FEMS Microbiology Letters 207 (2001) 21^27
www.fems-microbiology.org
Genetic relationships of Bacillus anthracis and closely related species
based on variable-number tandem repeat analysis and
BOX-PCR genomic ¢ngerprinting
Wonyong Kim a , Yeon-pyo Hong b , Jae-hyung Yoo c , Won-bok Lee d ,
Chul-soon Choi a , Sang-in Chung a; *
b
a
Department of Microbiology, Chung-Ang University College of Medicine and Institute of Medical Research, Seoul 156-756, South Korea
Department of Preventive medicine, Chung-Ang University College of Medicine and Institute of Medical Research, Seoul 156-756, South Korea
c
Department of Pathology, Chung-Ang University College of Medicine and Institute of Medical Research, Seoul 156-756, South Korea
d
Department of Anatomy, Chung-Ang University College of Medicine and Institute of Medical Research, Seoul 156-756, South Korea
Received 14 August 2001 ; received in revised form 16 November 2001; accepted 23 November 2001
First published online 13 December 2001
Abstract
Variable-number tandem repeats (VNTR) analysis and BOX-repeat-based PCR (BOX-PCR) genomic fingerprinting were performed on
25 Bacillus strains to investigate the genetic relatedness of Bacillus anthracis to the closely related species. Based on VNTR analysis, all
B. anthracis strains could be assigned to (VNTR)4 , which is the most commonly found type in the world. Interestingly, a (VNTR)2 was also
observed in Bacillus cereus KCTC 1661 and with an exact match to the tandem repeats found in B. anthracis. This finding has never been
reported before in the closely related species. According to the BOX-PCR, B. anthracis strains clustered together and separated reliably from
the closely related species. However, B. cereus KCTC 1661 was linked to the B. anthracis cluster and showed close relationships with
B. anthracis strains. These results indicated that there was a strong correlation between VNTR analysis and BOX-PCR genomic
fingerprinting. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Variable-number tandem repeats; BOX-PCR genomic ¢ngerprinting; Bacillus anthracis; Bacillus cereus group
1. Introduction
Bacillus anthracis, the etiological agent of anthrax, has
been taxonomically classi¢ed into the B. cereus group also
comprising the closely related species such as Bacillus cereus, Bacillus thuringiensis and Bacillus mycoides [1].
Although the pathogenesis and ecological manifestation
may be di¡erent among the members of the B. cereus
group, B. anthracis shares a strong degree of DNA sequence similarity with the closely related species [2,3]. Furthermore, these species are indistinguishable by the ribosomal RNA analysis, which is widely used for taxonomic
studies [4^6]. For these reasons, it has been proposed that
the members of B. cereus group have evolved from a common ancestor [7].
* Corresponding author. Tel. : +82 (2) 820 5664;
Fax: +82 (2) 812 5495.
E-mail address : [email protected] (S.-i. Chung).
Repetitive DNA that is ubiquitous in eukaryotic cell has
also been identi¢ed in microbes. It is observed as multiple
copies of the same sequence copies throughout the genome
and classi¢ed into two major classes : the short sequence
repeats (SSRs) and the interspersed repeats [8]. Because
both classes have been shown to mutate faster and to be
more diverse than other genomic regions, the approaches
based on these repeats could provide functional and evolutionary information of genetic relationships in microbial
species [9].
Some SSRs representing a single locus and showing in
the number of sequence units were so-called variable-number tandem repeats (VNTR) loci [10]. Recently, VNTR
were found in the vrrA gene of B. anthracis. The gene
encodes a putative 30 kDa glutamine-rich protein. They
comprised ¢ve polymorphisms, which di¡ered by the number of copies of 12 bp tandem repeats, thus facilitating the
identi¢cation of B. anthracis from the closely related species or di¡erentiation among isolates [11^13].
