Rhodobacter Capsulatus SHAFIQUE Fidait SHIVAYOGEPPA B

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Identification of the PufQ Protein in Membranes of Rhodobacter capsulatus SHAFIQUE FIDAIt SHIVAYOGEPPA B. HINCHIGERI,4 THOR J. BORGFORD, AND WILLIAM R. RICHARDS* Department of Chemistry and Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia, Canada V5A 1S6 Received 18 January 1994/Accepted 20 September 1994

The PufQ protein has been detected in vivo for the first time by Western blot (immunoblot) analyses of the chromatophore membranes of Rhodobacter capsulatus. The PufQ protein was not visible in Western blots of membranes of a mutant (ARC6) lacking the puf operon but appeared in membranes of the same mutant to which the pufQ gene had been added in trans. It was also detected in elevated amounts in a mutant (CB1200) defective in two bch genes and unable, therefore, to make bacteriochlorophyll. The extremely hydrophobic nature of the PufQ protein was also apparent in these studies since it was not extracted from chromatophores by 3% (wt/vol) n-octyl-p-D-glucopyranoside, a procedure which solubilized the reaction center and light- harvesting complexes. During adaptation of R. capsulatus from aerobic to semiaerobic growth conditions (during which time the synthesis of bacteriochlorophyll was induced), the PufQ protein was observed to increase to the level of detection in the developing chromatophore fraction approximately 3 h after the start of the adaptation. The enzyme, S-adenosyl-L-methionine:magnesium protoporphyrin methyltransferase, also increased in amount in the developing chromatophore fraction but was present in a cell membrane fraction at the start of the adaptation as well.

The classic study by Cohen-Bazire et al. (14) first demon- light-harvesting II (LH II) complex encoded by thepuc operon strated the regulatory role of oxygen and light on pigment (13, 37, 43). synthesis in purple nonsulfur bacteria. In heterotrophic cul- In addition to its effect on enzyme induction and/or repres- tures of Rhodobacter sphaeroides grown with vigorous aeration sion, oxygen has a direct effect on Bchl synthesis. In cells of R. for several successive transfers, bacteriochlorophyll (Bchl) was sphaeroides grown phototrophically, the introduction of oxygen barely detectable (14). Even so, Gorchein et al. (26) showed caused an immediate cessation of Bchl synthesis (14), even that an enzyme of the magnesium branch of Bchl synthesis, though the activity of the MT was only slowly reduced under S-adenosyl-L-methionine:magnesium protoporphyrin methyl- these conditions (26). This inhibition was reversible, since a transferase (MT), was still present in low but measurable subsequent lowering of the oxygen partial pressure allowed an amounts in oxygen-grown cultures. After subsequent incuba- immediate resumption in Bchl synthesis, with no period of tion under anaerobic or semiaerobic conditions in the light, the adaptation (14). The effect of oxygen may be mediated by the cells underwent a period of adaptation during which time no oxidation state of a redox coenzyme, or other redox sensor, growth occurred, but the level of the MT increased approxi- which may cause the reversible inactivation of an enzyme of mately sevenfold, followed by the appearance of Bchl and Bchl synthesis, or there may be a reversible inhibition of a phototrophic growth (26). Gorchein (25) found a similar effect biosynthetic reaction by molecular oxygen itself. Biel (7) has on the induction of magnesium chelatase activity in whole cells R recently obtained evidence that the conversion of protopor- of sphaeroides during adaptation from aerobic to phototro- phyrin IX to magnesium protoporphyrin monomethyl ester phic growth conditions. and the conversion 5-aminolevulinic Although no assays for any other enzymes of the magnesium of acid to porphobilino- branch of Bchl synthesis have been developed, genes for these gen are sites of oxygen regulation, but the exact nature of the enzymes (the bch genes) have been mapped and cloned in both regulation was not investigated in either case. Rhodobacter Genetic studies have indicated that thepujfQ gene, located at capsulatus (38, 44) and R. sphaeroides (15). High the extreme 5' end of the puf operon in both R. capsulatus (1, oxygen tension resulted in a two- to fourfold repression of the 3, 4, 22, 30) and R. sphaeroides (16, 17, 29), plays a regulatory transcription of bch genes (2, 8, 28, 33, 43), whereas high light role in the biosynthesis of Bchl. Strains of both bacteria from intensity had no such effect (5). However, a much more severe which the entire had repression by oxygen occurred on the transcription of the puf operon been deleted (but which still genes of Bchl-binding proteins, including the a and 13 subunits contained intact bch genes and an intact puc operon) formed of the B870 light-harvesting I (LH I) complex and L and M drastically reduced amounts of the Bchl-containing LH II subunits of the reaction center (RC) complex encoded by the complex (3, 16, 22, 30). However, when a plasmid-borne pufQ puf and the ao and 13 subunits gene was reintroduced into these strains, a normal level of LH operon, of the B800-850 II complexes was observed. Forrest et al. (22) determined that thepufQ gene product (the PufQ protein) had no effect on the or * Corresponding author. Phone: (604) 291-4355. Fax: (604) 291- transcription translation of puc operon genes. They con- 5583. cluded that it most likely exerted its effect either on the t Present address: Department of Microbiology, University of Brit- assembly of LH II complexes or on the biosynthesis of Bchl. ish Columbia, Vancouver, B.C., Canada V6T 1Z4. They also determined that any regulation by the PufQ protein t Permanent address: Department of Chemistry, Karnatak Univer- of the tetrapyrrole biosynthetic pathway must occur on that sity, Dharwad-580003, India. part of the pathway specific to Bchl, since the level of cyto- 7244 PROTEIN 7245 VOL. 176, 1994 IDENTIFICATION OF THE PufQ A and also examined by chromes was unaffected by the deletion of the puf operon. fraction) was washed once in buffer and Marrs (3) constructed a fusion of the promoter for Western blotting. One-liter cultures of mutant strains CB1200, Bauer in the dark the nifHDK operon to thepufQ and lacZ genes so that