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Properties of ribonucleic acids from photosynthetic and heterotrophic R hodospirillum rubrum1
Accepted October 28. 1975
CHOW,C . T . 1976. Properties of ribonucleic acids from photosynthetic and heterotrophic Rlrodospirillirtn rirhrirtn. Can. J. Microbiol. 22: 228-236. Rifampicin causes an initial rapid breakdown of about 30% of the pulse-labeled RNA in both photosynthetic and heterotrophic Rlrodospirillrrtn rirhrirtn. An additional degradation of RNA has been observed in the heterotrophic, but not in the photosynthetic cells at a later stage of rifampicin treatment. This secondary RNA degradation is probably caused by breakdown of ribosomal RNA. especially of the 23s species, a s shown by polyacrylamide gel electrophoresis analyses. Noqualitative difference between the pulse-labeled RNA species inphotosynthetic and heterotrophic cells can be detected by the DNA-RNA hybridization competition technique. CHOW,C . T. 1976. Properties of ribonucleic acids from photosynthetic and heterotrophic Rlrodo.spirillrrt~trrtbr~on.Can. J . Microbiol. 22: 228-236. La rifampicine cause un bris initial rapide d'environ 30% de I'ARN marque par pulsation chez Rl~orlo.spirillrrt~ir~rhrrrttlphotosynthetique e t heterotrophe. Une degradation additionnelle de I'ARN a ete observee dans le systeme heterotrophe, mais non dans celui photosynthetique, chez des cellules h un stade ~ l l t e r i e ~de l r traitement B la rifampicine. Cette degradation secondaire de I'ARN est probablement causee par un brisde I'ARN ribosomal, specialement chez I'espece 23s. tel que dkmontre par analyses electrophoretiques sur gel de polyacrylamide. Aucune difference qualitative ne peut Otre detectee entre les especes d'ARN marque e n pulsation chez les cellules photosynthitiques et celles heterotrophes, par la technique d'hybridisation a competition de ADN-ARN. [Traduit par le journal]
Introduction Rhodospirilluin rubrum, a facultative photosynthetic bacterium, can grow either anaerobically in the light or aerobically in the dark. Under the former conditions organic compounds serve as hydrogen donors for photosynthesis, whereas they are used as substrates for oxidative phosphorylation under the latter conditions. Comparisons between photosynthetically and heterotrophically grown R. rubrum cells have been made in regard to their physiology, biochemistry, and ultrastructures by a large number of investigators (for review, see refs. 10, 13, 14, 19). It has been shown that the rate and extent of synthesis of many structural components, such as photosynthetic pigments and certain enzymes, change significantly during the transition from heterotrophic to photosynthetic growth (2,5, 14, 15). These changes were suspected to be caused by synthesis of some specific messenger ribonucleic acids (mRNA), because, according to the current models, protein synthesis in bacteria is believed to be regulated primarily at the level 'Received August 7, 1975.
of transcription. Investigations on R. rubrurn mRNA, in respect to its stability and qualitative difference between photosynthetic and heterotrophic cells, have been carried out by Yamashita and Kamen (27, 28). The stability of mRNA in photosynthetic, but not in heterotrophic R. rubrum has been examined by treating pulselabeled cells with proflavin, and about 65% of the RNA is found to be unstable. By using a DNA-RNA hybridization competition technique under saturated RNA conditions, these authors have concluded that the qualitative difference between the mRNA species of photosynthetic and heterotrophic R. rubrum cells is very small, if it exists a t all. The R N A examined in the studies cited above was extracted from cells labeled during a 3-min period, and its stability was examined by using a non-specific inhibitor of RNA synthesis (proflavin). Since the half-life of mRNA in many other bacteria, including Rhoclopseuclomonas spheroides (another member of the photosynthetic bacteria Athiorhodaceae) is less than 3 min (12, 21, 24), a relatively long labeling period may have obscured the results. In the present communication, a detailed
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examination on the stability and quality of R. Long-labeled cells were prepared by labeling with [3H]urirubrum labeled RNA was undertaken. Cells were dine (2 pCi/ml) for three to four generations, chased by uridine (50 pg/ml) for one generation and labeled either for 30 s or for several generations unlabeled treated with rifampicin (200 pg/ml). The time at which during photosynthetic or heterotrophic growth rifampicin was added was defined as the zero time. and during the transition between them. The Labeled cultures (0.5 ml) were removed in triplicate at stability of labeled RNA was measured by rifam- 5-min (for pulse-labeled cultures) or 10-min (for longpicin, a specific RNA synthesis inhibitor (7, 17, labeled cultures) intervals into 5 ml of ice cold 5% trichloroacetic acid (TCA) and left at O°C for a t least 30 23), and the qualitative and quantitative changes min. T h e TCA-insoluble material was collected by centriin various RNA species in rifampicin-treated, fugation and the alkaline-labile, the alkaline-resistant, long-labeled R. rubrum was examined by poly- and the total TCA-insoluble radio-activities were deteracrylamide gel electrophoresis. The pulse-labeled mined (24). RNA, either before or after fractionation in Pho/osyn/he/ic + He/ero/rophic Transi/ions Photosynthetic R. rirbriirn was labeled with [3H]uridine sucrose gradients, was used to carry out hybridization competition experiments under both (2 pCi/ml) for 90 min and then divided into two equal parts; one was left under the photosynthetic growth conDNA and RNA saturated conditions. ditions and the other was gassed with a mixture of 95% air-5% CO, for 10 min in the dark, wrapped in aluminum Materials and Methods foil, and incubated on a reciprocal shaker in a 28 "C inCul/ure of Bacteria Rhodospirill~imrubrum was obtained from Dr. D. N. Burton of this department. The organism was maintained as stab cultures in a medium containing in every litre 3.50 g of dl-malic acid, 4.0 g of I-glutamic acid, 0.8 g of sodium citrate, 0.12 g of KH2P0,, 0.18 g of K2HP0,, 0.2 g of MgS0,.7H20, 33.0 mg of CaCI2, 5.0 pg of biotin, 2.0 g of Difco yeast extract, and 15.0 g of Difco Bacteriological agar, and the pH of the medium was adjusted to 6.8 with saturated NaOH solution before autoclaving. Inoculated agar tubes were incubated at 28 "C in a Conviron model E7 incubator with maximal illumination until a heavy growth was obtained which usually took 2-3 days. The stab cultures were kept at 4 "C and subcultured every 4-6 weeks. T o obtain fresh liquid cultures, a loopful of the stab culture was used to inoculate a large test tube filled to the neck with the same medium wlthout agar, and incubated under the same conditions u n t ~ lthe cells had reached early stationary phase of growth This culture was used as the inoculum for growing large quantities of cells. For photosynthetic cultures, cells were cultivated in rubber-stoppered Erlenmeyer flasks equipped with gas inlet, gas outlet, and sampling tubes (3.8 litres of medium in 4-litre flasks). Immediately after inoculation (1% inoculum), the culture was aerated with a mixture of 95% N2-5% C 0 2 for 15 min and incubated in the illuminated, 28 "C Conviron incubator. Heterotrophic cultures were cultivated in aluminum-foil-wrapped, cotton-stoppered Erlenmeyer flasks (I litre of medium in 2-litre flasks) on a reciprocal shaker in a 28 "C incubation room. Bacterial growth was measured by following absorbance in a Klett photometer, using a Corning No. 66 filter. The doubling time has been found to be about 5.2 h under either the photosynthetic o r the heterotrophic conditions. Rqampicin Treattnerlt of Labeled Cells Rhodospirilliim riibrurtl cultures were grown in Klett flasks (30 ml of culture per flask) and bubbled with mixtures of 95% N2-5Z C 0 2 and 9 5 z air-5% C 0 2 t o achieve anaerobiosis and aerobiosis, respectively. For pulse-label experiments, cells were labeled for 30 s with [5-3H]uridine (27.8 Ci/mmol, 20 pCi/ml for photosynthetic and 30 pCi/ ml for heterotrophic cultures), and then treated with rifampicin (200 pg/ml) and unlabeled uridine (50 pglml).
cubation room. Complementary procedures were used for heterotrophic to photosynthetic transition.
