OF BACTERIOLOGY, May 1978, p. 606-611 0021-9193/78/0134-0606$02.00/0 Copyright © 1978 American Society for Microbiology
JOURNAL
Vol. 134, No. 2 Printed in U.S.A.
Structure, Function, and Regulation of Escherichia coli rRNA in Proteus mirabilis E. A. MORGANt AND S. KAPLAN*
Department ofMicrobiology, University of Illinois, Urbana, Illinois 61801
Received for publication 7 September 1977
Escherichia coli rRNA genes have been introduced into Proteus mirabilis on F-prime factor (F'14). A portion of the ribosomes in the resulting merodiploid consist of E. coli rRNA and P. mirabilis ribosomal proteins. These ribosomes are structurally similar to normal P. mirabilis or E. coli ribosomes and exhibit many or all of the functional properties of normal ribosomes. The accumulation of E. coli rRNA in the merodiploid is regulated in a way similar to the regulation of P. mirabilis rRNA. an
Escherichia coli F-prime factor, F'14, which contains two E. coli rRNA operons, has been introduced into Proteus mirabilis (1). We have previously shown that E. coli 16S, 23S, and 5S rRNA are synthesized in P. mirabilis and that each rRNA species is incorporated into ribonucleoprotein particles which consist of E. coli rRNA and P. mirabilis ribosomal proteins (14). All three E. coli rRNA species can be distinguished from P. mirabilis rRNA by differences in the primary sequence of the rRNA of the two species (14), with the rRNA of the two species differing in approximately 20% of their base residues. Each E. coli-derived rRNA species comprises between 20 and 28% of the total of each rRNA species in the merodiploid. Employing oligonucleotide mapping procedures, the E. coli rRNA in the hybrid-derived ribosomes has been shown to be indistinguishable from mature E. coli rRNA; additionally, these hybrid ribosomes have sedimentation properties similar to normal P. mirabilis ribosomes (14). In this paper we further report on the structural properties of the ribosomes derived from the merodiploid. In addition, we have investigated the functional properties of these ribosomes as well as the regulatory responses of E. coli and P. mirabilis rRNA operons present in the merodiploid. MATERIALS AND METHODS Bacterial strains. The bacterial strains used have been previously described (1, 13, 15; E.A. Morgan, Ph.D. thesis, University of Illinois, Urbana, 1976). Preparation of ribosomal subunits and rRNA. The procedures for preparation of ribosomal subunits and rRNA have been previously described (14). rRNA was isolated from separated ribosomal subunits and t Present address: Enzyme Institute, University of Wisconsin, Madison, WI 53703.
further purified by sedimentation through sucrose gradients or by gel electrophoresis. Analysis of rRNA. Detection of E. coli rRNA in the bacterial hybrids was done as follows. The purified [32P]rRNA species from the hybrid was mixed with purified [3H]uracil-labeled E. coli rRNA species (from E. coli K-12 strain LMUR), digested with ribonuclease Ti or ribonuclease Ti and alkaline phosphatase, and fingerprinted as described previously (14). The amount of E. coli rRNA in the [:2P]rRNA was determined by analysis of the 'H-32P ratios of individual oligonucleotides, as previously described (14). Measurements of RNA content in cells. RNA was determined by the orcinol method of Fraenkel and Neidhart (4). ATP standards were processed with experimental samples and constituted a standard curve. Optical density of samples was determined in a Zeiss PMQ spectrophotometer at 420 nm. Cell numbers were determined by diluting cell samples to 40,000 to 90,000 cells per ml in 0.85% NaCl-3% formaldehyde, which had been twice filtered through a 0.1-ftm membrane filter (Millipore Corp.). The cells were counted in a model ZBI Coulter Counter with a 50-jil sample volume and 30-,um aperture. Low growth rate conditions and labeling. P.
mirabilis F14/PM14IV-4 was labeled with 32p; for several generations in GRCLP medium (13) containing 2 jig of nicotinic acid per ml, 10 ,ug of thymine per ml, and 50 yg each of methionine, tryptophan, isoleucine, and valine per ml. Aspartate (0.2%) was added as the sole carbon and nitrogen source. The doubling time in this medium is 26 h. For all other experiments, cells were labeled in standard low-phosphate medium as described (14). Preparation of polysomes. Polysomes were prepared as described by Randall and Hardy (18), with minor modifications. Cells for polysome preparations were grown in a 24-ml culture in minimal low-phosphate media (14).