different ARC6, and ARC6(pA4) grown in RCV+ medium of expression of both genes could be attained by varying were also harvested and disrupted as described above. Each of levels X was the nitrogen source. When this gene fusion was introduced into the supernatants from the 17,000 g centrifugation zonal gradient formed a strain of R. capsulatus lacking the puf operon, it was found layered directly onto a discontinuous the level of PufQ protein expression, inferred from the from 3.5 ml each of 10, 20, and 35% (wt/wt) sucrose and that 200,000 X g for 90 measured P-galactosidase activity, was directly proportional to centrifuged in a Beckman SW40 rotor at of Bchl biosynthesis. One possibility to account for min. Disruption of the mutants does not yield a true chromato- the amount is re- these results was that the PufQ protein functions as a carrier phore fraction; hence, the gradient fraction collected protein for intermediates of the magnesium branch of Bchl ferred to below as the lower pigmented band (LPB) fraction. It biosynthesis (3). is located at the interface between the 20 and 35% zones in a have recently reported the construction of a vector position in the gradient normally occupied by mature or We at the containing the pufQ gene of R capsulatus fused to the C- developing chromatophores. The pellet fraction located of the maltose-binding domain of the malE gene, bottom of the tube was also collected, and both membrane terminal end for the a factor Xa protease recognition sequence (21). fractions were analyzed by Western blotting and separated by adapta- The vector was successfully overexpressed in Escherichia coli, presence of the MT. Cell extracts obtained during the the fusion protein was purified, and the PufQ protein was tion were fractionated identically except that the soluble liberated by factor Xa proteolysis (21). We report herein the fraction located above the 10% zone, in addition to the two raised against the fusion protein membrane fractions, was collected. preparation of antibodies chromato- which we have used to detect the PufQ protein both in the Protein dissociation studies of R. capsulatus intracytoplasmic membranes (ICM) of phototrophically grown phores. The crude chromatophore pellet derived from pho- R. capsulatus and in developing ICM during an adaptation totrophically grown strain B10 was purified by application to a from aerobic to semiaerobic growth conditions. linear density gradient of 10 to 50% (wt/wt) sucrose and centrifuged in a Beckman SW40 rotor at 200,000 X g for 2 h. Purified chromatophores were collected from a band centered MATERIALS AND METHODS at about 32% sucrose. A series of extractions analogous to Bacterial strains and growth conditions. E. coli TB1 was those used by Farchaus et al. (19) during localization studies of the transformation and expression of vectors pMal-c the PufX protein in R sphaeroides was then carried out. The used for above, and pSF3 and was grown in 2X TY medium (21) at 370C. R. purified chromatophores were sedimented as described capsulatus wild-type strain B10 was grown phototrophically and the pellet was resuspended (to a final concentration of 0.3 under high light intensities in RCV medium (40) at 30'C. R mg of protein ml-') in buffer A containing 3 M NaBr and capsulatus mutant strain CB1200 was grown in closed bottles in incubated on ice for 30 min. The sample was then diluted with in RCV+ medium (42) at 30'C, while strains ARC6 an equal volume of 50 mM glycylglycine (pH 7.8) and centri- the dark X ARC6(pA4) were grown similarly in the presence of fuged in a Beckman Ti75 rotor at 300,000 g for 60 min. The and of 10 kanamycin (10 pLg ml-') and in the presence of kanamycin (10 resulting pellet was resuspended (to a final concentration (pH 7.8) contain- tig ml-') plus tetracycline (0.5 tig mnl-), respectively (1). mg of protein ml-') in 50 mM glycylglycine Adaptation of R. capsulatus from aerobic to semiaerobic ing 1% (wt/vol) n-octyl-,3-D-glucopyranoside (OG) and incu- growth conditions. A 15-liter culture of R. capsulatus B10 was bated on ice for 20 min. Following recentrifugation as above, grown to mid-log phase (A680 = 0.64) in a Chemap fermentor the procedure was repeated with 3% (wt/vol) OG. All of these at 30'C in the dark under highly aerobic conditions. The flow supernatants and pellets were analyzed by Western blotting; rate of air was then reduced from 6 to 8 liters min' to 1 to 2 supernatants from the 1 and 3% OG extractions were purified liters min-', and the stir rate was reduced from 300 rpm to 200 by the method of Gabellini et al. (24) by application to linear rpm. During the next 34 h, the synthesis of Bchl was induced density gradients of 10 to 50% (wt/wt) sucrose and centrifuga- while the cell density increased only slightly. One-liter samples tion in a Beckman SW40 rotor at 200,000 X g for 19 h. Upper were taken every hour up to 4 h, as well as 34 h after the start and lower pigmented bands were collected and examined by of adaptation. Cell extracts of each sample were separated by visible spectroscopy (measured with a Beckman model DU 640 zonal sucrose density gradient ultracentrifugation into soluble spectrophotometer) and Western blotting. and two membrane fractions as described below. Construction of expression vector pSF3. The source of the Cell fractionation methods. One-liter cultures of pho- pufQ gene used in the construction of vector pSF3 was plasmid totrophically grown R. capsulatus B10 were harvested at the pA4, originally constructed by Adams et al. (1). The assembly end of the exponential growth phase = 1.2) by centrifu- of expression vector pSF3, which carries a malE-pufQ gene (A600 Expression of the gation in a Sorvall GS3 rotor at 13,500 X g for 20 min and fusion, has been previously described (21). washed twice with buffer A (0.05 M potassium phosphate [pH inducible coding region of the vector produces a fusion protein 7.5]). These and all subsequent centrifugations were per- containing a maltose-binding protein (MBP) domain fused to formed at 40C. The washed cells were resuspended in 10 ml of a factor Xa protease recognition sequence (FX) engineered to buffer A, cooled on ice, and sonicated three to four times at release the full-length PufQ protein when treated with the maximum power for 30 s (with 1-min intervals between