Factors affecting conjugative transfer rates of plasmids in batch and continuous cultures

JOURNAL OF FERMENTATIONAND BIOENGINEERING VOI. 69, NO. 4, 215-219. 1990

Factors Affecting Conjugative Transfer Rates of Plasmids in Batch and Continuous Cultures PEI-XIN YAO,§ HISAO OHTAKE, AND KIYOSHI TODA*

Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Yayoi 1-1-1, Tokyo 113, Japan Received 13 November 1989/Accepted 16 January 1990 Factors affecting the rates of plasmid transfer were investigated using Escherichia coli LC102 bearing a conjugative plasmid R100-1 and E. coli DH1. The rate constant of transconjugant increase, kti, was used for presenting the degree of plasmid transmissibility instead of the plasmid transfer efficiency (pte). The rate constant was defined as the specific rate of transconjugant increase (srti, the number of transconjugants per donor per h) divided by the recipient cell concentration. The kti values ranged between 10-10 and 10-is ml cells-i h-I, when estimated under various conditions. Moderate liquid agitation had a favorable effect on ktf but agitation rates higher than 33 s - t (intergrated shear force) greatly decreased the value of kti. The transconjugant-forming activity of the cells growing in continuous culture did not significantly change with the dilution rate, except those growing at dilution rates less than 0.1 h - i . The rate constant kti at temperatures of 10-15°C was as low as the detection limit (10 -15 ml cells -t h-I).

used as the donor strain. The plasmid R100-1 (7) is a conjugative plasmid conferring on its host cells the resistant to tetracycline (Tc) and chloramphenicol (Cm). E. coli ME8569 (E. coli DH1) was used as the recipient strain. It is resistant to nalidixic acid. These two strains were obtained from the National Institute of Genetics, Mishima, Japan. Medium composition L broth consisted of (in g-l-1) Bactotryptone (Difco Laboratories, Detroit, USA) 10, yeast extract (Difco) 5, and NaC1 5 (pH 7.5). L broth was supplemented with 0.1 g.l -~ Adekanol (an antifoamer; Asahi Denka Co., Tokyo, Japan). L agar was prepared by adding 15 g ' l -~ agar to L broth. In the agar plates for selecting transconjugants, the following antibiotics were added (in mg.l-~): nalidixic acid (Nal) 20, tetracycline (Tc) 15, and chloramphenicol (Cm) 30. Membrane mating Overnight cultures of donor and recipient cells and L broth were mixed in the volume ratio of 1, 10, and 50. The mixture was filtered through a membrane filter (pore size, 0.45 ~m). The filter with the cake was placed on L agar for 1 h at 30°C, and then the cake on the filter was suspended in sterile saline solution (0.85~ NaCI). The number of donors, recipients, and transconjugants were counted on selective L agar containing appropriate antibiotics. Plate mating Two kinds of experiments were conducted. Small volumes (0.05 ml) of the cultures of donor and recipient cells were spread separately on L agar plates containing antibiotics for selecting transconjugants. Matings were conducted by the surface-to-surface contact of the two L agars (30°C, 16 h). The transconjugants appeared in the interface of the agar plates. In the other experiment 0.1 ml each of donor and recipient suspensions was dotted on L agar for transconjugant selection. The droplets of the cell suspensions on the plate was mixed by a bent glass rod for a fixed time and then incubated for colony formation of transconjugants. Broth mating Broth mating experiments in batch culture were done in test tubes, shake flasks, spinner flasks with both aeration and stirring, and a 5-I mini-jar fermentor (Model KMJ, Mitsuwa Co., Tokyo). A 500-ml spinner

