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RESEARCH ARTICLE
Evidence for particle-induced horizontal gene transfer and serial
transduction between bacteria
Hiroshi Xavier Chiura1, Kazuhiro Kogure1, Sylvia Hagemann2, Adolf Ellinger2 & Branko Velimirov2
1
Marine Microbiology Laboratory, Department of Marine Ecosystems Dynamics, Atmosphere and Ocean Research Institute, University of Tokyo,
Kashiwa, Chiba, Japan; and 2Centre of Anatomy and Cell Biology, Medical University of Vienna, Vienna, Austria
Correspondence: Branko Velimirov, Centre
of Anatomy and Cell Biology, Medical
University of Vienna, Währingerstraße 10,
1090 Vienna, Austria. Tel.: 1431 427 760
630; fax: 143 427 761 233; e-mail:
[email protected]
Received 9 November 2010; revised 7 February
2011; accepted 14 February 2011.
Final version published online 23 March 2011.
DOI:10.1111/j.1574-6941.2011.01077.x
MICROBIOLOGY ECOLOGY
Editor: Julian Marchesi
Keywords
virus-like particles; membrane vesicles; marine
bacteria; horizontal gene transfer (HGT).
Abstract
Incubation of the amino acid-deficient strain Escherichia coli AB1157 with
particles harvested from an oligotrophic environment revealed evidence of
horizontal gene transfer (HGT) with restoration of all deficiencies in revertant
cells with frequencies up to 1.94 105. None of the markers were preferentially
transferred, indicating that the DNA transfer is performed by generalized
transduction. The highest gene transfer frequencies were obtained for single
markers, with values up to 1.04 102. All revertants were able to produce
particles of comparable size, appearing at the beginning of the stationary phase.
Examination of the revertants using electron microscopy showed bud-like
structures with electron-dense bodies. The particles that display the structural
features of membrane vesicles were again infectious to E. coli AB1157, producing
new infectious particles able to transduce genetic information, a phenomenon
termed serial transduction. Thus, the o 0.2-mm particle fraction from seawater
contains a particle size fraction with high potential for gene transfer. Biased
sinusoidal field gel electrophoresis indicated a DNA content for the particles of
370 kbp, which was higher than that of known membrane vesicles. These findings
provide evidence of a new method of HGT, in which mobilizable DNA is trafficked
from donor to recipient cells via particles.
Introduction
It is well documented that viruses in aquatic environments
play an important role in regulating bacterial biomass and
transferring genetic elements between bacteria (Fuhrman,
1999). Considering narrow host specificity, the latter contribution may be meaningful only among closely related
strains or species (Schicklmaier & Schmieger, 1995). However, there are many indications that gene transfer may also
occur among more phylogenetically divergent bacteria
(Jiang & Paul, 1998; DeLong et al., 2006), which significantly
increases the magnitude of the transferred genetic information within the prokaryotes. Prokaryotes are known to be
unique in their ability to react to environmental changes by
the rapid acquisition of the necessary genetic traits for
continued survival. This genetic flexibility is, besides their
very short generation time, mainly due to the natural
existence of seemingly efficient means for horizontal gene
transfer (HGT) among bacteria, as well as between bacteria
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and other organisms (Lengeler et al., 1999). Virus-mediated
transfer of genetic elements between bacteria has become a
major research topic in the last two decades, whereby
conjugation, transduction and transformation are wellinvestigated mechanisms resulting in HGT between prokaryotic organisms. However, considering viral host specificity,
the relatively short length of transferable DNA segments and
the lack of de novo particle production from the transductants (Ochman et al., 2000; Brüssow et al., 2004), one may
conclude that viruses are not always the major players in
gene transfer, given that recent developments in microbial
genomics and metagenomic approaches revealed the existence of reminiscent genes from exotic origins (Lawrence
et al., 2002). Novel modes of lateral gene transfer have been
described in the past decades. The particle sizes of gene
transfer agents (GTAs) range between 30 and 80 nm (Marrs,
1974; Wall et al., 1975; Barbian & Minnick, 2000; Lang &
Beatty, 2000, 2002, 2007; Biers et al., 2008; Zhao et al., 2009),
and a report of McDaniel et al. (2010) states that GTA gene
FEMS Microbiol Ecol 76 (2011) 576–591
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Evidence for particle-induced horizontal gene transfer
transfer frequencies are a thousand to a hundred million
fold higher than prior estimated for HGT in the oceans. It
was presumed that some 47% of the culturable natural
microbial community might be seen as gene recipients.
Similarly, vesicle trafficking was mentioned as a possible
mechanism for the transfer of DNA between prokaryotes.
However, this was rather considered as a sideline of
investigations dealing with interspecies communication
(Kadurugamuwa & Beveridge, 1999; Marketon et al., 2002;
Mashburn & Whiteley, 2005; Mashburn-Warren & Whiteley,
2006), delivery of toxins (Kuehn & Kesty, 2005; McBroom &
Kuehn, 2005) or antibiotic resistance determinants (Ciofu
et al., 2000; Wai et al., 2003). It is therefore plausible that
other unknown, but dynamic mechanisms function in
aquatic systems to promote lateral gene exchange. Within
the framework of a series of studies on HGT between
bacteria in marine (Chiura, 1997) and thermal environments (Chiura, 2002; Chiura & Umitsu, 2004), it was shown
that virus-like particles (VLPs) that were infectious to
Escherichia coli are spontaneously produced by budding
and released in the stationary phase from the following:
Alphaproteobacteria such as Ahrensia kieliensis and Flavobacterium spp. 16-04, ubiquinone-possessing marine bacteria
(Q10MB) and Aquificales cells (Chiura et al., 2000, 2002). A
number of these experiments led to the observation that the
recipient E. coli cells acquire the ability to produce new
particles in the size range between 100 and 130 nm, and
molecular biological analyses revealed that the nucleic acid
species encapsulated in such particles was DNA (Chiura,
2004). In the context of the above observations, it was
postulated that such particle production may also take place
in the ocean’s water column and would possibly reinforce
the currently known mechanisms of HGT between bacteria.
Because it is impossible to identify these particles in water
samples by normal epifluorescence microscopy, we decided
to work with a concentrated VLP fraction (o 0.2 mm
diameter particles) and test whether one would obtain
recipient bacteria that could produce new particles.
Particle sizes between 100 and 130 nm were selected from
this size fraction in order to eliminate possible interferences
with large and tailless GTA particle types such as those
derived from Silicibacter pomeroyi DSS-3 cultures (Biers
et al., 2008) from Bartonella spp. (Barbian & Minnick,
2000) or from Methanococcus voltae (Bertani, 1999), which
all have particle diameters in the size range of 80 nm.
Furthermore, the size class chosen was consistent with the
previously investigated particles obtained from Aquificales
(Chiura, 2002). It should be emphasized that particle
production in recipient bacterial cells is a feature that is
characteristic of infections by bacteriophages, but not for
VLPs in the abovementioned size range, which strengthens
the assumption that a nonviral transfer of genetic material
may occur.
