Is Transposition Really Random?
Is Transposition Really Random?
(Dwi Suryanto)
IS TRANSPOSITION REALLY RANDOM?
Dwi Suryanto
Departemen Biologi FMIPA
Universitas Sumatera Utara
Jl. Bioteknologi No. 1 Kampus USU Medan 20155
Abstract
To characterize bacteria, transposon mutagenesis is still one of the most extensively utilized techniques
available. These elements were believed to insert at random location. In this study, transposition was done by
diparental mating technique to transfer pJFF350 carrying Omegon-Km to a Gram-negative Serratia marcescens
DS-8. The result showed that diparental mating was successfully transfer pJFF350 into DS-8 cells. Interestingly,
Southern hybridization analyses showed that transposon was inserted not randomly, but tended to insert into
limited targets. It also indicated that duplication occurred on the target sequences upon insertion.
Keywords: Omegon-Km, Transposition Mutagenesis, Serratia Marcescens
INTRODUCTION
Transposition is a recombination process
in
which
DNA
sequences
termed
transposable elements move from an original
site on a DNA molecule to a new site on the
same or on different DNA molecule. In
addition, transposable elements can cause,
and are associated with, other types of
genetic rearrangement such as deletions,
inversions,
and
chromosome
fusion
(Reznikoff, 1993).
To characterize bacteria, transposon
mutagenesis is still one of the most
extensively utilized techniques available.
This technique is especially useful for
bacterial species with poorly described
genetic systems or when existing molecular
techniques are insufficient (Dennis and
Zylstra, 1998). These elements have been
extremely valuable as insertional mutagens
because they were believed to insert at
random locations (Scott, 1991).
In this study, Omegon-Km (pJJF350)
were used to determine whether it insert
randomly or tend to insert into specific
sequences. Omegon-Km was designed to
carry the artificial interposon Omegon-Km
flanked by two synthetic inverted 28-bp
repeats of IS1. The reason using these
transposons is that inserted fragment could
be cloned easily and derived plasmids were
stable (Fellay et al., 1989; Dennis and
Zylstra, 1998; Civolani et al. 2000; Downing
et al., 2000).
MATERIALS AND METHOD
Strains and Plasmids
Escherichia coli S17-1 was used to
promote a transfer of plasmid pJFF350
(Omegon-Km) to DS-8. Bacterial strain and
plasmids are listed below.
Bacterial strains and plasmids used in
this study.
Diparental Mating
S17-1 (pJFF350) and DS-8 were grown
in LB-kanamycin and LB-ampicillin
overnight, respectively. A 1-ml sample of
DS-8 was mixed with a 200-µl sample of
S17-1 (pJFF350) and centrifuged for 5000
rpm for 5 minutes. Pellet was washed once
with 1 ml of 0.85% NaCl solution,
resuspended with 40 µl LB broth, and spotted
into a microtube containing 500 µl LB agar.
After 1-day incubation at 30°C, culture were
resuspended with 400 µl of 0.85% NaCl
solution and spread on LB-kanamycin and
ampicillin agar. After 1-day incubation,
single transconjugant colonies were isolated
on the same medium.
5
Jurnal Sains Kimia
Vol. 11, No.1, 2007: 5-8
Bacterial strains or plasmid
Relevant genotype/phenotype
Strains
E. coli S17-1
recA thi pro hdsR4 (rK- mK+) (RP4-2Tc-Mu-Km-Tn7) Tpr Smr
E. coli DH5α
supE44 ΔlacU169 (Φ80 lacZΔM15) hsdR17 recA1 endA1
gyrA96 thi-1 relA1
Serratia marcescens DS-8
wildtype Ampr
Plasmid
pJFF350
Kmr (Omegon-Km)
Transformation of Flanking DNA
Suspected colonies of transposition were
grown in LB kanamycin and ampicillin broth
overnight in 30°C at 200 rpm. Modified
phenol-chloroform-isoamylalcohol treatment
and ethanol precipitation were used to extract
the genomic DNA as described previously.
The DNA were digested with KpnI and
transformed to DH5α using method as
described by Sambrook et al. (1989).