Many research projects have been intensively applying genetically engineered organisms (GEO) to environmental and agricultural problems (1). These include biodegradation of spilt petroleum (2) and recalcitrant organic pollutants (3), biological pest control (4, 5), and the improvement of agricultural production by expression on nitrogen fixing genes in transformed plants (6). Although the vector plasmid carrying a foreign gene should be nonconjugative, it cannot be denied that the recombinant plasmid may transfer to indigenous microbes through mobilization (7) after being released deliberately or accidentally into a natural environment. To predict the fate of microorganisms bearing a plasmid in a natural environment, it is essential to have a good understanding of physical and chemical factors affecting the rate of plasmid transfer. In laboratory experiments plasmid transmissibility is usually tested under its optimum conditions. In nature, however, the environmental condition is not necessarily favorable for plasmid transfer. Therefore investigation of factors affecting the rate of plasmid transfer must contribute to a rational assessment of the safety of GEO which might be released to a natural environment. In this work, we investigated rates of transconjugant increase in a mixture of E. coli cells with and without a conjugative plasmid in batch and continuous cultures. First we measured the transconjugant increase rate in a closed system of batch culture by changing the conditions of liquid mixing and concentrations of both donor and recipient cells. Second, the effects of dilution rate, temperature, and nutrient concentration of the feed medium on transconjugant increase were examined in an open system of continuous culture. MATERIALS AND METHODS Bacterial

strains

and

plasmid

Escherichia

coli

ME6045 (E. coli LC102 bearing the R-factor, R100-1) was * Corresponding author. § On leave from Shaanxi Institute of Microbiology,Xian, China. 215

216

YAO ET AL.

J. FERMENT. BIOENG.,

flask was used to measure rates o f conjugal transfer o f the plasmid in a continuous culture. Recipient cells were grown overnight in a spinner flask (working volume, 200 ml) at a defined dilution rate. Overnight culture o f d o n o r cells in the shake flask was added to the continuous culture o f the recipients (50/00 inoculum). Courses o f transconjugant increase were traced at a p p r o p r i a t e times, and the concentrations o f donors, recipients, and transconjugants were measured at steady state. Rate of transconjugant increase In this work, two parametric quantities for plasmid transfer were defined: the specific rate o f transconjugant increase (srti) and the rate constant o f t r a n s c o n j u g a n t increase (kti). Srti was defined as the increase o f t r a n s c o n j u g a n t numbers per h per average number o f donors during the mating; s r t i = i n c r e a s e o f transconjugant c o n c e n t r a t i o n / d o n o r cell c o n c e n t r a t i o n / m a t i n g time = p t e / m a t i n g time. where pte denotes the plasmid transfer efficiency which has been defined by Willets (8) as the n u m b e r o f transconjugants generated per number of donors. However, since this parametric quantity, srti, greatly changed with the recipient cell concentration (see Table 2 later), the rate constant o f transconjugant increase (kti) was defined as srti divided by the concentration o f recipients, kti = srti/recipient cell concentration The other parameter, kpt, was defined as the rate constant in the mass action equation for plasmid transfer (9). If the mass action law is applied to conjugal plasmid transfer between d o n o r and recipient cells, the rate o f increase o f t r a n s c o n j u g a n t concentration (NT) with mating time t m a y be written as,

dNT/ dt = kptNDNR+ flTNT

(1)

where ND and NR denote the concentrations of donors and recipients; /ZT is the specific growth rate o f transconjugants; kpt is the rate constant for plasmid transfer. By the definition o f srti, srti = (1/ND)dNT/dt = kptN R+

[JTNT/ND

(2)

By dividing srti with the recipient concentration, the rate constant o f transconjugant increase, kt~, is defined as, kt~= srti/NR = kpt +IaTNT/(ND NR)

(3)

If the contribution o f the growth term is negligible, kt~ becomes equal to the rate constant of plasmid transfer, kpt.