The interspersed repeats are larger than VNTRs and
0378-1097 / 01 / $22.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII: S 0 3 7 8 - 1 0 9 7 ( 0 1 ) 0 0 5 4 4 - 4
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W. Kim et al. / FEMS Microbiology Letters 207 (2001) 21^27
2. Materials and methods
BIOTECH GmbH, Ebersberg, Germany), 0.2 mM of each
deoxynucleoside triphosphate, 10 Wl of 10UPCR bu¡er
with 1.5 mM MgCl2 and 5 U of Taq DNA polymerase
(Roche MB, Mannheim, Germany). Each reaction mixture
was denatured at 94³C for 2 min and run for 35 cycles
using a 480 DNA thermal cycler (Perkin-Elmer, Norwalk,
CN, USA) with the following temperature pro¢les ; denaturation at 94³C for 1 min, annealing at 55³C for 1 min,
and extension at 72³C for 1 min. The ¢nal cycle included
for 5 min at 72³C to complete extension. 10 ml of each
PCR product was analyzed by electrophoresis on a 3.5%
Metaphor agarose gel (FMC Bioproducts, Rockland, ME,
USA) in 0.5UTBE bu¡er (45 mM Tris^borate, 1.0 mM
EDTA, pH 8.3). DNA molecular mass marker V (Roche
MB) was used as a size standard. Gel was stained with
ethidium bromide (1 mg ml31 ).
2.1. Bacterial strains
2.4. Direct sequencing of VNTR
A total of 25 bacterial strains were used in this study. B.
anthracis ATCC 14578T , ATCC 14185 and ATCC 14186
were obtained from the American Type Culture Collection
(Manassas, VA, USA). B. anthracis Pasteur #2, Stern
34F2, and BC were provided from the National Veterinary
Research and Quarantine Service (Anyang, Kyunggi, Korea). B. anthracis CAU 1, CAU 2 and CAU 3 were isolated in our laboratory from patients. B. anthracis closely
related species of B. cereus (KCTC 3624T , KCTC 1012,
KCTC 1014, KCTC 1661), B. thuringiensis (KCTC 3452T ,
KCTC 1034), Bacillus mycoides (KCTC 3453T , KCCM
40260) and other Bacillus reference strains such as
B. megaterium KCTC 3007T , B. subtilis KCTC 3135T ,
B. pumilus KCTC 3348T , B. licheniformis KCTC 1918T ,
B. circulans KCTC 3347T , B. coagulans KCTC 3625T ,
B. sphaericus KCTC 3346T and B. stearothermophilus
KCTC 1752T were obtained from Korean Collection for
Type Cultures (Taejon, Korea) and Korean Culture
Center of Microorganisms (Seoul, Korea).
About 1.1 kb of VNTR regions were ampli¢ed by PCR
using the £anking primers GPR-1 (5PCGTAGTTCACGAACTGCATCT-3P) and GPR-2 (5P-ATGATGTATCTAATGCGGCGT-3P) [12]. The PCR products were puri¢ed using Qiaquick PCR puri¢cation kit (Qiagen,
Valencia, CA, USA). The VNTR sequences were determined by using BigDye Terminator Cycle Sequencing
v2.0 kit and ABI 310 automatic DNA sequencer (Applied
Biosystems, Norwalk, CN, USA). The primes GPR-4 (5PACAACTACCACCGATGGC-3P) and GPR-5 (5P-TTATTTATCATATTAGTTGGATTCG-3P) were used as internal primers for DNA sequencing. The sequences were
aligned using CLUSTAL W (1.7) software [17].
appear to be dispersed evenly all around the chromosome.
At present, there are three families, which have been well
established in numerous bacterial species. Two repeats,
namely the enterobacterial repetitive intergenic consensus
sequences and repetitive extragenic palindrome sequence
have been found in di¡erent members of Enterobacteriaceae [14]. Another motif of BOX-repeat was ¢rst identi¢ed
in Streptococcus pneumoniae and has been used for molecular typing purpose of many Gram-positive bacteria [15].
The objectives of this study were to compare the VNTR
analysis and BOX-PCR genomic ¢ngerprinting of B. anthracis and closely related species for molecular genetic
study and their applicability for typing.
