JOURNAL OF BACTZRIOLOGY, Mar. 1976, p. 780-786
Vol. 125, No. 3 Printed in U.S.A.
Copyright © 1976 American Society for Microbiology
Suppressor Mutations in Pseudomonas aeruginosa J. M. WATSON
AND
B. W. HOLLOWAY*
Department of Genetics, Monash University, Clayton, Victoria 3168, Australia Received for publication 10 October 1975
Suppressor mutations of Pseudomonas aeruginosa have been identified. An isolate of strain PAT, initially selected as being temperature sensitive for growth, was found to suppress two different auxotrophic mutations. A suppressor locus, designated sup-i, has been mapped and is co-transducible with three closely linked thr loci. The suppressor mutation has been used to isolate suppressor-sensitive (sus) mutants of the virulent phage E79 and the R factor R18. By selecting for revertants of auxotrophic markers, other sup mutants have been isolated and are found to be of two types, either temperature sensitive for growth like the original mutant or showing wild-type growth at 43 C. The mutations giving rise to both these classes of suppressor are very closely linked. One of the sup-i alleles of strain PAT also shows suppressor activity when transferred into P. aeruginosa strain PAO. Escherichia coli strains carrying the nonsense suppressors supC, supD, or supF do not suppress the sus mutant of R18. This suggests that sup-1 is different from the amber and ochre suppressors of the enterobacteria. agar was Vogel and Bonner minimal medium (11) solidified with 1.2% Oxoid agar no. 1. Minimal agar was supplemented with L-amino acids at a final concentration of 1 mM except isoleucine, which was used at 0.5 mM. Layer agar was NB containing 0.8% Oxoid agar no. 1. TNM buffer, for preparation of bacteriophage lysates and dilutions, was 0.01 M tris(hydroxymethyl)aminomethane plus 0.15 M sodium chloride and 0.01 M magnesium sulfate (pH 7.4). Bacterial mutagenesis and carbenicillin enrichment. Bacterial mutants were obtained by EMS mutagenesis as follows. Two drops (ca. 0.05 ml) of EMS was added to 5 ml of an exponential-phase culture in NB, vortexed vigorously, and incubated at 37 C for 1 h without shaking. One-milliliter aliquots of the MATERIALS AND METHODS treated culture were diluted 1 in 30 in NB and Bacterial and bacteriophage strains. The bacte- incubated at 37 C overnight with shaking to allow rial strains used in this study are shown in Table 1. expression of mutations. This culture was diluted 1 in 30 in prewarmed NB All PAT recipient (FP-) strains were derived from PAT964 (10) by ethyl methane sulfonate (EMS) mu- and incubated at 43 C for 1 h with shaking. Carbenitagenesis. The PAO strains used were derived from cillin was then added to a final concentration of 2 PA0242 (7) by EMS mutagenesis. PT0226 is an mg/ml, and the culture was incubated at 43 C for a interstrain recombinant constructed by transducing further 6 h with shaking. Residual carbenicillin was PA0225 with F116C4 (V. Krishnapillai, unpub- inactivated by adding penicillinase (300 Levy units/ lished data) propagated on PAT2145. Phage F116L ml) and incubating for 1 h at 43 C with shaking. The (7) was used for all transductional analyses. Sup- culture was then incubated overnight at 37 C with pressor-sensitive derivatives of the virulent phage shaking. The culture was diluted appropriately in saline, and 0. 1-ml aliquots were spread on HIA E79 (5) are designated E79 sus-1 and E79 sus-2. Media. Nutrient broth (NB) was 2.5% Oxoid nu- plates and scored for mutants which grew at 37 C trient broth no. 2 supplemented with 0.5% Oxoid but not at 43 C by replica plating. Bacteriophage mutagenesis. The following proceyeast extract. Heart infusion agar (HIA) was Difco heart infusion broth solidified with 1.2% Oxoid agar dure was used to isolate suppressor-sensitive (sus) no. 1. The antibiotics streptomycin (Sigma), rifam- mutants of phage E79. A 5-ml amount of an expopin (Ciba-Geigy), carbenicillin (Beecham), kanamy- nential-phase culture of PAT2145 was mixed with cin (Sigma), and tetracycline (Lederle) were added an equal volume of phage E79 (at a multiplicity of to HIA at the concentrations indicated. Minimal infection of approximately 2) and incubated at 37 C 780
The characterization of suppressor mutations in bacteria has contributed much to the understanding of the expression of genetic information. Equally important, they have been a valuable experimental tool for microbial geneticists. This applies particularly to the translational suppressor mutations as detailed in a recent review (4). This paper describes the isolation of a suppressor mutation in Pseudomonas aeruginosa and the genetic evidence that supports this view. Complete identification of the suppressor mechanism awaits the biochemical investigation of this mutant.