(3')

kti = kpt

However, it must be stressed that the growth effect can not be neglected except in special conditions. In the case of continuous culture Eq. 1 can be rewritten as follows,

dNT/ dt = kptNDNR +,//TNT - - DNT

( 1")

where D is the dilution rate, h i. A t a steady state,

DNT

(4)

srti = (I/ND)(kptNDNR+ BTNT) = D N T / N D

(5)

kptND/VR -~-flTNT :

Consequently,

The value o f kti in continuous culture is given by, kti =

sr ti/NR

(6)

The values o f srti and kti in a continuous culture were estimated by Eqs. 5 and 6. RESULTS AND DISCUSSION Effects o f m a t i n g m e t h o d

on transconjugant

increase

Three mating procedures including m e m b r a n e mating, broth mating, and plate mating were used to measure the apparent rate o f plasmid transfer (Table 1). In the membrane mating almost all the d o n o r cells were considered to be in contact with recipient cells in the cake on the membrane filter. Therefore the srti ( 2 . 2 x 10 2 h - l ) may indicate the probability rate o f transconjugant increase after the mating pairs were established. The srti values in the broth mating were a r o u n d 1 x 10 3 h-X when the mating time was short (0.33 rain) or the mating was conducted in a spinner flask with a rather high degree o f liquid mixing. The value of srti (6.1 x 10-2h -1) obtained in the broth mating for 1 h using a shake test tube with gentle liquid mixing was threefold that measured by the membrane mating. It has been considered that the srti measured by m e m b r a n e mating would give the m a x i m u m value o f the transconjugant increase rate (8). This discrepancy m a y be ascribed partly to the higher growth activity of transconjugant cells in the b r o t h mating than in the membrane mating. W i t h respect to srti for the plate mating, it could not be measured in the short mating time (1 h) as done in the membrane or broth matings because the mating time on an agar plate could not be separated from the incubation time (overnight) of the plate. Therefore the transconjugant increase efficiency (tie) defined as the frequency of transconj u g a n t increase per d o n o r cell was measured and is shown

TABLE 1. Effects of mating method on transconjugant increase Method Membrane mating Broth mating in shake test tube in shake test tube in spinner flask Plate mating Plate mating Plate mating

Mating time (min) with without mixing mixing

Cell density per surface (cm-z) per volume (cm 3) donor recipient donor recipient

--

60

2.3 x 10 u

1.2 x 1012

0.33 60 60 0 0.5 1.5

---17 × 60 17 × 60 17 × 60

1.2x 108 3.4 x 107 1.6x 107

1.1xl09 1.5 x 108 3.8×107

a Specific rate of transconjugant increase. b Rate constant of transconjugant increase. c Transconjugant increase efficiency (transconjugants/donors).

2.3 × 10 7

6.3 x 105 6.3 x 105 6.3 x 105

srti (h i)a or tiec

(mlcells lh i)

1.2 × 10s

2.2 × 10 2

1.8 x 10 ~4

1.3 x 10 3

1.2x 10 u 4.1 x 10 to 3.2×10 ~

2.5 × 106 2.5 z 106 2.5 x 106

6.1 x 10 1.2x 10 1.2 x 10 2.7 x 10 2.9 x 10

2 3 6c ~c sc

ktib

VOL. 69, 1990 TABLE 2.

RATE OF CONJUGATIVE PLASMID TRANSFER

Effects of cell concentration on transconjugant increasea

Recipient (R) Donor (D) (ml 1) (ml 1)

Ratio (R/D) ( )

1.5xlO ~ 2.5×10 6 3.0xlO 5 5.0×10 4

1.6×10 8

1.8×10 7 1.5xlO 6 4.5x10 5

10.7 7.2 5.0 8.8

srti (h J)

kti

(mlcells-lhb

6.3×10 5.3×10 5.3x10 <2.4x10

2 3 4 4

3.9x10 2.9x10 3.5x10 ~5.5x10

m

E f f e c t s o f cell c o n c e n t r a t i o n s o n t r a n s c o n j u g a n t i n c r e a s e in b r o t h m a t i n g The effects o f concentrations o f d o n o r

and recipient cells on srti were examined in the range o f 104 to l0 s cells ml ~, while the ratio o f the number o f recipients to that o f donors was 5.0-10.7. The value o f srti decreased with the decrease o f recipient cell concentration (Table 2). However, the kti values calculated f r o m Eq. 3 were rather constant, 3 - 4 x 10 l ° m l c e l l s - ~ h - ~ for the matings with more than 1.Sz 106 recipients per ml. Therefore it appears appropriate to express the degree of plasmid transmissibility by kti rather than by srti or pte. of