FEMS Microbiol Ecol 76 (2011) 576–591
The purpose of the present investigation was to test
whether particles within the mentioned size fraction from
natural seawater trigger the abovementioned features in
recipient cells. Furthermore, we attempted to ultrastructurally characterize the previously mentioned particles and
subsequently quantify the postulated induced gene transfer
using the auxotrophic enteric bacterial mutant E. coli
AB1157 strain as the recipient cell. The VLP fraction was
harvested from samples of the oligotrophic waters of the
Western Mediterranean Sea (i.e. distant from punctual or
diffuse sources of pollution).
Materials and methods
Seawater samples (170 L total) were obtained at 42136 0 N,
8156 0 E, from a 5 m depth (19.8 1C) near the marine station
STARESO at Calvi, Corsica, France. Samples were collected
during the summer and autumn and transported to the
laboratory for further treatment within 1 h.
Preparation of VLPs
The sea water was passed through a 0.2-mm Durapore
membrane filter (Millipore, Billerica, MA) and concentrated
to c. 20 mL by successively using a Pelicon cassette (Millipore) system and a Minitan Ultrafiltration System (Millipore) with a 30-kDa cut-off filter. The final average
concentration rate was 2774-fold. Concentrated VLPs were
treated with 10 mg mL1 each of DNase I and RNase A at
25 1C overnight with 100 mM phenylmethylsulphonyl fluoride (Sigma) to exclude the possibility of gene transfer by
transformation. The concentrate was filtered again through
0.45- and 0.22-mm membrane filter, and then it was centrifuged at 80 000 g for 30 min using a Beckman Preparative
Ultracentrifuge L8M with a 55.2Ti rotor to pellet VLPs. The
supernatant of this ‘final concentrate’ was recovered and
used as a negative control in the subsequent experiments.
The pellet was resuspended overnight at 4 1C in 200 mL TBT
buffer (100 mM Tris-HCl, 100 mM NaCl and 10 mM
MgCl2) by gentle rotation with a Slow Rotator (TAAB,
UK). VLPs were purified by CsCl-density equilibrium ultracentrifugation at 174 400 g for 18 h. Purified particles were
harvested after ultracentrifugation from the density fraction
1.2–1.6 g cm3 (Chiura, 1997), and the resulting bands were
separately recovered by the side puncture technique using
2.5 mL syringes (Terumo, Japan). CsCl was removed via
dialysis using Spectra/Por 4 tubing (Spectrum, molecular
weight cut-off, MCWO = 12 000–14 000 Da) against five
changes of 100 volumes of TBT buffer. VLP abundance and
particle size distribution in the respective bands were
examined using electron microscopy as described below.
The protein and nucleic acid concentrations were determined photometrically by reading A260 nm and A280 nm using
a Shimadzu Spectrophotometer Type UV260 (Shimadzu
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578
Corp., Kyoto, Japan). Additionally, the protein content was
also determined using the Protein Assay Kit (Bio-Rad
Laboratories, Hercules, CA) following the specifications for
microassays.
H.X. Chiura et al.
For electron microscopic evaluation, cells were pelleted as
described above and fixed in 3% glutaraldehyde (electron
microscopy grade; Serva) in 0.1 M sodium cacodylate buffer
for 60 min, osmificated in veronal-acetate-buffered OsO4 for
60 min, dehydrated in a sequential series of ethanol solutions and embedded in Epon. Thin sections (80 nm) were
stained with uranyl acetate and lead citrate, and they were
examined using a Tecnai-20 electron microscope (FEI
Company, Eindhoven, the Netherlands) at 80 kV. Pictures
were taken using a slow-scan CCD camera (Gatan, MSC
794).
mid-exponential phase at 30 1C. Then, they were centrifuged
at 5000 g in a refrigerated centrifuge (Kubota TR 20000); the
pellet was resuspended in 5 mL TBT buffer. This suspension
yielded a viable count of 2 108 CFU mL1. One millilitre
of this suspension was mixed with an aliquot volume of the
harvested VLP fraction to obtain the various multiplicities
of infection (MOIs) of 0.12, 1, 5.12, 20 and 200. The tubes
were left undisturbed at 30 1C for 15 min. After incubation,
cells were washed with a Davis salt solution and finally
suspended in 1 mL of the same solution. The mixture was
plated in triplicate on appropriate selection media and
incubated for 2 days at 30 1C. The following controls were
included in the assays: (1) UV-irradiated VLPs added to
recipient cells; (2) recipient cells with Davis buffer instead of
VLPs to determine spontaneous reversion rates; (3) autoclave-inactivated VLPs added to recipient cells; (4) recipient
cells with the supernatant of the respective final VLP
concentrate; (5) UV-irradiated VLPs without recipient cells
to control for possible colony formation within the VLP
fraction; and (6) untreated VLPs without recipient cells to
control for possible colony formation from within the VLP
fraction.
Escherichia coli AB1157 colonies that reverted for one or
more amino acid markers were designated as revertants. All
generated revertants were also subjected to examination
for unselected marker transfer. For this purpose, positive
colonies for one selection medium were transferred to the
remaining three selection media to test whether colony
formation would occur.
Gene transfer assay
Lethal effect of particles on E. coli AB1157
An auxotrophic mutant strain, E. coli AB1157 (F; thr-1
leuB6 thi-1 lacY1 galK2 ara-14 xyl-5 mtl-1 proA2 his-4 argE3
rpsL31 tsx-33 supE44), was used as the recipient bacterium
for testing VLP-mediated gene transfer (Chiura, 1997). We
focused on the following markers: leucine (Leu), proline
(Pro), histidine (His) and arginine (Arg). Threonine deficiency was not considered to be a useful marker due to its
considerably high spontaneous reversion frequency of
107 per cell. For the other four markers, spontaneous
reversion frequencies were below the level of detection
(Chiura, 1997). The strain was obtained from the National
Institute of Genetics (Shizuoka, Japan). To ensure reproducible physiological conditions of the recipient E. coli
AB1157 for all subsequent experiments, the bacterium was
cultured at 30 1C by shaking (120 r.p.m.) until a cell density
of 4 108 CFU mL1 was reached. After the addition of
glycerine (7% final concentration), the culture was dispensed in 2-mL aliquot, frozen in liquid nitrogen and stored
at 85 1C until further use. For the gene transfer assays, 2mL aliquot of the frozen seed culture were mixed with 3 mL
of a fresh LB broth in L-shaped test tubes and grown to the
Recipient E. coli AB1157 cells from frozen seed cultures were
grown at 30 1C in LB broth, harvested in the mid-exponential
phase (5000 g) and resuspended in 5 mLTBT. The viable count
of the cell suspension corresponded to 2 108 CFU mL1.
