Vol. 1:37, No. 2
JOURNAL OF BACTERIOLOGY, Feb. 1979, p. 1013-1016 0021-9193/79/02-1013/04$02.00/0
Levallorphan-Tolerant Mutants of Escherichia coli with Altered Morphologies GERARD FRELAT AND PAUL L. BOQUET* Service de Biochimie, Departement de Biologie, CEN Saclay, 91190 Gif-sur- Yvette, France Received for publication 6 November 1978
The penetration of levallorphan, a synthetic morphinan known to interact with the cytoplasmic membrane of E. coli, is seriously limited by the outer membrane. To select target-resistant mutants rather than outer membrane mutants, a twostep procedure was developed, which involved the selection by penicillin of an "intermediate" parental strain with a decreased penetration barrier and a subsequent positive selection of levallorphan-tolerant pseudo-revertant clones. Unlike the direct selection, this technique yielded various types of mutants in which the morphology, the septation ability, and the growth rate were greatly affected.
Levallorphan, a synthetic analog of morphine, interacts mainly with the cytoplasmic membrane of Esherichia coli (2, 4-6, 10). It has not been possible however, to define with precision its molecular target within the cytoplasmic membrane in vivo. The direct selection of a levallorphan-resistant strain of E. coli has been reported (3). In most strains of E. coli, however, the penetration of the morphinan across the outer membrane was found to be severely restricted (G. Frelat and P. L. Boquet, manuscript in preparation). It follows that the screening of mutations increasing the level of resistance to levallorphan has a fairly good chance of selecting strains with modifications of the outer membrane itself. To increase the probability of selection of cytoplasmic membrane mutants, a two-step procedure was used. The first step consisted of the selection by penicillin enrichment of an intermediate strain more sensitive to levallorphan. Since, according to its concentration, this compound may either have bacteriostatic or bactericidal properties, the conditions had to be chosen so that the enrichment would only proceed via the penicillin-induced suicide of growing bacteria and not by a levallorphan-promoted cell death. E. coli CR 341 (thr leu metB thyA thi lacYl Str supE, A-), previously mutagenized with N,N'-nitrosoguanidine and grown for expression, was used for the selection in a growth medium previously described (2) containing levallorphan (0.84 mM) and penicillin (105 U/ml). A mutant (SBS 110) displaying an increased sensitivity to levallorphan on this medium was selected (Fig. 1). A survey of the new properties simultaneously acquired by this mutant (and also lost in a singlestep reversion with a frequency of 2 x 10-8)
indicated that an outer membrane mutant had been selected. It was found sensitive to bacteriophage C 21, a property related to the loss of a part of the outer core of the lipopolysaccharide (7, 8). Gas chromatography analysis of the sugar composition of the lipopolysaccharide revealed the absence of both glucosyl and galactosyl residues (data not shown). SBS 110 was unable to ferment galactose, and transduction experiments with Plkc, with SBS 110 as donor and CSH 27 (trpA) as recipient, indicated that the Gal phenotype of SBS 110 was 53% cotransducible with trpA. Consequently, the mutation was located in
-,
.
1 00-
7
A
.50-
.6
-f.5
' 20-
4
10
0\
.3
.2-
\1
.1 0 \o
0
°
5'
1 -
1.0 1.5
IevaIIorphan (mM)
a
]
0
10
20
30 min utes
FIG. 1. Compared sensitiv,ity to levallorphan of the parent strain CR 341 and the inter mediate stra .in SBS 110. (A) Growth rates of CR 341 and of SBS 110 were measured by the increase in absorbancy at o46 nm in minimal medium in the presence of levallorphan at various concentrations; (B) surv,ival of CR 341 and SBS 110 at a dilution of 1:101 in salinc, Mg2+-free CRS medium containing 1.4 mM levallorphan. The experimental conditions ar e as prev iouisly described (2). Symbols: (0) CR 341; (0) SBS 110; (U) CR 341, no addition; (A) SBS 110, no addlition.