liquid

mixing

on

transconjugant

increase

Rates o f conjugal transfer o f R100-1 plasmid were also measured using test tubes (18 m m in diameter; 22 cm in length), and 500-ml shake flasks with rotary, reciprocal, or vibratory shaking (Table 3). The volumes o f mixtures in a test tube or a shake flask were 6 ml and 50 ml, respectively. The values of srti were about 10 2 h - l , and the mode o f

10 . 9

: '

I

!u

'

'

I

'

I

'

I

I

,

10-1o

.-

10

A

Test tube

Standstill Rotary shaker a Reciprocal shaker b Vibratory shaker c Rotary shaker" Stirred, aerated a

kti (mlcells ~h 1)

2.6 x l0 5.6 x 10 6.1 × 10 3.1 × 10 3.8 × l0 1•2 × 10

2 2 2 2 2 3

1.9 x l 0 5.7 x l0 4.1 x l0 7.4x l0 2.2 × 10 3.1 x 10

m io io 1o m u

a 23 mm pitch x 108 rpm, (angle of test tube to horizontal) 45 °.

b 70mm pitchx 104spm, 45 ° . c 24mm pitchx 174spin, 15° . d 2.35 vvm, 196 rpm.

shaking did not significantly affect the value. The srti measured in test tubes without shaking was somewhat smaller than those obtained with shaken vessels. This may indicate that liquid mixing had a favorable effect on conjugal transfer o f plasmids. The frequency of cell collision might be increased by liquid movement. However, the value o f srti was apparently smaller in a stirred and aerated vessel (10 -3 h -1) than those measured in a shaking vessel ( 1 0 - Z h - ) . The excess liquid m o t i o n generated by mechanical stirring seemed inhibitory to transconjugant increase. The effects o f the shear rates on the rate constant of transconjugant increase (kti) in the stirred vessel were also assessed using a jar fermentor (working liquid volume, 1.0/). The values of kti were measured under various rotationary rates of an impeller for mating times o f 0.5, 1.0, and 1.5 h (Fig. 1). In parallel with each run o f the mating experiments, broth mating for 1 h in a gently shaken test tube was done using the same mixture of d o n o r and recipient cells. The ratio o f the values o f k, obtained in broth mating for 1 h in the jar fermentor to those in the test tubes are plotted against impeller speed in Fig. 1. It is believed that the ratio, not kti itself, can express the liquid mixing effect better, because kt~ value changes depending not only on liquid agitation but on the other factors (e.g., culture condition and concentrations o f d o n o r and recipient cells). The values o f kti was not significantly affected by the rotational rate of an impeller, when the rate was in the range o f 6 0 - 3 0 0 r p m (Reynold number, R e = N D i 2 p / p , o f 5,000-26,000). However, when the rotational rate

1

,_

-

10~°

)

i

,

i

i

[

l

l °

T__o_m 1 0 - 1 1

,~

srti (h J)

Culture vessel Liquid mixing

Shake f l a s k Spinner flask

in Table 1 ( 1 . 2 x 10 6 - 2 . 9 x 10 5). In one o f the experiments o f plate mating where the time for mixing the cell suspensions o f d o n o r and recipient on the plate was omitted, tie was 1.2 × 10 6. Mixing o f the d o n o r and recipient cell suspensions on a plate with a bent glass rod (0.5 to 1.5 min) increased srti from 10 -6 to 10 5 (Table 1). As was mentioned earlier, mixing o f the d o n o r and recipient cells in a test tube by a reciprocal shaker for 0.33 min enhanced srti up to 10 3. These indicate that the m o d e and degree o f liquid mixing had significant effects on the transconjugant increase rate.

Effects

TABLE 3. Effectsof liquid mixing on transconjugant increase

io lo to

In broth matings.

