Proc. Nat. Acad. Sci. USA Vol. 72, No. 8, pp. 2900-2904, August 1975 Biochemistry
Very stable prokaryotic messenger RNA in chromosomeless Escherichia coli minicells (outer membrane protein/lipoprotein/R plasmid)
STUART B. LEVY Departments of Molecular Biology and Microbiology and of Medicine, Tufts University School of Medicine, and Tufts-New England Medical Center, Boston, Massachusetts
Comnmunicated by Boris Magasanik, May 2, 1975 E. coli minicells lack DNA, yet they make ABSTRACT protein, the synthesis of which is sensitive to chloramphenicol but insensitive to rifamycin. This protein is coded for by very stable cellular mRNA with an estimated half-life of 40-80 min. In an R factor-containing minicell, two very different species of mRNA are observed: (i) R factor-specific mRNA with a short half-life whose synthesis is rifamycinsensitive and (if) cellular mRNA with a long half-life whose synthesis is rifamycin-insensitive. These findings indicate that minicells contain normal degradative mechanisms for mRNA and point out the existence of a unique class of very stable cellular mRNA. Greater than 80% of the rifamycininsensitive protein synthesized goes into the outer minicell membrane. Relatively stable mRNA, half-life 5.5-11.5 min, for outer membrane protein in whole cells has been reported [Hirashima et aL (193) 1. Mol. BioL 79, 373-389]. The stability of minicell mRNA is significantly greater. This and other observations suggest that there are two functional species of mRNA for outer membrane protein, perhaps in different sites in the cell. Furthermore, these studies suggest that a class of cellular proteins is synthesized in bacteria without concomitant transcription and in the absence of association with chromosomal DNA.
In prokaryotic cells, gene transcription and subsequent translation to protein appear to occur coordinately (1, 2). Electron microscopic visualization has confirmed the coupling of these processes, showing protein synthesis occurring on polyribosomes attached to messenger RNA (mRNA) transcripts which are still in association with the template DNA (3). Biochemical studies have shown that prokaryotic mRNA is labile, with an average half-life of less than 3 min (4-8). More stable species of mRNAs have been described, e.g., for certain T7 phage proteins (9), for penicillinase production (10) and sporulation (11) in Bacillus cereus. Recently a group of outer membrane proteins in Escherichia coli has been identified which appear synthesized from mRNA species with half-lives of 5.5-11.5 min (12, 13). The reason for the mRNA stability is not yet known. In each of these examples DNA was present in the cell, and a continued association of the mRNA with the DNA cannot be excluded. In this paper, I shall present evidence, however, that remarkably stable species of functional E. coli mRNA exist in E. coli minicells in the absence of DNA. MATERIALS AND METHODS E. coli x925 and X984 [the original Adler strain (14) and a derivative received from R. Curtiss III, designated R- (plasmid-lacking)] and D1-7 [a derivative of X984 containing R factor 222 (15), designated R+] were used. Media, growth conditions, minicell purification, and radioactive labeling of cell and minicell proteins with Abbreviation: NaDodSO4, sodium dodecyl sulfate.
[s5S]methionine were as described (16). Minicell preparations contained one viable cell per 105 minicells. The RNA in cells and minicells was labeled by growing fresh X984 cells in phosphate-deficient medium (17), in the presence of 3% L broth and 32PO402 (0.02 ,gCi/5 gg per ml). Extractions of RNA followed the procedure of Penman (18). Sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis was performed in 10% gels for analysis of proteins (19) and in 5% gels for analysis of RNA (20). E. coli lipoprotein was determined by chromatography on Whatman 3 paper in a solvent system consisting of isobutyric acid/i M ammonium hydroxide (5:3) (21). Samples of radioactively labeled cells and minicells were boiled for 15 min to release the soluble material. After several washes in distilled water, the samples were spotted onto paper and chromatographed for 15 hr. This initial chromatography was used to remove free lipoprotein (22) as well as other proteins with the same relative mobility (S. Torti and J. T. Park, personal communication). The chromatographed origins were cut out, treated overnight with Ivsozvme (750,gg/ml) in 1 M ammonium acetate buffer to free up lipoprotein bound to murein, washed, and dried before being sewn onto fresh Whatman 3 paper for repeat chromatography. Diaminopimelate-labeled E. coli W7 cells were used to identify the chromatographic position of the released lipoprotein (21). After lysozyme treatment, the only diaminopimelate remaining is that attached to lipoprotein (21). Therefore, the diaminopimelate label identified the lipoprotein (S. Torti and J. T. Park, personal communication). Separation of inner and outer membranes followed a modification of the procedure of Osborn et al. (23). In order to get effective separation of minicell envelope, samples were carefully lysed in 0.01 M EDTA by treating with lysozyme (500 gg/ml) (24). After sonication (19), MgCl2 was added to a concentration of 0.01 M, and the material was layered onto 25-60% linear sucrose gradients in the presence of 0.5 mM EDTA (M. J. Osborn, personal communication). After centrifugation at 46,000 rpm in a SW 50.1 rotor (Beckman) for 12 hr, the fractions were collected and radioactivity was determined on trichloroacetic acid-treated paper discs (25). Extraction of membranes with sodium lauryl sarcosinate (Sarkosyl) was as described (19, 26). EXPERIMENTAL Minicells, about one-tenth the size of whole cells, are produced during abnormal cell division of an E. coli mutant (14). Although the minicells contain the appropriate amount of RNA and protein for their size, they essentially lack DNA (14, 15). When cell cultures are labeled overnight with [3H]thymidine, small amounts of trichloroacetic acid-precipitable label, presumably DNA, are detected in minicell preparations (15, 27). An average of three such experiments 2900
