Molecular basis of agrobacterium mediated gene transfer into mammalian cells 3

Chapter 3. Agrobacterium-mediated DNA delivery into
mammalian cells
3.1. Introduction
Horizontal gene transfer (HGT) might play an important role in the evolution of
both eukaryotic and prokaryotic genome (Syvanen, 1994; Nelson et al., 1999;
Syvanen and Kado, 2001). Although HGT in prokaryotic cells has been established,
the role of HGT in eukaryotic genome evolution is not well elucidated because the
existence of a possible pathway or mechanism for HGT involved eukaryotes is often
questioned. A. tumefaciens is often used as a vector to generate transgenic plants.
The ability of Agrobacterium to transfer part of its DNA (T-DNA) into plant cells and
integrate such foreign genetic material into the plant genome, followed by the
consequent gene expression to induce the tumors on plant, is the best understood case
of horizontal gene transfer between bacteria and eukaryotic cells. The recent findings
that Agrobacterium could also transform other eukaryotes including mammalians,
yeast and fungi might shed light on the exact mechanism of HGT involving
eukaryotes. Studies on the interaction of Agrobacterium and mammalian cells can
provide information about the mechanism of gene transfer between bacterium and
mammalian cells and help us to understand the role that HGT might play in eukaryote
evolution.
3.2. Results
3.2.1. Attachment of Agrobacterium to mammalian cells
An early step in the plant tumor induction by Agrobacterium is the attachment
of bacterial cells to the host cells. Bacterial attachment to host cells is often an

72
essential step to initiate internalization because this process allows pathogens to locate
the appropriate target tissues. This attachment appears to be required for DNA
transfer from A. tumefaciens into the host plant cells. A. tumefaciens with mutations
in the chromosomal genes chvA and chvB are defective in both the efficient
attachment and the subsequent transformation of plant cells. To study the interaction
between Agrobacterium and mammalian cells, a plasmid pQM49 was constructed

(Fig. 3.1). This plasmid can express gfp constitutively in Agrobacterium. A.
tumefaciens strains harboring pQM49 exhibited bright green fluorescence when
illuminated with

UV light and could

be visualized easily under the confocal
microscope. Then the

ability of Agrobacterium to adhere to mammalian cells
EcoPack2-293 was examined. Fig. 3.2 (panel B) showed that the wild type strain
A348(pQM49) could specifically attach to the surface of mammalian cells and this
result is consistent with the previous report (Kunik et al., 2001).
To investigate whether the binding of Agrobacterium to mammalian cells
requires the same bacterial factors as its attachment to plant

cells, various
Agrobacterium mutant strains harboring pQM49 were tested for their attachment
ability to the EcoPack2-293 cells as described in the Materials and Methods. As
shown in Fig. 3.2 (panel D), At12513, a chvB mutant, showed reduced ability to attach
the mammalian cells after co-incubation. This indicates that the chvB gene is
important for the attachment of Agrobacterium cells to both mammalian and plant
cells. This is consistent with the previous study (Kunik et al., 2001). Interestingly,
the results also showed that the virB mutant strain, MX243, was less efficient in
binding to the mammalian cells (Fig. 3.2, panel C). Thus, this data suggest that unlike
what was observed in the interaction between Agrobacterium and plant cells, in which

73

pQM49

Fig. 3.1. Plasmid used for attachment study. For complete
construction details see Materials and Methods. Abbreviations:
P
lac
,LacZ promoter sequence; atpE, the efficient translation region of
atpE gene; gfp
uv
, green fluorescent protein; tetA, tetracycline
resistance gene; OriV, R6K origin of replication.



74
C D
A B
Fig. 3.2. Confocal microscopy analysis of Agrobacterium attachment to
EcoPack2-293 Packaging Cells. A. tumefaciens cells were incubated with
EcoPack2-293 Packaging Cells as described in Material and Methods and
observed under confocol microscopy at the excitation wavelength of 488
nm. A, 293 cells alone. B, A348 (pQM49) co-incubated with 293 cells. C,
MX243 (pQM49) co-incubated with 293 cells. D, At12513 (pQM49) co-
incubated with 293 cells.

