JOURNAL OF BACTERIOLOGY, Apr. 1976, p. 222-224 Copyright © 1976 American Society for Microbiology
Vol. 126, No. 1 Printed in U.SA.
Temperature Dependence of Mating-Pair Formation in Escherichia coli ROGER H. WALMSLEY
Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania 19174 Received for publication 12 January 1975
The temperature dependence of mating-pair formation by Escherichia coli has been studied from 2 to 45 C by means of a physical assay. No mating-pair formation was found below 24 C. Between 30 and 41 C pair formation increased very rapidly, followed by an equally sharp decline between 41 and 45 C. The results are interpreted in terms of the pilus adsorption process of recipient cells and pili production by donor cells. The first step in the complex process of sexual conjugation in Escherichia coli is the formation of a firmly attached pair of donor and recipient cells. The effects of temperature variation on pair formation are of interest for two reasons. Novotny and Lavin have reported a very sharp decrease in donor cell pilation between 30 and 24 C (3). If pili are an absolute requirement not only for recombinant production (2) but also for pair formation, then pair formation should show a similarly rapid decrease with decreasing temperature. In addition, Novotny and Lavin (3) showed that if cells are cooled rapidly from 37 to 0 C there is only a partial loss in piliation by the donor cells. This makes it possible to see if the pair formation process takes place spontaneously at 0 C even though recombinants cannot be formed at that temperature. MATERIALS AND METHODS Bacterial strains. The donor strain was HfrH (thi-) and the recipient was PA309 (thr-leu- trphis arg- thi- lac- gal- man- xyl- mal-tonrstrr). Media. Tryptone broth contained, per liter: tryptone (Difco), 10 g; nutrient broth (Difco), 6 g; and NaCl, 5 g. Preparation of cells. Overnight cultures were diluted 1:500 into 20-ml culture tubes that contained 10 ml of fresh broth. The cultures were grown at 37 C to exponential phase with moderate agitation on a shaker. Matings. To avoid disturbing the growth of the primary parental cultures, matings were made from secondary parental cultures that were maintained at the same temperature as that at wh>ich the mating was carried out. To do this, a small plastic bucket was clamped to a bar mounted on the shaker above the primary parental cultures in the 37 C water bath. Three culture tubes were placed in the bucket, to which was added water of the desired temperature. The temperature was checked periodically and maintained to within 1 C ofthe average. A 4-ml sample was drawn from each primary parental
tube and transferred to the corresponding secondary parental tube in the bucket, where it achieved the desired mating temperature after 3 min. The angle of inclination of the tubes in the bucket and the amplitude of shaking were identical to those of the primary parental cultures. After temperature equilibration, 2-ml samples were drawn from each of the secondary parental cultures and gently mixed in the third tube in the bucket. The remaining procedures were identical to those described previously (4). Briefly, mating was allowed to proceed for 7 min, at which time culture concentrations were measured with a Coulter counter. Parental cell concentrations in the mating mixture were equal and were typically 108 per ml. The standard deviation of all concentration measurements for all experiments was 5%.
RESULTS The mating mixture pairing fraction, f, is defined (4) as f = c2/(cf + c,,,), where C2, Cf, and c,,, are the concentrations in the mating mixture of mating pairs, recipient cells, and donor cells, respectively. In extended matings the cells eventually form clumps of various sizes, as observed with a microscope (1). In pulsed matings at 108 per ml, however, only donor-recipient pairs are formed to any appreciable extent. Hence the concentration of single cells plus mating pairs in the mating mixture is given by cl. = Cf + C,,, - C2.
The mating mixture pairing fraction is therefore related to the measured concentrations by
f=
1
-
CJ(Cf + c,,,).
