Microb Ecol (1984) 10:1-13

MICROBIAL ECOLOGY 9 1984 Springer-Verlag

Transfer and Stability of Drug Resistance Plasmids in Escherichia coli K12 Peter C. Gowland* and J. Howard Slater** Department of Environmental Sciences,Universityof Warwick, CoventryCV4 7AL, UK Abstract. Mating experiments between pairs of strains o f E s c h e r i c h i a coli containing either the compatible plasmids TP 120 (Inc N) and R 1 (Inc FII) or the incompatible plasmids TP125 (Inc B) and TP113 (Inc B) were undertaken in mixed continuous-flow cultures and in dialysis sacs suspended in pond water. Plasmid transfer was readily demonstrated between strains carrying compatible plasmids T P 120 and R1 in both continuous-flow culture and pond water. In mixed cultures of strains carrying plasmids TP125 and T P 113, transfer was only observed in continuous-flow culture systems. Strains o f E. coli containing aggregates o f plasmids T P I 2 0 and R1 were shown to be stable over 5 months continuous cultivation under carbonlimited conditions at a growth rate o f 0.1 hours-t in the presence of drugs which select for the maintenance of both plasmids. In the strains containing plasmid aggregates, a gene dosage effect was observed with respect to the levels o f resistance to drugs whose resistance was encoded by both plasmids. Chemostat experiments showed that no cointegrate plasmids were found from the strains o f E. coli initially containing both plasmid TP120 and plasmid R 1.

Introduction

The growth environment is an important factor in promoting significant evolutionary changes in the genetic constitution of microorganisms [12]. This is clearly demonstrated in the selection of bacteria which contain antibiotic resistance plasmids growing in response to the presence of an antibiotic challenge [1]. Conversely, the removal o f drugs from the growth environment may result in the displacement o f the resistant, plasmid-containing populations by sensitive, plasmid-free populations [2, 14]. It has been argued that "higher ordered and controlled processes," such as gene transfer and genetic rearrangements, are more significant than simple point mutations as the major source of genetic variation [13]. Evolution based on polynucleotide exchange is comparatively efficient since the participating genes have already evolved through the process of natural selection. Plasmid transfer * Present address: Department of Biochemistry,UMIST, P.O. Box 88, Manchester M60 1QD. **Present address: Departmentof Applied Biology,UWIST, P.O. Box 13, CardiffCF1 3XF, Wales,

UK.

2

P . c . Gowland and J. H. Slater

is o n e o f t h e m o s t efficient m e a n s o f t r a n s f e r r i n g p o l y n u c l e o t i d e s e q u e n c e s b e t w e e n b a c t e r i a . T h e r a t e o f t r a n s f e r o f genes, l i k e t h e b a s i c D N A m u t a t i o n rate, can be manipulated by appropriate selection pressures, one of which could b e a d e c r e a s e in t h e o r g a n i s m s ' g r o w t h r a t e a n d / o r t h e s u r v i v a l o f a p l a s m i d c a r r y i n g o r g a n i s m u n d e r n o n c h a l l e n g e c o n d i t i o n s [5, 7, 8, 10, 1 1, 15, 17, 18]; t h a t is, w h e r e t h e f u n c t i o n s c o d e d for b y t h e p l a s m i d g e n e s a r e d i s p e n s i b l e a n d d o n o t affect t h e s u r v i v a l o f t h e h o s t cell. P l a s m i d s c a n affect t h e s u r v i v a l o f t h e h o s t b a c t e r i u m . C l e a r l y , in t h e p r e s e n c e o f a n t i b i o t i c s for w h i c h t h e p l a s m i d c a r r i e s r e s i s t a n c e genes, t h e p l a s m i d c o n f e r s a d i s t i n c t a d v a n t a g e t o t h e o r g a n i s m b y a l l o w i n g s u r v i v a l w h e n its p l a s m i d minus counterparts are eliminated by the antibiotics. However, under nonchallenge conditions, resistance plasmids are unnecessary components of the b a c t e r i a l cell a n d m a y e i t h e r b e c o m e u n s t a b l e a n d f r a g m e n t o r b e c o m p l e t e l y e l i m i n a t e d f r o m t h e cell [5, 9]. I n d e e d , b a c t e r i a l cells w h i c h h a v e l o s t r e s i s t a n c e to o n e o r m o r e a n t i b i o t i c s a r e c a p a b l e o f h i g h e r m a x i m u m specific g r o w t h r a t e s t h a n t h e p a r e n t s t r a i n [5]. I t h a s b e e n s u g g e s t e d t h a t t h e loss o f e x t r a c h r o m o s o m a l D N A in a d r u g - f r e e e n v i r o n m e n t is t h e r e s u l t o f d i s c r i m i n a t i o n a g a i n s t plasmid-containing organisms since synthesis and replication of the plasmid u t i l i z e e l e m e n t a l a n d e n e r g y r e s o u r c e s w h i c h m i g h t o t h e r w i s e b e d i v e r t e d to h i g h e r b i o m a s s p r o d u c t i o n a n d a n i n c r e a s e d specific g r o w t h r a t e [5]. I f t h i s is t h e c a s e w i t h a single p l a s m i d , t h e n o n e m i g h t e x p e c t t h e p r e s e n c e o f a s e c o n d p l a s m i d in t h e s a m e cell to b e e v e n m o r e u n s t a b l e , e v e n u n d e r s e l e c t i v e c o n ditions. T h e p u r p o s e o f t h i s s t u d y w a s to i n v e s t i g a t e t h e f i ' e q u e n c y o f p l a s m i d t r a n s f e r b e t w e e n p o p u l a t i o n s o f Escherichia coli i n t h e l a b o r a t o r y a n d in n a t u r e a n d to investigate the stability of plasmid aggregates under selective conditions.