Es/rac/ior~ond Pi~rifico/ionof Labeled R N A Labeled RNA was extracted and purified by the techniques described before (4). Purified RNA was dissolved in a small volume of an acetate buffer containing 20 miM sodium acetate, 20 m M KCI, and 10 m M MgCI,, pH 5.2. The concentration of R N A was determined from the ab= 25 and its TCAsorbance at 260 nm, taking El ,.,".'% insoluble radioactivity was measured. Siicrose Gr.odierr/ Froc/io11a/iortof Labeled R N A Labeled RNA was fractionated by laying 0.2ml of RNA on top of a linear sucrose gradient (4.7 ml, 15-30%) and centrifuging at 35 000 rpm for 15 h in a n SW50 rotor at 4 'C in a Beckman L2-65 ultracentrifuge. After centrid fourfugation, the bottom of the tube was p ~ ~ n c t u r eand drop fractions were collected in tubes containing 1 ml of 6 x SSC (I x SSC = 0.15 M NaCI + 0.01 5 M sodium citrate, pH 7.0). The absorbanceofeach fraction was measured at 260 nm in a Beckman model DU spectrophotometer. An aliquot of the sample was used for determination of TCA-insoluble radioactivity. Prepoi.a/iorr orlrl Derlo/irra/iorr of DNA The techniques previously dexribed (4) were used to purify R. riibrwir DNA, and the amount of DNA was determined by the diphenylamine method (3). Purified DNA samples wel-e denatured either by heating at 100°C for 15 min o r by treatment with 0.5 N NaOH for 60 min, depending on the final D N A concentration desired. Both procedures were found to be equally effective for denaturing D N A . DNA-RNA H)~bridizo/ior~ Corrrpe/i/iorr This was carried out either by the method of Nygaard and Hall (18), with D N A free in solution, o r by that of Gillespie and Spiegelman (11). with D N A affixed onto nitrocellulose membrane filters (Schleicher and Schnell, No. B-6). Each annealing mixture, with a final volume of 1 ml, contained 6 x SSC, 0.1% SDS, various amounts of [3H]labeled and unlabeled RNA and denatured DNA (free o r membrane-bound). After incubation at 66°C for 16 h, the hybridization mixtures were cooled rapidly in an ice-water bath. If the Nygaard and Hall method was used,
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the samples were first collected on 25-nim nitrocellulose membrane filters. The remaining steps were identical with both hybridization methods. The membrane filters were washed in 100 ml of 2 x SSC, digested in 1 ml of 2 x SSC containing preboiled ribonuclease (20 pg/ml) for 1 h at room temperature, washed again with 100 ml of 2 x SSC, and counted in 10 rnl of Aquasol. Electt~opl~oresis Labeled R N A was electrophoresed in a 2.4%-10% double polyacrylamide gel (20). Twenty microlitres of unlabeled E. coli and L-cell ribosomal R N A was added to each sample and coelectrophoresed as molecular weight markers. The gels were scanned at 260 nm in a JoyceLoebl UV Scanner to determine the position of the markers and cut into 50 slices with a Bio.Rad gel slicer. The gel slices were hydrolyzed in 0.1 ml of H,O, for 2 h at 80°C and counted in 10 ml of Aquasol. Cl~e~nicnls The following chemicals were used: crystalline pancreatic deoxyribonuclease and ribonuclease (Worthington Biochemical Corp.), 3H-labeled uridine and Aquasol (New England Nuclear Corp.), and rifampicin (Sigma Chemical Co.).
labeled R. rubrum was found t o be more than 99% alkaline-labile showing specific labeling of R N A under the experimental conditions. The R N A degradation patterns of rifampicin-treated cells are shown in Fig. 1. Both photosynthetic and heterotrophic cultures showed an initial rapid decrease in radioactivity during the first 10 min of rifampicin treatment, after which time the radioactivity remained a t a constant level in the photosynthetic culture whereas a secondary degradation was observed in the heterotrophic culture starting a t 20 min after addition of rifampicin.
Photosynthetic =$ Heterotrophic Transitions Synthesis of R N A in R. rubrum during transition was measured, and the results are shown in Fig. 2. After transition from the heterotrophic t o the photosynthetic condition, R N A synthesis continued a t a decreased rate for at least 5.5 h (Fig. 2a) whereas a sudden halt in net RNA syn-
Results Degradation of Pulse-labeled RNA The amount of [3H]uridine incorporated into the cold TCA-insoluble fraction of the pulse-
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TIME TIME lmin/ FIG.1. Degradation of pulse-labeled R. rubrutn RNA. Photosynthetic (0) and heterotrophic (0) cultures were labeled with [3H]uridine for 30 s, and treated with rifampicin and unlabeled uridine as described in Materials and Methods. Samples were removed at 5-min intervals for determination of cold TCA-insoluble radioactivity.
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FIG.2. R N A synthesis during transitions. Rllodospirillum rubr~tmcultures were labeled with [3H]uridine at a n early exponential phase of growth for 90 min. Transitions of growth conditions were made a t the time indicated (arrow). Samples were removed a t 30-min intervals for determination of cold TCA-insoluble radioactivity. ( a ) Heterotrophic t o photosynthetic transition, and (b) photosynthetic to heterotrophic transition. (0) photosynheterotrophic. thetic, (0)
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CHOW: RNA'S FROM R H O D O S P l R l L L U M RUBRUM
thesis was observed immediately after transition from the photosynthetic to the heterotrophic condition (Fig. 2b). N o net RNA synthesis could be detected for 2.5 h and after which time RNA synthesis resumed at a rate similar to that of the photosynthetic cultures. Degradation of pulse-labeled RNA in R. r~rbrumat 1, 2, and 3 h after transition was also determined. During the photosynthetic to heterotrophic transition, a gradual change in the RNA degradation pattern was observed (Fig. 3) and 3 h after transition the RNA degradation pattern was almost identical with that of the heterotrophic culture (compare Figs. 3 and I). When the reverse transition was carried out, a complementary phenomenon was observed. he above results have clearly indicated the presence of an additional unstable RNA fraction in the rifampicin-treated heterotrophic, but not photosynthetic R. rubrum. T o characterize this RNA fraction, a qualitative difference between pulse-labeled photosynthetic and heterotrophic RNA (P- and H-RNA) was measured by DNA-
RNA hybridization competition technique, and the R N A from rifampicin-treated, long-labeled R. rubrum was analyzed by polyacrylamide gel electrophoresis. Competition under Saturated DNA Conditions Hybridization competition was carried out by incubating a DNA-bound membrane (100 pg DNA per membrane) (4), 1 pg of pulse-labeled RNA, and up to 300 pg of unlabeled RNA in 1 ml of an annealing mixture (1 1). The results are shown in Fig. 4. Increasing amount of unlabeled RNA caused a rapid decrease in the amount of DNA-3H-RNA hybrid formed. The amount of hybrids formed was reduced to the same extent by the unlabeled P- or H-RNA in all
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FIG.3. Degradation of pulse-labeled R. rrrbrum RNA during photosynthetic to heterotrophic transition. Photo2 (A), synthetic cultures were labeled at 0 (V), 1 and 3 ( 0 ) h after transition to heterotrophic conditions, q.nd treated with rifampicin as described in Fig. 1. Samples were removed at 5-min intervals for determination of cold TCA-insoluble radioactivity.
(a),
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RNA
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FIG. 4. Hybridization competition between labeled and unlabeled RNA on D N A sites under saturated DNA conditions. Purified R. rubrum labeled R N A (I pg) was hybridized with 100 pg of R. rubrum D N A affixed on a nitrocellulose membrane filter in the presence of indicated amount of unlabeled P- ( 0 ) or H- ( x ) RNA. Conditions for hybridization and determination of radioactivity were described in Materials and Methods. (a) Labeled PRNA P-DNA, ( b ) labeled P-RNA H-DNA, (c) labeled H-RNA + P-DNA, and ( d ) labeled H-RNA H-DNA.
