FEMS MicrobiologyLetters72 (1990)97-102
Publishedby Elsevier
97
FEMSLE04178
A plasmid vector for an extreme thermophile,
Thermus thermophilus
Y. Koyama t, y . Afikawa 2 and K. Furukawa t
t Fermentation ResearchInstitute. 41ST. MITL TaukubaScience Clff+Ibarakiand : NaganoState Laboratoryof FoodTechnology;
205.1 Nishibanbo.Kunta. NaganoCiO~Japan
Received27 February 1990
Re'asion received30 May 19qo
Acccptc¢l I June 1990
Key words: Thermus; Plasmid vector; Transformation; "l'ryptophan synthetase gene
1. SUMMARY
The host-vector system for an extreme thermophile. Thermus thermophilus HB27, was developed. The host strain has a mutation in tryptophan
synthetase gene (trpB). and the mutation was determined to be a missense mutation by DNA
sequence analysis. A Thermus.E. cob shuttle vector pYKI09 was constructed, pYKI09 consists of
Thermus cryptic plasmid p'I'T8, tryptophan synthetas¢ gene (trpB) of Thermus T2 and E. coh
plasmid vector pUCI3, pYK109 t-~nsformed T.
thermophilus HB27 trpB5 to Trp , a :. {uency
of 106 transformants per ~.g DNA.
2. INTRODUCTION
Thermus sp. are extremely thermophilie Gramnegative bacteria which can grow at over 75°C.
Since no antibiotic resistance plasmid has been
Correspondenceto."YoshinoriKoyama.Fermcl~tatlonResearch
Institute, AIST, MITt, Tsukuba Science City, Ibaxaki 305.
Japan+
igc,!ated from Thermus bacteria and antibiotic resistance g'mes from mesophilic bacteria do not
function at high temperature, a Thermus plasmid
vector has not yet been constructed.
We reported previously natural DNA transformation events in T. thermophilus HB27 [1]. Auxotrophic strains of T. thermophilus HB27 were
transformed 1o prototrophy at high frequencies of
10 -2 to 10 ~ when cells were incubated at 70°C
with wild-type chromosomal DNA. Cells of T.
thermophilus HB27 require no chemical treatment
to induce competence. Introduction of a cryptic
plasmid pTT8 into T. rhermophilus HB27 was also
demonstrated.
We have cloned tryptophan synthetase genes
(trpBA) from T. thermophilus HB27 for a selection
marker in Thermus [2]. However. T. thermophilus
HB27 trpBA genes were not suitable for a selection marker in T. thermophilus HB27 hosts, because the cloned DNA fragment of T. thermophilus HB27 trpBA genes recombined with the
chromosomal counterpart at high frequency.
We describe here the analysis of the trpB5
mutation of the host strain and the construction of
a Thermus-E. colt shuttle vector plasmid which
contains Thermus T2 trpB gene as a selection
marker.
0378.1097/90/S03,50 © 1990Federationof European MicrobiologicalSocieties
98
3. MATERIALS A N D METHODS
3. L Bacterial strains and plasmids
E. coli strains MC1009, MC1061 [3], JM83 [41
and E. coil plasmid vector pUC13, pUC19 [5]
were purchased from Pharmacia. E. colt plasmid
vector pUCII8 [61 was purchased from Takara
Shuzo Co. lhermus strains, T caldophilus GK24
[7], T. flavus AT62 [8], T aquaticus YTI [9] Ther.
mus T2 [10], T thermophilus HB8 [11], T thermophilus HB27 [12], T. thermophilus HB27 trpB5 [2]
and Thermus cryptic plasmid pTT8 [13] were described previously.
nucleotide sequence (315 bp) between the BamHl
and Sinai restriction sites of the insert D N A on
the plasmid was determined.
3.5. Determination of nucleotide sequence
The D N A fragment to be sequenced was cloned
in both orientation in pUCIIS. Single stranded
plasmid D N A was obtained by the method of
Vieira and Messing [6]. Nucleotide sequences were
determined with a D N A sequencer (Applied Bio.
systems model 370A),
4. RESULTS A N D DISCUSSION
3.2. Media and growth conditions
TM broth medium [1] was used for routine
cultivation and transformation experiments o~' T
thermophilus. Minimal medium for T. thermophilus
HB27 was described previously []4]. Liquid and
agar culture of :E thermophilus were incubated at
70°C.
