Regulation of Ribulose Bisphosphate Carboxylase Expression in ...

Vol. 170, No. 9

JOURNAL OF BACTERIOLOGY, Sept. 1988, p. 4065-4071 0021-9193/88/094065-07$02.00/0 Copyright © 1988, American Society for Microbiology

Regulation of Ribulose Bisphosphate Carboxylase Expression in Rhodospirillum rubrum: Characteristics of mRNA Synthesized In Vivo and In Vitro THOMAS LEUSTEK, ROMANA HARTWIG, HERBERT WEISSBACH, AND NATHAN BROT* Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110 Received 17 February 1988/Accepted 8 June 1988 The synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in Rhodospirillum rubrum was regulated by the CO2 concentration in the culture medium. The specific activity of RuBPCase in cells grown photolithotrophically in low concentrations of CO2 (1.5%) was five to ten times higher than that in cultures grown at high concentrations of CO2 (10%). Increased enzyme activity was reflected by an increase in both RuBPCase mRNA and RuBPCase protein. RuBPCase expression was also studied in vitro with a plasmid-borne genomic clone (pRR117) as the template in a partially defined Escherichia coli system containing either E. coli or R. rubrum RNA polymerase. With both enzymes there was excellent synthesis of RuBPCase mRNA, but no signfficant synthesis of RuBPCase was detected. The promoter region of the RuBPCase gene was sequenced, and mRNA start sites were mapped. A single major in vivo transcriptional start site was detected in RuBPCase mRNA extracted from R. rubrum. However, transcripts synthesized from pRR117 in vitro or from E. coli transformed with pRR117 started at upstream sites that were different from the in vivo transcription site. Two major features of the RuBPCase promoter region are three 6-base-pair direct repeats and a 31-base-pair region of dyad symmetry. The enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) is widespread among photosynthetic organisms. It catalyzes the first reaction in the assimilation of carbon dioxide. When the photosynthetic bacterium Rhodospirillum rubrum is grown under photolithotrophic conditions, RuBPCase expression is regulated by the CO2 concentration in the medium (20). Thus, when grown at high CO2 concentrations (6 to 10%), low levels of RuBPCase are present, whereas at low concentrations of CO2 (1 to 2%), there is high expression of RuBPCase, presumably owing to carbon limitation. In fact, under the latter conditions, RuBP Case can account for as much as 50% of the cellular soluble protein (20). Synthesis of large amounts of RuBPCase during carbon dioxide limitation is not unique to R. rubrum. In the closely related photosynthetic bacterium Rhodobacter sphaeroides, RuBPCase expression is also similarly controlled by CO2 concentration when the bacterium is grown under photolithotrophic conditions (9). However, the mechanism by which enzyme synthesis is regulated by CO2 concentration in these organisms is unknown. In the present study, we investigated the expression of the RuBPCase from R. rubrum in vivo and in vitro.

0.4% (wt/vol) malic acid as the carbon source (19). Photolithotrophic growth was initiated by transferring 10 ml of a stationary-phase malate-grown culture into 1 liter of the same medium without malic acid. These cultures were bubbled with a gas mixture of either 1.5% C02-98.5% H2 or 10% C02-90% H2 (20). Photoheterotrophic and photolithotrophic growths were carried out at 28°C under illumination by four 150-W incandescent plant lights positioned 1 m from the culture flasks. E. coli GM119 was used to express pRR117, and E. coli RR1 was used to propagate pRR117. Both strains were grown aerobically in Luria broth (supplemented with 50 jig of ampicillin per ml) at 37°C. Preparation of RNA polymerase. E. coli RNA polymerase was purified by the method of Burgess and Jendrisak (4). R. rubrum RNA polymerase was purified from mid-log-phase cells grown on malic acid. Frozen cells (150 g) were thawed in 400 ml of buffer containing 50 mM Tris hydrochloride (pH 7.5), 10 mM MgCl2, 200 mM KCI, 0.1 mM dithiothreitol, 0.1 mM EDTA, and 5% (vol/vol) glycerol, and the cells were lysed by two passages through a French pressure cell at 16,000 lb/in2. The lysate was blended at low speed in a Waring blender for 60 s to shear DNA, and then 5 mg of DNase (dissolved in the lysate buffer) was added. The lysate was incubated for 30 min at 4°C and then centrifuged at 20,000 x g for 30 min. The supernatant was then centrifuged at 200,000 x g for 2 h. This supematant was fractionated by ammonium sulfate precipitation followed by DEAE-cellulose chromatography (3). RNA polymerase was then purified from pooled DEAE fractions by DNA-cellulose affinity chromatography (4). The subunit profile of R. rubrum RNA polymerase determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was identical to that reported previously (24). Preparation of cell extracts and RuBPCase assay. R. rubrum cells were disrupted by sonication in a buffer containing 10 mM Tris acetate (pH 8.0), 14 mM magnesium acetate,