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C A N . J . MICROBIOL. VOL. 22, 1976
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FIG.5. Sedimentation of pulse-labeled R. t.l/br~rt~r R N A in sucrose gradients. Pulse-labeled RNA from photosynthetic and heterotrophic cells was centrifuged in sucrose gradients as described in Materadioactivity in P-RNA, (a) radioactivity in H - R N A . rials and Methods. ( x ) , 4 2 6 0 nm, (0)
combinations of D N A and R N A tested. Since a relatively small difference between the labeled P- and H-RNA might be masked when total labeled-RNA was used, the pulse-labeled R N A was fractionated in sucrose gradients. The sedimentation profiles of the P- and H-RNA are superimposed and shown in Fig. 5. Three ultraviolet-light absorbing peaks, corresponding to ribosomal and transfer RNA, were well separated under these conditions. The distribution of the radioactivity was very similar for the Pand the H - R N A ; both were heterogenous and exhibited a region of relatively high specific radioactivity with sedimentation constants between 6 and 12 S (fractions 25-39, Fig. 5). These fractions were pooled and used to perform hybridization colnpetition experiments. Results obtained by using these pooled R N A fractions were essentially identical with those described in Fig. 4, except that a higher concentration of unlabeled RNA was required to obtain the same reduction level. Competition under Saturated RNA Conditions m affixed Eight micrograms of R. r ~ ~ b r r i DNA on a nitrocellulose membrane filter was used to hybridize with 400 pg of unfractionated labeled RNA in the absence o r the presence of unlabeled
R N A (up to 6 mg). The competitive ability of the unlabeled P- and H-RNA was essentially identical (Fig. 6). Sucrose gradient fractionated RNA had also been used for this type of competition. Because only a limited amount of fractionated RNA was available, 25 pg of fractionated labeled RNA was used to hybridize with 0.5 pg of D N A in the presence of (up to) 500 p g of unlabeled R N A (18). The results (not shown) were essentially the same as that presented in Fig. 6. Degrad~tionof Long-labeled RNA About 90% of the radioactivity incorporated into the untreated long-labeled R. rubrutw was alkaline-labile (Fig. 7). After rifampicin treatment, the total TCA-insoluble counts as well as the ratio between the alkalin-labile and the alkaline-resistant fractions remained unchanged in the photosynthetic cells. I n contrast, a rapid breakdown of labeled RNA was observed in the heterotrophic culture, and about 30 and 4 0 z o f the RNA was degraded after 30 and 60 min, respectively (Fig. 7b). No breakdown of D N A was detectable. Labeled R N A was also analyzed by polyacrylamide gel electrophoresis. I n the untreated cultures, both photosynthetic a n d heterotrophic
CHOW: RNA'S FROM RHODOSPIRILLUM RUBRUM
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UNLABELED RNA /mg) FIG.6. Hybridization competition between labeled and unlabeled R N A on D N A sites under saturated RNA conditions. Purified R. rubrutn labeled R N A (400 pg) was hybridized with 8 pg of R . rubrum D N A affixed on a nitrocellulose membrane filter in the presence of indicated amount of unlabeled P- (0) o r H- (m) R N A . Conditions for hybridization and determination of radioactivity were described in Materials and Methods. (a) Labeled P-RNA P-DNA, ( b ) labeled P-RNA + H-DNA, (c) labeled H-RNA P-DNA, and ( d ) labeled H-RNA + H-DNA.
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+
samples showed four distinct RNA species and their radioactivity content in the 23S, 16S, 5S, and 4 s species was about 57%, 28%, 3%, and 1277, respectively (Fig. ga). This distribution remained unchanged in the photosynthetic cells during the period tested. On the other hand, a rapid decrease in the 23s peak, accompanied by the appearance of a new peak (about 2 0 ~ 1 was , observed in the 30-min heterotrophic sample, and the percentage of radioactivity in the 23S, 20S, 16S, 5S, and 4 s species were found to be 14, 24, 42, 5, and 15, respectively (Fig. 86). At 60 min, a further reduction in the 23s peak accompanied by a prominent increase in the 20s peak occurred whereas no significant change was detectable in the remaining RNA species (Fig. 8 4 . Discussion As it is widely accepted, bacterial mRNA is
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FIG.7. Degradation of long-labeled R. rrtbrum RNA. The cold TCA-insoluble radioactivity in 0.5 ml of longlabeled photosynthetic (a) and heterotrophic (b) R. rubrum was determined. (0) Total counts, (0) alkalinelabile counts, and ( A ) alkaline-resistant counts.
and to acid-so1ub1e nucleotides after antibiotic inhibition of RNA ( 9 , '2, 16, 21). It is, therefore, not 'nto assume that the RNA degraded during the first 10 min of rifampicin treatment is if not mRNA. most likely for the RNA degradation are (a) there are two distinct frattions of mRNA in the heterotrophic R. rubrum. One them (which is absent in the photosynthetic cells) has a longer half-life than the other, and (b) the RNA degraded is some RNA Other than mRNA, and its degradation is induced by rifampicin treatment. Our DNA-RNA hybridization competition results have clearly shown that there is no detectable qualitative dif-
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CAN. J .
MICROBIOL. VOL. 22, 1976
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FIG.8. Electrophoretic analysis of long-labeled R. t.~~brli,n R N A . Labeled R N A extracted from R. rltbr~ituat 0 ( o ) , 30 (b),and 60 min ( c ) after rifampicin treatment was analyzed in a 2.4%-10z double polyacrylamide gel (20)with added ~lnlabeledE. coli and L-cell ribosomal R N A as molecular weight markers. T h e positions of the nlolecular weight markers were determined in a Joyce-Loebl U V scanner a n d the radioactivity in the gel slices was determined after HzOzdigestion. The broken line a t position 27 indicates the boundary of the 2.4% and the 10% gel. (8-a) P - R N A , (0---0) H-RNA.