2.2. Genomic DNA preparation
Each bacterial strain was grown on brain heart infusion
agar at 28³C for 16 h. Genomic DNA of bacteria was
prepared by the cetyltrimethylammonium bromide method
[16]. Puri¢ed DNA was dissolved in sterile water with 40
Wg31 ml of RNase and quanti¢ed by MBA 2000 spectrophotometer (Perkin-Elmer, Norwalk, CN, USA) at a
wavelength of 260 nm.
2.3. VNTR ampli¢cation
VNTR ampli¢cation was performed with the EWA-1
(5P-TATGGTTGGTATTGCT-3P) and EWA-2 (5P-ATGGTTCCGCCTTATCG-3P) primers described by Anderson et al. [11]. PCR reaction was done in 100 Wl containing
50 ng of template DNA, 20 pmol of each primer (MWG-
2.5. BOX-PCR genomic ¢ngerprinting
BOX-PCR was carried out as described by Versalovic et
al. [18] with 50 ng of template DNA per reaction. The
BOX A1R (5P-ACGTGGTTTGAAGAGATTTTCG-3P)
was used as a single primer. Ampli¢cation reaction was
done in 25-Wl reaction mixtures containing 20 pmol of
primer, 1.25 mM of each deoxynucleoside triphosphate,
2.5 Wl of 10UPCR bu¡er with 4 mM MgCl2 , 10%
DMSO and 2 U of Taq DNA polymerase using a 480
DNA thermal cycler with the following temperature pro¢les: initial cycle at 95³C for 7 min; 4 cycles at 95³C for
5 min, at 40³C for 5 min, at 72³C for 5 min; 30 cycles at
94³C for 1 min, at 55³C for 1 min, at 72³C for 2 min, and
¢nal extension at 72³C for 10 min. Each test was repeated
three times to examine the reproducibility.
2.6. Analysis of BOX-PCR genomic ¢ngerprints
10 ml of BOX-PCR products were separated by electrophoresis on a 1.5% SeaKem LE agarose gel (FMC Bioproducts) in 0.5UTAE bu¡er (40 mM Tris^acetate, 2 mM
EDTA, pH 8.0) for 5 h. The 1-kb plus DNA ladder (Gib-
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23
co BRL, Rockville, MD, USA) was used as a size standard. Gel was stained with ethidium bromide and image
was stored as a TIFF ¢le with the Gel Doc 2000 system
(Bio-Rad, Hercules, CA, USA). Computer-assisted analysis of the ¢ngerprint was performed by using the GelCompar II software (version 1.5; Applied Maths, Kortrijk,
Belgium). The similarity of BOX-PCR ¢ngerprint was calculated with the Pearson coe¤cient. Cluster analysis of the
similarity matrix was performed by unweighted pair group
method using arithmetic averages (UPGMA) algorithm
[19].
be of the type (VNTR)4 . On the contrary, B. anthracis
ATCC 14578T appeared to have two copies of tandem
repeats. VNTR-like categories were also found in the
closely related species. Of those, B. cereus KCTC 1661
presented a (VNTR)2 , and with exactly matching tandem
repeats. In addition, B. cereus KCTC 1014, B. thuringiensis KCTC 3452T and B. mycoides KCTC 3453T exhibited
(VNTR)2 -like type. However, substantial variations were
found in three to four positions on their sequences. Sequences obtained from B. cereus KCTC 3452T , KCTC
1012, B. thuringiensis KCTC 1034 and B. mycoides
KCCM 40260 were not matched to those of B. anthracis.
3. Results
3.3. BOX-PCR genomic ¢ngerprinting
3.1. VNTR polymorphism
The ¢ngerprint patterns generated by BOX-PCR consisted of 4^17 fragments ranging in size of about 0.18^5.20
kb (Fig. 3). The size and distribution found in B. anthracis
strains were very similar. The patterns obtained from
B. anthracis strains revealed eight to nine fragments with
sizes between 0.39 and 2.0 kb. B. cereus strains appeared
to have broad patterns ranging ¢ve to six bands with size
between 0.39 and 3.0 kb. The ¢ngerprints obtained from
B. thuringiensis strains consisted of six bands with sizes
between 0.29 and 2.75 bp. The patterns of B. mycoides
strains yielded four to six bands with size between 0.45
and 1.9 kb. Of the reference species, B. stearothermophilus
KCTC 1752T showed the most multiple bands patterns
than those of other strains.