MATERIALS AND METHODS Plasmid, bacterial strains, and growth conditions. The gene for RuBPCase from R. rubrum (rbcR) was obtained from C. Somerville (Michigan State University) as a 6.6-kilobasepair EcoRI genomic fragment cloned into pBR325 (21). The 6.6-kilobase-pair fragment was subcloned into the EcoRI site of pBR322 (pRR117) and used to transform Escherichia coli GM119 and RR1. A partial restriction map of pRR117 is shown in Fig. 1. R. rubrum S1 was obtained from the American Type Culture Collection (Rockville, Md.). Stock cultures were maintained photoheterotrophically on medium containing *

Corresponding author. 4065

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FIG. 1. Restriction map of pRR117. The approximate positions of genes and restriction sites are indicated. Known genes are indicated by bold lines, and the direction of RuBPCase transcription is indicated by the arrow. The 6.6-kb R. rubrum fragment carrying the rbcR gene was cloned into the EcoRI site of pBR322.

60 mM potassium acetate, and 1 mM dithiothreitol. The lysate was centrifuged for 10 min in a microcentrifuge at 4°C, and the supernatant was used for measurement of RuBPCase activity (8). Protein concentration was determined by the method of Bradford (2) with bovine serum albumin as the standard. In vitro gene expression system. The preparation and characteristics of the partially defined in vitro E. coli protein synthesis system have been described previously (10, 25). The standard in vitro system was modified by the addition of 30 to 50 ,g of protein from a 0.25 M DEAE salt eluate (11) which was used as the source of aminoacyl-tRNA synthetases. The [35S]methionine-labeled polypeptides synthesized with pRR117 as the template were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For RNA synthesis, the [35S]methionine was omitted and after incubation for various times, 3 U of DNase (RQ1; Promega Biotec, Madison, Wis.) were added to each 35-1.d reaction mixture followed by incubation at 37°C for 30 min to digest the pRR117 template. RNA isolation and blot analysis. Samples (50 ml) of growing cultures of R. rubrum or E. coli were added to an equal volume of ice slurry containing 80 mM Tris chloride (pH 7.5), 10 mM MgCl2, 10 mM P-mercaptoethanol, 25 mM NaN3, and 200 ,g of chloramphenicol per ml, and the cells were pelleted by centrifugation. Total RNA was isolated from the cells by lysis in sodium dodecyl sulfate, extraction with phenol-chloroform, and centrifugation through CsCl (13). The RNA was then treated with 30 U of DNase (RQ1), extracted with phenol-chloroform, precipitated with ethanol, and stored at -70°C prior to use. In vitro-synthesized RNA was isolated by extracting the DNase-digested incubation mixtures (see above) twice with an equal volume of phenolchloroform (1:1) and recovering the aqueous phase. For dot blots, RNA was first denatured by heating to 65°C for 15 min in 5 M formaldehyde containing 1.5 M each sodium chloride and sodium citrate. The sample was then applied to nitrocellulose (0.4-,u.m pore size) and baked for 2 h at 80°C under reduced pressure (23). For Northern blot (RNA blot) analy-

