Molec. gen. Genet. 157, I 4 9 - I 5 3 (1977) © by Springer-Verlag 1977
Amber Mutations in Escherichia coli Essential Genes: Isolation of Mutants Affected in the Ribosomes Genevi6ve Delcuve, Teresa Cabez6n, Alain Ghysen, Albert Herzog, and Alex Bollen Laboratory of Genetics, University of Brussels, 67, rue des Chevaux, B-1640 Rhode St Gen6se, Belgium
Summary. A method to obtain amber mutations in ribosomal protein genes is described. It relies on the Pl-mediated localized mutagenesis (Hong and Ames, 1971) and on the fact that the recipient strain contains (a) an efficient but genetically unstable suppressor, (b) a particular thermoinducible 2 prophage which kills suppressor hosts at 42 ° C. Exposure of these bacteria to the high temperature yields frequent suppressor-free derivatives while none will be found if the strain carries an amber mutation in an essential gene. Eleven mutants have been isolated by this method, of which at least six appear to carry amber mutations. All of them map close to, and to the right of spcA, in a region which codes mostly for ribosomal proteins. Three mutants were studied biochemically; all three show defective ribosomal assembly in vivo upon loss of suppression.
ribosomal proteins are organized in at least four transcriptional units, clustered around the strA gene (72' on the genetic map). The analysis of the functional organization of ribosomal genes, and the mechanism which modulates their expression, would be greatly facilitated by the existence of polar amber mutations. However, nonsense mutations affecting essential genes cannot be readily isolated in haploid cells like Escherichia coli where they are lethal. In this paper, we describe a method which allows the isolation of amber mutations of vital genes in any localized region of the genome. This method has been used to obtain several mutations in the aroE-strA region. We provide biochemical information for three such mutants which show defective ribosomal assembly in vivo.
Material and Methods
Introduction Understanding the mechanisms which regulate the expression of ribosomal protein genes will depend on the previous knowledge of their organization. Earlier genetic studies relied essentially on the isolation and characterization of antibiotic resistant mutants or took advantage of particular features of various bacterial strains, species or genera (for a review, see Jaskunas et al., 1974; De Wilde et al., 1977). More recently an original approach has been developped by Nomura and his collaborators; it consists of the isolation of ribosome genes on transducing phages in order to study their organization using various genetic and biochemical techniques (for a review, see Nomura, 1976). From these studies and related works, it seems clear that many genes coding for For offprints contact: A. Bollen
a) Escherichia coli Strains Table 1. b) Phages: 2N7N53029cI857 (constructed in this laboratory); 080sus2sulII (Andoh and Ozeki, 1968). c) P1 transductions were performed as described by Cannon et al. (1974). d) P1 mutagenesis with hydroxylamine follows the procedure described by Hong and Ames (1971). e) In vivo Ribosomal Assembly. Cell extracts were prepared as follows : overnight cultures were diluted 25 fold in 20 ml tryptone broth (1% tryptone, 0.5% NaC1) and grown at 30 ° to a cell density of about 108/ml. One half of the culture was then incubated at 42 ° C for one hour to allow inactivation of the thermolabile tRNA suppressor molecules, then supplemented with 14C- or 3H-labelled uracil and allowed to grow for another half hour (14C-uracil: 1 gCi and 1 ~tg/ml; 3H-uracil: 5 ~tCi and i lag/ml). Non radioactive uracil was then added (300 gg/ml) and growth continued for 15 rain. The other half of the culture was treated the same way but at 30 ° C Cells were centrifuged, washed and resuspended in 2 mI TMAII (Tris-HCI 10 mM, pH 7.4; N H , C1 30 m M ; Mg acetate 0.3 m M and mercapto-ethanol 6 mM). Mixtures of 3H- and ~ C labelled cells were broken up in a French pressure cell, centrifuged twice at 12,000 g for 10 min. Appropriate portions of the supernatant were layered immediately on 7-25% sucrose gradients (in
150
G. Delcuve et al. : A m b e r Ribosomal Mutations
Table 1. Bacterial strains Designation
C h r o m o s o m a l markers
Source (method of construction
or reference) RH2569 (K12S)
prototroph
Appleyard, R.