soni- protease. cations) with a FisherSonic-Dismembrator model 300 sonica- Expression, purification, and hydrolysis of the recombinant tor fitted with a medium-size probe. The crude homogenate MBP-FX-PufQ fusion protein. Recombinant MBP-FX-PufQ of E. coli TB1 was centrifuged in a Sorvall SS34 rotor at 17,500 X g for 20 min fusion protein was expressed in a 15-liter culture to remove the unbroken cells and cell debris. The resulting transformed with vector pSF3 as previously described (21). supernatant was centrifuged in a Beckman Ti75 rotor at After growth at 37°C to a cell density of 0.6 at A600, gene the addition of 0.3 mM isopropyl- 110,000 X g for 90 min. Portions of the supernatants before expression was induced by and after this centrifugation were examined by Western blot- P-D-thiogalactopyranoside (IPTG) followed by growth for an ting (immunoblotting). The pellet (the crude chromatophore additional 2 h. Crude cell lysates were purified by affinity 7246 FIDAI ET AL. J. BACTERIOL. chromatography on an amylose column as previously described freeze-dried before being dissolved in SDS sample buffer (21). After elution with 10 mM maltose in 10 mM sodium concentrated 1.5 times (31). Samples were then separated by phosphate (pH 7.0) containing 0.5 M NaCl, 0.25% (vol/vol) SDS-PAGE on 15% polyacrylamide gels (31) and electro- Tween 20, 10 mM P-mercaptoethanol, 1 mM ethylene glycol- eluted onto polyvinylidene difluoride (Immobilon P) mem- bis(,-aminoethyl ether)-NNN',N'-tetraacetic acid, and 1 mM branes, using an LKB multipore transfer apparatus at 2.5 mA sodium azide, the purified MBP-FX-PufQ fusion protein was cm-2 for 6 h. It was found that the PufQ protein successfully dialyzed extensively against buffer B (20 mM Tris-Cl [pH 8.0], transferred to the membranes when the direction of migration 0.1 M NaCi). It was hydrolyzed by digestion for 12 h at 230C in was toward the cathode. The membranes were treated as buffer B plus 10 mM sodium cholate (necessary for maintain- previously described (21), and the blots were probed with ing the PufQ protein in solution), using an 80:1 (wt/wt) ratio of FPLC-purified anti-MBP-FX-PufQ rabbit IgG fraction (dilut- fusion protein to factor Xa prepared from bovine serum by the ed either 2 X 103-fold for crude chromatophores or 5 x method of Esnouf and Williams (18). The digestion was 104-fold for purified sucrose gradient fractions). The proteins terminated by the addition of 1 mM phenylmethylsulfonyl were detected by incubation of the membranes with a 4 x fluoride from a 100 mM stock solution in 2-propanol. Analysis 103-fold dilution of goat anti-rabbit serum conjugated to of fractions separated by reverse-phase high-performance liq- horseradish peroxidase as previously described (21) and devel- uid chromatography (21) by sodium dodecyl sulfate (SDS)- oped with 1.0 mM 3,3'-diaminobenzidine plus 0.01% (vol/vol) polyacrylamide gel electrophoresis (PAGE) confirmed the hydrogen peroxide in 50 mM Tris-Cl (pH 7.6). release of the MBP. Two additional peaks having Mr values MT and protein assays. The specific activity of the MT in (7,600 and 8,700) similar to the anticipated molecular weight of various subcellular fractions was assayed by the method of the PufQ protein (8,556) were also detected and further Hinchigeri et al. (27), using 10 nmol of magnesium protopor- characterized by five cycles of amino-terminal sequence anal- phyrin IX and 6.9 nmol of S-[methyl-14C]adenosyl-L-methio- ysis (21). One possessed an amino terminus corresponding nine (58 Ci mol-1) in a final volume of 1.0 ml of 0.2 M Tris-Cl exactly to PufQ protein, and the other contained a sequence buffer (pH 7.8) containing 0.05% (vol/vol) Triton X-100. identical to PufQ protein beginning seven amino acids from Incubation was for 2 h at 370C. Radioactivity was determined the starting methionine residue (termed A1l7PufQ). with a Beckman series LS6000 liquid scintillation counter. Isolation of rabbit anti-MBP-FX-PufQ IgG. Antibodies Protein content was determined by the method of Bradford raised against the MBP-FX-PufQ fusion protein were elicited (11). in a rabbit by subcutaneous injections of a thoroughly mixed Materials. S-[methyl-'4C]adenosyl-L-methionine (58 Ci solution consisting of 100 pug of purified fusion protein com- mol-1) was supplied by DuPont NEN Research Products, bined with either Freund's complete or Freund's incomplete Lachine, Quebec, Canada. Magnesium protoporphyrin IX was adjuvant (23). Injections were performed over approximately a purchased from Porphyrin Products, Inc., Logan, Utah; OG 10-week period. Typically, about 25 ml of blood was collected, and phenylmethylsulfonyl fluoride were from Sigma Chemical and the immunoglobulin G (IgG) fraction was purified from it Co., St. Louis, Mo.; IPTG was from Bethesda Research by fast protein liquid chromatography (FPLC) in a Pharmacia Laboratories, Gaithersburg, Md.; Mono Q anion-exchange model LCC-500 apparatus by the following procedure. Anti- resin was from Pharmacia LKB Biotechnology Inc., Baie serum proteins were precipitated overnight at 4°C with an d'Urf6, Quebec, Canada; Immobilon P filters were from equal volume of saturated ammonium sulfate. The precipitate Millipore, Bedford, Mass.; the protein fusion and purification was recovered by centrifugation at 3,000 X g for 30 min, kit (containing the pMal-c vector and amylose affinity column) resuspended in 50 ml of buffer C (10 mM Na2HPO4, 1.8 mM was from New England Biolabs, Beverly, Mass.; protein Mr KH2PO4 [pH 7.2], 2.7 mM KCl, 10 mM NaCl), and dialyzed standards were from Bio-Rad Laboratories Inc., Hercules, against 4 liters of buffer C (with three changes of buffer), using Calif.; and goat anti-rabbit serum conjugated to horseradish dialysis membranes having a molecular weight cutoff in the peroxidase was from Mandel Scientific, Rockwood, Ontario, range of 12,000 to 14,000. The dialysate was then applied to an Canada. All other chemicals and reagents were of the highest FPLC column (2.6 by 31 cm) of the anion-exchange resin grade commercially available. Mono Q, which had been previously washed with 20 ml of buffer C containing 1 M NaCl. The column was developed with RESULTS a 500-ml linear gradient from 10 mM to 1 M NaCl at a flow rate of 2 ml min-1. A major component, which eluted at a salt Western blot analysis of membranes from