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Bacterial fitness and plasmid loss: the
importance of culture conditions and plasmid
size
M. Alex Smith and Michael J. Bidochka
Abstract: Several pBluescript-derived plasmids of various sizes were constructed to study the effects of multicopy plasmid
size on bacterial fitness and plasmid loss. Transformed and untransformed bacterial clones were grown in media with or
without ampicillin. Bacterial fitness (measured by growth rate), plasmid presence or absence, and plasmid copy number were
assessed during successive subculturings. In selective media (minimal medium or Luria Broth plus ampicillin), the clone
transformed with the largest plasmid (pBluescript with a 9000-bp insert) had a significantly longer lag phase than all other
clones. In nonselective media the rate of plasmid loss during successive subculturings was greatest in the clone with the
largest insert. The clone with the largest insert displayed a lower plasmid copy number than clones with a small insert or no
insert at all. Plasmid loss in the form of segregational instability and plasmid copy number reduction in nonselective
environments are important to the understanding of the evolution of the bacteria–plasmid associations and the appreciation of
the potential for altering the genetic properties of a clone maintained or subcultured on a standard medium.
Key words: pBluescript, plasmid, stress, fitness, starvation.
Résumé : Nous avons construit quelques plasmides de diverses tailles dérivés de pBluescript en vue d’étudier les effets de la
taille d’un plasmide en plusieurs copies sur la compétence de la bactérie et la perte de plasmides. Des clones de bactéries
transformées et non-transformées ont été cultivés dans des milieux avec ou sans ampicilline. La compétence des bactéries
(évaluée par le taux de croissance), la présence ou l’absence de plasmides et le nombre de copies d’un plasmide ont été
mesurés lors de repiquages successifs. Dans les milieux sélectifs (milieux minimaux de culture ou bouillon de culture Luria
avec ampicilline), le clone transformé avec le plus gros plasmide (pBluescript plus une insertion de 9000 pb) affichait une
phase de latence significativement plus longue que tous les autres clones. Dans des milieux non-sélectifs, le taux le plus
élevé de perte plasmidique lors des repiquages successifs a été observé avec le clone portant la plus lourde séquence
d’insertion. Le clone contenant la plus lourde séquence d’insertion présentait un nombre de copies de plasmides inférieur à
celui de clones ayant une petite séquence d’insertion ou aucune insertion du tout. La perte de plasmides à la période
d’instabilité de division et la diminution du nombre de copies plasmidiques dans des environnements non-sélectifs sont des
facteurs importants pour comprendre l’évolution des associations bactérie–plasmide et pour évaluer la capacité d’altérer les
propriétés génétiques d’un clone maintenu ou sous-cultivé sur un milieu standard.
Mots clefs : pBluescript, plasmide, stress, compétence, jeune.
Introduction
Experimental studies in molecular biology with transformed
Escherichia coli assume that the recombinant plasmids are
stable. However, differing growth and storage conditions can
evoke changes in the transformed population. Environmental
conditions can influence segregational instability or plasmid
deamplification (Gerdes et al. 1986; Rohde 1995; GuerrierTakada et al. 1997). Furthermore, bacteria containing plasmids
of different sizes or copy number may have differing fitnesses
in different environments (Kues and Stahl 1989; Valenzuela
et al. 1996). Effects of plasmid maintenance on the fitness of
Received June 26, 1997. Revision received January 21,
1998. Accepted January 27, 1998.
M.A. Smith1 and M.J. Bidochka. Biology Department,
Trent University, Peterborough, ON K9J 7B8, Canada.
1
Author to whom all correspondence should be addressed
([email protected]).
