growth temperature of Pseudomonas aeruginosa. J. Bacteriol. 92:43-48. 1966. ... aeruginosa if the FP- parent is grown at 43 C prior to mating. This occurs where.
Vol. 92, No. 1 Printed in U.S.A.
JOURNAL OF BACTERIOLOGY, July, 1966 Copyright © 1966 American Society for Microbiology
Alterations in Host Specificity of Bacterial Deoxyribonucleic Acid After an Increase in Growth Temperature of Pseudomonas aeruginosa B. ROLFE AND B. W. HOLLOWAY Department of Microbiology, University of Melbourne, Victoria, Australia
Received for publication 18 March 1966
ABSTRACT B. of ROLFE, (University Melbourne, Victoria, Australia), AND B. W. HOLLOWAY.
Alterations in host specificity of bacterial deoxyribonucleic acid after an increase in growth temperature of Pseudomonas aeruginosa. J. Bacteriol. 92:43-48. 1966.Increased recombination frequency is found with some crosses of Pseudomonas aeruginosa if the FP- parent is grown at 43 C prior to mating. This occurs where there is a difference in host specificity between the FP- and FP+ parents, as is found in strains 1 and 2. Differences in recovery of unlinked markers are found in such crosses, and the results can be explained in terms of an alteration of the restriction and modification mechanisms by growth at the higher temperature, similar to that previously shown to occur with host-modified bacteriophage. As with that system, persistence of the temperature effect occurs for a number of generations after the strains are returned for growth at 37 C. It has been previously been shown (7) that when Pseudomonas aeruginosa is grown at 43 C, and subsequently infected with phage at 37C, there is no restriction of host-modified phage. The host specificity of the phage particles which have multiplied in these strains grown at 43 C prior to infection is different from that of phage particles propagated in bacteria grown only at 37 C. This can be shown by the different host range of phage particles released from infected bacteria previously grown at 43 C. Furthermore, these characteristics are retained for more than 60 generations when bacteria grown at 43 C are returned to growth at 37 C. It was shown (3) that the host specificity of a phage particle resides on the deoxyribonucleic acid (DNA). Differences in bacterial host specificity have been demonstrated in crosses between Escherichia coli K-12 and E. coli B (2, 4, 5). Similarly, it was shown (11) that phage-controlled restriction due to the phage P1 has considerable effects on genetic linkage in bacterial crosses. Therefore, if the above effects of temperature on the Pseudomonas system involved alterations in DNA specificity, then it should be possible to investigate any effects of restriction on bacterial recombination by growth at 43 C.
Genetic recombination has been shown to occur between strains 1 and 2 of P. aeruginosa (6), and these strains also show differences in host specificity (9). Crosses were made between strains 1 and 2 with the parental strains grown at 37 or 43 C, and the effects on prototroph formation and recombination of unselected markers were observed. The results supplement those obtained previously with phage infection, and indicate that the observed effects can be explained in terms of restriction and DNA specificity. MATER1ALS AND METHODS Bacterial strains. The strains used in this study are listed in Table 1. Media. The Minimal Medium of Vogel and Bonner (12), solidified where necessary with 1.5% Difco agar, was used. Nutrient broth contained 1% peptone (Evans), 1% beef extract (Bonox), and 0.5% sodium chloride. Nutrient agar was nutrient broth solidified with 1.2% agar (Davis Gelatin Ltd., New Zealand). Cultural procedures. Cultural procedures were the same as previously described (7, 9). Mating procedure on plates. The two auxotrophic parental strains were grown separately overnight in nutrient broth at 37 or at 43 C without shaking. Both parental cultures were then centrifuged and resuspended in saline to a concentration of between 2 X 109 43
