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Mkmbio/ogy
(1998), 144,2791-2801
Printed in Great Britain
A
PCR
technique based on the Hipl
interspersed repetitive sequence distinguishes
cyanobacterial species and strains
J.
K.
Smith,’
J.
D. Parry,’
J.
G.
Day2
and
R.
J.
Smith’
Author
for
correspondence:
R.
J.
Smith. Tel:
+44
1524 65201
ext.
93515.
Fax:
+44
1524 843854.
e-mail
:
r.smith@lancaster.ac.uk
1
IENS,
Division
of
Biological
Sciences, Lancaster
University, Bailrigg,
Lancaster
LA1
4YQ,
UK
Culture Collection
of
Algae
and Protozoa (CCAP), IFE,
Windermere Laboratory,
The Ferry House, Far
Sawrey, Ambleside,
Cumbria
LA22
OLP,
UK
The use of primers based on the Hipl sequence as a
typing
technique
for
cyanobacteria has been investigated. The discovery
of
short repetitive
sequence structures
in
bacterial DNA
during
the last decade has led to the
development of PCR-based methods
for
typing,
i.e. distinguishing and
identifying, bacterial species and strains. An octameric palindromic sequence
known
as Hipl has been shown to be present
in
the chromosomal DNA of
many species of cyanobacteria as a highly repetitious interspersed sequence.
PCR
primers were constructed that extended the Hipl sequence at the 3’ end
by
two
bases. Five of the 16 possible extended primers were tested. Each of
the five primers produced a different set of products when used to prime
PCR
from cyanobacterial genomic DNA. Each primer produced a distinct set of
products for each of the
15
cyanobacterial species tested. The ability of Hipl-
based
PCR
to
resolve taxonomic differences was assessed by analysis
of
independent isolates of
Anabaena flos-aquae
and
Nostoc
ellipsosporum
obtained from the CCAP (Culture Collection of Algae and Protozoa,
IFE,
Cumbria,
UK).
A PCR-based RFLP analysis
of
products amplified from the
235-165 rDNA intergenic region was used
to
characterize the isolates and to
compare
with
the Hipl typing data. The RFLP and Hipl
typing
yielded similar
results and both techniques were able
to
distinguish different strains.
On
the
basis of these results
it
is suggested that the Hipl PCR technique may assist
in
distinguishing cyanobacterial species and strains.
Keywords
:
cyanobacteria, typing, identification, Hipl, repetitive sequence primed
PCR
INTRODUCTION
The ability to distinguish species and strains of bacteria
is essential for environmental and epidemiological
studies. In recent years molecular genetic techniques
have been developed to supplement or supplant con-
ventional methods that are based on techniques such as
immunological (serotyping) and bacteriophage-suscep-
tibility characteristics (Tenover
et
al.,
1995). The newer
molecular genetic techniques all seek to detect
differences in DNA sequence structure present either in
the chromosomal DNA of a species or in a contained
plasmid. One common approach assesses differences,
often in the presence of restriction endonuclease sites,
that are present in a small segment of the genome such
.............................................................................................
.
...............
.
...............................
....
Abbreviations:
ITS,
internal transcribed spacer; RAPD, randomly ampli-
fied polymorphic DNA; STRR, short tandemly repetitive repeat.
as a particular gene. Methods based on the intergenic
regions within the rRNA cistrons (rDNA sequencing
and ribotyping) have been particularly popular (Stull et
al.,
1988; Vila
et
al.,
1996). Another, well-regarded
technique assesses RFLPs generated by restriction of
chromosomal DNA with rare-cutting restriction endo-
nucleases and the use
of
pulsed-field gel electrophoresis
to separate the fragments (Arbeit
et
al.,
1990; Frey
et
al.,
1996; Le-Bourgeois
et
a/.,
1993).
A third method employs the PCR reaction to show
differences between species by analysing the size of the
DNA products amplified from a genomic DNA template
by a variety of primers. In higher organisms (Welsh
&
McClelland, 1990), sets
of
random primers have been
used to generate randomly amplified polymorphic
DNA
(RAPD)-PCR products, which produce banding
patterns, when separated on agarose gels, that are
characteristic of species and even individual organisms,
279
1
0002-2605
0
1998
SGM
J.
K.
SMITH
and
OTHERS
The minimum size
of
a primer which is capable
of
specific interaction with
a
DNA sequence
(10-12
bp)
and the much smaller size
of
the bacterial genome as
compared
to
that
of
higher organisms limit the general
application
of
RAPD techniques
to
bacteria, although
several RAPD-PCR techniques for distinguishing bac-
teria have been described (Lipman
et
al.,
1996; Mahen-
thiralingam
et
al.,
1996; Schmidt
et
al.,
1991). Similar
techniques have been developed that are based on
primers designed
to
hybridize with repeated sequence
structures present in bacterial DNA. The primers permit
amplification
of
the DNA sequences between those
adjacent repeated sequences which are present in a
suitable orientation and distance apart.