Can. J. Microbiol. 44: 351–355 (1998)
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the bacterial host have been documented in several clones of
E. coli (Godwin and Slater 1979; Seo and Bailey 1985; Boe et
al. 1987; de Taxis du Poet et al. 1987; Lenski and Bouma 1987;
Caldwell et al. 1989; Modi and Adams 1991; Rhee et al. 1994)
and Bacillus subtilis (Bron et al. 1988; Leonhardt and Alonso
1991).
pBluescript (Stratagene Cloning Systems, La Jolla, Calif.)
is a commercially available multicopy plasmid used in many
experimental studies as a cloning vector. This plasmid has,
among other features, a gene encoding ampicillin resistance
and a multiple cloning site (MCS). Escherichia coli transformed with pBluescript are conventionally grown in a selective media containing ampicillin to maintain the plasmid. In
this investigation we asked (i) whether plasmid size
(pBluescript plus an insert in the MCS) affected bacterial fitness in a selective environment and (ii) whether larger plasmids are lost and (or) deamplified (copy number reduced) at a
greater rate in nonselective environments compared with
pBluescript itself.
Previous work has indicated that plasmid copy number and
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Can. J. Microbiol. Vol. 44, 1998
Table 1. Clones used in this study: designated nomenclature,
plasmids contained, and overall plasmid size.
culated with the following equation: %CFU with plasmid = CFU with
ampicillin / CFU without ampicillin × 100.
Clones used
Plasmid contained
XL1-Blue
XL1-BB
MB-700
MB-9000
None
pBluescript
pBluescript and 700 bp
pBluescript and 9000 bp
Effects of starvation on plasmid copy number
Three clones that resumed growth in starvation situations were
checked for copy number differences. Copy number was determined using two methods and three calculations. In the first method,
cultures from each of the clones of MB-9000, which resumed growth
in the second experiment (MB-9000, MB-9000-2, MB-9000-4),
pBluescript, MB-700, and MB-9000 were standardized using a
haemocytometer with respect to the number of bacteria per millilitre
of culture. Plasmids were isolated using methods described in Berghammer and Auer (1993). The 1-mL samples (equal number of bacteria, balanced to 1 mL total volume with Tris–EDTA buffer) were
centrifuged at 13 200 rpm for 5 min. A Gene-Quant II spectrophotometer (Pharmacia, Biotech) was used to quantify DNA in solution. Ten readings were taken for each sample. The second method
was the “Saran Wrap” method for DNA estimation (Sambrook et al.
1989). In this method, plasmid DNA was added to Tris–EDTA buffer
and ethidium bromide (0.2 mg/mL) and the suspensions were serially
diluted. The dilutions were compared with dilutions of a known
standard, in this case, pBluescript (1 mg/mL). A photo was taken of
these 10-mL dilution drops under shortwave UV light and the irradiance of each drop was compared with the most similar droplet from
the known standard solution. The estimations of plasmid DNA (ng/mL)
from either method were then employed to determine plasmid copy
number as follows:
Plasmid size (kb)
0.0
2.961
3.661
11.961
segregation can influence bacterial host fitness (Godwin and
Slater 1979; Moser and Campbell 1983; Bron and Luxen 1985;
Hakkaart et al. 1985; Griffiths et al. 1990). Much of that work
was conducted in rich (Luria–Bertani) media with antibiotics
present to select for the phenotypic marker of each individual
plasmid. Here we report significant impacts of plasmid size
and copy number on bacterial host fitness in starvation media
with, and without, the presence of antibiotics. Bacterial growth
rate is reduced when maintaining larger plasmids. The metabolic stress of maintaining plasmids in nonselective environments may be alleviated through plasmid segregational
instability and (or) plasmid deamplification.
Methods and materials
Plasmid constructs and clones
Genomic DNA from the fungus Metarhizium anisopliae was digested
with EcoRV and ligated with the plasmid pBluescript and transformed into E. coli XL1Blue using standard techniques (Sambrook
et al. 1989). Several clones were isolated using blue–white selection
and standard techniques. Transformants had fungal inserts of 700 and
9000 bp. The DNA was sequenced and contained no open reading
frames. Four clones (Table 1) were used in experimental studies: a
control with no plasmid (XL1Blue), only the plasmid pBluescript
(XL1-BB, 2.961 kb), pBluescript with a 700-bp insert (MB-700,
3.661 kb), and pBluescript with a 9000-bp insert (MB-9000, 11.961 kb).