75
mutation in the virB gene does not affect the attachment to the plant cells, virB gene
product does affect the bacterial attachment to mammalian cells. This might imply
that the mechanism of Agrobacterium attachment to human cells is different from that
to the plant cells.
3.2.2. Development of a system to detect the horizontal gene transfer between
the bacteria and mammalian cells

To observe inter-kingdom horizontal gene transfer directly, a sensitive system
that could detect a few copies of gene transfer from the bacterial cells into human
cells was developed. In this system, EcoPack2-293 Packaging Cell was used as the
host for Agrobacterium gene transfer. This Packaging Cell Line is a human
embryonic kidney (HEK 293)-derived cell line designed for the rapid and transient
production of high-titer retrovirus. It can stably express the viral gag, pol and env
genes, which are necessary for viral production. Then two gene transfer vectors,
pQM52 (with T-borders) and pQM54 (without T-borders) (Fig. 3.3) were constructed.
Both vectors contain an egfp gene under the control of a CMV immediate early
promoter, a 5' and 3' long-terminal repeats (LTRs) which contain promoter, poly-
adenylation and a sequence termed Ψ
+
which is essential for virus packaging.
Agrobacterium harboring these plasmids were used to infect EcoPack2-293 cells and
the expression of egfp in these cells would indicate the successful transfer of bacterial
DNA into the host cells. In this system, the expression of egfp could not be attributed
to contaminated bacteria, because CMV immediate early promoter is only functional
in the eukaryotic cells. In this system, once foreign genes as well as the transcript
containing Ψ
+
are delivered into EcoPack2-293 cells, the viral elements harbored on

76

ϕ
+

pQM52
ϕ
+

pQM54
Fig. 3.3. Plasmids used for transfection study. For complete
construction details see Materials and Methods. Abbreviations: LTR,
long terminal repeat sequence; Ψ
+
, extended packaging signal; Neo
r
,
Neomycin resistance gene; P
CMV
, immediate early CMV promoter;
egfp, enhanced green fluorescent protein; LB and RB, A. tumefaciens
left and right T-DNA borders, respectively; nptIII, kanamycin-
resistance gene; tetA, tetracycline resistance gene; OriV, R6K origin of
replication

77
the mammalian cell chromosome will facilitate the amplification and transcription of
foreign gene in host cells, which will result in expression of the egfp gene. Therefore,
only a few copies of the egfp gene need to be transferred in order to detect green
fluorescence protein expression in host cells. The integration of the foreign gene into
the recipient genome, which would result in stable transformant, is not required in this
process.
3.2.2.1. A. tumefaciens can deliver its DNA into human cells
First of all, whether A. tumefaciens could transfer T-DNA into mammalian cells
such as EcoPack2-293 cells or not was determined. Co-incubation of human
EcoPack2-293 cells (1 × 10
6
) with Agrobacterium strain LBA4404(pQM52) (4 × 10

7
)
resulted in the expression of egfp in mammalian cells, indicating that the
A.tumefaciens could deliver its plasmid DNA containing the egfp reporter gene into
human cells (Fig. 3.4, panel B). The multiplicity of infection (MOI) in this
experiment is about 40. On the contrary, when EcoPack2-293 cells (1 × 10
6
) were
incubated with DH5α(pQM52) (4 × 10
7
), in which E. coli cells were used as the
donor strain, no expression of egfp in those cells was observed in this experimental
condition (Fig. 3.4, panel C). Similarly, no transformants were observed when 50 µg
of the plasmid pQM52 was incubated directly with the recipient cells, suggesting that
DNA transfer is not due to a direct uptake of free plasmid DNA released by the dead
bacteria. Then the same Agrobacterium strain (LBA4404(pQM52) (4 × 10
7
) was
incubated with normal mammalian cell line such as HeLa (1 × 10
6
), which does not
encode viral gag, pol and env genes. As expected, no expression of egfp in these host
cells was observed under the same experimental conditions (data not shown). In order
to investigate whether the viral elements on vector (such as LTR sequence and Ψ
+
)

78
Fig. 3.4. Expression of egfp in human Cells ( EcoPack2-293). The
mammalian cells were incubated with different A. tumefaciens or E. coli

strains and the infected cells were viewed with blue light excitation (480
nm). A, 293 cells alone; B, LBA4404(pQM52); C, DH5α(pQM52); D,
LBA4404(pQM54); E, AG6(pQM52); F, A348::Tn5; G, A136::Tn5; H;
GMI::Tn5.

