoculation loop (Simport L200-1).
2. Insert loop into 25 ml
PCR mix and whisk to resuspend the cells.
Comments: Up to
4 and even 12 weeks old plates have been used successfully (Schneider
and de Bruijn 1996; M.H. Schultz, J.L.W. Rademaker and F.J. de
Bruijn unpublished results). Relatively few cells yield enough
DNA for a rep-PCR reaction; in fact, using too many cells results
in the generation of a background smear.
- Whole cell after alkaline lysis.
Whole cells of microbial species that are difficult
to lyse and do not early release DNA during the PCR cycles may
be pretreated by the alkaline lysis method.
1. Take 10 µl from a cell suspension (103 - 107 bacteria) or a portion of one colony.
2. Add 100 µl of 0.05 M NaOH and incubate at 95°C for 15 min.
3. Centrifuge for 2 min. at 14,000 rpm.
4. Use 1 µl of the supernatant per rep-PCR reaction.
- Purified genomic DNA.
The procedure described is based on Pitcher et
al. (1989), and modified by Dr. Luc Vauterin (personal communication).
This method describes the extraction of DNA from solid media (agar
plates), instead of liquid media. Best results are obtained using
young and fresh cells.
Materials and reagents
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