ference between the untreated photosynthetic and heterotrophic pulse-labeled R N A (Figs. 4 and 6) and, therefore, render the first explanation less plausible. Consequently, the R N A degraded was assumed to be one or more species of the stable RNA, probably in its matured form because a 20-min lag exists between the time of rifampicin addition and the starting time of the secondary degradation (Fig. 1). It is known that R N A chain elongation and maturation can proceed normally in the presence of rifampicin (7, 17,23,24) and a recent report by Yuan and Shen
has shown that rifampicin causes degradation of matured E. coli23S and 16s r R N A in their corresponding ribosomal subunits (29). This conciusion that the R N A degraded during the secondary degradation is matured stable RNA is supported by the degradation and electrophoresis results observed in the rifampicin-treated, longlabeled, heterotrophic R. rubrurn (Figs. 7b and 8). It is, however, still unclear whether only the 23s or the 23s plus other stable R N A species is degraded. The 30-min heterotrophic RNA contained about 70% of the TCA-insoluble radio-
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CHOW: RNA'S FROM R
activity originally incorporated and 42% of the in R. rubrum and in Rhodopseudomonas spherlabel appeared in the 16s peak. Considering oides is at the level of translation. The RNA these two figures, the label in the 30-min 16s degradation and hybridization competition reRNA species would be 29.4% (42% x 70%) of sults presented in this report have further supthe original counts, a value very close to that in ported their conclusion. the untreated cells (28%). This finding may be Acknowledgments interpreted as n o degradation of the 16s RNA, although the possibility of a specific breakdown This investigation was supported by a grant of 23s to 16s accon~panying 16s degradation A6722 from the National Research Council of cannot be eliminated. The same argument can Canada. also be applied for the 5 s and the 4 s RNA I thank B. Rerie and H. S. Wang for technical species. The exact mechanism of the 23s RNA assistance. degradation is at present unknown. It seems I. ARONSON, A. 1. 1965. Characterization of messenger likely that both endonuclease (appearance of a RNA in sporulating Btr~.illrr.sc ~ r e r rJ.~ .Mol. Biol. 11: 20s peak) and exonuclease activities (reduction 576-588. in TCA-insoluble counts) are involved. The 2. BOTORAD, L. 1966. 117 Chlor.ophyll. Eclirc,tl I?! L . P. breakdown of rRNA may be a consequence of Vernon and G. R. Seely. Academic Press, Inc., New release of ribosomal proteins from matured York. ribosomal subunits by treatment of rifampicin, 3. BURTON,K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colas shown by Yuan and Shen (29). The structural orimetric estimation of deoxyribonucleic acid. changes in rifampicin-treated ribosomal particles Biochem. J. 62: 315-323. and the fate of these ribosomes during transitions 4. CHOW, C . T.. and I. T A K A H A S H1972. I. Deoxyribonucleic acid of an asporogenous mutant of are currently being investigated by the doubleBocillri.~srrbrilis. Irl Spores V. Erlired Ay H. 0. Hallabeling technique. vorson. R. Hanson. and L. L. Campbell. American In conclusion, a difference in sensitivity to Society for Microbiology. Fontana, Wisconsin. pp. rifampicin between the photosynthetic and 133-139. R Eand , W. R. SISTROM. 1966. 111 heterotrophic R. rubrum rRNA has been de- 5. C O H E N - B A ~ ~G., Chlorophyll. Edirecll>y L. P. Vernon and G. R. Seely. tected. Whether this difference is related t o their Academic Pless, Inc.. New York. protein synthesis control mechanism is open to 6. COLD S P R I N HARBOR G SYMPOSIA ON QUANTITATIVE speculation. As mentioned earlier, the primary BIOLOGY, XXXV. 1970. Cold Spring Harbor Laboraregulation of protein synthesis in bacteria is betory, Cold Spring Harbor, New York. E., L. S N Y D E RP., MARINO, A. LAMPERT, lieved to be effected at the transcriptional level. 7. DIMAURO, A. COPPO,and G. P. T O C C H I N I - V A L E N T1969. INI. Synthesis of new mRNA species after a change Rifarnpicin sensitivity of the components of DNAin physiological conditions has been demondependent RNA polymerase. Nature (London), 222: strated in a number of microorganisms. For in533-537. stance, bacteriophage-infected cells synthesize 8. DOI, R. H., and T. J. LEIGHTON. 1972. Regulation during initiation and subsequent stages of bacterial mainly, or completely, phage-specific mRNA sporulation. Itt Spores V. Edirerl hy H. 0 . Halvorson, (6), and distinct mRNA species exist in cells at R. Hanson, and L. L. Campbell. American Society for different stages of cell differentiation (6, 22). Microbiology, Fontana, Wisconsin. pp. 225-232. Furthermore, sporulation-specific mRNA is 9. F A N ,D. P., A. HIGA,and C . LEVINTHAL. 1964. Messenger RNA decay and protection. J. Mol. Biol. 8: produced in Bacillus spp. shortly after the initia2 10-222. tion of sporulation process (1, 8, 26). In the H., A. SANPIETRO,and L. P. VERNON.1963. photosynthetic bacteria, at least in the case of R. 10. GEST, Bacterial photosynthesis. The Antioch Press, Yellow r~rblwt?~ and Rhodopseudornonas spheroides, no Springs, Ohio. D., and S . SPIEGELMAN. 1965. A quantitadetectable qualitative difference in mRNA can l I. GILLESPIE, tive assay for DNA-RNA hybrids with DNA imthus far be demonstrated in cells grown under mobilized on a membrane. J. Mol. Biol. 12: 829-842. completely different physiological conditions, 12. G 1970. The R A YW. , J. H., and J . E. M. MIDGLEY. although many structural changes have been obcontrol of ribonucleic acid synthesis in bacteria. served. Because of the lack of a qualitative difSteady state content of messenger ribonucleic acid in Esc11~1.ic~hicr coli M.R.E. 600. Biochern. J. 120: ference in mRNA between photosynthetic and heterotrophic cultures, Yamashita and Kamen 13. 279-288. KONDRAT'EVA. E. N. 1963. Photosynthetic bacteria. (27, 28) and Witkin and Gibson (24, 25) have Israel Program for Scientific Translations, Jerusalem, suggested that the regulation of protein synthesis Israel.
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14. LASCELLES, J. 1973. Microbial photosynthesis. Dowden, Hutchingson and Ross. Inc., Stroudsburg, Pennsylvani:~. 15. LASCELLES, J. 1964. Tetrapyrrole biosynthesisand its regulation. Benjamin Company Inc., New York. 16. L E V I N T H A L C., , D. P. F A N , A. H I G A ,and R. A. Z I M M E R M A N1963. N . The decay and protection of messenger RNA in bacteria. Cold Spring Harbor Symp. Q~rant.Biol. 28: 183-190. , N I H A . and H. 17. M I Z U N O ,S., H. Y A M A Z A K IK. U M A / A W A1968. . Inhibition of DNA-dependent RNA polymerase reaction of E.sc~lrc~ricllicr c,oli by an antibiotic, streptoval-icin. Biochim. Biophys. Acta, 157: 322-332. 18. NYGAARD, A,. and B. HALL.1963. A method for the detection of RNA-DNA complexes. Biochem. Biophys. Res. Commun. 12: 98-104. 19. PARSON,W. W. 1974. Bacterial photosynthesis. Annu. Rev. Microbial. 28: 41-59. 20. PEACOCK, A. C., and C . W. D I N G M A N1967. . Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry, 6 : 1818-1827. 21. SALSER,W . , J . J A N I N and , C . L E V I N T H A L1968. . Measurement of the unstable RNA in exponentially growing cultures of Bncillrrs sr~htilisand E.schericl7irr coli. J . Mol. Biol. 31: 237-266. 22. SUSSMAN, M. 1971. Molecular genetics and developmental biology. Prentice-Hall, Inc., Englewood Cliffs, New Jersey.
23. W E H R LW., ~ , and M. STAEHELIN. 1971. Actionsofthe rifamycins. Bacteriol. Rev. 35: 290-309. 24. WITKIN,S. S . , and K. D. GIBSON. 1972. Changes in ribonucleic acid turnover during aerobic and anaerobic growth in Rlloclopserrcio~not~c~s splleroides. J. Bacteriol. 110: 677-683. 25. W I T K I NS. , S., and K. D. GIBSON.1972. Ribonucleic acid from aerobically and anaerobically grown Rlrorlopse~rciornonnsspheroides: Comparison by hybridization to chromosomal and satellite deoxyribonucleic acid. J . Bacteriol. 110: 684-690. 26. Y A M A G I S HH., I , and 1. T A K A H A S H1968. I. Genetic transcription in asporogenous mutants of Bncillrrs sirbrilis. Biochim. Biophys. Acta, 155: 150-158. 27. YAMASHITA, J . , and M. D. K A M E N .1968. Observations on the nature of pulse-labeled RNA's from photosynthetically or heterotrophically grown Rlrodo.spirillrrm rrrbrrr~~l. Biochim. Biophys. Acta, 161: 162-169. 28. YAMASHITA. J., and M. D. K A M E N .1969. Uracil incorporation and photopigment synthesis in Rhoclo.spirill~r~rr~.rrbrrrtt~. Biochim. Biophys. Acta, 182: 322-333. 29. Y U A N D., , and V. S H E N .1975. Stability of ribosomal and transfer ribonucleic acid in E.sclrerichicr coli B/r after treatment with ethylene-dinitrilotetraacetic acid and rifampin. J . Bacteriol. 122: 425-432.