RH2358 (M6)
araam, lacJ25am, galUK2am, galE, trpam, tSXamSUIIIA81
Donachie, W.
(thermolabile suppressor t R N A ) RH2855
araam, lac125,m, galUK2,m, galE, trp~m, tSX, m, suIIIA81 malA, spcA, aroE353
Cabez6n et al. (1977)
RH3126
araam, lac125am, galUK2~m, galE, trpam, tSX~m, suIIIA81 spcA, aroE353
obtained by transduction of real ÷ in RH2855
RH3127
ara,m, lac125am, galUK2am, galE, tsx ....
obtained by transduction of trp ÷ suIII ÷ in RH2358
RH3134
ara .... lac125am, galUK2am, galE, ,tS~Cam, spcA, aroE353
obtained by transduction of spcRaroE + in RH3127
RH3149
RH3134 ( 2N7 N5 3029cI 8 57) ( ?980sus2suII1)
obtained by lysogenisation for each phage
RH3184
araam, lac125am, galUK2am, galE, trpa m, tSXam, aroE353 spcA, suIIA81
spontaneous derivative of RH3126 which shows increased efficiency of suppression, probably due to a second mutation in suIIIA81 (Delcuve, G., unpublished results)
RH3161 RH3162 RH3194
RH2358am3161 " RH3184am3162 RH3184am3194
TMAII) and centrifuged for 16 h at 21,000 rpm in a Spinco SW27 rotor. 0.5 ml fractions were collected in glass vials to which 5 ml of scintillation fluid were then added (Aqua Luma, Lumac).
Results
1. Description of the Method A nonsense mutation in an essential gene will be lethal unless the bacterium also carries a suppressor gene. It would be possible to screen for such mutations as conditional lethals if one could inactivate the suppressor at will. An easy way to achieve that result would be to use a thermosensitive suppressor. However the efficiency of suppression of such suppressors is low, even at 32 ° C, so that mutations in genes coding for proteins necessary in large quantities would probably not be recovered. This may well be the case for ribosomal protein genes as will be shown below. Therefore we developed a system where the bacteria carry an efficient but genetically unstable suppressor. This is the case of su III as carried on the transducing phage, d~80 suIII. In this phage, the bacterial genes are frequently excised because they are sandwiched between homologous viral regions. The transduced suppressor is then frequently lost by recombination between the flanking regions (Andoh and Ozeki, 1968). Thus lysogens for this phage "rev e r t " to non suppression at a frequency of about 10 -4 (Ghysen and Celis, 1974). In order to achieve
see text for construction
a temperature dependent selection against suppression, we used a thermoinducible prophage. This prophage 2NTN53029cI857 is induced at 42 ° and will kill the host bacterium only if the N and O amber mutations are suppressed. Thus the double lysogen (~80sus2suIII) ()~N7N53029cI857) will be viable at 30°C and lethal at 42°; derivatives which have lost the suppressor will be viable at both temperatures. The frequency of spontaneous suppressor loss is such that thermoresistant derivatives will always be present in velvet replicated clones. However, if the double lysogen carries an amber mutation in an essential gene, the derivatives which have lost the suppressor will be lethal, and no thermoresistant bacteria will be found (Table 2). Using this procedure, it is also possible to find mutants whose thermosensitive phenotype is due to missense mutation. These mutants can be distinguished from the amber ones by looking for the recovery of the thermosensitive phenotype after transducing their mutations into a bacterium without any suppressor.