A
number of
different examples
of
the technique have been published,
including ERIC (Enterobacterial Repetitive Interspersed
Consensus) (Hulton
et
al.,
1991) and REP (Repetitive
Extragenic Palindrome) (Versalovic
et
al.,
1991
;
Giesendorf
et
al.,
1993; Georghiou
et
al.,
1995).
Classically the distinguishing
of
different species
of
cyanobacteria has relied essentially upon identifying
morphological and developmental characteristics by
light microscopy (Rippka, 1988
;
Castenholz
et
al.,
1992).
Considerable expertise is required
to
identify species
since both morphological and developmental character-
istics can vary with the growth conditions (Evans
et
al.,
1976; Doers
&
Parker, 1988). In some instances it is not
too
difficult
to
identify cyanobacterial isolates
to
the
genus level, particularly where morphological character-
istics are significantly different from most other genera,
e.g.
Calothrix.
However, for many genera, including
Oscillatoria, Lyngbya
and
Phorrnidium,
it is often
difficult for the non-expert
to
be confident
of
their
diagnosis. The problems
of
identification increase
further at the species level and little is known about
subspecies variation, i.e. strains.
Although they would be useful in distinguishing cyano-
bacterial isolates and in maintaining laboratory and
collection cultures, only a few molecular genetic tech-
niques for typing cyanobacteria have been developed.
The cloning and sequencing of rRNA cistrons has most
frequently been used
to
identify cyanobacteria in en-
vironmental samples and
to
investigate phylogeny
(Britschgi
&
Giovannoni, 1991; Ferris
et
al.,
1996;
Fuhrman
et
al.,
1993; Nelissen
et
al.,
1994; Neilan,
1996; Schmidt
et
af.,
1991). Lotti
et
al.
(1996) used the
restriction endonucleases BfaI and
HpaI
to
create unique
RFLP banding patterns
of
chromosomal DNA
to
dis-
tinguish symbiotic
Nostoc
isolates. Neilan
et
al.
(1995)
found RFLPs, in PCR-amplified products
of
the phyco-
cyanin gene, that distinguished between toxic
Micro-
cystzs
and
Anabaena
strains. Neilan (1996) reported the
use of a RAPD technique
to
distinguish
Anabaena
and
Microcystis
isolates responsible for producing nuisance
blooms in freshwater.
Filamentous heterocystous cyanobacteria contain STRR
(short tandemly repetitive repeat) sequences (Maze1
et
al.,
1990) and LTRR (long tandemly repetitive repeat)
sequences, that have been used as Southern blot
RFLP
hybridization probes (Rouhiainen et
al.,
1995) and PCR
typing (Rasmussen
&
Svenning, 1998)
to
distinguish
cyanobacteria. Another interspersed repeated sequence
known
to
be common
to
many, but not
all,
cyano-
bacterial species is an 8 bp, highly iterated palindromic
sequence known as Hipl (Gupta
et
al.,
1993). Robinson
et
al.
(1995) have shown that the use
of
Hipl as a primer
in PCR amplification from genomic DNA generated
product banding patterns that were characteristic of
species. However, large numbers
of
products are gener-
ated with some species, producing complex banding
patterns. We demonstrate here that DNA amplification
from cyanobacterial DNA templates using primers
based on
the
Hipl sequence, but extended by an
additional two bases at the
3'
end, yields reproducible
banding patterns that distinguish different species and
isolates (strains)
of
cyanobacteria.
METHODS
Cyanobacteria and culture conditions.
A
range
of
cyano-
bacteria (Table
l),
maintained
in
the CCAP collection
(http
:
//www.ife.ac.uk/ccap/) and varying
in
morphology
from simple unicells to forms with branched filaments,
heterocysts and akinetes, were examined in
this
study. Species
cultured in JM medium or
BGll
medium (Tompkins
et
al.,
1995)
were grown under
'
environmental
'
conditions
(15
"C,
a
12
:
12
h light
:
dark cycle, irradiance
25
pmol
m-2
s-l).
Species
cultured
in
AA/4 medium (Allen
&
Arnon,
1955)
were grown
at
29
OC under
a
24
h
light regime
(30
pmol
m-2
s-l).
Isolates
used to assess
the
resolution of subspecific differences are
listed
in
Table
2.
Exponential or early stationary phase cultures
(10
ml) were
harvested by centrifugation
(3500
r.p.m.,
10
min) and washed
twice in
the
appropriate sterile medium and/or twice
in
sterile
TE
buffer
(10
mM
Tris/HCl, pH
7.2,
1.0
mM EDTA), with
a
final resuspension
in
750
p1
TLES buffer
(50
mM Tris/HCl,
pH
9.5, 150
mM LiCl,
5
mM EDTA,
5%
SDS).
DNA
isolation.
The resuspended material was added
to
a
screw-capped Eppendorf tube filled with
glass
microbeads
(acid washed, 425-600 nm, Sigma)
to
the
500
p1
mark.
The
tubes were attached
by
screw clamps to a Griffin flask shaker
and shaken at maximum revolutions (approximately
300
r.p.m.) for
7
min to break open cells. The release
of
phycoerythrin and phycocyanin pigments from some species
allowed cell breakage
to
be monitored by eye. Cell debris was
removed by centrifugation
in
a microfuge
(13
200 r.p.m.,
8
min). The supernatant was removed
to
a
fresh
tube
containing
an
equal volume
(500
pl)
of
phenol/chloroform/
isoamyl alcohol (25
:
24
:
1,
by vol.