Properties of ribonucleic acids from photosynthetic and heterotrophic R hodospirillum rubrum1
Accepted October 28. 1975
CHOW,C . T . 1976. Properties of ribonucleic acids from photosynthetic and heterotrophic Rlrodospirillirtn rirhrirtn. Can. J. Microbiol. 22: 228-236. Rifampicin causes an initial rapid breakdown of about 30% of the pulse-labeled RNA in both photosynthetic and heterotrophic Rlrodospirillrrtn rirhrirtn. An additional degradation of RNA has been observed in the heterotrophic, but not in the photosynthetic cells at a later stage of rifampicin treatment. This secondary RNA degradation is probably caused by breakdown of ribosomal RNA. especially of the 23s species, a s shown by polyacrylamide gel electrophoresis analyses. Noqualitative difference between the pulse-labeled RNA species inphotosynthetic and heterotrophic cells can be detected by the DNA-RNA hybridization competition technique. CHOW,C . T. 1976. Properties of ribonucleic acids from photosynthetic and heterotrophic Rlrodo.spirillrrt~trrtbr~on.Can. J . Microbiol. 22: 228-236. La rifampicine cause un bris initial rapide d'environ 30% de I'ARN marque par pulsation chez Rl~orlo.spirillrrt~ir~rhrrrttlphotosynthetique e t heterotrophe. Une degradation additionnelle de I'ARN a ete observee dans le systeme heterotrophe, mais non dans celui photosynthetique, chez des cellules h un stade ~ l l t e r i e ~de l r traitement B la rifampicine. Cette degradation secondaire de I'ARN est probablement causee par un brisde I'ARN ribosomal, specialement chez I'espece 23s. tel que dkmontre par analyses electrophoretiques sur gel de polyacrylamide. Aucune difference qualitative ne peut Otre detectee entre les especes d'ARN marque e n pulsation chez les cellules photosynthitiques et celles heterotrophes, par la technique d'hybridisation a competition de ADN-ARN. [Traduit par le journal]
Introduction Rhodospirilluin rubrum, a facultative photosynthetic bacterium, can grow either anaerobically in the light or aerobically in the dark. Under the former conditions organic compounds serve as hydrogen donors for photosynthesis, whereas they are used as substrates for oxidative phosphorylation under the latter conditions. Comparisons between photosynthetically and heterotrophically grown R. rubrum cells have been made in regard to their physiology, biochemistry, and ultrastructures by a large number of investigators (for review, see refs. 10, 13, 14, 19). It has been shown that the rate and extent of synthesis of many structural components, such as photosynthetic pigments and certain enzymes, change significantly during the transition from heterotrophic to photosynthetic growth (2,5, 14, 15). These changes were suspected to be caused by synthesis of some specific messenger ribonucleic acids (mRNA), because, according to the current models, protein synthesis in bacteria is believed to be regulated primarily at the level 'Received August 7, 1975.
of transcription. Investigations on R. rubrurn mRNA, in respect to its stability and qualitative difference between photosynthetic and heterotrophic cells, have been carried out by Yamashita and Kamen (27, 28). The stability of mRNA in photosynthetic, but not in heterotrophic R. rubrum has been examined by treating pulselabeled cells with proflavin, and about 65% of the RNA is found to be unstable. By using a DNA-RNA hybridization competition technique under saturated RNA conditions, these authors have concluded that the qualitative difference between the mRNA species of photosynthetic and heterotrophic R. rubrum cells is very small, if it exists a t all. The R N A examined in the studies cited above was extracted from cells labeled during a 3-min period, and its stability was examined by using a non-specific inhibitor of RNA synthesis (proflavin). Since the half-life of mRNA in many other bacteria, including Rhoclopseuclomonas spheroides (another member of the photosynthetic bacteria Athiorhodaceae) is less than 3 min (12, 21, 24), a relatively long labeling period may have obscured the results. In the present communication, a detailed
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examination on the stability and quality of R. Long-labeled cells were prepared by labeling with [3H]urirubrum labeled RNA was undertaken. Cells were dine (2 pCi/ml) for three to four generations, chased by uridine (50 pg/ml) for one generation and labeled either for 30 s or for several generations unlabeled treated with rifampicin (200 pg/ml). The time at which during photosynthetic or heterotrophic growth rifampicin was added was defined as the zero time. and during the transition between them. The Labeled cultures (0.5 ml) were removed in triplicate at stability of labeled RNA was measured by rifam- 5-min (for pulse-labeled cultures) or 10-min (for longpicin, a specific RNA synthesis inhibitor (7, 17, labeled cultures) intervals into 5 ml of ice cold 5% trichloroacetic acid (TCA) and left at O°C for a t least 30 23), and the qualitative and quantitative changes min. T h e TCA-insoluble material was collected by centriin various RNA species in rifampicin-treated, fugation and the alkaline-labile, the alkaline-resistant, long-labeled R. rubrum was examined by poly- and the total TCA-insoluble radio-activities were deteracrylamide gel electrophoresis. The pulse-labeled mined (24). RNA, either before or after fractionation in Pho/osyn/he/ic + He/ero/rophic Transi/ions Photosynthetic R. rirbriirn was labeled with [3H]uridine sucrose gradients, was used to carry out hybridization competition experiments under both (2 pCi/ml) for 90 min and then divided into two equal parts; one was left under the photosynthetic growth conDNA and RNA saturated conditions. ditions and the other was gassed with a mixture of 95% air-5% CO, for 10 min in the dark, wrapped in aluminum Materials and Methods foil, and incubated on a reciprocal shaker in a 28 "C inCul/ure of Bacteria Rhodospirill~imrubrum was obtained from Dr. D. N. Burton of this department. The organism was maintained as stab cultures in a medium containing in every litre 3.50 g of dl-malic acid, 4.0 g of I-glutamic acid, 0.8 g of sodium citrate, 0.12 g of KH2P0,, 0.18 g of K2HP0,, 0.2 g of MgS0,.7H20, 33.0 mg of CaCI2, 5.0 pg of biotin, 2.0 g of Difco yeast extract, and 15.0 g of Difco Bacteriological agar, and the pH of the medium was adjusted to 6.8 with saturated NaOH solution before autoclaving. Inoculated agar tubes were incubated at 28 "C in a Conviron model E7 incubator with maximal illumination until a heavy growth was obtained which usually took 2-3 days. The stab cultures were kept at 4 "C and subcultured every 4-6 weeks. T o obtain fresh liquid cultures, a loopful of the stab culture was used to inoculate a large test tube filled to the neck with the same medium wlthout agar, and incubated under the same conditions u n t ~ lthe cells had reached early stationary phase of growth This culture was used as the inoculum for growing large quantities of cells. For photosynthetic cultures, cells were cultivated in rubber-stoppered Erlenmeyer flasks equipped with gas inlet, gas outlet, and sampling tubes (3.8 litres of medium in 4-litre flasks). Immediately after inoculation (1% inoculum), the culture was aerated with a mixture of 95% N2-5% C 0 2 for 15 min and incubated in the illuminated, 28 "C Conviron incubator. Heterotrophic cultures were cultivated in aluminum-foil-wrapped, cotton-stoppered Erlenmeyer flasks (I litre of medium in 2-litre flasks) on a reciprocal shaker in a 28 "C incubation room. Bacterial growth was measured by following absorbance in a Klett photometer, using a Corning No. 66 filter. The doubling time has been found to be about 5.2 h under either the photosynthetic o r the heterotrophic conditions. Rqampicin Treattnerlt of Labeled Cells Rhodospirilliim riibrurtl cultures were grown in Klett flasks (30 ml of culture per flask) and bubbled with mixtures of 95% N2-5Z C 0 2 and 9 5 z air-5% C 0 2 t o achieve anaerobiosis and aerobiosis, respectively. For pulse-label experiments, cells were labeled for 30 s with [5-3H]uridine (27.8 Ci/mmol, 20 pCi/ml for photosynthetic and 30 pCi/ ml for heterotrophic cultures), and then treated with rifampicin (200 pg/ml) and unlabeled uridine (50 pglml).
cubation room. Complementary procedures were used for heterotrophic to photosynthetic transition.