P. AERUGINOSA SUPPRESSOR MUTATIONS
VOL. 125, 1976
TABLE 1. Bacterial strainsa Genotype and locus
Strain PAT2 PAT2127
PAT2141 PAT2143
PAT2145 PAT2151 PAT2152 PAT2153 PAT2154 PAT2155
PAT2177 PAT2205 PAT2206 PAT2207 PAT2208 PAT2209 PAT2210 PA0222 PA0225 PA0230 PT0226
Prototroph, FP2+ lys-1115, trp-3114, pur-1118, leu-2104, arg-1130, FPlys-1115, trp-3114, pur-1118, leu-2104, thr1104, FPlys-1115, trp-3114, pur-1118, leu-2104, pro2108, FPlys-1115, trp-3114, pur-1118, leu-2104, pro2108, sup-1100, FPlys-1115, trp-3114, pur-1118, leu-2104, pro2108, str-1114, FPPrototroph, str-1105, sup-1100, FP2+ Iys-1115, trp-3114, pur-1118, leu-2104, thr2106, FPlys-1115, trp-3114, pur-1118, leu-2104, thr3107, FPlys-1115, trp-3114, pur-1118, leu-2104, thr1108, FPlys-1115, trp-3114, pur-1118,1eu-2104, pro2108, his-2117, ilv-1118, thr-1109, FPSame at PAT2177 but sup-1103 Same as PAT2177 but sup-1104 Same as PAT2177 but sup-1105 Same as PAT2177 but sup-1106 Same as PAT2177 but sup-1107 Same as PAT2177 but sup-1108 met-28, trp-6, lys-12, his-4, pro-82, ilv-226, FPmet-28, trp-6, Iys-12, his-4, pro-82, ilv-226, thr-48, FPSame as PA0225 but str-44 met-28, trp-6, lys-12, his-4, pro-82, ilv-226, sup-1O1, FP- (see Materials and Meth-
ods) PT0231
Same
as
PT0226 but rif-7
Abbreviations: arg, Arginine requirement; his, histidine requirement, ilv, isoleucine and valine requirement; leu; leucine requirement; Iys, lysine requirement; pro, proline requirement; pur, adenine requirement; rif, rifampin resistance; str, streptomycin resistance; sup, suppressor activity; thr, threonine requirement; trp, tryptophan requirement. For PAT strains, the first number following each gene symbol indicates the arbitrary locus designation, whereas the following three numbers refer to the allele number; e.g., lys-1115 indicates allele 115 of the Iys-1 locus. a
for 20 min without shaking. The cells were then deposited by centrifuging for 5 min. The pellet was resuspended in the same volume of prewarmed citrate buffer (pH 5.6) containing 50 ,ig of N-methylN'-nitro-N-nitrosoguanidine per ml and incubated at 37 C for 15 min without shaking. The cells were then centrifuged, washed once in citrate buffer, and finally resuspended in 2 volumes of prewarmed NB. The treated cells were incubated at 37 C overnight with shaking to allow liberation of progeny phage. The overnight culture was centrifuged, and the supernatant was sterilized by passage through a type HA membrane filter (pore size, 0.45 ,um; Millipore Corp.). Plaques were isolated by overlaying HIA plates with 1 ml of appropriately diluted supernatant and 2 ml of layer agar seeded with 0.1 ml of an overnight culture of PAT2145. Individual plaques were then picked and spotted, first to a layer seeded with PAT2145 (permissive host) on HIA and then to a layer seeded with PAT2151 (restrictive host) on HIA containing 1 mg of streptomycin per ml.