44
ROLFE AND HOLLOWAY
J. BACTERIOL.
TABLE 1. Bacterial strains used Strain
1 1 1 1 1 1
ilva FPleu FPade-I FP-
ser FPade-2 FPmet try str-r FP1 ade leu FP+
1 leu str-r FP+ 2 2 2 2
try-3bi FP+ thr FP+ pro-1 FP+ ilva FP+
Description
Reference
Isoleucine plus valine-requiring mutant of strain 1 Leucine-requiring mutant of strain 1 Adenine-requiring mutant of strain 1 Serine-requiring mutant of strain 1 Adenine-requiring mutant of strain 1 Methionine and tryptophan-requiring, streptomycin-resistant double mutant of strain 1 Adenine and leucine-requiring double mutant of strain 1 Leucine-requiring, streptomycin-resistant mutant of strain 1 Tryptophan-requiring mutant of strain 2 Threonine-requiring mutant of strain 2 Proline-requiring mutant of strain 2 Isoleucine plus valine-requiring mutant of strain 2
and 3 X 109 organisms per milliliter. In all crosses, the density of the different recipient cells was calibrated by means of Burroughs Wellcome opacity tubes. Viable-cell counts were also recorded. The two parental suspensions were mixed, and 0.2 ml of the mixture was immediately plated onto the surface of minimal or supplemented minimal plates and spread evenly with a glass spreader. Controls of each parent were treated in the same manner to detect backmutation. The number of recombinant colonies per plate was recorded after 48 hr of incubation at 37 C. Mating in broth. A 1-ml amount of an overnight broth culture of each of the parental strains was added to 5 ml of nutrient broth supplemented with 1% glucose. This suspension was grown without shaking in a 100-ml flask for 90 min at 37 C. The cells were then centrifuged, resuspended in 2 ml of saline, and 0.2 ml was plated onto the surface of the appropriate plates. All interstrain crosses were made by the first method described above, because strain 1 is aeruginocinogenic for strain 2, and mating in broth gives very low recombination frequencies for 1 X 2 crosses. In general, both these techniques give good day-to-day reproducibility. To examine the segregation of nonselected markers, the recombinants were streaked for single colonies on the same medium on which the cross was performed, and then were spot tested (or replica plated) onto different supplemented and minimal medium plates. Resistance or sensitivity to streptomycin was determined with the use of nutrient agar plates plus 250 ,ug ml of streptomycin.
RESULTS
Early experiments on conjugation in P. aeruginosa (6) had shown that, whereas the unrelated strains 1 and 2 were interfertile, only some combinations of markers produced prototrophs. It was subsequently shown that when the same markers were crossed in 1 FP- X 1 FP+ crosses, prototrophs were readily formed. This suggested that some characteristic of the strain 2 chromo-
Holloway Holloway Holloway Holloway Holloway Holloway
(6) (6) (6) (6) (6) and Fargie (8)
Holloway and Fargie (8)
Holloway and Fargie (8)
Holloway Holloway Holloway Holloway
(6) (6) (6) (6)
some was involved, possibly the different host specificities of strains 1 and 2. Knowing that the manifestations of host specificity and restriction in Pseudomonas phage system can be modified considerably after growth at 43 C, crosses were set up between strains 1 and 2 in which the female or recipient strain 1 was grown at either 43 or 37 C before mating. It should be stressed that in all these experiments mating and incubation of plates after plating of the conjugating cells is carried out at 37 C. Preliminary experiments have shown that the recovery of prototrophic recombinants is greatly reduced when mating mixtures are grown at 43 C. The strain 1 FP- auxotrophs were grown for approximately 13 generations at either 37 or 43 C and then crossed to the appropriate male strain grown at 37 C. The results of a number of such matings are shown in Table 2. It is seen that more prototrophic recombinants are recovered when the female parent is grown at 43 C, rather than at 37 C. The effect is not uniform for all markers. For example, where selection for leu+ is made, there is at the most only a fivefold increase for the parent grown at 43 C, in contrast to the selection for ser+ where the increase in recovery of recombinants is particularly marked. Three possible explanations of these results can be suggested. First, growth at 43 C may enhance conjugation and transfer of chromosome from male to female parent. Under these circumstances, we would expect little or no difference between linkage values of unselected markers for parents grown at 37 and 43 C. Alternately, growth at 43 C may result in a reduced restriction mechanism (as occurs in phage infection), and thus a greater probability of integration of
VOL. 92, 1966
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TEMPERATURE EFFECTS ON PSEUDOMONAS
TABLE 2. 1 X 2 crosses showing the effect of growing the recipient strain at 43 C on the frequency of recombinationa Strain 1 FP auxotrophic cells
Selection for
No. of recombinants per 109 female cells after mating with strain 2 FP+ auxotrophic cells 2 ilva FP+
1 1 1 1 1 1 1 1 a
ser (37 C) ser (43 C) leu (37 C) leu (43 C) met try (37 C) met try (43 C) ilva (37 C) ilva (43 C)
ser+
serf leu+ Ieu+ try+ try+ i1va+ ilva +
5.3 1.5 2.3 5.0 1.1
19 X 103 X 104 X 104 X 103 X 104
2 try 3bi FP+
2 thr FP+
2pro- 1 FP+
0 50 2.8 X 103 6.6 X 103
0 250 358
0 450 372 2.6 X 103 10 95 3 264
40 99
3.0 X 103 8 58 5 462
All donor strains were grown at 37 C.
