Journal of Environmental Biotechnology
Vol. 9, No. 1, 31–36, 2009

Original paper (regular paper)

Examination of Integrated pML4 DNA Transfer from Genetically
Modified Zebrafish to Bacteria
AKIKO H. HASHIMOTO, KIMIKO AMANUMA*, KAZUHIRO IWASAKI and YASUNOBU AOKINational Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305–8506, Japan* TEL: +81–(0)29–850–2390 FAX: +81–(0)29–850–2588* E-mail: amanuma.kimiko@nies.go.jp(Received; 29 September, 2008/Accepted; 11 November, 2008)
The increased use of genetically modified (GM) organisms has raised concerns that the transgenes could be transferred toorganisms in the wild. We examined the transfer of a foreign gene integrated into the chromosomal DNA of rpsL transgenic
zebrafish to coexisting bacteria. This GM fish carries pML4 plasmid DNA containing a kanamycin (Km)-resistance gene as
the foreign DNA. We isolated Km-resistant bacteria from feces, gut, and putrescent tissues and examined pML4 DNA transfer to them by colony direct PCR to amplify specific pML4 sequences. Km-resistant bacteria were grown from feces, but
pML4 sequences were not detected in a stored sample of the colonies. We isolated 751 Km-resistant anaerobic bacteria fromthe gut, but none of them contained the pML4 sequence. To isolate Km-resistant bacteria from putrescent tissues, a GM fish
euthanized on ice was incubated in rearing water at 28°C for 7 days. The number of Km-resistant bacteria increased withputrefaction, to about 10 times more at 7 days than that at day 0, but the pML4 sequence was not detected in approximately
900 colonies isolated from the putrescent tissues at day 7. These findings suggest that the Km-resistant bacteria isolated inthis study were not produced by pML4 gene transfer from the GM fish.
Key words: transgenic fish, GMO, Km-resistant, gene transfer, bacteria

1. Introduction
Genetically modified (GM) organisms are increasingly
generated for use as agricultural, bio-industrial, and foodproducts. Their release into the environment has raised
public concern about the potential risk of horizontal genetransfer, including the integrated gene transfer from GM
organisms to bacteria in natural environments.
Gene transfer among bacteria occurs in natural environments10,12). Recombinant DNA, such as antibiotic resistancemarker genes, is also transferred from GM bacteria to otherbacteria in natural environments8,18,20). Since several kinds ofGM plants useful for agriculture have been developed andcommercialized, gene transfer from GM plants to bacteria
in the field has been examined extensively as is seen elsewhere17). Occurrence of such events in the field has not been
demonstrated so far14), however, problems and diﬃculties
have been pointed out in detecting such events with extremely low frequencies7,14). Occurrence of gene transferfrom GM plants to bacteria in gut microbiota of animals
has not been demonstrated, either3,6,11,19).
Various kinds of GM fish have been developed, mainly as
tools for biological and environmental research. GM fish
have also been developed for food, although no commercially valuable GM food fish have been obtained yet9). To
our knowledge, however, there are no reports dealing withgene transfer from GM fish to bacteria in fish flora and/or
surrounding aquatic environments.
We previously developed GM rpsL zebrafish containing a
shuttle vector plasmid (pML4) for detecting mutagens inwater1). pML4 contains the rpsL gene as a target gene fordetecting mutations and a kanamycin (Km)-resistance geneas a marker for transformation. Before we can use this rpsL
GM zebrafish to monitor environmental mutagens outside
the laboratory (i.e., in the river), we have to clarify in advance the level of integrated DNA transfer from the GM
fish to environmental bacteria. Therefore, we tried to determine the frequency of DNA transfer from GM fish tobacteria existing in or near the fish. Here, we showed thatthe amounts of pML4 DNA in environmental bacteria
cultured from feces, gut, and putrescent tissues of GM fish
were lower than the detection limit in all samples.

