Cyanobacterium Trichodesmium thiebautiit

Transcription

1 APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Oct. 1993, p /93/ $02.00/0 Copyright ) 1993, American Society for Microbiology Vol. 59, No. 10 Cytochrome Oxidase: Subcellular Distribution and Relationship to Nitrogenase Expression in the Nonheterocystous Marine Cyanobacterium Trichodesmium thiebautiit B. BERGMAN,1* P. J. A. SIDDIQUI,2t E. J. CARPENTER,2 AND G. A. PESCHEK3 Department of Botany, Stockholm University, S Stockholm, Sweden'; Marine Science Research Center, State University of New York at Stony Brook, Stony Brook, New York 11794S50002; and Biophysical Chemistry Group, Institute of Physical Chemistry, University of Vienna, Vienna, Austria Received 21 December 1992/Accepted 29 April 1993 Immunochemical labeling was used to study the subcellular distribution of cytochrome oxidase, a respiratory protein, in Trichodesmium thiebautii. The protein was found associated with both cytoplasmic and thylakoid membranes. About a sixfold variation in the protein content (gold particle count) was found among Trichodesmium cells within a single colony. Double labeling was performed with cytochrome oxidase and nitrogenase antisera. Regression analysis of gold particle counts per unit of cell area of cytochrome oxidase and nitrogenase showed a positive correlation (r2 = 0.911); cells with higher nitrogenase levels also had higher levels of cytochrome oxidase. The parallel expression of two proteins suggests that respiratory oxygen uptake may be involved in nitrogenase protection (respiratory protection) in Trichodesmium spp. The location of the respiratory electron transport chain in cyanobacteria has been a controversial issue. The thylakoid membranes have been considered an exclusive site for both respiratory and photosynthetic electron transport (for a review, see reference 22). Only recently, with the development of separation and purification methods for cytoplasmic and thylakoid membranes (10, 11, 15), has it been established that both membranes have respiratory activity based on an aa3-type cytochrome c oxidase (10-13, 19, 20, 28). These studies were carried out on a number of cyanobacteria including unicellular, filamentous heterocystous, and filamentous nonheterocystous species. However, some variations in these results reflect the possibility that cyanobacteria differ in the organization and location of respiratory electron transport (22). The respiratory electron acceptor is an aa3-type cytochrome oxidase in both cytoplasmic and thylakoid membranes, whereas the photosynthetic electron acceptor in thylakoid membranes is the P700 component of photosystem I (6, 13, 20). In both cases the electron carrier protein is a c-type cytochrome which is identical to cytochrome c6 or C553, the photosynthetic redox protein (13, 23). During respiration, electrons are ultimately transferred to oxygen and generate water with concomitant oxidation of the reductants NADPH and NADH (17). Nitrogenase is a highly oxygen-labile protein, and therefore aerobic diazotrophic cyanobacteria either have nitrogenase (protein/activity) confined to specialized cells (the heterocysts) or restrict nitrogen fixation mostly to dark periods to avoid oxygenic photosynthesis (5, 27). Despite such spatial and temporal segregation of nitrogen fixation and oxygenic photosynthesis, cells of aerobic cyanobacterial diazotrophs are still exposed to ambient oxygen, which diffuses into the cells. Therefore, a need for additional nitrogenase-protecting mechanisms still remains (19). In contrast to other cyanobacteria, natural populations of * Corresponding author. t Marine Science Research Center contribution 913. :t Present address: Department of Biology, Rensselaer Polytechnic Institute, Troy, NY nonheterocystous species in the genus Trichodesmium Ehrenberg perform both nitrogen fixation