FEMS Microbiology Letters 82 (1991) 73-78
© 1991 Federation of European Microbiological Societies 0378-1097/91/$03.50
Published by Elsevier
ADONIS 037810979100352Q
73
FEMSLE 04547
Isolation of strains of Thermus aquaticus from the Australian
Artesian Basin and a simple and rapid procedure
for the preparation of their plasmids
S. Denman, K. H a m p s o n and B.K.C. Patel
DiL,ision of Science and Technology, Griffith UniL,ersity, Nathan, Brisbane, Australia
Received 28 February 1991
Revision received and accepted 25 April 1991
Key words: Thermophile; Thermus; Plasmid; Australian Artesian Basin; Volcanic hot spring
1. SUMMARY
2. INTRODUCTION
Thirty four Thermus aquaticus strains have
been isolated from the non-volcanically naturally
heated waters of the Australian Artesian Basin
which extends the known ecological habitat of
this group of organisms. A simple and rapid
method developed for isolation of plasmids indicated that considerable variation in numbers and
molecular sizes existed within the 23 strains that
were investigated. Dissimilar plasmid profiles
were obtained from the strains that been isolated
from the source waters and those that had been
isolated from the runoff channels formed by these
source waters.
During the past 20 years there have been
dramatic advances in the isolation of thermophilic microorganisms with as many as 76 new
genera-species being described [1,2]. Amongst
them, Thermus aquaticus has been the most widely
investigated. The microbiology and growth characteristics have been described in detail and more
recently the question of their habitat, taxonomy
and phylogeny have been under close scrutiny
[3-5]. The reasons for the thermostability of enzyme and cell membrane has also been intensively studied [6,7]. However, the genetic apparatus of this group of organisms is still poorly
understood mainly due to lack of suitable investigating techniques. For example, the methods described for isolating plasmids are either time consuming or require expensive equipment and
reagents.
We report here the isolation of Thermus
aquaticus strains from the non-volcanically heated
Correspondence to: B.K.C. Patel, Division of Science and
Technology, Griffith University, Nathan, Brisbane, Queensland 4111, Australia.
74
waters of the Australian Artesian Basin, extending their known habitat. The use of a simple,
rapid and reproducible procedure for isolating
plasmids from these organisms is also reported.
3. M A T E R I A L S A N D M E T H O D S
3.1. Sampling site and sample collection
Samples from the source of 16 flowing Australian Artesian Basin bores situated in Ilfracombe, Barcaldine, Isisford, Blackall and Longreach areas of the State of Queensland were
collected by completely filling sterile glass containers directly with the flowing bore water before the water fell onto the ground and could be
contaminated by soil organisms. Other samples
(sediment and water) were collected at various
points of the runoff channel that had been formed
after the bore water had fallen onto the ground.
The temperature of the collected samples ranged
from the very hot (source samples) to the ambient
(runoff channel samples). The samples were
transported to the laboratory and stored at ambient temperature before use. Data on the samples
collected during the investigation is provided in
Table 1. T e m p e r a t u r e and p H were measured in
situ during sampling.
3.2. Enrichment and isolation of T. aquaticus
strains
T. aquaticus strains were enriched by inoculating the appropriate sample into Medium D [3]
and incubating at 6 8 ° C for up to 3 days. Pure
cultures were obtained by streaking positive enrichment cultures onto Medium D agar (2.5%),
wraping the plates in polyethylene bags to prevent evaporation and incubating them at 6 8 ° C
for 2 - 3 days. Pure cultures so obtained were
stored as bead cultures at - 7 0 ° C essentially as
described [8] until required.
3.3. Plasmid isolation
Cultures for plasmid analysis were grown (from
preserved bead cultures) as shaken-flask cultures
in 100 ml of Medium D, at 6 8 ° C for 18 h. The
cells were centrifuged at 8000 rpm and resuspended in sterile distilled water to give the de-
sired absorbance at 660 nm (see 4.3). Aliquots of
1 ml of the suspension were pipetted into microfuge tubes briefly centrifuged at 8000 rpm and
resuspended in 200/zl of lysis buffer (8% sucrose,
0.5% Triton X-100, 50 mM E D T A , 10 mM Tris.
HC1, p H 8.0) to which was added 15 txl lysozyme
(10 m g / m l prepared in 10 mM Tris. HCl, p H
8.0). The suspension was then incubated at 85 ° C,
90 ° C and 95 ° C for different time intervals between 30 and 90 s, cooled in an icebath and
centrifuged at 14000 rpm for 5 min.
3.4. Plasmid sizing
For sizing of the plasmids a supercoiled D N A
ladder from 2 to 16 kb was used (BRL, MD). The
product was aliquoted into 5 tzl on receipt and
stored at - 2 0 ° C until used. Escherichia coil
V517 was grown in Nutrient Broth (Difco Labs,
Detroit, MI) at 37 ° C. The procedure described
in section 3.3 was used to prepare plasmids and
subsequently used as a reference size standard as
described [9].
