Abstract. In the cyanobacterium Synechococcus UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon ...
Arch Microbiol (1993) 159:21-29
Archives of
Microbiology
9 Springer-Verlag1993
Effect of dissolved inorganic carbon on the expression of carboxysomes, localization of Rubisco and the mode of inorganic carbon transport in cells of the cyanobacterium SynechococcusUTEX 625 R. Michael L. M cKay 1,3, Sarah P. Gibbs 1, and George S. Espie 2. ,
1 Department of Biology, McGill University, 1205 ave. Docteur Penfield, Montr6al, Qu6bec, H3A 1B1, Canada 2 Department of Botany, Erindale College, University of Toronto, Mississauga, Ontario, L5L 1C6, Canada Received September 5, 1991/Accepted May 21, 1992 Abstract. In the cyanobacterium S y n e c h o c o c c u s UTEX 625, the extent of expression of carboxysomes appeared dependent on the level of inorganic carbon (CO2 + H C O f ) in the growth medium. In cells grown under 5% CO2 and in those bubbled with air, carboxysomes were present in low numbers (< 2 9longitudinal section- 1) and were distributed in an apparently random manner throughout the centroplasm. In contrast, cells grown in standing culture and those bubbled with 30 gl CO2 91 - 1 possessed many carboxysomes (>8.longitudinal section-I). Moreover, carboxysomes in these cells were usually positioned near the cell periphery, aligned along the interface between the centroplasm and the photosynthetic thylakoids. This arrangement of carboxysomes coincided with the full induction of the HCO;- transport system that is involved in concentrating inorganic carbon within the cells for subsequent use in photosynthesis. Immunolocalization studies indicate that the Calvin cycle enzyme ribulose bisphosphate carboxylase was predominantly carboxysome-localized, regardless of the inorganic carbon concentration of the growth medium, while phosphoribulokinase was confined to the thylakoid region. It is postulated that the peripheral arrangement of carboxysomes may provide for more efficient photosynthetic utilization of the internal inorganic carbon pool in cells from cultures where carbon resources are limiting. Key words: Cyanobacteria - S y n e c h o c o c c u s - Carboxysomes - Ribulose bisphosphate carboxylase - Immunogold localization - C O 2 / H C O 3 - transport
Depending on the conditions under which they are grown, cyanobacteria may possess an array of intracellular Institute of Marine Science, University of Alaska, Fairbanks. AK 99775-1080, USA
* Present address:
Correspondence to:
G.S. Espie
Chl, chlorophyll; DIC, dissolved inorganic carbon (CO2 + HCO~ + CO~ ); PRK, phosphoribulokinase; RuBP, ribulose 1,5-bisphosphate; Rubisco LS, large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase Abbreviations:
inclusions (Allen 1984; Shively et al. 1988). Among the various inclusions, polyhedral bodies have attracted a great deal of attention in recent years, mainly as a result of biochemical and immunocytochemical evidence indicating the presence of the Calvin cycle enzyme ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; E.C. 4.1.1.39) in these structures. Rubisco catalyzes the fixation of CO 2 to ribulose 1,5-bisphosphate (RuBP) in the initial reaction of photosynthetic carbon reduction. In recognition of this potential function of polyhedral bodies, the term "carboxysome" was coined by Shively and colleagues (1973) to designate these unique prokaryotic inclusions. Despite evidence linking Rubisco to carboxysomes, the role of these structures in cyanobacterial photosynthesis remains to be fully elucidated. Numerous investigations have shown a portion of the cell's complement of Rubisco to be soluble rather than carboxysome-associated (Cossar et al. 1985; Hawthornthwaite et al. 1985; references in Codd 1988), thereby fuelling speculation that carboxysome-localized Rubisco may function as a photosynthetic reserve. Still other investigators have suggested that carboxysomal Rubisco may be a general cellular nitrogen reserve (Duke and Allen 1990 and references within). However, other studies detailing the effect of nitrogen-deprivation on cyanobacterial fine structure do not support this function (Turpin et al. 1984; Wanner et al. 1986). The possibility that carboxysomes are active sites of CO2 fixation in vivo has been explored in a recent series of mathematical models (Reinhold et al. 1989, 1991). Cyanobacterial photosynthetic efficiency is regulated in part by a mechanism for the active transport of CO a and H C O j which acts to concentrate these species of inorganic carbon intracellularly to levels beyond those found in the external medium (Miller et al. 1990). The carboxysome models proposed by Reinhold and colleagues place the carboxysome as an integral component of this inorganic carbon concentrating mechanism, identifying it as the exclusive site where high levels of CO2 are generated by carbonic anhydrase for use by Rubisco. Evidence in support of this hypothesis has recently been provided by Friedberg et al. (1989) and Pierce et al. (1989)