;
Sigma) and mechanically
mixed
for
1
min followed by centrifugation (microfuge,
13200
r.p.m.,
15
min) and removal of aqueous supernatant
to
a fresh tube. The
phenol/chloroform/isoamyl
alcohol ex-
traction was repeated.
A
full
15
min centrifugation for
both
extractions decreased protein and polysaccharide contami-
nation of the final product.
A
one-tenth volume
(50
pl)
of
5
M
potassium acetate
(3
M
potassium acetate in
2M
acetic acid) was added
to
the
supernatant. The sample was placed
on
ice
for
10
min, during
which time a turbid suspension appeared
in
some samples.
This suspension was pelleted by centrifugation (microfuge,
13200
r.p.m.,
15
min) and the supernatant retained. Two
volumes
(1
ml) of absolute ethanol were added and the sample
2792
Hipl-based
PCR
for typing cyanobacteria
rable
I.
Test species of cyanobacteria
1
Species Isolate identification Medium
Anabaena cylindrica
Oscillatoria amoena
Calothrix
sp.
Nos
t
oc flagell
if0
rme
Anabaenopsis circularis
Fischerella muscicola
Symploca muscorum
Synechocystis
sp.
Nostoc muscorum
Anabaena
sp.
Anabaena flos-aquae
Nostoc ellipsosporum
Anabaena
Anabaena
Nostoc
Mac
R1
CCAP 1403/2a
CCAP 1459/39
CCAP 1429/1
CCAP 1553/33
CCAP 1402/1
CCAP 1427/1
CCAP 1478/1
CCAP 1480/4
CCAP 1453/20
CCAP 1446/1C
CCAP 1403/13A, /13B, /13D
to
/13H
CCAP 1453/2, /11,
/15
to
/19
ATCC 27892
PCC 7120
P.
Bisen (Barkatullah University, India)
JM
JM
BGll
BGll
BGll
BGll
BGll
BGll
BGll
BCll
BGll
BGll
AA/4
AA/4
AA/4
Table
2.
Isolates used
to
assess resolution of subspecific differences
See the
CCAP
on-line database [http
:
//www.ife.ac.uk/ccap/] for further information on these
strains.
Species Isolate identification Origin, year
of
isolation and other
characteristics
Anabaena
sp.*
Anabaena flos-aquae
Anabaena flos-aquae
Anabaena flos-aquae
Anabaena flos-aquae
Anabaena flos-aquae
Anabaena flos-aquae
Anabaena flos-aquae
Anabaena flos-aquaet
Nostoc ellipsosporum
Nostoc ellipsosporum
Nostoc ellipsosporum
Nostoc ellipsosporum
Nostoc ellipsosporum
Nostoc ellipsosporum
Nostoc ellipsosporum
CCAP 1403/13A
CCAP 1403/13B
CCAP 1403/13C
CCAP 1403/13D
CCAP 1403/13E
CCAP 1403/13F
CCAP 1403/13G
CCAP 1403/13H
CCAP 1446/1C
CCAP 1453/2
CCAP 1453/11
CCAP 1453/15
CCAP 1453/16
CCAP 1453/17
CCAP 1453/18
CCAP 1453/19
Sewage oxidation pond, Mississippi,
Windermere, Cumbria, England;
1964
Blelham Tarn, Cumbria, England;
1972
Wales;
1976;
mutant, no gas vacuoles
Wales;
1976;
mutant, helical filaments
Windermere, Cumbria, England
;
1976;
reisolation of CCAP
1403/13B
Windermere, Cumbria, England
;
1976;
reisolation of CCAP
1403/13B
Windermere, Cumbria, England
;
1964
Unknown
Freshwater ditch, Utrecht, Netherlands
Fresh water, Sweden;
1950
Fresh water;
USA
Fresh water;
1966
Fresh water;
1967
Fresh water, Wisconsin,
USA;
1968
Fresh water
USA;
1964
*Formerly designated
A. flos-aquae.
t
Formerly designated
A. inaequalis.
placed at
-20 "C
for at least
20
min. DNA, heavily con-
taminated by polysaccharide in samples derived from some
species, was recovered by centrifugation (microfuge, prior
to
use in PCR.
13200
r.p.m.,
8
min) and washed with
70%
ethanol prior to
drying
of
the sample in an airflow cabinet at room temperature
and resuspension in
TE
buffer containing DNase-free RNase
A
(25
pg ml-'; Sigma). DNA recovered by this technique
varied from
0.2
to
1.2
pg per
10
ml of culture. DNA prepar-
ations were stored at
-20
"C
and further diluted in
TE
buffer
DNA concentrations were estimated directly from ethidium
bromide fluorescence in agarose gel images against standard
quantities
of
1
bacteriophage DNA, either by using a
Pharmacia gel documentation system and associated software
2793
J.
K.