~ R-MINCELS
Proc. Nat. Acad. Sa. USA 72(1975)
Biochemistry: Levy r
7IC
61.c
5CD Q x
L43 z
RMINICELLS
a-
LJ130 2
20
R4'MINICELLS+ RIFy
/
i
60
=
90
120
O
0 +
RIf
150
18
TIME (MIN)
FIG. 1. Protein synthesis by R+ and R- minicells. Minicells (4 X 109/ml), purified from freshly growing x984 (R-) and D1-7 (R+, 222 R factor) minicell-producing strains were incubated in methionine assay medium with [35S]methionine (50 ,Ci/jg of methionine per ml) in the presence or absence of rifamycin (RIF) (200 ;ig/ml). At intervals during incubation duplicate 25-ul samples were removed and precipitated with trichloroacetic acid (cold, then hot) onto paper filter discs (14). Another sample of the Rminicell preparation was labeled in the presence of chloramphenicol (Cm) (25 ug/ml). Cultures were incubated 15 min with or without drug before [35S]methionine was added. A fourth sample of Rminicells was incubated in assay medium, but label was not added until 90 min after incubation. Viable cell counts in each preparation: R+ minicell, 6.7 X 104/ml; R- minicell, 6.0 X 104/ml. to 8
Ikf_
e.i
+ Cm * R- MIINICELLS
30
J
e-
R- MINICELLS
/
CELLS
b-
R+ MINICELLS
a.
R+
MINICELLS
2901
0.02% precipitable label per minicell as compared to whole cell (unpublished data; range 0.01-0.03). This figure
gave
represents an upper limit since it may include contaminating dead cells or free DNA. Plasmid DNA that segregates into minicells is transcribed and translated (16, 25, 29-31). Plasmid-less minicells do not synthesize DNA or RNA (precursor incorporation is
Very stable prokaryotic messenger RNA in chromosomeless Escherichia coli minicells (outer membrane protein/lipoprotein/R plasmid)
STUART B. LEVY Departments of Molecular Biology and Microbiology and of Medicine, Tufts University School of Medicine, and Tufts-New England Medical Center, Boston, Massachusetts
Comnmunicated by Boris Magasanik, May 2, 1975 E. coli minicells lack DNA, yet they make ABSTRACT protein, the synthesis of which is sensitive to chloramphenicol but insensitive to rifamycin. This protein is coded for by very stable cellular mRNA with an estimated half-life of 40-80 min. In an R factor-containing minicell, two very different species of mRNA are observed: (i) R factor-specific mRNA with a short half-life whose synthesis is rifamycinsensitive and (if) cellular mRNA with a long half-life whose synthesis is rifamycin-insensitive. These findings indicate that minicells contain normal degradative mechanisms for mRNA and point out the existence of a unique class of very stable cellular mRNA. Greater than 80% of the rifamycininsensitive protein synthesized goes into the outer minicell membrane. Relatively stable mRNA, half-life 5.5-11.5 min, for outer membrane protein in whole cells has been reported [Hirashima et aL (193) 1. Mol. BioL 79, 373-389]. The stability of minicell mRNA is significantly greater. This and other observations suggest that there are two functional species of mRNA for outer membrane protein, perhaps in different sites in the cell. Furthermore, these studies suggest that a class of cellular proteins is synthesized in bacteria without concomitant transcription and in the absence of association with chromosomal DNA.
In prokaryotic cells, gene transcription and subsequent translation to protein appear to occur coordinately (1, 2). Electron microscopic visualization has confirmed the coupling of these processes, showing protein synthesis occurring on polyribosomes attached to messenger RNA (mRNA) transcripts which are still in association with the template DNA (3). Biochemical studies have shown that prokaryotic mRNA is labile, with an average half-life of less than 3 min (4-8). More stable species of mRNAs have been described, e.g., for certain T7 phage proteins (9), for penicillinase production (10) and sporulation (11) in Bacillus cereus. Recently a group of outer membrane proteins in Escherichia coli has been identified which appear synthesized from mRNA species with half-lives of 5.5-11.5 min (12, 13). The reason for the mRNA stability is not yet known. In each of these examples DNA was present in the cell, and a continued association of the mRNA with the DNA cannot be excluded. In this paper, I shall present evidence, however, that remarkably stable species of functional E. coli mRNA exist in E. coli minicells in the absence of DNA. MATERIALS AND METHODS E. coli x925 and X984 [the original Adler strain (14) and a derivative received from R. Curtiss III, designated R- (plasmid-lacking)] and D1-7 [a derivative of X984 containing R factor 222 (15), designated R+] were used. Media, growth conditions, minicell purification, and radioactive labeling of cell and minicell proteins with Abbreviation: NaDodSO4, sodium dodecyl sulfate.