Methods Bacterial Strains a n d Plasmids a n d Growth Escherichia coli strains and plasmids used in the experiments are shown in Table 1. The E. coli strains were maintained on nutrient agar (Lab M) slopes containing the appropriate drugs each at 50 ug ml -~ except tetracycline (Tc) which was supplied at 20 ug ml -~. For the continuous-flow studies, the strains were grown in Davis and Mingioli [3] minimal medium containing 0.5% (w/ v) glucose. For mixed cultures involving E. coli KI 2 J5-3, the medium was supplemented with proline and methionine each to a concentration of 0.1 mg ml- ~. Continuous-flow fixed-bed column fermenters were packed with 4-5 mm diameter glass Ballontini beads and had an aerated working volume of approximately 100 ml (Fig. 1). All cultures were incubated at 37~

Procedure f o r R e c o m b i n a n t Selection in M i x e d Continuous-Flow Cultures The possible transfer of plasmids between 2 pairs of E. coli strains was examined. For the first pair, 17,.coli K12 (TP120) and E. coli K12 (RI), I0 ml of each of an overnight culture grown in Davis and Mingioli minimal medium [3] with the appropriate drugs were mixed and immediately inoculated into the fermenter. There was no batch growth and the fresh medium flow was initiated

Transfer and Stability of Plasmids Table 1.

E s c h e r i c h i a coli strains used in this study

Host E. coli

Plasmid

K I 2 IR713 K12.15-3 p r o - m e t K I 2 lacK12 lacK12 711 Nal R

Incompatibility group

TP120 RI TP113 TP125 None

N

FII B B

Plasmid markers Tra § Tra* Tra t Tra +

Ap R Sm R Su R Tc R Ap R Sm R Su ~ Cm R K m R Km R Sm R Su R Cm R Tc R

T r a § = ability to p r o m o t e self-transfer; lac- = does not ferment lactose; p r o = proline; m e t = methionine

Media Out Media In Sample Port

Air In

L I

.

~ Water Out

Wafer Jacket Battontini Beads

Water In

Fig. 1. Continuous-flow fixed-bed c o l u m n fermenter.

immediately at a flow rate o f 50.0 ml h o u r s - ' giving a dilution rate o f 0.5 h o u r s - L Samples (10 ml) were r e m o v e d at 0 h o u r s and 24 h o u r s and thereafter at 48 h o u r intervals. After appropriate dilution in 0.1 M phosphate buffer p H 7.0, 0.1 ml samples were spread plated onto Davis and Mingioli m i n i m a l m e d i u m [3] agar supplemented with 0,5% (w/v) glucose, the appropriate drugs each at 50 ug ml -~ except Tc at 20 gg ml -~ and proline and methionine each at 0.1 mg ml t. Viable counts were determined after 2 days at 37~ For the second mating pair, E. coli K I 2 (TP113) and E . coli K12 (TPI25), the basic procedure

4

P . C . Gowland and J. H. Slater

, , ~ . _ _ _ . _ . _ _ - ~ Syringe Needle ~_.__._.__----- Rubber Bu ncj ~51 ~-

-

Schuco CLip

-

131aLysis Tubing

,%

Grass l~d

Fig. 2. Dialysis sac system used for studying possible recombination in the natural environment.