and oxygenic photosynthesis exclusively in the light (2). However, these activities may be spatially separated in Trichodesmium colonies, since nitrogenase is confined to a limited number of cells (1). The colony configuration may be helpful in developing low-oxygen niches (2, 16), but it is not always essential for the activity and expression of nitrogenase (14, 21). Oxygen-scavenging processes that might be involved in the success of Trichodesmium species as potential diazotrophs in an aerobic environment are not well understood. Respiratory consumption of oxygen in aerobic diazotrophs has been proposed as a major nitrogenase-protecting mechanism (i.e., respiratory protection mechanism) in cyanobacteria (19). An increase in the cytochrome oxidase activity (19) and a high rate of oxygen consumption during the peak nitrogen fixation period (7, 8) have been found in many cyanobacterial species. The respiratory oxygen uptake therefore appears to be functionally related to the nitrogenase activity. Here we investigated the presence and distribution of the respiratory protein, aa3-type cytochrome oxidase, in Trichodesmium thiebautii by using in vivo immunochemical tagging, together with transmission electron microscopy. We also attempted to relate the expression of cytochrome oxidase to nitrogenase by using double immunochemical tagging of the two enzymes. The possibility that cellular respiration is an important nitrogenase protectant is discussed. MATERIALS AND METHODS T. thiebautii colonies were collected from a 15-m depth in the southwestern Sargasso Sea with a 1-m-diameter, 202-,um-mesh plankton net (24). The colonies were transferred to sterile seawater and then prepared for Western immunoblotting and immunochemical tagging experiments. SDS-PAGE and Western blotting. Thirty colonies were lyophilized immediately on collection and later dissolved in 30 ml of sample buffer at 90 C for 5 min for use in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- 3239

2 3240 BERGMAN ET AL. APPL. ENvIRON. MICROBIOL. Downloaded from FIG. 1. Electron micrograph of a transversely sectioned Tichodesmium cell postfixed with osmium tetroxide and stained with uranyl acetate and lead citrate. Subcellular inclusions which can be seen are the gas vesicle bundle (G), thylakoid membrane network (T), vacuole-like spaces (V), cyanophycin granules (C), cytoplasmic membrane (CM), and scroll-like membranes (S). Scale bar, 1,um. PAGE) and subsequently in Western blotting by previously described methods (26). The antiserum used was rabbit anti-aa3-type cytochrome oxidase subunit I from Anacystis nidulans (9). Immunochemical tagging and transmission electron microscopy. Fixation, embedding, and direct immunogold tagging of cytochrome oxidase on thin sections held on gold grids were carried out by methods described earlier (1, 24). The double gold tagging for cytochrome oxidase and nitrogenase was performed as detailed by Siddiqui et al. (24). The primary antibodies, used at a 1:100 dilution, were anticytochrome oxidase (same as described above) and mouse anti-nitrogenase (Fe protein) from T. thiebautii (kindly provided by J. P. Zehr [29]). Unstained sections were observed on a Zeiss 10 transmission electron microscope at 60 kv. The controls were run by replacing primary antibodies with 1% bovine serum albumin to detect nonspecific labeling with secondary antibodies and by omitting one of the primary antibodies at a time in the double-labeling experiment to check whether the nonspecific secondary antibody interfered with the distribution of the other antibodies. Controls showed no nonspecific binding of gold particles to the cell components, and the two primary antibodies used showed no nonspecific interference with the secondary antibodies. Gold particles were counted on magnified micrographs, FIG. 2. Polypeptides in a crude protein extract of T. thiebautii were separated by SDS-PAGE and blotted on nitrocellulose membranes. Western blot exhibits a polypeptide band of about 50 kda, specifically recognized by an antiserum against cytochrome oxidase from Anacystis sp. Molecular weight markers (in thousands) are on the left. on September 17, 2018 by guest