Computer-assisted analysis of the BOX-PCR ¢ngerprint
showed that all investigated strains could be grouped into
four distinct clusters (Fig. 4). Cluster A represented the
B. anthracis group. All B. anthracis strains appeared to
have close relationships with the type strain of B. anthracis
ATCC 14578T . In particular, B. cereus KCTC 1661 was
much more related to B. anthracis than other B. cereus
strains. Cluster B comprised of the closely related species
of B. cereus KCTC 3642T , KCTC 1012, KCTC 1014 ;
B. thuringiensis KCTC 3452T , KCTC 1034, and B. mycoides KCTC 3453T , KCCM 40260. Of this cluster,
B. mycoides strains form a subcluster. By contrast, B. cereus KCTC 1012 showed to be more closely related to the
The 25 Bacillus strains were analyzed to determine the
VNTR length variation (Fig. 1). Two polymorphisms were
observed in B. anthracis strains. B. anthracis ATCC 14185,
ATCC 14186, Pasteur #2, Stern 34F2, BC, CAU 1, CAU
2 and CAU 3 shared an identical pro¢le with 167 bp in
size. By contrast, B. anthracis ATCC 14578T yielded a
143-bp amplicon. VNTR-like amplicons were also found
in the closely related species with di¡erent size variations.
An amplicon of the size of 101 bp was obtained from
B. cereus KCTC 3624T and KCTC 1012. Amplicons of
unique sizes were produced from B. cereus KCTC 1014,
140 bp; B. cereus KCTC 1661, 137 bp; B. thuringiensis
KCTC 3452T , 143 bp; B. thuringiensis KCTC 1034, 101
bp; B. mycoides KCTC 3453T , 128 bp and B. mycoides
KCCM 40260, 95 bp. Other reference strains produced
non-speci¢c amplicons or did not amplify the products.
3.2. VNTR sequences
DNA sequencing was carried out on the VNTR regions
from 17 Bacillus strains. The result is shown in Fig. 2. All
B. anthracis strains contained a consensus sequence motif
(5P-CAATATCAACAA-3P) with tandem repeats. B. anthracis ATCC 14185, ATCC 14186, Pasteur #2, Stern
34F2, BC, CAU 1, CAU 2 and CAU 3 were shown to
Fig. 1. VNTR polymorphisms in B. cereus group strains and other Bacillus species. Lane M, molecular size marker ; 1, B. anthracis ATCC 14578T ;
2, B. anthracis ATCC 14185; 3, B. anthracis ATCC 14186; 4, B. anthracis Pasteur #2; 5, B. anthracis Stern 34F2; 6, B. anthracis BC; 7, B. anthracis
CAU 1; 8, B. anthracis CAU 2; 9, B. anthracis CAU 3; 10, B. cereus KCTC 1012 ; 11, B. cereus KCTC 1014 ; 12, B. cereus KCTC 1661 ; 13, B. cereus
KCTC 3624T ; 14, B. thuringiensis KCTC 1034; 15, B. thuringiensis KCTC 3452T ; 16, B. mycoides KCTC 3453T ; 17, B. mycoides KCCM 40260; 18,
B. megaterium KCTC 3007T ; 19, B. subtilis KCTC 3135T ; 20, B. pumilus KCTC 3348T ; 21, B. licheniformis KCTC 1918T ; 22, B. circulans KCTC
3347T ; 23, B. coagulans KCTC 3625T ; 24, B. sphaericus KCTC 3346T ; 25, B. stearothermophilus KCTC 1752T .
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Fig. 2. Multiple alignment of the nucleotide sequences corresponding to VNTR regions ampli¢ed with EWA-1 and EWA-2 primers from B. anthracis
and closely related species. VNTR were highlighted in box. ^ indicates a gap.