SMITH
and
OTHERS
or from scanned images
of
gel photographs analysed using
NIH Image software, version 1.59, on a Macintosh computer.
NIH Image software is available by ftp transfer protocols
from Info-mac archives (e:g. ftp
:
//src.doc.ic.ac.uk/
packages/info-mac/) that are mirrored worldwide.
PCR
conditions.
PCR reactions were either run as
50
pl
volumes in 300
p1
thin-walled PCR tubes (Perkin Elmer) or
2.5 pl volumes in 96-tube plates (skirted Thermo-Fast 96)
sealed with Thermo-Seal (heat-sealing foil) using a Combi
Thermo-Sealer (Advanced Biotechnologies). Reactions nor-
mally contained approximately 20-30 pg DNA template
(samples shown in Fig.
3
contained up to
3
ng DNA template),
one primer (0.6 pM), deoxynucleoside triphosphate mixture
(40 nM each), 1.0 unit
Tag
DNA polymerase (Gibco-BRL) or
other heat-stable DNA polymerase, one-tenth volume
of
the
appropriate 10
x
buffer supplied by the manufacturer, sup-
plemented where appropriate to give a final MgCl, con-
centration
of
2.0 mM, in a total volume
of
50
pl,
or half these
amounts in 25
pl
volumes.
Reactions were cycled on an Ericomp Deltacycler I1 thermal
cycler using a temperature profile of one cycle
of
95 "C,
5
min;
30 cycles
of
95 "C, 30
s;
30 "C, 30
s;
72 "C, 60
s;
one cycle of
72 "C,
5
min.
There are 16 possible variants of primers based on the Hipl
sequence GCGATCGC and containing two additional nucleo-
tides at the
3'
end. Four of the 16 possible primers were chosen
arbitrarily and used for this study: HipCA (GCGATCGCCA),
HipAT (GCGATCGCAT), HipTG (GCGATCGCTG) and
HipGC (GCGATCGCCC). The fifth primer used in this
study, HipTC (GCGATCGCTC), corresponds
to
a sequence
that is found upstream
of
nifH
genes and other genes
transcribed during heterocyst development in
Anabaena
species (Beesley
et al.,
1994). PCR products were separated by
agarose gel electrophoresis in TBE buffer according
to
standard protocols (Sambrook
et al.,
1989) by loading 2-10 pl
of the reaction mixture mixed with loading buffer onto an
agarose gel containing 1.5% (w/v) agarose (NBL Gene
Sciences) and 1.S
'/o
(w/v) NuSieve3
:
1
(FMC Bioproducts). In
some instances (Fig. 3) a deliberate overloading
of
the gel is
shown in order that minor products (light bands) are visible.
Typically a loading
of
2-3 pl
of
reaction mixture was sufficient
to show banding patterns suitable for typing. All the extended
Hipl PCR profiles shown were repeated at least five times.
The method
of
Lu
et al.
(1997) was used to provide an
alternative means
of
characterizing the
Anabaena flos-aquae
and
Nostoc ellipsosporurn
isolates. This technique employs
PCR
to
amplify products from the 16s-23s rDNA using the
primer pairs Rl/R18, R14/R18 or R17/R18, as described by
Wilmotte
et al.
111993). Rl/Rl%primed PCR amplifies the 16s
rDNA, the internal transcribed spacer (ITS) region, tDNA"",
or tDNA"" and tDNAA'", and the
5'
end
of
23s
rDNA.
R14/R18-primed PCR amplifies the same rDNA region, but
starting from the 3' end of 16s rDNA. R17/R18-primed PCR
amplifies part of the ITS region, starting at tDNA"". Products
formed in R17/R18-primed PCR were analysed for restriction
polymorphisms using the restriction endonucleases
Hinfr,
DdeI
and
AluI,
identified as discriminating cyanobacterial
species by Lu
et al.
(1997). The rDNA PCR and RFLP analysis
was repeated three times.
The results presented were scanned from the photographic
images obtained from a Pharmacia gel documentation system
(Pharmacia Biotech) and the digitized images were inverted,
corrected for brightness and contrast, and labelled using
Adobe Photoshop version 2.5 prior
to
printing. The size of
PCR products was determined using software associated with
the gel documentation system and a 123 bp ladder DNA
standard (Gibco-BRL).
RESULTS
Optimization
of
the extended Hipl
PCR
The template DNA preparation procedure used for
extended Hip
1
typing yields relatively small quantities
of impure DNA, but was adopted for the attributes of
simplicity, rapidity and general application to diverse
cyanobacterial species.
To
assess whether this crude
method allowed reproducible PCR, cultures
of
Ana-
baena
ATCC 27892 were grown and extracted sep-
arately. No significant difference was observed in
products obtained by extended Hipl PCR from these
templates (Fig. la). Extended Hipl PCR was achieved
directly from cultures concentrated 10-fold, resuspended
in
TE
buffer and
3
pl used in place
of
template DNA.
Rasmussen
&
Svenning (1998) achieved PCR using an
STRR primer from cyanobacterial cells (filaments).