RESULTS Structure of the hybrid ribosomes. Strain F14/PM14IV-4 is a P. mirabilis strain carrying E. coli F-prime F14, which carries two E. coli 606
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E. COLIIP. MIRABILIS HYBRID RIBOSOMES IN VIVO
rRNA operons (1, 3, 14). Previous results (14) indicated that E. coli rRNA was present in strain F14/PM14IV-4 in ribonucleoprotein particles whose gross sedimentation properties were indistinguishable from bulk ribosomal subunits. Fingerprinting techniques demonstrated that the E. coli rRNA in this strain had been processed to yield rRNA species similar or identical to mature E. coli rRNA species (14). The chromosomal location of E. coli ribosomal protein genes (16) indicates that the ribosomal particles in strain F14/PM14IV-4 must consist largely, if not solely, of P. mirabilis ribosomal proteins. We have analyzed the ribosomal proteins of these subunits by comparing the two-dimensional gel electrophoresis patterns of purified ribosomal proteins from E. coli, P. mirabilis, and the hybrid strain F14/PM14IV-4, using the method of Kaltschmidt and Wittman (10). The ribosomal proteins of strain F14/PM14IV-4 showed no obvious differences or additional spots when compared with those of PM14IV-4 (Fig. 1). CsCl core particles prepared from ribosomal subunits derived from strain F14/PM14IV-4 by the method of Traub et al. (19) contain E. coli rRNA (Morgan, Ph.D. thesis; data not shown); it is, therefore, likely that these particles represent a specific and functional complex (see below) of E. coli rRNA and P. mirabilis ribosomal proteins. Functional properties of the hybrid ribosomes. Lacking the ability to separate hybrid frota homologous ribosomes in strain F14/PM14IV-4, it is difficult to unambiguously test the activity of the hybrid ribosomes by use of conventional methods. However, if ribosomes which contain E. coli rRNA are active, then they should be present on polysomes derived from strain F14/PM14IV-4. Polysome profiles from strain F14/PM14IV-4 were found to be identical to those prepared from strain PM14IV4 (Fig. 2). E. coli 16S, 23S, and 5S rRNA species were present in approximately 20 to 30% of all ribosomes derived from polysomes prepared from strain F14/PM14IV-4 (Table 1). This is approximately the same level of each E. coliderived rRNA species found in the total ribosomal subunit pool of this strain (14). To further reveal functional activity of the hybrid ribosomal population of strain F14/PM14IV-4, we have measured polysome disappearance under conditions where normal polysome breakdown requires puromycin-induced polypeptide release and subsequent translocation of the ribosomes (2, 17). Assuming the random distribution of E. coli rRNA species among subunits, 47% of all the 70S ribosomes would contain at least one E. coli rRNA species, and 38% would contain at least one E. coli
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species if 50S subunits were composed solely of either E. coli or P. mirabilis rRNA species. If the ribosomes which contain E. coli rRNA were refractory to dissociation, then no less than 20% or as high as 47% of the polysomal-bound ribosomal subunits would remain as free 70S ribosomes, since active ribosomes are converted to free ribosomal subunits following puromycin treatment of polysomes. However, all ribosomes from strain F14/PM14IV-4 were present as free ribosomal subunits after puromycin-induced subunit release (Morgan, Ph.D. thesis; data not shown). This release is largely inhibited by the addition of chloramphenicol and requires the presence of K+ (Morgan, Ph.D. thesis; data not shown), which is further evidence that this assay measures functional ribosomes. In addition, we have found that both strains PM14IV-4 and F14/PM14IV-4 grow at identical rates in similar media. If hybrid ribosomes were inactive and, therefore, capable of blocking polysome movement of active ribosomes, then it is likely that this defect would manifest itself in the growth rate of the F14-containing strain. Therefore, we feel the evidence indicates that the hybrid ribosomes in this strain are fully active. Accumulation of E. coli rRNA in P. mirabii& Evolutionary divergence of P. mirabilis and E. coli has resulted in divergence of the rRNA sequences of these two organisms (14), and, therefore, differences in the regulatory sequences affecting rRNA synthesis might be anticipated between these two species. The level of RNA per cell during steady-state exponential growth in an M9 salts defined medium was determined for both strains PM14IV4 and F14/PM14IV-4. Under these growth conditions, both organisms have identical doubling times. Strain F14/PM14IV-4 was determined to have approximately 98% of the RNA per unit of absorbance at 420 nm with respect to strain PM14IV-4 (Morgan, Ph.D. thesis; data not shown). Additionally, direct cell counts by use of a Coulter Counter indicated that strain F14/PM14IV-4 had 96% of the RNA per cell of PM14IV-4 (Morgan, Ph.D. thesis; data not shown). About 80% of the RNA in cells is rRNA (12). Therefore, although rRNA gene dosage has been increased 20 to 30% in strain F14/PM14IV4 as compared to- PM14IV-4 (1, 14), no large increase in rRNA accumulation or decrease in cell growth rate was observed. This is evidence for both the proper function of E. coli rRNA in this strain and for a regulatory system which maintains proper levels of ribosome accumulation by a mechanism insensitive to rRNA gene dosage. Previous studies (12) have revealed that cells
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FIG. 1. Ribosomal protein profiles of P. mirabilis and F14/P. mirabilis merodiploid. Cultures of strains AB1206, PM)41IV-4, and F14/PM14IV-4 were grown aerobically in M9 minimnal salts medium appropriately supplemented and harvested in mid- to late exponential growth. Ribosomes were prepared fr-om these cells by the ammonium sulfate fr-actionation method (11). Subunits were prepared as described and repeatedly recentrifuged through sucrose gradients to insure their purity. Proteins were extracted fr-om the purified subunits by the acetic acid method (6). Proteins were dialyzed against several changes of TSM buffer and then exhaustively dialyzed against distilled water before lyophilizing to dryness. Two-dimensional gelprofiles were obtained according to the procedure of Kaltschmidt and Wittman (10) using a pH 9.5 first dimension. (A) Strain PM14IV-4 30S ribosomal proteins; (B) strain F14/PM14IV-4 30S ribosomal proteins; (C) E. coli AB1206 and PM14IV-4 30S ribosomal protein mixture; (D) strain PM14IV-4 50S ribosomal proteins; (E) strain F14/PM14IV-4 505 ribosomal proteins; (F) E. coli AB1206 and PMJ4IV-4 5OS ribosomal protein mixture.