Fig. 3. Fingerprint patterns generated from BOX-PCR in B. cereus group strains and other Bacillus species. Lane M, molecular size marker; 1, B. anthracis ATCC 14578T ; 2, B. anthracis ATCC 14185; 3, B. anthracis ATCC 14186; 4. B. anthracis Pasteur #2; 5, B. anthracis Stern 34F2; 6, B. anthracis BC; 7, B. anthracis CAU 1; 8, B. anthracis CAU 2; 9, B. anthracis CAU 3; 10, B. cereus KCTC 1012; 11, B. cereus KCTC 1014; 12, B. cereus
KCTC 1661; 13, B. cereus KCTC 3624T ; 14, B. thuringiensis KCTC 1034; 15, B. thuringiensis KCTC 3452T ; 16, B. mycoides KCTC 3453T ; 17, B. mycoides KCCM 40260; 18, B. megaterium KCTC 3007T ; 19, B. subtilis KCTC 3135T ; 20, B. pumilus KCTC 3348T ; 21, B. licheniformis KCTC 1918T ;
22, B. circulans KCTC 3347T ; 23, B. coagulans KCTC 3625T ; 24, B. sphaericus KCTC 3346T ; 25, B. stearothermophilus KCTC 1752T .
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25
Fig. 4. Dendrogram representing genetic relationships among B. cereus group strains and other Bacillus species based on BOX-PCR ¢ngerprint. Similarity (%) between patterns was calculated by using the Pierson coe¤cient. The data were sorted by using UPGMA clustering method.
B. thuringiensis strains than to the type strain of B. cereus
KCTC 4624T . Cluster C comprised B. subtilis KCTC
3135T , B. pumilus KCTC 3348T as well as B. stearothermophilus KCTC 1752T . Cluster D consisted of genetically
distinct lines each represented by strains of the ¢ve species
of B. coagulans KCTC 3625T , B. subtilis KCTC 3135T ,
B. sphaericus KCTC 3346T , B. licheniformis KCTC
1918T and B. megaterium KCTC 3007T at 64% genetic
similarity level.
4. Discussion
In eukaryotes, VNTRs may be involved in nucleosome
organization, recombination and regulation of gene expression or production activity [20]. On the contrary, the
presence and function of VNTRs in prokaryotes are not
well known so far. Only molecular switches controlling
gene expression have been described in several microbial
species [21,22]. Variability observed in microbial VNTRs
is thought to be caused by polymerase slipped-strand mis-
pairing, which may occur in combination with inadequate
DNA repair pathways [23].
VNTRs have presented low diversity among B. anthracis strains with (VNTR)2 to (VNTR)6 as the epidemiologically relevant types worldwide [12]. This indicates that
they are slowly evolving or recently derived from a common ancestor [24]. Distribution and occurrence of anthrax
in Korea was highly limited in numbers. In this study,
Korean isolates BC, CAU 1, CAU 2 and CAU 3 belonged
to the type (VNTR)4 , which is the most commonly found
type in the world. This is in accord with the results of
previous studies [12,13].
The type strain of B. anthracis ATCC 14578T originates
from B. anthracis Vollum. Therefore, these two strains are
identical [25]. However, a previous ¢nding demonstrated
that these strains assigned distinct categories of (VNTR)3
and (VNTR)2 , respectively [12]. In this study, B. anthracis
ATCC 14578T belonged to the type (VNTR)2 , which was
observed from B. anthracis Vollum.
VNTR-like categories have been reported in some
closely related species such as the type strains of B. cereus
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W. Kim et al. / FEMS Microbiology Letters 207 (2001) 21^27
and B. mycoides, and non-virulent bacilli related to
B. anthracis isolated from soil [11,13]. Their sequences
were dissimilar to those of B. anthracis or not determined.
