Vol. 138, No. 2
JOURNAL OF BACTERIOLOGY, May 1979, p. 383-396
0021-9193/79/05-0383/14$02.00/0
Expression of Ribosomal Protein Genes Cloned in a Hybrid Plasmid in Escherichia coli: Gene Dosage Effects on Synthesis of Ribosomal Proteins and Ribosomal Protein Messenger Ribonucleic Acidt ANN M. FALLON,: C. SUE JINKS, MASAYUKI YAMAMOTO,§ AND MASAYASU NOMURA* Institute for Enzyme Research, Departments of Genetics and Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
Received for publication 5 January 1979
Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (rprotein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 420C; in recA strains, the degradation took place at 42°C but was very slow or absent at 300C. The recA strains carrying pNO1001 failed to forn colonies at 300C, Wresumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process. In exponentially growing Escherichia coli, synthesis of ribosomal proteins (r-proteins) and rRNA is under coordinate control (for reviews, see references 18 and 27). The rate of r-protein accumulation is balanced with that of rRNA accumulation, and no significant pool of free rproteins or rRNA exists in the cell. How this regulation is achieved, however, is not well understood. We wished to examine whether increased gene copies for some r-proteins had an effect on coordinate expression of ribosomal components. For example, E. coli might have some regulatory mechanism which involves detection of excess free r-proteins followed by feedback inhibition of the expression of the corresponding genes at the level of transcription or translation. In this event, increased gene dosage for r-proteins t Paper no. 2320 from the Laboratory of Genetics. t Present address: Texas A&M University, Department of Entomology, College Station, TX 77843. § Present address: Department of Biophysics, Faculty of Science, Kyoto University, Kyoto, Japan. 383
would not lead to overproduction of those proteins. We have constructed a hybrid plasmid, pNO1001, which carries several r-protein genes together with their promoter. Expression of genes carried by pNO1001 was investigated by measuring mRNA and r-protein synthesis in cells carrying the plasmid. Although the mRNA for all the promoter-distal r-protein genes appeared to be synthesized in large excess relative to mRNA's from other chromosomal r-protein genes, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. We have also found that degradation of overproduced L14 and L24 is probably essential for normal cell growth in strains carrying pNO1001 and that the recA gene product appears to be involved in the degradation of excess r-proteins. MATERIALS AND METHODS Construction of strains. The bacterial strains used in the present work are listed in Table 1. To
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TABLE 1. E. coli strains used Strain C600 N01216 N01967 N01884 N01885
N01887 N01981 FEJ-1 (= N01976) N01983 JC1553 (= N01933) AB2462 (= N01936) N01986 N01988 N01961
Relevant genotype I.- - and comments -
F- thr leu lac thi F- trkA401 kdpABC5 lac gal thi (see reference 9) Same as N01216, but pro trkA+ rplX165 spcr mutant of N01967 F- trkA401 kdpAC5 lac gal thi spcr (rpsE) rplX165 recA' recAl derivative of N01885 recA+ derivative of N01887 Hfr leu mtl srlCl rpsL
Hfr leu mtl srlCl spcr rplX165 argG6 metBI his-I leu-6 recAl mtl-2 xyl- 7 malAI gal-6 lacY) rpsL104 tonA2 tsx-l supE144 thr-l leu-6 thi-l argE3 his-4proA2 recA12 lacYI galK2 mtl-l xyl-5 ara-14 rpsl3l tsx-33 supE44 Same as N01983, except srlC+ recAl Same as N01983, except srlC+ recA12 AprecA+ cI+ lysogen of N01887
construct a strain with a temperature-sensitive mutation in an r-protein gene, purified Afis3 transducing phage was mutagenized in vitro with hydroxylamine. Strain N01216 was then infected with the mutagenized phages, and trkAh transductants were isolated at 30°C. They were then screened for ability to grow at 42°C. One of the resulting temperature-sensitive strains, N01967, was found to have a mutation (rplX165) which altered the electrophoretic mobility of L24 (see Results and Fig. 3). Although rigorous experimental proof is lacking, the temperature-sensitive phenotype and the L24 alteration appear to be due to the same mutation, and the mutant resembles that described by Cabez6n and his co-workers (3). The full description of the mutation will be given elsewhere. An SpCr mutant of N01967 (= N01884) was obtained by mutagenesis with ethylmethane sulfonate. N01885 was constructed by introducing rplX165 and spcr from N01884 into N01216 by P1 transduction. A thy mutant of N01885 was isolated by trimethoprim selection (25). This strain (N01886) was crossed with a derivative of KL16-99 (Hfr ser recAl thi), and a thy' recAl recombinant (N01887) was obtained. A recA+ derivative (N01981) of N01887 was again constructed by isolating a thy mutant (N01980) of N01887 and mating it with an Hfr strain (KL16) which injects thy' recA+ genes as early markers. Thy' Spcr recombinants were screened for the presence of recA+. UV light sensitivity was used to distinguish RecA+ from RecAphenotypes. Another set of isogenic strains carrying rplX165 and various recA alleles was constructed in the following way. The rplX165 mutation was first introduced from N01884 into FEJ-1 by P1 transduction; one of the sper transductants which also received the rplX165 allele was named N01983. The recAl and recA12 genes were transferred to N01983 by a second P1 transduction from AprecA+cI857 lysogens of JC1553 and AB2462, respectively. (Lysogenization of the recA strains with AprecA+cI857 greatly improved the growth of P1 in these strains; since the prophage tended to segregate, the lysogens were usually grown in the presence of
Source
W. Epstein
W. Epstein B. Bachmann
B. Bachmann
nitrofurantoin [2 ug/ml, Sigma Chemical Co.] [24].) srl+ transductants were selected for, and these were screened for RecA- phenotype, N01986 and N01988 were thus isolated. AprecA+cI857 was made cI+ for experiments at 420C by the following crosses. Xh80cI857 was crossed with Xpapa (wild type), and lysogens carrying recombinant Ah8OcI+ were selected in a malA strain at 420C. The Ah8OcIW phage was then crossed with XprecA+cI857, and lysogens carrying recombinant AprecA+cI+ were isolated on a tonB strain at 420C. Phages were induced from the lysogens and checked for the ability to transduce recA+ into recA strains. Nitrofurantoin (see above) was used to facilitate detection of recA+ colonies and to prevent segregation of the prophage. N01887 was lysogenized with AprecA+cI+ (= N01961) and was used for various experiments. Construction of hybrid plasmids containing rprotein genes. Preparation of plasmid and transducing phage DNA has been described previously (17). Plasmid RSF2124, a ColEl derivative carrying an ampicillin resistance transposon (TnA), was used as a cloning vector (32). Xfs3 DNA (see references 14 and 21) and RSF2124 plasmid DNA were digested to completion with EcoRI restriction endonuclease, and the reaction was terminated by heating at 650C for 8 min. Digests were ligated as described by Tanaka and Weisblum (33). For transformation, exponentially growing E. coli K-12 strains were treated with cold 50 mM CaC12 as described by Mandel and Higa (23). Plasmid pNO1001, which carries the Afus3 EcoRI 10% fragment, was isolated by direct selection at 420C, in the absence of ampicillin, using temperature-sensitive N01967 as the recipient strain. Plasmid pNO1002, which carries the Afus3 EcoRI 9% fragment, was isolated by ampicillin selection, using C600 as the recipient strain. In this case, the DNA-treated cells were first diluted with rich medium, grown at 370C for 1 h, and then plated on ampicillin plates (100 jug/ml) at 370C. These hybrid plasmids were introduced into various strains by transformation, and the resulting strains were used in the present work (see below).
