1. APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1991, p. 480-485 Vol. 57, No. 2
0099-2240/91/020480-06$02.00/0
Copyright ©) 1991, American Society for Microbiology
Biolistic Transformation of a Procaryote, Bacillus megaterium
KATHERINE B. SHARK,' FRANZINE D. SMITH,2* PETER R. HARPENDING,' JEANETTE L. RASMUSSEN,3
AND JOHN C. SANFORD2
Department of Horticultural Sciences, Cornell University, Geneva, New York 144562; School of Biological Sciences,
University of Nebraska, Lincoln, Nebraska 68588-0118'; and Department of Biological Sciences,
Plattsburgh State University College, Plattsburgh, New York 129013
Received 16 July 1990/Accepted 25 November 1990
We present a simple and rapid method for introducing exogenous DNA into a bacterium, Bacillus
megaterium, utilizing the recently developed biolistic process. A suspension of B. megaterium was spread onto
the surface of nonselective medium. Plasmid pUB110 DNA, which contains a gene that confers kanamycin
resistance, was precipitated onto tungsten particles. Using a biolistic propulsion system, the coated particles
were accelerated at high velocities into the B. megaterium recipient cells. Selection was done by use of an agar
overlay containing 50 ,Ig of kanamycin per ml. Antibiotic-resistant transformants were recovered from the
medium interface after 72 h of incubation, and the recipient strain was shown to contain the delivered plasmid
by agarose gel electrophoresis of isolated plasmid DNA. All strains of B. megaterium tested were successfully
transformed by this method, although transformation efficiency varied among strains. Physical variables of the
biolistic process and biological variables associated with the target cells were optimized, yielding >104
transformants per treated plate. This is the first report of the biolistic transformation of a procaryote.
A great deal of effort has gone into the development of tion of B. megaterium. Some biological and physical param-
transformation technology, resulting in a large body of eters affecting the efficacy of this technique are elucidated
knowledge and diverse methodologies. Some methods are with respect to this species.
very simple but are not widely applicable. Other methods
may be more widely useful but are more complex or difficult. MATERIALS AND METHODS
Many gram-negative bacteria, such as Escherichia coli, are
transformed easily by pretreatment with divalent metal ions Bacterial strains and plasmid DNA. B. megaterium 7A17
before the addition of plasmid DNA (6). Conversely, many (metA4), 7A1, 7A2, 7A16, 7A24 (leuBI), and B. subtilis 1E6
gram-positive bacteria are notoriously difficult to transform. [thr-5 trpC2(pUB110)] were provided by the Bacillus Ge-
Natural transformation of gram-positive bacteria is only netic Stock Center, Ohio State University, Columbus (Table
known to occur among Bacillus subtilis and a few species of 1). B. megaterium pv. cerealis Hosford (ATCC 35075), the
Streptococcus. With considerable effort, protoplasting, con- causal agent of white blotch of wheat, was obtained from
jugation, electroporation, and injured-cell techniques have American Type Culture Collection, Rockville, Md. (11). All
made possible the genetic manipulation of various gram- cultures were maintained on Schaeffer's sporulation medium
positive genera, including Bacillus, Lactobacillus, Listeria, at 4°C (22).
Staphylococcus, Streptococcus, Streptomyces, and coryne- Plasmid pUB110, a 4.5-kb plasmid that confers kanamycin
form bacteria (15, 16, 23, 24). However, some recalcitrant resistance, was isolated from B. subtilis 1E6. Strain 1E6 was
species remain untransformable even after exhaustive test- incubated with aeration (150 rpm) for 24 h in Luria-Bertani
ing of currently available techniques. Also, many of the (LB) broth plus 50 ,ug of kanamycin sulfate per ml at 37°C,
protocols now in use must be painstakingly adjusted for and plasmid DNA was isolated by the boiling lysis method
individual strains to achieve satisfactory results. It is appar- (17). Plasmid DNA was purified by CsCl density gradient
ent that a widely applicable, rapid method of plasmid trans- ultracentrifugation. Purified DNA was resuspended in TE
fer for procaryotic cells is still highly desirable. buffer (1 mM Tris [pH 7.8], 0.1 mM EDTA), and the con-
The genus Bacillus contains species that are both scientif- centration was determined spectrophotometrically.