Therefore, VNTRs have so far been considered to be speci¢c only for B. anthracis strains. In this study, some
strains of the closely related species exhibited VNTR-like
categories with point mutations in their sequences. Interestingly, B. cereus KCTC 1661 was found to have a
(VNTR)2 , and with the exact tandem repeats as the
B. anthracis strains. This ¢nding has not been reported
for the closely related species.
At present, B. mycoides is considered to be genetically
distinct from other members of the B. cereus group and
regarded as a separate taxon [26]. On the other hand,
B. cereus and B. thuringiensis have been regarded as one
species [27]. Our BOX-PCR results correlated well with
those obtained from VNTRs analysis and supported previous taxonomic studies. From the dendrogram generated
from BOX-PCR, B. cereus KCTC 1661 was clearly linked
to the B. anthracis cluster. Of the closely related species,
B. mycoides strains clustered together and distinctly from
B. cereus and B. thuringiensis. By contrast, it was not
possible to reliably discriminate between the B. cereus
and B. thuringiensis strains.
According to these ¢ndings, the members of the B. cereus group may be separated into four di¡erent niches
from a common ancestor. However, a speci¢c clone such
as B. cereus KCTC 1661 is predicted to be transfered
horizontally and shares close genetic relationships to the
B. anthracis strains. In conclusion, we have shown that
two DNA repeat-based molecular genetic approaches of
VNTR analysis and BOX-PCR genomic ¢ngerprinting
could be used as highly discriminatory techniques to determine the genetic relatedness and diversity between
B. anthracis and closely related species.
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
[15]
[16]
[17]
Acknowledgements
This work was supported by the Research Grant of
Chung-Ang University, 1999.
[18]
[19]
References
[20]
[1] Ash, C., Farrow, J.A.E., Wallbanks, S. and Collins, M.D. (1991)
Phylogenetic heterogeneity of the genus Bacillus revealed by comparative analysis of small-subunit-ribosomal RNA sequences. Lett.
Appl. Microbiol. 13, 202^206.
[2] Kaneko, T.R., Nozaki, R. and Aizawa, K. (1978) Deoxyribonucleic
acid relatedness between Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis. Microbiol. Immunol. 22, 639^641.
[3] Seki, T., Chang, C.K., Mikami, H. and Oshima, Y. (1978) Deoxyribonucleic acid homology of the genus Bacillus. Int. J. Syst. Bacteriol. 28, 182^189.
[4] Ash, C., Farrow, J.A.E., Dorsch, M., Stackebrandt, E. and Collins,
M.D. (1991) Comparative analysis of Bacillus anthracis, Bacillus ce-
[21]
[22]
[23]
reus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int. J. Syst. Bacteriol. 41, 343^346.
Ash, C. and Collins, M.D. (1992) Comparative analysis of 23S ribosomal RNA gene sequences of Bacillus anthracis and emetic Bacillus
cereus determined by PCR-direct sequencing. FEMS Microbiol. Lett.
94, 75^80.
Harrell, L.J., Andersen, G.L. and Wilson, K.H. (1995) Genetic variability of Bacillus anthracis and related species. J. Clin. Microbiol.
33, 1847^1850.
Turnbull, P.C.B. (1999) De¢nitive identi¢cation of Bacillus anthracis
^ a review. J. Appl. Microbiol. 87, 237^240.
Van Belkum, A., Scherer, S., van Alphen, L. and Verbrugh, H. (1998)
Short-sequence DNA repeats in prokaryotic genomes. Microbiol.
Mol. Biol. Rev. 62, 275^293.
Van Belkum, A. (1999) Short sequence repeats in microbial pathogenesis and evolution. Cell. Mol. Life Sci. 56, 729^734.
Nakamura, Y., Leppert, M., O'Connel, P., Wol¡, R., Holm, T.,
Culver, M., Martin, C., Fujimoto, E., Ho¡, M., Kumlin, E. and
White, R. (1987) Variable number of tandem repeat (VNTR) markers
for human gene mapping. Science 235, 1616^1622.
Andersen, G.L., Simchock, J.M. and Wilson, K.H. (1996) Identi¢cation of a region of genetic variability among Bacillus anthracis strains
and related species. J. Bacteriol. 178, 377^384.