VOL. 138, 1979
DOSAGE EFFECTS ON r-PROTEIN GENE EXPRESSION
Growth conditions. For labeling experiments, cells were grown in a synthetic minimal medium (AB medium; see reference 4) supplemented with 0.4% glucose, 50 jAg of all amino acids except the one used for labeling per ml (or 0.2% Casamino Acids), thiamine at 2 Ag/ml, and, for trkA strains, 0.1 M KCI. KCl (0.1 M) was also included in the medium for trkA+ cells when they were used as controls for experiments with trkA strains. Uracil (5 Ag/ml) was added for steadystate labeling of mRNA. The details of the labeling conditions are given in the footnotes to the tables and in the figure legends. In all cases, ampicillin (100 ,ug/ ml) was used for overnight cultures of plasmid-carrying strains, and nitrofurantoin (2 ,ug/ml) was included for lysogens of XprecA . Overnight cultures of strains carrying pNO1001 were always grown at 42°C (see below). For direct analysis of unlabeled r-proteins by two-dimensional gel electrophoresis, cells were grown in rich medium (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 7.4 g of KCI per liter, pH 7.0). Ampicillin (100 yg/ml) was included whenever the strain carried a hybrid plasmid to prevent segregation of the plasmid. We note that the strains carrying pNO1001 were stable as long as the strains were grown in the presence of ampicillin or at 420C. Agar plates contained 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl (pH 7.0), and, where appropriate, 7.4 g of KCI per liter. Preparation and electrophoresis of r-proteins. In labeling experiments, 1 volume of labeled cells was added to 1.5 volumes of ice-cold acetone. This procedure (1) was used to rapidly stop the incorporation of labeled amino acids and to prevent the possible intracellular degradation of protein. After storage overnight at -20°C, samples were centrifuged at 12,000 x g for 20 min and decanted, and the pellets were lyophilized to dryness. All of the trichloroacetic acid-precipitable radioactivity was recovered in the pellet. For one-dimensional electrophoresis on urea gels (see reference 34), pellets were suspended in 50 jul of 50 mM Tris-hydrochloride (pH 8.0), 6.5 Al of 0.1 M EDTA (pH 7.1), and 5 pl of lysozyme (0.4 mg/ml). Preparations were subjected to, four cycles of freezethawing, and carrier 70S ribosomes were added (15 units of absorbance at 260 nm). Samples were made 0.1 M with MgCl2, and r-proteins were extracted with two volumes of glacial acetic acid (12). After addition of acetic acid, samples were mixed thoroughly, and portions were removed for determination of total radioactive protein. For two-dimensional gel electrophoresis (16), 100 units of absorbance at 260 nm of carrier ribosomes were used per gel. To analyze unlabeled, carriei-free r-proteins from various strains, cells were grown in rich medium (2 liters) at 42°C. At a cell density of about 108/ml, half of the culture was shifted to 30°C and the remaining half continued to incubate at 42°C. After two to four doublings in cell mass, cells were collected by centrifugation and stored frozen. After thawing, pellets were suspended in 10 ml of 10 mM Tris-hydrochloride (pH 7.5), 30 mM NH4Cl, 10 mM MgCl2, and 6 mM ,Bmercaptoethanol (TMAI) and were broken by sonic oscillation. Debris was removed by centrifugation at 12,000 x g for 1 h, and the supernatant was centrifuged at 60,000 x g for 4.5 h. The crude ribosome pellet was
385
resuspended in 0.3 ml of TMAI. For electrophoresis, r-proteins were extracted with 67% acetic acid from 100 units of absorbance at 260 nm of these crude ribosomes. "Salt washed" ribosomes were prepared by centrifuging the crude ribosomes through a 38% sucrose solution containing 20 mM Tris-hydrochloride (pH 7.4), 10 mM MgCl2, 500 mM NH4C1, 0.5 mM EDTA, and 1 mM dithiothreitol. The ribosome pellets were rinsed twice with TMAI, and the loose brown material lying over the clear ribosome pellet was discarded. Ribosomes were resuspended in 0.2 ml of TMAI, and protein was extracted from 100 units of absorbance at 260 nm of ribosomes for electrophoresis. Preparation of [2PJRNA. [3P]RNA was used as an internal standard to measure the relative efficiency of hybridization of 3H-labeled RNA samples to various DNAs. The [3P]RNA was prepared by in vitro transcription, using purified restriction enzyme fragments as DNA templates. The templates used were the 10% EcoRI fragment from pNO1001 and the 4.4%-L EcoRI fragment from Xrifdi8 (see the text; 22). The 4.4%-L fragment carries genes for r-proteins Li, L10, Lll, and L7/L12 (22). This fragment was prepared from hybrid phage Ch3R11, which is a derivative of the charon 3 vector phage and carries a cloned 4.4%-L fragment (L. Post and M. Nomura, unpublished data; for the charon 3 phage, see reference 2). In vitro transcription was done as described previously (29). After the reaction, carrier RNA (E. coli 16S rRNA; 50 ,g/ml) was added, and the mixture was treated with phenol. RNA in the aqueous phase was precipitated with two volumes of ethanol, washed twice by reprecipitation, lyophilized, and dissolved in 2x SSC (SSC is 0.15 M NaCl-0.015 M sodium citrate). Extraction and purification of [3HJRNA and RNA-DNA hybridization. Labeled celLs were added to an equal volume of boiling lysis solution containing 1% sodium dodecyl sulfate, 0.1 M NaCl, 8 mM EDTA (pH 7.0), and 100 Ag of carrier yeast tRNA per ml. The mixture was heated in a boiling-water bath for an additional 2 min and then treated with phenol at room temperature. The aqueous phase was made 0.3 M NaCl and precipitated with two volumes of ethanol. The samples were washed twice by reprecipitation, lyophilized to dryness, and dissolved in 1 ml of 2x SSC. Appropriate portions were hybridized to filters containing excess DNA in the presence of constant amounts of in vitro-labeled [32P]RNA to monitor hybridization efficiency. Input radioactive RNA was determined by precipitation with 5% trichloroacetic acid followed by membrane (Millipore Corp.) filtration. Hybridization was done in 2x SSC at 670C for 15 to 18 h as described previously (8). The amounts of [3H] RNA hybridized were determined, and corrections were made for the small variation of hybridization efficiency observed with [32P]RNA. If the hybridization efficiency is the same in all the reaction mixtures in a given experiment, the amounts of [32P]RNA hybridized to DNA should be the same in all the samples. The average of the values for the amounts of [32p] RNA hybridized was calculated in each experiment and used for correction according to the following
[3H] equation: [3H]RNA hybridized (corrected) RNA hybridized (observed) x (average of [32P]RNA hybridized/[32P]RNA hybridized). Corrections made
386
FALLON ET AL.
J. BACTERIOL.
in this way were usually within 10% of the observed values. Other methods. Methods for restriction enzyme digestion, separation of restriction enzyme fragments on analytical gels, and preparative separation of DNA fragments have been described previously (22). The cell densities of bacterial cultures were measured with a Klett-Summerson photoelectric colorimeter, using a blue filter (no. 42). Fifty Klett units corresponded to about 2 x 108 cells per ml with ordinary E. coli K-12 strains (e.g., strain N01216) growing in AB-glucose minimal medium.