A 1-ml overnight culture of DH5α was
sub-cultured in LB broth for 3 h. The culture
was harvested by centrifugation at 5000 rpm
for 2 minutes at 4oC. The supernatant was
discharge. Pellet was resuspended in 200 ml
of ice-cold 50 mM CaCl2 + 50 mM Tris and
incubated on ice for 20 minutes. The cells
were pelleted by centrifugation at 5000 rpm
for 2 minutes at 4oC. The supernatant was
discharged. Pellet was resuspended in 250 ml
of ice-cold 0.1 M CaCl2 and reincubated on
ice for 10 minutes. KpnI-digested DNA was
put into the microtube and gently mixed by
swirling. The tube was heated at 42oC for 4560 seconds. The tube was rapidly placed on
ice to cool for 60 minutes. The cells were
transferred into 2 ml of SOC broth. The
culture was incubated for 45-60 minutes at
37oC to allow the cell to recover. A 50-100
ml of the transformation mix were plated
onto LB-kanamycin agar and incubated
overnight.
Plasmid Preparation
In
general,
DNA
plasmid
minipreparation was done with Quantum
6
PrepTM Plasmid Miniprep Kit (Bio-Rad,
Hercules, CA). The preparation was done as
specified by the manufacturer.
Southern Hybridization
Total bacterial DNA was extracted as
previously described (Sambrook et al. 1989).
After digested with KpnI, DNA was
fractionated on 1.5% agarose gel in 1x TAE
buffer. The gel was stained with EtBr and
photographed under UV illumination. DNA
was denatured by soaking the gel into
denaturing solution (1.5 N NaCl and 0.5 N
NaOH) for 30 minutes at room temperature
with constant, gentle agitation and then
rinsed
briefly
in
deionized
water.
Neutralization was done by soaking the gel
for 15 minutes 2 times into the neutralization
solution pH 7.5 (1 M Tris and 1.5 N NaCl) at
room temperature with constant, gentle
agitation.
DNA was transferred in 20x SSC to a
nylon Zeta-Probe (Bio-Rad Laboratories,
CA) following NEBlot Phototope Kit
protocol (New England Biolabs, Inc. MA).
Hybridization of biotynilation labeled probes
using to the blot was performed as described
in Phototope Detection Kit protocol. Random
biotynilated octamers were used to prime
DNA synthesis in vitro from denaturated
double-stranded template DNA as described
by NEBlot Phototope Probe Labelling
protocol.
Is Transposition Really Random?
(Dwi Suryanto)
RESULTS AND DISCUSSION
Diparental mating was successfully
transfer pJFF350 into S. marcescens DS-8
cells. Mating of S. marcescens DS-8 with E.
coli (pJFF350) was obtained at a frequency
of 5x10-7 to 2x10-6. Downing et al. (2000)
and Fellay et al. (1989) reported that the
mutations caused by this transposable
element were random. The data in this study
showed that the artificial interposon
Omegon-Km has specific site preferences. It
also showed duplication on the target
sequence upon insertion. Berg et al. (1983),
Scott (1991), and Wall et al. (1996) showed
that many transposons have specific sites of
transposition either in Gram-negative or
Gram-positive bacteria.
Southern-blot analysis of total cellular
DNA DS-8 and its Omegon-Km mutants
digested with KpnI. The DNA of lane 1 was
marker, lanes 2-5 were mutants, lane 6 was
pJFF350 digested with EcoRI, and lane 7
was DS-8.
Site preferences were reported in Tn5
transposition in tet genes of pBR322 (Berg et
al.,
1983),
Tn7
in
Desulfovibrio
desulfuricans (Wall et al., 1996), and in B.
subtilis (Scott, 1991). Furthermore, the DNA
sequence terminations indicated that GC base
pairs occupied the first and ninth positions in
some target sequence duplication at each of
the five Tn5 insertion hotspots suggested
GC-cutting
preference
during
Tn5
transposition (Berg et al., 1983). The GCcutting preference was proposed earlier to
guide IS1 and Tn9 insertion (Galas et al.,
1980).
DAFTAR PUSTAKA
Berg, D.E., M.A. Schmandt, and J.B. Lowe. 1983.
Specificity of Transposon Tn5 Insertion.
Genetics 105: 813-828.
Civolani, C., P. Barghini, A.R. Roncetti, M. Ruzzi,
and A. Schiesser. 2000. Bioconversion of
Ferulic Acid Into Vannilic Acid by Means of a
Vannilate-Negative Mutant of Pseudomonas
Fluorescens Strain BFB. Appl. Environ.
