Two mutants of Rhodobacter sphaeroides defective in formation of light-harvesting spectral complexes were examined in detail. Mutant RS103 lacked the B875 spectral complex despite the fact that substantial levels of the B875-oa polypeptide (and presumably the ,( polypeptide) were present. The B800-850 spectral complex was derepressed in RS103, even at high light intensities, and the growth rate was near normal at high light intensity but decreased relative to the wild type as the light intensity used for growth decreased. Mutant RS104 lacked colored carotenoids and the B800-850 spectral complex, as well as the cognate apoproteins. This strain grew normally at high light intensity and, as with RS103, the growth rate decreased as the light intensity used for growth decreased. At very low light intensities, however, RS104 would grow, whereas RS103 would not. Structural analysis of these mutants as well as others revealed that the morphology of the intracytoplasmic membrane invaginations is associated with the presence or absence of the B800-850 complex as well as of carotenoids. A low-molecular-weight intracytoplasmic membrane polypeptide, which may play a role in B800-850 complex formation, is described, as is a 62,000-dalton polypeptide whose abundance is directly related to light intensity as well as the absence of either of the light-harvesting spectral complexes. These data, obtained from studies of mutant strains and the wild type, are discussed in light of photosynthetic membrane formation and the abundance of spectral complexes per unit area of membrane. Finally, a method for the bulk preparation of the B875 complex from wild-type strain 2.4.1 is reported.
A reaction center H-strain (RCH-) of Rhodobacter sphaeroides, PUHAl, was made by in vitro deletion of an Xhol restriction endonuclease fragment from the puhA gene coupled with insertion of a kanamycin resistance gene cartridge. The resulting construct was delivered to R. sphaeroides wild-type 2.4.1, with the defective puhA gene replacing the wild-type copy by recombination, followed by selection for kanamycin resistance. When grown under conditions known to induce intracytoplasmic membrane development, PUHAl synthesized a pigmented intracytoplasmic membrane. Spectral analysis of this membrane showed that it was deficient in B875 spectral complexes as well as functional reaction centers and that the level of B800-850 spectral complexes was greater than in the wild type. The RCH-strain was photosynthetically incompetent, but photosynthetic growth was restored by complementation with a 1. 45-kilobase (kb) Photoheterotrophic growth in Rhodobacter sphaeroides is dependent upon photosynthetic pigment-protein complexes which capture light energy and initiate its conversion into chemical potential energy (7,25).The photopigments are organized within the intracytoplasmic membrane (ICM) into photosynthetic units consisting of two different light-harvesting (LH) complexes and a reaction center (RC) complex (20). The LH complexes are classified by their spectral absorption maxima, 800 to 850 nm and 875 nm, respectively (7). B800-850 (LHII) complexes harvest incident light energy and transfer it as excitation energy to B875 (LHI) complexes which are arranged peripherally to the RC. In the RC, an electron located in the special pair of bacteriochlorophyll (Bchl) molecules is excited to a higher energy state. Energy from this excited state is passed via a series of membrane-bound or -associated carrier molecules along the photosynthetic electron transport chain, generating reducing equivalents and ATP (16,17,25).There are two different pairs of polypeptides associated with the photopigments in the LHI and LHII complexes, known as B875a and B875P and B85Ooa and B8500, respectively (4, 31). The RC consists of three polypeptides; these are the RCL, RCM, and RCH polypeptides, so called for their apparent molecular weights on sodium dodecyl sulfatepolyacrylamide gels (light, medium, and heavy) (26). In a photosynthetic ICM, the three RC polypeptides are in the ratio 1:1:1, but in total membranes of photosynthetic cells RCH is in a 33% molar excess over RCL and RCM (20). Immunological studies in this laboratory have shown that polypeptide RCH is present in the cytoplasmic membrane of * Corresponding author. aerobically growing R. sphaeroides cells, whereas RCL and RCM are not (5,14).The role of polypeptide RCH in the RC as assayed by flash spectroscopy has not been clearly established; there are conflicting reports in the literature from in vitro reconstitution of RCH with RCL and RCM Bchl-containing complexes (1,3,10,11). Previously published data from this laboratory (5) suggest that RCH acts as a focus for RC assembly and to co...
Cells of Rhodopseudomonas sphaeroides grown in a 25% 02 atmosphere were rapidly subjected to total anaerobiosis in the presence of light to study the progression of events associated with the de novo synthesis of the inducible intracytoplasmic membrane (ICM). This abrupt change in physiological conditions resulted in the immediate cessation of cell growth and whole cell protein, DNA, and phospholipid accumulation. Detectable cell growth and whole cell protein accumulation resumed ca. 12 h later. Bulk phospholipid accumulation paralleled sell growth, but the synthesis of individual phospholipid species during the adaptation period suggestedthe existence of a specific regulatory site in phospholipid synthesis at the level of the phosphatidylethanolamine methyltransferase system. Freeze-fracture electron microscopy showed that aerobic cells contain small indentations within the cell membrane that appear to be converted into discrete ICM invaginations within 1 h after the imposition of anaerobiosis. Microscopic examination also revealed a series of morphological changes in ICM structure and organization during the lag period before the initiation of photosynthetic growth. Bacteriochlorophyll synthesis and the formation of the two light-harvesting bacteriochlorophyll-protein complexes of R. sphaeroides (B800-850 and B875) occurred coordinately within 2 h after the shift to anaerobic conditions. Using antibodies prepared' against various ICM-specific polypeptides, the Synthesis of reaction center proteins and the polypeptides associated with the B800-850 complex was monitored. The reaction center H polypeptide was immunochemically detected at low levels in the cell membrane of aerobic cells, which contained no detectable ICM or bacteriochlorophyll. The results are discussed in terms of the oxygendependent regulation of gene expression in R. sphaeroides and the possible role of the r,eaction center H polypeptide and the cell membrane indentations in the site-specific assembly of ICM pigment-protein complexes during the de novo synthesis of the ICM.