RESULTS Cloning of r-protein genes with the Spc promoter. The Afus3 transducing phage carries 27 r-protein genes and the genes for elongation factor Tu (tufA), elongation factor G (fus), and RNA nucleotidyltransferase (RNA polymerase) subunit a (rpoA) (13-15, 21; see Fig. 1). To examine the effects of high dosages of r-protein genes, we cleaved Xfus3 DNA with EcoRI restriction endonuclease and tried to clone EcoRI fragments carrying r-protein genes and their promotor into a plasmid cloning vector. We first used ColEl-TnA hybrid plasma RSF2124 as the cloning vector and strain C600 as the recipient. Random cloning procedures were carried out, that is, ligation of a mixture of vector and Afus3
Il?7
0
1~ ~~~~~~~~~t S4 S13 S5 SlI
P
(LI5
L30)
L18
Xfus3
-"_ 1
5 131
4.6
L6S8
"I
27
10
EcoRI fragments, transformation, selection of ampicillin-resistant transformants, screening of transformants for inability to produce colicin El, and analysis of plasmid DNA carried by the transformants. We failed to clone any fragment with a promoter, but we were able to clone other fragments (1.2,4.6,8.5, and 9%) with sizes similar to those of the promoter-containing fragments (2.2, 5, 10, and 11%) of Xfus3. (Sizes are given in percentage-of-X units; 1% of X = 465 base pairs.) These observations suggest that promoter-containing fragments were probably ligated into RSF2124, but, for some reason, the presence of the resulting plasmids was harmful to the growth of the recipient cells. (The same conclusion was also obtained by J. M. Zengel [Ph.D. thesis, University of Wisconsin-Madison, 1976].) Strain NO1967, which has a temperature-sensitive mutation (rplX165) in (or very close to) the gene for L24 (rpLX), was then used as a recipient for cloning experiments; selection of the transformants was done at 42°C in the absence of ampicillin. We expected that transformants which grew at 42°C would harbor hybrid plasmids carrying the 10% EcoRI fragment of Xfus3, since this fragment carries the wild-type L24 gene and its promoter, p., (cf. Fig. 1). This expectation was, in fact, realized. Over 99% of
5S14 L5 L24 L141SL729LC6)S3 (S19 L22)(L2
1.0 2.2 114.61 _-9-1
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L23)
2.2 L3 50
2
19
VA II I 10 lo
j
8.5
j
Tu
G S7 S12
11
P...
EcoRI(M) P,s,
EcoRI (J)
pNO 1001
Smo (1) BomHI (*) HindII (t)
FIG. 1. Map of Afus3 and ofpNO1001. The locations of bacterial genes and cleavage sites of restriction endonucleases are shown. The hatched parts represent A DNA. The circular pNO1001 plasmid molecule is shown with two horizontal bars, one representing vector RSF2124 DNA (crosshatched bar) and the other representing the cloned EcoRI 10% fragment. The locations of bacterial DNA fragments A, B, C, and D, used for hybridization experiments, are shown. Fragments A and B were prepared from hybrid plasmids pNO2003 and pNO2002, respectively (29). Fragments C and D were prepared from similar plasmids, pNO2014 and pNO2015, respectively (Post and Nomura, unpublished data). The orientation of the cloned 10% fragment relative to the Sma restriction endonuclease site in RSF2124 (see reference 5) is shown. Restriction enzyme sites for BamHI and HindII are shown for the 10% fragment only; sites for Sma are shown for the 10% fragment and RSF2124. Distances between restriction sites are in percentage-of-A units. It should be noted that the spc operon promoter (p.,) is not included in fragment B (29). Fragments A, B, C, and D are about 820, 440, 840, and 1,000 base pairs long, respectively.
DOSAGE EFFECTS ON r-PROTEIN GENE EXPRESSION
VOL. 138, 1979
387
the temperature-resistant colonies analyzed the spc operon in this study) in RecA- cells was ampicillin resistant, and, of these, 95% responsible for this apparent "cold sensitivity." contained a cloned fragment. Plasmid DNA was We used hybrid plasmid pNO1002, which has isolated from 20 independent transformants, and the 9% EcoRI fragment cloned into RSF2124, as restriction enzyme analyses with EcoRI and a control for pNO1001. The 9% EcoRI fragment Sma confirmed that the 10% EcoRI fragment of XAfus3 does not carry genes for any r-proteins carrying p., had been cloned. In all 20 cases, the and is similar in size (9% of A, or about 4,200 10% fragment was in the same orientation rela- base pairs) to the 10% fragment (about 4,700 tive to the RSF2124 vector DNA. One of the base pairs) cloned in pNO1001. It was found that hybrid plasmids thus constructed, pNO1001, was the same recAl strain (NO1887) carrying used in the experiments described in this paper pNO1002 grew normally and formed colonies at (see Fig. 1 and Table 2, group 1). (We have not 30°C (Table 2). In addition, isogenic recA+ parstudied the reason why we failed to isolate a ent strain N01885 and strain N01981, which hybrid plasmid carrying the 10% EcoRI frag- was derived from N01887 and was made recA+ by mating, did not show cold sensitivity when ment in the opposite orientation.) Growth characteristics of strains harbor- carrying pNO1001. Similarly, lysogenization of ing the pNO1001 plasmid. To prevent possible the original recAl strain (NO1887) with recombination between chromnosomal genes and AprecA+cI+ abolished this pNO1001-induced cold sensitivity (Table 2, group 2). genes on the plasmid, hybrid plasmid pNO1001 We also constructed a strain (NO1983) carrywas introduced by transformation and selection ing the temperature-sensitive rplX165 mutation on ampicillin plates at 42°C into recAl strain N01887, which carries rplX165. It was found and subsequently introduced recA mutations by that the resultant strain, N01887(pNO1001), P1 transduction. Both the recAl derivative and the recA12 derivative were cold sensitive when was not able to form colonies when incubated on plates at 30°C, in contrast to the parent, NO1887 carrying the pNO1001 plasmid, although the (Table 2). Systematic studies showed that the phenomenon was less striking with the recA12 strain (Table 2, group 3). presence of multiple copies of r-protein genes The cold sensitivity caused by the presence of with their promoter (specifically, the genes in
were also
TABLE 2. Colony-forming abilities ofplasmid-carrying strains Colony formation at: Group'
1
Strain
Plasmid
300C
N01967
pNO1001 pNO1002 2
RecA phenotype
N01885
pNO1001 pNO1002 N01887 pNO1001 pNO1002
N01887 (AprecA+cI+) pNO1001 pNO1002
N01981
pNO1001
420C
Ratio of colonies at 30'C/ colonies at
42"C
+ +
+
-
+
+
+
+
-
+ + + - (recAI) - (recAI) - (recAI) +
+ + + + + +
+ -
1.0
+
0.001
+ +
+ +
+
+
+
+
+ +
1.0
-
1.0 1.0
+ + 1.0 + + + + - (recAI) N01986 0.0007 + - (recAI) pNO1001 + - (recA12) N01988 + 0.02 - (recA12) (+)b pNO1001 aStrains within a group are isogenic except for recA alleles and possibly some genes linked to recA (see the text). b Small colonies appeared at 30°C after longer incubation. 3
N01983
PNO1001
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FALLON ET AL.