781
Plaques showing lysis on PAT2145 but not on PAT2151 were purified by three consecutive singleplaque isolations on PAT2145. R-factor mutagenesis. An exponential-phase culture of PT0231 (R18) was centrifuged, and the pellet was resuspended in prewarmed citrate buffer (pH 5.6) containing 50 ,ug of N-methyl-N'-nitro-N-nitrosoguanidine per ml. After incubating at 37 C for 15 min without aeration, the cells were deposited by centrifugation, washed once in citrate buffer, and resuspended to original volume in NB. The cells were then diluted 1 in 30 in NB and incubated at 37 C overnight with aeration to allow expression of mutations. The mutagenized R factors were then transferred into a nonsuppressor host by mixing 2 ml of this culture with 2 ml of an overnight culture of PA0230 and incubating at 37 C without shaking. After 2 h the mixture was vortexed and diluted, and 0.1-ml aliquots were plated on HIA containing 1 mg of streptomycin per ml and 250 jAg of either carbenicillin, kanamycin, or tetracycline per ml. After overnight incubation, the colonies were replica plated to HIA containing streptomycin and either of the nonselective drugs at the above concentrations. Transduction. The procedure used for transduction has been described previously (7).
RESULTS Isolation of the suppressor mutant. Strain PAT2145 was isolated as a temperature-sensitive derivative of strain PAT2143 after EMS mutagenesis and carbenicillin enrichment, using 43 C as the restrictive temperature. PAT2145 grows very poorly on HIA at 37 C relative to the parent strain PAT2143, and, in addition, it was found that PAT2145 no longer required either lysine or adenine. The loss of these two auxotrophies by simultaneous independent reversion of the original mutations seemed improbable. A likely alternative hypothesis was that the two auxotrophic lesions were suppressed either by the newly introduced temperature-sensitive mutation or by mutation at another site introduced during the mutagenesis. Evidence of suppressor activity. Support for the suppressor mutation hypothesis was sought by looking for recombinants in which such a mutation had been separated from the adenine and lysine alleles. PAT2145 was transduced with phage F116L propagated on the wildtype strain PAT2, and selection made for temperature-resistant transductants on HIA at 43 C. Approximately 1,500 transductants selected in this way were tested, and all were found to be both lysine- and adenine-requiring and thus, in this respect, had the same phenotype as the parent strain PAT2143. Independent mapping experiments using interrupted mating (J. Watson, unpublished data) have shown that lys-
782
WATSON AND HOLLOWAY
J. BACTERIOL.
1115 andpur-1118 are more than 20 min apart markers is significantly lower than the exon the PAT chromosome. Thus, the above pected value of about 13% obtained in the contransductants could not have arisen by cotrans- trol transduction using PAT2 as the donor. The duction of the wild-type alleles of the pur, lys, apparent anomaly in these results was resolved and temperature-sensitive loci. The most likely by further examination of the cotransductants explanation for these results is that the temper- found to grow poorly on HIA at 37 C and to be ature-sensitive mutation acts as a suppressor of phenotypically Lys' and temperature sensitive. These observations indicated that the three thrthe Iys-1115 and pur-1118 alleles. Evidence that the same mutation determines 1104+ transductants had coinherited sup-1100 suppressor activity and temperature sensitivity from PAT2145 to give them their Pur+ phenowas obtained from an examination of tempera- type. The above results indicate that PAT2145 ture-insensitive revertants. Strain PAT2145 re- still carries the pur-1118 mutation and, furverts to temperature insensitivity at a fre- ther, that the sup-1 1 00 mutation is cotransduquency of approximately 10-8 on HIA at 43 C. cible with the thr-1104 mutation. Mapping of the suppressor locus. The useWhen 100 such revertants were tested, all were found to have regained the lysine and adenine fulness of any suppressor locus is enhanced by auxotrophies. In view of the above results, we the ability to move it into a variety of genetic have decided