79
B
A
D
C
F
E
F
H
G

80
are required for this gene transfer, another vector pQM45 that contains only an egfp
gene under the control of a CMV promoter (Fig. 3.3) was constructed. Compared to
pQM52, pQM45 lacked the viral components. When EcoPack2-293 cells (1 ×10
6
)
was incubated with LBA4404(pQM45) strain (4 ×10
7
), no expression of egfp in
mammalian host cells was observed. These results showed that this system is much
more sensitive in detecting DNA transfer due to the amplifying function of the viral
elements.
To further verify that EGFP was indeed synthesized by the host cells, RT-PCR
was carried out to detect the egfp transcription in the infected human cells. The host

cells (2 × 10
7
)

were treated as described in Material and Methods and total RNA of
the host cells was extracted. RT-PCR was performed with a pair of egfp primers
(EGFPF, 5’-CTAACGCAGTCAGTGCTTCTG-3’; EGFPR, 5’-
CAGTCATAGCCGAATAGCCTCTC-3’). One step RT-PCR Kit (QIAGEN) was
used to conduct the standard RT-PCR. According to the instructions of the
manufacturer, approximately 10-100 of target molecules should be sufficient for the
detection. Forty cycles of the reaction was necessary to detect the egfp transcription
in the host cells infected by Agrobacterium in this system. As shown in Fig. 3.5, a
clear band of 700 bp RT-PCR product, which matched well with the predicted egfp
transcript product, could be amplified from the total RNA extract of host cells co-
incubated with wild-type A. tumefaciens strain LBA4404(pQM52). On the contrary,
no amplification product could be detected when RNA extract from cells incubated
with E.coli strain DH5α(pQM52) was used. These data are consistent with our visual
observations of the EGFP fluorescence. Taken together, these results suggest that A.
tumefaciens could deliver genes carried on its plasmids into the mammalian cells,
while E.coli could not mediate such DNA transfer in similar experimental conditions.

81

1 2 3 4 5 6 7 8 9 10
700 bp
dimer
NC
DH5
α(
pQ

Μ52)

LBA4404(pQM52)
LBA4404(pQM54)
GMI9023(pQM52)
A6880(pQM52)
A6340(pQM6340)
A348::Tn5
A136::Tn5
GMI::Tn5
actin
Fig. 3.5. Transcription of egfp in human cells. EcoPack2-293 Packaging Cells
were incubated with different Agrobacterium strains as indicated. Total RNA
extracted from mammalian cells after 48 hr infection was used for RT-PCR
analysis. Lane 1, 293 cells; lane 2, DH5α(pQM52); lane 3,
LBA4404(pQM52); lane 4, LBA4404(pQM54); lane 5, GMI9023(pQM52);
lane 6, A6880(pQM52); lane 7, A6340(pQM52); lane 8, A348::Tn5; lane 9,
A136::Tn5; lane 10, GMI::Tn5.

82
Then flow cytometry analysis was conducted to estimate the DNA transfer efficiency
of Agrobacterium–mediated gene delivery by counting host cells with high green
fluorescence, as a result of egfp expression. A total of 100,000 EcoPack2-293 cells
incubated with LBA4404(pQM52) were counted, while same number of the host cells
incubated with DH5α(pQM52) were also counted as a negative control. Each
analysis was repeated for three times independently. As shown in Table 3.1, the
average efficiency of LBA4404(pQM52) mediated gene transfer was 9 × 10
-5
per
recipient cell.

3.2.2.2. A. tumefaciens can also transfer DNA into mouse cells
To test whether a specific genetic background of the recipient mammalian cells
was required for the gene transfer, PT67 cells were incubated with
LBA4404(pQM52). PT67 is a NIH 3T3-derived mouse cell line having been
integrated of viral gag, pol and env genes into its chromosome. High green
fluorescence was observed in infected PT67 cells, suggesting that Agrobacterium
could also deliver its DNA into the mice cells (Fig. 3.6). This result indicates that the
gene transfer mediated by Agrobacterium could occur not only in human cells but
also in mouse cells. Thus, this observed DNA transfer might occur widely in
mammalian cells and it does not require specific genetic background of the host cells.
3.2.2.3. R. meliloti could not deliver its DNA into mammalian cells
As Rhizobium is closely related to Agrobacterium in many aspects during their
interactions with plants, the ability of Rhizobium to transfer DNA into EcoPack2-293
cells was test. R. meliloti strain RCR2011(pQM52) was used to incubated with 293
cells under the same experimental conditions. However, no green fluorescence was