Harvested particles were added to the cell suspensions to
obtain MOIs of approximately 0.1, 1, 5, 20 and 200 and left
undisturbed for 15 min at 30 1C. Subsequently, cells were
washed with Davis solution and then resuspended in 1 mL of
the same solution. After dilution with Davis salt solution, the
cells were plated in triplicate on LB medium and incubated for
2 days at 30 1C.
The following controls were included: (1) recipient cells
plus TBT buffer, but without particles, to determine the
viable cell count; (2) UV-treated particles (15-min irradiation with a 15 W sterilizing lamp; Phillips, the Netherlands)
plus recipient cells; (3) autoclaved particles (121 1C, 200 kPa,
20 min) plus recipient cells; and (4) recipient cells with the
supernatant of the respective final concentrate of particles.
The supernatant was obtained by ultracentrifugation at
55 000 g for 45 min to precipitate particles and subsequently
filtered through a 0.2-mm membrane. Control experiments
Enumeration of cells and VLPs
Viable cell counts were determined on Luria–Bertani (LB)
solid medium incubated at 30 1C. The number of VLPs was
determined according to Børsheim et al. (1990). After staining
for 30 s with 2% uranyl acetate, grids were examined at
75 000 magnification at a voltage of 80 kV using a Zeiss
EM902 (Zeiss Inc., Germany) or a JEM-1200EX transmission
electron microscope (Jeol Inc., Japan). At least 50 fields were
selected for counting. The average burst was derived from the
number of VLPs observed in a cell (n = 2000).
Transmission electron microscopy (TEM)
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FEMS Microbiol Ecol 76 (2011) 576–591
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Evidence for particle-induced horizontal gene transfer
were performed using 20 mL coliphage T4 under the above
conditions.
The lethal effect of particles on recipient cells was expressed as efficiency of plating (EOP) calculated as % of
CFU formed as compared with the number of CFU in TBT
control plates.
Detection of VLP production during incubation
of revertants
A revertant grown on a minimal agar plate (no amino acidsupplemented minimal agar after Davis) at an MOI of 5.1
was incubated at 30 1C for 370 h in 3.4 L of leu1 medium
(leucine-supplemented minimal broth after Davis) to examine its growth profile and compare it with that of the
recipient E. coli AB1157. For this purpose, 0.5-mL sample
were intermittently withdrawn to determine the cell numbers and the number of free particles using epifluorescence
microscopy. Samples filtered through Anodisc filters were
stained with SYBR Gold (Invitrogen Corp., Carlsbad, CA)
and mounted according to Noble & Fuhrman (1988). The
proportion of particle-bearing cells and the number of
particles per cell (burst size) were determined from 0.1-mL
sample using electron microscopy.
Free particles produced during prolonged incubation
were harvested, purified and tested for gene transfer capability using the same method described above.
Purified particles that were newly generated from the 370h culture of the first revertant were subjected to a second
gene transfer experiment with E. coli AB1157 in the same
manner as described above. The revertants obtained from
the second transduction experiment were again cultured in
the same medium (leu1) at 30 1C for 167 h to determine the
growth profiles, particle production and burst size.
Nucleic acid extraction from particles produced
by revertants in agarose gel plugs (in situ
lysis gel)
The nucleic acid species of purified particles from a leu1
revertant were examined electrophoretically using in situ
lysis. For this purpose, samples were embedded in an
agarose gel block. The procedures for the preparation of
agarose gel plugs followed the recommendations of the
agarose manufacturer (InCert Agarose).
For nucleic acid molecular type estimation, in situ lysis
gel plugs were embedded in 1.0% agarose ME (SeaKem,
UK). Before embedding, the gel plug was sliced using a
microscope cover slip to obtain a 3.0 3.0 1.0 mm block.
The separating agarose gel, liquefied 1.0% agarose in TBE
buffer (89 mM Tris-HCl, 89 mM boric acid and 0.5 M
EDTA, pH 8.0), was poured into a casting mould to prepare
a gel that was 6 mm thick, to which the sliced gel plug
(attached to a slot-making comb) was applied. The cast gel
FEMS Microbiol Ecol 76 (2011) 576–591
was incubated at ambient temperature for 30 min, and then
it was placed in a refrigerator overnight to solidify completely.
Biased sinusoidal field gel electrophoresis (BSFGE) was
used to run gels in 0.5 TBE buffer at room temperature
for 30 h under the following conditions: 1.6 V cm1 DC,
9.6 V cm1 AC, a start frequency of 0.01 Hz, an end frequency of 0.3 Hz and a logarithmic ramp. The molecular
mass standards used were Lambda ladder (48.5 kb–1.2 Mbp,
FMC) and l/HindIII (0.13–23.13 kbp, Nippon Gene, Japan). Nucleic acid species were visualized by staining the gel
with a 1/10 000 SYBR Green II stock solution (Molecular
probe) at room temperature for 1 h, followed by illumination with a Spectroline Trans Illuminator Model TC-365A
(Spectronics) at 360 nm. The results were recorded using a
Canon PowerShot Pro 70 digital camera. The concentration
of nucleic acid was determined from the pictures obtained
using the public domain NIH IMAGE program (developed at
the U.S. National Institute for Health and available online at
http://rsb.info.nih.gov/nih-image/).
Determination of nucleic acid type content in
the particle
After BSFGE, bands from the leu1 revertant-produced
particles were excised from the gel, and each gel slice was
placed in a Falcon tube. DNase I (10 mg mL1, Sigma) and
RNase A (10 mg mL1, Sigma) were then added to the tubes.
Four gel slices from the particles were treated with DNase I,
RNase A, distilled water and TM buffer overnight at 37 1C.
The gel slices with and without treatment were again
examined with BSFGE for 16 h and the results were recorded
as described above.
DNA-antibody labelling of TEM thin sections
For DNA detection, bacterial samples were fixed and embedded as indicated above. Epon sections (80 nm) were
etched with 3% sodium metaperiodate for 40 min, rinsed
in A. dest. and preincubated with blocking solution B1 (1%
bovine serum albumin and 1% goat serum in phosphatebuffered saline–Tween 20, pH 7.4), followed by a 2-h
incubation with a monoclonal anti-DNA antibody (Ac-3010; Progen, Heidelberg, Germany) in B1. Detection was
performed using an anti-mouse colloidal gold-labelled secondary antibody. Control incubations were performed by
excluding the anti-DNA antibody.
Results
Size distribution of VLPs and ultracentrifugation
The average number of bacteria and VLPs in the three samples were 1.07 106 cells mL1 (SD = 0.43) and 1.17 108
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580
256
4.15 1.64
42.9 17.0
12.38
20.0
309
4.36 0.73
45.1 7.5
10.39
11.6
Middle band was used for gene transfer experiments. n = 6.
particles mL1, respectively. The size distribution of VLPs in
the untreated oligotrophic sample and the 0.2-mm filtered
seawater (Table 1) revealed that there was no significant
difference in particle number or size. The concentration of
the particles in the 0.2-mm filtrate by tangential flow filtration yielded a particle recovery ranging from 44% to 78%,
while the use of ultracentrifugation resulted in the complete
recovery of particles.