J. BACTERIOL.

sis, RNA from R. rubrum was electrophoresed in a 1.2% agarose-2.2 M formaldehyde gel (12), blotted onto nitrocellulose (0.4-p,m pore size) (22), and baked as described above. All RNA blots were prehybridized, hybridized, washed, and exposed to X-ray film as described previously (27). The levels of in vivo-synthesized RuBPCase mRNA were quantitated by scanning the dot blots with an Ultroscan laser densitometer (LKB Instruments, Inc., Rockville, Md.) and integrating the area under the curve. The DNA probes used for RNA blot analysis included an 845-base-pair (bp) ApaI fragment from the 3' end of the rbcR gene or a 692-bp DraI fragment from the 3' end of the P-lactamase gene (Fig. 1). These fragments were isolated from restriction digests of pRR117 or pBR322 after electrophoresis in a low-melting-temperature agarose gel (15). The DNA probes were labeled with [a-32P]dGTP and [a32P]dCTP by nick translation (nick translation kit; Bethesda Research Laboratories, Inc., Gaithersburg, Md.) according to the instructions of the manufacturer. The probes were purified by the spun column technique (15). Si protection assay. The DNA probe used for Si analysis of RuBPCase mRNA was an AvaII fragment extending from the rbcR translation start site to 190 bp upstream (Fig. 1). This fragment was isolated from pRR117 by restriction digestion and electrophoresis in a polyacrylamide gel (15). The fragment was dephosphorylated with calf intestinal phosphatase and end labeled with [,y-32P]ATP by using polynucleotide kinase. To specifically detect transcription occurring in the opposite direction from RuBPCase transcription, we cut the end-labeled AvaII probe with restriction enzyme Hinfl which removes the 32p labeled proximal to the rbcR (Fig. 1), so that RuBPCase transcripts will not be detected. RNA (20 ,ug) was heated to 75°C for 5 min with 20 to 80 fmol of labeled DNA probe and then allowed to hybridize at 49°C overnight. Si nuclease (90 U) was added, and digestion was allowed to proceed for 1 h at 37°C (1). Protected DNA fragments were denatured and electrophoretically fractionated on an 8% polyacrylamide-8 M urea sequencing gel (16). Sequencing of rbcR promoter region. The rbcR promoter region was sequenced with the M13 sequencing kit (Amersham Corp., Arlington Heights, Ill.) according to the instructions of the manufacturer. Two 24-base oligonucleotides used as sequencing primers were synthesized homologous to the known gene sequence located, respectively, 50 bp 3' and 136 bp 5' from the translational start site. RESULTS RuBPCase enzyme activity and mRNA levels in R. rubrum. The derepression of RuBPCase synthesis in R. rubrum grown photolithotrophically in low concentrations of CO2 has been reported previously (20) and verified in the present studies. R. rubrum cultures grown photoheterotrophically were inoculated into photolithotrophic medium and gassed with either 1.5% CO2 or 10% Co2. Fully derepressed R. rubrum cultures grown for 6 days on a photolithotrophic medium with 1.5% CO2 consistently showed RuBPCase enzyme activities 5- to 10-fold higher than those of repressed cultures grown on 10% CO2 (Table 1). It should be noted that no further increase in RuBPCase activity was observed beyond 6 days after subculture in photolithotrophic medium. The increase in the specific activity of the enzyme correlated well with increased amounts of RuBPCase protein as shown by immunoblot analysis (data not shown). The increase in RuBPCase at low CO2 concentration could be due to either transcriptional or translational regulation.

RIBULOSE BISPHOSPHATE CARBOXYLASE IN R. RUBRUM

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TABLE 1. Comparison of RuBPCase enzyme activity and mRNA levels in cultures grown in 1.5% and 10% CO2a RuBPCase activity and mRNA levelsb Culture

Malate 1.5% CO2

10% CO2

Days in culture

0 2 4 6 2 4 6

Expt 3

Expt 2

Expt 1

RuBPCase

mRNA

RuBPCase

mRNA

RuBPCase

mRNA

30 180 425 940 120 59 105

1 1.7 6.9 5.2 2.3 3.8 2.1

172 639 1,336 209 104 64

1 1.8 8.8 5.5 2.2 2.2 2.1

70 170 640 1,120 210 110 120

1 1.5 6.3 4.6 1.5 1.3 1.5

a Stationary-phase malate-grown cultures were inoculated into medium lacking malate and grown in the presence of either 1.5% or 1O0% C02. Samples were removed as indicated for measurement of enzyme activity or RNA levels. RNA (10 Fg) was blotted and probed with a 32P-labeled 845-bp ApaI fragment (Fig. 1). Blots were then subjected to autoradiography, and the levels of RuBPCase mRNA were quantitated by densitometry (see Materials and Methods). b mRNA values are presented relative to the level found in the malate-grown culture. RuBPCase activity is expressed as nanomoles of CO2 fixed per minute per milligram of protein.