22 who demonstrated that structurally intact carboxysomes were i m p o r t a n t for the efficient o p e r a t i o n o f t h e i n o r g a n i c c a r b o n c o n c e n t r a t i n g m e c h a n i s m . F u r t h e r m o r e , Price a n d B a d g e r (1989a, b) h a v e s h o w n t h a t e q u i l i b r a t i o n o f the c y t o s o l i c p o o l of H C O f with C O 2 results in a d r a m a t i c increase in the efflux of C O 2 a n d a n i n a b i l i t y o f the cells to a c c u m u l a t e D I C . Since the e q u i l i b r a t i o n o f c y t o s o l i c HCO~- was b r o u g h t a b o u t b y the i n d u c t i o n of p l a s m i d b o r n e h u m a n c a r b o n i c a n h y d r a s e , the clear i m p l i c a t i o n o f these e x p e r i m e n t s was t h a t n a t i v e c a r b o n i c a n h y d r a s e is n o r m a l l y a b s e n t f r o m the c y t o s o l b u t p r e s e n t in c a r b o x y s o m e s . As w i t h o t h e r c y a n o b a c t e r i a l i n c l u s i o n bodies, the m o r p h o l o g i c a l e x p r e s s i o n of c a r b o x y s o m e s is often dep e n d a n t u p o n the n u t r i e n t status o f the cell. S u p p o r t for this c o m e s f r o m n l t r a s t r u c t u r a l i n v e s t i g a t i o n s of c y a n o p h y t e s u n d e r g o i n g v a r i o u s forms o f n u t r i e n t - l i m i t a t i o n . I n v e s t i g a t o r s h a v e c o m m e n t e d o n the e x p r e s s i o n of c a r b o x y s o m e s in cells of cultures l i m i t e d in n i t r o g e n ( T u r p i n et al. 1984; W a n n e r et al. 1986), p h o s p h o r u s ( T u r p i n et al. 1984), s u l p h u r ( W a n n e r et al. 1986), i r o n ( S h e r m a n a n d S h e r m a n 1983), a n d c a r b o n (Miller a n d H o l t 1977; P r i c e a n d B a d g e r 1989b; T u r p i n et al. 1984). U n d e r s t a n d a b l y , studies of the l a t t e r n a t u r e m a y be e x t r e m e l y v a l u a b l e in e l u c i d a t i n g a p o t e n t i a l role for c a r b o x y s o m e s in c y a n o b a c t e r i a l p h o t o s y n t h e s i s . I n this r e p o r t , we p r e s e n t a d e t a i l e d a n a l y s i s o n the effect o f v a r y i n g levels o f d i s s o l v e d i n o r g a n i c c a r b o n ( D I C ) in the g r o w t h m e d i u m o n the e x p r e s s i o n of c a r b o x y s o m e s , the s u b c e l l u l a r l o c a l i z a t i o n of R u b i s c o a n d the m o d e o f D I C t r a n s p o r t in the u n i c e l l u l a r c y a n o b a c t e r i u m Synechococcus U T E X 625 (PCC6301).
Materials and methods
Organism and growth conditions The unicellular cyanobacterium Synechococcus UTEX 625, also known as Synechococcus PCC6301 (Pasteur Culture Collection), was obtained from the Culture Collection of Algae at the University of Texas at Austin, USA. Cells were grown in batch culture in a modified Allen's medium (Espie and Canvin 1987) and, when desired, buffered to the appropriate pH with 25 mM BTP. The pH of unbuffered cultures quickly rose to about 10 and then remained constant. Growth was at 29 ~ and continuous light was provided at a photon flux rate of 50 ~tmol (photons), m - 2 , S - 1 for cells grown with air-bubbling through the culture. For cells growing in standing culture (no air-bubbling) light was provided at 25 gmol (photons) 9m - z. s- 1 All cultures were inoculated to an initial Chl a concentration of 0.2 I-tg9ml- 1 with cells grown in standing culture. In the case of cultures bubbled with 5% CO2 in air, cells were grown to late tog phase and then used to start a second 5% CO2-grown culture which was used for analysis. Inorganic carbon was supplied by bubbling the cultures with air containing various concentrations of COz (Table 1). The DIC concentration in the growth medium at harvest was determined by gas chromatography (Birmingham and Colman 1979). Chlorophyll a was determined spectrophotometrically at 665 nm following extraction in methanol (Espie and Canvin 1987).