Growth rates
Bacterial growth rates were monitored by measuring the turbidity in
culture at 610 nm or by monitoring the colony-forming units (CFU)
after plating culture aliquots. The optical densities at 610 nm were
recorded at 3-h intervals. At 6-h intervals, a 1:10 000 dilution was
made (in phosphate-buffered saline and plated onto Luria–Bertani
(LB; 10 g tryptone, 5 g yeast extract, 0.5 g NaCl, and 2 mL of 1 M
NaOH per litre of distilled water) agar plates containing 75 µg ampicillin/mL. Plates were incubated for approximately 18 h at 37°C
and the CFU on each plate for each clone were counted. The experiments were repeated five times.
Effects of plasmid sizes on bacteria in a starvation situation
Three clones were used: control (no insert; XL1Blue), 700-bp insert
(MB-700), and 9000-bp insert (MB-9000). All clones were grown in
minimal media (M9) broth (6.0 g Na2HPO4, 3.0 g KH2PO4, 5 g NaCl,
1.0 g NH4Cl plus 10 mL of 0.01 M CaCl2 after autoclaving.) This
broth constitutes a starvation situation as only a limited supply of
glucose and salts are available. Supplemental amino acids were not
added to further enhance the creation of a starvation environment.
Cultures were grown until (and if) an optical density (OD) of 0.6 at
610 nm was reached. This value represents a mid-log phase of
growth. Once an OD610 of 0.6 had been reached that culture was
subcultured again into M9 broth. At every third subculturing a 1:10 000
dilution was plated onto LB agar and grown at 37°C for 18 h. The
resultant bacterial colonies were replica plated onto LB agar with
ampicillin (Lederberg and Lederberg 1952). The CFU from each
plate were compared and the percentage plasmid retention was cal-
[1]
1 mole
plasmids 6.02×1023 plasmids
=
×
bacteria
single plasmid mass (ng)
1 mole
amount of plasmid DNA (ng) no. of bacteria
×
×
1 mL of solution
1 mL of culture
Statistical comparisons of bacterial growth profiles
The statistical significance of the OD of the growth curves for each
clone was determined using a Tukey multiple comparison test performed on the length of time each clone took to exceed an OD610 of
0.1. (An OD610 of 0.1 was chosen to represent the end of the lag phase
of the bacterial growth profile.) The Tukey test was also employed to
determine interclone differences in exponential growth rates.
Results
Effects of plasmid sizes on bacterial growth profiles
In M9 broth, the following lag phases and exponential growth
rates (µ) were observed for the various clones: lag phase =
12.6 h and µ = 0.21 for XL1Blue, lag phase = 12.6 h and µ =
0.20 for XL1-BB, lag phase = 12.9 h and µ = 0.22 for MB-700,
and lag phase = 19.8 h and µ = 0.2 for MB-9000. The lag
phases and the exponential growth rates for XL1Blue, XL1BB, and MB-700 were statistically similar (Fig. 1). However,
the lag phase of MB-9000 was statistically longer than the lag
phase of XL1Blue, XL1-BB, and MB-700 (F = 34.87, n = 10,
df = 3, p < 0.005) (Fig. 1). The exponential growth rate of
MB-9000 was similar to the other three clones (F = 2.00, n =
8, df = 3, p > 0.10) (Fig. 1).
In LB broth the following lag phases and expontential
growth rates were observed for the various clones: lag phase
= 1.5 h and µ = 0.31 for XL1Blue, lag phase = 2.1 h and µ =
0.35 for XL1Blue BB, lag phase = 2.5 h and µ = 0.35 for
MB-700, and lag phase = 5.1 h and µ = 0.32 MB-9000. The
lag phases and exponential growth rates of XL1Blue, XL1BB, and MB-700 were statistically similar (p < 0.005) (Fig. 1).
However, as in M9 media, the lag phase of MB-9000 was
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Smith and Bidochka
Fig. 1. Growth of four clones of E. coli as measured by the change in OD610 in M9 containing 75 µg ampicillin/mL (closed symbols) or LB
containing 75 µg ampicillin/mL (open symbols). An OD610 of 0.1 represented the end of the lag phase. d and s, XL1-Blue; r and e,
XL1-BB; j and h, MB-700; m and n, MB-9000.