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TABLE 2. E. coli rRNA sequences present in rRNA species prepared from ribosomal subunits of strain F14/PM14IV-4 grown with a doubling time of 26 h Oligonucleotidea Fraction' 16S rRNA AUUAACG ................. 0.33 pAAAUUG ................. 0.35 ACCUUCG ................. 0.40 UCUG ..................... 0.31 Bottom
Distance Migrated
Top
FIG. 2. Polysomes prepared from strain F14/PM14IV-4. The portion of the gradient included within the arrows was pooled to determine whether E. coli rRNA was in ribosomes on polysomes isolated from strain F14/PM14IV-4. Polysome profiles prepared from strain F14/PM14IV-4 are undistinguishable from those of strain PM14IV-4. A2Mnm, Absorbance
at 254 nm.
TABLE 1. E. coli rRNA sequences present in rRNA species prepared from ribosomal subunits derived from polysomes of strain F14/PM14IV-4 Oligonucleotidea
Fraction
16S rRNA AUUAACG
.................
pAAAUUG ACCUUCG
UCUG
.................
.....................
23S rRNA 12 ....................... 14 15
....................... .......................
5S rRNA UCUCCUCAUG ............ ACCCCAUG ............... CCAUG
0.24 0.22
....................
5S rRNA UCUCCUCAUG ............ 0.22 c ACCCCAUG ............... 0.14 CCUG ..................... 0.29 CAG ....................... 0.28c a Sequence of the E. coli unique oligonucleotides examined. b Fraction of E. coli rRNA in the hybrid, calculated using the 32P-3H ratio of the oligonucleotide. c The oligonucleotide is not present on all 5S rRNA molecules coded by F14 or the E. coli genome (8, 14). As a result, the value given has been calculated according to the suggested frequency of occurrence of this oligonucleotide, as previously described (14; Table 1, footnote b). The values for these fractional E. coli oligonucleotides compare favorably with the values resulting from oligonucleotides present in all E. coli 5S rRNA molecules, which strongly supports the conclusion that synthesis of rRNA takes place from both rRNA operons on F14 (see 14).
0.30
0.20
0.23 0.23 0.28
(0.62) 0.19b 0.12 0.17
CCUG ..................... 0.13 CAG ....................... (0.23) 0.11b E. coli unique oligonucleotides produced by ribonuclease Ti digestion of the rRNA. For 16S and 5S, the sequence is given. For 23S rRNA, the sequence is not known; the numbers refer to designations used previously (14). b The particular oligonucleotide is not present in all copies of 5S rRNA of F14 or in E. coli (8, 14). Therefore, the values given have been calculated according to the suggested frequency of occurrence of these oligonucleotides. The logic and evidence for this procedure have been previously described (14) and are consistent with equal expression of both rRNA operons on F14 in strain F14/PM14IV-4 as well as the presence of rRNA from both operons on polysomes. a
growing with different growth rates possess different rates of rRNA synthesis as well as having characteristically different rRNA-protein ratios.
Direct comparison of the differential accumulation of E. coli and P. mirabilis rRNA in strain F14/PM14IV-4 has been made at high and low growth rates. Many individual determinations (here; 14) place the E. coli rRNA content in strain F14/PM14IV-4 at near 20 to 30% in a medium in which this bacterium doubles every 80 min (14). Strain F14/PM14IV-4 was grown in medium where aspartate served as the sole carbon and nitrogen source with a doubling time of 26 h. From Table 2, it is apparent that the fraction of E. coli rRNA in strain F14/PM14IV4 grown with a doubling time of 26 h is near 20 to 30%, similar to that of a culture grown with a doubling time of 80 min (14). These estimates are most strongly supported from studies of the 16S rRNA in E. coli-specific oligonucleotide UCUCG, which we estimate is inflated by no more than 2 to 4% due to imperfect resolution of neighboring oligonucleotides (14). The well-resolved oligonucleotides of 5S rRNA also agree with the conclusion. Certain 5S rRNA oligonucleotides may derive from only one, or more, of the rRNA operons present, due to sequence heterogeneity in 5S rRNA. Oligonucleotide UCUCCUCAUG is present on only one copy of the 5S rRNA genes in E. coli and is present on F14 (8, 14). It is apparent that both copies of 5S rRNA on F14 contrib-
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J. BACTrERIOL.
TABLE 3. Relative molarities of fractional oligonucleotides from P. mirabilis 5S rRNA sequences in strain F14/PM14IV-4' Sequence b Molarity Glucose (80 min) CCAUAG ...... 1.08 CCAACG .................. 0.92 0.10 U,C,A,A,Gc ................. 0.11 U,U,C,G ...................
Aspartate (26 h) CCAUAG CCAAG ...... U,C,A,A,GC .......... .......
0.91 1.09 0.11
U,U,C,GC ...................
0.10 a Values for 32P counts per minute per total number of bases were determined for each oligonucleotide, then were normalized to values obtained by the formula (32P counts per minute of CCAUAG + 32P counts per minute of CCAAG)/11. This procedure gives the molarity of each oligonucleotide relative to the CCAUAG and CCAAG standards. CCAUAG and CCAAG occur in most or all 5S rRNA cistrons of P. mirabilis. b Sequence of oligonucleotides derived from 5S rRNA from strain F14/PM14IV-4 grown on indicated medium. Parentheses indicate doubling time. c U,C,A,A,G and U,U,C,G are proposed to be fractional oligonucleotides in P. mirabilis 5S rRNA. Base composition is suggested on the basis of chromatographic mobility of the oligonucleotide.
ute equally to rRNA synthesis at doubling times of 80 min or 26 h (Table 2). P. mirabilis 5S rRNA-derived fractional oligonucleotides, which we have previously identified (14), exhibit similar fractional values at doubling times of 80 min or 26 h (Table 3). Therefore, all rRNA operons in strain F14/PM14IV-4, whether of E. coli or P. mirabilis origin, very probably contribute in the same proportion to the accumulation of rRNA at both high and low growth rates. We have previously observed that individual rRNA operons in E. coli contribute equally to rRNA accumulation in E. coli under a variety of conditions (13).