to designate the temperature-sen- backgrounds, and for this to be done its map sitive locus of PAT2145 as sup-i rather than ts. position must be known. The cotransducibility Additional evidence that PAT2145 still car- of sup-1100 and thr-1104, the previously demries the Iys-i15 and pur-1118 mutations was onstrated cotransducibility of thr-1104 and obtained from transduction experiments simi- pur-1108, and the previous observations that lar to the external suppressor test used by Eg- sup-1 100 and pur-1 108 are less than 0.1% cogertson and Adelberg (3) in their studies of transducible indicate that the most likely order suppressor mutations of E. coli. Table 2 shows of these three markers is sup-i1 00 thr-1 104 that, using PAT2 as a donor, lys + transductants pur-1 108. Confirmation of this marker order can be were recovered, whereas no Iys+ transductants made by using the known location of three were recovered using PAT2145 as the donor. These results indicate that PAT2145 still pos- different thr loci (J. Watson, unpublished data) sesses the Iys-1115 allele. The lack of Iys+ co- (Fig. 1). Reciprocal transductions were carried transductants when selection is made for arg- out using PAT2145 and three strains each 1130+ with PAT2145 as the donor supports this carrying a mutation in one of these thr loci. The results of these transductions are shown in Taview. Similar results are shown in Table 2 with ble 3. If the cotransduction frequencies obrespect to the pur-1118 allele in PAT2145, with tained in reciprocal transductions are averaged, then the provisional marker order, based one additional point of interest. The pur-1 118 and thr-1 104 markers are cotransducible, and, only on these two factor crosses, is found to be although no pur-1 118 + transductants were ob- sup-1100 thr-1108 - thr-2106 - thr-3107. To confirm the relative order of the three thr tained directly, three of the thr-1104+ transductants were found to be phenotypically Pur+ loci, transductions were carried out using when PAT2145 was used as donor. The ob- F116L propagated on PAT2 to transduce the served 3% cotransduction of the thr and pur three thr mutants used in the previous trans-
-
TABLE 2. Transductional tests for suppression of the Iys-15 and pur-1118 alleles Doept et Transductants Selected Donor Recipient perper 0.2 marker
mln
PAT2145
PAT2127
lys-1115+ arg-1130+
PAT2
PAT2127
lys-1115+ arg-1130+
PAT2145
% Cotransduction witha:
lys-
plated
1115+
0 138 180 134
Vol. 125, No. 3 Printed in U.S.A.
Copyright © 1976 American Society for Microbiology
Suppressor Mutations in Pseudomonas aeruginosa J. M. WATSON
AND
B. W. HOLLOWAY*
Department of Genetics, Monash University, Clayton, Victoria 3168, Australia Received for publication 10 October 1975
Suppressor mutations of Pseudomonas aeruginosa have been identified. An isolate of strain PAT, initially selected as being temperature sensitive for growth, was found to suppress two different auxotrophic mutations. A suppressor locus, designated sup-i, has been mapped and is co-transducible with three closely linked thr loci. The suppressor mutation has been used to isolate suppressor-sensitive (sus) mutants of the virulent phage E79 and the R factor R18. By selecting for revertants of auxotrophic markers, other sup mutants have been isolated and are found to be of two types, either temperature sensitive for growth like the original mutant or showing wild-type growth at 43 C. The mutations giving rise to both these classes of suppressor are very closely linked. One of the sup-i alleles of strain PAT also shows suppressor activity when transferred into P. aeruginosa strain PAO. Escherichia coli strains carrying the nonsense suppressors supC, supD, or supF do not suppress the sus mutant of R18. This suggests that sup-1 is different from the amber and ochre suppressors of the enterobacteria. agar was Vogel and Bonner minimal medium (11) solidified with 1.2% Oxoid agar no. 1. Minimal agar was supplemented with L-amino acids at a final concentration of 1 mM except isoleucine, which was used at 0.5 mM. Layer agar was NB containing 0.8% Oxoid agar no. 1. TNM buffer, for preparation of bacteriophage lysates and dilutions, was 0.01 M tris(hydroxymethyl)aminomethane plus 0.15 M