the male chromosome into the female parent would occur. Hence, not only will prototroph recovery be higher, but linkage values for unselected markers will be higher. The remaining possibility is that both these mechanisms may be operating. In the cross, 1 met try str-r FP- X 2 ilva str-s FP+ plated on methionine, with selection for try+, we can examine the recovery of the unselected markers met and str where the female parents were grown at either 37 or 43 C (Table 3). It is seen that, although this is one cross where the number of prototrophic recombinants does not differ much when the female parent is grown at either 37 or 43 C, there is a significant difference in the linkage values of met, try, and str for the parent grown at each temperature. This result thus favors the view that restriction is operating in crosses of 1 X 2 grown at 37 C, which is abolished or modified when the female parent is grown at 43 C. Further evidence supporting this view comes from the results of Table 2. If the increase in recombination frequencies was due solely to effects on the efficiency of formation of mating pairs, we would expect that it would be independent of the marker being selected, and that more or less equivalent rises would be found when a range of markers is tested. In Table 2,
however, selection for two markers, try+ and leu+, gives much the same recombination frequency for female parents grown at 37 or 43 C, in contrast to the 100-fold or greater increases in prototroph recovery found with the parents grown at 43 C, for other marker combinations. An important consideration in this respect is the oriented transfer of the male chromosome, and reference to this will be made in the Discussion. I X I crosses. Previous experiments with phage suggested that, not only did growth at 43 C pro-
duce aberrations in the restriction mechanism, but it also imposed a new host specificity upon the DNA of phage liberated from bacteria grown at 43 C. It seems logical to assume that the DNA of these bacteria has a characteristic specificity, and it should be possible to demonstrate this by crossing experiments similar to that described above. If the DNA specificity of bacteria grown at 43 C is different from that of cells grown at 37 C, then we can best detect it by growing the male at 43 C and the female at 37 C and comparing this cross with one in which both parents are grown at 37 C. In the former cross, the restriction mechanisms of the female grown at 37 C should act on the DNA of different specificity from that of the male grown at 43 C. Experiments of this type are shown in Table
TABLE 3. Influence of parental incubation temperature on the recovery of unselected FP+ markers in a 1 X 2 cross Cross Cross
1 met try str-r FP- (37 C) X 2 ilva str-s FP+ (37 C) 1 met try str-r FP- (43 C) X 2 ilva str-s FP+ (37 C)