DISCUSSION All E. coli rRNA species derived from F'14 are synthesized in P. mirabilis and packaged into ribonucleoprotein particles which have many physical and functional properties characteristic of normal ribosomes. These ribosomes consist of E. coli rRNA and P. mirabilis proteins. Therefore, the sequence divergence does not seem to affect the function of E. coli rRNA species in ribosome assembly or in protein synthesis, when present in P. mirabilis. In addition, E. coli rRNA appears to be properly regulated in P. mirabilis under several distinct physiological conditions.
Previous in vitro studies have shown that ribosome components of distantly related bacterial species can form functional heterologous ribosomes in the highly artificial conditions of ribosome reconstitution (5, 7). The present results reveal that this compatibility extends to ribosome assembly and rRNA maturational pathways which occur in vivo. It is possible that other types of hybrid ribosomes may be constructed in vivo. For example, if E. coli ribosomal proteins can be synthesized and incorporated into functional ribosomes in P. mirabilis, it should be possible to construct E. coli-P. mirabilis hybrids diploid for ribosomal protein genes, and then to inactivate the P. mirabilis ribosomal protein genes totally by simple genetic selections similar to those previously employed with partial diploids of E. coli (9). The resulting ribosomes would contain certain protein(s) entirely of E. coli origin. With recent genetic engineering techniques, it may be possible to construct hybrid ribosomes in vivo using quite distantly related species. The influence of ribosome components on translational specificity may perhaps be fruitfully investigated by this type of in vivo approach. ACKNOWLEDGMENTIS This research was supported by Public Health Service grant HD-03521 from the National Institute of Child Health and Human Development. E.M. was supported by Public Health Service predoctoral training grant GM-00510 from the National Institute of General Medical Sciences. This work is in partial fulfillment of the Ph.D. degree by E.M.
LITERATURE CITED 1. Birnbaum, L. S., and S. Kaplan. 1971. Localization of a portion of the ribosomal RNA genes in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 68:925-929. 2. Blobel, G., and D. Sabatini. 1971. Dissociation of mammalian polysomes into subunits by puromycin. Proc. Nat. Acad. Sci. U.S.A. 68:390-394. 3. Deonier, R. C., E. Ohtsubo, H. J. Lee, and N. Davidson. 1974. Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. VII. Mapping the ribosomal RNA genes of plasmid F14.
J. Mol. Biol. 89:619-629. 4. Fraenkel, D. G., and F. C. Neidhart. 1961. Use of chloramphenicol to study control of RNA synthesis in bacteria. Biochim. Biophys. Acta 53:96-110. 5. Goldberg, M. L., and J. A. Steitz. 1974. Cistron specificity of 30S ribosomes heterologously reconstituted with components from Escherichia coli and Bacillus stearothermophilus. Biochemistry 13:2123-2129. 6. Hardy, S. J. S., C. G. Kurland, P. Voynow, and G. Mora. 1969. The ribosomal proteins of Escherichia coli. I. Purification of the 30S ribosomal proteins. Biochemistry 8:2897-2905. 7. Held, W. A., S. Mizushima, and M. Nomura. 1973. Reconstitution of Escherichia coli 30S ribosomal subunits from purified molecular components. J. Biol. Chem. 248:5720-5730. 8. Jarry, B., and R. Rosset. 1973. Further mapping of 5S RNA cistrons in Escherichia coli. Mol. Gen. Genet. 126:29-35. 9. Jaskunas, S. R., L. LiAndahl, and M. Nomura. 1975.
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10.
11. 12. 13.
14.
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Isolation of polar insertion mutations and the direction of transcription of ribosomal protein genes in E. coli. Nature (London) 266:183-187. Kaltschmidt, E., and H. G. Wittman. 1970. Ribosomal proteins. VII. Two dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins. Anal. Biochem. 36:401-412. Kurland, C. G. 1966. The requirements for specific sRNA binding by ribosomes. J. Mol. Biol. 18:90-108. Maaloe, O., and N. 0. Kjeldgaard. 1966. Control of macromolecular synthesis. W. A. Benjamin, New York. Morgan, E. A., and S. Kaplan. 1976. Coordinate regulation of the individual ribosomal RNA operons in Escherichia coli. Biochem. Biophys. Res. Commun. 68:969-974. Morgan, E. A., and S. Kaplan. 1976. Transcription of Escherichia coli ribosomal DNA in Proteus mirabilis. Mol. Gen. Genet. 147:179-188.
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15. Morgan, E. A., and S. Kaplan. 1977. Expression and stability of Escherichia coli F-prime factors in Proteus mirabilis. Mol. Gen. Genet. 151:41-47. 16. Nomura, M. 1977. Organization of bacterial genes for ribosomal components: studies using novel approaches. Cell 9:633-644. 17. Pestka, S. 1970. Studies on the formation of transfer ribonucleic acid-ribosome complexes. VII. Survey of the effect of antibiotics on N-acetylphenylalanyl-puromycin formation: possible mechanisms of puromycin action. Arch. Biochem. Biophys. 136:80-88. 18. Randall, L. L, and S. J. S. Hardy. 1975. Analysis of the ribosomes engaged in the synthesis of the outer membrane proteins of Escherichia coli. Mol. Gen. Genet. 137:151-160. 19. Traub, P., S. Mizushima, C. U. Lowry, and M. Nomura. 1971. Reconstitution of ribosomes from subribosomal components. Methods Enzymol. 10:391-407.