Es/rac/ior~ond Pi~rifico/ionof Labeled R N A Labeled RNA was extracted and purified by the techniques described before (4). Purified RNA was dissolved in a small volume of an acetate buffer containing 20 miM sodium acetate, 20 m M KCI, and 10 m M MgCI,, pH 5.2. The concentration of R N A was determined from the ab= 25 and its TCAsorbance at 260 nm, taking El ,.,".'% insoluble radioactivity was measured. Siicrose Gr.odierr/ Froc/io11a/iortof Labeled R N A Labeled RNA was fractionated by laying 0.2ml of RNA on top of a linear sucrose gradient (4.7 ml, 15-30%) and centrifuging at 35 000 rpm for 15 h in a n SW50 rotor at 4 'C in a Beckman L2-65 ultracentrifuge. After centrid fourfugation, the bottom of the tube was p ~ ~ n c t u r eand drop fractions were collected in tubes containing 1 ml of 6 x SSC (I x SSC = 0.15 M NaCI + 0.01 5 M sodium citrate, pH 7.0). The absorbanceofeach fraction was measured at 260 nm in a Beckman model DU spectrophotometer. An aliquot of the sample was used for determination of TCA-insoluble radioactivity. Prepoi.a/iorr orlrl Derlo/irra/iorr of DNA The techniques previously dexribed (4) were used to purify R. riibrwir DNA, and the amount of DNA was determined by the diphenylamine method (3). Purified DNA samples wel-e denatured either by heating at 100°C for 15 min o r by treatment with 0.5 N NaOH for 60 min, depending on the final D N A concentration desired. Both procedures were found to be equally effective for denaturing D N A . DNA-RNA H)~bridizo/ior~ Corrrpe/i/iorr This was carried out either by the method of Nygaard and Hall (18), with D N A free in solution, o r by that of Gillespie and Spiegelman (11). with D N A affixed onto nitrocellulose membrane filters (Schleicher and Schnell, No. B-6). Each annealing mixture, with a final volume of 1 ml, contained 6 x SSC, 0.1% SDS, various amounts of [3H]labeled and unlabeled RNA and denatured DNA (free o r membrane-bound). After incubation at 66°C for 16 h, the hybridization mixtures were cooled rapidly in an ice-water bath. If the Nygaard and Hall method was used,
230
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the samples were first collected on 25-nim nitrocellulose membrane filters. The remaining steps were identical with both hybridization methods. The membrane filters were washed in 100 ml of 2 x SSC, digested in 1 ml of 2 x SSC containing preboiled ribonuclease (20 pg/ml) for 1 h at room temperature, washed again with 100 ml of 2 x SSC, and counted in 10 rnl of Aquasol. Electt~opl~oresis Labeled R N A was electrophoresed in a 2.4%-10% double polyacrylamide gel (20). Twenty microlitres of unlabeled E. coli and L-cell ribosomal R N A was added to each sample and coelectrophoresed as molecular weight markers. The gels were scanned at 260 nm in a JoyceLoebl UV Scanner to determine the position of the markers and cut into 50 slices with a Bio.Rad gel slicer. The gel slices were hydrolyzed in 0.1 ml of H,O, for 2 h at 80°C and counted in 10 ml of Aquasol. Cl~e~nicnls The following chemicals were used: crystalline pancreatic deoxyribonuclease and ribonuclease (Worthington Biochemical Corp.), 3H-labeled uridine and Aquasol (New England Nuclear Corp.), and rifampicin (Sigma Chemical Co.).
labeled R. rubrum was found t o be more than 99% alkaline-labile showing specific labeling of R N A under the experimental conditions. The R N A degradation patterns of rifampicin-treated cells are shown in Fig. 1. Both photosynthetic and heterotrophic cultures showed an initial rapid decrease in radioactivity during the first 10 min of rifampicin treatment, after which time the radioactivity remained a t a constant level in the photosynthetic culture whereas a secondary degradation was observed in the heterotrophic culture starting a t 20 min after addition of rifampicin.
Photosynthetic =$ Heterotrophic Transitions Synthesis of R N A in R. rubrum during transition was measured, and the results are shown in Fig. 2. After transition from the heterotrophic t o the photosynthetic condition, R N A synthesis continued a t a decreased rate for at least 5.5 h (Fig. 2a) whereas a sudden halt in net RNA syn-
Results Degradation of Pulse-labeled RNA The amount of [3H]uridine incorporated into the cold TCA-insoluble fraction of the pulse-
0
1
2
3
TIME TIME lmin/ FIG.1. Degradation of pulse-labeled R. rubrutn RNA. Photosynthetic (0) and heterotrophic (0) cultures were labeled with [3H]uridine for 30 s, and treated with rifampicin and unlabeled uridine as described in Materials and Methods. Samples were removed at 5-min intervals for determination of cold TCA-insoluble radioactivity.
4
5
6
7
l hours 1
FIG.2. R N A synthesis during transitions. Rllodospirillum rubr~tmcultures were labeled with [3H]uridine at a n early exponential phase of growth for 90 min. Transitions of growth conditions were made a t the time indicated (arrow). Samples were removed a t 30-min intervals for determination of cold TCA-insoluble radioactivity. ( a ) Heterotrophic t o photosynthetic transition, and (b) photosynthetic to heterotrophic transition. (0) photosynheterotrophic. thetic, (0)
23 1
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CHOW: RNA'S FROM R H O D O S P l R l L L U M RUBRUM
thesis was observed immediately after transition from the photosynthetic to the heterotrophic condition (Fig. 2b). N o net RNA synthesis could be detected for 2.5 h and after which time RNA synthesis resumed at a rate similar to that of the photosynthetic cultures. Degradation of pulse-labeled RNA in R. r~rbrumat 1, 2, and 3 h after transition was also determined. During the photosynthetic to heterotrophic transition, a gradual change in the RNA degradation pattern was observed (Fig. 3) and 3 h after transition the RNA degradation pattern was almost identical with that of the heterotrophic culture (compare Figs. 3 and I). When the reverse transition was carried out, a complementary phenomenon was observed. he above results have clearly indicated the presence of an additional unstable RNA fraction in the rifampicin-treated heterotrophic, but not photosynthetic R. rubrum. T o characterize this RNA fraction, a qualitative difference between pulse-labeled photosynthetic and heterotrophic RNA (P- and H-RNA) was measured by DNA-
RNA hybridization competition technique, and the R N A from rifampicin-treated, long-labeled R. rubrum was analyzed by polyacrylamide gel electrophoresis. Competition under Saturated DNA Conditions Hybridization competition was carried out by incubating a DNA-bound membrane (100 pg DNA per membrane) (4), 1 pg of pulse-labeled RNA, and up to 300 pg of unlabeled RNA in 1 ml of an annealing mixture (1 1). The results are shown in Fig. 4. Increasing amount of unlabeled RNA caused a rapid decrease in the amount of DNA-3H-RNA hybrid formed. The amount of hybrids formed was reduced to the same extent by the unlabeled P- or H-RNA in all
0
100
200
300
Unlabe/ed
FIG.3. Degradation of pulse-labeled R. rrrbrum RNA during photosynthetic to heterotrophic transition. Photo2 (A), synthetic cultures were labeled at 0 (V), 1 and 3 ( 0 ) h after transition to heterotrophic conditions, q.nd treated with rifampicin as described in Fig. 1. Samples were removed at 5-min intervals for determination of cold TCA-insoluble radioactivity.
(a),
0
RNA
100
200
300
f yg)
FIG. 4. Hybridization competition between labeled and unlabeled RNA on D N A sites under saturated DNA conditions. Purified R. rubrum labeled R N A (I pg) was hybridized with 100 pg of R. rubrum D N A affixed on a nitrocellulose membrane filter in the presence of indicated amount of unlabeled P- ( 0 ) or H- ( x ) RNA. Conditions for hybridization and determination of radioactivity were described in Materials and Methods. (a) Labeled PRNA P-DNA, ( b ) labeled P-RNA H-DNA, (c) labeled H-RNA + P-DNA, and ( d ) labeled H-RNA H-DNA.