However, in this study both the productivity and the
reproducibility
of
the Hipl PCR reaction, particularly in
formation
of
larger PCR products, was decreased when
whole-cell suspensions were employed instead of a DNA
template. This may be a consequence of the different
PCR conditions employed.
Concentration
of
template.
The concentration
of
DNA
template that was required for extended Hipl PCR was
determined empirically. Most DNA preparations ad-
equately supported the PCR reaction at a concentration
of
20 pg per
SO
pl reaction (Fig. lb), but others, notably
those from species in which substantial polysaccharide
contamination was apparent, required up to
3
ng per
50
pl
reaction to achieve good productivity, presumably
due to some inhibition
of
the PCR reaction. However
3
ng quantities are not excessive. Various quantities
of
cyanobacterial templates have been employed in PCR
reactions reported in the literature; for example, 100 ng
(Lu
et
af.,
1997),
50
ng (Nelissen
et
af.,
1994; Rasrnussen
&
Svenning, 1998),
10
ng (Bolch
et
af.,
1996) and
5
ng
(Wilmotte
et
al.,
1994) of template DNA per reaction.
Reaction with contaminant bacteria.
To
assess the effect of
bacterial contamination, DNA preparations
of
purified
freshwater coliform and streptococcal isolates were used
in extended Hipl PCR and were shown to form products
(Fig. lc).
Concentration
of
primer.
Primer concentration was de-
termined empirically. Concentrations between 100 and
600
nM did not affect the number of bands obtained, but
productivity of the reaction was improved at the higher
primer concentration. Primer concentrations above
600
nM and enzyme activities above 2 units per
50
pl
reaction increased nonspecific background relative to
discrete products, the two factors being interdependent
(data not shown).
Concentration
of
Mg2*.
The MgC1, concentration of PCR
reactions directly affects product formation through the
Hipl-based PCR for typing cyanobacteria
Fig.
I.
(a) Assessment of variation due to template preparation. DNA prepared separately from different cultures of
Anabaena
ATCC
27892
was purified and used
as
template in
a
PCR
reaction employing HipCA primer. Four examples are
shown. (b) Assessment of DNA template concentration. An
Anabaena
ATCC
27892
DNA template was serially diluted
10-
fold and
3
pl
used
as
template in
a
PCR
reaction employing HipTG primer under standard conditions. Lanes:
1,
123
bp
ladder;
2,
3
ng template DNA;
3, 300
pg template DNA;
4,
30
pg template DNA;
5,
3.0
pg template DNA. (c) Hip primer
products from freshwater bacterial isolates. A
PCR
reaction employing DNA templates prepared from coliform (lanes
2
and
3)
and streptococcal (lanes
4
to
7)
freshwater bacterial isolates and HipTG primer was performed under standard
conditions. Lane
1,
123
bp ladder. (d) Effect of varying Mg2+ concentration. A
PCR
reaction employing
an
A.
cylindrica
DNA template and HipCA primer was performed under standard conditions. Lanes:
1, 123
bp ladder;
2,
no Mg2';
3,
0.5
mM Mg2+;
4, 1.0
mM Mg2+;
5,
1.5
mM Mg2+;
6, 2.0
mM Mg2+;
7,
2.5
mM Mg2+;
8, 3.0
mM Mg2+. (e) Effect of using
different DNA polymerase preparations. A
PCR
reaction employing an
A.
circularis
DNA template and HipTG primer was
performed under standard conditions. The DNA polymerase preparations used were: lane
1,
Taq
polymerase, HT
Biotechnologies,
UK;
lane
2,
Taq
polymerase, Applied Biotechnologies,
UK;
lane
3,
Tag
polymerase, Perkin Elmer,
UK;
lane
4,
Taq
polymerase, Gibco-BRL; lane
5,
Tbr
polymerase, Northumbria Biochemicals,
UK;
lane
6, 123
bp ladder.
(f)
Effect of annealing temperature.
PCR
reactions employing
a
Nostoc
Mac DNA template and HipGC primer were
performed under standard conditions, but at different annealing temperatures. Lanes:
1,
30
"C;
2, 32
"C;
3, 34
"C;
4,
36
"C;
5,
38
"C;
6,40
"C;
7,
50
"C;
8, 60
"C.
stringency
of
primer-template interaction.
A
minimum
of
1.5
mM MgCI, was required to obtain products, but
no significant difference was observed in products
obtained from reactions containing between
2.0
and
3.0
mM MgCl, (Fig. Id).
Number
of
cycles.
Although
30
PCR reaction cycles were
used in this work,
20
cycles were sufficient
to
yield ample
product with most templates (data not shown).
A
minimum of
30
s
denaturation was sufficient, but
reduction
of
the extension step below
30
s
decreased
productivity of longer products, particularly with some
template preparations. Use
of
a stepped programme in
which the time taken
to
raise the annealing temperature,
30
"C,
to the extension temperature,
72
"C, was delayed,
in order
LO
encourage extension and stabilization
of
the
short Hipl primers, yielded products that did not differ
in number or quantity from those obtained with the
standard profile (data not shown).
Taq
DNA polymerase has been reported (Meunier
&
Grimont,
1993).