Absorbance at 610 nm
2
1.5
1
0.5
0
0
3
6
9
12
15
Culture A ge (h)
significantly longer (p < 0.005) than the lag phase of the other
clones (Fig. 1). Similar between clone patterns were exhibited
in M9 and LB broth.
A 10-mL sample of each culture was diluted 10 000 fold,
and plated on LB agar every 6 h to determine the number of
CFU that corresponded with the OD readings. Patterns similar
to those of the OD readings were observed in the CFU comparison (data not shown).
Plasmid size and segregation (continuous subculture on
M9 with ampicillin)
The OD did not change for all clones (XL1Blue, XL1-BB,
MB-700, and MB-9000) after reinoculating the culture grown
in M9 onto M9. XL1Blue (with no plasmid) subcultures did
reach the an OD610 of 0.6 but only after 5 days of growth. This
was then subcultured five times during the 500-h duration of
this experiment. Two replicates of clone MB-9000 did eventually reach an OD610 of 0.6 after approximately 3 days of no
apparent growth (i.e., OD did not change). Observations of
colony morphology and colour (on LB and MacConkey’s
agar) and a microscopic examination of these MB-9000 colonies indicated that they were not extraneous contaminants. No
replicate of the clone MB-700 grew to reach an OD610 of 0.6
and therefore, no between clone statistical comparisons were
possible, as all clones did not reach an OD610 of 0.6 (when
subculturing would have occurred). For each of the two MB9000 replicates that did grow, three additional replica plate
repetitions were made. The proportion of the colonies that
contained the plasmid decreased drastically after the initial
growth to an OD610 of 0.6 (Fig. 2).
18
21
24
Plasmid size and amplification (continuous subculture
on M9 with ampicillin)
Two different methods and two different equations for copy
number determination produced different values for the plasmid copy number (Fig. 3). Amounts of plasmid DNA did not
differ because of a differential ability to isolate DNA, since the
amount of chromosomal DNA extracted from each sample
was constant as observed by agarose gel electrophoresis. Two
equations with the Gene Quant method gave similar results.
Two patterns are evident in Fig. 3. There is a negative correlation between the insert size in a multicopy plasmid such as
pBluescript and the number of plasmids per bacterium after
successive subculture on nonselective media. There were no
significant differences between the original MB-9000 clone
and the MB-9000 clones that grew following multiple subculturings (500 h) in M9 starvation media (F = 0.02, p = 0.890).
Discussion
The principal findings of this study were (i) plasmid size was
negatively correlated with plasmid copy number (MB-9000
copy number was less than either XL1-BB or MB-700, (ii)
continual subculture in minimal media did not affect copy
number in plasmids of any size tested, and (iii) any growth
observed during continual subculture in minimal media without ampicillin was due to segregational loss of the plasmid (as
demonstrated through replica-plating). It has been hypothesized that when bacterial cells are starved they retain any plasmids present but lose the enzymes required to detoxify the
antibiotic (Griffiths et al. 1990). The work reported here indi© 1998 NRC Canada
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Can. J. Microbiol. Vol. 44, 1998
Fig. 2. The proportion of colony MB-9000 that retained the
plasmid through subculturing in M9 without ampicillin. Subculture
intervals occurred over 500 h of growth in a starvation
environment with no antibiotic present. Results are means of four
replicates with standard error bars.
100
Fig. 3. Different methods of copy number determination using the
“Saran Wrap” method (Sambrook et al. 1989) (h), a calculation
utilized in Modi and Adams (1991) with data collected using Gene
Quant ( ), and an equation determined here with data collected
using Gene Quant (j).
600
u
Copy Number
% Colonies Retaining Plasmid
500
80
60
u
40
400
300
200
100
u
20
u
u
u
u
0
0
1
2
3
4
5
6
7
8
9 10 11 12 13 14 15
0
2.96
3.66
11.96
Plasmid Size (kb)
11.96
(Post 500 h
starvation)
Subculture No.
cates that when E. coli XL1-Blue cells are starved, the plasmid
is lost and the rate of loss is positively correlated with the size
of the plasmid.