+
+
+
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C A N . J . MICROBIOL. VOL. 22, 1976
Fraction
Number
FIG.5. Sedimentation of pulse-labeled R. t.l/br~rt~r R N A in sucrose gradients. Pulse-labeled RNA from photosynthetic and heterotrophic cells was centrifuged in sucrose gradients as described in Materadioactivity in P-RNA, (a) radioactivity in H - R N A . rials and Methods. ( x ) , 4 2 6 0 nm, (0)
combinations of D N A and R N A tested. Since a relatively small difference between the labeled P- and H-RNA might be masked when total labeled-RNA was used, the pulse-labeled R N A was fractionated in sucrose gradients. The sedimentation profiles of the P- and H-RNA are superimposed and shown in Fig. 5. Three ultraviolet-light absorbing peaks, corresponding to ribosomal and transfer RNA, were well separated under these conditions. The distribution of the radioactivity was very similar for the Pand the H - R N A ; both were heterogenous and exhibited a region of relatively high specific radioactivity with sedimentation constants between 6 and 12 S (fractions 25-39, Fig. 5). These fractions were pooled and used to perform hybridization colnpetition experiments. Results obtained by using these pooled R N A fractions were essentially identical with those described in Fig. 4, except that a higher concentration of unlabeled RNA was required to obtain the same reduction level. Competition under Saturated RNA Conditions m affixed Eight micrograms of R. r ~ ~ b r r i DNA on a nitrocellulose membrane filter was used to hybridize with 400 pg of unfractionated labeled RNA in the absence o r the presence of unlabeled
R N A (up to 6 mg). The competitive ability of the unlabeled P- and H-RNA was essentially identical (Fig. 6). Sucrose gradient fractionated RNA had also been used for this type of competition. Because only a limited amount of fractionated RNA was available, 25 pg of fractionated labeled RNA was used to hybridize with 0.5 pg of D N A in the presence of (up to) 500 p g of unlabeled R N A (18). The results (not shown) were essentially the same as that presented in Fig. 6. Degrad~tionof Long-labeled RNA About 90% of the radioactivity incorporated into the untreated long-labeled R. rubrutw was alkaline-labile (Fig. 7). After rifampicin treatment, the total TCA-insoluble counts as well as the ratio between the alkalin-labile and the alkaline-resistant fractions remained unchanged in the photosynthetic cells. I n contrast, a rapid breakdown of labeled RNA was observed in the heterotrophic culture, and about 30 and 4 0 z o f the RNA was degraded after 30 and 60 min, respectively (Fig. 7b). No breakdown of D N A was detectable. Labeled R N A was also analyzed by polyacrylamide gel electrophoresis. I n the untreated cultures, both photosynthetic a n d heterotrophic
CHOW: RNA'S FROM RHODOSPIRILLUM RUBRUM
233
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80
*
60
.P 40 2
::
.a 20
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3 loo
-
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C 0
3
9 80
-
P ae
60 I 0
40 20 0
2
4
6
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2
4
6
UNLABELED RNA /mg) FIG.6. Hybridization competition between labeled and unlabeled R N A on D N A sites under saturated RNA conditions. Purified R. rubrutn labeled R N A (400 pg) was hybridized with 8 pg of R . rubrum D N A affixed on a nitrocellulose membrane filter in the presence of indicated amount of unlabeled P- (0) o r H- (m) R N A . Conditions for hybridization and determination of radioactivity were described in Materials and Methods. (a) Labeled P-RNA P-DNA, ( b ) labeled P-RNA + H-DNA, (c) labeled H-RNA P-DNA, and ( d ) labeled H-RNA + H-DNA.
+
+
samples showed four distinct RNA species and their radioactivity content in the 23S, 16S, 5S, and 4 s species was about 57%, 28%, 3%, and 1277, respectively (Fig. ga). This distribution remained unchanged in the photosynthetic cells during the period tested. On the other hand, a rapid decrease in the 23s peak, accompanied by the appearance of a new peak (about 2 0 ~ 1 was , observed in the 30-min heterotrophic sample, and the percentage of radioactivity in the 23S, 20S, 16S, 5S, and 4 s species were found to be 14, 24, 42, 5, and 15, respectively (Fig. 86). At 60 min, a further reduction in the 23s peak accompanied by a prominent increase in the 20s peak occurred whereas no significant change was detectable in the remaining RNA species (Fig. 8 4 . Discussion As it is widely accepted, bacterial mRNA is
40
50
60 TIME
o
10
20 X)
40
50
60
(rn~n)
FIG.7. Degradation of long-labeled R. rrtbrum RNA. The cold TCA-insoluble radioactivity in 0.5 ml of longlabeled photosynthetic (a) and heterotrophic (b) R. rubrum was determined. (0) Total counts, (0) alkalinelabile counts, and ( A ) alkaline-resistant counts.
and to acid-so1ub1e nucleotides after antibiotic inhibition of RNA ( 9 , '2, 16, 21). It is, therefore, not 'nto assume that the RNA degraded during the first 10 min of rifampicin treatment is if not mRNA. most likely for the RNA degradation are (a) there are two distinct frattions of mRNA in the heterotrophic R. rubrum. One them (which is absent in the photosynthetic cells) has a longer half-life than the other, and (b) the RNA degraded is some RNA Other than mRNA, and its degradation is induced by rifampicin treatment. Our DNA-RNA hybridization competition results have clearly shown that there is no detectable qualitative dif-
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CAN. J .
MICROBIOL. VOL. 22, 1976
(b)
30 m i n
I
FIG.8. Electrophoretic analysis of long-labeled R. t.~~brli,n R N A . Labeled R N A extracted from R. rltbr~ituat 0 ( o ) , 30 (b),and 60 min ( c ) after rifampicin treatment was analyzed in a 2.4%-10z double polyacrylamide gel (20)with added ~lnlabeledE. coli and L-cell ribosomal R N A as molecular weight markers. T h e positions of the nlolecular weight markers were determined in a Joyce-Loebl U V scanner a n d the radioactivity in the gel slices was determined after HzOzdigestion. The broken line a t position 27 indicates the boundary of the 2.4% and the 10% gel. (8-a) P - R N A , (0---0) H-RNA.