of try+ recombi~~~~~~~~~No. nants per 109 FP+ cells
5.0 X 103 1.1 X 104
Per cent try+ recombinants that have the FP+ alleles
try+met+
try+str-s
8 56
41 81
46
J. BACTERIOL.
ROLFE AND HOLLOWAY
4. It is seen that the prototroph-forming capacity of males grown at 43 C is very much less than the corresponding males grown at 37 C, in agreement with the above argument. However, an objection could be raised here similar to that used with the 1 X 2 crosses, namely, that the ability of the strain 1 males grown at 43 C to form mating pairs or to transfer chromosome is lower than that of males grown at 37 C. That this is not so is shown first by the crosses between organisms both grown at 43 C (Table 4), where it is seen that the recombination frequency per input male is as high as in the males grown at 37 C. Furthermore, if the effects of temperature are limited to the formation of mating pairs and chromosome transfer, then we should see no effects on linkage, assuming both these stages of the conjugation process have no effect on the recombination event. In Table 5, the segregation of unselected markers is followed, and a comparison is made between 37 C X 37 C and 37 C X 43 C crosses. It is seen thai the latter show considerable perturbations of linkage values, consistent with the breaking down of the chromosome by a restriction
process as suggested by Arber and Morse (1), Boyer (2), and Pittard (11). Similar results to the crosses between organisms both grown at 37 C were obtained for crosses between females grown at 43 C and males grown at 37 C. Persistence of temperature-induced mating effects. One of the most curious and as yet unexplained features of the differences caused by growth at 43 C is the persistence of the effect for up to 60 generations or more when the bacteria grown at 43 C are returned to growth at 37 C (7). If the above effects in bacterial conjugation found with cells grown at 43 C are another manifestation of the same effect, then we would expect to observe a persistence of the increased recovery of prototrophs even after the female cells grown at 43 C were returned to 37 C. It is shown in Table 6 that the effect persists for at least 30 generations. Similar experiments were applied to 1 x 1 crosses after the segregation of the unselected markers. The results in Table 7 show that the alteration of linkage values, found when strain 1 males grown at 43 C are crossed with strain 1 female cells grown at 37 C, persisted for at least
TABLE 4. Differences in host specificity of bacterial DNA emerging from straint I FP+ strains grown at 37 and 43 C No. of recombinants per i0' FP+ cells
Cross
Selection for
1 ilva str-r FP- (37 C) X 1 ade leu FP+ (37 C) 1 ilva str-r FP- (37 C) X 1 ade leu FP+ (43 C) 1 ilva str-r FP- (43 C) X 1 ade leu FP+ (43 C)
ilva+ ilva+ ilva+
740 78 592
1 ser FP- (37 C) X 1 ade leu FP+ (37 C) 1 ser FP- (37 C) X 1 ade leu FP+ (43 C) 1 ser FP- (43 C) X 1 ade leu FP+ (43 C)
ser+ ser+ ser+
2,300
1 met try FP- (37 C) X 1 leu str-r FP+ (37 C) 1 met try FP- (37 C) X 1 leu str-r FP+ (43 C) 1 met try FP- (43 C) X 1 leu str-r FP+ (43 C)
met+ met+ met+
2,170
1 ser FP- (37 C) X 1 leu str-r FP+ (37 C) 1 ser FP- (37 C) X 1 leu str-r FP+ (43 C) 1 ser FP- (43 C) X 1 leu str-r FP+ (43 C)
ser+ ser+ ser+
5,000
420
1,100 350
1,880 910
2,100
TABLE 5. Influence of parental incubation temperature on the recovery of unselected FP+ markers in I X I crosses Selected marker