3.3. DNA isolation, transformation and cloning
E. colt transformation and plasmid isolation
were performed as described [15]. T. thermophilus
HB27 trpB was transformed to Trp + as described
previously [1].
Thermus T2 trpBA genes were cloned as follows. Thermus T2 chromosomal D N A was partially digested with Mbol and 4-10 kbp D N A
fragments were isolated. After ligation of the D N A
fragments to BamH! digested pUCI3, E. ¢~li
MC1009 was transformed. The E. colt recombinant colony bank was screened by colony-hybridization with a 1,8 kbp BgllI-$acl D N A fragment
which contained trpB and the N-ternfinal region
of trpA of 7". thermophil~' HB27 121.
3.4. Location of the trpB5 mutation
Chromosomal DNA of T. thermophilus HB27
trpB5 mutant was digested with Sinai and 1.1-1.2
kbp DNA fragments were isolated. The D N A
fragments were ligated to Smal digested p U C I I 8
and E. colt JM83 was transformed. A colony
containing the trpB5 mutant gene was selected
from the colony bank by colony hybridization
with the 1.15 kbp Sinai fragment of wild type T~
thermophilus HB27 trpB geae [2] as a probe. The
4.L Location of the T. thermophilus HB27 trpB3
mutation
trpB5 mutation is known to be in a 315 bp
SmaI-BamH1 fragment which contains the Nterminal part of the trpB gent and the 5" flanking
region (Fig. 1) [2]. This region was cloned from
tile T. thermophilus HB27 trpB5 mulant. The
nuclcotide sequence of the mutant D N A revealed
one base change (G to A) at position 234 in Fig 1.
The glycine at position 20 was replaced with
glutamate in the trpB5 mutant enzyme,
4.Z Transformation of T, thermophilus HB27 trpB5
with heterospeeific chromosomal DNA
We have tried to construct a Thermus plasmid
vector containing the previously cloned tryplophan
synthetasc genes (trpBA)of T. thermophilus HB27
[2] as a selection marker. However, T, thermophilus HB27 trpBA genes were not suitable for a
selection marker, because these genes were found
to recombine with their chromosomal counterpart
at high frequency.
So we screened several Thermus strains for
tryptophan synthetase genes which would not recombine with T, thermophilas HB27 chromosomal
DNA, Z thermophilus HB27 trpB5 mutant was
transformed 1o Trp+ with DNA from various
Thermus strains (Table 1). The results suggested
that Thermus T2 tryptophan synthetase gene can
be used as a selection marker in T, thermophilus
HB27 trpB5 host, because the transformation
frequency with Thermus T2 chromosomal D N A
was lowest, with a frequency of about 10 -a corn-
SmaX
CCCGGGCCGCCTGGACGATGGCGCGGTCGTCCCCCATCTGGTCCACCAGGCGGACGAAGC
60
CC~TGTCCAAGACCGOGATCGTGAGAGGCCCTTCCATCCCCCCGAGTTTACCGGGAGGCC
120
CCTCCGGGGTAGUATCGGAGTTGTCTTGGCOCGAGGCGCCTTTAGG~AGCGAAGCATGCT
180
MerLe
CACCCTACCCGACTTTCCCTTGCCCGACGCGACGGGGCGGTTCGG~CCCTACGGGGGGCG
uThrLeuP~oAspPheProLeuPzoAspAlaArgG1y~rgPheGIyProTzrGlyGlyAr
24Q
GAG
G1u
CZ'pB5 m u t a Z l o n
GTACGTGCCCGAGACCCTGATCCCCGCCCTGGAGGAGTTGGAGGCCGCCTACCGGGAGGC
300
gTyrvelProG!uThrLeufleProAlaleuGluGluLeuGZuA1aAlaTyrArgGluA1
m~m~Z
CAAGAAG~ATCC
312
,~L/SZySASp
Fig, l. NudL~tidusequcu~c~ofthere~on
¢ofllalnins;hctrpB) lnu|allon cf l[Ihermophilll~HB27.
"Iabl¢ 1
pared with the parental I-iB27 chromosomal DNA.
Thermus T2 and T. thermopllilus are distantly
related taxonomically and were classified in differ¢nt groups [16].