ference between the untreated photosynthetic and heterotrophic pulse-labeled R N A (Figs. 4 and 6) and, therefore, render the first explanation less plausible. Consequently, the R N A degraded was assumed to be one or more species of the stable RNA, probably in its matured form because a 20-min lag exists between the time of rifampicin addition and the starting time of the secondary degradation (Fig. 1). It is known that R N A chain elongation and maturation can proceed normally in the presence of rifampicin (7, 17,23,24) and a recent report by Yuan and Shen
has shown that rifampicin causes degradation of matured E. coli23S and 16s r R N A in their corresponding ribosomal subunits (29). This conciusion that the R N A degraded during the secondary degradation is matured stable RNA is supported by the degradation and electrophoresis results observed in the rifampicin-treated, longlabeled, heterotrophic R. rubrurn (Figs. 7b and 8). It is, however, still unclear whether only the 23s or the 23s plus other stable R N A species is degraded. The 30-min heterotrophic RNA contained about 70% of the TCA-insoluble radio-
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CHOW: RNA'S FROM R
activity originally incorporated and 42% of the in R. rubrum and in Rhodopseudomonas spherlabel appeared in the 16s peak. Considering oides is at the level of translation. The RNA these two figures, the label in the 30-min 16s degradation and hybridization competition reRNA species would be 29.4% (42% x 70%) of sults presented in this report have further supthe original counts, a value very close to that in ported their conclusion. the untreated cells (28%). This finding may be Acknowledgments interpreted as n o degradation of the 16s RNA, although the possibility of a specific breakdown This investigation was supported by a grant of 23s to 16s accon~panying 16s degradation A6722 from the National Research Council of cannot be eliminated. The same argument can Canada. also be applied for the 5 s and the 4 s RNA I thank B. Rerie and H. S. Wang for technical species. The exact mechanism of the 23s RNA assistance. degradation is at present unknown. It seems I. ARONSON, A. 1. 1965. Characterization of messenger likely that both endonuclease (appearance of a RNA in sporulating Btr~.illrr.sc ~ r e r rJ.~ .Mol. Biol. 11: 20s peak) and exonuclease activities (reduction 576-588. in TCA-insoluble counts) are involved. The 2. BOTORAD, L. 1966. 117 Chlor.ophyll. Eclirc,tl I?! L . P. breakdown of rRNA may be a consequence of Vernon and G. R. Seely. Academic Press, Inc., New release of ribosomal proteins from matured York. ribosomal subunits by treatment of rifampicin, 3. BURTON,K. 1956. A study of the conditions and mechanism of the diphenylamine reaction for the colas shown by Yuan and Shen (29). The structural orimetric estimation of deoxyribonucleic acid. changes in rifampicin-treated ribosomal particles Biochem. J. 62: 315-323. and the fate of these ribosomes during transitions 4. CHOW, C . T.. and I. T A K A H A S H1972. I. Deoxyribonucleic acid of an asporogenous mutant of are currently being investigated by the doubleBocillri.~srrbrilis. Irl Spores V. Erlired Ay H. 0. Hallabeling technique. vorson. R. Hanson. and L. L. Campbell. American In conclusion, a difference in sensitivity to Society for Microbiology. Fontana, Wisconsin. pp. rifampicin between the photosynthetic and 133-139. R Eand , W. R. SISTROM. 1966. 111 heterotrophic R. rubrum rRNA has been de- 5. C O H E N - B A ~ ~G., Chlorophyll. Edirecll>y L. P. Vernon and G. R. Seely. tected. Whether this difference is related t o their Academic Pless, Inc.. New York. protein synthesis control mechanism is open to 6. COLD S P R I N HARBOR G SYMPOSIA ON QUANTITATIVE speculation. As mentioned earlier, the primary BIOLOGY, XXXV. 1970. Cold Spring Harbor Laboraregulation of protein synthesis in bacteria is betory, Cold Spring Harbor, New York. E., L. S N Y D E RP., MARINO, A. LAMPERT, lieved to be effected at the transcriptional level. 7. DIMAURO, A. COPPO,and G. P. T O C C H I N I - V A L E N T1969. INI. Synthesis of new mRNA species after a change Rifarnpicin sensitivity of the components of DNAin physiological conditions has been demondependent RNA polymerase. Nature (London), 222: strated in a number of microorganisms. For in533-537. stance, bacteriophage-infected cells synthesize 8. DOI, R. H., and T. J. LEIGHTON. 1972. Regulation during initiation and subsequent stages of bacterial mainly, or completely, phage-specific mRNA sporulation. Itt Spores V. Edirerl hy H. 0 . Halvorson, (6), and distinct mRNA species exist in cells at R. Hanson, and L. L. Campbell. American Society for different stages of cell differentiation (6, 22). Microbiology, Fontana, Wisconsin. pp. 225-232. Furthermore, sporulation-specific mRNA is 9. F A N ,D. P., A. HIGA,and C . LEVINTHAL. 1964. Messenger RNA decay and protection. J. Mol. Biol. 8: produced in Bacillus spp. shortly after the initia2 10-222. tion of sporulation process (1, 8, 26). In the H., A. SANPIETRO,and L. P. VERNON.1963. photosynthetic bacteria, at least in the case of R. 10. GEST, Bacterial photosynthesis. The Antioch Press, Yellow r~rblwt?~ and Rhodopseudornonas spheroides, no Springs, Ohio. D., and S . SPIEGELMAN. 1965. A quantitadetectable qualitative difference in mRNA can l I. GILLESPIE, tive assay for DNA-RNA hybrids with DNA imthus far be demonstrated in cells grown under mobilized on a membrane. J. Mol. Biol. 12: 829-842. completely different physiological conditions, 12. G 1970. The R A YW. , J. H., and J . E. M. MIDGLEY. although many structural changes have been obcontrol of ribonucleic acid synthesis in bacteria. served. Because of the lack of a qualitative difSteady state content of messenger ribonucleic acid in Esc11~1.ic~hicr coli M.R.E. 600. Biochern. J. 120: ference in mRNA between photosynthetic and heterotrophic cultures, Yamashita and Kamen 13. 279-288. KONDRAT'EVA. E. N. 1963. Photosynthetic bacteria. (27, 28) and Witkin and Gibson (24, 25) have Israel Program for Scientific Translations, Jerusalem, suggested that the regulation of protein synthesis Israel.
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14. LASCELLES, J. 1973. Microbial photosynthesis. Dowden, Hutchingson and Ross. Inc., Stroudsburg, Pennsylvani:~. 15. LASCELLES, J. 1964. Tetrapyrrole biosynthesisand its regulation. Benjamin Company Inc., New York. 16. L E V I N T H A L C., , D. P. F A N , A. H I G A ,and R. A. Z I M M E R M A N1963. N . The decay and protection of messenger RNA in bacteria. Cold Spring Harbor Symp. Q~rant.Biol. 28: 183-190. , N I H A . and H. 17. M I Z U N O ,S., H. Y A M A Z A K IK. U M A / A W A1968. . Inhibition of DNA-dependent RNA polymerase reaction of E.sc~lrc~ricllicr c,oli by an antibiotic, streptoval-icin. Biochim. Biophys. Acta, 157: 322-332. 18. NYGAARD, A,. and B. HALL.1963. A method for the detection of RNA-DNA complexes. Biochem. Biophys. Res. Commun. 12: 98-104. 19. PARSON,W. W. 1974. Bacterial photosynthesis. Annu. Rev. Microbial. 28: 41-59. 20. PEACOCK, A. C., and C . W. D I N G M A N1967. . Resolution of multiple ribonucleic acid species by polyacrylamide gel electrophoresis. Biochemistry, 6 : 1818-1827. 21. SALSER,W . , J . J A N I N and , C . L E V I N T H A L1968. . Measurement of the unstable RNA in exponentially growing cultures of Bncillrrs sr~htilisand E.schericl7irr coli. J . Mol. Biol. 31: 237-266. 22. SUSSMAN, M. 1971. Molecular genetics and developmental biology. Prentice-Hall, Inc., Englewood Cliffs, New Jersey.
23. W E H R LW., ~ , and M. STAEHELIN. 1971. Actionsofthe rifamycins. Bacteriol. Rev. 35: 290-309. 24. WITKIN,S. S . , and K. D. GIBSON. 1972. Changes in ribonucleic acid turnover during aerobic and anaerobic growth in Rlloclopserrcio~not~c~s splleroides. J. Bacteriol. 110: 677-683. 25. W I T K I NS. , S., and K. D. GIBSON.1972. Ribonucleic acid from aerobically and anaerobically grown Rlrorlopse~rciornonnsspheroides: Comparison by hybridization to chromosomal and satellite deoxyribonucleic acid. J . Bacteriol. 110: 684-690. 26. Y A M A G I S HH., I , and 1. T A K A H A S H1968. I. Genetic transcription in asporogenous mutants of Bncillrrs sirbrilis. Biochim. Biophys. Acta, 155: 150-158. 27. YAMASHITA, J . , and M. D. K A M E N .1968. Observations on the nature of pulse-labeled RNA's from photosynthetically or heterotrophically grown Rlrodo.spirillrrm rrrbrrr~~l. Biochim. Biophys. Acta, 161: 162-169. 28. YAMASHITA. J., and M. D. K A M E N .1969. Uracil incorporation and photopigment synthesis in Rhoclo.spirill~r~rr~.rrbrrrtt~. Biochim. Biophys. Acta, 182: 322-333. 29. Y U A N D., , and V. S H E N .1975. Stability of ribosomal and transfer ribonucleic acid in E.sclrerichicr coli B/r after treatment with ethylene-dinitrilotetraacetic acid and rifampin. J . Bacteriol. 122: 425-432.