was the same except that amino acids were omitted from the growth medium and the agar plates and a batch period of several hours elapsed before medium flow was initiated. After 192 hours, growth in the absence of antibiotics streptomycin (Sin) and kanamycin (Km) were added to the medium reservoir each at a concentration of 50 ug ml-L

Procedure for R e c o m b i n a n t Selection in the Natural E n v i r o n m e n t (Dialysis Sacs) Recombination in the natural environment was examined with mating pairs E. coli K 12 (TP 120) with E. coli K12 (R1) and E. coli K12 (TP113) with E. coli K12 (TPI25). Cultures were grown separately for 48 hours at 37~ in 1 liter of minimal medium supplemented with 0.5% (w/v) glucose and the appropriate antibiotics and amino acids. Total cell counts were determined using a Thoma counting chamber. For each mating pair, 2.5 ml of overnight cultures were centrifuged in a bench centrifuge and resuspended in 2.5 ml sterile pond water (autoclaved at 15 p.s.i, for 30 min). The resuspended cultures were introduced aseptically into sterile dialysis tubing (Fig. 2) using a sterile syringe and gently mixed. The ratio of different cells was approximately 1:1 and termed experiment A1 for the TPI20 and R1 cross and B1 for the TP113 and TP125 cross. The procedure was repeated for 250 ml of overnight cultures which were centrifuged and resuspended in 2.5 ml sterile pond water. The ratio of cells was also 1:1 but the density was 100 times greater than in the first pairs and these experiments were designated A2 for the TP120 and R1 cross and B2 for the TP113 and TP125 cross. For both series of mating, the mixtures were prepared in triplicate. The dialysis sacs were weighted and submerged in a freshwater pond on the University of Warwick campus. One bag from each mating pair was removed after 16 hours, 192 hours and 360 hours and sampled on appropriate antibiotic supplemented minimal agar medium to detect putative transconjugants. Total viable counts were determined on minimal agar medium containing no antibiotics.

Transfer and Stability of Plasmids

5

Plasmid Transfer in Membrane-Mating Experiments The formation oftransconjugants, whether as cointegrates (i.e., fused parent plasmids) or aggregates (i.e., independent parent plasmids) was demonstrated in the E. coli K12 (R1) and E. coil K12 (TP120) cross by showing transfer of the expected markers to a plasmid-minus host, namely, E. coli K 12 711 Nal R(nalidixic acid) (strain J-62 nal r of Harden & Meynell [7]) as previously described [51. For the E. coli KI2 (TP113) and E. coli (TPI25) cross putative transconjugants were checked for by the co-transfer of markers other than those used in the selection process, that is Sm (sulphanilamide), Su and Tc, by plating transconjugants onto minimal agar plates containing each antibiotic singly.

Procedure for Stability Studies of Recombinants ofE. c o l i K12 (TP120) and E. c o l i K12 (R1) in Chemostat Culture For stability studies, 10 ml o f an overnight mixed culture o f E . coli KI2 strains (TP120 and RI) was grown in batch for several hours in minimal medium containing all the appropriate drugs to select for the presence of the 2 plasmids. Proline and methionine were each added to a concentration of 0.1 mg m1-1 and glucose to a growth-limiting concentration of 0.2 g carbon liter-t. Medium flow was initiated to give a dilution rate of 0.1 hours-1 and samples were taken at weekly intervals and plated onto minimal plates containing all the antiobiotics. The culture vessels had a working volume of I liter and were aerated at the rate of 1 volume of culture per rain. Viable counts were made after 48 hours at 37~

Organism Screening for Plasmids Organisms in the culture effluent were screened for the presence of plasmids by a modification of the method of Eckhardt [4]. Culture effluent (1.5 ml) was pipetted into Eppendorf tubes and centrifuged for 5 min in a Beckman microfuge B. The supernatant was discarded and the pellet dried, resuspended in 15 ~1 lysozyme mixture [4] and left at room temperature for 5 min. A volume of this suspension (15/d) was put into a slot of a previously prepared 0.7% (w/v) horizontal agarose slab gel made up in Tris-borate electrophoresis buffer (89 m M Trizma base, 2.5 mM disodium EDTA, and 89 m M boric acid), and overlayed with enough o f the SDS mixture for gram-negative bacteria [4] to fill the well. No mixing was required. Electrophoresis was performed on a horizontal gel apparatus at 60 mA for 75 min. Following electrophoresis, the gel was stained in 0.4 ug ml -~ ethidium bromide for 10 min and photographed under short-wave UV light using Polaroid type 665 film, 2 UV filters, and an orange filter.

