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Expanding and understanding the genetic toolbox of the hyperthermophilic genus
Sulfolobus
Wagner, Michaela; Berkner, Silvia; Ajon, Malgorzata; Driessen, Arnold; Lipps, Georg; Albers,
Sonja-Verena
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Biochemical Society Transactions
DOI:
10.1042/BST0370097
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Wagner, M., Berkner, S., Ajon, M., Driessen, A. J. M., Lipps, G., & Albers, S-V. (2009). Expanding and
understanding the genetic toolbox of the hyperthermophilic genus Sulfolobus. Biochemical Society
Transactions, 37(1), 97-101. DOI: 10.1042/BST0370097
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Molecular Biology of Archaea
Expanding and understanding the genetic toolbox
of the hyperthermophilic genus Sulfolobus
Michaela Wagner*, Silvia Berkner†, Malgorzata Ajon‡, Arnold J.M. Driessen‡, Georg Lipps§ and
Sonja-Verena Albers*‡1
Biochemical Society Transactions
www.biochemsoctrans.org
*Max-Planck-Institute for Terrestrial Microbiology, Karl-von Frisch-Strasse 1, 35043 Marburg, Germany, †Department of Biochemistry, University of Bayreuth,
95447 Bayreuth, Germany, ‡Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute and the Zernike Institute for
Advanced Materials, University of Groningen, Kerklaan 30, 9751 NN Haren, The Netherlands, and §University of Applied Sciences Northwestern Switzerland,
School of Life Sciences, Gründenstrasse 40, 4132 Muttenz, Switzerland
Abstract
Although Sulfolobus species are among the best studied archaeal micro-organisms, the development and
availability of genetic tools has lagged behind. In the present paper, we discuss the latest progress in
understanding recombination events of exogenous DNA into the chromosomes of Sulfolobus solfataricus
and Sulfolobus acidocaldarius and their application in the construction of targeted-deletion mutant strains.
Background
Manipulation of the genetic information of organisms is a vital
tool to investigate the role and function of genes. In model
organisms from all three domains of life, methods have been
developed to integrate engineered DNA site-specifically into
the genome. These techniques rely mainly on homologous
recombination and allow new genes to be introduced into
a genome, parts of a genome to be deleted or specific
mutations to be established in the genome. After changing
the genotype, it is possible to analyse the phenotype (reverse
genetics). For eukarya and bacteria, these methods are very
well established and continue to be extremely powerful for
the elucidation of the functions of genes. However, archaea
are lagging behind [1]. The main problem was and is the
availability and development of efficient selectable markers.
Most bacterial antibiotics are ineffective in archaea, although
some examples have been used successfully. Novobiocin, an
inhibitor of the DNA gyrase, has been used to develop a
vector for halophilic archaea [2,3], and puromycin is the most
widely used antibiotic for methanogens [4]. Owing to the
elevated temperatures of the habitats of hyperthermophilic
archaea, it has proven even more difficult to find suitable
marker systems. However, for Thermococcus kodakaraensis,
a genetic system has been established successfully recently
using uracil and tryptophane auxotrophic mutants [5].
The thermoacidophilic Sulfolobus species belong to the
most studied archaea, but, so far, studies have been greatly
hampered by the lack of genetic tools. However, recently,
considerable progress has been made in the improvement
of plasmid systems and also protocols for the generation of
deletion mutants have been developed for different Sulfolobus
species [6–10]. The plasmid systems available, including
self-spreadable virus vector systems and other Escherichia
Key words: archaeon, deletion mutant, genetics, plasmid, recombination, Sulfolobus.
Abbreviations used: 5-FOA, 5-fluoro-orotic acid; GOI, gene of interest; SSOGOI, Sulfolobus
solfataricus GOI.
1
To whom correspondence should be addressed (email [email protected]).
Biochem. Soc. Trans. (2009) 37, 97–101; doi:10.1042/BST0370097
coli–Sulfolobus shuttle vectors, have been reviewed recently
by Berkner and Lipps [7] in detail and will not be discussed
further here.