217

8

_

a

10-1

10 -12

10 -2

o 10-13

10 . 3

_.~

"•""

10-14 0

,

I,

200

J, 400

.

I, 600

[

l

l

l

l

o

o

8 10e

°~

0 U

104

(..)

1,4', 800

l

-~10 8

A D

l

10-4

1000

(rpm)

FIG. 1• Effectsof liquid agitation in a jar fermentor on kti. Right side ordinate indicates the ratio of kti values measured in a jar fermentor to those measured in a gently shaken test tube (mating time, lh). Mating time: O,0.5h;zx, A, 1.0h; [], 1.5h.

102

. . . .

0

t

5

. . . .

,

10

. . . .

15

Mating time (h) FIG. 2. Course of mating in a continuous culture. A shake culture of donor cells was added to a continuous culture of recipienl cells at steady-state• Symbols: o , donor cells; o , recipient cells; ±, transconjugant cells.

218

YAO ET AL.

J. FERMENT. B1OENG.,

10 . 9 r

T A B L E 4. Transconjugant increase in stage 2 of two-stage continuous culture with step feed of donor cells from stage 1

10-1o

Medium concentration~

10-11

1 0.1 0.001 1

Ic-

• -I

Dilution rate (h ~) stage 1 stage 2 0.34 0.33 0.36 0.076

0.34 0.33 0.36 0.076

srti (h ~) 5.6×10 9.2× 10 7.1 '~10 4.4 × 10

R~, (mlcells ~h ~) 4 s 5 6

1.0×10 1.4×10 2.5×10 4.0 × 10

I2 iz i~ ~5

Factor based on L-medium.

10-12 u)

O

G

O

1 0-13

E

"-~ 10-14 o _

10-15 10-16 L

0

0.2 0.4 0.6 0.8 Dilution rate (h -1)

.0

FIG. 3. Effects of dilution rate on kti in continuous culture at 30°C (~, [] ), 25°C ( o ), 20°C ( • ) and 10°C (A). The donor cells were cultivated overnight (±, ©, I and A) or until the logarithmic growth phase ( [] ), and added to the continuous culture of the recipient cells. exceeded 300 rpm, kti decreased with the increase of rotational rate. C r o u g h a n et al. (10) defined the integrated shear factor (ISF) to express the degree of liquid agitation pertaining to its effect on cell inactivation as follows, I SF = 2 IrNDi/(Dt-Di)

(6)

where Dt and Di denote diameters of the tank and the impeller, respectively. They observed that inactivation of animal cells occurred at critical ISF values of 2.5-3.5 (s ]) for FS-4 cells and 7.0 for chicken embryo fibroblasts grown on microcarriers, respectively. In comparison with these, it may be said that the mating pairs in this experiment were stable up to 33 ( s i) at N = 3 0 0 rpm. Effects of dilution rate, temperature, and cell state on transconjugant increase in continuous culture The result of transconjugant increase in a continuous culture conducted at 25°C at a dilution rate of 0.24 h ] is shown in Fig. 2. The concentrations of the donor, recipient, and transconjugant cells attained a steady state 5 h after a pulse input of donor cells with an inoculation ratio of about 10% into a chemostat culture of recipient cells. With various dilution rates and temperatures, kt~ was measured and correlated with the dilution rate (Fig. 3). The values of kt~ did not depend on the dilution rate, except those at very low dilution rates (less than 0.1 h ]). The effect of temperature on kc~ was significant. The values of kt~ at 20 and 25°C were two or three orders of magnitude smaller than that at 30°C. At 10°C the/qa value was 10 ~5ml cells ~h l, which was as low as the detection limit for t r a n s c o n j u g a n t cells. In practice plasmid transfer was negligible at 10°C. Trevors and Oddie reported that transfer of a conjugative plasmid (60 Md) occurred among E. coli strains within 24 h at 22°C, but not at 15°C (11). Mercury resistance plasmids of some marine bacteria were