Determination o f Minimal Inhibitory Concentration (MIC) Dilution series o f the appropriate antibiotics were prepared in triplicate in minimal medium supplemented with 0.5% (w/v) glucose and 0.1 mg ml-1 each of proline and methionine in 10 ml quantities in sterile test tubes. The tubes were inoculated with 0.1 ml of an overnight culture grown on minimal medium in the absence of antibiotics. The tubes were incubated for 24 hours or 48 hours at 37~ Following incubation, the tubes were vortex-mixed and examined for growth. The MIC (#g ml 1) was recorded as the lowest concentration of antibiotic that prevented growth of the organism.

P. C. Gowland and J. H. Slater

6 10'o

109

708

107

lo6 ;,,.-

~I0 S "6 elo ~.

1@

102

!0~

--

[]

J

0

I

2~,

]

I

~8

72

}

%

I

Fig. 3, Transfer of plasmids between E. coli K12 (R1) and E. coli K12 (TP120). O, total viable cells; [], E. coli resistant to Ap, Sm, Su, Tc, Cm, Kin.

120

T~meh)

Results

Selection of Recombinants in Continuous-Flow Culture Transfer o f plasmids between E. coli K12 (RI) and E. coli K12 ( T P I 2 0 ) was e x a m i n e d in the absence o f any selection pressure u n d e r continuous-flow culture conditions in a fixed-bed c o l u m n fermenter at a flow rate o f 50 ml hours -1 which a p p r o x i m a t e d to a dilution rate o f 0.5 h o u r s - L Figure 3 shows that r e c o m b i n a t i o n took place readily under these conditions, increasing from 0% at time 0 to 100% after 72 hours. Experiments at lower flow rates, and in the presence o f Ap (ampicillin), Sm, and Su, also showed r e c o m b i n a t i o n between E. coli K12 (R1) and E. coli K I 2 (TP120) occurred readily. T w o isolates, X and Y, were retained for further study and e x a m i n e d for co-transfer o f all the expected markers o f both T P 120 and R 1 into a Nal R recipient strain o f E. coil K I 2 . When selected on Nal and Sm only, all the recipients expressed the R1

Transfer and Stability of Plasmids

7

Table 2. Transfer ofplasmids from isolate X into the nalidixic acid resistant strain of Escherichia coli K 12 711 NalR Original selection pressure

Percentage growth of transconjugants

Selection for marker transfer

Nal and Sm

Nal, Cm + Tc

Ap Ap Ap Ap Ap Ap

Sm Sm Sm Sm Sm Sm

Su Su Su Su Su Su

Tc Cm Tc Cm Tc Cm Tc Cm

0.0 100.0 0.0 100.0 100.0 100.0

Nal = nalidixic acid; Ap = ampicillin; Sm = streptomycin; Su = sulphanilamide; Tc = tetracycline; Cm = chloroamphenicol

Table 3. Minimum inhibitory concentrations conferred on host strains by plasmids Observed minimum inhibitory conCalculated sum of centrations for minimum inhibitory E. coli strain X concentrations for containing plasE. coil containing both mids TP120 plasmids TP120 and RI and RI

Minimum inhibitory concentrations ug ml -t for:

E. coli

E. coil

Antibiotic

containing TPI20

containing RI

Ap Cm Km Sm Su Tc

1,200 --200 3,500 50

3,200 200 2,000--3,000 150 5,000 --

3,200 200 2,000-3,000 350 8,500 a 50

3,000 200 2,500 300 5,000 70

Theoretical value which exceeds maximum solubility value and therefore cannot be confirmed