The lack of suitable selectable markers again posed a major
problem in the progress of the development of methods
for directed gene deletion in Sulfolobus species. Although
auxotrophic strains for uracil biosynthesis of Sulfolobus
acidocaldarius [11] and Sulfolobus solfataricus [12] have been
isolated, these have not been used as selectable markers. One
problem is that Gelrite, the polymer used for solid media
in hyperthermophiles, contains traces of uracil, so that the
background after selection can be quite high. A more severe
problem is that the widely used S. solfataricus strains P1 and
P2 do not recombine foreign plasmid DNA into their chromosome [10]. However, an S. solfataricus strain PBL2025,
originating from S. solfataricus 98/2, has been shown to be
capable of homologous recombination and is used for the generation of deletion mutants [6]. This strain lacks the gene that
encodes a β-galactosidase, lacS, that is essential for growth of
S. solfataricus on lactose minimal medium and can therefore
be used as a selective marker. Details on the progress of
constructing directed gene-deletion mutants in S. solfataricus
PBL2025 and S. acidocaldarius are described below.
Recombination by single- and
double-cross-over events into the
chromosome of S. solfataricus
The first successful targeted-deletion mutant in S. solfataricus
PBL2025 was constructed by Schelert et al. in 2004 [6]. For
integration of foreign DNA into the chromosome, a plasmid
was used containing the up- and down-stream flanking
region of the target gene and a marker cassette encompassing
the lacS gene with its original promoter and terminator
region. Positive clones are first selected by growth on lactose
minimal medium. After plating, blue colonies can be analysed
for correct integration of the construct. We optimized the
post-electroporation conditions and found that a 10 min
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Figure 1 Recombination of plasmid DNA via single cross-over into the S. solfataricus PBL2025 chromosome
(A) PCR analysis of pET401 and genomic DNA of PBL2025, a semi-knockout strain and a SSOGOI strain using either a
primer for the ampicillin cassette (amp) or the GOI. M, markers (sizes in kb). (B) Cartoon of a recombination event via single
cross-over. Amp, ampicillin cassette; lacS, lacS cassette.
incubation step in demineralized water yielded the largest
amount of positive clones after selection on lactose minimal
medium [9]. Moreover, methylation of the plasmid DNA,
which is necessary for electroporation of plasmids in S.
acidocaldarius, is obsolete for S. solfataricus PBL2025 [9].
By using linear DNA for recombination, double-crossover events can be obtained. Therefore either linearized
plasmid DNA or a PCR product covering the flanking
region and the lacS cassette of the gene-targeting construct
were transformed into PBL2025. After selection, single blue
colonies were obtained that were shown to contain the
C The
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Authors Journal compilation expected deletion-mutant genotype by PCR and Southern
blot analysis. In three cases tested, recombination of the linear
DNA fragment occurred in 90% of the colonies tested at the
correct position. This showed that linear DNA fragments
containing the up- and down-stream flanking regions of
a GOI (gene of interest) and the lacS cassette can be used
efficiently for the deletion of genes in S. solfataricus PBL2025.
Using plasmid DNA for electroporation resulted in
blue colonies that exhibited the expected deletion-mutant
product as well as the wild-type gene product after PCR
analysis of their genomic DNA (Figure 1B). Only after a
Molecular Biology of Archaea
Figure 2 Tandem insertion of plasmid DNA into the chromosome of S. solfataricus
(A) Southern blot analysis of genomic DNA of four different semi-knockout strains of SSOGOI digested with HpaI and using a
lacS probe for detection. M, markers. As shown in the cartoon of tandem insertion (B), a third band is only present if more
than one copy of the gene-targeting construct is inserted.
few more selection rounds on solid media were true deletion
strains isolated [9,13]. PCR and Southern blot analysis were
performed on strains exhibiting both the wild-type and
deletion-mutant PCR products. Primers directed against the
ampicillin cassette of the gene-targeting constructs were used
for PCR analysis of integration strains (strains containing
both the wild-type and gene-deletion PCR products)
and correct deletion mutants. As shown in Figure 1(A),
the integration-strain SSOGOI (S. solfataricus GOI) gave the
same specific band as found in the plasmid control pET401,
which is the backbone plasmid for gene-targeting constructs.
However, this band was absent from the correct knockout
strain SSOGOI, as it was from the PBL2025 wild-type
strain. This indicated that the recombination of plasmid
DNA occurred via single-cross-over events and resulted
in strains in which the whole gene-targeting plasmid was
integrated into the chromosome (Figure 1B). Southern blot
analysis confirmed further the presence of the plasmid DNA
in the semi-knockout chromosome (results not shown).
Further Southern blot analysis showed that the integration
strains in many cases did not contain only one copy of the
gene-targeting construct, but tandem insertion occurred
as has also been observed in Methanococcus voltae [14]
(Figure 2). However, the number of tandem insertions has
yet to be determined.