The products formed in extended Hipl-
primed PCR from cyanobacterial genomic DNA tem-
plates did not vary significantly when any of four
different
Taq
polymerase preparations, obtained from
different manufacturers, or
T6r
polymerase was used
to
catalyse the reactions, even when the reactions were
constructed with the different manufacturers' buffers
and MgCl, solutions (Fig. le). However, it was necessary
to
compare equivalent enzyme activities per PCR
reaction and/or equivalent loading
of
PCR products on
agarose gels. Comparison
of
different quantities
of
products may be complicated by low-band-intensity
products, which,
in
extremis,
are visible at high loading,
but not at low loading. In this limited study, comparison
of
banding patterns
of
different intensities could not give
rise
to
confusion between species. Even
so,
recognition
and compensation for low yield in the PCR reaction is
recommended. Extended Hipl PCR yielded the same
Source
of
polymerase and
PCR
machine.
Variation in PCR
typing techniques due to the use
of
different supplies
of
products when cycling was performed with three dif-
ferent PCR machines (Deltacycler
I1
system, Ericomp;
2795
J.
K.
SMITH
and
OTHERS
Fig.
2.
Comparison of the
PCR
products formed in reactions
primed with different extended Hipl primers. Template DNA
isolated from
Anabaena
PCC
7120 (a) and
A. flos-aquae
CCAP
1403/13B
(b).
DNA thermal cycler, Perkin Elmer; GeneAmp
2400
PCR system, Perkin Elmer). Nor was variation en-
countered in the use of PCR tubes or sealed 96-tube
plates or due to the position of a tube in a heating block
(data not shown).
Annealing temperature.
The use
of
annealing temper-
atures in the PCR cycle between
30
"C and
40
"C did not
significantly alter the products obtained with extended
Hip1 PCR except that a few products increased or
decreased in yield (Fig.
lf).
At higher temperatures
(50-60
"C) some products were decreased in intensity/
productivity, while other products, which formed low-
intensity bands on agarose gel electrophoresis when
obtained from
low-annealing-temperature PCR (30 "C),
increased in productivity and appeared as high-intensity
bands on agarose gel electrophoresis.
Comparison
of
the five primers
Each
of
the five extended Hip1 primers reproducibly
yielded a distinct set of products when used individually
to prime
PCR
from the same cyanobacterial genomic
DNA template (Fig. 2). Each of the five extended Hipl
primers reproducibly yielded a distinct set
of
products
from each
of
15
cyanobacterial species tested. Examples
of HipTG-, HipGC- and HipCA-primed PCR reactions
.................................................................................................................................................
Fig.
3.
Comparison of the
PCR
products obtained in extended
Hipl
PCR
from
ten different cyanobacterial DNA templates
using HipTG (a), HipGC (b) and Hip-
(c).
with ten different cyanobacterial species are shown in
Fig.
3.
Resolution
of
strains within species
To
assess the resolution of subspecies differences
afforded by extended Hipl primer typing, two separate
sets
of
isolates of cyanobacterial species, which have
been maintained in the CCAP, were used. These sets
of
isolates were taken as likely to represent different strains
of cyanobacterial species. One set,
of
eight isolates,
originally classified
as
Anabaena
eos-aquae,
originated
2796
Hipl-based PCR for typing cyanobacteria
.
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.
Fig.
4.
Comparison of the R14/R18-primed rDNA PCR products
obtained for the
A.
flos-aquae
(a) and
N.
ellipsosporum
(b)
isolates.
Fig.
5.
Comparison of the R17/R18-primed rDNA
PCR
products
obtained for the
A.
flos-aquae (a) and
N.
ellipsosporum
(b)
isolates.
predominantly from
UK
sources. Isolate CCAP
1403/
13A
was originally recorded as
A.
flos-aquae,
but
has since been reclassified as
Anabaena
sp. It was
included in the experiment as a sample that was likely to
be distinct. The other set,
of
seven isolates classified as
Nostoc ellipsosporum,
originated from a variety of
European and North American sources (Table
2).
The rDNA analysis
of
the
A.
flos-aquae
isolates showed
that isolates CCAP 1403/13A
to
/13H
produced similar
PCR products and restriction products except that
isolate 1403/13D showed an additional product in
R14/Rl8-primed PCR (Fig. 4). Isolate 1446/1C was
distinct from the other
A.
flos-aquae
isolates (Figs 4 and
5).
Hipl typing confirmed the differences between
isolates identified by rDNA analysis. The CCAP
1403/13B,
/13E,
/13F, /13G and
/13H
isolates yielded
indistinguishable products by rDNA analysis and in all
five extended Hipl-primed PCR reactions. Isolate CCAP
1446/1C was clearly distinguished by both rDNA- and
Hipl-primed PCR. Examples
of
HipCA- and HipAT-
primed PCR products are shown in Fig.
7.
However,
although distinguished by an additional product in
R14/R18-primed PCR (Fig. 4), CCAP 1403/13D was
not distinguished by Hipl-primed PCR (Fig.
7).
In
contrast, isolate CCAP 1403/13A was clearly dis-
tinguished from the majority
of
CCAP 1403 isolates by
Hipl PCR (Fig.