3 VOL. 59, 1993 CYTOCHROME OXIDASE IN TRICHODESMIUM THIEBAUTII 3241 Downloaded from FIG. 3. Portion of a T. thiebautii cell labeled for cytochrome oxidase. Gold spheres are distinctly localized along thylakoid membranes (T) surrounding the vacuolated spaces (V) and the gas vesicle bundles (G). Some gold particles also appeared to be associated with the cytoplasmic membrane (CM). Compare this cell without osmium fixation with the cell in Fig. 1 for locating cell inclusions. Scale bar, 1 pm. and the numbers obtained per unit of cell area on micrographs were converted to the actual cell area. Whole cells were counted for cytochrome oxidase labels, whereas for nitrogenase, since the particles were small and abundant, gold spheres were enumerated in 15 to 20 grids (area, 2,um2) per cell. A total of 30 cells were randomly selected for the average count for cytochrome oxidase, and 20 cells were enumerated for cytochrome oxidase and nitrogenase in the double-labeling experiments for determining averages and regression analysis. Sections were obtained from five different preparations. All values were corrected for the background (if any) by subtracting the count per unit area, adjacent to and outside the cell, from the cell counts. RESULTS AND DISCUSSION T. thiebautii has large cells (about 15,um in diameter), and the photosynthetic (thylakoid) membranes have an unusual configuration compared with those of other cyanobacteria. Thylakoids form a loose network throughout the cell (Fig. 1), and the thylakoid network surrounds vacuole-like spaces. The detailed ultrastructure of T. thiebautii has been reported earlier (24, 25). Western blotting experiments showed that subunit I of aa3-type cytochrome oxidase was present in T. thiebautii (Fig. 2). The antibody specifically recognized a polypeptide band with a molecular mass of about 50 kda, and this was in the range previously given for several other cyanobacteria on September 17, 2018 by guest

4 3242 BERGMAN ET AL. APPL. ENvIRON. MICROBIOL. (18). The specificity of the anti-nitrogenase serum for Tnchodesmium species has been shown earlier (29). The immunochemical tagging of cytochrome oxidase clearly demonstrated that the protein was predominantly associated with both the cytoplasmic and thylakoid membranes in T. thiebautii (Fig. 3). Our results were in agreement with those of recent studies demonstrating cytochrome oxidase activity in isolated cytoplasmic and thylakoid membranes from cyanobacteria (10-13, 20, 28). A considerable variation was, however, observed in the protein levels (gold particle counts) of cytochrome oxidase from one Trichodes- mium cell to another within the same colony. The average gold count was (standard deviation) gold spheres,um-', but the density of gold particles varied manyfold (4 to 25 spheres p.m-2). To resolve whether the increase in the cytochrome oxidase protein in some Trichodesmium cells was correlated with the variable expression of nitrogenase (1), we localized both proteins simultaneously (Fig. 4). The gold markers localizing nitrogenase showed a distribution similar to that previously observed; i.e., the protein was present in the cytoplasmic area throughout the cell (1, 24). Also, in agree- FIG. 4. Double gold labeling for cytochrome oxidase (10-nm gold particles) and nitrogenase (5-nm gold particles) in two adjacent T. thiebautii cells. Note the high label density for both proteins in the cell on the right compared with the cell on the left. See the legends to Fig. 1 and 3 for abbreviations. Scale bar, 0.5 plm.