Recombination by single- and
double-cross-over events into the
chromosome of S. acidocaldarius
The introduction of foreign DNA into genomic DNA by
recombination has already been studied in quite some detail
in S. acidocaldarius by Dennis Grogan and co-workers,
and they could demonstrate that even oligonucleotides
of 14 bp in length can be incorporated efficiently by
OMT (oligonucleotide-mediated transformation) [15,16].
However, a genetic system had not yet been established for
the construction of targeted-deletion mutants.
In contrast with S. solfataricus strains, uracil selection is
more efficient in S. acidocaldarius. Uracil auxotrophic strains
contain mutations in the pyrEF gene sequences, encoding
orotate phosphoribosyltransferase and orotidine-5 -monophosphate decarboxylase respectively. PyrEF catalyse the
last two steps of the de novo uridine monophosphate
synthesis pathway [17]. Using 5-FOA (5-fluoro-orotic acid),
which is converted into toxic 5-fluorouracil in wild-type
cells, several uracil auxotrophic strains of S. acidocaldarius
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Figure 3 Scheme of the construction of a markerless deletion mutant in S. acidocaldarius using the p2pyrEF construct
After integration into the chromosome by single cross-over, a second recombination step can lead to either a wild-type
genotype (A) or the desired deletion-mutant genotype (B). ampR , ampicillin cassette; pyrEF, pyrEF cassette of S. solfataricus.
were isolated [11,18]. We used the S. acidocaldarius strain
MR31 that has an 18 bp deletion in the pyrE gene as a host
for gene-targeting constructs. The gene-targeting plasmid
p2pyrEF was constructed using pBluescript as a backbone
for replication in E. coli and the pyrEF cassette from S.
solfataricus was cloned into this vector. The pyrEF cassette of
S. solfataricus was used to avoid homologous recombination
with the endogenous S. acidocaldarius pyrEF cassette. This
vector was used to construct plasmids for single- and doublecross-over recombination events into the S. acidocaldarius
chromosome. For double-cross-over recombination events,
the up- and down-stream flanking regions of the target gene
were cloned up- and down-stream of the pyrEF cassette in
the gene-targeting vector p2pyrEF. Because of the presence
of the restriction endonuclease SuaI in S. acidocaldarius,
the plasmid, when transformed unmethylated, is restricted
after DNA uptake and yields linear DNA fragments which
can serve as substrates for a double-cross-over integration
event. The E. coli part of the shuttle vector contained numerous recognition sites for SuaI, whereas the remainder of
the construct has no restriction site for SuaI. After transformation, cells were selected directly on plates or first
selected for two to three rounds in liquid medium and then
on plates which did not contain uracil. All colonies tested
by PCR and/or Southern blot showed correct replacement
of the GOI. In this manner, we constructed two deletion
mutants of S. acidocaldarius, a tryptophane auxotroph mutant
lacking the trpA gene and a Saci_1494 strain.
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single-cross-over events leading to a markerless deletion
strain, the up- and down-stream flanking regions were cloned
consecutively upstream of the pyrEF cassette (Figure 3).
Integrants were obtained by uracil selection on plates and
integration of the gene-targeting plasmid was confirmed by
Southern blot analysis. To remove the GOI from the chromosome, the plasmid has to be removed by a second recombination step, leading either to the wild-type situation or to
the deletion-mutant genotype. This process was not observed
spontaneously, but occurred very efficiently after counterselection for uracil auxotrophs on 5-FOA-containing plates.
Concluding remarks
In our work, we have shown that exogenous DNA
recombines into the chromosomes of S. solfataricus and in
the genetically more stable S. acidocaldarius via single- and
double-cross-over events. In S. solfataricus, tandem insertion
of plasmid DNA was observed after recombination via single
cross-over. We demonstrated that directed deletion mutants
can be constructed in both Sulfolobus strains, which will give
a starting point for the development of more elaborate and
efficient genetic tools for these species.
Acknowledgements
We thank Chris van der Does for inspiring discussions.
Molecular Biology of Archaea
Funding
This work was supported by a Vidi grant of the Dutch
Science Organization (Nederlandse Organisatie voor Wetenschappelijk Onderzoek) to S.-V.A., and M.A. was supported by the
Earth and Life Science Foundation subsidized by the Dutch Science
Organization.
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Received 30 July 2008
doi:10.1042/BST0370097
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