JOURNAL
Vol. 134, No. 2 Printed in U.S.A.
Structure, Function, and Regulation of Escherichia coli rRNA in Proteus mirabilis E. A. MORGANt AND S. KAPLAN*
Department ofMicrobiology, University of Illinois, Urbana, Illinois 61801
Received for publication 7 September 1977
Escherichia coli rRNA genes have been introduced into Proteus mirabilis on F-prime factor (F'14). A portion of the ribosomes in the resulting merodiploid consist of E. coli rRNA and P. mirabilis ribosomal proteins. These ribosomes are structurally similar to normal P. mirabilis or E. coli ribosomes and exhibit many or all of the functional properties of normal ribosomes. The accumulation of E. coli rRNA in the merodiploid is regulated in a way similar to the regulation of P. mirabilis rRNA. an
Escherichia coli F-prime factor, F'14, which contains two E. coli rRNA operons, has been introduced into Proteus mirabilis (1). We have previously shown that E. coli 16S, 23S, and 5S rRNA are synthesized in P. mirabilis and that each rRNA species is incorporated into ribonucleoprotein particles which consist of E. coli rRNA and P. mirabilis ribosomal proteins (14). All three E. coli rRNA species can be distinguished from P. mirabilis rRNA by differences in the primary sequence of the rRNA of the two species (14), with the rRNA of the two species differing in approximately 20% of their base residues. Each E. coli-derived rRNA species comprises between 20 and 28% of the total of each rRNA species in the merodiploid. Employing oligonucleotide mapping procedures, the E. coli rRNA in the hybrid-derived ribosomes has been shown to be indistinguishable from mature E. coli rRNA; additionally, these hybrid ribosomes have sedimentation properties similar to normal P. mirabilis ribosomes (14). In this paper we further report on the structural properties of the ribosomes derived from the merodiploid. In addition, we have investigated the functional properties of these ribosomes as well as the regulatory responses of E. coli and P. mirabilis rRNA operons present in the merodiploid. MATERIALS AND METHODS Bacterial strains. The bacterial strains used have been previously described (1, 13, 15; E.A. Morgan, Ph.D. thesis, University of Illinois, Urbana, 1976). Preparation of ribosomal subunits and rRNA. The procedures for preparation of ribosomal subunits and rRNA have been previously described (14). rRNA was isolated from separated ribosomal subunits and t Present address: Enzyme Institute, University of Wisconsin, Madison, WI 53703.
further purified by sedimentation through sucrose gradients or by gel electrophoresis. Analysis of rRNA. Detection of E. coli rRNA in the bacterial hybrids was done as follows. The purified [32P]rRNA species from the hybrid was mixed with purified [3H]uracil-labeled E. coli rRNA species (from E. coli K-12 strain LMUR), digested with ribonuclease Ti or ribonuclease Ti and alkaline phosphatase, and fingerprinted as described previously (14). The amount of E. coli rRNA in the [:2P]rRNA was determined by analysis of the 'H-32P ratios of individual oligonucleotides, as previously described (14). Measurements of RNA content in cells. RNA was determined by the orcinol method of Fraenkel and Neidhart (4). ATP standards were processed with experimental samples and constituted a standard curve. Optical density of samples was determined in a Zeiss PMQ spectrophotometer at 420 nm. Cell numbers were determined by diluting cell samples to 40,000 to 90,000 cells per ml in 0.85% NaCl-3% formaldehyde, which had been twice filtered through a 0.1-ftm membrane filter (Millipore Corp.). The cells were counted in a model ZBI Coulter Counter with a 50-jil sample volume and 30-,um aperture. Low growth rate conditions and labeling. P.
mirabilis F14/PM14IV-4 was labeled with 32p; for several generations in GRCLP medium (13) containing 2 jig of nicotinic acid per ml, 10 ,ug of thymine per ml, and 50 yg each of methionine, tryptophan, isoleucine, and valine per ml. Aspartate (0.2%) was added as the sole carbon and nitrogen source. The doubling time in this medium is 26 h. For all other experiments, cells were labeled in standard low-phosphate medium as described (14). Preparation of polysomes. Polysomes were prepared as described by Randall and Hardy (18), with minor modifications. Cells for polysome preparations were grown in a 24-ml culture in minimal low-phosphate media (14).