2. Isolation of Amber Mutants in the aroE-strA Region Phage P1 grown on the prototrophic strain RH2569 was mutagenized with hydroxylamine (Hong and Ames, 1971). The survival of P1 after mutagenesis was 0.5%. The stock, after concentration, was used to transduce the aroE-spcA region into the recipient
151
G. Delcuve et al. : Amber Ribosomal Mutations Table 2. Genotype and phenotype of amber mutants in the dilysogen recipient RH3149 Strain
Relevant a genotype
Distribution of dp80sus2suIII and ~?80sus2 lysogens within one transductant clone
32 ° C recipient (RH3149) or transductant not carrying an amber mutation
x+
transductant carrying an amber mutation
Xam
a x
99.99% (~p8Osus2suII1) (2NTN53029cI857) 0.01% (do8Osus2) (2N7N53029cI857)
99.99% (~?80sus2suII1) (2N7N53029cI857) 0.01% (qbSOsus2) (2NTN53039cI857)
UJ
t
r',- 00
99.7 q
~,
I
1
°I
-
+
+
-
-
ts +
ts
-
Mutation
97
am3161 am3162 am3194 am3157 am3158 am 3163
Recipient RH3126 (suIIIAS1)
RH3184 (suIIIASI*)
RH3149 (suIII)
+ nt -
nt + + nt nt -
+ + + + + +
RH3134 (su +)
69 91
d
+
+
Table 3. PI-mediated transduction of amber mutations in various recipients (selection for aroE +)
m
C
42 ° C
complete genotype of strain RH3149 is listed in material and methods refers to an essential gene, localised in the aroE-strA region, for instance the gene coding for a ribosomal protein
50-60"~ 70-90 ~
at
Resulting phenotype
Growth at
86 51
97
84 7717
Fig, l a - e . Figures on the map refer to % cotransduction; small size numbers refer to deduced values. -~- cotransduction frequencies aroE-spcA and spcA-aroE as compiled from the litterature (Weisblum and Davies, 1968; selection for spectinomycin resistance usually yields lower figures for the spcA-aroE cotransduction frequency, unpublished observations), a selection for aroE + (RH3157:261 transductants), (RH3158:210 transductants), b selection for aroE + (189 transductants) and reciprocal transduction with selection for spcR (133 transductants), e selection for aroE ÷ (200 transductants), d selection for aroE + (187 transductants) and reciprocal transduction with selection for spcR (153 transductants). e selection for aroE + (193 transductants)
strain RH3149 lysogen for tp8Osus2suIII and 2N7N53 O29ci857. After selection for aroE + at 32 °, we screened for complete thermosensitivity at 42 °. Eleven mutants were obtained out of 850 transductants; all of them retained the ability to suppress the ara~m lac~m and lrpa m mutations in RH3149. Five mutations map close to, and to the left of spcA; they were not further characterized. Six map to the right of spcA and cotransduce with this marker at frequencies ranging
+ , survival of the recipient after transduction; - , no survival (120 aroE + transductants were examined for the unselected temperature sensitive mutations); nt, not tested
from 93 to 99.0% (Fig. 1). All six mutants harbor an amber mutation; this was shown by transduction of the aroE-spcA region into a recipient strain, RH3134, which does not carry any suppressor. Selection was made for aroE ÷ and in all cases it was impossible to recover thermosensitive transductants (Table 3). The fact that we did not find any missense mutation among the 6 mutants we studied is not really surprising: the frequency of missense thermosensitive mutations in a localized mutagenesis is about 1 out of 1000 (Cabez6n et al., unpubl, data) In order to carry out the biochemical analysis of these mutations, we transduced them into receptors which carry either a thermosensitive suppressor (suIH A81) or a spontaneous derivative of suIIIA81 which gives a higher level of suppression (suIII A81 *). Table 3 gives the results of these transductions. It should be noted that, although the mutations can be readily transduced in a suIII receptor, some cannot be transduced in bacteria that carry suIII A81 or suIII A81*, suggesting that the efficiency of these thermosensitive suppressors is too low to allow survival.
152
G. Delcuve et al. : Amber Ribosomal Mutations
4I--
42 °C
42°C 20
~0
4
20
50S 50S
lip
II
~!