3.5. Electrophoresis
For plasmid analysis, 18 tzl of the supernatant
was mixed with 2 izl of × 10 loading buffer [10]
and loaded on a 4 m m thick 0.8% agarose gel; for
plasmid sizing, 10 txl of the frozen stock supercoiled D N A ladder that had been diluted fifty-fold
with x 1 loading buffer, was used. The gel was
run at 5 - 6 V / c m for 12-14 h in × 1 T B E buffer
[10], stained with ethidium bromide (0.5 / z g / m l )
for 30 rain and photographed over ultraviolet
light.
4. R E S U L T S
4.1. Isolation of T. aquaticus strains
A total of 34 enrichment cultures were obtained from the 48 Australian Artesian water
samples. All enrichments gave rise to yellow pigmented colonies on agar medium on incubation
for 2 - 3 days at 68 °C. Of the 34 pure cultures
obtained from the initial enrichment cultures, six
strains were isolated from the water samples collected directly from the source of 15 bores and
the remaining 28 strains were isolated from 33
75
Table 1
D i s t r i b u t i o n of
Bore
No.
Thermus aquaticus strains, isolated f r o m hot w a t e r s of the A u s t r a l i a n A r t e s i a n Basin a n d t h e i r p l a s m i d profiles
Sampling
site
Laboratory
No.
A
B
AB53
AB54
365
65
58
+
+
ND
ND
A
B
C
Av
AB31
AB32
AB33
AB2
914
60
57
53
63
+
+
+
+
0
ND
10.8, 14.8, 46.1
0
AB3
AB42
AB1
AB28
AB29
AB30
AB35
AB36
A B 16
AB17
AB18
ABI9
AB20
AB10
ABllA
ABllB
AB12
A B 13
AB14
AB15
AB55
ND
506
762
1205
4381
B
Av
Av
Av
B
C
Av
B
Av
B
C
D
E
Av
B
C
Av
B
C
D
Av
ND
32
42
53
70
60
53
57
52
73
70
64
53
47
75
70
65
88
73
62
60
74
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
0
0
0
0
ND
10.6, 12, 15.1, 15.5, 54.4
ND
ND
ND
15.1, 48.3
0
10.5, 15.1
0
48.3
ND
ND
4892 *
B
Av
AB56
AB4
1086
56
75
+
ND
0
B
C
D
A
B
Av
B
AB5
AB6
AB7
AB8
AB9
AB21
AB22
74
57
31
76
66
80
74
+
+
+
+
+
0
0
0
10.5, 48.3
19.7
C
D
E
F
Av
Av
B
C
D
E
Av
Av
AB23
AB24
AB25
AB26
AB43
AB37
AB38
AB39
AB40
AB41
AB27
AB34
70
70
65
48
59
88
87
66
75
60
46
+
+
+
+
+
+
-
15.5, 20.4, 20.8
0
0
0
0
0
3
94
135
317
371
376 *
1494
3034
4164 *
4165
4893 *
5142
14588
17263
22981
22985
Bore Depth
(m)
765
1235
1268
1312
ND
ND
843
1614
46
403
Temperature
( o C)
Isolation of
Thermus
aquaticus
P l a s m i d size
(kb)
-
p H of all s a m p l e s was b e t w e e n 8 to 8.5. A l p h a b e t s ( A to F) d e n o t e the s a m p l i n g sites. V = Virgin b o r e w a t e r collected f r o m b o r e
outlet; N D = N o t d e t e r m i n e d ; 0 = N o b a n d s d e t e c t e d ; - = N e g a t i v e ; Thermus aquaticus strains not isolated.
* S a m p l e d twice at two d i f f e r e n t t i m e s o f t h e year; similar results w e r e o b t a i n e d .
76
samples obtained from the runoff channels of
these bores. Samples collected from bores or
their runoff channels with temperature below
57 ° C and 53 ° C respectively and above 80 ° C did
not yield T. aquaticus strains.
1
2
3
--54.4
--chr
4.2. Characteristics of T. aquaticus isolate AB17
The cells from all the 34 isolates were found to
be Gram-negative non-sporulating rods to filaments which are characteristic features of 7".
aquaticus group (Table 1). Further characterisation of isolate AB17, regarded as representative
of the Australian T. aquaticus strains, was found
to have a pH growth range between 6.0 and 9.5,
and a temperature range between 45 and 78 °C
with an optimum pH between 7 and 7.5 and an
optimum temperature of approximately 70 °C.
Isolate AB17 produced rotund bodies, possessed
a typical annelid cell wall structure [3], had a
predominance of iC15 and iC17 fatty acid [11]
and was extremely sensitive to the following antibiotics at 10 /,tg/ml: penicillin, streptomycin,
chloramphenicol, novobiocin, polymyxin B and
tetracycline. Growth was also inhibited in the
presence of 1% NaC1 or 3% agar. Scanning of
acetone extracted pigments from isolate AB17
produced a typical absorption spectrum profile
with a peak at 452 nm and is similar to that
reported for T. aquaticus [2,3]. The G + C content of isolate AB17 was 66% and is within the
range described for other T. aquaticus strains
[2,3].