1013
1014
NOTES
J. BACTERIOL.
FIc. 2. Motphological cha a(c te istics of the mutants obtailnedl b,y leuallorphan toleran ce. All bacteria awecre groan in br-oth medium pluis th,vniinc ()50 ,otg/'ml). (A) Intertfer-en7ce contrast micrograph of SBS 40 001 at 2t3°C; (B) same culture 2 h after- a shift at 37°C( (C atnd1 D) phcise andl interference contrast micrographs of SBS 40 183 ait 23GC; (E and F) same stiiln ia/fteh I h at 37^C; (GC) phase contrast micrograph of SBS 41 .300 at 230°C; (H) samine cuilturi-e after 2 h at i37(C (I) interlferenec contrast aiicrograph of SBS 40 1 11 at 23°C: (J) .sam2e .strainl after 3 h at 37° C (niote the disposition of the uesicles on each side of the septumi (duriing their fornmation): (K) interference contrast microgrciph of stcain SB,S; 43 102 groan cit 300 C: (I) interference contrast micrograph of .SBKS 40 500 ait 300 C.
VOL. 137, 1979
NOTES
1015
but showed blunt septations and no cell separation at 37°C (Fig. 2A and B). Strain SBS 40 183, selected in the same conditions, had a similar behavior, and monster cells with vacuoles and Minimal inhibitory concentration polar tips, but without chains, were formed at 37°C (Fig. 2C to F). Strain 41 300 had a slightly (pg/mnl) for strains: Inhibitor different morphology at 37°C, and daughter cells CR 341 SBS 110 SBS 255 were formed in spite of the aberrant septation Sodium deoxychol- >5,000 >5,000 >5,000 (Fig. 2G and H). ate The morphology of strains selected in the Sodium dodecyl >2,000 1,500 >2,000 same way but at 30 or 37°C was different. Strain sulfate SBS 40 111 was apparently normal at 30°C (Fig. 450 Cetyl-trimethyl75 450 2I). At 37°C and above, a large number of small ammonium brovesicles of variable diameters appeared in the mide Rifampin 40 5 40 medium. Those vesicles seemed to be manufacAcridine orange 30 2.5 30 tured symmetrically during septation, on both Lhrehdiamine A 7.5 2.5 daughter cells, near the septum itself (Fig. 5J). Levallorphan n-tar250 20 200 Strain SBS 43 102 had a coccoid appearance trate (Fig. 2K), and strain 40 500 (Fig. 2L) was a Erythromycin 16 0.12 15 minicell-forming strain. Virginiamycin 12 0.75 12 The existence of various possibilities of overTetracycline 2 2 2 coming the action of levallorphan by mutations Chloramphenicol 5 4 5 affecting the morphology of the cell in so many different ways may have at least three possible galU at 26 minutes of the recalibrated linkage explanations. First, the morphinan might have a number of map (1). A measure of the sensitivity of SBS 110 to different molecular targets within the cytoplasmic membrane. Second, levallorphan could insome growth inhibitors known to penetrate poorly into gram-negative bacteria showed that teract in vivo with only one particular comthe effects of the mutation were not restricted pound, which itself might be involved in differto levallorphan (Table 1). Gal' transductants (as ent physiological processes. In both cases, each SBS 255) recovered normal levels of resistance of the mutants observed could compensate for to those compounds and were C 21". The growth only one of the final functions altered. Alternately, there might exist a number of rate of SBS 110 was not affected, and microscopic examination did not reveal any morpho- different possible mutations leading unspecifically to a greatly reduced growth rate. This logical abnormality. When bacteria of strain SBS 110 were layered reduction would allow a compensation of the unbalanced growth state induced by levalloron minimal plates containing 1 mmol of the initial selective agent levallorphan per liter and phan. In the absence of the morphinan, the incubated at 23 or 30°C, two types of colonies selected mutations could themselves generate specific morphological abnormalities by unbalwere observed, true revertants and small, slowgrowing colonies. These pseudo-revertants had anced envelope synthesis. The detailed physiological and genetic study conserved the main phenotypic characters of the intermediate strain SBS 110 (Gal- and C 21'), of each of these mutants may help the underexcept for their decreased sensitivity to levallor- standing of both the action of levallorphan on phan. They also exhibited unusually slow the cytoplasmic membrane and the relationships growth rates. In the absence of levallorphan, between this membrane and the outer memmost of the strains selected at 23°C were found brane in the maintenance of rod-shaped morto be thermosensitive on rich media only. Under phology. these restrictive conditions, they displayed very LITERATURE CITED aberrant morphologies and/or perturbations of 1. Bachmann, B. J., K. Low, and A. L. Taylor. 1976. the septation process. The detailed physiological Recalibrated linkage map of Escherichia coli K- 12. and genetic properties of some of these strains Bacteriol. Rev. 40:116-167. will be reported elsewhere. 2. Boquet, P. L., M. A. Devynck, H. Aurelle, and P. An example of the morphology changes obFromageot. 1971. On the bactericidal action of levallorphan. Irreversible alterations of the plasmic nmenmtained by a temperature shift of bacteria selected brane. J. Biochem. 21:536-541. by this procedure is given in Fig. 2. Strain SBS 3. Dame, J.Eur. B., and B. M. Shapiro. 1976. UJse of polyvmyxin 40 001 was unable to form colonies at 37°C on B, levallorphan, and tetracaine to isolate novel envelope broth medium. It grew as small rods at 23°C, mutants of Escherichia coli. J. Bacter iol. 127:96197 ). TABLE 1. Compared sensitiities of the intermediate strain SBS 110, the parent strain CR 341, and a Gal' transductant (SBS 255) to some growth inhibitors