Immunoelectron microscopy Cells were harvested by centrifugation and washed in ice-cold 100 mM sodium phosphate, pH 7.2. Cell pellets were fixed at 4 ~
for 2 h in a solution containing I% glutaraldehyde in sodium phosphate buffer. The pellets were washed with cold buffer and the ceils dehydrated through a graded ethanol series and embedded in L.R. White medium grade resin (J.B. EM Services, Montrbal, P.Q., Canada) as described previously (Lichtl6 et al. 1992). For immunolabelling, pale gold-coloured sections were cut with a diamond knife and mounted on formvar-coated nickel grids. The grids were floated section-side down on drops of the following solutions: 1% bovine serum albumin (albumin; fraction V) in PBS, 30 min; antiserum, 30 rain; 1% albumin in PBS, 4 x 3 rain; protein A-gold (15 nm; Intermedico, Markham, Ont., Canada) diluted 1 : 25 in 1% albumin in PBS, 25 rain; PBS, 4 x 3 rain; de-ionized water rinse. Rabbit antiserum raised against Rubisco LS isolated from tobacco was kindly provided by Dr. J. Fleck (IBMC-CNRS, Strasbourg, France). Immunoblot analysis indicated that the antiserum was monospecific and recognized a 54 kilodalton polypeptide from a crude protein extract of Synechococcuswhich corresponded to the Rubisco LS (data not shown). Antiserum directed against phosphoribulokinase (E.C. 2.7.1.19) purified from the cyanobacterium Chlorogloeopsisfritschii was kindly provided by Dr. G.A. Codd (Univ. of Dundee, Dundee, U.K.). Its specificity has been demonstrated previously (Marsden et al. 1984). Both antisera were diluted 1 : 500 in 1% albumin in PBS. Immunolabelled sections were post-stained with 2% aqueous uranyl acetate prior to viewing in a Philips (Eindhoven, The Netherlands) EM 410 electron microscope at an operating voltage of 80 kV. In control experiments, the antibody was replaced with rabbit non-immune immunoglobulin G, or with PBS alone, prior to protein A-gold incubation.
Quantitative evaluation Cell volume was determined from measurements of individual cells on micrographs. Only longitudinal sections, through cells whose shape approximated that of a cylinder, were used for analysis. The relative volume of carboxysomes was determined from measurements of surface areas of both carboxysomes and whole cells. That area measurements can be used to determine relative volumes has been shown previously (Gibbs 1968). The density of labelling over various cell compartments was obtained by determining the number of gold particles per square micrometer of compartment sectioned. All area determinations were made using a Zeiss (New York, U,S.A.) MOP-3 digital analyzer.
Transport of inorganic carbon The active transport and intracellular accumulation of COz and/or HCO;- by Synechococeus results in a characteristic quenching of Chl a fluorescence which was used to indirectly indicate the occurrence of these transport events in subsamples of cells used in the immunolabelling studies (Espie et al. 1991; Miller et al. 1991). Changes in Chl a fluorescence yield were measured with a pulse amplitude modulation fluorometer (PAM 101, H. Walz, Effeltrich, FRG). White light to drive photosynthesis and transport was provided at a photon flux of 100 gmol (photons). m -z .s 1 by a quartz-halogen projector lamp. Simultaneously, a weak pulse modulated red light beam (4 gmol (photons). m -2 - s-1, ~,~,~x= 650 nm at 100 kHz) was provided by a light-emitting diode. Fluorescence was detected with a photodiode shielded by a long-pass filter, but only the pulsed fluorescence signal was amplified. The pulsed signal was subsequently processed to yield a continuous signal which was recorded on a strip chart recorder. Changes in fluorescence yield are expressed as a percentage of the variable fluorescence, F*, where F* = F* - Ft. Near-maximum fluorescence yield, F*, was achieved after a few minutes of exposure to white light in the complete absence of DIC, while Fo was determined with dark-adapted (5 rain) cells as the fluorescence arising from cells illuminated with the pulse modulated light beam alone, For fluorescence measurements, cells (1.5 ml) were washed 3 times by centrifugation, resuspended in DIC and Na+-free BTP
23
,
+l +1 +1 +1
+1 +1 +1
+1 +1 +1 +1
+1 +1 +l
buffer (pH 8) and placed in the clear plexiglass chamber of an oxygen electrode (Hansatech, UK). A fiberoptics bundle, which optically connected the cell suspension to the pulsed red light source, and the fluorescence detector were placed at the surface of the chamber so that fluorescence yield and photosynthetic 02 evolution (not shown) could be measured simultaneously. Fluorescence yield of cells illuminated with both white light and the pulse modulated beam was allowed to rise to F* and the transport assay was initiated by adding either 10 gM HCO~ or 10 gM CO2 to the suspension in the absence or presence of 25 mM NaC1.
r
r~
~
6
.,.a
0
+,-
+1
oO "~" '~" 0-,
+,
,.
+'
Results
+1 +1
tt~ e~ oo
Subcellular location of Rubisco
& ~
=
~
~ c,.)=
4