ically and commercially important (18). Strains of B. mega- Preparation of cells and microprojectiles for bombardment.
terium developed for industry are used in the production of B. megaterium strains were grown in LB broth for 15 h at
vitamin B12, penicillin amidase, nucleotides, and other 35°C with aeration (250 rpm). A 50-ml sample of each culture
chemicals. Some strains of B. megaterium are also signifi- was centrifuged for 10 min at 8.93 x 102 x g at room
cant as plant pathogens. The technique currently employed temperature. The bacterial pellet was resuspended in 5 ml of
for plasmid transformation in this species is protoplasting (4, supernatant, and then the cell density was determined spec-
25, 26). Although this method has resulted in successful trophotometrically. Except where noted, 108 CFU were
plasmid transfer, preparation and regeneration of protoplasts spread on the surface of each plate, containing LB medium
is an onerous process, often resulting in low efficiency and with methionine (50 ,ug/ml), D-sorbitol (1.0 M), D-mannitol
inability to achieve reproducible results. In this report, we (0.75 M), and agar (15 g/liter). The surface of the plate was
demonstrate the use of biolistic technology (formerly used as slowly dried before bombardment to remove all visible
a method of gene transfer only in eucaryotic cells) as an water.
expeditious means for high-efficiency plasmid transforma- M5 tungsten particles (Sylvania, GTE Products Corp.,
Towanda, Pa.) were used as microprojectiles. These parti-
cles were characterized by a mean distribution size of 0.771
*
Corresponding author. pLm, with a median of 0.362 p.m and a mode in the range of
480

2. VOL. 57, 1991 BIOLISTIC TRANSFORMATION OF BACILLUS MEGATERIUM 481
TABLE 1. Bacterial strains threaded aluminum sleeve, 2 cm deep and 2 cm in internal
Strain . Source or
diameter. Into this sleeve are screwed brass rings which are
(BGSC' code) Original name reference used to hold the launch surfaces (membranes or screens) in
the direct path of the gas shock. In the flying disk treatment,
Bacillus megaterium a stainless steel stopping screen is fixed within the sleeve 1
7A1 899 10a cm below the launch site of the flying disk. All other physical
7A2 ATCC 19213 19 aspects of the system are the same or comparable to the
7A16 QM B1551 24a
7A17 PV2 24b commercially available PDS-1000 gunpowder-driven sys-
7A24 JV75 9 tem.
Particle accelerator conditions were optimized for strain
Bacillus megaterium pv. WB28 (ATCC 35075) 11 7A17 and were then used without modification on other
cerealis strains. To arrive at the optimum biolistic conditions for B.
Bacillus subtilis 1E6 BD366 10 megaterium 7A17, we tested several physical variables of
a
Bacillus Genetic Stock Center, Ohio State University, Columbus. particle acceleration with respect to transformation effi-
ciency. Those variables included helium pressure (600, 900,
1,200, and 1,500 lb/in2), distance between the helium shock
source and launch site (1.6, 1.0, and 0.55 cm), distance
0.1 to 0.2 Rm. pUB110 DNA was precipitated onto the between launch site and target cells (4.1, 6.1, and 8.1 cm),
tungsten particles as previously described (7) with minor and launch mechanism (flying disk, ruptured membrane, and
modifications. Spermidine was used at 0.1 M (10 ,ul) instead gas entrainment).
of 1.0 M (5 ,ul). Coated particles were washed after precip- Bombardment protocol and selection of transformants and
itation with 70% ethanol (to remove the free CaC12 and controls. A plate inoculated with B. megaterium was placed
spermidine) and were then resuspended in 100% ethanol to inside the bombardment chamber (with the lid removed),
facilitate rapid drying of the coated particles onto the launch- and a partial vacuum was drawn (29 in. [ca. 74 cm] of
ing surface. After a brief water bath sonication to disperse mercury). The plate was bombarded with the DNA-coated
the particles, 3 ,ul of suspended particles was loaded in the microprojectiles, the vacuum was released, and the plate
central portion of the launch surface. Hence, approximately was covered and then incubated for 2 h at 35°C. After
600 ,ug of tungsten coated with 0.8 jig of pUB110 was used to incubation, each plate was overlaid with 15 ml of LB
bombard each plate containing 108 CFU. medium containing methionine (50 jig/ml), kanamycin (50
Particle accelerator. A new helium-driven biolistic device ,ug/ml), and agar (15 g/liter) and was returned to the 35°C
(20a) was used in this study to transform B. megaterium. The incubator. Transformants were visible after 24 h, and final
new acceleration device is driven by a shock wave of transformation counts (colonies per plate) were determined
compressed helium. This shock wave is used to accelerate after 72 h of incubation.