1016
NOTES
4. Devynck, M. A., P. L. Boquet, P. Fromageot, and E. J. Simon. 1971. On the nmode of action of levallorphan
Escherichia coli: effects on cellulai maginesiumi. Mol. Pharmacol. 7:605-610. 5. Holland, M. J. C., and E. J. Simon. 197 5. Inhibition by levorphanol and related druLgs of aminao acid transport bv isolated nmemnbrane vesicles fronm Escherichia co/li. Antimicrob. Agents Chemiother. 7:530-537. 6. Knape, H., P. L. Boquet, and R. Roschenthaler. 1972. Inhibition of amiiino acid transport in Escher-ichia coli cells and its cell nmembranies. FEBS Lett. 19:311-314. Lindberg, A. A. 1973. Bacteriophage receptors. Annu. on
J. BACTERIOL. Rev. Microbiol. 27:205-241. 8. Miller, J. H. 1972. Experiments in nmolecular genetics. Cold Spring Harbor Laboratory, Col(I Spring Harbor, New York. 9. Rapin, A. M. C., and H. M. Kalckar. 1971. The relation of bacteriophage attachment to lipopolysaccharide structure, p. 267-307. In G. Weinbatumii, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, Vol. 4. Academic Press, New York. 10. Simon, E. J., L. Schapira, and N. Wurster. 1970. Effect of levorphanol on putrescine transport in Escherichia coli. Mol. Pharmacol. 6:577-587.
JOURNAL OF BACTERIOLOGY, Feb. 1979, p. 1013-1016 0021-9193/79/02-1013/04$02.00/0
Levallorphan-Tolerant Mutants of Escherichia coli with Altered Morphologies GERARD FRELAT AND PAUL L. BOQUET* Service de Biochimie, Departement de Biologie, CEN Saclay, 91190 Gif-sur- Yvette, France Received for publication 6 November 1978
The penetration of levallorphan, a synthetic morphinan known to interact with the cytoplasmic membrane of E. coli, is seriously limited by the outer membrane. To select target-resistant mutants rather than outer membrane mutants, a twostep procedure was developed, which involved the selection by penicillin of an "intermediate" parental strain with a decreased penetration barrier and a subsequent positive selection of levallorphan-tolerant pseudo-revertant clones. Unlike the direct selection, this technique yielded various types of mutants in which the morphology, the septation ability, and the growth rate were greatly affected.
Levallorphan, a synthetic analog of morphine, interacts mainly with the cytoplasmic membrane of Esherichia coli (2, 4-6, 10). It has not been possible however, to define with precision its molecular target within the cytoplasmic membrane in vivo. The direct selection of a levallorphan-resistant strain of E. coli has been reported (3). In most strains of E. coli, however, the penetration of the morphinan across the outer membrane was found to be severely restricted (G. Frelat and P. L. Boquet, manuscript in preparation). It follows that the screening of mutations increasing the level of resistance to levallorphan has a fairly good chance of selecting strains with modifications of the outer membrane itself. To increase the probability of selection of cytoplasmic membrane mutants, a two-step procedure was used. The first step consisted of the selection by penicillin enrichment of an intermediate strain more sensitive to levallorphan. Since, according to its concentration, this compound may either have bacteriostatic or bactericidal properties, the conditions had to be chosen so that the enrichment would only proceed via the penicillin-induced suicide of growing bacteria and not by a levallorphan-promoted cell death. E. coli CR 341 (thr leu metB thyA thi lacYl Str supE, A-), previously mutagenized with N,N'-nitrosoguanidine and grown for expression, was used for the selection in a growth medium previously described (2) containing levallorphan (0.84 mM) and penicillin (105 U/ml). A mutant (SBS 110) displaying an increased sensitivity to levallorphan on this medium was selected (Fig. 1). A survey of the new properties simultaneously acquired by this mutant (and also lost in a singlestep reversion with a frequency of 2 x 10-8)
indicated that an outer membrane mutant had been selected. It was found sensitive to bacteriophage C 21, a property related to the loss of a part of the outer core of the lipopolysaccharide (7, 8). Gas chromatography analysis of the sugar composition of the lipopolysaccharide revealed the absence of both glucosyl and galactosyl residues (data not shown). SBS 110 was unable to ferment galactose, and transduction experiments with Plkc, with SBS 110 as donor and CSH 27 (trpA) as recipient, indicated that the Gal phenotype of SBS 110 was 53% cotransducible with trpA. Consequently, the mutation was located in
-,
.