83
Table 3.1. Frequency of DNA transfer from Agrobacterium to EcoPack2-293
Packaging Cells
Strain Genetic
background
Location of
reporter gene
Total number of
egfp expressing
cells
Gene transfer
efficiency**
(x10
-5

)
No bacterium NC* NC* 0 0
Naked DNA NC*
plasmid with T-
borders
0 0
DH5α(pQM52)
E. Coli
plasmid with T-
borders
0 0
RCR2011(pQM52)
Rhizobium wild
type
plasmid with T-
borders
0 0
LBA4404(pQM52)
Agrobacterium
wild type
plasmid with T-
borders
28
9 ± 2
LBA4404(pQM54)
Agrobacterium
wild type
plasmid without
T-borders
20

7 ± 2
GMI9023(pQM52)
Agrobacterium
without Ti and
cryptic plasmid
plasmid with T-
borders 18
6 ± 1
A6880(pQM52) chvH
-
plasmid with T-
borders
7
2 ± 1
A6340(pQM52) chvG
-
plasmid with T-
borders
2
1 ± 1
AG6(pQM52) katA
-
plasmid with T-
borders
88
29 ± 6
A6340-
AG6(pQM52)
chvG
-

, katA
-

plasmid with T-
borders
6
2±1
A6880-
AG6(pQM52)
chvH
-
, katA
-

plasmid with T-
borders
4
1±1
A348::Tn5
Agrobacterium
wild type
Random insertion
without T-borders
25
8 ± 3
A136::Tn5
Agrobacterium
without Ti plasmid
Random insertion
without T-borders

31
10 ± 3
GMI::Tn5
Agrobacterium
without Ti and
cryptic plasmid
Chromosome
without T-borders 19
6 ± 3
* NC: Negative Control
**The data were analyzed using Excel (Miscrosoft Software, USA)

84
A
B
Fig. 3.6. Expression of egfp in mouse cells. The PT67 cells were incubated
with A. tumefaciens and the infected cells were viewed with blue light
excitation (480 nm). A, PT67 cells alone; B, PT 67 cells infected with
LBA4404(pQM52);

85
detected(data not shown). This result suggests that the delivery of a foreign gene into
the mammalian cells might require some unique factor(s) from A. tumefaciens.
3.2.3. Roles of Agrobacterium genes and T-DNA border sequences in horizontal
gene transfer to mammalian cells
In Agrobacterium-mediated transformation of plant cells, three genetic
components of Agrobacterium are required: T-DNA border sequences, virulence (vir)
genes located on the Ti plasmid and chromosomal virulence (chv) genes. Therefore
the effect of these factors on Agrobacterium-mediated gene delivery into the
mammalian cells was investigated.

3.2.3.1. Agrobacterium vir genes are not required for DNA transfer into
mammalian cells
Phenolics and monosaccharides, which serve as inducers of vir genes, are
required for T-DNA transfer into the plant cells. To determine if they are also
required for A. tumefaciens-mediated mammalian cell transformation, Agrobacterium
cells were cultured the in the absence of Acetosyringoue (AS) and incubated with
EcoPack2-293 cell. Under such condition, the expression of vir genes is minimal.
The data showed that the gene transfer efficiency of these A. tumefaciens cells was
similar to that of the induced cells, indicating that vir gene induction is not necessary
for mammalian cell DNA delivery. Then the gene transfer ability of different
virulence gene mutant strains was tested, including MX226 (virA
-
), MX363 (virG
-
),
MX306 (virD2
-
) and MX358 (virE
-
) harboring pQM52. All these strains have
retained their ability to deliver DNA into mammalian cells and they showed similar
gene transfer efficiency as the wild type strain, LBA4404(pQM52) (Table 3.1). Thus,