plasmid pNO1001 was measured by growing strains at 42°C, plating cells both at 30 and at 420C in the presence of ampicillin, and calculating the ratio of the colonies formed at 300C to those formed at 420C (the last column in Table 2). About 1,000-fold and 50-fold reductions were observed in the numbers of colony formers measured at 300C when plasmid pNO1001 was carried by recAl and recA12 strains, respectively. [Several colonies from N01887(pNO1001) which grew on plates at 300C were analyzed for the plasmids they carried. Many of them had plasmids with altered sizes, so it seems that most of these survivors represented mutants formed during residual growth of these cold-sensitive cells on plates at 300C. The nature of these mutations has not been further examined.] The observed cold sensitivity of recA strains carrying plasmid pNO1001 was probably due to the excess accumulation of certain r-proteins at 300C. This will be described below. Synthesis of r-proteins in N01887 (pNO1001). Since N01887(pNO1001) carried multiple copies of several r-protein genes in the spc operon, we examined the question of whether r-proteins coded for by these genes were overproduced in this strain. Because of their temperature-sensitive character, N01887 cells could not be used as control cells at 420C. Therefore, we used C600(pNO1002) cells as control cells, as described above. Another control strain, N01216, had a genetic background closely related to that of N01887 (see Table 1). Similar results were obtained with either control strain. In the first experiments, cells were grown exponentially at 420C and labeled with [35S]methionine. The incubation temperature, 420C, was chosen to prevent the segregation of the plasmid. It should be noted that N01887 cells do not grow at 420C unless they harbor the plasmid carrying the wild-type L24 gene (rpLX). Radioactive r-proteins were analyzed by onedimensional polyacrylamide-urea gel electrophoresis, followed by autoradiography (Fig. 2). Marked overproduction of one protein relative to other methionine-containing r-proteins was observed after a short pulse-labeling period. This protein had the mobility of L14. Analysis by two-dimensional gel electrophoresis (see below) confirmed that the overproduced protein was in fact L14, the protein coded for by the first gene in the spc operon. Most of the overproduced protein was degraded within 30 min at 420C (Fig. 2, slot 11). Degradation was either very slow or did not take place at 300C in this strain (data not shown; see also below). Visual inspection of the autoradiogram given in Fig. 2
J. BACTERIOL. .,o. .4.6
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2
2
3
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FIG. 2. Expression of r-protein genes on pNO1001. Cells (5 ml) were labeled at 42°C with 65 ,uCi of [3SJmethonine, and 1-mi samples were taken at various times after addition of label. Total acid-precipitable counts indicated complete uptake of isotope by 2 min. Lanes 1, 4, 7, and 10 are C600(pNO10(X2); 2, 5,8, and 11 are N01887(pNO1001); 3,6,9, and 12 are N01216 at 2, 4, 6, and 30 min after addition of isotope, respectively. Samples were analyzed by polyacrylamide-urea gel electrophoresis. An autoradiogram of a dried gel is shown, and the position of L14 is indicated. The protein band shown with an arrow was seen only in short-pulse-labeled samples from control cells, C600(XpNO1002) and N01216, but not in samples from N01887(pNO1001). The nature of this protein has not been studied. In this experiment, N01887(pNO1001) was grown in the absence of ampicillin. Examination after the experiment showed that most or all of the cells retained the pNO1001 plasmid during the experiment.
shows that no marked overproduction of other methionine-containing r-proteins took place in N01887(pNO1001). L24, whose gene on the plasmid was used for the original selection (see above), does not contain methionine and could not be examined by these autoradiograms. To determine whether L24 was overproduced in N01887(pNO1001), we analyzed the rates of r-protein synthesis in cells labeled with [3H]lysine. The synthesis rates of several r-proteins in the plasmid-carrying strains, including those coded for by genes carried by pNO1001, were compared with those in the control strain as described in the footnote to Table 3. It was found that the amounts of L14 and L24 synthesized during a 2-min labeling time were five- to sevenfold higher in NO1887(pNO1001) cells than in control (NO1216) cells when normalized to L1 (Table 3). Overproduction of other proteins coded for by genes on pNO1001 which are distal to the L24 gene in the spc operon was small (L5, S14, and L6) or insignificant (S8 and L18). Proteins L16, L29, and S17, whose genes precede
DOSAGE EFFECTS ON r-PROTEIN GENE EXPRESSION
VOL. 138, 1979
TABLE 3. Rates of individual r-protein synthesis in N01887(pNO1001) relative to those in control strain N01216a
Relative syntheClass Cre Protein rate
1
L16 L29 S17
0.95 1.03 0.95
2
L14 L24 L5 S14 S8 L6 L18
6.7 5.5 1.80 2.04 1.23 1.51 1.34
3
S5 (+L11) L15 L30
1.09 0.81 1.14
Li L3 1.11 L10 0.99 a Genes for the class 1 proteins are carried by the pNO1001 plasmid, but their promoter is not present on the plasmid. Genes for the class 2 proteins and their promoter are carried by pNO1001. Genes for the class 3 proteins are in the spe operon at 72 min, but are not carried by pNO1001. Chromosomal locations of genes for the class 4 proteins are known; these genes are definitely not carried by the plasmid. Both experimental [NO1887(pNO1001), doubling time of 110 min] and control (NO1216, doubling time of 40 min) cells were grown at 420C to approximately 2 x 108/ml. [3H]lysine (490 pmol/ml; 61 Ci/mmol) was added to experimental cells for 2 min and to control cells for 1 min, followed by a 1-min "chase" with excess nonradioactive lysine. Celis were rapidly chilled and collected. [3H]lysine-labeled cells were then mixed with a suitable amount of ['4C]lysine-labeled carrier cells. r-Proteins were extracted and separated by two-dimensional gel electrophoresis. After staining the gels, the spots corresponding to the indicated r-proteins were cut, the gels were oxidized with a Packard sample oxidizer, and 3H and '4C were counted separately. The 3H/14C ratio in each protein in the experimental cells was then compared with that in the control cells; that is, the values were first calculated with the following equation: 3H/'4C in protein i in experimental cells + 3H/'4C in protein i in control cells. The ratios obtained were then normalized to the ratio obtained with protein Li. The gene for Li is not carried by Xfus3 and maps near rpoB at 88 min (20, 35); hence, the value for protein Li was used as a reference for normalization and is boxed in the table. The values given are averaged from three experiments. 4
the spc operon promoter (ppC) on pNO1001, were not overproduced. This suggests that the p8pc promoter was functioning as the main promoter for the L14 and L24 genes on the plasmid
389
and that readthrough transcription of cloned rprotein genes from a promoter(s) on the vector was small or nonexistent. The experiments described above showed that N01887(pNO1001) overproduced L14 and L24 but that the overproduced L14 was unstable and was gradually degraded at 420C. As in the case of L14, the overproduced L24 was gradually degraded at 420C. This was demonstrated by pulse-labeling cells with [3H]leucine and chasing with nonradioactive leucine at 420C (data not shown). Degradation of the overproduced L14 and L24 was also apparent when the amounts of r-proteins in N01887(pNO1001) were directly visualized by staining two-dimensional gels of the extracted r-proteins (see Fig. 3H compared with Fig. 3A through D). Details of the experiments shown in Fig. 3 will be described later. Synthesis of r-protein mRNA in NO 1887(pNO1001). We were surprised that only L14 and L24 were strongly overproduced in N01887(pNO1001). To determine whether transcription also drastically decreased after the L24 gene, we prepared DNA fragments representing various segments of the 10% fragment (Fig. 1) and examined the syntheses of the mRNA's corresponding to these various DNA segments. Cells were pulse-labeled with [3H]uracil, and radioactive RNA was analyzed by the DNARNA hybridization method. We used the EcoRI 4.4%-L fragment carrying four r-protein genes (the genes for Li and L10 and parts of the genes for Lii and L7/L12; see reference 22 and our unpublished data cited in reference 36) in the rif region at 88 min as a reference DNA. As mentioned above, these genes are not carried by plasmid pNO1001, and, therefore, the amounts of the plasmid-coded mRNA's relative to the 4.4%-L DNA-coded mRNA could be used to compare the syntheses of the pertinent mRNA's in N01887(pN01001) with those in the control haploid strain. Cells carrying the pNO1001 plasmid overproduced mRNA corresponding to segment B (14- to 19-fold higher than in the control strain) which carries the L14 gene and a part of the L24 gene (Table 4). In addition, the results showed that even the mRNA distal to the L14 and L24 genes was overproduced, although the more distal portions of the message showed a lesser degree of overproduction. In contrast, the message corresponding to the genes for L16, L29, and S17 was only 2-fold higher in N01887(pNO1001) than in the control strain (N01216). This weak overproduction may reflect transcription from a promoter(s) on the vector. It should be noted that the degree of relative overproduction of mRNA corresponding to seg-
390
FALLON ET AL.
