FEMS Microbiology Letters 49 (1988) 267-271 267 Published by Elsevier

FEM 03074

Two different genes and gene products for the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase

(RuBisCOase) in Nitrobacter hamburgensis

Stephanie Harr i s a, Axel Ebe r t b, Eva Schfitze b, M a r e n Diercks b, E. Bock b and J.M. Shively a

a Department of Biological Sciences, Clemson University, Clemson, SC 29634-1903, U.S.A. and ~' A bteilung Mikrobiologie, lnstitut ,ear A llgemeine Botanik, D-2000 Hamburg 52, 1~ R. G.

Received 7 September 1987 Accepted 10 September 1987

Key words: Gene; Gene product; Ribulose-l,5-bisphosphate carboxylase/oxygenase; Nitrobacter hamburgensis; Anacystis nidulans

1. SUMMARY

Heterologous DNA hybridization using a ribu° lose- l ,5-b isphosphate ca rboxy la se /oxygenase (RuBisCOase) large subunit gene (rbcL) probe from Anacystis nidulans revealed the presence of two rbcL in Nitrobacter hamburgensis. One gene is located on a plasmid, the other on the chro- mosome. The genes appear to be very similar since both hybridized strongly to the A. nidulans probe. However, restriction endonuclease digestions re- vealed differences.

Two different RuBisCOase enzymes were iso- lated from N. hamburgensis. The Mr of the native enzymes were 520000 and 480000. Sodium dode- cyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed the presence of both LSU and small subunits (SSU) for both enzymes. The M r were 53 000 and 16 000, and 49 000 and 13 500,

Correspondence to: J.M. Shively, Department of Biological Sciences, Clemson University, Clemson, SC 29634-1903, U.S.A.

respectively. A hexadecameric structure is sug- gested for both enzymes.

2. I N T R O D U C T I O N

Ribulose-l ,5-bisphosphate ca rboxylase /oxy- genase (RuBisCOase) has been isolated and characterized from numerous bacteria [1,2]. In nearly all of the bacteria examined there is only a single type of RuBisCOase. However, Rhodobacter sphaeroides and Rhodobacter capsulatus produce two different enzymes [3-6]. Form I RuBisCOase in these bacteria is a hexadecamer of 8LSU and 8SSU and is very similar to the enzyme of most bacteria, cyanobacteria, green algae, and higher plants [1,2]. Form II RuBisCOase is a hexamer of only LSU and is closely related to the dimeric enzyme of Rhodospirillum rubrum [7-11]. The LSU of these two forms are encoded by two different rbcL [7-12]. Preliminary evidence suggests that in R. capsulatus both genes are located on the chro- mosome (JMS, unpublished data). There are also two rbcL in Alcaligenes entrophus [13,14]. In this

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organism one gene is located on a plasmid and the other on the chromosome. The restriction patterns of the two genes are totally different, but hybridi- zation experiments indicate a high degree of se- quence homology [13]. Isolation and characteriza- tion studies imply that both enzymes are of the hexadecameric type, i.e. no other structure was identified [15,16]. Purohit and McFadden [16] re- ported two types of LSU in their purified hexadecameric enzyme preparation. The chro- mosomal rbcL and small subunit gene (rbcS) as well as the protein products have been char- acterized, but examination of the plasmid encoded system has not been reported [14].

Herein we describe the presence of two differ- ent rbcL as well as the isolation of two different RuBisCOase enzymes in Nitrobacter hamburgen- sis.

3. MATERIALS AND METHODS

3.1. Cultivation N. hamburgensis strain X14 (culture collection

of the Botanical Institute, Hamburg) was grown mixotrophically in 18-1 batch cultures with aera- tion at 30°C as previously described [17]. Cells were harvested when the nitrite was consumed.

3.2. DNA isolation and analysis Total DNA and plasmid DNA were isolated

from 1 g and 2 g (wet weight) of cells, respectively. Plasmid DNA was isolated by the method of Hansen and Olsen [181 as modified by Kraft and Bock [19]. Total DNA was isolated by the method of Marmur [20] as modified by Koops and Harms [21]. Cell lysis was described by Kraft and Bock [19]. Restriction endonuclease digestion, electro- phoresis, Southern blotting, probe preparation, and hybridization were accomplished as previ- ously described [11]. The heterologous probe used in these studies was a 1.5-kbp fragment from pANP1155. This fragment represents nearly the complete rbcL from A. nidulans [22,23].