sodium chloride and 0.01 M magnesium sulfate (pH 7.4). Bacterial mutagenesis and carbenicillin enrichment. Bacterial mutants were obtained by EMS mutagenesis as follows. Two drops (ca. 0.05 ml) of EMS was added to 5 ml of an exponential-phase culture in NB, vortexed vigorously, and incubated at 37 C for 1 h without shaking. One-milliliter aliquots of the MATERIALS AND METHODS treated culture were diluted 1 in 30 in NB and Bacterial and bacteriophage strains. The bacte- incubated at 37 C overnight with shaking to allow rial strains used in this study are shown in Table 1. expression of mutations. This culture was diluted 1 in 30 in prewarmed NB All PAT recipient (FP-) strains were derived from PAT964 (10) by ethyl methane sulfonate (EMS) mu- and incubated at 43 C for 1 h with shaking. Carbenitagenesis. The PAO strains used were derived from cillin was then added to a final concentration of 2 PA0242 (7) by EMS mutagenesis. PT0226 is an mg/ml, and the culture was incubated at 43 C for a interstrain recombinant constructed by transducing further 6 h with shaking. Residual carbenicillin was PA0225 with F116C4 (V. Krishnapillai, unpub- inactivated by adding penicillinase (300 Levy units/ lished data) propagated on PAT2145. Phage F116L ml) and incubating for 1 h at 43 C with shaking. The (7) was used for all transductional analyses. Sup- culture was then incubated overnight at 37 C with pressor-sensitive derivatives of the virulent phage shaking. The culture was diluted appropriately in saline, and 0. 1-ml aliquots were spread on HIA E79 (5) are designated E79 sus-1 and E79 sus-2. Media. Nutrient broth (NB) was 2.5% Oxoid nu- plates and scored for mutants which grew at 37 C trient broth no. 2 supplemented with 0.5% Oxoid but not at 43 C by replica plating. Bacteriophage mutagenesis. The following proceyeast extract. Heart infusion agar (HIA) was Difco heart infusion broth solidified with 1.2% Oxoid agar dure was used to isolate suppressor-sensitive (sus) no. 1. The antibiotics streptomycin (Sigma), rifam- mutants of phage E79. A 5-ml amount of an expopin (Ciba-Geigy), carbenicillin (Beecham), kanamy- nential-phase culture of PAT2145 was mixed with cin (Sigma), and tetracycline (Lederle) were added an equal volume of phage E79 (at a multiplicity of to HIA at the concentrations indicated. Minimal infection of approximately 2) and incubated at 37 C 780
The characterization of suppressor mutations in bacteria has contributed much to the understanding of the expression of genetic information. Equally important, they have been a valuable experimental tool for microbial geneticists. This applies particularly to the translational suppressor mutations as detailed in a recent review (4). This paper describes the isolation of a suppressor mutation in Pseudomonas aeruginosa and the genetic evidence that supports this view. Complete identification of the suppressor mechanism awaits the biochemical investigation of this mutant.
P. AERUGINOSA SUPPRESSOR MUTATIONS
VOL. 125, 1976
TABLE 1. Bacterial strainsa Genotype and locus
Strain PAT2 PAT2127
PAT2141 PAT2143
PAT2145 PAT2151 PAT2152 PAT2153 PAT2154 PAT2155
PAT2177 PAT2205 PAT2206 PAT2207 PAT2208 PAT2209 PAT2210 PA0222 PA0225 PA0230 PT0226
Prototroph, FP2+ lys-1115, trp-3114, pur-1118, leu-2104, arg-1130, FPlys-1115, trp-3114, pur-1118, leu-2104, thr1104, FPlys-1115, trp-3114, pur-1118, leu-2104, pro2108, FPlys-1115, trp-3114, pur-1118, leu-2104, pro2108, sup-1100, FPlys-1115, trp-3114, pur-1118, leu-2104, pro2108, str-1114, FPPrototroph, str-1105, sup-1100, FP2+ Iys-1115, trp-3114, pur-1118, leu-2104, thr2106, FPlys-1115, trp-3114, pur-1118, leu-2104, thr3107, FPlys-1115, trp-3114, pur-1118, leu-2104, thr1108, FPlys-1115, trp-3114, pur-1118,1eu-2104, pro2108, his-2117, ilv-1118, thr-1109, FPSame at PAT2177 but sup-1103 Same as PAT2177 but sup-1104 Same as PAT2177 but sup-1105 Same as PAT2177 but sup-1106 Same as PAT2177 but sup-1107 Same as PAT2177 but sup-1108 met-28, trp-6, lys-12, his-4, pro-82, ilv-226, FPmet-28, trp-6, Iys-12, his-4, pro-82, ilv-226, thr-48, FPSame as PA0225 but str-44 met-28, trp-6, lys-12, his-4, pro-82, ilv-226, sup-1O1, FP- (see Materials and Meth-
ods) PT0231
Same
as
PT0226 but rif-7