J. BACTERIOL.
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JOURNAL OF BACTERIOLOGY, May 1979, p. 383-396
0021-9193/79/05-0383/14$02.00/0
Expression of Ribosomal Protein Genes Cloned in a Hybrid Plasmid in Escherichia coli: Gene Dosage Effects on Synthesis of Ribosomal Proteins and Ribosomal Protein Messenger Ribonucleic Acidt ANN M. FALLON,: C. SUE JINKS, MASAYUKI YAMAMOTO,§ AND MASAYASU NOMURA* Institute for Enzyme Research, Departments of Genetics and Biochemistry, University of Wisconsin, Madison, Wisconsin 53706
Received for publication 5 January 1979
Using ColE1-TnA hybrid plasmid RSF2124 as the cloning vector, we constructed a hybrid plasmid, pNO1001, which carried seven ribosomal protein (rprotein) genes in the spc operon together with their promoter. The plasmid also carried three r-protein genes which precede the spc operon, but did not carry the bacterial promoter for these genes. Expression of r-protein genes carried by pNO1001 was studied by measuring messenger ribonucleic acid and r-protein synthesis in cells carrying the plasmid. It was found that the messenger ribonucleic acid for all the promoter-distal r-protein genes was synthesized in large excess relative to messenger ribonucleic acid from other chromosomal r-protein genes which are not carried by the plasmid. However, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. The absence of large gene dosage effects on the synthesis of other distal proteins appeared to be due, at least in part, to preferential inactivation and/or degradation of the distal message which codes for these proteins; in addition, some preferential inhibition of translation of the distal message might also have been involved. Overproduced L14 and L24 were found to be degraded in recA+ strains at both 30 and 420C; in recA strains, the degradation took place at 42°C but was very slow or absent at 300C. The recA strains carrying pNO1001 failed to forn colonies at 300C, Wresumably because of overaccumulation of r-proteins. The results suggest that degradation of excess r-proteins is an important physiological process. In exponentially growing Escherichia coli, synthesis of ribosomal proteins (r-proteins) and rRNA is under coordinate control (for reviews, see references 18 and 27). The rate of r-protein accumulation is balanced with that of rRNA accumulation, and no significant pool of free rproteins or rRNA exists in the cell. How this regulation is achieved, however, is not well understood. We wished to examine whether increased gene copies for some r-proteins had an effect on coordinate expression of ribosomal components. For example, E. coli might have some regulatory mechanism which involves detection of excess free r-proteins followed by feedback inhibition of the expression of the corresponding genes at the level of transcription or translation. In this event, increased gene dosage for r-proteins t Paper no. 2320 from the Laboratory of Genetics. t Present address: Texas A&M University, Department of Entomology, College Station, TX 77843. § Present address: Department of Biophysics, Faculty of Science, Kyoto University, Kyoto, Japan. 383
would not lead to overproduction of those proteins. We have constructed a hybrid plasmid, pNO1001, which carries several r-protein genes together with their promoter. Expression of genes carried by pNO1001 was investigated by measuring mRNA and r-protein synthesis in cells carrying the plasmid. Although the mRNA for all the promoter-distal r-protein genes appeared to be synthesized in large excess relative to mRNA's from other chromosomal r-protein genes, only the two promoter-proximal r-proteins, L14 and L24, were markedly overproduced. We have also found that degradation of overproduced L14 and L24 is probably essential for normal cell growth in strains carrying pNO1001 and that the recA gene product appears to be involved in the degradation of excess r-proteins. MATERIALS AND METHODS Construction of strains. The bacterial strains used in the present work are listed in Table 1. To
384
FALLON ET AL.
J. BACTERIOL.
TABLE 1. E. coli strains used Strain C600 N01216 N01967 N01884 N01885
N01887 N01981 FEJ-1 (= N01976) N01983 JC1553 (= N01933) AB2462 (= N01936) N01986 N01988 N01961
Relevant genotype I.- - and comments -
F- thr leu lac thi F- trkA401 kdpABC5 lac gal thi (see reference 9) Same as N01216, but pro trkA+ rplX165 spcr mutant of N01967 F- trkA401 kdpAC5 lac gal thi spcr (rpsE) rplX165 recA' recAl derivative of N01885 recA+ derivative of N01887 Hfr leu mtl srlCl rpsL
Hfr leu mtl srlCl spcr rplX165 argG6 metBI his-I leu-6 recAl mtl-2 xyl- 7 malAI gal-6 lacY) rpsL104 tonA2 tsx-l supE144 thr-l leu-6 thi-l argE3 his-4proA2 recA12 lacYI galK2 mtl-l xyl-5 ara-14 rpsl3l tsx-33 supE44 Same as N01983, except srlC+ recAl Same as N01983, except srlC+ recA12 AprecA+ cI+ lysogen of N01887
construct a strain with a temperature-sensitive mutation in an r-protein gene, purified Afis3 transducing phage was mutagenized in vitro with hydroxylamine. Strain N01216 was then infected with the mutagenized phages, and trkAh transductants were isolated at 30°C. They were then screened for ability to grow at 42°C. One of the resulting temperature-sensitive strains, N01967, was found to have a mutation (rplX165) which altered the electrophoretic mobility of L24 (see Results and Fig. 3). Although rigorous experimental proof is lacking, the temperature-sensitive phenotype and the L24 alteration appear to be due to the same mutation, and the mutant resembles that described by Cabez6n and his co-workers (3). The full description of the mutation will be given elsewhere. An SpCr mutant of N01967 (= N01884) was obtained by mutagenesis with ethylmethane sulfonate. N01885 was constructed by introducing rplX165 and spcr from N01884 into N01216 by P1 transduction. A thy mutant of N01885 was isolated by trimethoprim selection (25). This strain (N01886) was crossed with a derivative of KL16-99 (Hfr ser recAl thi), and a thy' recAl recombinant (N01887) was obtained. A recA+ derivative (N01981) of N01887 was again constructed by isolating a thy mutant (N01980) of N01887 and mating it with an Hfr strain (KL16) which injects thy' recA+ genes as early markers. Thy' Spcr recombinants were screened for the presence of recA+. UV light sensitivity was used to distinguish RecA+ from RecAphenotypes. Another set of isogenic strains carrying rplX165 and various recA alleles was constructed in the following way. The rplX165 mutation was first introduced from N01884 into FEJ-1 by P1 transduction; one of the sper transductants which also received the rplX165 allele was named N01983. The recAl and recA12 genes were transferred to N01983 by a second P1 transduction from AprecA+cI857 lysogens of JC1553 and AB2462, respectively. (Lysogenization of the recA strains with AprecA+cI857 greatly improved the growth of P1 in these strains; since the prophage tended to segregate, the lysogens were usually grown in the presence of
Source
W. Epstein
W. Epstein B. Bachmann
B. Bachmann
nitrofurantoin [2 ug/ml, Sigma Chemical Co.] [24].) srl+ transductants were selected for, and these were screened for RecA- phenotype, N01986 and N01988 were thus isolated. AprecA+cI857 was made cI+ for experiments at 420C by the following crosses. Xh80cI857 was crossed with Xpapa (wild type), and lysogens carrying recombinant Ah8OcI+ were selected in a malA strain at 420C. The Ah8OcIW phage was then crossed with XprecA+cI857, and lysogens carrying recombinant AprecA+cI+ were isolated on a tonB strain at 420C. Phages were induced from the lysogens and checked for the ability to transduce recA+ into recA strains. Nitrofurantoin (see above) was used to facilitate detection of recA+ colonies and to prevent segregation of the prophage. N01887 was lysogenized with AprecA+cI+ (= N01961) and was used for various experiments. Construction of hybrid plasmids containing rprotein genes. Preparation of plasmid and transducing phage DNA has been described previously (17). Plasmid RSF2124, a ColEl derivative carrying an ampicillin resistance transposon (TnA), was used as a cloning vector (32). Xfs3 DNA (see references 14 and 21) and RSF2124 plasmid DNA were digested to completion with EcoRI restriction endonuclease, and the reaction was terminated by heating at 650C for 8 min. Digests were ligated as described by Tanaka and Weisblum (33). For transformation, exponentially growing E. coli K-12 strains were treated with cold 50 mM CaC12 as described by Mandel and Higa (23). Plasmid pNO1001, which carries the Afus3 EcoRI 10% fragment, was isolated by direct selection at 420C, in the absence of ampicillin, using temperature-sensitive N01967 as the recipient strain. Plasmid pNO1002, which carries the Afus3 EcoRI 9% fragment, was isolated by ampicillin selection, using C600 as the recipient strain. In this case, the DNA-treated cells were first diluted with rich medium, grown at 370C for 1 h, and then plated on ampicillin plates (100 jug/ml) at 370C. These hybrid plasmids were introduced into various strains by transformation, and the resulting strains were used in the present work (see below).