86
these results suggest that virulence genes are not required for A. tumefaciens-mediated
gene delivery into mammalian cells under our assay system.
3.2.3.2. T-DNA border sequences are not required for DNA transfer into
mammalian cells
T-DNA border sequences are necessary for the transfer of T-DNA from A.
tumefaciens to plant cells but are not required for the conjugative transfer of the Ti

plasmid between A. tumefaciens strains. In order to find out whether T-DNA border
sequence is essential for mammalian cell gene transfer, a binary-vector pQM54,
which lacks the T-DNA border sequences that presented in pQM52 (Fig. 3.3), was
conducted. Agrobacterium strain LBA4404 harboring pQM54 was incubated with
mammalian cells. The efficiency of mammalian cell gene transfer was quite similar,
7×10
-5
for LBA4404(pQM54) and 9×10
-5
for LBA4404(pQM52) (Table 3.1). No
significant reduction of mammalian cell gene transfer efficiency was observed,
indicating that T-border sequences are not required for the DNA transfer.
Furthermore, the gene transfer efficiency of Agrobacterium strain
GMI9023(pQM52) was tested, as GMI9023 contains neither the Ti plasmid nor the
cryptic plasmid (Rosenberg and Huguet, 1984). The DNA delivery efficiency of
GMI9023(pQM52) was 6×10
-5
, which is similar to that observed with
LBA4404(pQM52) (Table 3.1). Thus the data demonstrated undoubtedly that the
DNA delivery into mammalian cells mediated by A. tumefaciens is independent of the
Ti plasmid, which indicate that the Agrobacterium factor(s) involved in mammalian
cell gene transfer should be located on its chromosome.

87
3.2.3.3. Roles of Agrobacterium chromosomal gene in DNA transfer into
mammalian cells
A. tumefaciens cells with mutations in certain chromosomal genes are defective
in the transformation of plant cells. Thus, several strains with mutation in the
chromosomal virulence genes, such as At12512 (chvA
-

), At12513 (chvB
-
), A6340
(chvG
-
), A6880 (chvH
-
), CGI1 (aopB
-
), B119 (acvB
-
) and AG6 (katA
-
), respectively
(Charles and Nester, 1993; Peng et al., 2001; Xu and Pan, 2000; Jia et al., 2002),
were tested for their ability to deliver DNA into mammalian cells. Agrobacterium
strains with chvA and chvB mutation (At12512 and At12513) could not synthesize or
transport β-1,2-glucan, which appears to be required for the efficient attachment to the
plant cells. However, when At12512(pQM52) or At12513(pQM52) was incubated
with EcoPack2-293 cells, no reduction in gene transfer efficiency was observed (data
not shown). This data suggested that the early steps of interaction, such as
attachment, might not be critical for DNA delivery into mammalian cells in our
system, in which relatively small percentage of bacterial attachment may be enough
for the occurrence of gene transfer due to the high MOI (~40) used.
3.2.3.3.1. chvG and chvH are involved in Agrobacterium-mediated gene transfer
into mammalian cells
Interestingly, two chromosomal mutants, A6880(pQM52) (chvH
-
) and
A6340(pQM52) (chvG

-
), showed attenuated ability to gene transfer to the human cells
(Table 3.1). The transient gene transfer efficiencies of these two mutants are 2 × 10
-5

and 1 × 10
-5
per recipient cells, respectively, which are five to nine times lower in the
DNA transfer frequency than that of the wild type. chvH encodes a homologue of an
elongation factor P (efp) involved in protein synthesis (Peng et al., 2001). The wild

88
type chvH locus is essential for the full expression

of not only vir genes but also some
chromosomal genes. These genes might

code for particular amino acid sequences,
perhaps near the

start site of translation that are exceptionally dependent on elongation

factor P for translation. ChvG encodes a global pH sensor protein that is important for
the virulence of Agrobacterium tumefaciens. It is involved in the regulation of many
acidic pH-inducible genes, including both the chromosomal genes and the Ti plasmid
encoded vir genes (see Chapter 4). In addition, both A6880 and A6340 are sensitive
to acidic extracellular pH. Therefore, the low gene delivery efficiency of these strains
might be due to the low expression of one or more important protein(s), which are
necessary for mammalian cell gene delivery. These results indicate that some
Agrobacterium chromosomal genes might play important role(s) in mammalian cell

gene transfer. Taken together, the data suggest that the DNA transfer mediated by
Agrobacterium is not a process of passive uptake of foreign DNA by mammalian cells.
3.2.3.3.2. Effect of katA on DNA delivery into mammalian cells
katA gene encodes a catalase, which is involved in the dismutation of hydrogen
peroxide to water and oxygen (Xu and Pan, 2000). It has been reported that the rapid
production and accumulation of H
2
O
2
might lead to the hypersensitive response (HR)
in the plant defense system. Mutation in this gene will highly attenuate the bacterial
ability to cause tumors on plants and to tolerate H
2
O
2,
but not the bacterial viability in
the absence of exogenous H
2
O
2
. To determine if the same defense response is utilized
in mammalian cells, the katA mutant strain AG6 was used to infect 293 cells (Table
3.1 and Fig. 3.4). Surprisingly, the transient gene transfer efficiency of AG6 was 2.9
× 10
-4
per recipient cell, based on flow cytometry analysis, which was four times
higher than that of wild type. As catalase plays an important role in protecting