Jackson, P.J., Walthers, E.A., Kalif, A.S., Richmond, K.L., Adair,
D.M., Hill, K.K., Kuske, C.R., Andersen, G.L., Wilson, K.H.,
Hugh-Jones, M.E. and Keim, P. (1997) Characterization of the variable-number tandem repeats in vrrA from di¡erent Bacillus anthracis
isolates. Appl. Environ. Microbiol. 63, 1400^1405.
Patra, G., Vaissaire, J., Weber-Levy, M., Le Doujet, C. and Mock,
M. (1998) Molecular characterization of Bacillus strains involved in
outbreaks of anthrax in France in 1997. J. Clin. Microbiol. 36, 3412^
3414.
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. 19, 6823^6831.
Martin, B., Humbert, O., Camara, M., Guenzi, E., Walker, J., Mitchell, T., Andrew, P., Prudhomme, M., Alloing, G. and Hakenbeck, R.
(1992) A highly conserved repeated DNA element located in the
chromosome of Streptococcus pneumoniae. Nucleic Acids Res. 20,
3479^3483.
Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman,
J.G. and Struhl, K. (1993) Current Protocols in Molecular Biology,
Section 2.4. John Wiely and Sons, New York.
Thompson, J.D., Higgins, D.G. and Gilbon, T.J. (1994) CLUSTAL
W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting position, speci¢c gap penalties and
weight matrix choice. Nucleic Acids Res. 22, 4673^4680.
Versalovic, J., Schneider, M., de Bruijin, F.J. and Lupski, J.R. (1994)
Genomic ¢ngerprinting of bacteria using repetitive sequence based
polymerase chain reaction. Methods Mol. Cell. Biol. 5, 25^40.
Saitou, N. and Nei, M. (1987) The neighbor-joining method : a new
method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4,
406^425.
Caskey, C.T., Pizzuti, A., Fu, Y.H., Fenwick Jr., R.G. and Nelson,
D.L. (1992) Triple repeat mutations in human disease. Science 256,
784^789.
Van der Ende, A., Hopman, C.T.P., Zaat, S., Essink, B.B., Berkhout,
B. and Dankert, J. (1995) Variable expression of class I outer membrane protein in Neisseria meningitidis is caused by variation in the
spacing between the 310 and 035 regions of the promoter. J. Bacteriol. 177, 2475^2480.
Van Ham, S.M., van Alphen, L., Mooi, F.R. and van Putten, J.P.M.
(1993) Phase variation of Heamophilus in£uenzae ¢mbriae: transcritional control of two divergent genes through a variable combined
promoter region. Cell 73, 1187^1196.
Strand, M., Prolla, T.A., Liskay, R.M. and Petes, T.D. (1993) Desta-
FEMSLE 10286 26-2-02
W. Kim et al. / FEMS Microbiology Letters 207 (2001) 21^27
bilization of tracts of simple repetitive DNA in yeast by mutations
a¡ecting DNA mismatch repair. Nature 365, 274^276.
[24] Smith, K.L., DeVos, V., Bryden, H., Price, L.B., Hugh-Jones, M.E.
and Keim, P. (2000) Bacillus anthracis diversity in Kruger National
Park. J. Clin. Microbiol. 38, 3780^3784.
[25] Gherna, R. and Pienta, P. (1992) Catalogue of Bacteria and Phages,
37 pp. American Type Culture Collection, Rockville, MD.
27
[26] Nakamura, L.K. and Jackson, M.A. (1995) Clari¢cation of the
taxonomy of Bacillus mycoides. Int. J. Syst. Bacteriol. 45, 46^49.
[27] Carlson, C.R., Caugant, D.A. and Kolsto, A.B. (1994) Genotypic
diversity among Bacillus cereus and Bacillus thuringiensis strains.
Appl. Environ. Microbiol. 60, 1719^1725.
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Variabüity of tobacco mosaic virus in relation to control of tomato

Bradyrhizobium viridifuturi sp. nov., encompassing nitrogen