r e s i s t a n c e p a t t e r n o n l y ( T a b l e 2), suggesting that R1 was t r a n s f e r r e d i n prefe r e n c e to T P 1 2 0 . H o w e v e r , w h e n the e x p e r i m e n t was r e p e a t e d b y c o u n t e r s e lecting o n Nal, C m ( c h l o r o a m p h e n i c o l ) , a n d T c ( c o n d i t i o n s selecting for the t r a n s f e r o f b o t h p l a s m i d s ) , the a n t i b i o t i c r e s i s t a n c e m a r k e r s o f b o t h T P 120 a n d R l were t r a n s f e r r e d i n t o all the t r a n s c o n j u g a n t s ( T a b l e 2). I s o l a t e X was t e s t e d to d e t e r m i n e w h e t h e r a gene dosage effect o c c u r r e d w i t h respect to the levels o f d r u g resistance. O n e w o u l d expect t h a t w h e r e r e s i s t a n c e genes are d u p l i c a t e d , c o r r e s p o n d i n g l y m o r e gene p r o d u c t m a y b e p r o d u c e d a n d the level o f d r u g r e s i s t a n c e m a y i n c r e a s e as a result. T a b l e 3 s h o w s t h a t for A p a n d Sin, the level o f r e s i s t a n c e o f isolate X was a p p r o x i m a t e l y the s u m o f the r e s i s t a n c e s o f the 2 p a r e n t s t r a i n s c a r r y i n g the m a r k e r s singly, w h e r e a s for Tc, C m , a n d K m , i s o l a t e X h a d a p p r o x i m a t e l y the s a m e level o f r e s i s t a n c e as the p a r e n t s t r a i n c a r r y i n g the o r i g i n a l m a r k e r . I n the case o f the i n c o m p a t i b l e p l a s m i d s T P 1 13 a n d T P 1 2 5 (Fig. 4), it c a n

8

P.C. Gowland and J. H. Slater

10B

--

109

_ o

10~

-

o

~g

-

,D []

z 10 ~.

l o3

--

!02

-

10'

I

I

I

96

192

I

I

2 %3 3%4 Time{t~

I t,~O

I

I

576

672

Fig. 4. Transferof incompatible plasmids TPll3 and TPI25 in E. coli. O, total viable cells; [3, E. coli resistant to Km and Sin. For the first 192 hours of growth there was no antibiotic selection. At 192 hours, Km and Sm (at 50 ~g ml-~ each) were added to the medium supply and the culture vessel.

be seen that, in the absence o f antibiotic selection pressure, r e c o m b i n a n t s accounted for only a b o u t 2.0% o f the population. However, when a suitable antibiotic selection pressure (Km and Sm at 50 m g ml-1) was applied (at 192 hours o f growth), despite an initial drop in population size, the proportion o f r e c o m b i n a n t organisms in the population increased until they accounted for 100% o f the population, although the total population size after addition o f the antibiotics was only 10% o f what it was before their addition. Transconjugants f r o m the fermenter were examined for co-transfer o f all the expected resistance markers (Table 4), f r o m isolates before and after the antibiotic selection pressure was applied. This showed that before the antibiotic selection pressure was applied, 90% o f the putative transconjugants had co-transferred all their markers, whereas after antibiotic selection was applied, only 70% o f the putative r e c o m b i n a n t s co-transferred all the expected resistance markers. In all cases it was the Tc R marker alone that was apparently not transferred. By comparison, the experiments with E . coli T P 1 2 0 and R1 transconjugants showed that in all cases, all the markers were transferred in all the cases tested (Table 4).

Transfer and Stability of Plasmids Table 4. Percentage co-transfer of markers in compatible and incompatible plasmids

Compatbility status

Source of transconjugants

TP 120 • R 1 TPI20 x R1 TP113 x TP 125

Compatible Compatible Incompatible

TP113 • TP125

Incompatible

Fermenter Dialysis sacs Fermenter (before antibiotic selection) Fermenter (after antibiotic selection)

Cross

% transfer of non- Markselected ers markers lost 100 100 90

70

None None Tc R

Tc ~

Tc = tetracycline

Table 5. Frequency of transfer of the compatible plasraids TPI20 and R1 in pond water comparing 2 different starting cell densities

Time (h) 0 16 192 360

Total number Cells resistof viable cells ant to Tc, (organisms Km, and Sm ml -~) (t~g ml -t ) 3.0 3.0 1.7 2.6 2.1 3.4 2.1 3.4