RESULTS Structure of the hybrid ribosomes. Strain F14/PM14IV-4 is a P. mirabilis strain carrying E. coli F-prime F14, which carries two E. coli 606
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rRNA operons (1, 3, 14). Previous results (14) indicated that E. coli rRNA was present in strain F14/PM14IV-4 in ribonucleoprotein particles whose gross sedimentation properties were indistinguishable from bulk ribosomal subunits. Fingerprinting techniques demonstrated that the E. coli rRNA in this strain had been processed to yield rRNA species similar or identical to mature E. coli rRNA species (14). The chromosomal location of E. coli ribosomal protein genes (16) indicates that the ribosomal particles in strain F14/PM14IV-4 must consist largely, if not solely, of P. mirabilis ribosomal proteins. We have analyzed the ribosomal proteins of these subunits by comparing the two-dimensional gel electrophoresis patterns of purified ribosomal proteins from E. coli, P. mirabilis, and the hybrid strain F14/PM14IV-4, using the method of Kaltschmidt and Wittman (10). The ribosomal proteins of strain F14/PM14IV-4 showed no obvious differences or additional spots when compared with those of PM14IV-4 (Fig. 1). CsCl core particles prepared from ribosomal subunits derived from strain F14/PM14IV-4 by the method of Traub et al. (19) contain E. coli rRNA (Morgan, Ph.D. thesis; data not shown); it is, therefore, likely that these particles represent a specific and functional complex (see below) of E. coli rRNA and P. mirabilis ribosomal proteins. Functional properties of the hybrid ribosomes. Lacking the ability to separate hybrid frota homologous ribosomes in strain F14/PM14IV-4, it is difficult to unambiguously test the activity of the hybrid ribosomes by use of conventional methods. However, if ribosomes which contain E. coli rRNA are active, then they should be present on polysomes derived from strain F14/PM14IV-4. Polysome profiles from strain F14/PM14IV-4 were found to be identical to those prepared from strain PM14IV4 (Fig. 2). E. coli 16S, 23S, and 5S rRNA species were present in approximately 20 to 30% of all ribosomes derived from polysomes prepared from strain F14/PM14IV-4 (Table 1). This is approximately the same level of each E. coliderived rRNA species found in the total ribosomal subunit pool of this strain (14). To further reveal functional activity of the hybrid ribosomal population of strain F14/PM14IV-4, we have measured polysome disappearance under conditions where normal polysome breakdown requires puromycin-induced polypeptide release and subsequent translocation of the ribosomes (2, 17). Assuming the random distribution of E. coli rRNA species among subunits, 47% of all the 70S ribosomes would contain at least one E. coli rRNA species, and 38% would contain at least one E. coli
607
species if 50S subunits were composed solely of either E. coli or P. mirabilis rRNA species. If the ribosomes which contain E. coli rRNA were refractory to dissociation, then no less than 20% or as high as 47% of the polysomal-bound ribosomal subunits would remain as free 70S ribosomes, since active ribosomes are converted to free ribosomal subunits following puromycin treatment of polysomes. However, all ribosomes from strain F14/PM14IV-4 were present as free ribosomal subunits after puromycin-induced subunit release (Morgan, Ph.D. thesis; data not shown). This release is largely inhibited by the addition of chloramphenicol and requires the presence of K+ (Morgan, Ph.D. thesis; data not shown), which is further evidence that this assay measures functional ribosomes. In addition, we have found that both strains PM14IV-4 and F14/PM14IV-4 grow at identical rates in similar media. If hybrid ribosomes were inactive and, therefore, capable of blocking polysome movement of active ribosomes, then it is likely that this defect would manifest itself in the growth rate of the F14-containing strain. Therefore, we feel the evidence indicates that the hybrid ribosomes in this strain are fully active. Accumulation of E. coli rRNA in P. mirabii& Evolutionary divergence of P. mirabilis and E. coli has resulted in divergence of the rRNA sequences of these two organisms (14), and, therefore, differences in the regulatory sequences affecting rRNA synthesis might be anticipated between these two species. The level of RNA per cell during steady-state exponential growth in an M9 salts defined medium was determined for both strains PM14IV4 and F14/PM14IV-4. Under these growth conditions, both organisms have identical doubling times. Strain F14/PM14IV-4 was determined to have approximately 98% of the RNA per unit of absorbance at 420 nm with respect to strain PM14IV-4 (Morgan, Ph.D. thesis; data not shown). Additionally, direct cell counts by use of a Coulter Counter indicated that strain F14/PM14IV-4 had 96% of the RNA per cell of PM14IV-4 (Morgan, Ph.D. thesis; data not shown). About 80% of the RNA in cells is rRNA (12). Therefore, although rRNA gene dosage has been increased 20 to 30% in strain F14/PM14IV4 as compared to- PM14IV-4 (1, 14), no large increase in rRNA accumulation or decrease in cell growth rate was observed. This is evidence for both the proper function of E. coli rRNA in this strain and for a regulatory system which maintains proper levels of ribosome accumulation by a mechanism insensitive to rRNA gene dosage. Previous studies (12) have revealed that cells
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FIG. 1. Ribosomal protein profiles of P. mirabilis and F14/P. mirabilis merodiploid. Cultures of strains AB1206, PM)41IV-4, and F14/PM14IV-4 were grown aerobically in M9 minimnal salts medium appropriately supplemented and harvested in mid- to late exponential growth. Ribosomes were prepared fr-om these cells by the ammonium sulfate fr-actionation method (11). Subunits were prepared as described and repeatedly recentrifuged through sucrose gradients to insure their purity. Proteins were extracted fr-om the purified subunits by the acetic acid method (6). Proteins were dialyzed against several changes of TSM buffer and then exhaustively dialyzed against distilled water before lyophilizing to dryness. Two-dimensional gelprofiles were obtained according to the procedure of Kaltschmidt and Wittman (10) using a pH 9.5 first dimension. (A) Strain PM14IV-4 30S ribosomal proteins; (B) strain F14/PM14IV-4 30S ribosomal proteins; (C) E. coli AB1206 and PM14IV-4 30S ribosomal protein mixture; (D) strain PM14IV-4 50S ribosomal proteins; (E) strain F14/PM14IV-4 505 ribosomal proteins; (F) E. coli AB1206 and PMJ4IV-4 5OS ribosomal protein mixture.
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TABLE 2. E. coli rRNA sequences present in rRNA species prepared from ribosomal subunits of strain F14/PM14IV-4 grown with a doubling time of 26 h Oligonucleotidea Fraction' 16S rRNA AUUAACG ................. 0.33 pAAAUUG ................. 0.35 ACCUUCG ................. 0.40 UCUG ..................... 0.31 Bottom
Distance Migrated
Top
FIG. 2. Polysomes prepared from strain F14/PM14IV-4. The portion of the gradient included within the arrows was pooled to determine whether E. coli rRNA was in ribosomes on polysomes isolated from strain F14/PM14IV-4. Polysome profiles prepared from strain F14/PM14IV-4 are undistinguishable from those of strain PM14IV-4. A2Mnm, Absorbance
at 254 nm.