2. Materials and Methods
2.1. Fish and transgene
A GM zebrafish line carrying approximately 350 copiesof the pML4 plasmid per haploid genome was establishedfrom the zebrafish AB line for a mutagenicity assay1). The
GM fish have been maintained as hemizygous GM fish inrearing water (0.1% Instant Ocean salts, Tomita Pharmaceutical Co., Ltd. Tokushima, Japan) at 26°C over 10 gen-

HASHIMOTO et al.

32

Fig. 1. Zebrafish and pML4 plasmid.

erations. The pML4 construct, which contains KanR, a
Km-resistance gene5), is shown in Fig. 1. The zebrafish AB
line was used as non-GM control fish.
2.2. Isolation of bacteria and preparation of PCR
samples
We used three kinds of media, Brain Heart Infusion (BHI;
BD, NJ, USA), Sabouraud Dextrose Broth (SDB; BD), andNutrient Broth (NB; BD), of which constituents and/or pH
are diﬀerent among them, for the cultivation of a wide
variety of microorganisms according to the methods ofRawls et al.15)
Feces collected from several GM fish and non-GM fish
(17.8 mg and 4.7 mg wet weight, respectively) were suspended in BHI medium and spread onto BHI, SDB, or NB agar
plates with or without Km (50 mg/l). Colonies were counted
after the plates were aerobically cultured at 28°C for 3 days.
Fifty colonies obtained from GM fish on Km-containing
plates were randomly and individually picked for PCR. All
the colonies from GM fish remaining on each Km plate
type (BHI-Km, SDB-Km, or NB-Km) were scraped andcombined into a pooled bacterial cell suspension in 10 mM
Tris-HCl-1mM EDTA (TE) buﬀer (pH 8.0) for PCR; eachpooled sample consisted of thousands of colonies grown oneach type of plate.
A gut collected from a GM fish (19.4 mg wet weight) was
immediately put into BHI medium, cut into pieces, andhomogenized. The gut suspension was spread onto BHI,
SDB, or NB plates with or without Km (50 mg/l) in ananaerobic globe box (Anaerorator model FEB-350, Sanki
Kagaku Kougei Co., Ltd, Tokyo). These plates were cultured
anaerobically in a BBL GasPak (BD) at 28°C for 3 days. A
total of 751 colonies of Km-resistant bacteria were isolated
individually. The 751 colonies were pooled into 76 bacterialcell suspensions containing 9 or 10 colonies each in TE
buﬀer for PCR.
To obtain putrescent fish, a GM fish was killed by anesthesia on ice and placed in 50 ml rearing water and incubated at 28°C for 7 days. Just before the incubation, part of
the abdomen (skin and intestine, 23.6 mg wet weight, Fig.
2a) was collected as control tissues (0 day), cut into pieces

Fig. 2. Putrescence of euthanized GM zebrafish.
A GM fish euthanized on ice was placed in rearing water andincubated at 28°C for 7 days.
a, GM fish after death. Red circle indicates the part that wasremoved immediately as control tissues; b, GM fish at 0 days
after removing control tissues; c, GM fish after 7 days incubation. Red circle indicates the part removed as putrescent tissue
at 7 days.

DNA transfer from GM fish to bacteria

and cultured immediately. At 7 days, 27 mg (wet weight) of
putrescent tissue around the abdomen (Fig. 2c) was collected. Each tissue (before and after putrescence) was suspended in BHI medium and spread onto BHI, SDB, or NB
plates with or without Km (50 mg/l). After the plates were
aerobically cultured at 28°C for 3 days, approximately 300
Km-resistant colonies were picked from each plate (6 plates
each of BHI-Km, SDB-Km, or NB-Km) cultured from control (0 day) or putrescent (7 days) tissues. The 300 colonies
were pooled into samples containing 10 colonies each, toobtain approximately 30 bacterial cell suspensions in TE
buﬀer for each kind of plate and PCR was performed.
2.3. Detection of pML4 sequence by PCR
Colony direct PCR was performed using primers specificfor pML4; primer-8 (5’-ATCGCGAGCCCATTTATACC-3’)
and primer-9 (5’-GGCACGTTGCTTTTCGCAAC-3’) (Fig.
1). We performed PCR in a final volume of 20 μl of Mg2+
plus Ex Taq buﬀer (Takara Bio Inc., Shiga, Japan) containing 0.2 mM dNTPs, 10 pmole of each primer, 0.5 unit of
Ex Taq polymerase (Takara Bio Inc.) and 0.05 OD660 unitsof the bacterial cell suspension. The PCR conditions were
94°C for 2 min; 35 cycles of 94°C for 30 sec, 63°C for
30 sec, and 72°C for 1 min; and 72°C for 5 min. PCR prod-33