phototrophically concentration of 55 mM NaCl, was identified as IgG by grown R. capsukitus. FPLC-purified rabbit polyclonal antibod- screening fractions for relative activity against both MBP-FX- ies raised against the MBP-FX-PufQ fusion protein were used PufQ fusion protein and MBP with enzyme-linked immunosor- in immunoblots to detect the presence of the PufQ protein in bent assays, using standard procedures (34). Followinf the phototrophically grown R. capsulatus. A band in Western blots addition to sample-containing wells of 100 RI of a 4 x 10 -fold corresponding to authentic PufQ protein liberated from the dilution of goat anti-rabbit serum conjugated to horseradish fusion protein was detected both in cell-free homogenates and peroxidase and 100 ,u of a solution of 1 mg of 1,2-diamino- in a crude chromatophore preparation (Fig. 1), although other benzene plus 0.03% (vol/vol) hydrogen peroxide per ml in 0.1 bands also cross-reacted with the purified IgG fraction. No M sodium acetate (pH 6.0), the A490 which developed was PufQ protein was detected in the supernatant following sedi- measured with a Bio-Tek model Ceres 900HDi plate reader. mentation of the chromatophore fraction (data not shown). A By this method, the IgG fraction demonstrated an approxi- series of extractions was then carried out on chromatophores mately four- to sevenfold increase in activity against the fusion purified by linear sucrose density gradient ultracentrifugation protein in comparison with the unconjugated MBP. to determine whether the PufQ protein could be removed from Immunoblotting of proteins. Western immunoblot assays the membrane under these conditions. As described in Mate- were performed by standard procedures (39). Sucrose gradient rials and Methods, the purified chromatophores were washed fractions were prepared for electrophoresis by extensive dial- with 3 M NaBr to remove extrinsic proteins and then washed ysis against 8 liters of buffer A (with three changes of buffer) in succession with 1 and 3% (wt/vol) OG to remove proteins of followed by dialysis against distilled water. The dialysate was a more intrinsic nature (including the LH and RC complexes). VOL. 176, 1994 IDENTIFICATION OF THE PufQ PROTEIN 7247

w z

0 C') C1= w

1 2 3 c: FIG. 1. Western immunoblot analysis of subcellular fractions of R 800 850 capsulatus. Samples were probed with an IgG fraction purified by WAVELENGTH (nm) FPLC from rabbit anti-MBP-FX-PufQ serum. Lane 1, crude chromatophores; lane 2, hydrolyzed MBP-FX-PufQ fusion protein; lane 3, FIG. 3. Absorption spectra of complexes extracted from purified crude cell lysate. The locations of the MBP (upper arrow) and PufQ chromatophores by 3% (wt/vol) OG. Following centrifugation in linear protein (lower arrow) are marked. density gradients formed between 10 and 50% (wt/wt) sucrose, bands containing primarily B800-850 LH II (upper band) and B870 LH I (lower band) were obtained. After each extraction, the suspensions were centrifuged and were analyzed by West- the resulting pellets and supernatants ARC6(pA4), which was constructed from ARC6 by addition in ern blotting. The PufQ protein was detected in the sediment- strain able fractions after each extraction, consistent with its highly trans of the pufQ gene on vector pM4 (1); and (iii) CB1200, which has a point mutation in the bchF gene and a hydrophobic nature. The results for each of the sedimentable make fractions were similar and are shown only following extraction directed disruption of the behA locus and is unable to Bchl (10). Although none of these three strains is able to make with 3% OG (Fig. 2, lane 2). No PufQ protein was ever isolated detected in the supernatant fractions (data not shown). The normal ICM, membrane fractions can nevertheless be supernatants from both the 1 and 3% OG extractions were from disrupted cells by zonal sucrose density gradient ultra- centrifuged in a linear sucrose density gradient, and two bands centrifugation. Membranes collected from the interface be- were obtained. Visible spectra of the 3% OG samples (Fig. 3) tween the 20 and 35% (wt/wt) sucrose zones (the LPB fraction) the were examined by Western blotting (Fig. 4). It can be seen that indicated that the upper band contained predominantly was LH II complex and the lower band contained predominantly a band identical in apparent Mr to the PufQ protein the LH I complex. The latter band was reported (24) to also detected in -20-2Og samples of the LPB membrane fractions contain the RC complex, although we did not specifically from strains ARC6(pA4) and CB1200 but not in membranes determine whether it was present in our preparation. Western from strain ARC6, which cannot make the PufQ protein. None of the pellet fractions, which were also examined by Western blot analyses of these two fractions from both the 1 and 3% This OG extracts failed to detect the PufQ protein in either fraction blotting (Fig. 4), gave a positive test for the PufQ protein. (Fig. 2, lanes 3 to 6). fraction, which sedimented through the 35% sucrose zone, Western blot analysis of membranes from mutants of R. probably contained the majority of the cytoplasmic membrane capsukitus. Membrane fractions from three mutants of R. (CM) together with components of the outer cell envelope. It capsulatus were used: (i) strain ARC6, which contains a can also be seen that in all strains, there is cross-reactivity to deletion of the puf operon from the Sall site in the middle of other higher-Mr proteins, perhaps due to nonspecific binding the pufQ gene to the XhoII site downstream of the puJX gene of the IgG. One in particular had an apparent Mr of 31,000, and is unable to make intact PufQ protein (12); (ii) strain similar to that of the outer membrane porin (36). Since the

kDu. kDa 200116. 97.4 * 45 66.* 45 * 42 *

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21.5 *