TABLE 1. E. coli rRNA sequences present in rRNA species prepared from ribosomal subunits derived from polysomes of strain F14/PM14IV-4 Oligonucleotidea
Fraction
16S rRNA AUUAACG
.................
pAAAUUG ACCUUCG
UCUG
.................
.....................
23S rRNA 12 ....................... 14 15
....................... .......................
5S rRNA UCUCCUCAUG ............ ACCCCAUG ............... CCAUG
0.24 0.22
....................
5S rRNA UCUCCUCAUG ............ 0.22 c ACCCCAUG ............... 0.14 CCUG ..................... 0.29 CAG ....................... 0.28c a Sequence of the E. coli unique oligonucleotides examined. b Fraction of E. coli rRNA in the hybrid, calculated using the 32P-3H ratio of the oligonucleotide. c The oligonucleotide is not present on all 5S rRNA molecules coded by F14 or the E. coli genome (8, 14). As a result, the value given has been calculated according to the suggested frequency of occurrence of this oligonucleotide, as previously described (14; Table 1, footnote b). The values for these fractional E. coli oligonucleotides compare favorably with the values resulting from oligonucleotides present in all E. coli 5S rRNA molecules, which strongly supports the conclusion that synthesis of rRNA takes place from both rRNA operons on F14 (see 14).
0.30
0.20
0.23 0.23 0.28
(0.62) 0.19b 0.12 0.17
CCUG ..................... 0.13 CAG ....................... (0.23) 0.11b E. coli unique oligonucleotides produced by ribonuclease Ti digestion of the rRNA. For 16S and 5S, the sequence is given. For 23S rRNA, the sequence is not known; the numbers refer to designations used previously (14). b The particular oligonucleotide is not present in all copies of 5S rRNA of F14 or in E. coli (8, 14). Therefore, the values given have been calculated according to the suggested frequency of occurrence of these oligonucleotides. The logic and evidence for this procedure have been previously described (14) and are consistent with equal expression of both rRNA operons on F14 in strain F14/PM14IV-4 as well as the presence of rRNA from both operons on polysomes. a
growing with different growth rates possess different rates of rRNA synthesis as well as having characteristically different rRNA-protein ratios.
Direct comparison of the differential accumulation of E. coli and P. mirabilis rRNA in strain F14/PM14IV-4 has been made at high and low growth rates. Many individual determinations (here; 14) place the E. coli rRNA content in strain F14/PM14IV-4 at near 20 to 30% in a medium in which this bacterium doubles every 80 min (14). Strain F14/PM14IV-4 was grown in medium where aspartate served as the sole carbon and nitrogen source with a doubling time of 26 h. From Table 2, it is apparent that the fraction of E. coli rRNA in strain F14/PM14IV4 grown with a doubling time of 26 h is near 20 to 30%, similar to that of a culture grown with a doubling time of 80 min (14). These estimates are most strongly supported from studies of the 16S rRNA in E. coli-specific oligonucleotide UCUCG, which we estimate is inflated by no more than 2 to 4% due to imperfect resolution of neighboring oligonucleotides (14). The well-resolved oligonucleotides of 5S rRNA also agree with the conclusion. Certain 5S rRNA oligonucleotides may derive from only one, or more, of the rRNA operons present, due to sequence heterogeneity in 5S rRNA. Oligonucleotide UCUCCUCAUG is present on only one copy of the 5S rRNA genes in E. coli and is present on F14 (8, 14). It is apparent that both copies of 5S rRNA on F14 contrib-
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J. BACTrERIOL.
TABLE 3. Relative molarities of fractional oligonucleotides from P. mirabilis 5S rRNA sequences in strain F14/PM14IV-4' Sequence b Molarity Glucose (80 min) CCAUAG ...... 1.08 CCAACG .................. 0.92 0.10 U,C,A,A,Gc ................. 0.11 U,U,C,G ...................
Aspartate (26 h) CCAUAG CCAAG ...... U,C,A,A,GC .......... .......
0.91 1.09 0.11
U,U,C,GC ...................
0.10 a Values for 32P counts per minute per total number of bases were determined for each oligonucleotide, then were normalized to values obtained by the formula (32P counts per minute of CCAUAG + 32P counts per minute of CCAAG)/11. This procedure gives the molarity of each oligonucleotide relative to the CCAUAG and CCAAG standards. CCAUAG and CCAAG occur in most or all 5S rRNA cistrons of P. mirabilis. b Sequence of oligonucleotides derived from 5S rRNA from strain F14/PM14IV-4 grown on indicated medium. Parentheses indicate doubling time. c U,C,A,A,G and U,U,C,G are proposed to be fractional oligonucleotides in P. mirabilis 5S rRNA. Base composition is suggested on the basis of chromatographic mobility of the oligonucleotide.
ute equally to rRNA synthesis at doubling times of 80 min or 26 h (Table 2). P. mirabilis 5S rRNA-derived fractional oligonucleotides, which we have previously identified (14), exhibit similar fractional values at doubling times of 80 min or 26 h (Table 3). Therefore, all rRNA operons in strain F14/PM14IV-4, whether of E. coli or P. mirabilis origin, very probably contribute in the same proportion to the accumulation of rRNA at both high and low growth rates. We have previously observed that individual rRNA operons in E. coli contribute equally to rRNA accumulation in E. coli under a variety of conditions (13).