ucts were separated by agarose gel electrophoresis andvisualized with ethidium bromide. A detection limit for
scraped and pooled samples from feces was estimated froman experiment employing, as a standard, E. coli RR1 transformed with pML4, and it was 2.6 bacterial cells per 20 μlof the PCR mixture (Fig. 3). A similar detection limit wasalso obtained by using another primer set specific for
pML4. For colonies isolated from the gut and putrescentabdomen, 10 colonies were combined into one bacterial cellsuspension for PCR, and therefore, the bacterial cell number
from each isolated colony in the PCR mixture was muchhigher than the 2.6 cells/20 μl detection limit describedabove.
2.4. Calculation of pML4 DNA transfer frequency
The pML4 DNA transfer frequency in Km-resistant bacteria
is calculated by the following formula:(pML4 DNA transfer frequency in Km-resistantbacteria)=(Number of bacteria with transferred pML4
DNA)/(Number of Km-resistant bacteria checked by PCR)The pML4 DNA transfer frequency in total bacteria cultured without Km was calculated by the following formula:(pML4 DNA transfer frequency in total bacteria)={pML4 DNA transfer frequency in Km-resistant bacteria
×Number of total bacteria (CFU/g dry sample)}/{Numberof Km-resistant bacteria (CFU/g dry sample)}

3. Results
3.1.

Fig. 3. Detection of pML4 DNA in bacterial samples preparedfrom feces by PCR.
Lanes 1 and 9, 100-bp ladders; lane 2, E. coli RR1 bearing noplasmid (negative control); lanes 3, 4, 5, mixture of colonies
pooled from Km-BHI plates, SDB-Km plates, and NB-Km
plates, respectively; lanes 6, 7, and 8, E. coli RR1 bearing thepML4 plasmid at 0.26, 2.6, and 2.6×106 cells/20 μL reaction
mixture, respectively (positive control).

pML4 DNA transfer from GM fish feces to bacteria

Table 1 shows the number of bacteria cultured from fecescollected from GM fish or non-GM fish using 3 kinds of
medium. After 3 days of culture, approximately 107 to 109
CFU/g were grown on each plate without Km from both
GM and non-GM fish. Considerable numbers of Km-resistant colonies grew from the feces of GM fish on BHI-Km
and NB-Km plates (8.5×108 and 2.0×105 CFU/g dry feces,respectively), and these numbers were very similar to thosecultured from feces of non-GM fish (8.5×108 and 2.1×105
CFU/g dry feces on BHI-Km and NB-Km plates, respectively). To examine whether the Km-resistant colonies origi-

Table 1. Km resistance and pML4 DNA transfer to bacteria from GM fish feces.
Km-resistantTotal bacteria
bacteria
Km resistance rate
(CFU/g dry feces) (CFU/g
dry feces)

Medium
BHI
SDB
NB

GM

2.3×109

Non-GM
GM
Non-GM
GM
Non-GM

1.8×109
2.2×108
4.4×107
2.5×109
1.6×109

8.5×108
8.5×108
1.5×107
6.4×105
2.0×105
2.1×105

3.7×10–1
4.7×10–1
6.8×10–2
1.5×10–2
8.0×10–5
1.3×10–4

Number ofbacteria used
for PCR
59645
n.d.
7677
n.d.
2879
n.d.