1 00-
7
A
.50-
.6
-f.5
' 20-
4
10
0\
.3
.2-
\1
.1 0 \o
0
°
5'
1 -
1.0 1.5
IevaIIorphan (mM)
a
]
0
10
20
30 min utes
FIG. 1. Compared sensitiv,ity to levallorphan of the parent strain CR 341 and the inter mediate stra .in SBS 110. (A) Growth rates of CR 341 and of SBS 110 were measured by the increase in absorbancy at o46 nm in minimal medium in the presence of levallorphan at various concentrations; (B) surv,ival of CR 341 and SBS 110 at a dilution of 1:101 in salinc, Mg2+-free CRS medium containing 1.4 mM levallorphan. The experimental conditions ar e as prev iouisly described (2). Symbols: (0) CR 341; (0) SBS 110; (U) CR 341, no addition; (A) SBS 110, no addlition.
1013
1014
NOTES
J. BACTERIOL.
FIc. 2. Motphological cha a(c te istics of the mutants obtailnedl b,y leuallorphan toleran ce. All bacteria awecre groan in br-oth medium pluis th,vniinc ()50 ,otg/'ml). (A) Intertfer-en7ce contrast micrograph of SBS 40 001 at 2t3°C; (B) same culture 2 h after- a shift at 37°C( (C atnd1 D) phcise andl interference contrast micrographs of SBS 40 183 ait 23GC; (E and F) same stiiln ia/fteh I h at 37^C; (GC) phase contrast micrograph of SBS 41 .300 at 230°C; (H) samine cuilturi-e after 2 h at i37(C (I) interlferenec contrast aiicrograph of SBS 40 1 11 at 23°C: (J) .sam2e .strainl after 3 h at 37° C (niote the disposition of the uesicles on each side of the septumi (duriing their fornmation): (K) interference contrast microgrciph of stcain SB,S; 43 102 groan cit 300 C: (I) interference contrast micrograph of .SBKS 40 500 ait 300 C.
VOL. 137, 1979
NOTES
1015
but showed blunt septations and no cell separation at 37°C (Fig. 2A and B). Strain SBS 40 183, selected in the same conditions, had a similar behavior, and monster cells with vacuoles and Minimal inhibitory concentration polar tips, but without chains, were formed at 37°C (Fig. 2C to F). Strain 41 300 had a slightly (pg/mnl) for strains: Inhibitor different morphology at 37°C, and daughter cells CR 341 SBS 110 SBS 255 were formed in spite of the aberrant septation Sodium deoxychol- >5,000 >5,000 >5,000 (Fig. 2G and H). ate The morphology of strains selected in the Sodium dodecyl >2,000 1,500 >2,000 same way but at 30 or 37°C was different. Strain sulfate SBS 40 111 was apparently normal at 30°C (Fig. 450 Cetyl-trimethyl75 450 2I). At 37°C and above, a large number of small ammonium brovesicles of variable diameters appeared in the mide Rifampin 40 5 40 medium. Those vesicles seemed to be manufacAcridine orange 30 2.5 30 tured symmetrically during septation, on both Lhrehdiamine A 7.5 2.5 daughter cells, near the septum itself (Fig. 5J). Levallorphan n-tar250 20 200 Strain SBS 43 102 had a coccoid appearance trate (Fig. 2K), and strain 40 500 (Fig. 2L) was a Erythromycin 16 0.12 15 minicell-forming strain. Virginiamycin 12 0.75 12 The existence of various possibilities of overTetracycline 2 2 2 coming the action of levallorphan by mutations Chloramphenicol 5 4 5 affecting the morphology of the cell in so many different ways may have at least three possible galU at 26 minutes of the recalibrated linkage explanations. First, the morphinan might have a number of map (1). A measure of the sensitivity of SBS 110 to different molecular targets within the cytoplasmic membrane. Second, levallorphan could insome growth inhibitors known to penetrate poorly into gram-negative bacteria showed that teract in vivo with only one particular comthe effects of the mutation were not restricted pound, which itself might be involved in differto levallorphan (Table 1). Gal' transductants (as ent physiological processes. In both cases, each SBS 255) recovered normal levels of resistance of the mutants observed could compensate for to those compounds