• • x • • x x x

108 10 '~ 108 10 l~ 108 10 ~o l0 s 10 ~0

0.0 0.0 0.0 0.0 0.0 1.5 x 104 0.0 1.6 x 103

Frequency of transfer of markers 0.0 0.0 0.0 0.0 0.0 4.4 • 10 -~ 0.0 4.7 x I0 -s

Tc = tetracycline; K m = kanamycin; S m = streptomycin

Frequency of Transfer of Plasmids in Pond Water M a t i n g s b e t w e e n E. coli s t r a i n s c o n t a i n i n g p l a s m i d s T P 120 a n d R 1 o r p l a s m i d s TP113 and TP125 were performed in pond water with the relevant pairs of p o p u l a t i o n s c o n f i n e d i n d i a l y s i s sacs. T h e m a t i n g s w e r e p e r f o r m e d a t 2 d i f f e r e n t p o p u l a t i o n d e n s i t i e s , o n e o f w h i c h w a s 100 t i m e s g r e a t e r t h a n t h e o t h e r . T r a n s conjugants were only obtained from strains containing TP120 and RI, and o n l y in t h e h i g h d e n s i t y p o p u l a t i o n s ( T a b l e 5). H o w e v e r , t r a n s f e r o c c u r r e d at v e r y l o w f r e q u e n c i e s a n d t r a n s c o n j u g a n t s w e r e o n l y i s o l a t e d a f t e r 92 h o u r a n d 3 6 0 h o u r g r o w t h i n p o n d w a t e r ( T a b l e 5). I n t h e c a s e o f t h e n o n c o m p a t i b l e p l a s m i d s T P 1 1 3 a n d T P 1 2 5 , n o t r a n s c o n j u g a n t s w e r e d e t e c t e d at e i t h e r c e l l d e n s i t y ( T a b l e 5).

10

P.C. Gowland and J. H. Slater

v~

B

o

E

I

I

0

1000

I 2000 Time(hi

[ 3000

t,000

Fig. 5. Stability of E. coli containing plasmids TP120 and R1. O, total viable count; H, E. coli

resistant to Ap, Sm, Su, To, Cm, and Kin.

Stability Studies o f a Transconjugant E. coli K12 Containing Plasmids T P 1 2 0 and R1 A long-term e x p e r i m e n t was carried out o v e r 5 m o n t h s to d e t e r m i n e h o w stable the 2 plasmids, T P 1 2 0 a n d R I , were u n d e r c a r b o n - l i m i t e d growth with a selection pressure for the m a i n t e n a n c e o f b o t h plasmids. Figure 5 shows that the total cell count a n d the count o f cells resistant to all 6 antibiotics were a p p r o x i m a t e l y the s a m e and fluctuated a b o u t a m e a n o f a p p r o x i m a t e l y 1.0 x 107 cells m l - 1. Screening o f the culture for p l a s m i d s showed that no change in the size o f the 2 p l a s m i d s occurred a n d that there was no f o r m a t i o n o f a single cointegrate p l a s m i d f r o m the 2 separate plasmids.

Discussion

T h e results reported in this p a p e r d e m o n s t r a t e d the ease with which c o m p a t i b l e p l a s m i d s can transfer between different E. coli p o p u l a t i o n s growing in a continuous-flow fixed-bed c o l u m n fermenter. T h e s e e v e n t s showed two interesting features. Firstly, the transfer o f p l a s m i d s occurred without specific antibiotic selection pressures forcing the survival o f an E. coli strain containing b o t h plasmids. Secondly, in the e x p e r i m e n t shown in Fig. 3, the c o m p l e t e p o p u l a t i o n (about 108 organisms m l -~) c o n t a i n e d the 2 p l a s m i d s (as indicated by the c o m b i n e d phenotypes) after only 72 hours o f m i x e d culture growth. Analysis o f s o m e o f the transconjugants produced, such as strains X a n d Y, showed that they contained b o t h parental p l a s m i d s and, f u r t h e r m o r e , an e x a m i n a t i o n o f the host