Microbiol. 66: 2311-2317.
Dennis, J.J. and G.J. Zylstra. 1998. Plasposons:
Modular
Self-Cloning
Minitransposon
Derivative for Rapid Genetic Analysis of
Gram-Negative Bacterial Genomes. Appl.
Environ. Microbiol. 64: 2710-2715.
Downing, K.J., G. Leslie, and J.A. Thomson. 2000.
Biocontrol of the Sugarcane Borer Elsana
Saccharina by Expression of the Bacillus
Thuringiensis Cryac7 and Serratia Marcescens
Chia Genes in Sugarcane-Associated Bacteria.
Appl. Environ. Microbiol. 66:2804-2810.
Fellay, R., H.M. Krisch, P. Prentki, and J. Fey. 1989.
Omegon-Km: a Transposable Element Designed
for in Vivo Insertional Mutagenesis and
Cloning of Genes in Gram-Negative Bacteria.
Gene 76: 215-226.
Galas, D.J., M.P. Calos, and J.H. Miller. 1980.
Sequence Analysis of Tn9 Insertions in the
LacZ Gene. J. Mol. Biol. 144: 19-41.
Reinkoff, W.S. 1993. The Tn5 Transposon. Annu.
Rev. Microbiol. 47: 945-963.
Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989.
Molecular
Cloning.
Cold
Spring
HarborLaboratory Press. Cold Spring Harbor.
New York.
Scott. J.R. 1991. Mechanism of Transposition of
Conjugative Transposons. In Genetics and
Molecular
Biology
of
Streptococci,
Lactococci, and Enterococci. Ed. G.M. Dunny,
P.P. Cleary, and L.L. McKay. ASM. Washington
D.C. pp. 28-33.
7
Jurnal Sains Kimia
Vol. 11, No.1, 2007: 5-8
Wall, J.D., T. Murman, J. Argyle, R.S. English, and B.J.
Rapp-Ciles. 1996. Transposon Mutagenesii in
Desulfovibrio Desulfuricans: Development of a
Random Mutagenesis Tool from Tn7. Appl.
Environ. Microbiol. 62: 3762-3767.
8
(Dwi Suryanto)
IS TRANSPOSITION REALLY RANDOM?
Dwi Suryanto
Departemen Biologi FMIPA
Universitas Sumatera Utara
Jl. Bioteknologi No. 1 Kampus USU Medan 20155
Abstract
To characterize bacteria, transposon mutagenesis is still one of the most extensively utilized techniques
available. These elements were believed to insert at random location. In this study, transposition was done by
diparental mating technique to transfer pJFF350 carrying Omegon-Km to a Gram-negative Serratia marcescens
DS-8. The result showed that diparental mating was successfully transfer pJFF350 into DS-8 cells. Interestingly,
Southern hybridization analyses showed that transposon was inserted not randomly, but tended to insert into
limited targets. It also indicated that duplication occurred on the target sequences upon insertion.
Keywords: Omegon-Km, Transposition Mutagenesis, Serratia Marcescens
INTRODUCTION
Transposition is a recombination process
in
which
DNA
sequences
termed
transposable elements move from an original
site on a DNA molecule to a new site on the
same or on different DNA molecule. In
addition, transposable elements can cause,
and are associated with, other types of
genetic rearrangement such as deletions,
inversions,
and
chromosome
fusion
(Reznikoff, 1993).
To characterize bacteria, transposon
mutagenesis is still one of the most
extensively utilized techniques available.
This technique is especially useful for
bacterial species with poorly described
genetic systems or when existing molecular
techniques are insufficient (Dennis and
Zylstra, 1998). These elements have been
extremely valuable as insertional mutagens
because they were believed to insert at
random locations (Scott, 1991).
In this study, Omegon-Km (pJJF350)
were used to determine whether it insert
randomly or tend to insert into specific
sequences. Omegon-Km was designed to
carry the artificial interposon Omegon-Km
flanked by two synthetic inverted 28-bp
repeats of IS1. The reason using these
transposons is that inserted fragment could
be cloned easily and derived plasmids were
stable (Fellay et al., 1989; Dennis and
Zylstra, 1998; Civolani et al. 2000; Downing
et al., 2000).