Three bands were obtained (Table 2) by CsCl-density
equilibrium ultracentrifugation of the 0.2-mm filtrate; the
middle band with the highest protein/nucleic acid ratio
and a corresponding particle diameter size range from
118 to 128 nm was used for all of the following assays
(Chiura, 2002).
Gene transfer experiments
Exposing the amino acid-deficient E. coli AB1157 cells to the
harvested particle fraction resulted in the restoration of the
genetic deficiencies for all MOIs (Table 3). In the assays with
MOIs of 5.1, 20 and 200, we obtained a restoration of all
four amino acid synthesis deficiencies. The lowest transfer
frequency of 1.39 107 was noted for MOI = 5.1, and the
highest frequency was 1.94 105 for MOI = 200. Exposure
of E. coli AB1157 at MOIs of 0.12 and 1.0 yielded no
comparable results. The reversion of either Leu, Pro, His or
Arg synthesis in E. coli AB1157 was observed for all MOIs
(Table 3). The highest gene transfer frequency for Leu and
His was 1.04 1.62 102 and 9.83 3.88 103, respec2011 Federation of European Microbiological Societies
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0
1.94 105 (12)
7.34 3.04 104 (443)
7.15 0.28 105 (43)
7.34 4.13 104 (443)
5.83 1.08 104 (352)
0
0
n = 6. UV, irradiation of particles with UV; aut., autoclaved VLPs; untr., untreated VLPs; UC supernat., ultracentrifugation supernatant. Numbers in parentheses represent the numbers of CFU obtained for
selected markers.
299
0.891 0.615
9.2 6.4
20.53
17.9
0
Lower
Volume (mL)
Particles SD 1012
Particle proportion (%)
Nucleic acids (mg)
Protein/nucleic acids
0
Middle
0
Upper
Control
Bands
1.46 10 (1)
1.04 1.62 102 (714)
7.42 1.12 103 (509)
9.83 3.88 103 (675)
6.03 0.92 103 (414)
0
Table 2. Characterization of bands obtained by CsCl-density equilibrium ultracentrifugation of VLPs derived from concentrate of 0.2-mm
seawater filtrate by tangential flow filtration
1.39 10 (2)
9.04 2.85 103 (13)
8.34 2.38 103 (12)
9.04 3.07 103 (13)
9.73 1.90 103 (14)
0
n, number of subsamples.
0
3.33 3.22 105 (9)
2.22 2.38 105 (6)
7.41 1.76 105 (2)
1.48 0.52 105 (4)
0
5.0 2.7
107.7 24.6
0
4.41 3.04 106 (10)
5.73 2.82 106 (13)
3.53 0.92 106 (9)
3.09 0.88 106 (7)
0
30.0 11.9
72.9 10.8
All amino acids reverted
Leu reverted
Pro reverted
His reverted
Arg reverted
VLPs1UV AB1157
VLPs aut.1AB1157
VLPs aut./untr. AB1157
Davis buffer1AB1157 or UC supernat.1AB1157
65.0 16.6
46.9 8.9
Experiment
6.8 7.6
111.8 25.4
200
36.0 12.5
71.3 11.0
5
57.2 17.4
49.8 8.7
20
4 100
7
60–100
5.1
Seawater (n = 16)
Proportion SD (%)
Size SD (nm)
0.2-mm filtrate (n = 15)
Proportion SD (%)
Size SD (nm)
30–60
1.0
Sample
0.12
Size classes (nm)
MOIs
Table 1. Proportion (%) and diameter size distribution of VLPs in
nanometres (nm) in seawater and 0.2-mm filtrate of seawater
Table 3. Gene transfer frequency SD from VLPs derived from 0.2 mm filtered seawater concentrate to Escherichia coli AB1157, designated as ‘reverted’ at five different MOIs and the corresponding
control experiments
H.X. Chiura et al.
FEMS Microbiol Ecol 76 (2011) 576–591
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Evidence for particle-induced horizontal gene transfer
Table 4. Survival of Escherichia coli AB1157, expressed as % EOP at various MOIs
MOIs
0.1
1
5
20
200
UV-treated VLPs
Native VLPs
Davis buffer
VLP autoclaved
UC Supernatant
EOP SD (%)
100.0 9.8
76.5 9.8
64.9 1.7
35.3 1.4
17.8 0.5
100.0 9.9
64.8 9.8
48.2 0.2
20.8 0.6
15.2 0.0
100.0 9.1
100.0 9.1
98.7 0.5
99.4 2.7
98.2 3.1
98.3 0.5
98.3 6.3
98.4 4.4
94.0 6.9
94.0 6.9
98.3 0.3
95.8 6.1
93.5 1.0
86.3 0.0
62.6 5.8
n = 6. UV, irradiation of particles with UV; native VLPs, particles sampled from the water column (see Materials and methods); UC, ultracentrifugation
supernatant; and 100%, number of CFUs formed with Davis buffer.
tively, and both were at MOI = 20. However, the transfer
frequency for the synthesis of Pro and Arg was always the
highest at MOI = 5.1, with 8.34 2.38 103 and 9.73 1.90 103, respectively. The overall trend of the data from
Table 3 indicates an increase in the transfer frequency of
selected markers with increasing MOI and maximum values
at MOIs of 5.1 and 20. At MOI = 200, the frequencies again
decreased. However, none of the markers was preferentially
transferred, indicating that the DNA transfer was performed
by generalized transduction.
To define the most favourable conditions for the gene
transfer, with respect to the number of particles to which a
recipient cell was exposed in our experiments, we calculated
the specific gene transfer frequencies (i.e. the ratio of the
average transfer frequency of the restored amino acids
divided by the respective MOI). The highest transfer frequency was obtained for MOI = 5.1, with a mean of
1.41 2.50 103. It should be noted that the effect of
particle exposure on the recipient cells (Table 4) indicated
an increase in cell mortality with increasing MOI. Additionally, exposing recipient cells to UV-treated particles had a
similar effect on mortality compared with the exposure to
native particles. At an MOI of 5, we still obtained an EOP
amounting to 48.2% and 64.9% for exposure to native
particles and UV-treated particles, respectively. However,
these values declined to 15.2% and 17.8% at an MOI of 200.
An increase in mortality was also observed when recipient
cells were exposed to the ultracentrifugation supernatant of
the respective final concentrate of particles. Notably, this
increase was less pronounced, and at an MOI of 200, we
recorded 62.6% CFUs. Only a marginal lethal effect was
caused by exposure to autoclaved VLPs; the EOP ranged
from 98.3% to 94.0% for MOIs of 0.1–200, respectively.