7),
but was distinguished by rDNA
analysis only by slightly smaller R17/R18 major PCR
product and restriction fragments (Figs
5
and
6).
The rDNA PCR analysis
of
the seven
N. ellipsosporum
isolates showed that they were diverse (Figs 4 and
5),
in
either the size
of
the PCR products or restriction
fragments formed. Extended Hipl-primed PCR also
yielded diverse products, confirming the differences
between species identified by rDNA analysis
;
examples
of
HipCA- and HipAT-primed PCR products are shown
in Fig.
8.
Major and minor PCR products were observed
for both the R14/R18-primed products and the
R17/RlS-primed products
of
both sets
of
isolates (Figs
4 and
5).
DISCUSSION
The discovery of the Hipl sequence and its presence in
many, though not all, cyanobacteria (Robinson
et al.,
1995) allowed an assessment
of
a cyanobacterial typing
technique which, akin
to
the repetitive-sequence-
2797
J.
K.
SMITH
and
OTHERS
Fig.
6.
RFLP analysis
of
the R17/Rl&primed rDNA PCR products
obtained
for
the
A.
flos-aquae isolates using
Ddel
(a),
Alul
(b)
and
Hinfl
(c).
Fig.
8.
Extended Hipl-primed PCR products obtained
for
the
N.
ellipsosporurn
isolates using HipCA primer (a) and HipAT primer
(b).
amplification of
DNA
between adjacent repeated Hipl
sequences in the chromosomal
DNA
of cyanobacteria.
However, the frequency of Hipl repeats in the
DNA
of
some cyanobacteria is high. For example, Robinson
et
al.
(1995) estimated from analysis
of
database sequence
that the Hipl sequence occurred on average every
320
bp
in the chromosomal
DNA
of
Synechococcus
PCC 6301.
Consequently the number
of
discrete products obtained
in a Hipl-primed PCR reaction is very large and not
conducive
to
distinguishing species by a simple com-
parison
of
agarose gel electrophoresis patterns.
To
decrease
the
number of PCR products obtained and
therefore provide a clearer and more distinctive banding
pattern, primers were constructed in which two ad-
ditional bases were added to the
3’
end
of
the Hipl
sequence.
DNA
polymerases extend the
3’
end of primers. Since
the complementarity of the
3’
end
of
the primer with the
template dictates efficient extension and PCR product
formation, extending the
3’
end
of
the palindromic Hipl
sequence by two nucleotides should restrict efficient
priming
to
those genomic Hipl sequences in which the
flanking sequences
complement
the
nucleotide
extension. This strategy was successful, as shown by the
different PCR products obtained from a cyanobacterial
genomic
DNA
template with each
of
the
5’
extended
Hipl primers that were tested (Fig.
2).
The prime objective of the work was
to
assess the ability
of
extended Hipl-primed PCR
to
clearly distinguish
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.
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,
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,
.
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,
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.
Fig.
7.
Extended Hipl-primed
PCR
products obtained
for
the
A.
flos-aquae
isolates using HipCA primer (a) and HipAT primer
(b).
orientated PCR techniques used with bacteria (e.g.
Versalovic
et
al.,
1991
;
Vila
et
al.,
1996) and cyano-
bacteria (Rasmussen
&
Svenning, 1998), is based on the
2798
Hipl-based
PCR
for typing cyanobacteria
species and strains of cyanobacteria. In the three
examples shown, ten species of cyanobacteria drawn
from a variety of genera were tested and yielded clearly
distinct extended Hipl-primed PCR products (Fig. 3).
The extended Hipl primers generated PCR products
and agarose gel electrophoresis banding patterns that
clearly distinguished all ten species of cyanobacteria.
These included the three
Nostoc
and two
Anabaena
species that present examples of closely related species
occupying the same genera.
One characteristic of the banding patterns on agarose
gel electrophoresis obtained from extended Hipl PCR
products is the presence of bands of widely different
intensities resulting from the different quantities of
products formed (Fig.
3).
This variation in productivity
was reproducible and was not affected by
Taq
poly-
merase activity, Mg2+ concentration or primer con-
centration. Such variation in the intensity of bands (i.e.
productivity of individual products) is a characteristic of
RAPD and repetitive-sequence PCR as shown by pub-
lished data (e.g. for bacteria, Meunier
&
Grimont, 1993
;
Gillings
&
Holley, 1997; Mahenthiralingam
et al.,
1996;
and cyanobacteria, Rasmussen
&
Svenning, 1998).
Experiments showed that some high-intensity products
declined in productivity as the PCR annealing tempera-
ture was increased, indicating that they resulted from
inexact priming from template sites that were not
completely complementary to the primer (Fig.
lf).
Consequently inexact priming does not provide a simple
explanation for the formation of low-intensity products.
Other low-intensity products increased in productivity
as the annealing temperature increased. This would be
consistent with the hypothesis that intramolecular
template secondary structure occludes some priming
sites, reducing productivity of some Hipl-primed
products. Since most typing reactions employ annealing
temperatures well below 65 "C, at which template
secondary structure is minimized, occlusion of primer
sites by secondary structure may explain the variation in
PCR product intensity which is common to PCR typing
techniques.