Transfer and Stabilityof Plasmids

11

organism showed that it was plasmid R 1 which had transferred into the original E. coli strain containing T P 120. There are at least 3 possible reasons for this pattern of plasmid transfer. One possibility is that plasmid R I transferred at much higher frequencies than plasmid TP 120. Alternatively, the E. coli containing plasmid R 1 might in some way, not associated with plasmid compatibility, prevent the entry o f plasmid T P I 2 0 into the cell. Finally, the transfer of plasmids TP120 and RI could equally occur but the original host strain carrying TP120 (i.e., E. coli K I 2 IR713) might be selected against in competition with the other strain. This explanation seems unlikely since no isolate of E. coli K l 2 IR713 containing both plasmids was ever detected and it seems highly unlikely that this transconjugant would be so rapidly selected against. This is especially true for a column fermenter which provides an extremely heterogeneous environment (unlike a conventional chemostat) which should enable noncompetitive populations to be retained, even at low densities. Indeed even in conventional chemostat cultures, uncompetitive populations are frequently retained at low, but detectable, levels [5, 16]. The second possibility indicated above is also unlikely to be valid since platemating experiments have demonstrated that plasmid TP120 can be transferred to the host containing plasmid R 1. The most probable explanation, therefore, is the relative frequency o f R 1 and T P 120 plasmid transfer, since experiments transferring TP120 or R1 separately into a Nal R strain o f E. coli (Table 1) showed that R 1 transferred about 200 times more frequently than TP120. An interesting observation is that the 2 plasmid-containing strains became the dominant population at least for the duration of these experiments (Fig. 3) and, indeed, the long-term stability experiment (Fig. 5) showed that the 2 plasmid-containing populations were maintained. This illustrated that under these nonselective conditions there was a competitive advantage to the presence o f 2 plasmids (compared with 1 or none). Elsewhere we have argued that under nonselective conditions, a plasmid represents a cellular burden which can be eliminated [5] but this clearly did not apply here. This may reflect the ease with which plasmid R1 can be dispersed throughout the population rather than a measure of the competitive advantage of strains such as X or Y. For the incompatible plasmids TP113 and TP125, 2 plasmid-containing transconjugants accounted for about 2% o f the total population under nonselective conditions (Fig. 4). This seems a high value and again must reflect the ease with which plasmids transfer since subsequent growth of 2 plasmid-containing cells must result in the elimination of 1 of the incompatible plasmids. That is, there is a dynamic relationship between the rate o f formation of 2 plasmid-containing populations and their removal from the mixed culture. As expected, however, application of a combined selection pressure resulted in the establishment o f a novel population containing both plasmids, notwithstanding their incompatibility problems. The initial drop in the population size (Fig. 4) after 192 hours was caused by the lethal action of drugs on the sensitive populations, and recovery only happened once plasmid transfer had occurred to produce a novel population carrying resistance to all the drugs. Nevertheless, recovery was not complete since after 480 hours the new steady state population size (1 x 108 organisms ml -~) was only 3% of the total population under non-

12

P.c. Gowland and J. H. Slater

selective conditions. This must reflect the difficulties encountered in maintaining 2 incompatible plasmids. Isolate X, a r e c o m b i n a n t from a c o l u m n fermenter containing both T P 1 2 0 and R 1, was tested to determine whether there was a gene dosage effect for the duplicated Ap, Sin, and Su resistance genes (Table 3). Sulphanilimide resistance o f isolate X could not be d e t e r m i n e d due to solubility limitations. However, for Ap and Sm, the MIC values o f isolate X were approximately the sum o f those for T P 120 and R 1 d e t e r m i n e d separately. The level o f resistance o f isolate X to C m and K m was approximately the same in R i as in X, and the level o f resistance o f X to Tc was approximately the same as in TP120. Transfer experiments for the 2 plasmids in isolate X into a Nal R plasmid-minus strain showed that unless both plasmids were specifically selected for, then plasmid R 1 was preferentially transferred. For the putative recombinants o f the incompatible plasmids T P 1 1 3 and T P 125, it was shown that 90% o f the recombinants isolated before the antibiotic selection was applied, co-transferred all o f the expected markers. It was not clear whether T P 113 transferred into the T P 125-containing strain or vice versa since there was no way o f distinguishing between the 2 hosts (Table 1). After the antibiotic selection pressure was applied, only 70% o f strains carried all the expected resistance markers. In all cases, it was only the Tc ~ gene that was lost; all the other genes were co-transferred (Table 4). The observation that there was a greater percentage loss o f the Tc resistance marker after application o f antibiotic selection pressures, could be a result o f partial fragmentation o f TP125, since complete segregation o f the 2 plasmids could not occur. The compatible plasmids R 1 and T P 120 always appeared to transfer all their markers in all the cases tested both before and after antibiotic selection pressure was applied. This is not entirely surprising since loss o f the Ap, Sm, and Su markers from either T P 1 2 0 or R1 would be masked by the presence o f these resistance genes on the other plasmid. In p o n d water, for strains containing the incompatible plasmids T P 1 1 3 and T P 125, no recombinants were isolated. For strains containing plasmids T P 120 and R 1, recombinants were isolated only in the high density mixed population at the low frequency o f 1:2.3 x 10-6-1:2.1 x 10 -7 after 192 hours and 360 hours respectively. N o recombinants were isolated from the less-dense mixed population. This very low transfer frequency, when c o m p a r e d with laboratory results, is probably a reflection o f the p o o r e r nutritional status and lower temperature o f the natural e n v i r o n m e n t , as under laboratory conditions transfer occurred at a frequency o f 2.6 x 10 -2. Long-term stability studies o f a T P 120-R1 plasmid aggregate under carbonlimited growth conditions, in the presence o f all the antibiotics to which the plasmids jointly conferred resistance on the host, showed that the plasmid aggregate was extremely stable under these conditions. Due to the extra energetic and metabolic burden on the cell o f carrying 2 plasmids [5], it might have been expected that a plasmid co-integrate would evolve from the aggregate, dispensing with nonessential genes from one o f the plasmids which had previously been coded for by both plasmids. Alternatively, it might have been expected that such loss o f nonessential genes would result in a decrease in size o f one or both o f the plasmids. However, after 5 m o n t h s continuous growth,