Unselected marker restoration was examined for revertants obtained at MOIs of 0.12, 1.0 and 5.1, while those for
MOIs of 20 and 200 were excluded. Additionally, Table 5
shows that for unselected marker restoration, the gene
transfer frequencies were unexpectedly high, especially at
an MOI of 5.1. Although the unselected marker transfer was
low for the selected marker (His) at an MOI of 0.12 and
absent at an MOI of 1.0, we obtained 84.6–92.3% of restored
amino acid synthesis at an MOI of 5.1. For the other three
FEMS Microbiol Ecol 76 (2011) 576–591
markers, unselected marker restoration accounted for
69.2–92.9% at this same MOI. At the lower MOIs, unselected marker restoration remained low, ranging from
10.0% to 42.9% at an MOI of 0.12 and between 0.00% and
33.3% at an MOI of 1.0. This implies that the presence of
five particles per cell results in unselected marker restoration
that is three times greater than one potentially infective
particle per cell.
To study and quantify the growth behaviour of the
obtained revertants in a liquid medium and to test whether
particle formation would take place, two revertants were
selected, and their growth behaviours were compared with
that of the parental strain.
Growth behaviour of revertants and particle
production
Two revertants obtained in minimal medium at an MOI of
5.1 in this study (arbitrarily named Calvi-E-trans-F1a and
Calvi-E-trans-F1b) were examined, and their growth characteristics were compared with that of the parental E. coli
AB1157 cells. Because both strains displayed similar growth
characteristics, the growth curves of the parental strain
E. coli AB1157 and of one transductant (Calvi-E-trans-F1a)
are presented in Fig. 1a, showing that there were only minor
differences in the profiles of these strains. AB1157 reached
the stationary phase after 50 h, while the revertant strain
required nearly 80–90 h, and its density remained slightly
above that of AB1157 during the 370 h of observation. The
appearance of particles, although at very low quantities, was
observed after only 12 h in the liquid medium, and the
maximum density was observed between 50 and 60 h when
the transcolony strain entered the stationary phase; the
maximum density reached 0.8 109 VLP mL1. Once the
stationary phase of the host cell was well established (after
130–140 h), the particles range remained low and varied
between 6.8 108 and 8.3 108 VLP mL1 (Fig. 1b). No
particle formation was observed in the liquid medium for
the original AB1157 strain (data not shown).
Inspection of revertant cells by electron microscopy
revealed that the number of infected cells, indicated by the
number of particle-bearing cells, ranged from 4% to 23%
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582
H.X. Chiura et al.
Table 5. Unselected marker restoration (%) as the result of VLP-mediated gene transfer (selected marker) to Escherichia coli AB1157 at various MOI
Selected marker
MOI: 0.12, total colonies formed: 39
Unselected marker
Leu reverted (10)
Leu1
Pro1
His1
Arg1
–
10 (1)
20 (2)
10 (1)
Pro reverted (13)
7.7 (1)
–
15.4 (2)
7.7 (1)
His reverted (9)
Arg reverted (7)
11.1 (1)
11.1 (1)
–
11.1 (1)
14.3 (1)
42.9 (3)
28.6 (2)
–
MOI: 1.0, total colonies formed: 22
Leu1
Pro1
His1
Arg1
Leu reverted (9)
Pro reverted (6)
His reverted (3)
Arg reverted (4)
–
22.2 (2)
33.3 (3)
22.2 (2)
16.7 (1)
–
33.3 (2)
16.7 (1)
0 (0)
0 (0)
–
0 (0)
0 (0)
25.0 (1)
25.0 (1)
–
MOI: 5.1, total colonies formed: 66
Leu1
Pro1
His1
Arg1
Leu reverted (13)
Pro reverted (12)
His reverted (13)
Arg reverted (28)
–
84.6 (11)
84.6 (11)
69.2 (9)
75.0 (9)
–
91.7 (11)
75.0 (9)
84.6 (11)
92.3 (12)
–
84.6 (11)
78.6 (22)
85.7 (24)
92.9 (26)
–
1, The reverted amino acid synthesis for the unselected marker. Numbers in parentheses represent the number of CFUs obtained for a selected marker
or resulting from an unselected marker transfer.
during the stationary phase (Fig. 1c), with the average burst
size per cell ranging from one to four particles (Fig. 1d).
Particle formation was observed both in the previously
mentioned strain and in all revertant strains that were
retained for later use.
CsCl-density equilibration centrifugation of the harvested
particles yielded five bands; of these five, we chose the
middle band for the second transduction experiment. The
size range of the corresponding particles was 124.91 11.5 nm (n = 69), which was comparable to the range of the
particles that were selected from the 0.2-mm seawater filtrate
for the production of the first revertants.
Molecular biological and ultrastructural
characterization of VLPs produced by Calvi-Etrans-F1a
Several Calvi-E-trans-F1a cultures were separately incubated
for 165 h in Minimal Medium (MM) (3.8 L) to allow the
harvest of both the particles produced for further analysis
and bacterial cells for TEM observation. Alternative treatment of revertant cells and VLPs with RNase and DNase,
followed by BSFGE demonstrated that the particles contained DNA (Fig. 2).
Figure 3 demonstrates that revertant E. coli cells (lane T
and lanes 6–12) are characterized by two distinct DNA
fractions: the E. coli genome and a DNA molecule of
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360 kbp. However, E. coli AB1157 (before and at the
beginning of the infection; lanes E1, E2, and 1 and 2) is
characterized only by the genomic DNA fraction. The
isolated VLPs contained the 360-kbp band. Thus, even
though the particles observed varied in size between 100
and 140 nm, they contained DNA with a generally uniform
molecular mass of 370 kbp.
TEM inspection of the E. coli AB1157 revertant cells
during growth over 165 h provided morphological evidence
of particle production by the revertant Calvi-E-trans-F1a; it
also revealed previously unseen structures in E. coli revertant
cells before and during particle production. Figure 4a and b
demonstrate that particle production is achieved by budding, which accounts for the observed low mortality of the
revertants during growth. This phenomenon is in agreement
with the fact that during the 370-h incubation of Calvi-Etrans-F1, the stationary phase was maintained without a
noticeable decrease in cell density, indicating that mortality
of the cultured cells was low. Also, the addition of the particles
from the concentrated sample of the seawater onto lawns of
AB1157 cells did not result in plaque formation compared
with the addition of coliphage T4, which produced clear
plaques, thus indicating virulence (data not shown).