VOL. 138, 1979
DOSAGE EFFECTS ON r-PROTEIN GENE EXPRESSION
Growth conditions. For labeling experiments, cells were grown in a synthetic minimal medium (AB medium; see reference 4) supplemented with 0.4% glucose, 50 jAg of all amino acids except the one used for labeling per ml (or 0.2% Casamino Acids), thiamine at 2 Ag/ml, and, for trkA strains, 0.1 M KCI. KCl (0.1 M) was also included in the medium for trkA+ cells when they were used as controls for experiments with trkA strains. Uracil (5 Ag/ml) was added for steadystate labeling of mRNA. The details of the labeling conditions are given in the footnotes to the tables and in the figure legends. In all cases, ampicillin (100 ,ug/ ml) was used for overnight cultures of plasmid-carrying strains, and nitrofurantoin (2 ,ug/ml) was included for lysogens of XprecA . Overnight cultures of strains carrying pNO1001 were always grown at 42°C (see below). For direct analysis of unlabeled r-proteins by two-dimensional gel electrophoresis, cells were grown in rich medium (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl, and 7.4 g of KCI per liter, pH 7.0). Ampicillin (100 yg/ml) was included whenever the strain carried a hybrid plasmid to prevent segregation of the plasmid. We note that the strains carrying pNO1001 were stable as long as the strains were grown in the presence of ampicillin or at 420C. Agar plates contained 10 g of tryptone, 5 g of yeast extract, 5 g of NaCl (pH 7.0), and, where appropriate, 7.4 g of KCI per liter. Preparation and electrophoresis of r-proteins. In labeling experiments, 1 volume of labeled cells was added to 1.5 volumes of ice-cold acetone. This procedure (1) was used to rapidly stop the incorporation of labeled amino acids and to prevent the possible intracellular degradation of protein. After storage overnight at -20°C, samples were centrifuged at 12,000 x g for 20 min and decanted, and the pellets were lyophilized to dryness. All of the trichloroacetic acid-precipitable radioactivity was recovered in the pellet. For one-dimensional electrophoresis on urea gels (see reference 34), pellets were suspended in 50 jul of 50 mM Tris-hydrochloride (pH 8.0), 6.5 Al of 0.1 M EDTA (pH 7.1), and 5 pl of lysozyme (0.4 mg/ml). Preparations were subjected to, four cycles of freezethawing, and carrier 70S ribosomes were added (15 units of absorbance at 260 nm). Samples were made 0.1 M with MgCl2, and r-proteins were extracted with two volumes of glacial acetic acid (12). After addition of acetic acid, samples were mixed thoroughly, and portions were removed for determination of total radioactive protein. For two-dimensional gel electrophoresis (16), 100 units of absorbance at 260 nm of carrier ribosomes were used per gel. To analyze unlabeled, carriei-free r-proteins from various strains, cells were grown in rich medium (2 liters) at 42°C. At a cell density of about 108/ml, half of the culture was shifted to 30°C and the remaining half continued to incubate at 42°C. After two to four doublings in cell mass, cells were collected by centrifugation and stored frozen. After thawing, pellets were suspended in 10 ml of 10 mM Tris-hydrochloride (pH 7.5), 30 mM NH4Cl, 10 mM MgCl2, and 6 mM ,Bmercaptoethanol (TMAI) and were broken by sonic oscillation. Debris was removed by centrifugation at 12,000 x g for 1 h, and the supernatant was centrifuged at 60,000 x g for 4.5 h. The crude ribosome pellet was
385
resuspended in 0.3 ml of TMAI. For electrophoresis, r-proteins were extracted with 67% acetic acid from 100 units of absorbance at 260 nm of these crude ribosomes. "Salt washed" ribosomes were prepared by centrifuging the crude ribosomes through a 38% sucrose solution containing 20 mM Tris-hydrochloride (pH 7.4), 10 mM MgCl2, 500 mM NH4C1, 0.5 mM EDTA, and 1 mM dithiothreitol. The ribosome pellets were rinsed twice with TMAI, and the loose brown material lying over the clear ribosome pellet was discarded. Ribosomes were resuspended in 0.2 ml of TMAI, and protein was extracted from 100 units of absorbance at 260 nm of ribosomes for electrophoresis. Preparation of [2PJRNA. [3P]RNA was used as an internal standard to measure the relative efficiency of hybridization of 3H-labeled RNA samples to various DNAs. The [3P]RNA was prepared by in vitro transcription, using purified restriction enzyme fragments as DNA templates. The templates used were the 10% EcoRI fragment from pNO1001 and the 4.4%-L EcoRI fragment from Xrifdi8 (see the text; 22). The 4.4%-L fragment carries genes for r-proteins Li, L10, Lll, and L7/L12 (22). This fragment was prepared from hybrid phage Ch3R11, which is a derivative of the charon 3 vector phage and carries a cloned 4.4%-L fragment (L. Post and M. Nomura, unpublished data; for the charon 3 phage, see reference 2). In vitro transcription was done as described previously (29). After the reaction, carrier RNA (E. coli 16S rRNA; 50 ,g/ml) was added, and the mixture was treated with phenol. RNA in the aqueous phase was precipitated with two volumes of ethanol, washed twice by reprecipitation, lyophilized, and dissolved in 2x SSC (SSC is 0.15 M NaCl-0.015 M sodium citrate). Extraction and purification of [3HJRNA and RNA-DNA hybridization. Labeled celLs were added to an equal volume of boiling lysis solution containing 1% sodium dodecyl sulfate, 0.1 M NaCl, 8 mM EDTA (pH 7.0), and 100 Ag of carrier yeast tRNA per ml. The mixture was heated in a boiling-water bath for an additional 2 min and then treated with phenol at room temperature. The aqueous phase was made 0.3 M NaCl and precipitated with two volumes of ethanol. The samples were washed twice by reprecipitation, lyophilized to dryness, and dissolved in 1 ml of 2x SSC. Appropriate portions were hybridized to filters containing excess DNA in the presence of constant amounts of in vitro-labeled [32P]RNA to monitor hybridization efficiency. Input radioactive RNA was determined by precipitation with 5% trichloroacetic acid followed by membrane (Millipore Corp.) filtration. Hybridization was done in 2x SSC at 670C for 15 to 18 h as described previously (8). The amounts of [3H] RNA hybridized were determined, and corrections were made for the small variation of hybridization efficiency observed with [32P]RNA. If the hybridization efficiency is the same in all the reaction mixtures in a given experiment, the amounts of [32P]RNA hybridized to DNA should be the same in all the samples. The average of the values for the amounts of [32p] RNA hybridized was calculated in each experiment and used for correction according to the following
[3H] equation: [3H]RNA hybridized (corrected) RNA hybridized (observed) x (average of [32P]RNA hybridized/[32P]RNA hybridized). Corrections made
386
FALLON ET AL.
J. BACTERIOL.
in this way were usually within 10% of the observed values. Other methods. Methods for restriction enzyme digestion, separation of restriction enzyme fragments on analytical gels, and preparative separation of DNA fragments have been described previously (22). The cell densities of bacterial cultures were measured with a Klett-Summerson photoelectric colorimeter, using a blue filter (no. 42). Fifty Klett units corresponded to about 2 x 108 cells per ml with ordinary E. coli K-12 strains (e.g., strain N01216) growing in AB-glucose minimal medium.