microprojectiles by three distinct mechanisms. In the gas Controls plates were included in all experiments. DNA-
entrainment mechanism, particles are loaded onto a nylon coated tungsten was resuspended in 0.01 M spermidine
mesh which transects the path of the shock wave, and the instead of 100% ethanol and then mixed with cells. The
particles are launched by and entrained into the shock wave mixture of cells and coated tungsten was spread over an agar
directly. In the ruptured membrane mechanism, particles are surface. The control plates were exposed to vacuum in the
loaded onto an anchored aluminum foil disk which transects vacuum chamber but not to helium bombardment. In all
the path of the shock wave and are launched when the shock experiments, there were no spontaneous kanamycin trans-
wave distends and then bursts the membrane. In the flying formants or mutants.
disk mechanism, particles are loaded onto an unanchored Optimization of biological variables affecting transforma-
2-mil (ca. 51-,um) Kapton membrane which transects the tion of strain 7A17. Four biological factors associated with
path of the shock wave and are accelerated as the flying disk the target cells were tested with regard to transformation
is accelerated and are launched when the flying disk is yield. These factors, growth phase of the target cells, con-
stopped by a brass ring with a steel screen insert. In all cases centration of osmoticum in the bombardment medium, cell
a vacuum chamber surrounds the source of the helium shock density, and cell strain, had previously been shown to affect
wave, the particle launch surface, and the target cells. transformation yield in Saccharomyces cerevisiae (1). To
The helium-driven apparatus we used is merely a retrofit test growth-phase effects, we spread cells from cultures in
of our previously described (14, 20) gunpowder-driven ap- the mid-logarithmic (6.5 h at 35°C), late logarithmic (15 h),
paratus. The firing mechanism and acceleration barrel have stationary (24 h), and late stationary (40 h) phases onto
been replaced by a mechanism which can generate a con- plates and bombarded them. Each treatment was replicated
trolled helium shock. Helium is delivered from a standard five times. Osmotic effects were tested by using various
gas tank through a regulator and high-pressure hose to a concentrations of sorbitol (0.5, 0.75, 1.0, and 1.25 M) and
brass tube which replaces the acceleration barrel. At the mannitol (0.75 M) and combined concentrations of these
lower end of the brass tube is a high-pressure cavity (ap- (0.75 M sorbitol plus 0.75 M mannitol, 1.0 M plus 0.75 M,
proximately 1 cm3), which is sealed at its lower end by one and 1.25 M plus 0.75 M) as osmotic agents in the bombard-
or more layers of Kapton membrane. Each layer of Kapton ment medium. The two best osmoticum concentrations were
is 2 mils thick and will hold 300 lb/in2 of pressure. When the then selected and were tested further at five different cell
pressure overlying the membranes exceeds their combined densities. The cell densities tested were S x 106, 1 X 107, 5
strength (or when the membranes are mechanically rup- X 107, 7.5 x 107, and 1 x 108 CFU/85-mm-diameter plate
tured), the membranes burst, releasing a sharply defined from 15-h-old cultures. Five replicates per treatment were
shock wave. This shock wave propagates downward toward bombarded, and the number of transformants per treatment
the partition (stopping plate platform) of the old gunpowder was determined. Last, strain differences of B. megaterium
system. Where the Lexan stopping plate was inserted in this were tested. The optimum physical and biological conditions
platform, a new insert is placed which holds an internally of strain 7A17 were used to transform B. megaterium 7A1,

3. 482 SHARK ET AL. APPL. ENVIRON. MICROBIOL.