and were C 21". The growth only one of the final functions altered. Alternately, there might exist a number of rate of SBS 110 was not affected, and microscopic examination did not reveal any morpho- different possible mutations leading unspecifically to a greatly reduced growth rate. This logical abnormality. When bacteria of strain SBS 110 were layered reduction would allow a compensation of the unbalanced growth state induced by levalloron minimal plates containing 1 mmol of the initial selective agent levallorphan per liter and phan. In the absence of the morphinan, the incubated at 23 or 30°C, two types of colonies selected mutations could themselves generate specific morphological abnormalities by unbalwere observed, true revertants and small, slowgrowing colonies. These pseudo-revertants had anced envelope synthesis. The detailed physiological and genetic study conserved the main phenotypic characters of the intermediate strain SBS 110 (Gal- and C 21'), of each of these mutants may help the underexcept for their decreased sensitivity to levallor- standing of both the action of levallorphan on phan. They also exhibited unusually slow the cytoplasmic membrane and the relationships growth rates. In the absence of levallorphan, between this membrane and the outer memmost of the strains selected at 23°C were found brane in the maintenance of rod-shaped morto be thermosensitive on rich media only. Under phology. these restrictive conditions, they displayed very LITERATURE CITED aberrant morphologies and/or perturbations of 1. Bachmann, B. J., K. Low, and A. L. Taylor. 1976. the septation process. The detailed physiological Recalibrated linkage map of Escherichia coli K- 12. and genetic properties of some of these strains Bacteriol. Rev. 40:116-167. will be reported elsewhere. 2. Boquet, P. L., M. A. Devynck, H. Aurelle, and P. An example of the morphology changes obFromageot. 1971. On the bactericidal action of levallorphan. Irreversible alterations of the plasmic nmenmtained by a temperature shift of bacteria selected brane. J. Biochem. 21:536-541. by this procedure is given in Fig. 2. Strain SBS 3. Dame, J.Eur. B., and B. M. Shapiro. 1976. UJse of polyvmyxin 40 001 was unable to form colonies at 37°C on B, levallorphan, and tetracaine to isolate novel envelope broth medium. It grew as small rods at 23°C, mutants of Escherichia coli. J. Bacter iol. 127:96197 ). TABLE 1. Compared sensitiities of the intermediate strain SBS 110, the parent strain CR 341, and a Gal' transductant (SBS 255) to some growth inhibitors
1016
NOTES
4. Devynck, M. A., P. L. Boquet, P. Fromageot, and E. J. Simon. 1971. On the nmode of action of levallorphan
Escherichia coli: effects on cellulai maginesiumi. Mol. Pharmacol. 7:605-610. 5. Holland, M. J. C., and E. J. Simon. 197 5. Inhibition by levorphanol and related druLgs of aminao acid transport bv isolated nmemnbrane vesicles fronm Escherichia co/li. Antimicrob. Agents Chemiother. 7:530-537. 6. Knape, H., P. L. Boquet, and R. Roschenthaler. 1972. Inhibition of amiiino acid transport in Escher-ichia coli cells and its cell nmembranies. FEBS Lett. 19:311-314. Lindberg, A. A. 1973. Bacteriophage receptors. Annu. on
J. BACTERIOL. Rev. Microbiol. 27:205-241. 8. Miller, J. H. 1972. Experiments in nmolecular genetics. Cold Spring Harbor Laboratory, Col(I Spring Harbor, New York. 9. Rapin, A. M. C., and H. M. Kalckar. 1971. The relation of bacteriophage attachment to lipopolysaccharide structure, p. 267-307. In G. Weinbatumii, S. Kadis, and S. J. Ajl (ed.), Microbial toxins, Vol. 4. Academic Press, New York. 10. Simon, E. J., L. Schapira, and N. Wurster. 1970. Effect of levorphanol on putrescine transport in Escherichia coli. Mol. Pharmacol. 6:577-587.