3
J; IO
15F
p
30S
30S
ji LO
10
~
5
5
i
O 0
10
30
50
0
10
30
15
50
0
10
30
'
0
50
Fig. 2. Sucrose gradient sedimentation analysis of extracts from cells incubated at 42 ° C in the presence of radioactive uracil. Abscissa indicate the n u m b e r of fractions from the top of the gradient. Ordinates are in cts/min × 10 3; left scales are for 14C (parent strain) and right scales for aH (mutants). a R H 2358 ( 1 4 C ) - e - e - x R H 3161 (3H)--©--o--. b R H 2358 ( 1 4 C ) - e - e - x R H 3162 (3H)--©--©--. e R H 2358 ( 1 4 C ) - e - e - x RH3194 (3H)--©--o--
Whenever transduction was possible, the resulting strain became thermosensitive, further indicating the amber nature of the mutations. The strains carrying a thermosensitive suppressor and the mutations am3161, am3162 and am3194 are refered to as RH3161, RH3162 and RH3194, respectively.
3. In vivo Assembly of Ribosomal Particles in Strains RH3161, RH3162 and RH3194 In conditions which inactivate the suppressor t R N A molecules, the ribosomal protein coded for by the mutated gene will not be synthesized and subsequently assembled in mature ribosomes. This will result in defective assembly of ribosomal particles. The extent of defectivity will depend on the nature of the mutated gene and on the possible polar effect of the mutation on genes distal to the mutated locus. We labelled the mutant strains with 14C-or 3Huracil at 42 ° C, as explained in Material and Methods, i.e., in conditions where the suIIIts suppressor t R N A is inactivated. We analyzed the extracts of the parent and mutant strains on sucrose gradients in conditions which allow observation of the ribosomal subunits (low magnesium salt). As seen in Figure 2, all three mutants are defective in ribosomal assembly in vivo
at 42 ° C. The strain RH3161 no longer synthesizes mature ribosomes, but accumulates precursors of 30S and 50S, whereas mutants RH3162 and RH3194 do not assemble 50S ribosomal particles nor detectable amounts of precursors. It should be noted that, even at 30 °, strain RH3161 accumulates incomplete particles, in lower amounts than at 42°C however. This is consistent with the fact that the sulII thermosensitive mutations have a relatively low suppression efficiency at 30 ° C. The same comment applies to the mutants RH3161 and RH3194 which, at 30 ° C, do not assemble normal amounts of 50S ribosomes (data not shown). Using classical techniques, the preliminary analysis of the ribosomal proteins isolated from mutants RH3161 and RH3162 did not yet reveal any clear modification of the patterns.
Discussion
In this report we describe a method to isolate amber mutations in any gene of E. coll. We have used this method in a search for mutations in ribosomal protein genes. Eleven mutations leading to complete thermosensitivity to the recipient strain have been recovered; all are cotransducible with spcA and all six
G. Delcuve et al.: Amber Ribosomal Mutations
153
we studied were shown to be lethal in the absence of suppression. Furthermore, the transduction data for the six mutations which map at the right of spcA are in good agreement with the localisations for ribosomal protein genes obtained by Nomura (1976). Thus, it appears that the selection procedure effectively yields amber mutations in the region of interest. The method was designed to allow the recovery of amber mutations in bacteria carrying an efficient suppressor. Indeed ribosomal proteins are necessary in large quantities and one may expect that a low efficiency of suppression, such as that provided by a thermosensitive suppressor, would severely restrict the recovery of such mutations. This expectation proved to be true as 4 out of 5 mutations could not be transduced in a strain carrying a thermosensirive suppressor, suIII A81. At least one could not even be transduced in a more efficient, spontaneous, derivative of suIII A81. Clearly, the use of a thermosensitive suppressor in the selection procedure would limit the selection to a small subgroup of mutations, at least in the spcA-strA region. On the other hand, the fact that different mutants require different efficiencies of suppression to be saved may be analyzed in terms of level of expression of the various genes. However, such an analysis requires the identification of the mutated genes and of their product. Among the six mutations which map in the region coding for ribosomal proteins, three have been studied biochemically in non-suppressing conditions. All three prevent normal ribosomes to be formed in vivo. One of them, am3161 prevents the formation of mature 30S and 50S subunits; this is consistent with the hypothesis that the mutation exerts a polar effect on genes coding for 30S and 50S ribosomal proteins which are distal to the mutated gene. Indeed, the map location of the mutation am3161 places it in the so-called spc operon (Nomura, 1976) which has the following structure:
(L15 L30) $5 L18 L6 $8 S14 L5 L24 L14
s ....