89
bacterial cells from damages caused by H

2
O
2
to cellular components, including
nucleic acids, proteins and cell membranes (Imlay and Linn, 1998; Storz and Imlay,
1999), thus it is possible that the incomplete bacterial membrane may facilitate the
Agrobacterium-mediated mammalian cell gene transfer.
3.2.3.3.3. Combinatorial effect of mutations in chromosomal genes on DNA
delivery into mammalian cells
The chromosomal genes chvG, chvH and katA were indicated to be involved in
the bacterial ability to deal with the stress condition of low pH, while the above
results showed that they are also involved in the Agrobacterium-mediated mammalian
cell gene transfer. These prompted studies on the functional relationship of these
genes in the gene delivery of mammalian cells. Thus, the gene transfer efficiency of
the chvG and chvH mutant strains in the absence of any functional katA gene was
tested. The double mutant strains were created by introducing the total DNA of AG6,
which is a katA
-
mutant (Xu et al, 2001), into A6880 and A6340 by electroporation.
(Charles et al, 1994). The resulting recombinants, A6880-AG6 and A6340-AG6,
lacked functional katA genes in the A6880 and A6340 background, respectively, as
verified by Southern analysis (Fig. 3.7). The gene transfer ability of these strains was
tested. The double mutant strains gave similar gene delivery efficiency as that of the
chvH or chvG single mutant strain (Table 3.1), instead of an increasing efficiencies in
mammalian cell gene transfer as that of katA mutant strain. These suggest that the
proteins regulated by chvG and chvH might play roles before the above proposed
bacterial membrane lysis. Again, these data agree with the hypothesis that
Agrobacterium play active roles in the gene transfer process instead of gene being
transfer from bacteria lysed passively.

90

1 2 3 4 5
Fig. 3.7. Southern blot analysis of homologous recombinants. The total
DNA of Agrobacterium strains were extracted and digested with PstI. gfp
uv

from mini-Tn5 was used as probe. Lane 1, A6340; lane 2, A6880; lane 3,
AG6; lane 4, A6880-AG6; lane 5, A6340-AG6.

91
3.2.4. Agrobacterium can deliver its chromosomal DNA into human cells
In order to investigate whether genes located on the A. tumefaciens chromosome
can also be transferred into human cells, the egfp gene as well as the viral LTR gene
were introduced into the A. tumefaciens chromosome by transposon mutagenesis.
The plasmid pQM61 was used as the transposon donor strain, which contains both the
egfp gene and the viral LTR gene located between the mini-Tn5 border sequences
(Lorenzo et al, 1990). Three Agrobacterium strains, wild type strain A348, A136
which lacks Ti-plasmid and GMI9023 which contains neither the Ti plasmid nor the
cryptic plasmid, were used as the recipients. The transposon mutants were selected
on AB plates containing 50 µg/ml of gentamicin and more than 100 targeted mutants
of GMI9023, A136 and A348 were obtained. The frequency of transposition was 10
-7
per recipient cell, which agrees with the previous report (Berg and Berg, 1996). The
structure of the transposon insert is shown in Fig. 3.8.
To confirm that the egfp as well as viral elements (LTR and Ψ
+
) were
integrated into the Agrobacterium genome via transposon mutagenesis, one colony for
each strain was picked up for Southern analysis. The total DNA of these strains were

extracted and digested with HindIII and probe with pQM61. The bands detected in
mutant strains were apparently different from the original transposon donor plasmid
pQM61 (Fig. 3.9), demonstrating that the egfp gene as well as transcript containing
Ψ
+
has been integrated into the recipient genome by random transposon mutagenesis
rather than existing on a free plasmid.
These mutant strains (A348::Tn5, A136::Tn5 and GMI::Tn5) were incubated
with the human cells. As shown in Fig. 3.4 (F)-(H), these strains could also express