4.3. Development of plasmid extraction procedure
for T. aquaticus
Three parameters were found to be most critical for obtaining reproducible plasmid extraction
from the T. aquaticus isolates under study. A
minimum of number of cells (A660n m of 5.0 per
ml representing 1.5 × 10 9 cells/ml) were essential for plasmid extraction. Plasmids were not
obtained if the numbers of cells were decreased.
If cell numbers were increased then interference
due to chromosomal D N A occurred. Plasmid extraction was found to be more efficient when cells
were incubated at 95 ° C for 45 s as opposed to
incubation at other temperatures and incubation
times. Lower incubation temperatures gave poor
--12.9
__5.6
--5.1
--3.9
--3.0
--2.7
I2.2
Fig. 1. Plasmids from T. aquaticus (ATCC 25104) showing 6
plasmid bands (lane 1), purified genomic D N A from "E
aquaticus AB31 (lane 2) and reference plasmids from E. cog
V517. Size estimation is in kb. Chr - indicates chromosomal
band.
lysis whereas increased incubation times released
greater amounts of chromosomal material from
the cells. Figs. 1 and 2 provide examples of the
1
54.4
-
chr
-
16.2
-
14.2
-
12.4
-
10
-
8.1
2
3
4
5
6
7
--
Fig. 2. Plasmid profiles ot the Australian Basin T. aquattcus
strains A B l l B , AB13, AB17, AB22, AB23, AB28, and AB33
in lanes 1, 2, 3, 4, 5, 6, and 7 respectively. Size estimation is in
kb. Chr = indicates chromosomal band. Note the variation in
the amounts of chromosomal DNA.
77
optimized extraction procedure described in this
paper.
4.4. Plasmid profiles from Thermus isolates
Only eight out of the 23 Australian Artesian
Basin T. aquaticus isolates investigated harboured plasmids (Table 1). The number of plasmids varied between 0 and 5 and the size ranged
from 10 to 55 kb. This is in contrast to the studies
by Raven and Williams [12] who demonstrated
the greater heterogeneity in size and numbers of
plasmids of T. aquaticus isolates of Yellowstone
National Park, U.S.A.
5. DISCUSSION
The isolation of T. aquaticus strains from the
naturally heated non-volcanic waters of the Artesian Basin, represents the first report of their
existence in the Australian environment. Their
isolation from source bore waters indicates that
these organisms were thriving in the depths of the
aquifer. The Artesian waters are alkaline (pH 7.6
to 9.0) and are different to that of volcanic hot
springs in which the pH varies from pH 1.0 to pH
10.0 and thereby influences the growth of T.
aquaticus in that environment. Therefore the upper and lower temperature niche recorded in the
Australian Artesian waters and their runoff channels for this bacterium is limited due to the
effects of temperature rather than pH. In general, however, the temperature range is similar to
that observed for the isolates obtained from the
volcanic hot waters of Iceland [13,14].
There appeared to be no correlation between
the plasmid size a n d / o r numbers amongst the
four of the six T. aquaticus strains isolated from
the water samples collected directly from bores.
This is an interesting finding since the aquifer,
unlike the volcanic hot springs, is made up of one
single huge reservoir of heated water and hence
the strains isolated from the water of bores represented the aquifer, and therefore these strains
should have had similar plasmid profiles. Also of
interest is that the plasmid profiles of T. aquaticus isolates from the runoff channel of bore number 4164 did not possess the same plasmid profile
as the isolates obtained from the source waters of
the bore (Table 1). This type of plasmid variation
may be similar to the phenotypic variation observed amongst T. aquaticus strains that had been
isolated from various sites of the runoff channel
of an Icelandic hot spring [14]. It has been suggested that the extraordinary degree of spontaneous phenotypic variability in thermophiles, including T. aquaticus, may be a genetic property of
organisms that are exposed to an extreme environmental stress not experienced by mesophilic
organisms. For example, loss of plasmids (e.g,
due to temperature fluctuations) may lead to the
evolution of new phenotypes through loss of plasmid-encoded functions [14].
The procedure described in this paper is more
rapid and simpler than the recent procedure described by Raven and Williams [12]. The method
will assist in plasmid profiling of global isolates,
and further studies on the role of plasmids and
plasmid instability may provide insights into diversity and evolution of genotypes and phenotypes in T. aquaticus isolates. The procedure will
also assist in the construction of plasmid vectors
and provide a rapid procedure for screening of
recombinant clones that would be required with
the development of an efficient host-vector
cloning system in this group of organisms.
ACKNOWLEDGEMENTS
The financial assistance of these studies from
U R G is gratefully acknowledged. We thank Mr.
John Hillier, Mr. John Palmer and field staff of
Queensland Water Resources Division for providing invaluable assistance in obtaining samples.
We are indebted to Dr. Ifor Beacham for providing Escherichia coli V517 and for helpful discussions during the preparation of this manuscript.
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