1 2 : 4 5 6 7 8 9 TABLE 2. Transformation of B. megaterium 7A17 at
different growth phasesa
Kb Growth phase Mean no. of No. of transformants/
SE
(h) transformants/plateb recipient cell
Expt 1
Late log (15) 436.8 92.3 5.80 x 10-6
Stationary (24) 87.7 31.1 1.16 x 10-6
Late stationary (40) 71.4 31.9 9.52 x 10-7
23- Expt 2
Mid-log (6.5) 832.4 454.9 8.32 x 10-6
9.4- Late log (15) 90.2 35.3 9.02 x 10-7
6.7- " 7.5 x 107 CFU per plate and 1 x 108 CFU per plate and 10 and 5 replicates
per treatment were used in experiments 1 and 2, respectively. Cells were
4.4- bombarded on medium containing 0.75 M sorbitol and 0.75 M mannitol.
b Each plate was bombarded with tungsten coated with 0.8 jLg of pUBllO
DNA.
2.3-
2.0-
more) than cells from stationary cultures (24 and 40 h) (Table
2). Cells from mid-log-phase (6.5-h) cultures yielded signifi-
cantly (P = 0.18) more transformants than cells from late-
log-phase (15-h) cultures.
FIG. 1. Agarose gel electrophoresis of plasmid DNA isolated Increasing the molar concentration of sorbitol in the
from B. megaterium transformants. Plasmid DNA was isolated from bombardment medium from 0 to 1.5 M increased the trans-
six Kmr transformants of strain 7A17 (lanes 4 to 9), and pUB110 was formation efficiency of B. megaterium 7A17 sevenfold (Fig.
isolated from its host, B. subtilis 1E6 (lane 3), restricted with BamHI 2). Bombardment medium with a saturated concentration of
for 1 h, subjected to electrophoresis, and stained with ethidium mannitol (0.75 M) yielded more transformants than the same
bromide. A Hindlll markers (lane 1) and undigested pUB110 from a concentration of sorbitol (Table 3). Increasing the overall
CsCl preparation (lane 2) were also included. concentration of osmoticum by combining 0.75 M mannitol
with 1 M sorbitol gave a significantly (P = 0.25) higher
transformation rate than medium containing the same sorbi-
7A2, 7A16, 7A17, and 7A24 and B. megaterium pv. cerealis tol concentration alone. The greatest number of transfor-
with plasmid pUB110. Each treatment was replicated five mants per plate was produced on medium with total osmotic
times, and the experiment was repeated five times. concentrations greater than a combined concentration of 1.5
Confirmation of transformation. Ten putative transfor- M (i.e., 1 M sorbitol plus 0.75 M mannitol).
mants were tested for authenticity by Gram stain reaction The number of target cells per plate also affected the
and isolation and visualization of plasmid DNA on ethidium transformation rate. The optimum cell density tested for
bromide-stained agarose gels. Plasmid DNA was isolated
from overnight cultures of putative transformants by a
mini-boiling preparation method (17), a 1-h digestion with
BamHI (Promega Biotec, Madison, Wis.) following the 100
manufacturer's directions, and agarose gel electrophoresis
(0.8% agarose, 90 V, 4 h). 80
I-
a.
RESULTS
Transformation of B. megaterium 7A17 was verified by z 60
growth in the presence of kanamycin, a positive Gram stain
reaction, and plasmid isolation from putative transformants 2
followed by sizing of plasmid DNA by agarose gel electro- 0
LL 40
phoresis (Fig. 1). BamHI-restricted plasmid DNA from C/3
z
putative transformants was identical in size to digested 20
pUB110 (4.5 kb), while nontransformed cells of the recipient
strain lacked any equivalent plasmid.
The efficiency of transformation of B. megaterium by the O '-
biolistic process is affected by numerous biological factors as 0.0 0.5 1.0 1.5
well as by physical factors associated with the acceleration
process. The growth phase of the target cells, cell density, SORBITOL (M)
and concentration of osmotic agent in the bombardment FIG. 2. Effect of sorbitol concentration in the bombardment
medium were all found to be important biological factors and medium on transformant yield in B. megaterium 7A17. A total of 108
drastically affected transformation efficiency. When equal CFU per plate were bombarded with tungsten particles coated with
numbers of cells from different growth phases were bom- 0.8 ,ug of pUB110. Cells were bombarded by the flying disk launch
barded with an equal amount of DNA-coated particles, cells mechanism at optimum pressure and distances. Transformants were
from late-logarithmic cultures of strain 7A17 yielded signif- selected with a 10-ml LB agar overlay containing 50 ±g of kanamy-
icantly (P = 0.05) more transformants (four to five times cin per ml.