Transfer and Stability of Plasmids

13

t h e r e w a s n o e v i d e n c e t h a t a n y c h a n g e in e i t h e r s i z e o r c o m p o s i t i o n o f t h e p l a s m i d s h a d o c c u r r e d . T h i s s h o w e d t h a t t h e p l a s m i d T P 1 2 0 - R 1 a g g r e g a t e is s t a b l e u n d e r t h e c o n d i t i o n s e m p l o y e d in t h i s s t u d y . Acknowledgments. PCG acknowledges receipt of a M.R.C. research studentship.

References 1. Anderson ES (1968) The ecology of transferable drug resistance in the Enterobacteriaceae. Ann Review Microbiol 22:131-180 2. Anderson JD (1974) The effect of R factor carriage on the survival of Escherichia coli in the human intestine. J Med Microbio! 7:85-90 3. Davis BD, Mingioli ES (1950) Mutants of Escherichia coli requiring methionine or vitamin B~. J Bacteriol 60:17-21 4. Eckhardl T (1978) A rapid method for the identification ofplasmid DNA in bacteria. Plasmid 1:584--588 5. Godwin D, Slater JH (1979) The influence of the growth environment on the stability of a drug-resistance plasmid in Escherichia coli K 12. J Gen Microbiol 111:201-210 6. Harden P, Meynell EW (1972) Inhibition ofgene transfer by antiserum and identification of serotypes by sex pill J Bacteriol 109:1067-1074 7. Helling, RB, Kinney T, Adams J (1981) The maintenance of plasmid containing organisms in populations of Escherichia coil J Gen Microbiol 123:129-141 8. Inselberg J (1978) Col E1 plasmidmutants affecting growth of an Escherichia coli rec B rec C sbs mutant. J Bacteriol 133:433-436 9. Jones IM, Primrose SB, Robinson A, Eltwood DC (1980) Maintenance of some Col El-type plasmids in chemostat culture. M01 Gen Genet 180:579-584 10. Lacey RW, Chopra T (1975) Effect of plasmid carriage on the virulence of Staphylococcus aureus. J Med Microbiol 8:137-147 11. Nakazawa T (1978) TOL plasmid in Pseudomonas aeruginosa PAO: thermo-sensitivity of self-maintenance and inhibition of host cell growth. J Bacteriol 133:527-535 12. Neijssel OM (1980) A microbiologist's view of genetic engineering. Trends Biochem Sci 5: 111-112 13. Reanney D (1976) Extrachromosomal elements as possible agents of adaptation and development. Bacteriol Reviews 40:422-590 14. Richmond MH (1977) The survival of R-plasmids in the absence of antibiotic selection pressures. In: Drews J, Hogenauer G (eds) Topics in infectious diseases. Springer-Verlag, Wien 15. Rubens CE, McGee ZA, Farrar WE (1980) Loss of an aminoglycoside-resistance plasmid by Serratia marcescens during treatment of meningitis with amikacin. J Infect Dis 141:346-350 16. Smith AL, Kelly DP (1979) Competition in the chemostat between an obligately and a facultatively chemolithotrophic thiobaci/lus. J Gen Microbiol I 15:377-384 17. Terawaki Y, Kakizawa Y, Takayasu H (1968) Temperature sensitivity of cell growth in E. coli associated with the temperature sensitive R (KM) Factor. Nature, London 219:284-285 18. Zund P, Lebek G (1980) Generation time-prolonging R plasmids: correlation between increases in the generation time of Escherichia coli caused by R plasmids and their molecular size. Plasmid 3:65-69