5 VOL. 59, 1993 CYTOCHROME OXIDASE IN TRICHODESMIUM THIEBAUTII con 0 co 60-0 z 30 -i y s x r 2 z a / Cytochrome Oxidase FIG. 5. Regression analysis between the levels of cytochrome oxidase and nitrogenase in T. thiebautii cells. Values on the x andy axes represent the number of gold particles counted in each cell in the double-immunolabeling experiment (see Materials and Methods for details). The positive correlation indicates coexpression of two proteins in T. thiebautii. ment with these reports, our data revealed that 20 to 40% of the cells had considerably higher levels of nitrogenase than did the others (compare cells in Fig. 4). The distribution of cytochrome oxidase was identical to that seen in cells labeled with the cytochrome oxidase antiserum only. Again, the average number of gold markers (diameter, 10 nm) representing cytochrome oxidase was 10 5 (standard deviation) jxm-2. The number of gold markers (diameter, 5 nm) labeling nitrogenase varied from 12 to 115 particles um-2, with an average of 36 particles p.m2. To investigate the cell-to-cell variations more closely on a quantitative basis, we performed regression analysis on the counts of gold particles localizing cytochrome oxidase and nitrogenase (Fig. 5). Regression analysis demonstrated that increases in the levels of the two proteins were positively correlated, with a regression coefficient (r2) of (see Fig. 5 for the regression equation). The ratio of cytochrome oxidase to nitrogenase label was maintained at about 1:4 irrespective of the level of nitrogenase. Our data demonstrated approximately 6- and 10-fold increases from the lowest to the highest levels of cytochrome oxidase and nitrogenase proteins, respectively. Interestingly, parallel increases in protein levels are in agreement with activity data of cytochrome oxidase reported for the unicellular cyanobacterium Gloeothece sp. and filamentous heterocystous Anabaena and Nostoc species (19). In Gloeothece spp., the activity of cytochrome oxidase under nitrogen-fixing conditions in the light was 8 to 12 times higher than that of nitrate-grown cells. In addition, high rates of oxygen uptake have been found in Synechococcus (8) and Trichodesmium (7) spp. during periods of nitrogen fixation in the light. Although respiratory activity in phototrophic prokaryotes is suppressed in the light, recent studies showed that cytochrome oxidase under nitrogen-fixing conditions is less inhibited by illumination (19). The precise mechanism for protecting oxygen-labile nitrogenase from oxygen inactivation is yet to be resolved. Previously it was proposed that a spatial separation of nitrogen fixation and oxygenic photosynthesis occurs in Trichodesmium cells (2). This concept has been contradicted by several recent findings: (i) nitrogenase is not confined to the center of the colony (17); (ii) photosynthetic proteins (phycobiliproteins and ribulose-1,5-bisphosphate carboxylase/oxygenase [Rubisco]) are present in all cells, including those carrying nitrogenase (24); and (iii) the photosynthetic ability is present in all cells (3). These studies show that all cells (with or without nitrogenase) are physiologically active and also lead to the conclusion that Trichodesmium spp. not only are devoid of a temporal segregation of the two incompatible processes but also apparently lack the spatial partitioning between nitrogen fixation and oxygenic photosynthesis which occurs in heterocystous species. The parallel expression of cytochrome oxidase and nitrogenase proteins found in T. thiebautii cells and the previous data on high rates of oxygen consumption (7) suggest that respiratory oxygen uptake is involved in lowering the intracellular oxygen tension, necessary for the optimal activity of nitrogenase, in this aerobic nonheterocystous diazotroph. The molecular aspects of parallel expression of the two proteins are, however, not known. A possible involvement of the oxygenase activity of Rubisco in oxygen scavenging in Tichodesmium spp. has recently been proposed (24). In addition, initial work on superoxide dismutase activity indicates that this enzyme is present in Trichodesmium spp. and may participate in nitrogenase protection by removing oxygen radicals (4). We believe that several mechanisms may be operating together to protect nitrogenase from oxygen deactivation. ACKNOWLEDGMENTS We gratefully acknowledge financial support from the Bank of Sweden Tercentenary Foundation and the C. Tryggers Fund to B.B.; the Ministry of Science and Technology, Government of Pakistan, to P.J.A.S.; and the U.S. National Science Foundation to E.J.C. REFERENCES 1. Bergman, B., and E. J. Carpenter Nitrogenase confined to randomly distributed trichomes in the marine cyanobacterium Tnchodesmium thiebautii (Cyanophyta) J. Phycol. 27: Carpenter, E. J Physiology and ecology of marine planktonic Oscillatoria (Trichodesmium). Mar. Biol. Lett. 4: Carpenter, E. J., J. Chang, M. Cottrel, D. G. Capone, H. Paerl, and B. Bebaut Reevaluation of nitrogenase oxygen protecting mechanisms in the planktonic marine cyanobacterium Trichodesmium. Mar. Ecol. Prog. Ser. 65: Cunningham, K., and D. G. Capone Superoxide dismutase as a protective enzyme against oxygen toxicity: an overview and initial studies in Trichodesmium, p In E. J. Carpenter, D. G. Capone, and J. 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