TEM observation of various stages of revertant cells
during particle formation revealed the appearance of electron-dense bodies (EDBs) in the cells (Figs 4b and 5). These
bodies were often in close vicinity to a connected electronFEMS Microbiol Ecol 76 (2011) 576–591
583
Evidence for particle-induced horizontal gene transfer
Fig. 1. (a) Growth profiles of Calvi-E-trans-F1 and
the parental strain Escherichia coli AB1157. (b)
Growth profiles of Calvi-E-trans-F1a and
related particle appearance. (c) Time course of
particle-bearing cells in the host population (%) in
Calvi-E-trans-F1a. (d) Time course of the mean
number of particles observed in a cell. SD: vertical
bars. All assays were run at 30 1C over 370 h.
dense network. Figure 5a shows EDBs in the recipient cells at
different degrees of electron density, Fig. 5b and c indicate
situations where the EDBs are in close contact with the inner
layer of the cell membrane and Fig. 5d shows a doublemembraned budding structure with an EDB and displays
the features of a membrane vesicle. To argue that these EDBs
are precursors of the observed budding particles, a basic
assumption is that these bodies contain DNA. Incubation of
gold-conjugated DNA-antibodies indicated discrete clusters
of DNA-antibody-binding sites in the EDBs in the revertant
cells (Fig. 6a–c). Revertants without EDBs were used as
controls (Fig. 6d) and showed no or sparse DNA-antibody
binding. Figures 5 and 6c provide evidence that the EDBs
that appear in the recipient cells vary not only in shape but
also in composition. While gold-conjugated DNA-antibody
complexes are densely clustered on and around the dark
structures within the cells (as seen in Fig. 6a and b), Fig. 6c
shows a concentration of gold grains aligned along half of
the circumference of the electron-dense material within the
cell. Thus, the data suggest that EDBs appearing in the
recipient cells vary in shape and in the composition of their
constituents.
FEMS Microbiol Ecol 76 (2011) 576–591
Reinfection experiments
For this experiment, harvested and purified particles derived
from Calvi-E-trans-F1a were added to the recipient E. coli
strain at an MOI of 5.5, which was assumed to yield the most
efficient gene transfer with respect to both the selected
marker and unselected marker restoration, as mentioned
above. Assessing the effect of particle exposure on recipient
cells at an MOI of 5.5 again showed a particle-induced
mortality of 33.8%, which is comparable to the mortality
obtained via UV-irradiated particles (32.2%). The results of
the second gene transfer experiments are presented in Table 6.
The generated revertants were arbitrarily named Calvi-E-transF2. Using a 101 dilution, revertants of Leu1 (42 colonies),
Pro1 (42 colonies), His1 (31 colonies) and Arg1 (46 colonies)
were generated. The highest gene transfer frequency was
obtained for the Leu marker (12.3 18.10 106), which was
two to three times higher than for the other markers. Revertants
where the synthesis of all amino acids was restored were
generated on MM plates at a frequency of 2.0 1.3 106.
The gene transfer frequency was reduced by three orders of
magnitude compared with the results obtained with the
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584
particles from the Calvi-seawater concentrate at nearly the same
MOI. However, the unselected marker restoration test again
Fig. 2. BSFGE of excised and with RNase- and DNase-treated gel bands
corresponding to Calvi-E-trans-F1 and VLPs. 1, Calvi-E-trans-F1 band
treated with RNase; 2, Calvi-E-trans-F1 band treated with DNase; 3, VLP
band untreated; 4, VLP band treated with RNase; 5, VLP band treated
with DNase. Size marker: Lambda ladder (M).
H.X. Chiura et al.
demonstrated that there is a strong linkage between selected
and unselected markers (Table 7). The highest unselected
marker transfer was observed for the His revertants, with an
unselected marker transfer of 96.8% for Pro; all other restorations of amino acid synthesis ranged from 67.4% to 90.5%.
Isolated Calvi-E-trans-F2 colonies and parental E. coli
AB1157 were cultured in MM medium supplemented with
the required amino acids at 30 1C for 167 h. We found that
all of the revertants acquired the ability to produce particles,
and the incubation profiles of the revertants were comparable to those of the parental strain. As a typical example, the
growth profile of the Leu1 revertants is presented in Fig. 7a.
Leu1 Calvi-E-trans-F2 revertants showed a growth profile
comparable to that of the parental E. coli AB1157 strain.
Both the revertant and the parental strains reached values of
2.9 109 cells mL1 in the stationary phase, and free particles in the MM medium reached 2.7 107 particles mL1, a
value that is much lower than for the particles produced by
the F1 particle generation (Fig. 7b). The number of infected cells over 167 h showed a different time-course profile
than that of Calvi-E-trans-F1a, with several values o 5% for
particle-bearing cells (Fig. 7c). The number of visible
particles per cell again ranged between one and five, but the
mean decreased to 2.6 particles per cell (Fig. 7d). Purified
particles from Leu1 revertants (Pt2) were found to be of
similar mean size and mode compared with the Pt1 particles
of the F1 generation (Table 8). However, the most frequently
occurring particle diameter of the Pt2 particles (128 nm) was
nearly twice as high as for Pt1 at 24.2%. Furthermore, the
range values indicate a more constrained diameter distribution, favouring particle production with a lower size limit of
70 nm (instead of 36 nm as for Pt1) and an upper size
diameter of 162 nm (instead of 190 nm).
Discussion
Fig. 3. BSFGE of inoculated Escherichia coli AB1157 before infection
(E1, E2), Calvi-E-trans-F1 (T), harvested VLPs (VP) and samples taken from
1 to 165 h after infection from a Calvi-E-trans-F2 culture (1–12). Size
markers: Lambda ladder (M), l/HindIII.
Because cell mortality increases with increasing MOI and
because UV treatment of particles primarily leads to DNA
damage, but does not seem to impact the envelope composition of the particles (Table 4), we assume that ‘lysis from
without’ (Heldal & Bratbak, 1991; Proctor & Fuhrman,
1992; Weinbauer, 2004) is the main process, in which
Fig. 4. Particle production by Calvi-E-trans-F1.
(a) Negative-stained total view of bacterial cell in
the process of budding. Expected burst size of 3.
(b) Ultrathin section of bacterial cell in the
process of budding.
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FEMS Microbiol Ecol 76 (2011) 576–591
585
Evidence for particle-induced horizontal gene transfer
Fig. 5. Thin sections of Calvi-E-trans-F1,
demonstrating the appearance of EDBs. (a) EDBs
of different electron densities and sizes.
(b) Magnification of an EDB in vicinity of an
electron-dense network (EDN) within the
recipient cell. (c) EDB in contact with the inner
layer of the cell membrane. (d) Budding
structure displaying features of a
double-membraned vesicle.
Fig. 6. Epon-etched thin sections of the
Calvi-E-trans-F1. (a–c) Sections with a central
EDB and a dense cluster of gold-coated
DNA-antibodies. (d) Control: thin section without
an EDB with sparse gold-coated DNA-antibodies.
All sections belong to the same assay.
FEMS Microbiol Ecol 76 (2011) 576–591
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586
H.X. Chiura et al.
Table 6. Gene transfer frequency SD from VLPs derived from Calvi-E-trans-F1 to Escherichia coli AB1157, designated as ‘reverted’ at MOI of 5.5 and
the corresponding control experiments
All amino acids
reverted
Leu reverted
Pro reverted
His reverted
Arg reverted
Experiment
Transcolonies
2.0 1.3 106 12.3 18.1 106 8.77 8.74 106 3.16 0.65 106 12.0 1.9 106
Control
VLPs1UV AB1157
0
0
0
0
0
VLPs aut.1AB1157
VLPs aut./untr. AB1157
Davis buffer1AB1157 or UC supernat.1AB1157 0
0
0
0
0
UV, irradiation of particles with UV; aut., autoclaved VLPs; untr., untreated VLPs; UC supernat., ultracentrifugation supernatant. n = 7.