3.3. RuBisCoase purification All steps were performed at 4 ° C. Cells (2 g wet

weight) were resuspended in 10 ml of the follow- ing buffer: Tris-HC1, 10 mM; MgC12, 25 mM; N a H C O 3, 10 m M ; D T E , 1 m M , /3-

mercaptoethanol, 5 mM; pH 8.0. The cells were passed twice through a chilled french pressure cell (Aminco) at 20000 psi. The pressate was centri- fuged for 1 h at 50000 × g. Ammonium sulfate was added to the supernatant to 50% saturation. The precipitate was removed by centrifugation at 25 000 × g for 20 rain, resuspended in 3.0 ml of buffer, and applied to 6 linear 0.2-0.8 M sucrose gradients. After centrifugation at 27000 rpm for 20 h in a SW27 rotor (Beckman) the gradients were fractionated using a ISCO Model 185 gradi- ent fractionator. The RuBisCOase-active fractions were pooled and the enzyme concentrated by centrifugation in a Ti75 rotor (Beckman) at 300000 × g for 3 h. The resulting preparation (about 1 ml) was subjected to (PAGE) in a 4--12% acrylamide linear gradient slab gel using a GE4 apparatus (Pharmacia) at 120 V for 20 h. The gels were scanned (ISCO gel scanner) at 280 nm, the bands removed, and the proteins eluted electro- phoretically. The isolated enzymes were dialyzed against buffer for 12 16 h.

Protein was determined by the method of Lowry et al. [24] with bovine serum albumin as the stan- dard. Enzyme assays were performed as previ- ously reported [25]. Radioactivity was measured in a Beckman scintillation counter using Insta Gel (Packard). All values were corrected for back- ground and quenching. Specific activities were calculated as #mol CO 2 f ixed/ ra in / rag protein. The RuBisCoase activity was determined in poly- acrylamide gels by homogenizing 2-ram slices (about 50/~1 volume) and assaying as above.

3.4. RuBisCOase characterization Both enzyme preparations were negatively

stained with uranyl acetate and examined in a Philips 201 electron microscope.

The molecular weight of the isolated enzymes was estimated using the same gradient gel em- ployed for purification. Reference proteins were: thyroglobin, 669000 MW; horse spleen ferritin, 440 000; and catalase, 232 000.

The subunit composition of the proteins and the molecular weight of the polypeptides were determined by S D S - P A G E according to the method of Laemmli [26]. The low-molecular weight kit (Pharmacia) provided reference proteins.

4. RESULTS

The A. nidulans rbcL probe gave positive heter- ologous hybridization with 3 fragments of EcoRI, SalI, and BamHI digested total (chromosomal plus plasmid) N. hamburgensis DNA (Fig. 1). Purified plasmid DNA showed only two positive fragments in each endonuclease digest. These two fragments (in each digest) were identical with two of the positive fragments in the total DNA. Each panel (A, B, C) in Fig. 1 came from the same agarose gel /Southern blot/hybridization. The lanes were reorganized to provide easy compari- son of total and plasmid DNA. These data suggest that N. hamburgensis possesses two rbcL: one on a plasmid, the other on the chromosome.

PAGE (gradient gel) of the pooled RuBisCOase fractions from the sucrose gradients revealed 3

269

major bands (Fig. 2). Band a and b showed RuBisCOase activity and were designated Forms Ia and Ib, respectively. Band c, possessing a red- brown color and no RuBisCOase activity has been identified as ferritin [27]. The specific activity for purified forms Ia and Ib were 0.18 and 0.46, respectively. These values are quite low for puri- fied RuBisCOase; the PAGE purification step re- sulted in considerable inactivation. Electron mi- croscopy revealed the presence of 10-nM particles with the typical ringshaped structure and dense core in both purified preparations. The M r of forms Ia and Ib were estimated to be 520000 and 480000, respectively. The gel was nearly identical to Fig. 2 (without ferritin and other minor con- taminants) and is consequently not shown.

S D S - P A G E demonstrated that both form Ia and Ib possess LSU and SSU (Fig. 3). However,

A B C

Fig. 1. Hybridization of the A. nidulans LSU probe to EcoRI (Panel A), Sal l (Panel B), and BamH1 (Panel C) digested DNA. Lanes 1 and 4 under each panel ethidium bromide stained agarose gels of total and plasmid D N A , respectively. Lanes 2 and 3 autoradiograms of Southern blots of total and plasmid DNA, respectively. Far left lane Hind l l I digest of bacteriophage DNA.