tor
Amber Mutations in Escherichia coli Essential Genes: Isolation of Mutants Affected in the Ribosomes Genevi6ve Delcuve, Teresa Cabez6n, Alain Ghysen, Albert Herzog, and Alex Bollen Laboratory of Genetics, University of Brussels, 67, rue des Chevaux, B-1640 Rhode St Gen6se, Belgium
Summary. A method to obtain amber mutations in ribosomal protein genes is described. It relies on the Pl-mediated localized mutagenesis (Hong and Ames, 1971) and on the fact that the recipient strain contains (a) an efficient but genetically unstable suppressor, (b) a particular thermoinducible 2 prophage which kills suppressor hosts at 42 ° C. Exposure of these bacteria to the high temperature yields frequent suppressor-free derivatives while none will be found if the strain carries an amber mutation in an essential gene. Eleven mutants have been isolated by this method, of which at least six appear to carry amber mutations. All of them map close to, and to the right of spcA, in a region which codes mostly for ribosomal proteins. Three mutants were studied biochemically; all three show defective ribosomal assembly in vivo upon loss of suppression.
ribosomal proteins are organized in at least four transcriptional units, clustered around the strA gene (72' on the genetic map). The analysis of the functional organization of ribosomal genes, and the mechanism which modulates their expression, would be greatly facilitated by the existence of polar amber mutations. However, nonsense mutations affecting essential genes cannot be readily isolated in haploid cells like Escherichia coli where they are lethal. In this paper, we describe a method which allows the isolation of amber mutations of vital genes in any localized region of the genome. This method has been used to obtain several mutations in the aroE-strA region. We provide biochemical information for three such mutants which show defective ribosomal assembly in vivo.
Material and Methods
Introduction Understanding the mechanisms which regulate the expression of ribosomal protein genes will depend on the previous knowledge of their organization. Earlier genetic studies relied essentially on the isolation and characterization of antibiotic resistant mutants or took advantage of particular features of various bacterial strains, species or genera (for a review, see Jaskunas et al., 1974; De Wilde et al., 1977). More recently an original approach has been developped by Nomura and his collaborators; it consists of the isolation of ribosome genes on transducing phages in order to study their organization using various genetic and biochemical techniques (for a review, see Nomura, 1976). From these studies and related works, it seems clear that many genes coding for For offprints contact: A. Bollen
a) Escherichia coli Strains Table 1. b) Phages: 2N7N53029cI857 (constructed in this laboratory); 080sus2sulII (Andoh and Ozeki, 1968). c) P1 transductions were performed as described by Cannon et al. (1974). d) P1 mutagenesis with hydroxylamine follows the procedure described by Hong and Ames (1971). e) In vivo Ribosomal Assembly. Cell extracts were prepared as follows : overnight cultures were diluted 25 fold in 20 ml tryptone broth (1% tryptone, 0.5% NaC1) and grown at 30 ° to a cell density of about 108/ml. One half of the culture was then incubated at 42 ° C for one hour to allow inactivation of the thermolabile tRNA suppressor molecules, then supplemented with 14C- or 3H-labelled uracil and allowed to grow for another half hour (14C-uracil: 1 gCi and 1 ~tg/ml; 3H-uracil: 5 ~tCi and i lag/ml). Non radioactive uracil was then added (300 gg/ml) and growth continued for 15 rain. The other half of the culture was treated the same way but at 30 ° C Cells were centrifuged, washed and resuspended in 2 mI TMAII (Tris-HCI 10 mM, pH 7.4; N H , C1 30 m M ; Mg acetate 0.3 m M and mercapto-ethanol 6 mM). Mixtures of 3H- and ~ C labelled cells were broken up in a French pressure cell, centrifuged twice at 12,000 g for 10 min. Appropriate portions of the supernatant were layered immediately on 7-25% sucrose gradients (in
150
G. Delcuve et al. : A m b e r Ribosomal Mutations
Table 1. Bacterial strains Designation
C h r o m o s o m a l markers
Source (method of construction
or reference) RH2569 (K12S)
prototroph
Appleyard, R.