Table 7. Unselected marker restoration (%) as the result of Calvi-E-trans-F1a derived VLP to Escherichia coli AB1157 at MOI of 5.1
Total colonies formed: 161
Selected marker
Unselected marker
Leu reverted (42)
Pro reverted (42)
His reverted (31)
Arg reverted (46)
Leu1
Pro1
His1
Arg1
–
72.4 (21)
82.7 (24)
72.4 (21)
90.5 (38)
–
90.5 (38)
85.7 (36)
90.3 (28)
96.8 (30)
–
90.3 (28)
67.4 (31)
71.7 (33)
82.6 (38)
–
1, The reverted amino acid synthesis for the unselected marker. Numbers in parentheses represent the number of CFUs obtained for a selected marker
or resulting from an unselected marker transfer.
multiple infections lead to the leakage of ions from the
cytosol of the recipient cell, resulting in cell death (Dreiseikelmann, 1994; Kivelä et al., 2004). This assumption is
partially supported by the fact that autoclaved particles
induce low mortality in the recipient cells. Surface molecules
of the envelope, which may act as potential contact receptors
(e.g. proteins or lipoproteins), experience conformation
changes via heat treatment. Therefore, successful binding to
the cells after the contact phase is rather restricted. In the
actual state of analysis, we have difficulties finding an
explanation for the cell mortality resulting from exposure
to the various ultracentrifugation supernatants. The material in the supernatant ( 4 30 kDa, but o 0.2 mm) is assumed
to consist of cell lysing substances, such as holins, endolysins
or bacteriocins, which could act in complement to the
postulated lysis and enhance cell mortality. A comparable
observation was recorded by Chiura (2006) for particles
from thermophiles that were shown to contain peptidase
and glycosidase. Thus, our experiments clearly indicate that
the control of the bacterial population is not only due to
phages but also concentration-dependent nonviral particles.
In contrast, the appearance of revertants at MOIs of 5, 20
and 200, where all marker deficiencies were repaired (i.e. all
amino acid synthesis deficiencies reverted), can be taken as
an indication that multiple infections occurred. This is also
supported by the coordinated marker transfer experiments,
where the percentage of revertants with unselected markers
increased from an MOI of 0.12–5.1, as shown in Table 5. It
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should be recalled that the coordinated marker transfer data
in Table 5 are due to transducing particles from the water
column, where their donor bacteria are unknown. However,
for the second set of transduction experiments (Tables 6 and
7), the particle DNA was provided by E. coli revertants.
Therefore, the data obtained from the coordinated marker
transfer allow a number of considerations: Leu and Pro
(90.5% linkage) should have the highest percentage of
coordinated marker transfer because the distance between
the loci of the biochemical markers amounts to only 185 kbp
(Dewitt & Adelberg, 1962; Rudd, 1998), which is shorter
than the length of the particle-bearing DNA (370 kbp).
Regardless, the highest percentage of coordinated marker
transfer was recorded for His and Pro (96.8%), which have a
mutual loci distance that exceeds 1762 kbp (far greater than
the length of the transferred particle DNA fragment). Also,
we recorded a 90.3% coordinated marker transfer for His
and Arg, for which the loci distance is 2134 kbp. Such a high
marker linkage can be explained by successful multiple
particle infection, providing enough DNA matrix for mismatch repair.
Because we have no information on the relative abundance
of the described particles within the viral fraction of the water
column, one may only speculate about the potential impact of
the observed gene transfer on the bacterial population. Burst
size, which can be equated with ‘budding size’ in our study,
indicates that the particle abundance may be well below that
of marine bacteriophages. Events where 20–200 particles are
FEMS Microbiol Ecol 76 (2011) 576–591
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Evidence for particle-induced horizontal gene transfer
Fig. 7. (a) Growth profiles of Leu1
Calvi-E-trans-F2 and the parental strain
Escherichia coli AB1157. (b) Growth profiles of
Leu1 Calvi-E-trans-F2 and related particle
appearance. (c) Time course of particle-bearing
cells in the host population (%) in Leu1
Calvi-E-trans-F2. (d) Time course of the mean
number of particles observed in a cell. SD: vertical
bars. All assays were run at 30 1C over 370 h.
available for one recipient cell would imply an abundance of
2 107–2 108 particles mL1 in the water column, but
this could not be verified experimentally. Experiments with
MOIs of 20 and 200 are probably not representative because
they enhance multiple infections in the recipient cell, which
may have led to the increase in gene transfer frequencies with
increasing MOI (Table 3). It is therefore assumed that lower
MOIs may be a better reflection of the in situ situation with
episodic events, where several bacterial strains of the population that simultaneously produce budding particles lead to
higher MOIs.
In the above investigation, we reconfirmed (Chiura, 2001;
Rohwer & Thurber, 2009) that there are particles among the
VLPs harvested from seawater that are able to induce intergenic HGT to the enterobacterium E. coli. The recorded gene
transfer frequencies ranging from 102 to 106 are among the
highest data published thus far. Comparison with published
information reveals that the observed frequencies range
between 1.5 108 and 4.7 107 (Amin & Day, 1988; Saye
et al., 1990; Jiang & Paul, 1998; Hertwig et al., 1999). Only the
frequencies recorded by McDaniel et al. (2010) were higher
than those obtained in the present study.
FEMS Microbiol Ecol 76 (2011) 576–591
The high frequencies of gene transfer obtained in the
present investigation suggest that this type of particle is
highly efficient in transferring genes. All of the above
experiments indicate that we observed nonspecific, VLPinduced gene transfer. One of the most surprising features of
this study is that the results of this gene transfer are revertant
cells, which are able to produce new particles. These
particles produced by the first cell generation (F1) are again
infectious and also possess the capability to transfer genes.
The resulting second-generation (F2) cells were observed to
manifest particle production again. These particles had a
mean diameter and mode that were similar to those
produced by the F1 cells (Table 8), were again infectious
and also induced the production of an F3 revertant population. It is noteworthy that a difference in the diameter size
range was recorded between the two particle generations, Pt1
and Pt2. One possible interpretation of this observation,
along with the fact that the most frequent particle diameter
was nearly twice as abundant for Pt2 (Table 8), is a trend
towards a higher frequency of packaging of larger DNA
molecules into the particles as serial reinfection processes
increase.
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588
H.X. Chiura et al.
Table 8. Mean particle size, mode and its frequency (%) and range of the particle fraction harvested from seawater (Pt0), of the particles produced by
the revertant generation F1 (Pt1), obtained by incubation of Escherichia coli AB1157 with harvested particles from seawater (West coast of Corsica,
Calvi/STARESO) and the particles produced by the revertant generation F2 (Pt2), obtained by incubation of E. coli AB1157 with particles Pt1 at an MOI of
5.1
Particle type
Source
n
Diameter (mean SD)
Mode (frequency)
Range
Pt0
Pt1
Pt2
Seawater
Calvi-E-trans-F1
Calvi-E-trans-F2
317
1526
598
78.5 35.8
115.6 43.5
110.5 28.7
60 (13.2)
130 (12.7)
128 (24.2)
32–186
36–190
70–162
n, number of randomly sized particles by EM.