Number of pML4
pML4 DNA
DNA transferred transfer frequency
bacteria
in total bacteria
<2.6
n.d.
<2.6
n.d.
<2.6
n.d.

<1.6×10–5
n.d.
<2.3×10–5
n.d.
<7.2×10–8
n.d.

n.d.: not determined.
The detection limit was estimated to be 2.6 bacterial cell per 20 μL for PCR of scraped and pooled samples. Therefore, the pML4 transfer frequency in Km-resistant bacteria used for PCR was calculated to be below 2.6 cells/number of Km-resistant bacteria used for PCR,
and the pML4 transfer frequency in total bacteria was calculated to be below 2.6 cells/number of Km-resistant bacteria for PCR×Kmresistance rate. (For example, for the BHI plate, the value was calculated to be below 2.6/59645×3.7×10–1=1.6×10–5.)

HASHIMOTO et al.

34

nating from GM fish contained pML4 DNA, we randomly
isolated 50 colonies each grown from GM fish feces on the
BHI, SDB, and NB plates. All the remaining colonies oneach plate were scraped and combined with colonies fromother plates containing the same medium into 3 pooled
samples and subjected to PCR along with individually isolated colonies. After gel electrophoresis analysis, no PCR
products were detected in the 50 individual colonies or in
the pooled samples (Fig. 3). The pML4 DNA transfer frequency in the 3 pooled samples was calculated to be below
10–5–10–8 (Table 1).
3.2.

pML4 DNA transfer from GM fish gut to anaerobic
bacteria

Anaerobic intestinal bacteria from GM fish gut were
cultured on BHI, SDB, or NB plates with or without Km(Table 2). Substantial numbers of Km-resistant bacteria
were detected only on BHI-Km plates. The Km-resistance
rate of bacteria cultured on BHI plates was 8.7×10–3 ascalculated from the number of Km-resistant colonies(9.6×104 CFU/g dry gut) divided by the total number ofcolonies (1.1×107 CFU/g dry gut). We did not scrape together all the colonies on the plates, but isolated 751 colonies
individually to avoid contamination of GM fish tissues in
the PCR samples. We looked for pML4 DNA in the 751
isolated Km-resistant intestinal bacteria by PCR but did
not detect any PCR products corresponding to pML4 DNA,
indicating that pML4 was transferred to less than one outof 751 isolated bacteria. From these results, we estimatedthe pML4 transfer frequency to be under 1.2×10–5 (1.2×
10–5=1/751×8.7×10–3).
3.3.

pML4 DNA transfer from putrescent GM fish tissue
to bacteria

We next examined whether the gene could be transferredfrom decomposing fish tissue. After the fish was killed byexposure to cold, we removed a part of abdomen containing skin and gut as day 0 control tissues (Fig. 2a, b). The
remainder of the fish was incubated in rearing water at
28°C. After 5 days of incubation, the outlines of the deadfish became unclear, likely due to the putrescence of thetissues. On day 7, we collected some of this tissue fromaround the abdomen (Fig. 2c). Both control and putrescenttissues were cultured aerobically on BHI, SDB, and NB
plates. Considerable numbers of Km-resistant bacteria grewfrom the control tissue harvested on day 0, but after 7 days
incubation, the numbers of Km-resistant bacteria increased
to 7.4–13.3 times that at day 0 (Table 3, Fig. 4). We isolated approximately 300 colonies from each plate, and PCR
was performed as described in the Materials and Methods.
No amplified products were detected in any of the bacterial
samples from BHI-Km, SDB-Km, or NB-Km plates at either
0 and 7 days, indicating that pML4 DNA transfer frequencies
were below 10–3.
3.4. DNA sequence homology to Km-resistance genes
deposited in GenBank
Considerable numbers of Km-resistant bacteria were
cultured even from feces of non-GM fish (Table 1). Km isan aminoglycoside antibiotic and resistance to Km is often
due to enzymatic inactivation of Km. The DNA sequencesof many bacterial genes encoding aminoglycoside-modifying
enzymes that confer Km-resistance have been already determined (Table 4).
We performed a homology search between KanR in
pML4 and all of the Km-resistance genes we found in the
DNA Data Bank of Japan and in the review of Shaw etal.16) (Table 4), and found only very low homologies between KanR and other bacterial genes, except for the uncultured eubacterium plasmid pIE1130 (99.1% homology) andTable 2. Km resistance and pML4 transfer to bacteria from GM fish gut.
MediumTotal bacteria(CFU/g dry gut)