The cytoplasmic membrane and the photosynthetic intracytoplasmic membranes of Rhodopseudomonas palustris are spatially differentiated into regions of extremely high intramembrane-particle density (4,400 to 9,800/[Lm2) and areas of lower intramembrane-particle density (2,700 to 5,900/Ium2). The high intramembrane-particle-density areas were always seen in association with photosynthetic membrane stacks. This differentiation was also seen in those areas of the cytoplasmic membrane which adhere to the underlying intracytoplasmic membranes, implying that the cytoplasmic membrane too is differentiated for photosynthesis in these regions. Changes in intramembrane-particle size distribution in response to changes in light intensity during growth were measured. We found that, as light levels were decreased from 8,500 to 100 lx, the average particle diameter in the protoplasmic face of stacked intracytoplasmic and cytoplasmic membranes increased from 8.6 to 10.3 nm. We also observed a distinct periodicity in the sizes of the intramembrane particles found in the stacked regions-7.5, 10.0, 12.5, and 15.0 nm-with the larger-size peaks becoming more pronounced as light intensity decreased. This suggests that, as light levels decrease, subunits of discrete size are being added to a core particle. A comparison of propane jetfrozen cells versus fixed, glycerinated, and then frozen cells indicated that ultrarapid freezing leads to a higher quality of fine-structure preservation than does chemical fixation followed by glycerination and conventional freezing in Freon-12 or propane. The intramembrane particles appeared to be more regular in size, lacking the deformed or jagged appearance displayed in fixed preparations.
We demonstrated the utility of Escherichia coli alkaline phosphatase, encoded by phoA, as a reporter molecule for genetic fusions in Rhodobacter sphaeroides. A portion of the R. sphaeroides cycA gene was fused to phoA, yielding a fusion protein comprising the putative signal sequence and first 10 amino acids of the cytochrome c2 apoprotein joined to the sixth amino acid of alkaline phosphatase. The fusion protein was efficiently transported to the periplasm of R. sphaeroides as determined by enzyme activity, Western immunoblot analysis, and immunogold electron microscopy. We also documented the ability of an R. sphaeroides mutant, RS104, with gross defects in photosynthetic membrane morphology to efficiently recognize and translocate the fusion protein to the periplasmic compartment. The inclusion of 500 base pairs of R. sphaeroides DNA in cis to the cycA structural gene resulted in a 2.5-fold increase in alkaline phosphatase activity in photosynthetically grown cells compared with the activity in aerobically grown cells, demonstrating that the fusion protein is regulated in a manner similar to that of cytochrome c2 regulation. We also constructed two pUC19-based plasmids suitable for the construction of translational fusions to phoA. In these plasmids, translational fusions of phoA to the gene under consideration can be made in all three reading frames, thus facilitating construction and expression of fusion protein systems utilizing phoA.Rhodobacter sphaeroides is an ideal procaryote for the study of protein targeting. Depending upon the growth conditions, it can have either two or three distinct membrane systems: the outer membrane, cytoplasmic membrane, and, under photosynthetic growth conditions, intracytoplasmic membrane (ICM) vesicles (8,20,33). Each of these membranes possesses a specific subset of proteins which must be properly targeted and inserted for normal cell growth (see references 12 and 22 for reviews). As (2,15,27).Recently the utility of Escherichia coli alkaline phosphatase, encoded by phoA, as a reporter for gene fusions was demonstrated (3,18,27,28 investigated in E. coli, both translocated proteins (3,18,27) and membrane proteins with regions exposed to the periplasmic side of the cytoplasmic membrane (28).In order to test the application of this particular experimental approach to phoA gene fusions in R. sphaeroides, cytochrome c2 (cyt c2), a known, periplasmically localized protein, was chosen for study. cyt c2 is expressed in both aerobically and photosynthetically grown R. sphaeroides cells and is localized to the periplasm (or ICM lumen) under both growth conditions (35). The DNA sequence of the R. sphaeroides cycA gene encoding cyt c2 is known (13), and the gene has been cloned and studied in detail (4,13,14). From the DNA sequence data, a 21-amino-acid N-terminal signal sequence has been proposed (13). Although processing of the signal sequence has not been directly demonstrated in vivo, in vitro transcription-translation studies have revealed a 15.5-kilodalton [kDa] polypeptide...
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