It is not known why T. thermophilus HB27
trpB5 was transformed to Trp* at higher frequencies with chromosomal DIN?, from T. thermophilus
HBS, T. flavus AT62 and T. ca/dophilus GK24
than with that of the parental strain. A similar
phenomenon was also obsgrved in the transformation of other auxotrophic strains (Leu-, Met-,
Lys-) of T. thcrmophilus HB27 (data not shown).
Combined with the fact that the transformation
Trans[otmation of T ihermophih~ HB27 trpB5 Io Tip ~ with
various DNAs
C h r o m o s o m a l DNAs of wild type Thermu~ slrains except for
pKA2 plasmid were used for transformation ¢xpcrim©nL pKA2
plasntid contains 7~ thermophdr~ HB27 trpBA genes in pljC13.
DNA source
Transformation
frequency (%)
pKA2 plasmid DNA
T Ihcrmophilu.t HB27
2~ thermophilas HB?
T flav~.~ AT62
2~ caldophilv,¢ GK24
T aquatlc~ YTI
Thermu~ "[2
4.1
6.3
14.1
9,8
12,4
0.00(~
f r e q u e n c y o f 7". ther mophilus
C.(~05
HB27 auxotrophic
}
pKA 07[ 0c,3 =.l
[
--
pKA210 1
~I,I
pKA216 ]
pUG13
.~1
pKA211 I
pUC19
~/I
I
t
i
II
i I
II II
I
I
I
II I
I i u
I
I
I-----1
Fig, 2. pKA207 containing Therm~ T'2 Iryptophan syn|helase gen¢~ and its derivative p]asnfids. Location of Ihe trpB,4 genes is
shown,
100
markers with HB27 wild type chromosomal DNA
varies from 1 to 15% [1], this suggests that T.
thermophilus HB27 may have a base excision repair system which is similar to "'hex" system in
Streptococcus pneumoniae [17].
4.3. Cloning of Thermus T2 tryptophan synthetase
genes
A Thermus "I"2 chromosomal DNA gene bank
was constructed in E, cell using pUC13 and the
resultant recombinant plasmid pKA207 containing the Thermus T2 trpBA genes was selected by
colony-hybridization with the previously isolated
T. thermophilus HB27 trpB gene as a probe (Fig.
2). Since preliminary hybridization suggested that
the trpBA genes ate located in a 3.1 kbp Bglll.
BamHI fragment, this fragment was subeloned
into pUC13 (making pKA210) and a detailed restriction map was constructed. The 1.8 kbp
BgllI-SacI fragment and the 0.95 kbp XhoI fragment on pKA210 insert DNA were also sub.
cloned. The nucleotide sequence of the 0.95 kbp
Xhol fragment indicated that it coded for a peptide
which is homologous to T. thermophilus HB2)
trpB peptide (40th to 353th amino acid; data not
shown). The possible location of Thermus T2
trpBA genes is shown in Fig, 2.
Pstl~
1
pKA216(4.SKb)
~
EcoRI
'St)
.Kpn)
Pstl
PSII
phi
18ell
Fig, 3. Constructionof Thermt~-E. coli shuttlereeler plasmid
pYKI09.
for use as a shuttle vector for Thermus and /~: coil,
using EcoRI as a cloning site.
4.4. Construction of a Thermus plasmid vector
REFERENCES
Vasquez et al, reported that the cryptic plasmid
pTT8 (9.7 kbp) is stably maintained in T. thermophilus HB8 and cannot easily be cured [18]. Stable
maintenance without selective pressure is a preferable feature of a vector plasmid. We chose this
pTTg plasmid as a base plasmid for the construelion of a Thermus vector,
The Thermus T2 trpB gene was inserted in the
BgllI site of the cryptic plasmld pTl"8 from T.
therulophilus HB8 as shown in Fig. 3. After the
ligation of BamHl digested pKA216 and Bglll
digested pTT8. T. thermophilus HB27 trpB5 was
transformed. The recombinant plasmld pYK109
was obtained from a Trp + transformant, E. co//
MC1061 was also transformed with pYK109 and
the same plasmid was isolated from Apr transformants, pYK109 transformed T, thermophilus HB27
trpB5 to Tip + at the frequency of 106 transfer.
mants per/~g DNA. pYK109 should be suitable
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