RESULTS Cloning of r-protein genes with the Spc promoter. The Afus3 transducing phage carries 27 r-protein genes and the genes for elongation factor Tu (tufA), elongation factor G (fus), and RNA nucleotidyltransferase (RNA polymerase) subunit a (rpoA) (13-15, 21; see Fig. 1). To examine the effects of high dosages of r-protein genes, we cleaved Xfus3 DNA with EcoRI restriction endonuclease and tried to clone EcoRI fragments carrying r-protein genes and their promotor into a plasmid cloning vector. We first used ColEl-TnA hybrid plasma RSF2124 as the cloning vector and strain C600 as the recipient. Random cloning procedures were carried out, that is, ligation of a mixture of vector and Afus3
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EcoRI fragments, transformation, selection of ampicillin-resistant transformants, screening of transformants for inability to produce colicin El, and analysis of plasmid DNA carried by the transformants. We failed to clone any fragment with a promoter, but we were able to clone other fragments (1.2,4.6,8.5, and 9%) with sizes similar to those of the promoter-containing fragments (2.2, 5, 10, and 11%) of Xfus3. (Sizes are given in percentage-of-X units; 1% of X = 465 base pairs.) These observations suggest that promoter-containing fragments were probably ligated into RSF2124, but, for some reason, the presence of the resulting plasmids was harmful to the growth of the recipient cells. (The same conclusion was also obtained by J. M. Zengel [Ph.D. thesis, University of Wisconsin-Madison, 1976].) Strain NO1967, which has a temperature-sensitive mutation (rplX165) in (or very close to) the gene for L24 (rpLX), was then used as a recipient for cloning experiments; selection of the transformants was done at 42°C in the absence of ampicillin. We expected that transformants which grew at 42°C would harbor hybrid plasmids carrying the 10% EcoRI fragment of Xfus3, since this fragment carries the wild-type L24 gene and its promoter, p., (cf. Fig. 1). This expectation was, in fact, realized. Over 99% of
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FIG. 1. Map of Afus3 and ofpNO1001. The locations of bacterial genes and cleavage sites of restriction endonucleases are shown. The hatched parts represent A DNA. The circular pNO1001 plasmid molecule is shown with two horizontal bars, one representing vector RSF2124 DNA (crosshatched bar) and the other representing the cloned EcoRI 10% fragment. The locations of bacterial DNA fragments A, B, C, and D, used for hybridization experiments, are shown. Fragments A and B were prepared from hybrid plasmids pNO2003 and pNO2002, respectively (29). Fragments C and D were prepared from similar plasmids, pNO2014 and pNO2015, respectively (Post and Nomura, unpublished data). The orientation of the cloned 10% fragment relative to the Sma restriction endonuclease site in RSF2124 (see reference 5) is shown. Restriction enzyme sites for BamHI and HindII are shown for the 10% fragment only; sites for Sma are shown for the 10% fragment and RSF2124. Distances between restriction sites are in percentage-of-A units. It should be noted that the spc operon promoter (p.,) is not included in fragment B (29). Fragments A, B, C, and D are about 820, 440, 840, and 1,000 base pairs long, respectively.
DOSAGE EFFECTS ON r-PROTEIN GENE EXPRESSION
VOL. 138, 1979
387
the temperature-resistant colonies analyzed the spc operon in this study) in RecA- cells was ampicillin resistant, and, of these, 95% responsible for this apparent "cold sensitivity." contained a cloned fragment. Plasmid DNA was We used hybrid plasmid pNO1002, which has isolated from 20 independent transformants, and the 9% EcoRI fragment cloned into RSF2124, as restriction enzyme analyses with EcoRI and a control for pNO1001. The 9% EcoRI fragment Sma confirmed that the 10% EcoRI fragment of XAfus3 does not carry genes for any r-proteins carrying p., had been cloned. In all 20 cases, the and is similar in size (9% of A, or about 4,200 10% fragment was in the same orientation rela- base pairs) to the 10% fragment (about 4,700 tive to the RSF2124 vector DNA. One of the base pairs) cloned in pNO1001. It was found that hybrid plasmids thus constructed, pNO1001, was the same recAl strain (NO1887) carrying used in the experiments described in this paper pNO1002 grew normally and formed colonies at (see Fig. 1 and Table 2, group 1). (We have not 30°C (Table 2). In addition, isogenic recA+ parstudied the reason why we failed to isolate a ent strain N01885 and strain N01981, which hybrid plasmid carrying the 10% EcoRI frag- was derived from N01887 and was made recA+ by mating, did not show cold sensitivity when ment in the opposite orientation.) Growth characteristics of strains harbor- carrying pNO1001. Similarly, lysogenization of ing the pNO1001 plasmid. To prevent possible the original recAl strain (NO1887) with recombination between chromnosomal genes and AprecA+cI+ abolished this pNO1001-induced cold sensitivity (Table 2, group 2). genes on the plasmid, hybrid plasmid pNO1001 We also constructed a strain (NO1983) carrywas introduced by transformation and selection ing the temperature-sensitive rplX165 mutation on ampicillin plates at 42°C into recAl strain N01887, which carries rplX165. It was found and subsequently introduced recA mutations by that the resultant strain, N01887(pNO1001), P1 transduction. Both the recAl derivative and the recA12 derivative were cold sensitive when was not able to form colonies when incubated on plates at 30°C, in contrast to the parent, NO1887 carrying the pNO1001 plasmid, although the (Table 2). Systematic studies showed that the phenomenon was less striking with the recA12 strain (Table 2, group 3). presence of multiple copies of r-protein genes The cold sensitivity caused by the presence of with their promoter (specifically, the genes in
were also
TABLE 2. Colony-forming abilities ofplasmid-carrying strains Colony formation at: Group'
1
Strain
Plasmid
300C
N01967
pNO1001 pNO1002 2
RecA phenotype
N01885
pNO1001 pNO1002 N01887 pNO1001 pNO1002
N01887 (AprecA+cI+) pNO1001 pNO1002
N01981
pNO1001
420C
Ratio of colonies at 30'C/ colonies at
42"C
+ +
+
-
+
+
+
+
-
+ + + - (recAI) - (recAI) - (recAI) +
+ + + + + +
+ -
1.0
+
0.001
+ +
+ +
+
+
+
+
+ +
1.0
-
1.0 1.0
+ + 1.0 + + + + - (recAI) N01986 0.0007 + - (recAI) pNO1001 + - (recA12) N01988 + 0.02 - (recA12) (+)b pNO1001 aStrains within a group are isogenic except for recA alleles and possibly some genes linked to recA (see the text). b Small colonies appeared at 30°C after longer incubation. 3
N01983
PNO1001
388
FALLON ET AL.