Maintenance of large plasmids causes a fitness reduction in
the bacterial host in nutrient-limiting culture conditions. The
maintenance of plasmids during subculture is important to
studies in molecular biology where transformants are used and
stored under different conditions. Plasmid maintenance is also
important in describing the evolution of the bacteria–plasmid
association. The segregational stability of the bacteria–plasmid
association and potential changes in plasmid copy number
must be considered when culturing or storing bacteria containing multicopy plasmids.
We monitored bacterial fitness when E. coli was grown
under the stress of subculture in minimal media. Although
each culture had initially grown to stationary phase after a
period of approximately 24 h, subculturing resulted in a decrease in fitness as measured by bacterial growth rate. To our
knowledge, no reference of such a response to multiple subculturing in M9 minimal media by E. coli has been reported.
Kolter et al. (1993) stated that when E. coli are starved, they
can become less metabolically active, more resistant to stress,
and smaller owing to cellular divisions with no increase in cell
mass. The rapid decrease in growth rate observed when colonies were repetitively subcultured in M9 might be explained
by such a reduction in size and metabolic activity. Lenski and
Bouma (1987) concluded that any increase in the growth rate
of bacterial transformants in selective media was due to selection against plasmid carriage. Directional selection of this type
was observed in our experiments.
Multicopy plasmids (such as pBluescript) are generally lost
from cultures due to the imperfect segregation of plasmids into
daughter cells at the time of cell division (Summers and Sherratt 1984; Lenski and Bouma 1987; Modi and Adams 1991).
This is characterized as segregational instability. Structural
instability is the rearrangement of plasmid DNA or changes in
the number of plasmids per bacterium (copy number) (Bron et
al. 1988). Therefore, any negative effect of the plasmid presence on bacterial fitness could be alleviated through the loss
of plasmids at segregation (and subsequent enhanced growth
of the plasmid-free cells) or a decrease in copy number. Analysis of plasmid and chromosomal DNA extracted from the various clones ruled out multimerization of the plasmids as a
potential method by which plasmid copy number could be
reduced. Seo and Bailey (1985) and de Taxis du Poet et al.
(1987) reported that growth in a minimal media caused a sharp
increase in plasmid copy number in E. coli. This contradicts
the findings of Warnes and Stephenson (1986) who reported
that 25 generations of culturing on selective media caused a
subsequent decrease in plasmid copy number. Seo and Bailey
(1985) found that maximum specific growth rates in Luria
broth and in M9 media were reduced with increasing plasmid
content. We found that a large plasmid size resulted in a significantly longer lag phase in either nutrient-limiting or
nutrient-rich environments.
Our experiments were conducted in a brief time period (24,
48, or 500 h). This may not have been sufficient time to implicate a genetic change that would reduce the negative effects
of plasmid carriage in the bacterial chromosome. Hakkaart et
al. (1985) reported that the effects of copy number are dominant over the effects of plasmid size relative to plasmid maintenance. In this work plasmids endured segregational loss
correlated with the size of plasmid through consecutive subculturings in starvation media, while copy number remained
constant. Such a finding indicates that with the E. coli XL1Blue – pBluescript association the effects of plasmid size appear to dominate the effects of copy number.
Understanding plasmid maintenance in a starvation environment is important when considering the utility of
E. coli and other bacteria containing plasmids in molecular biology. It is also important in understanding how the
plasmid–bacteria association has endured in a natural envi© 1998 NRC Canada
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Smith and Bidochka
ronment, for clearly the environment in which bacteria are
cultured affects plasmid maintenance. Differing growth and
storage conditions can evoke and maintain changes in subsequent generations. Environmental conditions influence segregation instability or plasmid copy number and bacteria
containing plasmids of different sizes have different fitness
levels in different environments. The findings reported here
have important implications in the use of plasmids in molecular biology and in understanding the bacteria–plasmid association in conditions of starvation and nutrient absence.
Maintenance of pBluescript is significantly improved by reductions in plasmid size. High copy number plasmids reduce
the fitness of the bacterial host.
Acknowledgements
Thanks to Michael Berrill for continued discussions regarding
the nature of these experiments and to an anonymous reviewer
for comments regarding an earlier manuscript.
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