observed to transfer at 20°C, but no transfer was detected at 16°C (12). Singleton and A n s o n also reported that transfer of Rldrd-19 was not observed at 15°C in 2-d matings (13). These previous findings also support the above result. In this paper most of the transfer experiments were done with a pulse input of an overnight culture of donor cells to the chemostat culture of recipient cells. In a few experiments donor cells growing at the logarithmic phase of growth were used as the inoculum. The kti values (open squares in Fig. 3) were almost the same as those of overnight cultures. In mating experiments in batch culture, bacterial cells in the logarithmic growth phase were recommended (8). However, in continuous culture, the history of the cells did not matter for the plasmid transfer activity of the cells at steady state. Effects of nutrient conditions on transconjugant increase Donor and recipient cells were grown separately in two continuous fermentors. The effluent of the overnight culture of d o n o r cells in the first fermentor was introduced into the second fermentor where the recipient cells had been growing. At appropriate times the viable cell concentrations of donors, recipients, and transconjugants were counted to estimate kti from Eq. 6. The kti values were in the order of 10 j~ to 10 12ml cells ~h ~at the dilution rates of 0.33-0.36 h -I, although the nutrient concentration in the feed medium varied by two orders of magnitude as shown in Table 4. As it was considered that the concentration of a growth-limiting nutrient in culture depended on the dilution rate and not on the nutrient concentration in the feed, the above result suggests that the rate of transconjugant increase was varied as a function of limiting substrate concentration. At dilution rates less than 0.1 h ] where the growth-limiting nutrient concentration was low, kti was as low as the detection limit (10 -]5 ml cells ~h - ]), even when nutrient-rich medium was fed. The kt~ values of 10-~2 mlcells ~h -] was much smaller than those (10 ]0 ml cells ] h ~) measured with the mating in a single-stage continuous culture with a pulse input of donor cells. The cause of the difference is left for further investigation. Altherr and Kasweck (14) conducted in situ studies of plasmid transfer and observed that the frequencies were one order of magnitude lower than those in vitro; transfer frequencies of the conjugative plasmid pKK1 were 3.2 x 10 5 to 1.0 x 10 -6 in 24-h matings in a m e m b r a n e diffusion chambers in the sewage effluent-receiving waters, but those observed in nutrient broth (Difco) were 1.6 x 10 4 to 4.4 x 10 5. Mach and Crimes reported that mean transfer frequencies of antibiotic resistances among the isolates from a sewage treatment plant and a hospital were 2.1 × 10 3 in laboratory matings and 4.9 × 10 5 and 7.5 × 10 5 in in situ matings in primary and secondary clarifiers, respectively (15).

VOL. 69, 1990

RATE OF CONJUGATIVE PLASMID TRANSFER

T h e s e studies suggests p l a s m i d t r a n s f e r f r e q u e n c i e s in situ were o n e or t w o o r d e r s o f m a g n i t u d e l o w e r t h a n t h o s e m e a s u r e d in vitro. In this study the rate o f t r a n s c o n j u g a n t increase was greatly r e d u c e d u n d e r n o n - o p t i m u m c o n d i t i o n s o f t e m p e r a t u r e , d i l u t i o n rate, and a g i t a t i o n rate. It m u s t be n o t e d that the m u l t i p l i c a t i o n rate o f t r a n s c o n j u g a n t s c o u l d n o t be i g n o r e d in the e s t i m a t i o n o f the p l a s m i d transmissibility at such p o o r c o n d i t i o n s for p l a s m i d t r a n s m i s s i o n . W h e n m a t i n g t i m e is long e n o u g h for the t r a n s c o n j u g a n t s to m u l t i p l y c o n s i d e r a b l y , the freq u e n c i e s o f p l a s m i d t r a n s f e r m a y be o v e r e s t i m a t e d . NOMENCLATURE D Di Dt ISF kpt kti N ND NR NT pte srti t tie p p