Km-resistantbacteria (CFU/g dry gut)

Km resistance rate

Number ofisolated bacteria
used for PCR

Number pML4
DNA transferredbacteria

pML4 DNAtransfer frequency
in total bacteria

BHI
SDB
NB

1.1×107
1.9×105
5.4×107

9.6×104
00

8.7×10–3
n.d.
n.d.

751
n.d.
n.d.

<1
n.d.
n.d.

<1.2×10–5
ND
ND

ND: Not detected, n.d.: not determined.
Table 3. Km-resistant bacteria and pML4 transfer to bacteria from dead GM fish.
MediumCulture days

Total bacteria(CFU/g dry tissue)

Km-resistantbacteria (CFU/g dry tissue)Kmresistance
rate

BHI

0

6.2×109

1.8×107

2.9×10–3

310<1<9.5×10–6

7
0
7
0
7

3.5×109
3.6×107
2.7×1082.0×109
2.4×109

2.4×108
2.7×1072.0×108
4.1×106
4.8×107

6.9×10–2
7.5×10–1
7.4×10–1
2.0×10–3
2.0×10–2

310
310
310
262
302

<1
<1
<1
<1
<1

<2.2×10–4<2.4×10–3<2.4×10–3<7.9×10–6<6.5×10–5

SDB
NB

Number of
Number of pML4
pML4 DNA
isolated bacteria DNA transferred transfer frequency
used for PCR
bacteria
in total bacteria

DNA transfer from GM fish to bacteria

Klebsiella pneumoniae (72.2% homology).

4. Discussion
As a first trial for investigating gene transfer from GM
fish to environmental bacteria, we cultured Km-resistantbacteria from feces, gut, and putrescent tissues of GM fishand examined by PCR whether they contained pML4 DNA
sequences. Appreciable numbers of Km-resistant bacteria
were cultured in all samples, but the pML4 sequences in

35

them were below the detection limit, suggesting that theseKm-resistant bacteria were not generated by pML4 DNA
transfer from the GM fish. The pML4 DNA transfer frequencies were calculated to be below 10–3 to 10–8 (Tables
1–3). Notably, similar numbers of total and Km-resistantbacteria were grown from GM and non-GM feces andsimilar frequencies of Km resistance were observed (Table1), suggesting that these Km-resistant bacteria are probably
indigenous to natural environments. These findings are consistent with the existence of many Km-resistant bacteria

Fig. 4. CFU of total and Km-resistant bacteria isolated from euthanized GM zebrafish before (day 0) and after (day 7) putrescence.
Table 4. Km resistance genes in bacteria.
Km-resistant bacteria

Enzymea (gene)

Escherichia coli

(Kanamycin resistance transposon Tn903; KanR)

Campylobacter coli
plasmid pIP1433
Pseudomonas aeruginosa
Staphylococcus aureus
Streptomyces albulus
Streptomyces kanamyceticus
Streptomyces kanamyceticus
Uncultured eubacterium
plasmid pIE1130
Vibrio cholerae
Pseudomonas aeruginosa PST-1
Pseudomonas aeruginosac
Pseudomonas aeruginosac
Unidentified bacteriumc
Escherichia coli plasmid RP4c
Klebsiella pneumoniaec
Escherichia colic

Streptococcus faecaliscAcinetobacter baumannii
Campylobacter jejunic

c

c

b

V00359Kanamycin resistance protein (aphA-3)