MATERIALS AND METHOD
Strains and Plasmids
Escherichia coli S17-1 was used to
promote a transfer of plasmid pJFF350
(Omegon-Km) to DS-8. Bacterial strain and
plasmids are listed below.
Bacterial strains and plasmids used in
this study.
Diparental Mating
S17-1 (pJFF350) and DS-8 were grown
in LB-kanamycin and LB-ampicillin
overnight, respectively. A 1-ml sample of
DS-8 was mixed with a 200-µl sample of
S17-1 (pJFF350) and centrifuged for 5000
rpm for 5 minutes. Pellet was washed once
with 1 ml of 0.85% NaCl solution,
resuspended with 40 µl LB broth, and spotted
into a microtube containing 500 µl LB agar.
After 1-day incubation at 30°C, culture were
resuspended with 400 µl of 0.85% NaCl
solution and spread on LB-kanamycin and
ampicillin agar. After 1-day incubation,
single transconjugant colonies were isolated
on the same medium.
5
Jurnal Sains Kimia
Vol. 11, No.1, 2007: 5-8
Bacterial strains or plasmid
Relevant genotype/phenotype
Strains
E. coli S17-1
recA thi pro hdsR4 (rK- mK+) (RP4-2Tc-Mu-Km-Tn7) Tpr Smr
E. coli DH5α
supE44 ΔlacU169 (Φ80 lacZΔM15) hsdR17 recA1 endA1
gyrA96 thi-1 relA1
Serratia marcescens DS-8
wildtype Ampr
Plasmid
pJFF350
Kmr (Omegon-Km)
Transformation of Flanking DNA
Suspected colonies of transposition were
grown in LB kanamycin and ampicillin broth
overnight in 30°C at 200 rpm. Modified
phenol-chloroform-isoamylalcohol treatment
and ethanol precipitation were used to extract
the genomic DNA as described previously.
The DNA were digested with KpnI and
transformed to DH5α using method as
described by Sambrook et al. (1989).
A 1-ml overnight culture of DH5α was
sub-cultured in LB broth for 3 h. The culture
was harvested by centrifugation at 5000 rpm
for 2 minutes at 4oC. The supernatant was
discharge. Pellet was resuspended in 200 ml
of ice-cold 50 mM CaCl2 + 50 mM Tris and
incubated on ice for 20 minutes. The cells
were pelleted by centrifugation at 5000 rpm
for 2 minutes at 4oC. The supernatant was
discharged. Pellet was resuspended in 250 ml
of ice-cold 0.1 M CaCl2 and reincubated on
ice for 10 minutes. KpnI-digested DNA was
put into the microtube and gently mixed by
swirling. The tube was heated at 42oC for 4560 seconds. The tube was rapidly placed on
ice to cool for 60 minutes. The cells were
transferred into 2 ml of SOC broth. The
culture was incubated for 45-60 minutes at
37oC to allow the cell to recover. A 50-100
ml of the transformation mix were plated
onto LB-kanamycin agar and incubated
overnight.
Plasmid Preparation
In
general,
DNA
plasmid
minipreparation was done with Quantum
6
PrepTM Plasmid Miniprep Kit (Bio-Rad,
Hercules, CA). The preparation was done as
specified by the manufacturer.
Southern Hybridization
Total bacterial DNA was extracted as
previously described (Sambrook et al. 1989).
After digested with KpnI, DNA was
fractionated on 1.5% agarose gel in 1x TAE
buffer. The gel was stained with EtBr and
photographed under UV illumination. DNA
was denatured by soaking the gel into
denaturing solution (1.5 N NaCl and 0.5 N
NaOH) for 30 minutes at room temperature
with constant, gentle agitation and then
rinsed
briefly
in
deionized
water.
Neutralization was done by soaking the gel
for 15 minutes 2 times into the neutralization
solution pH 7.5 (1 M Tris and 1.5 N NaCl) at
room temperature with constant, gentle
agitation.
DNA was transferred in 20x SSC to a
nylon Zeta-Probe (Bio-Rad Laboratories,
CA) following NEBlot Phototope Kit
protocol (New England Biolabs, Inc. MA).
Hybridization of biotynilation labeled probes
using to the blot was performed as described
in Phototope Detection Kit protocol. Random
biotynilated octamers were used to prime
DNA synthesis in vitro from denaturated
double-stranded template DNA as described
by NEBlot Phototope Probe Labelling
protocol.