14.5 * * 6.5. FIG. 2. Western immunoblot analysis (probed as in Fig. 1) of soluble and insoluble proteins of R. capsulatus during extraction experiments. Lane 1, hydrolyzed MBP-FX-PufQ fusion protein; lane 2, the insoluble sediment following extraction of purified chromato- FIG. 4. Western immunoblot analysis (probed as in Fig. 1) of phores with 3 M NaBr, 1% (wtlvol) OG, and 3% (wt/vol) OG; lanes 3 subcellular fractions of mutant strains of R. capsulatus. Lane 1, to 6, pigmented sucrose density gradient bands (see Fig. 3) of extracts hydrolyzed MBP-FX-PufQ fusion protein; lanes 2 and 3, LPB and solubilized by 1% (wt/vol) OG (lower band, lane 4; upper band, lane 6) pellet fractions, respectively, of strain ARC6(pA4); lanes 4 and 5, LPB or 3% (wt/vol) OG (lower band, lane 3; upper band, lane 5); lane 7, and pellet fractions, respectively, of strain ARC6; lanes 6 and 7, LPB prestained protein standards. The location of the PufQ protein is and pellet fractions, respectively, of strain CB1200. The molecular marked with an arrow on the left, and the molecular mass values of masses of protein standards are indicated on the left, and the location protein standards are indicated on the right. of the PufQ protein is marked with a thick arrow. 7248 FIDAI ET AL. J. BACTERIOL.

A 116 97.4-: 66 - w I-- CD 45 : < 42 w 31 - cL O z 215 - Z m 14.5 - <: E alo 65 - mIt Ia: C')0 B 0 i-- E C _ -5 0 5 TIME (h) FIG. 5. Adaptation of R capsulatus from aerobic to semiaerobic growth conditions. The A680 (0) and A860 (l) of the culture were used as measures of the cell density and Bchl content, respectively; MT 1 2 3 4 5 activities were measured in the LPB (developing chromatophore) FIG. 6. SDS-PAGE (A) and Western immunoblot fraction (0) as described in Materials and Methods. (B) analyses (probed as in Fig. 1) of subcellular fractions of R. capsulatus during adaptation from aerobic to semiaerobic growth conditions. Protein standards and their molecular mass values are indicated on the cells were disrupted by sonication, this hydrophobic protein leftmost lane of panel A. Lane 1, hydrolyzed MBP-FX-PufQ fusion may have been redistributed into membrane fractions derived protein; lanes 2 to 5, the LPB (developing chromatophore) fractions from the CM and ICM. It is interesting that in analyzed at 0 h (lane 2), 3 h (lane 3), 4 h (lane 4), and 34 h (lane 5) the LPB fraction following the switch to semiaerobic conditions (see Fig. 5). Locations of strain in which an CB1200, increased amount of the PufQ of the PufQ protein are marked with arrows. protein was detected (Fig. 4, lane 6), the putative porin band also exhibited a much higher reactivity toward the antibody, possibly indicating a strong hydrophobic association between the porin and the PufQ protein. The mechanism of excretion had remained at about the same level observed at 3 h, while its of pigments accumulated by bch mutants may involve specific activity in the LPB fraction was approximately five transport times via the outer membrane porin (6, 9). However, we were unable higher. No MT activity was ever observed in the soluble to detect the presence of the PufQ protein by Western blotting fraction. The MT activity was also assayed in the LPB and of a pigment-protein complex isolated as previously described pellet fractions of the ARC6 and ARC6(pA4) mutant strains of (35) from the growth medium of the CB1200 mutant (data not R. capsulatus grown under anaerobic conditions in RCV+ shown). medium and compared with MT activity measured in the Western blot analysis of membranes from R. capsulatus chromatophore fraction isolated from phototrophically grown adapting from aerobic to semiaerobic growth conditions. A wild-type R. capsulatus. The results (Table 1) indicated that culture of R capsulatus, grown under highly aerobic conditions whereas both mutants possessed a level of MT activity in the fraction to that found to mid-log phase, was adapted in the dark for 10 h under pellet comparable in wild-type chro- semiaerobic conditions. During this time, the synthesis of Bchl matophores, only in the case of ARC6(pA4), which contains was induced while the cell density increased only slightly (Fig. 5). The adaptation was continued for a total of 34 h, by which time a significant amount of Bchl had accumulated (Fig. 5). 50 However, since the adaptation was conducted in the dark, the cells were unable to grow phototrophically. Cell extracts were w prepared from 1-liter samples collected every hour up to 4 h 40 and after 34 h of adaptation. Following zonal sucrose density <: gradient ultracentrifugation, -10-jig samples of the purified LU ILO LPB fractions were analyzed by SDS-PAGE and Western C/) C. 30- Z blotting. Results for samples from 1 and 2 h of adaptation were 0) similar to the 0-h sample and are not included. The PufQ protein was not detected by Western blotting of the LPB 7-20- fraction until after 3 h of adaptation (Fig. '6B). It was also W-6 visible in a 32-jig sample of the LPB fraction 34 h after the start of adaptation, when the LPB had become recognizable as 10 a true chromatophore fraction as a result of the incorporation of RC and LH complexes (Fig. 6A). Analysis of MT in activity membranes isolated from R. 0 1 2 3 4 34 capsulats adapting from aerobic to semiaerobic growth conditions and from mutants of R. capsulatus. The soluble, LPB, TIME (h) and pellet were fractions analyzed for MT activity during the FIG. 7. early stages and after 34 h of The Analysis of MT activities of subcellular fractions of R adaptation (Fig. 7). specific capsulatus during adaptation from aerobic to semiaerobic growth activity of the MT increased in parallel in the pellet and LPB conditions. Enzyme activities in the soluble fraction (0), pellet fraction fractions during the first 3 h of adaptation. After 34 h of (l), and LPB (developing chromatophore) fraction (-) were analyzed adaptation, the specific activity of the MT in the pellet fraction as described in Materials and Methods. VOL. 176, 1994 IDENTIFICATION OF THE PufQ PROTEIN 7249