M26832

Aminoglycoside acetyltransferase (aac(6')-Iae)
Kanamycin nucleotidyltransferase (aadD)
Kanamycin acetyltransferase (kat)16S rRNA methylase (kmr)Aminoglycoside 6'-N-acetyltransferase (kac)AF181950
AB116646
AB164229
AB164230

Aminoglycoside phosphotransferase (aph(3')-I)

AJ271879

Aminoglycoside adenylyltransferase (aadB)

AF221902

Aminoglycoside-(3)-N-acetyltransferase (aac(3)-IIIa)
n.d.d (aac(3)-IIIb)
Aminoglycoside 3’-N-acetyltransferase (aac(3)-IIIc)
Aminoglycoside adenylyltransferase (ant(2’’)-Ia)
Aminoglycoside-3’-phosphotransferase (aph(3’)-Ib)
Aminoglycoside-3’,5’’-phosphotransferase-I (aph(3’)Ic)

X55652
L06160
L06161
X04555
M20305

Neomycin phosphotransferase (aph(3’)-IIa)
3’,5’’-Aminoglycoside phosphotransferase type III(aph(3’)-IIIa)
3’-Aminoglycoside phosphotransferase (aph(3’)-VIa)
Kanamycin phosphotransferase (aph(3’)-VIIa)

V00618, Registered name in DDBJ/GenBank.
, The transgene integrated in the GM fish used in this study carries this gene, KanR.
c, Reported in the review of Shaw et al.16)d, n.d. not described in Shaw et al.16)a

DDBJ/EMBL/GenBank accession no.
b

AB104852

M37910

V01547
X07753
M29953

HASHIMOTO et al.

36

confirmed by the survey of the previously identified bacteria;almost all Km-resistant bacterial genes listed (Table 4) haveno homology with KanR in pML4. Boon reported that a
considerable percentage of bacteria isolated from river waterin rural, semi-rural, and urban sites show resistance to several
antibiotics, including Km2).
The variation in the DNA transfer frequency (below 10-3
to 10-8) depended on the percentage of Km-resistant bacteria
among the total cultured bacteria and the number of colonies isolated for PCR. To obtain more precise data, we mayneed to isolate much higher numbers of Km-resistant colonies for PCR, for example, from putrescent tissues cultured
in SDB medium (Table 3). The transformation frequencies
of naturally transformable bacteria were reported to be 10–2
to 10–9 12), and the transformation frequencies of a bacterial
strain specialized to receive a GM plant transgene were atthe 10–8 level13). Considering these reports and the non-conjugative nature of the pML4 plasmid, the frequency oftransformation of the bacteria surrounding GM fish by
pML4 might be lower than natural bacterial transformation
and GM plant DNA transfer. To detect such rare events using the strategy employed here, we might need to isolate
much higher numbers of Km-resistant colonies and to culture them to create much more bacterial colonies. This
strategy, however, contains serious problems; bacteria receiving pML4 may not always become Km-resistant because
the Km-resistance gene in pML4 may not always be expressed in a heterologous bacterial host. Moreover, only asmall proportion of the bacteria present in the feces, gut,
and fish environment are culturable. To estimate more
precisely the possibility of DNA transfer from GM fish to
surrounding bacteria, new strategies might be required, suchas the recently developed gnotobiotic zebrafish technique15),
and/or the establishment of a new transgenic fish line
bearing a marker gene that is eﬃciently selectable and functional in a broad range of bacteria.
Despite the limitations to the experiments describedabove, we showed at least that an integrated transgene ofGM fish, pML4, did not cause transfer of culturable Km
resistance to bacteria existing with the GM fish under theexperimental conditions, suggesting that using rpsL GM
fish1) for monitoring aquatic environments is unlikely tocause a serious problem with pML4 transfer.

Acknowledgments
We thank Yukari Sakashita for her excellent technicalcontributions.
This study was supported by the Global Environment Research Fund from the Ministry of the Environment of Japan.

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