4. VOL. 57, 1991 BIOLISTIC TRANSFORMATION OF BACILLUS MEGATERIUM 483
TABLE 3. Effect of osmoticum concentration on transformation of B. megaterium 7A17'
Mean no. of No. of transformants/
transformants/plateS recipient cell
Sorbitol 0.75 235C 154.1 3.13 x 10-7
Sorbitol 1.25 328C 225.8 4.37 x 10-7
Mannitol 0.75 584C 204 7.79 x 10-7
Sorbitol + mannitol 0.75 + 0.75 1,085c.d 847 1.44 x 10-5
Sorbitol + mannitol 1.00 + 0.75 5,693d 1,852.4 7.59 x 10-5
Sorbitol + mannitol 1.25 + 0.75 3,584 .d 1,943.5 4.77 x 10-5
a 7.5 x 107 CFU per plate of a 15-h-old culture were spread on bombardment medium containing various concentrations of osmotic agents. Five replicates were
used per treatment.
b Each plate was bombarded with tungsten coated with 0.8 pLg
of pUBllO DNA.
c.d Means not followed by a common letter are significantly different from one another.
strain 7A17 was 108 CFU/85-mm-diameter plate (Table 4). and 4.1 cm between microprojectile launch site and target
However, there appeared to be some interaction between cells. Use of these optimum conditions often resulted in such
osmoticum concentration and cell density. At 5 x 107 CFU a large number of transformants (>20,000) that they some-
per plate, there were more transformants on medium con- times appeared as a lawn of confluent colonies too numerous
taining 1.0 M sorbitol plus 0.75 M mannitol, but at the higher to count except when counted with a binocular dissecting
cell densities tested (7.5 x 107 and 1.0 x 108 CFU per plate), scope at 48 to 65 h of incubation (Fig. 3).
there were more transformants on 1.25 M sorbitol plus 0.75
M mannitol. DISCUSSION
In addition to growth phase, cell density, and osmoticum
concentration, strain differences were observed to affect Since its inception in the early 1980s (14, 20, 21), biolistic
transformation efficiency. The relative ranking among the technology has been successfully used to genetically trans-
strains remained constant in five experiments, and the re- form a wide variety of eucaryotic cells and their organelles
sults of one experiment are presented in Table 5. Note that and has gained recognition and credibility as an effective
three of six strains had higher transformation rates than methodology. MonoFpts including corn (12), rice, and wheat
strain 7A17, the principal strain used in most studies. How- (27) as well as dicots such as tobacco (13) and soybean (5, 27)
ever, only strain 7A1 yielded significantly (P = 0.075) more have all been transformed by DNA introduction through
transformants per plate than the other five strains. There was particle bombardment. Cultured animal cells have been
no statistically significant difference in transformant yield transformed utilizing this technology (28). Also, nuclear
among the remaining five strains. transformation ; f eucaryotic microorganisms including
Pressures and distances were empirically optimized for yeasts and filamentous fungi (1) has been shown. Biolistic
the three launch mechanisms. The resulting optimized treat- technology has been unique in its capability to directly and
ments (flying disk [900 lb/in2, 4.1 cm], gas entrainment [1,200 reproducibly transform both mitochondria (8) and chloro-
lb/in2, 6.1 cm], and ruptured membrane [900 lb/in2, 6.1 cm]) plasts (2, 3).
were then compared. The flying disk mechanism yielded 20 The previous success of biolistic technology in transform-
and 40 times more transformants per plate than gas entrain- ing a diversity of cell types, together with the need for a
ment and ruptured membrane, respectively. Pressure (900, rapid, easily adaptable method for the transformation of
1,200, and 1,500 lb/in2), distance between helium source and procaryotic cells, led to our desire to determine whether
macroprojectile (1.6, 1.0, and 0.55 cm), and distance be- bacteria could be transformed via particle bombardment. It
tween microprojectile launch site and target cells (4.1, 6.1, was not clear that the use of high-velocity microprojectiles
and 8.1 cm) were varied in different combinations to further would be effective in transformation of procaryotic cells.