: d i l u t i o n rate, h-1 : impeller d i a m e t e r , m : tank diameter, m : i n t e g r a t e d shear f a c t o r , s - t : rate c o n s t a n t o f p l a s m i d t r a n s f e r , ml cells -~ h -1 : rate c o n s t a n t o f t r a n s c o n j u g a n t increase, ml c e l l s h 1 : r o t a t i o n a l speed o f impeller, s : c o n c e n t r a t i o n o f d o n o r cells, cells, ml : c o n c e n t r a t i o n o f recipient cells, cells m1-1 : c o n c e n t r a t i o n o f t r a n s c o n j u g a n t cells, cells m l - I : p l a s m i d t r a n s f e r efficiency, d i m e n s i o n l e s s : specific rate o f t r a n s c o n j u g a n t increase, ml cells- x h : time of mating, h : t r a n s c o n j u g a n t increase efficiency, d i m e n s i o n l e s s : specific g r o w t h rate o f t r a n s c o n j u g a n t cells, h-~; viscosity o f b r o t h , kg s m 2 : density o f b r o t h , kg m 3 REFERENCES

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Acad. Sci. USA, 75, 3109-3112 (1978). 3. Furukawa, K. and Chakrabarty, A. M.: Involvement of plasmids in total degradation of chlorinated biphenyls. Appl. Environ. Microbiol., 44, 619-626 (1982). 4. Lindow, S.E.: Competitive exclusion of epiphytic bacteria by Ice Pseudomonas syringae mutants. Appl. Environ. Microbiol., 53, 2520-2527 (1987). 5. Ward, E. S., Ellar, D. J., and Todd, J. A.: Cloning and expression in Escherichia coli of the insecticidal gammaendotoxin gene of Bacillus thuringiensis var. israelensis. FEBS Lett., 175, 377382 (1984). 6. Pankhurst, C.E., MacDonald, P . E . , and Reeves, J.M.: Enhanced nitrogen fixation and competitiveness for nodulation of Lotus pedunculatus by a plasmid-cured derivative of Rhizobium loti. J. Gen. Microbiol., 132, 2321-2328 (1986). 7. Gealt, M . A . , Chai, M.D., AIpert, K.B., and Boyer, J.C.: Transfer of plasmids pBR322 and pBR325 in wastewater from laboratory strains of Escherichia coil to bacteria indigenous to the waste disposal system. Appl. Environ. Microbiol., 49, 836841 (1985). 8. Willetts, N.: Conjugation, p. 49-77. In Grinsted, J. and Bennett, P. M. (ed.), Methods in microbiology, vol. 21. Academic Press, New York (1988). 9. Levin, B.R., Stewart, F.M., and Rice, V.A.: The kinetics of conjugative plasmid transmission: fit of a simple mass action model. Plasmid, 2, 247-260 (1979). 10. Croughan, M.S., Hamel, J.-F., and Wang, D . I . C . : Hydrodynamic effects on animal cells grown in microcarrier cultures. Biotechnol. Bioeng., 29, 130-141 0987). 11. Trevors, J. T. and Oddie, K. M.: R-plasmid transfer in soil and water. Can. J. Microbiol., 32, 610-613 (1986). 12. Gauthier, M. J., Cauvin, F., and Breittmayer, J-P.: Influence of salts and temperature on the transfer of mercury resistance from a marine pseudomonad to Escherichia coli. Appl. Environ. Microbiol., 50, 38-40 (1985). 13. Singleton, P. and Anson, A. E.: Conjugal transfer of R-plasmid Rldrd-19 in Escherichia coli below 22°C. Appl. Environ. Microbiol., 42, 789-791 (1981). 14. AItherr, M.R. and Kasweck, K.L.: In situ studies with membrane diffusion chambers of antibiotic resistance transfer in Escherichia coli. Appl. Environ. Microbiol., 44, 838-843 (1982). 15. Mach, P . A . and Crimes, D.J.: R-Plasmid transfer in a wastewater treatment plant. Appl. Environ. Microbiol., 44, 1395-1403 (1982).