RH2358 (M6)
araam, lacJ25am, galUK2am, galE, trpam, tSXamSUIIIA81
Donachie, W.
(thermolabile suppressor t R N A ) RH2855
araam, lac125,m, galUK2,m, galE, trp~m, tSX, m, suIIIA81 malA, spcA, aroE353
Cabez6n et al. (1977)
RH3126
araam, lac125am, galUK2~m, galE, trpam, tSX~m, suIIIA81 spcA, aroE353
obtained by transduction of real ÷ in RH2855
RH3127
ara,m, lac125am, galUK2am, galE, tsx ....
obtained by transduction of trp ÷ suIII ÷ in RH2358
RH3134
ara .... lac125am, galUK2am, galE, ,tS~Cam, spcA, aroE353
obtained by transduction of spcRaroE + in RH3127
RH3149
RH3134 ( 2N7 N5 3029cI 8 57) ( ?980sus2suII1)
obtained by lysogenisation for each phage
RH3184
araam, lac125am, galUK2am, galE, trpa m, tSXam, aroE353 spcA, suIIA81
spontaneous derivative of RH3126 which shows increased efficiency of suppression, probably due to a second mutation in suIIIA81 (Delcuve, G., unpublished results)
RH3161 RH3162 RH3194
RH2358am3161 " RH3184am3162 RH3184am3194
TMAII) and centrifuged for 16 h at 21,000 rpm in a Spinco SW27 rotor. 0.5 ml fractions were collected in glass vials to which 5 ml of scintillation fluid were then added (Aqua Luma, Lumac).
Results
1. Description of the Method A nonsense mutation in an essential gene will be lethal unless the bacterium also carries a suppressor gene. It would be possible to screen for such mutations as conditional lethals if one could inactivate the suppressor at will. An easy way to achieve that result would be to use a thermosensitive suppressor. However the efficiency of suppression of such suppressors is low, even at 32 ° C, so that mutations in genes coding for proteins necessary in large quantities would probably not be recovered. This may well be the case for ribosomal protein genes as will be shown below. Therefore we developed a system where the bacteria carry an efficient but genetically unstable suppressor. This is the case of su III as carried on the transducing phage, d~80 suIII. In this phage, the bacterial genes are frequently excised because they are sandwiched between homologous viral regions. The transduced suppressor is then frequently lost by recombination between the flanking regions (Andoh and Ozeki, 1968). Thus lysogens for this phage "rev e r t " to non suppression at a frequency of about 10 -4 (Ghysen and Celis, 1974). In order to achieve
see text for construction
a temperature dependent selection against suppression, we used a thermoinducible prophage. This prophage 2NTN53029cI857 is induced at 42 ° and will kill the host bacterium only if the N and O amber mutations are suppressed. Thus the double lysogen (~80sus2suIII) ()~N7N53029cI857) will be viable at 30°C and lethal at 42°; derivatives which have lost the suppressor will be viable at both temperatures. The frequency of spontaneous suppressor loss is such that thermoresistant derivatives will always be present in velvet replicated clones. However, if the double lysogen carries an amber mutation in an essential gene, the derivatives which have lost the suppressor will be lethal, and no thermoresistant bacteria will be found (Table 2). Using this procedure, it is also possible to find mutants whose thermosensitive phenotype is due to missense mutation. These mutants can be distinguished from the amber ones by looking for the recovery of the thermosensitive phenotype after transducing their mutations into a bacterium without any suppressor.