optimize the flying disk configuration. The flying disk treat- The procaryotic cells provided a markedly smaller target
ment that gave the greatest yield Qf transformants was 900 than eucaryotic cells; outer cell structures and the genetic
lb/in2, 1.6 cm between helium source and macroprojectile,
TABLE 5. Transformation of six strains of B. megateriuma
TABLE 4. Effect of cell density and osmoticum on
No. of transformants/
transformation of B. megaterium 7A17a Strain Mean no. of SE
S
transformants/plate recipient cell
Mean no. of transformants/plateb 526.1 1.56 10-5
Cell density/plate 7A1 1,568.2 x
Osmoticum A Osmoticum B 7A2 4.2 1.6 4.20 x 10-8
7A16 0.8 0.8 8.00 x 10-9
5x106 0 0 7A17 36.8 9.3 3.68 x 10-8
1 X 107 0.2 0.2 7A24 217.2 114.7 2.17 x 10-6
5 x 107 251.6 4.6 pv. cerealis 253.4 160.3 2.53 x 10-6
7.5 x 107 261.2 >20,000
x 108 1,009.8 >20,000 a Cells from 15-h-old cultures were bombarded on medium containing 1.0 M
sorbitol plus 0.75 M mannitol. Rates were generally low in this experiment, as
a
Cells from a 15-h-old culture were bombarded on medium containing reflected by only 37 colonies for 7A17, which often gave >104 colonies per
either 1.0 M sorbitol plus 0.75 M mannitol (A) or 1.25 M sorbitol plus 0.75 M plate. However, relative ranking of these strains was consistent over five
mannitol (B). experiments.
b Each plate was bombarded with tungsten coated with 0.8 Fig of pUB110 b Each plate was bombarded with tungsten coated with 0.8 ,ug of pUBllO
DNA. DNA.

5. 484 SHARK ET AL. APPL. ENVIRON. MICROBIOL.
associated with DNA uptake and it was previously recog-
nized as a hard-to-transform species needing improved
transformation methodology (25). Recently, an efficient pro-
toplasting transformation method for B. megaterium has
been published (26), although this method is relatively te-
dious and time-consuming compared with the biolistic
method. In our experiments, transformation of B. megate-
rium was initially accomplished by using a commercially
available gunpowder-driven device (PDS-1000; Dupont).
The rates were, however, too low to be workable (less than
one colony per plate). The improved helium-driven biolistic
device described here and elsewhere (20a) was found to be
dramatically more effective than the PDS-1000 unit, so all
the work shown in this report employed that device.
B. megaterium 7A17 was easily transformed with plasmid
DNA via the biolistic process. Under optimum biological
and physical conditions, greater than 1 x 104 transformants
per plate were often produced, equivalent to 1 x 10-4
transformants per recipient cell or 8 x 103 transformants per
,ug of DNA. For B. megaterium 7A17, an osmoticum con-
centration of >1.5 M was optimum and cells in logarithmic
growth were more efficiently transformed than cells of
stationary cultures. As in fungal systems, osmoticum in the
bombardment medium affects transformation rates (1). Un-
like yeasts, B. megaterium cells in the log phase are more
efficiently transformed than stationary cells. Although all B.
megaterium strains tested were successfully transformed,
transformation rates varied between some strains. For max-
imum numbers of transformants, biological factors such as
growth phase, osmoticum concentration, and cell density
should be optimized for each species. After optimum condi-
tions are determined, cell density or DNA load can be
reduced to decrease the number of transformants per plate
so that colonies are well separated and easily quantified.
High-efficiency biolistic transformation of a gram-positive
bacterium was described here. Similar results have been
found for a gram-negative bacterium (E. coli) (23a). These
results and the relative ease with which this technology can
be employed suggest a potential for use of the biolistic
process in the transformation of procaryotic cells.
FIG. 3. Typical shotgun pattern of B. megaterium 7A17 (A) and ACKNOWLEDGMENTS
7A1 (B) transformants 72 h after bombardment.
We thank R. Marrero, S. Zahler, and P. Van der Horn for
valuable information at the inception of this study.
This work was supported by a grant from DuPont Co. F.S. was
supported by Public Health Service grant ROI-GM 41426-01 from
structure of the cells are fundamentally different. For many the National Institutes of Health.
years, it was believed the biolistic process might only be
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