Genetic exchange plays a key role in the evolution of
prokaryotes. In the last few years, additional mechanisms
responsible for horizontal DNA transfer (in addition to the
well-known mechanisms, e.g., conjugation, transduction
and transformation) were discovered, including GTAs
(Marrs, 1974; Wall et al., 1975; Lang & Beatty, 2002; Stanton,
2007) and the transfer of DNA via membrane vesicles
(Kolling & Matthews, 1999; Yaron et al., 2000; Renelli et al.,
2004; Mashburn-Warren & Whiteley, 2006). GTAs, occurring among Alphaproteobacteria, resemble tailed phages with
heads of 30 nm diameter, short spikes and a tail varying
between 30 and 50 nm. They are smaller than any morphologically similar virus and package random fragments of
chromosomal DNA from the donor bacterium and have
incomplete copies of their own genome, ranging between 4.5
and 13.6 kbp. The small DNA fragments can be taken up by
recipient cells and recombine with chromosomal DNA.
Concerning gene transfer via formation and shedding of
membrane vesicles during the growth of Gram-negative
bacteria, it was shown that these membrane vesicles are
proficient in mediating the transfer of DNA from one strain
to another. It is also worth mentioning that the formation
and shedding of membrane vesicles in Gram-negative
bacteria is a well-known and common phenomenon. Membrane vesicles usually contain only small DNA molecules
between 3.3 and 36 kbp (Dorward et al., 1989; Yaron et al.,
2000). Notably, none of the characteristics of these additional mechanisms are comparable to the traits of the
particles in the above investigation.
The present study allows us to assume that in addition to
the previously described mechanisms responsible for horizontal DNA transfer, a thus far undescribed vector enforces
the flow of genetic information between prokaryotes. The
particle type described shows an analogy to membrane
vesicles, but their DNA content is a multiple ( 4 350 kbp),
the host range is broader and the transferred material
induces the production of new particles in the recipient cells
(i.e. serial transduction). The particles studied are larger
than any known GTA particle, tailless, are released by
budding from the transduced cell and characterized by a
double membrane (Fig. 5d). Their DNA is more than
77 larger than in RcGTA from Rhodobacter capsulatus
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(Lang & Beatty, 2000), which has the smallest DNA content,
and 26 larger than in Dd1 from Desulfovibrio desulfuricans
(Rapp & Wall, 1987) or BLP from Bartonella spp. (Barbian &
Minnick, 2000), which has the largest known DNA content.
Thus, we have evidence of a new HGT type in which we
observed the packaging of mobilizable DNA into particles
and its trafficking from donor to recipient cells. This gives
rise to a number of pertinent questions that are related to
the peculiarity of the observed particles and that concern
their potential as GTAs.
Although we have no information about the original
particle type, which was harvested from the sea and obtained
by ultracentrifugation, it is highly plausible that in analogy
to the observations from the reinfection experiments, we are
faced with a naturally occurring particle that cannot be
clearly assigned to viruses or so far described membrane
vesicles. The experimentally induced particles are larger
than the classical membrane vesicles, and the DNA, which
is larger than for all reported membrane vesicles, is associated with an unidentified moiety and forms an electrondense particle that distinguishes itself from all other particles. Furthermore, none of the investigated membrane
vesicles (Dorward et al., 1989; Beveridge, 1999; Kolling &
Matthews, 1999; Yaron et al., 2000) or viral particles
(Lengeler et al., 1999) are known to trigger new particle
production after cell infection has taken place.
All of these specific features [especially particle production and subsequent release via budding during the stationary phase, particle-related transfer of large amounts of DNA
(up to 370 kbp) to recipient cells and the potential of these
particles for serial gene transfer] are an indication that we
are confronted with a new gene transferring particle that
induces horizontal gene flow between prokaryotic cells.
Although we lack information about the as yet undetected
original particle that induced the observed gene transfer to
the first recipient cell, speculation on the evolutionary
importance of particle formation by prokaryotes should
continue. Experimental observations indicated that the
recipient cells always undergo particle formation in the
stationary phase. In aquatic environments, most bacteria
are assumed to live under conditions of a nutrient shortage
(Haller et al., 1999; Denner et al., 2002), except for the
FEMS Microbiol Ecol 76 (2011) 576–591
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Evidence for particle-induced horizontal gene transfer
periods of planktonic blooms or seasons of allochthonous
organic inputs from various sources. The starvation survival
reaction (Kjelleberg, 1993) is one possibility to bridge the
periods of limitation between two events of nutrient input.
However, each bacterial species does not have the same
capability to survive over long periods of nutrient limitations, and any mechanism leading to the conservation of
specific genetic information may be an advantage for this
specific bacterial population. One such strategy to ensure a
backup of genetic information is the transfer of bacterial
DNA to recipient cells; the possibility of perpetuating this
information is by a sustained cell-to-cell transfer.
Because transfected cells produce particles with roughly
370 kbp of DNA in the stationary phase, 13 particles (each
carrying a unique piece of the E. coli chromosome) would be
sufficient to save the entire bacterial genome. It seems
obvious that one may expect species-specific processes of
this particle formation after the stationary phase is induced,
but it is also highly probable that the overlying mechanisms
that determine the formation of these backup particles are
similar.
Acknowledgements
We thank the STARESO S.A. for providing laboratory
facilities and the requested ship service for in situ sampling.
Thanks are also due to H. Naito, D. Koketsu, H. Soma, T.
Takemura, M. Imada, Y. Suzuki and other Lab members at
ICU, as well as I. Gerstl, M. Fliesser, B. Mallinger, E. Scherzer
and R. Wegscheider from the Medical University of Vienna
for their excellent assistance during the experiments and
electron microscopic analysis. We sincerely thank R.W.
Ridge, S. Suzuki, K. Matsumoto and L. Stöger for critical
reading, advice, useful comments and suggestions on the
manuscript. This research was supported in part by the
International Joint Research Programme ‘Horizontal gene
transfer by marine virus-like particles’ Grant No. 10044199,
Grant-in-Aid for Scientific Research No. 10490012 and No.
12490009 from the Ministry of Culture, Science, Sports and
Education, Tokyo, Japan to K.K. The study was also supported by the Fonds zur Förderung wissenschaftlicher Forschung, Wien, Austria (Project No. P11936-MOB and No.
and P17246-BO3) to B.V. This research was additionally
funded in part by the Japan Society of the Promotion of
Science, Grant-in-Aid for Scientific Research No. 16310031,
and Donations to Encourage Research by Kyowa Hakko
Kogyo and S.I.C. to H.X.C.
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