For each mating f is calculated. Each point in the figure is the average value of f for all the matings carried out at that temperature. 222
VOL. 126, 1976
MATING-PAIR FORMATION
223
A total of 62 matings were carried out at 20 eight temperatures from 2 to 45 C, the results of which are shown in Fig. 1. The slightly negative value of f at 2 C is due either to an un0 15 _ traced small systematic error or to an unlikely statistical fluctuation. In either case it is clear z that significant pair formation does not take 0 place in a 7-min mating at 2 C. As the tempera- U 1O0ture is raised to 24 C the data indicate the possibility of a very slight degree of pair forma- ILL tion taking place, but, if so, it is just barely 5_ significant statistically. The pairing fraction z rises very rapidly with increasing temperature from 30 C to a maximum at 41 C, and then falls O_ precipitously. The narrowness of the temperature range over which pairing occurs can be ~~I characterized by noting that the full width in 0D _ I temperature at half-maximum pairing is only 50 40 30 10 20 0 8 C. TEMPERATURE (0C) A series of experiments was done to see if inhibition of pairing by low temperatures is FIG. 1. Temperature dependence of specific matreversible. The secondary parental cultures ing-pair formation. Parental cells were mixed at were maintained at 2 C for 10 min which corre- equal concentrations for 7 min. and then assayed for sponds to the same temperature treatment as the concentration of mating pairs, relative to the total that used for pairing at 2 C. The cultures were cell concentration. Each point is the average of the then returned to 37 C for 3 min of temperature values obtained from five to eleven separate matings equilibration, followed by a standard 7-min at each temperature. mating. The value off observed was 2.9 + 1.7%, which is half or less of the normal value at 37 C, but still significantly greater than zero. complete in 5 min (3). Alternatively, it is conceivable that reversible changes in the molecuDISCUSSION lar configuration of pili or receptors occur at The lack of pair formation at 2 C confirms an low temperatures. The rise in pair formation between 24 and observation by Brinton (2) who counted the number of cell pairs that appeared to be con- 37 C follows very closely the results of Novotny nected by F pili as seen in a Petroff-Hauser and Lavin (3) on piliation of the donor cells. In counting chamber. Cells mixed at 0 C showed fact, this detailed agreement may be the no such pairs. This could be caused either by a strongest evidence to date, if more were needed, loss of pili or by an inhibition of pili attachment that pili are an absolute requirement for pair to the female cell. Novotny and Lavin (3) found formation. The precipitous drop above 41 C, on a partial loss of piliation in donor cells that the other hand, is not as easily understood. were shifted from 37 to 0 C, but it was too small There seem to be no data on the melting of F to account for the total lack of pair formation pili, but other types of pili are known to retain shown by my data. It seems likely, therefore, their integrity to much higher temperatures. that pili attachment to the female cell requires As with the situation at 2 C, we may suppose a process with a positive activation energy, pre- either that the donor cells lose their pili or that sumably a chemical reaction. This is supported the pili cannot attach to the female cell. This is by the observation that pairing ability is re- the temperature range in which typical enzycovered as soon as the cells are returned to matic activities and the growth rate of E. coli 37 C. The quantitative loss in pair formation decline sharply with increasing temperature. due to cycling between the two temperatures Consequently, either explanation is a possibilcan be attributed to partial loss in piliation. ity. The possibility cannot be ruled out, however, The results of this work, when taken tothat cellular pairing functions are irreversibly gether with the study of Novotny and Lavin on impaired at 0 C and that the cells generate new the temperature dependence of piliation (3), pili or receptors when returned to 37 C. For show that under normal physiological condiexample, the recovery of pairing ability at 37 C tions the limiting step in pair formation is the after blending the donors at 0 C is virtually production of pili by the donors. 1-
c
I
224
WALMSLEY LITERATURE CITED
1. Achtman, M. 1975. Mating aggregates in Escherichia coli conjugation. J. Bacteriol. 123:505-515. 2. Brinton, C. C. 1965. The structure, function, synthesis
and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram-negative bacteria. Tran. N.Y. Acad. Sci., Ser. III. 27:1003-
J. BACTERIOL. 1054. 3. Novotny, C. P., and K. Lavin. 1971. Some effects of temperature on the growth of F pili. J. Bacteriol. 107:671-682. 4. Walmsley, R. H. 1973. Physical assay of competence for specific mating-pair formation in Escherichia coli. J. Bacteriol. 114:144-151.