2. Isolation of Amber Mutants in the aroE-strA Region Phage P1 grown on the prototrophic strain RH2569 was mutagenized with hydroxylamine (Hong and Ames, 1971). The survival of P1 after mutagenesis was 0.5%. The stock, after concentration, was used to transduce the aroE-spcA region into the recipient
151
G. Delcuve et al. : Amber Ribosomal Mutations Table 2. Genotype and phenotype of amber mutants in the dilysogen recipient RH3149 Strain
Relevant a genotype
Distribution of dp80sus2suIII and ~?80sus2 lysogens within one transductant clone
32 ° C recipient (RH3149) or transductant not carrying an amber mutation
x+
transductant carrying an amber mutation
Xam
a x
99.99% (~p8Osus2suII1) (2NTN53029cI857) 0.01% (do8Osus2) (2N7N53029cI857)
99.99% (~?80sus2suII1) (2N7N53029cI857) 0.01% (qbSOsus2) (2NTN53039cI857)
UJ
t
r',- 00
99.7 q
~,
I
1
°I
-
+
+
-
-
ts +
ts
-
Mutation
97
am3161 am3162 am3194 am3157 am3158 am 3163
Recipient RH3126 (suIIIAS1)
RH3184 (suIIIASI*)
RH3149 (suIII)
+ nt -
nt + + nt nt -
+ + + + + +
RH3134 (su +)
69 91
d
+
+
Table 3. PI-mediated transduction of amber mutations in various recipients (selection for aroE +)
m
C
42 ° C
complete genotype of strain RH3149 is listed in material and methods refers to an essential gene, localised in the aroE-strA region, for instance the gene coding for a ribosomal protein
50-60"~ 70-90 ~
at
Resulting phenotype
Growth at
86 51
97
84 7717
Fig, l a - e . Figures on the map refer to % cotransduction; small size numbers refer to deduced values. -~- cotransduction frequencies aroE-spcA and spcA-aroE as compiled from the litterature (Weisblum and Davies, 1968; selection for spectinomycin resistance usually yields lower figures for the spcA-aroE cotransduction frequency, unpublished observations), a selection for aroE + (RH3157:261 transductants), (RH3158:210 transductants), b selection for aroE + (189 transductants) and reciprocal transduction with selection for spcR (133 transductants), e selection for aroE ÷ (200 transductants), d selection for aroE + (187 transductants) and reciprocal transduction with selection for spcR (153 transductants). e selection for aroE + (193 transductants)
strain RH3149 lysogen for tp8Osus2suIII and 2N7N53 O29ci857. After selection for aroE + at 32 °, we screened for complete thermosensitivity at 42 °. Eleven mutants were obtained out of 850 transductants; all of them retained the ability to suppress the ara~m lac~m and lrpa m mutations in RH3149. Five mutations map close to, and to the left of spcA; they were not further characterized. Six map to the right of spcA and cotransduce with this marker at frequencies ranging
+ , survival of the recipient after transduction; - , no survival (120 aroE + transductants were examined for the unselected temperature sensitive mutations); nt, not tested
from 93 to 99.0% (Fig. 1). All six mutants harbor an amber mutation; this was shown by transduction of the aroE-spcA region into a recipient strain, RH3134, which does not carry any suppressor. Selection was made for aroE ÷ and in all cases it was impossible to recover thermosensitive transductants (Table 3). The fact that we did not find any missense mutation among the 6 mutants we studied is not really surprising: the frequency of missense thermosensitive mutations in a localized mutagenesis is about 1 out of 1000 (Cabez6n et al., unpubl, data) In order to carry out the biochemical analysis of these mutations, we transduced them into receptors which carry either a thermosensitive suppressor (suIH A81) or a spontaneous derivative of suIIIA81 which gives a higher level of suppression (suIII A81 *). Table 3 gives the results of these transductions. It should be noted that, although the mutations can be readily transduced in a suIII receptor, some cannot be transduced in bacteria that carry suIII A81 or suIII A81*, suggesting that the efficiency of these thermosensitive suppressors is too low to allow survival.
152
G. Delcuve et al. : Amber Ribosomal Mutations
4I--
42 °C
42°C 20
~0
4
20
50S 50S
lip
II
~!