TABLE 1. MT activities in isolated membrane fractions from wild- fractions isolated from strains ARC6 and ARC6(pA4) con- type and mutant strains ofR. capsulatus tained the MT at similar levels of specific activity (Table 1). strain not strain contained Membrane MT activity (pmol h- However, only ARC6(pA4), ARC6, Strain fraction mg of protein-1) additional MT activity in the LPB fraction. It may be significant, therefore, that while the PufQ protein could not be B10 Chromatophore 95 detected in the pellet fraction isolated from either strain (Fig. ARC6 LPB -- it was detected in the LPB fraction of strain Pellet 78 4), easily ARC6(pA4) LPB 81 ARC6(pA4) (Fig. 4). Pellet 54 The PufQ protein was also detected in the LPB (developing chromatophore) fraction approximately 3 h after the start of an adaptation of the wild-type strain from aerobic to semiaerobic growth conditions (Fig. 6B). We could not detect the PufQ the pufQ gene added in trans, was MT activity also found in protein in the LPB fraction isolated from aerobically grown membranes isolated in the LPB fraction. cells at the start of the adaptation. Although the entire puf operon would be under oxygen repression during aerobic DISCUSSION growth, R. capsulatus is still able to transcribe reduced levels of the puf operon by read-through transcription from upstream Western blot analysis of crude chromatophores of pho- crtEF and bchCA operons, which are not as severely repressed totrophically grown R. capsulatus demonstrated the presence by oxygen as is thepufoperon. These three operons have been of a low-Mr antigenic polypeptide located in a position in the shown to compose one of two superoperons found in this gel identical to that of authentic PufQ protein (Fig. 1). bacterium (41). A demonstration of the read-through mecha- Unfortunately, the polyclonal antibodies contained in the nism in operation was recently provided by Sganga and Bauer purified IgG fraction were not specific for this protein only; a (37), who have isolated a mutant of R. capsulatus incapable of number of other higher-Mr proteins that also were antigenic inducingpufoperon transcription under low-oxygen conditions were present. Therefore, Western blots of membranes of the from the oxygen-regulated puf promoter. The mutant was still puf deletion (ARC6) strain of R. capsulatus were compared able to grow phototrophically under high (but not low) light with blots of membranes of strain ARC6(pA4) (in which the intensities as a result of the formation of low levels of puf pufQ gene had been added in trans to mutant ARC6 on operon gene products by read-through transcription of the plasmid pA4). Membrane fractions located in sucrose density upstream operons. Therefore, while the PufQ protein could gradients in the region of the LPB fraction were isolated from potentially have been present at the start of the adaptation, it both of these strains; however, only in the case of ARC6(pA4) was below the level of detection of our analyses, given the were they visibly pigmented. The results (Fig. 4) clearly amounts of protein that we used. However, our results did demonstrated that the low-Mr polypeptide was the only anti- demonstrate a clear increase of the PufQ protein in the LPB genic component present in the LPB fraction of the pufQ- fraction during the course of the adaptation. containing strain ARC6(pA4) which was not also present in the Several cell fractions were also examined for MT activity LPB fraction of strain ARC6, thus identifying it as the PufQ during the adaptation from aerobic to semiaerobic conditions protein. (Fig. 7). As had been observed for R sphaeroides by Gorchein Farchaus et al. (19) have recently prepared a polyclonal et al. (26), a low level of MT activity was easily detected in antibody raised against a synthetic oligopeptide based on the aerobically grown R. capsulatus and was found to be present at pufX sequence ofR. sphaeroides. Using this antibody, they have approximately equal levels of specific activity in both the LPB identified the PufX protein in chromatophores of R. spha- and pellet fractions. The MT appears, therefore, to be local- eroides. They also found that the PufX protein copurified with ized generally throughout the CM of aerobically grown R. an RC-LH I complex extracted from the chromatophore capsulatus. Subsequently, a large increase in MT activity membrane by 1% OG. The association of the PufX protein occurred in the LPB fraction, where 34 h after the start of the with the RC-LH I complex supports experimental observations adaptation (when the LPB had become a recognizable chro- in R. capsulatus by Lilburn et al. (32) that the PufX protein matophore fraction), its specific activity had increased 17-fold. plays a role in the transfer of electrons between the RC and From the results of this study, we are not able to say where other component(s) of cyclic electron transfer (e.g., the cyto- the PufQ protein exerts its effect on the synthesis of Bchl. It chrome bc1 complex). When we applied the fractionation was found in significant quantities in the developing chromato- methods used by Farchaus et al. (19), we could not detect the phore fraction (which may be derived from ICM or partially PufQ protein in either the LH II or RC-LH I complex; invaginated regions of the CM) and in the membrane fraction however, it was readily detectable in the insoluble membrane which gives rise to the LPB upon cellular disruption of the sediment following treatment of purified chromatophores with ARC6(pA4) mutant. This location is consistent with a role for both 1 and 3% OG (Fig. 2). Hence, the PufQ protein appears it as a carrier protein of Bchl intermediates from magnesium to be a much more intrinsic membrane protein than the PufX protoporphyrin monomethyl ester onward. Other than the protein and does not appear to be intimately associated with MT, nothing is known about the location of any of the enzymes LH and/or RC complexes, consistent with the view that it has of the magnesium branch required for the synthesis of Bchl. some function in Bchl biosynthesis. However, if Bchl synthesis does occur in this fraction, newly It is known that cells of both R. capsulatus (3) and R. synthesized Bchl would be in the correct location for immedi- sphaeroides (16) can form some Bchl in the complete absence ate incorporation into RC and LH complexes during ICM of the PufQ protein. For example, the ARC6 strain of R. development. We have recently obtained evidence for the capsulatus makes a small amount of the Bchl-containing LH II association of the tetrapyrrole intermediate, protochlorophyl- complex; however, possession of the pufQ gene in trans allows lide, with the PufQ protein in reconstituted liposomes (20). it to form several times more Bchl (22, 30). Hence, the role of However, we have not yet been able to demonstrate that the the PufQ protein appears to be to increase Bchl synthesis (or PufQ-associated intermediate was employed for Bchl synthesis LH II complex assembly) to an optimum level. The pellet in cell-free systems. 7250 FIDAI ET AL. J. BACT1ERIOL.