Vol. 126, No. 1 Printed in U.SA.
Temperature Dependence of Mating-Pair Formation in Escherichia coli ROGER H. WALMSLEY
Department of Physics, University of Pennsylvania, Philadelphia, Pennsylvania 19174 Received for publication 12 January 1975
The temperature dependence of mating-pair formation by Escherichia coli has been studied from 2 to 45 C by means of a physical assay. No mating-pair formation was found below 24 C. Between 30 and 41 C pair formation increased very rapidly, followed by an equally sharp decline between 41 and 45 C. The results are interpreted in terms of the pilus adsorption process of recipient cells and pili production by donor cells. The first step in the complex process of sexual conjugation in Escherichia coli is the formation of a firmly attached pair of donor and recipient cells. The effects of temperature variation on pair formation are of interest for two reasons. Novotny and Lavin have reported a very sharp decrease in donor cell pilation between 30 and 24 C (3). If pili are an absolute requirement not only for recombinant production (2) but also for pair formation, then pair formation should show a similarly rapid decrease with decreasing temperature. In addition, Novotny and Lavin (3) showed that if cells are cooled rapidly from 37 to 0 C there is only a partial loss in piliation by the donor cells. This makes it possible to see if the pair formation process takes place spontaneously at 0 C even though recombinants cannot be formed at that temperature. MATERIALS AND METHODS Bacterial strains. The donor strain was HfrH (thi-) and the recipient was PA309 (thr-leu- trphis arg- thi- lac- gal- man- xyl- mal-tonrstrr). Media. Tryptone broth contained, per liter: tryptone (Difco), 10 g; nutrient broth (Difco), 6 g; and NaCl, 5 g. Preparation of cells. Overnight cultures were diluted 1:500 into 20-ml culture tubes that contained 10 ml of fresh broth. The cultures were grown at 37 C to exponential phase with moderate agitation on a shaker. Matings. To avoid disturbing the growth of the primary parental cultures, matings were made from secondary parental cultures that were maintained at the same temperature as that at wh>ich the mating was carried out. To do this, a small plastic bucket was clamped to a bar mounted on the shaker above the primary parental cultures in the 37 C water bath. Three culture tubes were placed in the bucket, to which was added water of the desired temperature. The temperature was checked periodically and maintained to within 1 C ofthe average. A 4-ml sample was drawn from each primary parental
tube and transferred to the corresponding secondary parental tube in the bucket, where it achieved the desired mating temperature after 3 min. The angle of inclination of the tubes in the bucket and the amplitude of shaking were identical to those of the primary parental cultures. After temperature equilibration, 2-ml samples were drawn from each of the secondary parental cultures and gently mixed in the third tube in the bucket. The remaining procedures were identical to those described previously (4). Briefly, mating was allowed to proceed for 7 min, at which time culture concentrations were measured with a Coulter counter. Parental cell concentrations in the mating mixture were equal and were typically 108 per ml. The standard deviation of all concentration measurements for all experiments was 5%.
RESULTS The mating mixture pairing fraction, f, is defined (4) as f = c2/(cf + c,,,), where C2, Cf, and c,,, are the concentrations in the mating mixture of mating pairs, recipient cells, and donor cells, respectively. In extended matings the cells eventually form clumps of various sizes, as observed with a microscope (1). In pulsed matings at 108 per ml, however, only donor-recipient pairs are formed to any appreciable extent. Hence the concentration of single cells plus mating pairs in the mating mixture is given by cl. = Cf + C,,, - C2.
The mating mixture pairing fraction is therefore related to the measured concentrations by
f=
1
-
CJ(Cf + c,,,).