3
J; IO
15F
p
30S
30S
ji LO
10
~
5
5
i
O 0
10
30
50
0
10
30
15
50
0
10
30
'
0
50
Fig. 2. Sucrose gradient sedimentation analysis of extracts from cells incubated at 42 ° C in the presence of radioactive uracil. Abscissa indicate the n u m b e r of fractions from the top of the gradient. Ordinates are in cts/min × 10 3; left scales are for 14C (parent strain) and right scales for aH (mutants). a R H 2358 ( 1 4 C ) - e - e - x R H 3161 (3H)--©--o--. b R H 2358 ( 1 4 C ) - e - e - x R H 3162 (3H)--©--©--. e R H 2358 ( 1 4 C ) - e - e - x RH3194 (3H)--©--o--
Whenever transduction was possible, the resulting strain became thermosensitive, further indicating the amber nature of the mutations. The strains carrying a thermosensitive suppressor and the mutations am3161, am3162 and am3194 are refered to as RH3161, RH3162 and RH3194, respectively.
3. In vivo Assembly of Ribosomal Particles in Strains RH3161, RH3162 and RH3194 In conditions which inactivate the suppressor t R N A molecules, the ribosomal protein coded for by the mutated gene will not be synthesized and subsequently assembled in mature ribosomes. This will result in defective assembly of ribosomal particles. The extent of defectivity will depend on the nature of the mutated gene and on the possible polar effect of the mutation on genes distal to the mutated locus. We labelled the mutant strains with 14C-or 3Huracil at 42 ° C, as explained in Material and Methods, i.e., in conditions where the suIIIts suppressor t R N A is inactivated. We analyzed the extracts of the parent and mutant strains on sucrose gradients in conditions which allow observation of the ribosomal subunits (low magnesium salt). As seen in Figure 2, all three mutants are defective in ribosomal assembly in vivo
at 42 ° C. The strain RH3161 no longer synthesizes mature ribosomes, but accumulates precursors of 30S and 50S, whereas mutants RH3162 and RH3194 do not assemble 50S ribosomal particles nor detectable amounts of precursors. It should be noted that, even at 30 °, strain RH3161 accumulates incomplete particles, in lower amounts than at 42°C however. This is consistent with the fact that the sulII thermosensitive mutations have a relatively low suppression efficiency at 30 ° C. The same comment applies to the mutants RH3161 and RH3194 which, at 30 ° C, do not assemble normal amounts of 50S ribosomes (data not shown). Using classical techniques, the preliminary analysis of the ribosomal proteins isolated from mutants RH3161 and RH3162 did not yet reveal any clear modification of the patterns.
Discussion
In this report we describe a method to isolate amber mutations in any gene of E. coll. We have used this method in a search for mutations in ribosomal protein genes. Eleven mutations leading to complete thermosensitivity to the recipient strain have been recovered; all are cotransducible with spcA and all six
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we studied were shown to be lethal in the absence of suppression. Furthermore, the transduction data for the six mutations which map at the right of spcA are in good agreement with the localisations for ribosomal protein genes obtained by Nomura (1976). Thus, it appears that the selection procedure effectively yields amber mutations in the region of interest. The method was designed to allow the recovery of amber mutations in bacteria carrying an efficient suppressor. Indeed ribosomal proteins are necessary in large quantities and one may expect that a low efficiency of suppression, such as that provided by a thermosensitive suppressor, would severely restrict the recovery of such mutations. This expectation proved to be true as 4 out of 5 mutations could not be transduced in a strain carrying a thermosensirive suppressor, suIII A81. At least one could not even be transduced in a more efficient, spontaneous, derivative of suIII A81. Clearly, the use of a thermosensitive suppressor in the selection procedure would limit the selection to a small subgroup of mutations, at least in the spcA-strA region. On the other hand, the fact that different mutants require different efficiencies of suppression to be saved may be analyzed in terms of level of expression of the various genes. However, such an analysis requires the identification of the mutated genes and of their product. Among the six mutations which map in the region coding for ribosomal proteins, three have been studied biochemically in non-suppressing conditions. All three prevent normal ribosomes to be formed in vivo. One of them, am3161 prevents the formation of mature 30S and 50S subunits; this is consistent with the hypothesis that the mutation exerts a polar effect on genes coding for 30S and 50S ribosomal proteins which are distal to the mutated gene. Indeed, the map location of the mutation am3161 places it in the so-called spc operon (Nomura, 1976) which has the following structure:
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