Abbreviations: arg, Arginine requirement; his, histidine requirement, ilv, isoleucine and valine requirement; leu; leucine requirement; Iys, lysine requirement; pro, proline requirement; pur, adenine requirement; rif, rifampin resistance; str, streptomycin resistance; sup, suppressor activity; thr, threonine requirement; trp, tryptophan requirement. For PAT strains, the first number following each gene symbol indicates the arbitrary locus designation, whereas the following three numbers refer to the allele number; e.g., lys-1115 indicates allele 115 of the Iys-1 locus. a
for 20 min without shaking. The cells were then deposited by centrifuging for 5 min. The pellet was resuspended in the same volume of prewarmed citrate buffer (pH 5.6) containing 50 ,ig of N-methylN'-nitro-N-nitrosoguanidine per ml and incubated at 37 C for 15 min without shaking. The cells were then centrifuged, washed once in citrate buffer, and finally resuspended in 2 volumes of prewarmed NB. The treated cells were incubated at 37 C overnight with shaking to allow liberation of progeny phage. The overnight culture was centrifuged, and the supernatant was sterilized by passage through a type HA membrane filter (pore size, 0.45 ,um; Millipore Corp.). Plaques were isolated by overlaying HIA plates with 1 ml of appropriately diluted supernatant and 2 ml of layer agar seeded with 0.1 ml of an overnight culture of PAT2145. Individual plaques were then picked and spotted, first to a layer seeded with PAT2145 (permissive host) on HIA and then to a layer seeded with PAT2151 (restrictive host) on HIA containing 1 mg of streptomycin per ml.
781
Plaques showing lysis on PAT2145 but not on PAT2151 were purified by three consecutive singleplaque isolations on PAT2145. R-factor mutagenesis. An exponential-phase culture of PT0231 (R18) was centrifuged, and the pellet was resuspended in prewarmed citrate buffer (pH 5.6) containing 50 ,ug of N-methyl-N'-nitro-N-nitrosoguanidine per ml. After incubating at 37 C for 15 min without aeration, the cells were deposited by centrifugation, washed once in citrate buffer, and resuspended to original volume in NB. The cells were then diluted 1 in 30 in NB and incubated at 37 C overnight with aeration to allow expression of mutations. The mutagenized R factors were then transferred into a nonsuppressor host by mixing 2 ml of this culture with 2 ml of an overnight culture of PA0230 and incubating at 37 C without shaking. After 2 h the mixture was vortexed and diluted, and 0.1-ml aliquots were plated on HIA containing 1 mg of streptomycin per ml and 250 jAg of either carbenicillin, kanamycin, or tetracycline per ml. After overnight incubation, the colonies were replica plated to HIA containing streptomycin and either of the nonselective drugs at the above concentrations. Transduction. The procedure used for transduction has been described previously (7).
RESULTS Isolation of the suppressor mutant. Strain PAT2145 was isolated as a temperature-sensitive derivative of strain PAT2143 after EMS mutagenesis and carbenicillin enrichment, using 43 C as the restrictive temperature. PAT2145 grows very poorly on HIA at 37 C relative to the parent strain PAT2143, and, in addition, it was found that PAT2145 no longer required either lysine or adenine. The loss of these two auxotrophies by simultaneous independent reversion of the original mutations seemed improbable. A likely alternative hypothesis was that the two auxotrophic lesions were suppressed either by the newly introduced temperature-sensitive mutation or by mutation at another site introduced during the mutagenesis. Evidence of suppressor activity. Support for the suppressor mutation hypothesis was sought by looking for recombinants in which such a mutation had been separated from the adenine and lysine alleles. PAT2145 was transduced with phage F116L propagated on the wildtype strain PAT2, and selection made for temperature-resistant transductants on HIA at 43 C. Approximately 1,500 transductants selected in this way were tested, and all were found to be both lysine- and adenine-requiring and thus, in this respect, had the same phenotype as the parent strain PAT2143. Independent mapping experiments using interrupted mating (J. Watson, unpublished data) have shown that lys-