Is Transposition Really Random?
(Dwi Suryanto)
RESULTS AND DISCUSSION
Diparental mating was successfully
transfer pJFF350 into S. marcescens DS-8
cells. Mating of S. marcescens DS-8 with E.
coli (pJFF350) was obtained at a frequency
of 5x10-7 to 2x10-6. Downing et al. (2000)
and Fellay et al. (1989) reported that the
mutations caused by this transposable
element were random. The data in this study
showed that the artificial interposon
Omegon-Km has specific site preferences. It
also showed duplication on the target
sequence upon insertion. Berg et al. (1983),
Scott (1991), and Wall et al. (1996) showed
that many transposons have specific sites of
transposition either in Gram-negative or
Gram-positive bacteria.
Southern-blot analysis of total cellular
DNA DS-8 and its Omegon-Km mutants
digested with KpnI. The DNA of lane 1 was
marker, lanes 2-5 were mutants, lane 6 was
pJFF350 digested with EcoRI, and lane 7
was DS-8.
Site preferences were reported in Tn5
transposition in tet genes of pBR322 (Berg et
al.,
1983),
Tn7
in
Desulfovibrio
desulfuricans (Wall et al., 1996), and in B.
subtilis (Scott, 1991). Furthermore, the DNA
sequence terminations indicated that GC base
pairs occupied the first and ninth positions in
some target sequence duplication at each of
the five Tn5 insertion hotspots suggested
GC-cutting
preference
during
Tn5
transposition (Berg et al., 1983). The GCcutting preference was proposed earlier to
guide IS1 and Tn9 insertion (Galas et al.,
1980).
DAFTAR PUSTAKA
Berg, D.E., M.A. Schmandt, and J.B. Lowe. 1983.
Specificity of Transposon Tn5 Insertion.
Genetics 105: 813-828.
Civolani, C., P. Barghini, A.R. Roncetti, M. Ruzzi,
and A. Schiesser. 2000. Bioconversion of
Ferulic Acid Into Vannilic Acid by Means of a
Vannilate-Negative Mutant of Pseudomonas
Fluorescens Strain BFB. Appl. Environ.
Microbiol. 66: 2311-2317.
Dennis, J.J. and G.J. Zylstra. 1998. Plasposons:
Modular
Self-Cloning
Minitransposon
Derivative for Rapid Genetic Analysis of
Gram-Negative Bacterial Genomes. Appl.
Environ. Microbiol. 64: 2710-2715.
Downing, K.J., G. Leslie, and J.A. Thomson. 2000.
Biocontrol of the Sugarcane Borer Elsana
Saccharina by Expression of the Bacillus
Thuringiensis Cryac7 and Serratia Marcescens
Chia Genes in Sugarcane-Associated Bacteria.
Appl. Environ. Microbiol. 66:2804-2810.
Fellay, R., H.M. Krisch, P. Prentki, and J. Fey. 1989.
Omegon-Km: a Transposable Element Designed
for in Vivo Insertional Mutagenesis and
Cloning of Genes in Gram-Negative Bacteria.
Gene 76: 215-226.
Galas, D.J., M.P. Calos, and J.H. Miller. 1980.
Sequence Analysis of Tn9 Insertions in the
LacZ Gene. J. Mol. Biol. 144: 19-41.
Reinkoff, W.S. 1993. The Tn5 Transposon. Annu.
Rev. Microbiol. 47: 945-963.
Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989.
Molecular
Cloning.
Cold
Spring
HarborLaboratory Press. Cold Spring Harbor.
New York.
Scott. J.R. 1991. Mechanism of Transposition of
Conjugative Transposons. In Genetics and
Molecular
Biology
of
Streptococci,
Lactococci, and Enterococci. Ed. G.M. Dunny,
P.P. Cleary, and L.L. McKay. ASM. Washington
D.C. pp. 28-33.
7
Jurnal Sains Kimia
Vol. 11, No.1, 2007: 5-8
Wall, J.D., T. Murman, J. Argyle, R.S. English, and B.J.
Rapp-Ciles. 1996. Transposon Mutagenesii in
Desulfovibrio Desulfuricans: Development of a
Random Mutagenesis Tool from Tn7. Appl.
Environ. Microbiol. 62: 3762-3767.
8