[s5S]methionine were as described (16). Minicell preparations contained one viable cell per 105 minicells. The RNA in cells and minicells was labeled by growing fresh X984 cells in phosphate-deficient medium (17), in the presence of 3% L broth and 32PO402 (0.02 ,gCi/5 gg per ml). Extractions of RNA followed the procedure of Penman (18). Sodium dodecyl sulfate (NaDodSO4)/polyacrylamide gel electrophoresis was performed in 10% gels for analysis of proteins (19) and in 5% gels for analysis of RNA (20). E. coli lipoprotein was determined by chromatography on Whatman 3 paper in a solvent system consisting of isobutyric acid/i M ammonium hydroxide (5:3) (21). Samples of radioactively labeled cells and minicells were boiled for 15 min to release the soluble material. After several washes in distilled water, the samples were spotted onto paper and chromatographed for 15 hr. This initial chromatography was used to remove free lipoprotein (22) as well as other proteins with the same relative mobility (S. Torti and J. T. Park, personal communication). The chromatographed origins were cut out, treated overnight with Ivsozvme (750,gg/ml) in 1 M ammonium acetate buffer to free up lipoprotein bound to murein, washed, and dried before being sewn onto fresh Whatman 3 paper for repeat chromatography. Diaminopimelate-labeled E. coli W7 cells were used to identify the chromatographic position of the released lipoprotein (21). After lysozyme treatment, the only diaminopimelate remaining is that attached to lipoprotein (21). Therefore, the diaminopimelate label identified the lipoprotein (S. Torti and J. T. Park, personal communication). Separation of inner and outer membranes followed a modification of the procedure of Osborn et al. (23). In order to get effective separation of minicell envelope, samples were carefully lysed in 0.01 M EDTA by treating with lysozyme (500 gg/ml) (24). After sonication (19), MgCl2 was added to a concentration of 0.01 M, and the material was layered onto 25-60% linear sucrose gradients in the presence of 0.5 mM EDTA (M. J. Osborn, personal communication). After centrifugation at 46,000 rpm in a SW 50.1 rotor (Beckman) for 12 hr, the fractions were collected and radioactivity was determined on trichloroacetic acid-treated paper discs (25). Extraction of membranes with sodium lauryl sarcosinate (Sarkosyl) was as described (19, 26). EXPERIMENTAL Minicells, about one-tenth the size of whole cells, are produced during abnormal cell division of an E. coli mutant (14). Although the minicells contain the appropriate amount of RNA and protein for their size, they essentially lack DNA (14, 15). When cell cultures are labeled overnight with [3H]thymidine, small amounts of trichloroacetic acid-precipitable label, presumably DNA, are detected in minicell preparations (15, 27). An average of three such experiments 2900
~ R-MINCELS
Proc. Nat. Acad. Sa. USA 72(1975)
Biochemistry: Levy r
7IC
61.c
5CD Q x
L43 z
RMINICELLS
a-
LJ130 2
20
R4'MINICELLS+ RIFy
/
i
60
=
90
120
O
0 +
RIf
150
18
TIME (MIN)
FIG. 1. Protein synthesis by R+ and R- minicells. Minicells (4 X 109/ml), purified from freshly growing x984 (R-) and D1-7 (R+, 222 R factor) minicell-producing strains were incubated in methionine assay medium with [35S]methionine (50 ,Ci/jg of methionine per ml) in the presence or absence of rifamycin (RIF) (200 ;ig/ml). At intervals during incubation duplicate 25-ul samples were removed and precipitated with trichloroacetic acid (cold, then hot) onto paper filter discs (14). Another sample of the Rminicell preparation was labeled in the presence of chloramphenicol (Cm) (25 ug/ml). Cultures were incubated 15 min with or without drug before [35S]methionine was added. A fourth sample of Rminicells was incubated in assay medium, but label was not added until 90 min after incubation. Viable cell counts in each preparation: R+ minicell, 6.7 X 104/ml; R- minicell, 6.0 X 104/ml. to 8
Ikf_
e.i
+ Cm * R- MIINICELLS
30
J
e-
R- MINICELLS
/
CELLS
b-
R+ MINICELLS
a.
R+
MINICELLS
2901
0.02% precipitable label per minicell as compared to whole cell (unpublished data; range 0.01-0.03). This figure
gave
represents an upper limit since it may include contaminating dead cells or free DNA. Plasmid DNA that segregates into minicells is transcribed and translated (16, 25, 29-31). Plasmid-less minicells do not synthesize DNA or RNA (precursor incorporation is