782
WATSON AND HOLLOWAY
J. BACTERIOL.
1115 andpur-1118 are more than 20 min apart markers is significantly lower than the exon the PAT chromosome. Thus, the above pected value of about 13% obtained in the contransductants could not have arisen by cotrans- trol transduction using PAT2 as the donor. The duction of the wild-type alleles of the pur, lys, apparent anomaly in these results was resolved and temperature-sensitive loci. The most likely by further examination of the cotransductants explanation for these results is that the temper- found to grow poorly on HIA at 37 C and to be ature-sensitive mutation acts as a suppressor of phenotypically Lys' and temperature sensitive. These observations indicated that the three thrthe Iys-1115 and pur-1118 alleles. Evidence that the same mutation determines 1104+ transductants had coinherited sup-1100 suppressor activity and temperature sensitivity from PAT2145 to give them their Pur+ phenowas obtained from an examination of tempera- type. The above results indicate that PAT2145 ture-insensitive revertants. Strain PAT2145 re- still carries the pur-1118 mutation and, furverts to temperature insensitivity at a fre- ther, that the sup-1 1 00 mutation is cotransduquency of approximately 10-8 on HIA at 43 C. cible with the thr-1104 mutation. Mapping of the suppressor locus. The useWhen 100 such revertants were tested, all were found to have regained the lysine and adenine fulness of any suppressor locus is enhanced by auxotrophies. In view of the above results, we the ability to move it into a variety of genetic have decided to designate the temperature-sen- backgrounds, and for this to be done its map sitive locus of PAT2145 as sup-i rather than ts. position must be known. The cotransducibility Additional evidence that PAT2145 still car- of sup-1100 and thr-1104, the previously demries the Iys-i15 and pur-1118 mutations was onstrated cotransducibility of thr-1104 and obtained from transduction experiments simi- pur-1108, and the previous observations that lar to the external suppressor test used by Eg- sup-1 100 and pur-1 108 are less than 0.1% cogertson and Adelberg (3) in their studies of transducible indicate that the most likely order suppressor mutations of E. coli. Table 2 shows of these three markers is sup-i1 00 thr-1 104 that, using PAT2 as a donor, lys + transductants pur-1 108. Confirmation of this marker order can be were recovered, whereas no Iys+ transductants made by using the known location of three were recovered using PAT2145 as the donor. These results indicate that PAT2145 still pos- different thr loci (J. Watson, unpublished data) sesses the Iys-1115 allele. The lack of Iys+ co- (Fig. 1). Reciprocal transductions were carried transductants when selection is made for arg- out using PAT2145 and three strains each 1130+ with PAT2145 as the donor supports this carrying a mutation in one of these thr loci. The results of these transductions are shown in Taview. Similar results are shown in Table 2 with ble 3. If the cotransduction frequencies obrespect to the pur-1118 allele in PAT2145, with tained in reciprocal transductions are averaged, then the provisional marker order, based one additional point of interest. The pur-1 118 and thr-1 104 markers are cotransducible, and, only on these two factor crosses, is found to be although no pur-1 118 + transductants were ob- sup-1100 thr-1108 - thr-2106 - thr-3107. To confirm the relative order of the three thr tained directly, three of the thr-1104+ transductants were found to be phenotypically Pur+ loci, transductions were carried out using when PAT2145 was used as donor. The ob- F116L propagated on PAT2 to transduce the served 3% cotransduction of the thr and pur three thr mutants used in the previous trans-
-
TABLE 2. Transductional tests for suppression of the Iys-15 and pur-1118 alleles Doept et Transductants Selected Donor Recipient perper 0.2 marker
mln
PAT2145
PAT2127
lys-1115+ arg-1130+
PAT2
PAT2127
lys-1115+ arg-1130+
PAT2145
% Cotransduction witha:
lys-
plated
1115+
0 138 180 134