To determine this, we determined the level of RuBPCase mRNA in cells after 2, 4, and 6 days of growth from repressed (10% CO2) and derepressed (1.5% C02) cultures. The RNA was subjected to dot-blot analysis with a 32p_ labeled DNA probe homologous to the 3' end of rbcR (see Materials and Methods). Table 1 also shows that in cells from 1.5% CO2 cultures, the level of RuBPCase mRNA paralleled the increase in enzymatic activity for the first 4 days and was approximately four- to fivefold higher than in cultures grown on 10% CO2. Although the level of RuBP Case mRNA decreased slightly from day 4 to day 6 in 1.5% CO2 cultures, the RuBPCase specific activity continued to increase. These results suggest that the synthesis of this enzyme is regulated at the level of translation as well as

transcription. Northern blot analysis verified that there was more RuBP Case mRNA in 1.5% C02-grown cultures than in 10%O CO2or malate-grown cultures and that under all culture conditions the size of the RuBPCase RNA is approximately 1.4 kilobases (kb) (data not shown). This value is in agreement with the size of the rbcR gene from R. rubrum (18). In vitro expression of rbcR gene. A partially defined in vitro system was used to express the rbcR gene with pRR117 as the template (10, 25). The advantage of this system is that it depends on added RNA polymerase and therefore both the E. coli and R. rubrum enzymes could be tested. Identical patterns of 35S-labeled in vitro proteins were obtained when either E. coli or R. rubrum RNA polymerase was added to the in vitro incubations (Fig. 2). The polypeptides synthesized include a band in the 55-kilodalton (kDa) region where RuBPCase migrates as well as a number of other minor bands, both higher and lower in molecular weight. In addition, there is a major product migrating at about 32 kDa corresponding to P-lactamase. Despite the presence of a protein at 55 kDa, none of the higher-molecular-weight polypeptides showed cross-reactivity with antibodies against purified RuBPCase (data not shown). In addition, we were not able to detect RuBPCase activity in the in vitro incubations. From the specific activity of purified RuBPCase, it was calculated that as little as 10 fmol of active enzyme, if synthesized, could have been measured. These results suggest that significant levels of the protein were not made in this coupled in vitro system. To understand whether the lack of synthesis of RuBPCase in vitro was at the level of transcription or translation, we measured the level of RuBPCase mRNA in these in vitro incubations using pRR117 as the template. Samples of the

incubations were subjected to dot-blot analysis with 32Plabeled DNA probes homologous to RuBPCase mRNA or, as an internal control, 1-lactamase mRNA (see Materials and Methods). The dot blots are shown in Fig. 3A. Quantitation of the results (Fig. 3B) showed a time-dependent increase in the levels of both RuBPCase and ,B-lactamase mRNA during the 40-min incubation. Normalizing for the difference in the specific activity of each DNA probe, it appears that the amount of RuBPCase mRNA is about 25% of the amount of 3-lactamase mRNA. Since p-lactamase is a major product synthesized in vitro, these results indicate that significant levels of RuBPCase mRNA are synthesized in these incubations despite the lack of detectable RuBP Case. Northern analysis of the in vitro-synthesized RNA from pRR117 showed that the RuBPCase mRNA was at least Kd 69

55 46

30

RR

EC

FIG. 2. In vitro-synthesized polypeptides directed by pRR117 with either R. rubrum or E. coli RNA polymerase. Samples (5 ,ul) from each in vitro reaction mixture (35 ,ul) were precipitated with 10% C13CCOOH, washed with 80%o acetone, and electrophoresed on a 10% polyacrylamide gel containing SDS. The incubations contained, in addition to other components required for protein synthesis, either R. rubrum RNA polymerase (RR) or E. coli RNA polymerase (EC). The migration positions of molecular size markers are indicated on the left. Purified RuBPCase from R. rubrum migrates at 55 kDa. Kd, Kilodaltons.

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