270

a b 280 ; C

10 ~ !

/ I I0i E~ . . . . . . I ]

L~ % Acrylemi de 12

Fig. 2. Polyaemylamide gel electrophoresis (4-]2% acrylamide gradient) of RuBisCOase fractions from sucrose gradients. Solid Fine is absorption profile at 280 nM. Interrupted line is RuBisCOase activity. (a) form Ia RuBisCOase; (b) form Ib RuBisCOase; (c) Ferritin.

the M r of both the LSU and SSU differed. Form Ia dissociated into two major polypeptides with M r of 53000 and 16000; form Ib into 49000 and 13500. The native and subunit M r suggests a hexadecameric structure for both enzymes. Two minor polypeptides ( M, 40 000-45 000) were noted in both preparations. In addition, some hetero- geneity of the LSU could be observed in both forms.

a b c d

53000 . i L - . .

zoOo- . . . . . . . . . . .

iilfii i~

1 6 0 0 0 - I 1 3 5 0 0 - ~

--67000

-. 3000

, - , . 1 - 1 4 4 0 0

Fig. 3. Sodium dodecyl sulfate polyacrylamide gel electro- phoresis (12.5% acrylamide) of purified RuBisCOases. (a) RuBisCOase fractions from sucrose gradient; (b) form Ia; (c) form lb; (d) molecular weight standards.

5. DISCUSSION

The enzymological and D N A hybridization data are entirely consistent, i.e. two different rbcL and two different RuBisCOase LSU are present in N. hamburgensis. The rbcL and consequently the gene products should be similar since both genes hy- bridized to the rbcL probe from A nidulans. How- ever, the two must be somewhat different; the plasmid gene has an obvious internal restriction site for EcoRI, SalI and BamHl, and the chro- mosomal gene does not. It should be noted that the chromosomal gene might also have internal EcoRI, SalI, and BamHI restriction site(s), but they were either not detectable under our experi- mental conditions or one of the chromosomal fragments in each digest is identical to one of the two positive plasmid fragments. Since the LSU proteins are similar the latter case appears possi- ble. Furthermore, one could propose the existence of two different plasmid genes; the restriction fragments are both large enough to code for a rbcL. However, this seems unlikely. We also can- not eliminate the existence of an rbcL gene in the chromosome identical to the plasmid rbcL. We have been unable to purify chromosomal DNA totally free of plasmid DNA.

Hybridization to the rbcL probe from A. nidu- lans suggests, but does not prove a LSU-SSU enzyme structure. Failure of a rbcL probe from R. rubrum, which possesses a dimeric enzyme, to hybridize to any fragments of N. hamburgensis D N A also supports an LSU-SSU structure [11].

The protein data also indicates that the en- zymes are similar; the M r of the native enzymes are close and both possess LSU and SSU. Fur- thermore, as suggested by the hybridization re- sults, the M, of the native enzymes and of their constituent polypeptides infers a hexadecameric structure for both. In addition, cyanogen bromide cleavage, isoelectric focusing, and proteolysis pro- tection experiments provided data (not shown) which are also consistent with the presence of two different LSU.

Thus, the genetic organization of rbcL as well as the LSU in N. hamburgensis appear to be similar to that found in A. eutrophus, not Rhodo- bacter sp. (see Section 2).

Harrison et al. [28] reported the presence of two types of LSU for RuBisCoase of Nitrobacter agilis. They proposed a (LSU; LSU')8 SSUs struc- ture. This is similar to the structure proposed for the A. eutrophus enzyme by Purohit and McFad- den [16]. In both instances this might be the result of proteolysis, but the two LSU could be products of two different rbcL.

Kraft and Bock [19] reported the presence of a 76-MDa plasmid in N. hamburgensis strain X14. More recently, Pohl [29] demonstrated the pres- ence of two additional plasmids of 124 MDa and 182 MDa. The three plasmids, representing ap- proximately 10% of the total genome of this organism have been designated pPB 11, pPB 12, and pPB 13, respectively. Preliminary evidence places one rbcL on the largest plasmid, pPB 13.

Cloning and expression of the two N. hambur- gensis rbcL in E. coli as well as experiments to determine the location of the rbcS genes are in progress.

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