ACKNOWLEDGMENTS requirement for photoheterotrophic growth in Rhodobacter spha- We thank J. T. Beatty for providing plasmid pA4 and R. capsulatus eroides. EMBO J. 11:2779-2788. B10, ARC6, and ARC6(pA4); C. E. Bauer for R. capsulatus CB1200; 20. Fidai, S., S. B. Hinchigeri, and W. R Richards. 1994. Association G. B. Kalmar for aid in preparation of the antibodies; and all for many of protochlorophyllide with the PufQ protein of Rhodobacter helpful discussions. We also acknowledge the work of David Huang in capsulatus. Biochem. Biophys. Res. Commun. 200:1679-1684. preparation of the LH II and RC-LH I complexes. 21. Fidai, S., G. B. Kalmar, W. R Richards, and T. J. Borgford. 1993. This work was supported by grants A5060 (to W.R.R.) and 42351 (to Recombinant expression of the pufQ gene of Rhodobacter capsu- T.J.B.) from the Natural Sciences and Engineering Research Council latus. J. Bacteriol. 175:4834-4842. of Canada. 22. Forrest, M. E., A. P. Zucconi, and J. T. Beatty. 1989. The pufQ gene product of Rhodobacter capsulatus is essential for formation REFERENCES of B800-850 light harvesting complexes. Curr. Microbiol. 19:123- 1. Adams, C. W., M. E. Forrest, S. N. Cohen, and J. T. Beatty. 1989. 127. Structural and functional analysis of transcriptional control of the 23. Freund, J. 1956. The mode of action of immunologic adjuvants. Rhodobacter capsulatus puf operon. J. Bacteriol. 171:473-482. Adv. Tuberc. Res. 7:130-148. 2. Armstrong, G. A., D. N. Cook, D. Ma, M. Alberti, D. H. Burke, and 24. Gabellini, N., Z. Gao, D. Oesterhelt, G. Venturoli, and B. A. J. E. Hearst. 1993. Regulation of carotenoid and bacteriochloro- Melandri. 1989. Reconstitution of cyclic electron transport and phyll biosynthesis genes and identification of an evolutionary photophosphorylation by incorporation of the reaction center, conserved gene required for bacteriochlorophyll accumulation. J. cytochrome bc, complex and ATP synthase from Rhodobacter Gen. Microbiol. 139:897-906. capsulatus into ubiquinone-10/phospholipid vesicles. Biochim. 3. Bauer, C. E., and B. L. Marrs. 1988. The Rhodobacter capsulatus Biophys. Acta 974:202-210. puf operon encodes a regulatory protein (pufQ) for bacteriochlo- 25. Gorchein, A. 1972. Magnesium protoporphyrin chelatase activity rophyll biosynthesis. Proc. Natl. Acad. Sci. USA 85:7074-7078. in Rhodopseudomonas spheroides. Biochem. J. 127:97-106. 4. Bauer, C. E., D. A. Young, and B. L. Marrs. 1988. Analysis of the 26. Gorchein, A., A. Neuberger, and G. H. Tait. 1968. Adaptation of Rhodobacter capsulatus puf operon. Location of the oxygen- Rhodopseudomonas spheroides. Proc. R. Soc. Lond. Ser. B 171: regulated promoter region and the identification of an additional 111-125. puf-encoded gene. J. Biol. Chem. 263:4820-4827. 27. Hinchigeri, S. B., J. C.-S. Chan, and W. R Richards. 1981. 5. Biel, A. J. 1986. Control of bacteriochlorophyll accumulation by Purification of S-adenosyl-L-methionine:magnesium protoporphy- light in Rhodobacter capsulatus. J. Bacteriol. 168:655-659. rin methyltransferase by affinity chromatography. Photosynthetica 6. Biel, A. J. 1991. Characterization of a coproporphyrin-protein 15:351-359. complex from Rhodobacter capsulatus. FEMS Microbiol. Lett. 28. Hunter, C. N., and S. Coomber. 1988. Cloning and oxygen- 81:43-48. regulated expression of the bacteriochlorophyll biosynthesis genes 7. Biel, A. J. 1992. Oxygen-regulated steps in the Rhodobacter bch E, B, A, and C of Rhodobacter sphaeroides. J. Gen. Microbiol. capsulatus tetrapyrrole biosynthetic pathway. J. Bacteriol. 174: 134:1491-1497. 5272-5274. 29. Hunter, C. N., P. McGlynn, M. K. Ashby, J. C. Burgess, and J. D. 8. Biel, A. J., and C. E. Bauer. 1983. Transcriptional regulation of Olsen. 1991. DNA sequencing and complementation-deletion several genes for bacteriochlorophyll biosynthesis in Rhodo- analysis of the bchA-pufoperon region ofRhodobacter sphaeroides: pseudomonous capsulatus in response to oxygen. J. Bacteriol. in vivo mapping of the oxygen-regulated puf promoter. Mol. 156:686-694. Microbiol. 5:2649-2662. 9. Bollivar, D. W., and C. E. Bauer. 1992. Association of tetrapyrrole 30. Klug, G., and S. N. Cohen. 1988. Pleiotropic effects of localized intermediates in the bacteriochlorophyll a biosynthetic pathway Rhodobacter capsulatus puf operon deletions on production of with the major outer membrane porin protein of Rhodobacter light-absorbing pigment-protein complexes. J. Bacteriol. capsulatus. Biochem. J. 282:471-476. 170:5814-5821. 