For each mating f is calculated. Each point in the figure is the average value of f for all the matings carried out at that temperature. 222
VOL. 126, 1976
MATING-PAIR FORMATION
223
A total of 62 matings were carried out at 20 eight temperatures from 2 to 45 C, the results of which are shown in Fig. 1. The slightly negative value of f at 2 C is due either to an un0 15 _ traced small systematic error or to an unlikely statistical fluctuation. In either case it is clear z that significant pair formation does not take 0 place in a 7-min mating at 2 C. As the tempera- U 1O0ture is raised to 24 C the data indicate the possibility of a very slight degree of pair forma- ILL tion taking place, but, if so, it is just barely 5_ significant statistically. The pairing fraction z rises very rapidly with increasing temperature from 30 C to a maximum at 41 C, and then falls O_ precipitously. The narrowness of the temperature range over which pairing occurs can be ~~I characterized by noting that the full width in 0D _ I temperature at half-maximum pairing is only 50 40 30 10 20 0 8 C. TEMPERATURE (0C) A series of experiments was done to see if inhibition of pairing by low temperatures is FIG. 1. Temperature dependence of specific matreversible. The secondary parental cultures ing-pair formation. Parental cells were mixed at were maintained at 2 C for 10 min which corre- equal concentrations for 7 min. and then assayed for sponds to the same temperature treatment as the concentration of mating pairs, relative to the total that used for pairing at 2 C. The cultures were cell concentration. Each point is the average of the then returned to 37 C for 3 min of temperature values obtained from five to eleven separate matings equilibration, followed by a standard 7-min at each temperature. mating. The value off observed was 2.9 + 1.7%, which is half or less of the normal value at 37 C, but still significantly greater than zero. complete in 5 min (3). Alternatively, it is conceivable that reversible changes in the molecuDISCUSSION lar configuration of pili or receptors occur at The lack of pair formation at 2 C confirms an low temperatures. The rise in pair formation between 24 and observation by Brinton (2) who counted the number of cell pairs that appeared to be con- 37 C follows very closely the results of Novotny nected by F pili as seen in a Petroff-Hauser and Lavin (3) on piliation of the donor cells. In counting chamber. Cells mixed at 0 C showed fact, this detailed agreement may be the no such pairs. This could be caused either by a strongest evidence to date, if more were needed, loss of pili or by an inhibition of pili attachment that pili are an absolute requirement for pair to the female cell. Novotny and Lavin (3) found formation. The precipitous drop above 41 C, on a partial loss of piliation in donor cells that the other hand, is not as easily understood. were shifted from 37 to 0 C, but it was too small There seem to be no data on the melting of F to account for the total lack of pair formation pili, but other types of pili are known to retain shown by my data. It seems likely, therefore, their integrity to much higher temperatures. that pili attachment to the female cell requires As with the situation at 2 C, we may suppose a process with a positive activation energy, pre- either that the donor cells lose their pili or that sumably a chemical reaction. This is supported the pili cannot attach to the female cell. This is by the observation that pairing ability is re- the temperature range in which typical enzycovered as soon as the cells are returned to matic activities and the growth rate of E. coli 37 C. The quantitative loss in pair formation decline sharply with increasing temperature. due to cycling between the two temperatures Consequently, either explanation is a possibilcan be attributed to partial loss in piliation. ity. The possibility cannot be ruled out, however, The results of this work, when taken tothat cellular pairing functions are irreversibly gether with the study of Novotny and Lavin on impaired at 0 C and that the cells generate new the temperature dependence of piliation (3), pili or receptors when returned to 37 C. For show that under normal physiological condiexample, the recovery of pairing ability at 37 C tions the limiting step in pair formation is the after blending the donors at 0 C is virtually production of pili by the donors. 1-
c
I
224
WALMSLEY LITERATURE CITED
1. Achtman, M. 1975. Mating aggregates in Escherichia coli conjugation. J. Bacteriol. 123:505-515. 2. Brinton, C. C. 1965. The structure, function, synthesis
and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram-negative bacteria. Tran. N.Y. Acad. Sci., Ser. III. 27:1003-
J. BACTERIOL. 1054. 3. Novotny, C. P., and K. Lavin. 1971. Some effects of temperature on the growth of F pili. J. Bacteriol. 107:671-682. 4. Walmsley, R. H. 1973. Physical assay of competence for specific mating-pair formation in Escherichia coli. J. Bacteriol. 114:144-151.