markLinkage of unselected marker1 ers to selected %
1 ilva str-r FP- (37 C) X 1 ade leu str-s FP+ (37 C) 1 ilva str-r FP- (37 C) X 1 ade leu str-s FP+ (43 C) 1 i/va str-r FP- (43 C) X 1 ade leu str-s FP+ (43 C)
ilva+ ilva+ i1va+
ade, 23; str-s, 16 ade, 9; str-s, 7 ade, 14; str-s, 20
1 ser str-s FP- (37 C) X 1 leu str-r FP+ (37 C)
ser+ serf
str-r, 38 str-r, 13
Cross
1 ser str-s FP- (37 C) X 1 leu str-r FP+ (43 C)
VOL. 92, 1966
TEMPERATURE EFFECTS ON PSEUDOMONAS
47
TABLE 6. Persistence of the temperature effect on recombination frequency in 1 X 2 crossesa No. of recom- Ratio of 1 ser FP- (43) binants per recombinants/1 ser FP109 FP- cells (37) recombinants
Cross
1 ser grown at 37 C for 15 generations 1 ser grown at 43 C for 13 generations
192
16
1 ser (37) grown at 37 C for 13 generations 1 ser grown at 43 C for 13 generations, then for 13 generations at 37 C
4 290
72
1 ser (37) grown at 37 C for 26 generations 1 ser grown at 43 C for 13 generations then for 26 generations at 37 C
12 142
11.8
1 ser (37) grown at 37 C for 41 generations 1 ser grown at 43 C for 13 generations then for 41 generations at 37 C
21 28
1.3
12
a The recipient strain 1 ser FP- was grown at 37 C and at 43 C prior to mating with the strain 2 ilva grown at 37 C. Samples from these recipient cultures, containing between 107 and 10' bacteria per milliliter, were inoculated into 100 ml of fresh nutrient broth and grown overnight with agitation at 37 C. Bacteria from this passage were used to inoculate another 100 ml of fresh nutrient broth, and the culture was grown in the same manner. This serial passage was carried out for a total of about 40 generations at 37 C; the inoculum at each transfer was always greater than 107 bacteria. After each passage of cells, the efficiency of recombinant formation was measured, by mating the passaged recipient cells with freshly grown cultures of strain 2 ilva FP+. The number of prototrophs formed was recorded, and the results were calculated as the number of recombinants per 109 FP- cells.
TABLE 7. Persistence of the temperature effect on recombination frequency and the unselected parenztal markers in I X I crosses No. of mnet+ recombinants per 10' FP+ cells
Cross
Per cent met+ recombinants that are FP- type FP+ type
try+str-r
try-str-s
350 1,880
92 59 73
2 30 11
Strain 1 leu str-r FP+ (43 C) returned to 37 C for 13 generations 1 met try str-s FP- (37 C) X 1 leu str-r FP+ (37 C) 1 met try str-s FP- (37 C) X 1 leu str-r FP+ (43 C/37 C)
2,060 400
89 56
2 28
Strain 1 leu str-r FP+ (43 C) returned to 37 C for 26 generations 1 met try str-s FP- (37 C) X 1 leu str-r FP+ (37 C) 1 met try str-s FP- (37 C) X 1 leu str-r FP+ (43 C/37 C)
2,430 1,430
88 70
3 16
1 met try str-s FP- (37 C) X 1 leu str-r FP+ (37 C) 1 met try str-s FP- (37 C) X 1 leu str-r FP+ (43 C) 1 met try str-s FP- (43 C) X 1 leu str-r FP+ (43 C)
20 generations. These experiments demonstrate that the temperature effect is not exhibited solely with bacteriophage infections. Bacterial DNA has a different host specificity if cells of the same strain are grown at 43 C rather than 37 C. It is probable that the mechanisms for restricting foreign nucleic acids derived from either phage infection or conjugation have a common genetic basis which can be altered by growing cells at 43 C. Apparently this altered condition of the host specificity has the property of persisting over a large number of generations at 37 C.