10. Bollivar, D. W., J. Y. Suzuki, J. T. Beatty, J. Dobrowlski, and C. E. 31. Laemmli, U. K. 1970. Cleavage of structural protein during the Bauer. 1994. Directed mutational analysis of bacteriochlorophyll a assembly of the head of bacteriophage T4. Nature (London) biosynthesis in Rhodobacter capsulatus. J. Mol. Biol. 237:622-640. 227:680-685. 11. Bradford, M. M. 1976. A rapid and sensitive method for the 32. Lilburn, T. G., C. E. Haith, R C. Prince, and J. T. Beatty. 1992. quantitation of microgram quantities of protein utilizing the Pleiotrophic effects of puJX gene deletion on the structure and principle of protein-dye binding. Anal. Biochem. 72:248-254. function of the photosynthetic apparatus of Rhodobacter capsula- 12. Chen, C. A., J. T. Beatty, S. N. Cohen, and J. G. Belasco. 1988. An tus. Biochim. Biophys. Acta 1100:160-170. intercistronic stem-loop structure functions as an mRNA decay 33. Ma, D., D. N. Cook, D. A. O'Brien, and J. E. Hearst. 1993. Analysis terminator necessary but insufficient for puf mRNA stability. Cell of the promoter and regulatory sequences of an oxygen-regulated 52:609-619. bch operon in Rhodobacter capsulatus by site-directed mutagene- 13. Clark, W. G., E. Davidson, and B. L. Marrs. 1984. Variation of sis. J. Bacteriol. 175:2037-2045. levels of mRNA for antenna and reaction center polypeptides in 34. Miles, L. E. M., and C. N. Hales. 1968. Labelled antibodies and Rhodopseudomonas capsulatus in response to changes in oxygen immunological assay systems. Nature (London) 219:186-189. concentration. J. Bacteriol. 157:945-948. 35. Richards, W. R., R B. Wallace, M. S. Tsao, and E. Ho. 1975. The 14. Cohen-Bazire, G., W. R Sistrom, and R Y. Stanier. 1957. Kinetic nature of a pigment-protein complex excreted from mutants of studies of pigment synthesis by non-sulfur purple bacteria. J. Cell. Rhodopseudomonas sphaeroides. Biochemistry 14:5554-5561. Comp. Physiol. 49:25-68. 36. Schiltz, E., A. Kreusch, U. Nestel, and G. E. Schulz. 1991. Primary 15. Coomber, S. A., M. Chaudhri, A. Connor, G. Britton, and C. N. structure of porin from Rhodobacter capsulatus. Eur. J. Biochem. Hunter. 1990. Localized transposon Tn5 mutagenesis of the 199:587-594. photosynthetic gene cluster of Rhodobacter sphaeroides. Mol. 37. Sganga, M. W., and C. E. Bauer. 1992. Regulatory factors control- Microbiol. 4:977-989. ling photosynthetic reaction center and light-harvesting gene 16. Davis, J., T. J. Donohue, and S. Kaplan. 1988. Construction, expression in Rhodobacter capsulatus. Cell 68:945-954. characterization, and complementation of a Puf- mutant of 38. Taylor, D. P., S. N. Cohen, W. G. Clark, and B. L. Marrs. 1983. Rhodobacter sphaeroides. J. Bacteriol. 170:320-329. Alignment of genetic and restriction maps of the photosynthesis 17. Donahue, T. J., P. J. Kiley, and S. Kaplan. 1988. The puf operon region of the Rhodopseudomonas capsulatus chromosome by a region of Rhodobacter sphaeroides. Photosyn. Res. 19:39-61. conjugation-mediated marker rescue technique. J. Bacteriol. 154: 18. Esnouf, M. P., and W. J. Williams. 1962. The isolation and 580-590. purification of a bovine-plasma protein which is a substrate for the 39. Tobin, H., T. Staehelin, and J. Gordon. 1979. Electrophoretic coagulant fraction of Russell's-viper venom. Biochem. J. 84:62-71. transfer of proteins from polyacrylamide gels to nitrocellulose 19. Farchaus, J. W., W. P. Barz, H. Grunberg, and D. Oesterhelt. sheets: procedure and some applications. Proc. Natl. Acad. Sci. 1992. Studies on the expression of the puJX polypeptide and its USA 76:4350-4354. VOL. 176, 1994 IDENTIFICATION OF THE PufQ PROTEIN 7251

40. Weaver, P. F., J. D. Wall, and H. Gest. 1975. Characterization of 43. Zhu, Y. S., D. N. Cook, F. Leach, G. A. Armstrong, M. Alberti, and Rhodopseudomonas capsulata. Arch. Microbiol. 105:207-216. J. E. Hearst. 1986. Oxygen-regulated mRNAs for light-harvesting 41. Wellington, C. L., C. E. Bauer, and J. T. Beatty. 1992. Photosyn- and reaction center complexes and for bacteriochlorophyll and thesis gene superoperons in purple non-sulfur bacteria: the tip of carotenoid biosynthesis in Rhodobacter capsulatus during the shift the iceberg? Can. J. Microbiol. 38:20-27. from anaerobic to aerobic growth. J. Bacteriol. 168:1180-1188. 42. Yang, Z., and C. E. Bauer. 1990. Rhodobacter capsulatus genes 44. Zsebo, K. M., and J. E. Hearst. 1984. Genetic-physical mapping of involved in early steps of the bacteriochlorophyll biosynthetic a photosynthetic gene cluster from Rhodobacter capsulatus. Cell pathway. J. Bacteriol. 172:5001-5010. 37:937-947.