92
egfp
3’ LTR
Gm
r


93
Fig. 3.8. Transposon insertion in mutant bacterial strains. For random
transposon mutagenesis details see Materials and Methods. Abbreviations: LTR,
long terminal repeat sequence; Ψ
+,
extended packaging signal; Neo
r
, Neomycin
resistance gene; P
CMV
, immediate early CMV promoter; egfp, enhanced green
fluorescent protein; Gm
R

, gentamycin-resistance gene; I and O, inverted repeats
of IS50;
O
5’ LTR
P
cmv
Νeo
r
Ψ
+
I
1 2 3 4
8kb
10kb
6kb
0.8kb
Fig. 3.9. Southern blot analysis of genomic DNA of transposon
mutants. Genomic DNA or plasmid was digested with HindIII and
separated on a 0.8% agarose gel. Hybridization was performed
using pQM61 as probe. The position and size in kilobase are
indicated on the left. Lane 1, pQM61; lane 2, A136::Tn5; lane 3,
A348::Tn5; lane 4, GMI::Tn5

94
the egfp gene, indicating that they could deliver the DNA located on their
chromosomes into the 293 cells. The RT-PCR results were consistent with the above
observation, as a clear band corresponding to the egfp gene could be produced by RT-
PCR from the RNA extracts of mammalian cells incubated with these strains (Fig 3.5).
The gene transfer efficiency based on flow cytometry analysis is approximately the
same as that observed for the cells infected by strains harbouring the reporter gene on

plasmid. These clearly show that the transfer of chromosomal DNA from
Agrobacterium to the host cells must have taken place. Thus, the DNA transferred by
Agrobacterium might be random or nonspecific.
Taken together, all results suggest that the mechanism of Agrobacterium-
mediated gene delivery into human cells is different from that of the transformation of
plant cells in many aspects. Therefore, it is likely that an unknown mechanism is
utilized by Agrobacterium to facilitate this inter-kingdom genetic information
exchange.
3.2.5. Internalization of A. tumefaciensinto mammalian cells
3.2.5.1. General characteristics of internalization
Unlike plant cells, mammalian cells do not have the structure of cell wall. Some
pathogenic bacterium, such as Listeria monocytogenes and Shigella flexneri can easily
invade mammalian cells (Goldberg, 2001). These prompted us to detect whether
Agrobacterium strains are able to invade mammalian cells. Human cells (1 ×10
6
)
were incubated

with A. tumefaciens (4 × 10
7
) for 4-72 h and tested the attachment and
invasion rate of Agrobacterium strains. The MOI is about 40, which is consistent
with gene transfer condition mentioned above. As shown in Table 3.2, the attachment

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Table 3.2 Attachment and invasion of bacteria to 293 cells
a
The number of bacteria added was used as 100%
Percentage of attachment or invasion
a

Time(h)
DH5α

RCR2011 LBA4404

AG6
b
A6340 A6880 A6340-AG6
b
A6880-AG6
b

Attachment

4 <0.01 <0.01 3.41 2.34 3.69 3.03 3.52 3.87
24 <0.01 <0.01






3.77 3.34 4.23 4.02 3.64 4.84
48 <0.01 <0.01 1.09 0.75 0.65 0.36 0.84 0.35
72 <0.01 <0.01 0.24 0.22 0.31 0.24 0.13 0.14
Invasion

4 <0.01 <0.01 0.63 1.48 0.46 0.88 2.68 2.71
24 <0.01 <0.01 1.22 2.24 1.42 1.39 2.84 2.91

48 <0.01 <0.01 0.42 0.62 0.38 0.33 0.61 0.27
72 <0.01 <0.01 0.11 0.18 0.19 0.12 0.12 0.09
Average number of bacteria per 293 cell
Attachment

4 <0.01 <0.01 7.6 6.8 9.6 10 8.8 12
24 <0.01 <0.01






8.3 9.7 11 14 9.1 15
48 <0.01 <0.01 2.4 2.2 1.7 1.2 2.1 1.1
72 <0.01 <0.01 0.5 0.64 0.86 0.8 0.33 0.42
Invasion

4 <0.01 <0.01 1.4 4.3 1.2 2.2 6.7 8.4
24 <0.01 <0.01 3.1 6.5 3.7 4.6 7.1 9.2
48 <0.01 <0.01 0.9 1.8 1 1.1 1.5 8.4
72 <0.01 <0.01 0.23 0.51 0.67 0.6 0.3 0.28
3
The bacterial strain with Gm
r


96