plasmid pNO1001 was measured by growing strains at 42°C, plating cells both at 30 and at 420C in the presence of ampicillin, and calculating the ratio of the colonies formed at 300C to those formed at 420C (the last column in Table 2). About 1,000-fold and 50-fold reductions were observed in the numbers of colony formers measured at 300C when plasmid pNO1001 was carried by recAl and recA12 strains, respectively. [Several colonies from N01887(pNO1001) which grew on plates at 300C were analyzed for the plasmids they carried. Many of them had plasmids with altered sizes, so it seems that most of these survivors represented mutants formed during residual growth of these cold-sensitive cells on plates at 300C. The nature of these mutations has not been further examined.] The observed cold sensitivity of recA strains carrying plasmid pNO1001 was probably due to the excess accumulation of certain r-proteins at 300C. This will be described below. Synthesis of r-proteins in N01887 (pNO1001). Since N01887(pNO1001) carried multiple copies of several r-protein genes in the spc operon, we examined the question of whether r-proteins coded for by these genes were overproduced in this strain. Because of their temperature-sensitive character, N01887 cells could not be used as control cells at 420C. Therefore, we used C600(pNO1002) cells as control cells, as described above. Another control strain, N01216, had a genetic background closely related to that of N01887 (see Table 1). Similar results were obtained with either control strain. In the first experiments, cells were grown exponentially at 420C and labeled with [35S]methionine. The incubation temperature, 420C, was chosen to prevent the segregation of the plasmid. It should be noted that N01887 cells do not grow at 420C unless they harbor the plasmid carrying the wild-type L24 gene (rpLX). Radioactive r-proteins were analyzed by onedimensional polyacrylamide-urea gel electrophoresis, followed by autoradiography (Fig. 2). Marked overproduction of one protein relative to other methionine-containing r-proteins was observed after a short pulse-labeling period. This protein had the mobility of L14. Analysis by two-dimensional gel electrophoresis (see below) confirmed that the overproduced protein was in fact L14, the protein coded for by the first gene in the spc operon. Most of the overproduced protein was degraded within 30 min at 420C (Fig. 2, slot 11). Degradation was either very slow or did not take place at 300C in this strain (data not shown; see also below). Visual inspection of the autoradiogram given in Fig. 2
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FIG. 2. Expression of r-protein genes on pNO1001. Cells (5 ml) were labeled at 42°C with 65 ,uCi of [3SJmethonine, and 1-mi samples were taken at various times after addition of label. Total acid-precipitable counts indicated complete uptake of isotope by 2 min. Lanes 1, 4, 7, and 10 are C600(pNO10(X2); 2, 5,8, and 11 are N01887(pNO1001); 3,6,9, and 12 are N01216 at 2, 4, 6, and 30 min after addition of isotope, respectively. Samples were analyzed by polyacrylamide-urea gel electrophoresis. An autoradiogram of a dried gel is shown, and the position of L14 is indicated. The protein band shown with an arrow was seen only in short-pulse-labeled samples from control cells, C600(XpNO1002) and N01216, but not in samples from N01887(pNO1001). The nature of this protein has not been studied. In this experiment, N01887(pNO1001) was grown in the absence of ampicillin. Examination after the experiment showed that most or all of the cells retained the pNO1001 plasmid during the experiment.
shows that no marked overproduction of other methionine-containing r-proteins took place in N01887(pNO1001). L24, whose gene on the plasmid was used for the original selection (see above), does not contain methionine and could not be examined by these autoradiograms. To determine whether L24 was overproduced in N01887(pNO1001), we analyzed the rates of r-protein synthesis in cells labeled with [3H]lysine. The synthesis rates of several r-proteins in the plasmid-carrying strains, including those coded for by genes carried by pNO1001, were compared with those in the control strain as described in the footnote to Table 3. It was found that the amounts of L14 and L24 synthesized during a 2-min labeling time were five- to sevenfold higher in NO1887(pNO1001) cells than in control (NO1216) cells when normalized to L1 (Table 3). Overproduction of other proteins coded for by genes on pNO1001 which are distal to the L24 gene in the spc operon was small (L5, S14, and L6) or insignificant (S8 and L18). Proteins L16, L29, and S17, whose genes precede
DOSAGE EFFECTS ON r-PROTEIN GENE EXPRESSION
VOL. 138, 1979
TABLE 3. Rates of individual r-protein synthesis in N01887(pNO1001) relative to those in control strain N01216a
Relative syntheClass Cre Protein rate
1
L16 L29 S17
0.95 1.03 0.95
2
L14 L24 L5 S14 S8 L6 L18
6.7 5.5 1.80 2.04 1.23 1.51 1.34
3
S5 (+L11) L15 L30
1.09 0.81 1.14
Li L3 1.11 L10 0.99 a Genes for the class 1 proteins are carried by the pNO1001 plasmid, but their promoter is not present on the plasmid. Genes for the class 2 proteins and their promoter are carried by pNO1001. Genes for the class 3 proteins are in the spe operon at 72 min, but are not carried by pNO1001. Chromosomal locations of genes for the class 4 proteins are known; these genes are definitely not carried by the plasmid. Both experimental [NO1887(pNO1001), doubling time of 110 min] and control (NO1216, doubling time of 40 min) cells were grown at 420C to approximately 2 x 108/ml. [3H]lysine (490 pmol/ml; 61 Ci/mmol) was added to experimental cells for 2 min and to control cells for 1 min, followed by a 1-min "chase" with excess nonradioactive lysine. Celis were rapidly chilled and collected. [3H]lysine-labeled cells were then mixed with a suitable amount of ['4C]lysine-labeled carrier cells. r-Proteins were extracted and separated by two-dimensional gel electrophoresis. After staining the gels, the spots corresponding to the indicated r-proteins were cut, the gels were oxidized with a Packard sample oxidizer, and 3H and '4C were counted separately. The 3H/14C ratio in each protein in the experimental cells was then compared with that in the control cells; that is, the values were first calculated with the following equation: 3H/'4C in protein i in experimental cells + 3H/'4C in protein i in control cells. The ratios obtained were then normalized to the ratio obtained with protein Li. The gene for Li is not carried by Xfus3 and maps near rpoB at 88 min (20, 35); hence, the value for protein Li was used as a reference for normalization and is boxed in the table. The values given are averaged from three experiments. 4
the spc operon promoter (ppC) on pNO1001, were not overproduced. This suggests that the p8pc promoter was functioning as the main promoter for the L14 and L24 genes on the plasmid
389
and that readthrough transcription of cloned rprotein genes from a promoter(s) on the vector was small or nonexistent. The experiments described above showed that N01887(pNO1001) overproduced L14 and L24 but that the overproduced L14 was unstable and was gradually degraded at 420C. As in the case of L14, the overproduced L24 was gradually degraded at 420C. This was demonstrated by pulse-labeling cells with [3H]leucine and chasing with nonradioactive leucine at 420C (data not shown). Degradation of the overproduced L14 and L24 was also apparent when the amounts of r-proteins in N01887(pNO1001) were directly visualized by staining two-dimensional gels of the extracted r-proteins (see Fig. 3H compared with Fig. 3A through D). Details of the experiments shown in Fig. 3 will be described later. Synthesis of r-protein mRNA in NO 1887(pNO1001). We were surprised that only L14 and L24 were strongly overproduced in N01887(pNO1001). To determine whether transcription also drastically decreased after the L24 gene, we prepared DNA fragments representing various segments of the 10% fragment (Fig. 1) and examined the syntheses of the mRNA's corresponding to these various DNA segments. Cells were pulse-labeled with [3H]uracil, and radioactive RNA was analyzed by the DNARNA hybridization method. We used the EcoRI 4.4%-L fragment carrying four r-protein genes (the genes for Li and L10 and parts of the genes for Lii and L7/L12; see reference 22 and our unpublished data cited in reference 36) in the rif region at 88 min as a reference DNA. As mentioned above, these genes are not carried by plasmid pNO1001, and, therefore, the amounts of the plasmid-coded mRNA's relative to the 4.4%-L DNA-coded mRNA could be used to compare the syntheses of the pertinent mRNA's in N01887(pN01001) with those in the control haploid strain. Cells carrying the pNO1001 plasmid overproduced mRNA corresponding to segment B (14- to 19-fold higher than in the control strain) which carries the L14 gene and a part of the L24 gene (Table 4). In addition, the results showed that even the mRNA distal to the L14 and L24 genes was overproduced, although the more distal portions of the message showed a lesser degree of overproduction. In contrast, the message corresponding to the genes for L16, L29, and S17 was only 2-fold higher in N01887(pNO1001) than in the control strain (N01216). This weak overproduction may reflect transcription from a promoter(s) on the vector. It should be noted that the degree of relative overproduction of mRNA corresponding to seg-
390
FALLON ET AL.
J. BACTERIOL.
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