DISCUSSION All E. coli rRNA species derived from F'14 are synthesized in P. mirabilis and packaged into ribonucleoprotein particles which have many physical and functional properties characteristic of normal ribosomes. These ribosomes consist of E. coli rRNA and P. mirabilis proteins. Therefore, the sequence divergence does not seem to affect the function of E. coli rRNA species in ribosome assembly or in protein synthesis, when present in P. mirabilis. In addition, E. coli rRNA appears to be properly regulated in P. mirabilis under several distinct physiological conditions.
Previous in vitro studies have shown that ribosome components of distantly related bacterial species can form functional heterologous ribosomes in the highly artificial conditions of ribosome reconstitution (5, 7). The present results reveal that this compatibility extends to ribosome assembly and rRNA maturational pathways which occur in vivo. It is possible that other types of hybrid ribosomes may be constructed in vivo. For example, if E. coli ribosomal proteins can be synthesized and incorporated into functional ribosomes in P. mirabilis, it should be possible to construct E. coli-P. mirabilis hybrids diploid for ribosomal protein genes, and then to inactivate the P. mirabilis ribosomal protein genes totally by simple genetic selections similar to those previously employed with partial diploids of E. coli (9). The resulting ribosomes would contain certain protein(s) entirely of E. coli origin. With recent genetic engineering techniques, it may be possible to construct hybrid ribosomes in vivo using quite distantly related species. The influence of ribosome components on translational specificity may perhaps be fruitfully investigated by this type of in vivo approach. ACKNOWLEDGMENTIS This research was supported by Public Health Service grant HD-03521 from the National Institute of Child Health and Human Development. E.M. was supported by Public Health Service predoctoral training grant GM-00510 from the National Institute of General Medical Sciences. This work is in partial fulfillment of the Ph.D. degree by E.M.
LITERATURE CITED 1. Birnbaum, L. S., and S. Kaplan. 1971. Localization of a portion of the ribosomal RNA genes in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 68:925-929. 2. Blobel, G., and D. Sabatini. 1971. Dissociation of mammalian polysomes into subunits by puromycin. Proc. Nat. Acad. Sci. U.S.A. 68:390-394. 3. Deonier, R. C., E. Ohtsubo, H. J. Lee, and N. Davidson. 1974. Electron microscope heteroduplex studies of sequence relations among plasmids of Escherichia coli. VII. Mapping the ribosomal RNA genes of plasmid F14.
J. Mol. Biol. 89:619-629. 4. Fraenkel, D. G., and F. C. Neidhart. 1961. Use of chloramphenicol to study control of RNA synthesis in bacteria. Biochim. Biophys. Acta 53:96-110. 5. Goldberg, M. L., and J. A. Steitz. 1974. Cistron specificity of 30S ribosomes heterologously reconstituted with components from Escherichia coli and Bacillus stearothermophilus. Biochemistry 13:2123-2129. 6. Hardy, S. J. S., C. G. Kurland, P. Voynow, and G. Mora. 1969. The ribosomal proteins of Escherichia coli. I. Purification of the 30S ribosomal proteins. Biochemistry 8:2897-2905. 7. Held, W. A., S. Mizushima, and M. Nomura. 1973. Reconstitution of Escherichia coli 30S ribosomal subunits from purified molecular components. J. Biol. Chem. 248:5720-5730. 8. Jarry, B., and R. Rosset. 1973. Further mapping of 5S RNA cistrons in Escherichia coli. Mol. Gen. Genet. 126:29-35. 9. Jaskunas, S. R., L. LiAndahl, and M. Nomura. 1975.
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Isolation of polar insertion mutations and the direction of transcription of ribosomal protein genes in E. coli. Nature (London) 266:183-187. Kaltschmidt, E., and H. G. Wittman. 1970. Ribosomal proteins. VII. Two dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins. Anal. Biochem. 36:401-412. Kurland, C. G. 1966. The requirements for specific sRNA binding by ribosomes. J. Mol. Biol. 18:90-108. Maaloe, O., and N. 0. Kjeldgaard. 1966. Control of macromolecular synthesis. W. A. Benjamin, New York. Morgan, E. A., and S. Kaplan. 1976. Coordinate regulation of the individual ribosomal RNA operons in Escherichia coli. Biochem. Biophys. Res. Commun. 68:969-974. Morgan, E. A., and S. Kaplan. 1976. Transcription of Escherichia coli ribosomal DNA in Proteus mirabilis. Mol. Gen. Genet. 147:179-188.
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15. Morgan, E. A., and S. Kaplan. 1977. Expression and stability of Escherichia coli F-prime factors in Proteus mirabilis. Mol. Gen. Genet. 151:41-47. 16. Nomura, M. 1977. Organization of bacterial genes for ribosomal components: studies using novel approaches. Cell 9:633-644. 17. Pestka, S. 1970. Studies on the formation of transfer ribonucleic acid-ribosome complexes. VII. Survey of the effect of antibiotics on N-acetylphenylalanyl-puromycin formation: possible mechanisms of puromycin action. Arch. Biochem. Biophys. 136:80-88. 18. Randall, L. L, and S. J. S. Hardy. 1975. Analysis of the ribosomes engaged in the synthesis of the outer membrane proteins of Escherichia coli. Mol. Gen. Genet. 137:151-160. 19. Traub, P., S. Mizushima, C. U. Lowry, and M. Nomura. 1971. Reconstitution of ribosomes from subribosomal components. Methods Enzymol. 10:391-407.