To explore the resolution afforded by extended Hip1
PCR, two sets of isolates present in the-CCAP collection
(Tables
1
and 2) were analysed. The Hip1 analysis
compared favourably with the rDNA analysis in dis-
criminating similar and distinct strains. Both techniques
found the
A.
flos-aquae
strains to be similar and the
N.
ellipsosporum
strains to be more diverse. However, the
two techniques differ in their discrimination
of
isolates
CCAP 1403/13A and /13D. Clearly there exists an
element of chance as to whether a chosen technique will
distinguish particular strains and it would be unwise to
rely upon the results of a single technique as evidence
that two isolates are identical.
The products obtained from Rl/RWprimed (data not
shown) and R14/R18-primed (Fig. 4) PCR contained
minor products as reported by
Lu
et al.
(1997). These
minor products were ascribed
by
Lu
et al.
(1997) to
heterogeneity for heterocyst differentiation or the for-
mation of heteroduplexes containing conserved
3'
and
5'
'
ends, but highly variable ITS sequences. Minor products
were also present in R17/RWprimed rDNA PCR (Fig.
5).
Such minor products were not reported by Lu
et al.
(1997). In addition to the explanations suggested by
Lu
et al.
(1997) for minor products, they may also be
attributed to a template at low concentration, e.g. a
contaminating bacterial DNA
-
although the R18
primer discriminates against bacterial rDNA amplifi-
cation (Lu
et al.,
1997; Nelissen
et al.,
1996) -or rare
cyanobacterial rDNA cistrons that retain the R17 primer
site within the tDNA"" gene, but are of a different
structure within the 3' end of the ITS region. Alter-
natively they may represent poor amplification of
products primed from sites outside the rRNA cistrons.
RFLP analysis of R17/R18-primed PCR products was
complicated by the presence of the minor products, but
the restriction fragments obtained for the
A.
flos-aquae
isolates were similar to those described by
Lu
et al.
(1997) for the
A.
flos-
aquae
isolate included in their
survey.
Extended Hipl PCR appears from this initial study to be
robust, contain less inherent variability than reported
for other PCR typing systems (Meunier
&
Grimont,
1993) and be applicable to a wide range of cyano-
bacterial species and strains. Hipl PCR produces
products with bacterial templates (Fig. lc). While fewer
than those normally found with cyanobacterial tem-
plates, this necessitates the purificiation of cyano-
bacterial isolates prior to PCR typing. Use of different
enzyme activities per PCR reaction and/or comparison
of different loads on agarose gels were identified as
potential sources
of
confusion, particularly where less
intense bands (low-productivity products) are included.
As with other enzyme-based assays, a consistent and
uniform construction of the assay and analysis of
products is required, with recognition of and com-
pensation for poor amplification of products.
The CCAP database contains information describing
the
A.
flos-aquae
and
N.
ellipsosporum
isolates used in
this study (compiled in Table 2). The six related strains
of
A.
flos-aquae
all originated from
UK
sources (Lake
Windermere and Wales) over a period of 12 years. The
two distinct strains are probably of American origin.
Note that CCAP 1446/1C was originally designated
Anabaena inaequalis
and later reclassified as
A.
flos-
aquae.
CCAP 1403/ 13A, originally designated
A.
flos-
aquae,
has been reclassified as
Anabaena
sp. (Tompkins
et al.,
1995). The
N.
ellipsosporum
isolates, which
appear to be more diverse than the
A.
flos-aquae
group,
have a more varied geographical origin. The number of
isolates tested is small, but would suggest that strain
variation is an aspect of major geographical location
and that ecotypes within a region are more closely
related.
Five of the 16 possible Hipl extended primers have been
shown to produce different PCR products (Figs 2 and 3)
for each species of cyanobacterium studied and to
distinguish cyanobacterial strains (Figs
7
and
8).
One
advantage of Hipl typing is the degree of resolution that
is afforded
by
possible use of all 16 extended Hipl
I
2799
J.
K.
SMITH
and
OTHERS
primers. Even
so,
as with other PCR-based techniques,
the need to assume that size
of
product is an indicator
of
identical products, and the relatively low information
content
of
the data, detract from the use
of
extended
Hipl PCR for phylogenetic studies. Extended Hipl PCR
typing may be
of
use in distinguishing cyanobacterial
strains and species where a large number of samples are
involved, e.g. the enumeration of purified isolates from
environmental samples, in the maintenance of stock
cultures or as a preliminary assay to distinguish between
multiple strains prior to rDNA sequence analysis.
ACKNOWLEDGEMENTS
We thank the Leverhulme Trust, who supported this work
under grant number F/185/W. We also wish
to
thank
N.
and
P.
Robinson (Newcastle upon Tyne University,
UK)
for kindly
providing a sample
of
Hipl primer and a
Calothrix
D253
DNA sample, and
P.
Bisen (Barkatullah University, India) and
P.
Rowel1 (Dundee University,
UK)
for providing cultures.
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Received
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April
1998; revised 15 May 1998; accepted
22
June 1998.
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