2,170
DIscussIoN We have shown that mating between strains of different native host specificity can be enhanced when the recipient strain is grown at 43 C. This result can be explained by an alteration in the restriction mechanisms of the recipient strain, similar to that shown previously with the acceptance of host-modified bacteriophage DNA by such bacteria (7). Furthermore, in crosses where no such native host specificity occurs, as in strain 1 X strain 1 crosses, we can impose a temperature-induced one by growing the male parent at
48
ROLFE AND HOLLOWAY
43 C. This is also analogous to the previously described situation where phage yielded from bacteria grown at 43 C had acquired a completely new host range. These results clearly confirm the results of earlier workers (2, 4, 10), who have shown that the restriction and modification mechanisms of bacteria apply equally well to phage and bacterial DNA. They further show that the temperature-induced alterations in Pseudomonas apply to both types of DNA. The observed modifications of linkage confirm our view that these alterations are compatible with a restriction mechanism rather than, in the case of the conjugation experiments, alterations in cell contact and chromosome transfer. The results for the 1 X 2 crosses have provided an explanation for the observation made early in the Pseudomonas genetic work that, with 1 x 2 crosses, certain combinations of markers failed to give any recombinants. It seems to us that the most likely explanation of these results is that DNA which is transferred from the strain 2 donor is recognized as foreign and is broken-down before integration by the process of recombination can occur in the FP (37 C) cells. Boyer (2) suggested that one way by which the restrictive mechanism of a recipient cell could be overcome would be by the introduction of large fragments of male DNA which could saturate the cell nucleases, so that some integration could occur. It has been demonstrated by interrupted mating techniques (Holloway, unpublished data) that an oriented transfer of male chromosome into the female parent occurs in FP- X FP+ crosses. The time of transfer found for various markers strongly supports the view that the variations in prototroph frequency for various 1 X 2 crosses (6) are related to the amount of male DNA transferred in each case. It has been found that ser and ilva are transferred relatively early, whereas transfer of try and leu occurs much later. These results are in harmony with the comparison of recombination frequencies shown in Table 2 for female strains grown at 37 and 43 C, whereas we would expect more nuclease saturation at 37 C with the late entry mutants, and hence less difference in recombination frequency between parents grown at 37 and 43 C. Just as the ability to accept and support multiplication of host-modified phage persists in cells grown at 43 C for up to 70 generations after they are returned to growth at 37 C, so persistence of these temperature-induced effects on conjugation
J. BAC-ERIOL.
occurs for nearly 30 generations. While clearly confirming the generality of these effects, we are still left with two major unanswered problems: first, the mechanism by which growth at 43 C affects restriction and modification, and second, the genetic control of this mechanism and the reason for its persistence at 37 C for so many
generations. ACKNOWLEDGMENTS This investigation was supported by a grant from the National Health and Medical Research Council of Australia. LITERATURE CITED 1. ARBER, W., AND M. L. MORSE. 1965. Host specificity of DNA produced by Escherichia coli. VI. Effects on bacterial conjugation. Genetics 51:137-148. 2. BOYER, H. 1964. Genetic control of restriction and modification in Escherichia coli. J. Bac-
teriol. 88:1652-1660. 3. Dussoox, D., AND W. ARBER. 1962. Host specificity of DNA produced by Escherichia coli. II. Control over acceptance of DNA from infecting phage X. J. Mol. Biol. 5:37-49. 4. GLOVER, S. W., AND C. COLSON. 1965. The breakdown of the restriction mechanism in zygotes of Escherichia coli. Genet. Res. 6:153-155. 5. HOEKSTRA, W. P. M., AND P. G. DE HAAN. 1965. The location of the restriction locus for X. K in Escherichia coli B. Mutation Res. 2:204-212. 6. HOLLOWAY, B. W. 1955. Genetic recombination in Pseudomonas aeruginosa. J. Gen. Microbiol. 13:572-581. 7. HOLLOWAY, B. W. 1965. Variations in restriction and modification of bacteriophage following increase of growth temperature of Pseudomonas aeruginosa. Virology 25:634-642. 8. HOLLOWAY, B. W., AND B. FARGIE. 1960. Fertility factors and genetic linkage in Pseudomonas aeruginosa. J. Bacteriol. 80:362-368. 9. HOLLOWAY, B. W., AND B. ROLFE. 1964. Host genome control in host-induced modification of Pseudomonas aeruginosa phages. Virology 23:
595-602. 10. LEDERBERG, S. 1965. Host-controlled restriction and modification of deoxyribonucleic acid in Escherichia coli. Virology 27:378-387. 1 1. PrTTARD, J. 1964. Effect of phage-controlled restriction on genetic linkage in bacterial crosses. J. Bacteriol. 87:1256-1257. 12. VOGEL, H. J., AND D. M. BONNER. 1956. Acetylornithase of Escherichia coli: partial purification and some properties. J. Biol. Chem. 218: 97-106.
